Retrospective Study of Clinicopathological Changes and Prediction Model for Canine Vascular Neoplasms.

Vascular neoplasms, including hemangiosarcoma (HSA) and hemangioma (HMA), are more common in dogs than other domestic animal species; however, comprehensive laboratory screening tests for early diagnosis are currently limited. The aims of this study were to investigate general signalments, anatomic locations, and clinicopathological abnormalities of dogs diagnosed with vascular neoplasms and to determine the diagnostic significance of these abnormalities. Retrospective data of dogs with HMA, HSA, and healthy dogs were analyzed. Dogs with HMA and HSA were seniors, with mixed breeds being most affected. HMA affected predominantly non-visceral sites, while HSA was more common in visceral sites, particularly the spleen. In multivariate model analyses, the odds of HMA diagnosis were 5.5 times higher in anemic dogs and 33.0 times higher in lymphopenic dogs compared to dogs without the abnormalities. The odds of HSA diagnosis were 42.5 times higher in anemic dogs, 343 times higher in lymphopenic dogs and 92.7 times higher in dogs with hyperfibrinogenemia compared to dogs without the abnormalities. The study suggested that these identified abnormalities were nonspecific and commonly observed in various chronic diseases, and hence their combination with clinical information, such as diagnostic imaging and histopathology, is important to facilitate a more precise diagnosis of canine vascular neoplasms.

Development and characterization of mouse anti-canine PD-L1 monoclonal antibodies and their expression in canine tumors by immunohistochemistry in vitro.

Immune escape is the hallmark of carcinogenesis. This widely known mechanism is the overexpression of immune checkpoint ligands, such as programmed cell death protein 1 and programmed death-ligand 1 (PD-1/PD-L1), leading to T cell anergy. Therefore, cancer immunotherapy with specific binding to these receptors has been developed to treat human cancers. Due to the lack of cross-reactivity of these antibodies in dogs, a specific canine PD-1/PD-L1 antibody is required. The aim of this study is to develop mouse anti-canine PD-L1 (cPD-L1) monoclonal antibodies and characterize their in vitro properties. Six mice were immunized with recombinant cPD-L1 with a fusion of human Fc tag. The hybridoma clones that successfully generated anti-cPD-L1 antibodies and had neutralizing activity were selected for monoclonal antibody production. Antibody properties were tested by immunosorbent assay, surface plasmon resonance, and immunohistochemistry. Four hybridomas were effectively bound and blocked to recombinant cPD-L1 and cPD-1-His-protein, respectively. Candidate mouse monoclonal antibodies worked efficiently on formalin-fixed paraffin-embedded tissues of canine cancers, including cutaneous T-cell lymphomas, mammary carcinomas, soft tissue sarcomas, squamous cell carcinomas, and malignant melanomas. However, functional assays of these anti-cPD-L1 antibodies need further investigation to prove their abilities as therapeutic drugs in dogs as well as their applications as prognostic markers.

Diagnostic utility of LDH measurement for determining the etiology of modified transudate pleural effusion in cats.

Fluid analysis is an initial approach for determining the underlying causes of body cavity effusions. Modified transudate is commonly diagnosed in pleural effusion in cats, however, it provides limited diagnostic information. Aims of this study were to investigate common etiologies causing different pleural fluid types and to evaluate the usefulness of lactate dehydrogenase (LDH) for differentiating the etiology in modified transudates in cats. Pleural effusion samples from 122 cats were analyzed and classified into three types: transudate, modified transudate, and exudate. Causes of pleural effusion were classified into four conditions: cardiac disease, neoplasia, feline infectious peritonitis (FIP), and pyothorax. The relationship of underlying etiology and fluid types was described. The LDH levels in pleural fluid and plasma were compared between the causes in the samples classified as modified transudate. The fluid analysis of pleural effusion showed that modified transudate was the most common fluid type (44.2%). Neoplasia was predominantly diagnosed (38.5%) as the etiology of pleural effusion. There was no significant correlation between pleural fluid and plasma LDH level in any type of pleural fluid, suggesting that pleural fluid LDH does not appear to be affected by plasma LDH. The occurrence of modified transudate was not associated to its etiologies, however, the LDH level in modified transudates showed significant differences between etiologic groups. The LDH level in modified transudate was excellent in separating cardiac from non-cardiac diseases with a cut-off value of <535 U/L and separating FIP from non-FIP diseases with a cut-off value of >641 U/L. Based on the current findings, pleural fluid LDH can be a useful adjunctive marker for differentiating some causes of modified transudate pleural effusion and should be added in the routine diagnostic work-up of feline patients with pleural effusions.

