Bovine mastitis is a widespread and costly disease that affects dairy farming globally, characterized by mammary gland inflammation. Bovine intramammary gland infection has been associated with more than 135 different pathogens of which Staphylococcus aureus is the main etiology of sub-clinical mastitis (SCM). The current study was designed to investigate the prevalence, antibiotic resistance pattern, and the presence of antibiotic resistance genes (mecA, tetK, aacA-aphD and blaZ) in S. aureus isolated from the raw milk of cows with subclinical mastitis. A total of 543 milk samples were collected from lactating cows such as Holstein Friesian (n = 79), Sahiwal (n = 175), Cholistani (n = 107), and Red Sindhi (n = 182) from different dairy farms in Pakistan. From the milk samples microscopic slides were prepared and the somatic cell count was assessed to find SCM. To isolate and identify S. aureus, milk was streaked on mannitol salt agar (MSA) plates. Further confirmation was done based on biochemical assays, including gram staining (+ coccus), catalase test (+), and coagulase test (+). All the biochemically confirmed S. aureus isolates were molecularly identified using the thermonuclease (nuc) gene. The antibiotic resistance pattern of all the S. aureus isolates was evaluated through the disc diffusion method. Out of 543 milk samples, 310 (57.09%) were positive for SCM. Among the SCM-positive samples, S. aureus was detected in 30.32% (94/310) samples. Out of 94 isolates, 47 (50%) were determined to be multidrug resistant (MDR). Among these MDR isolates, 11 exhibited resistance to Cefoxitin, and hence were classified as methicillin-resistant Staphylococcus aureus (MRSA). The S. aureus isolates showed the highest resistance to Lincomycin (84.04%) followed by Ampicillin (45.74%), while the least resistance was shown to Sulfamethoxazole/Trimethoprim (3.19%) and Gentamycin (6.38%). Polymerase chain reaction (PCR) analysis revealed that 55.31% of the isolates carried blaZ gene, 46.80% carried tetK gene, 17.02% harbored the mecA gene, whereas, aacA-aphD gene was found in 13.82% samples. Our findings revealed a significant level of contamination of milk with S. aureus and half (50%) of the isolates were MDR. The isolated S. aureus harbored various antibiotic resistance genes responsible for the absorbed phenotypic resistance. The alarmingly high prevalence of MDR S. aureus isolates and MRSA strains in these cases possess a serious risk to public health, emphasizes the urgent need to address this issue to protect both human and animal health in Pakistan.

The present research was planned to assess the occurrence of intestinal parasites in small ruminants of Upper Dir of Khyber Pakhtunkhwa Province of Pakistan. For this purpose, the faecal material was collected randomly with gloved fingers directly from the rectum region of sheep and goats and the faecal materials were then put in hygienic plastic bottles with 10% formalin. The overall 315 (n=184 sheep and n= 131 goats) faecal samples were collected out of 315 samples, 281 were found positive for different parasites. Patterns-wise prevalence of GI parasites of the study area was found. Overall Single parasitism 89.20% (281/315) with 94.0% (173/184) in sheep and 82.43% (108/131) in goats. Double parasitic infection in small ruminant recorded in which Fasciola+ Haemonchus. contortus in sheep were found their prevalence was 25.54% (47/184). While in goats, the double parasitic infection in which Haemonchus contortus+Trichuris spp were found and their prevalence were 23.43% (30/131). The species found in the sample of sheep were includes, i.e., Strongyloides papillosus (41.30%), Heamonchus controtus (21.73%), Trichuris ovis (17.39%), and Fasciola hepatica (13.58%), the corresponding value for goat were Strongyloides spp 33.33% (36/108), Haemonchus spp 28.70%, (27/108), Trichuris spp 25.20% (27/184) and Fasciola spp 10.68% (14/184). The sheep of the study area are more infected as compared to goats. This study suggested that gastrointestinal parasites are major health problems of small ruminants in the study area. Therefore, a comprehensive study on species of gastrointestinal parasites circulating in the area, control options, cost-effective strategies and awareness about gastrointestinal parasites among the farmers in the study area should be instituted.

