A Novel Real-Time PCR-Based Screening Test with Pooled Fecal Samples for Bovine Johne’s Disease
| Autor: | Yasuyuki Mori, Reiko Nagata, Yasutaka Minegishi, Shingo Kishizuka, Satoko Kawaji, Akiko Mita, Yumi Saruyama, Masahiro Saito |
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| Rok vydání: | 2020 |
| Předmět: |
Microbiology (medical)
Veterinary medicine 040301 veterinary sciences Cattle Diseases Paratuberculosis Enzyme-Linked Immunosorbent Assay Real-Time Polymerase Chain Reaction Sensitivity and Specificity Clinical Veterinary Microbiology Serology law.invention 0403 veterinary science Feces 03 medical and health sciences law medicine Animals Polymerase chain reaction 030304 developmental biology 0303 health sciences biology 04 agricultural and veterinary sciences biology.organism_classification medicine.disease Mycobacterium avium subsp. paratuberculosis Real-time polymerase chain reaction Infectious disease (medical specialty) Herd Cattle Mycobacterium |
| Zdroj: | J Clin Microbiol |
| ISSN: | 1098-660X 0095-1137 |
| Popis: | Johne’s disease (JD) is an economically important infectious disease in livestock farming caused by Mycobacterium avium subsp. paratuberculosis. As an alternative to serological tests, which are used mainly for the screening of whole herds, we developed a novel ResoLight-based real-time PCR (RL-PCR) assay with pooled fecal samples for the detection of fecal shedders in cattle herds. The RL-PCR assay included an internal amplification control (IC) which was amplified using the same primer pair as the target molecule M. avium subsp. paratuberculosis IS900 and differentiated based on melting temperatures. Individual fecal suspensions were pooled and concentrated by centrifugation to avoid a loss of sensitivity by the dilution effect. Combined with a DNA extraction kit (Johne-PureSpin; FASMAC), no inhibition of PCR amplification was observed with up to 15 fecal samples in a pool. The detection limit of RL-PCR at a pool size of 10 was 10 M. avium subsp. paratuberculosis organisms per gram of feces, which was comparable to that of individual testing. A total of 2,654 animals in 12 infected herds were screened by individual antibody-enzyme-linked immunosorbent assay (ELISA) and the RL-PCR assay using pooled feces. Fifty animals were diagnosed with JD through the screening by RL-PCR, compared with only 5 by ELISA (which were also positive in RL-PCR). In 7 JD-free herds, the results of 4 out of 327 pools (1.2%) were invalid due to the lack of IC amplification, and then animals were confirmed negative individually. Our results suggest that implementation of herd screening by pooled RL-PCR would advance the monitoring and control of JD in cattle herds. |
| Databáze: | OpenAIRE |
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