UNIVERSITATEA DE ŞTIINŢE AGRICOLE ŞI MEDICINĂ VETERINARĂ A BANATULUI TIMIŞOARA FACULTATEA DE MEDICINĂ VETERINARĂ LUCRĂRI ŞTIINŢIFICE

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1 UNIVERSITATEA DE ŞTIINŢE AGRICOLE ŞI MEDICINĂ VETERINARĂ A BANATULUI TIMIŞOARA FACULTATEA DE MEDICINĂ VETERINARĂ LUCRĂRI ŞTIINŢIFICE MEDICINĂ VETERINARĂ TIMIŞOARA VOLUMUL L (3) SCIENTIFICAL PAPERS VETERINARY MEDICINE

2 EDITORIAL BOARD Prof. VIOREL HERMAN, PhD, DVM - Faculty of Veterinary Medicine BUASVM Timisoara Prof. ILEANA NICHITA, PhD, DVM Faculty of Veterinary Medicine BUASVM Timisoara Prof. MARIUS PENTEA, PhD, DVM - Faculty of Veterinary Medicine BUASVM Timisoara Lecturer DORU MORAR, PhD, DVM - Faculty of Veterinary Medicine BUASVM Timisoara Prof. ION OPRESCU, PhD, DVM Faculty of Veterinary Medicine BUASVM Timisoara Prof. EMIL TIRZIU, PhD, DVM - Faculty of Veterinary Medicine BUASVM Timisoara EDITOR-IN-CHIEF: Assoc. Prof. NARCISA MEDERLE, PhD, DVM - Faculty of Veterinary Medicine BUASVM Timisoara Editorial assistants: Prof. SORIN MORARIU, PhD, DVM - Faculty of Veterinary Medicine BUASVM Timisoara Assoc. Prof. VIOLETA IGNA, PhD, DVM - Faculty of Veterinary Medicine BUASVM Timisoara Lecturer CORINA PASCU, PhD, DVM - Faculty of Veterinary Medicine BUASVM Timisoara Lecturer MIRELA AHMADI, PhD, DVM - Faculty of Veterinary Medicine BUASVM Timisoara SCIENTIFIC ADVISORY COMMITTEE Prof. DUSAN ORLIC, PhD, DVM - Scientific Veterinary Institute Novi Sad, Serbia Prof. JOVAN BOJKOVSKI, PhD, DVM - Faculty of Veterinary Medicine, Belgrade, Serbia Prof. IOVAN PAVLOVIĆ, PhD, DVM - Scientific Veterinary Institute, Belgrade, Serbia Prof. MANFRED GAREIS, PhD, DVM - Ludwig-Maximilians-Universität München, Germany Prof. associate HANS WERNER KUTSCH, PhD, DVM Institute of Meet Science, Nuremberg, Germany Prof. NICOLAE MANOLESCU, PhD, DVM, Dr. HC Oncologic Institute Prof. dr. Al. Trestioreanu Bucharest, Corresponding member of Romanian Academy, Titular member of Romanian Academy of Medical Science, Honorific member of Romanian Academy of Agricultural and Forestry Science Prof. MIHAI DECUN, PhD, DVM Faculty of Veterinary Medicine BUASVM Timisoara, Titular member of Romanian Academy of Agricultural and Forestry Science Prof. HORIA CERNESCU, PhD, DVM, Dr. HC - Faculty of Veterinary Medicine BUASVM Timisoara, Titular member of Romanian Academy of Agricultural and Forestry Science, Member of BASeVA Prof. GHEORGHE DARABUS, PhD, DVM Faculty of Veterinary Medicine BUASVM Timisoara, Titular member of Romanian Academy of Agricultural and Forestry Science Prof. IOAN GROZA, PhD, DVM - Faculty of Veterinary Medicine UASVM Cluj Napoca Prof. CORNEL CATOI, PhD, DVM - Faculty of Veterinary Medicine UASVM Cluj Napoca Prof. VASILE COZMA, PhD, DVM - Faculty of Veterinary Medicine UASVM Cluj Napoca Prof. GABRIEL PREDOI, PhD, DVM - Faculty of Veterinary Medicine UASVM Bucuresti Prof. LIVIU MIRON, PhD, DVM - Faculty of Veterinary Medicine UASVM Iasi Prof. SĂVUTĂ GHEORGHE, PhD, DVM - Faculty of Veterinary Medicine UASVM Iasi Prof. BOGDAN LIVIU, PhD, DVM - Faculty of Veterinary Medicine UASVM Bucuresti Prof. ALEXANDRA TRIF, PhD, DVM, Dr. HC. - Faculty of Veterinary Medicine BUASVM Timisoara Prof. NICOLAE CĂTANĂ, PhD, DVM - Faculty of Veterinary Medicine BUASVM Timisoara Prof. ROMEO CRISTINA, PhD, DVM - Faculty of Veterinary Medicine BUASVM Timisoara Prof. CORNEL IGNA, PhD, DVM - Faculty of Veterinary Medicine BUASVM Timisoara To be cited: LUCRARI STIINTIFICE: MEDICINA VETERINARA TIMISOARA (SCIENTIFICAL PAPERS: VETERINARY MEDICINE TIMISOARA), vol. L (3), 2017 Available online at: Indexed and/or abstracted in: CABI Full Text, CAB Abstracts, Ulrich's Periodicals Directory Editor: AGROPRINT TIMISOARA ISSN: Printed by: IMPRIMERIA MIRTON TIMISOARA

3 UNIVERSITATEA DE ŞTIINŢE AGRICOLE ŞI MEDICINĂ VETERINARĂ A BANATULUI TIMIŞOARA FACULTATEA DE MEDICINĂ VETERINARĂ LUCRĂRI ŞTIINŢIFICE MEDICINĂ VETERINARĂ TIMIŞOARA VOLUMUL L (3) SCIENTIFICAL PAPERS VETERINARY MEDICINE TIMIŞOARA 2017

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5 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA IDENTIFICATION OF METHICILLIN RESISTANT STAPHYLOCOCCUS STRAINS BY THE DETECTION OF MEC STAPHYLOCOCCAL CHROMOSOME CASSETTE N. CĂTANĂ 1, M. NECȘULESCU 2, VIRGILIA POPA 3, V. HERMAN 1, IULIA BUCUR 1 1 Banat s University of Agricultural Sciences and Veterinary Medicine King Michael I of Romania from Timisoara, Faculty of Veterinary Medicine, Calea Aradului, 119, Timisoara , Romania 2 S.N. Pasteur Institute S.A. 3 Military Medical Research Center epirovet_tm@yahoo.com Summary Methicillin resistance was first reported in S. aureus strains, but lately methicillin-resistant strains, included in other staphylococci species, have been isolated from animals and humans. Methicillin resistance is encoded by large genetic elements known as "mec staphylococcal chromosome cassette " (SCCmec). The research was made on a total of 50 strains resistant to methicillin and oxacillin and susceptible to cefoxitin and included in 6 staphylococcal species. The resistance to these three betalactams was studied using the Kirby-Bauer disk diffusion method. The Seeplex rapid MRSA Ace detection Seegene kit was used for the detection of this cassette, using multiplex PCR technique. Staphylococcal chromosome cassette mec was detected in a variable number of strains included in 6 species as follows: S. aureus subsp. aureus: 18 positive strains and 2 negative strains; S. epidermidis: one positive strain and 4 negative strains; S. hycus: 7 positive strains; S. xylosus: 4 positive strains and one negative strain; S. intermedius/pseudintermedius: 4 positive strains and one negative strain; S. sciuri: 5 positive strains and 3 negative strains. The results show the presence of mec staphylococcal chromosome cassette in a number of 39 strains (positive), and its absence from a total of 11 strains (negative). The presence of this genetic element in coagulase negative and coagulase positive staphylococci strains demonstrates the expansion of methicillin resistance, a phenotypic character with zoonotic risk. Key words: mec gene, MRSA strains S. aureus subsp. aureus is an infectious pathogenic agent for both humans and animals, which may cause localized infection or septicemia. To this bacterial species the phenomenon of multiple antibiotic resistance is very common, especially to beta-lactams, due to some enzymes called beta-lactamases, whose synthesis is encoded by extrachromosomal genetic elements (2, 3). During the last, years, strains of S. aureus subsp. aureus resistant to methicillin were isolated, generically referred to as MRSA strains (methicillin resistant S. aureus). Genetic basis of resistance to methicillin are represented by mec staphylococcal chromosome cassette, which are mobile genetic elements made of: mec complex gene (meca, mecr1 and meco), a complex which encodes 5

6 6 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA the recombinases enzymes (crra and crrb), the transposon Tn544, a copy of pub110 plasmid and the insertion element IS431. These mec complexes can be either type A (1), type B (2), type C (3) or type D (4) (2, 3). In the last years, methicillin resistance has also been reported in coagulase positive and negative staphylococci of other species, demonstrating thus the expansion of this phenomenon, due to the acquisition of the mentioned genetic elements. The research was made in order to detect the mec chromosome cassette in coagulase positive and negative staphylococci strains, included in six species. Materials and methods The research was made on a total of 50 strains resistant to methicillin and oxacillin and susceptible to cefoxitin, classified in six species of staphylococci and definitive typified based on the glucidolytic activity. The resistance to these three beta-lactams was studied using the Kirby-Bauer disk diffusion method with methicillin (5 µg), oxacillin (1 µg) and cefoxitin (30 µg) biodiscs. In this research only meca1 and meca2 genes were detected. The bacterial DNA was obtained from isolated colonies, from each strain, using the rapid Seeplex MRSA ACE Detection Kit Seegene. One colony was taken, from each strain and resuspended in the DNA extraction solution, present in this kit (2, 3). The detection of meca1 and meca2 genes was performed using the primers: - meca-1: 5'-GGGATCATAGCGTCATTATTC-3': 50 nmol; - meca-2: 5'-AACGATTGTGACACGATAGCC-3': 50 nmol (2, 3). For the amplification of bacterial DNA, the DreamTaq Green PCR Master Mix (2X) kit was used. The amplification conditions were 95 C-3 minutes; 95ºC-30 seconds, 55ºC-30 seconds, 72ºC-1 minute (35 cycles) and the final extension of the reaction lasted 1 minute at 72 C (2, 3). The detection of amplicons specific for the two genes, was performed by horizontal electrophoresis in 2% agarose gel, followed by immersing the gel in TAE 1X electrophoresis buffer. This buffer contains ethidium bromide as a fluorescent agent, which becomes visible in UV light, allowing the visualization of the formed amplicons. The presence of amplicons with the size of 448 bp bands indicated the presence of the meca1 and meca2 genes (2, 3). For correct results, 100 pb weight markers were used, namely a methicillin resistant S. aureus subsp. aureus strain as the positive control and ultrapure sterile water as negative control (2, 3). Results and discussions The results of the research on the detection of meca1 and meca2 genes are shown in Table 1. The analysis of these results shows that meca1 and meca2

7 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA genes were present in 39 strains, representing 78% of the tested strains. Most of the strains, on which were detected these two genes, were included in S. aureus subsp. aureus species. Furthermore, it is noted that these genes were also present in the most of the tested strains, included in other staphylococci species. In all strains of S. hycus, the two genes were detected governing methicillin resistance, which confirms that the pigs, through the staphylococcal species that carry them, are sources of infection with professional risk on the phenomenon of methiciliin resistance. The results confirm that even coagulase negative staphylococci included in S. xylosus and S. sciuri species, may be carriers of such genes, thus demonstrating the existence of methicillin resistance phenomenon in nonpathogenic staphylococci. Presence of meca1 and meca2 genes in strains of S. intermedius/s. pseudintermedius species shows that the phenomenon of methicillin resistance is also present in opportunists staphylococci adapted to dogs and cats, from which, through direct contact, can reach out to humans. The results of the duplex PCR Crt. Result Species no S. aureus subsp. aureus 18/20 2/20 2. S. epidermidis 1/5 4/5 3. S. hycus 7/7 0/7 4. S. xylosus 4/5 1/5 5. S. intermedius/pseudintermedius 4/5 1/5 6. S. sciuri 5/8 3/8 TOTAL 39/50 11/50 Table 1 The duplex PCR detected these two genes encoding the phenomenon of methicillin resistance both in pathogenic and non-pathogenic staphylococci, demonstrating that these genes can be transmitted intra and interspecific between the staphylococci strains and the existence of the phenomenon of methicillin resistance points out the zoonotic risk of the carrying strains. After visualizing the 2% agarose gel, bands with length of 981 bp for the internal control and 448 bp for the detected genes were observed. Figure 1 illustrates some of the products, namely for the internal control, positive control, negative control and 9 strains of staphylococci. Also, in this figure a negative strain of the detected genes is present. 7

8 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA 8 Fig. 1. The presence of amplicons in some strains of staphylococci Line1-100pb marker weight, Line 2 S. aureus MRSA positive control, Line 3 S. hycus MRSH, Line 4 - S. hycus MRSH, Line 5 - S. hycus MRSH, Line 6 - S. aureus MRSA, Line 7 - S. aureus MRSA, Line 8 - S. aureus MRSA, Line 9 - nonspecific amplification, Line 10 - S. aureus MRSA, Line 11 - S. aureus MRSA Through this technique of molecular biology, 11 strains did not detect the meca1 and meca2 genes, strains that, were resistant to methicillin and oxacillin at the disk diffusion method, thus demonstrating the existence of some differences between molecular biology techniques and phenotypic method. These results prove that the duplex PCR technique, used to detect meca1 and meca2 genes, has a higher degree of specificity and sensitivity compared to the phenotypic disk diffusion technique and allows the elimination of false negative or false positive results. It is a rapid method that can be taken as the current laboratory technique for the study of staphylococci strains searched for the phenomenon of methicillin resistance. Also, this technique eliminates the false results for staphylococci strains which has other resistance mechanism to methicillin than the mechanism encoded by the mec cassette gene. In recent years, the prevalence of methicillin resistant staphylococci strains isolated from animals and food increased, which is why there are screening studies on these strains both in Romania and in other countries. In Romania, Bicheru Simona et al. (2013, 2014) adapted a variant of the PCR duplex technique to detect the meca1, meca2 and nuc genes in strains of S. aureus isolated from humans. For this purpose, 54 strains included in S. aureus species were tested and the frequency of mec gene was 83.33% (2, 3). In order to study the methicillin resistant strains of staphylococci isolated from animals, Vanghelie Monica et al. (2012) optimized a variant of PCR multiplex for the detection of the meca, nuc gene and 16S rrna. Authors used the reference strains: S. aureus resistant to methicillin, S. aureus susceptible to methicillin and S. epidermidis (negative control). Based on the results, the authors recommend this

9 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA technique as a method of rapid detection, specific and sensitive of coagulase positive and coagulase negative staphylococci strains isolated from animals (7). The phenomenon of methicillin resistance is studied and monitored due to the zoonotic risk. In literature there is many data on the existence of this phenomenon in S. aureus subsp. aureus and other staphylococcal species, known as "non-s. aureus staphylococci ". Feng Y. et al. (2012) in China, focused on the prevalence of methicillin resistant strains of S. pseudintermedius isolated from pets in Guangdong province. During two years, the authors collected 898 samples of pathologic material from 785 dogs and cats from this province. The authors isolated 144 strains of S. pseudintermedius, at which they observed the presence of SCCmec (mec staphylococcal chromosome cassette). Authors detected this cassette at a total of 69 strains, namely 47.9% and also established the existing types of the mec gene (4). In 2013, Gindonis V. et al., in Finland, aimed the presence of mec genes in 135 strains of S. aureus and in 324 coagulase negative staphylococci isolated from cows with mastitis. The presence of mec gene was detected in 1.5% strains of S. aureus and in 5.2% coagulase negative staphylococci. A total of 18 strains included in S. epidermidis species showed the SCCmec type IV (13/18) and SCCmec type V (1/18). Based on these results, the authors consider that the phenomenon of methicillin resistance was rare in S. aureus, but more frequent in coagulase negative staphylococci strains (5). In Canada, in 2013, Ameet Singh et al. followed the frequency of methicillin resistance in strains of S. aureus and S. pseudintermedius isolated from animals and the personnel from a veterinarian hospital. The authors studied 114 staphylococci strains included in these two species, of which 20 strains (17.5%) were methicillin resistant. Among them, 4 strains (3.5%) were included in S. aureus species and 16 strains (14%) were included in S. pseudintermedius species. Based on these results, the authors found that methicillin resistant staphylococci strains are a risk factor in veterinary hospitals (1). Pletinckx L. J. et al., in 2013, in Belgium, conducted a study on methicillin resistance in S. aureus strains isolated from pigs from two farms. The presence of SCCmec were detected by multilocus sequence typing technique (MLST) and MRSA ST398 strain was used as a positive control. The authors found that the prevalence of MRSA strains was 86.3%, thus demonstrating that the staphylococci isolated from swine are a reservoir of mec gene for humans (6). These results show the presence of the methicillin resistant phenomenon in staphylococci strains included in several species, from which the mechanisms for transfer and the genetic elements, governing this phenomenon, can be transmitted to humans. 9

10 10 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA Conclusions PCR duplex technique detected the presence of meca1 and meca2 genes to 39 staphylococcal strains, included in six species. This molecular biology technique used as a test for detecting the methicillin resistant staphylococci strains is rapid, sensitive and specific, and eliminates the false positive and false negative results provided by phenotypic tests. MecA1 and meca2 genes were detected in both strains of S. aureus subsp. aureus and in low pathogenic or non-pathogenic staphylococci. Acknowledgements This study was realised using the support and infrastructure project Dezvoltarea infrastructurii de cercetare, educaţie şi servicii în domeniile medicinei veterinare şi tehnologiilor inovative pentru RO 05, cod SMIS-CSNR References 1. Ameet, S., Walker, M., Rousseau, J., Monteith, G., J., Weese, J., S., Methicillin-resistant staphylococcal contamination of clothing worn by personnel in a veterinary teaching hospital, Veterinary Surgery, 2013, 42(6), Bicheru, Simona, Ionescu, Lucia, Necșulescu, M., Popescu, Diana, Dumitrescu, Gabriela, Ordeanu, V., Identification of Staphylococcus aureus strains resistant to methicillin through molecular biology techniques, Rev. Rom. Med. Vet., 2013, 23(1), Bicheru, Simona, Necșulescu, M., Cumpănășoiu, Bianca, Tîrziu, E., Popescu, Diana, Ionescu, Lucia, Dumitrescu, Gabriela, Necșulescu, A., PCR for the identification of methicillin resistant Staphylococcus aureus (MRSA) strains using primers specific for SCCmec elements, Lucr. Șt. Med. Vet. Timișoara, 2014, 47(2), Feng, Y., Tian, W., Lin, D., Luo, Q., Zhou, Y., Yang, T., Deng, Y., Liu, Y. H., Liu, J., H., Prevalence and characterization of methicillin-resistant Staphylococcus pseudintermedius in pets from South China, Vet. Microbiol., 2012, 160(3-4), Gindonis, V., Taponen, S., Myllyniemi, A., L., Pyörälä, S., Nykäsenoja, S., Salmenlinna, S., Lindholm, L., Rantala, M., Occurence and characterization of methicillin-resistant staphylococci from bovine mastitis milk samples in Finland, Acta Veterinaria Scandinavica, 2013, 55 (61). 6. Pletinckx, L., J., Verhegghe, M., Crombé, F., Dewulf, J., Bleecker, Y., De, Rasschaert, G., Butaye, P., Goddeeris, B., M., Man, I., De, Evidence of possible methicillin-resistant Staphylococcus aureus ST398 spread between pigs and other animals and people residing on the same farm, Preventive Veterinary Medicine, 2013, 109(3/4),

11 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017 TIMISOARA FIRST REPORT OF RED BLOOD CELL INDICES OF THE ENDARNGERED DANUBE SALMON (HUCHO HUCHO) REARED IN A MOUNTAIN LAKE C. G. CERBU, MARINA SPÎNU*, CARMEN DANA ŞANDRU, EMOKE PALL, MIHAELA NICULAE, R.M. GIUPANĂ, DIANA IOANA OLAH, G. F. BRUDAȘCĂ University of Agricultural Sciences and Veterinary Medicine Cluj- Napoca, , 3-5 Mănăştur Street, Cluj-Napoca, Romania marinaspinu@gmail.com Summary Danube salmon is the largest representative of the Salmonidae family. The population, severely fragmented, has been listed as an endangered species by IUCN. Extinct in many parts of its habitat, data concerning the huchen is more than necessary for a better understanding of this phenomenon. The aim of the study is to describe, for the first time, the main blood parameters such as: red blood cell counts (ery mil/mm 3 ), haemogoblin concentration (Hb g/dl) and hematocrit values (Hct %) of huchen reared in a mountain lake culture. Healthy fish were anesthetized with tricaine methane-sulfonate (MS-222) and blood sampling was done from both males (n=15) and females (n=15) by puncturing the caudal vessels. In males, Hct (%) values averaged 46,6 while for females Red blood cells count (mil/mm 3 ) averaged 3,60 in males and 3,27 for females. Haemoglobin concentration (g/dl) averaged 15,11 for males and for females. The reference (physiological) parameters can be defined by an upper and lower limit that covers the majority of the values obtained for healthy fish. Keywords: endangered, Danube salmon, huchen, blood parameters The Danube salmon is the best known representative of the genus Hucho, Günther, 1866, Salmonidae family (4). Currently, the species occupies only about 25-30% of its original range in Europe, primarily because of river regulation, dam and reservoir construction, major water consumption by industry and agriculture, river pollution, and accelerated eutrophication (2, 4, 5). Extinct in many parts of its native habitat across its original range, the huchen is still present in Romania. The species was encountered in rivers like Tisa, Vișeu, Crișul Repede, Mureș, Bistrița and several lakes: Izvorul Muntelui (Bicaz), Pecineag, Vidraru and Brădișor (1). The species is endangered (EN) according to IUCN (3) and listed under Annexes II and V of the European Habitat Directive, as well as Appendix III of the Bern Convention (8). Management strategies will be most effective if they simultaneously combine knowledge of the genetic structuring of H. hucho populations and of the species biology (6). Establishing the physiological blood parameters for this 11

12 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA species represents a very important step forward in understanding and evaluating its capacity to adapt to environmental disturbances. The aim of the study is to describe, for the first time, the main blood parameters such as: red blood cell count (ery mil/mm 3 ), haemogoblin concentration (Hb g/dl) and hematocrit values (Hct %) of huchen reared in a mountain lake culture. Materials and methods The research was carried out on a number of 30 mature, healthy Danube salmons (Hucho hucho), aged between 4 and 11 years (males: n=15, females: n=15) reared in a mountain lake in South-Central Romania, in 32 m 2 special nets at a maximum depth of 4 m. The fish were anesthetized with tricaine methanesulfonate (MS-222, Western Chemical Inc., USA), and sampling was done by puncturing the caudal vessels. The blood was placed immediately in plastic EDTA tubes (BD Franklin Lakes, USA), and stored at 5 o C for 3 hours, until processing. Water temperature when sampling was done was 5 o C. Haemoglobin concentration (g/dl) was evaluated using an end-point method. For each sample, a quantity of 10 μl of blood was mixed with a 1000 μl of a 0,4% ammonia solution. The reading was made at room temperature, using a UV-vis spectrophotometer (Screen master touch, Hospitex Diagnostics, Italy) at a wavelength of 546 nm. For red blood cells count (ery mil/mm 3 ), a quantity of 2,5 μl of blood was mixed with 1250 μl of Govers reagent. The reading was done at room temperature, using a UV-vis spectrophotometer (Screen master touch, Hospitex Diagnostics, Italy) at a wavelength of 546 nm. Hematocrit values (Hct %) were obtained using the micro hematocrit tubes (Vitrex medical, Denmark), after centrifugation at rpm (Hettich, Germany). Statistical analysis was performed using Minitab Results and discussions Following one-way ANOVA analysis, hematocrit levels (Table 1) averaged 46.6 % for males and 46.4 for females, with no significant difference (p > 0.05) between the two. The maximum and minimum values obtained for both males and females were % and % respectively. 12

13 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017 TIMISOARA Table 1 Hematocrit levels (Hct %) (mean, min, max, and standard deviation) of males and females included in the study Hct(%) Males Females M+F Mean 46.6 a 46.4 a Min Max St dev * Same lower-case superscripts within rows indicate no significant difference (p > 0.05) Red blood cells values (Table 2) averaged 3.60 mil/mm 3 for males and 3.27 mil/mm 3 for females, with no significant difference (p > 0.05) between the two. The maximum and minimum values obtained for both males and females were 5.38 % and 1.49 % respectively. Table 2 Reb blood cells values (mil/mm 3 ) (mean, min, max, and standard deviation) of males and females included in the study Ery (mil/mm3) Males Females M+F Mean 3.60 a 3.27 a 3.44 Min Max SD * Same lower-case superscripts within rows indicate no significant difference (p > 0.05) Haemoglobin levels (Table 3) averaged g/dl for males and g/dl for females, with significant statistical difference (p < 0.05) between the two. The maximum and minimum values obtained for males were g/dl and g/dl respectively. For females, the maximum value was g/dl and the minimum was g/dl. 13

14 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA Table 3 Haemoglobin levels (g/dl) (mean, min, max, and standard deviation) of males and females included in the study Hb(g/dl) Males Females M+F Mean a b Min Max SD * Same lower-case superscripts within rows indicate no significant difference (p > 0.05) Conclusions The study represents an important contribution to the current knowledge of Hucho hucho. From a veterinary point of view it is important to have a database with blood parameters of a species that is currently endangered. The environmental factors must be taken into consideration when evaluating the main blood parameters. Any change of these factors would be first translated into blood parameters modification. These current results can be used a starting point for further research related to huchen in different environments and conditions. Any comparison with further studies would help us to better understand the way the huchen adapts and how the environment and rearing system may influence its physiology. Also, an important element, as stated before by Rehulka et. al (2004) (7), is that the most thoroughly constructed haematological profile of fish requires repeated measurements is some cases. References 1. Cerbu, C. G., Brudașcă, G.F., Giupană, R.M., Guranda, S., Niculae Mihaela, Paștiu, Anamaria-Ioana, Șandru, Carmen Dana, Spînu, Marina, A Theoretical Approach Of The Immune System Of Danube Huchen (Hucho hucho), Lucrări Ştiinţifice Medicină Veterinară Timişoara, 2015, 48(2), Formicki, K., Szulc, Joanna, Tañski, A., Korzelecka-Orkisz, Agata, Witkowski, A., Kwiatkowski, P., The effect of static magnetic field on Danube huchen, Hucho hucho (L.) sperm motility parameters, Arch. Pol. Fish., 2013, 21,

15 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017 TIMISOARA 3. Freyhof, J., Kottelat, M., Hucho hucho In: IUCN 2013.IUCN Red List of Threatened Species. Version \ Downloaded on September, 12 th Holĉík, J., Conservation of the huchen, Hucho hucho (L.), (Salmonidae) with special reference to Slovakian rivers, J Fish Biol., 1990, 37, Holèík, J., Hensel, K., Nieslanik, J. Skácel, L., The Eurasian huchen, Hucho hucho, largest salmon of the Word, DrW. Junk Publisher., Dordrecht, Boston, Lancaster, Ihuţ, Andrada, Zitek, A., Weiss, S., Ratschan, C., Holzer, G., Kaufmann, T., Cocan, D., Constantinescu, R., Mireşan, Vioara, Danube Salmon (Hucho hucho) in Central and South Eastern Europe: A Review for the Development of an International Program for the Rehabilitation and Conservation of Danube Salmon Populations, Bulletin UASVM Animal Science and Biotechnologies, 2014, 71, Rehulka, J., Adamec, V., Red Blood Cell Indices for Rainbow Trout (Oncorhynchus mykiss Walbaum) Reared in Cage and Raceway Culture, Acta Vet. Brno, 2004, 73, Weiss, S., Schenekar, T., Genetic evaluation of the self-sustaining status of a population of the endangered Danube salmon, Hucho hucho. Hydrobiologia, 2016, 775,

16 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA 16 EVALUATION OF BRUCELLA OVIS SEROPREVALENCE IN SICILIAN FARMS (ITALY) G. CHIARENZA, S. VILLARI, P. GALLUZZO, M. ALFANO, V. PILATO, D. VICARI, A. STANCANELLI Istituto Zooprofilattico Sperimentale della Sicilia A. Mirri - 3, via G. Marinuzzi, Palermo, Italy giuseppina.chiarenza@izssicilia.it Summary Brucella ovis infection is one of the major causes of subfertility or infertility in rams. It is clinically characterized by unilateral or bilateral epididymitis and orchitis. There is no evidence of infection for goats and cattle and no cases of transmission to humans have been reported so far. B. ovis spreads ubiquitously and it is passively transmitted among rams by venereal contact with sheep, which act as mechanical vectors hosting the bacterium in the vagina. However, there could be also direct infection among males, especially in extensive breeding, in which rams are housed together. Diagnosis of B. ovis infections is usually based on clinical examination, serology and bacterial isolation from semen samples. During 2015 B. ovis was isolated from sheep milk and from urine and epididymis samples of rams in Sicily. Therefore, it was decided to investigate the spread of this pathogen in our region. The goal of this study was to estimate the seroprevalence of B. ovis in Sicily. Samples from 608 rams farmed in five different Sicilian districts were tested for B. ovis antibodies by ELISA test. We found 68 sample (11.2 %) seropositive for antibodies to B. ovis. The highest prevalence was found in Caltanissetta district (16%), followed by Enna (9.6%) and Palermo (7.9%). This is the first screening in Sicily for this pathogen and the results here obtained will be useful to increase the attention to this disease and to take preventive measures useful to reduce the prevalence in Sicilian farms. Keywords: ovine, seroprevalence, Brucella ovis Brucella ovis is a gram-negative coccobacillus, the causative agent of unilateral or bilateral epididymitis, orchitis and infertility in rams, and just occasionally associated with abortion in sheep and stillbirth. There is no evidence of infection for goats and cattle and no cases of transmission to humans have been reported so far (1). B. ovis and B. canis are the only species of Brucella naturally in the rough phase. B. ovis spreads ubiquitously and is passively transmitted among rams by venereal contact with sheep, which act as mechanical vectors hosting the bacterium in the vagina (2). However, there could be also direct infection among males, especially in extensive breeding, in which rams are housed together. Just occasionally, even females can be infected and eliminate the pathogen with vaginal discharge and milk, thus passing on the infection even to newborn lambs. Rams generally develop persistent infection and eliminate B. ovis intermittently with the semen and urine for at least 2-4 years and 30-50% of those infected has palpable

17 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA epididymis lesions. Infection with B. ovis should always be taken into account in presence of ram s epididymitis, testicular atrophy or poor quality of semen. B.ovis can be identified by cultural, serological (mostly ELISA and CFT) methods (3) or PCR. Repeated sampling is required because excretion is often intermittent. In order to reduce the likelihood of false negatives, the best approach would be a combination of serology, specific genital examination and, where possible, semen cultivation (4). The outbreak of the disease in a pathogen - free herd is related to acquisition of infected animals or semen and sometimes the fertility can remain low even after its eradication. Following the confirmation of serological circulation of B.ovis in some flocks of Sicily, also confirmed by isolation from sheep milk and positivity for the presence of genital diseases in rams, it seems essential to verify the extent of the potential risk of infection in regional animal husbandry and better investigate the clinical and pathogenesis of this disease. Materials and methods A total of 608 serum samples were examined from rams belonging to 114 Sicilian farms. Blood samples were collected from the jugular vein into a 10ml vacuum tube, stored in a refrigerated bag and conferred to Serology department at Istituto Zooprofilattico Sperimentale della Sicilia. Sera were then removed by centrifugation and stored at -20 C until tested by ELISA. Antibodies to B. ovis were detected by a commercial ELISA test (Brucella ovis Antibody test kit - IDEXX) according to the manufacturer s instructions. As recommended by the manufacturer, any sample was considered as positive if the OD percent was 45, while any sample with an OD percentage under 45 was considered as negative. Results and discussions The results are presented in Figure 1. Thus, we found 68 samples (11.2 %) seropositive for antibodies to B. ovis. The highest prevalence was found in Caltanissetta district (16%), followed by Enna (9.6%) and Palermo (7.9%). This is the first screening in Sicily for this pathogen and the information obtained on the circulation of B. ovis in ovine of central and western Sicilian farms will help to establish appropriate strategies to control and eradicate the disease, resulting in reduction of economic losses and livestock damages associated with it (e.g. infertility, abortions and stillbirth). 17

18 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA Fig. 1. Seropositive samples for antibodies to B. ovis in different districts of Italy Conclusions In conclusion, it could be very useful and important to increase the knowledge and diagnostic attention toward such an infectious disease which, although less significant in terms of public health, may decrease in a significant way the farming economic performances. References 1. Blasco, J.M., Brucella ovis infection. In: Infectious and Parasitic Diseases of Livestock, Lefèvre PC, Blancou J, Chermette R & Uilenberg G. eds. Lavoisier, Paris, France, 2010, 2,

19 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA 2. Picard-Hagen, N., Xavier, B., Champoin, Eon, L., Lyazrhi, F., Marois, M., Peglion, M., Schuster, A., Trouche, C., Garin-Bastuji, B., Contagious epididymitis due to Brucella ovis: relationship between sexual function, serology and bacterial shedding in semen, BMC Vet. Res., 2015, 11, Praud, A., Champion, J.L., Crode, Y., Drapeau, A., Meyer, L., Garin- Bastuji, B., Assessment of the diagnostic sensitivity and specificity of an indirect ELISA kit for the diagnosis of Brucella ovis infection in rams, BMC Veterinary Research, 2012, 8, Ridler, A.L., Smith, S.L., West, D.M., Seroconversion and semen shedding in rams experimentally infected with Brucella ovis, N.Z. Vet. J., 2014, 62(1),

20 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA CTX-M GROUPS CHARACTERIZATION IN ESCHERICHIA COLI PRODUCING CTX-M EXTENDED-SPECTRUM BETA- LACTAMASES (ESBL), ISOLATED FROM DOGS FECES IN TWO DOG SHELTERS FROM MOLDOVA ANDREEA-PAULA COZMA 1*, M. CARP-CĂRARE 1, C. CARP-CĂRARE 1, ELEONORA GUGUIANU 1, CRISTINA RÎMBU 1, IULIA ELENA MĂCIUCĂ 2, DORINA TIMOFTE 1,2 1 Ion Ionescu de la Brad" University of Agricultural Sciences and Veterinary Medicine Iaşi, Faculty of Veterinary Medicine, Aleea Mihail Sadoveanu, nr. 8, Iaşi , Romania. 2 Institute of Veterinary Sciences, University of Liverpool, Leahurst Campus Neston Cheshire CH64 7TE, Liverpool, United Kindom cozmaandreeapaula@yahoo.com Summary The increasing prevalence of bacteria producing extended-spectrum beta-lactamases (ESBL) asociated with human and animal infections is an alarming and growing phenomenon and undestanding the reservoirs and mechanisms that facilitate their emergence is key to preventing their spread. Although previous reports have shown pets, especially dogs as source of ESBL, there is still insufficient information regarding the role they may play in mainatining and transmission of ESBL positive bacteria. The aim of this study was to detect the prevalence of CTX-M groups in a population of Escherichia coli resistant to third generation cephalosporins obtained from the dogs faeces from two dog shelters from Moldava. For this, 88 fecal samples were collected using rectal swabs. ESBL screening method included a first enrichment step with overnight cultivation in nutrient broth, followed by transfer of a small aliquote onto the chromogenic medium (Oxoid Brilliance ESBL). Isolates presumptively identified as ESBL producers based on their appearance on the ESBL chromogenic media, were confirmed as ESBL producers with the combination disc test (CDT) and sub-cultivated onto blood agar medium; fresh 24h colonies were used for bacterial DNA extraction by boiled preps method. The identification of E. coli isolates and of the CTX-M genes was performed by PCR following the specific protocols. ESBL screening on chromogenic medium identified 30 Enterobacteriaceae (30/88, 34%) presumvtive ESBL and which were confirmed as producing ESBL by combined disc method. The species identification was performed by detection of blauida and blauspa genes, which identified 28 E. coli isolates, which were subsequently selected for molecular testing. Molecular investigations to identify the genetic substrate of ESBL phenotype showed that 17 (60.71%) of E. coli isolates carried blactx-m gene whilst specific PCR for CTX-M groups showed that 15 (88.23%) of the E. coli isolates carried blactx-1 and 2 isolates (11.76%) carried blactx -9. None of the E. coli investigated harboured blactx-m-2, blactx-m-8 or blactx-m-25. This study identified a high prevalence of fecal carriage of CTX-M extended-spectrum betalactamases producing E. coli in dogs from the two dog shelters investigated. Key words: ESBL, CTX-M, pets, antibioresistance, E. coli 20

21 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA The bacterial strains of Escherichia coli take part of the normal microflora of the alimentary duct not only at human beings but also at animals. The increased prevalence of these resistant bacterial strains at the antimicrobial therapy is an alarm signal and a researched subject on a large scale. The resistence mechanism through the extended spectrum beta-lactamases enzymes is the most frequently met at the Gram negative bacteria and especially at E.coli not only of human origin but also of animal one. The relations and the direct contact between the human being and the pets have evolved, today existing a close connection between these. E.coli strains of ESBL positive isolated type from the pets are of interest for the public health in terms of the sending potential of the bacterial strains of ESBL between animals and the human being (1). The E.coli strains of the CTX-M-15 genotype the clonal group of ST131, O25 serogroup, are considered pandemic and they are associated with the multiple resistance at antibiotics. Such strains were isolated especially from the human beings, but they were also identified at pets (4;11). The mobile genetics elements that are involved in the mechanism of sending the resistance genes which encodes the extended spectrum beta-lactamases enzymes and close cohabitation between the animals and the human beings are factors which favor the transfer of the resistance markers intra and interspecies. The previous arguments underline the need of supervision of the antimicrobial resistance at pets through the molecular characterization of the strains of E.coli ESBL for establishing the role that these play in spreading the resistance at antibiotics. From the point of view of the concept One Health, the fecal carriage with multidrug resistance in bacteria at pets is essential through the impact and the risk that they can have on the public health the propagation of these bacteria. The purpose of our study was to establish the prevalence of the groups CTX-M identified at strains of E.coli isolated from the faces of the dogs from two shelters from Moldova. Materials and methods The study was carried out on 88 samples of faeces were collected from clinically healthy dogs from two shelters from Moldova. The collection of the samples was carried out with sterile swab from the rectal level. A first phase in the primary processing of the samples consisted of enriching the rectal samples through the cultivation on the nutritive broth, and then the screening of ESBL was carried out by using a chromogenous medium Brilliance ESBL Oxoid that contains Cefpodoxime, cephalosporin of the 2 nd generation at which in accordance with the specialized literature all the strains of Enterobacteriaceae ESBL are resistant (8). The molecular biology tests that were carried out for the identification of the strains and the genetics characterization of the resistance profile were carried out 21

