Comparison of bacteriological culture and a multiplex real-time PCR kit for the detection of mastitis pathogens in small ruminants

Abstract

This study aimed to evaluate the applicability of a multiplex real-time PCR (rPCR) kit, originally developed for bovine mastitis, for detecting mastitis pathogens in small ruminants and reducing bacterial identification time compared with conventional bacteriological culture (BC). Milk samples were obtained from 175 sheep and 124 goats and included cases of clinical mastitis (n = 57) and subclinical mastitis (n = 242). With BC, bacterial growth was detected in 125 of 299 milk samples (41.8%), yielding a total of 169 bacterial isolates. The pathogens most frequently isolated by BC were coagulase-negative staphylococci (58.0%), followed by Streptococcus spp. (9.4%), Staphylococcus aureus (7.1%), and Escherichia coli (4.1%). Using the multiplex rPCR assay, which targeted 14 bacterial species, one yeast, and one beta-lactamase gene, 210 milk samples (70.2%) tested positive, yielding a total of 459 pathogen detections. The pathogens detected by rPCR were predominantly Staphylococcus spp. (31.1%), followed by beta-lactamase-positive isolates (19.8%), E. coli (13.9%), S. aureus (7.1%), S. uberis (8.0%), Mycoplasma spp. (7.1%), and Enterococcus spp. (2.8%). Among the 174 BC-negative samples, 77 were also negative by rPCR. Among the 97 samples that were positive only by rPCR, Staphylococcus spp. (15.4%), beta-lactamase positive isolates (23.7%), Mycoplasma spp. (15.4%), S. aureus (5.1%), E. coli (13.4%), and S. uberis (8.2%) were detected. This study demonstrated that the bovine rPCR kit yielded reliable results for the diagnosis of mastitis in small ruminants compared with BC. These findings highlight the value of molecular diagnostic tools for supporting early mastitis detection and improving control strategies in small ruminant herds.