Morphometric and histopathological evaluation of modified Elnady's plastinated tissue compared to non-plastinated tissue: Highlighting its relevance for teaching and research.

The present study aims to evaluate the morphometric and histopathological properties of Modified Elnady's plastinated tissue after a period compared to non-plastinated tissue. The plastination technique is utilized in research and teaching due to the potential health risks associated with prolonged exposure to formalin. The tissues and organs are permanently dried during plastination and can be used for further anatomical, histopathological and surgical educational purposes. This method involves drying tissue and allowing synthetic materials like glycerin to permeate it. The study compared non-plastinated and plastinated tissue post-plastination to determine if structural alterations differed from those linked to plastination. The study examined the histopathological examination of dogs' skin, muscles, liver, lung, and intestine using formalin-fixed organs for paraffin embedding and previously plastinated organs for a plastinated group. The study examined non-plastinated and plastinated tissues, their histological composition and biometric parameters revealing typical structures in the non-plastinated group. Plasmodiumted tissues exhibited a compacted appearance, volume changes, nuclear clarity, and cytoplasmic hypereosinophilia, with statistical differences between the two groups. The study reveals that plastinated tissues, after 5 years of plastination, maintain their histological architecture well, with some exceptions. Plastinated tissues can be utilized in future microscopic and immunological studies and will be beneficial for teaching and research.

Canine urothelial carcinoma: a pilot study of microRNA detection in formalin-fixed, paraffin-embedded tissue samples and in normal urine.

We assessed the effects of fixation time in formalin and inclusion of surrounding tissue on microRNA (miRNA) cycle quantification (Cq) values in formalin-fixed, paraffin-embedded (FFPE) urothelial carcinoma (UC) tissue (n = 3), and the effect of conditions on miRNAs in urine from 1 healthy dog. MiRNAs were extracted using commercial kits and quantified using miRNA-specific fluorometry in normal bladder tissue scrolls, UC tissue cores, and bladder muscularis tissue cores from 4 FFPE bladder sections (3 UCs, 1 normal), plus 1 UC stored in formalin for 1, 8, 15, and 22 d before paraffin-embedding. Urine was collected from a healthy dog on 4 occasions; 1-mL aliquots were stored at 20, 4, -20, and -80°C for 4, 8, 24, and 48 h, and 1 and 2 wk. For both FFPE tissue and urine, we used reverse-transcription quantitative real-time PCR (RT-qPCR) to quantify miR-143, miR-152, miR-181a, miR-214, miR-1842, and RNU6B in each tissue or sample, using miR-39 as an exogenous control gene. The Cq values were compared with ANOVA and t-tests. The time of tissue-fixation in formalin did not alter miRNA Cq values; inclusion of the muscularis layer resulted in a statistically different miRNA Cq profile for miR-152, miR-181a, and RNU6B in bladder tissue. MiRNAs in acellular urine were stable for up to 2 wk regardless of the storage temperature. Our findings support using stored FFPE and urine samples for miRNA detection; we recommend measuring miRNA only in the tissue of interest in FFPE sections.

Immunohistochemical identification of T and B lymphocytes in formalin-fixed, paraffin-embedded tissues of 53 avian species using commercial antibodies.

