Veterinary Research Forum

Abstract The purpose of this study was to elucidate some factors affecting the growth of Salmonella typhimurium. These factors included Carum copticum essential oil (0%, 0.015%, 0.03% and 0.06%), temperature (25 ˚C and 35 ˚C), pH (5, 6 and 7) and inoculum size (10³ and 10⁵
 CFU mL^-1). Brain heart infusion broth was used as the growth medium. There were 48 treatment combinations and the experiment was carried out in triplicate. Growth was monitored by visible turbidity over a 30 days period. A parametric survival model based on the log-normal distribution was used to estimate the most influential factors on the time to detection of Salmonella growth. According to our results, the selected factors significantly affected the growth of Salmonella typhimurium. Furthermore, the final graph demonstrated good agreement between the values predicted by predictive model and the results which were observed in this study. So that a parametric survival model can be a useful and practical tool to predict how the parameters will influence the bacterial growth.

Abstract Foods with animal origins play a substantial role in the transmission of Helicobacter pylori. The present investigation was carried out to study the vacA and cagA genotypes status of H. pylori isolated from various types of meat samples. Two hundred and twenty meat samples were collected and cultured. H. pylori-positive strains were analyzed for the presence of vacA and cagA genotypes. Eleven out of 220 (5.00%) samples were positive for H. pylori. Findings were confirmed by nested PCR. Prevalence of H. pylori in the meat samples of slaughterhouses and butcheries were 72.20% and 27.70%, respectively. The most commonly detected genotypes in the meat samples of slaughterhouses and butcheries were vacA m1a (66.66%) and vacA s1a (37.50%), respectively. The S1am1a was the most commonly detected genotype. Meat sampled from butcheries had the higher prevalence of H. pylori and its genotypes than those of slaughterhouses (p < 0.05). Results showed that meat samples could be the potential sources of virulent strains of H. pylori. Application of sanitary measures in the storage, transportation and sale of meat is essential for reducing the levels of H. pylori cross contamination.

Abstract Polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) and phylogenetic analysis were used for molecular identification of lactic acid bacteria (LABs) isolated from Apis mellifera. Eighteen honeybee workers were collected from three different apiaries in West Azerbaijan. LABs from the gut of honeybees were isolated and cultured using routine biochemical procedures. Genomic DNA was extracted from LABs and a fragment of 1540 bp in size of 16S rRNA gene was amplified. PCR products were digested using HinfI endonuclease and digested products with different RFLP patterns were subjected to nucleotide sequencing and phylogenetic analysis. The results revealed that Lactobacillus and Bifidobacteria spp. are were the most abundant LABs in honeybee gut. Phylogenetic analysis showed that both Lactobacillus and Bifidobacterium were closely clustered with high similarity percentage with the same bacteria isolated from honeybees’ gut elsewhere. It was concluded that LABs isolated from honeybees had low sequence divergence in comparison with LABs isolated from other sources such as dairy products.

Abstract The aim of this study was to determine antibiotic resistance genes, phylogenetic groups and anti-microbial resistance patterns of Escherichia coli isolates from fecal samples of healthy pet cats in Kerman city. Ninety E. coli isolates were recovered from obtained rectal swabs. Antibiotic resistance pattern of the isolates against seven selected antibiotic was determined using disc diffusion method. Phylogenetic background of the isolates was determined according to the presence of the chuA, yjaA and TspE4C2 markers. Theisolates were examined to determine a selection of antibiotic resistance genes including tetA, tetB, aadA, sulI and dhfrV by polymerase chain reaction. Forty two isolates (46.6%) were positive at least for one of the examined genes. Phylotyping revealed that the isolates are segregated in phylogenetic groups A (66.7%), B1 (1.2%), B2 (13.4%) and D (18.9%). Among 90 isolates, 26.6% were positive for tetB gene, 10.0% for cqnrS gene, 12.3% for sulI and aadA genes, 8.9% for tetA and 2.2% for dhfrVgene. None of the E. coli isolates were positive for qnrA and qnrB genes. Sixteen combination patterns of antibiotic resistance genes were identified which belonged to four phylogroups. Maximum and minimum resistant isolates were recorded against to tetracycline (82.3%) and gentamycin (1.2%), respectively. Fifteen antibiotic resistance patterns were determined in different phylo-genetic groups. In conclusion, feces of healthy pet cat in Kerman could be a source of antibiotic resistant E. coli isolates, whereas these isolates were distributed all over the main phylogroups.

