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Development of a novel detection system for microbes from bovine diarrhea by real-time PCR.

ABSTRACT: Diarrhea in cattle is one of the most economically costly disorders, decreasing milk production and weight gain. In the present study, we established a novel simultaneous detection system using TaqMan real-time PCR designed as a system for detection of microbes from bovine diarrhea using real-time PCR (referred to as Dembo-PCR). Dembo-PCR simultaneously detects a total of 19 diarrhea-causing pathogens, including viruses, bacteria and protozoa. Specific primer-probe sets were newly designed for 7 pathogens and were synthesized on the basis of previous reports for 12 pathogens. Assays were optimized to react under the same reaction conditions. The PCR efficiency and correlation coefficient (R(2)) of standard curves for each assay were more than 80% and 0.9766, respectively. Furthermore, the sensitivity of Dembo-PCR in fecal sample analysis was measured with feces spiked with target pathogens or synthesized DNA that included specific nucleotide target regions. The resulting limits of detection (LOD) for virus-spiked samples, bacteria and DNA fragments were 0.16-1.6 TCID50 (PFU/reaction), 1.3-13 CFU/reaction and 10-100 copies/reaction, respectively. All reactions showed high sensitivity in pathogen detection. A total of 8 fecal samples, collected from 6 diarrheic cattle, 1 diarrheic calf and 1 healthy cow, were tested using Dembo-PCR to validate the assay's clinical performance. The results revealed that bovine coronavirus had infected all diarrheic adult cattle and that bovine torovirus had infected the diarrheic calf. These results suggest that Dembo-PCR may be a powerful tool for diagnosing infectious agents in cattle diarrhea.

SUBMITTER: Tsuchiaka S

PROVIDER: S-EPMC4829504 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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Development of a novel detection system for microbes from bovine diarrhea by real-time PCR.

Tsuchiaka Shinobu S Masuda Tsuneyuki T Sugimura Satoshi S Kobayashi Suguru S Komatsu Natsumi N Nagai Makoto M Omatsu Tsutomu T Furuya Tetsuya T Oba Mami M Katayama Yukie Y Kanda Shuhei S Yokoyama Tadashi T Mizutani Tetsuya T

The Journal of veterinary medical science 20151130 3

Diarrhea in cattle is one of the most economically costly disorders, decreasing milk production and weight gain. In the present study, we established a novel simultaneous detection system using TaqMan real-time PCR designed as a system for detection of microbes from bovine diarrhea using real-time PCR (referred to as Dembo-PCR). Dembo-PCR simultaneously detects a total of 19 diarrhea-causing pathogens, including viruses, bacteria and protozoa. Specific primer-probe sets were newly designed for 7 ...[more]

PMID: 26616156

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Project description:BACKGROUND:As a pestivirus of the Flaviviridae family, bovine viral diarrhea virus (BVDV), has imposed a large burden on animal husbandry worldwide, and such virus can be transmitted mainly through direct contact with other infected animals and probably via aerosols. In the present study, we aimed to develop a real-time RT-PCR method for detection of BVDV-1 in aerosol samples. METHODS:A pair of primers specific for highly conserved regions of the BVDV-1 5'-UTR was designed. The standard curve and sensitivity of the developed assay were assessed based on 10-fold serial dilutions of RNA molecular standard. The specificity of the assay was evaluated with other pestiviruses and infectious bovine viruses. The clinical performance was examined by testing 169 aerosol samples. RESULTS:The results showed that a good linear relationship existed between the standard curve and the concentration of template. The lowest detection limit was 5.2 RNA molecules per reaction. This assay was specific for detection of BVDV-1, and no amplification was found for other pestiviruses such as classical swine fever virus (CSFV), border disease virus (BDV), and common infectious bovine viruses, including BVDV-2, infectious bovine rhinotracheitis virus (IBRV), bovine parainfluenza virus type 3 (BPIV-3), bovine respiratory syncytial virus (BRSV), bovine ephemeral fever virus (BEFV) and bovine coronavirus (BcoV). The assay was highly reproducible with low variation coefficient values (CVs) for intra-assay and inter-assay. A total of 169 aerosol samples collected from six dairy herds were tested using this method. The results showed that the positive detection rate of BVDV-1 was 17.2% (29/169), which was significantly higher compared with the conventional RT-PCR. Additionally, the positive samples (n = 29) detected by real-time RT-PCR were verified by BVDV RPA-LFD, and a concordance rate of 100% was obtained between them. CONCLUSIONS:Taken together, we developed a real-time RT-PCR assay for quantitative analysis of BVDV-1 in aerosol samples, and our finding provided valuable insights into the risk on aerosol transmission of BVDV-1.

Project description:Bovine respiratory and enteric diseases have a profound negative impact on animal, health, welfare, and productivity. A vast number of viruses and bacteria are associated with the diseases. Pathogen detection using real-time PCR (rtPCR) assays performed on traditional rtPCR platforms are costly and time consuming and by that limit the use of diagnostics in bovine medicine. To diminish these limitations, we have developed a high-throughput rtPCR system (BioMark HD; Fluidigm) for simultaneous detection of the 11 most important respiratory and enteric viral and bacterial pathogens. The sensitivity and specificity of the rtPCR assays on the high-throughput platform was comparable with that of the traditional rtPCR platform. Pools consisting of positive and negative individual field samples were tested in the high-throughput rtPCR system in order to investigate the effect of an individual sample in a pool. The pool tests showed that irrespective of the size of the pool, a high-range positive individual sample had a high influence on the cycle quantification value of the pool compared with the influence of a low-range positive individual sample. To validate the test on field samples, 2,393 nasal swab and 2,379 fecal samples were tested on the high-throughput rtPCR system as pools in order to determine the occurrence of the 11 pathogens in 100 Danish herds (83 dairy and 17 veal herds). In the dairy calves, Pasteurella multocida (38.4%), rotavirus A (27.4%), Mycoplasma spp. (26.2%), and Trueperella pyogenes (25.5%) were the most prevalent pathogens, while P. multocida (71.4%), Mycoplasma spp. (58.9%), Mannheimia haemolytica (53.6%), and Mycoplasma bovis (42.9%) were the most often detected pathogens in the veal calves. The established high-throughput system provides new possibilities for analysis of bovine samples, since the system enables testing of multiple samples for the presence of different pathogens in the same analysis test even with reduced costs and turnover time.

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Development of a novel detection system for microbes from bovine diarrhea by real-time PCR.

Publications

Development of a novel detection system for microbes from bovine diarrhea by real-time PCR.

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