Dataset Information

Development of an indirect ELISA for bovine mastitis using Sip protein of Streptococcus agalactiae.

ABSTRACT: The sip gene encoding for a conserved highly immunogenic surface protein of Streptococcus agalactiae was amplified using polymerase chain reaction (PCR) and subcloned into prokaryotic expression vector pET32a (+) and expressed as a recombinant protein in E. coli BL21 (DE3). An indirect enzyme linked immunosorbent assay (ELISA) was developed using the purified Sip protein as a coating antigen, which could identify S. agalactiae specific antibody in sera. The coating antigen at a concentration of 3.125 ?g/ml, serum diluted to 1:160, and HRP-conjugated secondary antibody concentration at 1:4000 was found to be most effective in exhibiting positive result. The ELISA was found to be highly specific for S. agalactiae that may be used for the detection of the pathogen in mastitis cases, for epidemiological studies and for surveillance.

SUBMITTER: Bu RE

PROVIDER: S-EPMC4782699 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Development of an indirect ELISA for bovine mastitis using Sip protein of Streptococcus agalactiae.

Bu R E RE Wang J L JL DebRoy C C Wu J H JH Xi L G W LG Liu Y Y Shen Z Q ZQ

Iranian journal of veterinary research 20150101 3

The sip gene encoding for a conserved highly immunogenic surface protein of Streptococcus agalactiae was amplified using polymerase chain reaction (PCR) and subcloned into prokaryotic expression vector pET32a (+) and expressed as a recombinant protein in E. coli BL21 (DE3). An indirect enzyme linked immunosorbent assay (ELISA) was developed using the purified Sip protein as a coating antigen, which could identify S. agalactiae specific antibody in sera. The coating antigen at a concentration of ...[more]

PMID: 27175190

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Project description:IntroductionStreptococcus agalactiae is a highly contagious pathogen that causes bovine mastitis, leading to significant economic losses. This study aimed to (1) identify and characterize S. agalactiae strains responsible for bovine mastitis by examining their phenotypic and genotypic characteristics in Thai dairy-intensive farming areas and (2) determine their susceptibility profiles to antimicrobial agents.Material and methodsIn total, 100 S. agalactiae isolates obtained from clinical and subclinical mastitis cases from 13 dairy herds located in the central region of Thailand were examined. To confirm the identity of the bacterial pathogens, conventional microbiological procedures recommended by the National Mastitis Council (NMC) and the VITEK® 2 system were employed.ResultsAll 100 isolates were successfully identified as S. agalactiae using the NMC procedure, whereas 94 isolates were identified as S. agalactiae using the VITEK® 2 system. Finally, the S. agalactiae-specific gene dlt S was identified in all the examined isolates using polymerase chain reaction. Capsular polysaccharide (CPS) typing revealed that all strains belonged to CPS type Ia. Multilocus sequence typing identified 33 selected isolates as sequence type 103. Random amplified polymorphic DNA (RAPD) typing yielded 43 RAPD types, with 6 RAPD clusters identified. These results demonstrated a high level of genetic diversity among S. agalactiae within the studied herds. RAPD analysis suggested that specific S. agalactiae strains could persist in dairy farms for 2-12 months. Furthermore, antimicrobial susceptibility testing was performed using the broth microdilution method. Most strains demonstrated susceptibility to ampicillin, penicillin, penicillin/novobiocin, cephalothin, oxacillin, ceftiofur, and erythromycin.DiscussionThis study revealed the phenotypic and genotypic characteristics of S. agalactiae isolates responsible for bovine mastitis in the central region of Thailand. The rapid identification of S. agalactiae and application of molecular typing methods can provide valuable epidemiological information regarding S. agalactiae causing mastitis in dairy farms. The antimicrobial susceptibility of S. agalactiae indicates that antimicrobial treatment for control and eradication could be a successful protocol. Our findings revealed that a single clonal strain of S. agalactiae affected the 13 studied farms. Further research is needed to explore the feasibility of vaccine development and application.

Project description:BackgroundMastitis is a disease of economic concern that affects dairy industry worldwide. This study aimed to investigate and identify possible etiologies encountered in an episode of acute gangrenous mastitis in lactating she-camels in Al Dhafra region, Abu Dhabi Emirate, United Arab Emirates (UAE). Beside the routine clinical examination, conventional bacteriological methods were used to isolate and identify possible aerobic/anaerobic bacterial or fungal pathogens from cultured milk samples collected from the mastitic she-camels. Moreover, quantitative real-time polymerase chain reaction (qPCR) was used for the detection of Mycoplasma agalactiae and Mycoplasma bovis strains, and the 16S rRNA gene was sequenced to confirm the isolation. The isolates were also tested for their susceptibility to antimicrobials.ResultsAcute gangrenous mastitis is reported in the dromedary camel herd with about 80% morbidity rate among lactating she-camels exhibited acute, painful hard swelling of affected teat, quarter or entire udder. About 41.7% of the infected animals were stamped out for culling due to complete or partial amputation of udder quarters. Streptococcus agalactiae was the sole isolated organism (6 isolates). The antimicrobial susceptibility testing revealed that, the Streptococcus agalactiae isolates were sensitive to both penicillin and ampicillin. Comparison of the 16S rRNA gene sequencing results by BLASTN confirmed the presence of Streptococcus agalactiae with high confidence (100% identity). Phylogenetic analysis indicated clustering of one isolate (CMAUAE accession number; MN267805.1) with Streptococcus agalactiae that infects multi-hosts including humans, while strains (CMBUAE to CMFUAE with accession numbers; MN267806.1 to MN267810.1 respectively) clustered with Streptococcus agalactiae that infects humans. No Mycoplasma spp was detected by qPCR analysis.ConclusionsIn the present study, the Streptococcus agalactiae was found to be the main cause of acute gangrenous mastitis in dromedary camels in UAE. More research should be done to investigate other possible causes of clinical or subclinical mastitis in dromedary camels in UAE.

Dataset Information

Development of an indirect ELISA for bovine mastitis using Sip protein of Streptococcus agalactiae.

Publications

Development of an indirect ELISA for bovine mastitis using Sip protein of Streptococcus agalactiae.

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