Development of monoclonal antibodies against Rhodococcus equi virulence-associated protein N and their application to pathological diagnosis.

IMPORTANCE: Rhodococcus equi can cause infection in ruminants, and its pathogenicity is suggested to be associated with VapN. Despite its wide distribution, no immunological diagnostic method has been developed for VapN-producing R. equi. Against this background, we attempted to develop monoclonal antibodies targeting VapN and assess their application in immunostaining. In the study, mice were immunized with recombinant VapN, and cell fusion and cloning by limiting dilution permitted the generation of three antibody-producing hybridomas. The utility of the antibodies produced from the hybridomas in immunostaining was demonstrated using an infected mouse model, and the antibodies were further applied to previously reported cases of R. equi infection in goats and cattle. Although the 4H4 antibody induced the strongest reactions, the reactivity of two other antibodies was improved by antigen retrieval. Our monoclonal antibodies will be utilized to support the definitive diagnosis of suspected R. equi infection, including cases that were previously missed.

Role of autopsy imaging in veterinary forensic medicine: experiences in 39 cases.

While numerous scientific studies have suggested the usefulness of autopsy imaging (Ai) in the field of human forensic medicine, the use of imaging modalities for the purpose of veterinary forensics is currently scant. The current study performed Ai on suspicious dead animals requested by the police department to determine their cause of death. Radiography and/or computed tomography and/or magnetic resonance imaging were performed on 39 suspicious dead animals before necropsy. After diagnostic imaging, pathological examination was performed, with drug testing added as needed. Among the 39 cases, 28, 6, 3, 1, and 1 involved cats, dogs, rabbits, a ferret, and a pigeon, respectively. The major Ai findings included skull and rib fractures, subcutaneous emphysema, pneumothorax, pneumoperitoneum, diaphragmatic hernia, and abdominal rupture. The leading causes of death, determined comprehensively via Ai and pathological reports and drug test results, included traumatic impact, blood loss, poisoning, suffocation, tension pneumothorax, starvation, and drowning, all of which have been strongly suspected to indicate animal abuse by humans. All eight cases of skull fractures and all five cases of poisoning, including suspected poisoning, were of cats. As the numbers of dogs and cats in Japan are almost equal, violence against cats may occur more frequently than dogs. Ai can be a valuable examination tool for veterinary forensic cases. As computed tomography is useful for ruling out fractures that screening computed tomography before necropsy is a more practical option for veterinary forensics.

First isolation and genomic characterization of bovine parechovirus from faecal samples of cattle in Japan.

A novel picornavirus was isolated from the faeces of a diarrhoeic cow using MA-104 cells at the third blind passage. This virus, named Den1/2021/JPN, was completely sequenced using total RNA from the cell culture supernatant by deep sequencing. The genome of Den1/2021/JPN had a standard picornavirus genome organisation with conserved picornaviral motifs. The 5' untranslated region harboured a type-II internal ribosomal entry site. Den1/2021/JPN was most closely related to a bovine parechovirus (Bo_ParV) named cow/2018/4, which has been recently identified in publicly available databases. Phylogenetic analyses and pairwise sequence comparison revealed that Den1/2021/JPN and Bo_ParV cow/2018/4 clustered with parechoviruses and were most closely related to Parechovirus E identified in birds of prey, exhibiting nucleotide sequence similarity of 64.2-64.5 %, 58.6-59.7 % and 66.3-66.4 % in the polyprotein, P1 and 2C+3 CD coding regions, respectively. This study presents the first report on the isolation of Bo_ParV. Den1/2021/JPN and Bo_ParV cow/2018/4, which are candidates for a novel species in the genus Parechovirus.

Genomic diversity and intragenic recombination of species C rotaviruses.

