Comparative analysis of serological tests and fecal detection in the diagnosis of Mycobacterium avium subspecies paratuberculosis infection

Abstract: Johne's disease (JD) caused by Mycobacterium avium subspecies paratuberculosis (MAP) is a chronic, wasting infectious disease in ruminants that causes enormous economic losses to the dairy and beef cattle industries. The most effective way to eradicate JD is to detect infected individuals as early as possible and remove them from the herd. However, it is difficult to detect infected individuals early with the currently using diagnostic methods. Two serological diagnostic kits commercially used worldwide and a fecal detection test were compared using 298 serum samples and feces of cattle in this study to present an efficient diagnostic method.Although there was a high correlation between the 2 serological diagnostic kits (R2 = 0.7473), kit A showed a higher serological positive rate. However, the correlation between fecal tests and serological diagnosis was very low. MAP was also detected in fecal tests in many serologically negative individuals. In the periodical diagnosis of JD, MAP was detected in the feces of only cows with the higher antibody titer to MAP. These results suggest that for effective eradication of JD, early detection of infected individuals by fecal tests together with the serological tests currently in use and by removal of infected individuals are needed.

Gene Expression Profiles of Th1-type Chemokines in Whole Blood of Mycobacterium avium subsp. paratuberculosis-Infected Cattle

Johne's disease (JD) is a chronic, debilitating disease of ruminants including cows, and is caused by Mycobacterium avium subsp. paratuberculosis (MAP). MAP is not only important in animal husbandry, but also in public health as it is associated with the onset of Crohn's disease, a chronic inflammatory bowel disease in humans. JD, like other mycobacterial diseases including tuberculosis, is classified into different stages based on the progression of infection. In addition, development of diagnostic assays that can distinguish between subclinical and clinical stages of JD is essential to control mycobacterial infection by providing an effective treatment. For the development of novel diagnostic methods of JD, it is important to investigate and understand the mRNA expression of the various immune markers in individuals at each stage of infection. In this study, we measured the levels of Th1-type chemokines, CXCR3, CCL4, CCL5, CXCL9, CXCL10, and CXCL11 in MAP-infected bovine blood by interferon (IFN)-γ release assay (IGRA) using IFN-γ as an alternative biomarker. The association of mRNA expression patterns of these chemokines with the MAP infection stages was analyzed and IFN-γ, CCL5, and CXCL10 were found to be significantly upregulated compared to IFN-γ, the biomarker used in IGRA. Our results further indicate that IFN-γ levels significantly increased in individuals with MAP-specific antibody, and CCL5 and CXCL10 levels significantly increased in those with MAP DNA. In particular, CCL5 was significantly upregulated in individuals, in which both MAP-specific antibody and MAP DNA were detected, but the expression of CXCL10 was specifically elevated in MAP DNA-detected individuals without MAP-specific antibody.

Genetic diversity of bovine Mycobacterium avium subsp. paratuberculosis discriminated by IS1311 PCR-REA, MIRU-VNTR, and MLSSR genotyping

The aim of this study was to describe the genetic diversity of Mycobacterium avium subsp. paratuberculosis (MAP) obtained from individual cows in Korea. Twelve MAP-positive fecal DNA samples and 19 MAP isolates were obtained from 10 cattle herds located in 5 provinces in Korea. In addition, 5 MAP isolates obtained from the Czech Republic and Slovakia and 3 isolates from Australia were genotyped for comparison with the domestic isolates. The most prevalent strains in Korea were of the “bison-type” genotype (23 of 31 fecal DNA/isolates) and were distributed nationwide. The remaining MAP isolates (8) and all of the foreign isolates were identified as “cattle-type”. The bison-type strains which were discriminated only as INMV 68 in variable-number tandem repeats of mycobacterial interspersed repetitive units (MIRU-VNTR) typing. Multilocus short sequence repeat (MLSSR) typing differentiated the bison-type strains into 3 different subtypes. The cattle-type strains were divided into 3 subtypes by MIRU-VNTR and 8 subtypes by MLSSR. The allelic diversities in the MIRU-VNTR and MLSSR results were calculated as 0.567 and 0.866, respectively. These results suggest that MIRU-VNTR typing cannot provide a sufficient description of the epidemiological situation of MAP. Therefore, an alternative method, such as MLSSR, is needed for typing of MAP strains to elucidate the molecular epidemiology of MAP infections. Overall, this study is the first epidemiological survey report in Korea using both MIRU-VNTR and MLSSR typing methods, and it has provided basic data necessary to elucidate the characteristics of MAP infections in Korea.

