Differences in susceptibility to Mycobacterium chelonae in zebrafish (Danio rerio) lines commonly used in scientific research.

Mycobacteriosis is one of the most common diseases encountered in laboratory zebrafish. These infections can present a problem to researchers using zebrafish because they may introduce unknown experimental variables. Whilst differences in severity of infections between species of Mycobacterium infecting zebrafish have been well documented, little is known about differences in susceptibility between zebrafish lines. Previous surveys have found higher prevalence in the TU zebrafish line relative to other lines, suggesting that there may be underlying genetic differences in susceptibility. This study investigates Mycobacterium chelonae H1E2-GFP infections in four different zebrafish lines commonly used in research (AB, 5D, casper and TU). Fish were exposed to a labelled (green-fluorescent protein (GFP)) strain of M. chelonae by intraperitoneal injection, and infection status was evaluated after 10 weeks. Visualization of GFP in euthanized fish and histology were used as endpoints. In GFP images, severity was assessed by image analysis, and in histological sections, counts of granulomas containing acid-fast bacteria were used. Results indicated differences in severity of infections between lines, but no significant differences in prevalence.

GENETIC DIVERSITY OF CYSTODISCUS SPECIES IN AMPHIBIANS IN THE SOUTHERN UNITED STATES.

Myxosporean species in the genus Cystodiscus are parasites of amphibians and have been reported from several continents. Typically used for the identification of myxozoans, the spores produced by these species are similar to one another, possessing 2 polar capsules and being ovoid. The number of transverse depressions on the spore can be useful for delineating species, but these can sometimes be difficult to distinguish. In North America, Cystodiscus serotinus and Cystodiscus melleni have been described, and for C. serotinus in particular, numerous reports and a wide range of hosts have been associated with this species. Given the challenges of identifying some of these species, we questioned whether all encounters of Cystodiscus species can be attributed to these 2 described species, or if there may be additional undescribed species or cryptic species. Over 7 yr, 383 amphibians representing 13 species of toads, frogs, and salamanders were collected from sites in Oklahoma and Arkansas. Cystodiscus infections were found in 56 individuals (14.6%). Tissues from these infected individuals were preserved in alcohol for genetic analysis. The small subunit (SSU) and large subunit (LSU) ribosomal RNA genes were partially sequenced and analyzed phylogenetically. Nine distinct SSU sequence types and 7 distinct LSU sequence types were identified. Phylogenetically, sequence types were attributable to C. serotinus, C. melleni, Cystodiscus axonis, and an undescribed species. For the previously described species, there were multiple SSU sequence types: 4 for C. serotinus and 2 for both C. melleni and C. axonis. Phylogenetic patterns were similar for the LSU sequence analysis using a shorter sequence than the SSU, and we propose that the LSU is useful for initial barcoding of Cystodiscus species in any future surveys. In our qualitative assessment of sequence types compared to geography and host species, SSU types C1 and C2 (C. axonis) were only found in Union County, Arkansas, and McCurtain County, Oklahoma, respectively. Also, salamanders were only infected with SSU types B or D (C. melleni), and type B was only found in salamanders. Our finding of C. axonis in North America is notable because this species was described in Australia and is associated with host pathology. Our work reveals that there are cryptic species of Cystodiscus in the United States, one of which may be a pathogen, highlighting the importance of genetic analysis for future surveys of these species.

A New Species of ThelohanellusKudo, 1933 (Myxozoa: Bivalvulida) Infecting Skeletal Muscle of Blacktail Shiner, Cyprinella venusta Girard, 1856 (Cypriniformes: Cyprinidae) in the Chattahoochee River Basin, Georgia.