Abstract Ornithobacterium rhinotracheale (ORT) is a bacterium associated with respiratory disease, growth retardation, decreased egg production and mortality in chickens and turkeys. The objective of this study was isolation, identification and evaluation of antimicrobial susceptibility of ORT bacterium in slaughtered broilers chicken flocks based on cultural and molecular tests in Khuzestan province, south-west of Iran. A total of 210 tracheal swab samples were collected from 21 broiler flocks slaughtered in abattoirs of the province. The results of cultural and biochemical tests showed that 23 (10.95%) isolates from tracheal swabs of 4 flocks (19.04%) were identified as ORT, but according to molecular characterization, 18 (8.57%) ORT isolates were positive in PCR assay and produced the predicted 784 bp amplification product. Finally, using the disk diffusion method, the drug resistance patterns of ORT isolates were determined against a panel of commonly used antimicrobial agents. Antimicrobial susceptibility test revealed that all isolates (100%) were sensitive to tetracycline, florfenicol and cephalexin. The highest antimicrobial resistance (89.00%) was seen for fosfomycin, sultrim and gentamicin. The results of present research showed that there was significant difference between the isolation rates of ORT from various areas of the province. As well, our findings indicated that the simultaneous use of both cultural and molecular techniques results in more comprehensive outcomes in the isolation and identification of the organismfrom understudy hosts.

Abstract Mycoplasma synoviae (MS) is a pathogen responsible for respiratory and locomotor disorders and causes major economic losses in poultry industry. Early and accurate diagnosis of MS infection plays a major role in control of the infection. This study was conducted to characterize Iranian field isolates of MS isolated from broiler chickens of West Azarbaijan province (Northwest of Iran), and differentiate them from vaccine strain MS-H. Two encoding genes, 16S rRNA and vlhA were employed. PCR results using primers related to 16s rRNA and vlhA genes were analyzed and compared. Out of 21 field samples, eight samples (38.0%) were positive using both sets of primers. Amplified products of vlhA gene were sequenced for MS strain identification. The results showed that Iranian field isolates of MS had high nucleotide and amino acid similarity. Iranian field isolates were distinct from vaccine strain MS-H. Results presented in this study showed that characterization of field isolates of MS by sequencing of vlhA gene and is beneficial for strain typing and differentiating them from vaccine strain. To our knowledge, this is the first study characterizing vlhA gene of MS isolates from broiler chickens in the West Azarbaijan province.

Abstract This study was conducted to reveal the phylogenetic background, to detect the genes encoding TEM, SHV and CTX-M-15 extended-spectrum β-lactamases (ESBL), and to analyze their distribution in phylo-groups of 150 Escherichia coli isolates from broiler chickens in Ahvaz (Southwest of Iran). Seventy- five cloacal swabs from healthy birds (fecal isolates), and 75 heart blood samples from birds with colibacillosis (septicemic isolates) were obtained. All isolates were phylotyped and screened for ESBL genes by polymerase chain reaction (PCR). The fecal isolates belonged to four main phylo-groups, including 41 isolates (54.67%) to A, 9 (12.00%) to B1, 5 (6.67%) to B2, and 20 (26.67%) to D. Of septicemic isolates, 37 isolates (49.33%) were classified as phylotype A, 5 (6.67%) as B1, 10 (13.33%) as B2, and 23 (30.67%) as D. In molecular analysis, a total of 72 isolates (35 fecal and 37 septicemic) were identified to harbor ESBL genes, which were distributed in phylo-groups A, B1, B2, and D. Regardless of the type of isolate, bla_CTX-M-15 gene was the most common genotype, followed by bla_TEM and bla_SHV genes. This study suggests that broiler chickens in Iran are infected to ESBL genes- harboring Escherichia coli strains which may be spread to the food chain through fecal contamination of carcasses during slaughtering.

Abstract Althoughpoultry meat is considered as the main source for human Campylobacter infections,there is limited information about non-poultry sources. The present study was aimed to investigate the prevalence and the antibiotic resistance of thermophilic Campylobacter spp. in fecal samples of the cattle and sheep in Shiraz, Iran. A total of 302fecal samples were obtained from clinically healthy, slaughtered cattle and sheep from Shiraz slaughterhouse. The animals were clinically healthy before being slaughtered. The samples were cultured according to the specific cultivation method under thermophilic conditions. The susceptibility of Campylobacter isolates were determined for 13 antimicrobial agents. All enriched samples and cultured isolates were targeted for polymerase chain reaction (PCR) detection of 16S rRNA and multiplex PCR for determining their species. Among 302 fecal samples, 65 (21.5%) and 205 (67.8%) samples were positive for the presence of Campylobacter species with the cultivation and PCR techniques, respectively. All 65 distinct isolates were susceptible to neomycin and colistin and the isolates showed high resistance to cephalotin (83.0%) and ciprofloxacin (67.7%). After the multiplex PCR, 78.5% of total positive samples showed the simultaneous presence of Campylobacter jejuni and Campylobacter coli. In conclusion, the results emphasized that non-poultry farms are important as a possible source of Campylobacter infections.

