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Bats belong to the second-largest order in a number of species diversity within the Mammalia class, containing 21 families and more than 1300 species. It is estimated that more than 200 viruses from 28 families have been isolated or detected in 37 different bat genera, many of them related to emerging infectious diseases with the potential to cross species barriers and infect other animals. The group of coronaviruses (CoV) is one of these viruses, which includes CoVs that can cause serious diseases in humans and animals, such as severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), porcine epidemic diarrhea (PED), swine acute diarrheal syndrome (SADS) and coronavirus disease 2019 (COVID-19). Some of the human and animal coronaviruses appear to be originated from bats. With the advent of new generation molecular techniques and increased surveillance of wild animal species, many new coronaviruses have been identified. The coronaviruses belong to the Nidovirales order and Coronaviridae family. The subfamily Coronavirinae is divided into four genera, Alphacoronavirus (alphaCoV), Betacoronavirus (betaCoV), Deltacoronavirus, and Gammacoronavirus. Since the first report of coronavirus in a Miniopterus pusillus bat, the coronavirus occurrence in a bats population of different regions in the world has been studied and, until now, both alphaCoV and betaCoV have been detected. The aim of this study was to perform a literature review regarding the detection of coronavirus (alphaCoV or betaCoV) in different bat species around the world and their immune response against coronavirus. This review reinforces the importance of these animals as hosts, reservoirs, or sources of viruses, including emerging viruses.

Equid alphaherpesvirus 1 (EHV-1) causes myeloencephalopathy in horses and occasionally in non-equid species. Although mouse models have been developed to understand EHV-1 pathogenesis, few EHV-1 strains have been identified as highly neurovirulent to mice. The aim of this study was to evaluate the pathogenesis of 2 neurovirulent EHV-1 strains in mice, and to characterize the inflammatory cells and expression of chemokines and the apoptosis marker caspase-3 in the brain of infected mice. C57BL/6J mice were inoculated intranasally with EHV-1 strains A4/72 or A9/92 and evaluated on 1, 2, and 3 days post inoculation (DPI). EHV-1-infected mice showed severe neurological signs at 3 DPI. Ultrastructural analysis revealed numerous viral nucleocapsids and fewer enveloped virions within degenerated and necrotic neurons and in the surrounding neuropil. Histologically, at 3 DPI, there was severe diffuse neuronal degeneration and liquefactive necrosis, prominent microgliosis, and perivascular cuffing composed of CD3+ cells (T cells) and Iba-1+ cells (macrophages), mainly in the olfactory bulb and ventral portions of the brain. In these areas, moderate numbers of neuroglial cells expressed CCL5 and CCL2 chemokines. Numerous neurons, including those in less affected areas, were immunolabeled for cleaved caspase-3. In conclusion, neurovirulent EHV-1 strains induced a fulminant necrotizing lymphohistiocytic meningoencephalitis in mice, with microgliosis and expression of chemokines and caspase-3. This model will be useful for understanding the mechanisms underlying the extensive neuropathology induced by these viral infections.

The genus Bartonella comprises gram-negative bacilli that possess tropism for erythrocytes and endothelial cells in animals of the orders Rodentia, Lagomorpha, Carnivora, Artiodactyla, Eulipotyphla, and Chiroptera. Bacterial infection may be associated with lymphadenitis, endocarditis, bacillary angiomatosis, and peliosis hepatis. Thirteen species of Bartonella are recognized as zoonotic and bats are considered to be their potential reservoirs. This study aimed to analyze the occurrence of Bartonella spp. in livers of neotropical bats belonging to the families Molossidae, Phyllostomidae, and Vespertilionidae, classified into 21 genera from the Atlantic Forest biome in São Paulo. A total of 341 (n = 341) chiropterans samples were tested for the presence of citrate synthase (gltA) gene of Bartonella by partial amplification using polymerase chain reaction (PCR). Samples of two bats (0.6%) of the species Glossophaga soricina from the municipality of São Roque were tested positive for gltA gene. Phylogenetic analysis indicated that sequences clustered in a clade that was close to the Bartonella sp. detected in G. soricina, which was collected from the Cerrado biome in Tocantins, Brazil. Despite a low prevalence of the detected infection, results indicated that neotropical bats from the Atlantic Forest were potential hosts of Bartonella spp., which might be related to the maintenance of a wild enzootic cycle of the bacterium. Additional studies, particularly on bats of genus Glossophaga, are required to elucidate the dynamics of intraspecific relationships between etiologic agent-vector-hosts.