Case report: BCL-2 and CD31 immunoexpression related to clinical and histopathological evaluation of renal dysplasia in a Welsh Corgi Puppy.

A case of renal dysplasia (RD) in the Welsh Corgi dog has been reported. Clinically, the affected 3-month-old, female, Welsh Corgi dog showed unclear symptoms of chronic kidney disease. Grossly, both left and right kidneys revealed cystic hypoplasia. Histologically, the primary lesions included immature or fetal glomeruli/tubules, proliferative arterioles, persistent metanephric ducts, persistent mesenchyme, and atypical tubular epithelium were presented. A group of degenerative and inflammatory lesions consisting of interstitial nephritis, interstitial fibrosis, and mineralization of tubules were found. Immunohistochemically, the epithelial cells of immature (fetal) tubules had BCL-2 labeling whereas CD31 (PECAM-1) was labeled in the endothelial cells of the proliferative arterioles. The immunohistochemical findings were confirmed and consolidated with the routine histopathological findings. This study was the first demonstration of the clinical, histopathological, and immunohistochemical features of RD disease in a Welsh Corgi puppy.

Evaluation of 41 single nucleotide polymorphisms in canine diffuse large B-cell lymphomas using MassARRAY.

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of lymphoma in dogs with a multicentric form. This study aimed to assemble 41 variants of the previously reported genes and to investigate these variants in canine DLBCL using the Agena MassARRAY platform. These variants were chosen based on the high prevalence observed in canine B- and T-cell lymphomas, their significance for target therapy, and compatibility for multiplex PCR amplification. Lymph node biopsy was performed from 60 dogs with B-cell lymphoma comprising 47 purebred and 13 crossbred dogs. All dogs presented single nucleotide polymorphisms (SNPs) at HYAL4 and SATB1 genes. The lesser mutual SNPs were observed at SEL1L, excluding a cocker spaniel, and c-Kit, with the exception of a pug and a French bulldog. Even though no statistical association was noted between each SNP and dog breed, purebreds were 3.88 times more likely to have a SNP at FLT3 rs852342480 (95%CI 0.50-45.03, p = 0.26), 3.64 times at TRAF3 F306X (95%CI 0.58-42.50, p = 0.43) and 2.66 times at TRAF3 E303EX (95%CI 0.56-13.12, p = 0.31). Also, DLBCL dogs (CHOP-based treatment) with c-Kit T425= had a poorer prognosis with shorter median overall survival times (OST) than dogs with the wild type. Dogs treated with COP chemotherapy and contained 3-5 variants at SEL1L were associated with decreased median OST. Therefore, this SNP's lymphoma panel provides valuable information that we can use to outline a prognosis and develop a treatment plan for the targeted therapy of each dog.

Novel clonality assays for T cell lymphoma in cats targeting the T cell receptor beta, T cell receptor delta, and T cell receptor gamma loci.

BACKGROUND: T cell clonality assays in veterinary medicine currently target only the T cell receptor gamma (TRG) locus. Existing assays have suboptimal sensitivity because of insufficient primer coverage of all possible rearrangements. OBJECTIVE: Develop higher sensitivity clonality assays targeting the TRG, delta (TRD), and beta (TRB) loci in cats. ANIMALS: Cats with histopathologically confirmed lymphoma (n = 89), non-lymphoma (n = 35), and possible hepatic small cell lymphoma (n = 31). METHODS: Molecular clonality assay development utilizing our recently reported topology and expressed repertoire data of the T cell receptor loci in cats. Determination of clonality status of lymphoma, non lymphoma, and possible hepatic small cell lymphoma samples, and calculation of assay sensitivity and specificity. RESULTS: The new multiplex TRG assay yielded the highest sensitivity (95.5%). All assays yielded 100% specificity except for the new multiplex TRG assay (97.3%). The combination of the new TRG and TRB assays yielded sensitivity of 98.9% and specificity of 97.0%. The new TRG assay detected clonality in 17/31 possible small cell lymphoma livers, whereas an existing TRG assay detected clonality in 6/31 livers. CONCLUSIONS AND CLINICAL IMPORTANCE: The assessment of multiple T cell loci compensates for the potential shortcomings of individual assays. Using a combination of molecular clonality assays will increase the overall sensitivity for the diagnosis of T-cell lymphoma in cats, especially intestinal, and hepatic small cell lymphoma. Hepatic small cell lymphomas detected by the new TRG assay utilized rarely expressed V and J genes not recognized by previous assays, likely indicating unique biology of hepatic small cell lymphoma in cats.