Abstract This study was conducted to investigate the prevalence of subclinical mastitis caused by Staphylococcus spp. in ewes in West-Azerbaijan province of Iran. Molecular characterization of isolated Staphylococcus spp. from diseased ewes were performed using polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) and DNA sequencing of glyceraldehyde-3-phosphate dehydrogenase (gap) gene. Also, antibiotic resistance of staphylococcal isolates against different antibiotics was investigated. A total number of 900 milk samples from 450 native ewes in their mid-lactation period were examined by the California mastitis test (CMT). The CMT positive samples were cultured and bacteria were isolated from 86 (9.50%) glands and 74 (16.40%) ewes. The prevalence of subclinical mastitis in the examined ewes was 16.40%. Microbiological analysis of milk samples revealed that 27 out of 74 sheep with subclinical mastitis were infected with Staphylococcus spp. Amplification of gap gene of 27 Staphylococcus isolates generated a single amplicon of 933 bp in size confirming that isolates were belonged to Staphylococcus genus. Digestion of PCR products by AluI endonuclease generated different RFLP patterns for each species. Nucleotide sequencing of gap gene followed by phylogenetic analysis showed that the most dominant Staphylococcusspecies were S. epidermidis, S. xylosus and S. chromogenes. Staphylococcal isolates showed the highest resistance to penicillin and ampicillin. In conclusion, Staphylococcus species, except for the southern parts of the province, play an important role in the development of subclinical mastitis in sheep in West-Azerbaijan province of Iran. Also, chloramphenicol, ciprofloxacin and neomycin are the most effective antibiotics for treatment of this disease.

Abstract Listeria monocytogenes is one of the major causes of infections in developing countries. In this study, chemical composition and anti-listerial effect of the essential oil of Zataria multiflora Boiss. alone and in combination with monolaurin were evaluated at different pH values (5, 6, and 7) and temperatures (5 ˚C and 30 ˚C). Chemical composition of Zataria multiflora Boiss. essential oil was evaluated by gas chromatography-mass spectrometry (GC-MS) analysis. Minimum inhibitory concentration (MIC) of the essential oil and monolaurin were determined using microbroth dilution method and the interactions of essential oil and monolaurinwere determined by the evaluation of fractional inhibitory concentrations (FIC) index.Carvacrol (63.20%) and thymol (15.10%) were found as the main components of the essential oil. The MIC values of the oil and monolaurin at pH 7 and 30 ˚C were measured as 312.50 µg mL^-1 and 125.00 µg mL^-1, respectively. Combination of monolaurin and Z. multiflora essential oil were found to act synergistically (FIC index < 0.5) against L. monocytogenes under different pH and temperature conditions. Decrease in the pH and temperature values have increased the anti-listerial activity of monolaurin and the essential oil. The lowest MIC value of monolaurin and essential oil was observed at pH 5 and 5 ˚C. According to our results, the oil alone or in combination with monolaurin at low pH and temperature conditions showed a promising inhibitory effect on L. monocytogenes.

Abstract Clostridium perfringens is an important cause of bacterial food poisoning worldwide. The disease is caused by C. perfringens enterotoxin (CPE) encoded by cpe gene. The aim of this research was to identify the different types of C. perfringens and the presence of cpe gene in isolated bacteria from broilers’ meat marketed in retail meat shops of Mashhad city in Northeastern of Iran. After isolation of C. perfringens using conventional culture method and confirmation by specific 16S rDNA gene, a multiplex polymerase chain reaction assay with specific primers, were performed for toxin typing of isolates. Clostridium perfringens was isolated from 31 broilers’ meat samples (15.50%) out of 200 samples and for toxin typing the results showed 9 isolates as type A (29.03%) and 22 isolates as type C (70.96%). In this study, cpe-positive C. perfringens were detected in eight isolates of type C (25.00%). Our results indicated that C. perfringens type C is the most common type in broiler chicken carcasses.

Abstract Staphylococcus aureus is generally regarded as a leading cause of mastitis in dairy cattle. The aim of this study was to investigate the pattern of agr groups and any possible relationship between agr groups and antibiotic resistance among S. aureus strains isolated from bovine mastitis in Northeast of Iran. For this purpose, a total of 300 bovine mastitic milk samples were taken from dairy industry farms of Khorasan Razavi Province, Iran. S. aureus were isolated and identified according to the standard methods. Antibiotic susceptibility testing was conducted by disk diffusion method. In this study a total of 31 isolates of S. aureus were evaluated for agrD gene polymorphism by specific primers. Most of the isolates belonged to agr group I (54.8%), followed by agr group III (25.8%) and agr group II (19.4%). There was not any isolates belonging to group IV. Resistance to methicillin in agr group I isolates was more than other groups. Agr groups II and III were quite susceptible to methicillin. Due to high prevalent of S. aureus isolates and high antibiotic resistance rate in bovine mastitic isolates, it is important to verify the characteristics of S. aureus strains in Iran.

