Veterinary Research Forum

Abstract Fungal contamination in drinking water has garnered considerable attention over the past few decades, especially considering the detrimental consequences of pathogenic fungal species on both human and animal health. The formation of biofilms by certain species is a considerable factor contributing to the emergence of severe fungal infections. This research was designed to isolate and identify fungi, particularly those capable of forming biofilms from 150 samples of drinking water sourced from various locations. The isolated fungal species were tested for them in vitro biofilm formation using a microtitration plate method and the crystal violet assay was applied to quantify the established biofilms. The effectiveness of three disinfectants, namely ozone, chlorine, and hydrogen peroxide, in preventing the formation of biofilms by the most isolated fungal species was monitored. The findings indicated that Aspergillus species were the most prevalent in drinking water, comprising 63.33% (95/150) of the total number of fungal species identified. Aspergillus fumigatus and Aspergillus flavus were identified as the primary contributors to biofilm formation in drinking water distribution systems with prevalence rates of 41.00 and 34.00%, respectively, among all Aspergillus species. The outcomes of the in vitro studies demonstrated that the ozone disinfectant exhibited promising results in inhibiting fungal biofilms compared to chlorine and hydrogen peroxide. In conclusion, these findings provided valuable insights for water distribution authorities to develop effective regimens for controlling biofilm-forming fungal species using suitable antifungal biofilm disinfectants.

Abstract Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis, an oncogenic deltaretrovirus that has emerged as a potential zoonotic infection. The BLV naturally infects cattle and causes economic losses through a slow persistent infection with various clinical symtoms following preleukosis. The main objective of this study was to determine the seroprevalence of BLV antibodies in cattle and buffaloes in the border provinces of the Eastern Anatolia region, Türkiye, using the agar gel immunodiffusion (AGID) assay and  enzyme-linked immunosorbent assay (ELISA). For this purpose, a total of 1,033 serum samples were collected from 982 cattle and 51 buffaloes from the provinces of Ağrı (n = 178), Iğdır (n = 252), Kars (n = 317), Van (n = 221), and Hakkari (n = 65) during 2021 - 2022. In AGID and ELISA tests, seropositivity for BLV-specific antibodies was not detected in cattle and buffaloes from the mentioned provinces. This study revealed that BLV was not circulating in cattle and buffaloes in the easternmost border provinces of Türkiye during the sampling period and contributed to determine the status of BLV in the mentioned region. Due to the presence of virus in other regions of Türkiye and neighboring countries, Iran and Iraq, it is recommended to control animal movements, continue efforts to combat the transmission of the virus, and maintain control measures.

Abstract Leptospirosis is a worldwide zoonotic disease caused by pathogenic Leptospira spp., often occurring in tropical and subtropical regions. Focusing on development of rapid diagnostic methods to facilitate early diagnosis and a universal vaccine are the main critical issues to overcome the burden of leptospirosis. Here, we have studied the immunogenic potential of prepared recombinant Loa22 protein (rLoa22) of local pathogenic Leptospira species in mice and its ability to induce humoral and cellular immunity, being further evaluated by analyzing the immunoglobulin G (IgG) subclasses and cytokines produced through immunization. Based on the results, mice immunized with rLoa22/adjuvant and a trivalent vaccine, induced high titers of total IgG. All immunized groups increased IgG1 almost on the same level; but, IgG2a level was significantly higher in the vaccine and rLoa22/adjuvant groups than rLoa22 alone group. Animals immunized with the vaccine produced more interleukin 4 than rLoa22/ adjuvant group. The results of evaluating interferon gamma level showed that the rLoa22/adjuvant and vaccine groups had a significant increase compared to the rLoa22 alone group. The results also demonstrated that the rLoa22 protein, in indirect enzyme-linked immunosorbent assay, was able to detect the anti-Leptospira antibodies in mice serum that can be used as a marker in assessing the seroprevalence of leptospirosis and/or in combination with other leptospiral antigens in development of an effective vaccine against leptospirosis.