Providing a method to detect avian lymphocytes by immunohistochemistry (IHC) would be helpful for analyzing immune function and diagnosing diseases in birds. In this study, we comprehensively examined the immunohistochemical identification of avian T and B lymphocytes in formalin-fixed, paraffin-embedded tissues from 53 avian species across 15 orders, using eight commercially available lymphocyte markers. T lymphocytes from all 53 avian species tested were specifically detected by IHC using the anti-CD3 antibody (clone F7.2.38). The appropriate antibody for detecting avian B lymphocytes in IHC varied depending on the avian species. B lymphocytes were specifically labeled by IHC in 46 of 53 avian species (86.8%) using any of seven B cell markers. The anti-PAX5 antibody (clone SP34) immunohistochemically detected B lymphocytes from the majority of avian species (41 out of 53 species), excluding those in the orders Falconiformes (falcons) and Passeriformes (oscines). The anti-BAFF-R antibody (clone 2C4) proved suitable for detecting B lymphocytes in the orders Galliformes (landfowls) and Anseriformes (waterfowls) in IHC. Caution is advised when using the anti-BLA36 (clone A27-42) and two anti-CD20 (clone L26 and product No. PA5-16701) antibodies, which are commonly used as B cell markers in mammals, for detecting avian B lymphocytes. These antibodies reacted with cells located in both T and B cell areas in certain avian species. The anti-Bu-1a/b (clone AV20) and anti-CD79a (clone HM57) antibodies were found not to bind to B lymphocytes in various avian species in IHC.

Development of a sensitive, high-throughput extraction protocol for qPCR detection of African swine fever virus in formalin-fixed, paraffin-embedded tissues.

African swine fever (ASF) causes fatal disease in pigs and is an escalating threat to the global swine industry. ASF has re-emerged from Africa as a transcontinental epidemic spreading through the Caucasus into Europe, Russia, China, numerous Asian countries, and the Caribbean. ASF virus (ASFV) is a U.S. select agent requiring handling in high-containment biosafety level 3 (BSL-3) laboratories for pathogen work. Formalin-fixation eliminates infectivity and preserves the genome, providing noninfectious specimens for BSL-2 work. Recovery of DNA from formalin-fixed, paraffin-embedded tissue (FFPET) is challenging and cumbersome. A reliable and easy-to-perform method for DNA recovery from FFPET would facilitate surveillance. To meet this objective, we developed a high-throughput protocol for the recovery of ASFV DNA from FFPET. Deparaffinization, tissue lysis, and reversal of cross-linking were performed in a single tube, followed by DNA purification via automated magnetic bead extraction. Quantitative PCR (qPCR) detection was used to determine the copy number of the B646L gene that encodes for the ASFV p72 protein in tissues (5 pigs, 4 tissues) from pigs with lesions consistent with acute ASF. Copy numbers obtained from FFPET were within one log of copy numbers obtained from fresh tissue, thus enabling ASF qPCR surveillance from formalin-inactivated and preserved tissues at BSL-2 at diagnostic sensitivity similar to fresh tissues tested at BSL-3.

Successful restoration of archived ovine formalin fixed paraffin-embedded tissue DNA and single nucleotide polymorphism analysis.

Archived formalin fixed paraffin-embedded (FFPE) tissues are powerful tools in medicine, capable of harboring diagnostic and genetic answers to challenging clinical questions. Successful utilization of DNA derived from FFPE samples is dependent upon repairing DNA damage generated from the fixation process. Methods to repair FFPE DNA have been successful in human medicine for a variety of research and clinical applications, yet remain underutilized in veterinary medicine. Despite the available technology, our study is the first to evaluate the repair of FFPE derived DNA from veterinary species for single-nucleotide polymorphism (SNP) analysis using the Illumina OvineSNP50 BeadChip and Illumina FFPE QC and DNA Restore kit. To accomplish this, 48 ovine FFPE samples were run using the Illumina OvineSNP50 BeadChip with and without restoration. Compared to pre-restore data, we found increased sample call rates, SNP call frequency, and assay metrics for all samples post-restoration. Further, we utilized four sheep with available parallel fresh DNA and FFPE DNA to compare assay metrics and genotype calls between the two starting sample types. Although fresh samples generated increased call rates, we found 99% concordance in allele calls between restored FFPE and fresh DNA for all four samples. Our results indicate successful restoration and genotyping of ovine FFPE samples using this technology, with potential for utilization in other veterinary species.

Viral replication site distribution for rabbit hemorrhagic disease virus 2 in formalin-fixed, paraffin-embedded tissues via in situ hybridization.

