Mycobacterium haemophilum infections of zebrafish (Danio rerio) in research facilities

In May 2005, a disease outbreak was investigated at a zebrafish (Danio rerio) research facility experiencing severe losses. Mycobacterium haemophilum was isolated from these fish and the disease was subsequently recreated in experimentally infected zebrafish. Fish exhibited signs characteristic of mycobacteriosis, including granuloma formation and severe, diffuse, chronic inflammation. Bacteria were observed in multiple tissues, including the central nervous system. Biofilm samples from the outbreak facility were PCR positive for M. haemophilum, suggesting biofilms might act as a reservoir for infection. Zebrafish appear to be particularly vulnerable to M. haemophilum, and measures such as quarantine and treatment of incoming water should be implemented to minimize the likelihood of introduction of this bacterium to zebrafish research facilities. Zebrafish are already a well-established laboratory animal model for genetics, toxicology and disease, their susceptibility to M. haemophilum may make them useful for the study of this bacterium in the future.     

Pathogenesis of Mycobacterium spp. in zebrafish (Danio rerio) from research facilities

One of the most common diseases that we have diagnosed in zebrafish is mycobacteriosis, caused by several Mycobacterium spp. The severity of the disease ranged from severe outbreaks to incidental infections. We conducted an in vivo study to evaluate the pathogenesis of six isolates of Mycobacterium from zebrafish with mycobacteriosis from four research facilities and one wholesale supplier of zebrafish in the United States: Mycobacterium abscessus, Mycobacterium peregrinum, Mycobacterium chelonae (2 isolates), and Mycobacterium marinum. We also included two isolates of M. marinum from other fishes. Fish were exposed by intraperitoneal injection at a target does of 5 x 10(4) bacteria/fish, and were held in static aquaria at 28 degrees C for 8 weeks. Fish were examined by histology and culture, and mortalities were recorded. The M. marinum isolates caused 100% infection and mortality between 30% and 100%. None of the other Mycobacterium species caused significant mortalities, but several of these fish had granulomatous lesions in visceral organs. Mycobacteria were consistently recovered in culture from fish exposed to M. marinum, and from only 9% of fish exposed to the other species. This study suggests that, of the isolates tested, only M. marinum is highly pathogenic and virulent to healthy zebrafish.     

Further studies of a new pathogenic mycobacterium (M. haemophilum sp. nov.).

Mycobacterium haemophilum is an acid-fast rod-shaped organism, originally isolated from deep subcutaneous granulomata of a patient with Hodgkin's disease. Like the other two mycobacterial skin-pathogens, M. ulcerans and M. marinum, M. haemophilum has a maximum temperature for growth below 37 degrees C. Mycobacterium haemophilum is distinguished from all other species examined by its requirement of haemin for growth and its complete lack of catalase activity. Extraneous catalase cannot replace haemin as a growth factor for this organism. Mycobacterium haemophilum can also be differentiated from other species by the patterns of electrophoresis of protein extracts and by gas-liquid chromatography of saponificated and methylated lipid extracts. A monospecific-agglutinating antiserum against M. haemophilum was obtained by adsorption of an immunoserum with M. intracellulare. A number of slow-growing mycobacterial species develop on monolayers of McCoy fibroblasts, and growth on these tissue cultures can be observed much earlier than on artificial media. Mycobacterium haemophilum is characterized by exclusively intracellular development.     

Proposal of Mycobacterium peregrinum sp. nov., nom. rev., and elevation of Mycobacterium chelonae subsp. abscessus (Kubica et al.) to species status: Mycobacterium abscessus comb. nov.

We studied the taxonomic positions of the rapidly growing organism Mycobacterium fortuitum and phenotypically related organisms. We confirmed that "Mycobacterium peregrinum" ATCC 14467T (T = type strain) is genetically independent of M. fortuitum ATCC 6841T by using various DNA hybridization conditions. Strains that were genetically identified as "M. peregrinum" were phenotypically differentiated from M. fortuitum ATCC 6841T. Thus, we propose that "M. peregrinum" should be revived as an independent species, Mycobacterium peregrinum sp. nov., nom. rev. The type strain is strain ATCC 14467. M. fortuitum subsp. acetamidolyticum ATCC 35931T exhibited a high level of DNA relatedness to M. fortuitum ATCC 6841T. The hybridized DNAs maintained stable heteroduplexity at high stringency; thus, we confirmed that M. fortuitum subsp. acetamidolyticum is identical to M. fortuitum ATCC 6841T. We found that M. chelonae subsp. abscessus ATCC 19977T is genetically different from M. chelonae subsp. chelonae NCTC 946T on the basis of the results of quantitative hybridization even under optimal conditions. There was no reason to maintain this organism as a subspecies of M. chelonae. Thus, we propose that M. chelonae subsp. abscessus should be elevated to species status as Mycobacterium abscessus (Kubica et al.) comb. nov. The type strain is strain ATCC 19977.     

