Mastitis is considered one of the most widespread infectious disease of cattle and buffaloes, affecting dairy herds. The current study aimed to characterize the Staphylococcus aureus isolates recovered from subclinical mastitis animals in Pothohar region of the country. A total of 278 milk samples from 17 different dairy farms around two districts of the Pothohar region, Islamabad and Rawalpindi, were collected and screened for sub clinical mastitis using California Mastitis Test. Positive milk samples were processed for isolation of Staphylococcus aureus using mannitol salt agar. The recovered isolates were analyzed for their antimicrobial susceptibility and virulence genes using disc diffusion and PCR respectively. 62.2% samples were positive for subclinical mastitis and in total 70 Staphylococcus aureus isolates were recovered. 21% of these isolates were determined to be methicillin resistant, carrying the mecA gene. S. aureus isolates recovered during the study were resistant to all first line therapeutic antibiotics and in total 52% isolates were multidrug resistant. SCCmec typing revealed MRSA SCCmec types IV and V, indicating potential community-acquired MRSA (CA-MRSA) transmission. Virulence profiling revealed high prevalence of key genes associated with adhesion, toxin production, and immune evasion, such as hla, hlb, clfA, clfB and cap5. Furthermore, the Panton-Valentine leukocidin (PVL) toxin, that is often associated with recurrent skin and soft tissue infections, was present in 5.7% of isolates. In conclusion, the increased prevalence of MRSA in bovine mastitis is highlighted by this study, which also reveals a variety of virulence factors in S. aureus and emphasizes the significance of appropriate antibiotic therapy in combating this economically burdensome disease.

Riaz A, et al. J Vet Sci. 2021 Jun;22(4):e51. https://doi.org/10.4142/jvs.2021.22.e51

The use of fetal bovine serum (FBS) in regenerative medicine raises serious ethical and scientific concerns. We have cultured and differentiated the canine mesenchymal stem cells (cMSCs) in five different media combinations of autologous platelet lysate (A-PL) and FBS; consisting of 0% A-PL and 10% FBS (M-1), 2.5% A-PL and 7.5% FBS (M-2), 5% A-PL and 5% FBS (M-3), 7.5% A-PL and 2.5% FBS (M-4), and 10% A-PL and 0% FBS (M-5). The cMSCs were evaluated for their doubling time, differentiation efficiency, and expression of CD73, CD90, CD105, and PDGFRα. The mRNA expression of NT5E, THY1, ENG, PPARγ, FABP4, FAS, SP7, BGLAP, and SPP1 was also assessed. The results indicated non-significant differences in cellular proliferation/viability; positive expression of surface markers, and PDGFRα with substantial adipo/osteogenic differentiation. The expression of adipogenic (PPARγ, FABP4, FAS), and osteogenic (SP7, BGLAP, SPP1) genes were higher (p < 0.05) in the M5 group. In conclusion, A-PL in cMSCs culture did not negatively affect cellular proliferation and viability but also enhanced their genetic potential for multilineage differentiation. Our results indicate that A-PL can be used as an alternative for FBS to develop potent cMSCs under good manufacturing practice protocol for regenerative medicine.

Shiga toxin-producing E. coli (STEC) are considered important zoonotic pathogens of great economic significance, associated with diarrhea, hemolytic uremic syndrome (HUS), hemorrhagic colitis (HC), and death in humans. This study aimed to investigate the distribution of various STEC virulence gene markers and antimicrobial susceptibility (AST) profiles associated within E. coli isolates from the recto-anal mucosal swabs (RAMSs) of slaughtered cattle and buffaloes in Islamabad, Pakistan. The RAMSs (n = 200) were analyzed using multiplex PCR for the presence of stx1, stx2, eae, and ehxA genes. Samples that were positive for one or more of the virulence genes were inoculated with Sorbitol MacConkey agar (SMAC) for isolation of STEC. The isolates were further analyzed for the presence of virulence genes using multiplex PCR. Of the 200 RAMS, 118 (59%) were positive for one or more virulence genes. E. coli isolates (n = 18) with one or more virulence genes were recovered from the 118 positive samples. The DNA of the isolates positive for one or more virulent genes was extracted and subjected to whole genome sequencing using Illumina. Analysis of the WGS data indicated that the E. coli isolates could be differentiated into 11 serotypes. Most E. coli isolates (13/18; 72.2%) carried five genes (stx1, stx2, Iha, iss, and IpfA) in various combinations. In addition to these five genes, other virulence genes identified in these isolates were espI, ireA, espP, exhA, epeA, mcmA, mch, ast, celB, eilA, katP, and capU. The AST was performed using the Kirby–Bauer disk diffusion test. The study indicated that all the isolates were resistant to rifampicin and a significant proportion of the isolates were MDR. A wide range of antimicrobial resistance genes (ARGs) were detected among the isolates, reflecting the complex nature of resistance mechanisms. The study results indicate that cattle and buffaloes slaughtered in Islamabad might be the carriers of antimicrobial resistant STEC of zoonotic significance, thus representing a source of human infection.