Potential biomarkers as an indicator of vertical transmission of Johne's disease in a Korean native cattle farm

Paratuberculosis (PTB) is caused by Mycobacterium avium subsp. paratuberculosis (MAP) and is one of the most widespread and economically important diseases in cattle. After birth, calves are raised with natural breast feeding without separation from their mothers in most Korean native cattle (Hanwoo breed) farms. Vertical transmission of PTB has been reported, but the exact PTB infection route has not been revealed in Hanwoo farms. Calves of MAP seropositive dams were tested for MAP presence and MAP antibodies in feces and tissues. MAP was detected in calf tissues by using polymerase chain reaction. Expressions of genes reported to be prognostic biomarkers of MAP infection changed in both calves and cows (p < 0.05). Expression of two genes (HGF and SERPINE1) were significantly decreased in MAP-infected cattle and their offspring (p < 0.01). The results suggest that biomarker gene expression profiles can be useful in detecting early stage MAP infection. Based on the results, complete eradication of MAP may be possible if accurate diagnostic methods to detect infected calves are added to the current PTB eradication strategy, which, because infected individuals are likely to develop into fecal MAP shedders at any time, includes isolation of new born calves and feeding sterilized colostrum.

Discovery of antigens for early detection of Mycobacterium avium subsp. paratuberculosis and analysis of characteristics using bioinformatics tools / 대한수의학회지

Johne's disease, caused by Mycobacterium avium subsp. paratuberculosis (MAP), is one of the most widespread and economically important diseases in cattle. Current diagnostic methods are based on the detection of anti-MAP antibodies in serum or isolation of the causative agent. However, these techniques are often not applicable for cases of subclinical infection due to relatively low sensitivity. Therefore, finding new antigen candidates that strongly react with the host immune system had been attempted. To effectively detect infection during the subclinical stage, several antigen candidates were selected based on previous researches. Characteristics of the selected antigen candidates were analyzed using bioinformatics-based prediction tools. A total of nine antigens were selected (MAP0862, MAP3817c, MAP2077c, MAP0860c, MAP3954, MAP3155c, MAP1204, MAP1087, and MAP2963c) that have MAP-specific and/or high immune responses to infected animals. Using a transmembrane prediction tool, five of the nine antigen candidates were predicted to be membrane protein (MAP3817c, MAP3954, MAP3155c, MAP1087, and MAP1204). Some of the predicted protein structures identified using the I-TASSER server shared similarities with known proteins found in the Protein Data Bank database (MAP0862, MAP1204, and MAP2077c). In future studies, the characteristics and diagnostic efficiency of the selected antigen candidates will be evaluated.

Virulence factors, antimicrobial resistance patterns, and genetic characteristics of hydrogen sulfide-producing Escherichia coli isolated from swine / 대한수의학회지

Escherichia (E.) coli is commensal bacteria found in the intestine; however, some pathogenic strains cause diseases in animals and humans. Although E. coli does not typically produce hydrogen sulfide (H2S), H2S-producing strains of E. coli have been identified worldwide. The relationship between virulence and H2S production has not yet been determined. Therefore, characteristics of H2S-producing isolates obtained from swine feces were evaluated including antibiotic resistance patterns, virulence gene expression, and genetic relatedness. Rates of antibiotic resistance of the H2Sproducing E. coli varied according to antibiotic. Only the EAST1 gene was detected as a virulence gene in five H2S-producing E. coli strains. Genes conferring H2S production were not transmissible although the seeA gene encoding 3-mercaptopyruvate sulfurtransferase was detected in all H2S-producing E. coli strains. Sequences of the seeA gene motif CGSVTA around Cys238 were also identical in all H2S-producing E. coli strains. Diverse genetic relatedness among the isolates was observed by pulsed-field gel electrophoresis analysis. These results suggested that H2S-producing E. coli strains were not derived from a specific clone and H2S production in E. coli is not associated with virulence genes.

Effective DNA extraction method to improve detection of Mycobacterium avium subsp. paratuberculosis in bovine feces / 대한수의학회지

Paratuberculosis caused by Mycobacterium avium subsp. paratuberculosis (MAP) has extended latent periods of infection. Due to this property, difficulties in the detection of fecal shedder have been raised. A newly designed method for DNA extraction from fecal specimens, mGITC/SC was evaluated in terms of diagnostic efficiency. The detection limit of IS900 real-time PCR was about 50 MAP (1.5 cfu) in 250 mg of feces (6 cfu per g). Also, this DNA extraction method was faster and cheaper than that using commercial kit or other methods. Consequently, the mGITC/SC is an economical DNA extraction method that could be a useful tool for detecting MAP from fecal specimens.