Mycobacterium marinum infections in humans and tracing of its possible environmental sources

The low frequency of nontuberculous mycobacterial infections, nonspecific symptoms for individual mycobacteria, and the lack of specific identification methods could alter correct diagnosis. This study presents a combined microbiology and molecular-based approach for Mycobacterium?marinum detection in four aquarists with cutaneous mycobacterial infection. Simultaneously, ecology screening for M.?marinum presence in the aquarists' fish tanks was performed. A total of 38 mycobacterial isolates originated from four human patients (n?=?20), aquarium animals (n?=?8), and an aquarium environment (n?= 10). Isolate identification was carried out using 16S?rRNA sequence analysis. A microbiology-based approach, followed by 16S rRNA sequence analysis, was successfully used for detection of M.?marinum in all four patients. Animal and environmental samples were simultaneously examined, and a total of seven mycobacterial species were isolated: Mycobacterium chelonae , Mycobacterium fortuitum , Mycobacterium gordonae , Mycobacterium?kansasii , Mycobacterium mantenii , Mycobacterium marinum , and Mycobacterium?peregrinum . The presence of M.?marinum was proven in the aquarium environments of two patients. Although M.?marinum is described as being present in water, it was detected only in fish.     

Mycobacterium marinum produces long-term chronic infections in medaka: a new animal model for studying human tuberculosis

Human infection by Mycobacterium tuberculosis is endemic, with approximately 2 billion infected and is the most common cause of adult death due to an infectious agent. Because of the slow growth rate of M. tuberculosis and risk to researchers, other species of Mycobacterium have been employed as alternative model systems to study human tuberculosis (TB). Mycobacterium marinum may be a good surrogate pathogen, conferring TB-like chronic infections in some fish. Medaka (Oryzias latipes) has been established for over five decades as a laboratory fish model for toxicology, genotoxicity, teratogenesis, carcinogenesis, classical genetics and embryology. We are investigating if medaka might also serve as a host for M. marinum in order to model human TB. We show that both acute and chronic infections are inducible in a dose dependent manner. Colonization of target organs and systemic granuloma formation has been demonstrated through the use of histology. M. marinum expressing green fluorescent protein (Gfp) was used to monitor bacterial colonization of these organs in fresh tissues as well as in intact animals. Moreover, we have employed the See-Through fish line, a variety of medaka devoid of major pigments, to monitor real-time disease progression, in living animals. We have also compared the susceptibility of another prominent fish model, zebrafish (Danio rerio), to our medaka-M. marinum model. We determined the course of infections in zebrafish is significantly more severe than in medaka. Together, these results indicate that the medaka-M. marinum model provides unique advantages for studying chronic mycobacteriosis.     

鲟分枝杆菌病及其病原研究

20092010年间,我国人工养殖的中华鲟（Acipenser sinensis）、史氏鲟（Acipenser schrencki）和杂交鲟（hybrid sturgeon:A. baeri-A. gueldenstaedtii）暴发了细菌性疾病。患病鲟通过组织切片观察,病原菌的分离、鉴定以及组织样品中病原菌的检测,结果显示从19条患病鲟中分离到49株分枝杆菌。病原菌经过多个保守基因的测序分析和部分生理生化特征的鉴定,共发现有7种分枝杆菌,分别为龟分枝杆菌（Mycobacterium chelonae）、海分枝杆菌（Mycobacterium marinum）、戈登氏分枝杆菌（Mycobacterium gordonae）、偶发分枝杆菌（Mycobacterium fortuitum）、苏尔加分枝杆菌（Mycobacterium szulgai）、猪分枝杆菌 （Mycobacterium porcinum）和Mycobacterium arpuense。在诊断过程中发现两种或三种分枝杆菌同时存在于同一样品中,分子生物学的诊断结果表明分枝杆菌复合感染十分常见,而海分枝杆菌是分枝杆菌复合感染中最为常见的分枝杆菌。分离的病原菌对斑马鱼的攻毒试验结果表明在以上7种分枝杆菌中海分枝杆菌的毒力最强。以上结果表明海分枝杆菌是鲟分枝杆菌病的主要致病菌,分枝杆菌复合感染是鲟分枝杆菌病的主要感染形式。研究中史氏鲟和中华鲟的分枝杆菌病,以及在病鱼体内分离的猪分枝杆菌和M. arupense在国内外均尚未见报道。       

