ISOLATION, IDENTIFICATION AND ANTIMICROBIAL SUSCEPTIBILITY TESTING OF SALMONELLA FROM SELECTED POULTRY FARMS IN DEBRE ZEIT

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1 ISOLATION, IDENTIFICATION AND ANTIMICROBIAL SUSCEPTIBILITY TESTING OF SALMONELLA FROM SELECTED POULTRY FARMS IN DEBRE ZEIT Elsabeth Solomon 1,Hailelule Aleme 2,Habtamu Tassew 1, Kassaw Amssalu 1, Filimon Mitiku 2, Haile Alemayehu 2 1. College of Veterinary Medicine, Mekelle University, Mekelle, Ethiopia 2. Dilla University College of Agriculture and Natural Resources, Department of Animal and range sciences, Dilla, Ethiopia 2. Aklilu Lemma Institute of Pathobiology, Addis Ababa University, Addis Ababa, Ethiopia ABSTRACT A cross sectional study was conducted from December 2013 to May 2014 with the objectives to isolate, identify and determine the antimicrobial resistance pattern of salmonella species from selected poultry farms in Debre zeit, Ethiopia. From a total of 196 cloacal swabs collected 50 (25.5%) were found to be positive for Salmonella organisms using culture method, and 15 (7.6%) Salmonella isolates were confirmed using biochemical tests. All culture and biochemical positive samples were further confirmed by Polymerase Chain Reaction (PCR) through amplification of histidine transport operon as a target gene for the presence of salmonella isolates. From culture and biochemical positive samples, gel electrophoresis of the PCR product revealed the presence of 496bp segments in 13 (6.7%) Salmonella isolates. The statistical analysis has revealed a significantly association between different age groups of chickens (X 2 = 10.56; P = 0.005) and farms (X 2 =10.74; P=0.013) with the percentage of Salmonella isolates. Most of the Salmonella isolates were found to be resistant against commonly used antimicrobials such as Sulfisoxazole, Chloramphenicol and Ampicillin followed by Tetracycline, Amoxicillin/Clavulanic acid and Cephalotin and more than half(69.3%) of the isolates were found to be multi-drug resistant. The high prevalence of resistance to antimicrobial agents found in this study might be attributed to uncontrolled use of antimicrobial agents as growth promoters in poultry farms. Therefore, proper treatment of chickens using appropriate antibiotics is then quite essential. Key words: Antimicrobials; Isolation; PCR; Poultry; Salmonella 1

2 1. INTRODUCTION Animal production in general and chicken production in particular plays important socioeconomic roles in developing countries. The importance of poultry production in the national economy of developing countries and its role in improving the nutritional status and incomes of many small scale farmers and womens has been recognized by various scholars and rural development agencies for the last few decades (Melesse et al., 2005; Moges et al., 2010; Melesse et al., 2011). Infectious diseases of various etiology and management problems are among the major problems in commercial poultry production. (Labago et al., 2003; Namata et al., 2008). Newcastle disease, Marek s disease and infectious bursal disease are the most devastating diseases of chickens (Lobago and Woldemeskel 2004 ; Zeleke et al., 2005). More over Bacterial disease like salmonellosis, mycoplasmosis, and collibacilosis causes stumbling block for commercial poultry production. Salmonellosis is a bacterial disease caused by Gram negative facultative rod shaped bacterium strains of Salmonella which is the family Enterobacteriaceae. Chickens are the natural hosts for the highly host adapted biovar S. gallinarum and S. pullorum, the causative agents of fowl typhoid and pullorum respectively. Fowl typhoid and pullorum disease are known as the major obstacle of poultry industry (Medina et al., 2013; kassaye et al., 2010; Chrysostome et al., 1995; Sato et al., 1997). Fowl typhoid is per acute, acute or chronic form of disease affecting mostly adult chickens, whereas pullorum disease affects the very young chickens of mostly 2 3 weeks of age. Confirmatory diagnosis depends on the bacteriological isolation and molecular characterization while serological tests can be used to detect the presence and estimate the prevalence of infection within a flock (OIE, 2008). Salmonellosis in poultry has been reported by various scholars from Ethiopia (Medina et al., 2013; Genet et al., 2014). As the poultry industry develops and become more intensified, the incidence of salmonellosis including pullorum disease and fowl typhoid become rising concern and bottleneck for the sector advancement (Genet et al., 2014). In addition to the direct economic impact of salmonellosis in poultry production, currently resistance of Salmonella to commonly used antimicrobials is rising concern both in the veterinary and public health sectors and has emerged as a global problem. There are reports of high prevalence of resistance in Salmonella isolates from countries such as Taiwan (Lauderdale et al., 2006), India (Mandal et al., 2004), The Netherlands (Duijkeren et al., 2003), France (Weill et al., 2006), Canada (Poppe et al., 2006), and Ethiopia (Molla et al, 2003). The increasing proportion of single and multiple antimicrobial-resistant Salmonella strains isolated from human salmonellosis cases has been associated with the widespread use of antimicrobial agents in food animal production. A considerable number of antimicrobials commonly used in the treatment of salmonellosis and other bacterial infections of humans are also used in veterinary practices (Wenger et al,. 1999; Gay et al,. 1994) This present a public health risk by the 2

