Veterinary Research Forum

Abstract This study, aimed to investigate Chlamydia abortus, C. pecorum, and C. psittaci in bovine, sheep, and goat abortions sent to the Konya Veterinary Control Institute diagnostic laboratory from various provinces in Central Anatolia and Mediterranean regions of Turkey between 2019 and 2022 via pathological and molecular methods. For this purpose, a total number of 150 abortions (from 58 cattle, 73 sheep, and 19 goats) were analysed via quantitative real-time polymerase chain reaction, histopathology and immunohistochemistry. The chlamydial DNA positivity rate was determined to be 24.00% via quantitative polymerase chain reaction analyses. According to the animal species, the positivity rate was 45.20% in sheep and 15.78% in goats. No cases positive for chlamydial DNA were found in the cattle abortions analyzed in the present study. When chlamydial agents were analysed on a species basis, C. abortus and C. pecorum were detected in 22.66and 1.33% of the cases, respectively. Among the animal species, 42.47% were C. abortus positive, whereas, 2.74% were C. pecorum positive in sheep. In the present study, the percentage of C. abortus-positive goats was 15.78%. and 22.66% of the Chlamydia spp. were positive according to immunohistochemical staining of aborted fetal lung, liver, heart, placenta and umbilical cord samples. This comparative study revealed that C. abortus was the most common chlamydial abortion agent in Türkiye. Chlamydial abortion agents cause abortion mostly in sheep in the region according to the animal species, and immunohistochemical staining contributes to the diagnosis of the agent at the Chlamydia spp. level.

Abstract The Q fever is a zoonotic bacterial infection caused by an obligate intra-cellular bacterium, Coxiella burnetii. Members of the Canidae family (Mammalia), including dogs and foxes, are potential reservoirs of C. burnetii, which has a wide host range from mammals and birds to arthropods (primarily ticks). Infected dogs can transmit the disease to other animals and humans. This study aimed to investigate the presence of C. burnetii in dogs and ticks collected from infested dogs in the Kars, Ardahan, and Iğdir provinces of Türkiye by serological and molecular methods. Three hundred canine serum samples were analyzed for phase I and phase II C. burnetii antibodies using indirect enzyme-linked immunosorbent assay. Whole blood samples (n = 300) from the dogs sampled for sera and 184 ticks randomly collected from these dogs were also analyzed for C. burnetii with touch-down polymerase chain reaction. The ticks were classified according to the taxonomic characteristics. In result, 107 tick DNA samples collected from individual females and pooled males were evaluated. The C. burnetii was detected in 3.73% (of the tick samples. However, C. burnetii was not detected in any of the canine blood samples by polymerase chain reaction. Out of the 300 dogs, 18.33% presented antibodies against C. burnetii in their blood serum. When assessed for location, C. burnetii seropositivity was found to be significantly high especially in the Northeastern Anatolia region (18.33%). Study data highlighted the zoonotic risk of ticks, demonstrating that ticks on dogs can carry C. burnetii.

Abstract Clostridial disease causes severe economic losses in livestock by rapidly killing ruminants. Therefore, implementing effective control approaches to prevent this fatal disease is importance. The causative agent of this disease is Clostridium spp. Accurate identification of this microorganism is crucial for effectively managing clostridial diseases in farm. There are conventional methods for detecting the disease, including microbiological and biochemical tests, many of these tests are time-consuming and exhibit low sensitivity. So, this study aims to use conventional and molecular approaches to identify Iranian isolates associated with animal infections. To achieve this, 61 samples were collected from1984 to 2024 and cultured on liver media, and subsequently subjected to microbiological and biochemical tests. For molecular identification, the DNA of isolates was extracted, and the isolates were confirmed by polymerase chain reaction (PCR) using specific primers. The results of the conventional analysis revealed that all Iranian isolates were identified as C. perfringens and its type determined by PCR assay. According to our findings, C. perfringens type A is the most prevalent strain in Iran, which predominantly found in ostriches and bird samples, followed by type D. This study underscores the presence of C. perfringens types across variety hosts and geographic locations in Iran. In conclusion, the combining conventional methods with PCR helps reliably detecting Clostridium spp. This information holds the potential to significantly contribute to the development of preventive strategies against clostridial diseases in Iran.