Rotavirus C (RVC) is a major cause of diarrhoea in swine, cattle, and humans worldwide. RVC exhibits sequence diversity in all 11 genes, especially in VP4 and VP7, and all segment-based genotyping has been performed similar to rotavirus A. To date, recombination events have been reported in rotavirus A and B. However, there are no reports describing gene recombination of RVC, except for recombination in NSP3 between RVC and rotavirus H. In this study, nine porcine RVC strains identified in Japanese pigs were completely sequenced and analysed together with RVC sequences from the GenBank database. The analyses showed that sequences of the VP4, VP2, and NSP1 of several porcine RVC strains did not branch with any of those of the RVC strains in the GenBank database, suggesting new genotypes. Several homologous recombination events, between or within genotypes, were identified in the VP4, VP7, VP2, NSP1, and NSP3 genes. Of these, nine, one, and one intergenotypic recombination events in the VP4, VP2, and NSP3 genes, respectively, were supported with sufficient statistical values. Although these findings suggest occurrences of the intragenic recombination events in the RVC genome, potential sequence errors and poor sequence assemblies in the databases should be watched with care. The results in this study present data about the important recombination events of the RVCs, which influence evolution of the virus by aiding them to gain genetic diversity and plasticity, although further sequence data will be necessary to obtain more comprehensive understanding of such mechanisms.

Pathogenicity and genomic features of vapN-harboring Rhodococcus equi isolated from human patients.

Rhodococcus equi is a saprophytic soil bacterium and intracellular pathogen that causes refractory suppurative pneumonia in foals and has emerged as a pathogenic cause of zoonotic disease. Several studies have reported human infections caused by R. equi harboring a recently described third type of virulence plasmid, the ruminant-associated pVAPN, which carries the vapN virulence determinant. Herein, we analyzed pathogenicity and genomic features of nine vapN-harboring R. equi isolated from human patients with and without HIV/AIDS. Four of these strains showed significant VapN production and proliferation in cultured macrophages. These strains were lethally pathogenic after inoculation with 1.0 × 108 CFU in mice and reproduced a necrotizing granulomatous inflammation in the liver and spleen similar to that observed in humans. Additionally, we determined entire genome sequences of all nine strains. Lengths of sequences were 5.0-5.3 Mbp, and GC contents were 68.7 %-68.8 %. All strains harbored a 120- or 125-kbp linear plasmid carrying vapN (Type I or Type II pVAPN) classified on the basis of differences in the distal sequences on the 3' side. Interestingly, VapN production differed significantly among strains harboring nearly identical types of pVAPN with variation limited to several SNPs and short base pair indels. The pVAPN sequences possessed by the VapN-producing strains did not retain any common genetic characteristics, and more detailed analyses, including chromosomal genes, are needed to further elucidate the VapN expression mechanism.

African pygmy hedgehog adenovirus: Virus replication, virus-induced cytopathogenesis and activation of mitogen-activated protein kinase signaling pathways in infected MDCK cells.

We examined several aspects of African hedgehog adenovirus (AhAdv-1) that was isolated from an African pygmy hedgehog, including: replication kinetics of, virus-induced cytopathic effect (CPE), activation status of mitogen-activated protein kinase (MAPK) signaling pathways, and possible roles of these signaling pathways in virus replication and virus-induced CPE in MDCK cells. AhAdv-1 efficiently replicated and induced CPE in infected cells and caused accumulation of cleaved caspase-3 at 24 h post-infection (p.i.), suggesting apoptosis induction. Analysis of several intracellular signal transduction pathways, which are involved in apoptosis, showed activation of p38 MAPK, Akt and ERK1/2 pathways at 3 h p.i., and upregulation of phosphorylated SAPK/JNK at 24 h p.i. Although p38 MAPK inhibitor and SAPK/JNK inhibitor suppressed activation of the respective pathways in infected cells, they did not inhibit virus-induced CPE. Treatment of infected cells with inhibitor of the Akt pathway, the p38 pathway, the SAPK/JNK pathway or the ERK pathway revealed that inhibitors of p38 pathway inhibited viral replication by real-time PCR and TCID50 assay in infected MDCK cells, suggesting that AhAdv-1 uses p38 pathway for multiplication in infected cells.

Equine sarcoid of the glans penis with bovine papillomavirus type 1 in a miniature horse (Falabella).

A 23-year-old Falabella gelding kept in Tochigi, Japan, for more than 20 years presented with a recurrent mass of the glans penis that was first noticed about a year earlier. Partial phallectomy was performed with no adjunctive therapy for local regrowth of the mass. The horse was euthanized 3 months after surgery for urinary retention due to suspected regrowth. The resected mass affected the genital and urethral mucosa of the glans penis, and was diagnosed as equine sarcoid by histopathology and identification of bovine papillomavirus (BPV) DNA. Phylogenetic analysis of the BPV genome of the sarcoid showed high sequence homology to BPV type 1 (BPV-1) from Hokkaido, Japan, suggesting a geographical relationship for BPV-1 in Japan.