Abstract Pasteurella multocida is a Gram-negative bacterium causing economically significant diseases in cattle. This study aimed to determine P. multocida susceptibility to different antibiotics and antibiotic alternatives. In this study, 246 samples (180 nasal swabs and 66 lung tissue specimens) were collected from cattle showing respiratory manifestations in Egypt. Suspected P. multocida colonies following culture were subjected to polymerase chain reaction (PCR) for molecular confirmation of the isolates. A multiplex PCR was employed to identify P. multocida capsular groups. Susceptibility of the isolated P. multocida to different antibiotics and nanoparticles as antibiotic alternatives including silver (AgNPs), chitosan (CNPs) and curcumin (CurNPs) were tested using broth microdilution method. Thirty-two P. multocida isolates were obtained, kmt1 gene was detected in these isolates, and molecular capsular types classification revealed that all isolates were belonged to the capsular type A. Based on broth microdilution method findings, 20 (62.50%) isolates were considered as multi-drug resistant (MDR); the isolates were most sensitive to danofloxacin and kanamycin, whereas they were most resistant to doxycycline and tilmicosin. Antibiotic alternatives showed high anti-microbial activity against tested isolates with minimum inhibitory concentrations ranging from 1.56 - 6.25 μg mL^-1, 156 - 625 μg mL^-1, and 128 - 512 μg mL^-1 for AgNPs, CNPs and CurNPs, respectively. Our finding demonstrated that MDR P. multocida was evident in cattle in Egypt. Although antibiotic alternatives showed promising in vitro anti-microbial effects against MDR isolates, additional studies are required to be actually applicable in veterinary practices.

Abstract The most serious problem in public health is salmonellosis, a common disease in horse. The aim of this study was to investigate the shedding of Salmonella serotypes in healthy Caspian pony. We examined 143 pony's fecal samples collected from the north of Iran belonging to different ages and sexes. Samples were cultured, then identification of isolates were performed by common bacteriological methods and polymerase chain reaction (PCR). The PCR was also used to explore the presence of fimA and salmonella secreted effector L (SseL) genes as virulence factors in the isolates and all were assigned to antibiotic susceptibility test via disc diffusion method. Results showed two fecal samples (1.39%) contaminated with Salmonella and further examination demonstrated the isolates belonging to S. enterica serotype typhimurium. Both serotypes were isolated from female and ˂6 years of age group of ponies and we detected fimA and SseL genes in the isolates. Observing multiple drug resistance and virulence genes in isolates is of utmost importance from both clinical and public health perspectives. It is highly likely that we face instances of salmonellosis in animals or humans that lead to severe infections and fail to respond to treatment in future. This study revealed that the occurrence of Salmonella was low in ponies, however, regarding the presence of virulence factors with multidrug resistant trend in this zoonotic bacterium, establishment of good hygienic measurement to prevent the transmission of bacteria between animal and human is necessary. 

Abstract The present study evaluated the Salmonella isolates obtained from various origins in Iran. Salmonella strains previously recovered and stored in the veterinary microbiology laboratory were serotyped and subjected to antibiotic susceptibility test, detection of the virulence and resistance genes by polymerase chain reaction (PCR), and genotyping by enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR). All Salmonella isolates showed resistance to erythromycin and the most resistance rates were detected for trimethoprim (86.66%), ampicillin (75.00%), and sulfamethoxazole-trimethoprim (63.33%), respectively. In total, 86.33% of the isolates were known as multi-drug resistant and none of the isolates showed resistance to cefepime, nalidixic acid, imipenem, ceftriaxone, and polymyxin B. The virulence genes, invA, sdiA, and hilA besides the tetA resistance gene were identified in all 60 Salmonella strains. The most prevalent resistance genes were respectively tetC (70.00%), sul2 (58.33%), and ereA (55.00%). Statistical analysis revealed a significant difference between Salmonella serotypes associated with the sul1 resistance gene. In ERIC-PCR analysis, 14 distinct clusters were obtained. Statistically, there were significant relationships between the source and ERIC’s genomic pattern and between the serotype of Salmonella isolates and genotypic pattern of ERIC. According to the results, Salmonella serotypes from non-human sources had considerable resistance to different antibiotics and carried significant virulence determinants and resistance genes. In addition, ERIC-PCR showed relevant results in discriminating Salmonella serotypes from other sources.

