ETIOLOGICAL RESEARCH OF MASTITIS IN COWS

Transcription

1 UNIVERSITY OF AGRICULTURAL SCIENCES AND VETERINARY MEDICINE OF BANAT TIMIŞOARA FACULTY OF VETERINARY MEDICINE TIMIŞOARA MIHAELA SMĂRĂNDIŢA POPESCU (BRÎNDA) ETIOLOGICAL RESEARCH OF MASTITIS IN COWS - ABSTRACT OF DOCTORAL THESIS - SCIENTIFIC SUPERVISOR, PROF. DR. VIOREL HERMAN TIMIŞOARA 2010

2 This doctoral thesis contains: Abstract (rom., eng.) General part (100 pages) Special part (175 pages) Tables: 68 Figures (photos, graphics): 152 Bibliography: 402 references ABSTRACT of the doctoral thesis: Etiological research of mastitis in cows This work was conceived as a study on determining the etiology of mastitis in dairy cows being studied different types of mastitis. From an evolutionary standpoint, mastitis were classified as: subclinical mastitis, if there were not marked local or systemic signs or organoleptic changes of milk, respectively clinical mastitis, when local and/or systemic reactions or any change in appearance of milk (clots, changes of color) were noted. After the character of the first culture, mastitis from which pathogens were isolated in pure culture were noted as monomicrobial and those from which have been isolated microorganisms belonging to at least two species were noted as polymicrobial. Data obtained by us reflect the results recorded over a period of four years of investigations in five dairy farms in three counties (Arad, Caraş-Severin and Timiş). Two of these farms asked us to support them by performing bacteriological examinations and antibiosusceptibility testing, because there were problems of resistance in bacterial agents to prior treatments and there was necessary a correct therapy. The rest of 3 farms were visited in order to detect mastitis with the purpose of the etiological research. In total, there were bacteriological examined 341 samples of milk sterile collected from 255 dairy cows, respectively from 341 mammary quarters. The thesis contains 275 pages and is structured in two parts extended in 11 chapters. The First Part (Chapter 1-5) summarizes the main bibliographic database of scientific literature on the classification of mastitis on different criteria, data on the different types of mastitis, depending on their etiology, and data on the diagnosis methods of mastitis in cows and on antibiosusceptibility testing of pathogens isolated from milk samples, this part representing "The state of knowledge". Part Two is divided into six chapters (Chapter 6-11). In chapters 6-10 are systematically rendered own research on the etiologic agents that caused mastitis in dairy cows in the studied farms, over the periods and Chapter 6 of Part 2, which is an overall assessment of the etiologic agents isolated from cows' mastitis in the study, includes the introduction and the purpose of the present study, the materials and methods applied to various isolated and studied pathogens, the obtained results, with their discussion and partial conclusions. Chapters 7-10 include results and discussion of different types of mastitis, depending on their etiology, being included here the results of epidemiological examination, bacteriological identification, antibiosusceptibility testing, the results on changes of milk secretion and mammary gland and partial conclusions, while Chapter 11 summarizes the general conclusions that have emerged from the investigations. The paper has a number of 152 figures (photos, graphics), 68 tables and it is based on 402 works of scientific literature. Part one is a summary of currently available data related to etiology, epidemiology characters, clinical and morphopathological signs in the major bacterial mastitis of dairy cows and in other types of mastitis. In this part are also summarized data on the etiologic diagnosis II

3 and other diagnosis tests of mastitis in cows, and also notions about antimicrobials susceptibility testing of germs isolated from mastitis. In Chapter 1 are reviewed general terms about mastitis on etiology, epidemiology (incidence, the interaction between microbial factors, host and environment, economic importance), on pathogenesis and classification of mastitis with regard to several criteria, namely as the etiologic agent nature, source of the etiologic agent, clinical evolution severity, duration and morphopathological criterion. In Chapter 2 are presented the etiology, epidemiology, clinical and morphopathological signs for major bacterial mastitis of cows, being included here staphylococcal mastitis, streptococcal mastitis, mastitis caused by Arcanobacterium pyogenes and coliform germs. In our country, the frequency of staphylococcal mastitis has been reported to range from 25.5 to 43.8%. From staphylococcal mastitis cases were isolated, along time, species of coagulase-positive staphylococci (CPS) and coagulase-negative staphylococci (CNS). CPS from cows with mastitis are classified in the following species: S. aureus that is the most frequently isolated, S. intermedius and S. hycus in smaller proportion, while CNS isolated from mastitic milk include over 10 species, of which the most common are: S. epidermidis, S. chromogenes, S. warneri, S. xylosus, S. simulans and S. sciuri. S. aureus is ubiquitous in dairy farms. Infected mammary gland is the main reservoir for S. aureus. In S. aureus mastitis are described more evolutive forms: subclinical, subacute, acute, superacute and chronic, clinical evolution being dependent of the pathogenicity of staphylococcal strains, the animals' resistance and the intensity of favoring factors, and concerning the morphopathological criterion the severe forms of S. aureus mastitis are the lightning superacute