Genomic Surveillance of Yellow Fever Virus Epizootic in São Paulo, Brazil, 2016 - 2018.

São Paulo, a densely inhabited state in southeast Brazil that contains the fourth most populated city in the world, recently experienced its largest yellow fever virus (YFV) outbreak in decades. YFV does not normally circulate extensively in São Paulo, so most people were unvaccinated when the outbreak began. Surveillance in non-human primates (NHPs) is important for determining the magnitude and geographic extent of an epizootic, thereby helping to evaluate the risk of YFV spillover to humans. Data from infected NHPs can give more accurate insights into YFV spread than when using data from human cases alone. To contextualise human cases, identify epizootic foci and uncover the rate and direction of YFV spread in São Paulo, we generated and analysed virus genomic data and epizootic case data from NHPs in São Paulo. We report the occurrence of three spatiotemporally distinct phases of the outbreak in São Paulo prior to February 2018. We generated 51 new virus genomes from YFV positive cases identified in 23 different municipalities in São Paulo, mostly sampled from NHPs between October 2016 and January 2018. Although we observe substantial heterogeneity in lineage dispersal velocities between phylogenetic branches, continuous phylogeographic analyses of generated YFV genomes suggest that YFV lineages spread in São Paulo at a mean rate of approximately 1km per day during all phases of the outbreak. Viral lineages from the first epizootic phase in northern São Paulo subsequently dispersed towards the south of the state to cause the second and third epizootic phases there. This alters our understanding of how YFV was introduced into the densely populated south of São Paulo state. Our results shed light on the sylvatic transmission of YFV in highly fragmented forested regions in São Paulo state and highlight the importance of continued surveillance of zoonotic pathogens in sentinel species.

Differential Yellow Fever Susceptibility in New World Nonhuman Primates, Comparison with Humans, and Implications for Surveillance.

A major outbreak of yellow fever (YF) occurred in Brazil during 2016-2018. Epizootics in New World nonhuman primates are sentinel events for YF virus circulation. However, genus-specific susceptibilities and suitability for YF surveillance remain poorly understood. We obtained and compared epidemiologic, histopathologic, immunohistochemical, and molecular results from 93 human and 1,752 primate cases submitted during the recent YF outbreak in Brazil (2017), with the support of the Brazilian National YF Surveillance Program. We detected heterogeneous YF-associated profiles among the various genera of primates we analyzed. Alouatta primates were the most reliable sentinel; Sapajus and Callicebus primates had higher viral loads but lower proportional mortality rates. Callithrix primates were the least sensitive, showing lower viral loads, lower proportional mortality rates, and no demonstrable YF virus antigen or extensive lesions in liver, despite detectable viral RNA. These differences in susceptibility, viral load, and mortality rates should be considered in strategic surveillance of epizootics and control measures for YF.

Yellow Fever as Cause of Death of Titi Monkeys (Callicebus Spp.).

From 2016 to 2018, an epidemic wave of yellow fever (YF) occurred in Brazil, affecting a large number of Platyrrhini monkeys. Titi monkeys (Callicebus spp.) were severely affected yet pathological characterizations are lacking. This study characterized epizootic YF in 43 titi monkeys (Callicebus spp.) with respect to the microscopic lesions in liver, kidney, spleen, heart, brain, and lung, as well as the distribution of immunolabeling for YF virus antigen, and the flaviviral load in the liver. Of 43 titi monkeys examined, 18 (42%) were positive for yellow fever virus (YFV) by immunohistochemistry or reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). Affected livers had consistent marked panlobular necrotizing hepatitis, lipidosis, and mild inflammation, with intense immunolabeling for YFV mainly in centrilobular hepatocytes (zone 1; P = .05). In the spleen, consistent findings were variable lymphoid depletion (10/11), lymphoid necrosis (lymphocytolysis; 4/11), and immunolabeling for YFV in histiocytic cells (3/16). The main finding in the kidney was multifocal acute necrosis of tubular epithelium (5/7) that was occasionally associated with intracytoplasmic immunolabeling for YFV (6/15). These data indicate that titi monkeys are susceptible to YFV infection, developing severe hepatic lesions and high viral loads, comparable to humans and Alouatta spp. Thus, Callicebus spp. may be reliable sentinels for YF surveillance.

Multiple trichoblastomas in a dog.

BACKGROUND: Hair follicle tumours generally present as benign, solitary masses and have a good prognosis following surgical resection. HYPOTHESIS/OBJECTIVES: This report describes a case of multiple trichoblastomas in a dog. ANIMAL: A 2-year-old crossbred dog presented with multiple soft cutaneous periocular, perilabial, submandibular and nasal nodules, between 2 and 9 cm in diameter, located on the right side of the face. New nodules were observed on the same side of the face at a second consultation 3 weeks later. METHODS: Surgical resection of all nodules was performed in two procedures. Three nodules were initially resected and submitted for histolopathology and immunohistochemistry. The diagnosis was trichoblastoma for all three. At the time of the second consultation, new and remaining nodules were biopsied and the diagnosis of trichoblastoma confirmed. The dog was treated with doxorubicin and piroxicam for 30 days prior to the second surgical procedure in an attempt to reduce new tumour growth and the size of present tumours. All nodules were resected and the defects closed using rotation flaps. RESULTS: No recurrence of the neoplasm was noted within 10 months after surgery. CONCLUSIONS AND CLINICAL IMPORTANCE: Trichoblastomas are generally benign but can present as multiple neoplasms that may require surgical resection and may respond to chemotherapy. To the authors' knowledge, this is the first report of multiple trichoblastomas in a dog.

Disseminated paracoccidioidomycosis in a captive western black-handed tamarin (Saguinus niger).

Paracoccidioidomycosis (PCM) is an endemic fungal disease that occurs in Latin America and primarily affects humans. The disease has been rarely documented in non-human primates. This report details a disseminated and fatal case of PCM caused by Paracoccidioides brasiliensis in a western black-handed tamarin (Saguinus niger) under human care. Histopathological examination revealed extensive pyogranulomatous inflammation in the lungs, spleen, liver, lymph nodes, kidneys, epididymis, right testicle, heart, adrenal gland and intestines, associated with characteristic yeast forms consistent with Paracoccidioides spp and confirmed by immunohistochemistry. Molecular analysis indicated a high nucleotide similarity with P. brasiliensis sequences for both the 18S rRNA and gp43 genes. This naturally occurring infection highlights the susceptibility of these animals to PCM and their role in ecoepidemiology warrants further investigation.

Felid herpesvirus 1 as a causative agent of severe nonsuppurative meningoencephalitis in a domestic cat.

Felid herpesvirus 1 is an important respiratory pathogen of domestic cats. This report presents the first case of severe nonsuppurative meningoencephalitis caused by this virus in a cat.

Intrahost diversity of feline coronavirus: a consensus between the circulating virulent/avirulent strains and the internal mutation hypotheses?

To evaluate the most controversial issue concerning current feline coronavirus (FCoV) virology, the coexisting hypotheses of the intrahost and interhost origins of feline infectious peritonitis virus (FIPV) in regard to the pathogenesis of feline infectious peritonitis (FIP), this study aimed to assess the molecular diversity of the membrane gene FCoVs in 190 samples from 10 cats with signs of FIP and in 5 faecal samples from cats without signs of FIP. All samples from the non-FIP cats and 25.26% of the samples from the FIP cats were positive for the FCoV membrane (M) gene. Mutations in this gene consisted of SNP changes randomly scattered among the sequences; few mutations resulted in amino acid changes. No geographic pattern was observed. Of the cats without FIP that harboured FECoV, the amino acid sequence identities for the M gene were 100% among cats (Cats 1-3) from the same cattery, and the overall sequence identity for the M gene was ≥91%. In one cat, two different lineages of FCoV, one enteric and one systemic, were found that segregated apart in the M gene tree. In conclusion, the in vivo mutation transition hypothesis and the circulating high virulent-low virulent FCoV hypothesis have been found to be plausible according to the results obtained from sequencing the M gene.

A rare case of primary hepatic poorly differentiated leiomyosarcoma in a goat.

A 12-year-old Saanen goat presented with a history of hyporexia and sudden recumbency. Euthanasia was indicated due to suspicion of hepatic neoplasia associated with senility. Necropsy revealed generalized oedema and increased liver size and weight (33 × 38 × 17 cm and 10.6 kg, respectively), with a firm, multilobular mass. Fusiform to polygonal neoplastic cells, with marked pleomorphism, anisocytosis and anisokaryosis, were seen on histopathological examination of the hepatic mass. The neoplastic cells were immunohistochemically positive for alpha-smooth muscle actin and vimentin but immunonegative for pancytokeratin. The Ki-67 index was 18.8%. A poorly differentiated leiomyosarcoma was diagnosed on the basis of the gross, histopathological and immunohistochemical findings and should be included in the differential diagnosis of liver disease in goats.

Infections by entomopathogenic fungi in common green iguanas (Iguana iguana) in captivity in Brazil.

Entomopathogenic fungi, widely available biological agents used to control agricultural pests, are sporadically reported to cause focal or disseminated infection in reptiles and mammals, including humans. This study summarizes the clinical presentation, histopathological and molecular findings by panfungal polymerase chain reaction and sequencing of four cases of hypocrealean fungal infections in captive common green iguanas (Iguana, iguana). One case of granulomatous pneumonia, hepatitis and serositis was related to Metarhizium flavoviride complex infection. Two disseminated fungal infection cases, with scarce inflammatory cell infiltration, were caused by Beauveria bassiana while there was one case of multifocal granulomatous and necrotizing pneumonia by Purpureocillium spp. To the best of our knowledge, this is the first report of fatal mycosis infection due to entomopathogenic fungi in captive common green iguanas.

Cytokine profile and parasite load in lymph nodes of dogs naturally infected with Leishmania infantum from distinct epidemiological scenarios in São Paulo State, Brazil.

Visceral leishmaniasis (VL) is an important zoonotic vector-borne disease and domestic dogs are considered the main domiciliary and peri-domiciliary reservoir of Leishmania (Leishmania) infantum in South America. Distinct eco-epidemiological scenarios associated to the prevalence of the disease, clusters of parasite genotypes and chemotypes of vectors population are described in Brazil, especially in the state of São Paulo (SP). In this context, the purpose of the present study is to evaluate the clinical signs, histopathological lesions, parasite load and cytokine profile by immunohistochemistry (IHC) in popliteal lymph nodes of canines naturally infected with L. infantum, from different municipalities of the state of SP. Eighty-three dogs with VL, 61 from northwest SP (NWSP) and 22 from southeast SP (SESP), were clinically classified in stage II, with no babesiosis and ehrlichiosis. Subcapsular inflammatory infiltration and histiocytosis were significantly higher in the SESP group (p = 0.0128; 0.0077, respectively). On the other hand, dogs from NWSP revealed 4.6-fold significantly higher parasite burden (p = 0.0004) and higher IHC scores of IL-1ß (p = 0.0275) and IL-4 (p = 0.0327) in the popliteal lymph node tissues, which may be associated with the susceptibility and progression of the disease in these dogs. Differences in immune response profile associated with higher parasite load in dogs can also contribute to explain the distinct eco-epidemiological patterns of VL in specific geographic regions.

Against All Odds: RNA Extraction From Different Protocols Adapted to Formalin-fixed Paraffin-embedded Tissue.

For the preservation of tissue samples, formalin fixation followed by paraffin embedding (FFPE) has been the method of choice for decades, mainly because it maintains the morphologic characteristics of the original tissue particularly preserved, as well as its genetic material. FFPE cells can be used to perform molecular tests, such as conventional (c) or quantitative (q) reverse transcriptase polymerase chain reaction (RT-PCR), in retrospective investigations. However, extracting RNA from archived FFPE tissues is a challenging procedure, as it requires time and the use of complex extraction methods. As specific FFPE extraction methods are not always available in the laboratories, the objective of this study was to evaluate the performance of a method based on phenol-chloroform (PC) and 2 commercial methods for RNA extraction, adapting their protocols for FFPE tissues. For this study, a pool of FFPE tissues underwent RNA extraction by PC, QIAmp Viral RNA Mini, and RNeasy Mini Kit. Both the RT-cPCR and the RT-qPCR results were favorable, demonstrating the viability of the RNA. As these results expanded the alternatives for low-budget FFPE extraction, the choice of the ideal method to be used will depend on the availability of reagents and kits.

Yellow fever surveillance challenge: Investigation of a marmoset non-autochthonous case.

Yellow fever (YF) surveillance in Brazil is focused mainly on the detection of epizootic events regarding New World non-human primates (NWNHP). We present a challenging case of a Callitrichidae (Callithrix spp) kept as a domiciliated pet that lived in the urban area of São Paulo municipality and was positive to YF virus by RT-qPCR and immunohistochemistry. After investigation, it was the first occurrence of non-autochthonous YF case of NWNHP described, with probable place of infection in the North shore of São Paulo state. This case illustrates the importance of coordinated laboratorial and field actions, and risks posed by transit of wildlife.

Age-based ultrasonographic criteria for diagnosis of autosomal dominant polycystic kidney disease in Persian cats.

OBJECTIVES: The aim of this study was to establish ultrasound criteria for the diagnosis of autosomal dominant polycystic kidney disease (ADPKD) in Persian cats. METHODS: Eighty-two Persian cats were assessed using renal ultrasound and genotyped for the C→A transversion in exon 29 of PKD1. The animals were also submitted to hematological characterization, serum biochemistry analyses and urinalysis. RESULTS: Age, sex and neutering status did not differ between ADPKD (n = 12) and non-ADPKD (n = 70) cats. After integrated molecular genetics/ultrasonographic analysis, the presence of at least one renal cyst was sufficient to establish a diagnosis of ADPKD in animals up to 15 months of age. Two or more cysts were required for diagnosis in cats aged 16-32 months, and at least three cysts warranted diagnosis of ADPKD in animals aged 33-49 months. Finally, four or more cysts led to diagnosis in cats aged 50-66 months. Although cats with ADPKD exhibited higher serum calcium levels than non-affected cats, hematological, urinalysis and other biochemical parameters did not differ between the two groups. CONCLUSIONS AND RELEVANCE: Integrated analyses of imaging and molecular genetics data enabled, for the first time, the establishment of age-based ultrasonographic criteria for the diagnosis of ADPKD in Persian cats. The development of imaging criteria is particularly relevant and useful in the clinical setting given the current limitations to access and the cost of molecular genetics-based diagnostic tests.

Feline Sporothrix spp. detection using cell blocks from brushings and fine-needle aspirates: Performance and comparisons with culture and histopathology.

BACKGROUND: Sporotrichosis is an emerging zoonotic mycosis that presents as a cutaneous lymphatic or disseminated disease, caused by fungi from the Sporothrix schenkii (S schenkii) clinical clade. Its importance is growing, primarily due to an outbreak that occurred in Brazil, affecting mainly cats and people. OBJECTIVES: In Brazil, an S schenkii diagnosis is often made using cultures, which allows genus identification and sufficient growth to perform molecular biology testing. Despite its advantages, fungal cultures are slow to develop and can delay public health measures, highlighting the importance of developing additional diagnostics techniques. METHODS: Cell block cytology (CBLC) is an older method that regained importance after liquid-based cytology (LBC) was introduced, and it has been previously and successfully applied to veterinary diagnostics. We aimed to standardize and compare CBLC from cervical brush exfoliation of open wounds and fine-needle aspirates with culture and immunohistochemistry of skin biopsies for sporotrichosis in cats, as a novel method. RESULTS: For this purpose, we selected 40 cats with skin lesions suspected of having sporotrichosis in Guarulhos city, São Paulo state, Brazil. We achieved 97.5% and 95% positivity using CBLC and culture, respectively, and 100% of feline skin biopsies were positive for Sporothrix spp on histopathology/immunohistochemistry. CONCLUSIONS: Cell block cytology is an efficient and rapid tool to diagnose sporotrichosis in cats, particularly during epidemics.

Sertraline Delivered in Phosphatidylserine Liposomes Is Effective in an Experimental Model of Visceral Leishmaniasis.

Liposomes containing phosphatidylserine (PS) has been used for the delivery of drugs into the intramacrophage milieu. Leishmania (L.) infantum parasites live inside macrophages and cause a fatal and neglected viscerotropic disease, with a toxic treatment. Sertraline was studied as a free formulation (SERT) and also entrapped into phosphatidylserine liposomes (LP-SERT) against intracellular amastigotes and in a murine model of visceral leishmaniasis. LP-SERT showed a potent activity against intracellular amastigotes with an EC50 value of 2.5 µM. The in vivo efficacy of SERT demonstrated a therapeutic failure. However, when entrapped into negatively charged liposomes (-58 mV) of 125 nm, it significantly reduced the parasite burden in the mice liver by 89% at 1 mg/kg, reducing the serum levels of the cytokine IL-6 and upregulating the levels of the chemokine MCP-1. Histopathological studies demonstrated the presence of an inflammatory infiltrate with the development of granulomas in the liver, suggesting the resolution of the infection in the treated group. Delivery studies showed fluorescent-labeled LP-SERT in the liver and spleen of mice even after 48 h of administration. This study demonstrates the efficacy of PS liposomes containing sertraline in experimental VL. Considering the urgent need for VL treatments, the repurposing approach of SERT could be a promising alternative.

Molecular, serological, pathological, immunohistochemical and microbiological investigation of Brucella spp. in marine mammals of Brazil reveals new cetacean hosts.

Brucella-exposure and infection is increasingly recognized in marine mammals worldwide. To better understand the epidemiology and health impacts of Brucella spp. in marine mammals of Brazil, molecular (conventional PCR and/or real-time PCR), serological (Rose Bengal Test [RBT], Competitive [c]ELISA, Serum Agglutination Test [SAT]), pathological, immunohistochemical (IHC) and/or microbiological investigations were conducted in samples of 129 stranded or by-caught marine mammals (orders Cetartiodactyla [n = 124], Carnivora [n = 4] and Sirenia [n = 1]). Previous serological tests performed on available sera of 27 of the 129 animals (26 cetaceans and one manatee), indicated 10 seropositive cetaceans. Conventional PCR and/or real-time PCR performed in cases with available organs (n = 119) and/or blood or swabs (n = 10) revealed 4/129 (3.1%) Brucella-infected cetaceans (one of them with positive serology; the remaining three with no available sera). Pathological, IHC and/or microbiological analyses conducted in PCR/real-time PCR and/or seropositive cases (n = 13) revealed Brucella-type lesions, including meningitis/meningoencephalitis, pneumonia, necrotizing hepatitis, pericarditis and osteoarthritis in some of those animals, and positive IHC was found in all of them (excepting two live-stranded animals without available organs). Brucella spp. culture attempts were unsuccessful. Our results demonstrated exposure, asymptomatic, acute and chronic Brucella sp. infection in several cetacean species in the Brazilian coast, highlighting the role of this pathogen in stranding and/or death, particularly in Clymene dolphin (Stenella clymene) and short-finned pilot whale (Globicephala macrorhynchus) off Ceará State. Novel hosts susceptible to Brucella included the franciscana (Pontoporia blainvillei), the Guiana dolphin (Sotalia guianensis) and the spinner dolphin (Stenella longirostris). Additionally, three coinfection cases involving Brucella spp. and cetacean morbillivirus, Edwarsiella tarda and Proteus mirabilis were detected. To the best of our knowledge, this is the first long-term and large-scale survey of Brucella spp. in marine mammals of South America, widening the spectrum of susceptible hosts and geographical distribution range of this agent with zoonotic potential.

Experimental Zika virus infection induces spinal cord injury and encephalitis in newborn Swiss mice.

A widespread epidemic of Zika virus (ZIKV) infection was reported in 2015 in South and Central America, with neurological symptons including meningoencephalitis and Guillain-Barré syndrome in adults, besides an apparent increased incidence of microcephaly in infants born to infected mothers. It is becoming a necessity to have a trustworthy animal model to better understand ZIKV infection. In this study we used newborn white Swiss mice as a model to investigate the ZIKV strain recently isolated in Brazil. ZIKV was inoculated via intracerebral and subcutaneous routes and analysed through gross histopathology and immunohistochemistry. Here we demonstrated first that the intracerebral group (ICG) displayed severe cerebral lesions, with neuronal death, presence of apoptotic bodies, white matter degeneration and neutrophil perivascular cuffing. In the subcutaneous group (SCG), we observed moderate cerebral lesions, morphologically similar to that found in ICG and additional myelopathy, with architectural loss, marked by neuronal death and apoptotic bodies. Interestingly, we found an intense astrogliosis in brain of both groups, with increased immunoexpression of GFAP (glial fibrillary acidic protein) and presence of hypertrophic astrocytes. The spinal cord of subcutaneous group (SCG) exhibited reduction of astrocytes, but those positive for GFAP were hypertrophic and presented prolonged cellular processes. Finally significant lesions in the central nervous system (CNS) were present in newborn mice inoculated by both routes, but SCG method led to an important neurological manifestations (including myelopathy), during a longer period of time and appears for us to be a better model for ZIKV infection.

Yellow fever surveillance challenge: Investigation of a marmoset non-autochthonous case

Yellow fever (YF) surveillance in Brazil is focused mainly on the detection of epizootic events regarding New World non-human primates (NWNHP). We present a challenging case of a Callitrichidae (Callithrix spp) kept as a domiciliated pet that lived in the urban area of São Paulo municipality and was positive to YF virus by RT-qPCR and immunohistochemistry. After investigation, it was the first occurrence of non-autochthonous YF case of NWNHP described, with probable place of infection in the North shore of São Paulo state. This case illustrates the importance of coordinated laboratorial and field actions, and risks posed by transit of wildlife.

Validation of chromogenic in situ hybridization reactions for DNA and RNA detection in formalin-fixed paraffin-embedded tissue / Validação das reações de hibridização in situ cromogênica para detecção de DNA e RNA em tecidos fixados em formalina e incluídos em parafina

ABSTRACT Introduction: Chromogenic in situ hybridization (CISH) is used alternatively to the traditional immunohistochemical methods for the diagnosis of infectious diseases in formalin-fixed paraffin-embedded samples, since it presents high sensitivity and specificity. This type of sample undergoes several chemical modifications during histological processing, and both poor and excessive fixation can impair sample quality, making it difficult to obtain good results. In CISH, it is common to use positive samples as quality control for the reactions; however, this practice does not provide any information regarding the preservation of the genetic material, nor does it avoid false-negative results. Objective: The objective of this study was to validate the deoxyribonucleic acid (DNA) (+) and (-), and ribonucleic acid (RNA) (+) and (-) control probes to be used as quality control for the samples, evaluating preservation of the genetic material. Materials and methods: Twelve histological sections were used (in quadruplicate, n = 48), prepared from a pool of tissues without microscopic changes related to infectious and/or inflammatory processes. The CISH protocol was conducted according to the manufacturer's instructions, standardized under the conditions of our laboratory, using commercial DNA and RNA probes chemically linked to digoxigenin. Results and conclusion: Our results were very satisfactory, showing high reproducibility, accuracy, sensitivity and analytical specificity, high predictive values for positive and negative assays and with zero ratio of false-positive and false-negative results, allowing the validation of this reaction.

RESUMEN Introducción: La hibridación in situ cromogénica (CISH) es una alternativa a los métodos tradicionales inmunohistoquímicos para el diagnóstico de enfermedades infecciosas en muestras fijadas en formoly embebidas enparafina, puesto que tiene alta sensibilidad y especificidad. Este tipo de muestra sufre diversas modificaciones químicas durante elprocesamiento histológico, y tanto la mala fijación cuanto la fijación excessiva pueden perjudicar la calidad de las muestras, impidiendo buenos resultados. En la CISH, es común el empleo de muestras positivas para control de calidad de reacciones; sin embargo, esta práctica no proporciona ninguna información acerca de la preservación del material genético. Objetivo: El propósito de este estudio ha sido realizar la validación de las sondas comerciales para ácido desoxirribonucleico (ADN) (+) y (-) y ácido ribonucleico (ARN) (+) y (-), para que sean utilizadas como control de calidad, evaluando la preservación del material genético en las muestras testadas. Material y métodos: Se incluyen en el estudio 12 cortes histológicos (en cuadruplicado, n = 48), confeccionados a partir de un pool de tejidos sin alteraciones microscópicas relacionadas con procesos infecciosos y/o inflamatorios. El protocolo de CISH se desarrolló de acuerdo a las instrucciones del fabricante y bajo las condiciones del nuestro laboratorio, haciendo uso de sondas comerciales de ADN y ARN quimicamente ligadas a digoxigenina. Resultados y conclusión: Nuestros resultados han sido muy satisfactorios, demostrando alta reproducibilidad, exactitud, sensibilidad, y especificidad analítica, así como altos valores predictivos para ensayos positivos y negativos, y con proporción nula de falsos negativos y falsos positivos, lo que ha permitido la validación de esa reacción.

RESUMO Introdução: A hibridização in situ cromogênica (CISH) é uma alternativa aos métodos tradicionais imuno-histoquímicospara diagnóstico de doenças infecciosas em amostras fixadas em formalina e incluídas em parafina, visto que apresenta grande sensibilidade e especificidade. Esse tipo de amostra sofre diversas modificações químicas durante o processamento histológico, e tanto a má fixação quanto a fixação em excesso podem prejudicar a qualidade das amostras, inviabilizando bons resultados. Na CISH, é comum a utilização de amostras positivas como controle de qualidade das reações; entretanto essa prática não fornece nenhuma informação a respeito da preservação do material genético, nem evita resultados falso-negativos nas amostras testadas. Objetivo: O objetivo deste estudo foi realizar a validação das sondas comerciais para ácido desoxirribonucleico (DNA) (+) e (-) e ácido ribonucleico (RNA) (+) e (-), para serem utilizadas como controle de qualidade, avaliando apreservação do material genético nas amostras testadas. Materiais e métodos: Foram utilizados 12 cortes histológicos (em quadruplicata, n = 48), confeccionados a partir de um pool de tecidos sem alterações microscópicas relacionadas com processos infecciosos e/ou inflamatórios. O protocolo de CISH foi conduzido de acordo com as instruções do fabricante e padronizado conforme as condições do nosso laboratório, utilizando sondas comerciais de DNA e RNA quimicamente ligadas à digoxigenina. Resultados e conclusão: Nossos resultados foram muito satisfatórios, demonstrando alta reprodutibilidade, acurácia, sensibilidade e especificidade analítica, bem como altos valores preditivos para ensaios positivos e negativos e com proporção nula de resultados falso-negativos e falso-positivos, o que possibilitou a validação dessa reação.

Hypervitaminosis A-induced hepatic fibrosis in a cat.

RATIONALE: The excessive intake of vitamin A in the form of vitamin concentrate, supplement or vitamin-rich liver can result in hypervitaminosis A in man and animals. Although osteopathologies resulting from chronic vitamin A intoxication in cats are well characterized, no information is available concerning feline hypervitaminosis A-induced liver disease. CLINICAL SUMMARY: We report the first case of hepatic stellate cell lipidosis and hepatic fibrosis in a domestic cat that had been fed a diet based on raw beef liver. Radiographic examination revealed exostoses and ankylosis between vertebrae C1 and T7, compatible with deforming cervical spondylosis. Necropsy showed a slightly enlarged and light yellow to bronze liver. Microscopic and ultrastructural analyses of liver tissues revealed diffuse and severe liver fibrosis associated with hepatic stellate cell hyperplasia and hypertrophy. These cells showed immunopositive staining for α-smooth muscle actin and desmin markers. The necropsy findings of chronic liver disease coupled with osteopathology supported the diagnosis of hypervitaminosis A. PRACTICAL RELEVANCE: As in human hepatology, if there is dietary evidence to support increased intake of vitamin A, then hypervitaminosis A should be considered in the differential diagnosis of chronic liver disease in cats.