Clinical evaluation of PCR diagnosis for detection of Mycobacterium avium subspecies paratuberculosis and epidemiological analysis of Korean isolates derived from cattle

Abstract

Johnes disease (JD) or paratuberculosis (PTB) is a chronic debilitating disease in ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). The disease causes significant economic losses in livestock industries worldwide. Because MAP is slow growing bacteria and it has very long incubation period, the clinical signs of disease can be produced in more than two years after initial infection. The gold standard for MAP diagnosis is isolation and identification of the bacteria directly from feces or tissues. However, visible colonies cannot be seen for 6 to 16 weeks using this method, and its sensitivity is relatively low. Serological tests such as ELISA are also commonly used, but these also have low sensitivity. Fecal PCR is a diagnostic method that can complement the shortcomings of the culture method. Specifically, PCR can detect the DNA of pathogens present in very small numbers with greater sensitivity and speed than culture methods. <br/> The purpose of this study was to investigate epidemiological situation of MAP infection in cattle based on fecal diagnosis to provide basic information, especially about subclinical infection which is essential for eradication of the disease. <br/> In this study, we designed new cost-effective DNA extraction method (mGITC/SC) to improve detection sensitivity of fecal PCR and evaluated in terms of diagnostic efficiency compared with other DNA extraction methods. The detection limit of IS900 real-time PCR was about 50 MAP (6 cfu) per g of MAP-spiked feces. The time taken per sample to extract DNA was about 60 minutes, and the cost was calculated to be $1 per sample. <br/> For the fecal diagnosis of MAP, a PCR targeting MAP-specific elements, IS900 and ISMap02 was performed using fecal samples collected from Korean cattle herds. Of the 1,562 fecal samples obtained from 37 herds, regardless of diarrhea, 35 samples were positive in both IS900 and ISMap02. At the herd level, 12 of the 37 herds were found to be positive for MAP. Thirty-five positive cows were considered to be in the subclinical stage because they did not show any clinical symptoms. In addition, the herd level prevalence investigated in this study was similar to those of the previous reports measured by ELISA-based methods. <br/> Molecular typing was conducted to describe the genetic diversity of MAP in Korea using twelve MAP-positive fecal DNA samples and 19 MAP isolates obtained from 10 cattle herds located in 5 provinces. The most prevalent strains in Korea were the bison-type genotype (23 of the 31 samples) and distributed nationwide. Rest of 8 samples were cattle-type, and all the foreign isolates were also cattle-type. The bison-type strains which were discriminated only as INMV 68 in MIRU-VNTR were divided into 3 different subtypes by MLSSR typing. Cattle-type was divided into 3 subtypes by MIRU-VNTR and 8 subtypes by MLSSR. The allelic diversities of MIRU-VNTR and MLSSR were calculated as 0.567 and 0.866, respectively. To the best of our knowledge, this is the first epidemiological survey report about the MAP isolated from cattle in Korea using MIRU-VNTR and MLSSR typing methods.<br/> In the present study, non-MAP mycobacteria were shown to be positive by ISMap02 targeting PCR. Two bacterial isolates (Sample ID: BO-038 and BO-042) were cultured from bovine fecal samples that produced positive results in three of two ISMap02 targeting PCR analyses with negative results in IS900 real-time PCR. Species identification using 16S rRNA gene sequencing and hsp65 gene partial sequencing revealed that strains BO-038 and BO-042 were M. virginiense and M. nonchromogenicum, respectively, which both belong to the M. terrae complex (MTC). Moreover, the two isolates shared a novel insertion sequence (IS) with high similarity to some parts of nucleotide sequences of ISMap02, and IS was presumed to be identical to that present in M. heraklionense. Both the novel IS and ISMap02 were characterized as IS1182 family members, and several sequences similar to ISMap02 were identified by BLAST analysis. The present study demonstrated the existence of ISMap02-like sequence for the first time. Moreover, this study found that two previously used ISMap02 PCR targets were not suitable for MAP detection.<br/> Based on the results, the fecal PCR diagnosis is an appropriate method to detect fecal shedder, which is important for understanding the MAP occurrence situation, and suggested that appropriate DNA extraction methods and MAP specific targets should be used to improve diagnostic efficiency. Moreover, the results of the molecular epidemiology survey of Korean MAP isolates can provide basic information for understanding of genetic diversity, evolutionary relationships, and inter and intra-species transmissions which are essential for establishing control strategy.<br/>