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.

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.

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.

Evaluation of weekly bathing in allergic dogs with methicillin-resistant Staphylococcal colonization.

We evaluated the efficacy of weekly bathing in reducing methicillin-resistant Staphylococcus (MRS) colonization in canine allergic dermatitis in a pilot clinical trial. Six dogs with allergic dermatitis controlled by prescription medications were treated with weekly bathing for 1 month. The Canine Atopic Dermatitis Extent and Severity Index version 3 (CADESI-03) and pruritus scores and frequency of mecA-positive Staphylococcus spp. isolated from three body sites between weeks 0 and 4 were compared. There was no significant difference in CADESI-03 scores with bathing, whereas the pruritus scores were significantly reduced (p < 0.05). Furthermore, MRS frequency was decreased in four of the six dogs (p < 0.05). In conclusion, weekly bathing should be considered for reducing MRS colonization in canine allergic dermatitis.

Diode laser coagulation for the treatment of epistaxis in a Scottish fold cat.

We report the case of a 4-year-old, castrated 4.2-kg Scottish fold cat with recurrent epistaxis that was unresponsive to medical therapy. Diathermocoagulation of the nasal mucosa with a diode laser controlled the epistaxis and there was no significant recurrence of epistaxis during 1 year of follow-up.

Coagulation à la diode laser pour le traitement de l'épistaxis chez un chat Scottish Fold. Nous signalons le cas d'un chat Scottish Fold castré âgé de 4 ans d'un poids de 4,2 kg atteint d'épistaxis récurrente qui n'a pas répondu au traitement médical. La diathermocoagulation de la muqueuse nasale à l'aide d'une diode laser a contrôlé l'épistaxis et il n'y pas eu de récurrence de l'épistaxis durant le suivi d'un an.(Traduit par Isabelle Vallières).

Lipomatosis of the canine parotid gland: case report with a literature review.

In this report, we describe a case of lipomatosis in the left parotid gland of an eight-year-old female Shetland sheepdog and review the relevant literature. Preoperative diagnosis of lipomatosis with Tru-Cut biopsy presented difficulties in this case. The postoperative diagnosis was based on the gross appearance of a non-infiltrative, circumscribed swollen mass in the parotid gland and the histological appearance of normal adipocytes that infiltrated into the parotid gland without involving surrounding structures. Additionally, flotation of the whole parotid gland in formalin solution together with well-maintained residual lobulation and a well-maintained configuration on the cut surface were identified as subsidiary characteristics of lipomatosis of the parotid gland. Lipomatosis of the parotid gland has been rarely reported in dogs and is a poorly understood condition.

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.

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.

Gingival overgrowth in a dog that received long-term cyclosporine for immune-mediated hemolytic anemia.

Gingival mass lesions developed when cyclosporine was administered for 600 days to a female, 7-year-old, longhaired dachshund diagnosed with intractable immune-mediated hemolytic anemia (IMHA). Histopathology indicated hyperplastic suppurative gingivitis. As the anemia improved, the dosage of cyclosporine A (CsA) was markedly decreased, and the mass lesions decreased in size and disappeared, thus suggesting that the mass lesions were an adverse reaction to CsA.

Retrospective study of canine nasal tumor treated with hypofractionated radiotherapy.

The object of this study was to evaluate hypofractionated multiportal field and two-portion (rostral and caudal portions divided by the eyelid) radiation therapy for canine nasal tumors. Sixty-three dogs underwent multiportal hypofractionated radiation therapy. The radiation field was divided into rostral and caudal portions by the eyelid. Treatments were performed four times for 57 dogs. The median irradiation dose/fraction was 8 Gy (range, 5-10 Gy); the median total dose was 32 Gy (10-40 Gy). Improvement of clinical symptoms was achieved in 53 (84.1%) of 63 cases. Median survival time was 197 days (range, 2-1,080 days). Median survival times with and without destruction of the cribriform plate before radiotherapy were 163 and 219 days, respectively. There was no significant difference between them. No other factors were related to survival according to a univariate analysis. All radiation side effects, except one, were grade I according to the VRTOG classification. It was not necessary to treat any dogs for skin side effects. One dog (1.6%) developed an oronasal fistula 1 year after completion of radiation therapy. This radiation protocol may be useful in reducing radiation side effects in dogs with cribriform plate destruction.

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.

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.

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.

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.

Peripheral acanthomatous ameloblasoma in a rabbit with review of previous submissions of the Armed Forces Institute of Pathology Wednesday Slide Conference.

A case of peripheral acanthomatous ameloblasoma of the angulus orisa in a 6-year-old female rabbit was described. The tumor composed of confluent follicles of stellate reticulum with peripheral cuboidal to reverse polarized columnar cells with nuclear palisading in the submucosa of the angulus orisa. The follicular cell elements stained positive for pan-cytokeratin. Additionally, there were multifocal keratinizing foci of the stellate reticulum with some mineralization. Calcified product such as enamel or dentin was not formed. Acanthomatous ameloblasoma in the submucosa of the angulus orisa has not been described and it may develop from an ectopic rest of odontogenic tissue. After excision, neither local recurrence nor metastasis has been observed.

Retrospective study of canine insulinomas: eight cases (2005-2008).

This retrospective study examined 8 cases of pathologically diagnosed insulinoma. Surgical intervention was performed in all cases, once insulinoma was suspected based on unique clinical signs, hypoglycemia, and/or findings on ultrasonography. At the same time, insulin and glucose concentrations were measured before surgery for clinical diagnosis. Although all cases displayed typical clinical signs and hypoglycemia, insulin concentrations varied with 3 of 8 cases showing levels within the reference interval. In addition, to confirm the reliability of measured values, we submitted serum samples from 4 cases to two commercial veterinary laboratories. Results differed considerably between laboratories, with no apparent correlations between the two. In Laboratory A, 3 of 4 cases were above the reference interval, and 1 case was in the middle of the reference interval. Conversely, in Laboratory B, 3 of 4 cases were above the reference interval, and 1 case was below the reference interval. Split decisions regarding the diagnosis of insulinoma were seen for 2 of the 4 cases.

Cutaneous clear cell adnexal carcinoma in a dog: special reference to cytokeratin expression.

A 5-year-old, male Bichon-Frise dog presented with a cutaneous mass in the basal region of the auricle. Histologically, the cutaneous neoplasm was comprised of lobules with solid cellular proliferation separated by thin fibrous septa. Neoplastic cells varied in size, with moderate to abundant amounts of PAS-positive cytoplasm, large nuclei and prominent nucleoli. Immunohistochemical examinations showed that tumor cells were positive for pan-cytokeratin (CK) (AE1/AE3 and CAM5.2), CK8 and CK18, but negative for pan-CK (KL1), CK7, CK14, CK16 and CK20. Double-labeled immunofluorescence testing indicated that neoplastic cells frequently co-expressed CK and vimentin, suggesting divergent differentiation of tumor cells. Based on these findings, the tumor was diagnosed as canine clear cell adnexal carcinoma.