We made 2 Z-based in situ hybridization (ISH) probes for the detection of rabbit hemorrhagic disease virus 2 (RHDV2; Lagovirus GI.2) nucleic acid in formalin-fixed, paraffin-embedded tissues from European rabbits (Oryctolagus cuniculus) that had died during an outbreak of RHD in Washington, USA. One probe system was made for detection of negative-sense RNA (i.e., the replicative intermediate RNA for the virus), and the other probe system was constructed for detection of genomic and mRNA of the virus (viral mRNA). Tissue sets were tested separately, and the viral mRNA probe system highlighted much broader tissue distribution than that of the replicative intermediate RNA probe system. The latter was limited to liver, lung, kidney, spleen, myocardium, and occasional endothelial staining, whereas signal for the viral mRNA was seen in many more tissues. The difference in distribution suggests that innate phagocytic activity of various cell types may cause overestimation of viral replication sites when utilizing ISH of single-stranded, positive-sense viruses.

An optimized workflow for microCT imaging of formalin-fixed and paraffin-embedded (FFPE) early equine embryos.

Here, we describe a workflow for high-detail microCT imaging of formalin-fixed and paraffin-embedded (FFPE) equine embryos recovered on Day 34 of pregnancy (E34), a period just before placenta formation. The presented imaging methods are suitable for large animals' embryos with intention to study morphological and developmental aspects, but more generally can be adopted for all kinds of FFPE tissue specimens. Microscopic 3D imaging techniques such as microCT are important tools for detecting and studying normal embryogenesis and developmental disorders. To date, microCT imaging of vertebrate embryos was mostly done on embryos that have been stained with an X-ray dense contrast agent. Here, we describe an alternative imaging procedure that allows to visualize embryo morphology and organ development in unstained FFPE embryos. Two aspects are critical for high-quality data acquisition: (i) a proper sample mounting leaving as little as possible paraffin around the sample and (ii) an image filtering pipeline that improves signal-to-noise ratio in these inherently low-contrast data sets. The presented workflow allows overview imaging of the whole embryo proper and can be used for determination of organ volumes and development. Furthermore, we show that high-resolution interior tomographies can provide virtual histology information from selected regions of interest. In addition, we demonstrate that microCT scanned embryos remain intact during the scanning procedure allowing for a subsequent investigation by routine histology and/or immunohistochemistry. This makes the presented workflow applicable also to archival paraffin-embedded material.

Development of monoclonal antibodies specific to Marek disease virus-EcoRI-Q (Meq) for the immunohistochemical diagnosis of Marek disease using formalin-fixed, paraffin-embedded samples.

Marek disease (MD) is a viral disease characterized by the development of lymphoma in poultry. Although morphologic confirmation of lymphoma is used to diagnose MD, immunohistochemical detection of MD virus-EcoRI-Q (Meq), which is a viral protein that is expressed exclusively in MD tumor cells, would further improve the accuracy of diagnosis. We developed monoclonal antibodies (mAbs) that specifically detect Meq by immunohistochemistry (IHC) using formalin-fixed, paraffin-embedded (FFPE) sections. We evaluated the sensitivity and specificity of 14 mAbs that we produced, using FFPE samples of MDCC-MSB1 cells, MD tumor tissues, and tissues of uninfected chickens. Four different antigen retrieval conditions were investigated. Thirteen mAbs reacted with Meq in FFPE sections, but immunohistochemical reactivity and specificity varied depending on the mAb and antigen retrieval condition; heat-induced antigen retrieval (HIAR) was more effective at detecting Meq than the other tested conditions. HIAR pH 9 tended to increase immunoreactivity and decrease specificity. Of the 5 mAbs that immunoreacted strongly with Meq without nonspecific reactions under the optimal antigen retrieval conditions, 3 mAbs (1C1-121, 3A3-112, 5F7-82) did not produce background staining of tumor or non-tumor tissues; 2 mAbs (2C5-11, 4A5-54) produced background staining. The mAb 6B5-128 reacted moderately with Meq without nonspecific reactions and background staining. The remaining mAbs showed weak immunoreactivity or problematic nonspecific reactions. Our results suggest that some of our developed mAbs can be used in IHC to detect Meq in FFPE sections with high specificity, and that the use of IHC may greatly improve the diagnosis of MD.