3 transfer of resistant Salmonella and other zoonotic bacterial pathogens or the resistant genes from food animals to humans through consumption of contaminated food and food products. In the current study area both small scale and large scale commercial poultry production are flourishing. As the poultry industry further develops and become more intensified, the avian Salmonellosis can cause serous damage on the poultry industry and human health. Hence documentation of the prevalence of the disease with confirmatory diagnosis; culture and molecular methods will have paramount importance to design a control or prevention program and moreover the identification of antimicrobial resistant serotypes of Salmonella will help in instituting an appropriate therapy and provide information on the auxiliary emergence of drug resistance. Therefore, the objectives of this study were to isolate Salmonella rganism from cloacal swab of chicken using culture, biochemical and polymerase chain reaction (PCR) and to determine the antimicrobial susceptiblity of Salmonella organisms isolated from cloacal swab samples of chickens. 2. MATERIALS AND METHODS 2.1. Study Area The study was carried out between November 2013 and April 2014 in Debre zeit, Ethiopia. Debre zeit is found in Oromia regional state 47 km south east of Addis Ababa. It is located at 9 0 N and 40 0 E, with human population of about 95,000.Its altitude is about 1850 m above sea level. It experience bimodal patterns of rainfall with the main rainy season extending from June to September. A short rainy season occurs between march and May with an average rainfall of about 80mm.The mean annual minimum and maximum temperature are and c respectively with an overall average of (CSA,2008). Highest temperatures are recorded in May and it has 61.3%mean relative humidity. The isolation of Salmonella, identification and antimicrobial resistance determination was done at Aklilu Lema institute of pathobiology microbiology laboratory Study Design and Sample Size Cross sectional study was conducted to isolate, identify and determine antimicrobial susceptibility testing of salmonella organisms from four selected poultry farms in Debre Zeit, Ethiopia. Farms were selected purposively based on willingness of the farm owners. A total of 196 chickens were sampled from four purposively selected commercial poultry farms. 2.3 Study animals and Sample collection The study animals were commercial chicken flocks that were purposively selected from four commercial poultry farms. A total of 196 Cloacal swab samples were collected from live chickens using sterile cotton tipped swabs moistened with buffered peptone water. The swabs were kept in properly plugged sterile test tubes and transported as soon as possible using icebox to Aklilu lema institute of pathobiology, microbiology laboratory. 3