mastitis, respectively the acute ones with pernicious or gangrenous evolution. Some authors consider that the mammary gland colonization with some of CNS species increases the resistance to other intramammary infections. Thus, CNS seem to have protective effect against the colonization of mammary duct and udder skin with CPS and other major pathogens, except E. coli and environmental streptococci. Generally the bovine mastitis caused by low pathogenic agents evolve subclinical or mild clinical, but there were also reported cases of CNS chronic mastitis. The most common of streptococcal mastitis is the one caused by Str. agalactiae, which is an enzootic infectious disease in lactating cows anatomopathological expressed by changes in mammary secretion and mammary gland parenchyma, with a tendency to become chronic. This mastitis may develop in 50-80% dairy cows of a herd. Usually, cows with mastitis caused by Str. agalactiae have an increased number of somatic cells in milk ( cells/ml, occasionally reaching cells/ml or more), but its appearance is normal, being noted a subclinical mastitis. There can be observed mastitis caused by Str. agalactiae with superacute, acute, subacute and chronic evolution, the latter two forms being the most common, the milk being easily coagulated and having alkaline ph and salted taste, and if the milk is left to stand for 24 hours, it separates into two layers, one lower formed by clots and leucocytes and one serous upper. Anatomical lesions primarily intereste the walls of the galactophor sinus, suffering a noticeable induration on palpation. Beside Str. agalactiae, environmental streptococci (ES) represent a common cause of clinical and subclinical mastitis in herds in which mastitis caused by major contagious pathogens were prevented. The environmental streptococci which can cause intramammary infections are: Streptococcus uberis (group E), Str. dysgalactiae (group C), Str. equi (group C), Str. bovis (group D), Str. pyogenes (group A), Str. pneumoniae (ungroupable), Str. canis (group G) and Enterococcus spp. The percentage of clinical mastitis caused by ES increased worldwide. Approximately 14-26% cases of clinical mastitis occurring in Canada, U.S.A., Netherlands and United Kingdom are caused by Str. uberis. Str. dysgalactiae behave both as contagious pathogen, as well as environmental pathogen and it can invade mammary epithelial cells, which may explain the persistence of intramammary infection. Str. uberis may cause mastitis of different evolution forms from subclinical to severe clinical ones, even toxic mastitis. A. pyogenes is rarely isolated from summer mastitis in pure culture because there is not a specific infection of this type of mastitis. The infection of mammary gland by contact III

4 with A. pyogenes occurs only when the mammary gland skin has lesions, being also involved anaerobic and microaerophilic bacteria. Mastitis caused by A. pyogenes is found in lactating and in dry period cows, generally in youth as calf heifers, but it can also affect unlactating heifers being reported cases from the age of 2-4 months. The frequency of mastitis cases caused by A. pyogenes is generally low, affecting less than 10% from the herd. There is characteristic the evolution of superacute form of pyobacillar mastitis, with severe systemic signs and watery secretion with floaters of fibrin and casein, which sooner or later becomes purulent, with fetid odor caused by the presence of Peptostreptococcus indolicus. If the animal survives there are forming abscesses in the mammary quarter, which becomes indurated. Abscesses subsequent break in the bottom of the udder, usually at the teat basis. This stage is described by some authors as a chronic form of summer mastitis. It may also be observed acute form, which takes the form of diffuse purulent mastitis. Mastitis caused by coliform germs is one of the main lethal mastitis in dairy cows, despite of intensive therapy, the superacute form of that mastitis reaching up to 80% lethality. From coliform germs that cause mastitis in cattle there are noted different serotypes of E. coli, some types of encapsulated Klebsiella spp. and Enterobacter aerogenes, which are within the conditional pathogenic Enterobacteriaceae. In the mammary gland, E. coli strains multiply and develop endotoxins, which cause exudative inflammation of mammary gland tissue and tissue necrosis. There was noted that an anaphylactic mechanism (Arthus phenomenon) would also interfere in the pathogenesis of colibacillar mastitis. While some authors have found that 30-40% of all clinical mastitis cases are caused by coliform germs (E. coli, Enterobacter aerogenes, Klebsiella pneumoniae and Klebsiella oxytoca) in some herds, particularly those where the contagious pathogens are controlled, others authors argued that the percentage of mammary quarters infected with coliform pathogens is generally low (2-4%). Mastitis caused by coliform germs evolves frequently as superacute and acute forms, but there are chronic and subclinical evolutions too. From morphopathological point of view, in severe cases (infections with strains high inducing TNF - tumor necrosis factor) at necropsy, outside the mammary gland lesions, there can be seen haemorrhages on serous and congestive-dystrophic changes of major organs and muscle, intestinal catarrh. Chapter 3 brings together bibliographic information about the etiology, epidemiology, clinical and morphopathological signs in other types of mastitis in cows, being included here the mastitis caused by other bacterial species (Mycoplasma spp., Pseudomonas aeruginosa, Corynebacterium spp., Pasteurella spp., Mannheimia haemolytica, Nocardia