Molecular analysis of bovine leukemia virus in early epidemic phase in Japan using archived formalin fixed paraffin embedded histopathological specimens.

Bovine leukemia virus (BLV) is an important pathogen associated with enzootic bovine leukosis. In this study, we performed PCR and sequencing analysis to characterize BLVgp51 sequences from formalin-fixed paraffin-embedded (FFPE) specimens made from 1974 to 2000 and successfully obtained BLV proviral genome sequences from 94% of the analyzed samples. Furthermore, from these samples, we reconstructed eight full-length and nearly full-length BLVgp51 sequences. These sequences were classified as BLV genotype 1, implying that genotype1 has already been circulating in Japan since the 1970s. In our results, the proviral DNA was detected in the 1970s, 1980s, and 1990s in the same manner, indicating that the detection of BLV proviral genome depends on storage conditions rather than storage period. The sequences obtained in this study provide direct insights into BLV sequences before 2000, which serves as a good calibrator for inferring ancient BLV diversity.

Assessment of panfungal PCR performance with formalin-fixed paraffin-embedded tissue specimens†.

We reviewed the performance of a panfungal ITS-2 PCR and Sanger sequencing assay performed on 88 FFPE specimens at The Hospital for Sick Children (Toronto, Canada) in 2019. A potential fungal pathogen was identified by ITS PCR in 62.7 and 2.9% of positive and negative direct slide examination of tissue specimens, respectively. ITS amplicons were detected in 87/88 specimens, with 53/88 (60.2%) considered as 'positive-contaminants' and 34/88 (38.6%) as 'positive-potential pathogen' upon sequencing. Potential pathogens included Blastomyces dermatitidis (17.1%), Cryptococcus neoformans (17.1%), Histoplasma capsulatum (14.3%) and Mucormycetes (11.4%). Laboratories should only perform ITS PCR on FFPE tissues if fungal elements have been confirmed on histopathology slides. LAY SUMMARY: In this study, we examined how well a DNA-based test could detect DNA from fungi in archived human biopsy tissues. The best performance was achieved if fungi were seen in the tissue under a microscope before being tested. Our results indicate that we should only use this test if these conditions are met.

Detection of rabbit hemorrhagic disease virus 2 in formalin-fixed, paraffin-embedded tissues via in situ hybridization.

Formalin-fixed, paraffin-embedded tissues from European rabbits (Oryctolagus cuniculus) that succumbed to rabbit hemorrhagic disease virus 2 (RHDV2; Lagovirus GI.2) during the 2019 outbreak in Washington, USA, were utilized for in situ hybridization via RNAscope (ACDBio). This detection method was both sensitive and specific, with no staining in tissues from RHDV- (Lagovirus GI.1) and RHDV2-negative rabbits, and only slight background staining of RHDV-positive rabbits; RHDV2-positive tissues had bright-red cytoplasmic staining. Although much of the viral mRNA detection was consistent with previously described antigen detection via immunohistochemistry of the liver, lungs, and spleen, there was also significant glomerular staining in the kidneys, and endothelial staining within blood vessels of almost all organs. We validated the RNAscope technique for detection of RHDV2 mRNA in formalin-fixed, paraffin-embedded tissues, with increased sensitivity from previous techniques, and identified additional affected cell types that may contribute to the understanding of pathogenesis.

The advanced paraffin-section preparation technique based on multiple cumulus-oocyte complexes rather than ovaries in ovine.

