Antibiotic treatment of zebrafish mycobacteriosis: tolerance and efficacy of treatments with tigecycline and clarithromycin.

Zebrafish (Danio rerio) are a popular model organism used in a growing number of research fields. Maintaining healthy, disease-free laboratory fish is important for the integrity of many of these studies. Mycobacteriosis is a chronic bacterial infection caused by several Mycobacterium spp. and is the second most common disease found in laboratory zebrafish. Current mycobacteriosis control measures recommend the removal of infected fish and in severe outbreaks, depopulation. These measures can be effective, but less disruptive measures should be assessed for controlling mycobacteriosis, particularly when valuable and rare lines of fish are affected. Here, the in vivo efficacy of two drug candidates, tigecycline (1 µg g-1 ) and clarithromycin (4 µg g-1 ), was tested in adult zebrafish experimentally infected with Mycobacterium chelonae. We assessed both short (14 day)- and long-term (30 day) treatments and evaluated fecundity and pathological endpoints. Fecundity and histology results show that zebrafish tolerated antibiotics. Antibiotic treatments did not significantly impact the prevalence of acid-fast granulomas; however, the severity of infections (acid-fast granuloma intensity) was significantly decreased following treatments.

REFERENCE AND BASELINE HEMATOCRIT MEASURES FOR THE THREATENED NEW ENGLAND COTTONTAIL (SYLVILAGUS TRANSITIONALIS) AND COMPARISON WITH SYMPATRIC EASTERN COTTONTAIL (SYLVILAGUS FLORIDANUS) RABBITS.

From June 2014 to June 2015, capillary tube collections of blood were obtained concurrently with ear clips of trapped free-ranging, globally vulnerable New England cottontails (NEC; Sylvilagus transitionalis) and eastern cottontail rabbits (EC; Sylvilagus floridanus) in the Hudson Valley region of New York, United States. Species identification (NEC, EC) and sex (NEC) were determined genetically using a mitochondrial DNA assay and Y chromosome marker, respectively. Hematocrit values were obtained using a microhematocrit centrifuge. We provide the reference values 35.15-49.55 (2.5 and 97.5 percentiles) and 90% confidence intervals (CI) [lower: 33.00, 36.08; upper: 46.95, 51.00], for hematocrit of NEC. The mean hematocrit for NEC was 42.35% (SE = 0.58, n = 47) and a comparative contemporaneous mean in the same area for EC [39.96 (SE = 0.81, n = 26)], which was significantly different from NEC (P = 0.02). There was a significant sex difference for NEC [male: 43.99 (SE = 1.02, n = 28); female: 39.92 (SE = 0.78, n = 19), P < 0.0001], though not for EC.

Myxobolus musseliusae (Myxozoa: Myxobolidae) from the gills of common carp Cyprinus carpio and revision of Myxobolus dispar recorded in China.

During a survey of myxozoan parasites of common carp Cyprinus carpio in Honghu Lake, Hubei Province, China, a parasite was collected that was identified as Myxobolus dispar based on an earlier description from China. However, the small subunit ribosomal DNA of this species shared only 90 % similarity with M. dispar, instead matching M. musseliusae with 100 % identity. To resolve this apparent taxonomic conflict, the validity of M. dispar reported from China was investigated. The species encountered here and in the earlier report from China both bear spores that are notably smaller than those of M. dispar in Europe. In the present study, a mucous envelope was adhered to the posterior of many fresh spores and was observed to expand and surround the spore. This structure has never been reported from fresh spores of M. dispar. Histology showed extravascular plasmodia in the gill filaments in close contact with the cartilaginous ray of the filament, which contrasts with the plasmodia of M. dispar which develop in the arteries of the gill filaments. Phylogenetically, the current species is distinct from M. dispar, instead forming a sister group with M. musseliusae. The data presented here allow us to conclude that the species isolated is M. musseliusae and that prior reports of M. dispar in China are unsubstantiated.

Disease risk surface for Coxiella burnetii seroprevalence in white-tailed deer.

Coxiella burnetii is considered a re-emerging zoonosis in many countries. The bacterium is enzootic in livestock and wildlife in the United States, and environmental contamination is widespread. Despite the potential for exposure, the estimated prevalence of Q fever in humans and animals is not well elucidated, and reported human infections in the United States are relatively rare. Zoonotic transmission of the bacterium is usually associated with abortions in domestic ruminants, but other modes of transmission, such as contact with infected blood and/or milk during field dressing of infected wildlife, have not been thoroughly investigated. Studies of zoonotic pathogen transmission between animal reservoir hosts and humans are usually established in response to documented emergence or re-emergence of a zoonosis in a particular locale, and, as such, the prevalence of infection in wildlife is largely unknown for many zoonotic pathogens, including C. burnetii. The objective of this study was to create a disease risk surface for C. burnetii seroprevalence in wild white-tailed deer (Odocoileus virginianus) in New York State. Blood samples were collected from hunter-harvested deer from across New York State in 2009 and 2010. The samples were processed and tested for the presence of anti-C. burnetii antibodies via indirect microimmunofluorescence assays using phase II C. burnetii strain RSA439. Overall, 14.50% of the tested white-tailed deer were C. burnetii phase II seropositive. The dual Kernel density estimation method was used to create a smoothed disease risk surface, which revealed variation in seroprevalence ranging from 0% to 32.0%. Areas of higher seroprevalence were detected in four discrete areas of Central New York and in one additional area in the southwest corner of the northern part of the state. This suggests certain locales where humans may be at increased risk for exposure to the bacterium secondary to contact with potentially infected deer.