Abstract Bovine clinical mastitis is an economically important disease in dairy industry worldwide resulting in reduction of milk yield and quality. Among mycotic mastitis, Candida spp. are commonly occurring opportunistic mycosis in immunocompromised animals. The micro-organism’s causing mastitis has high zoonotic potential and has been linked with rapid growth and introduction of antimicrobial resistance between animals and humans. The present study was conducted to isolate and identify the common pathogenic Candida spp. from bovine mastitis cases in India. The isolates were phenotypically characterized by culturing on Sabouraud’s dextrose agar, Hichrome Candida differential agar and germ tube production test. Antibiogram was also performed to determine their antifungal activities. The phenotypically positive isolates were confirmed by polymerase chain reaction (PCR) and genetically analyzed by targeting 18S-ITS1-5.8S-ITS2-28S region specific for Candida spp. and identified the yeast at the species level. The antibiogram showed the isolates were highly sensitive with ketoconazole, clotrimazole and miconazole. The PCR assay identified C. lusitaniae and C. tropicalis based on the two distinctive amplicon sizes (592bp and 737bp) respectively. Also, the sequence analysis and phylogeny confirmed C. lusitaniae in six sequences and C. tropicalis in one sequence. It is worth noting that in this study, the species identification was consistent among PCR and genetic analysis. Therefore, the PCR based identification system of the fungal species performed in this study could be an efficient and time saving tool for early diagnosis of clinical mastitis in milch animal, which allows prompt control and application of speedy effective treatment.

Abstract This study aims to detect Escherichia coli which encodes beta-lactamase Cefotaxime (blaCTX-M) gene from the reproductive tract of Bali cattle with repeat breeder cases. This research was conducted from June to August 2021 using 16 Bali cattle with repeat breeder cases. The reproductive fluids were taken using a plastic sheet gun which was inserted into a Brain Heart Infusion medium, isolated in eosin-methylene blue agar (EMB) and identified using biochemical tests. Antibiotic susceptibility testing of E. coli was carried out using the disc diffusion method. Double-disk approximation test was used to screen the presence of E. coli which produces Extended-spectrum beta-lactamase. The polymerase chain reaction (PCR) method was used to detect the blaCTX-M gene of E. coli and sequences of the blaCTX-M gene were phylogenetically analyzed. The research results obtained three E. coli isolates from 16 reproductive tract fluids of Bali cattle. Antibiotic sensitivity tests showed that 100% of E. coli was resistant to penicillin G and oxytetracycline. 66.66% of E. coli was resistant to cefotaxime (CTX) and gentamicin, and 33.33% of E. coli was resistant to tetracycline. Escherichia coli isolates that were resistant to penicillin and CTX showed positive results in the double-disk approximation test. The results of E. coli detection using PCR showed that three E. coli isolates encoded the blaCTX-M gene located at 370 bp on gel electrophoresis. The results of the phylogenetic analysis showed that E. coli from the reproductive tract of Bali cattle was related to E. coli that encoded blaCTX-M-14 isolated from humans.

Abstract The bovine leukemia virus (BLV) is an important infectious agent transmitted from cattle to humans. It is considered one of the oncogenic viruses in breast cancer, so an accurate detection of this virus is important. The study aimed to design a specific and sensitive method based on TaqMan^® real-time polymerase chain reaction (RT-PCR) for BLV detection. Probes and primers were designed using bioinformatics software for a 108 pairs region of the BLV tax gene. Criteria employed for determining analytical sensitivity were prepared using in-vitro RNA transcriptions. The National Center for Biotechnology Information (NCBI), basic local alignment search tool (BLAST) databases various viral panels and genomic samples from healthy individuals (Qom Province, Iran in 2023) were used to verify analytical specificity and clinical specificity, respectively. This method can measure a minimum of 10 copies of DNA and RNA mL^-1. Moreover, the assay is linear in the range of 10⁰ - 10⁹ copies mL^-1. By testing negative specimens, the method specificity was 100%. The reproducibility results of the reaction were examined at the intra- and inter-assay comparison. In fact, 10 technical replicates of each concentration of the control sample were analyzed in each working reaction. Due to the locally made kit, exact sensitivity and specificity, rapid analysis, and relatively low cost, as compared to commercial kits of other countries, the method introduced in the present study could be suitable for accurate detection of the BLV. Also, the TaqMan^® real-time PCR method could be detected in cattle and human and before malignant changes of breast cancer which could reduce infection and breast cancer.