Immunohistochemical staining is the important method for the identification of protein expression in mammal ovaries, in particular in the follicles with the potential to develop into cumulus-oocyte complexes (COCs), which are able to support oocyte maturation regardless of in vivo or in vitro. Here, we reported an advanced immunohistochemical method based on an artificial structure gathering multiple COCs by paraffin embedding for rapid and highly sensitive detection of co-expressed proteins in ovine COCs rather than ovaries. Compared with the conventional immunohistochemistry on ovine ovaries, the advanced COC paraffin sectioning technique showed the better immunostaining effect and featured the higher generation rate for COCs, the distincter cumulus layers, and the more simplified procedures. These results indicate that the COC paraffin sectioning technique is highly effectively applied for identification of protein expression in ovine COC.

Detection and Phylogenetic Analyses of Taura Syndrome Virus from Archived Davidson's-Fixed Paraffin-Embedded Shrimp Tissue.

Taura syndrome is a World Organization for Animal Health (OIE)-listed disease of marine shrimp that is caused by Taura syndrome virus (TSV), a single-stranded RNA virus. Here we demonstrate the utility of using 15-year-old archived Davidson's-fixed paraffin-embedded (DFPE) shrimp tissues for TSV detection and phylogenetic analyses. Total RNA was isolated from known TSV-infected DFPE tissues using three commercially available kits and the purity and ability to detect TSV in the isolated RNA were compared. TSV was successfully detected through RT-qPCR in all the tested samples. Among the TSV-specific primers screened through RT-PCR, primer pair TSV-20 for the RNA-dependent RNA polymerase (RdRp), primers TSV-15 and TSV-16 for the capsid protein gene VP2 and primers TSV-5 for the capsid protein gene VP1 amplified the highest number of samples. To assess the phylogenetic relation among different TSV isolates, the VP1 gene was amplified and sequenced in overlapping segments. Concatenated sequences from smaller fragments were taken for phylogenetic analyses. The results showed that the TSV isolates from this study generally clustered with homologous isolates from the corresponding geographical regions indicating RNA derived from DFPE tissues can be used for pathogen detection and retrospective analyses. The ability to perform genomic characterization from archived tissue will expedite pathogen discovery, development of diagnostic tools and prevent disease spread in shrimp and potentially other aquaculture species worldwide.

Bovine Babesiosis Diagnosed in Formalin-Fixed, Paraffin-Embedded Tissues by Using In Situ Hybridization.

Bovine babesiosis, caused by Babesia divergens, is in general a rare disease in Europe. Nonetheless, local outbreaks can cause severe economic damage, and postmortem identification represents a diagnostic challenge. During a recent outbreak in May 2018 in northern Germany, 21 animals of a herd of 150 cattle died within 40 days having had clinical signs of fever and hemoglobinuria. Gross examination of 4 of the 21 deceased animals revealed a tick infestation, jaundice, and dark brown staining of urine and kidneys. Histologically, there were iron-positive deposits, hyperplasia of the red pulp of the spleen, and centrilobular necrosis of hepatocytes. In several locations, small basophilic granules suggestive of intraerythrocytic parasites were visible in hematoxylin-eosin- and Giemsa-stained sections. Peripheral blood smears from a living cow from the herd and polymerase chain reaction (PCR) of feeding ticks revealed B. divergens infection. In situ hybridization (ISH) was applied on formalin-fixed, paraffin-embedded (FFPE) tissue of the necropsied cattle to confirm babesiosis in these animals postmortem. Digoxigenin-labeled DNA probes were generated based on a specific nucleotide sequence for B. divergens, obtained by PCR and sequencing of DNA isolates from infected Ixodes ricinus ticks from deceased cattle. ISH using these probes allowed postmortem diagnosis of B. divergens infection in routinely fixed FFPE tissues.

Immunohistochemical identification of T and B lymphocytes in formalin-fixed paraffin-embedded chicken tissues using commercial antibodies.

