Veterinary Research Forum

Abstract Bovine ephemeral fever (BEF) is a re-emerging arbovirus infection, causing substantial economic losses primarily attributed to decreased milk production. BEF is a frequently reported disease in Pakistan, however, there is a lack of scientific data, particularly regarding gene sequencing and phylogenetic analysis of this viral infection in bovines. This research was planned for the molecular detection of the BEF virus in dairy animals of Sindh province, Pakistan. The association of various risk factors, including seasonal variation, species, breed, sex, age, lactation and feeding pattern was also analyzed. A total of 470 blood samples from cattle and buffaloes were collected across different topographical regions of Sindh from 2021-2022; and subjected to molecular detection through RT-PCR. Sequencing of positive RT-PCR amplicons and phylogenetic analysis were then performed. A questionnaire was designed to get comprehensive information about each animal before sample collection to develop an association between various risk factors. RT-PCR amplification of the glycoprotein gene yielded a 420 bp product and the phylogenetic tree revealed that the Pakistani BEF virus isolates clustered closely with strains from Iran, India and Australia indicating a strong genetic resemblance. The overall prevalence was 13.83% (65/470) which significantly higher in cattle (15.49%) than in buffaloes (3.70%), and cattle had significantly higher chance of infection than buffaloes (odds ratio = 4.31, confidence interval = 1.33 - 13.95). This study provided the first comprehensive gene sequencing data on BEF in Pakistan, significantly enhancing the global understanding of the virus through detailed molecular and phylogenetic analysis.

Abstract Bovine tuberculosis is a chronic bacterial disease primarily caused by Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex (MTBC), with significant zoonotic implications. This study aimed to detect MTBC in wildlife species, specifically nilgai (Boselaphus tragocamelus) and sambar deer (Rusa unicolor), using gross pathology, histopathology, acid-fast staining, and molecular confirmation. Necropsied tissue samples were collected during post-mortem examination of a nilgai and a sambar deer from the Assam State Zoo, Guwahati, India. Macroscopically, multiple granulomatous tubercles of varying sizes were observed in the lungs and liver, with creamy white caseous material marked upon sectioning. Ziehl-Neelsen staining of the tissue smears from granulomatous lesions confirmed the presence of acid-fast bacilli. Microscopic examination of tuberculosis granulomas revealed a central necrotic mass surrounded by inflammatory cell infiltration, including Langerhans-type giant cells. Molecular confirmation of MTBC infection was achieved by amplifying hsp65 and IS1081 in tissue samples, further validated by Basic Local Alignment Search Tool for nucleotide analysis following Sanger dideoxy sequencing. In conclusion, this study confirmed the presence of tuberculosis in these wildlife species through an integrated approach combining pathology, microbiology, and molecular diagnostics, highlighting the need to understand pathogen entry into the herd and prevent potential spillover.

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.