Abstract Methicillin-resistant Staphylococcus (MRS) bacteria, including methicillin-resistant S. aureus and methicillin-resistant S. pseudintermedius (MRSP), pose a significant threat in veterinary medicine, given their potential for zoonotic transmission and their implications for companion animals and humans’ health. This study aimed to assess the prevalence of MRS and anti-microbial resistance patterns at a university clinical hospital in Madrid, Spain. Samples were collected from both the environment and hospital staff at Veterinary Clinical Hospital of Alfonso X el Sabio University. Anti-microbial susceptibility assays, molecular detection of mecA gene and genetic relationships among the identified bacterial strains were performed. The study revealed an MRS prevalence of 1.50% in environmental samples, with MRSP accounting for 0.75% of the cases. Genetically related MRSP strains were found in different hospital areas. Among hospital staff, there was a MRS prevalence of 14.03%, including S. pseudintermedius and S. epidermidis strains. Antibiogram tests revealed multi-drug resistance among MRSP strains. Additionally, methicillin-resistant coagulase-negative staphylococci were isolated, suggesting potential cross-species transmission. This study underscores the presence of MRS in a veterinary clinical hospital, highlighting the significance of infection control through the implementation of protective measures, stringent hygiene practices among personnel and in the environment and responsible use of antibiotics. Further research is necessary to assess MRS incidence in animal patients and explore geographical variations, enhancing our understanding of MRS in veterinary medicine and addressing its challenges.

Abstract Caprine paratuberculosis (PTB) is a progressive, debilitating and production-limiting disease that causes significant economic losses and raises public health concerns. The goal was to study active surveillance and associated epidemiological risk factors of caprine PTB in selected district of Odisha, India. The 818 goats of various ages, sexes and breeds were randomly screened in ten different districts for a year based on history, clinical signs and fecal smear examination using the Ziehl-Neelsen stain, yielding an overall prevalence of 38.75%, with clinical and sub-clinical PTB at 8.06 and 30.68%, respectively. A molecular tool, IS900 polymerase chain reaction, was also used to confirm the disease. With Mycobacterium avium subsp. paratuberculosis (MAP) bacilli and endoparasite infections, the majority of affected goats (69.08%) were low shedders. Puri coastal district had the highest prevalence (52.29%) followed by Sambalpur (48.61%), while Khordha had the lowest prevalence (26.41%). Caprine PTB was more common in goats over 2 years old (51.23%), in the Ganjam breed (42.30%), in females (39.17%) and in goats housed on earthen floors (55.83%) according to chi-square analysis. The current study concluded that higher (30.68%) observations of subclinical PTB were cause of real concern due to its insidious spread as well as its zoonotic significance with potential human consequences, which requires immediate attention at all levels. Because of the public health importance of this hidden killer disease, the current findings would be useful in developing a roadmap for implementing prevention and control policies, prompting provision for adequate funding with elaborative research.

Abstract Foot-and-mouth disease virus (FMDV) cripples livestock by imparting devastating effects to economy. A good vaccine is the key to stopping it, but due to instability of 146S of FMDV, it is becoming difficult. This is bad because only 146S can fight against disease and its dissociation ultimately leads to decreased potency of vaccine. This study aimed to preserve the integrity of 146S in vaccine using different inactivators and preservatives. Foot-and-mouth Disease virus type ‘O’ was propagated on baby hamster kidney 21 cell lines and inactivated using formalin or binary ethylenimine (BEI). Size exclusion high performance liquid chromatography analysis revealed minimal 146S loss after double inactivation with formalin and BEI. This inactivated virus was further formulated into oil-based vaccine with sodium thiomersal or chloroform as a preservative. Our findings demonstrated that chloroform outperformed thiomersal in maintaining shelf life of vaccine. This claims that the combined approach of double inactivation with formalin and BEI followed by chloroform as preservative offered a promising strategy for developing efficacious FMDV.