Evaluation of antimicrobial susceptibilities of rapidly growing mycobacteria by Sensititre RAPMYCO panel

This study used Sensititre RAPMYCO to test the activities of amikacin, cefoxitin, ciprofloxacin, clarithromycin, doxycycline, imipenem, linezolid, sulfamehoxazole, tigecycline and tobramycin against 25 clinical isolates of rapidly growing mycobacteria (RGM), including the common disease producing species Mycobacterium abscessus, Mycobacterium chelonae, Mycobacterium fortuitum and Mycobacterium peregrinum. Analysis of the four different RGM species showed that isolates of M. fortuitum and M. peregrinum were more susceptible than M. abscessus and M. chelonae. Different antimicrobials showed a variable sensitivity in all strains. Therefore, each species and strain must be individually evaluated, and it is always advisable to perform in vitro sensitivity tests before the treatment of infections due to RGM.     

Pseudoloma neurophilia n. g., n. sp., a new microsporidium from the central nervous system of the zebrafish (Danio rerio)

An unusual xenoma-forming microsporidium was discovered in the central nervous system of moribund zebrafish from a laboratory colony in Eugene, Oregon. Infected fish were often emaciated and lethargic, and histological examination commonly revealed severe myelitis and myositis associated with the infection. Based on its structure, development, and small subunit ribosomal DNA sequence it is unique among fish microsporidia. Spores are uninucleate, ovoid to pyriform, with a prominent posterior vacuole. Spores average 5.4 x 2.7 microm with 13-16 coils of the polar filament. The microsporidium produces xenomas within the spinal cord and hindbrain of fish, and xenomas contained sporophorous vesicles with up to 16 spores. Sporoblasts and presporoblast stages (probably sporonts) are found occasionally in small aggregates dispersed randomly throughout xenomas. It clustered in the "Ichthyosporidium group" along with other fish microsporidian genera based on rDNA sequence analysis. The rDNA sequence of the zebrafish microsporidium was most similar to that of Ichthyosporidium, but showed only 12.1% similarity and therefore this microsporidium can be considered a distinct genus and species, which we have named Pseudoloma neurophilia n. g., n. sp.     

Detection of mycobacteria in aquarium fish in Slovenia by culture and molecular methods

Thirty-five aquarium fish were investigated for the presence of mycobacteria by culture and molecular methods. The following species were examined: goldfish Carassius auratus auratus, guppy Poecilia reticulata, 4 three-spot gourami Trichogaster trichopterus, dwarf gourami Colisa lalia, Siamese fighting fish Betta splendens, freshwater angelfish Pterophyllum scalare, African cichlid fish Cichlidae spp., cichlid fish Microgeophagus altispinosus, cichlid fish Pseudotropheus lombardoi, blue streak hap Labidochromis caeruleus, sterlet Acipenser ruthenus, southern platyfish Xiphophorus maculatus, and catfish Corydoras spp. Isolates of mycobacteria were obtained in 29 cases (82.9%). Two specimens were positive using Ziehl-Neelsen (ZN) staining, but the cultivation failed. Four specimens were both ZN- and culture-negative. On the basis of GenoType Mycobacterium assay (Hain Life-science) and restriction enzyme analysis of the amplified products (PCR-RFLP), 23 isolates (79.3%) were identified: 7 as Mycobacterium fortuitum, 6 as M. gordonae, 6 as M. marinum, 3 as M. chelonae, and 1 as M. peregrinum. Five isolates remained unidentified (Mycobacterium spp.). One case probably represented a mixed infection (M. marinum/M. fortuitum). Since M. marinum infections are also detected in humans, the significance of mycobacteria in aquarium fish should not be overlooked.     

Identification of a repetitive DNA sequence specific to Mycobacterium paratuberculosis

Abstract A 0.2-kb DNA sequence specific to Mycobacterium paratuberculosis , the causative organism of Johne's disease, was isolated from a partial genomic library. The sequence was part of a larger repetitive DNA element and was present in strains of M. paratuberculosis from cattle, sheep, goat, deer and also a woman with Crohn's disease but not in M. paratuberculosis strain 18. The sequence was not present in strains of 19 other mycobacterial species including 31 reference serotype strains of the M. avium-M. intracellular-M. scrofulaceum (MAIS) complex, some strains of which are closely related to M. paratuberculosis . The sequence may be useful for developing a diagnostic test for Johne's disease.     

