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.

Core single nucleotide polymorphism analysis reveals transmission of Mycobacterium marinum between animal and environmental sources in two aquaria.

Mycobacterium marinum is a slow-growing, photochromogenic nontuberculous mycobacterium, which can cause mycobacteriosis in various animals, including humans. Several cases of fish mycobacteriosis have been reported to date. Mycobacterium marinum has also been isolated from aquatic environmental sources such as water, sand, biofilms, and plants in the natural environments. Hence, we hypothesized that a wide variety of sources could be involved in the transmission of M. marinum. In this study, we tested this hypothesis by isolating M. marinum from various sources such as fish, invertebrates, seagrass, periphytons, biofilms, sand, and/or water in two aquaria in Japan and conducting a phylogenetic analysis based on single-nucleotide polymorphisms (SNPs) using whole-genome sequences of the isolated strains. The analysis revealed that the strains from animal and environmental sources belonged to the same clusters. This molecular-based study epidemiologically confirmed that various sources, including fish, invertebrates, and environmental sources, could be involved in transmission of M. marinum in a closed-rearing environment. This is the first report where M. marinum was isolated from different sources, and various transmission routes were confirmed in actual cases, which provided essential information to improve the epidemiology of M. marinum.

Draft Genome Sequence of a Mycobacterium chelonae subsp. bovis Strain Isolated from a Baikal Seal (Pusa sibirica) in Captivity.

Mycobacterium chelonae is a nontuberculous mycobacterium that causes infections in various animals, including humans. In this study, we report the draft genome sequence of M. chelonae subsp. bovis strain NJB1701, which was isolated from a Baikal seal (Pusa sibirica) in captivity in Japan.

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.

Complete Genome and Partial Megaplasmid Sequences of Mycobacterium pseudoshottsii Strain NJB1907-Z4, Isolated from an Aquarium-Reared Japanese Sardine (Sardinops melanostictus) in Japan.

Mycobacterium pseudoshottsii, a slow-growing nontuberculous mycobacterium, has been isolated from wild and cultured fish. We report here the complete genome and partial megaplasmid sequences of a strain isolated from an aquarium-reared Japanese sardine (Sardinops melanostictus) in Japan, M. pseudoshottsii NJB1907-Z4.

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.

Draft Genome Sequences of 25 Mycobacterium marinum Strains Isolated from Animals and Environmental Components in Aquaria and an Aquaculture Farm.

Mycobacterium marinum is a ubiquitous nontuberculous mycobacterium that causes infections in various animals. Here, we report the annotated draft genome sequences of 25 strains isolated from vertebrates, invertebrates, and environmental components in aquaria and an aquaculture farm in Japan, sampled between 2015 and 2020.

Metronidazole enhances steatosis-related early-stage hepatocarcinogenesis in high fat diet-fed rats through DNA double-strand breaks and modulation of autophagy.

Nonalcoholic fatty liver disease is a hepatic disorder with deposition of fat droplets and has a high risk of progression to steatosis-related hepatitis and irreversible hepatic cancer. Metronidazole (MNZ) is an antiprotozoal and antimicrobial agent widely used to treat patients infected with anaerobic bacteria and intestinal parasites; however, MNZ has also been shown to induce liver tumors in rodents. To investigate the effects of MNZ on steatosis-related early-stage hepatocarcinogenesis, male rats treated with N-nitrosodiethylamine following 2/3 hepatectomy at week 3 were received a control basal diet, high fat diet (HFD), or HFD containing 0.5% MNZ. The HFD induced obesity and steatosis in the liver, accompanied by altered expression of Pparg and Fasn, genes related to lipid metabolism. MNZ increased nuclear translocation of lipid metabolism-related transcription factor peroxisome proliferator-activated receptor gamma in hepatocytes, together with altered liver expression of lipid metabolism genes (Srebf1, Srebf2, Pnpla2). Furthermore, MNZ significantly increased the number of preneoplastic liver foci, accompanied by DNA double-strand breaks and late-stage autophagy inhibition, as reflected by increased levels of γ-H2AX, LC3, and p62. Therefore, MNZ could induce steatosis-related hepatocarcinogenesis by inducing DNA double-strand breaks and modulating autophagy in HFD-fed rats.

Differential impacts of mineralocorticoid receptor antagonist potassium canrenoate on liver and renal changes in high fat diet-mediated early hepatocarcinogenesis model rats.