Abstract Staphylococcus aureus is one of the most common causes of mastitis worldwide. This study aimed to determine the prevalence and antimicrobial resistance (AMR) patterns of S. aureus in mastitic milk samples collected from camel farms in Matrouh Governorate, Egypt. A total of 200 mastitic camel milk samples were evaluated for S. aureus using both conventional culture-based and molecular-based methods. Antibiotic susceptibility testing of S. aureus isolates was conducted using disc diffusion and agar dilution methods, with antibiotic resistance genes identified through polymerase chain reaction with specific primers. Out of samples tested, 60 (30.00%) were positive for S. aureus. The isolates displayed the highest of resistance against piperacillin-tazobactam (55.00%) followed by trimethoprim- sulfamethoxazole (45.00%) and amoxicillin (40.00%). Half of the isolates were multidrug-resistant (MDR). The AMR genes included methicillin-resistant gene (mecA), β-lactamase gene (blaZ), tetracycline resistance gene (tetK), erythromycin resistance gene (ermB) and vancomycin resistant gene (vanA) were detected in 100%, 100%, 95.00%, 90.00% and 20.00% of the isolates, respectively. In conclusion, the presence of MDRS aureus as a cause of clinical camel mastitis is a significant veterinary and public health concern. These findings highlight the importance of proper antibiotic use in Egyptian camel farms and the need for molecular techniques to fully understand the genetic profile of antimicrobial-resistant S. aureus isolates.

Abstract The aim of this study was to investigate the probiotic potential of autochthonous Lactobacillus species isolated from buffalo calves against multidrug-resistant Escherichia coli. A total of 252 rectal swabs were collected from healthy neonatal buffalo calves under 30 days old from six districts of Andhra Pradesh, India in a completely randomized design from August 2019 to August 2021, of which 190 Lactobacillus strains were isolated based on cultural, morphological, biochemical and molecular tests. Out of 190 isolates, 57 showed high levels of auto-aggregation (> 80.00%) and hydrophobicity (> 60.00%) and 51 of the 57 isolates had a zone of inhibition greater than 15.00 mm in diameter against multidrug-resistant E. coli in an Agar well diffusion assay. Among the 51 isolates, 36 were found to be acid and bile tolerant and showed varying levels of sensitivity to antibiotics such as erythromycin, clindamycin, tetracycline, chloramphenicol, and ampicillin. Among the 36 isolates, Limosilactobacillus reuteri 178, L. reuteri 209, L. fermentum 182, L. fermentum 211, and Lactiplanti-bacillus plantarum 34 were non-hemolytic, and none of the isolates were able to hydrolyse gelatine. Therefore, these five autochthonous Lactobacillus species may be used in probiotic or synbiotic formulations against multidrug resistant E. coli in buffalo calves.

Abstract Staphylococcus aureus is gaining worldwide attention because of its substantial impact on public health. The current study aimed to characterize S. aureus strains isolated from wild birds in the Kasur district of Punjab, Pakistan from 2021 to 2022. A total of one hundred samples were collected from five wild bird species. The samples were enriched, inoculated on selective agars and cultured for 24 hr at 37.00 ˚C. All isolates were verified by matrix-assisted laser desorption ionization–time-of-flight mass spectrometry (MALDI-TOF MS) and polymerase chain reaction (PCR) after Gram staining. Positive isolates were screened for phenotypic (Kirby-Bauer disk diffusion and minimum inhibitory concentration s), genotypic antibiotic resistance, and virulence genes. These samples yielded 30 (30.00%) S. aureus isolates, confirmed by polymerase chain reaction utilizing the 16S rRNA gene. Staphylococcus aureus was more prevalent in cloacal samples (16.00%) than oral samples (14.00%). Various S. aureus isolates showed varying degrees of resistance to three different antibiotics. Oxacillin (56.66%; n = 17) and tetracycline (33.33%; n = 10) showed the highest resistance rates with the lowest susceptibility (43.33%; n = 13). In contrast, vancomycin, rifampicin, linezolid, and daptomycin were 100% susceptible. Further disc diffusion study revealed resistance to tetracycline (33.33%), erythromycin (16.66%), and gentamicin (10.00%). The tetK gene was found in 33.33% of wild bird samples, while the ermA gene was found in 16.66% of samples. The aacA-D gene was only found in three (10.00%) isolates. None of the isolates tested positive for virulence genes. In conclusion, S. aureus is carried by wild birds in this area, posing a potentail threat to both humans and animals.

