Molecular epidemiology, characterization of virulence factors and antibiotic resistance profile of Streptococcus agalactiae isolated from dairy farms in China and Pakistan

Streptococcus agalactiae is one of the most common pathogens that cause bovine mastitis worldwide. Identifying pathogen prevalence and virulence factors is critical for developing prevention and control approaches. Herein, 1 161 milk samples from various dairy farms in China(n=558) and Pakistan(n=603) were collected between 2019–2021 and were subjected to S. agalactiae isolation. Prevalence, serotyping, virulence genes, and antibiotic-resistant genes of S. agalactiae were evaluated by PCR assay. All isolates were characterized for haemolysis, biofilm production, cytotoxicity, adhesion, and invasion on bovine mammary epithelial cells. The prevalence of S. agalactiae-induced mastitis in cattle was found to be considerably higher in Pakistan than in China. Jiangsu and Sindh provinces had the highest area-wise prevalence in China and Pakistan, respectively. Serotypes Ia and II were prevalent in both countries, whereas serotype III was found only in Pakistan. Moreover, all isolates tested positive for PI-2b gene but negative for PI-1 and PI-2a genes. All isolates harboured cfb, cylE, hylB, and fbsB virulent genes, whereas many of them lacked bibA, rib and bca. However, the absence of bac and scp genes in Chinese isolates and cspA in Pakistani isolates was noted, while spb1 and lmb were not detected in isolates of both countries. Pakistani isolates, particularly serotype Iapositive, had a considerably higher ability to produce biofilm, haemolysis, cytotoxicity, adhesion, and invasion than Chinese isolates. Most of the isolates were phenotypically resistant to tetracycline, erythromycin, and clindamycin and genotypic resistance was confirmed by the presence of ermA, ermB, tetM and tetO genes. Our study highlights the antimicrobial resistance profile and virulence-related factors contributing to the epidemiological spread of mastitis-causing S. agalactiae in China and Pakistan. The findings may facilitate future studies designed to develop improved treatment and control strategies against this pathogen.

Hapten design to prepare monoclonal antibodies and establishment of immunoassay for direct screening of oxamichydrazide in chicken,the metabolite of nifuraldezone

In this study,two novel haptens of oxamichydrazide(OXZ),the metabolite of nifuraldezone,were designed and synthesized by derivatization with ethyl-4-bromobutanoate and ethyl-2-bromoacetate for the first time.The rationality of the haptens was enlightened by conformational alignment and electronic evaluations based on computional chemistry.The synthesized haptens were efficient in generating antibody response to OXZ.A monoclonal antibody(mAb)named 2D9 with high affinity was obtained and used to establish an indirect competitive enzyme-linked immunosorbent assay(icELISA)for the direct determination of OXZ in chicken samples without the need for derivatization step.Under optimum conditions,the developed icELISA showed an IC50 value of 0.66 ng mL^(−1),and limit of detection of 0.11μg kg^(−1)in chicken.The average recoveries were found to be 80.4-91.3%with a coefficient of variation less than 9.0%.In conclusion,we have successfully desigened haptens,prepared mAbs and developed an immunoassay for the direct determination of OXZ in chicken samples without the need for a derivatization step for the first time.This provides a potentially rapid,accurate,and sensitive screening tool for nifuraldezone residue in animal-derived food.