22 22 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA within the Microbiology laboratory of the Veterinary Medicine Faculty from the University of Liverpool. The taxonomic classification of the isolates obtained after the screening of ESBL was carried out molecularly through the identification of the gene and. In this way, in order to extract the bacterial DNA through the method of boiled preps the strains were previously cultivated on the blood agar. The design of the primers used for the identification of the two genes characteristic for the strains of E.coli is given in the table 1. The used protocol for the PCR technique was the one recommended by Anastasi and col. for the gene and by Mc. Daniels and col. for the gene (2, 9). Table 1 The design of primers and the temperatures of alignment used at PCR for the taxonomic classification of the isolated strains and the detection of the main codant genes that codify the ESBL enzymes Primers uspa uida Alignment temperature CTX- MU Fw: 5 -CCGATACGCTGCCAATCA GT-3 Rev:5 -ACGCAGACCGTAGGCCAGAT C Fw: 5 -CCAAAAGCCAGACAGAGT-3 Rev: 5 -GCACAGCACATCCCCAA AGAG C Fw: 5 -ATGTGCAGYACCAGTAARGTKATGGC-3 Rev: 5 - TGGGTRAARTARGTSACCAGAAYCAGCGG-3 CTX- M-1 CTX- M-2 CTX- M-9 CTX- M-8 CTX- M-25 Nucleotide sequences (5-3 ) Length (bp) Fw: 5 - TGGGTRAARTARGTSACCAGAAYCAGCGG-3 Rev: 5 -AGC TTA TTC ATC GCC ACG TT-3 Fw: 5 -CGA CGC TAC CCC TGC TAT T-3 Rew : 5 - CCA GCG TCA GAT TTT TCA GG-3 Fw: 5 -CAA AGA GAG TGC AAC GGA TG-3 Rev : 5 -ATT GGA AAG CGT TCA TCA CC-3 Fw: 5 -TCG CGT TAA GCG GAT GAT GC CTX-M-8/25R: 5 -AAC CCA CGA TGT GGG TAG C-3 Fw: 5 -GCA CGA TGA CAT TCG GG-3 CTX-M-8/25R : 5 - AAC CCA CGA TGT GGG TAG C-3 References Anastasi & col.,2010 McDaniels & col., C Wedley & col., C Wedley & col., C Wedley & col., C Wedley & col., C Wedley & col., C Wedley & col., 2011 The phenotypic confirmation of the isolated strains after the screening of ESBL was carried out through the combination disc test (CDT) recommended and accepted by CLSI (5). This technique is based on the diffusimetric antibiogram, and the used antibiotics were Cefpodoxime (10), Cefpodoxime/Clavulanic acid (10/1), Cefotaxime (30), Cefotaxime/Clavulanic acid (30/10), Ceftazidime (30) and Ceftazidime/ Clavulanic acid (30/10). The isolated colonies after the screening

23 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA were cultivated on the blood agar. For every bacterial strain were carried out dilutions of whose turbidity was similar with the turbidity of 0,5McFarland. The plates with Müller Hinton were inoculated through clearing, and the used antibiotics were placed at distances of 25-30mm between them. A difference of 5mm between the diameters of the areas of inhibition of a cephalosporin and the same cephalosporin potentiated confirms phenotypically the presence of the extended spectrum beta-lactamases enzymes within the tested strain. The strain of E.coli ATCC was used as a control strain. The molecular investigations for the identification of the genetic substrate of the phenotype of ESBL were centred on the identification of the genes that encode the extended spectrum beta-lactamases enzymes from the five groups of CTX-M: CTX-M-1, CTX-M-9, CTX-M-2, CTX-M-25 and CTX-M-8. The amplification conditions and also the primers design respected the work protocol recommended by Wedley and col. (12). In the table number 1 is given the model of the nucleotide sequences for each analysed gene of bla CTX-M. Results and discussions The study was carried out on 88 samples of feces collected from clinically healthy dogs. After the screening of ESBL were isolated 30 (34%) of strains of Enterobacteriaceae presumtively ESBL (Figure 1). The molecular investigations that were carried out for the taxonomic classification of the isolated strains showed that 28 (93,33%) from these belong to the species of Escherichia coli, gene being identified at 25 (89,28%) among the strains and the gene at 3 (10,72%) among the strains of E.coli (fig.1). Fig.1. The strains taxonomic classification of Enterobacteriaceae obtained after the screening of ESBL 23

24 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA All the isolated E.coli strains after ESBL screening were phenotypically confirmed through the combination disc test (CDT). The molecular investigations for the characterization of the genetic substrate of the confirmed strains from the phenotypical point of view followed the identification of the genes that codify ehe extended spectrum beta-lacatamases enzymes from the groups of CTX-M. From those 28 of isolated E.coli, 17 (60,71%) carried the bla CTX-M gene. The positive strains for the group of CTX-M were investigated for the identification of those 5 groups of CTX-M enzymes. Out of the total number of 17 strains, 15 (88,23%) carried the bla CTX-M-1 gene and 2 (11,76%) carried bla CTX-M-9 gene. None of the E. coli investigated harboured bla CTX-M-2, bla CTX-M-8 or bla CTX-M-25 (fig. 2). 24 Fig. 2. The percentage of the genes of CTX-M identified at the analyzed E. Coli strains In this study was for the first time characterized the molecular substrate of the strains of E.coli CTX-M isolated producers from clinically healthy dogs. The prevalence of 19,31% of the dogs carried E.coli CTX-M strains obtained by us is close to the one obtained in France, 18,5% within the biggest study that was carried out for the determination of the faecal carriage with strains of ESBL/AmpC carried out on 368 of clinically healthy dogs (7). A bigger prevalence, of 24% of the clinically healthy dogs which are bearers of strains that produce CTX-M was

25 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA reported in China in 2010 (10). Also, the prevalence obtained by us is increased if we analyze the importance of the feacal carriage with strains which are resistent for the public health. The direct contact with the human being favors the conveyance of these strains. More than that, the strains which produce the extended spectrum betalactamases enzyme are many times associated and with the resistance at other classes of antibiotics: fluoroquinolone, aminoglycoside and trimethoprim + sulfamethoxazole (6). The multiple resistence determined by the strains of ESBL can be explained through the presence of plasmids which carried the genes of bla CTX-M and which frequently carry also gnes which confer resistence at antibiotics classes previously mentioned (3; 13). The enzyme of ESBL CTX-M-15 takes part of the group of CTX-M-1. Although with a much more reduced frequency, infections determined by the strains of E.coli CTX-M-15, the clonal group of ST131:O25, were reported also in the veterinary medicine at dogs (11). In this way, the prevalence of the faecal carriage with strains of CTX-M-1 is even more important, the pets could be considered a source of bacteria of ESBL. This study shows that the faeces of the pets constitute a risk in the conveyance of the strains of E.coli CTX-M positive also in Romania and it is a starting point for the molecular analysis of these isolates for their characterization regarding the resistance to other classes of antibiotics and the identification of the plasmids on which there are found distributed the genes of ESBL. Conclusions This study is the first carried out in Romania for the characterization of the molecular substrate of E.coli ESBL positive strains of pets origine and for the molecular identification of the genes which determine CTX-M enzymes. The prevalence of 19, 31% of the bacterial strains carried bla CTX-M gene is increased and alarming if we analyze in terms of the impact on the public health. The strains of E. coli ESBL positive are molecularly characterized through the presence of the genes that take part of the group of CTX-M-1 (88,23%) and CTX-M-9 (11,76%). There were not identified strains of E.coli carried of the genes that determine the extended spectrum beta-lactamases enzymes from the group of CTX-M-2, CTX-M-8 & CTX-M-25. References 1. Albrechtova, K., Dolejska, M., Cizek, A., Tausova, D., Klimes, J., Bebora, L., Literak, I., Dogs of nomadic pastoralists in northern Kenya are reservoirs of plasmid-mediated cephalosporin and quinolone-resistant Escherichia coli, including pandemic clone B2-O25-ST131., Antimicrobial 25

26 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA Agents and Chemotherapy, 2012, 56, Anastasi, E.M., Matthews, B., Gundogdu, A.,Vollmerhausen, T.L., Ramos, N.L., Stratton, H., Ahmed, W., Katouli, M., Prevalence and Persistence of Escherichia coli Strains with Uropathogenic Virulence Characteristics in Sewage Treatment Plants, Applied and Environmental Microbiology, 2010, 76(17), Ben-Sallem, R., Ben, Slama, K., Rojo-Bezares, B., Porres-Osante, N., Jouni, A., Klibi, N., Boudabous, A., Sáenz, Y., Torres, C., IncI1 plasmids carrying blactx-m-1 or blacmy-2 genes in Escherichia coli from healthy humans and animals in Tunisia. Microbial Drug Resistance, 2014, 20, Cantón, R., Novais, A., Valverde A., Machado E., Peixe L., Baquero F., Coque T.M., Prevalence and spread of extended-spectrum β-lactamaseproducing Enterobacteriaceae in Europe., Clinical Microbiology and Infection, 2008, 1, Clinical and Laboratory Standards Institute (CLSI), Performance Standards for Antimicrobial Susceptibility Testing, Coque, T.M., Baquero, F., Canton, R., Increasing prevalence of ESBLproducing Enterobacteriaceae in Europe., Euro Surveillance, 2008, 13, Haenni, M., Saras, E., Métayer, V., Médaille, C., Madec, J.Y., High Prevalence of blactx-m-1/inci1/st3 and blacmy-2/inci1/st2 Plasmids in Healthy Urban Dogs in France., Antimicrobial Agents and Chemotherapy, 2014, 58(9), Huang, T.D., Bogaerts, P, Biren, C, Guisset, A, Glupczynski, Y., Evaluation of Brilliance ESBL agar, a novel chromogenic medium for detection of extended-spectrum-beta- lactamase-producing Enterobacteriaceae, J Clin Microbiol, 2010, 48, McDaniels, A.E.., Rice, E.W., Reyes, A.L., Johnson, C.H., Haugland, R.A., Stelma, G.N.Jr., Confirmational Identification of Escherichia coli, a Comparison of Genotypic and Phenotypic Assays for Glutamate Decarboxylase and b-d-glucuronidase, Applied and Environmental Microbiology, 1996, 62 (9), Sun, Y., Zeng, Z., Chen, S., Ma, J., He, L., Liu, Y., Deng, Y., Lei, T., Zhao, J., Liu, JH., High prevalence of blactx-m extended-spectrum betalactamase genes in Escherichia coli isolates from pets and emergence of CTX-M-64 in China. Clin. Microbiol. Infect., 2010, 16, Timofte, D., Măciucă, I.E., Kemmett, K., Wattret, A.,Williams, N.J., Detection of the human-pandemic Escherichia coli B2-O25b-ST131 in UK dogs., Veterinary Record, 2014, 174(14), Wedley, A.L., Maddox, T.W., Westgarth, C., Coyne, K.P., Pinchbeck, G.L., Williams, N.J., Dawson, S., Prevalence of antimicrobial-resistant Escherichia coli in dogs in a cross-sectional, community-based study, Veterinary Record, 2011, 168(13),

27 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA 13. Wieler, L.H., Ewers, C., Guenther, S., Walther, B., Lübke-Becker, A., Methicillin-resistant staphylococci (MRS) and extended-spectrum β- lactamases (ESBL)- producing Enterobacteriaceae in companion animals: Nosocomial infections as one reason for the rising prevalence of these potential zoonotic pathogens in clinical samples, International Journal of Medical Microbiology, 2011, 301,

28 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA 28 CHARACTERIZATION OF STAFILOCOCI STRAINS ISOLATED FROM AGREMENT HORSES J. DÉGI, IONICA IANCU, DIANA MARIA DÉGI, V. HERMAN Banat s University of Agricultural Sciences and Veterinary Medicine King Michael I of Romania from Timisoara, Faculty of Veterinary Medicine, Calea Aradului, 119, Timisoara , Romania janos.degi@gmail.com Summary Colonization with methicillin resistant Staphylococcus aureus (MRSA) has been recently identified in horses and that the people working in these sectors, and the role of horses as reservoirs of MRSA infections in humans are little known. The research covered the work were performed in order to establish the frequency of staphylococcal strains isolated from horses herds and methicillin-resistant strains identify. In this study they were isolated 38 staphylococci strains, including coagulase positive strains 25 (CoP, represented by S. hyicus and S. aureus) strains of coagulase negative and 13 (CoN, represented by S. haemolyticus, S. sciuri, respectively S. epidermidis) isolated from recreational horses in different anatomical areas. Antibiotic sensitivity was variable depending on the group of antibiotics. If antibiotics: novobiocin, rifampicin, pristinamycin, ciprofloxacin, vancomycin, ceftriaxone, cefoxitin, cefaclor and ampicillin/sulbactan considered antibiotic of choice for staphylococci, number of sensitive strains was 100%. Against used Β-lactams (methicillin, ceftriaxone, cefoxitin, cefaclor, ampicillin with sulbactan), sensitivity was highest, except methicillin, the three resistant strains isolated. Keywords: Staphylococus, antibiotics, horses, infections Staphylococci strains not exposed to antibiotic pressure are susceptible to these substances; instead, staphylococcal strains isolated from animals and humans with various conditions and subjected to antibiotic pressure due to unreasonable therapy may exhibit multiple resistances. Resistance to antibiotics is based on genetic determinants located in plasmids R and chromosomes and can be transmitted by conjugation or other mechanisms, both intraspecific ally and interspecifically (5, 7). The development of resistance to various antibiotics, to staphylococci, is a consequence of the non-rational use in the therapy of both antibiotics in animals and humans without prior sensitivity testing. The non-rational use of antibiotics creates a selection pressure whose consequence is the selection and transmission of genetic determinants (genes) by R- type plasmids or the occurrence of chromosomal mutations. Antibiotic resistance to staphylococci is exerted differently depending on the classes of antibiotics (5, 10, 14). Colonization with methicillin-resistant staphylococcus aureus (MRSA) has recently been identified in horses and humans working in these sectors and the

29 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA role of horses as MRSA reservoirs for human infections has been reported more and more frequently in the literature (3, 4, 5). Materials and methods The samples required for bacteriological examinations were taken from a 34 clinically healthy horses (different in age, sex and race), with samples from two anatomical areas: cutaneous and nasal, totaling 68 samples. Samples were taken using sterile wool buffers attached to a plastic rod and placed in sterile tubes (standard product). Pathological materials were seeded on Columbia agar with 5% sheep blood. After incubation, the plates were examined with a stereoscopic magnifier, and Gram stained smears were made from each colony type. From characteristic morphological colonies, from which Gram positive, spherical, equals and hemispherical cubes were found, were sown with bacteriological anesthesia, by exhaustion, also on Columbia agar with 5% sheep blood. The subcultures obtained were bacterioscopically controlled to verify purity, and then, from the characteristic colonies, sowings were made on inclined agar and broth, these final cultures being for subsequent examinations.isolated and purified staphylococcus strains have been biochemically tested for pathogenicity. The controlled biochemical properties were: fermentation of mannitol on the Chapman (mannitol salt) agar. For testing the biochemical properties and identification of staphylococci species, using the Staph API system. Catalase determination was performed after by the two standard techniques. To highlight the bound coagulase (clumping factor) and/or protein A, was used the Prolex Staph Latex Rapid Kit (Pro-Lab Diagnostics). Results and discussions During the study, were isolated 38 strains belonging to the Staphylococcus genus. All bacterial strains, Gram-stained were Gram-positive bacteria, and clustering and morphology of bacterial cells was specific to staphylococci (cocci and predominant grouping in the form of bunches, occasionally in pair or solitary bacterial cells). In this study, 38 strains of staphylococci were isolated, of which on the basis of biochemical properties, the presence of coagulase types, staphylococci strains were included in coagulase positive, 25 strains (CoP, represented by S. hyicus and S. aureus) and coagulase negative, 13 strains (CoN, represented by S. haemolyticus, S. sciuri and S. epidermidis, respectively), isolated from recreational horses, from different anatomical areas. The results of the bacteriological examinations are shown in Table 1. 29

30 30 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA Table 1 Distribution of Staphylococcus strains isolated from recreational horses Location of horses / anatomical area No. of samples collected Positive samples S. aureus Isolated staphylococcal strains S. hycus S. epidermidis S. sciuri S. haemolyticus Herneacova Equestrian Center Skin Nose Sânandrei Equestrian Club Victoria Skin Nose Pony for children Dragoieşti Skin Nose Total (55, 89%) 15 (39, 47%) 11 (28, 95%) 5 (13, 16%) 3 (7, 9%) 4 (10, 53%) The results obtained from the testing of antibiotic susceptibility of strains of staphylococci isolated from the recreational horses are shown in Table 2. Table 2 Results of antibiotic susceptibility of strains of Staphylococcus isolated from horses Antimicrobial substance S. aureus (no=15) S. hyicus (no=11) No. of sensitive strains S S. epidermidis. haemolyticus (no=5) (no=4) S. sciuri (no=3) Methicillin - ME - 30µg Gentamicin CN 10µg Tetracycline TE 30µg Ciprofloxacin CIP 30 µg Kanamycin K 30 µg Novobiocin NV 30 µg Doxycycline DO 30 µg Eritromicin E 15 µg Vancomycin VA 30 µg Ceftriaxone CRO 30 µg Cefoxitin FOX - 10µg Polymyxin B PB 50UI Rifampicin RA 30 µg Lincomycin L 30 µg Cefaclor CEC 30 µg Pristinamycin PT 15 µg Ampicillin/sulbactan SAM 30 µg One of the main objectives of this study was to determine the types of resistance to different classes of antibiotics. The results on resistance and

31 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA resistance to methicillin in the 38 staphylococcus strains included in this study compared to the 17 antibiotics are shown in Table 3. Table 3 Resistance model of Staphylococcus strains isolated from horses Staphylococcus species No. resistant The antimicrobial resistance model (no. strain) antibiotics Staphylococcus aureus 8 ME (1) CN (7), K (9), DO (8), E (9), PB (15), L (10), TE (8) Staphylococcus hycus 8 ME (2) CN (5), K (4), DO (4), E (5), PB (11), L (7), TE (6) Staphylococcus 5 TE (3), DO (4), E (4), PB (4), L (3) epidermidis Staphylococcus 3 DO (3), E (3), PB (3) haemolyticus Staphylococcus sciuri 1 PB (3) Analyzing the results in the table it can be seen that the antibiotic sensitivity was variable depending on the antibiotic groups. In the case of antibiotics: novobiocin, rifampicin, pristinamycin, ciprofloxacin, vancomycin, ceftriaxone, cefoxitin, cefaclor and ampicillin / sulbactan, considered staphylococcal selection antibiotics, the number of susceptible strains was 100%, all strains isolated. This suggests that isolated and tested strains originated from animals where these antibiotics were not used. It can also be said that all these antibiotics constitute the kit for staphylococci or are usually used in humans in the therapy of staphylococcal and animal infections, respectively. In comparison to the beta-lactamins used (methicillin, ceftriaxone, cefoxitin, cefaclor, ampicillin with sulbactan), antibiotic sensitivity was maximal, except for methicillin, where 3 resistant strains were isolated. Of these, two strains of methicillin resistant to S. hycus and a strain of S. aureus. The strains tested were largely sensitive to the other β-lactam as a result of previous correct treatments. As compared to aminoglycosides (gentamicin, kanamycin) and macrolides (erythromycin and vancomycin), antibiotic sensitivity was different, being maximal for vancomycin. In the case of gentamicin, 12 resistant strains, 13 strains versus kanamycin and 21 erythromycin resistant strains were isolated. Most strains were resistant to polymyxin B (36 strains) due to the use of locally applied preparations containing this antibiotic. Antibiotic sensitivity to tetracycline (tetracycline, doxycycline) was reduced, with 36 strains resistant to this group of antibiotics, with plasmid and chromosomal resistance (17 tetracycline strains and 19 doxycycline strains). All strains tested were sensitive to ciprofloxacin because this quinolone is not used in horses' drug therapy. Staphylococcus aureus resistant to methicillin (MRSA) is an emerging pathogen in horses. Initial reports of MRSA infections in horses were considered to 31

32 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA be largely sporadic, associated with veterinary hospitals (6, 8). However, the number of MRSA colonization reports and horse infections is steadily rising, in fact associated with private practices and the community (11). The prevalence of MRSA colonization to date ranges between 0% and 4.7% in European, Canadian and North American (1, 2, 9, 12) horses, between 2.9% and 10.9% in horses presented In horses clinics (13) and 16% in horses in specialized hospitals (1) respectively. In hospitalized horses, surgeries due to surgery are predominant (11). In Canada and North America, most MRSA infections in horses are caused by an ST8 (MRSA canadian-5 or USA500) multilocus sequential human cloned (11). It has been suggested that this strain is adapted to horse because of its preponderance to horses and staff working in this field (11). Recently there have been reports of colonization and infection of horses with MRSA ST398 (15), a MRSA clone associated with domestic animals (4). Published studies on isolated isolates of staphylococci from the Netherlands and another study of 200 healthy horses (2), respectively, did not reveal the presence of MRSA. The reason for the sudden increase in the number of MRSA strains in horses since 2006 is currently unknown. The high prevalence of ST398 strains in pigs and calves in the Netherlands (4) and the fact that horses are often kept on farms with other animals and are sometimes treated by veterinarians who are also involved in the treatment of domestic animals are possible explanations for increased cases with strains ST398 in horses. MR8 ST8 strains of spa t064 have been identified and isolated in horses and staff at a veterinary hospital in Ireland (7), and strains of MRSA ST398 spa t011 have been isolated in horses and staff from a veterinary hospital in Vienna (3). Also, international horses and trades could favor the release of MRSA strains. Changes in the use of certain antimicrobial substances are responsible for the continuous increase in resistance phenomena. Conclusions From the clinically healthy recreational horses, were isolated 38 strains of staphylococci, of which 25 positive coagulase strains (CoP, represented by S. hyicus and S. aureus) and coagulase negative, 13 strains (CoN, represented by S. haemolyticus, S. sciuri and S. epidermidis, respectively). All isolates of staphylococcus strains showed 100% sensitivity for antibiotics: novobiocin, rifampicin, pristinamycin, ciprofloxacin, vancomycin, ceftriaxone, cefoxitin, cefaclor and ampicillin / sulbactan, considered antibiotics of choice for these bacteria. In contrast to the beta-lactamins used (methicillin, ceftriaxone, cefoxitin, cefaclor, ampicillin with sulbactan), antibiotic sensitivity was maximal, except methicillin, where 3 resistant strains, two strains of methicillin resistant to S. hycus and one S. aureus strain. 32

33 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA Following testing of strains of staphylococci isolated from recreational horses, methicillin-resistant strains and several resist types were identified against 17 antibiotics compared to β-lactam, tetracycline, macrolide and polymyxin B. Acknowledgements This study was realised using the support and infrastructure project Dezvoltarea infrastructurii de cercetare, educaţie şi servicii în domeniile medicinei veterinare şi tehnologiilor inovative pentru RO 05, cod SMIS-CSNR References 1. Baptiste, K., Williams, K., Willams, N., Wattret, A., Clegg, P., Dawson, S., Corkill, J., O Neill, T., Hart, C., Methicillin-resistant staphylococci in companion animals, Emerg. Infect. Dis., 2005, 11, Busscher, J., Van Duijkeren, E., Sloet Van Oldruitenborgh-Oosterbaan, M., The prevalence of methicillin-resistant staphylococci in healthy horses in the Netherlands, Vet. Microbiol., 2006, 113, Cuny, C., Strommenger, B., Witte, W., Stanek, C., Clusters of Infections in Horses with MRSA ST1, ST254, and ST398 in a Veterinary Hospital, Microb. Drug Resist., 2008, 14, Graveland, H., Wagenaar, J.A., Bergs, K., Heesterbeek, H., Heederik, D., Persistence of livestock associated MRSA CC398 in humans is dependent on intensity of animal contact, PLoS One, 2011, 6, e Guardabassi, L., Courvalin P., Modes of antimicrobial action and mechanisms of bacterial resistance, În Antimicrobial resistance in bacteria of animal origin. Ed. ASM Press, Washington, D.C., Hartmann, F.A., Trostle, S.S., Klohnen, A.A., Isolation of methicillinresistant Staphylococcus aureus from a postoperative wound infection in a horse. J. Am. Vet. Med. Assoc., 1997, 211, Moodley, A., Stegger, M., Bagcigil, A.F., Baptiste, K.E., Loeffler, A., Lloyd, D.H., Williams, N.J., Leonard, N., Abbott, Y., Skov, R., Guardabassi, L., Spa typing of methicillin-resistant Staphylococcus aureus isolated from domestic animals and veterinary staff in the UK and Ireland, J. Antimicrob. Chemother., 2006, 58, Seguin, J.C., Walker, R.D., Caron, J.P., Kloos, W.E., George, C.G., Hollis, R.J., Jones, R.N., Pfaller, M.A., Methicillin-resistant Staphylococcus aureus outbreak in a veterinary teaching hospital: potential humanto-animal transmission, J. Clin. Microbiol., 1999, 37, Vengust, M., Anderson, M., Rousseau, J., Weese, J., Methicillin resistant staphylococcal colonization in clinically normal dogs and horses in the community, Lett. Appl. Microbiol., 2006, 43,

34 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA 10. Wan Mintao, Lauderdale T. L., Chou Chincheng, Characteristics and vrirulence factors of livestock associated ST9 methicillin-resistant Staphylococcus aureus with a novel recombinant staphylocoagulase type, Veterinary Microbiology, 2013, 162(24), Weese, J.S., Van Duijkeren, E., Methicillin-resistant Staphylococcus aureus and Staphylococcus pseudintermedius in veterinary medicine, Veterinary Microbiology, 2010, 140, Weese, J., Archambault, M., Willey, B., Hearn, P., Kreiswirth, B., Said- Salim, B., McGeer, A., Likhoshvay, Y., Prescott, J., Low, D., Methicillin resistant Staphylococcus aureus in horses and horse personnel, Emerg. Infect. Dis., 2005, 11, Weese, J., Rousseau, J., Traub-Dargatz, J., Willey, B., McGeer, A., Low, D., Community-associated methicillin-resistant Staphylococcus aureus in horses and humans who work with horses, J. Am. Vet. Med. Assoc., 2005, 226, Wertheim, H. F., Vos, M. C., Boelens, H.A., Low prevalence of methicillin resistant Staphylococcus aureus (MRSA) at hospital admission in the Netherlands: the value of search and destroy and restrictive antibiotic use, J. Hosp. Infect., 2004, 56, Witte, W., Strommenger, B., Stanek, C., Cuny, C., Methicillin-resistant Staphylococcus aureus ST398 in humans and animals, Central Europe, Emerg. Infect. Dis. 2007, 13,

35 LUCRĂRI ŞTIINŢIFICE MEDICINĂ VETERINARĂ VOL. L (3), 2017, TIMIŞOARA CHANGES IN THE MICROBIOME OF FISH FROM THE DANUBE DELTA BY HABITAT AND FEEDING BEHAVIORS G. GATI, MIHAELA NICULAE, EMÖKE PÁLL, CARMEN DANA ŞANDRU, F. BRUDAȘCĂ, A. VASIU, SILVANA POPESCU, MARINA SPÎNU University of Agricultural Sciences and Veterinary Medicine Cluj- Napoca, , 3-5 Mănăştur Street, Cluj-Napoca, Romania vlad_negrutiu@yahoo.com Summary Bacterial pathogens such as E. coli/coliforms, Salmonella spp., Vibrio spp. could be a cause of food and water born infections and pose a health risk to consumers and tourists in wetlands. We hypothesized that the number of bacterial strains and therefore the infectious risk will be higher in benthic fish due to the higher number of potential sources than in the more active, pelagic group in the Danube Delta. The research aimed to identify the potentially pathogenic bacteria from both benthic and pelagic fishes and evaluate their zoonotic potential as a consumption item. A number of 20 samples of gills and hepatopancreas from each perch (Perca fluviatilis), wels catfish (Silurus glanis), sander (Sander lucioperca), pike (Esox lucius), hardtail (Alosa immaculate), gibel carp (Carassius gibelio) and common carp (Cyprinus carpio) were collected from the Danube river mouth (Melea, Turcesc, Flamanda, Ciobanesc channels) and subjected to classical isolation techniques using Chromogenic UTI medium, Brilliance TM E. coli/ coliform selective medium and TCBS Cholera Medium (Oxoid). The results were analyzed by Statistica program. Bacteria of the Vibrio genus (V. cholerae, V. fluvialis, V. alginolyticus, V. metschnikovii, V. mimicus, V. vulnificus, V. parahaemolyticus) dominated the microbiome structure, along with E. coli/coliforms and Pseudomonas spp. There were no significant differences in the distribution frequency of the bacteria on the gills between benthic, plankton eating fish (0.36±0.06) and pelagic, raptor (0.42±0.02) fish. The microflora overall frequency was higher in the hepatopancreas of pelagic raptor fish (0.432±0.06) than in plankton eating, benthic (0.35±0.00) fish species. The results indicated that both benthic and pelagic fish could be a source of pathogenic bacteria with zoonotic potential, independently on feeding behavior or habitat. Key words: fish, benthic, pelagic, microbiome, Danube Delta Fecal pathogens, such as bacteria, viruses and protozoa, strongly resistant to water environment and disinfectants, sometimes to antibiotics as well, are able to cause disease in humans and animals (6). Amongst those, a genus commonly found in fish and water, Aeromonas is also involved in human pathology (intestinal disease, skin injuries, joint, bone, respiratory, urinary tract and ocular infections. Researches also noted high mortality in A. hydrophila and A. veronii species in immunocompromised patients (1). Other fresh water fish, such as lean lake trout (Salvelinus namaycush), and walleye (Sander vitreus), and seeforellen brown trout (Salmo trutta) (3) host Shewanella, also involved in human pathology (10). 35

36 36 LUCRĂRI ŞTIINŢIFICE MEDICINĂ VETERINARĂ VOL. L (3), 2017, TIMIŞOARA Some of the bacteria found in fish, but also in water, although potentially pathogenic for mammalian hosts, have a beneficial effect on the immune system of fish. In this group could be included Aeromonas veronii BA-1, Vibrio lentus BA-2, and Flavobacterium sasangense BA-3 from the intestinal tract of the common carp (Cyprinus carpio) (2, 11). Studies carried out in a fish rearing environment on both water and fish intestines indicated counts as high (>4 log cfu ml -1 or 8 log cfu g -1 ) of total aerobic bacteria in pond waters and fish intestines, where the bacteriome was dominated by Salmonella spp., Vibrio spp., Staphylococcus spp. and Escherichia coli, all of these being recorded as animal and human pathogens (4, 7). Thus, investigations on bacteriome carriage in fish and its presence in their environment is important for animal, consumer and environmental health equally. The research aimed to identify the potentially pathogenic bacteria from both benthic and pelagic fishes and evaluate their zoonotic potential as a consumption item. Materials and methods A number of 20 samples of gills and hepatopancreas from each perch (Perca fluviatilis), wels catfish (Silurus glanis), sander (Sander lucioperca), pike (Esox lucius), hardtail (Alosa immaculate), gibel carp (Carassius gibelio) and common carp (Cyprinus carpio) were collected from the Danube river mouth (Melea, Turcesc, Flamanda, Ciobanesc channels). The fish species with indication of their habitat and feeding behavior were presented in Table 1. Most of the studied fish, due to the investigated area, were from pelagic and predatory species. Table 1 Feeding and habitat peculiarities of tested fish Feeding Habitat Latin name English name behavior predatory pelagic Perca fluviatilis Perch predatory benthic Silurus glanis Wels catfish predatory pelagic Sander lucioperca Sander predatory pelagic Esox lucius Pike predatory pelagic Alosa immaculata Hardtail herbivorous benthic Carassius gibelio Gibel carp herbivorous benthic Cyprinus carpio Common carp Similarly, water samples were collected from the habitats of the tested fish. All samples from fish and water were then subjected to classical isolation techniques using Chromogenic UTI medium, Brilliance TM E. coli/ coliform selective medium and TCBS Cholera Medium (Oxoid). The colonies were identified by color, according to the producer s instructions. The frequency of the bacteria found in all

37 LUCRĂRI ŞTIINŢIFICE MEDICINĂ VETERINARĂ VOL. L (3), 2017, TIMIŞOARA sampled hepatopancreas and gills were calculated. When the bacteria were found in both fish and water, the sample was scored 1, when it was present only in the fish, the score was 0. The average values of the scores were established and expressed as percentages of the total. The results were analyzed by Statistica program. Results and discussion Worldwide researchers consider that the infectious waterborne diseases are highly underestimated globally. These pathogens could easily reach increased levels of endemic transmission (caliciviruses, Cryptosporidium) since they are ubiquitous in aqueous environment (rivers, springs, drinking water) and also could be transferred by inhabitans of this habitats (6). More and more bacterial strains, including those in aqueous habitats, share antibiotic resistance or MDR and of these, mainly Beta-lactamase producing ones pose the highest health risk. Four ecological compartments were considered as sources for the resistome transfer, and these include mammalian host (humans, animals) and also environmental sources (plant, soil and water). Surprisingly, bacteria from the environment, with no pathogenic significance and no direct contact with antibiotics, could remain a permanent source of R genes for pathogenic bacteria. One best example is Aeromonas, present in the aqueous environment, posing risk to those fishing, surfing, swimming, diving (1). In another study, P. aeruginosa was found in natural waters in concentrations of 10/100 ml to >1,000/100 ml (9). Nevertheless, very few researchers investigated the reciprocal relationships of fish and their habitat microbiota, in connection with various environmental factors (8). In this study, bacteria of the Vibrio genus (V. cholerae, V. fluvialis, V. alginolyticus, V. metschnikovii, V. mimicus, V. vulnificus, V. parahaemolyticus) dominated the microbiome structure, along with E. coli, coliforms and Pseudomonas spp. The overall bacterial frequency was somewhat higher in the hepatopancreas of the fish as opposed to the gills. There were no significant differences between the predatory and herbivorous fish in this respect (Table 2, Table 3). The highest values of the frequency were found in the gills and hepatopancreas of sander, pearch and hardtail. The distribution of Vibrio genus was lesser in the hepatopancreas than in the gills, no significant differences being recorded between the fish species based on their feeding behaviour or habitat. The "dilution effect" is being defined by the diminishment of the infection prevalence in a host, where the species are numerous and vary in susceptibility to infection by a pathogen. Competitors and predators may (1) alter host behavior to reduce pathogen transmission or (2) reduce host density (5). 37

38 LUCRĂRI ŞTIINŢIFICE MEDICINĂ VETERINARĂ VOL. L (3), 2017, TIMIŞOARA Table 2 The frequency of bacteria from branchiae of fish that differ by habitat and feeding habits Total Perch Wels catfish Sander Pike Hardtail Gibel carp Common carp Legend: 1-V. cholerae, V. fluvialis, 2- V. alginolyticus, V. metschnikovii, 3- V. mimicus, V. vulnificus, 4- V. parahaemolyticus, 5- E.coli, 6- Coliformi, 7- Pseudomonas spp. Table 3 The frequency of bacteria from hepatopancreas of fish that differ by habitat and feeding habits Total Perch Wels catfish Sander Pike Hardtail Gibel carp Common carp Legend: 1-V. cholerae, V. fluvialis, 2- V. alginolyticus, V. metschnikovii, 3- V. mimicus, V. vulnificus, 4- V. parahaemolyticus, 5- E.coli, 6- Coliformi, 7- Pseudomonas spp. 38

39 LUCRĂRI ŞTIINŢIFICE MEDICINĂ VETERINARĂ VOL. L (3), 2017, TIMIŞOARA Fig. 1. The frequency of bacterial isolates by species, found in branchiae and also the habitat water Fig. 2. The frequency of bacterial isolates by species, found in hepatopancreas and also the habitat water 39

40 LUCRĂRI ŞTIINŢIFICE MEDICINĂ VETERINARĂ VOL. L (3), 2017, TIMIŞOARA In this case, the prevalence of bacteria might have been influenced by the feeding behaviour of the analysed fish species, but certainly the dilution effect occurred also due to the intervention of non-host species of the habitat. Comparing the frequency of the simultaneous isolation of bacteria species from the water and the fish species, it could be seen that no significant differences were recorded neither for branchiae (Fig. 1) nor for the hepatopancreas (Fig. 2). The values were quite low, this indicating that the bacterium was not necessarily transferred to the habitat, but increased the risk of transmission to consumers by handling the fish or consuming it. Conclusions The results indicated that both benthic and pelagic fish could be a source of pathogenic bacteria with zoonotic potential, independently on feeding behavior or habitat. Aknowledgement This research was supported by grant PNII PCCE 61/2012. References 1. Balotescu, C., Israil, A., Radu, R., Alexandru, I, Dobre, G., Aspects of constitutive and acquired antibioresistance in Aeromonas hydrophila strains isolated from water sources, Roum. Arch. Microbiol. Immunol., 2003, 62(3-4), Chi, C., Jiang, B., Yu, X.B., Liu, T.Q., Xia, L., Wang, G.X., Effects of three strains of intestinal autochthonous bacteria and their extracellular products on the immune response and disease resistance of common carp, Cyprinus carpio, Fish Shellfish Immunol., 2014, 36(1), Dailey, F.E., McGraw, J.E., Jensen, B.J., Bishop, S.S., Lokken, J.P., Dorff, K.J., Ripley, M.P., Munro, J.B., The Microbiota of Freshwater Fish and Freshwater Niches Contain Omega-3 Fatty Acid- Producing Shewanella Species, Appl. Environ. Microbiol., 2015, 82(1), Kaktcham, P.M., Temgoua, J.B., Ngoufack, Zambou, Diaz-Ruiz, G., Wacher, C., Pérez-Chabela, M.L., Quantitative analyses of the bacterial microbiota of rearing environment, tilapia and common carp cultured in earthen ponds and inhibitory activity of its lactic acid bacteria on fish spoilage and pathogenic bacteria, World J. Microbiol. Biotechnol., 2017, 33(2),