Immunohistochemical method to detect avian lymphocytes is an efficient and reliable tool for accurate diagnosis, and immunological analysis of avian diseases. However, there are scarce studies reporting immunohistochemistry (IHC) using commercially available antibodies in formalin-fixed paraffin-embedded (FFPE) chicken tissues. In the present study, we established an immunohistochemical method to identify chicken T and B lymphocytes in FFPE chicken tissues using commercial antibodies against chicken or human antigens. For this IHC method, the five tested anti-T lymphocyte antibodies reacted with chicken T lymphocytes on the FFPE sections. Further, 10 commercial anti-B lymphocyte antibodies were tested; of these, three successfully detected chicken B lymphocytes for IHC. In particular, anti-human CD3 (clone F7.2.38) antibody was most suitable for the detection of chicken T lymphocytes, whereas anti-chicken B cell activating factor receptor (BAFF-R) antibody (clone 2C4) was most suitable for the detection of chicken B lymphocytes under our IHC staining conditions. These two antibodies reacted with numerous lymphocytes of all representative lymphoid tissues without problematic background staining and nonspecific reactions. Our results indicate that T and B lymphocytes in FFPE chicken tissues can be immunohistochemically detected using commercial antibodies.

Quality of DNA extracted from formalin-fixed, paraffin-embedded canine tissues.

Veterinary pathology tissue banks are valuable resources for genetic studies. However, limited data exist as to whether quality DNA can be extracted from these tissues for use in canine genotyping studies. We extracted DNA from 44 formalin-fixed, paraffin-embedded (FFPE) tissue blocks from dogs; 9 of these dogs had DNA available from whole blood samples that had been banked. We genotyped DNA from 30 of 44 tissue blocks and 9 whole blood samples on the Illumina CanineHD BeadChip; DNA quality was insufficient in 14 of 44 samples from tissue blocks. There was significant correlation between the 260/280 ratio and single-nucleotide variation (SNV) call rate (p = 0.0276; r2 = 0.162); 23 of 30 samples from FFPE were genotyped with > 65% call rates. Median pairwise identical-by-state (IBS) analysis was 0.99 in 8 pairs of dogs with call rates > 65%. Neither age of tissue block nor specific tissue types were associated with significant differences in DNA concentration, 260/280 ratio, or SNV call rate. DNA extracted from tissue blocks can have variable quality, although comparable levels of homozygosity suggest that extracts from FFPE with call rates > 65% might provide similar results to samples from whole blood when analyzed on the Illumina CanineHD BeadChip.

Immunohistochemical evidence of canine morbillivirus (canine distemper) infection in coatis (Nasua nasua) from Southern Brazil.

The pathological and immunohistochemical (IHC) findings associated with infection due to canine morbilivírus (canine distemper virus, CDV) are described in coatis (Nasua nasua). Tissue fragments of coatis (n = 13) that died at the Bela Vista Sanctuary, Paraná, Southern Brazil, were routinely processed for histopathology to identify the main histopathologic patterns as compared to that of the domestic dog. Selected formalin-fixed paraffin-embedded (FFPE) tissue fragments of the lungs, liver, urinary bladder and small intestine were used in IHC assays designed to identify the antigens of CDV, canine adenovirus (CAdV-1 and CAdV-2) and canine parvovirus type 2 (CPV-2). The main histopathologic patterns identified were interstitial pneumonia (n = 9), interstitial nephritis (n = 6), atrophic enteritis (n = 4) and ballooning degeneration of the uroepithelium (n = 3). Positive immunolabelling for intralesional antigens of CDV was identified in the lung with interstitial pneumonia (n = 3), in the intestine (n = 2) and in the degenerated epithelium of the urinary bladder (n = 2). Antigens of CPV-2, CAdV-1 and CAdV-2 were not identified in any FFPE tissue sections evaluated. These findings indicate that these wild carnivores were infected by a viral disease pathogen common to the domestic dog and develop similar histopathologic findings. Collectively, these findings suggest that these coatis were infected by CDV and can serve as a potential host for this infectious disease pathogen.

Parathyroid hormone receptor 1 (PTHR1) is a prognostic indicator in canine osteosarcoma.

