Non-tuberculous mycobacterial disease associated with Mycobacterium montefiorense in salamanders.

Introduction: Mycobacterium montefiorense is one of the causes of non-tuberculous mycobacterial infections in moray eels and salamanders. Although M. montefiorense infection could be a threat to salamanders, little information is available regarding this pathogen and associated infection. This study aimed to provide fundamental information regarding M. montefiorense and its infection in salamanders. Methods: Nine M. montefiorense strains isolated from three species of salamanders, namely, Japanese black salamander (Hynobius nigrescens), Hakuba salamander (H. hidamontanus), and Tohoku hynobiid salamander (H. lichenatus), between 2010 and 2018, were characterized based on phenotypic and genetic examination. We also pathologically observed salamanders infected with the M. montefiorense strains, including Hakuba salamanders and Tohoku hynobiid salamanders. Results: The microbiological and chemical characteristics of the M. montefiorense salamander and an eel strain (reference strain) matched. Susceptibility testing for antimicrobials suggested that clarithromycin may be effective. Regarding disinfectants, phtharal, peracetic acid, glutaral, sodium hypochlorite, and benzalkonium chloride may be effective. Phylogenetic analyses revealed that the strains isolated from salamanders in 2014 and 2018 were genetically closely related, which could indicate an outbreak. The main gross findings in infected salamanders include skin ulcerative lesions or nodules in the enlarged liver. Microscopically, multifocal to coalescent granulomatous lesions composed of massive macrophages containing numerous acid-fast bacilli were prominently observed in the liver. Conclusion: This study contributes to our understanding of the genetic diversity and phenotypic characteristics of M. montefiorense, as well as the pathology of the infection.

Genital mycobacteriosis caused by Mycobacterium marinum detected in two captive sharks by peptide nucleic acid-fluorescence in situ hybridization.

Mycobacterium marinum is a prevalent nontuberculous mycobacterium (NTM)-infecting teleosts. Conversely, little is known about mycobacteriosis in elasmobranchs, and M. marinum infection has never been reported from the subclass. This study investigated the histopathological characteristics and localization of this mycobacterium through molecular analysis of two captive sharks, a scalloped hammerhead Sphyrna lewini and a Japanese bullhead shark Heterodontus japonicus, exhibited in the same aquarium tank. We detected genital mycobacteriosis caused by M. marinum infection using molecular analyses, including polymerase chain reaction (PCR) and DNA sequencing targeting the 60 kDa heat-shock protein gene (hsp65), and peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH) targeting the 16S rRNA gene. Both sharks showed granulomas in connective tissues of the gonads without central necrosis or surrounding fibrous capsules, which is unlike the typical mycobacterial granulomas seen in teleosts. This study reveals that elasmobranchs can be aquatic hosts of M. marinum. Because M. marinum is a representative waterborne NTM and a potential zoonotic agent, cautious and intensive research is needed to overcome a lack of data on the relationship between NTM and the aquatic environment in association with this subclass of Chondrichthyes.

A case of mycobacteriosis associated with Mycobacterium pseudoshottsii in aquarium-reared fish in Japan.

In 2019, several aquarium-reared fish died at a sea life park in Japan. Necropsy revealed micronodules on the spleen in the dotted gizzard shad (Konosirus punctatus). Seven of 16 fish exhibited microscopic multifocal granulomas associated with acid-fast bacilli in the spleen, kidney, liver, alimentary tract, mesentery, gills, and/or heart. Bacterial cultures yielded isolates from the dotted gizzard shad and a Japanese sardine (Sardinops melanostictus). Microbiological and molecular biological examinations revealed the isolates as Mycobacterium pseudoshottsii. To our knowledge, this is the first isolation of M. pseudoshottsii from aquarium-reared fish.

Seroprevalence of Antibodies Against Paracoccidioides Spp. in Captive Dolphins from Three Aquaria in Japan.

The skin disease paracoccidioidomycosis ceti occurs in several dolphin species globally. Infection by the unculturable fungi Paracoccidioides brasilensis or other Paracoccidioides spp. results in chronic cutaneous and granulomatous lesions. In this study we used immunohistochemistry to investigate the seroprevalence of antibodies to Paracoccidioides spp. in captive dolphins from three aquaria in Japan. We had previously reported that there were serological cross-reactions for Paracoccidioides spp. with related species in the order Onygenales. We hypothesized that the degree of serological cross-reactions for Paracoccidioides spp. might be lower in areas, such as Japan, where the fungal diseases coccidiodomycosis and paracoccidiodomycosis are not endemic. Sera from 41 apparently healthy dolphins, including 20 Atlantic bottlenose dolphins (BD: Tursiops truncatus), 6 Indo-Pacific bottlenose dolphins (IPBD: Tursiops aduncus), 2 F1 generation of a cross between BD and IPBD (F1), 3 Pacific white-sided dolphins (PWD: Lagenorhynchus obliquidens), 2 pantropical spotted dolphins (PSD: Stenella attenuata), 6 false killer whales (FKW: Pseudorca crassidens), and 2 rough-toothed dolphins (RTD: Steno bredanensis) were investigated. Sera from three dolphins with paracoccidioidomycosis ceti were used as a positive control. The yeast-form cells of Paracoccidioides spp. in the cutaneous tissue sample derived from the first Japanese paracoccidioidomycosis ceti case were used as the antigen for the immunohistochemistry. Of the 41 dolphins tested, 61.0% had antibodies against Paracoccidioides spp. This indicates that dolphins of several species in Japanese aquaria have likely been exposed to the pathogen Paracoccidioides spp.