41 LUCRĂRI ŞTIINŢIFICE MEDICINĂ VETERINARĂ VOL. L (3), 2017, TIMIŞOARA 5. Khalil, H., Ecke, F., Evander, M., Magnusson, M., Hörnfeldt, B., Declining ecosystem health and the dilution effect, Sci. Rep., 2016, 8(6), Leclerc, H., Schwartzbrod, L., Dei-Cas, E., Microbial agents associated with waterborne diseases, Crit. Rev. Microbiol., 2002, 28(4), Li, K., Petersen, G., Barco, L., Hvidtfeldt, K., Liu, L., Dalsgaard, A., Salmonella weltevreden in integrated and non-integrated tilapia aquaculture systems in Guangdong, China Food Microbiol., 2017, 65, Li, T., Li, H., Gatesoupe, F.J., She, R., Lin, Q., Yan, X., Li, J., Li, X., Bacterial Signatures of "Red-Operculum" Disease in the Gut of Crucian Carp (Carassius auratus), Microb. Ecol., 2017 Apr 1. doi: /s Mena, K.D., Gerba, C.P., Risk assessment of Pseudomonas aeruginosa in water, Rev. Environ. Contam. Toxicol., 2009, 201, Muñoz-Gallego, I., Chaves, F., Orellana, M.A., Epidemiological and clinical characteristics of Shewanella spp. infections in a tertiary hospital in Madrid, Infect. Dis (Lond)., 2016, 48(10), Rusin, P.A., Rose, J.B., Haas, C.N., Gerba, C.P., Risk assessment of opportunistic bacterial pathogens in drinking water, Rev. Environ. Contam. Toxicol., 1997, 152,

42 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA LEPTOSPIROSIS AND BARTONELLOSIS SEROPREVALENCE IN STRAY CATS CIRCULATING IN PALERMO (SICILY, ITALY) F. GRIPPI 1, P. GALLUZZO 1, M. ARNONE 1, V. GARGANO 1, S. LOMBARDO 2, F. SANTANGELO 2, S. VILLARI 1, G. CHIARENZA 1 1 Istituto Zooprofilattico Sperimentale della Sicilia A. Mirri - 3, via G. Marinuzzi, Palermo, Italy 2 U.O. Igiene Urbana ASP 6, Palermo - Canile Municipale - 5, Piazza Tiro a Segno, Palermo, Italy francesca.grippi@izssicilia.it Summary A serological survey was conducted to determine the occurrence of Leptospira spp. and Bartonella henselae in stray cats circulating in Palermo (Sicily, Italy). Samples from 721 cats were tested for Leptospira spp. antibodies by serological Microagglutination Test (MAT) and for B. henselae antibodies by Indirect Immunofluorescence Antibody assay (IFA). We found only one sample (0.1%) seropositive for antibodies to Leptospira spp. with a title of 1:100 for Leptospira australis. Concerning B. henselae, 513 samples analyzed by IFA were positive (71%). This is the first screening in Sicily for these two pathogens and the results here obtained will be useful to take preventive measures to reduce the prevalence of such diseases in pets Key words: cat, seroprevalence, Leptospira spp., Bartonella henselae Feline diseases can be caused by a range of pathogens and many of these infections have zoonotic implications, so stray cats are potential sentinels for both human and pet health. Stray cats roam freely and often form colonies which live and feed in close proximity to humans (10). This study investigated the prevalence of Leptospira spp. and Bartonella henselae in stray cats circulating in Palermo (Sicily, Italy). Leptospirosis is a zoonotic disease with a worldwide distribution affecting most mammalian species (3). Clinical leptospirosis is rare in cats (2) but they could shed leptospires in their urine without showing any sign of the disease (11). This is problematic, as it could lead to human exposure. Bartonella spp. is a vector-borne Gram-negative bacterium able to infect the erythrocytes and endothelial cells of mammalian hosts (4, 5). B. henselae is transmitted among cats by the cat flea (Ctenocephalides felis), and this vector is essential for maintaining the infection within cat populations. Most cats infected with B. henselae are asymptomatic or have mild self-limiting symptoms, such as fever or lymphadenopathy (9, 12). 42

43 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017 TIMISOARA The control of leptospirosis and bartonellosis, therefore, is important not only for animals but also for public health concerns. Materials and methods For this project, 721 serum samples from stray cats circulating in Palermo were collected and tested for both anti-leptospira spp. and B. henselae antibodies. All cats belonged to the European breed. The diagnosis for Leptospira spp. was based on antibodies detection by serological Microagglutination Test (MAT). The analysis was conducted towards eight common serogroups circulating in Italy (Leptospira australis, Leptospira ballum, Leptospira canicola, Leptospira grippotyphosa, Leptospira icterohaemorrhagiae, Leptospira pomona, Leptospira serjroe, Leptospira tarassovi). A commercial immunofluorescence antibody assay (IFA) (MegaFLUO BARTONELLA henselae - MEGACOR Diagnostik GmbH) was used to conduct a screening for the presence of antibodies against B. henselae. Results and discussions The results demonstrated that only one of 721 samples analyzed (0,1%) was seropositive for antibodies to Leptospira spp., with a title of 1:100 for Leptospira australis. Concerning B. henselae, 513 out of the 721 samples analyzed by IFA resulted positive (71%). Our data showed that cats are not likely reservoirs of Leptospira for the eight strains searched, while the analysis conducted for the detection of antibodies against B. henselae revealed a higher prevalence compared to other Countries or also other Italian regions (1, 6, 7, 8, 13). Conclusions Our study is the first large-scale screening for the detection of antibodies against Leptospira spp. and B. henselae conducted in stray cats circulating in Sicily. This work thus represents a first, useful piece of information to be implemented by further analysis. References 1. Alamán Valtierra, M., Simón Valencia, C., Fuertes Negro, H., Unzueta Galarza, A., Flores Somarriba, B., Halaihel Kassab, N., Molecular Epidemiology of Bartonella henselae in Stray and Sheltered Cats of Zaragoza, Spain, Rev Esp Salud Publica, 2016, 90,E5. 43

44 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA 2. Arb Our, J., Blais, M. C., Carioto, L., Sylvestre, D., Clinical leptospirosis in three cats ( ), Journal of the American Animal Hospital Association, 2012, 48, Bharti, A.R., Nally, J.E., Ricaldi, J.N., Matthias, M.A., Diaz, M.M., Lovett, M.A., Levett, P.N., Gilman, R.H., Willig, M.R., Gotuzzo, E., Vinetz, J.M., Leptospirosis: a zoonotic disease of global importance, Lancet Infect Dis, 2003, 3(12), Chomel, B.B., Boulouis, H.J., Maruyama, S., Breitschwerdt, E.B., Bartonella spp. in pets and effect on human health, Emerg Infect Dis, 2006, 12, Chomel, B.B., McMillan-Cole, A.C., Kasten, R.W., Stuckey, M.J., Sato, S., Maruyama, S., Diniz, P.P., Breitschwerdt, E.B., Candidatus Bartonella merieuxii, a potential new zoonotic Bartonella species in canids from Iraq, PLoS Negl Trop Dis, 2012, 6: e Ebani, V.V., Bertelloni, F., Fratini, F., Occurrence of Bartonella henselae types I and II in Central Italian domestic cats, Res Vet Sci, 2012, 93(1), Fabbi, M., De Giuli, L., Tranquillo, M., Bragoni, R., Casiraghi, M., Genchi, C., Prevalence of Bartonella henselae in Italian stray cats: evaluation of serology to assess the risk of transmission of Bartonella to humans, J Clin Microbiol, 2004, 42(1): Maia, C., Ramos, C., Coimbra, M., Bastos, F., Martins, A., Pinto, P., Nunes, M., Vieira, M.L., Cardoso, L., Campino, L., Bacterial and protozoal agents of feline vector-borne diseases in domestic and stray cats from southern Portugal, Parasit Vectors, 2014, 7, Mosbacher, M.E., Klotz, S., Klotz, J., Pinnas, J.L., Bartonella henselae and the potential for arthropod vector-borne transmission, Vector Borne Zoonotic Dis, 2011, 11, Robertson, S.A., A review of feral cat control, J Feline Med Surg, 2008, 10, Rodriguez, J., Blais, M.C., Lapointe, C., Arsenault, J., Carioto, L., Harel, J., Serologic and Urinary PCR Survey of Leptospirosis in Healthy Cats and in Cats with Kidney Disease., Journal of Veterinary Internal Medicine, 2014, 28, Stutzer, B., Hartmann, K., Chronic Bartonellosis in cats: what are the potential implications? J Feline Med Surg, 2012, 14, Switzer, A.D., McMillan-Cole, A.C., Kasten, R.W., Stuckey, M.J., Kass, P.H., Chomel, B.B., Bartonella and Toxoplasma infections in stray cats from Iraq, Am J Trop Med Hyg, 2013, 89(6),

45 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA QUORUM SENSING AN OVERVIEW OF COORDINATED BACTERIAL SOCIAL BEHAVIORS S. GURANDA, ELENA MARIAN, MARINA SPÎNU University of Agricultural Sciences and Veterinary Medicine, , Mănăştur Street 3-5, Cluj-Napoca, Romania, phone , fax , silvian.guranda@usamvcluj.ro Summary Bacteria are a domain that shows the complexity of microscopic life. They don t live separately, but in populations that seldom are pure. Researchers found that complex multibacterial organization implies cell to cell communication. This communication process known as quorum sensing ensures bacteria to decide whether it is worthwhile to invest energy in various processes such as biofilm formation, DNA uptake, gene expression of virulence, production of exoenzymes and substances having antimicrobial effects. Since bacterial language is of a chemical nature, environmental conditions may hinder it. This process known as quorum quenching is the result of the action of different inhibitors that can destroy signal molecules, block their production or prevent bacteria from detecting signal molecules. Autocrine and paracrine signaling are both based on signaling molecules reaching a certain threshold which leads to changes in population behavior by triggering changes in gene expression. Though many bacterial species have their own language, their chemical messages can reach other species as well and can determine changes in bacterial population behavior. The aim of this review is to show the complexity of bacterial communication. Keywords: quorum sensing, quorum quenching, bacterial communication With the discovery of the microscopic forms of life and the shift of the scientific understanding from spontaneous generation (abiogenesis) to biogenesis, microbiology and medicine entered a new era. Once Alexander Flemming ( ) opened the era of antibiotics a new hope seemed to flicker at the horizon and the historical fierce battle of humanity with infectious diseases seemed to get with rapid steps closer to the dawns of victory. Though Flemming had warned on the phenomenon of antibiotic resistance, it took a few decades to scientific world to understand from clinical practice that bacteria are not simple living creatures encapsulated in some simple rigid mechanisms, but they are adapting to environment and develop ways to survive. Their organization in complex communities, such as biofilm and mixed populations from different bacterial types, strains and even molds, led to the conclusion that cells might have been communicating one with another, since a homeostatic tendency and a coordinated response toward the environment was observed. That made the researchers to admit that an organized social behaviour can be seen in bacteria. Meanwhile, they were forced to make a shift from the idea that bacteria are simple cells living 45

46 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA as individuals, to a new paradigm that admitted there must be a communication system between bacteria, either they are of the same type or of distant genres. That opened a new field to experiences that eventually led to the discovery of a system of stimuli and response depending on bacterial population density (1,6). What is quorum sensing? Various experiments proved that bacteria communicate with one another, and they have a common language regardless the type they belong to. Information is provided and delivered using chemical signal molecules. These signal molecules are produced by bacteria and designed to be understood by cells of the same type and even by different genres cells. This means the bacteria membranes are equipped with sensors receptors that signal molecules can reach to. In response to that, when the receptors get saturated, they become activated. Then, they can trigger a response, so that bacterial population change its behavior, via gene transcript modulation. This mechanism through which the information supplied by signal molecules triggers a feedback in bacteria s behavior is critical for synchronizing the activities of large of cells and is called Quorum-sensing (QS) Fig. 1 (1,6). 46 Fig.1. Quorum-sensing has a correspondence in a threshold signal cells density (Fuqua et al., 1994) Quorum-sensing is a term that was first used in a review by Fuqua et al. in 1994 (5) and referred to a stimuli-response system correlated to a threshold population density. It showed that there is a minimum threshold level of individual cell mass required to produce a change in bacterial population s behavior. This concerted population response seemed to depend of a critical number of active cells that form that bacterial group. In other words, the bacterial population senses

47 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA how large is the cells number and synchronizes its actions when a threshold value is reached (figure 1) (5, 8). In order to sense the minimum quorum of members, bacteria need to give information to one another about their presence and their active state. That is reached by a common bacterial language, that must be both spoken and understood by all of the population members. The language used by bacteria to transmit information is a chemical language, in other words it is based on signal molecules. Each bacterial cell is able to produce and deliver in the environment signal molecules, which represent the material support for information. The signal molecules spread within the environment by diffusion. As signal molecules diffuse in the environment, their density gradient decreases with the distance from the emitting cell. That would be equivalent to the concept of power of the signal which is stronger around the emitter and weaker as the distance from the source increases (1,3,7). Fig. 2. Quorum-sensing schematic process (Li and Tian, 2012) The system would not be complete unless each bacterial cell could hear the spoken chemical words emitted by surrounding bacteria. This means that every cell must have on its membrane sensors that can sense the presence of the signal molecules. In fact, these membrane receptors work together with the signal cells on the principle of lock and key. This theory that uses the analogy of lock and key was first postulated in 1894 by Nobel laureate in chemistry Hermann Emil Fischer ( ), to explain how the substrate and enzyme work together in enzymatic reactions. Thus, the architecture of the enzyme corresponds to the negative spatial form of the substrate. In a similar manner the receptors from the bacterial cells membranes are accessed by the signal molecules and bind together. That determines the receptor to become activated. This, in turn, triggers other 47

48 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA mechanisms that influence the gene transcription process. A new gene product, such as a virulence protein or an extracellular enzyme, is then released. As a results, a change in the social behavior of the bacterial population becomes evident (figure 2) (10,11). Quorum sensing is kind of a social feedback mechanism that is used by bacterial populations to optimize, synchronize and coordinate their behavior. Since the signal molecule produced by the bacterial cell can be detected by both the receptors of same type cells and by the cell itself, it was called autoinducer. Similarly, the process through which the desired behavioral response is attained is called autoinduction. The chemical signal is provided by low molecular mass molecules that are produced inside the bacteria and then delivered outside the outer membrane of the cell. The gradient of the extracellular concentration grows with the population density that is producing the signal molecules. The bacteria are able to sense the signal molecules and to reimport them into the cells, where they trigger the modulation of gene transcription. Thus all the bacterial cells become able to coordinate their response to the changing environment once a critical concentration has been reached due to the increasing of cell density to a threshold value. The quorum-sensing concept refers to the number of bacteria in the population, that is to the cell density, which triggers a modulation in gene transription (5, 13, 6, 14). 48 Fig. 3. Quorum-sensing switching the behavior of bacterial population (Li and Tian, 2012) The main requirements of quorum-sensing is that bacteria may produce, release and detect chemical signals (autoinducers). When there are just a few bacteria, the autoinducers level is low. The more bacteria grow and multiply, the more autoinducers are produced. They spread outside the cell emitting the signal in

49 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA all directions through diffusion. Thus, the further the autoinducers are from the cell, the lower their concentration and the weaker the signal. When a threshold value of the gradient of autoinducers is reached, the cell membrane receptors are activated due to saturation. The activated receptors induce the transcription of some specific genes on which new bacterial behavior is based, such as the production of a particular gene product (for instance, a virulence protein or an extracellular enzyme) (figure 3) (10,11). Bacterial language signal molecules Communication between bacteria can be very complex. Signaling can be autocrine the chemical agent binds to the receptors on that same cell, leading to changes in that cell or paracrine the signal molecule is produced to induce changes and to alter the behavior of nearby cells (12). As a mechanism of changing bacterial social behavior, quorum-sensing allows bacteria to modulate the expression of specific genes namely to restrict the gene expression at high cell densities and expand it at low cell densities. The purpose of quorum-sensing is to obtain some collective benefits through biofilm formation, increased antibiotic resistance or higher virulence, based on the local density of the bacterial population, either it is formed from a single type of bacteria or from diverse species. It will be of a little value for a few bacteria to make a gene product because its concentration would be too low to become effective. The benefit of quorum-sensing mechanism is to make use of such a regulatory process that ensures the bacterial density is high enough before a specific gene product is made and released in the environment, otherwise the effort of the cells to change their behavior would be useless. Quorum-sensing allows the bacterial population to increase in number before starting to produce a specific gene product (1, 3). There are various types of molecules that can be used as signaling agents between individual bacterial cells and different types of bacterial populations. Gram-negative and Gram positive bacteria use specific signal molecules, though a common chemical language is available for both. Signaling molecules that work in both Gram-negative and Gram-positive bacteria comes from a family of autoinducers known as autoinducer-2 (AI-2). Autoinducers are produced as a response to the modification of cell-population density. The concentration of the autoinducer increases proportionally with the density of quorum sensing bacterial cells. The quorum-sensing phenomenon allows both Gram-negative and Grampositive bacteria to sense one another, once a threshold number of bacterial cells is reached. As a result, the bacterial community may regulate a variety of physiological activities, such as virulence, symbiosis, biofilm formation, motility, antibiotic production. Thus, when bacteria detect these signal molecules, a stimulation effect, produced by altered gene expression, can be seen (2, 9). The most common signaling molecules for the two Gram types of bacteria are: 49

50 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA In Gram-negative bacteria the signal molecule is acyl homoserine lactone (AHL). When it reaches a threshold concentration in the environment due to a quorum of bacterial cells that produce it it binds and activates regulatory protein which then binds to a specific site of the cell DNA. The binding of this regulatory protein transcription activator results in production of the specific quorum-dependent protein as well as more enzymes to make AHL (figure 4) (13, 14, 15) Fig. 4. Schematic representation of quorum-sensing in Gram-negative bacteria (Waters and Bassler, 2005) In Gram-positive bacteria a precursor oligopeptide is cleaved into functional signal molecules of aminoacids. These molecules are actively transported out of the cell through a special transporter protein. When the signal oligopeptides reach a thershold concentration on the outisde of the cell, they are detected by a sensor protein on the surface of the cell. When the oligopeptide reacts with the sensor protein, the protein becomes phosphorylated on the inside of the cell membrane. The phosphate is then tranferred to a response regulator protein which allows it to bind to a specific site on the DNA. This binding results in alteration in the transcription of target genes. Quorum-dependent proteins such as virulence factors are produced (figure 5) (13, 14, 15) 50

51 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA Fig. 5. Schematic representation of quorum-sensing in Gram-positive bacteria (Waters and Bassler, 2005) Quorum-sensing circuits Parallel and arranged in series QS circuits The idea that bacteria might make use of various quorum-sensing circuits was first investigated and proved in Vibrio harveyi, a bioluminescent marine Gramnegative bacterium. Research showed that Gram-negative bacteria could communicate with multiple quorum-sensing signals, since Vibrio harveyi produces and responds to three distinct autoinducers. In a similar way to the mentioned bacteria works Vibrio cholerae, which is a human pathogen responsible for the endemic diarrheal disease cholera. Both V. harveyi and V. cholerae posses a quorum-sensing network made from three parallel quorum-sensing circuits (15). Pseudomonas aeruginosa, which is a common soil organism, can seriously affect cystic fibrosis (CF) patients and those having chronic respiratory infection. Using quorum-sensing, P. aeruginosa can control adhesion, biofilm formation, and virulence factor expression, making it difficult to combat the lungs infection. In contrast with the quorum-sensing circuits found in vibrios, P. aeruginosa uses regulatory systems which are arranged in series. Both the above quorum-sensing networks bases their activity on multiple signals acting synergistically (4, 9). Other types of QS circuits There are also quorum-sensing networks whose architecture is such that the signals antagonize one another. In all the above presented quorum-sensing circuits bacteria can move from a set of low cell density behaviors to a different set characteristic for a high cell density level. However, there are circuits that permit reversion to the original set of 51

52 52 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA behaviors, the so called quorum-sensing circuits with on-off switches (found, for instance, in Gram-positive bacterium such as Streptococcus pneumoniae) (4, 9). Agrobacterium tumefaciens is the causal agent of crown gall disease formation of tumours in plants. The bacteria can do that through transducing (transfer and integration) of a tumor-inducing (TI) plasmid into plant cells. The plant tumors produce molecules called opines, which are a good nutrient for the A. tumefaciens. The quorum-sensing circuit is responsive to host cues, since it is activated only by simultaneous signals produced by both plant and bacteria. The mobilization of the tumor-inducing plasmid is possible only in the proximity of the plant because it requires detection of opines by a cytoplasmic receptor (4, 9). Quorum-quenching In their struggle for existence and dominance, bacteria can develop strategies to intercept and hijack the quorum-sensing mechanisms of their bacterial neighbors. Quorum-quenching (QQ) can be done by any mechanism that can effectively interfere with any main processes in quorum-sensing. By disrupting the quorum-sensing processes of another bacterial species give an advantage to the bacteria that uses quorum-quenching mechanisms. Such mechanisms are present in many prokaryotic and eukaryotic organisms (4). The general purpose of using quorum-quenching is the global control of the physiology and activity of bacterial populations, especially where is a competition for limited resources, such as nourishment or vital space. Researchers found that secondary metabolites and degrading enzymes produced by various organisms, such as algae, and microorganisms can interfere with quorum-sensing. Findings showed that various plants can exert and inhibitory activity on quorum-sensing. Extracts from plants used as spices can inhibit quorum-sensing activity. For instance, Curcuma longa L. (turmeric) produces curcumin, which inhibits the expression of virulence genes of Pseudomonas aeruginosa, while extracts from fruits, such as raspberry (Rubus idaeus L.), blueberry (Vaccinium angustifolium Aiton), and grape (Vitis sp.), can inhibit AHL activity (8, 7). Conclusion According to a global report of World Health Organization from 2014, humanity entered in a post antibiotic era. Bacterial microorganisms develop resistance toward antibiotics. It is a common fact that many bacterial strains are multi-drug resistant (MDR). It seems that bacteria are enough well equipped to take on all the battles. Given this context, quorum-sensing inhibitors could be a promising new generation of drugs. Virulence, multiplication rate, biofilm formation, antibiotic resistance mechanisms could be targeted by simply intercepting and hijacking the main quorum-sensing processes. Therefore, the main purpose would be to develop non-competitive inhibitors of quorum-sensing systems (7, 8, 16).

53 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA References 1. ***, Bacterial Communication Systems, _communications.html 2. Davies, D.G., Parsek, M.R., Pearson, J.P., Iglewski, B.H., Costerton, J.W., Greenberg, E.P., The involvement of cell-to-cell signals in the development of a bacterial biofilm, Science, 1998 April De Kievit, T.R., Iglewski, B.H., Bacterial Quorum Sensing in Pathogenic Relationships, American Society for Microbiology, Infection and immunity, May 2017, 85(5), 4. Dong, Y.H., Wang, L.H., Zhang, L.H., Quorum-quenching microbial infections: mechanisms and implications, Philos Trans R Soc Lond B Biol Sci Jul 29; 362(1483), , PMC / 5. Fuqua, W.C., Winans, S.C., Greenberg, E.P., Quorum sensing in bacteria: the LuxR LuxI family of cell density-responsive transcriptional regulators. J. Bacteriol., 1994, 176, Gera, C., Srivastava, S., Quorum-sensing: The phenomenon of microbial communication, Current Science, 2006, 90(5), 7. Hong, Kar-Wai, Koh, Chong-Lek, Sam, Choon-Kook, Yin, Wai-Fong, Chan, Kok-Gan., Quorum Quenching Revisited From Signal Decays to Signalling Confusion, Sensors 2012, 12, Kalia, Vipin Chandra, Quorum sensing vs Quorum Quenching: a Battle with No and in Sight, Springer India, Kaufmann, G.F., Sartorio, R., Revisiting quorum sensing: discovery of additional chemical and biological functions for 3-oxo-N-acylhomoserine lactones, Proc. Natl Acad. Sci. USA, 2005, 102, Li, Y.H., Tian, X., Quorum Sensing and Bacterial Social Interactions in Biofilms, 2012, 12(3), Miller, M.B., Bassler, B.L., Quorum sensing in bacteria, Annu. Rev. Microbiol., 2001, 55 (1), Pandit, N.K., Soltis, R.P., Introduction To The Pharmaceutical Sciences: An Integrated Approach, Lippincot Williams & Wilkins, Vijayalakshmi, M., Quorum Sensing The Language of Bacteria, lecture at School of Chemical and Biotechnology, SASTRA University, Vijayalakshmi, M., Quorum Sensing in Gram-negative and Gram-positive bacterial systems, lecture at School of Chemical and Biotechnology, SASTRA University, Lecture%202.pdf 53

54 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA 15. Waters, C.M., Bassler, B.L., Quorum Sensing: Cell-to-Cell Communication in Bacteria, Annu. Rev. Cell. Dev. Biol., 2005, 21, , * * * WHO, Antimicrobial Resistance. Global Report on Surveillance, World Health Organization, 2014, _eng.pdf 54

55 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA SANGUINE BIOCHEMICAL ASPECTS OF PORCINE PROLIFERATIVE ENTEROPATHY ANCA SOFIANA HULEA, ANDREEA IONELA GARTNER, ANDREEA IOANA MERNEA, CORINA PASCU, C.I. HULEA, V. HERMAN Banat s University of Agricultural Sciences and Veterinary Medicine King Michael I of Romania from Timisoara, Faculty of Veterinary Medicine, Calea Aradului, 119, Timisoara , Romania viorel.herman@fmvt.ro Summary To highlight blood biochemical aspects of porcine proliferative enteropathy, it was taken blood from swine with clinical sign of disease and after randomized selected, 12 samples were subject to examination. Were determinate total protein, albumin, the albumin/globulin ratio, GOT, GPT, GGT, total bilirubin, creatinine, urea, potassium, calcium, magnesium. Of all studied parameters it was observed a significant modification of serum protein and hepatic transaminase values, compared to the physiological limits, in most cases. Decrease of total serum protein and albumin/globulin ratio observed in more than 50% of the cases can be the results of malabsorption causing weigh losses. These protein values modification were been accompanied by increase of hepatic transaminase values, more than 66% of the samples had GOT values higher than the normal one and 75 % of the studied samples presented a greater values of GPT than the physiological one. Keywords: porcine proliferative enteropathy, biochemical aspects, Lawsonia intracellularis The infection with intestinal bacterium Lawsonia intracellularis, the aetiological agent of porcine proliferative enteropathy (5, 6), it is known that can produce significance economic losses through young breeding and growingfinishing pigs especially due the lower average daily gain, but the pathogenesis of the disease remain unclear. It is believed that the bacterium is capable to infect mitotically active epithelial cells of intestinal crypts and then multiply and spread in these cells as it divide (1, 4, 9). However, the implications of infection on the whole organism are not elucidated yet. Although, porcine proliferative enteropathy, currently, raises numerous questions whose response has not been yet elucidated, it is known that the prevalence of the infection is higher through worldwide farms (3, 7, 10), present of disease being proved also in Romania (8). Materials and methods To highlight sanguine biochemical aspects of proliferative enteropathy, blood samples were taken by auricular and jugular vein puncture from all swine that presented clinical sign of disease. Of all collected samples, 12 samples were randomized selected to perform biochemical examination. 55

56 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA Biochemical examination of blood was carried out in Bioclinica laboratories. Were determinate only some of biochemical parameters, reminding about total protein, albumin, the albumin/globulin ratio, GOT, GPT, GGT, total bilirubin, creatinine, urea, potassium, calcium, magnesium. The examined samples were grouped into several categories depending of modified biochemical parameters that were found. Total protein values allowed classification of samples into 5 categories: Swine category that had total protein values between 3,50-4,00 g/dl; Swine category that had total protein values between 4,01-4,50 g/dl; Swine category that had total protein values between 4,50-5,00 g/dl; Swine category that had total protein values between 5,01-5,50 g/dl; Swine category with normal total protein values. Depending of serum albumin values, studied samples were grouped into 4 categories: Swine category with values of serum albumin between 1,00-1,50 g/dl; Swine category with values of serum albumin between 1,51-2,00 g/dl; Swine category with values of serum albumin between 2,01-2,40 g/dl; Swine category that had normal vales of serum albumin. Based on serum globulin values, studied samples were framed into 3 categories: Category with serum globulin between 2,00-2,50 g/dl; Category with serum globulin values between 2,51-2,80 g/dl; Category with normal serum globulin values. Using the albumin/globulin ratio, studied samples were grouped into 3 categories: Swine category with albumin/globulin ratio values between 0,1-0,5; Swine category with albumin/globulin ratio values between 0,51-1; Swine category with normal values of albumin/globulin ratio. According to GOT obtained values, serum samples were categorized into 4 categories: Swine group with normal GOT values; Swine group with GOT values between 49,8-70, 0 UI/l; Swine group with GOT values between 70,01-80,0 UI/l; Swine group with GOT values more than 80,01 UI/l/. GPT obtained values allowed grouping serum samples into 3 categories: Group of samples with normal values of GPT; Group of samples with values of GPT between 50,01-70 UI/l; Group of samples with values of GPT between 70,01-82,00UI/l. 56

57 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA Results and discussions Biochemical exam of serum samples revealed no values modification of GGT, creatinine, urea, calcium, potassium and magnesium. Regarding to total bilirubin values it was observed that 3 cases presented modification of this parameter, reaching to values of 1,71 µmol/l in two cases and 1,24 µmol/l in one case, compared to normal values of 0-0,85 µmol/l. From all studied biochemical parameters, it seems to serum protein (table 1) and hepatic transaminase (GOT and GPT) (table 2) suffered significant modification. Serum protein values of studied samples Total protein g/dl No. of samples Percentage of samples 3,50-4,00 g/dl 1 8,3% 4,01-4,50 g/dl 3 25,0% 4,51-5,00 g/dl 7 58,4% 5,01-5,5 g/dl 1 8,3% Normal values (5,51-6,50 g/dl) 0 0% Albumin g/dl No. of samples Percentage of samples 1,00-1,50 g/dl 2 16,7% 1,51-2,00 g/dl 6 50,0% 2,01-2,40 g/dl 4 33,3% Normal values (2,41-3,00 g/dl) 0 0% Globulin g/dl No. of samples Percentage of samples 2,00-2,50 g/dl 5 41,7% 2,51-2,80 g/dl 7 58,3% Normal values (2,8-3,8 g/dl) 0 0% Albumin/globulin ratio No. of samples Percentage of samples 0,1-0,5 2 16,7% 0, ,3% Normal values 1,1-1,5 0 0% Table1 57

58 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA Hepatic transaminase values of serum tested samples GOT UI/l No. of samples Percentage of samples Normal values ( UI/l) % UI/I % UI/l 1 8.3% over 80.1 UI/l 1 8.3% GPT UI/I No. of samples Percentage of samples Normal values (31-58 UI/) 0 0% UI/l % UI/l % Table 2 Decreased serum protein can be explained by malabsorption and existing of an enteropathy that cause protein losses, as the nephrotic syndromes are excluded based on normal values of creatinine and urea. As it observed in fig. 1, more than a half of tested samples presented values of total protein between g/dl and 25% of studied samples had values of total protein between g/dl. Only 1 sample, representing 8.3%, reaching values of total protein close to the normal one. Fig. 1. Percentage distribution of total protein values on categories Serum albumin values were also lower than the normal limits, for all tested samples. A half (6 samples) of studied samples presented values of albumin between 58

59 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA g/dl, 2 samples (16.7%) had values of this parameter between g/dl and 4 samples (33.3%) reaching values between g/dl (fig. 2a). Such as albumin values, globulin values were lowers the physiological limits, phenomenon observed in case of all tested samples. As it presented in fig 2b, more than a half (7 samples or 58.3%) had globulin values no more than 2.80 g/dl and 5 samples (41.7%) revealed values lower than 2.50g/dl. Because albumin and globulin values were decrease compared to the physiological limits, it is understood that albumin/globulin ratio was subunit also. a b Fig. 2. Distribution of albumin values (a) and globulin values (b) on categories Hepatic transaminase values were also modified, being increase compared to the physiological limits. Thus as showed in fig. 3a, from the 12 studied samples, only 2 (16.7%) had values of GOT within normal limits, while most of them (8 samples or 66.7%) were more than 20 UI/l increase compared to physiological values and one (8.3%) of the sample reaching high level (414 UI/l). About GPT values, limits were slightly increase compared to the physiological one. Most of the samples (75%) had values of GPT between UI/l, being with 10 UI/l higher than normal limits (fig. 3b). Only 3 samples (25%) presented values of GPT greater than 70.00UI/l, but have not reached worrying levels. These results demonstrate that the changes caused by Lawsonia intracellularis infection are digestive. Bilirubin is produced in macrophages by the enzymatic catabolism of the hem fraction of hemoproteins, and increased beyond normal limits may suggest hemolysis, hepatocellular jaundice, extrahepatic biliary obstruction, and even viral hepatitis (2). For the differential diagnosis of these conditions, it is necessary to determine the direct and indirect bilirubin, the results being corroborated with those of the hemoleucogram in the case of hemolysis suspicion or liver transaminase 59

60 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA values in case of suspicion of hepatocellular jaundice, viral hepatitis or extrahepatic biliary obstruction. a b 60 Fig. 3. Percentage distribution of GOT (a) and GPT (b) values on categories Although blood counts were not possible based on hepatic transaminases, which were increased compared to the normal values, it is suspected that increased bilirubin for the 3 samples is due to an extrahepatic biliary obstruction. Decreased of serum protein caused by malabsorption/maldigestion justifies the low of daily average gain. Physiologically, GOT is found in tissues such as heart, skeletal muscles, liver and encephalus, so an increase of serum value does not always mean a liver disease. Instead, GOT correlation with increased GPT, given that GPT high concentrations are found in the liver and reduced amounts in the heart and kidney, may suggest pathological processes of varying degrees of hepatocytes (2, 11). So, the proliferation occurs in small intestine as consequence of Lawsonia intracellularis infection cause slightly hepatocytes alteration, translated by increase serum transaminase values, probably due the inappropriate drainage of the bile. Conclusions The biochemical examination of tested serum revealed total protein changes, 58,33% of the examined samples revealed total protein values between 4,51-5,00 g/dl, on average with 0,75 g/dl lower compared to normal values. All studied samples revealed values of serum albumin and globulin lower than normal values, so the albumin/globulin ratio had a subunit value, a phenomenon due to malabsortion and maldigestion, which explains body weakening; even the food intake was considered adequate. Hepatic transaminase values were increased for the most of the studied samples, so 66.67% of investigated GOT values were higher by 20UI/l compared to

61 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA normal limits, whereas 75% of serum samples the GPT values was higher by 10UI/l compared to physiological one. Acknowledgements This study was realised using the support and infrastructure project Dezvoltarea infrastructurii de cercetare, educaţie şi servicii în domeniile medicinei veterinare şi tehnologiilor inovative pentru RO 05, cod SMIS-CSNR References 1. Boutruop, T.S., Boesen, H.T., Bove, M., Agerholm, J.S., Jemsen, T.K., Early pathogenesis in porcine proliferative enteropathy caused by Lawsonia intracellularis, Journal of Comparative Pathology, 2010, 143 (2-3), Falcă, C., Ciorba, Gh., Tehnici de examinare clinică și paraclinică la animale, Ed. Mirton, Timișoara, Hardge, T., Wang, J., Yang, H., Liu, P., Song, C.H., Keller, C.H., - The prevalence of Lawsonia intracellularis infection in Chinese pig farms, Proceedings of the 19th IPVS Congress, Copenhagen, Denmark, 2006, Jacobson, M., Felstrom, C., Jensen-Waern, M., Porcine proliferative enteropathy: An important disease with questions remaining to be solved, The Veterinary Journal, 2009,184(3), Lawson, G.H., McOrist, S., Jasni, S., Mackie, R.A., Intracellular bacteria of porcine proliferative enteropathy: cultivation and maintenance in vitro, J. Clin. Microbiol.,1993, 31(5), McOrist, S., Gebhart, C,J., Boid, R., Barns, S.M., Characterization of Lawsonia intracellularis gen. nov., sp., the obligately intracellular bacterium of porcine proliferative enteropathy, Int. J. Syst. Bacteriol., 1995, 45(4), Paradis, M.A, Gottschalk, M., Rajic, A., Ravel, A., Wilson, J.B., Aramini, J., McClure, C.A., Dick, C.P., Seroprevalence of Lawsonia intracellularis in different swine populations in 3 provinces in Canada, Can. Vet. J., 2007, 48(1), Surpat, Anca Sofiana, Pascu, Corina, Degi, J., Brezovan, Diana, Savici, Jelena, Morariu, S., Herman, V., Seroprevalence of Lawsonia intracellularis infection in fattening pigs, Scientific Works. C Series.Veterinary Medicine, București, 2013, 19(4), Vannucci, F.A., Gebhart, C.J., Recent advances in understanding the pathogenesis of Lawsonia intracellularis infections, Vet. Pathol., 2014, 51(2), Wattanaphansak, S., Rurgkhum, S., Assavacheep, P., Luengyosluechakul, S., Gebhart, C., Seroprevalence of Lawsonia intracellularis infection in swine breeding herds in Thailand, Proceedings of the 5 Asian Pig Veterinary Society Congress, Pattaya, Thailand, visited in

