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:BACKGROUND:Mastitis 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. RESULTS:Acute 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. CONCLUSIONS:In 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.

Project description:BackgroundStreptococcus agalactiae (S. agalactiae) is a contagious pathogen of bovine mastitis. It has financial implications for the dairy cattle industry in certain areas of the world. Since antimicrobial resistance increases in dairy farms, natural antimicrobials from herbal origins and nanoparticles have been given more attention as an alternative therapy. Hence, this study reported the antimicrobial and antibiofilm potentials of cinnamon oil, silver nanoparticles (AgNPs), and their combination against multidrug-resistant (MDR) S. agalactiae recovered from clinical bovine mastitis in Egypt.ResultsOur findings revealed that 73% (146/200) of the examined milk samples collected from dairy cows with clinical mastitis were infected with Streptococci species. Of these, 9.59% (14/146) were identified as S. agalactiae and categorized as MDR. S. agalactiae isolates expressed four virulence genes (Hyl, cylE, scpB, and lmb) and demonstrated an ability to produce biofilms. Cinnamon oil showed high antimicrobial (MICs ≤0.063 μg /mL) and antibiofilm (MBIC50 = 4 μg/mL) potentials against planktonic and biofilms of S. agalactiae isolates, respectively. However, AgNPs showed reasonable antimicrobial (MICs ≤16 μg/mL) and relatively low antibiofilm (MBIC50 = 64 μg/mL) activities against screened isolates. Synergistic antimicrobial or additive antibiofilm interactions of cinnamon oil combined with AgNPs were reported for the first time. Scanning electron microscope (SEM) analysis revealed that biofilms of S. agalactiae isolates treated with cinnamon oil were more seriously damaged than observed in AgNPs cinnamon oil combination. Moreover, reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) showed that cinnamon oil exerted a remarkable down-regulation of pili biosynthesis genes (pilA and pilB) and their regulator (rogB) against S. agalactiae biofilms, meanwhile the AgNPs cinnamon oil combination demonstrated a lower efficacy.ConclusionsThis is an in vitro preliminary approach that documented the antibiofilm potential of cinnamon oil and the inhibitory activity of cinnamon oil and its combination with AgNPs against MDR S. agalactiae recovered from clinical mastitis. Further in vivo studies should be carried out in animal models to provide evidence of concept for implementing these alternative candidates in the treatment of dairy farms infected by streptococcal mastitis in the future.

Project description:The precision by which animal diseases are diagnosed affects our ability to make informed decisions with regards to animal health management, from a clinical and economic perspective. Lameness is a major health condition in dairy cattle. The underlying causes of lameness include bovine digital dermatitis (BDD), which is reported as one of the main causes of infectious lameness in dairy cattle. Presently, the gold standard for BDD diagnosis in dairy cattle is visual inspection of lifted hooves-a labour intensive and subjective method. Research has suggested that Treponema spp. are the main pathogens associated with the establishment of BDD. We explored the potential of indirect enzyme-linked immunosorbent assay (ELISA) as a diagnostic serological tool in the identification of cows at different stages of BDD. Additionally, we evaluated the predictive power of this diagnostic tool on the future occurrence of BDD lesions. A total of 232 cows from three farms were used in the study. Serum samples and hoof health data were collected at three time points: ~ 30 days pre-calving, around calving, and approximately 30 days post-calving. The mean absorbance from the ELISA test was compared across different clinical presentations of BDD as assessed by visual inspection of the hooves according to the M-stage classification system. A transition model was developed to estimate the probability of lesion occurrence in time t + 1 based on the spectrophotometer (absorbance) reading in time t. The mean absorbance reading for both IgG1 and IgG2 anti-Treponema antibodies was associated with disease presence-apart from M4.1 lesions, animals with no lesions had a lower mean when compared to animals with lesions regardless of the score. Additionally, the mean absorbance reading of animals with active lesions was higher when compared to animals with no lesions. However, the anti-Treponema antibody assays failed to identify disease presence in a consistent manner. Moreover, indirect ELISA readings were not a predictor of the future occurrence of BDD lesions. In conclusion, although the levels anti-Treponema antibodies were associated with disease presence, the ELISA test failed to detect disease unequivocally and had no predictive value in the future occurrence of BDD lesions.

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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