Staphylococcus aureus is a prominent cause of food-borne diseases worldwide. Enterotoxigenic strains of this bacteria are frequently found in raw milk, and some of these strains are resistant to antimicrobials, posing a risk to consumers. The main objectives of this study were to determine the antimicrobial resistance pattern of S. aureus in raw milk and to detect the presence of mecA and tetK genes in it. A total of 150 milk samples were obtained aseptically from lactating cattle, including Holstein Friesian, Achai, and Jersey breeds, maintained at different dairy farms. The milk samples were checked for the presence of S. aureus, and it was detected in 55 (37%) of them. The presence of S. aureus was verified by culturing on selective media, gram staining, and performing coagulase and catalase tests. Further confirmation was performed through PCR with a species-specific thermonuclease (nuc) gene. Antimicrobial susceptibility testing of the confirmed S. aureus was then determined by using the Kirby–Bauer disc diffusion technique. Out of the 55 confirmed S. aureus isolates, 11 were determined to be multidrug-resistant (MDR). The highest resistance was found to penicillin (100%) and oxacillin (100%), followed by tetracycline (72.72%), amikacin (27.27%), sulfamethoxazole/trimethoprim (18.18%), tobramycin (18.18%), and gentamycin (9.09%). Amoxicillin and ciprofloxacin were found to be susceptible (100%). Out of 11 MDR S. aureus isolates, the methicillin resistance gene (mecA) was detected in 9 isolates, while the tetracycline resistance gene (tetK) was found in 7 isolates. The presence of these methicillin- and tetracycline-resistant strains in raw milk poses a major risk to public health, as they can cause food poisoning outbreaks that can spread rapidly through populations. Our study concludes that out of nine empirically used antibiotics, amoxicillin, ciprofloxacin, and gentamicin were highly effective against S. aureus compared to penicillin, oxacillin, and tetracycline.

The present study was designed to evaluate the gene expression and DNA methylation level in the promoter region of the CD4 and the JAK-STAT-pathway-related genes. A total of 24 samples were deployed in the gene expression and 118 samples were used in the DNA methylation study. Student’s t-tests were used to analyze the gene expression and DNA methylation. The evaluation of DNA methylation in promoter regions of JAK2 and STAT5A revealed hypo-methylation levels of CpG sites and higher gene expression in cows diagnosed with mastitis as compared to the healthy control, and vice versa in those with CD4. DNA methylation was negatively correlated with gene expression in JAK2, STAT5A, and CD4 genes. Six, two, and four active transcription factors were identified on the CpG sites in the promoter regions of JAK2, STAT5A, and CD4 genes, respectively. Regarding correlation analysis, the DNA methylation levels of CD4 showed significantly higher positive correlations with somatic cell counts (p < 0.05). Findings of the current study inferred that aberrant DNA methylation in the CpG sites at the 1 kb promoter region in JAK2, STAT5A, and CD4 genes due to mastitis in cows can be used as potential epigenetic markers to estimate bovine mastitis susceptibility in dairy cattle.

Background and Aim: Anaplasmosis, a tick-borne disease affecting livestock caused by the bacteria Anaplasma, poses a global concern. This study aimed to estimate the prevalence, spatiotemporal variation, and associated risk factors of anaplasmosis in cattle from the Bannu and Lakki Marwat districts of Khyber Pakhtunkhwa, Pakistan. Materials and Methods: This study used 197 cattle exhibiting clinical symptoms of anaplasmosis in natural settings. Microscopic examination was used to estimate the prevalence. Potential risk factors, such as sampling regions and months, gender, breed, and age were studied. Results: The study revealed an overall anaplasmosis prevalence of 19.79%. Bannu district exhibited a higher occurrence at 22.10%, compared to Lakki Marwat district at 17.64%. Young cattle (5 years), which had a prevalence of 12.35% (p < 0.05). Female cattle (22.36%) were more susceptible than male cattle (11.11%). Prevalence peaked in June (45.71%) and was lowest in February (3.57%). Crossbred cattle had a higher prevalence (23.52%) than purebred cattle (11.47%). Conclusion: Anaplasmosis can be effectively controlled using a comprehensive approach encompassing selective breeding for resilience, targeted care of young calves and females, effective tick control during warmer months, consistent use of insecticides, and proactive risk factor management. Raising awareness among farmers through diverse channels, including media, is pivotal to bolster tick-borne disease management strategies. Keywords: anaplasmosis, biotic risk factors, cattle, Khyber Pakhtunkhwa, Pakistan, spatiotemporal variation.

Objective: Abnormally increased somatic cell counts (SCCs) in milk is usually a sign of bovine subclinical mastitis. Mutual interaction between the host and its associated microbiota plays an important role in developing such diseases. The main objective of this study was to explore the difference between cows with elevated SCCs and healthy cattle from the perspective of host-microbe interplay.Methods: A total of 31 milk samples and 23 bovine peripheral blood samples were collected from Holstein dairy cattle to conduct an integrated analysis of transcriptomic and metagenomics.Results: The results showed that Ralstonia and Sphingomonas were enriched in cows with subclinical mastitis. The relative abundance of the two bacteria was positively correlated with the expression level of bovine transcobalamin 1 and uridine phosphorylase 1 encoding gene. Moreover, functional analysis revealed a distinct alternation in some important microbial biological processes.Conclusion: These results reveal the relative abundance of Ralstonia and Sphingomonas other than common mastitis-causing pathogens varied from healthy cows to those with subclinical mastitis and might be associated with elevated SCCs. Potential association was observed between bovine milk microbiota composition and the transcriptional pattern of some genes, thus providing new insights to understand homeostasis of bovine udder.