spp., Fusobacterium necrophorum, Mycobacterium spp., Bacillus spp., Serratia spp., Listeria monocytogenes, Clostridium perfringens type A, Campylobacter jejuni and Haemophilus somnus), mycotic mastitis and mastitis caused by Prototheca spp. In other types of mastitis in cows are noted all bacterial species rarely involved in the etiology of mastitis in cows. Some mastitis in cows have been sporadically reported, this is also the case of mycoplasmal mastitis, that can evolve also as endemic, being sometimes preceded by unusual clinical signs (lameness, distal forearm edema or polyarthritis). Mastitis caused by Pseudomonas aeruginosa rarely develops in cattle, and only as sporadic form following intramammary infusions of contaminated material. Mastitis caused by Mannheimia haemolytica and Pasteurella spp. are more common in sheep, but is rarely reported also in cattle and, when encountered, usually is sporadic. Another mastitis that evolves sporadically in a herd, affecting one or two cows, is caused by Nocardia spp. This may cause problems in the herd, unless occurs accidental inoculation of bacteria in the mammary gland by intramammary infusions application. In cows mastitis caused by Nocardia spp. frequently evolve as chronic mastitis in Cuba. Also, most cases of mastitis caused by Serratia spp. are sporadic, with subclinical or clinical evolution, outbreaks may occur due to contaminated sawdust used for bedding and also in the case of unadecvate milking hygiene. In cattle mastitis caused by mycobacteria develops sporadic and can be generated by Mycobacterium bovis, which is the etiologic agent of bovine tuberculosis, and other mycobacteria (M. lacticola, M. fortuitum, M. phlei, M. smegmatis, M. chelonei and M. tuberculosis). Tuberculosis mastitis is not an independent nosologic entity but almost always is a secondary location, metastased from lung or other primary sites localizations. Bacillus cereus and IV

5 Bacillus subtilis are saprophytic organisms, occasionally causing acute hemorrhagic mastitis in cattle. Although generally coryneform bacteria are frequently isolated from bovine mastitis, the prevalence of mastitis caused by C. bovis being low in the herds where milking hygiene is respected and there is carried out treatment in the dry period. In addition to increasing the total number of somatic cells in milk, C. bovis that is also classified as low pathogenic germ increases the resistance to invasion by major pathogens of the affected mammary quarters. Fusobacterium necrophorum is part of the normal anaerobic flora of animals and can cause acute mastitis in cattle, being commonly isolated from cases of summer mastitis, with Arcanobacterium pyogenes and Peptostreptococcus indolicus. Mastitis caused by Listeria monocytogenes are important because the zoonotic risk due to the consumption of contaminated dairy products, 6.8% of cows with subclinical and clinical mastitis caused by this organism removing Listeria by milk. The risk of this mastitis is more prominent if subclinical evolution occurs, only with lower milk production, mammary secretion being apparently normal. Also, mastitis caused by Clostridium perfringens type A, very rare in cattle, has zoonotic risk and can cause food poisoning in humans. But this type of mastitis is easily diagnosed through clinical signs, with systemic signs (fever) and local ones (swelling, then gangrene) with mammary secretion changes (thickened, brown, containing gas bubbles due to subcutaneous emphysema). In 1985, it was reported the only case of mastitis caused by Campylobacter jejuni, with subclinical evolution, but it is worth mentioning because of its zoonotic impact. Mastitis caused by Haemophilus somnus has been reported in an animal at 10 days after calving, the mammary gland showing moderate inflammation without systemic signs reported. The species of fungi that may be involved in the pathology of mycotic mastitis in cows are: Cryptococcus neoformans, Trichosporon spp., Candida spp., Saccharomyces spp., Pichia spp., Torulopsis spp., Aspergillus spp., Rhodotorula spp., Geotrichum candidum, Trichoderma koningii, Trichotecium roseum. Fungi are widespread in nature, being noted in bedding and gear from the stables, on milking machines. Mycotic mastitis generally progress due to bacterial infections treated with antibiotics for a long time. Cryptococcus neoformans, the yeast which produces cryptococcosis in humans, cause acute mastitis in cattle. The sources of infection are the most likely represented by contaminated infusions intramammary administered or other cows with mastitis caused by this germ. No epidemiological risk is present that the milk is heat treated before being consumed, as this yeast does not tolerate the pasteurization temperature of milk. Candida spp. infection is often acute and transient, the organism disappearing in about three weeks. In the infections with Aspergillus fumigatus and Aspergillus nidulans numerous abscesses surrounded by granulation tissue are forming in the mammary quarters, but the lactipherous ducts are not generally affected. Of the algae involved in the etiology of mastitis in cattle have been reported the following species: Prototheca zopfii, Prototheca wickerhamii, Prototheca trispora and Prototheca blaschkeae. Prototheca spp. is considered opportunistic germ and to cause mastitis require the intervention of predisposing factors (immunodeficiency, abusive treatment with antibiotics). Illness caused by these algae is generally sporadic, mastitis prevalence being approximately 0.1%. In cows, Prototheca spp. may cause clinical and subclinical mastitis, accompanied by mammary gland tissue necrosis, thrombosis