Detection of Multiple Budding Yeast Cells and a Partial Sequence of 43-kDa Glycoprotein Coding Gene of Paracoccidioides brasiliensis from a Case of Lacaziosis in a Female Pacific White-Sided Dolphin (Lagenorhynchus obliquidens).

Lacaziosis, formerly called as lobomycosis, is a zoonotic mycosis, caused by Lacazia loboi, found in humans and dolphins, and is endemic in the countries on the Atlantic Ocean, Indian Ocean and Pacific Ocean of Japanese coast. Susceptible Cetacean species include the bottlenose dolphin (Tursiops truncatus), the Indian Ocean bottlenose dolphin (T. aduncus), and the estuarine dolphin (Sotalia guianensis); however, no cases have been recorded in other Cetacean species. We diagnosed a case of Lacaziosis in a Pacific white-sided dolphin (Lagenorhynchus obliquidens) nursing in an aquarium in Japan. The dolphin was a female estimated to be more than 14 years old at the end of June 2015 and was captured in a coast of Japan Sea in 2001. Multiple, lobose, and solid granulomatous lesions with or without ulcers appeared on her jaw, back, flipper and fluke skin, in July 2014. The granulomatous skin lesions from the present case were similar to those of our previous cases. Multiple budding and chains of round yeast cells were detected in the biopsied samples. The partial sequence of 43-kDa glycoprotein coding gene confirmed by a nested PCR and sequencing, which revealed a different genotype from both Amazonian and Japanese lacaziosis in bottlenose dolphins, and was 99 % identical to those derived from Paracoccidioides brasiliensis; a sister fungal species to L. loboi. This is the first case of lacaziosis in Pacific white-sided dolphin.

Discrimination of Mycobacterium abscessus subsp. massiliense from Mycobacterium abscessus subsp. abscessus in clinical isolates by multiplex PCR.

The rapidly growing mycobacterium M. abscessus sensu lato is the causative agent of emerging pulmonary and skin diseases and of infections following cosmetic surgery and postsurgical procedures. M. abscessus sensu lato can be divided into at least three subspecies: M. abscessus subsp. abscessus, M. abscessus subsp. massiliense, and M. abscessus subsp. bolletii. Clinical isolates of rapidly growing mycobacteria were previously identified as M. abscessus by DNA-DNA hybridization. More than 30% of these 117 clinical isolates were differentiated as M. abscessus subsp. massiliense using combinations of multilocus genotyping analyses. A much more cost-effective technique to distinguish M. abscessus subsp. massiliense from M. abscessus subsp. abscessus, a multiplex PCR assay, was developed using the whole-genome sequence of M. abscessus subsp. massiliense JCM15300 as a reference. Several primer sets were designed for single PCR to discriminate between the strains based on amplicons of different sizes. Two of these single-PCR target sites were chosen for development of the multiplex PCR assay. Multiplex PCR was successful in distinguishing clinical isolates of M. abscessus subsp. massiliense from samples previously identified as M. abscessus. This approach, which spans whole-genome sequencing and clinical diagnosis, will facilitate the acquisition of more-precise information about bacterial genomes, aid in the choice of more relevant therapies, and promote the advancement of novel discrimination and differential diagnostic assays.

Lymphocytes with T-cell-like properties express the Fas ligand in the Japanese flounder Paralichthys olivaceus.

In this study, we aimed to identify the leukocyte population that expresses Fas ligand (FasL) in the Japanese flounder (Paralichthys olivaceus). The transcriptional activity of FasL was examined for the first time in the fish leukocytes. Transcription of the FasL gene in flounder leukocytes was significantly increased by phytohemagglutinin (PHA) treatment. All the leukocyte populations we tested possessed binding activity for PHA, but this was especially high in the lymphocyte population. However, the lymphocytes consisted of two subsets showing heterogeneity with respect to PHA binding, with the high-binding subset being surface IgM-negative. We also found that only the lymphocyte population showed a significant increase in the expression of the FasL gene after stimulation with PHA. In addition, only the lymphocyte subset showing high binding to PHA showed conspicuous expression of the FasL gene. This subset also had a CD3γ/δ+, CD8α+ and IgM heavy-chain (-) phenotype. These results suggested that lymphocytes with T-cell-like properties are FasL-expressing cells in the Japanese flounder.