Thelohanellus magnacysta n. sp. (Bivalvulida: Myxobolidae) infects the skeletal muscle of blacktail shiner, Cyprinella venusta Girard, 1856 (Cypriniformes: Cyprinidae) in Bull Creek, Chattahoochee River Basin, eastern Georgia. Although numerous members of ThelohanellusKudo, 1933 have overlapping myxospore dimensions with the new species, it differs from all nominal congeners by polar filament coil number and polar capsule width as well as by lacking a mucous envelope, iodinophilic vacuole, and sutural markings. With the use of novel primers for Myxozoa, a phylogenetic analysis of the small subunit ribosomal DNA (SSU rDNA) suggests that the new species shares a recent common ancestor with a clade of cyprinid-infecting species of Myxobolus Bütschli, 1882 (Bivalvulida: Myxobolidae) and Thelohanellus. Consistent with other published research concerning the systematics of Thelohanellus, this result suggested that Thelohanellus and Myxobolus are polyphyletic and need revision. Histological sections of infected blacktail shiners confirmed that myxospores were only found within a plasmodium and only infected skeletal muscle and that plasmodia were encapsulated by a granuloma comprising varying degrees of acute granulomatous inflammation. The new species is the fourth of Thelohanellus reported from North America and the first reported from Cyprinella, as well as the first myxozoan described from the blacktail shiner.

Gastrointestinal parasites of the New England cottontail rabbit (Sylvilagus transitionalis) and eastern cottontail rabbit (Sylvilagus floridanus) in the Hudson Valley, New York.

The New England cottontail rabbit (NEC, Sylvilagus transitionalis) population has decreased dramatically in New York, USA, and the role of parasites in limiting the population has never been examined. The closely related and sympatric eastern cottontail rabbit (EC, Sylvilagus floridanus) was introduced into the range of NEC by humans and is currently thriving. This study aimed to investigate gastrointestinal parasites of the NEC and the EC and compare their parasite communities. Fecal pellets from 195 NEC and 125 EC were collected from the Hudson Valley, New York, in the winter of 2013-2014. Centrifugal fecal floats were performed in Sheather's sugar solution, and parasite ova and cysts were examined microscopically to identify gastrointestinal parasites present. For all pellets combined (n = 320), 91% were found to harbor at least 1 parasite species, with Eimeria species being the most common. Genetic analysis of pellets using microsatellite DNA identified 248 individual rabbits, with parasite prevalence (94%) similar to the prevalence estimate based on all pellets (91%). EC samples had a significantly higher (p < 0.05) parasite species richness (1.73, range 0-4) than NEC (1.20, range 0-3). EC and NEC shared 3 moderate to high (9-89%) prevalence parasites, in which EC prevalence was consistently higher. One parasite species was only found in NEC, and two were only found in EC, but the majority of these were of low abundance, precluding further statistical analyses.

Multiple evolutionary routes of the single polar capsule in Thelohanellus species (Myxozoa; Myxobolidae).

Thelohanellus Kudo, 1933 is a species rich genus of Myxosporea, sharing many morphological similarities with species of Myxobolus but the former possesses a single polar capsule, and the latter has two. Based on molecular phylogenetic analyses, this single distinguishing feature is not monophyletic, and members of Thelohanellus are intermixed with Myxobolus species, calling into question the validity of genus Thelohanellus. The occurrence of two polar capsules in a small proportion of Thelohanellus spores as observed in this study suggests that these species have the capacity to express this Myxobolus-like trait, clouding the distinction of these two genera further. Herein, using the most comprehensive data set to date, we explored the phylogenetic relationships of Thelohanellus to other myxobolids, to investigate the evolutionary history of the genus Thelohanellus and the origins of single polar capsule in this group. The phylogenetic analyses and statistical tests of topology revealed Thelohanellus as a strongly supported polyphyletic lineage, clustering in five distinct branches within Myxobolus clade. Ancestral state reconstruction for polar capsule number showed that Thelohanellus species have evolved from myxosporean species with two polar capsules at least four times, which could be classified in three possible evolutionary pathways. The polyphyly of Thelohanellus and the multiple evolutionary origins of single polar capsule of Thelohanellus demonstrate that the distinction of this genus from Myxobolus is largely for convenience, and does not reflect their evolutionary history.

RISK FACTORS FOR AND SPATIAL DISTRIBUTION OF LYMPHOPROLIFERATIVE DISEASE VIRUS (LPDV) IN WILD TURKEYS (MELEAGRIS GALLOPAVO) IN NEW YORK STATE, USA.