Rapid Detection and Identification of Nontuberculous Mycobacterial Pathogens in Fish by Using High-Resolution Melting Analysis

Mycobacterial infections in fish are commonly referred to as piscine mycobacteriosis, irrespectively of the specific identity of the causal organism. They usually cause a chronic disease and sometimes may result in high mortalities and severe economic losses. Nearly 20 species of Mycobacterium have been reported to infect fish. Among them, Mycobacterium marinum, M. fortuitum, and M. chelonae are generally considered the major agents responsible for fish mycobacteriosis. As no quick and inexpensive diagnostic test exists, we tested the potential of high-resolution melting analysis (HRMA) to rapidly identify and differentiate several Mycobacterium species involved in fish infections. By analyzing both the melting temperature and melting profile of the 16S-23S rRNA internal transcribed spacer (ITS), we were able to discriminate 12 different species simultaneously. Sensitivity tests conducted on purified M. marinum and M. fortuitum DNA revealed a limit of detection of 10 genome equivalents per reaction. The primers used in this procedure did not lead to any amplification signal with 16 control non-Mycobacterium species, thereby demonstrating their specificity for the genus Mycobacterium.     

Diagnosis and management of atypical Mycobacterium spp. infections in established laboratory zebrafish (Brachydanio rerio) facilities

Two established zebrafish colonies experienced increased mortality and decreased reproductive performance. Initial examination of several fish from one facility revealed hyperemic gills, petechia around the opercula, abdominal distention, and emaciation. Affected fish had congested liver with inflammation and multifocal hepatic necrosis. Large numbers of acid-fast-positive, rod-shaped bacteria were evident in multiple tissues and the blood. Mycobacterium fortuitum was subsequently isolated from several fish. Zebrafish from the second facility had skin erosions and ulceration along the flank just caudal to the pectoral fins. Large numbers of acid-fast-positive, rod-shaped bacteria were observed within the necrotic centers of well-demarcated, multifocal granulomas in gonads, liver, and peritoneum from affected fish. Mycobacterium abscessus and M. chelonae were isolated and identified biochemically. Definitive diagnosis in these outbreaks was obtained by culture on selective media. Because Mycobacterium spp. grow extremely slowly and positive confirmation may require 45 to 60 days, Mycobacterium species-specific polymerase chain reaction analysis was used to provide a rapid screening assay for Mycobacterium spp. as well as for verification of culture results. To our knowledge, this is the first documentation of mycobacterial infection in laboratory-maintained zebrafish and provides guidelines for diagnosis, management, and prevention of atypical mycobacteriosis in laboratory zebrafish colonies.     

Disseminated mycobacteriosis affecting a prosthetic aortic valve: first case of Mycobacterium peregrinum type III reported in Colombia

Rapidly growing mycobacteria are non-tuberculous mycobacteria amply present in the environment. Although they are not usually pathogenic for humans, they are opportunistic in that they can cause disease in people with disadvantageous conditions or who are immunocompromised. Mycobacterium peregrinum, an opportunistic, rapidly growing mycobacteria, belongs to the M. fortuitum group and has been reported as responsible for human cases of mycobacteriosis. A case of M. peregrinum type III is herein reported as the first in Colombia. It presented as a disseminated disease involving a prosthetic aortic valve (endocarditis) in a seventeen-year-old girl with a well-established diagnosis of prosthetic aortic valve endocarditis who was referred for a surgical replacement. Due to a congenital heart disease (subaortic stenosis with valve insufficiency), she had two previous aortic valve implantation surgeries. One year after the second implantation, the patient presented with respiratory symptoms and weight lost indicative of lung tuberculosis. A chest X-ray did not show parenchymal compromise but several Ziehl-Neelsen stains were positive. An echocardiography showed a vegetation on the prosthetic aortic valve. In blood and sputum samples, M. peregrinum type III was identified through culture, biochemical tests and hsp65 gene molecular analysis (PRA). The patient underwent a valve replacement and received a multidrug antimycobacterial treatment. Progressive recovery ensued and further samples from respiratory tract and blood were negative for mycobacteria.     

A characteristic phenolic glycolipid antigen from Mycobacterium haemophilum

A characteristic phenolic glycolipid was detected, by thin layer chromatography, in non-polar lipid extracts of nine representative strains of Mycobacterium haemophilum . The lipid was a specific antigen that reacted strongly with serum raised against whole cells of M. haemophilum. Sera from six other mycobacterial strains were tested but only that from Mycobacterium kansasii give a weak reaction.     