Topology and expressed repertoire of the Felis catus T cell receptor loci.

BACKGROUND: The domestic cat (Felis catus) is an important companion animal and is used as a large animal model for human disease. However, the comprehensive study of adaptive immunity in this species is hampered by the lack of data on lymphocyte antigen receptor genes and usage. The objectives of this study were to annotate the feline T cell receptor (TR) loci and to characterize the expressed repertoire in lymphoid organs of normal cats using high-throughput sequencing. RESULTS: The Felis catus TRG locus contains 30 genes: 12 TRGV, 12 TRGJ and 6 TRGC, the TRB locus contains 48 genes: 33 TRBV, 2 TRBD, 11 TRBJ, 2 TRBC, the TRD locus contains 19 genes: 11 TRDV, 2 TRDD, 5 TRDJ, 1 TRDC, and the TRA locus contains 127 genes: 62 TRAV, 64 TRAJ, 1 TRAC. Functional feline V genes form monophyletic clades with their orthologs, and clustering of multimember subgroups frequently occurs in V genes located at the 5' end of TR loci. Recombination signal (RS) sequences of the heptamer and nonamer of functional V and J genes are highly conserved. Analysis of the TRG expressed repertoire showed preferential intra-cassette over inter-cassette rearrangements and dominant usage of the TRGV2-1 and TRGJ1-2 genes. The usage of TRBV genes showed minor bias but TRBJ genes of the second J-C-cluster were more commonly rearranged than TRBJ genes of the first cluster. The TRA/TRD V genes almost exclusively rearranged to J genes within their locus. The TRAV/TRAJ gene usage was relatively balanced while the TRD repertoire was dominated by TRDJ3. CONCLUSIONS: This is the first description of all TR loci in the cat. The genomic organization of feline TR loci was similar to that of previously described jawed vertebrates (gnathostomata) and is compatible with the birth-and-death model of evolution. The large-scale characterization of feline TR genes provides comprehensive baseline data on immune repertoires in healthy cats and will facilitate the development of improved reagents for the diagnosis of lymphoproliferative diseases in cats. In addition, these data might benefit studies using cats as a large animal model for human disease.

Genetic and evolutionary analysis of a new Asia-4 lineage and naturally recombinant canine distemper virus strains from Thailand.

Canine distemper virus (CDV), a pathogen causing fatal disease in a wide range of carnivores, can be classified into several geographically-related lineages. It is unclear how genetic recombination contributed to the evolution and emergence of the novel CDV strains and the evolutions of these strains are not fully yet investigated. In this study, the complete genome sequences of eight CDV viruses, isolated from domestic dogs in Thailand, were investigated. Interestingly, most of the identified CDV strains (CDV1-3, -5, -8 TH/2014) clustered as a novel Asia-4 lineage, while the CDV4, -6, -7 TH/2014 belonged to the Asia-1 lineage. Recombination analysis revealed that the CDV4 TH/2014 is a putative recombinant virus from the Asia-1 and America-2 parent viruses. In contrast, no recombination events were detected in the Asia-4 lineage, indicating that it is a distinctive lineage. Evolutionary analysis suggested that the CDV Asia-4 lineage had emerged since 1924 and shared common ancestor with the America-2 lineage. Pressure analysis revealed that CDV nucleotides were under negative selection pressure for its rapid adaptation. These findings demonstrate the evolution of CDV Asia-4 lineage and identified the Asia-1 recombination event. The information regarding genetic diversity of CDVs is essential for further CDV's research and monitoring.

Expression of canine distemper virus receptor nectin-4 in the central nervous system of dogs.