Lymphoproliferative disease virus (LPDV) is an oncogenic avian retrovirus that was previously thought to exclusively infect domestic turkeys but was recently shown to be widespread in Wild Turkeys ( Meleagris gallopavo ) throughout most of the eastern US. In commercial flocks, the virus spreads between birds housed in close quarters, but there is little information about potential risk factors for infection in wild birds. Initial studies focused on distribution of LPDV nationally, but investigation of state-level data is necessary to assess potential predictors of infection and detect patterns in disease prevalence and distribution. We tested wild turkey bone marrow samples (n=2,538) obtained from hunter-harvested birds in New York State from 2012 to 2014 for LPDV infection. Statewide prevalence for those 3 yr was 55% with a 95% confidence interval (CI) of 53-57%. We evaluated a suite of demographic, anthropogenic, and land cover characteristics with logistic regression to identify potential predictors for infection based on odds ratio (OR). Age (OR=0.16, 95% CI=0.13-0.19) and sex (OR=1.3, 95% CI=1.03-1.24) were strong predictors of LPDV infection, with juveniles less likely to test positive than adults, and females more likely to test positive than males. The number of birds released during the state's 40-yr translocation program (OR=0.993, 95% CI=0.990-0.997) and the ratio of agriculture to forest cover (OR=1.13, 95% CI=1.03-1.19) were also predictive of LPDV infection. Prevalence distribution was analyzed using dual kernel density smoothing to produce a risk surface map, combined with Kulldorff's spatial scan statistic and the Anselin Local Moran's I to identify statistically significant geographic clusters of high or low prevalence. These methods revealed the prevalence of LPDV was high (>50%) throughout New York State, with regions of variation and several significant clusters. We revealed new information about the risk factors and distribution of LPDV in New York State, which may be beneficial to game bird managers and producers of organic or pasture-raised poultry.

DIAGNOSING LYMPHOPROLIFERATIVE DISEASE VIRUS IN LIVE WILD TURKEYS (MELEAGRIS GALLOPAVO) USING WHOLE BLOOD.

Lymphoproliferative disease virus (LPDV) is a retrovirus that infects wild and domestic turkeys ( Meleagris gallopavo ). The first cases of LPDV in the United States were diagnosed in 2009, and subsequent surveillance has revealed the virus to be widespread in wild turkey populations throughout the eastern half of the country. More research is needed to determine whether LPDV is having a negative effect on turkey populations, but progress has been impeded by the lack of a simple method for diagnosing the virus in living birds. Infected animals may appear asymptomatic, and diagnostics currently rely on tissue or bone marrow, which can be difficult to obtain. This study investigated the reliability of polymerase chain reaction (PCR) to detect LPDV in whole blood, compared with previous methods using buffy coat (concentrated white blood cells) and bone marrow. Paired samples of whole blood and buffy coat were collected from 137 live turkeys and paired samples of whole blood and bone marrow were collected from 32 turkeys postmortem. Compared with buffy coat, whole blood had 97% sensitivity and 100% specificity. When compared with bone marrow, whole blood had 100% sensitivity and 89% specificity. Both comparisons had a high degree of agreement using Cohen's kappa statistic. Based on these results, PCR of whole blood provides detection of LPDV in living birds that is on par with both buffy coat and bone marrow.

Mycobacterium saopaulense sp. nov., a rapidly growing mycobacterium closely related to members of the Mycobacterium chelonae--Mycobacterium abscessus group.