62 LUCRĂRI ŞTIINŢIFICE MEDICINĂ VETERINARĂ VOL. L (3), 2017, TIMIŞOARA CHARACTERISATION OF AN EPISODE WITH CANINE HERPESVIRUS INFECTION IN A KENNEL SHEPHERD MIORITIC DOG IONICA IANCU, J. DÉGI, CORINA PASCU, N. CĂTANĂ, V. HERMAN Banat University of Agricultural Sciences and Veterinary Medicine King Michael I of Romania from Timisoara, Faculty of Veterinary Medicine, , Calea Aradului 119, Timişoara, Romania Ifodor2001@yahoo.com Summary Canine herpesvirus infections (CHV) is caused by an alpha herpesvirus, which causes reproductive disorders, infertility, abortion and neonatal mortality. This study was conducted in a kennel Shepherd Mioritic dog, the puppies resulting from two females, died in a proportion of 66% in the first 48 hours after birth. The adult dogs did not show clinical signs of disease. Serological examination by reacting immunofluorescence, confirmed the presence of antibodies antiherpesvirus canine, to females with reproductive problems. Key words: herpesvirus canin, immunofluorescence, antibody. Canine herpesvirus is produced by an alpha herpesvirus, which is part of the Herpesviridae family, and produces a number of clinical manifestations: respiratory, ocular, reproductive, and neonatal mortality in puppies (5). The disease may evolve as such or in combination with other bacterial infections (Bordetella bronchiseptic) or viral ones such as Parainfluenza. The disease can be transmitted by respiratory, ocular and transplacentar way (4). In the case of pregnant females, the virus may pass from the fetal annexes to the fetus and may produce, depending on the stage of gestation, embryonic resorption, fetal resorption, abortion, mummification of the offspring, infertility, premature births, in proportion of 100% (2, 4, 6). In the anatomopathological examination of the puppies that were born dead, necrotic lesions can be seen in several organs: liver, spleen, lung, intestines, thymus, brain, stomach, myocardium, pancreas, adrenal glands and kidneys. Also, haemorrhages in the chest and abdominal cavity can be observed (3). Seroprevalence in the case of pet dogs varies from one country to another, in England 94%, in the Netherlands 40%, and in the US and Switzerland 6% of the dogs examined were seropositive to CHV infection (1). A little amount of data on CHV infection is reported in Romania. Considering these aspects, the purpose of this study was the confirmation of the diagnosis of canine herpesvirus, in a Mioritic Romanian Shepherd's kennel, where breeding problems were recorded. 62

63 LUCRĂRI ŞTIINŢIFICE MEDICINĂ VETERINARĂ VOL. L (3), 2017, TIMIŞOARA Materials and methods In a Mioritic Romanian Shepherd's kennel, two of the breeding females had breeding problems in the period between February December 2015, as follows: Case 1. Female A was paired with male M1, and at first offspring she had 3 puppies who lived and were healthy. During the year, several dogs from the kennel participated in the country's chinological exhibitions with international participation. Case 2. Female A was paired again with male M1 the following year, and the second time she gave birth she had 5 live puppies and one was born dead. The five live puppies immediately started feeding and after a few hours they became apathetic, they refused to feed and died after 48 hours. Case 3. Female A was subsequently paired with a male M2 from another kennel, but in this case, on the ultrasound fetal resorption was observed. Case 4. Female A was paired with another male M3 from the same kennel and she gave birth to 4 puppies, which after the first feeding refused to feed anymore and died in the first two days. Case 5. Female B was paired with a male M4 and the first time she gave birth she had 4 puppies that lived. Case 6. Female B was paired with the same male M2 and the second time she gave birth she had 4 live puppies, which died within 2-3 days. The dead puppies were subjected to the anatomopathological examination. From the two females, blood samples were collected 3 weeks after giving birth for the serological examination. 5 ml of blood from the cephalic vein were harvested, placed in tubes, left at room temperature for one hour, then centrifuged, separated, processed and examined. In order to highlight the anti-brucella canis antibodies, an etiological agent that may be involved in breeding problems, the serums were examined using the rapid diagnostic kit C. Brucella Ab. Using the Mega Fluo CHV kit, the serums were tested to detect IgG, anti CHV antibodies and examined by indirect immunofluorescence reaction. The lamellae were read under fluorescence microscope. Results and discussions On the clinical examination of the adult dogs from the kennel, during the course of the outbreak, there were no clinically obvious signs that would draw the attention of the breeder, possibly some faint respiratory symptoms, which occurred in the cold season. Newborn pappies, immediately after the first suckle refused to feed anymore, became anorexic, refused to suck, were initially agitated and crying, later became lethargic, presented hypothermia, were rejected by their mother, then became rigid, lost weight and died. 63

64 LUCRĂRI ŞTIINŢIFICE MEDICINĂ VETERINARĂ VOL. L (3), 2017, TIMIŞOARA From the 5 birthings, 21 puppies resulted, of which 14 puppies (66, 66%) died in the first days after they were born. Clinical signs found by us are similar to those described in the literature, which also mentions nervous signs, deafness and blindness in puppies that have been infected after 2-3 weeks of age (4). During the anatomopathological examination, of all the puppies, severe haemorrhagic lesions were found in the thoracic and abdominal cavity (Fig 1). Internal necrotic haemorrhagic lesions in the liver (Fig. 3) kidney and spleen (Fig 4) have been observed during the internal organ examination. Pulmonary lesions and hemorrhage were observed during the examination of the lungs (Fig 2). The presence of necrosis outbreaks and multifocal haemorrhages in the internal organs found in the necropsy examination are in correlation with those described in the literature (3). The serological exam performed on the serum samples collected for the detection of Brucella canis antibodies was negative. To confirm CHV infection in the kennel, serum samples were tested by indirect immunofluorescence reaction using the Mega Fluo CHV kit according to the manufacturer's recommendations. In this study, the serums were tested only at a 1:80 (cut off) dilution. According to the kit manufacturer's recommendation, IgG titers of 1: 80 and higher are considered positive and reflect the infection in an undetermined time. The lamellae revealed antibody titers of anti-herpesvirus canine antibodies at all serum samples examined. Our study confirmed the presence of CHV infection in the Mioritic Romanian Shepherd's kennel studied. The large number of seropositive dogs in CHV infection has been reported by several authors worldwide (1, 3). Dogs older than 3 years, those participating in exhibitions and those used for breeding purpose have a higher infection exposure rate than younger ones because the sexual path is considered the main route of infection (7). Based on anamnesis, clinical signs and anatomopathological lesions, the clinician may suspect the disease, but the etiological confirmation is not always performed. This study contributes to the identification and raising awareness of the existence of a common cause of neonatal deaths in puppies and recommends the introduction of control measures and vaccination, which is currently not practiced in Romania. For kennels with a history of CHV infection, preventive vaccination of breeding females is recommended. 64

65 LUCRĂRI ŞTIINŢIFICE MEDICINĂ VETERINARĂ VOL. L (3), 2017, TIMIŞOARA Fig. 1. Severe haemorrhagic lesions in the thoracic and abdominal cavity. Fig. 2. Haemorrhagic lesions in the lungs 65

66 LUCRĂRI ŞTIINŢIFICE MEDICINĂ VETERINARĂ VOL. L (3), 2017, TIMIŞOARA Fig. 3. Haemorrhagic lesions in the liver Fig. 4. Haemorrhagic lesions in the kidney and spleen Conclusions The results of this study have shown the presence of CHV infection in a Romanian Mioritic Shepherd's kennel. Neonatal mortality in puppies was 66.66% The serological test confirmed the presence of canine herpesvirus infection at titers of 1:80 by indirect immunofluorescence reaction. 66

67 LUCRĂRI ŞTIINŢIFICE MEDICINĂ VETERINARĂ VOL. L (3), 2017, TIMIŞOARA Acknowledgements This study was realised using the support and infrastructure project Dezvoltarea infrastructurii de cercetare, educaţie şi servicii în domeniile medicinei veterinare şi tehnologiilor inovative pentru RO 05, cod SMIS-CSNR References 1. Babaei, H., Akhtardanesh, B., Ghanbarpour, R., Namjoo, A., Serological Evidence of Canine Herpesvirus 1 in dogs of Kerman City, South east of Iran, Transboundary and emerging diseases, 2010, 57, Cargnelutti, J.F., Masuda, E.K., Neuls, M.G., Weiblen, R., Flores, E.F., Outbreaks of canid herpesvirus 1 disease in puppies in southern Brazil. Pesquisa Veterinária Brasileira, 2015, 35(6), Carmichael, L. E., Greene, C., Canine herpesvirus infection. In: Greene, C. E. (ed.), Infectious Diseases of the Dog and Cat, WB Saunders, Philadelphia Decaro N., Martella V. & Buonavoglia C., Canine adenoviruses and herpesvirus, Vet. Clin. North Am., Small. Anim. Pract. 2008, 38, Evermann, J.F., Ledbetter, E.C., Maes, R.K., Canine reproductive, respiratory, and ocular diseases due to canine herpesvirus, Vet. Clin. North. Am., Small. Anim. Pract., 2011, 41, Hashimoto, A., Hirai, K., Suzuki, Y., Fujimoto, Y., Experimental transplacental transmission of canine herpesvirus in pregnant bitches during the 2nd trimester of gestation, Am. J. Vet. Res., 1983, 44, Ronsee, V., Verstegen, J., Onclin, K., Farnir, F. Poulet, H., Risk factors and reproductive disorders associated with canine herpesvirus-1 (CHV-1), Theriogenology, 2004, 61,

68 68 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017 TIMISOARA INFLUENCE OF ACID ENVIRONMENT ON ANTIBIOTIC SUSCEPTIBILITY OF E. COLI STRAINS ISOLATED FROM TURKEYS I. IONUŢ, L. KÖBÖLKUTI, MARINA SPÎNU University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, , 3-5 Mănăştur Street, Cluj-Napoca, Romania ionut.v.ionut@gmail.com Summary E. coli is a major pathogen in the animal health industry, and one of the bacteria that very rapidly develop resistance to antibiotics. The presence of multi-drug-resistant strains is becoming more common, and the discovery of new antibiotics by researchers is not able to keep up with the rate of evolution. The present study aims at trying to explore the possibility that susceptibility to antibiotics is augmented by an acidified living environment. The starting point of this prospect is the fact that feed acidifiers used prior or in the same as antibiotics increase the efficiency of therapy. A number of 50 E. coli samples isolated from intensive growth systems turkeys where tested. After a 24 h and 72 h exposure time to ph values ranging from 4 to 6.5, and performing susceptibility tests by Kirby-Bauer method prior and after the exposure time. The antibiotics used are of both clinical importance and experimental ones: florfenicol, lincomycin + neomicyn, nitrofurantoin, ciprofloxacin, enrofloxacin, doxycycline, amoxiciline + clavulanic acid and furazolidone. Results show a small variation in susceptibility to antibiotics, the greatest difference seen being of 6 mm in inhibition diameters between original sample strain and acid exposed strain. Occurrence of such differences do not vary with ph nor antibiotic tested, and can be seen as positive or negative variations from initial strain inhibition diameters. Keywords: acid environment, antibiotic susceptibility, E. coli, turkeys Escherichia coli is a commensal bacterium of the intestinal tract of various animal species and humans. Among the many harmless strains, pathogenic isolates exist; and such strains can be harmful, especially for young animals but even for fully immunocompetent adults as well as for humans (6, 10). Virulent E. coli strains can be classified into two major groups, intestinal and extraintestinal pathogens, with the latter represented mainly by those variants causing neonatal meningitis and uropathogenic ones. The first category comprises various pathotypes, including enterotoxigenic, enteropathogenic, enteroaggregative, enteroinvasive and Shigatoxin producing E. coli (STEC), with the last group including the subgroup of enterohemorrhagic E. coli (2, 7). Especially strains in the STEC category have the capacity to inflict major damage on different organs, depending on the presence of several virulence factors, and STEC pathotypes are of interest for epidemiological surveillance. Some STEC strains have zoonotic potential, with a reservoir mainly in ruminants (3, 4, 10).

69 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017 TIMISOARA Antibiotic resistance is a commonly seen problem with this bacteria, being one of the most studied in this regard. Most recently the development of multi drug resistant strains have taken the focus of studies due to the negative implications on animal and human health. Resistance appears after exposure to sublethal doses of drugs after which bacteria develop general or specific coping mechanisms (excretion pumps, antibiotic action site alteration, synthesis enzymes directed against the chemical structures of drug molecules) (5). Dealing with the phenomenon in practice is difficult because after acquiring resistance the bacterial strain maintains and diffuses this characteristic onto its descendants and other bacteria, even from other species. The bacteria that was capable of developing resistance to one antibiotic is genetically favored to do so with others thus acquiring multi drug resistance and in extreme cases pan drug resistance, E. coli being one of the candidates for these achievements. Therefore antibiotic resistance in the antibiotic era is an important problem that is rapidly evolving to a crisis if methods of control and limitation are not found and implemented. Such measures are reducing and avoiding, when possible, the use of antibiotics and using them as directed by an antibiogram. There are few alternatives to antibiotics, the more rational and feasible approach being focusing more on prevention rather than treating in advance. One way for preventing bacterial disease of intestinal origin is diet acidifiers (9). The use of feed acidifiers in poultry is a common practice among farmers in the industry. It seems the method works as a growth promoter by inhibiting bacterial development in the gut of the birds. The general view upon the topic and its mechanism of action is that organic acids added in the drinking water reduce the bacterial load, primarily in the drinking water itself and consequently reduces the bacterial load in the intestinal tract, thus minimizing exposure of the animal to them (2, 5). This strategy is the foundation for reducing antibiotic use in food animals and only implementing antibiotic therapy when necessary. The use of other feed additives in animals mostly follows the same principle in the global effort to reduce and if possible to avoid antibiotic drugs in the diet of food production animals. In addition to organic acids as feed additives for growth promotion, enzymes and oligosaccharides have been implemented (9). In one study it was determined that lactic acid treatment reduces bacterial count of six nstec serogroups of E.coli, antibiotic-susceptible and multidrug-resistant Salmonella (5). In 2000, Alakomi et al. (1), utilizing a fluorescent-probe uptake assay and sensitization to bacteriolysis, studied the effect of lactic acid, EDTA and hydrochloric acid on the outer membrane permeability of Escherichia coli, Pseudomonas aeruginosa, and Salmonella enterica. They found that a ph value of 4 lactic acid caused prominent permeabilization in each species, the effect being stronger than that of HCl or EDTA. The permeabilization by hydrochloric and lactic acid was partly suppressed by MgCl 2. Lactic acid sensitized E. coli and Salmonella to the lytic action of sodium dodecyl sulfate (SDS) more efficiently than hydrochloric acid did, whereas both acids sensitized P. aeruginosa to SDS and to Triton X-100. P. aeruginosa was 69

70 70 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017 TIMISOARA effectively sensitized to lysozyme by lactic acid and by hydrochloric acid. Considerable proportions of lipopolysaccharide were liberated from Salmonella by these acids; analysis of liberated material by fatty acid analysis and by electrophoresis showed that lactic acid was more active than HCl or EDTA in liberating outer membrane lipopolysaccharide. Thus, lactic acid, in addition to its antimicrobial property due to the lowering of the ph, also functions as a permeabilizer of the gram-negative bacterial outer membrane and may act as a potentiator of the effects of other antimicrobial substances (1,6). The present study aims at trying to explore the possibility that susceptibility to antibiotics is augmented by an acidified living environment. The starting point of this prospect is the fact that feed acidifiers used prior or in the same as antibiotics increase the efficiency of therapy. Materials and methods The study was performed using E.coli isolates from turkey meat farms in central Romania, 50 strains were taken from 3 rounds of sampling corresponding to 3 production cycles. Sampling was performed during the necropsy of young dead birds showing hemorrhagic-necrotic enteritis, peritonitis, airsacculitis, pericarditis, severe dehydration, unabsorbed vitellus, polyserositis. The presence of E. coli in the majority of cases in the intestine, abdominal airsacks, spleen, liver and bone indicated a coli-septicemia. The isolation protocol used was using sterile technique to harvest samples from internal organs (liver, lung/airsack, pericardium, femur medula) and plaiting on MacConkey agar (OXOID CM0115). Lactose positive colonies were then plated on Levine agar (eosin methylene blue agar, OXOID CM 0069) and Brilliance E. coli / coliform selective agar (OXOID CM 1046). The characteristic green sheen/ purple colonies were then plated on nutrient agar and a oxidase test was performed. The oxidase negative presumptive E. coli strains were confirmed by API 20E (biomérieux, France) multi-test system. After the biochemical confirmation for each strain an antibiogram was carried out using the Kirby-Bauer disk diffusion susceptibility test (8). Antibiotics used were selected to be of both practice importance and some for experimental purposes, thus the following antibiotic discs were implemented: florfenicol (30μg), lincomycin + neomicyn (75μg), nitrofurantoin (300g), ciprofloxacin (30μg), enrofloxacin (5μg), doxycycline (30μg), amoxiciline + clavulanic acid (30μg) and furazolidone (50μg). The next step was preparing the acidified medium using standard liquid broth (neutral ph) and a commercial feed acidifier: VerSal Liquid (formic acid (E 236) 61%, lactic acid (E 270) 8%, propionic acid (E 280) 5%, citric acid (E 330) 3%, acid acetic (E 260)). Using sterile glassware and a ph-meter the liquid broth was titrated to ph values of 6.5, 6, 5.5, 5, 4.5 and 4 with the commercial acid mix. The acid broth solutions were then sterilized in the autoclave 15 minutes at 121ºC, in stock solution jars. For each strain tested a series of 6 acid broth test tubes (ph 6.5 to 4) containing 4ml solution was inoculated and incubated for 72h at 37ºC in aerobic

71 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017 TIMISOARA conditions. At the 24h mark a subculture from each test tube was carried out onto normal broth and from these a Kirby-Bauer disk diffusion susceptibility test was made using the same antibiotics as described before. After the 72h elapse period another susceptibility test was conducted, all three assays were recorded and compared. Results and discussions The strains were codified using the roman letters I, II, III for first, second and third batch of samples and for each of them in consecutive order, each strain was given a number. Then each Kirby-Bauer set of assays was coded with letters: no letter for initial test, a) for first test (after 24h exposure time) and b) for the second test (after 72h exposure time). The antibiotics used are abbreviated as follows: Florfenicol= FFC, Lincomycin + Neomycin = LCN, Nitrofurantoin = F, Ciprofloxacin = CIP, Enrofloxacin = ENF, Doxycycline = DO, Amoxicillin Clavulanic acid = AMC, Furazolidone = FX50. Comparison between initial, first and second susceptibility tests were performed according to the example in Table 1, for each isolate, showing the diameter of inhibition in millimeters. The initial antibiogram for each strain is shown in table 2 for the first batch, table 3 for the second batch and table 4 for the third batch. There were minimal differences between initial strain susceptibility and the one observed after the exposure to the acidified medium. In most cases the growth was completely inhibited by the ph value of 4, showing no growth in the subculture test tube after 24h. Similarities in susceptibility to antibiotics were seen between isolates from the same batch, pointing towards either selection under pressure of the same antimicrobials or maybe the same strain. Differences between initial and acid medium exposed strains in the following cases: I1 initial susceptibility to ciprofloxacin 27 to a max increase of 3 for various ph levels in I1a) and I1b), AMC from 16 to +3(19mm) and -3(13mm), FX 17 to +4(21mm) for I1a)pH4.5 and I1b)pH4.5. Significant differences (>5mm) were observed in only 4 strains II6 CIP from 30mm to II6a)pH5-24mm, II6a)pH mm, II6b)pH5.5 25mm; II7 from ENF 15mm to II7 a)ph6 20mm, II7a)pH5 20mm, II7b)pH5.5 21mm; III1 from FFC 21mm to III1b)pH6.5-15mm and III5 from LCN 12mm to III5a)pH4.5 16mm, III5a)pH4-16mm, III5b)pH5 15mm, III5b)pH4.5 16mm. 71

72 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017 TIMISOARA Strain Table 1 Comparison between the three susceptibility tests for strain i1 (first of batch one, diameters in mm) I1 I1a) ph 6.5 I1a) ph 6 I1a) ph 5.5 I1a) ph 5 I1a) ph 4.5 I1a) ph 4 I1b) ph 6.5 I1b) ph 6 I1b) ph 5.5 I1b) ph 5 I1b) ph 4.5 Antibiotic FFC N.G N.G. LCN N.G N.G. F N.G N.G. CIP N.G N.G. ENF N.G N.G. DO N.G N.G. AMC N.G N.G. FX N.G N.G. Table 2 Initial kirby-bauer assay for first batch of isolates (diameters in mm) FFC LCN F CIP ENF DO AMC FX I I I I I I I I I I I I I I I I I I1b) ph 4 72

73 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017 TIMISOARA Table 3 Initial kirby-bauer assay for second batch of isolates (diameters in mm) FFC LCN F CIP ENF DO AMC FX II II II II II II II II II II II II II II II II II Table 4 Initial kirby-bauer assay for third batch of isolates (diameters in mm) FFC LCN F CIP ENF DO AMC FX III III III III III III III III III III III III III III III III

74 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017 TIMISOARA Conclusions In the overview on the results of the experiment by exposure of E. coli clinical isolates to acid environments, the statistically significant change in susceptibility to antimicrobials could not be linked to a specific ph value. Significant differences ( 5mm) were observed at 8% of the isolates for a total of four antibiotics. Statistically not significant differences (2-4mm) were observed in 100% of the isolates for at least one antibiotic, probably due to slight differences in inoculum concentrations. Changes in antibiotic susceptibility do occur, but the causative factor or factors could not be identified, advocating the need for further study. References 1. Alakomi, H.L., Skytta, E., Saarela, M., Mattila-Sandholm, T., Latva- Kala, K., Helander, I. M., Lactic Acid Permeabilizes Gram-Negative Bacteria by Disrupting the Outer Membrane, Applied And Environmental Microbiology, 2000, Blanco, J., M. Blanco, J.E. Blanco, A. Mora, E.A. Gonzalez, M.I. Bernardez, M.P. Alonso, A. Coira, A. Rodriguez, J. Rey, J.M. Alonso, Usera M.A., Verotoxin-producing Escherichia coli in Spain: prevalence, serotypes, and virulence genes of O157:H7 and non-o157 VTEC in ruminants, raw beef products, and humans, Exp. Biol. Med. (Maywood), 2003, 228, Boerlin, P., McEwen, S.A., Boerlin Petzold, F., Wilson, J.B., Johnson, R.P., Gyles C.L., Associations between virulence factors of Shigatoxinproducing Escherichia coli and disease in humans. J. Clin. Microbiol. 1999, 37, Ethelberg, S., Olsen, K.E., Scheutz, F., Jensen, C., Schiellerup, P., Enberg, J., Petersen, A.M., Olesen, B., Gerner-Smidt, P., Molbak, K., Virulence factors for hemolytic uremic syndrome, Denmark. Emerg. Infect. Dis. 2004, 10, Fouladkhah, A., Geornaras, I., Yang, H., Belk, K.,, Nightingale, K.K., Woerner, D., Smith, G.C., Sofos, J., Sensitivity of Shiga toxin-producing Escherichia coli, multidrug-resistant Salmonella, and antibiotic-susceptible Salmonella to lactic acid on inoculated beef trimmings. J Food Prot. 2012, 75(10), Kaper, J.B., Nataro, J.P., Mobley, H.L., Pathogenic Escherichia coli. Nat. Rev. Microbiol. 2004, 2, Karin, Ballmer, Bozena, M., Korczak, P., Kuhnert, P., Slickers, R., Ehricht, Ha Chler, H., Fast DNA Serotyping of Escherichia coli by Use of 74

75 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017 TIMISOARA an Oligonucleotide Microarray, Journal of Clinical Microbiology, 2007, Markey, E.K., Leonard, F.L., Archambault, M., Cullinane A., Maguire D. Clinical Veterinary Microbiology, 2013, Elsevier, pg Owens, B., Tucker, L., Collins, M.A., McCracken, K.J., Effects of different feed additives alone or in combination on broiler performance, gut microflora and ileal histology, British Poultry Science, 2008, 49:2, Zweifel, C., Schumacher, S., Blanco, M., Blanco, J.E., Tasara, T., Blanco, J., Stephan, R., Phenotypic and genotypic characteristics of non- O157 Shiga toxin-producing Escherichia coli (STEC) from Swiss cattle. Vet. Microbiol., 2005, 105,

76 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA TRUEPERELLA PYOGENES ASSOCIATED LUNG ABSCESSES IN CALVES A CASE REPORT C.I. MATES, EMÖKE PÁLL, MARINA SPÎNU, MIHAELA NICULAE University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Faculty of Veterinary Medicine, Discipline of Infectious Diseases, , Cluj-Napoca, Calea Mănăștur Street no 3-5, România niculaemihaela1@gmail.com Summary Trueperella pyogenes (formerly known as Arcanobacterium pyogenes) is described as an opportunistic bacterium incriminated in miscellaneous pyogenic infections (mastitis, abscesses, pneumonia, lymphadenitis, septicemia, encephalitis, pyometra, prostatitis, orchitis, seminal vesiculitis, pericarditis, omphalitis). Previous case reports indicated this pathogenic ability in several domestic animals: ruminant, pigs, horses and dogs. This case report describes the etiological role of Trueperella pyogenes in calves pneumonia based on the isolation and identification of the bacterium from lungs and lymph nodes samples referred to a veterinary laboratory by a fattening calves farm. The outbreak was characterized by elevated morbidity and mortality, limited response to antimicrobial treatment, with animals presenting chronic respiratory signs and severe extended lung abscesses. The strain was found to be resistant towards several antimicrobial products: penicillin/streptomycin combination, ampicillin, amoxicillin and clavulanic acid, doxycycline, oxytetracycline, gentamicin and florfenicol; in vitro susceptibility was recorded only in case of cefquinome, marbofloxacin and enrofloxacin. Keywords: Trueperella pyogenes, calves, pneumonia Trueperella pyogenes (formerly known as Arcanobacterium pyogenes) is regarded as an opportunistic bacterium incriminated in miscellaneous pyogenic infections described in animals (3,4,7,9,11,14,15), but also human (6). The most commonly reported pathologies in animals are the following: mastitis (1,2,3,14), abscesses, pneumonia (5,12), lymphadenitis, septicemia, encephalitis, pyometra (15), prostatitis, orchitis, seminal vesiculitis, pericarditis, omphalitis) (3,4,7,9). Previous case reports indicated this pathogenic ability in several domestic animals: ruminant, pigs, horses and dogs (3,4,8,9). This study was aimed to describe the isolation of Trueperella pyogenes from calves presenting chronic pneumonia. The outbreak was characterized by elevated morbidity and mortality, limited response to antimicrobial treatment, with animals presenting chronic respiratory signs and severe extended lung abscesses. 76

77 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017 TIMISOARA Materials and methods Samples were submitted for examination to the diagnostic laboratory of the Infectious Diseases Discipline, Faculty of Veterinary Medicine Cluj-Napoca, Romania. The referred organs were represented by lungs and lymph nodes collected from calves. The animals originated from a fattening farm with relatively poor hygienic and welfare conditions were confirmed with repeated outbreaks of bovine respiratory diseases complex. The latest outbreak involved about 90% of the calves, with the animals displaying fever, lethargy, anorexia or reduced appetite, serous to mucopurulent nasal discharge, cough, and dyspnea. The animals were administrated at least two antimicrobial treatments (tulatromycin and marbofloxacin, enrofloxacin), with variable clinical efficacy. The samples were cultured on Blood agar (Oxoid Ltd., Hampshire, UK) for 48 hours at 37 C in 5% CO 2. Trueperella pyogenes identification was based on conventional bacteriological methods (Gram staining, colony morphology, haemolysis, catalase test, oxidase test, CAMP test. The in vitro antimicrobial susceptibility testing was performed using the standard Kirby-Bauer disc diffusion method according to CLSI guidelines. The following antimicrobials were tested: penicillin (P, 1IU), streptomycin (S, 10 µg), ampicillin (AMP, 10 µg), amoxicillin/ clavulanic acid (AMC, 20/10 µg), tetracycline (TE, 30 µg), doxycycline (DO, 30 µg), gentamycin (CN, 10 µg), enrofloxacin (ENR, 5 µg), marbofloxacin (MAR, 5 µg), cefquinome (30 µg), florfenicol (FFC, 30 µg) (susceptibility discs, Oxoid Ltd., Cambridge/UK). Results and discussions All samples (lungs and lymph nodes) were found microbiologically positive for the isolation of Trueperella pyogenes. The results of the in vitro antimicrobial susceptibility testing indicated resistance towards the following antimicrobial agents: penicillin/streptomycin combination, ampicillin, amoxicillin and clavulanic acid, doxycycline, tetracycline, gentamicin and florfenicol; in vitro susceptibility was observed only for cefquinome and two quinolones - marbofloxacin and enrofloxacin. The use of antimicrobials is a requirement given the nature of the infection, but the limited clinical efficacy can be explained based on several aspects; among these, the lesion characteristics - purulent and extended (Figure 1) suggesting a delayed administration and a possible reduced penetration, together with a limited antimicrobial activity in pus. Also, the laboratory results indicated in vitro antimicrobial resistance towards seven products commonly used to treat bovine respiratory disease: penicillin/streptomycin combination, ampicillin, amoxicillin and clavulanic acid, doxycycline, oxytetracycline, gentamicin and florfenicol. 77

78 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA Fig.1. Lesional aspects associated with Trueperella pyogenes The occurrence and patterns of antimicrobial resistance found in Trueperella pyogenes strain isolated from calves are comparable with literature data (1,10,11,13). Trueperella pyogenes strains isolated from domestic animals were found susceptible to tested beta-lactams, but resistant to enrofloxacin, tetracycline, macrolides, and clindamycin (10). A Chinese study evaluating the strains responsible for bovine mastitis reported biofilm production ability and high occurrence of multidrug resistance, with low susceptibility to trimethoprimsulphamethoxazole (10%) and bacitracin (2%) and susceptibility to rifampin (96%), ampicillin (94%), ciprofloxacin (94%), and penicillin (92%) (1). Also, bovine strains isolated from metritis cases were found resistant to amoxicillin (56.9%); ampicillin (86.1%), chloramphenicol (100%), florfenicol (59.7%), oxytetracycline (54.2%), penicillin (86.1%) and tetracycline (50%), with 88.9% of the isolates resistant to at least 3 of the antimicrobial agents tested (11). 78

79 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017 TIMISOARA Since this bacterium is described as an opportunistic pathogen (3,4,7,9), the measures should also target the detection and control of the triggering factor(s) for the immunosuppressive state (stress factors, concurrent diseases, poor welfare and hygiene conditions). Conclusions This case report described the etiological role of Trueperella pyogenes in calves chronic pneumonia based on the isolation and identification of the bacterium from lungs and lymph nodes samples. The bacterial strain was found to be resistant towards several antimicrobial products: penicillin/streptomycin combination, ampicillin, amoxicillin and clavulanic acid, doxycycline, oxytetracycline, gentamicin and florfenicol; in vitro susceptibility was recorded only in case of cefquinome, marbofloxacin and enrofloxacin. These results underline the Trueperella pyogenes potential to develop antimicrobial resistance, thus the need to evaluate and monitor its trends, but also point out the need of the prudent use of antimicrobial agents. References 1. Alkasir, R., Wang, J., Gao, J., Ali, T., Zhang, L., Szenci, O., Bajcsy, Á.C., Han, B., Properties and antimicrobial susceptibility of Trueperella pyogenes isolated from bovine mastitis in China, Acta Vet. Hung., 2016, 64(1), Bi, Y., Wang, Y.J., Qin, Y., Guix Vallverdú, R., Maldonado García, J., Sun, W., Li, S., Cao, Z., Prevalence of Bovine Mastitis Pathogens in Bulk Tank Milk in China, PLoS One, 2016,11(5),e Hijazin, M., Ulbegi-Mohyla, H., Alber, J., Lämmler, C., Hassan, A.A., Abdulmawjood, A., Prenger-Berninghoff, E., Weiss, R., Zschöck, M., Molecular identification and further characterization of Arcanobacterium pyogenes isolated from bovine mastitis and from various other origins, J. Dairy Sci., 2011, 94(4), Jost, B.H., Billington, S.J., Arcanobacterium pyogenes: molecular pathogenesis of an animal opportunist, Antonie Van Leeuwenhoek, 2005, 88(2), Kishimoto, M., Tsuchiaka, S., Rahpaya, S.S., Hasebe, A., Otsu, K., Sugimura, S., Kobayashi, S., Komatsu, N., Nagai, M., Omatsu, T., Naoi, Y., Sano, K., Okazaki-Terashima, S., Oba, M., Katayama, Y., Sato, R., Asai, T., Mizutani, T., Development of a one-run real-time PCR detection system for pathogens associated with bovine respiratory disease complex, J. Vet. Med. Sci., 2017, 79(3),

80 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA 6. Levy, C.E., Pedro, R.J., Von Nowakonski, A., Holanda, L.M., Brocchi, M., Ramos, M.C., Arcanobacterium pyogenes Sepsis in Farmer, Brazil, Emerging Infectious Diseases, 2009, 15(7), Ozturk, D., Turutoglu, H., Pehlivanoglu, F., Guler, L., Virulence Genes, Biofilm production and antibiotic susceptibility in Trueperella pyogenes isolated from cattle, Israel Journal of Veterinary Medicine, 2016, 71 (3), Ponnusamy, P., Ronald, B.S.M., Ranjith Kumar, M., Ananda Chitra, M., Manickam, R., A rare case of bovine abortion due to Trueperella pyogenes, International Journal of Science, 2017, 6(1), Ribeiro, M.G., Risseti, R.M., Bolaños, C.A., Caffaro, K.A., de Morais, A.C., Lara, G.H., Zamprogna, T.O., Paes, A.C., Listoni, F.J., Franco, M.M., Trueperella pyogenes multispecies infections in domestic animals: a retrospective study of 144 cases (2002 to 2012), Vet Q., 2015, 35(2), Rzewuska, M., Czopowicz, M., Gawryś, M., Markowska-Daniel, I., Bielecki, W., Relationships between antimicrobial resistance, distribution of virulence factor genes and the origin of Trueperella pyogenes isolated from domestic animals and European bison (Bison bonasus), Microb. Pathog., 2016, 96, Santos, T.M., Caixeta, L.S., Machado, V.S., Rauf, A.K., Gilbert, R.O., Bicalho, R.C., Antimicrobial resistance and presence of virulence factor genes in Arcanobacterium pyogenes isolated from the uterus of postpartum dairy cows, Vet. Microbiol., 2010,145(1-2), Scott, P.R., Clinical presentation, ascultation recordings, ultrasonographic findings and treatment response of 12 adult cattle with chronic suppurative pneumonia: case study, Irish Veterinary Journal, 2013, 66(1), Yoshimura, H., Kojima, A., Ishimaru, M., Antimicrobial susceptibility of Arcanobacterium pyogenes isolated from cattle and pigs, Infect. Dis. Vet. Public Health, 2000,47(2), Zastempowska, E., Lassa, H., Genotypic characterization and evaluation of an antibiotic resistance of Trueperella pyogenes (Arcanobacterium pyogenes) isolated from milk of dairy cows with clinical mastitis, Vet. Microbiol., 2012,161(1-2), Zhang, D., Zhao, J., Wang, Q., Liu, Y., Tian, C., Zhao, Y., Yu, L., Liu, M., Trueperella pyogenes isolated from dairy cows with endometritis in Inner Mongolia, China: Tetracycline susceptibility and tetracycline-resistance gene distribution, Microb. Pathog., 2017, 105,

81 LUCRĂRI ŞTIINŢIFICE MEDICINĂ VETERINARĂ VOL. L (3), 2017, TIMIŞOARA PROLIFERATIVE BACTERIAN ENTERITIS OF THE SWINE. EXPERIMENTAL STUDY O. MEDERLE 1, ANCA HULEA 2, MILANA MIUCA 1, NARCISA MEDERLE 2, V. HERMAN 2 1 Victor Babes University of Medicine and Pharmacy, Faculty of Medicine, Eftimie Murgu, 2, Timisoara , Romania 2 Banat s University of Agricultural Sciences and Veterinary Medicine King Michael I of Romania from Timisoara, Faculty of Veterinary Medicine, Calea Aradului, 119, Timisoara , Romania mederle.ovidiu@umft.ro Summary To describe the lesions of the porcine proliferative enteritis, we used the morphological and histopathological examinations. We applied the immunohistochemical technique for identification of bacterial antigens. We used the working system NovoLink Max Polymer detection with antibody Lawsonia intracelularis (Novocastra, Newcastle UponTyne, UK). We performed imunohistochemical method using DakoCytomation Autostainer. We used chromogen 3,3-diamino-benzidine and Lille haematoxylin for counterstain. We examined mophopathologically 520 gastrointestinal samples taken from the slaughtered pigs. There were investigated 120 ileum fragments with lesions and 26 intestinal fragments without lesions by histopathological technique. Immunohistochemical technique has been used to examine 135 intestinal samples (109 samples with lesions, 26 samples without lesions). Intestinal adenomatosis lesions have been identified in 71,1% (370) out of 520 gastro-intestinal pig samples, by morphopathological exam.the histological examination of 120 intestinal samples has releved the pathognomonic lesion. A cellular infiltrate in the lamia propria of the mucosa with mastocytes, lymphoplastocytes, eosinophils and, in a smaller proportion, macrophages could be due to an allergic reaction to a protein on the surface of Lawsonia intracellularis.by IHC technique, we have highlighted bacterial antigens Lawsonia intracellularis in lamina propria of the intestinal samples with or without lesions. Keywords: Lawsonia intracellularis, lesions, immunohistochemistry Intracellular infection by Lawsonia intracellularis, the aetiological agent of proliferative enteritis, occurs all over the world, in different types of production systems that affect young pigs. This infection determines significance economic losses through young breeding and growing-finishing pigs especially due the lower average daily gain. Unfortunatly, the pathogenesis of this disease has an aspects unknown yet (1, 2, 5, 10). Materials and methods To describe the lesions of the porcine proliferative enteritis, we used the morphological and histopathological examinations. We applied the immunohistochemical technique for identification of bacterial antigens. We used the 81