Metagenomic identification, sequencing, and genome analysis of porcine hepe-astroviruses (bastroviruses) in porcine feces in Japan.

Recently, hepe-astrovirus-like RNA viruses named bastroviruses (BastVs), have been found in human, pig, bat, and rat fecal samples. In this study, we determined nearly complete genome sequences of four BastVs in the feces of healthy pigs. Genetic characterization revealed that these porcine BastVs (PBastVs) and BastVs from other animals including humans, had the same genome organization, that is, they contained three predicted conserved domains of viral methyltransferase, RNA helicase, and RdRp in the nonstructural ORF1 and the astrovirus capsid domain in the structural ORF2. Phylogenetic analyses using RNA-dependent RNA polymerase and the capsid region revealed that PBastVs branched with bat and rat BastVs; however, the groups formed by each host were distantly related to human BastVs. Pairwise amino acid sequence comparison demonstrated that PBastVs shared 95.2-98.6% and 76.1-95.5% sequence identity among each other in the ORF1 and ORF2 regions, respectively; the sequence identities between PBastVs and BastVs from other animals were 21.4-42.5% and 9.1-20.6% in the ORF1 and ORF2 regions, respectively. This suggested that BastVs were derived from a common ancestor but evolved independently in each host population during a prolonged period. Putative recombination events were identified in the PBastV genome, suggesting that PBastVs gain sequence diversity and flexibility through recombination events. In an analysis of previously obtained metagenomic data, PBastV sequence reads were detected in 7.3% (23/315) of fecal samples from pigs indicating that PBastVs are distributed among pig populations in Japan.

Multiple genotypes of enterovirus G carrying a papain-like cysteine protease (PL-CP) sequence circulating on two pig farms in Japan: first identification of enterovirus G10 carrying a PL-CP sequence.

Two and three genotypes of enterovirus G (EV-G) carrying a papain-like cysteine protease (PL-CP) sequence were detected on two pig farms and classified into genotypes G1 and G10, and G1, G8, and G17, respectively, based on VP1 sequences. A G10 EV-G virus bearing a PL-CP sequence was detected for the first time. Phylogenetic analysis of the P2 and P3 regions grouped the viruses by farm with high sequence similarity. Furthermore, clear recombination break points were detected in the 2A region, suggesting that PL-CP EV-G-containing strains gained sequence diversity through recombination events among the multiple circulating EV-G genotypes on the farms.

Systemic amyloid A (AA) amyloidosis in the Bengalese finch (Lonchura striata var. domestica).

The incidence of systemic amyloid A (AA) amyloidosis was high in Bengalese finches (Lonchura striata var. domestica), as 150 of 235 birds from 5 breeding colonies (63.8%) tested positive for it. The incidence was statistically significantly higher in one colony. The liver was the most frequently affected organ (150 cases), followed by the spleen and then the kidneys. The degree of amyloid deposition was high in the liver and spleen and low in the kidneys. The histological distribution of amyloid deposits was similar in all finch species. Species-specific or colony-specific predisposing factors for systemic AA amyloidosis in Bengalese finches remain to be elucidated. As far as we know, this is the first report of amyloidosis in Bengalese finches.

Pancreatic adenosquamous carcinoma with invasion to the spleen in a cat.

A four-and-a-half-year-old female Scottish Fold cat underwent partial pancreatectomy with en-bloc splenectomy. The resected specimen was a biphasic tumor that was diagnosed histologically and immunohistochemically as pancreatic adenosquamous carcinoma (ASC), a ductal carcinoma variant according to the WHO classification of tumors in humans. There was a gradual transition between the adenocarcinoma component and the squamous cell carcinoma component. The squamous cell carcinoma component comprised approximately 30-40% of the tumor. A pancreatic tumor infiltrated into the gastrosplenic ligament and spleen with regional lymph node and mesenteric metastases. Pancreatic ASC has not been reported in animals. This is a case report of feline pancreatic ASC with splenic involvement.