Supplemental diagnosis of a myxozoan parasite from common carp Cyprinus carpio: synonymy of Thelohanellus xinyangensis with Thelohanellus kitauei.

Thelohanellus kitauei Egusa et Nakajima, 1981, was described from common carp Cyprinus carpio L. in Japan. In China, a myxosporean infecting the intestinal tissue of the same host species was described as Thelohanellus xinyangensis Xie, Gong, Xiao, Guo, Li et Guo, 2000, despite many similarities to T. kitauei. To examine the potential conspecificity of these species, a morphological and molecular investigation of T. xinyangensis was conducted. Comparing myxospore morphology, the mean spore length and width of each species is not identical between species, but ranges of dimensions overlap. These data are more suggestive of intraspecific variation than distinct species. Comparison of relative ratios of spore length to polar capsule length and spore width to polar capsule width of T. xinyangensis and T. kitauei reveal no differences and scanning electron microscopy reveals a smooth spore surface of T. xinyangensis, which is consistent with that of T. kitauei. Most convincingly, DNA sequences of the small subunit ribosomal RNA (ssrRNA) gene of the two species were identical. From the morphological and molecular biological data, we propose T. xinyangensis from the intestine of common carp is not a distinct species and is synonymous with T. kitauei.

Biochemical, molecular, and virulence characteristics of select Mycobacterium marinum isolates in hybrid striped bass Morone chrysops x M. saxatilis and zebrafish Danio rerio.

A panel of 15 Mycobacterium marinum isolates was characterized by biochemical tests, sequencing the ribosomal DNA intergenic spacer (ITS) region and the heat shock protein 65 gene (hsp65) and pulsed-field gel electrophoresis (PFGE). The biochemical characteristics of all isolates were similar, except for Tween 80 hydrolysis. DNA sequence of hsp65 for a subset of isolates were identical; however, at position 5 of the ITS rDNA, a single nucleotide polymorphism was identified. Isolates possessing a guanine residue at this position (G strains) were unable to hydrolyze Tween 80, while isolates that contained an adenine residue at this position (A strains) were positive for Tween 80 hydrolysis. PFGE successfully discriminated between the G and A strains; all G strains had identical AseI restriction enzyme-cutting patterns while the A strains exhibited a variety of cutting patterns. Eight isolates (4 G and 4 A strains) were further characterized for virulence by experimental infection of hybrid striped bass (HSB) Morone chrysops x M. saxatilis and zebrafish Danio rerio. Seven of the 8 strains produced cumulative mortality ranging from 13.3 to 83.3% in the HSB virulence trial. The M. marinum reference strain ATCC 927T did not produce mortality in HSB. HSB exposed to the G strains had significantly higher cumulative mortality than those exposed to the A strains. When these same isolates were tested in zebrafish, 6 of the 8 strains caused 100% cumulative mortality, with 2 of the A strains being the most pathogenic. In zebrafish, however, ATCC 927T was virulent and produced 28.5% mortality. Collectively, we conclude that the M. marinum G strains are unique and may represent a distinct virulence phenotype in HSB, but this trend was not consistent in zebrafish.

Renal myxozoanosis in crowned river turtles Hardella thurjii: description of the putative agent Myxidium hardella n. sp. by histopathology, electron microscopy, and DNA sequencing.

Chelonian myxozoanosis is rarely reported and has previously not been documented to cause disease. This report describes myxozoanosis associated with significant renal disease in two Crowned River turtles (Hardella thurjii). One turtle presented with emaciation and died. The cage mate presented with emaciation and was euthanized. Histologically, renal intratubular myxozoan spores were associated with renal tubular necrosis, tubular mineralization, and chronic interstitial nephritis, with membranoproliferative and mes-angioproliferative glomerulopathy. Both turtles also had disseminated metastatic mineralization. On the basis of these findings, chronic renal insufficiency from myxozoanosis and subsequent metastatic mineralization were considered the primary problems. By light and electron microscopy, the myxozoan spores had features of the genus Myxidium. Maximum parsimony analysis of small-subunit rDNA sequences placed the turtle myxozoan basal to a clade containing Myxidium truttae and a Myxidium sp. with strong bootstrap support. This myxozoan agent appears to be a significant pathogen in H. thurjii on the basis of morphologic changes in the kidneys of in the infected turtles.

Phylogeny of the multivalvulidae (Myxozoa: Myxosporea) based on comparative ribosomal DNA sequence analysis.