82 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA working system NovoLink Max Polymer detection with antibody Lawsonia intracelularis (Novocastra, Newcastle UponTyne, UK). We performed imunohistochemical method using DakoCytomation Autostainer. We used chromogen 3,3-diamino-benzidine and Lille haematoxylin for counterstain. We examined mophopathologically 520 gastrointestinal samples taken from the slaughtered pigs. There were investigated 120 ileum fragments with lesions and 26 intestinal fragments without lesions by histopathological technique. Immunohistochemical technique has been used to examine 135 intestinal samples (109 samples with lesions, 26 samples without lesions) (3, 4, 6, 7, 8, 9). Results and discussions The mophopathological exam of the gastro-intestinal pig samples has shown that 370 (71, 1%) out of 520 subjects presented enteritis. Only cases of intestinal adenomatosis have been observed (table 1). Table1 The results from the macroscopic examination hemorrhagic intestinal necrotic without lesions enteropathy adenomatosis enteritis ileum ileum and jejunum ileum, jejunum and colon number of positive probes total number of examined probes

83 LUCRĂRI ŞTIINŢIFICE MEDICINĂ VETERINARĂ VOL. L (3), 2017, TIMIŞOARA We emphasized in the intestinal samples, the folowing morphopatological aspects: edema of the serous membrane, deep proliferation with folds of the mucosa, narrowing of the lumen and cerebriform aspect of the intestine. We did not identify hemorrhagic,ulcerative and necrotic lesions; this eliminates the possibility of an acute form of the disease. The pathognomonic lesion is the epithelial proliferation accompanied by the growing in thickness of the intestinal mucosa (10). This lesion has been found in this study, the characteristic lesions have been evidenced in the ileum and jejunum in 219 cases (59,1%) out of 520 examined samples. The extension of the lesions at the level of the proximal colon has been remarked in 83 cases (22, 4%), and regional ileitis has been found in 68 cases (18, 3%) (table 1). The swine s proliferative enteritis generates changes within every layer of the mucosal tunic (epithelial, lamina propria and muscular). The histopathological exam of the colored sections with Hematoxylin-Eosin allowed us to analyze the intestinal mucosa s structure. The atrophy of the intestinal villi was evidenced on the intestinal samples which are contracted and covered in poorly differentiated cells. Histological, the small intestine s epithelium is simple, columnar, made of goblet cells and enterocytes (4). Lawsonia intracellularis stimulates the proliferation of enterocytes and stops them maturation, the result will be an epithelial hyperplasia caused by the immature enterocytes villi mobility at the surface or in the intestine s glands (1,2, 5, 10). In our study, the epithelium seems stratified with the presence of numerous immature enterocytes; goblet cells are absent. The presence of a moderate, or sometimes, abundant lymphocyte infiltrate has been observed at the level of the lamina propria, reaching out to the muscular tunic of the small intestine (Fig.1). An alternation between the areas with epithelial hyperplasia and those with cellular desquamation has been observed; and sometimes, intestinal vilii without histopathological changes near the lesions. The goblet cells are missing in the areas affected by hyperplasia, and the desquamated cells and the detritus are eliminated in the intestinal lumen. By IHC technique, we have highlighted bacterial antigens Lawsonia intracellularis in lamina propria enterocytes and macrophages (Fig. 2, 3). 83

84 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA Fig. 1. Leucocytes infiltrate - lamina propria and muscular tunic The antigens have been evidenced in macrophages and enterocytes in 62,8% (68) out of the 109 samples of lesions (Fig. 2). In 19,2% (21) out of total samples, the antigens were present only within the macrophages (Fig. 3) and in 11,0% (12) out of total samples, within the enterocytes, macrophages and free in the lumen of intestinal glands. Despite the presence of lesions, in 7,3% (2,9) out of 109 samples, the antigens have not found. This aspect can be the result of terminal phase of intestinal adenomatosis. 12 cases out of 26 samples without the characteristic macroscopic lesions, have emphasized the antigens Lawsonia intracellularis by IHC technique. 84 Fig. 2. Ileum, the presence of the antigen (brown color)

85 LUCRĂRI ŞTIINŢIFICE MEDICINĂ VETERINARĂ VOL. L (3), 2017, TIMIŞOARA Fig. 3. Antigens present within the macrophages (transversal section) Conclusions Intestinal adenomatosis lesions have been identified in 71,1% (370) out of 520 gastro-intestinal pig samples, by morphopathological exam. The histological examination of 120 intestinal samples has releved the pathognomonic lesion: epithelial proliferation accompanied by the growing in thickness of the intestinal mucosa. A cellular infiltrate in the lamia propria of the mucosa with mastocytes, lymphoplastocytes, eosinophils and, in a smaller proportion, macrophages could be due to an allergic reaction to a protein on the surface of Lawsonia intracellularis. By IHC technique, we have highlighted bacterial antigens Lawsonia intracellularis in lamina propria of the intestinal samples with or without lesions. References 1. Boutruop, T.S., Boesen, H.T., Bove, M., Agerholm, J.S., Jemsen, T.K., Early pathogenesis in porcine proliferative enteropathy caused by Lawsonia intracellularis, Journal of Comparative Pathology, 2010, 143 (2-3), Chouet, S., Prieto, C., Mieli, L., Patterns of exposure to Lawsonia intracellularis infection on European pig farms, Veterinary Record, 2003, 15, Cîmpean, A. M., Curs de imunomorfologie, Ed. Eurobit, Timisoara, Eurell, J. A., Veterinary histology, Ed. Teton New Media, Iowa,

86 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA 5. Jacobson, M., Felstrom, C., Jensen-Waern, M., Porcine proliferative enteropathy: An important disease with questions remaining to be solved, The Veterinary Journal, 2009,184(3), Lazăr, E., Dema, A., Tăban, S. et al., Morfopatologie-Ghid practic, Ed. Universității, București, Lin, F., Prichard, J., Liu, H., Handbook of Practical Immunohistochemistry, Ed. Springer, New York, Olariu Jurca, I., Diagnostic necropsic și medicină veterinară legală, Ed. Brumar, Timișoara, 2006; 9. Șincai, M., Citohistologie și tehnici de specialitate, Ed. Mirton, Timișoara, Vannucci, F.A., Gebhart, C.J., Recent advances in understanding the pathogenesis of Lawsonia intracellularis infections, Vet. Pathol., 2014, 51(2),

87 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA CASE REPORT RELATED TO LOCAL EFFICACY OF PROPOLIS AND BEESWAX ANDREEA-IOANA MERNEA, ANCA HULEA, ANDREEA GARTNER, CORINA PASCU, V. HERMAN Banat s University of Agricultural Sciences and Veterinary Medicine King Michael I of Romania from Timisoara, Faculty of Veterinary Medicine, Calea Aradului, 119, Timisoara , Romania viorel.herman@fmvt.ro Summary This report describes a case of pruriginous interscapular cutaneous lesion, rebel to antiseptic treatment frequency used in veterinary medicine. First examination diagnostic it was wet gangrene and after local and general treatment, even the necrotizing skin was removed, behind that remaining an extended area of cutaneous lake and an intense pruriginous lesion. In the followed month the lesions persisted even local antiseptic treatment was applied. Skin scratching and mycological exam revealed no parasitic or fungal skin diseases. From all local naturals applied treatment, it was observed that the only efficacy ointment contained beeswax and propolis. Keywords: propolis, beeswax, cutaneous lesion, wet gangrene Actual medicine practice tends to treat many disease, if exist the possibility, using natural products to the detriment of chemical drugs. One of these natural products refer to propolis, a substance that contains flavonoid compounds, caffeic acid esters and diterpenic, conferring it bacteriostatic, bactericidal, antiviral and antifungal properties, therapeutic characteristics applied both in human and veterinary medicine. In veterinary medicine, therapeutic effectiveness of propolis was demonstrated to treat large animals remind about cow mastitis (2) and companion animals mention about neoplastic diseases like Sticker tumor (1) and osteosarcoma (3), bacterial infection (6), mycotic infection (7), dermatophytosis (4) and some of the parasitological disease (8, 9). Recently, was demonstrated that beeswax has antimicrobial properties too (5), being use especially for treatment of skin diseases. Materials and methods The study was carried out on a 1 year and 6 month old male cat, with a interscapular cutaneous lesion, hospitalized for one year to veterinary hospital of University Veterinary Clinics Timișoara. Owner discussion revealed that it was a week old traumatic lesion appearing after an altercation with another cat. The lesion it was oval shape having the length with 9 cm and width 6 cm, more extended on the right sides of the neck. 87

88 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA First clinical examination revealed wet gangrene, the reason why we choose to applied local and general antibiotherapy. First it was performed clinical examination and because the situation asked, was administrated general and local therapy. In the first 14 th days was administrated Enroxil 5% (Enrofloxacin) by sc route. Applied medical treatments as well the duration are presented in Table 1. Table 1 Local medical treatments administration used for cutaneous lesion No. crt. Commercial name 1. Germostop 2. Baneocin 3. Cicatrisol 4. Herba sol Active substance Time Observations Neomycin, Clorhexidine Bacitracin zinc, Neomycin Phenol Tannin Iodoform Gentian violet Gentian violet Oxytetracycline 60 days 60 days 60 days 60 days 240 days Because the results of this therapy are not satisfying will chose to apply the natural treatments. Applied local naturals treatments as well the duration are presented in Table 2. Local naturals treatments means used for cutaneous lesion No. Active Commercial name crt. substance Time 1. Atrauman Ag Silver ions 7 days Bismuthi subgallas Olivae oleum 2. Biotitus Helianti oleum Rinici oleum 30 days Cera Colophonium Camphora 3. Beeswax Steuart s natural wound Propolis cream (9). Olivae oleum 80 days Observations 117 days Table 2 Because of intense itching it was performed skin scratching for identification of possible mites and mycological exam using Sabouraud medium which was incubated 7 days at 37ºC in aerobic condition. 88

89 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA Results and discussions Even the itching was always present and sometimes was so intense that cause skin auto-mutilation, dermatological exam revealed no sign of mites and fungal disease. Results after local medical treatments administration After 240 days of local antiseptic therapy, based of usual drugs even the lesion sometimes it seemed to shrink, it always relapse because of intense itching aggravating the situation (Figure 1). Fig. 1. Skin appearance lesion after local Germostop administration (60 days of treatment) no clinical sign of recovery Results after local naturals treatments Of all local treatment, the use of silver ions had the shortest duration of administration, only 7 days, because the plaque became a suppurate lesion. Using Biotitus, it was observed for the first time an improvement in the first 19 days of treatment, but the following days although itching decreased in intensity, there was so sign of reducing the affected area (Figure 2a, fig 2b). 89

90 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA a b Fig. 2. Lesion appearance before application of Biotitus (a) and after 30 days of treatment (b) Using Steuart s Natural Wound Cream (9) with propolis, after 19 days it was observed that the itching was almost absent and granular tissue area was significant increased (Figure 3a). After 50 days of treatment, the lesion dimension halved (Figure 3b) and at the end of the 80 days of treatment the wound was completely cured (Figure 4). a b Fig. 3. Lesion appearance after 19 days of Steuart s Natural Woond Cream application (a) and after 50 days of treatment (b) 90

91 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA Fig. 4. Lesion after 80 days of treatment with Steuart s Natural Wound Cream Antimicrobial activity of beeswax and propolis was demonstrated by many researches (6, 7, 5), the reason why skin treatment effectiveness of bees products reach high levels. This study demonstrated that the propolis and beeswax can be used not only as a complementary therapy to treat traumatic skin lesion, but also as a primary treatment, being useful where other medical treatments have no results. Conclusions Steuart s Natural Wound Cream contains a natural antiseptic (propolis from bee hives), that promotes healing wounds, after 80 days the lesion it was totally healing. Also relieves itching, after 19 days, accelerating granulation tissue formation. Acknowledgements This study was realised using the support and infrastructure project Dezvoltarea infrastructurii de cercetare, educaţie şi servicii în domeniile medicinei veterinare şi tehnologiilor inovative pentru RO 05, cod SMIS-CSNR References 1. Bassani-Silva, S., Sforcin, J.M., Amaral, A.S., Gazpar Luiz, F.J., Rocha Noeme, S., Propolis effect in vitro on canine transmissible venereal tumor cells, Revista Portuguesa de Ciências Veterinárias, 2007, 102, Bogdanov, S., Propolis: composition, health, medicine: A Review, Bee Product Science,

92 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA 3. Costa Cinegaglia, N., Oliveira Bersano, P.R., Mendes Araújo, M.J.A., Cristiane Búfalo, M., Sforcin, J.M., Anticancer effects of geopropolis produced by sting less bees on canine osteosarcoma cells in vitro, Evidence-Based Complementary and Alternative Medicine, 2013, Article ID: Cruz Sánchez, T.A., Estrada García, P.A., López Zamora, C.I., Autran Martínez, M., Pérez Valencia, V., Londoño Orozco, A., Use of propolis for typical treatment of dermatophytosis in dog, Open Journal of Veterinary Medicine, 2014, 4, Ghanem, N., Study on the antimicrobial activity of honey products and some Saudi Folkloric substances, Res J Biotech, 2011, 6, Gross, G.S., Carvajal, I.L.C., Principal, J., Perfil de flavonoides e índices de oxidación de algunos propó- leoscolombianos, Zootecnia Tropical, 2007, 25, Lozina, L.A., Peichoto, M.E., Boehringer, S.I., Koscinczuk, P., Granero, G.E., Acosta, O.C., Efficacy of argentine propolis formulation for topical treatment of canine otitis extern, Arquivo Brasileiro de Medicina Veterinária e Zootecnia, 2010, 62, Lucas, T.G., Da Silva, A.S., Machado, G., Dalla Rosa, L., Dorneles, F., Gressler, T.L., Oliveira, M.S., Zanette, R.A., Agueda de Vargas, C.P., Monteiro, S.G., Susceptibility of Trypanosoma evansi to propolis extract in vitro and in experimentally infected rats, Research in Veterinary Science, 2012, 93, * * * Propolis-1-oz_p_53.html 92

93 LUCRĂRI ŞTIINŢIFICE MEDICINĂ VETERINARĂ VOL. L (3), 2017, TIMIŞOARA MICROBIAL DIVERSITY INFLUENCED BY MIGRATORY AND FEEDING BEHAVIOR IN BIRDS FROM THE DANUBE DELTA V. NEGRUȚIU, MIHAELA NICULAE, EMÖKE PÁLL, CARMEN DANA ŞANDRU, F. BRUDAȘCĂ, A. VASIU, SILVANA POPESCU, MARINA SPÎNU University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, , 3-5 Mănăştur Street, Cluj-Napoca, Romania vlad_negrutiu@yahoo.com Summary The microbiome of the digestive tract in birds is inhabited by a diverse community of bacteria, separated by taxonomy, of which several carry pathogenic/zoonotic potential. Evidence has been provided that social contact between different individuals or species in the same habitat mediates horizontal transfer of such bacteria, contributing to the enlargement of various epidemiological chains. This study aimed at investigating the microbiome in sedentary versus migratory and insectivorous/grain eating versus raptor birds from the Danube Delta. Pharingeal and cloacal samples (n=30) were collected from sedentary insectivorous (Passer montanus, tree sparrow, n=6) and raptor (Corvus cornix, Corvus coronae cornix, hooded grow, n=6) birds as well as from migratory insectivorous (Lanius collurio, red-backed shrike, n=8) and raptor birds (Falco subbuteo, Eurasian hobby, n=4, Accipiter nisus, Eurasian sparrowhawk, n=6). These samples were subject to classical microbiology and cultivation on chromogenic culture media (UTI, TCBS, API 20E) for identification. The bacteria identified from all categories of birds belonged to the same genera (Vibrio spp., Staphylococcus spp., Pseudomonas spp., Klebsiella spp., E. coli, Salmonella spp.), all of them with pathogenic/zoonotic potential. A total of 10 species of bacteria were identified in sedentary birds while 25 were isolated in migratory ones. In the latest category, the number of Vibrio spp. (V. cholerae, V. mimicus, V. alginolitycus, V. fluvialis) was higher than in the others. V metschikovii S. typhimurium and S. enteritidis were found in addition in migratory insectivorous birds. The broad variety of potentially pathogenic/zoonotic species isolated from migratory birds support the hypothesis of their participation in broadening epidemiological cycles that include human hosts. Key words: microbiome, birds, migratory behavior, feeding, Danube Delta The bacteriome of vertebrates could not only exert pathogenic effects but it is well known that some of the bacteria with intestinal habitat are beneficial for the host. Due to difficulties connected with access and welfare issues, in wildlife these interactions and their outcomes are less studied (3, 6, 7).The microbiome of the digestive tract in birds is inhabited by a diverse community of bacteria, separated by taxonomy, of which several carry pathogenic/zoonotic potential. Evidence has been provided that social contact between different individuals or species in the same habitat mediates horizontal transfer of such bacteria, contributing to the enlargement of various epidemiological chains, including humans (9). Wild birds have been considered to be reservoirs of enteric human pathogens and vectors of resistance dissemination to the environment (4). 93

94 94 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017 TIMISOARA This study aimed at investigating the microbiome in sedentary versus migratory and insectivorous/grain eating versus raptor birds from the Danube Delta. Materials and methods Birds from the Danube Delta were subjected to pharingeal and cloacal sampling. A total of 30 samples were collected from sedentary insectivorous (Passer montanus, tree sparrow, n=6) and raptor (Corvus cornix, Corvus coronae cornix, hooded grow, n=6) birds as well as from migratory insectivorous (Lanius collurio, red-backed shrike, n=8) and raptor birds (Falco subbuteo, Eurasian hobby, n=4, Accipiter nisus, Eurasian sparrowhawk, n=6). The samples were collected during bird ringing, with care for the bird welfare. All birds were released after sampling. The swabs were subject to classical microbiology and cultivation on usual and of Chromogenic UTI medium Brilliance TM and TCBS Cholera medium (Oxoid) and API 20E for identification. The data were processed by Excel program to interpret the statistical relevance. Results and discussions Researches concerning the gastrointestinal flora in wild birds are limited due to difficult sampling access, unless the outcome of the bacteria-host interaction is lethal. Mainly zoonotic bacteria hosted by wild birds were given attention lately, based on the potential risk represented by migratory birds to habitat and human health. Similarly, the habitat could serve as source for pathogenic bacteria, which this way will close the epidemiological loop. Paths used by various bacteria to spread and find new susceptible hosts could include migratory birds and could serve as models for clarification of epidemiology of other pathogens (1, 2). In a study concerning the gut bacterial flora of common European wild birds in a rescue center in Sicily, the bacterial isolated were: Escherichia coli, Proteus mirabilis, Proteus vulgaris, Citrobacter freundii, Enterobacter cloacae, Klebsiella oxytoca, Salmonella Typhimurium, Escherichia vulneris, Enterobacter amnigenus biogroup 2, Salmonella Duesseldorf and Hafnia alvei All these species were, in different clinical studies, identified to bear pathogenic effect for other animal species or humans (4). Another source for zoonotic bacteria could be the illegal wildlife trade, which could also augment the proportion of infectious disease transmission. The final hosts could be not only people, but also in livestock, native wild populations, and ecosystem. In a study on illegally sold birds, Hamer et al., 2012 (5) identified most frequently Escherichia coli, followed by Enterobacter spp., Klebsiella pneumoniae and other enteric bacteria. In a Temminck's seedeater (Sporophila

95 LUCRĂRI ŞTIINŢIFICE MEDICINĂ VETERINARĂ VOL. L (3), 2017, TIMIŞOARA falcirostris) the authors found Salmonella ser. Typhimurium, while chestnut-capped blackbirds (Chrysomus ruficapillus) hosted Salmonella ser. Panama. Although wild birds have been suggested to carry bacteria and other microbial agents to large distances, thus enhancing their spreading and causing new outbreaks, in a study conducted by Tsiodras et al., 2008 (8) it has been considered that the evidence available in literature limited the wild birds role in human infectious diseases, at least by direct transmission. A total of 35 strains were isolated belonging to Vibrio, Klebsiella, Pseudomonas, Proteus, Escherichia, Staphylococcus and Enterococcus genera. In sedentary birds, paradoxically, the total number of strains was higher (n=7) than in predatory ones (n=3). Their genera were indicated in Fig no of strains SIG SP 0 Salm Pseudo Vibrio Klebsiella E coli Staph Fig. 1. Distribution of bacteria strains isolated from sedentary insectivorous/grain eating (SIG) and predatory (SP) birds The total number of bacterial strains isolated from migratory birds (n=25) (Fig. 2) was significantly higher that in sedentary birds (n=10)(p<0.05). The isolated genera were similar to those cited in the literature. While E. coli dominated the bacterial population in sedentary birds, Vibrio was the genus most encountered in migratory birds, equally in insect/grain eaters (MIG) and predatory (MP) ones (n=6). Numerous Vibrio species were identified in migratory birds, i.e. V. metschikovii, V. alginolyticus, V. fluvialis, V. vulnificus, V. mimicus, while only V. cholerae and V. fluvialis were isolated in SP and SIG birds respectively. Salmonella typhimurium was present, of Salmonellae in both sedentary and migratory birds. 95

96 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017 TIMISOARA 6 5 no of isolated strains MIG MP 0 Ent Vib Prot Pseudo salm Staph Klebs E.coli/colif Fig. 2. Distribution of bacteria strains isolated from migratory insectivorous/grain eating (MIG) and predatory (MP) birds Our results indicated that the distribution of potentially zoonotic bacteria is higher in migratory birds and that in Danube Delta birds, independently on their habitat and feeding habits, besides bacteria commonly found in the gut, aquatic bacteria were also present. Nevertheless, these results should be connected with microbiological research for evaluating the antibiotic resistance and also beta lactamase spectrum of the isolates, to better interpret their pathogenic potential. Conclusions The broad variety of potentially pathogenic/zoonotic bacteria species isolated from migratory birds support the hypothesis of their participation in broadening epidemiological cycles that include human hosts. Aknowledgement This research was supported by grant PNII PCCE 61/

97 LUCRĂRI ŞTIINŢIFICE MEDICINĂ VETERINARĂ VOL. L (3), 2017, TIMIŞOARA References 1. Bengis, R.G., Leighton, F.A., Fischer. J.R., Artois, M., Mörner, T., Tate, C.M., The role of wildlife in emerging and re-emerging zoonoses, Rev Sci Tech., 2004, 23(2), Benskin, C.M., Wilson, K., Jones, K., Hartley, I.R., Bacterial pathogens in wild birds: a review of the frequency and effects of infection, Biol Rev Camb Philos Soc., 2009, 84(3), Costello, E.K., Lauber, C.L., Hamady, M., Fierer, N., Gordon, J.I., Knight, R., Bacterial Community Variation in Human Body Habitats Across Space and Time, Science, 2009, 326, Giacopello, C., Foti, M., Mascetti, A., Grosso, F., Ricciardi, D., Fisichella, V., Lo Piccolo, F., Antimicrobial resistance patterns of Enterobacteriaceae in European wild bird species admitted in a wildlife rescue centre, Vet Ital., 2016, 52(2), Hamer, S.A., Lehrer, E., Magle, S.B., Wild birds as sentinels for multiple zoonotic pathogens along an urban to rural gradient in greater Chicago, Illinois, Zoonoses Public Health, 2012, 59(5), Kreisinger, J., Čížková, D., Kropáčková, L., Albrecht, T., Cloacal Microbiome Structure in a Long-Distance Migratory Bird Assessed Using Deep 16sRNA Pyrosequencing, PLoS ONE, 2015, 10(9), e Muegge, B.D., Kuczynski, J., Knights, D., Clemente, J.C., González, A., Fontana, L., Henrissat, B., Knight, R., Gordon, J.I., Diet drives convergence in gut microbiome functions across mammalian phylogeny and within humans, Science, 2011, 332, Tsiodras, S., Kelesidis, T., Kelesidis, I., Bauchinger, U., Falagas, M.E., Human infections associated with wild birds, J Infect., 2008, 56(2), Wu, B., Wang, X,C., Dzakpasu, M., Genetic characterization of fecal impacts of seagull migration on an urban scenery lake, Water Res., 2017, 117,

98 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA 98 COMPARATIVE EVALUATION OF THE ANTIMICROBIAL RESISTANCE IN E. COLI OF HUMAN AND FARM ANIMAL ORIGIN MIHAELA NICULAE University of Agricultural Sciences and Veterinary Medicine, Faculty of Veterinary Medicine, , Calea Mănăștur Street no 3-5, Cluj-Napoca, România niculaemihaela1@gmail.com Summary Escherichia coli is described as a common normal flora organism in the gastrointestinal tract of animals and human, but also as pathogenic bacterium with a demonstrated tendency to develop antimicrobial resistance. This retrospective study was aimed to evaluate the antibiotic resistance in human and farm animal clinical E. coli strains. Samples obtained from finisher pigs (edema disease, n=65), dairy bovine (clinical and sub-clinical mastitis, n=52), chicken (polyserositis, n=47) and human (recurrent cystitis, n=35) during were processed by classical microbiological methods: isolation of bacterial strains on selective agar, biochemical properties testing, with in vitro susceptibility patterns of a total of 199 strains towards 11 antimicrobials determined using the standard Kirby- Bauer disc diffusion method according to CLSI guidelines. Overall, E. coli isolates were resistant to at least one antimicrobial, with decreased susceptibility towards: trimethoprim+sulphamethoxazole> colistin>tetracycline>ampicillin>amoxicillin+ clavulanic acid. The resistance level varied widely among clinical isolates depending on the species, with the most reduced susceptibility recorded in case of bovine and poultry. Multi-drug resistance (MAR) was recorded also in case of these isolates, with strains simultaneously resistant to two or more antimicrobial groups. The results of the in vitro antimicrobial susceptibility testing from the study indicate that animal origin strains of E. coli display resistance to various antimicrobials commonly used in veterinary medicine and with relevance for human medicine. MAR detection in case of E. coli reflects the importance of the rational use of antimicrobials and recommends continuous monitoring of the trends and features of resistance given the role of enteric bacteria as potential reservoir of resistance genes. Key words: E. coli, multi-drug antimicrobial resistance, farm animals Escherichia coli is described as a common normal flora organism in the gastrointestinal tract of animals and human, but also as pathogenic bacteria responsible for several enteric and non-enteric pathologies (1, 2, 9). The increasing level of the antibiotic resistance reported worldwide in both commensal and pathogenic Escherichia coli animal strains is of particular concern because it is the one of the most common Gram-negative pathogen in human (1, 2, 9). In addition, the transfer of antibiotic resistance determinants not only to other strains of E. coli, but also to other bacteria residing in the gastrointestinal tract

99 LUCRĂRI ŞTIINŢIFICE MEDICINĂ VETERINARĂ VOL. L (3), 2017, TIMIŞOARA was demonstrated in case of these resistant E. coli strains, complicating further the consequences of the phenomenon (1, 2, 4, 6, 9). Antibiotics are a major group of therapeutic products commonly used in food producing animals, but their intense and irrational use for prophylaxis and growth promotion was incriminated for the emergence, spread and augmentation of the antimicrobial resistance (1, 2, 5, 6, 9). The aim of this retrospective study was to evaluate and analyze the antibiotic resistance in human and animal clinical E. coli strains considering the impact of antimicrobials practices, therapeutic and prophylactic, applied especially in farm animals on the antimicrobial resistance emergence. Materials and methods Clinical samples obtained from finisher pigs (edema disease, n=65), dairy bovine (clinical and sub-clinical mastitis, n=52), chicken (polyserositis, n=47) and human (recurrent cystitis, n=35) during were processed by classical microbiological methods: isolation of bacterial strains on a selective chromogenic agar (Brilliance E. coli/coliform Selective Agar, Oxoid Ltd., Cambridge/UK) after incubation under aerobic conditions at 37 C for 24 hours, followed by biochemical properties testing using API 20E system (BioMerieux SA, Marcy Etoile, France) or Rapid ID system, ERIC (Eric Web, Remel). The in vitro evaluation of susceptibility patterns for a total number of 199 strains towards 11 antimicrobials was determined using the standard Kirby-Bauer disc diffusion antimicrobial susceptibility testing method according to CLSI guidelines. The following antimicrobials were included: ampicillin (AMP, 10 µg), amoxicillin/clavulanic acid (AMC, 20/10 µg), tetracycline (TE, 30 µg), gentamycin (CN, 10 µg), colistin (CS, 10 µg), enrofloxacin (ENR, 5 µg), marbofloxacin (MAR, 5 µg), ciprofloxacin (CIP, 5 µg), cefquinome (30 µg), trimethoprim + sulphamethoxalone (SXT, 1.25/23.75 µg), florfenicol (FFC, 30 µg) (susceptibility discs, Oxoid Ltd., Cambridge/UK). Escherichia coli ATCC (Oxoid Ltd., Cambridge/UK) was also tested as a quality control organism. Results were statistically analyzed using the chi-square test to compare the antimicrobial resistance rates (%) to antimicrobials and a minimum p value of <0.05 was considered to indicate statistically significant differences. Results and discussions Overall, E. coli isolates recovered from clinical samples were resistant to one or more of 11 tested antimicrobials, with decreased susceptibility towards: trimethoprim + sulphamethoxazole > colistin > tetracycline > ampicillin > amoxicillin + clavulanic acid (Figure 1). 99

100 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA. Fig. 1. Antibiotic sensitivity pattern of E. coli strains Fig. 2. Antibiotic resistance profile of E. coli isolates The most commonly recorded multidrug-resistance patterns were represented by: trimethoprim + sulphamethoxazole/colistin, trimethoprim + sulphamethoxazole/colistin/tetracycline, trimethoprim+sulphamethoxazole/colistin/ ampicillin, trimethoprim + sulphamethoxazole/colistin/ ampicillin/ amoxicillin /clavulanic acid, trimethoprim + sulphamethoxazole/colistin/ ampicillin/tetracycline. 100

101 LUCRĂRI ŞTIINŢIFICE MEDICINĂ VETERINARĂ VOL. L (3), 2017, TIMIŞOARA The resistance level vary widely among clinical isolates depending on the species, with the most reduced susceptibility recorded in case of bovine, poultry and swine isolates and also on the antimicrobial, suggesting the intense use of the product. A significant difference (p<0.05) in cefquinome and quinolones (enrofloxacin, marbofloxacin and ciprofloxacin) susceptibility compared to the other antimicrobials was noticed, suggesting differences in use regimens of these drugs in case of the farm species and human (Figure 2). Florfenicol was also active against E. coli with the same level for all farm animals and 100% susceptibility in case of human strains. Multi-drug resistance (MAR) was exhibited by bovine, poultry and swine isolates, with strains simultaneously resistant to two or more antimicrobial groups. As stated before, antibiotics are the main tool to treat and control bacterial infections in farmed animals especially in intensive breeding system and (1, 2, 5, 6, 7, 8, 9), therapeutic, metaphylactic and prophylactic practices are considered in order to reduce the economic losses associated with animal mortality and condemnation of carcasses (1, 2, 3, 4, 5, 7, 8). Unfortunately, such intensive use increased the antibiotic selection pressure and the consequent occurrence of multidrug resistant bacteria (9, 10, 11). Conclusions The results of the in vitro antimicrobial susceptibility testing from the study indicated that animal origin strains of E. coli displayed resistance to various antimicrobials commonly used in veterinary medicine and with relevance for human medicine. MAR detection in case of E coli underlines the importance of the rational use of antimicrobials and recommends continuous monitoring of the trends and features of resistance given the role of enteric bacteria as potential reservoir of resistance genes. Acknowledgements The work was done under the frame of European Social Fund, Human Resources Development Operational Programme , project no. POSDRU/159/1.5/S/ References 1. Bhoomika, S.S., Patyal, A., Gade, N.E., Occurrence and characteristics of extended-spectrum β-lactamases producing Escherichia coli in foods of animal origin and human clinical samples in Chhattisgarh, India, Vet World, 2016, 9(9), Boonyasiri, A., Tangkoskul, T., Seenama, C., Saiyarin, J., Tiengrim, S., Thamlikitkul, V., Prevalence of antibiotic resistant bacteria in healthy 101

102 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA adults, foods, food animals, and the environment in selected areas in Thailand, Pathog Glob Health, 2014,108(5), de Been, M., Lanza, V.F., de Toro, M., Scharringa, J., Dohmen, W., Du, Y., Hu, J., Lei, Y., Li, N., Tooming-Klunderud, A., Heederik, D.J., Fluit, A.C., Bonten, M.J., Willems, R.J., de la Cruz, F., van Schaik, W., Dissemination of cephalosporin resistance genes between Escherichia coli strains from farm animals and humans by specific plasmid lineages, PLoS Genet, 2014, 10(12):e Fluckey, W.M., Loneragan, W.G., Warner, R., Brashears, M.M., Antimicrobial drug resistance of Salmonella and Escherichia coli isolates from cattle feces, hides, and carcasses, J Food Prot., 2007, 70(3), Kar, D., Bandyopadhyay, S., Bhattacharyya, D., Samanta, I., Mahanti, A., Nanda, P.K., Mondal, B., Dandapat, P., Das, A.K., Dutta, T.K., Bandyopadhyay, S., Singh, R.K., Molecular and phylogenetic characterization of multidrug resistant extended spectrum beta-lactamase producing Escherichia coli isolated from poultry and cattle in Odisha, India, Infect Genet Evol., 2015, 29, Koo, H.J., Woo, G.J., Characterization of antimicrobial resistance of Escherichia coli recovered from foods of animal and fish origin in Korea, J Food Prot, 2012, 75(5), Mora, A., Blanco, J.E., Blanco, M., Alonso, M.P., Dhabi, G., Echeita, A., González, E.A., Bernárdez, M.I., Blanco, J., Antimicrobial resistance of Shiga toxin (verotoxin)-producing Escherichia coli O157:H7 and non-o157 strains isolated from humans, cattle, sheep and food in Spain, Res Microbiol., 2005, 156(7), Platteel, T.N., Leverstein-Van Hal, M.A., Cohen Stuart, J.W., Voets, G.M., van den Munckhof, M.P., Scharringa, J., van de Sande, N., Fluit, A.C., Bonten, M.J., ESBL National Surveillance Working Group, Differences in the antibiotic susceptibility of human Escherichia coli with poultry-associated and non-poultry-associated extended-spectrum betalactamases, Eur J Clin Microbiol Infect Dis, 2013, 8, Rasheed, M.U., Thajuddin, N., Ahamed, P., Teklemariam, Z., Jamil, K., Antimicrobial drug resistance in strains of Escherichia coli isolated from food sources, Rev Inst Med Trop Sao Paulo, 2014, 56(4), Tadesse, D.A., Zhao, S., Tong, E., Ayers, S., Singh, A., Bartholomew, M.J., McDermott, P.F., Antimicrobial Drug Resistance in Escherichia coli from Humans and Food Animals, United States, , Emerging Infectious Diseases, 2012, 18(5), Thorsteinsdottir, T.R., Haraldsson, G., Fridriksdottir, V., Kristinsson, K.G., Gunnarsson, E., Prevalence and genetic relatedness of antimicrobial-resistant Escherichia coli isolated from animals, foods and humans in Iceland, Zoonoses Public Health, 2010, 57(3),

103 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA STUDY OF INFECTIOUS CAUSES OF BOVINE INFECTIOUS MASTITIS IN LACTATING COWS IN A FARM FROM WESTERN ROMANIA CORINA PASCU, V. HERMAN, IONICA IANCU, J. DEGI, SORINA MERISANU Banat s University of Agricultural Sciences and Veterinary Medicine King Michael I of Romania from Timisoara, Faculty of Veterinary Medicine, Calea Aradului, 119, Timisoara , Romania corina_pascu_ro@yahoo.co.uk Summary A cross-sectional study was conducted from January 2016 to March 2016 on a total of 45 lactating (Holstein and Red Holstein) dairy cows randomly selected from a dairy farm from western Romania. The aim of this study was to determine the prevalence of mastitis and isolate the bacteria that cause mastitis. Milk samples was collected and bacteriological culture was done. Confirmation was done by API system. The prevalence of mastitis in the farm was 62.2% (n = 28). From these cases 28,8% (n = 13) were clinical and 51.1% (n =23) were subclinical mastitis. Among the isolated bacterial genera, the isolate were Staphylococcus (54.2%), Streptococcus (25.7%), E. coli (8.5%), Enterococcus (5.7%) and Pseudomonas (5.7%). There were characterized the follow species: Staphylococcus aureus, S. intermedius and S. hycus, Streptococcus agalactiae, Enterococcus faecium and E.durans. Other objective of study was to determine isolated bacteria susceptibility at antimicrobials, knowing the resistance present in a large number of bacteria isolated from mastitis cases. This study shows that mastitis is significant problem of dairy cows in the study farm and the isolated bacteria were contagious pathogens. Keywords: lactating cows, mastitis, infectious causes. Mastitis represents the main causes of economical losses in cows, in lactating cows specially (Holstein, Romanian black Pied, Romanian spotted). Etiology of these mastitis is grouped in two categories: pathogens that produce contagious mastitis (S. aureus, Str. agalactiae, Mycoplasma bovis), and pathogens that produce environmental mastitis - Str. uberis, Str. dysgalactiae, Enterococcus spp., coliforms (1, 3, 8). Pathogens isolate from clinical mastitis were changed in time as a result of control measures from a mastitis produced by S. aureus and Str. agalactiae high incidence to a higher incidence of environmental or minor pathogens coagulasenegative staphylococci. Antimicrobials susceptibility in vitro determined was considered a condition for treatment; however in vitro antimicrobial sensitivity is not a guarantee for their efficiency in vivo (2). In this condition is necessary a treatment program, as a part of farm management, based on testing of sensitivity microorganisms at antibiotics. 103