of arteries, veins and lymphatic vessels and the presence of Prototheca spp. in the acines and mammary gland ducts, these algae being able even to diffuse in the body by lymphatic pathway, producing a systemic infection. In Chapter 4 are presented the data on diagnostic methods of mastitis in cows as detailed following categories: etiologic diagnosis, including here the isolation and identification of bacterial etiologic agents of mastitis (Staphylococcus spp., Streptococcus spp., Enterococcus spp., Arcanobacterium pyogenes and coliform germs), the diagnostic technique in fungal infections and other diagnostic tests, respectively organoleptic examination, chemical examination, total mesophilic aerobic bacteria count, total number of somatic cells count, cytological examination - polymorphonuclear neutrophil leukocytes, immunological and molecular methods of diagnosis. V

6 Chapter 5 summarizes antimicrobials susceptibility testing of germs isolated from mastitis in cows being included general terms, information relating to the diffusimetric antibiogram on standardized culture and specific methods for antibiotic susceptibility testing of some pathogens. Part two, entitled "Own research", is written in six chapters (Chapter 6-11). In Chapters 6-10 are presented the investigations realized in this study, in order to isolate and identify etiologic agents that caused clinical and subclinical mastitis, mono- and polymicrobial, being also monitored their frequency during the four years of observations in the five dairy cows farms in the study from three counties in western side of Romania, while the Chapter 11 summarizes the general conclusions. Chapter 6 includes: introduction and purpose of the study, materials and methods that have been applied to various isolated and studied pathogens in Chapters 6-10, results and discussion - with reference to the correlation between the evolutionary forms of mastitis (subclinical and clinical mastitis), the character of first culture (mono- and polymicrobial mastitis) and frequency of bacterial and fungal agents involved in the studied mastitis - and partial conclusions. Chapters 7-10 of the second part includes results and discussion on staphylococcal and streptococcal mastitis, mastitis caused by Gram-negative germs and mycotic mastitis, being presented: the results of epidemiological examination, results concerning the bacteriological identification, antibiosusceptibility testing - Chapters 7-9 (for some strains of S. aureus, CNS, Str. agalactiae, Enterococcus spp. and E. coli), the changes of mammary secretion and mammary gland and partial conclusions. In the present research there were carried out the following exams and tests: epidemiological (epidemiological investigation, the reveal of the possible source of infection and predisposing factors), organoleptic examination of milk, clinical exam (general symptoms, mammary gland changes), indirect field tests for the detection of subclinical mastitis (R-Mastitest, Mast-O-Test), bacteriological examination (isolation of etiological agent, phenotypic characterization of the etiological agent and antimicrobials susceptibility testing of some bacterial strains isolated). Epidemiological investigations, clinical ones and laboratory tests performed to elucidate the infectious causes of mastitis in dairy cows resulted in drawing the following conclusions: Chapter 6 Epidemiological studies of mastitis in cows There were studied 255 lactating dairy cows, from the breeds Bălţată cu negru românească, Bălţată cu roşu and also Holstein crossbreed. Overall 341 milk samples were collected from the studied cows of five farms, as follows: a farm in Arad County, a farm in Caraş-Severin County and 3 farms in Timiş County. For bacteriological examination there were used the following usual and special culture mediums: agar (Blood Agar Base - Biolab) with added sheep defibrinated blood or horse inactivated sterile serum 5%, for staphylococci - Mannitol salt phenol-red agar (Merck), hiperchlorinated medium, Baird-Parker medium, for enterococci - Azide Dextrose Broth (Biomedics), for Gram-negative - MacConkey, Levine, SS agar and politrope mediums (TSI and MIU) and for mycological examination, Sabouraud dextrose agar type II and Sabouraud agar with 2% glucose, both with added 0.05% gentamicin. The bacterial cultures and the mycotic ones were incubated at 37 C in aerobic conditions. The organisms were identified by using standard procedures associated with rapid tests for identification, such as Staphytect Plus (Oxoid) and Patorex Strep (Bio-Rad Laboratoires), respectively API Staph, API 20 Strep and API 20 E multitest systems, all research materials being provided by the Department of Infectious Diseases, from the FMV Timişoara. It also was tested the antibiotic susceptibility by diffusimetric method in some bacterial strains isolated. Etiology for most of mastitis in dairy cows in the study proved to be bacterial, the bacterial strains having a isolation frequency of 92.1%, being mainly Gram-positive, the difference of 7,9% being fungal strains. In bacterial mastitis, the strains of Gram-positive isolates had a significantly higher proportion (91.5%) versus Gram-negative bacteria (8.5%). VI