Abstract Tropical or Mediterranean theileriosis in dairy cattle is widely distributed in many tropical regions of the world. The purpose of this study was to evaluate the proliferation status of mononuclear cells infected with Theileria annulata schizonts in different tissues and its relationship with the pathogenesis of the parasite in cattle by histopathology, immuno-histochemistry and polymerase chain reaction (PCR). Blood and tissue samples of eight Holstein cattle that had been lost due to theileriosis and eight healthy slaughtered cattle of the same breed were collected as a control group after necropsy. The piroplasms in the blood smears and the schizonts in the cytoplasm of the lymphocytes and macrophages of the lymph nodes were microscopically detected. Histopathologically, the proliferation of macrophages, lymphocytes, and plasma cells in lymph nodes and the heart, congestion, and bleeding in the red pulp of the spleen, portal tracts of the liver, interstitial tissue of the kidneys, multifocal necrosis and ulceration in the abomasum together with hyperemia and hemorrhages and lymphoblastic infiltration in the submucosa and lamina propria adjacent to these lesions and emphysema with ecchymotic hemorrhage in the lungs were evident. Immunohistochemistry identified the proliferated cells as mostly Cluster of Differentiation 3- Positive T lymphocytes and macrophage marker antibody 387- positive macrophages. Positive results of PCR for the Tams1 30.00 kDa gene were observed in lymph nodes, liver, lung and abomasum. It was concluded that the pathological changes were the result of schizont-infected macrophage proliferation leading to severe uncontrolled proliferation of uninfected T lymphocytes.

Abstract Salmonellosis is one of the most important bacterial diseases in human and animals. Rapid diagnosis and sub sequence accurate treatment of Salmonella carriers help reduce the salmonellosis in human and livestock animals. In this study, 420 fecal samples were taken during year 2019 from buffalo in the Urmia, Khoy and Piranshahr regions in west Azerbaijan province, Iran. Samplings were carried out in different seasons. Presence of Salmonella invasion genes (FimA, Stn and InvA) were evaluated by polymerase chain reaction. The bacterial culture and biochemical tests were performed on feces samples for isolation of bacterium Salmonella; however, all samples were negative in culture method. PCR findings showed that, 50 (11.90%) fecal samples were positive to the genes. The analysis of results showed that frequency of salmonellosis outbreak in different parts of west Azerbaijan province followed a similar pattern and the incidence of salmonellosis according to forecast in the warm seasons (spring and summer) was more than in cold seasons (autumn and winter). The prevalence of Salmonella in buffalo’s feces based on warm and cold seasons were 32 (64.00%) and 18 (36.00%), respectively. The results showed significant difference between cold and warm season in the prevalence of salmonellosis. Therefore, the application of molecular technics is essential for the prevention and treatment of salmonellosis. The results also showed that specificity of PCR method was better than culture method for detection of Salmonella in feces sample.

Abstract Brucellosis is a costly contagious disease of human, domestic and wild animals. It is a serious health problem in Iran causing significant economic losses therefore, control approaches to prevent its spread are of great importance. In Iran, the species and biovars of virulent Brucella species are still under-reported due to the inadequate diagnostic protocols and insufficient laboratory facilities. The objective of this study was to characterize Brucella isolates obtained from passive animal and human surveillance in Iran from 2011 to 2018 in order to understand the current epidemiological situation of the disease. A total of 419 samples (milk, blood, cerebrospinal fluid, abomasum content and aborted fetus tissues) were collected from 65 cases/case series (human and animals) and examined bacteriologically. The initially identified Brucella isolates were further characterized using phenotypic and molecular approaches. All recovered isolates were either B. abortus or B. melitensis. The infection in sheep appeared to be exclusively associated with B. melitensis, but both B. abortus and B. melitensis were common in bovine samples. Samples from one sheep and one goat were confirmed to be infected by the B. melitensis vaccine strain Rev1. In spite of B. abortus burden in animals (14 cases in cattle and camel), brucellosis in human was predominantly associated with B. melitensis (15 cases). The results confirmed that B. melitensis biovar 1 and B. abortus biovar 3 remain the most prevalent biovars in Iran. This report builds a picture of the significance of different Brucella species in different hosts in Iran and provides applicable information for the healthcare professionals about the public health risks of brucellosis and relevant preventive strategies.

Abstract Genus Brachyspira,as Gram negative anaerobic bacteria, colonize in dogs intestine. The aim of the current study was to determine the prevalence of Brachyspira spp. for the first time in Iran and rapid identification of Brachyspira spp. in dogs by a new designment of a species-specific primer set for B. canis. One hundred fifty-one fecal samples were obtained from dogs by rectal swab. Twenty dogs suffered from diarrhea and 131 of them were healthy. In 9.27% (14/151) of samples, spirochaetes were detected on primary cultures by weak hemolysis and positive Gram staining and then Brachyspira genus was confirmed by NADH oxidase (nox) gene via polymerase chain reaction. Among 14 isolates, twelve isolates were B. canis, one isolate was B. intermedia and another one was non-typeable. From 12 B. canis, only eight isolates were detected by designed specific primers. Ten Brachyspira spp. were isolated from dogs ≤ 1 year old (10/67, 14.92%) and 4 isolates were from > 1 year old dogs (4/84, 4.76%). The isolation rates from healthy and diarrheic dogs were (12/131, 9.16%) and (2/20, 10.00%), respectively. A statistically significant association was observed between the presence of Brachyspira spp. and the age under one year. Based on our findings, the nox gene in B. canis might have more sequence variability compared to other Brachyspira spp.