4 2.4. Isolation and Identification of Salmonella The study was conducted utilizing the conventional methods for the detection of Salmonella following the standard guide lines given by ISO 6579: (2002). Cloacal swab samples pre-enriched in buffered peptone water were incubated for 24 hours at 37 0 C and the pre-enrichment broth after incubation was mixed and 0.1 ml of the broth was transferred into a tube containing 10 ml of Rappaport-Vassiliadis medium with soya (RVS broth). Another 1 ml of the pre-enrichment broth was transferred into a tube containing 10 ml of Muller-Kauffmann tetrathionate novobiocin broth (MKTTn broth). The inoculated RVS broth was incubated at 42 o C for 24 hours and the inoculated MKTTn broth at 37 o C for 24 hours. After incubation for 24 hours, a loop-full of material from the RVS broth and MKTTn was transferred and streaked separately onto the surface of Xylose lysine deoxycholate agar (XLD agar). The plates were incubated at 37 o C for 24 hours. The plates were incubated in an inverted position and after incubation; the plates were checked for growth of typical Salmonella colonies. Characteristic Salmonella colonies, having a slightly transparent zone of reddish Color and a black center, were sub-cultured onto the surface of XLD agar for purification. This step was repeated until the colonies in the plate showed morphological homogeneity. All suspected colonies were streaked on the surface of nutrient agar plates, in a manner which allowed well isolated colonies to develop. The inoculated plates were incubated at 37 o C for 24 hours. Thus the pure culture obtained was used for biochemical and molecular confirmation Biochemical Confirmation Biochemical identification of isolates were performed using Triple sugar iron agar (TSI), Christensen's urea agar, Simmon Citrate Agar, Lysine iron agar (LIA) and motility test was also done For separation of motile and non-motile Salmonella according to Cown (1985) Polymerase Chain Reaction (PCR) Few Salmonella colonies from each biochemical positive culture were taken for DNA isolation using the procedure described by Welson. (1990) and ultimately for further confirmation with PCR. DNA extracted from Salmonella culture was amplified by using primers specific to the gene encoding histidine transport operon. Oligonucleotide primers of 25bp (base pair) (FF: 5 -ACTGGCGTTATCCCTTTCTCTGGTG -3 ; Rev: 5 -ATGTTGTCCTGCCCCTGGTAAGAGA-3 ) were used to amplify a region of 496bp segment, histidine transport operon, of Salmonella (Rychlik and Rhoades, 1989). A PCR assay was performed with a final volume of 25µl mixture and the reaction was allowed to run for 30 sec at 94 0 C, 30 sec at 60 0 C, and 45 sec at 72 0 C for denaturation, annealing and extension, respectively for about 30 cycles in a Thermocycler. The PCR products then were electrophoresed on a 1% agarose gel containing Ethidium bromide and photographed. Positive results were indicated by the presence of a 496-bp band seen on the gel with an ultraviolet transilluminator. 4

5 2.5. Antimicrobial susceptibility testing Table 1: Antimicrobial drugs and concentrations used Antimicrobial disks Amikacin 30 Amoxicillin and clauvlonic acid 20/10(30) Ampicillin 10 Cefoxitin 30 Ceftriaxone 30 Cephalothin 30 Chloramphenicol 30 Ciprofloxacin 5 Gentamycin 10 Kanamycin 30 Naldixic acid 30 Neomycin 30 Nitrofurantoin 100 Streptomycin 10 Sulfisoxazole 1000 Strength in microgram Sulphamethoxazole and trimethopirim and 1.25 Tetracycline 30 Trimethoprim 5 The antimicrobial susceptibility testing was done by the agar disk diffusion method as described by Clinical and Laboratory Standards institute (CLSI, 2013). In brief, a 0.5 Mac-Farland standardized suspension of the bacteria was prepared in 0.8% sterile saline and swabbed over the entire surface of Mueller Hinton agar (Oxoid) with a sterile cotton swab. A ring of disks of each (Mast Diagnostics, UK) containing single concentrations of each antimicrobial agent was then placed onto the inoculated surface. After overnight incubation at 37 C, clear zones of inhibition was produced by bacterial growth and these was measured in mm using a straight line ruler. Reading of the diameter of the zone of inhibition was done by using an interpreting chart for zone sizes and interpreted as susceptible, intermediate, and resistant (CLSI, 2013). 5