Genetic diversity of enterovirus G detected in faecal samples of wild boars in Japan: identification of novel genotypes carrying a papain-like cysteine protease sequence.

The genetic diversity of enterovirus G (EV-G) was investigated in the wild-boar population in Japan. EV-G-specific reverse transcription PCR demonstrated 30 (37.5 %) positives out of 80 faecal samples. Of these, viral protein 1 (VP1) fragments of 20 samples were classified into G1 (3 samples), G4 (1 sample), G6 (2 samples), G8 (4 samples), G11 (1 sample), G12 (7 samples), G14 (1 sample) and G17 (1 sample), among which 11 samples had a papain-like cysteine protease (PL-CP) sequence, believed to be the first discoveries in G1 (2 samples) or G17 (1 sample) wild-boar EV-Gs, and in G8 (2 samples) or G12 (6 samples) EV-Gs from any animals. Sequences of the non-structural protein regions were similar among EV-Gs possessing the PL-CP sequence (PL-CP EV-Gs) regardless of genotype or origin, suggesting the existence of a common ancestor for these strains. Interestingly, for the two G8 and two G12 samples, the genome sequences contained two versions, with or without the PL-CP sequence, together with the homologous 2C/PL-CP and PL-CP/3A junction sequences, which may explain how the recombination and deletion of the PL-CP sequences occured in the PL-CP EV-G genomes. These findings shed light on the genetic plasticity and evolution of EV-G.

Complete Genome Sequence of an Adenovirus-1 Isolate from an African Pygmy Hedgehog (Atelerix albiventris) Exhibiting Respiratory Symptoms in Japan.

This study reports the complete genome sequence of an African pygmy hedgehog adenovirus-1 isolate from an African pygmy hedgehog which displayed respiratory symptoms that included nasal discharge, sniffling, coughing, and respiratory distress. The viral genome is 31,764 bp long and shows four deletion sites compared to that of skunk adenovirus-1.

Disseminated nontuberculous mycobacterial disease in a cat caused by Mycobacterium sp. strain MFM001.

Mycobacterium sp. strain MFM001 (MFM001), a new strain of slow-growing nontuberculous mycobacteria not associated with the Mycobacterium avium complex, led to disseminated mycobacterial disease characterized by transmural granulomatous gastroenterocolitis of lepromatous type in a cat treated with immunosuppressive medication. MFM001 was found to be closely related to M. kyorinense or M. celatum by partial sequence of 16S rDNA, and identical with an unspecified M. sp. strain B10-07.09.0206 isolated from a human pulmonary infection in Germany. MFM001 should be considered as a pathogenic organism in cats, especially in immunocompromised animals.

Increased expression of the stromal fibroblast-secreted periostin in canine squamous cell carcinomas.

Canine squamous cell carcinoma (SCC) shows highly invasive and locally destructive growth. In animal models and human cancer cases, periostin plays a critical role in the enhancement of cancer growth; however, the mechanism of involvement in canine cancers remains unknown. The aim of this study was to examine the involvement of periostin in the pathophysiology of SCC in dogs. We examined the localization of periostin and periostin-producing cells in 20 SCC and three squamous papilloma specimens. Furthermore, we focused on transforming growth factor (TGF)-ß1, which was assumed to be an inducing factor of periostin, using culture cells. By immunohistochemistry, limited periostin expression in the stroma was observed in all squamous papillomas. In SCC, periostin protein diffusely expressed at the tumor invasion front of cancer growth. In situ hybridization revealed that periostin mRNA was expressed in the stromal fibroblasts in SCC. In vitro analysis determined that canine SCC cells expressed significantly higher levels of TGF-ß1 mRNA compared with canine keratinocytes. In addition, recombinant TGF-ß1 induced secretion of periostin from cultured dermal fibroblasts. These data suggest that periostin produced by stromal fibroblasts may be involved in the pathophysiology of canine SCC. TGF-ß1 derived from SCC cells may stimulate fibroblasts to produce periostin.

Genetic diversity and recombination of enterovirus G strains in Japanese pigs: High prevalence of strains carrying a papain-like cysteine protease sequence in the enterovirus G population.