Osteosarcoma (OS) is the most common malignant primary bone tumour in humans and dogs. Several studies have established the vital role of parathyroid hormone-related protein (PTHrP) and its receptor (PTHR1) in bone formation and remodeling. In addition, these molecules play a role in the progression and metastasis of many human tumour types. This study investigated the expression of PTHR1 and PTHrP in canine OS tissues and assessed their prognostic value. Formalin-fixed, paraffin-embedded tissue samples from 50 dogs diagnosed with primary OS were immunolabeled with antibodies specific for PTHR1 and PTHrP. The immunostaining intensity of tumours from patients with OS was correlated with survival time. Both PTHR1 and PTHrP were detected in all OS samples (n = 50). Dogs with OS tumours showing high immunostaining intensity for PTHR1 (n = 36) had significantly shorter survival times (p = 0.028, Log Rank; p = 0.04, Cox regression) when compared with OS that had low immunostaining intensity for PTHR1 (n = 14).PTHrP immunostaining intensity did not correlate with survival time (p > 0.05). The results of this study indicate that increased expression of PTHR1 antigen in canine OS is associated with poor prognosis. This suggests that PTHR1 may be useful as a prognostic indicator in canine OS.

Immunohistochemical identification of antigens of canine distemper virus in neotropical felids from Southern Brazil.

The pathologic and immunohistochemical findings associated with infections due to canine distemper virus (CDV) are described in the cougar (Puma concolor), margay (Leopardus wiedii) and jaguarundi (Herpailurus yagouaroundi) from Southern Brazil. Tissue sections of the neotropical felids (n = 3) that died at the Bela Vista Sanctuary, Paraná, Southern Brazil were routinely processed for histopathology to identify possible histopathologic patterns associated with infections due to CDV. Selected formalin-fixed paraffin embedded tissue sections of the lungs and urinary bladder were used in immunohistochemical assays designed to identify the antigens of CDV. The main histopathologic patterns identified were interstitial pneumonia in the margay and jaguarundi, while ballooning degeneration of the transitional epithelium of the urinary bladder was observed in the cougar. Positive immunoreactivity to antigens of CDV was identified within intralesional sections of the lungs of the two wild felids with interstitial pneumonia and in the degenerated urothelium of the cougar. These findings indicate that these neotropical cats were infected by a viral infectious disease pathogen common to the domestic dog and add to the few documented descriptions of CDV-induced infections in wildlife from Brazil.

MicroRNA Expression in Formalin-Fixed, Paraffin-Embedded Samples of Canine Cutaneous and Oral Melanoma by RT-qPCR.

MicroRNAs (miRNAs) are a class of small, noncoding RNA that post-transcriptionally regulate protein expression. miRNAs are emerging as clinical biomarkers of many diseases, including tumors. The aim of this study was to investigate whether miRNA expression could vary in melanoma samples derived from formalin-fixed, paraffin-embedded (FFPE) tissues. The study included 4 groups: (1) 9 samples of oral canine malignant melanoma, (2) 10 samples of cutaneous malignant melanoma, (3) 5 samples of healthy oral mucosa, and (4) 7 samples of healthy skin. The expression levels of 6 miRNAs-miR-145, miR-146a, miR-425-5p, miR-223, miR-365, and miR-134-were detected and assessed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) using TaqMan probes. Cutaneous canine malignant melanoma showed a decrease of the expression level of miR-145 and miR-365 and an increase of miR-146a and miR-425-5p compared to control samples. MiR-145 was also downregulated in oral canine malignant melanoma. The miRNAs with decreased expression may regulate genes involved in RAS, Rap1, and transforming growth factor ß (TGF-ß) signaling pathways, as well as upregulated genes associated with phosphatidylinositol signaling system, adherens junction, and RAS signaling pathways. In conclusion, miR-145, miR-365, miR-146a, and miR-425-5p were differentially expressed in canine malignant melanoma and healthy FFPE samples, suggesting that they may play a role in canine malignant melanoma pathogenesis.