104 104 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA Materials and methods A cross-sectional study was conducted from January 2016 to March 2016 on a total of 45 lactating (Holstein and Red Holstein) dairy cows randomly selected from a dairy farm from western Romania. The aim of this study was to determine the prevalence of mastitis and isolate the bacteria that cause mastitis. For detection mastitis cases were performed some exams and tests: organoleptic aspect of milk, clinical exam of udder, both to detect clinic mastitis, and to detect subclinical mastitis California Mastitis Test was used. All milk samples were bacteriological tested to isolate and identified pathogens. The sensitivity test to antibiotics was made using diffusimetric Kirby Bauer method, with the next antibiotics: enrofloxacin (ENF 5µg), gentamicin (G 10µg), ceftiofur (EFT 30µg), cefquinome (CEQ 30µg), amoxicillin-clavulanic acid (AMC 30µg), penicillin (P 10IU), lincomycin+neomycin (15+60µg), novobiocin (5µg), tetracicline (TE 30µg). Results and discussions Milk samples was collected and bacteriological culture was done. Confirmation was done by API system. The prevalence of mastitis in the farm was 62.2% (n = 28). From a clinical point of view mastitis cases were classified in: clinical mastitis and subclinical mastitis. From these cases 37.1% (n = 13) were subclinical and 62.8% (n =22) were subclinical mastitis. Depending on cultural character, mastitis was classified as monomicrobial and polimicrobial mastitis. From all milk samples were isolated 35 bacterial strains belonging to several bacterial genera: Staphylococcus (54.2%), Streptococcus (25.7%), E. coli (8.5%), Enterococcus (5.7%) and Pseudomonas (5.7%). There were characterized the follow species: Staphylococcus aureus, S. intermedius and S. hycus, Streptococcus agalactiae, Enterococcus faecium and E.durans. Table 1 Correlation between microorganism type and mastitits type Mastitis No Bacterial genera Clinical Subclinical strains No strains % No strains % Staphylococcus spp Streptococcus spp Escherichia coli Enterococcus sp Pseudomonas sp TOTAL

105 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA Antimicrobials were chosen watching more aspects: high efficiency, easy to administrate, quick absorbance and, not in the last, the antimicrobial`s resistance phenomena present to a large number of antimicrobials. Generally, it could be observed a variable behavior, resistant, intermediate and sensitive strains were found. Results are presented in figure 1. No of strains Fig. 1. Antimicrobials testing results Each strain was resistant at least one antibiotic. At enroxil were obtained the most sensitive strains 22 from 35 tested, 7 were intermediate and 6 strains were resistant. Resistant strains were streptococci and the two enterococci strains. From cephalosporines, ceftiophur was more efficiency, 20 strains were sensitive, comparing with cefquinome with only 13 sensitive strains. This is probably the result of overuse of this antibiotic in that farm. Similar behavior has been reported to penicillin, with 17 sensitive strains, but 16 strains were resistant. At amoxiciline-clavulanic acid and lincomycin-neomycin were sensitive 15 strains each. At amoxiciline-clavulanic acid were resistant 11 strains, and at lincomycin-neomycin 9 strains were resistant. Neomycin resistance, alone or in association with other antibiotic, was mentioned in other studies, in which 41% from strains were resistance to neomycin (5, 6). The lowest number of sensitive strains has been obtained at tratraciclline and novobiocine, 6 and 4 strains respectively. Our results about E. coli strains were comparable with other results from literature; increasing resistant E. coli strains isolate from mastitis was mentioned by a number of authors (7, 8). 105

106 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA Conclusions From all 45 milk samples which came from cows with/without mastitis symptoms has been isolated 35 bacterial strains belonging to several genera as it follows: 19 strains of staphylococci, 9 strains of streptococci, 3 strains of E. coli, 2 strains of Pseudomonas sp. and 2 strains of Enterococcus sp. From cows with clinical mastitis were isolated 22 strains, and from subclinical mastitis were isolated 13 strains, staphylococci predominating in both mastitis types. Enrofloxacin and cephtiofur were the most efficient antibiotics for all tested bacterial strains. Amoxicillin-clavulanic acid and cefquinome, use frequent in mastitis treatment, had generated the most resistant strains, as tetracycline also. Acknowledgements This study was realised using the support and infrastructure project Dezvoltarea infrastructurii de cercetare, educaţie şi servicii în domeniile medicinei veterinare şi tehnologiilor inovative pentru RO 05, cod SMIS-CSNR References 1. Bakr, E. M., Abd El-Tawab, M., Elshemey, T. M., Abd-Elrhman. A. H., Diagnostic and therapeutic studies on mycotic mastitis in cattle, Alexandria Journal of Veterinary Sciences, 2015, 46, Buiuc D., Neguţ, M., Tratat de microbiologie clinică, ed. a II-a, Ed. Medicală, Bucureşti, Drugociu, D., Mastita subclinică la vacă, Veterinaria, 2011, 2, Quinn, P. J., Markey, B., Carter, M. E., Donnely, W. J., Leonard, F. C., Veterinary Microbiology and Microbial Disease, Blackwell Science Ltd., TJ International Ltd., Padstow, Cornwall, Great Britain, Pyörälä, S., Pyörälä, E., Efficacy of parenteral administration of three antimicrobial agents in treatment of clinical mastitis in lactating cows: 487 cases ( ), J.A.V.M.A., 1998, 212, Rabello, R. F., Susceptibilidade aos antimicrobianos e diversidade genética de amostras de Staphylococcus aureus e Streptococcus agalactiae isoladas de casos de mastite subclínica no Estado do Rio de Janeiro, Universidade Federal do Rio de Janeiro, Rio de Janeiro, 2003, Makovec, J. A., Ruegg, Pamela L., Antimicrobial resistance of bacteria isolated from dairy cow milk samples submitted for bacterial culture: 8,905 samples ( ), J.A.V.M.A., 2003, 222(11), Prescott, J. F., Baggot, J. D., Antimicrobial therapy in Veterinary Medicine, vol. II, Ames: Iowa State University Press, USA,

107 LUCRĂRI ŞTIINŢIFICE MEDICINĂ VETERINARĂ VOL. L (3), 2017, TIMIŞOARA CHANGES IN CELL- MEDIATED IMMUNITY DUE TO ANTI- ANTHRAX VACCINATION AND IN VITRO VEGETAL EXTRACT TREATMENT IN GOATS CARMEN DANA ŞANDRU, Z. INCZE, F. BRUDAȘCĂ, R. GIUPANĂ, C. CERBU, I. IONUȚ, SILVANA POPESCU, I.S. GROZA, MARINA SPÎNU University of Agricultural Sciences and Veterinary Medicine Cluj- Napoca, , 3-5 Mănăştur Street, Cluj-Napoca, Romania sandranac@gmail.com Summary Anti-anthrax vaccination, one of the most relevant preventive measures in infectious disease control, is a procedure that could be invasive for goats, a species subject to acute stress. Cell-mediated immunity in these animals is therefore at crossroads between vaccination stress and immune enhancing antigen priming. The experimental goats were divided into two age groups, under (n=10) and above three (n=20) years of age and were vaccinated with the Romanian R 1190 anthrax strain. Blood was sampled before and 14 days after the vaccination. The blood picture and calculation of the N/L ratio was used as an indicator of stress. Total Ig and circulating immune complexes levels were performed to monitor the humoral immune response, while the blast transformation test aimed to investigate the changes in the cell mediated immunity. N/L ratio remained unchanged in both age categories in spite of stressful conditions (vaccination). The total Ig levels decreases in young animals, the level of complexation being strongly increased after the vaccination as well as the synthesis of circulating immune complexes clearance. The antigen priming led to a booster type response in the age category over three years, standing for their prolonged contact with this type of vaccine over the years of their economic life in the blast transformation test. Tymus serpyllum alcoholic extract was the most effective in vitro in both age categories, before and also after the vaccination. The Juniperus communis extract had a modulating character, augmenting and diminishing the cell-mediated response before and after the vaccination, respectively. These results supported the use of certain extracts as to improve the post-vaccination cell-mediated immunity, depending on their immunological activity and the age category. Keywords: systemic immunity, goats, total Ig, anthrax vaccination Anthrax is a bacterial disease, common in several animal species and humans, caused by Bacillus anthracis, a large Gram positive bacterium defined by an antiphagocytic polyglutamic capsule, ensuring resistance against the immune system and and spores, securing survival in adverse environment (12). The bacteria possesses toxins, namely the edema factor (EF), the lethal factor (LF), and the protective antigen (PA) sharing polypeptidic structure. Their effect is alteration of cellular signaling pathways in the host and subsequent interference with innate immune responses. Later during the clinical course, vascular collapse occurs (6). 107

108 108 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA The disease is best prevented by vaccination in animals. Nevertheless, current anthrax vaccines can cause considerable local and general secondary reactions such as erythema, induration, soreness, fever or even, sometimes, clinical disease (10, 11). The immunity subsequent to vaccination lasts for about 6 month, and not all animals are protected different strains of B. anthracis (11). Lately, due to Bacillus anthracis bioterrorism threat, next-generation vaccines are needed (3). Modern vaccines including recombinant live bacteria and recombinant sub-unit antigens, free of side effects, require multiple vaccinations and inclusion of adjuvants, increasing the difficulties of vaccination programs (1). In Romania, vaccination against anthrax is included in the National stategic program for disease prevention and goats are subject to it, depending on the infectious pressure of the habitat. Nevertheless, this vaccination is extremly stressfull for goats, the immune mechanisms involved participating in immunogenesis but also in the adverse reactions (4). This study aimed at monitoring the changes caused by the vaccinarion with the live attenuated vaccine R 1190 Stamatin in goats, based on their age, investigating the interferance of immune responses at cellular and humoral levels and the stressfull effect of the vaccination. Materials and methods The research was carried out in a herd of 300 Carpathian goats, two groups being selected, one of animals older than 3 years (n=20), subjected to several vaccinations up to the testing and the other of animals below 3 years (n=10), vaccinated fewer times. The animals were sc injected with 0.2 ml each of the R1190 Stamatin vaccine (Romvac, Romania) on the farm. Blood was sampled on EDTA and heparine (50IU/ml) and on clothing gell from all groups before and two weeks after the vaccination. Changes of N/L ratios as stress indicators, changes in total Ig levels and circulating immune complexes and also the in vitro adaptive cell mediated response were monitored to evaluate the changes caused by vaccination. The blood smears from blood preserved on EDTA were stained with Dia quick Panoptic kit and leukocyte subpopulations were counted. The N/L ratio was calculated and interpreted as stress indicators according to Davis et al., 2008 (2). Circulating immune complex (CIC) measurements allow an evaluation of the level of molecular clearance capacity of the body. Serum was separated from the clot by centrifugation at 3,000 rpm for 10 min and kept at -80 C till testing. A 4.2% polyethylene glycol (PEG) 6000 solution in borate buffer was used as precipitating agent, while buffer-treated samples served as controls for borateinduced precipitation. For each sample, 6.6µl aliquots of the serum were mixed with µl of borate buffer or PEG solution in parallel wells. The immune complexes precipitate in 60 min at C. Spectrophotometrical reading of the results was performed at a wavelength of 450 nm in the test plate (d=0.5 cm; multichannel

109 LUCRĂRI ŞTIINŢIFICE MEDICINĂ VETERINARĂ VOL. L (3), 2017, TIMIŞOARA spectrophotometer SUMAL PE2, Karl Zeiss, Jena). CIC concentrations, expressed in optical density units (ODU), were calculated by subtracting the value of the control serum+buffer from that of the PEG precipitate. Total immunoglobulins are precipitated by concentrations as low as 24 mg/l of heavy metal salts. Volumes of 3.3µl of the sera were diluted in µl of a 0.024% barbital-buffer zinc sulfate solution, and allowed to precipitate for 30 min at room temperature (22 23 C). The levels of total immunoglobulins were quantified in optical density units (ODU) after spectrophotometrical readings (λ=475 nm, d=0.5 cm; multichannel spectrophotometer SUMAL PE2, Karl Zeiss, Jena). The leukocyte blast transformation was performed to estimate the spontaneous, mitogen and vegetal extract induced transformation on lymphomonocytes. Blood collected from the experimental animals was processed within 4 hours after sampling. Each blood sample (1 ml) was diluted with four times the amount of RPMI 1640 supplemented with 5% FCS and antibiotics, ph 7.4, (Sigma- Aldrich, USA). The mixture was distributed in 96-sterile-well plate (200 μl per well). Eight in vitro experimental variants were tested in duplicate for each individual animal, namely: 1) untreated control culture, 2) phytohaemagglutinin-m (PHA), 3) concanavalin (ConA) and 4) lipopolisacharid (LPS)(1μ per well) treated culture, 5) 70 alcohol treated culture and (6 11) alcoholic vegetal extracts of Allium sativum, Tymus serpyllum, Hypericum perforatum, Juniperus communis, Echinacea purpurea and Calendula officinalis produced by Plantextract, Romania according to the German Homeophatic Pharmacopeia and subject to company quality control, were used to treat the whole blood cultures (1,5 μl /well).the cultures were incubated for 72 h at 37.5 C and 5% CO 2 atmosphere. Glucose concentrations, as a growth indicator, were measured in the initial culture medium and in all variants at the end of the incubation period, using a glucose standard (100 mg%), by means of an orto-toluidine colorimetric test. For this, 12.5 μl of the cultural supernatant were transferred to 0.5 ml of orto-toluidine reagent, boiled for 8 min, cooled suddenly in cold water and read in a spectrophotometer at 610 nm wavelength (SUMAL PE2, Karl Zeiss, Jena, Germany), using the reagent as a blank. The cell stimulation/inhibition index (S/I) was calculated as follows: S/I %=[(IG GR)/MG]x100, where S/I =blast transformation index, IG= the initial glucose concentration in the culture medium and GR=glucose residue in the sample after incubation (5, 9). Student t-test was used to calculate the significance of differences and Excel program was used to calculate the average and standard error. Results and discussions Goats are very sensitive to anti-anthrax vaccination, given their severe reaction to stress of any kinds. In case of numerous animals showing side effects subsequent to anti-anthrax vaccination, the problem consisted mainly of outbreak control (8). In the outbreak described, 5 of 10 deaths occurred 3, 5, 11, 68 and

110 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA days after vaccination and, in another case, 37 days after revaccination. OIE, WHO, FAO indicated in 2008, that the potential use of a double vaccination, the booster being included one month after the primo-vaccination could solve the problem (12). Bibliography on the immunogenicity of Sterne 34F2 vaccine in ruminant hosts is scarce. Ndumnego et al., in a study in 2016, looking at the humoral response to the Bacillus anthracis protective antigen against various toxic compounds concluded that the crucial second vaccination could be done even after 3 month from the previous one (7). The results of our study indicated that the N/L ratio did not undergo major changes after the vaccination compared to the initial sampling, in neither of the groups (Fig. 1) % 30 N/L N/L <3 20 > Neutroph Eosinoph Basoph Limpho Mono N/L Neutroph Eosinoph Basoph Limpho Mono N/L Before vaccination After vaccination Fig. 1. There was no increase in the N/L ratio, expected to indicate postvaccination stress in goats injected with an R1190 anti-anthrax vaccine As oposed to that, the sharp increase in eosinophiles could suggest an alergic type reaction in the animals under 3 years of age, less exposed to this kind of vaccination before than the adults. In this case, the N/L ratio could not be used as a stress indicator, as suggested by Davies et al., 2008 (2), in spite of the wellknow responsiveness to stress in this species. There was a decrease of total Ig 110

111 LUCRĂRI ŞTIINŢIFICE MEDICINĂ VETERINARĂ VOL. L (3), 2017, TIMIŞOARA levels in young animals, as opposed to the older ones, subjected to numerous booster operations by the moment of the experiment (Fig. 2). This parameter should be interpreted only in conection to the CIC levels in both groups (Fig. 3) before vacc post vacc < 3 years > 3 years Fig. 2. Increased total Ig levels in older animals as opposed to the younger ones, possibly stand for the more numerous booster vaccines the first were subjected to in time before vacc post vacc < 3 years > 3 years Fig. 3. CIC values before and after the vaccination in both age groups: there was an increase in younger animals and little or no change in the older ones 111

112 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA M PHA LPS ConA Vac Before After Alc Ust Cim Sun Ien E.pur Gal Fig. 4. Blast transformation indices in the younger animals:cic values before and after the vaccination in both age groups: there was an increase in younger animals and little or no change in the older ones Legend: M-control, PHA- phytohaemagglutinin, LPS- lipopolisacharid, ConA-concanavalin A, V-vaccine, Alc- alcohol, Ust-garlic, Cim- Tymus serpyllum, Sun Hioppericum perforatum, Ien - Juniperus communis, E.pur-Echinacea purpurea, Gal- Calendula officinalis M PHA LPS ConA Vac Inainte de vacc dupa vacc Alc Ust Cim Sun Ien E.pur Gal Fig. 5. Blast transformation indices in the younger animals:cic values before and after the vaccination in both age groups: there was an increase in younger animals and little or no change in the older ones 112

113 LUCRĂRI ŞTIINŢIFICE MEDICINĂ VETERINARĂ VOL. L (3), 2017, TIMIŞOARA As mentioned before, the sharp decrease of total Ig in younger animals and relatively high CIC levels, stand for the mild complexation and eliminationof the complexes, in spite of the fact that vaccine stimulation should activate all immune processes. The blast transformation indices were different (Fig. 4 and 5), although non significantly, between the two age groups prior to vaccination. In older animals, the indices were somewhat higher before the vaccination than in the youger group, but subsequent to the vaccination, in youn ynimals there was a sharo increase in cellmediated immunity. The Juniperus extract was the one who acted inhibiting in both groups. This could have happened because of the single in vitro dose that was used. It is obvious that the vegetal extracts enhance the response to the vaccine, thus opening perspectives for their use as adjuvants. Conclusions The anti-anthrax vaccination in goats is immunological activating not only the antibody synthesis, but also other, non-specific and specific mechjanisma, showing a systemic effect. Towards this vaccine, the overall immunological activity was more intense in the younger animals, in spite of the numerous boosters the older ones received before the experiment. This stands for a more pronounced overall immunological adaptability to this type of antigenic stimulation in the younger animals. The blast trasformation response indicated the immune stimulating but meanwhile potentially modulating activity of certain vegetal extracts. References 1. Baillie, L.W., Is new always better than old?: The development of human vaccines for anthrax., 2009, 5(12), Davis, A.K., Maney, D.L., Maerz, J.C., The use of leukocyte profiles to measure stress in vertebrates: a review for ecologists, Functional Ecology, 2008, Friedlander, A.M., Little, S.F., Advances in the development of nextgeneration anthrax vaccines., 2009, 27(4), D Ghergariu, S., Pop, A., Kadar, L., Spînu, Marina, Ghid de laborator clinic veterinar, Ed. ALL, Bucureşti, Khokhlova, Irina S., Spinu, Marina, Krasnov, B.R., Degen, A.A., Immune response to fleas in a wild desert rodent: effect of parasite species, parasite burden, sex of host and host parasitological experience, The Journal of Experimental Biology, 2004, 207, Moayeri, M., Leppla, S.H., Vrentas, C., Pomerantsev, A.P., Liu, S., Anthrax Pathogenesis, Ann. Rev. Microbiol., 2015, 69,

114 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA 7. Ndumnego, O.C., Köhler, S.M., Crafford, J., Van Heerden, H., Beyer, W., Comparative analysis of the immunologic response induced by the Sterne 34F2 live spore Bacillus anthracis vaccine in a ruminant model, Vet. Immunol. Immunopathol., 2016, 178, Salmon, D.D., Ferrier, G.R., Post-vaccination occurrence of anthrax in cattle, Vet. Rec., 1992, 130, Spînu, Marina, Niculae, Mihaela, Paştiu, Anamaria Ioana, Şandru, Carmen Dana, Pall, Emoke, Vasiu, A., Vegetal extracts influence in vitro on the cell-mediated immunity in carnivores depending on health status, target species and plant taxonomy, Industrial Crops and Products, 2016, 88, Splino, M., Patocka, J., Prymula, R., Chlibek, R., Anthrax vaccines, Ann. Saudi. Med., 2005, 25(2), Zakowska, D., Kocik, J., Bartoszcze, M., Selected research problems of anthrax vaccine development., Przegl Epidemiol., 2009, 63(4), *** OIE, WHO, FAO, Anthrax in humans and animals Fourth edition, 2008, WHO Press, Geneva, Switzerland. 114

115 LUCRĂRI ŞTIINŢIFICE MEDICINĂ VETERINARĂ VOL. L (3), 2017, TIMIŞOARA INCREASE OF ANTIBIOTIC RESISTANCE OF MICROFLORA FROM MASTITIC SOWS SUBSEQUENT TO UNDISCRIMINATORY GENERAL ANTIBIOTIC THERAPY MARINA SPÎNU, C. CERBU, DELIA-ANDREEA DIȚU, F. BRUDAȘCĂ, I. IONUȚ, R. GIUPANĂ, A. VASIU, SILVANA POPESCU, CARMEN DANA ŞANDRU University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca , 3-5 Mănăştur Street, Cluj-Napoca, Romania marina.spinu@gmail.com Summary The bacterial load of the colostrum and milk as well as that of the mammary gland in sows exerts a strong influence on the growth rate of the piglets. Regular testing by the Kirby-Bauer disc diffusion method proves to be valuable in designing the antibiotic therapy in sows, thus avoiding the antibiotic - resistance phenomena that could lead to the economic losses. The mastitic sow milk microbiome and its antibiotic - resistance were evaluated in this study in order to establish an appropriate protocol for general therapy on the farm. The research was conducted on 38 sows, of which 9 with clinical mastitis signs while the others were clinically healthy, previously subject to unsystematic treatment with antibiotics for various health problems. Milk was sampled and examined by classical microbiological techniques. Bacterial strains were identified and tested for antibiotic sensitivity/resistance in two steps: the resistant colonies identified in all samples during the first step were subjected to testing by Kirby Bauer method using a second batch of antibiotics. In both mastitic and healthy milk samples Streptococcus, Staphylococcus, coliforms were present. Clostridium, Corynebacterium and others were observed as well, all these being included in the other bacteria category. Colonies resistant to florfenicol, doxicycline, tetracycline, amoxicillin, streptomycine, ampicilline, enrofloxacine were identified in all isolates. Cefquinome, then Tetra-delta and tulathromycin were the most active during the second testing, with resistant colonies or total resistance to Neomicine/Bacitracine/ Tetracycline, Lincomycin/ Spectinomycin, Trimethoprim/ Sulfametoxazole. The results supported the increased antibiotic resistance due to irrational use of antibiotics, compromising the sow in the case of a bacterial re-infection due to lack of antibiotic treatment efficacy. Key words: sow, mastitis, KB testing, antibiotic-resistance Modern swine production all over the world confronts nowadays the Postpartum Dysgalactia Syndrome (PDS). Within this framework, the coliform mastitis (CM) is known as a multi-factorial infectious disease postpartum. The animals refuse to eat and drink, the mammary glands are red and painfull, the rectal temperature is increased over 39.5 C, sometimes vaginal discharge. The most important symptom is a severe drop in the milk production, which fails to appropriately feed the piglets. Husbandry- and management were incriminated earlier as the most important factors inducing mastitis in sows, but lately, the role of infectious agents was stressed. Ubiquitous agents such as E. coli and S. aureus were mostly isolated from the milk of the animals, but other Enteriobacteriaceae 115

116 116 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017 TIMISOARA could also be present (2, 6). Mastitis due to Trueperella pyogenes, if present, could be a major cause of losses in piglets (4). Frequency and severity of this complex disease depends on the immune status and reactive capacity of the sows, some remaining healthy, while others develop clinical or sub-clinical mastitis (3). In a study carried out on 56 sows after farrowing the prevalence of different bacterial species in the anterior and posterior mammary glands of sows was monitored, in connection with mastitis and agalactia, but the results did not support any differences between the healthy and diseased sows. It was concluded that the development of clinical infection was dependant on the individual resistance of single sows (5). Resistance to antibiotics became lately one of the most important issues in human and animal therapy. The coliform bacteria are sensitive to a broad spectrum of antibiotics especially macrolides (azithromycin, clarithromycin, erythromycin, spiramycin, tylosin/tylocine, roxithromycin, etc.), tetracyclines or sulphamides (12). 50% of the mastitis resistant to antibiotics, mainly amoxicillin and oxitetracyclin were caused by E. coli. These bacteria were though sensitive to enrofloxacine (1). Resistance to antibiotics in swine is also an increasing phaenomenon. A research conducted to establish the resistance/sensitivity of the coliform bacteria to antibiotics in order to establish an appropriate treatment protocol showed that their sensitivy to different drugs was very low: to ampicillin only 19.2%, to tetracycline %, to neomycin %, while to streptomycin - 0.4% and to β-lactams (cephalosporins, monobactams, carbapenems) of 3.5% (14). The mastitic sow milk microbiome and its antibiotic - resistance were evaluated in this study in order to establish an appropriate protocol for general therapy on the farm. Materials and methods The research was carried out on 38 sows on an intensive breeding farm. Out of the total, 47.37% were aged 2 years, 50 % were 3 years old and only 2.63% were four years old. Before the farrowing, the animals were moved to individual boxes in the maternity shelter. All the treatments on the farm occasioned by infections of any kind were done with antibiotics, marbofloxacin being frequently used. No sensitivity tests were carried out, the protocol including a double dose of any available antibiotic, done once. All sows were lactating at the moment of the research. At the clinical examination, the animals showed acute or chronic mastites with nodules, cutaneous lesions or ulcers on the teats skin. The vaccination protocol included two vaccines, ie Gletvax 6 (Zoetis, Belgia) against Escherichia coli strains K88ab, K88ac, K99, 987P and Clostridium perfringens C, Porcilis Parvo (Intervet Olanda) against swine parvovirus. Milk was sampled from each sow once the piglets fed, in healthy animals and before the treatment in the clinically diseased ones. Before the sampling, the mammary gland was washed with warm water and soap, rinsed thoroughly with

117 LUCRĂRI ŞTIINŢIFICE MEDICINĂ VETERINARĂ VOL. L (3), 2017, TIMIŞOARA warm water, dried with a clean towel and disinfected with 9% medical alcohol. The samples were harvested and kept in sterile recipients at +4 C till processing. The milk samples were transfered to simple broth, incubated for 24h at 37 C and then transfered to Mueller Hinton and to MacConkey agar. After 24 h of incubation and preliminary microscopy by Gram stain (11), API 20 Strep, API Staph, API 20 E selective tests were applied to isolated colonies, depending on their type. The Kirby Bauer antibiotic sensitivity test on Muller Hinton agar was performed, using enrofloxacin (ENF), amoxicillin (AX), neomicin/bacitracin/ tetracycline (NBT), oxytetracycline (O), ampicillin (AM), Tetra Delta (TD95), a typical antimastitic combination efficient against Streptococcus aureus, S. uberis, S. dysgalactie, S. agalactiae şi Escherichia coli, florfenicol (FLO), streptomycine (S), tetracycline (T), doxicicyclin (DO), tulathromycin (TUL), Lincomycin/ Spectinomycin (LCS 109), Cefquinome (CEQ), Trimethroprime/Sulphametho-xazole (SXT 25). The inhibition diameters were measured in mm and resistant colonies or total resistance recorded. Resistant colonies were retested with another batch of antibiotics. Results and discussions Swine farming expanded worldwide in the last decades, the number of animals increased due to the increased need for pork. Sow mastitis represented a study object for at least half a century, but the interest in this topic has not diminished (10). Once diagnosed, antibiotic resistance explained the failure of various treatments and posed numerous new questions to practicing vets and microbiologists. The starting point in designing mastitis research in sows concerning antibiotic resistance was represented by mastitis and their treatment in cows. There, more numerous experiments were carried out due to the importance of milk quality for the consumers. In an experiment aiming at establishing the efficacy of cephalonium or cloxacillin within Australian dairy herds, the results indicated that there was no significant difference between treatments for infections with Staphylococcus aureus, Streptococcus agalactiae and Streptococcus uberis. Cephalonium was more efficient than cloxacillin against Corynebacterium bovis and Staphylococcus epidermids (80.3% versus 70.7%)(13, 15). Similarly, in cow mastitis, epidemiological studies indicated bacteriological cure rates vary between 0% and 80% when antibiotics were used. There are researches that support the idea of a biofilm formation and significant genetic and physiological changes in bacteria after the antibiotic treatment, rather than the loss of activity of the major classes of antibiotics used for the treatment of bovine mastitis (9). In sows, a study assessing the effects of oral administration of chlortetracycline (CTC) to prevent and minimize reproductive infections in sows and gilts indicated that reproductive failure of bacterial origin can be controlled with in-feed use of broad spectrum antimicrobials (7,8). 117

118 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017 TIMISOARA During the first isolation step (16 animals), coliforms were present in 6 (37.50%) while other bacteria including Streptococcus/Staphylococcus were present in 15 samples (93.75%). In the second group, the other bacteria were more numerous, present in all 11 samples (100%), while Streptococcus/Staphylococcus was found in 10 samples (90.90%), and coliforms were the (36.36%). The results indicated that the vaste majority of etilogical agents in these animals were Streptococcus/Staphylococcus. They could be transfered via the water, the fodder, suckling piglets to their mothers, indicating fecal pollution of the environment on the farm. Figure 1 includes the overall percentages of the isolated flora. 120,00% 100,00% 80,00% 60,00% 55,26% 76,31% 97,36% 40,00% 20,00% 0,00% Fig. 1. The distribution by genera of the microflora isolated from mastitic sows. The overlaps are explicable due to the isolation of similar, but also several different strains from various animals After the readings of the antibiograms, the colonies were mostly sensitive to florfenicol. The colonies resistant to this antibiotic were further tested, supplementing the number of antibiotics used. G- bacilli were also constantly present, with E. coli traits. 118

119 LUCRĂRI ŞTIINŢIFICE MEDICINĂ VETERINARĂ VOL. L (3), 2017, TIMIŞOARA Fig. 2. The antibiotic susceptibility to the first series of antibiotics in mastitic sows Table 1 Sensitivity of the colonies resistant to florfenicol against the second round of antibiotics Antibiotic Sample no Tulathromycin RC Neomicin/bacitraci n/ tetracycline RC Tetra-Delta RC RC 20 + RC RC RC 15 Lincomycin/Specti 16 + TR TR TR nomycin RC TR 15 + RC Cefquinome Trimethroprime/ Sulphametho- xazole 17 TR 18 TR 18 TR 11 + RC 12 + RC 19 + RC RT 8 RT Legend: TR- total resistance, RC-resistant colonies. The most efficient antibiotic was florfenicol followed by ampicillin and amoxicillin. Tetracycline was the least active (Fig. 2). After re-insemination, the cultures showed furtherr resistance, tulathromycin and cephquinome being the most 119

120 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017 TIMISOARA active while lincomycin/spectinomycin and trimethroprime/sulphamethoxazole were the least active. Total resistance and resistant colonies were present in case of the latest antibiotics. Conclusions The microbiome in sows with clinical and subclinical mastitis was selected towards resistance under the inappropriate antibiotic treatment pressure. The irrational use of antibiotics at the hand, without testing the bacteria sensitivity as well as the unjust protocol used created a high level of antibiotic resistance at herd level. Not only Gram negative bacteria (E. coli, coliforms) were highly antibiotic resistant, but also Gram positive ones such as Streptococcus/Staphylococcus, Clostridium and Corynebacterium were insensitive to most of the antibiotics, the microbiome changing in a sense where most antibiotics are useless in the treatment of any of the current infections on that farm. References 1. Chandrasekaran, D., Venkatesan, P., Tirumurugaan, K. G., Nambi, A.P., Thirunavukkarasu, P.S., Kumanan, K., Vairamuthu, S., Ramesh, S., Pattern of antibiotic resistant mastitis in dairy cows, Veterinary World, Gerjets, I, Kemper, N., Coliform mastitis in sows: A review. J Swine Health Prod., 2009, 17(2), Jaeger, Alexandra, Bardehle, D., Oster, M., Günther, Juliane, Muráni, E., Ponsuksili, S., Wimmers, K., Kemper, Nicole, Gene expression profiling of porcine mammary epithelial cells after challenge with Escherichia coli and Staphylococcus aureus in vitro, Veterinary Research 2015, 46, Jarosz, ŁS, Gradzki, Z, Kalinowski, M., Trueperella pyogenes infections in swine: clinical course and pathology. Pol J Vet Sci., 2014, 17(2), Kemper, Nicole, Gerjets, Imke, Bacteria in milk from anterior and posterior mammary glands in sows affected and unaffected by postpartum dysgalactia syndrome (PPDS), Acta Veterinaria Scandinavica, 2009, 51, Kemper, N., Bardehle, D., Lehmann, J., Gerjets, I., Looft, H., Preissler, R., The role of bacterial pathogens in coliform mastitis in sows, Berl. Munch. Tierarztl. Wochenschr., 2013, 126(3-4), Korudzhijski, N., Bozhkova, G. Gulubinov, G.V., Dzhurova, I., Georgiev, S., Drug resistance of bacterial strains isolated from sows with the clinical picture ofmastitis-metritis-agalactia, Vet Med Nauki., 1987, 24(7),

121 LUCRĂRI ŞTIINŢIFICE MEDICINĂ VETERINARĂ VOL. L (3), 2017, TIMIŞOARA 8. Martineau, G.P., Smith, B.B., Doizé, B., Pathogenesis, prevention, and treatment of lactational insufficiency in sows, Vet Clin North Am Food Anim Pract., 1992, 8(3), Melchior, M.B., Vaarkamp, H., Fink-Gremmels, J., Biofilms: a role in recurrent mastitis infections?, Vet J., 2006, 171(3), Ponomarova, M.I., Effect of antibiotics on cocci that are mastitis agents in sows, Mikrobiol Zh., 1965, 27(3), Răducanescu, H., Bica-Popii, Valerica, Bacteriologie veterinară, Ed. Ceres, Bucureşti, Răpuntean, Gh., Răpuntean S., Bacteriologie Veterinară Specială, Ed. AcademicPres, Cluj-Napoca, Shephard, R.W., Burman, S., Marcun, P., A comparative field trial of cephalonium and cloxacillin for dry cow therapy formastitis in Australian dairy cows. Aust Vet J., 2004, 82(10), Zanella, G.N., Mikcha, J.M.G, Bando, E., Siqueira, V.L.D., Machinski, M., Occurrence and Antibiotic Resistance of Coliform Bacteria and Antimicrobial Residues in Pasteurized Cow's Milk from Brazil, Journal of Food Protection, 2010, 9, , and * * * WHO Antimicrobial Resistance Global Report on Surveillance, A Summary, 2014,

122 122 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017 TIMISOARA ANTIBIOTIC RESISTANCE PROFILE OF THE MASTITIC BACTERIOME IN DAIRY COWS COULD BE INFLUENCED BY CHANGES IN ph MONICA SUĂTEAN, MARINA SPÎNU, F. BRUDAŞCĂ, R. GIUPANĂ, C. CERBU, A. VASIU, SILVANA POPESCU, CARMEN DANA ŞANDRU, I. IONUŢ University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, , 3-5 Mănăştur Street, Cluj-Napoca, Romania sandranac@gmail.com Summary Mastitis, mainly localized but sometimes generalized infections occurring in dairy animals are initiated by microflora associated with chemical and physical factors. Not only clinical, but also subclinical mastitis represent causes of severe economic loss at farm level. The research was conducted on Romanian Spotted dairy cows raised under semi-intensive farming conditions, aged three to five years, showing clinical or subclinical mastitis diagnosed by California mastitis test and somatic cell counts. The milk samples were colected before the morning milking in sterile containers and processed by classical microbiological methods: cultivation on classic and selective media, performing sensitivity testing by Kirby-Bauer disc diffusion method to ampicillin, enrofloxacin, gentamicin, oxytetracycline, ciprofloxacin, amoxicillin clavulanic acid and commercial product Tetra delta containing novobiocin, neomycin, penicillin procaine, dihydrostreptomycin and prednisolone. To estimate the potential changes in antibiotic sensitivity induced by changes in the environmental ph, the Kirby- Bauer method was repeated on isolated bacteria after their pre-treatment with acid (4-6.5) or alcaline ( ) ph.the dominant bacteria (Enterococcus - 45% and Staphylococcus - 55%), were sensitive to ciprofloxacine and partially or totally resistant to other antibiotics. Both alcaline (8.0) and acid (5.0) ph proved to be useful in increasing the sensitivity to ciprofloxacine for all isolates. The behaviour of bacteria in presence of antibiotics was highly variabble after pre-treatment with with other ph values, ranging from low sensitivity to total resistance. We concluded that changing the ph could be of help in treating mastitis in dairy cows, while associated with the appropriate antibiotics. Key words: ph, mastitis, antibiotic resistance, staphylococci, enterococci Mastitis represent one of the most important diseases which occur in dairy farms, causing significant economical losses due to the high costs of the treatment, as well as the decrease of the milk quantity along the changes of its components (8). This pathology is frequent in bovines due to the breeding conditions on one hand, and due to the association of the microflora with physical and chemical factors, on the other hand. It is characterised by the increase of the somatic cells in the milk along with pathological alterations of the mammary gland tissue (10). Mastitis are classified in clinical, subclinical, they are contagious and enviromental. When the mastitis is subclinical there are no local signs of inflamation, nor systemic, but once the pathogen enteres the cows blood stream it

123 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA can persist throught the whole lactation period. In clinical mastitis we encounter inflamatory responses along side organoleptic changes of the milk. Currently the therapy used in treating this pathology is based on the use of antibiotics, but using them without testing them we allowed the appereance of antibioticoresistance fenomena. This creates difficulties in the therapy protocols (1). Types of mastitis Staphylococi mastitis emerges when the bacteria acts on the mammary gland causing its inflammation, being one of the most encountered types of mastitis. It commonly affects the cows with high milk production, Staphylococcus aureus and Staphylococcus albus being the most frequent strains isolated. Staphylococcus aures is coagulase positive and haemolytic. Other possible strains causing this pathology are: Staph. epidermitis, S. chromogenes, Staph.hominis, Staph. hycus,staph. Sciuri, Staph. Cohnii, Staph. wörneri alongside Staph. xylosus (4). Serous mastitis represent an inflamation of the interstitial connective tissue of the mamary gland. The microflora is comprised of Streptococcus spp.; Staphylococcus spp. (11). The etiological factor in pyobacilar mastits is Corynebacterium pyogenes, bacteria that comes from enteritis and uterus infections causing the inflamation of the mamary gland. This bacteria recieved recently the name of Trueperella pyogenes and it spreads mostly in the residual milk, when the milking is poorly done. The affected udder is enlarged, hard on palpation and frequently there are absseces that become fluctuent and open. The milk is fibrous then purulent (9). Another type of mastitis frequently encountered is that produced by Escherichia coli, and the clinical signs are represented by two types. First there can be a mastitis with paraplegia or without it. The second one manifest through the alteration of the general status, pyrexia, anorexia, and muscular weakness. The milk has a pink-yellowish colour and it is serous. With proper treatment it heals in about 10 days. When discussing the paraplegia mastitis adding to the before mentioned symptoms the cows are paralyzed and can t get up from the ground, they are down on the opposite side of the affected udder (2). A productive inflamation of the udder, characterized by the appearance of granulation nodules with localization under the skin or in the glandular parenchyma as well as the increase in the volume of the mammary gland. In the center of these nodules we can find a small cavity filled with creamy white or yellow puss, that is extremly rich in Actinomices israeli. This type of mastitis is hard to treat because of its antibioticresistance (7). The first case of mycotic mastitis was discovered by Fleischer in This type is caused by the following germs: Pichia fariosa, Candida parapsilosis, Trichosporum cutaneum, Aspergillus spp. or Criptococus neoformans. Udder lesions are a big part of this pathology because the infection with these fungi is made possible through the sorroundings, of the milking machine, the lack of a propper hygene being the most important risk factor (6). 123