Mineralocorticoid receptor (MR)/NADPH oxidase (NOX) signaling is involved in the development of obesity, insulin resistance, and renal diseases; however, the role of this signaling on steatotic preneoplastic liver lesions is not fully elucidated. We determined the effects of the MR antagonist potassium canrenoate (PC) on MR/NOX signaling in hepatic steatosis and preneoplastic glutathione S-transferase placental form (GST-P)-positive liver foci. Rats were subjected to a two-stage hepatocarcinogenesis model and fed with basal diet or high fat diet (HFD) that was co-administered with PC alone or in combination with the antioxidant alpha-glycosyl isoquercitrin (AGIQ). PC reduced obesity and renal changes (basophilic tubules that expressed MR and p22phox) but did not affect blood glucose tolerance and non-alcoholic fatty liver disease activity score (NAS) in HFD-fed rats. However, the drug increased the area of GST-P-positive liver foci that expressed MR and p22phox as well as increased expression of NOX genes (p22phox, Poldip2, and NOX4). PC in combination with AGIQ had the potential of inhibiting the effects of PC on the area of GST-P-positive liver foci and the effects were associated with increasing expression of an anti-oxidative enzyme (Catalase). The results suggested that MR/NOX signaling might be involved in development of preneoplastic liver foci and renal basophilic changes in HFD-fed rats; however, the impacts of PC were different in each organ.

Developmental exposure of citreoviridin transiently affects hippocampal neurogenesis targeting multiple regulatory functions in mice.

To investigate the developmental exposure effect of citreoviridin (CIT) on postnatal hippocampal neurogenesis, pregnant ICR mice were dietary exposed to CIT at 0, 1, 3 and 10 ppm from gestation day 6 to postnatal day (PND) 21 on weaning. Offspring were maintained through PND 77 without CIT exposure. Male offspring were analyzed. At 10 ppm on PND 21, weak changes suggestive of neural stem cell reduction and progenitor cell proliferation were observed. Number of hilar CALB1+ interneurons reduced, suggesting an influence on neurogenesis. In contrast, number of hilar SST+ interneurons increased and Bdnf and Ntrk2 transcripts upregulated in the dentate gyrus, suggesting a facilitation of BDNF-TRKB signaling for progenitor cell proliferation. Transcript expression changes of an outside regulatory system suggested suppressed function of GABAergic interneurons, especially of PVALB+ interneurons for compensation on neural stem cell reduction. At ≥ 3 ppm, number of ARC+ mature granule cells increased, and at 10 ppm, number of hilar GRIA1+ cells increased and Gria2 and Gria3 upregulated, suggesting an operation of AMPA receptor membrane trafficking on the increase of ARC-mediated synaptic plasticity. On PND 77, all the transcript expression changes of the neurogenesis regulatory system except for Grin2d were inverted, suggesting an operation of a homeostatic mechanism on CIT-induced disruptive neurogenesis. Simultaneous downregulation of Grin2a and Grin2d suggests suppression of GABAergic interneuron function to adjust neurogenesis at the normal level. The no-observed-adverse-effect level of CIT for offspring neurogenesis was determined to be 1 ppm, translating to 0.13-0.51 mg/kg body weight/day of maternal oral exposure.

A case of rapid recurrence of apocrine ductal carcinoma originating from the oral scent gland of a Richardson's ground squirrel (Urocitellus richardsonii).

A 3-year-old female Richardson's ground squirrel developed a subcutaneous mass at the left oral angle. Seven days after removal of the mass, the mass recurred and metastasized to the cervical lymph node. Histologically, the primary mass was subdivided by fibrous trabeculae into various-sized neoplastic cell lobules showing a solid growth pattern with frequent mitoses and sometimes forming intracytoplasmic lumina. Large to medium-sized lobules formed a central cyst plugged by comedo necrosis. Neoplastic cells showed infiltrative subcutaneous growth. In the recurrent tumor, tubular structures lacking apparent apocrine secretion appeared within the solid growth portion. Neutrophil infiltration was evident within the tubules and intracytoplasmic lumina. Neoplastic cells were diffusely immunopositive for AE1/AE3 pan-cytokeratin (CK) in all lobules and focally positive for CAM5.2 CK in the lobules forming a central cyst and/or tubular structures, but they entirely lacked positivity for the periodic acid Schiff reaction. Ki-67-positive proliferating neoplastic cells were higher in numbers with the recurrent tumor than with the primary tumor. In addition, phosphorylated c-MYC immunoreactivity was observed in neoplastic cell nuclei, distinctly at the portion of invasive growth. Thus, the present case was diagnosed as apocrine ductal carcinoma originating from the oral scent gland, which typically shows highly aggressive biological behavior.

Developmental Exposure to Aluminum Chloride Irreversibly Affects Postnatal Hippocampal Neurogenesis Involving Multiple Functions in Mice.