6 2.5. Data Management and Analysis Different statistical models were employed to analyze the data collected using STATA version 11 software. Descriptive statistics was used to describe the frequency and percentage of the results. Chi-sequare test was used to check the association of potential risk factor with the occurrence of the organism. The association was taken as significant when p- value is less than 0.05 and not significant when p-value is greater than Antimicrobial susceptibility test result was analyzed using WHONET version RESULTS 3.1. Isolation and Identification of Salmonella Isolation and identification of Salmonella organisms were conducted on cloacal swab samples taken from chicken flocks using conventional culture methods, Biochemical analysis and PCR technique Culture and Biochemical Characteristics Typical Salmonella colonies with a slightly transparent zone of reddish color and a black center were observed on XLD agar medium in an overnight culture (Fig.1). From a total of 196 cloacal swabs collected, 50 (25.5%, 50/196) were found to be positive for Salmonella organisms by culture, and 15 (7.6%) Salmonella isolates were confirmed using biochemical tests (Table 2 and Fig.2). All Salmonella confirmed isolates were checked for their motility and have been found to be motile. Figure 1: Salmonella Culture on XLD agar medium Figure 2: Biochemical test results; A) TSI B) LIA C) Urease test D) Citrate 6

7 Table 2: Result Summary on Biochemical Characteristics of Salmonella Isolates Sample TSI LIA Citrate Urease Slant/Butt H 2 S Gas 1 K/A K/A K/A K/A K/A K/A K/A K/A K/A K/A K/A K/A K/A K/A K/A Polymerase Chain Reaction (PCR) All culture and biochemical positive samples were further confirmed by PCR through amplification of histidine transport operon as a target gene for the presence of Salmonella isolates. Gel electrophoresis of the PCR product on 1% agarose gel revealed the presence of 496bp segments in 13 (6.6%) Salmonella isolates out of 15 culture and biochemical positive samples (Fig.2). Figure 3: Polymerase chain reaction for detection of Genus Salmonella Lane M: 100bp ladder; Lane 1, 3-10, 12-15: Positive isolates (496bp); Lane 2 and 11: Negative; Lane 16: Negative control 7

8 3.3. Risk Factors and Existence of the Organism In this study, the occurrence of the organism has been significantly associated with different age groups of chickens (X 2 = 10.56; P = 0.005) (Table 3) and between farms (X 2 =10.74; P=0.013) (Table 4). The highest salmonella level was isolated from the 3 rd age category (Table 3) which is 40 weeks of age (12.5%) and no salmonella was isolated in first age category. The percentage of isolation of salmonella was increased with the advancement of age in this study. Among all different farms of cloacal swabs analyzed, Genesis and Elere farms revealed maximum isolation of Salmonella (Table 4) whereas Salmonella was not detected in Alema farm. Table 3: Salmonella isolates in different age groups of cloacal swab sample. PCR Age category Number of tested No of positive (%) No of Negative (%) 45 days 60 0 (0%) 60 (100%) 26 weeks 40 1 (2.5%) 39 (97.5%) 40 weeks (12.5%) 84(87.5%) Total (6.7%) 183(93.3%) X 2 = P=0.005 Table 4: Description of Salmonella in cloacal swab by their farm origin PCR Farm name Number of tested No positive (%) No Negative (%) Alema 60 0 (0%) 60 (100 %) Efrem 40 1(2.5%) 39 (97.5%) Elere 36 5(13.9%) 31(86.1%) Genesis 60 7(11.7%) 53(88.3%) Total (6.7%) 183(93.3%) X 2 =10.74 P=0.013 The present finding revealed, the association between different breeds and sex of chickens with the existence of the organism were not statistically significant (X 2 = 3.53; P= 0.06 and X 2 =2.61; P =0.106), respectively (Table 5 and 6). 8

9 Table 5: Description of Breed associated with Salmonella isolates PCR Breed type Number of tested No of positive (%) No of negative (%) Bovans 60 7 (11.7%) 53 (88.3%) Cobb (4.4%) 130 (95.6%) Total (6.7%) 183(93.3%) X 2 = 3.53 P= Table 6: Description of sex associated with Salmonella isolates. PCR Sex category Number of tested No of positive (%) No of negative (%) Female (8.8%) 114(91.2%) Male 71 2(2.8%) 69(97.2%) Total (6.7%) 183(93.3%) X 2 =2.61 P = Antimicrobial Susceptibility Test A total of 13 isolates (6.6%) were tested against 18 antimicrobial discs following CLSI guidelines. Salmonella isolates were found to be resistant to Sulfisoxazole, Chloramphenicol and Ampicillin and followed by Tetracycline, Amoxicillin/Clavulanic acid and Cephalotin antibiotics (Fig. 2). In the other way, all of the Salmonella isolates had been 100 % susceptible to Amikacin, Cefoxitin, Ceftriaxone, Gentamicin, Naldixic acid, Trimethoprim/Sulfamethoxazole, and Trimethoprim and were the only antimicrobials not resistance to any of the isolates tested (Table 7). Resistance to Kanamicin, Ciprofloxacin, Neomicin, and Nitrofurantion was not observed although the isolates had intermediate sensitivity to these antimicrobials. Figure 4: Percentages of antimicrobial resistance 9