7 By microbiological examinations, bacteriological and mycological ones, of the samples of milk collected from dairy cows with subclinical and clinical mastitis, with mono- and polymicrobial etiology there were noted varying frequencies of the following pathogens: Staphylococcus spp. (46.7%), Streptococcus spp. (25.5%), Enterococcus spp. (6.5%), Corynebacterium spp. (5.6%), Escherichia coli (2.3%), Klebsiella pneumoniae (1.4%), Candida spp. (1.4%), Aspergillus fumigatus (0.9%), Mannheimia haemolytica (0.5%). Of all strains of microorganisms isolated from mastitis predominated the strains derived from monomicrobial mastitis (59.3%) compared with those of isolates from polymicrobial mastitis (40.7%), microorganisms of the genus Staphylococcus being the most frequently isolated from both monomicrobial (47.6%) and polymicrobial (45.5%) mastitis. Polymicrobial mastitis of dairy cows studied were both bacterial and mixed (bacterial and mycotic). Fungi were mainly isolated from polymicrobial mastitis and from evolutionary point of view from clinical mastitis. Strains belonging to the genera Staphylococcus, Streptococcus, Enterococcus, and the species E. coli and K. pneumoniae predominated in monomicrobial mastitis. Corynebacterium spp. was predominantly isolated from polymicrobial mastitis and the only one strain of Mannheimia haemolytica originated from an animal with polymicrobial intramammary infection. Overall, most strains were isolated from subclinical mastitis (63.9%), in which the highest percentage was represented by the staphylococcal strains (53.6%). Isolates from clinical mastitis cases accounted only 36.1% of all strains of microorganisms. From these mastitis the strains belonging to the genera Staphylococcus and Streptococcus were isolated with equal frequency, 34.6% respectively. Staphylococcus spp., Enterococcus spp., Corynebacterium spp. and "other coliforms" strains were isolated mainly from subclinical mastitis, unlike the strains of Streptococcus spp., E. coli, K. pneumoniae, M. haemolytica and fungi were isolated more frequently or exclusively from clinical mastitis. Chapter 7 Staphylococcal mastitis is divided into two subsections, where are presented the results on mastitis caused by coagulase-positive staphylococci (CPS), respectively, on mastitis caused by coagulase-negative staphylococci (CNS). Also in this chapter were also included the results of bacteriological identification of coryneform germs which were mostly isolated in association with other germs. Of the total strains isolated in this study, in the genus Staphylococcus were identified most frequently coagulase-positive species, totally 57 strains (26.4%) from which 54 strains of S. aureus (25.0%), two strains of S. intermedius (0.9%) and one strain of S. hycus (0.5%). There were isolated 44 strains (20.4%) identified and classified into ten coagulase-negative species as follows: 8 strains of S. xylosus (3.7%), 7 strains of S. chromogenes (3.2%), 6 strains of S. sciuri (2.8%), 6 strains of S. lentus (2.8%), 4 strains of S. epidermidis (1.9%), 4 strains of S. haemolyticus (1.9%), 3 strains of S. hominis (1.4%), 2 strains of S. hycus (0.9%), 2 strains of S. simulans (0.9%) and 2 strains of S. saprophyticus (0.9%). S. aureus was the most frequently isolated species (54 strains %) of all staphylococcal strains isolated in this study (101 strains), but also the most common (25.0%) of all microbial strains isolated from the examined mastitic milk samples (216 strains). Of the 101 staphylococcal strains isolated CNS had frequencies ranging from 2.0 to 7.9%. Of the 44 CNS strains isolated from mastitis in cows, the most frequently isolated species was S. xylosus (18.3%), followed by nine other species: S. chromogenes (16.0%), S. sciuri (13, 6%), S. lentus (13.6%), S. epidermidis (9.1%), S. haemolyticus (9.1%), S. hominis (6.8%), S. hycus (4.5%), S. simulans (4.5%) and S. saprophyticus (4.5%). From the mastitis caused by S. aureus, this bacterial species had the frequency equal to 61.1% in the case of subclinical and monomicrobial mastitis, and 38.9% frequency in clinical and polymicrobial mastitis. In polymicrobial mastitis, S. aureus was most frequently isolated in association with strains of Streptococcus spp., followed in frequency by the association with fungi in mixed culture, and bacterial association with coagulase-negative staphylococci, Enterococcus spp. and Corynebacterium spp. VII

8 In the case of clinical mastitis caused by S. aureus there were observed different forms of evolution: acute, subacute and chronic. S. intermedius was isolated only in two cases of monomicrobial mastitis, one subclinical and one clinical. Regarding the mastitis from which we have isolated the species coagulase-negative of staphylococci, there was a notable difference between the proportion of isolates from subclinical mastitis (88.6%) and isolates from clinical mastitis (11.4%). Monomicrobial clinical mastitis, from which were isolated strains of coagulasenegative staphylococci, evolved as subacute mastitis in the case of S. lentus and acute mastitis in the case of S. haemolyticus. In tube coagulase test, realized to highlight the free coagulase (staphylocoagulase), most coagulase-positive strains had a rapid reaction within the first 4 hours. In the bacteriological examinations of mastitic milk samples only one coagulasepositive strain of S. hycus was isolated and identified. Bacteriological examination on blood-agar revealed few S. aureus strains with α- haemolysis (complete haemolysis) or lake of haemolysis, while the most of S. aureus strains produced β-haemolysis (incomplete haemolysis or "hot-cold" haemolysis). Both strains of S. intermedius isolated were β-haemolytic, while the strain of S. hycus was unhaemolytic. In the case of coagulase-negative staphylococci on blood-agar, haemolysis varied depending on bacterial species such: α-haemolysis in some strains of S. chromogenes and S. haemolyticus, β-haemolysis in S. lentus and some strains of S. xylosus, S. sciuri and S. haemolyticus, the lack of haemolysis in S. epidermidis, S. hominis, S. hycus, S. simulans, S. saprophyticus