Transmission, diagnosis, and recommendations for control of Pseudoloma neurophilia infections in laboratory zebrafish (Danio rerio) facilities

The microsporidium Pseudoloma neurophilia represents a considerable challenge for laboratory zebrafish (Danio rerio) facilities. In 2010, P. neurophilia infections were diagnosed in zebrafish from 74% of the facilities that submitted fish to the Zebrafish International Resource Center (ZIRC) pathology service, and this organism remains the most commonly diagnosed pathogen in submitted fish. Accordingly, many of the ZIRC pathology service consultations deal with control and prevention of microsporidiosis. Here we describe observations and experiments performed at the ZIRC elucidating aspects of P. neurophilia transmission in zebrafish colonies. We then review current knowledge about P. neurophilia transmission and diagnosis. Considering this information, we present recommendations for control of P. neurophilia in zebrafish facilities.     

Parvicapsula pseudobranchicola (Myxosporea) in farmed Atlantic salmon Salmo salar. Tissue distribution, diagnosis and phylogeny

Parvicapsula pseudobranchicola infections in farmed Atlantic salmon in Norway are associated with low-grade to significant mortalities. The parasite is found as mature spores in pseudobranchs, but has also been detected in the gills, liver and kidney. Diagnosis has relied on the detection of Parvicapsula spores, with the pseudobranch being the preferred organ. A better understanding of the epizootiology of this myxosporean is a prerequisite for appropriate management and control. Hence, early detection of infections and life cycle studies are needed. We sequenced the small subunit (ssu) rDNA (18S) from P. pseudobranchicola and developed a sensitive diagnostic PCR protocol. This allowed us to (1) identify appropriate tissues for diagnostic assays, (2) examine the intraspecific variation in ssu rDNA in the parasite's Norwegian range, (3) examine annelid potential primary hosts and (4) obtain additional ssu rDNA sequences of marine Parvicapsula species to perform a phylogenetic study. Primers were constructed targeting the ssu rDNA from P. minibicornis. With these we obtained a partial ssu sequence of the P. pseudobranchicola type isolate. A new set of primers (PCF3/PCR3) was constructed for diagnostic purposes. These were tested against DNA from the host and several myxozoan species infecting Norwegian salmon. The primers give a positive product of 203 bp and pick out P. pseudobranchicola in salmnonids. They also amplify the congeners P. unicornis and P. asymmetrica infecting unrelated fish. The PCR protocol developed showed a greater sensitivity than light microscopy. The pseudobranchs were always positive and are the recommended organ for PCR diagnostics. There was no sequence variation between geographic isolates from farmed salmon. Preliminary examinations of marine polychaetes and oligochaetes collected from farm sites with parvicapsulose problems were negative. A comparison of the sequence of the ssu rDNA from P. pseudobranchicola with that of other myxozoans shows that it groups closely together with P. unicornis and P. asymmetrica. The closest relative to this group is P. minibicornis.     

Mycobacterium ulcerans causes minimal pathogenesis and colonization in medaka (Oryzias latipes): an experimental fish model of disease transmission

Mycobacterium ulcerans causes Buruli ulcer in humans, a progressive ulcerative epidermal lesion due to the mycolactone toxin produced by the bacterium. Molecular analysis of M. ulcerans reveals it is closely related to Mycobacterium marinum, a pathogen of both fish and man. Molecular evidence from diagnostic PCR assays for the insertion sequence IS2404 suggests an association of M. ulcerans with fish. However, fish infections by M. ulcerans have not been well documented and IS2404 has been found in other mycobacteria. We have thus, employed two experimental approaches to test for M. ulcerans in fish. We show here for the first time that M. ulcerans with or without the toxin does not mount acute or chronic infections in Japanese Medaka "Oryzias latipes" even at high doses. Moreover, M. ulcerans-infected medaka do not exhibit any visible signs of infection nor disease and the bacteria do not appear to replicate over time. In contrast, similar high doses of the wild-type M. marinum or a mycolactone-producing M. marinum "DL" strain are able to mount an acute disease with mortality in medaka. Although these results would suggest that M. ulcerans does not mount infections in fish we have evidence that CLC macrophages from goldfish are susceptible to mycolactones.     

Distribution of a novel mycolic acid in species of the genus Mycobacterium

We found that Mycobacterium porcinum ATCC 33776T (T = type strain) contains a new kind of mycolic acid with a methoxy group at the omega-1 position. This mycolic acid was identified by comparing it with the previously described methoxymycolic acids. The patterns of mycolic acid methyl esters from 418 strains belonging to 44 species of mycobacteria were studied by using thin-layer chromatography. In addition to M. procinum ATCC 33776T, representative strains of M. porcinum, Mycobacterium fortuitum, "Mycobacterium peregrinum," Mycobacterium senegalense, and a recently isolated Mycobacterium sp. contained appreciable amounts of the newly described mycolic acid.