Abstract The poultry products are known as a source of zoonotic and multi-drug resistant pathogens, especially Salmonella spp. The objective of this study was using bacteriophages as an alternative anti-microbial agent against Salmonella typhimurium isolate from turkey poults. The antibiotic susceptibility test was used to identify the antibiotic resistance pattern of the isolates. The bacteriophage was purified, enhanced and titrated using the Spot test and double layer agar (DLA) techniques after being isolated from a chicken slaughterhouse and sewage treatment facility. By determining the morphological characteristics of resulting plaque, the specificity and host range of the phage were studied on S. typhimurium isolates. A total number of 22 suspected Salmonella isolates were confirmed biochemically positive in sample by cultures method. Nine of these isolates (40.90%) were identified as S. typhimurium by polymerase chain reaction. All of isolates (100%) were resistant to chloramphenicol, doxycycline, kanamycin, florfenicol, rifampin, and erythromycin. Seven isolates (77.77%) were resistant to amoxicillin and nalidixic acid. The plaques were present with 3.00 ± 0.22 mm in diameter on the culture of 6 out of 9 (66.66%) isolates of S. typhimurium on brain heart infusion broth using DLA method. The amount of phage titer was 7.60 × 10⁷ phage forming unit mL^-1 and its multiplicity of infection value was calculated as 5.06 × 10^-2 based on obtained results. In place of antibiotics, the multi-drug resistant (MDR) S. typhimurium was successfully destroyed by the isolated bacteriophage from wastewater. In vitro settings were used in this investigation to identify the efficient bacteriophages against MDR S. typhimurium.

Abstract Since decades, Newcastle disease (ND) has become endemic in the poultry population of the Indian subcontinent. ND is a highly contagious disease of poultry and other avian species. However, the genetic nature of ND viruses circulating in the rock pigeons is unraveled. The present investigation is a part of Newcastle disease virus (NDV) surveillance in wild birds. Two velogenic NDV strains could be isolated from apparently healthy rock pigeons, thus establishing the status of carrier/reservoir host. The fusion protein cleavage site in the fusion protein has multiple basic amino acid (RRRKRF) motifs similar to velogenic isolates. Phylogenetic analysis based on complete fusion gene sequences confirmed that the isolates belong to NDV sub genotype XIII 2.2. Further analysis revealed several amino acid substitutions in the hypervariable region, heptad repeat regions and neutralizing epitopes of the fusion protein and heptad repeat regions and antigenic sites of the hemagglutinin-neuraminidase (HN) protein that are critical for fusion. A unique D170A substitution in the neutralizing epitope is identified that is critical for structure and function of the fusion protein. Mutations within the virulence determinants including fusion (F) and HN, elucidate continuous evolution of the viruses among the rock pigeons. Accidental spillover of these mutated viruses into commercial poultry operations may result in disease outbreaks with economic breakdown.

Abstract Glanders caused by Burkholderia mallei is one of the most dangerous zoonotic diseases in solipeds. Clinical diagnosis of this disease in its early stages in horses, is difficult. This study investigated serological and molecular identification of B. mallei in East Azerbaijan province. In the third and fourth quarters of 2020, throughout 2021, and in the first and second quarters of 2022, the complement fixation test (CFT) was performed on 350 horses. The malleination was used to confirm the positive CFT cases. Blood samples were taken for culture and for preparing serums to perform the enzyme-linked immunosorbent assay (ELISA). Deep eye discharge, nostril, cutaneous ulcers and lymph fluid swabs were cultured, and polymerase chain reaction (PCR) was carried out. Eleven horses were CFT-positive. Based on the malleination on the 11 horses, six were affected by glanders, five were not affected (false positive), and one horse was CFT-negative despite exhibiting clinical signs. It was positive by malleination, ELISA and PCR. A total number of seven positive cases of glanders were diagnosed. The B. mallei could not be isolated, but the Burkholderia cepacia complex was isolated in one case. Except for three cases, the results of the CFT, mallein and ELISA tests were consistent. The amount of confidence interval was 95.00%. It is suggested that ELISA could be used as a complement to CFT in screening and, if positive results are observed in one of the tests, the entire herd must be examined more accurately using the mallein and western blot confirmatory tests.