Hepatitis B virus (HBV) is the prototype of the Orthohepadnavirus genus and represents an important cause of chronic hepatitis, liver cirrhosis, and hepatic cancer in humans worldwide. To verify the occurrence and genetic variability of orthohepadnavirus among neotropical bats, we tested 81 liver samples of New World bats from Sao Paulo State, Southeastern Brazil, collected during 2012. PCR, sequencing, and phylogenetic analysis of Surface/Polymerase and Core viral genes confirmed the occurrence of the first isolate of bat orthohepadnavirus detected in South America. These results may contribute to subsequent studies of the origin, variability, host species, and evolution of bat orthohepadnaviruses in South America.

The rabies virus (RABV) has been isolated in several bats species in the world, and among them, hematophagous, frugivorous and insectivorous species. Bats found in Brazil are small, which can lead to situations in which there are limitations in the collection of the central nervous system (CNS) and the amount of material may be insufficient to carry out laboratory diagnostic techniques for rabies. The objective of this work was to evaluate an alternative sample collection for the diagnosis of rabies in bats. A total of 92 bat samples, 82 positives and 10 negatives were selected. The cranial cavity was scraped with the aid of sterile tips and a virus diluent was added to create a suspension. All samples were submitted to Rabies Tissue Culture Infection Test (RTCIT) and reverse transcription polymerase chain reaction (RT-PCR). The diagnostic sensitivity and specificity of the RTCIT and RT-PCR using the cranial cavity lavage were calculated in comparison with the results of the laboratory routine (DFAT and RTCIT) performed with the CNS (considered gold standard). The results of the RTCIT show that the cranial cavity lavage is not an adequate sample for viral isolation, since the diagnostic sensitivity was low (37.8 %) when compared with the tests with the CNS. However, the RT-PCR of the cranial cavity lavage may be a tool to assist in the diagnosis, since it presented a sensitivity of 76.8 %. The results of this study suggest that cranial cavity lavage is an interesting alternative to enable the diagnosis of rabies in bats and increases the possibility of diagnosis contributing to rabies surveillance and control.

Equine herpesvirus type 1 (EHV-1) is an emerging pathogen that causes encephalomyelitis in horses and non-equid species. Several aspects of the immune response in the central nervous system (CNS), mainly regarding the role of inflammatory mediators during EHV-1 encephalitis, remain unknown. Moreover, understanding the mechanisms underlying extensive neuropathology induced by viruses would be helpful to establish therapeutic strategies. Therefore, we aimed to evaluate some aspects of the innate immune response during highly neurovirulent EHV-1 infection. C57BL/6 mice infected intranasally with A4/72 and A9/92 EHV-1 strains developed a fulminant neurological disease at 3 days post-inoculation with high viral titres in the brain. These mice developed severe encephalitis with infiltration of monocytes and CD8+ T cells to the brain. The inflammatory infiltrate followed the detection of the chemokines CCL2, CCL3, CCL4, CCL5, CXCL2, CXCL9 and CXCL-10 in the brain. Notably, the levels of CCL3, CCL4, CCL5 and CXCL9 were higher in A4/72-infected mice, which presented higher numbers of inflammatory cells within the CNS. Pro-inflammatory cytokines, such as interleukins (ILs) IL-1α, IL-1β, IL-6, IL-12β, and tumour necrosis factor (TNF), were also detected in the CNS, and Toll-like receptor (TLR) TLR2, TLR3 and TLR9 genes were also upregulated within the brain of EHV-1-infected mice. However, no expression of interferon-γ (IFN-γ) and IL-12α, which are important for controlling the replication of other herpesviruses, was detected in EHV-1-infected mice. The results show that the activated innate immune mechanisms could not prevent EHV-1 replication within the CNS, but most likely contributed to the extensive neuropathology. The mouse model of viral encephalitis proposed here will also be useful to study the mechanisms underlying extensive neuropathology.