Fish parasites of the Multivalvulida (Myxozoa, Myxosporea) are widespread and can be associated with mortality or poor flesh quality in their commercially important marine hosts. Traditional classifications divide members of this order into families based on spore valve and polar capsule numbers. Analyses of the small-subunit (SSU) ribosomal DNA (rDNA) sequences from all representative families in the order (Trilosporidae, Kudoidae, Pentacapsulidae, Hexacapsulidae, and Septemcapsulidae) indicate that a revision of the taxonomy and nomenclature is warranted. In our phylogenetic analysis of (SSU and large subunit) rDNA sequences, members of Pentacapsula, Hexacapsula, and Septemcapsula root within a clade of Kudoa species with Unicapsula (Trilosporidae) as an outlier to these genera. Therefore, we propose to synonymize Pentacapsulidae, Hexacapsulidae, and Septemcapsulidae with Kudoidae alter the diagnosis of Kudoidae and Kudoa to accommodate all marine myxozoan parasites having 4 or more shell valves and polar capsules.

A new species of Sphaeromyxa (Myxosporea: Sphaeromyxina: Sphaeromyxidae) in devil firefish, Pterois miles (Scorpaenidae), from the northern Red Sea: morphology, ultrastructure, and phylogeny.

Sphaeromyxa zaharoni n. sp. (Myxosporea) is described from the gallbladder of devil firefish, Pterois miles (Scorpaenidae), from coral reefs of the Gulf of Eilat, Israel, northern Red Sea. The parasite was found also in bearded scorpionfish, Scorpaenopsis barbata, from the same area. This is the first report on Sphaeromyxa sp. from this zoogeographical region. The plasmodia are amoeboid when young, becoming disc-shaped and elongated when mature. In paraffin sections, the plasmodium periphery appears as a finely granulated, strongly eosinophilic layer with an intricate surface membrane network. Sphaeromyxa zaharoni n. sp. is polysporous, disporoblastic, and has asynchronous sporogenesis. The mature spore is elongated and fusiform, has delicately ridged valves, and contains a single, binucleated sporoplasm. In valvular view, the tips are truncated. The mean spore size is length 14.5 microm, width 4.8 microm, and polar capsule 4.8 x 3.4 microm. The 2 equally sized ovoid polar capsules are positioned at opposite ends of the spore, each containing a filament loosely folded in 2 loops. The fine structure of the sporoblast and spore corresponded with previously studied Sphaeromyxa species. According to small-subunit ribosomal DNA gene sequence analysis, S. zaharoni n. sp. is most closely related to 2 Myxidium spp. The close phylogenetic relatedness of Sphaeromyxa and Myxidium and similar spore morphology raises the question whether these 2 genera should be maintained in separate families and suborders.

Two unusual myxozoans, Kudoa quadricornis n. sp. (Multivalvulida) from the muscle of goldspotted trevally (Carangoides fulvoguttatus) and Kudoa permulticapsula n. sp. (Multivalvulida) from the muscle of Spanish mackerel (Scomberomorus commerson) from the Great Barrier Reef, Australia.

Two unusual myxozoan parasites are described from the somatic muscle of 2 reef fishes from Australia's Great Barrier Reef. Kudoa quadricornis n. sp. from the somatic muscle of Carangoides fulvoguttatus is morphologically consistent with other Kudoa sp., having 4 polar capsules and 4 shell valves. Kudoa quadricornis n. sp. is unique in that it has a pyriform spore body with a greater length than width (7.82-9.95 and 5.94-8.66 microm, respectively) and distinct posterolateral projections. Spores of Kudoa permulticapsula n. sp. observed within pseudocysts of the somatic muscle tissue of Scomberomorus commerson are different from those of all other myxozoans. The ovoid spores (length, 4.69-6.65 microm; width, 8.42-9.92 microm; thickness, 6.36-8.33 microm) contain 13 polar capsules with an equal number of shell valves. Phylogenetic analysis using small subunit ribosomal DNA sequences of K. quadricornis n. sp. and K. permulticapsula n. sp. showed that these parasites cluster within a clade comprised of Kudoa species. This brings into question the division of parasites of the Multivalvulida into genera based solely on polar capsule numbers.

Experimental and natural host specificity of Loma salmonae (Microsporidia).

The microsporidian Loma salmonae (Putz, Hoffman & Dunbar, 1965) Morrison & Sprague, 1981 has caused significant gill disease in Pacific salmon Oncorhynchus spp. Host specificity of the parasite was examined experimentally by per os challenge of selected salmonids and non-salmonids with infective chinook salmon O. tshawytscha gill material. Pink Oncorhynchus gorbuscha and chum salmon O. keta, brown Salmo trutta and brook trout Salvelinus fontinalis, and chinook salmon (controls) were positive, whereas Atlantic salmon Salmo salar and Arctic char Salvelinus alpinus were negative. In addition, no non-salmonids were susceptible to experimental exposure. Wild Pacific salmon species in British Columbia, Canada, were examined for L. salmonae during their freshwater life history stages (smolts, prespawning, spawning). All stages were infected, although infections in smolts were only detectable using a L. salmonae-specific PCR test. Many previous Loma spp. described from Oncorhychus spp. are likely L. salmonae based on host, parasite morphology, and site of infection.