Five isolates of non-pigmented, rapidly growing mycobacteria were isolated from three patients and,in an earlier study, from zebrafish. Phenotypic and molecular tests confirmed that these isolates belong to the Mycobacterium chelonae-Mycobacterium abscessus group, but they could not be confidently assigned to any known species of this group. Phenotypic analysis and biochemical tests were not helpful for distinguishing these isolates from other members of the M. chelonae­M.abscessus group. The isolates presented higher drug resistance in comparison with other members of the group, showing susceptibility only to clarithromycin. The five isolates showed a unique PCR restriction analysis pattern of the hsp65 gene, 100 % similarity in 16S rRNA gene and hsp65 sequences and 1-2 nt differences in rpoB and internal transcribed spacer (ITS) sequences.Phylogenetic analysis of a concatenated dataset including 16S rRNA gene, hsp65, and rpoB sequences from type strains of more closely related species placed the five isolates together, as a distinct lineage from previously described species, suggesting a sister relationship to a group consisting of M. chelonae, Mycobacterium salmoniphilum, Mycobacterium franklinii and Mycobacterium immunogenum. DNA­DNA hybridization values .70 % confirmed that the five isolates belong to the same species, while values ,70 % between one of the isolates and the type strains of M. chelonae and M. abscessus confirmed that the isolates belong to a distinct species. The polyphasic characterization of these isolates, supported by DNA­DNA hybridization results,demonstrated that they share characteristics with M. chelonae­M. abscessus members, butconstitute a different species, for which the name Mycobacterium saopaulense sp. nov. is proposed. The type strain is EPM10906T (5CCUG 66554T5LMG 28586T5INCQS 0733T).

Occurrence of a myxozoan parasite Myxidium streisingeri n. sp. in laboratory zebrafish Danio rerio.

Over several years of screening diagnostic cases, the Zebrafish International Resource Center Health Services has encountered a myxozoan parasite of the ducts associated with the kidney in zebrafish, Danio rerio , from an average of 21% of facilities submitting specimens over 5 yr. The parasite is coelozoic and is associated with no appreciable histological changes. Plasmodia bear ovoid spores with 3 sutural ridges. Spores are consistent with the genus Myxidium, but they are distinct from any known species and are thus described as Myxidium streisingeri n. sp. Phylogenetically, this parasite is a member of the polyphyletic urinary bladder clade, which is consistent with the site of infection. The common occurrence of a myxozoan in this closed husbandry system is unexpected because these parasites are known to have complex life cycles, alternating between a vertebrate and invertebrate host. It may be that biofilters provide habitat for suitable invertebrate hosts or perhaps M. streisingeri n. sp. can be transmitted directly. Future control of this parasite in zebrafish research laboratories depends on a better understanding of this life cycle.

Comparison of fixatives and fixation time for PCR detection of Mycobacterium in zebrafish Danio rerio .

Mycobacteriosis is a common disease of laboratory zebrafish Danio rerio. Different infection patterns occur in zebrafish depending on mycobacterial species. Mycobacterium marinum and M. haemophilum produce virulent infections associated with high mortality, whereas M. chelonae is more widespread and is not associated with high mortality. Identification of mycobacterial infections to the species level provides important information for making management decisions. Observation of acid-fast bacilli in histological sections or tissue imprints is the most common diagnostic method for mycobacteriosis in fish, but only allows for diagnosis to the genus level. Mycobacterial culture followed by molecular or biochemical identification is the traditional approach, but DNA of diagnostic value can also be retrieved from paraffin blocks. Here we investigated the type of fixative, time in fixative before processing, species of mycobacteria, and severity of infection as parameters to determine whether the hsp gene PCR assay (primer set HS5F/hsp667R) could detect and amplify mycobacterial DNA from paraffin-embedded zebrafish. Whole zebrafish were experimentally infected with either M. chelonae or M. marinum, and then preserved in 10% neutral buffered formalin or Dietrich's fixative for 3, 7, 21, and 45 d. Subsequently, fish were evaluated by hematoxylin and eosin and Fite's acid-fast stains to detect mycobacteria within granulomatous lesions. The PCR assay was quite effective and obtained PCR product from 75 and 88% of the M. chelonae- and M. marinum-infected fish, respectively. Fixative type, time in fixative, and mycobacterial species showed no statistical relationship with the efficacy of the PCR test.

Myxobolus turpisrotundus (Myxosporea: Bivalvulida) spores with caudal appendages: investigating the validity of the genus Henneguya with morphological and molecular evidence.