10 Table 7: Level of antimicrobial resistance of Salmonella isolates Code Antibiotic name Breakpoint %R %I %S %R 95%C.I. AMC_ND20 Amoxicillin/Clavulanic acid AMK_ND30 Amikacin AMP_ND10 Ampicillin CEP_ND30 Cephalothin CHL_ND30 Chloramphenicol CIP_ND5 Ciprofloxacin CRO_ND30 Ceftriaxone FOX_ND30 Cefoxitin GEN_ND10 Gentamicin KAN_ND30 Kanamycin NAL_ND30 Nalidixic acid NEO_ND30 Neomycin NIT_ND100 Nitrofurantoin SOX_ND200 Sulfisoxazole STR_ND10 Streptomycin SXT_ND1.2 Trimethoprim/Sulfametho xazole TCY_ND30 Tetracycline TMP_ND5 Trimethoprim About 69.3% of Salmonella isolates were found to be multi-drug resistant as they were resistant to more than one antimicrobial (Table 8). Table 8: Distribution of multidrug resistance profile Resistance profile Number of isolates Isolates (%) AMC CEP CHL CIP AMC AMP CHL CIP AMC AMP CEP CHL AMC AMP CCEP CHL CIP Total

11 4. DISCUSSION Salmonella is the major cause of foodborne illness in poultry and its products, preliminary data on isolation and identification are needed to inform public health authorities about the antimicrobial resistance and magnitude of the problem and to monitor trends over time. Genus-specific detection of Salmonella has pronounced application to clinical situations in which detection of Salmonella would support or confirm a clinical diagnosis and would provide important information regarding treatment, control, and prevention. In the present study, the overall percentage of Salmonella isolates in the total examined cloacal swabs using conventional culture and PCR method was 6.632% which is higher as compared to the study by Kassaye et al. (2010) who reported 0.8% from cloacal swab samples in chickens and 2.6% by Mdegela et al. (2000). On the other way, significantly higher percentage of Salmonella isolates were reported by Yeliz et al. (2011), Alebachew and Mekonnen (2013) and Islam et al. (2006), who reported 34% from turkey, 41.9% from Jimma and 47.7% from Tehran respectively. This difference might be attributed to the variation of the test techniques employed (pre-enrichment steps), the origin of sample or geographical difference and difference in management practice. In this study, the ages of the poultries were grouped in three categories and tested for their association with the occurrence of the organism. The highest Salmonella level was isolated from the 3 rd age category, 40 weeks of age (12.5%) and no Salmonella was isolated in first age category, 45 days (0%). The percentage of isolation of Salmonella was increased with the advancement of age in this study. The statistical analysis has revealed a significantly associated different age groups of chickens (X 2 = 10.56; P = 0.005). This result is in agreement with the finding of Sikder et al. (2005) who reported the highest (30.76%) of Salmonella infection at 39 weeks of age and lowest (13.33%) at 32 weeks of age. The occurrence of the organism has been also significantly associated with different farms (X 2 =10.74; P=0.013). The significant difference in Salmonella isolates between farms might be due to the existence of different management system and feeding practice in different farms. The sex specific percentage of Salmonella isolates in the present study were found to be 8.0% in females and 2.0% in males and statistical analysis revealed there was no significant difference (X 2 =2.61; P =0.106). The current finding was in agreement with the study of Alebachew and Mekonnen (2013) in Jimma town who reported 42.2% in females and 39.1% in males, Sikder et al. (2005) who reported 40.5% and 41% prevalence of Salmonella among chicken flocks examined in Bangladesh. Kang et al. (2002) explained that there could not be any sexual impact on the prevalence of Salmonella infection in male and female poultry. Even though the statistical analysis result showed no significant association between sex groups, the slight difference in the percentage might be due to the fact that female chickens are physiologically stressed during egg production and molting which significantly depresses the immune response of female chickens and increase the susceptibility to Salmonella infection (Lynch et al., 2006). 11