Abstract In the present study, Mycobacterium avium subsp. paratuberculosis (MAP) was investigated in goats slaughtered in Shiraz abattoir using histopathological examinations and polymerase chain reaction (PCR). Ilium and mesenteric lymph node samples from 66 suspected goat carcasses to Johne’s disease were collected. Among 66 examined slaughtered goats, nine (13.63%) goats were positive for MAP in both histopathological and PCR examinations. Eight goats were positive in PCR method while no lesion related to Johne’s disease was observed in their histopathological sections. All positive goats in histopathological examination were also positive in PCR. Based on the results of PCR, the detection rate of MAP in Shiraz abattoir was 25.80% (17 goats). According to the present findings, although both histopathological and PCR methods are appropriate for detecting Johne’s disease, PCR is more sensitive than histopathological examination.

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 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 Bovine venereal campylobacteriosis, caused by Campylobacter fetus subsp. venerealis (Cfv), is regarded as one of the major threats to the cattle industry around the world. Abortion and infertility are two important reproductive problems in cows infected with C. fetus subsp. venerealis. Reports on the presence of Cfv are scarce in the cattle, in Iran. Therefore, the present study was designed to examine the presence of Cfv in the reproductive tract of dairy cattle either slaughtered in Shiraz abattoir or dairy herds with a history of infertility and abortion, and further to identify and differentiate this micro-organism in dairy cattle in Fars, south of Iran. A total of 95 smegma samples from the preputial cavity and the fornix of the cervical opening were collected using scraping method from bulls (n = 34) and cows (n = 61) in addition to eight samples of commercially bull frozen semen. Smegma samples were then cultured for isolation of Cfv and then the extracted DNA was examined for the presence of Cfv using an optimized multiplex PCR assay. None of the frozen semen samples examined were positive for Cfv. However, out of 95 smegma samples, thirteen animals (12.6%) were found positive for Cfv consisting of 3 males and 10 females. In conclusion, the results of the current study clearly confirmed the presence of Cfv using PCR in the slaughtered cattle and dairy farms with a history of poor fertility and abortion in Fars, Iran.

Abstract The Booroola fecundity gene (FecB) and growth differentiation factor 9 (GDF9) gene belong to the transforming growth factor β (TGF-β) superfamily. The mutations of these genes have additive effects on the prolificacy in sheep. The aim of the present study was to determine the possible mutations of FecB and FecG^H genes in Lory sheep breed of the Lorestan province, Iran. Sixty blood samples were collected and DNA was extracted from whole fresh blood. For detection of FecB and FecG^H mutations, the PCR products were incubated with AvaII and DdeI restricted enzymes. Based on the results we did not find the FecB and FecG^H mutations in this sheep breed population, so these mutations cannot the cause of the high prolificacy of Lory sheep breed and more study are needed to determine the genetic or environmental causes of high prolificacy of this sheep breed.

Abstract Torque teno virus (TTV) is prevalent worldwide and has been extensively studied in human and some wild and domestic animals. As the studies on TTV in chickens was rare and there was no information about the infection of domestic village chickens with TTV and also structural resemblance of this virus to chicken anemia virus, the frequency of the infection in domestic village chickens in different villages in Isfahan (Iran) was investigated. Sera were collected from 50 chickens. Viral DNA was extracted and subjected to polymerase chain reaction (PCR) using the previously described T801 and T935 primers that were used for amplification of a highly conserved non-coding region (UTR) of the viral genome in a single round of PCR and Set B primers of conserved region in a nested PCR reaction. Using T801 and T835 primers TTV or viruses of TTV family were detected in 16 out of 50 sera tested (32%). Fourteen out of the same 50 sera (28%) were positive for TTV using Set B primers. Totally 20 sera were positive using both primers (40%). Ten sera were detected with both sets of primers, six sera with T801 and T935 primers and only four sera were positive using Set B primers for TTV. Different patterns of the detection of the virus with the two different sets of primers suggests the possibility of the presence of different genotypes of TTV in domestic village chickens and the possibility of the transmission of the virus from human to village chickens and vice versa. This necessitates further investigations.

Abstract Bovine Leukemia ProVirus: Evidence of Presence of Part of Gag Gene in Seminal Plasma of Naturally Infected Bulls It is of critical importance to understand the modalities of BLV presence in semen, especially with regard to artificial insemination (AI). Presence of bovine leukemia provirus was demonstrated in fresh and frozen semen samples by researchers. In this study paired blood and semen samples from 45 bulls were assessed for the presence of part of gag gene and antibodies to BLV in blood, semen and cell-free fraction of the semen (seminal plasma). Proviral DNA was detected in 5 out of 45 seminal plasma samples. PCR products were sequenced and submitted to gene bank. This data strongly suggested that seminal plasma of seropositive bulls can be positive in PCR.