Spores of the myxozoan parasite Myxobolus turpisrotundus Zhang 2009 were observed for the first time bearing caudal appendages. Most spores had the typical Myxobolus spp. morphology, but approximately 10% of spores possessed a spore body that was slightly elongated with a short tail projecting from the spore valve. In other spores, the tail was much more clearly visible and elongate. The spore body of these unusual spores is consistent in morphology and dimension to the normal spores of M. turpisrotundus. Both spore types were found within individual cysts, and the small subunit ribosomal RNA (ssrRNA) gene sequence from parasite cysts of this type was nearly identical to the previously published sequence of M. turpisrotundus from allogynogenetic gibel carp Carassius auratus gibelio (Bloch). The phenomenon of Myxobolus spores with caudal appendages provides additional evidence that the use of this character to separate Myxobolus and Henneguya into distinct genera is not reflective of an evolutionarily accurate classification scheme. Phylogenetic analysis of ssrDNA sequence from Myxobolus and Henneguya species showed clustering of species in some locations of the tree, but ultimately these genera are intermixed. The use of a single character to delineate species in the two most species-rich myxozoan genera has been consistently challenged where DNA analyses are used. The present finding of a single species bearing both Myxobolus-type and Henneguya-type spores emphasizes the inadequacy of this classification scheme, and highlights the need for careful consideration of these variable characteristics when describing myxozoan species.

Distribution and genetic characterization of Mycobacterium chelonae in laboratory zebrafish Danio rerio.

During routine screening of zebrafish at a research facility, histological changes consistent with mycobacteriosis were observed, prompting an investigation to determine the background prevalence and distribution of Mycobacterium species throughout the facility. Infection status was evaluated in 240 zebrafish representing 9 genetic lines, using histology, culture and PCR. Environmental sources were also tested for the presence of mycobacteria. Prevalence in zebrafish by culture and PCR was 10% (24/240), 21 of which were TU line fish. All isolates from fish were identified as M. chelonae by hsp65 DNA sequencing; subsequent DNA fingerprinting delineated 3 strains, designated H1E1 (1/24), H1E2 (22/24), and H1E3 (1/24). From external sources, tank or tubing surface biofilms were positive by culture (13/32) with multiple species and strains isolated including M. neoaurum, M. phocaicum, and identical strains of M. chelonae that were found in zebrafish: H1E1 (2/13) and H1E2 (8/13). Comparing diagnostic methods, acid-fast stained histological sections showed substantial agreement with plate culture and PCR for detection of mycobacteria in fish. Observation of granulomas in hematoxylin and eosin-stained sections was a less reliable predictor of mycobacteriosis, as uninfected females with egg-associated inflammation and hyperplasia were misdiagnosed. These data revealed background levels of mycobacteriosis in a healthy and well-managed facility. Infected populations were removed, although the apparent ability for M. chelonae to remain viable in environmental reservoirs may make it difficult to eradicate completely. This highlights the importance of an animal-health monitoring program and good husbandry practices to prevent disease in zebrafish research laboratories.

Myxidium scripta n. sp. identified in urinary and biliary tract of Louisiana-farmed red-eared slider turtles Trachemys scripta elegans.

During a necropsy investigation of a mortality event occurring at a turtle farm in Assumption Parish, Louisiana, spores of a myxozoan were identified in the renal tubules in 3 of 6, the gall bladder lumen in 2 of 6, and the bile ductule in 1 of 6 red eared slider turtles Trachemys scripta elegans. In total, myxozoa were identified in 4 of 6 turtles. In 1 turtle, renal tubules contained numerous mature spores, had epithelial hyperplasia, granulomatous transformation, compression of adjacent tubules and interstitial lymphocytic nephritis. The genus of myxozoan was Myxidium, based on spore morphology in cytological preparations, in histologic section, and by electron microscopy. In cytological preparation the spores had mean dimensions of 18.8 x 5.1 microm and a mean polar capsule dimension of 6.6 x 3.5 microm. Electron microscopy showed renal tubules contained plasmodia with disporoblasts with spores in various stages of maturation. Ultrastructure of mature spores demonstrated a capsule containing 2 asymmetrical overlapping valves and polar capsules containing a polar filament coiled 6 to 8 times and surrounded by a membrane composed of a double layer wall. The small subunit rDNA gene sequence was distinct from all other Myxidium species for which sequences are available. Additionally, this is the first Myxidium species recovered from a North American chelonian to receive genetic analysis. Although T. s. elegans is listed as a host for Myxidium chelonarum, this newly described species of Myxidium possessed larger spores with tapered ends; thus, we described it as a new species, Myxidium scripta n. sp. This report documents a clinically significant nephropathy and genetic sequence from a Myxidium parasite affecting a freshwater turtle species in North America.