124 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017 TIMISOARA Mastits provoked by germs from the Nocardia genus are as well trasmited like the one caused by fungi, and it is characterised by the appearance of fybrosis and nodules on the udder. This type has a chronic evolution and its almost always refractory to antimicrobial therapy (12). A less documented mastitis is the one caused by algae, specifically Prototecha spp. (Prototecha zopfii), an unicelular organism (4). The milk production decreases, it has a watery consistance with clots of milk in it. Histologically, we encounter the pyogenic mastitis with the presence of algae within the alveolar lumen, interstitial or macrophages between epithelial cells (5). Materials and methods The biological material used in this research was milk. The milk samples were collected in sterile containers before the morning milking and after California Mastitis Test was performed. The exams used in this thesis were microbiological exams, consisting in bacteriological exams. The bacteriologic exams consists in a bacterioscopic exam using the Gram stain technique, alongside a cultivation on broth and simple agar and as well on blood agar. For the biochemical exams we used API test, more exactly API Staph and API Strep tests. The antibiograms used for testing the susceptibility/resistance to certain antibiotics, we used the Kirby-Bauer method. A specific test we used a protocol involving a in vitro treatment protocol of the isolated bacterial strains using acidifiers at different ph values such as (4.0, 4.5, 5.0, 5.5, 6.0 and 6.5). Similar we used a in vitro treatment protocol of the isolated bacterial strains but this time we used alkalinizing agents at ph values of 7.8, 8.0 and 8.2. Results and discussions After the before mentiones test we obtained different results. On cultural aspects on broth, we observed the degree of turbidity going from mild to a opac aspect of the broth. As well we need to observe the presence or absence of the deposit in the broth. we obtained a procent of 50% of samples with the presence of a white, adesive deposit. On simple agar we observed round colonies, both smooth and rugous, all the colonies were white, excepting two which were slightly yellow. The colonies varied in dimensions between small to medium colonies. On the Gram stain tehnique we observed coci and diplococci, gram positive.after using the biochemical tests, API test we obtained Staphylococcus xylosus, Staphylococcus scirui and two strains of Enterococcus faecium (Fig.1, Fig. 2 and Fig. 3). 124

125 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA Fig. 1. API Staph Test Staphylococcus xylosus (original) Fig. 2. API Staph Test Staphylococcus sciuri ( original) Fig. 3. API 20 STREP Test Enterococcus faecium (original) 125

126 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017 TIMISOARA The antibiotics used for testing in this thesis were: Tetracicline, Amoxilicillin with clavulanate acid, Enrofloxacin. Gentamicin, Ciprofloxacin and Ampicilin obtaining for the specific tests were we used acidifing and alkalinizing agents we obtained the following results explained in Fig. 4., Fig. 5, Fig. 6, and Fig. 7. Fig. 4. Antibiogramm results of the varying ph for the Enterococcus Faecium strain using alkalinizing agents Fig. 5. Antibiogramm results of the varying ph for the Staphylococcus spp. strains using alkalinizing agents 126

127 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA Fig. 6. Antibiogramm results of the varying ph for the Enterococcus faecium strain using acidifier agents Fig. 7. Antibiogramm results of the varying ph for the Staphylococcus spp. strains using acidifier agents Conclusionss The varying ph determinedd a different behaviour of the antibiotics for all the isolated strains, therefore ciprofloxacin proved efficient at a ph of 5 as well at a ph of 8. It generated a 21 mm diameter of inhibition for the Staph. sciuri strain, while for the Staph. xylosus strains it was more efficient the use of gentamicin at a 7.8 ph. On the other hand, Enterococcus faecium, can be treated with ciprofloxacin at a alkaline ph of 8.0 as well as amoxicillin with clavulanate acid at a ph of

128 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017 TIMISOARA In conclusion the association of various antibiotics has proven to be usefull in treating mastitis in cows, while considerating the varying values of the ph. REFERENCES Bortolami, A., Fiore, E., Gianesella, M., Corro, M., Catania, S., Morgante, M., Evaluation of the udder health status in subclinical mastitis affected dairy cows through bacteriological culture, somatic cell count and thermographic imaging, Polish Journal of Veterinary Sciences, 2015, 18(4), Burvenich, C., Van Merris, V., Mehrzad, J., Araceli, Diez-Fraile, Duchateau, L., Severity of E. coli mastitis is mainly determined by cow factors, Vet Res., 2003, 34, Corbellini, L.G., Driemeier, D., Cruz, C., Dias, M.M., Ferreiro, L., Bovine mastitis due to Prototheca zopfii: clinical, epidemiological and pathological aspects in a Brazilian dairy herd, Tropical animal health and production, 2001, 33(6), De Visscher, A., Piepers, S., Haesebrouck, F., De Vliegher, S., Intramammary infection with coagulase-negative staphylococci at parturition: Species-specific prevalence, risk factors, and effect on udder health, J Dairy Sci. 2016, 99(8), Jagielski Tomasz, A., Henryka Lassa, B., Jennifer Ahrholdt, C., Malinowski, E.B., Uwe, R.C., Genotyping of bovine Prototheca mastitis isolates from Poland, Journal of Veterinary Microbiology, 2010, 149(1-2), Khaled, A., Abd, El-Razic., Sherein, I., Abd, El Moez., Enas, S. Danial, New approach in diagnosis and treatment of bovine mycotic mastits Egypt, African Journal of Microbiology Research, 2011, Kirk, J., Mellenberger, R., Mastitis control program for Pseudomonas mastitis in dairy cows, Kumar, R., Yadav, B.R., Singh, R.S., Genetic determinants of antibiotic resistance in Staphylococcus aureus isolates from milk of mastitic crossbred cattle, Current Microbiology, 2010, 6, Morin, E.D., Smith, G.W., Constable P.., Ability of hematologic and serum variabilities to differentiate Gram-negative and Gram-positive mastitis in dairy cows, Journals of Veterinary Medicine, 2001,15(4), Ranjan, R., Gupta, M.R., Sinhg, S., Kumar, S., Curent trend of drug sensitivity in bovine mastitis, Vet World, 2010, 3(1), Pyorala, S., Taponen S., Coagulase-negative staphylococci emerging mastitis pathogens, Vet. Microbiol., 2009, 134, Shahee, M., Tantary, H.A., Nabi, S.U., A treatise on bovine mastitis: Disease and Disease economics, Etiology basis, Risk factors, Impact on Human Health, Therapy, Management, Prevention and control strategy, Journal Advances in Dairy Research, 2016, 4,1.

129 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA OZONE EFFECT ON SOME SALMONELLA STRAINS E. TÎRZIU 1, R. LAZĂR 2, OLIMPIA COLIBAR 1, IONELA HOTEA 1, DANIELA MOŢ 3, R.V. GROS 1, K. IMRE 1, ALEXANDRA CUCERZAN 1, ILEANA NICHITA 1 1 Banat s University of Agricultural Sciences and Veterinary Medicine King Michael I of Romania from Timisoara, Faculty of Veterinary Medicine, Calea Aradului, 119, Timisoara , Romania 2 DSVSA Constanţa, , Mangaliei Street No. 78, Constanta, Romania 3 Faculty of Animal Science and Biotechnologies Timisoara, Timisoara, Romania emiltarziu@yahoo.com Summary Chilled fresh meat in general, and poultry and turkey in particular is an extremely important source of pathogens for consumers, taking into account the particular E. coli and Salmonella spp., which are the main causative agents of food poisoning in both the European Union and other countries on other continents. Most research of nowadays have highlighted the importance of reducing the number of microorganisms on the surface of the carcass at the slaughterhouse. One way of eliminating these pathogens is represented by the end product decontamination. Thus, poultry slaughterhouses and processing plants for poultry apply several methods of decontamination, according to H.A.C.C.P. principles. In this research we aimed the disinfecting action of ozone, in different concentrations, ozone used to treat water for turkey slaughtering processes and also to treat the air from these spaces, the Salmonella strains and total number of germs. Key words: ozone, decontamination, enterobacteriaceae, meat. Fresh meat is an important nutrient substrate for microbial growth, due to the fact that it has a high water activity and contains low molecular weight substances, easily digestible, it is also a source of carbon and energy, containing carbohydrates, lactic acid, amino acids, minerals, soluble phosphorus etc. (1, 2). Some countries apply two types of decontamination methods: physical or chemical. Physical methods involve treatment with steam and water at high temperatures, UV exposure, high pressure processing and irradiation. Chemical methods relate to the use of chlorine and chlorine-based compounds, trisodium phosphate, organic acids, electro-activated (electrolyzed) water and ozone. We note that the European Union legislation allows only lactic acid decontamination (6). Among the chemical agents, ozone is characterized by a very high oxidative potential, which is considered to be about 1.5 times higher than that of other disinfectants used in food processing (1, 7, 9). As a non-toxic disinfectant, ozone may be used for the control of multiplication of undesirable microorganisms in food processing establishments, as well as on surface of various equipment and utensils used in the food industry. Also, ozone is considered to be the ideal disinfectant for water used in food 129

130 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA processing, having the main advantage in relation to most disinfectants, that it does not decompose in chemical products unfit for subsequent food consumption (3, 7, 9, 10). Materials and methods Biological samples were collected from a turkey slaughterhouse in which, in 2013, was detected the presence of Salmonella serovar Typhimurium. Sampling was performed once / week for two weeks, during and targeted, from the turkey carcasses: neck skin at detachment (n = 20); neck skin after ozone treated water bath (n = 20); neck skin after drying (exposure to ozone treated air) (n = 20), and from organs: liver after evisceration (n = 20); liver after ozone treated water bath (n = 20); gizzards after evisceration (n = 20); gizzards after ozone treated water bath (n = 20). Laboratory analysis targeted the detection/determination (isolation and identification) of Salmonella spp., E. coli, the total number of germs by total plate count (TPC) and colonies of other species from Enterobacteriaceae family. Decontamination was performed with GRYPHON FILTER GF3XO-101 A10.5KT ozone disinfection equipment. We mention that the equipment (the generator) runs 15 minutes/hour and delivers ozonized water with a concentration of 0.3 ppm. Ozone is also introduced in the air of storage chambers for carcass drying, where the ozone generating system operates for 20 minutes, every 40 minutes, producing ozone at a concentration of 0.2 ppm. The slaughterhouse also has other equipments for air ozonation, installed in workplaces and corridors. These devices are operating for 15 minutes/hour and generate ozone at a concentration of 0.1 ppm. For the determination of Salmonella spp. colonies the following stages were covered: Pre-enrichment: mixture of 25 g sample ml of buffered peptone water were introduced into the STOMACHER 400 CIRCULATOR (Seward Limited, UK) homogenizer and subjected to a speed of 230 rpm for 30 seconds; the obtained suspension was incubated at 37 C for 18 hours. Selective enrichment: after incubation, 0.1 ml of obtained culture was transferred in a test tube with 10 ml Rappaport-Vasiliadis broth (RVS), and incubated at 41.5 C for 24 hours; in another test tube was transferred 1 ml culture in 10 ml of Muller-Kauffman-tetrathionate-novobiocin (MKTTn) broth and incubated at 37 C for 24 hours. Isolation: from the RVS broth culture, using an inoculation loop, was inoculated the surface of a Petri plate with xylose-lysine deoxycholate agar (XLD), and the MKTTn broth culture was inoculated in RAMBACH agar; the incubation was performed at 37 C for 24 hours. Confirmation: the colonies of Salmonella grown on XLD agar have a black center and a transparent bright pink-reddish color area due to the color indicator change. For confirmation, from each plate were taken, at least one colony 130

131 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA considered to be typical or four if the former is negative and were inoculated on plates with nutrient agar, to be incubated at 37 C for 24 hours; this step was followed by biochemical confirmation. To determine the E. coli β-glucuronidase positive species the following steps were done: using a pipette or a micropipette 1 ml of the initial suspension was transferred into two Petri plates; inoculation was repeated in other plates containing decimal dilutions of the original solution; in each Petri plate was poured about 15 ml of TBX medium (tryptone-ball-glucuronidase), previously cooled at 44 C in a water bath; inoculums were homogenized thoroughly and allowed to solidify by placing the plates on a flat, cold surface; after solidification, the plates were incubated at 44 C for hours, after incubation, the typical β- glucuronidase-positive E. coli (blue colonies) colony forming units (CFU) were counted on each plate containing less than 150 typical CFU. For a valid result, only the plates that contained at least 15 blue CFU were considered. The number of CFU of E. coli β-glucuronidase-positive (N) present in analyzed samples per milliliter or per gram, was calculated based on the average of two successive dilutions using the following formula:, in which: a - the sum of CFU counted on all plates retained from two successive dilutions, at least one of which contains a minimum of 15 blue CFU; n1 - number of plates retained for the first dilution; V - volume of inoculums on each plate; n2 - number of plates retained for the second dilution; d - dilution factor corresponding to the first dilution retained. To determine the TPC, two Petri plates were inoculated with a sterile pipette, 1 ml of the original suspension, then, over the inoculums was added ml of agar cooled at C, after homogenization of the content, the plates were allowed to stand on a cold surface until solidification. After incubation at 30 C ± 1 C for 72 hours, colonies counting were performed in diffused light. Colonies belonging to bacteria from Enterobacteriaceae family were highlighted by the following steps: using a sterile pipette, 1 ml of the initial suspension was transferred in two Petri plates and then were made serial dilutions using a new pipette for each dilution; in each Petri plate was added approximately 10 ml of medium with violet red bile glucose (VRBG), cooled in a water bath at C; the inoculated medium was carefully homogenized by horizontal movement, and the resulting mixture was allowed to solidify at laboratory temperature, the plates were placed on a cold surface; after complete solidification of the mixture about 15 ml VRBG medium was added to the surface to ensure semianaerobe conditions, after which the plates were again allowed to stand for solidification; 131

132 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA incubation was carried out at 37 C for 24 hours; confirmation of the presence was performed by counting typical colonies (pink to red-purple, with / without precipitation haloes) on plates that contained less than 150 colonies. Results and discussions In laboratory tests carried out on turkey carcasses and their organs, Salmonella was detected in 25 g of sample, in a batch of a thousand turkeys with an average weight of about ten kilograms. Following these findings, the carcasses were routed to S.C. Protan S.A. for high thermal processing and for obtaining meat flour for pet food. As a corrective action and for improving the effectiveness of sanitation actions, the unit has ceased operations, has sanitized the whole slaughter line and has purchased and installed ozone disinfection equipment GRYPHON FILTER GF3XO-101 A10.5KT. As it can be seen in Table 1 and Fig. 1, Salmonella spp. were found in five of the ten samples of neck skin detachment, sampled for the first determination, specifying that they were taken before exposure to ozone treated water. If the neck skin samples were sampled after ozone treated water bath Salmonella spp. were absent. Table 1 Microbiological exam results after the first determination No. Sample Enterobacteri Salmonella E. coli TPC aceae / 25 g / 10 g / 10 g* / 25 g* 1 Neck skin at detachment 5 samples (+) Absent samples (+) 2 Neck skin after ozone treated water bath Absent Absent 252 Absent 3 Neck skin after drying (exposed to ozone treated air) Absent Absent 210 Absent 4 Liver after evisceration Absent Absent 57 Absent 5 Liver after ozone treaded water bath Absent Absent 39 Absent 6 Gizzards after evisceration Absent Absent Absent 7 Gizzards after ozone treated water bath Absent Absent 192 Absent This means that ozone used for water treatment used in the process of turkey slaughter at a concentration of 0.3 ppm, has bactericidal effects on strains belonging to the Salmonella genus. The disinfecting effect of ozone was manifested also upon the total number of germs (total plate count), the number of colony forming units was significantly reduced after exposure of carcasses and organs to ozone treated water or ozone treated air (Table 1, Fig. 2). 132

133 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA Fig. 1. Ozone influence on Salmonella spp. from turkey carcasses Simultaneously, bacteria belonging to Enterobacteriaceae present in samples from turkey carcasses were removed after contact with treated water, issue that was maintained after exposure of carcasses to ozonized air, during the drying step (Table 1). Fig. 2. Ozone effect on number of colony forming units in turkey carcasses and organs 133

134 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA In the second measurement, Salmonella spp., E. coli and Enterobacteriaceae were absent in samples taken both before and after treatment with ozone treated water or air, the effect of ozone can only be documented in the case of TPC (Table 2, Fig. 3). The positive effects of ozone, respectively reduction and elimination of bacterial species of interest for this study, are not surprising, literature states that exposure of poultry carcasses to ozonized water leads to a reduction of up to 99% of bacteria and fungi contaminants depending on the concentration used (5, 6). Eliminating all Salmonella strains can be explained by the fact that it was shown that such bacteria are sensitive to ozone treatment, Salmonella were killed completely after washing chicken carcasses with ozonized water for 20 minutes (5). Microbiological exam results after the second determination Table 2 No. Sample Salmonella / 25 g E. coli / 10 g TPC / 10 g* Enterobacter iaceae / 25 g* 1 Neck skin at detachment Absent Absent 391 Absent 2 3 Neck skin after ozone treated water bath Neck skin after drying (exposed to ozone treated air) Absent Absent 293 Absent Absent Absent 292 Absent 4 Liver after evisceration Absent Absent 98 Absent 5 Liver after ozone treaded water bath Absent Absent 15 Absent 6 Gizzards after evisceration Absent Absent 518 Absent 7 Gizzards after ozone treated water bath Absent Absent 145 Absent Thus, Sheldon Brown (8), using different concentrations (0.15 to 1.5 ppm) ozone treated water for cooling poultry carcasses noticed a reduction of 7-8 times of TPC and the disappearance of coliforms, E. coli and Salmonella spp. 134

135 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA Fig. 3. Effect of exposute to ozone treated water and air on numbers of colony forming units in assessed turkey carcasses and organs Jindal et al. (4) obtain similar effects on both NTG's, and the coliforms, E. coli, Salmonella spp. and Pseudomonas aeruginosa at a concentration of about 0.5 ppm ozone in water for cooling poultry carcasses. Conclusions Ozone treated water used in the slaughtering of turkeys at a concentration of 0.2 ppm, eliminates Salmonella spp. present on the carcasses. Ozone treated water used to wash the turkey carcasses and organs, has negative effects on bacteria from Enterobacteriaceae family, reducing at the same time, the total number of germs. Exposure to ozone treated air in a concentration of 0.15 ppm, while drying, leads to a further reduction of the total number of germs on the surface of carcasses. Acknowledgements This study was realised using the support and infrastructure project Dezvoltarea infrastructurii de cercetare, educaţie şi servicii în domeniile medicinei veterinare şi tehnologiilor inovative pentru RO 05, cod SMIS-CSNR

136 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L (3), 2017, TIMISOARA References 1. Dunne, P., Farkas, C., Yuan, T. J., Ozone Fact Sheet for Agri-Food Processors, J.Nonthermal Processing Technologies for Food, Blackwell Publishing Ltd, 2011, ISBN: Hinton, M., Meat spoilage and its control. in: Microbial Control in the Meat Industry, sub red. Gormley, R., The National Food Centre, Teagasc, Dublin, Jaksch, D., Margesin, R., Mikoviny, T., Skalny, J. D., Hartungen, E., Schinner, F., Mason, N. J., Märk, T. D., The effect of ozone treatment on the microbial contamination of pork meat measured by detecting the emissions using PTR-MS and by enumeration of microorganisms. International Journal of Mass Spectrometry, 2004, 239(2-3), Jindal, V., Waldroup, A.L., Forsythe, R.H., Ozone and improvement of quality and shelflife of poultry products, Journal of Applied Poultry Research, 1995, 4, Kim, J.G., Ozone as an antimicrobial agent in minimally processed foods, Teză de doctorat, Ohio State University, Columbus, SUA, Pichpol, D., Experimental Reduction of Salmonella in Raw Chicken Breasts, Teză de doctorat, Universitatea din Berlin, Germania, Rice, R., Netzer, A., Handbook of ozone technology and applications, Ozone for drinking water treatment, Butterworth, Stoneham, Sheldon, B.W., Brown, A.L., Efficacy of ozone as a disinfectant for poultry carcasses and chill water, Journal of food Science, 1986, 51(2), Wysok, B., Uradziñski, J., Ozone as an alternative disinfectant, Polish Journal of Food and nutrition sciences, 2006, 15/56(1), Yousef, A., Vurma, M., Rodriguez-Romo, L., Basics of Ozone Sanitization and Food aplications, J.Nonthermal Processing Technologies for Food,

137 LUCRĂRI ŞTIINŢIFICE MEDICINĂ VETERINARĂ VOL. L (3), 2017, TIMIŞOARA SEASONAL VARIATION OF ANTIBIOTIC RESISTANCE OF THE FECAL FLORA ISOLATED FROM FISH IN THE DANUBE DELTA G. GATI, MIHAELA NICULAE, EMÖKE PÁLL, CARMEN DANA ŞANDRU, F. BRUDAȘCĂ, A. VASIU, SILVANA POPESCU, MARINA SPÎNU University of Agricultural Sciences and Veterinary Medicine Cluj- Napoca, , 3-5 Mănăştur Street, Cluj-Napoca, Romania vlad_negrutiu@yahoo.com Summary Bacterial pathogens such as E. coli/coliforms, Salmonella spp., Vibrio spp. From aquatic sources or fish could cause food and/or water born infections, thus posing a health risk to consumers and tourists in areas such as the Danube Delta, even more in case the carried species are antibiotic resistant. Meanwhile, pelagic fish being active and itinerant, they could carry around bacteria, thus creating new epidemiological cycles. The research aimed to investigate the presence the antibiotic resistance in fecal microflora species isolated from benthic and pelagic fish and its variation depending on the season. For that, a number of 27 samples of gills and hepatopancreas from each perch (Perca fluviatilis), wels catfish (Silurus glanis), sander (Sander lucioperca), pike (Esox lucius), gibel carp (Carassius gibelio) and common carp (Cyprinus carpio) were collected from the Danube Delta and subjected to classical isolation techniques using simple culture media and afterwards Chromogenic UTI medium and Brilliance TM E. coli/ coliform selective medium. Antibiograms were done by the Kirby Bauer method, using ciprofloxacin, penicillin, streptomycin, erithromycine, oxitetracycline, marbofloxacin, akamicine, enrofloxacine and ampicilline. The selected microflora consisted of E. coli, coliforms and E. fecalis. There were no significant differences between the diameters of inhibition areas by season. Nevertheless, the number of resistant colonies was the highest in the cold season and lower in the warm season. The most active antibiotic was ciprofloxacine (20.8±2.3 mm), with no resistance or resistant colonies, but its activity declined with the temperature during the cold seasons. The inhibition values were highly variable in each species. The results showed changes in antibiotic resistance depending on season, rather than on fish species, their feeding habits or habitat. Key words: fish, fecal bacteria, antibiotic resistance, Danube Delta Antimicrobial resistance is one of the most complex global health challenges, threatening to reverse the substantial progress against infectious diseases made since the golden era of antibiotic discovery during the second half of the previous century. These miracles of modern medicine, and their tremendous gains for health, have long been taken for granted. said Dr. Margaret Chan, Director-general WHO in her Report on April 17, 2017 (3). She also mentioned that, after decades of overuse and missuse in human medicine and veterinary medicine, some antibiotics are no longer effective. As WHO reports show, antimicrobial resistance is on the rise in every region of the world. WHO, Food and Agriculture Organization of the United Nations (FAO) and the World Organisation for Animal Health (OIE), recognizes that a crisis of this magnitude 137

138 LUCRĂRI ŞTIINŢIFICE MEDICINĂ VETERINARĂ VOL. L (3), 2017, TIMIŞOARA requires an effective One Health approach involving coordination among many sectors at national and international levels (4). Four ecological compartments were considered as sources for the resistome transfer, and these include mammalian host (humans, animals) and also environmental sources (plant, soil and water). As shown, bacteria with no pathogenic significance and no direct contact with antibiotics originating from the environment, aquatic environment included, could remain a permanent source of R genes for pathogenic bacteria. One best example is Aeromonas, present in the aqueous environment, posing risk to those fishing, surfing, swimming, diving (1). Nevertheless, numerous niches where these bacteria could be present still remain undeinvestigated. This research aimed to investigate the presence the antibiotic resistance in fecal microflora species isolated from predatory and herbivorous, benthic and pelagic fish in the Danube Delta and its variation depending on the season. Materials and methods To monitor the fish species, the feeding behavior and habitat as well as the seasonal influence on antibiotic resistance, a number of 27 samples of gills and hepatopancreas from each perch (Perca fluviatilis), wels catfish (Silurus glanis), sander (Sander lucioperca), pike (Esox lucius), gibel carp (Carassius gibelio) and common carp (Cyprinus carpio) (Table 1) were collected from the Danube Delta. To monitor the seasonal changes in the antibiotic ressitance, sampling was performed during March 2013 and May 2013, from two locations, Canalu Central and Canal Turcesc. Five more sampling locations were added in September 2013 and February 2014 (Table 2). Feeding and habitat peculiarities of tested fish Feeding Latin name English name behavior pelagic predatory Perca fluviatilis Perch predatory Sander lucioperca Sander predatory Esox lucius Pike benthic predatory Silurus glanis Wels catfish herbivorous Carassius gibelio Gibel carp herbivorous Cyprinus carpio Common carp Table 1 The samples were subjected to classical isolation techniques. For that, the cultivation was performed using simple culture media and afterwards Chromogenic UTI medium and Brilliance TM E. coli/ coliform selective medium, for identification. 138

139 LUCRĂRI ŞTIINŢIFICE MEDICINĂ VETERINARĂ VOL. L (3), 2017, TIMIŞOARA The selected microflora consisted of E. coli, coliforms and E. fecalis. Antibiograms on these isolates were done by the Kirby Bauer diffusion method, the discs containing 9 antibitics (ciprofloxacin, penicillin, streptomycin, erithromycine, oxitetracycline, marbofloxacin, akamicine, enrofloxacine and ampicilline). Table 2 Experimental protocol (seasons, sampling sites, sampled organs, fish species, isolated bacteria) March 2013 May 2013 Place Species Bacteria Place Species Bacteria Canal central Pike Br E.coli Canal Tataru Gibel carp Hp E.fecalis Canal Central Pike Br E.coli Canal Tataru Gibel carp Br E.coli Canal Tataru Pike Br E.coli Canal Tataru Gibel carp Hp E.coli Canal central Pike Hp E.coli Canal Tataru Gibel carp Hp E.coli Canal Central Perch Hp E.coli Canal central Perch Hp Coliforms Canal central Sander Br E.coli Canal central Sander Hp E.coli Septembrie 2013 Februarie 2014 Place Species Bacteria Place Species Bacteria Gibel Intrare Canal Gibel carp Bazin Ciotica carp Br E.coli Flamanda Br E.coli Bazin Ciotica Gibel carp Br E.coli Intrare Canal Flamanda Gibel carp Br E.coli Canal Belciug Pike Br E.coli C. Turcesc spre vărsare în Melea Wels catfish Br E.coli Canal Belciug Pike Hp E.coli C. Turcesc spre vărsare în Melea Wels catfish Br Coliforms Canal Belciug Perch Br E.coli Bucla Km 19 Wels catfish Br E.coli Canal Belciug Perch Br E.coli Bucla Km 19 Carp Br E.coli Canal Belciug Perch Br E.coli Bucla Km 19 Carp Br Coliforms Canal Belciug Perch Br E.coli Inhibition diameters were read, and the presence of total resistance (R) or resistant colonies (partial resistance, CR) were recorded. The results were statistically interpreted by the use of Excel program. 139

140 LUCRĂRI ŞTIINŢIFICE MEDICINĂ VETERINARĂ VOL. L (3), 2017, TIMIŞOARA Results and discussions Raw fish as part of human ready-to-eat foods represents a source for microbial contamination, becoming an important public health issue. The number of coliform bacteria in raw fish attained 5.1x10 4 (CFU/g) while enterobacteriaceae were at 6.8x10 4 (CFU/g), stressing the importance of these bacteria in fish and also their potential pathogenic impact for consumers (5). Enterobacteriaceae, Escherichia coli, Salmonella, Staphylococcus aureus and Listeria monocytogenes were the most frequently reported organisms in a meta-analysis based on literature published in a 15 year interval, in several African countries standing for the length of persistence (6). E.coli populations, depending on their provenance, can show multi-drug resistance, which seems to be the highest in food producing animals and therefore represent a risk for consumers by food products of animal origin (7). mm March May September February Inhibition Fig. 1. Changes in overall inhibition (average all species, all antibiotics) diameters (mm) by season Escherichia coli and enterococci are used as indicator organisms in monitor antibiotic resistance in fish. When 44 fish and shrimp samples were tested against a panel of 29 antimicrobial agents, Enterococcus faecalis (59%) and E. coli (55%) were the most common. Multidrug-resistant (MDR) E.coli and E. coli, E. fecalis and S. aureus strains were found in shrimps (64%) and pangasius (17%). Thus, E. coli and E. faecalis in fish and seafood, could be used for antimicrobial resistance monitoring programs (2). In the present research, the inhibition diameters varied from strain to strain, being different from 11 to 26 mm; nevertheless, the average diameters calculated 140

141 LUCRĂRI ŞTIINŢIFICE MEDICINĂ VETERINARĂ VOL. L (3), 2017, TIMIŞOARA by antibiotic type, differ significantly for May and February (p<0.05)(14 to 19 mm in March, 11 to 21.5 in May, 12 to 19 in September and 11.5 to 20.4 in February). The highest overall sensitivity was recorded in March, with a slight subsequent decrease, the values for May, September and February being close. The onlz antibiotic with no R or CR in any of the seasons was the ciprofloxacin. There were no R and CR to enrofloxacin in February (Fig. 1). The variation of total resistance and number of samples showing resistant colonies were presented in Fig. 2. The highest total resistance was present in March and February, at similar levels, while May had the least R and CR R CR March May Sept Feb Fig. 2. Variation in cumulated total resistance and resistant colony numbers by season The isolated E. coli, coliform and E. fecalis strains showed multidrug resistance, to 7 or 8 of the tested antibiotics, at different levels. The presence of resistant colonies stands for the partial inefficacy of the antibiotics used against these strains. This phenomenon is further posing the risk of selective pressure towards MDR in these locations in case of their use and increasing the consumer risk. Conclusions The results indicated the presence of antibiotic resistance indicator bacteria in the tested samples and also showed that the changes in antibiotic resistance depended on the season, rather than on fish species, their feeding habits or habitat. 141

142 LUCRĂRI ŞTIINŢIFICE MEDICINĂ VETERINARĂ VOL. L (3), 2017, TIMIŞOARA Aknowledgement This research was supported by grant PNII PCCE 61/2012. References. 1. Balotescu, C., Israil, A., Radu, R., Alexandru, I., Dobre, G., Aspects of constitutive and acquired antibioresistance in Aeromonas hydrophila strains isolated from water sources, Roum. Arch. Microbiol. Immunol., 2003, 62(3-4), Boss, R., Overesch, G., Baumgartner, A., Antimicrobial resistance of Escherichia coli, Enterococci, Pseudomonas aeruginosa, and Staphylococcus aureus from Raw Fish and Seafood Imported into Switzerland, J. Food Prot., 2016, 79(7), Chan, Margaret (1), A global health guardian: climate change, air pollution, and antimicrobial resistance. Antimicrobial resistance: now a political priority, Report by Dr. Margaret Chan, Director-General, WHO, April 2017, publications/10-year-review/healthguardian/en/ index3. html 4. Chan, Margaret (2), A global health guardian: climate change, air pollution, and antimicrobial resistance. Antimicrobial resistance: the need for urgent action, Report by Dr. Margaret Chan, Director-General, WHO, April 2017, 5. Eromo, T., Tassew, H., Daka, D., Kibru, G., Bacteriological Quality of Street Foods and Antimicrobial Resistance of Isolates in Hawassa, Ethiopia, Ethiop. J. Health Sci., 2016, 26(6), Paudyal, N., Anihouvi, V., Hounhouigan, J., Matsheka, M.I., Sekwati- Monang, B., Amoa-Awua, W., Atter, A., Ackah, N.B., Mbugua, S., Asagbra, A., Abdelgadir, W., Nakavuma, J., Jakobsen, M., Fang, W., Prevalence of foodborne pathogens in food from selected African countries - A meta-analysis, Int. J. Food Microbiol., 2017, 249, Szmolka, Ama, Nagy, B., Multidrug resistant commensal Escherichia coli in animals and its impact for public health, Frontiers in Microbiology, 2013, 4,

143 LUCRĂRI ŞTIINŢIFICE MEDICINĂ VETERINARĂ VOL. L (3), 2017, TIMIŞOARA A NOTIFICATION OF POSSIBLE PEMS (POULT ENTERITIS AND MORTALITY SYNDROME) EPISODE IN TURKEYS CARMEN DANA ŞANDRU, H. PUHO, F. BRUDAŞCĂ, L. KÖBÖLKUTI, R. GIUPANĂ, C. CERBU, A. VASIU, SILVANA POPESCU, MARINA SPÎNU University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, , 3-5 Mănăştur Street, Cluj-Napoca, Romania sandranac@gmail.com Summary Turkey meat gains an increasing importance due to its particular nutritious value and also low colesterol content, therefore genetic studies tend to improve the phenotype by selecting lines which better convert the fodder and are also higly productive. Nevertheless, the epidemiology of specific or non-specific infectious diseases is continuously changing in spite of optimal technology farming technologies being provided (feeding, nutrition, prevention of infectious and parasitic diseases). Therefore, the early and precise diagnosis is essential for establishing the most efficient control and prevention programmes. The investigations were carried out to clarify the etiology and potential therapies during an episode of turkey chick enteritis and mortality syndrome, producing consistent loss at the farm level. For this, clinical, pathological and labotarory (bacteriological and molecular) investigations were conducted on a turkey farm affected by PEMS. The clinical picture included: numbness, anorexia, ruffled feathers and diarrhoea, accompanied by fibrine and puss deposits in the pericardium and abdomen, lesions of air saks, phytobezoar in the stomach, congestive mesenteric blood vessels, hemorhages in the intestine. Shiga2 positive, antibiotic sensitive E. coli was present in the samples colected from the chicks, also indicated by the literature as one of the agents in PEMS. The results confirmed the development of PEMS on the selected farm, severe protective measures, such as evaluation of the chick source, desinfections, as well as the improvement of the parent fenotype being necessary. This study represented the first disease alert for PEMS in Romania. Key-words: turkey chicks, E.coli, molecular diagnosis, PEMS Poultry production is increasing year by year. Since turkey meat is highly valued because its low colesterol content and its nutritious qualities, studies carried out by geneticists tend to improve the phenotype by selecting the most productive, best feed converting lines. Albeit on intensive farms optimal technology (feeding, nutrition, prevention of infectious and parasitic diseases) are provided, according to the highest standards and norms, the epidemiology of specific or non-specific infectious diseases is continuously changing.the relatively high number of influential factors condition the appearance of episodes with compex etiology. Thus, the precociuos and precise diagnosis is essential for establishing the most efficient control and preventive program (4). Subsequent to the appearance of a disease in a turkey flock, the consequences for both health and economy are severe, therefore appropriate management and technology as well as preventive vaccinations are compulsory (1). 143

144 144 LUCRĂRI ŞTIINŢIFICE MEDICINĂ VETERINARĂ VOL. L (3), 2017, TIMIŞOARA Poult enteritis complex (PEC) is an infectious intestinal syndrome of young turkeys. In fact, it comprises several microbial agents, and the outcome of their interaction in association with environmental factors is considered to be PEC. Some of its components (coronavirus enteritis) are well defined, while others (PEMS) are still under research. As a multifactorial disease, PEC is infectious and also transmissible, with a devastating economic impact (5). The investigations carried out in this research adress the etiological clarifications during an episode of turkey chick enteritis and mortality syndrome (possibly PEMS?), which has never been described in our country and which, nevertheless, already caused consistent loss. Materials and methods Clinical, pathological and laboratory (bacteriological and molecular) investigations were conducted on a turkey farm affected by PEMS, located in central Romania. The farm capacity is of turkeys per year, the birds being raised for 6 weeks and then comercialised. The birds are imported from Hungary, on day one and the first cycle is initiated in February, comprising individuals. The breed is a novel one, with valuable qualities, called Converter. The turkey house is of m 2, the permanent bedding being of straw. The inside temperature is 30-32ºC during the first two weeks, and heating is stopped after the first month of life. The temperature was regulated by four ventilators and automatically positioned windows. The chicks are fed with a started fodder the first 12 days, then it is changed to started II and fatening fodder after 28 days of age. A very strict vaccination protocol is implemented to prevent infectious diseases, including Necastle disease and rinotracheitis (day 1), vitamin C for the first five days, a vitamin complex from day 9 onewards for 5 days in the drinking water and booster vaccinations against newcastle disease (days 14 and 28), rinotracheitis (day 35) and antibody checks on day 38 to monitor the immune status against Newcastle disease. At the end of the productive cycle the chicken house is thoroughly cleaned, all the ventilators, feeders and waterers are removed, the premises are desinfected with 7,5% formaline and it is closed for 24 h. The next day another disinfection with 2,5% virocid is performed and the chickenhouse is closed for 10 days. Sanitation samples are taken before introduction of the new series of birds. The caretakers are only allowed to own dogs and cats and are checked periodically for E.coli and Salmonella. The study was carried out on 252 birds, diseased or dead. Clinical examination of the sick birds and necropsies were performed. Samples were taken to identify the causes of the disease, transfered to simple broth and incubated for 24h at 37 C. Further, the samples were transfered to simple and to MacConkey, Brilliance, XLD agar. After 24 h of incubation and preliminary microscopy by Gram stain (6), API 20 Strep, API Staph, API 20 E I selective tests were applied to

