Comparative Evaluation of Indirect-Elisaand IS900 PCR Assay for Diagnosis of Ovine Johne's Disease

Mycobacterium avium subspecies paratuberculosis (MAP), causative agent of Johne’s disease (JD) is chronic granulomatous enteritis affecting domestic and wild ruminants. Since, MAP is not killed by pasteurization, it has been isolated from commercially pasteurized milk and milk products resulting exposure of human population to this pathogen through milk. Control and eradication of JD is considered difficult because of its insidious nature and lack of early, rapid and accurate diagnostic tests. Therefore in present study, a visual loop-mediated isothermal amplification (LAMP) assay method has been developed using a total of six primers including 2 outer (F3 and B3), 2 inner (FIP and BIP) and 2 loop (LF and LB) primers specific for MAP for the first time on ‘S 5’ strain of Mycobacterium avium subsp. paratuberculosis ‘Indian Bison type’ biotype. After laboratory standardization, final optimized reaction performed at 65°C for 45 min was achieved after titration of incubation time, temperature conditions and the reporter dye calcein. Sensitivity and specificity of the LAMP assay was optimized and compared with traditional IS900 PCR. The sensitivity of LAMP assay was found to detect 10fg (100%) of DNA and 95.7% specificity was recorded with respect to traditional IS900 PCR. Comparison showed that LAMP had 98.6% and 96.1% sensitivity and specificity of 96.1% and 92.3%, with respect to microscopy and culture exhibiting ‘Almost perfect’ strength of agreement. The study concluded that LAMP assay was a reliable and sensitive diagnostic test to detect MAP infection in feces and can also be used for the ‘mass screening’ of the milk samples with the help of less expertise.

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A Novel Real-Time PCR-Based Screening Test with Pooled Fecal Samples for Bovine Johne’s Disease

Journal of Clinical Microbiology ◽

10.1128/jcm.01761-20 ◽

2020 ◽

Vol 58 (12) ◽

Author(s):

Satoko Kawaji ◽

Reiko Nagata ◽

Yasutaka Minegishi ◽

Yumi Saruyama ◽

Akiko Mita ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Johne's Disease ◽

Johne’S Disease ◽

Enzyme Linked Immunosorbent Assay ◽

Pcr Assay ◽

Serological Tests ◽

Fecal Samples ◽

Content Type ◽

Cattle Herds

ABSTRACT 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.

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