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.

Kudoa alliaria in flesh of Argentinian hoki Macruronus magellanicus (Gadiformes; Merlucciidae).

Myxozoans of the genus Kudoa are widespread parasites of marine fishes and primarily infect the body musculature of their hosts. Although Kudoa species are not usually associated with host mortality, some do form macroscopic cysts in the tissue and some are associated with post mortem tissue degradation. This is of concern to commercial fisheries as fillets may be unmarketable due to these infections. Because different species of Kudoa have different effects on their hosts, it is important to correctly identify species with epidemiological relevance, distinguishing those that are benign from those that are associated with these detrimental effects. Using morphological and molecular analyses, we identified K. alliaria infecting Argentinian hoki Macruronus magellanicus. Comparisons of the small subunit ribosomal DNA sequence revealed that K. alliaria is genetically very similar to K. rosenbuschi. Furthermore, there is significant overlap in myxospore dimensions between descriptions of these 2 Kudoa species as well as those of other Patagonian fishes. Thus, without careful examination of the myxospore dimensions, it may be difficult to identify these species on a routine basis. It is critical to accurately identify K. alliaria as, unlike K. rosenbuschi, it is not associated with tissue degradation. Ambiguities in some species descriptions highlight the need for thorough morphological analyses accompanied by molecular comparisons to clarify the species boundaries between Kudoa parasites of Patagonian fishes.

Kudoa hypoepicardialis n. sp. (Myxozoa: Kudoidae) and associated lesions from the heart of seven perciform fishes in the northern Gulf of Mexico.

Kudoa hypoepicardialis n. sp. infects the space between the epicardium and the compact myocardium and, in intense infections, the pericardial chamber of man-of-war fish (Nomeus gronovii) (Nomeidae) (the type host), blue runner (Caranx crysos) (Carangidae), Warsaw grouper (Epinephelus nigritus) (Serranidae), Atlantic tripletail (Lobotes surinamensis) (Lobotidae), northern red snapper (Lutjanus campechanus) (Lutjanidae), black drum (Pogonias cromis) (Sciaenidae), and bluefish (Pomatomus saltatrix) (Pomatomidae) in the northern Gulf of Mexico. This is the first report of a Kudoa sp. from the heart of a fish in the Gulf of Mexico, and of these hosts, only the bluefish was previously identified as a host for a species of Kudoa. Spores of the new species varied slightly in size among these hosts but were regarded as conspecific based on their nearly identical (99.9%) small-subunit (SSU) ribosomal DNA (rDNA) sequence. The new species differs both from the 4 nominal species of Kudoa reported from fishes in the Gulf of Mexico and from K. pericardialis, an allopatric species that infects the pericardial cavity, by the combination of having a large spore, a small polar capsule, and a polar filament with a single coil. The new species is morphologically and genetically most similar to K. shiomitsui, an allopatric species that infects the heart and pericardial cavity, but is distinguished from it based on a 4.2% difference in the SSU rDNA sequence. Heart lesions primarily were restricted to the vicinity of plasmodia and included a layer of fibrinous inflammation characterized by lymphocytes, macrophages, and granulomas as well as epithelioid encapsulations around plasmodia. Heavily infected hosts had melanin-like deposits and adipose cells beneath the epicardium. and the epicardium was discontinuous and apparently breached by plasmodia in some regions. Cardiac muscle, gill, liver, spleen, intestine, and kidney were normal.