and in some strains of S. xylosus, S. chromogenes and S. sciuri. In CAMP test applied in order to pursue the staphylococcal haemolysins and their interaction with the staphylococcal β-haemolysin, S. intermedius strains produced β- haemolysin, giving a clear zone of haemolysis, in contact with β-haemolysin of S. aureus streak, against which the test was performed. S. aureus strains showed multiple haemolysins, including δ-haemolysin, and in the strain of coagulase-positive S. hycus δ-haemolysin was not observed. Of CNS, in CAMP test, δ-haemolysin was observed in the species S. xylosus, while S. haemolyticus presented α-haemolysin. On Mannitol salt phenol-red agar (type Chapman hyperchlorinated medium), all strains of S. aureus were manito-positive, with intense positive reaction within 24 h of incubation at 37 C, while S. intermedius presented a weak and delayed manito-positive reaction and the strain of coagulase-positive S. hycus was manito-negative, giving an alkaline reaction expressed by turning the color of the culture medium in hot pink. On that culture medium, the CNS reactions varied, thereby being observed the following reactions: positive reaction in the species S. xylosus (delayed), S. sciuri, S. lentus (delayed) and S. simulans, negative reaction in S. chromogenes, S. epidermidis, S. hominis and S. hycus and variable reaction in species S. haemolyticus and S. saprophyticus. At the resistance testing on hyperchlorinated medium, some strains of S. aureus presented varied growth, in terms of incubation time and degree of colonies development, while the coagulase-positive strain of S. hycus grew on agar with 5%, 6.5% and 12% NaCl, with no longer grow at concentrations of 15%, 18% and 20% NaCl. The identification of staphylococcal strains was also based on the use of Baird-Parker medium by forming bright black colonies surrounded by a clear halo in S. aureus species. Also, the correct identification of S. aureus strains was confirmed by using the kit "Staphytect Plus". In the API Staph test, only 75% of S. aureus strains produced alkaline phosphatase, this percent being below the level from the identification table of API Staph system (97%) and S. intermedius strains behaved on API Staph like a standard strain of S. aureus, except the negative reaction to urease production. On API Staph system one S. lentus strain gave negative reaction to the acidification of D-trehalose, this property being 100% positive in the identification table for that staphylococcal species. Regarding antimicrobial susceptibility testing S. aureus has shown the high sensitivity to cephalotin (92.0%), this one being also the second antibiotic that has induced sensitivity to coagulase-negative staphylococci (62.4%). Staphylococcal strains isolated were also sensitive VIII

9 in varying proportions to: oxacillin, gentamicin, tetracycline, kanamycin, novobiocin, ampicillin and erythromycin. If amoxiclave showed the high sensitivity (65.6%) in CNS strains, S. aureus strains showed reduced sensitivity (4.0%) to this antibiotic. All S. aureus strains tested were found to be resistant to methicillin, unlike CNS that were sensitive, and to rifampicin all staphylococcal strains tested showed sensitivity. In the staphylococcal strains tested, resistance was observed to several groups of antibiotics used in the treatment of mastitis in cattle: β-lactams (ampicillin, oxacillin), aminoglycosides (gentamicin, kanamycin), macrolides (erythromycin) and tetracyclines (tetracycline). It was noted the phenomenon of multiple resistance to some antimicrobial substances tested. In the scientific literature, multiple antibiotic resistance is defined as the resistance of the same bacterial strain to four or more antibiotics. In the present research, of the 25 S. aureus strains tested, 23 representing 92% had revealed this phenomenon as follows: 8 strains (32%) were resistant to four antibiotics, 4 strains (16%) to 5 antibiotics, 5 strains (20%) to 6 antibiotics, 4 strains (16%) strains to 7 antibiotics and 2 strains (8%) to 8 antibiotics of all the 11 antibiotics tested. Any S. aureus strain showed resistance only to an antibiotic, 2 strains (8%) being resistant to 2 antibiotics. Chapter 8 Streptococcal mastitis consists of two subsections, being shown the results on mastitis caused by Streptococcus agalactiae, respectively, by environmental streptococci (ES). Of the 216 strains of microorganisms isolated in this study, strains belonging to the genus Streptococcus accounted for 25.5% (55 strains), being identified two streptococcal species: Str. agalactiae (46 isolates %) and Str. dysgalactiae (9 isolates - 4.2%). The last species was classified in the category of environmental streptococci (ES), with Enterococcus spp., isolated and identified with a frequency of 6.5% (14 strains). In streptococcal mastitis, including environmental ones, the highest isolation frequency was observed for Str. agalactiae (66.7%), followed by the strains of Enterococcus spp. (20.3%) and Str. dysgalactiae (13.0%). Concerning the mastitis caused by Str. agalactiae, the ratio of the strains isolated from monomicrobial and polymicrobial mastitis was about 2:1, the isolation frequency from monomicrobial mastitis being of 67.4%, while the isolation frequency from polymicrobial mastitis was 32.6%. In polymicrobial mastitis Str. agalactiae was frequently isolated in association with S. aureus and coagulase-negative staphylococci, while Str. dysgalactiae was isolated only in association with coagulase-negative staphylococci. Enterococcus spp. was isolated from polymicrobial mastitis cases in bacterial association with S. aureus, coagulasenegative