12 Emerging drug resistance in the foodborne bacteria isolates is of great public health concern. Although most intestinal Salmonella infections don t require treatment, antimicrobials may be life-saving in persons with immune suppressing conditions or invasive illness (Lynch et al., 2006). In studies performed worldwide, there is remarkable variation in resistance of Salmonella species from chickens to wide range of antimicrobial agents. In the present study, 6.63% of cloacal samples were found to be infected with Salmonella and more than half of the Salmonella isolates (84.7%) tested exhibited resistance to antimicrobials. The resistance against sulfisoxazole and chloramphenicol were found to be (69.2%) followed by Ampicillin (61.5%), tetracyclin and Amoxicillin/Clavulanic acid (30.8%) and cephalotin (23.1%). Molla et al. (2003) also reported (51.2%) of Salmonella strains were resistant to sulfisoxazole, (45.0%) to Amoxicillin/Clavulanic acid and ampicillin, (41.2%) to tetracyclin and (30.0%) to chloramphenicol in their study. High resistance to penicillin (100.0%) and (85.2%) ampicillin resistant Salmonella isolates were also reported by Yeliz et al. (2011). Resistance to tetracycline and cephalotin were also evident in Yeliz et al. (2011) and Zhao et al. (2005). In this study, Amikacin, Cefoxitine, Ceftriaxone, Gentamycin, Nladixic acid, trimethoprim/sulfamethoxazole, and trimethoprim showed maximum (100.0%) antimicrobial activity against Salmonella isolates. This result is in agreement with Molla et al. (2003) who reported all strains of Salmonella susceptible to antimicrobial effects of Naldixic acid, Amikacin, and Gentamycin. Sensitivity of Salmonella isolates to Gentamycin has also been reported by Cardso et al. (2006). Multidrug resistance has been reported in many isolates of Salmonella identified from poultry farms. In the present study, about 69.3% of Salmonella isolates were found to be multi-drug resistant as they were resistant to more than one antimicrobial. A relatively similar finding of multidrug resistant isolates has been reported by Bayleyegn et al. (2003), Mohammed et al. (2009) and Yeliz et al. (2011) as 63.7%, 23.5%, and 97.0 %, respectively. The presence and variation of multidrug resistance in Salmonella isolates may be associated with time, the serovar of Salmonella, broilers versus layer, one farm versus another and the particular antimicrobial agent (Yeliz et al,. 2011). 5. CONCLUSION In the present study, Most of the Salmonella isolates were found to be resistant against commonly used antimicrobials such as Sulfisoxazole, Chloramphenicol and Ampicillin followed by Tetracycline, Amoxicillin/Clavulanic acid and Cephalotin. More than half of the Salmonella isolates were found to be multi-drug resistant as they were resistant to more than one antimicrobial. The high prevalence of resistance to antimicrobial agents found in this study might be attributed to uncontrolled use of antimicrobial agents as growth promoters in poultry farms which leads to resistant strains and can be passed to human through food products. Therefore, proper treatment of chickens using appropriate antibiotics is then quite essential. 12