Abstract Bovine brucellosis, an infectious disease transmitted by Brucella melitensis and Brucella abortus, presents a significant zoonotic risk for agricultural economics and animal health. The primary objective of this study was to present a comprehensive understanding of the prevalence and features of Brucella strains within the industrial dairy farming sector in Iran. Rose Bengal plate test, standard agglutination test, and indirect enzyme linked immunosorbent assay tests were used to confirm all seropositive animals. A total number of 1,311 bovine samples from seropositive animals including were collected from 224 farms in 21 provinces of different regions of Iran and examined. The discovered Brucella isolates were phenotyped and molecularly characterized. The isolates were all B. abortus or B. melitensis. Bacteria analysis revealed that 70.53% of seropositive farms were tested positive for Brucella strains, predominantly B. melitensis biovar 1 (43.42%) and B. abortus biovar 3 (27.11%). Geographical distribution revealed that B. melitensis biovar 1 was the most common in dairy cow farms (16 provinces), followed by B. abortus biovar 3 (six provinces). Also, the prevalence of B. melitensis biovar 2, B. melitensis biovar 3, B. abortus biovar 1, B. abortus biovar 2 and RB51 vaccine were restricted to certain provinces. AMOS (abortus melitensis ovis suis)-polymerase chain reaction and Bruce-ladder PCR confirmed species identification. These results highlighted the complexity of bovine brucellosis in Iran and illustrated that B. melitensis was spread from small ruminants to cattle. This study provided important epidemiological insights for targeting future brucellosis control programs in the Iranian dairy farms.

Abstract Staphylococcus aureus is an important pathogen causing a wide range of diseases in both humans and animals. The aim of this research was to screen the vancomycin resistance-associated genes in methicillin-resistant Staphylococcus aureus (MRSA) isolates from animals. A total of 400 nasal swab samples were collected from cattle, goats and sheep between February and August 2022 from both industrial and traditional livestock farms in West Azerbaijan province, Iran. Then, nasal swabs were cultured on mannitol salt agar and molecular analysis was performed after bacteriological examination to confirm the presence of S. aureus. The MecA gene was used to detect MRSA isolates, and two important vancomycin resistance-associated genes, namely vanA and vanB, were searched in the isolates. Out of 400 nasal swabs, 69 samples had S. aureus; of which seven isolates were resistant against methicillin. No vancomycin resistance-associated genes were detected in the MRSA isolates. Based on these findings, vancomycin could be used to treat infections caused by this bacterium.

Abstract Mastitis associated Klebsiella pneumoniae species were isolated from bovine milk to characterize virulence genes (wabG and kfuBC) and extended spectrum β-lactamase (ESBL) genes (bla_CTX-M-1, bla_CTX-M-2, bla_CTX-M-9, bla_TEM, bla_SHV and bla_OXA). A total number of 325 bovine milk samples (195 raw and 130 mastitic milk specimens) collected from Banaskantha, a milk-shed district of Gujarat, India, were included in the study. A total number of 27 K. pneumoniae isolates were recovered, consisting of 17 (62.96%) isolates from raw milk and 10 (37.03%) isolates from mastitic milk samples, giving an overall prevalence of 8.31%. Antibiotic sensitivity patterns revealed that 20 out of 27 isolates were found to be multi-drug resistant. Based on combination disc diffusion test and HiCrome ESBL agar method, 20 (74.07%) and 25 (92.59%) isolates were detected as ESBL producers, respectively. Among virulence genes studied, presence of wabG (25/27; 92.59%) was higher than kfuBC (5/27; 18.51%). Beta-lactamase genes viz., bla_SHV, bla_TEM and bla_CTX-M-1 were detected in 23/27 (85.18%), 3/27 (11.11%) and 2/27 (7.40%) of isolates, respectively; while, none of the isolates was found to be positive for bla_CTX-M-9 and bla_OXA-1 genes. Outcome of the study provided an insight into virulence genes and ESBL producing K. pneumoniae isolated from bovine milk samples in India.