staphylococci and Corynebacterium spp. Unlike the Str. agalactiae strains, that were more frequently isolated from clinical mastitis in cows, environmental streptococcal strains (Str. dysgalactiae and Enterococcus spp.) were predominant in subclinical intramammary infections. The evolution of clinical mastitis caused by Str. agalactiae varied as acute, subacute and chronic, that of mastitis caused by Str. dysgalactiae was subacute, while the mastitis caused by Enterococcus spp. was chronic. In bacteriological examination on blood-agar, Str. agalactiae strains expressed all types of haemolysis: β (complete), α (incomplete) and α' (incomplete haemolysis, bordered by a narrow margin of complete haemolysis). In general, the colonies without pigment (translucent) were α'-haemolytic, and the pigmented ones (white-gray) were β-haemolytic. All strains of Str. agalactiae isolated were haemolytic. In CAMP test, all 46 strains of Str. agalactiae tested gave positive reaction by forming a arrow-shaped head zone of complete haemolysis. On blood-agar, all strains of Str. dysgalactiae were α-haemolytic (incomplete), giving colonies surrounded by a zone of greening, character that allowed the classification of Str. dysgalactiae strains as subsp. dysgalactiae. Enterococcus spp. strains tested were both haemolytic, causing α-haemolysis with trend of greening the culture medium, and unhaemolytic. On API 20 Strep system, 6 of 7 Str. agalactiae strains tested (86%) gave negative reactions to β-glucuronidase, alkaline phosphatase and leucine-aminopeptidase, characters IX

10 that in the standard strain of Str. agalactiae are positive in proportions of 79%, 96% and respectively 99%. In addition, amidon fermentation was positive in most strains of Str. agalactiae tested (71%), having a double amount of the percentage of positive reactions in the standard strain from the identification table. Biochemical characteristics of Str. dysgalactiae strains were tested by using API 20 Strep system and there were observed reactions less frequently reported in this streptococcal species, as positive reaction to aesculin hydrolysis and positive reaction to mannitol acidification. Concerning the antimicrobial susceptibility of tested Str. agalactiae strains these showed equal sensitivity (64.3%) to three antimicrobial substances a.s. (penicillin G, cephalotin and ampicillin) and in varying proportions to tylosin, lincomycin, erythromycin, the lowest sensitivity (14.3%) being to amoxiclave. In contrast, Enterococcus spp. strains tested were susceptible to: ampicillin, oxacillin, lincomycin, bacitracin, erythromycin, tylosin and tetracycline, and resistance was noted in varying proportions to all 10 a.s. tested. Regarding resistance to antimicrobial substances, all strains of Str. agalactiae tested were resistant to: novobiocin, tetracycline, kanamycin, bacitracin and trimethoprimsulfamethoxazole and in varying proportions to: gentamicin, oxacillin, tylosin, lincomycin, erythromycin, amoxiclave, penicillin G and ampicillin, unless cephalotin. As in staphylococci, also in the tested Str. agalactiae and Enterococcus spp. strains was observed the phenomenon of multiple resistance to antimicrobial substances. In the present research, all 14 strains of Str. agalactiae tested proved this phenomenon as follows: 6 strains (42.9%) to 5 a.s., 3 strains (21.4%) to 6 a.s., 2 strains (14.3%) to 8 a.s., 2 strains (14.3%) to 9 a.s. and one strain (7.1%) to 10 a.s. of the total of 14 a.s. tested. In the case of Enterococcus spp. of 9 strains tested, 7 strains representing 77.7% presented this phenomenon as follows: one strain (11.1%) was resistant to 4 a.s., 3 strains (33 3%) to 6 a.s., one strain (11.1%) to 7 a.s., and 2 strains (22.2%) to all 10 a.s. tested. Two strains were sensitive to only one a.s. (one strain to trimethoprim-sulfamethoxazole and one to tetracycline). The strains that were resistant to 6 a.s. proved the resistance to the same six a.s., namely erythromycin, tylosin, kanamycin, tetracycline, gentamicin and trimethoprimsulfamethoxazole. The strain that showed resistance to 7 a.s. was resistant to all these six, and in addition to lincomycin. Chapter 9 Mastitis caused by Gram-negative germs includes results on mastitis from that were isolated strains of Escherichia coli, Klebsiella pneumoniae and one strain of Mannheimia haemolytica. Gram-negative germs (17 strains) had an isolation frequency of 7.9% of total isolates (216 strains) studied, of which 7.4% (16 strains) were environmental coliform germs. Coliform germs included E. coli (5 isolates - 2.3%), K. pneumoniae (3 isolates - 1.4%) and "other coliforms" (8 strains - 3.7%). Of the two identified species of environmental coliform germs, E. coli (2.3%) had the highest frequency of isolation from milk samples. Of the total strains isolated from milk samples in cows one strain of M. haemolytica has been identified, representing an isolation rate of 0.5%. Of the 138 strains of microorganisms isolated from subclinical mastitis, E. coli represented 1.5% (2 strains), and of the strains isolated from clinical mastitis cases (78 strains), E. coli had 3.8% isolation frequency (3 strains). All three strains of K. pneumoniae and the one of M. haemolytica were isolated from clinical mastitis. Clinical mastitis caused by E. coli and M. haemolytica had an acute evolution, and those caused by K. pneumoniae had subacute and chronic evolutions. Concerning the coliform germs, monomicrobial mastitis were more frequently observed. Thus, the E. coli strains isolated from all milk samples predominated in monomicrobial mastitis (3 strains - 1.4%) compared with polymicrobial mastitis (as bacterial associations: 0.5% - with Corynebacterium spp. and 0.5% - with other bacteria), while K. pneumoniae was isolated only in monoculture (1.4%). The only M. haemolytica strain was isolated from a case of polymicrobial mastitis, in association with unidentified bacteria. At haemolysis testing on blood-agar, any E. coli strain isolated from mastitic milk was haemolytic. On politrope mediums the strains of E. coli have fermented glucose and lactose with gas formation, did not produce hydrogen sulfide, were mobile, produced indole and did X