To study the genetic diversity of enterovirus G (EV-G) among Japanese pigs, metagenomics sequencing was performed on fecal samples from pigs with or without diarrhea, collected between 2014 and 2016. Fifty-nine EV-G sequences, which were >5,000 nucleotides long, were obtained. By complete VP1 sequence analysis, Japanese EV-G isolates were classified into G1 (17 strains), G2 (four strains), G3 (22 strains), G4 (two strains), G6 (two strains), G9 (six strains), G10 (five strains), and a new genotype (one strain). Remarkably, 16 G1 and one G2 strain identified in diarrheic (23.5%; four strains) or normal (76.5%; 13 strains) fecal samples possessed a papain-like cysteine protease (PL-CP) sequence, which was recently found in the USA and Belgium in the EV-G genome, at the 2C-3A junction site. This paper presents the first report of the high prevalence of viruses carrying PL-CP in the EV-G population. Furthermore, possible inter- and intragenotype recombination events were found among EV-G strains, including G1-PL-CP strains. Our findings may advance the understanding of the molecular epidemiology and genetic evolution of EV-Gs.

Primary Histiocytic Sarcoma of the Brain in an African Hedgehog (Atelerix albiventris).

A 2.5-year-old African hedgehog (Atelerix albiventris) with signs of progressive paresis/paralysis for approximately 6 months was diagnosed with 'wobbly hedgehog syndrome' (WHS). Post-mortem examination revealed a primary central nervous system (CNS) histiocytic sarcoma (HS) associated with neurodegenerative changes of the CNS and skeletal muscle atrophy. Grossly, a spherical mass infiltrated the left forebrain and was located between the frontal lobe and thalamus in coronal sections. Microscopically, the neoplastic cells had eccentrically located anisokaryotic nuclei and abundant eosinophilic cytoplasm. Immunohistochemically, they were positive for calcium-binding adapter molecule 1, lysozyme, CD68 and vimentin. Ultrastructurally, the neoplastic cells had varying amounts of bundles and whorls of intermediate filaments and scattered lysosomes. This report represents the first documentation of primary CNS HS in this species. Primary CNS HS should be considered as a differential diagnoses for hedgehogs that present with neurological signs of WHS.

A nasal osteoma with an acute course in a Japanese Black heifer.

A 14-month-old Japanese Black heifer presented with unilateral epistaxis and mild swelling of the right face. Radiography revealed a mass with increased radiopacity on the right side of the nasal bridge, extending to the left side. Intranasal endoscopy confirmed a large tumor-like structure protruding into the nasal cavity. Following euthanasia, cranial computed tomography (CT) was performed, revealing a tumor 24.3 × 17.5 × 14.8 cm in size. The tumor occupied the entire right nasal cavity and the frontal and sphenoid sinuses. Histopathological examination revealed that the tumor consisted of well-differentiated trabecular bones and loose connective tissue. Based on these findings, a diagnosis of osteoma was established. This report describes a case of osteoma with an acute course in a Japanese Black heifer.

Congenital anterior staphyloma associated with Peters' anomaly and aphakia in a Holstein calf.

A Holstein calf was born with a large protruding right eye and a central corneal opacity. Enucleation was the first choice of treatment. The calf had a good prognosis and was raised for milking purposes. Macroscopically, the enucleated eye was characterized by the protruded cornea, adherence of the iris to the central posterior cornea and aphakia. Microscopically, central corneal thickening and a defect in the endothelium and Descemet's membrane were observed. These data suggest that this represents a case of unilateral anterior segment dysgenesis consistent with congenital corneal staphyloma with Peters' anomaly and aphakia. Neither a mutation of the WFDC1 gene in multiple ocular defects nor any other identifiable cause for ocular anomalies was detected.

Mycobacterium avium subsp. hominissuis menigoencephalitis in a cat.

A 33-month old, neutered female Abyssinian cat died. The cat had sudden onset of widespread neurologic signs about half a year after birth. Mycobacterium avium subsp. hominissuis (MAH) group was isolated and identified from the brain of a cat affected with pyogranulomatous meningoencephalitis. The central nervous system (CNS) was involved in the disseminated MAH infection. MAH infection should be considered in cats with neurologic signs in regard to zoonotic aspects. Comparatively, this is a first case of MAH infection observed in the brain in either humans or animals.