Abstract Borrelia species are spirochetes transmitted by ticks that are important in human and animals. In most countries, there is still no molecular epidemiology of borreliosis in ruminants. This study was aimed to evaluate the existence of Borrelia spp. DNA in the blood samples of small ruminants using polymerase chain reaction (PCR) method in West Azerbaijan Province, Iran. To detect Borrelia spp. DNA, about 1,018 ruminants (456 goats and 562 sheep) blood samples were examined from different bioclimatic regions in West Azerbaijan province, Iran. The DNA extracting and PCR were conducted. In sheep, the following prevalence rates were respectively obtained for the 16S rRNA, 5S - 23S rRNA and ospA genes: 3.55% (20/562), 2.13% (12/562) and 0.88% (5/562). And so, the prevalence rates of the genes in goats were 0.87% (4/456) for 5S - 23S rRNA gene, 1.75% (8/456) for 16S rRNA gene and 0.65% (3/456) for ospA gene. The prevalence of Borrelia spp. was significantly different in small ruminants based on the farms and localities. The sheep and goats in humid areas (north of West Azerbaijan) were infected statistically more than those in sub-humid areas (south of West Azerbaijan). It is demonstrated that host species like sheep and goats may have a key role in natural Lyme disease cycles and other borreliosis diseases in Iran.

Abstract Viral and bacterial gastroenteritis and diarrhea have long been a problem in livestock with devastating effects on animal health and production causing a heavy financial burden on producers. Therefore, the bead-based multiplex detection assay was created for simultaneous detection of three livestock viral diarrheic agents viz. bovine rotavirus (BRV), bovine coronavirus (BCoV) and bluetongue virus (BTV). The primers and probes for triplex MAGPIX assay for simultaneous detection of three enteric viruses were designed and the assay was optimized for hybridization temperature, primer-probe and bead concentrations. The newly developed MAGPIX assay was used to determine the prevalence of these diarrhea-associated viruses by testing 200 fecal samples collected from Haryana state of India during 2018-2019. The limit of detection of the developed triplex assay was 1 × 10⁵, 1 × 10⁴, and 1 × 10⁵ RNA copies for BRV, BCoV, and BTV, respectively, being lower than the reverse transcription-quantitative polymerase chain reaction (RT-qPCR). However, it was higher than the conventional RT-PCR, showing it to be more sensitive. The newly developed MAGPIX assay was a rapid, cost-effective and high throughput diagnostic tool for identification of three major entero-pathogenic diarrhea associated viruses, either alone or in tandem, with the aim to prevent and control viral diarrhea in animals.

Abstract World Organization for Animal Health has listed bluetongue (BT) under notifiable diseases. The BT is an arboviral infectious disease of domestic and wild ruminants caused by the bluetongue virus (BTV). Southern states of India had remained the point of attention for BT since first presence in 1964 in Maharashtra. Recently, northern states of India have also been reported positive for BTV in small ruminants. The present study reported the dual infection of BTV serotypes, BTV-12 and -16 in sheep population from Sirsa district of Haryana in the year 2016. After detection and serotyping with Seg-2 specific real time polymerase chain reaction (PCR), the Seg-2 and Seg-6 of BTV were PCR amplified and sequenced. On phylogenetic analysis it was detected to be clustered in nucleotype G and nucleotype B specific for BTV-12 and BTV-16, respectively. This was the first report of BTV-16 from Haryana. The results signified the co-infection of two different serotypes in an animal from a single outbreak.

Abstract The genetic diversity of Brucella strains has not been fully understood. To investigate this, the genetic characteristics of 64 isolates of Brucella melitensis from sheep and goats’ milk were studied using random fragment length polymorphism (RFLP) and multiple locus variable-number tandem repeat analysis (MLVA-16) methods developed in Orsay, France (MLVA-16_Orsay). The RFLP analysis revealed that all 64 isolates were of biovar one. The MLVA-typing showed that one sample was simultaneously infected with two strains of B. melitensis and the genotype of 65 isolate was analyzed. Four genotypes (47, 42, 43, and 63) were identified using MLVA-8 (panel 1), whereas six genotypes (138, 125, 116, 108, and two unknown genotypes) were identified using MLVA11 (panels 1 and 2A). From the review of MLVA-16 (panels 1, 2A, and 2B), panel 2B showed a very high discriminatory power. Two loci of Bruc04 and Bruc30 from this panel had diversity index values higher than 0.71 and the average diversity index was 0.619. So MLVA-16_Orsay 34 showed the genotype indicating a low genetic homogeneity among the isolates. The findings of MLVA genotyping of the isolates suggest that strains of B. melitensis isolated from the milk of small ruminants in Iran are most closely related to the isolates from neighboring countries of the Eastern Mediterranean group. To the best of our knowledge, this is the first study to indicate the potential use of MLVA genotyping for simultaneous detection of specimen contamination using two different B. melitensis biovars.