11 not decompose urea, giving variable reactions of sucrose fermentation. K. pneumoniae strains fermented glucose, lactose and sucrose with production of gas, did not produce hydrogen sulfide, were not mobile, were indole-negative and produced urease. Following the identification by API 20 E system, based on biochemical characteristics outlined in the tested strains, all E. coli strains were type 1 and those of Klebsiella pneumoniae were subsp. pneumoniae. Regarding the antimicrobial susceptibility of tested E. coli strains the highest sensitivity (80.0%) was noted to enrofloxacin and gentamicin, followed by sensitivity to florfenicol (60.0%) and cephalotin (20.0%). Any E. coli strain showed sensitivity to erythromycin, tetracycline and ampicillin. The resistance of tested E. coli strains was noted in large amounts to tetracycline and erythromycin (80.0%), ampicillin (60.0%) and cephalotin (40.0%). Any strain of E. coli showed resistance to gentamycin, enrofloxacin and florfenicol. Chapter 10 Mycotic mastitis reflects aspect of mastitis caused by two types of fungi identified in this study, namely Candida spp. and Aspergillus fumigatus. Of the 216 strains of microorganisms isolated in this study, fungi were noted with the frequency of 7.9% (17 strains), of which 1.4% Candida spp. (3 strains), 0.9% A. fumigatus (2 strains) and 5.6% other unidentified fungi (12 strains). The proportion of fungi isolated from studied mastitis cases was higher than in other studies, most likely due to uncontrolled antibiotic treatments applied in studied farms of dairy cattle. Of the 17 fungal strains isolated from examined mastitic milk samples, the frequency of fungi belonging to the genus Candida (17.6%) was higher compared with the species A. fumigatus (11.8%). Strains of Candida spp. and A. fumigatus were isolated from mono- and polymicrobial mastitis cases, the polymicrobial mastitis being represented by mixed association with bacterial species S. aureus for both fungi. All Candida spp. strains were isolated from clinical mastitis cases with acute evolution and the clinical mastitis from that has been isolated A. fumigatus had chronic evolution, one strain of A. fumigatus being isolated from a subclinical mastitis. In case of the identified fungi, both mycological culture medium used (Sabouraud dextrose agar - Oxoid and Sabouraud agar with glucose 2%), added with 0.05% gentamicin, have proved to be effective for rapid isolation both of Candida spp. and A. fumigatus strains from mastitic milk samples. By examining Gram-stained smears from cultures of Candida spp. spores were observed (blastospores/blastoconidies), that are characteristic of the budding stage, without being observed the filamentous form of the yeast (pseudohyphae and true hyphae). Observing overall epidemiological situation on dairy farms in the study, the probable causes of contagious mastitis (caused by S. aureus and Str. agalactiae) were sometimes common with those of environmental mastitis (caused by environmental streptococci and coliform germs), being represented mainly by inadequate management of the studied farms, refering to the shelters, dairy cows breeding and to milking system. Regarding the main sources and transmission mode, in the two types of mastitis (contagious and environmental) those ones could be different. One example is that S. aureus and Str. agalactiae were more frequent isolated in those farms where it was mainly observed inadequate milking hygiene, encouraging the spread of microorganism from one animal to another, particularly during milking, while environmental streptococci and coliform germs were more frequently isolated from the farms where inadequate hygiene conditions of livestock were mainly observed, the animals were overcrowded, dejections were not evacuated, there were inadequate water sources, all of these favoring the infection from the environment of the mammary gland during rest periods of the animals. In this study, diagnosis with a relatively high frequency of mycotic mastitis can be considered the consequence of the presence and development of a large number of mastitis with different etiologies in the dairy farms, mastitis that have been uncontrolled and excessive treated with antimicrobials, in many cases, without an advice from a laboratory concerning the antibiotic susceptibility of etiological agents. XI

12 Making an overall assessment of the etiological agents isolated from mastitis in dairy cows during the observation periods, we can enumerate the potential pathogens, in descending order of their isolation frequency in the studied herds, as follows: S. aureus (54 strains %), Str. agalactiae (46 strains %), CNS (44 strains %), Enterococcus spp. (14 strains - 6.5%), Corynebacterium spp. (12 strain - 5.6%), "other fungi" (12 strains - 5.6%), Str. dysgalactiae (9 strains - 4.2%), "other coliforms" (8 strains - 3.7%), E. coli (5 strains - 2.3%), K. pneumoniae (3 strains - 1.4%), Candida spp. (3 strains - 1.4%), Aspergillus fumigatus (2 strains - 0.9%), S. intermedius (2 strains - 0.9%), S. hycus coagulase-positive (one strain - 0.5%), M. haemolytica (one strain - 0.5%). XII