Estimation of the spread of pathogenic mycobacteria in organic broiler farms by the polymerase chain reaction

Organic poultry breeding allows for increased exposure of birds to soil, faeces, and wildlife, which have been associated with the transmission of mycobacterial infections. Therefore the aim of this study was to investigate the spread of the major pathogenic mycobacteria in organically reared broilers in Greece using a diagnostic algorithm that relied on a combination of the polymerase chain reaction (PCR) and the restriction fragment length polymorphism analysis (RFLP). Liver, spleen and gonads from 81 to 150 days old broilers were aseptically collected post-mortem. 500 broilers from a population of 35,370, reared in the 25 registered as organic farms in Greece for the 2005 were used. DNA was isolated and incorporated to PCR targeted to 16S-rRNA gene (for Mycobacterium spp.), IS6110 (for Mycobacterium tuberculosis complex-MTBc), IS1245 (for Mycobacterium avium complex-MAC), IS901 (for M. avium subsp. avium-MAA) and hsp65 (for Mycobacterium genavense, by PCR-RFLP). The mean prevalence of mycobacteria detected by PCR with a 95% confidence interval was estimated to 4.4-8.8%. The relevant percentage with regard to the mycobacterial species that were included in this study was 0.17-2.03% for MAC, 2.11-3.39% for MTBc and 0.66-3.08% for mycobacteria not belonging to any of the above groups. None of the mycobacteria detected were identified as MAA or M. genavense. Considering that avian tuberculosis has been eradicated from conventional farms, the level and the pattern of positivity recorded here, indicates that our results may be associated with the specific conditions that apply to organic breeding.     

大菱鲆脾肾结节病病原菌的分离和鉴定

2017年夏季，江苏省连云港市某养殖场大菱鲆出现大规模死亡，发病鱼体表无明显症状，解剖可见脾脏、肾脏出现白色散在结节，从发病鱼的内脏中分离得到1株优势菌SM-Myco001。人工感染实验结果显示，SM-Myco001可以引起大菱鲆内脏结节症状并造成鱼类死亡，且在高养殖水温(22℃)条件下发病更为剧烈。生理生化鉴定和16S rRNA基因分析结果显示，SM-Myco001属于海分枝杆菌(Mycobacterium marinum)。本研究首次报道了我国养殖大菱鲆感染海分枝杆菌的病例，可为大菱鲆养殖过程中的疾病防控提供参考。     

Strategy for the detection and differentiation of Mycobacterium avium species in isolates and heavily infected tissues

The members of Mycobacterium avium species, comprising M. avium subsp. paratuberculosis, M. a. hominissuis, M. a. avium, M. a. silvaticum, are currently the most prevalent opportunistic pathogenic mycobacteria causing mycobacterial infection in animals and humans. The ability to distinguish between these subspecies is of relevance for proper diagnosis and control programmes of the diseases. The aim of this study was to design a fast and specific PCR strategy for the detection and differentiation of M. avium subspecies from the solid plate cultures for use in routine veterinary diagnosis. We have developed a multiplex PCR based on IS900, IS901, IS1245 and the dnaJ gene. This method allows the detection of M. a. paratuberculosis, M. a. hominissuis and M. a. avium/M. a. silvaticum in one PCR reaction and theoretically enables mixed infections of M. a. paratuberculosis and M. a. avium or M. a. paratuberculosis and M. a. hominissuis to be revealed. The sensitivity of this multiplex PCR is 10(3)CFU for each bacterial strain in one PCR reaction, which also enabled the use of this test directly for DNA isolated from the tissue of the heavily infected sheep.     

IS1245 RFLP-based genotyping study of Mycobacterium avium subsp. hominissuis isolates from pigs and humans

Mycobacterium avium subsp. hominissuis, ubiquitous environmental mycobacterium, is an opportunistic pathogen that is regularly isolated from pigs and humans in Slovenia. Genetic diversity of 114 isolates from pigs (n = 57) and humans (n = 57), identified by means of bacteriology, DNA–RNA hybridization techniques, IS901 PCR and IS1245 PCR, was investigated in this study, using IS1245 restriction fragment length polymorphism (RFLP) analysis.Identical IS1245 RFLP profiles were found in isolates from pigs, isolates from humans and isolates from both origins. The proportion of clustered isolates varied as it depended on the similarity level (100% and 75%) chosen for the cluster limits. Using IS1245 RFLP, it was possible to detect monoclonal, polyclonal and recent infections and to monitor the genetic variability of the strains from individual patients.Our findings indicate the environment as the source of infection for both pigs and humans. The questions about the possibility of intra- and inter-species transmission of infection remain to be answered.     

Pathological and PCR detection of mycobacteriosis in pond-cultured Chinese soft shell turtles, Trionyx sinensis

Mycobacteriosis due to infection of Mycobacterium marinum is a common disease in pond-cultured Chinese soft shell turtles, especially in those surviving beyond their first year. The infected turtles independently showed either heterophilic or histiocytic granulomas in various organs such as the spleen, liver, lungs, intestine, kidneys, stomach and pancreas. The heterophilic granuloma contained many acid-fast unbranching bacilli intracellularly in macrophages and extracellularly in the necrotic center. The histiocytic granuloma had only a few bacteria, mainly in the cytoplasm of Langhan's giant cells. The organisms were rarely observed in the advanced lesions of both types. Based on PCR assays for partial hsp65 gene of Mycobacterium spp., all of our strains were identified as M. marinum which can be divided into two groups. The strains of the first group induced heterophilic granulomas and had very high nucleotide sequence identities (99.8%-100%) to the reference strains of M. marinum (AF456471) and M. pseudoshottsii (AY550226). Those strains of the second group caused histiocytic granulomas and also showed very high identities (99.8%-100%) to the reference strains of M. marinum ATCC 927 (AF456470) and M. shottsii (AY550225). However, when we compared the partial sequence of the hsp65 gene from group one and two strains the identities between the two groups range from 98.8% to 99.3%, therefore we can not assert that these two belong to the same species.