13 Competing Interests The author(s) declare that they have no competing interests. Author s Contributions Hailelule Aleme,Habtamu Tassew, Filimon Mitiku, Haile Alemayehu conceived of the study, and participated in its design and coordination and helped to drafted the manuscript. Elsabeth Solomon carried out the Laboratory work. Kassaw Amssalu participated in the design of the study and performed the statistical analysis. All authors read and approved the final manuscript. Author's Information On behalf of the authors, I take the full responsibility for the article during submission and peer review. Acknowledgement The authors would like to thank the Mekelle University College of Veterinary Medicine for Financial Support and the Aklilu Lemma Institute of Pathobiology, Addis Ababa University for unreserved assistance during data and sample collection and Laboratory work. REFERENCES Alebachew, k. and Mekonnen, A. (2013): A survey on Salmonella infection among chicken flocks in Jimma town, Ethiopia. W. Appli. Sci. J. 21 (10): Cardoso, M. O., Ribeiro, A. R., Santos, L. R., Pilotto, F., Moraes, H. L. S. and Salle, C. T. P. (2006): Antibiotic resistance in Salmonella Enteritidis isolated from broiler carcasses. Brazilian J.Microbiol. 37(3): Chrysostome, C. A. A.M., Bell, J.G., Demey F. and Verhulst, A. (1995): Sero prevalence to three diseases in village chickens in Benin. Prev. Vet. Med. 22: CLSI, (2013): Performance standards for antimicrobial susceptibility testing; Twenty- third informational supplement. M100-S 23. Cown, S.T. (1985): Cown and Steel's Manual for the Identification of Medical Bacteria. 2 nd ed. Cambridge University Press, Cambridge, UK. CSA, (2008): Central Statistical Agency: Report on monthly average retail prices of goods and services. Stat. Bulltn. Pp Duijkeren, E.V., Wannet, W.J.B., Houwers, D.J., and Pelt, W.V. (2003): Antimicrobial susceptibilities of Salmonella strains isolated from humans, cattle, pigs and chickens in The Netherlands from 1984 to J. Clin. Microbiol. 41: Gay, J.M., Rice, D.H. and Steiger, J.H. (1994): Prevalence of faecal Salmonella shedding by cull dairy cattle marketed in Washington State. J. Food Prot. 57: Tadele, G., Asrade, B., Bayleyegn, G, Ali, and M. S. (2014): Sero-prevalence of Fowl Typhoid and Pullorum Disease from Apparently Healthy Chickens in Eastern Ethiopia. J. Vetrinar. Sci. Technol. 5:

14 Islam, M.M., Haider, M.G., Chowdhury, E.H., Kamruzzaman, M. and Hossain, M.M. (2006): Seroprevalence and pathological study of Salmonella infections in layer chickens and isolation and identification of causal agents. Bangl. J. Vet. Med. 4: ISO, (2003): Microbiology of food and animal feeding stuffs-horizontal method for the detection and identification of Salmonella Part 3: Geneva, Switzerland. Kang, H., Srinivasan, J. and Curtiss, R. (2002): Immune response to recombinant pneumococcal PspA antigen delivered by live attenuated Salmonella Typhimurium serovar vaccine. J. Infect. Immun. 4: Kassaye, A., Lencho, T. and Mesele, A. (2010): Prevalence of Salmonella infection in intensive poultry farms in Hawassa and isolation of Salmonella species from sick and dead chickens. Ethio. Vet. J. 14(2): Lauderdale, T.L., Aarestrup, F.M., Chen, P.C., Lai, J.F., Wang, H.Y., Shiau, Y.R., Huang, I.W. and Hung, C.L. (2006): Multidrug resistance among different serotypes of clinical Salmonella isolates in Taiwan. Diagno. Microbiol. Infect. Dis. 55: Lobago, F. and Weldemeskel, M. (2004): An outbreak of Marek s disease in central Ethiopia. Trop. Anim. Hlth. and Prod. 36: Lobago, F., Nigussie, D., Wossene, A. and Ashenafi, H. (2003): Study on the majour diseases of chickens in Debre Zeit, Central Ethiopia. Bullet. Anim. Hlth. Prod. Afr. 51: Lynch, M., Painter, J., Woodruff, R., Braden, C. and Centers for disease control and prevention. (2006): Surveillance for foodborne-disease outbreaks United States, MMWR Surveill Summ. 55(10): Mandal, S., Mandal, M.D. and Pal, N.K. (2004): Reduced minimum inhibitory concentration of chloramphenicol for Salmonella enterica serovar Typhi. Ind. J.Med. Sci. 58: Mdegela, R.H., Yongolo, M.G.S., Minga, U.M. and Olsen, J.E. (2000): Molecular epidemiology of Salmonella gallinarum in chickens in Tanzania. Avian Pathol. 29: Medina, E., Fanos T., Mesula G., Teferi D. and Tariku J. (2013): Sero and media culture prevalence of Salmonellosis in local and exotic chicken, Debre Zeit, Ethiopia. African J. Microbiol. Res. 7(12): Melesse, A., Maak, S. and vonlengerken, G. (2005): The performance of naked neck and their F1 crosses with Lohmann White and New Hampshire chicken breeds under long-term heat stress conditions. Ethio. J. Anim. Prod. 5 (1): Melesse, A., Tiruneh, W. and Negesse, T. (2011): Effects of feeding Moringa stenopetala leaf meal on nutrient intake and growth performance of Rhode Island Red chicks under tropical climate. Trop. Subtrop. Agroecosyst. 14: Moges, F., Melesse, A. and Dessie, T. (2010): Assessment of village chicken production system and evaluation of the productive and reproductive performance of local chicken ecotype in Bure district, North West Ethiopia. Afr. J. Agric. Res. 5(13): Mohammad, M.S.D., Mahnaz, T., Latif G., Shabnam M., Mariam, S., Rounak B., Mohammad K.S.Y. and Mohammad R.Z. (2009): Characterization of antibiotic resistant patterns of salmonella serotypes isolated from beef and chicken sample in Tehran. Jundi. J. Microbiol. 2(4): Molla, B., Mesfin. A. and Alemayehu, D. (2003): Multiple antimocrobial-resistant Salmonella serotypes isolated from chicken carcass and giblets in Debre Zeit and Adis Ababa, Ethiopia. Ethio. J. Hlth Develop. 17 (2): Nwanta, J. A., Egege, S.C., Alli-Balogun, J. K. and. Ezema, W. S. (2008): Evaluation of prevalence and seasonality of Newcastle disease in chicken in Kaduna, Nigeria. World. Pltr. Sci. J. 64: OIE, (2008): Fowl typhoid and pullorum disease. In: Terrestrial Manual. Office International des Epizooties (OIE), Paris, France. Pp Poppe, C., Martin, L., Muckle, A., Archambault, M., McEwen, S. and Weir, E. (2006): Characterization of antimicrobial resistance of Salmonella Newport isolated from animals, the environment, and animal food products in Canada. Canad. J. Vet. Res. 70: Rychlik, W. and Rhoades, R.E. (1989): A computer program for choosing optimal oligonucleotides for filter hybridization, sequencing, and in vitro amplification of DNA. Nucleic Acids Res. 17: Sato, Y., Sato, G., Tuchili, L., Pandey, G.S., Nakajima, A., Chimana, H. and Sinsungwe, H. (1997): Status of Salmonella Gallinarum-Pullorum infections in poultry in Zambia. Avian Dis. 41: Sikder A, Islam M, Rahman M. and Rahman M. (2005): Seroprevalence of Salmonella and Mycoplasma gallisepticum infection in the six model breeder chicken flocks farms at Patuakhali District in Bangladesh. Int. J. Chicken flocks Sci. 4(11):

15 Wegner H.C. and Aarestrup, F.M., Gerner-Smidt P. and Bager, F. (1999): Transfer of antibiotic resistant bacteria from animals to man. Acta. Vet. Scand. Suppl. 92: Weill, F.X., Guesnier, F., Guibert, V., Timinouni, M., Demartin, M., Polomack, L. and Grimont, P.A.D. (2006): Multidrug resistance in Salmonella enterica serotype Typhimurium from human in France ( ). J. Clin.Microbiol. 44: Yeliz, Y., Zafer, G., Sebnem, P. and Nurhan, E. (2011): Incidence and antibiotic resistance of Salmonella spp. on raw chicken carcasses. Food. Res. Inter. 44: Zeleke, A., Sorri, T., Gelaye, E. and Ayelet, G. (2005): Newcastle disease in village chickens in the southern and rift valley district in Ethiopia. Inter. J. pltr. Sci. 4(7): Zhao, S., Fedorka-Cray, P. J., Friedman, S., Mcdermott, P. F., Walker, R. D., Qaiyumi, S., et al. (2005): Characterization of Salmonella Typhimurium of animal origin obtained from the National Antimicrobial Resistance Monitoring System. Foodborne Pathog. Dis. 2: