RESUMO
INTRODUCTION: The novel Porcine circovirus 3 (PCV-3) has been associated in the past years to different porcine diseases, including reproductive failure. The potential occurrence of PCV-3 in abortions from Swiss pig herds has not been investigated so far. Thus, we conducted a retrospective study on pig aborted cases submitted to our laboratory in the University of Bern during the last 10 years with the main aim of investigating the possible presence of PCV-3 in foetal and/or placental tissue. Twelve out of the 53 studied cases showed mild histopathological changes as previously described in PCV-3 positive cases. However, in none of the cases, PCV-3 genetic material could be detected in the examined formalin-fixed, paraffin-embedded tissues. In only one third of the cases, a cause for the abortion was found, which is similar to other studies. Our survey suggests that PCV-3 was not involved in the porcine abortion cases submitted over the last decade at our institution in Switzerland.
INTRODUCTION: Le nouveau Circovirus porcin 3 (PCV-3) a été associé ces dernières années à différentes maladies porcines, y compris des troubles de la reproduction. La présence potentielle du PCV-3 dans les avortements de porcs en Suisse n'a pas été étudiée jusqu'à présent. Nous avons donc mené une étude rétrospective sur les cas d'avortements de porcs soumis à notre laboratoire de l'Université de Berne au cours des 10 dernières années, dans le but principal d'étudier la présence éventuelle du PCV-3 dans les tissus fÅtaux et/ou placentaires. Douze des 53 cas étudiés présentaient des changements histopathologiques légers, tels que décrits précédemment dans les cas positifs au PCV-3. Cependant, dans aucun des cas, le matériel génétique du PCV-3 n'a pu être détecté dans les tissus examinés fixés au formol et inclus en paraffine. Dans un tiers des cas seulement, une cause d'avortement a été trouvée, ce qui est similaire à d'autres études. Notre étude suggère que le PCV-3 n'a pas été impliqué dans les cas d'avortements porcins soumis au cours de la dernière décennie dans notre institution en Suisse.
Assuntos
Aborto Animal , Infecções por Circoviridae , Circovirus , Inclusão em Parafina , Doenças dos Suínos , Animais , Suíça/epidemiologia , Circovirus/isolamento & purificação , Circovirus/genética , Suínos , Doenças dos Suínos/virologia , Doenças dos Suínos/patologia , Feminino , Aborto Animal/virologia , Infecções por Circoviridae/veterinária , Infecções por Circoviridae/virologia , Estudos Retrospectivos , Inclusão em Parafina/veterinária , Gravidez , Formaldeído , Placenta/virologia , Placenta/patologiaRESUMO
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.
Assuntos
Fígado , Pulmão , Plastinação , Animais , Cães , Plastinação/métodos , Pulmão/patologia , Fígado/patologia , Pele/patologia , Pele/anatomia & histologia , Intestinos/anatomia & histologia , Intestinos/patologia , Inclusão em Parafina/veterinária , Formaldeído , Anatomia Veterinária/educaçãoRESUMO
RNAscope in situ hybridization (ISH) detects target RNA in formalin-fixed, paraffin-embedded (FFPE) tissues. Protocols suggest that prolonged FFPE storage and formalin fixation may impact signal detection, potentially limiting the utility of RNAscope ISH in retrospective studies. To develop parameters for RNAscope use with archived specimens, we evaluated the effect of formalin-fixation time by measuring the signal of a reference gene (16srRNA) in selected tissues fixed in 10% neutral-buffered formalin for 1, 2, 3, 5, 7, 10, 14, 21, 28, 60, 90, 180, and 270 d. The signal intensity and percent area of signal decreased after 180 d. Tissues had detectable signal at 180 d but not at 270 d of formalin fixation. To assess target detection in paraffin blocks, we qualitatively compared the signal of canine distemper virus (CDV) antigen via immunohistochemistry and CDV RNA via RNAscope ISH in replicate sections from blocks stored at room temperature for 6 mo, 1, 3, 6, 8, 11, 13, and 15 y; RNA was detected in FFPE tissues stored for up to 15 y. Our results demonstrate that RNAscope ISH can detect targets in tissues with prolonged paraffin storage intervals and formalin-fixation times.
Assuntos
Formaldeído , Hibridização In Situ , Inclusão em Parafina , Fixação de Tecidos , Inclusão em Parafina/veterinária , Hibridização In Situ/veterinária , Hibridização In Situ/métodos , Animais , Fixação de Tecidos/veterinária , Fixação de Tecidos/métodos , Cães , RNA Viral/análise , Fatores de TempoRESUMO
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.
Assuntos
Carcinoma de Células de Transição , Doenças do Cão , MicroRNAs , Neoplasias da Bexiga Urinária , Cães , Animais , MicroRNAs/genética , MicroRNAs/análise , Projetos Piloto , Carcinoma de Células de Transição/genética , Carcinoma de Células de Transição/veterinária , Neoplasias da Bexiga Urinária/veterinária , Inclusão em Parafina/veterinária , Formaldeído , Fixação de Tecidos/veterinária , Fixação de Tecidos/métodos , Doenças do Cão/diagnóstico , Doenças do Cão/genética , Doenças do Cão/patologiaRESUMO
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.
Assuntos
Anticorpos Monoclonais , Linfócitos B , Animais , Inclusão em Parafina/veterinária , Inclusão em Parafina/métodos , Formaldeído , Aves , MamíferosRESUMO
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.
Assuntos
Vírus da Febre Suína Africana , Febre Suína Africana , Doenças dos Suínos , Suínos , Animais , Vírus da Febre Suína Africana/genética , Febre Suína Africana/diagnóstico , Febre Suína Africana/epidemiologia , Inclusão em Parafina/veterinária , Reação em Cadeia da Polimerase/veterinária , Formaldeído , Doenças dos Suínos/diagnósticoRESUMO
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.
Assuntos
Formaldeído , Polimorfismo de Nucleotídeo Único , Humanos , Animais , Ovinos/genética , Fixação de Tecidos/veterinária , Inclusão em Parafina/veterinária , DNA/genéticaRESUMO
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.
Assuntos
Infecções por Caliciviridae , Vírus da Doença Hemorrágica de Coelhos , Animais , Coelhos , Vírus da Doença Hemorrágica de Coelhos/genética , Inclusão em Parafina/veterinária , Sondas RNA , Infecções por Caliciviridae/epidemiologia , Infecções por Caliciviridae/veterinária , Hibridização In Situ/veterinária , Replicação Viral , Formaldeído , RNA , RNA Mensageiro/genéticaRESUMO
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.
Assuntos
Fluxo de Trabalho , Microtomografia por Raio-X , Animais , Formaldeído , Cavalos , Inclusão em Parafina/veterinária , Fixação de Tecidos/veterinária , Vertebrados , Microtomografia por Raio-X/métodos , Microtomografia por Raio-X/veterináriaRESUMO
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.
Assuntos
Herpesvirus Galináceo 2 , Doença de Marek , Animais , Anticorpos Monoclonais , Galinhas , Formaldeído , Doença de Marek/diagnóstico , Inclusão em Parafina/veterináriaRESUMO
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.
Assuntos
Doenças dos Bovinos , Leucose Enzoótica Bovina , Vírus da Leucemia Bovina , Animais , Bovinos , Leucose Enzoótica Bovina/diagnóstico , Formaldeído , Japão/epidemiologia , Vírus da Leucemia Bovina/genética , Inclusão em Parafina/veterinária , Carga Viral/veterináriaRESUMO
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.
Assuntos
Formaldeído , Histoplasma , Animais , DNA Fúngico/genética , Histoplasma/genética , Inclusão em Parafina/veterinária , Reação em Cadeia da Polimerase/veterinária , Sensibilidade e EspecificidadeRESUMO
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.
Assuntos
Vírus da Doença Hemorrágica de Coelhos , Animais , Formaldeído , Vírus da Doença Hemorrágica de Coelhos/genética , Imuno-Histoquímica , Hibridização In Situ/veterinária , Inclusão em Parafina/veterináriaRESUMO
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.
Assuntos
Células do Cúmulo/citologia , Oócitos/citologia , Inclusão em Parafina/veterinária , Ovinos/fisiologia , Animais , Conexinas/genética , Conexinas/metabolismo , Células do Cúmulo/metabolismo , Feminino , Regulação da Expressão Gênica , Oócitos/metabolismo , Inclusão em Parafina/métodosRESUMO
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.
Assuntos
Decápodes/virologia , Dicistroviridae/classificação , Dicistroviridae/isolamento & purificação , Inclusão em Parafina/métodos , Inclusão em Parafina/veterinária , Filogenia , Animais , Aquicultura , Crustáceos , Dicistroviridae/patogenicidade , Doenças dos Peixes , RNA Viral/genética , Estudos Retrospectivos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterináriaRESUMO
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.
Assuntos
Babesiose , Doenças dos Bovinos , Animais , Babesiose/diagnóstico , Bovinos , Doenças dos Bovinos/diagnóstico , Europa (Continente) , Feminino , Formaldeído , Alemanha , Hibridização In Situ/veterinária , Inclusão em Parafina/veterináriaRESUMO
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.
Assuntos
Linfócitos B , Separação Celular/veterinária , Galinhas/anatomia & histologia , Imuno-Histoquímica/veterinária , Inclusão em Parafina/veterinária , Linfócitos T , Animais , Anticorpos/imunologia , Feminino , Formaldeído , Humanos , Imuno-Histoquímica/instrumentação , Imuno-Histoquímica/métodos , Masculino , Fixação de Tecidos/veterináriaRESUMO
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.
Assuntos
DNA/análise , Cães/genética , Genótipo , Animais , Formaldeído/química , Especificidade de Órgãos , Inclusão em Parafina/veterináriaRESUMO
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.
Assuntos
Antígenos Virais/imunologia , Vírus da Cinomose Canina/imunologia , Cinomose/virologia , Procyonidae/virologia , Animais , Brasil/epidemiologia , Cinomose/epidemiologia , Cinomose/patologia , Vírus da Cinomose Canina/isolamento & purificação , Feminino , Imuno-Histoquímica/veterinária , Intestino Delgado/patologia , Intestino Delgado/virologia , Fígado/patologia , Fígado/virologia , Pulmão/patologia , Pulmão/virologia , Masculino , Inclusão em Parafina/veterinária , Bexiga Urinária/patologia , Bexiga Urinária/virologiaRESUMO
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.