Canine distemper virus (CDV) exhibits lymphotropic, epitheliotropic, and neurotropic nature, and causes a severe systemic infection in susceptible animals. Initially, signaling lymphocyte activation molecule (SLAM) expressed on immune cells has been identified as a crucial cellular receptor for CDV. Currently, nectin-4 expressed in epithelia has been shown to be another receptor for CDV. Our previous study demonstrated that neurons express nectin-4 and are infected with CDV. In this study, we investigated the distribution pattern of nectin-4 in various cell types in the canine central nervous system and showed its relation to CDV infection to further clarify the pathology of disease. Histopathological, immunohistochemical and immunofluorescent analyses were done using formalin-fixed paraffin-embedded tissues of CDV-infected dogs. Dual staining of nectin-4 and CDV antigen or nectin-4 and brain cell markers was performed. Nectin-4 was detected in ependymal cells, epithelia of choroid plexus, meningeal cells, neurons, granular cells, and Purkinje's cells. CDV antigens were detected in these nectin-4-positive cells, further suggesting contribution of nectin-4 for the CDV neurovirulence. On the other hand, astrocytes did not express nectin-4, although they were frequently infected with CDV. Since astrocytes are negative for SLAM expression, they must express an unidentified CDV receptor, which also contributes to CDV neurovirulence.

Determination phase at transition of gonocytes to spermatogonial stem cells improves establishment efficiency of spermatogonial stem cells in domestic cats.

The development of germ cells has not been entirely documented in the cat especially the transition phase of the gonocyte to the spermatogonial stem cell (G/SSC). The aims of study were to examine testicular development and to identify the G/SSC transition in order to isolate and culture SSCs in vitro. Testes were divided into 3 groups according to donor age (I, < 4 months; II, 4-6 months; and III, > 6 months). In Exp. 1, we studied testicular development by histology, transmission electron microscopy and immunohistochemistry. In Exp. 2, we determined the expression of GFRα-1, DDX-4 and c-kit and performed flow cytometry. The SSCs isolated from groups II and III were characterized by RT-PCR and TEM (Exp. 3). Chronological changes in the G/SSC transition were demonstrated. The size, morphology and ultrastructure of SSCs were distinguishable from those of gonocytes. The results demonstrated that group II contained the highest numbers of SSCs per seminiferous cord/tubule (17.66 ± 2.20%) and GFRα-1(+) cells (14.89 ± 5.66%) compared with the other groups. The findings coincided with an increased efficiency of SSC derivation in group II compared with group III (74.33 ± 2.64% vs. 23.33 ± 2.23%). The colonies expressed mRNA for GFRA1, ZBTB16, RET and POU5F1. Our study found that the G/SSC transition occurs at 4-6 months of age. This period is useful for isolation and improves the establishment efficiency of cat SSCs in vitro.

Genotypic lineages and restriction fragment length polymorphism of canine distemper virus isolates in Thailand.

Canine distemper virus (CDV) is known to cause multisystemic disease in all families of terrestrial carnivores. Attenuated live vaccines have been used to control CDV in a variety of species for many decades, yet a number of CDV infections in vaccinated dogs are still observed. The aims of this study were to investigate the genetic diversity of CDV lineages based on phosphoprotein (P), hemagglutinin (H) and fusion protein (F) genes and to develop the restriction fragment length polymorphism (RFLP) technique for effective differentiation among individual wild-type and vaccine lineages in Thailand. Four commercial vaccine products, thirteen conjunctival swabs and various tissues from 9 necropsied dogs suspected of having CDV infections were included. Virus isolation was performed using Vero cell expressing canine signaling lymphocyte activation molecules (Vero-DST cells). Reverse-transcription polymerase chain reaction (RT-PCR) on 3 gene regions from the dog derived specimens and the vaccines were carried out, then RFLP analysis upon F-gene amplified fragments was developed. Nucleotide sequence and phylogenetic analysis were compared with other CDV lineages in Genbank. Phylogenetic relationships revealed that CDV field isolates were separated from the vaccine lineage and could be divided into two clusters; one of which belonged to the Asia-1 lineage and another, not related to any previous recognized lineages was proposed as 'Asia-4'. RFLP patterns demonstrating concordance with phylogenetic trees of the distemper virus allowed for differentiation between the Asia-1, Asia-4 and vaccine lineages. Thus, RFLP technique is able to effectively distinguish individual wild-type canine distemper virus from vaccine lineages in Thailand.