Abstract The importance of cats in the Leptospira epidemiology is due to the possibility of transferring leptospirosis to wild and domesticated animals. The purpose of this survey was to determine the prevalence of Leptospira infection in shorthair cats in different location of Mashhad, Iran. Totally, 147 blood samples were taken from 42 (28.57%), 52 (35.37%) and 53 (36.05%) households, stray and cats which lived in industrial dairy cattle herds of Mashhad, Iran, respectively. Sera were tested with seven live Leptospira antigens using microscopic agglutination test (MAT). Sera with 50.00% agglutination at the dilution of ≥ 1/100 were considered as positive samples. Agglutination at dilutions of < 1/100 considered as suspected to Leptospira infection. Overall, 19 (12.92%) out of 147 cats showed reaction in MAT. The seroprevalence at a titer ≥ 1:100 and < 1:100 were 10 (6.80%) and 9 (6.12%), respectively. Serum samples showed positive reaction against Leptospira intterogans hardjo (no = 10; 52.63%), pomona (no = 5; 26.31%) and icterohaemorrhagiae (no = 4; 21.05%). Eight cats (42.10%) belong to dairy cattle herds had the most infection only by L. I. hardjo with 1:200 titer. There were no significant differences among the weight‚ age and sex of infected cats. However, there were significant differences between the infected cats in dairy cattle herds and the cats in the urban area (p < 0.05). It is concluded that cats can be infected by Leptospira spp. especially in commercial dairy cattle herds. Cats can be considered as a sanitation hazards in the area for this zoonotic disease.

Abstract Paratuberculosis (John’s disease) is infectious and chronically progressive granulomatous disease which affects domestic and wild ruminants. The causative agent is Mycobacterium avium paratuberculosis (MAP), a slow growing mycobactin dependent acid-fast bacillus. We investigated the detection and frequency of MAP in apparently healthy dromedary and Bactrian camels by insertion sequence 900 (IS900) polymerase chain reaction (PCR) and acid fast staining of fecal samples in Iran. Acid fast staining results showed that 6/50 (12.0%) samples of dromedary camels and 4/26 (15.3%) samples of Bactrian camels were suspected to MAP. Although the percentage of positivity for PCR assay of fecal dromedary camel was 8.0%, no bands corresponding to MAP detect in all samples of Bactrian camels. In conclusion, Although the incidence of MAP infection was low, further studies should be conducted to get more information on MAP infection in camel population, especially in areas where camels are close to other ruminants such as dairy cow, sheep and goat.

Abstract Clostridial myositis, considered to be rare in pet animals, is an acutely fatal toxaemic condition. Some species of clostridia are responsible for necrotic myositis. A 2-year-old male German shepherd dog was admitted with non-weight bearing lameness and massive swelling of the left hind limb. Clostridium perfringens type A with alpha toxin was diagnosed as a pathogenic agent. Based on the history, the bacteria were introduced inside the tissue via contaminated needle following intramuscular injection. Urgent medical therapy followed by surgical intervention was performed. The dog was discharged completely healthy after hospitalization for four weeks. The objective of this report was to describe necrotic myositis in a dog with an emphasis on clinical signs and treatment as well as bacteriological and molecular identification of the micro-organism. Because of the fatal entity of the disease, prompt diagnosis as well as proper and urgent treatment is very important for successful therapy.

Abstract Listeria monocytogenes can be found throughout the environment and in many foods. It is associated primarily with meat and animal products. Listeria monocytogenes has become increasingly important as a food-borne pathogen. The aim of this study was to evaluate the effect of microwave (MW) treatment of chicken meat samples which were inoculated with L. monocytogenes. Drumettes of broiler carcasses were soaked in fully growth of L. monocytogenes in Brain-Heart Infusion broth. The swab samples were taken from the inoculated samples, after various times of radiation (10, 20, 30, 40, 50, 60, 70 and 80 sec), using a domestic MW oven at full power. Following exposures, viable counts and surface temperaturemeasurements were performed. The bacterial counts were performed on Oxford agar. The results indicated that equal or longer than 60 sec exposures of chicken portions to MW heating which enhances the median surface temperature more than 74 ˚C could eliminate the superficial contamination of chicken meat with L. monocytogenes. Statistical analysis showed samples with equal or longer than 60 sec exposures to MW heating had significant decrease in population of inoculated bacteria compared with positive control group(p < 0.05). Pearson correlation showed a significant correlation between the bacterial population and temperature of samples due to MW exposure (p < 0.001, r = – 0.879 and r² = 0.773).