검색 상세

Diagnostic and epidemiological approach for paratuberculosis and bovine tuberculosis

  • 김나래 (대학원 의생명융합과학과 임상병리학전공)

초록/요약

Paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis (MAP), is a chronic incurable infection of the intestinal tract of animals. In this study, a rapid and sensitive nanoparticle-based method for directly detecting MAP by magnetic capture using anti-MAP chicken IgY-conjugated magnetic nanoparticles (MNPs), followed by IS900 polymerase chain reaction (PCR), was developed. 105, 104, 103, 102, 101 or 1 MAP cells were spiked in pooled stool samples then detected using antibody-conjugated MNPs and confirmed using PCR. The binding ratio of antibody–MNPs was 96.44 ± 0.77%, and one MAP could be detected using the IS900 primer set, even in the pooled stool environment. The detection limit was 20 MAP cells per gram of feces. The effects of MNPs and commercially available paramagnetic beads conjugated to different concentrations of antibody were evaluated, with MNPs showed better performing than that of paramagnetic beads. Moreover, the use of MNPs is cost-effective. Antibody-conjugated MNPs did not react with other bacteria, showing that this immunomagnetic separation method has good specificity. With the development of a new tool using antibody-conjugated MNPs and PCR, MAP was detected in cattle feces; the confirmation time was reduced from 1216 weeks to 23 days. This antibody-based immunocapture PCR method provides a useful platform for rapid screening for MAP in fecal samples in one step. Mycobacterium bovis is a pathogen that is well known for causing bovine tuberculosis to animals and humans. In this study, an immunochromatographic test (ICT) strip using recombinant MPB70 protein was evaluated for detecting naturally occurring tuberculosis in cattle. The study included 249 cattle from populations known to be free from M. bovis and 119 cattle with M. bovis infection, confirmed by postmortem examination. Compared to reference standards (culture isolation and/or visible lesion), the sensitivity of ICT was 94.12% (95% CI: 89.89–98.35%) while the specificity was 96.80% (95% CI: 94.62–96.82%). The findings indicate that the ICT strip is efficient for diagnosing bovine tuberculosis in cattle from Korea. Moreover, molecular and genomic experiments for M. bovis Korean isolates were performed to reveal the characteristics of Korean isolates. M. bovis Korean isolates showed SB0140 and SB1040 in spoligotyping and various profiles in mycobacterial interspersed repetitive units-variable numbers of tandem repeats (MIRU-VNTR) typing. Among these, M. bovis B-1595, B-3222, and W-1171 were sequenced consequently using next-generation sequencing methods. They showed similar sizes of genome, GC contents, and protein coding sequences. In addition, a brief comparative genomic analysis with genomes of Mycobacterium tuberculosis complex strains was performed. It also reveled that all genomes of the strains have similar genome size and GC content. In phylogenetic analysis, all strains showed 99.9% similarity in average nucleotide identity value. Genome comparison was based on BLASTP using M. bovis AF2122/97 as a reference sequence, the most similar strain with M. bovis B-1595. The genomes sequenced in this study will serve as a valuable reference for improving the understanding of the disparity in the virulence and epidemiologic traits between M. bovis genotypes in South Korea.

more

목차

Chapter 1. Johne’s disease
1. Introduction ………………………………………………………………………….… 1
2. Materials and methods
2.1 Immunomagnetic separation method for MAP ………………………………… 3
2.1.1 Bacterial strains and culture condition …………………………………….… 3
2.1.2 Bacterial confirmation …………………………………………………….… 4
2.1.3 Preparation of antibody-conjugated MNPs or PMBs ………………………… 5
2.1.4 Bacterial detection using antibody-conjugated MNPs or PMBs ……………… 6
2.1.5 Dose-effect of antibody …………………………………………………….… 7
2.1.6 Statistical analysis ………………………………………………………….… 7
3. Results
3.1 Immunomagnetic separation method for MAP ………………………………… 8
3.1.1 Characteristics of MNPs and combination with MAP ………………………… 8
3.1.2 Bacterial detection and dose effect of antibody ……………………………… 11
4. Discussion ……………………………………………………………………………… 14


Chapter 2. Bovine tuberculosis
1. Introduction ……………………………………………………………………….…. 17
2. Materials and methods
2.1 General characteristics of M. bovis isolates ……………………………………… 22
2.1.1 Bacterial detection …………………………………………………………… 22
2.1.2 M. bovis-specific PCR ………………………………………………………… 22
2.1.3 Histological examination and microscopy …………………………………… 23
2.2 Immunochromatographic test for detection of M. bovis ………………………… 24
2.2.1 Animals and sample treatment …………………………………………………24
2.2.2 Principle and procedure of the ICT …………………………………………… 25
2.2.3 Data analysis ………………………………………………………………… 25
2.3 Molecular identification of M. bovis isolates …………………………………… 26
2.3.1 Bacterial isolation …………………………………………………………… 26
2.3.2 Genomic DNA extraction …………………………………………………… 26
2.3.3 Spoligotyping ………………………………………………………………… 27
2.3.4 MIRU-VNTR ………………………………………………………………… 28
2.4 Genomic investigation of M. bovis isolates ……………………………………… 30
2.4.1 Genome sequencing …………………………………………………………… 30
2.4.2 Genome sequence assembly and annotation ………………………………… 31
2.4.3 Comparative genomic analysis ……………………………………………… 32
3. Results
3.1 General characteristics of M. bovis isolates ……………………………………… 34
3.1.1 M. bovis-specific PCR ………………………………………………………… 34
3.1.2 Histological examination and microscopy …………………………………… 34
3.2 Immunochromatographic test for detection of M. bovis ………………………… 39
3.2.1 Sensitivity and specificity of the ICT strip …………………………………… 39
3.2.2 Association between ICT results and bTB status …………………………… 41
3.3 Molecular identification of M. bovis isolates …………………………………… 41
3.4 Genomic investigation of M. bovis isolates ……………………………………… 45
3.4.1 M. bovis B-1595 ……………………………………………………………… 45
3.4.2 M. bovis B-3222 ……………………………………………………………… 47
3.4.3 M. bovis W-1171 ……………………………………………………………… 49
3.4.4 Comparative genomics ……………………………………………………… 52
4. Discussion
4.1 Immunochromatographic test for detection of M. bovis ………………………… 61
4.2 Molecular and genomic investigations …………………………………………… 63

List of reference …………………………………………………………………………… 69
국문요약 ………………………………………………………………………….….….… 86

more
반출 Meta View 목록