Aluminum (Al) is neurotoxic to adults and also to infants. In this study, we investigated the developmental exposure effect of AlCl3 on postnatal hippocampal neurogenesis. Pregnant mice were administered 0-, 900-, or 1800-ppm AlCl3 via drinking water from gestational day 6 to postnatal day (PND) 21, with their offspring examined on PND 21 and PND 77. On PND 21, GFAP-immunoreactive (+) neural stem cells (NSCs) and p21Cip1/Waf1+ cells were decreased in number in the subgranular zone at 900 and ≥900 ppm, respectively. Pcna transcript level examined at 1800 ppm was decreased in the dentate gyrus. These results suggest induction of compromised cell quiescence that caused impaired self-renewal capacity of NSCs accompanying slowing down of cell cycling, which ultimately resulted in exhaustion of the NSC pool. At 1800 ppm, Reelin+ hilar GABAergic interneurons were also decreased, suggesting a contribution to the NSC reduction. At this dose, TBR2+ or DCX+ progenitor and immature granule cells and PVALB+ interneurons were increased. Moreover, COX-2+ granule cells were increased at ≥900 ppm. These results suggest facilitation of transient progenitor cell proliferation and differentiation during exposure. Moreover, TUNEL+ or Morin-stained granule cells were increased, together with Casp12 transcript upregulation, suggesting induction of Al accumulation-related endoplasmic reticulum stress-mediated granule cell apoptosis. Transcript expression changes on cholinergic and glutamatergic signals and synaptic plasticity suggested contribution to disruptive neurogenesis. The NSC-targeting effects sustained through the adult stage despite no sustained Al-accumulation. These results suggest that developmental AlCl3-exposure irreversibly affects postnatal hippocampal neurogenesis involving multiple functions in mice.

Fluorescence tumor imaging by i.v. administered indocyanine green in a mouse model of colitis-associated colon cancer.

Fluorescence tumor imaging using exogenous fluorescent tumor-targeting agents has potential to improve early tumor detection. The fluorescent contrast agent indocyanine green (ICG) is used in medical diagnostics. The aim of the present study is to investigate the tumor imaging capability and the imaging mechanism of i.v. administered ICG in a mouse model of colitis-associated colon cancer. To do this, an azoxymethane/dextran sodium sulfate-induced colon cancer mouse model was used. Ex vivo imaging experiments were carried out 1 hour after i.v. injection of ICG. The ICG fluorescence was observed in the colon tumor tissues, with sufficient tumor to normal tissue ratio, correlating with tumor malignancy. In the tumor tissues, ICG fluorescence was localized in the vascular interstitial tissue. Immunofluorescence microscopy revealed that tumor cells formed tight junctions normally, suggesting an inability of tumor cellular uptake of ICG. In contrast, tumor tissues increased the CD31-immunoreactive endothelial cell area, and accumulated stromal cells immunoreactive for COX-2 and tumor cell population immunoreactive for inducible nitric oxide synthase. In vivo vascular permeability assay revealed that prostaglandin E2 promoted the endothelial cell permeability of ICG. In conclusion, our data indicated that fluorescence contrast-enhanced imaging following i.v. administered ICG can be applied to the detection of colon tumors in a mouse colitis-associated colon cancer model. The tumor tissue preference of ICG in the present model can be attributed to the enhanced vascular leakage of ICG involving inflammatory mediators, such as COX-2 and inducible nitric oxide synthase, in conjunction with increased tumor vascularity.

Choroid plexus carcinoma with neuronal and glial differentiation in a 7-week-old male Sprague-Dawley rat.

We describe a case of choroid plexus carcinoma arising in the cerebrum of a 7-week-old male Sprague-Dawley rat. The tumor mass occupied the right lateral ventricle of the cerebrum. Histological analyses revealed that the epithelial tumor cells had proliferated in tubular, cribriform, papillary and solid growth patterns in the vicinity of the choroid plexus, with slight invasion into the cerebrum parenchyma. We divided the tumor cells into cuboidal, elongated and intermediate cells. Immunohistochemical studies showed that these tumor cells expressed relatively high levels of cytokeratin AE1/AE3, vimentin and glial fibrillary acidic proteins, and low levels of nestin, oligodendrocyte transcription factor and doublecortin proteins. The present case was diagnosed as a choroid plexus carcinoma with neuronal and glial differentiation.

Intermediate-grade mammary gland adenocarcinoma in an 18-year-old female black leopard (Panthera pardus) with acute pancreatic necrosis and chronic interstitial nephropathy.

An 18-year-old female black leopard (Panthera pardus) showed renal failure, leukocytosis and presence of subcutaneous masses in the lower abdominal region and right shoulder; she eventually died. Histopathological observations included a mammary gland carcinoma with comedo, solid and tubulopapillary patterns in subcutaneous tissue, and highly proliferated tumor cells in systemic organs. The tumor cells were positive for cytokeratin AE1/AE3. The mammary gland tumor was diagnosed as intermediate-grade adenocarcinoma, based on a previously reported histological grading system of feline mammary carcinomas. Chronic interstitial nephritis was estimated to have been ongoing for 5 years, whilst acute necrotic pancreatitis in relation to tumor metastasis could have been the cause of death.