145 LUCRĂRI ŞTIINŢIFICE MEDICINĂ VETERINARĂ VOL. L (3), 2017, TIMIŞOARA isolated colonies, depending on their type, for identification by use of the specific software. The PCR (polymerase chain reaction) was carried out to identify the bacteria involved in the etiology of the disease (7). The only enzyme used in this method is a DNA polimerase (8). Since the presence of E. coli was suspected based on the gross pathology, the specific primers for stx1 and sxt2 genes were: F CAG TTA ATG TGG TGG GGA AG/ stx1r CTG TCA CAG TTA CAA CCG T and F TTC TTC GGT ATC CTA TTC CC/ stx2r ATG CAT CTC TGG TCA TTG TA, respectively. The device used was a PCR Bio-Rad C1000TM, and the test also involved a DNA extraction kit ( G- Dex IIC kit). The protocol was applied according to the producers instructions. Method: During the first phase the primers were heated in the PCR machine at 95ºC - 3 min, after that at 95ºC for 30 s. This stage, called DNA denaturation, the H bonds are ruptured. Further, the extracted DNA is added and heated to 65ºC for 30 s. Due to the decreased temperature, the primers bind with the extracted DNA. The mixture is kept at 72ºC for 7 min. The segments obtained by PCR were identified by electrophoresis and the specific segment was examined under UV light (Bio-Red BioDoc-It imagine system). Results and discussions The clinical picture included: numbness, anorexia, ruffled feathers and diarrhoea. Salient clinical features include stunting and poor feed utilisation that result from enteritis. In the more severe forms, runting, immune dysfunction and mortality are reported (4, 5). The gross lesions included fibrine and puss deposits in the pericardium and abdominal air sacs (Fig. 1), phytobezoar in the glandular stomach, congestion in mesenteric blood vessels (Fig. 2), hemorhages in the intestine. Microbiological examinations: The results of the microbiological tests from the pericardium and bone marrow of the turkey poults indicated the presence of average size colonies, smooth and yellowish in color on simple agar, while on Brilliance agar the color was blue, and on XLD yellow. On MacConkey, the colonies had a red color, that stood for lactozo positive bacteria. This bacteria was sensitive to antibiotics, the pathogenicity being induced by the toxine synthesis. In the literature, gross and microscopic lesions of enteritis are present in all forms but tend to be non-specific, due to various microbial agents. Similarly, these agents could induce various other lesions, according to their pathogenic pattern. The basic pathogenesis leans on: a) destruction of the intestinal mucosa, initiated by viruses and then subject to secondary infections and b) inflammation; c) proliferation of secondary agents, usually bacteria (5). The results confirmed the presence of E. coli and the development of PEMS on the selected farm, the severe microbiological protection measures being 145

146 LUCRĂRI ŞTIINŢIFICE MEDICINĂ VETERINARĂ VOL. L (3), 2017, TIMIŞOARA compulsory (evaluation of the chick source, desinfections, etc.) as well as the improvement of the parent fenotype, the disease being transmitted by eggs (vertically or in the incubation station). This study represented the first disease alert for PEMS in Romania. In the case of PEMS, the treatment is unefficient due to the complex etiology. Most of the agents are viruses (Astrovirus, Coronavirus) associated to bacteria (Escherichia coli, Salmonella) and eventually fungi, causing alltogether a very severe enteritis in poults (2, 3). The results of a study conducted to investigate the importance of Coronavirus of turkeys in inducing PEMS in chicks indicated that TCV can be associated with PEMS but is neither necessary nor sufficient to cause PEMS (5). Fig. 1. Fibrine deposits in the pericardium (original, Puho, 2016) Fig. 2. Oedema and congestion of the mesenteric blood vessels (original Puho, 2016) The actual requirements on bacterial isolation impose also the establishment of the pathogenicity. Since E. coli is an epiphitic bacteria, in this case, it is possible that new environments transfer, by genetic material, new traits to such bacteria (2). Once it became obvious that the isolated E. coli is involved in PEMS on the farms, PCR, was performed as mentioned in Materials and methods to confirm its pathogenicity. The samples tested positive for Shiga2 positive E. coli, indicated by the literature as one of the agents in PEMS. All strains tested negative for sxt 1 but all were positive for sxt 2 (Fig. 3, as indicated by the arrows). 146

147 LUCRĂRI ŞTIINŢIFICE MEDICINĂ VETERINARĂ VOL. L (3), 2017, TIMIŞOARA Linia de pozitivitate pentru stx2 Fig. 3. PCR for the establishment of the pathogenicity of the isolated Escherichia coli (original, Puho, 2016) These data were supported by other research (9), where investigations aiming at clarifying the role of EPEC in PEMS, concluded that EPEC were associated with 10 of 12 (83%) naturally occurring PEMS cases Such data supported the involvement of EPEC în poult enteritis mortality syndrome. Conclusionss The results of this research confirmed the presence of PEMS on a Romanian turkey farm, indicated by the clinical picture, lesions, presence of E. coli and its pathogenicity and positivity for sxt 2. Furthermore, these strains were pathogenic causing septicemia in poults, based on gross pathology. In spite of the diminishedresistance to antibiotics, thse e strains are difficult to eliminate due to their toxinogenesis. References 1. Húsvéth, F., Termeleselettan, A madarak emésztésenek jellegzetességei.p1, 2011, tamop 425/0010_1A_Book_12_Termeleselettan/pr01.html.. 2. Dren, N.C., Beszámoló a pulykabetegségek 3 Nemzetközi Szimpóziumáról, Berlin,

148 LUCRĂRI ŞTIINŢIFICE MEDICINĂ VETERINARĂ VOL. L (3), 2017, TIMIŞOARA 3. Barnes, H.J., Guy, J.S., Vaillancourt, J.P., Poult enteritis complex, Rev. Sci. Tech., Off. int. Epiz, 2000,19(2), Carver, D.K., Vaillancourt, J.P., Stringham, M., Risk factors associated with poult enteritis mortality syndrome-positive turkey flocks, Avian Dis., 2002, 46(4), Carver, D.K., Vaillancourt, J.P., Stringham, M., Guy, J.S., Barnes, H.J., Mortality patterns associated with poult enteritis mortality syndrome (PEMS) and coronaviral enteritis in turkey flocks raised in PEMS-affected regions, Avian Dis., 2001, 45(4), Răducanescu, H., Bica-Popii, Valerica, Bacteriologie veterinară, Ed. Ceres, Bucureşti, Bryan, M., Leonard, Finola, Archambault, Marie, Cullinnae, Ann, Maguire, Dores, Clinical Veterinary Microbiology, Ed. 2, Ed. Elsevier, Tőzsér, J., Kappelmayer, J., Balogh, I., Molekuláris Diagnosztika 12 Fejezet, Ed. Debreceni kiadó, Pakpinyo, S., Ley, D.H., Barnes, H.J., Vaillancourt, J.P., Guy, J.S., Prevalence of enteropathogenic Escherichia coli in naturally occurring cases of poult enteritis-mortality syndrome, Avian Dis., 2002, 46(2),

149 LUCRĂRI ŞTIINŢIFICE MEDICINĂ VETERINARĂ VOL. L (3), 2017, TIMIŞOARA THE EVALUATION OF THE SYSTEMIC BLOOD PRESSURE THROUGH THREE METHODS IN MIXOMATOUS MITRAL VALVE DISEASE I. SCURTU, C. PESTEAN University of Agricultural Sciences and Veterinary Medicine Cluj- Napoca, , 3-5 Mănăştur Street, Cluj-Napoca, Romania iuliu.scurtu@usamvcluj.ro Summary In this paper we evaluated the systemic blood pressure of dogs with mixomatous mitral valve disease. In this study we included 32 dogs with mitral regurgitation due to mixomatous mitral valve disease. Systemic blood pressure was evaluated using three methods: the evaluation of the mitral regurgitation peak velocity using continuous wave Doppler and calculation of the systemic blood pressure with modified Bernoulli equation, using a high definition oscillometry device and through Doppler ultrasonography. All of these three techniques of evaluation of the systemic blood pressure proved a moderate correlation between them. The highest systemic blood pressure was recorded by high definition oscillometry and the lowest measurements using Doppler ultrasonography. Further investigations must be done in order to identify the best indirect method for systemic blood pressure evaluation. Key words: systemic blood presure, HDO, mitral regurgitation, echocardiography Systemic hypertension is one of the most under diagnosed condition in dogs and cats (3). Systemic hypertension is categorized into three types: (1) stress induced or white-coat hypertension, (2) in association with other conditions (secondary hypertension) and (3) idiopathic hypertension, when none process that could trigger hyperetnsion is identified (3). Chronic systemic hypertension causes injury to tissues, being rationale the treatment of hypertension for prevention of these injuries. The organs most frequently cited to be affected in case of hypertension are the kidneys, the eyes, the brain, the heart and the vessels (3). Blood pressure can be evaluated through direct method, which consists of using an intra-arterial catheter to obtain a measurement (8), but it is invasive, and is technically challenging in awake animals (4) and indirect (Doppler ultrasonographic or oscillometric) methods (8). In case of heart pathology, with mitral regurgitation, Doppler echocardiography can be also used to estimate systolic blood pressure based on mitral regurgitation velocity using the Bernoulli equation (pressure gradient 4 x velocity 2 ) (4). The pressure estimation using Bernoulli equation plus left atrial pressure is equal to arterial systolic pressure in the absence of aortic stenosis (4). 149

150 LUCRĂRI ŞTIINŢIFICE MEDICINĂ VETERINARĂ VOL. L (3), 2017, TIMIŞOARA Material and methods In this study we included 32 client-owned dogs. Informed consent was obtained from each owner prior to enrollment. To all dogs we measured systolic blood pressure through three methods: Doppler ultrasonography, high definition oscillometry, and Continous wave Doppler echocardiography. Inclusion criteria: left apical systolic murmur that was documented through Color Doppler and class B or C patients according to ACVIM classification (2). Exclusion criteria: any other heart abnormality. Doppler ultrasonography (DU) Records were performed with a flow detector (Model 811-B Doppler ultrasonic Flow Detector with flat infant probe; Parks Medical Electronics, Inc., Aloha, OR, USA). For reliable measurements, in examination room remained only the owner and the doctor who assessed the blood pressure. Ten minutes of accommodation were given to each dog to acclimate to the environment and measurement personnel (7). We used dedicated cuffs, with a width of 40% of the limb circumference that was connected to a manometer, placed over the midantebrachium (4). The Doppler signal was captured over the digital artery, after clipping and applying of ultrasonographic gel. The cuff was inflated until the loss of pulse signal, and later deflated until the return of the pulse wave. We performed 3 5 measurements to all dogs and the average was recorded. Doppler echocardiography For the assessment of mitral regurgitation peak velocity we used apical four or two chambers apical view (fig. 1B). The alignment of ultrasound beam and regurgitant mitral flow it was less than 20º. The peak velocity was recorded with CW Doppler and for the estimation of the systolic pressure we used Bernoulli equation (to obtain the pressure gradient between left atrium and left ventricle) and we added also 10 mmhg (the estimation of left atrium pressure). Three to five measurements were done in all dogs and an average was performed. High-definition oscillometry (HDO) All measurements were done in a quiet room after 10 minutes of accommodation. We use a Vet HDO MD pro device designed for cats and dogs (Fig. 1A). The cuffs were chosen according to producer recommendations. The cuff was placed on the tail or on the midantebrachium. Three to five measurements were run and an average was performed. 150

151 LUCRĂRI ŞTIINŢIFICE MEDICINĂ VETERINARĂ VOL. L (3), 2017, TIMIŞOARA Fig. 1. A. The image presents Vet HDO MD Pro device. B. CW Doppler from a dog with a peak mitral velocity of 6.28 m/s Results and discussions Eighteen out of 32 dogs were male and 14/32 were female. The dog breeds were Chihuahua (2/32), Caniche (7/32), Bichon (7/32), Miniature Schnauzer (3/32), Beagle (2/32), mixed (6/32), Yorkshire terrier (5/32). The majority of dogs were weighing less than 12 kg (22/32). The mean weight was 8.9 kg (4 16 kg). The median age was 12.5 years old (6 18 years old). Mean systolic pressure measured by oscillometric method was mmhg ( mmhg). When the systemic blood pressure was evaluated by the estimation of regurgitant mitral flow peak velocity, the median value was mmhg ( mmhg), and when we used Doppler ultrasonography for the estimation of the systemic blood pressure, mean arterial pressure was mmhg ( mmhg). In this study we evaluated the systemic blood pressure by three methods: Doppler ultrasonography, high definition oscillometry and measuring mitral regurgitation peak velocity. In our study the highest blood pressure was obtained by high definition oscillometry ( mmhg) and the lowest by Doppler ultrasonography ( mmhg). The highest difference, in the same dog was 53 mmhg, and the lowest it was 12 mmhg. The same results were obtained in other study (4), demonstrating that these indirect methods cannot be used interchangeably in the same dog (4). More interestingly, were noted differences in the same dog when the sites of recording were different (7). Radial systolic arterial pressure measurements were higher than coccygeal measurements (mean difference 9 mmhg) (7). In other study was emphasized the importance of body position in Doppler ultrasonography measurements of systolic blood pressure (8). Differences between DU and oscillometric method were noted in other study (p<0.001) (6). The mean arterial blood pressure in sitting position (172.1 ±33.3 mmhg) was higher versus recumbent position (147±24.6 mmhg) (8). It was also proved 151

152 152 LUCRĂRI ŞTIINŢIFICE MEDICINĂ VETERINARĂ VOL. L (3), 2017, TIMIŞOARA that systolic arterial pressure depends on site location when records were made by direct method, also (1). According to ACVIM consensus statement systemic hypertension is classified in four classes based on the risk for future target organ damage (3). The animals that have a systolic blood pressure between mmhg are characterized as having mild hypertension, whereas animals that have a systolic blood pressure higher than 180 present a severe one. In our study, we estimated the systemic blood pressure by CW Doppler measurement of mitral regurgitation. Mean blood pressure by CW Doppler was between the mean pressure measured by Doppler ultrasonography and high definition oscillometry. When we speak about specific cases, we had situations when blood pressure estimation by CW Doppler were higher than measurements performed by DU and HDO and also cases when the estimation was the lowest compared to other two methods of estimations. It is very important to measure cuff size when measurements are performed. When the width of the cuff was too small, we overestimated systemic blood pressure. As a rule, cuff width must be 40% of site measurement circumference (5). Our study has some limitations. The measurements by HDO were recorded from tail or midantebrachium. The patients included in the study were even in class B or C, and this is possible to affect measurements by CW. As we did not measured exactly left atrium pressure, for the estimation of systemic blood pressure by CW we added 10 mmhg to the pressure gradient calculation obtained using Simpson s formula. Other limitation is related to the limb used for measurements by DU; we were not consistent in using all the time the same leg. Conclusions We noticed differences in the results between the methods that we used for systemic blood pressure estimation. Further investigations must be performed in order to identify the best indirect method for systemic blood pressure estimation in dogs with mixomatous mitral valve disease. References 1. Acierno, M.J., Domingues, M.E., Ramos, S.J., Shelby, A.M., Cunha, F.A., Comparison of directly measured arterial blood pressure at various anatomic locations in anesthetized dogs, Am J Vet res, 2015, 76, Atkins, C., Bonagura, J., Ettinger, S., Fox, P., Gordon, S., Haggstrom J., Hamlin, R., Keene, B., Fuentes, Virginia Luis, Stepien, R., Guidelines for the diagnosis and treatment of canine chronic valvular heart disease, j Vet Intern Med, 2009, 23,

153 LUCRĂRI ŞTIINŢIFICE MEDICINĂ VETERINARĂ VOL. L (3), 2017, TIMIŞOARA 3. Brown, S., Atkins, C., Bagley, R., Carr, A., Cowgill, L., Davidson, M., Egner, B., Elliot, J., Henik, R., Labato, M., Littman, M., Polzin, D., Ross, L., Snyder, P., Stepien, R. Guidelines for the identification, evaluation, and management or systemic hypertension in dogs and cats. Journal of Veterinary Internal Medicine, , Hanzlicek, A.S., Baumwart, R.D., Payton, M.E., Systolic arterial blood pressure estimated by mitral regurgitation velocity, high definition oscillometry, and Doppler ultrasonography in dogs with naturally occurring degenerative mitral valve disease, J Vet Cardio, 2016, 18, Henik, R.A., Dolson, M.K., Wenholz, J.L., How to obtain a blood pressure measurement, Clin Tech Small Anim Pract, 2005, 20, Hsiang, T.Y., Lien, H.Y., Huang, H.P., indirect measurement of systemic blood pressure in conscious dogs in a clinical setting, J Vet Med Sci, 2008, 70 (5), Mooney, A.P., Mawby, D.I., Price, J.M., Whittemore, J.C., Effects of various factors on Doppler flow ultrasonic radial and cocygeal artery systolic blood pressure measurements in privately-owned, conscious dogs. PeerJ, 2017, Rondeau, A.D., Mackalonis, M.E., Hess, R.S., Effect of body position on indirect measurement of systolic arterial blood pressure in dogs, JAVMA, 2013, 242,

154 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L(3), 2017, TIMISOARA WELFARE ASSESSMENT OF BREEDING HORSES BY HEALTH AND BEHAVIORAL INDICATORS EVA ANDREA DIUGAN 1, MARINA SPINU 2, SILVANA POPESCU 2 1 Beclean Studfarm, Department for Horse Breeding, Exploitation and Amelioration, The National Forest Administration Romsilva, Petricani street, no. 9A, , Bucuresti, Romania 2 University of Agricultural Sciences and Veterinary Medicine, , Manastur street, no. 3-5, Cluj-Napoca, Romania lazarevaandrea@yahoo.com Summary The welfare assessment of reproduction horses is important as it leads to the recognition of existing problems and their rapid remedial. This study assessed comparatively the welfare of two breeding horse categories: stallions and broodmares. The assessment included 27 stallions and 35 broodmares and it was based on health indicators (hair coat condition, hair quality in the mane/tail, body lesions, lower leg lesions, swollen tendons/joints, hoof horn quality, hoof walls length, quality of horseshoes, gait, dyspnea, nasal discharge, diarrhea) and behavioral parameters (behavioral response towards humans). The data were analyzed using the SPSS statistical software. The value of minimal significance was considered at P < The prevalence of stallions with dyspnea, tendon and joint swellings, abnormal gait and abnormal hoof horn quality was significantly (P < 0.05) higher than that of the broodmares. No significant difference was found in the behavioral response of the two categories of breeding horses, although the prevalence of indifference was higher in the breeding stallions in all of the three tests used. The welfare of the broodmares seems to be better than that of the breeding stallions, probably because of the different conditions they were kept in. Keywords: breeding horses, health indicators, behavioral indicators, horses welfare The welfare assessment of reproduction horses is important as it leads to the recognition of existing problems and their rapid remedial for improve the welfare of horses. In the past 15 years the scientific research focused more than before on the welfare of working horses (1, 3, 10, 11) besides of those horses used by other means (5,14). In this moment there is no widely accepted assessment protocol to evaluate the welfare of horses. During the past years a few researchers (1, 3, 4, 10, 11, 13) used animal-linked indicators (health and behavioural parameters), but also resource-based, indirect indicators, to assess considerably large horse populations. Recently the AWIN welfare assessment protocol for horses (2) was published, being based on the Welfare Quality principles and criteria. 154

155 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L(3), 2017, TIMISOARA The studies about breeding horses welfare are limited in the scientific literature (12). The aim of this study was to assess comparatively the welfare of two breeding horse categories (stallions and broodmares) using health and behavioral parameters. Materials and methods The study was performed on a sample formed by 27 breeding stallions and 35 broodmares kept in a breeding farm in Transylvania. The stallions were housed tethered, in closed barns, with ground flooring, natural and artificial illumination, using mainly straw as bedding (and occasionally wood shaving) and having permanent access to water (automatic waterers). The cleaning of the barns and feeding of the horses was made manually, using manpower. The access for free exercise in this category was limited, because of the need to provide individual space for each animal (to avoid fighting) and human supervision. The mares were housed in the same type of barns but having daily access to free exercise, in groups, in the paddocks of the barns and also in the pasture (all day long in the warm season). In the day of arrival, half of the horses of the studfarm were assessed, their selection being done randomly. The welfare assessment was made based on health and behavioral parameters by the methods described by Popescu and Diugan (10). Each horse was assessed by two experimented assessors. Within the health related parameters the followings were assessed: hair coat condition, hair quality in the mane/tail, body lesions, lower leg lesions, swollen tendons/joints, hoof horn quality, hoof walls length, quality of horseshoes, gait, dyspnea, cough, nasal discharge and diarrhea. The general attitude of the breeding horses (apathetic or alert) was assessed and their reactions (aggressiveness, fear/avoidance, indifference, friendliness) were evaluated in response to the assessors approach, walking besides and the attempt of touching the animal. The data were analyzed using the SPSS statistical software. The prevalence of the assessed health and behavioral parameters was calculated in the stallions and broodmares. For comparison of data the Mann-Witney U test was used. The value of minimal significance was considered at P < Results and discussions The prevalence of health parameters assessed in stallions and broodmares and the significance of differences between the two breeding horse categories is shown in Table 1. In this study the most health problems were recorded in the stallions, probably because their tethered housing system. Similar results are reported by Sanmartín Sanchez et al. (12) in their study performed in Spain. The prevalence of 155

156 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L(3), 2017, TIMISOARA stallions with tendon and joint swellings, dyspnea, abnormal gait and abnormal hoof horn quality was significantly (P < 0.05) higher than that of the broodmares. Based on the investigation of interrelations between the welfare indicators assessed in Romanian working horses, Popescu et al. (9) states that the good quality of the haircoat seems to be a valuable parameter of improved welfare. In the present study the prevalence of normal haircoat was lower in the stallions than in the broodmares, even if the difference was not statistically significant (Table 1). The cause could be the lack of access to free exercise, including the natural behaviour of rolling which has a certain role in cleaning the skin and haircoat and maintaining a good health of these. Table 1 The prevalence of health parameters assessed in stallions and broodmares and the significance of differences between two breeding horse categories Parameter Stallions Broodmares P value (n= 27) (n=35) Hair coat condition (Abnormal) Hair quality in the mane/tail (Abnormal ) Body lesions Lower leg (foot) lesions Swollen tendons/joints Hoof horn quality (Abnormal) Hoof walls too long or too short Inadequate horseshoes Gait (Abnormal) Dyspnea (Present) Cough (Present) Nasal discharge (Present) Diarrhoea (Present) If P value is less than 0.05 the difference between stallions and broodmares is significant The normal quality of hair in the mane and tail was more frequent in the stallions than in the mares. Even if the general aspect of the hairs is strongly related with the systemic health of the animal, the partial distruction of mane and tail could be a consequence of some management factors or behaviours. The lesions, both on the bodies and on the lower legs of the horses, were more frequent in the breeding stallions than in the mares. The explanation lies in the fact that when physical contact was possible (accidental untethering in the barn, for example), the stallions were attacking each other, producing wounds and lesions by biting or by hitting each other with their legs and heads. 156

157 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L(3), 2017, TIMISOARA The swellings of tendons and/or joints were identifiend only in the stallions. Insufficient movement and tethered housing could represent important risk factors for these problems. The prevalence of this parameter was lower than that reported in working horses (10,11). The abnormal quality of hoof horn was observed only in the stallions, in which the frequency of too long hoof walls was higher than in the mares. In this study the proportion of stallions that presented abnormal gait was significantly higher than that of the mares (P < 0.05). The main cause of this result is represented by tethered housing of the stallions. In the working horses higher prevalence of abnormal gait is reported (3, 11). The indicators of the presence or absence of dyspnea, caughing and nasal discharge had the role of airway health assessment. As regards dyspnea, the differences were significant (P < 0.05) between the two categories of breeding horses. A respiratory system dysease affecting many horses is the recurrent airway obstruction, which involves both genetical and environmental factors (7). An important risk factor in occurence of this disease is the exposure to airborne allergens and specific aero-irritsnts, such as ammonia, molds and inhalable dust particles from the barn, especially in the absence of adequate ventilation. Of the horse categories assessed, the stallions spent the most time inside the barn, tethered. It is remarcable the fact that in none of the breeding horse categories assessed had any signs of diarrhoea, probably because of regular deworming. Table 2 shows the results of behavioural parameters assessed in stallions and broodmares and the significance of differences between two breeding horse categories. Table 2 The prevalence of behavioural parameters assessed in stallions and broodmares and the significance of differences between two breeding horse categories Parameter Stallions (n= 27) Broodmares (n=35) P value General alertness Apathetic/depressed Alert Response to the approach of the assessor Aggressiveness Fear/avoidance Indifference Friendliness Response to the assessor walking besides Aggressiveness Fear/avoidance Indifference

158 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L(3), 2017, TIMISOARA Friendliness Response to the touch of the assessor Aggressiveness Fear/avoidance Indifference Friendliness If P value is less than 0.05 the difference between LHS and THS is significant No significant difference was found in the behavioral response towards humans of the two categories of breeding horses in none in the three tests performed. In this study, similar to the results of another recent research done in Romania, no apathetic mare was identified. These findings are in agreement with those obtained by Sanmartín Sanchez et al. (12). Aggressiveness was observed only in the mares in the test of the touch of the assessor. The number of horses showing fear increased from one test to the other, both in the stallions and in the mares. The fear reaction of animals towards people is produced probably as effect of improper human attitude in the relations with the animal, leading to previous negative experiences. Recognizing this negative mental state is important, because fear and stress, both acute or chronic, can jeopardises the health and welfare of animals (6). The frequency of indifference responses was higher in the stallions than in the mares, probably because of the housing management, tethered in closed barns. Similar to other studies performed in Romania (8), aproximately half of the breeding horses, in both categories, showed friendly response to the human presence in the three behavioural tests. As general welfare considerents, the friendly reaction of horses to humans is the most wanted behavioual response. jumatate din caii de reproductie din ambele categorii au manifestat răspuns Conclusions The welfare of the broodmares seems to be better than that of the breeding stallions, probably because of the different conditions they were kept in. References 1. Ali, A.B.A., El Sayed, M.A., Matoock, M.Y., Fouad, M.A., Heleski, C.R., A welfare assessment scoring system for working equids - A method for identifying at risk populations and for monitoring progress of welfare enhancement strategies (trialed in Egypt), Appl Anim Behav Sci, 2016, 176,

159 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L(3), 2017, TIMISOARA 2. AWIN, AWIN welfare assessment protocol for horses, Università degli Studi di Milano, 2015, Milan, Italy. 3. Burn, C.C., Dennison, T.L., Whay, H.R., Environmental and demographic risk factors for poor welfare in working horses, donkeys and mules in developing countries, Vet J, 2010a, 186, Burn, C.C., Dennison, T.L., Whay, H.R., Relationships between behaviour and health in working horses, donkeys, and mules in developing countries, Appl Anim Behav Sci, 2010b, 126, Dalla Costa, E., Dai, F., Lebelt, D., Scholz, P., Barbieri,S., Canali, E., Minero, M., Initial outcomes of a harmonized approach to collect welfare data in sport and leisure horses, Animal, 11, 2017, Hemsworth, P.H., Ethical stockmanship, A review, Aust Vet J, 2007, 85, Moran, G., Folch, H., Recurrent airway obstruction in horses an allergic inflammation: a review, Vet Med - Czech, 2011, 56, Popescu, S., Borda, C,. Oros, D., Sandru, C.D., Spinu, M., Giupana, R., Diugan, E.A., Human-Animal Relationship: A Comparative Study in Working and Breeding Horses, Bulletin UASVM Veterinary Medicine, 2016, Popescu, S., Diugan, E.A., Spinu, M., The interrelations of good welfare indicators assessed in working horses and their relationships with the type of work, Res Vet Sci, 2014, 96, Popescu, S., Diugan, E.A., The relationship between behavioral and other welfare indicators of working horses, J Equine Vet Sci, 2013, 33, Pritchard, J.C., Lindberg, A.C., Main, D.C.J., Whay, H.R., Assessment of the welfare of working horses, mules and donkeys, using health and behaviour parameters, Prev Vet Med, 2005, 69, Sanmartín Sanchez, L., Perea, J., Blanco-Penedo, I., Perez-Rico, A., Vega-Pla, J.L., Animal welfare in breeding horses (Equus Caballus): a comparative assessment in southern Spain, Rev Cient FCV-LUZ, 2015, XXV, Tadich, T., Escobar, A., Pearson, R.A., Husbandry and welfare aspects of urban draught horses in the South of Chile, Arch Med Vet, 2008, 40, Visser, E.K., Neijenhuis, F., de Graaf-Roelfsema, E., Wesselink, H.G.M.H., de Boer, J., van Wijhe-Kiezebrink, M.C.M., Engel, B., van Reenen, C.G., Risk factors associated with health disorders in sport and leisure horses in the Netherlands, J Anim Sci, 2014, 92,

160 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L(3), 2017, TIMISOARA TRANS ARTICULAR FIXATION OF HIP DISLOCATION IN DOGS M. MUSTE, I. PAPUC, R. LĂCĂTUȘ, R.C. PURDOIU, F. BETEG, A. MUSTE University of Agricultural Sciences and Veterinary Medicine, Faculty of Veterinary Medicine, , Manastur street, no. 3-5, Cluj-Napoca, Romania Summary Research and our observations were followed up on a total of 19 dogs, clinical cases, of different race, gender and age, diagnosed with hip dislocation. In 18 cases dislocation was caused by traffic accidents and in one patient the injury was caused by falling. In our study, hip dislocation was diagnosed only unilateral. The direction of movement of the femoral head in our study were as follows; in 12 subjects (63%) was identify a craniodorsal displacement of the femoral head, 5 cases (26.31%) presented a caudodorsal dislocation and 2 cases (10.52%) had cranioventral dislocation. In 14 cases, surgery was performed between hours from the accident and in 5 cases surgery was performed after 72 hours. Trans articular fixation was carried out by using the cortical screw and Steinmann pins. Cortical screw was inserted following two procedures; in 7 cases the cortical screw was inserted from fovea capitis toward the bone wall of greater trochanter, repositioning and run through the conduct of the ischium, this corresponding to open surgery procedure; in 6 dogs the screw has been introduced by tunneling with the appropriate drill bit (1.6 to 3.5 mm) depending on the dog size, through the greater trochanter, femoral neck and through the fovea capitis and insert in the ischium through the cavity of acetabular fossa. We have achieved good results for cortex screw through open surgery procedure. The method of using the Steinmann pin was unsatisfactory because in 4 subjects, after a period of 6 to 8 months, we found an abnormal mobility. Keywords: dislocation of the hip, stabilizing, repositioning, dogs Dislocation of the hip is a disease entity in which the breaking of normal and natural components of the hip (round ligament, joint capsule) is followed by the movement of the femoral head in the cavity. The causes are varied, the most important being the traumas caused by road accidents, data configured by other authors like Whitelock R., (2010); McLanghlin R.M., (1995), which mentions an incidence of 85%, Trostel C.T., (2000) stipulates other causes including severe dysplasia of the hip, severe trauma. Repositioning the dislocation is more difficult after 3-5 days, view shared by authors such as Evers P, (1997); McLaughlin RM (1995). The reduction and stabilization surgery is much higher (approximately 85%) than for reduction and stabilization through closed process (1,7). Repositioning and stabilization, are done through numerous known processes such as: capsuloraphy (9), capsular prosthesis, transposition of the great trochanter, transarticular fixing, toggle rod stabilizing (5, 12), stabilization with 160

161 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L(3), 2017, TIMISOARA the help of the fascia lata, iliofemoral transarticular suture, sacrotuberal ligament rearrangement using triple pelvic osteotomy (4) the total hip artoplastia, and combinations (11) or using gluteal muscle tendon (8). The variety of methods, unparalleled diversity of races, severity of injuries, complications and injuries initiative, individual characteristics in postoperative response consecutive implementing a process gives very different results. In this context, we wanted to approach hip stabilization using transarticular pinning. Material and method Observations and our study were conducted over a period of five years from 2011 to 2016 on a casuistry consists of 19 dogs, of different race, gender and age, diagnosed with hip dislocation. In 18 cases dislocation was caused by traffic accidents and in one patient the injury was caused by falling. In our study, hip dislocation was diagnosed only unilateral. Surgery was performed within hours post traumatic as shown and recording in consultation and treatment setting. The direction of movement of the femoral head in our study were as follows; in 12 subjects (63%) was identify a craniodorsal displacement of the femoral head, 5 cases (26.31%) presented a caudodorsal dislocation and 2 cases (10.52%) had cranioventral dislocation. In 14 cases surgery was performed in 12 to 20 hours and in 5 cases the intervention was performed in 72 hours. In or cases reducing and stabilizing was carried out by transarticular pinning, method assess differently according to the different authors. Anesthesia was induced by injection of atropine sulphate (Nycomed R Nicomed Austria GmbH. Linez, Austria) (0.04 mg / kg im) and diazepam (DiazepamR, Terapia Cluj-Napoca, Romania) (0.2 mg / kg intramuscularly), injection of Ketamine 10% (Ketamin R Protulab Pharma BV, Rdamsdonkszeer, the Neterlands) (3 mg / kg intramuscularly), and then they were intubated with Isoflurane 2% (Lunan Pharmaceuticol Group Corporation, Shandong, China) in the oxygen mixture. Local anesthesia was performed with lidocaine 2% solution with epinephrine 1: 100,000 (Septanest, Septodont, Quetigny, France). In this way, the patient lies on the operating table in the lateral position with the sprained member above. An assistant puts a small towel or rope in the inguinal area to prevent shrinkage and to secure the pelvis during remediation. Transarticular pinning procedure can be practiced in closed or open surgical procedure. In this sense, for any of the procedures, surgical management meets certain common surgical time, because depending on the procedure, at one point the technique differentiate. For the open technique, after preparation and sanitizing the place, a semicircular incision is carried out in the skin above coxo-femoral articulation, through subcutaneous connective tissue, and superficial, middle and deep gluteal muscle. In cases with cranial-dorsal dislocation, I detached the femoral head of the sprained position by pulling the hock or stifle and external rotating the limb. The 161

162 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L(3), 2017, TIMISOARA member was then pulled caudodistal to position the femoral head to the acetabular cavity. In cranioventral displacement, the femoral head has been manipulated into craniodorsal position and then fixed and stabilized by the method described above. For this procedure, the affected limb was fixed with one hand and with the other we have created a counter pressure on the pelvis. In this way, the femoral head was brought in lateral plane and craniodorsal in acetabular cavity. For caudoventral dislocation, I detached the femoral head from the obturator foramen by pulling on the member and pushing on the ischial tuberosity. In the small and medium sized dogs, detachment of the femoral head was performed by applying femur pressure in the ventral direction and introduction of a finger protected by the glove into the rectum. After highlighting the acetabular cavity, the soft tissue, round ligament, hematoma and remains on the femoral head is removed. On this occasion are evaluated destruction of the femoral head, the acetabular cavity and the joint capsule. Transarticular stabilization was carried out by using the cortex screw and Steinmann pin. The use of cortex screw has been tested in two ways in relation to the direction of luxation of the femoral head and the degree of tissue destruction. In subjects with craniodorsal luxation, with large displacement, with significant tearing and muscle rupture, we opted for open surgical procedure. This method was applied to a number of 7 subjects. In this way, after the isolation of the femoral head (Fig. 1), an appropriate diameter drill is directed to the fovea capitis (Fig. 2) through the femoral neck at the level of the third trochanter. Fig. 1. Isolation of the femoral head 162

163 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L(3), 2017, TIMISOARA Fig. 2. Tunelization of the head, neck and great trochanter of the femur From the large trochanter, through the hole created by the drill, insert cortex screw to the fovea capitis. The femoral head is properly positioned in the acetabular cavity with a slight abduction member after the screw is passed through the wall of the acetabular cavity in such a way that the end of the screw to penetrate the acetabular cavity wall together with all the coils in the pelvic channel (fig.3, fig. 4). Fig.3. Inserting the cortex screw 163

164 LUCRARI STIINTIFICE MEDICINA VETERINARA VOL. L(3), 2017, TIMISOARA 164 Fig.4 The correct possition of the screw The second method was tested on a total of 6 subjects out of 19 dogs, by closed intervening. In this way prepares the place of choice, makes the incision of the skin, subcutaneous connective tissue, muscle and do the tuneling using a suitable drill, from the side of the femur in the third trochanter. The drill is directed towards the fovea capitis and inserted through the femoral neck and head. Pull the drill and the tunnel created by the cortex screw is inserted by screwing. Member is properly positioned in the acetabular cavity in with a slight abduction, when the screw cavity of the acetabular fossa and inserted into the acetabular cavity in the medial wall of the pelvic canal. The Steinmann pin procedure is identical we can use or we can opt out of using the drill. Excess pin is removed from the compact bone of the greater trochanter to prevent sliding thereof in the direction of neighborhood media or tissue trauma. During surgical maneuvers, a nurse assesses penetration of stabilizing materials (rod and screw) in the wall of the medial acetabular cavity by rectal examination. Results and discussions Troubleshooting the hip dislocation through many therapeutic methods, each with advantages and disadvantages in postoperative evolution. The methods used in this paper, they are part of an attempt to find a methodology with enhanced efficacy in the context of more limited surgical maneuvers, less traumatic. Choosing the transarticular method have primarily immediate advantage in that it can call immediately after trauma or accident, surgical trauma is minimal, which promotes faster healing. Transarticular pinning can be use both in large dogs and small dogs, also in case of fracture of the femoral neck, in which we have less possibilities of preserving the femoral head. The use of cortex screw for remedy of dislocation is justified by the fact that it is acting as a lever, stable in the horizontal