This research reports the use of different diagnostic tests in cattle, naturally infected by Rabies lyssavirus (RABV), and correlates the positivity of the tests with the clinical moment of euthanasia, the intensity of the inflammatory lesion and viral load. It also highlights the possibility of euthanasia in early stages of the disease as a way to improve animal welfare. For that, samples of 34 bovine brains were collected for analysis, preserved in 10% buffered formaline and refrigerated with subsequent freezing. The samples were subjected to direct immunofluorescence antibody technique (DFAT) tests, viral isolation in cell culture (VICC), histopathology with hematoxylin and eosin staining (HE), immunohistochemistry (IHC), Shorr stainied neural tissue smears (DSS), Reverse transcription polymerase chain reaction (RT-PCR) and polymerase chain reaction by quantitative reverse transcriptase (qRT-PCR). The areas used for analysis were the cerebellum, parietal telencephalon and thalamus. Samples with Negri bodies (NBs) or immunostaining in at least one of the analyzed areas were considered positive. For the study of the intensity of histological lesions, the lesions were classified into grades 0, 1, 2 and 3 and the positivity of the test in the presence or absence of NBs in one of the three areas analyzed. To verify the influence of the disease clinical evolution, 4-four groups of analysis were created according to the animal’s clinical status at moment of the euthanasia, being: M1 = animal euthanized while standing, M2 = euthanized when in sternal recumbence, M3 = euthanized when in lateral recumbence, M4 = animal with natural death. Of the 34 brains evaluated, IHC was positive in 100% of cases, DFAT was positive in 97.05% of them, and in this negative sample the presence of RABV was confirmed by VICC. NBs ere seen in 88.23% of the cases, and the DSS test was positive in 82.35% of them. All diagnostic techniques showed positive cases in all groups analyzed. Each case was positive in at least two diagnostic methods. All cases that contained NBs were positive for rabies in the other tests. In this study, it was observed that the variables analyzed (intensity of injury and clinical evolution at the moment of euthanasia) had an influence only on HE and DSS techniques, which are based on NB research to form the diagnosis, but did not interfere with the effectiveness of the diagnosis performed by detecting the viral antigen performed by DFAT and IHC. All isolated RABV samples included in the present study have a genetic lineage characteristic of hematophagous bats Desmodus rotundus. The evaluation of qRT-PCR showed that the amount of virus did not interfere in the positivity of the tests. This work shows that IHC and DFAT are safe diagnostic techniques. They are capable of detecting RABV even in euthanized animals in the early stages of clinical evolution with mild intensities of histological lesions. RESUMO: Esta pesquisa relata a utilização de diferentes testes de diagnóstico em bovinos, naturalmente infectados pelo Rabies lyssavirus (RABV), e correlaciona a positividade dos testes com o momento clínico da eutanásia, a intensidade da lesão inflamatória, e a carga viral. Salienta também a possibilidade da eutanásia em estágios precoces da doença como forma de melhorar o bem-estar animal. Para isso amostras de 34 encéfalos bovinos foram coletados para análise, conservadas em formol tamponado 10% e sob refrigeração com posterior congelamento. As amostras foram submetidas aos testes de imunofluorescência direta (IFD), isolamento viral em cultivo de células (IVCC), histopatologia com coloração de hematoxilina e eosina (HE), imuno-histoquímica (IHQ), esfregaço direto com coloração de Shorr (EDS), reação da polimerase em cadeia por transcriptase reversa (RT-PCR) e reação da polimerase em cadeia por transcriptase reversa quantitativo (qRT-PCR). As áreas utilizadas para análise foram o cerebelo, telencéfalo parietal e tálamo. Foram consideradas positivas as amostras que apresentaram Corpúsculo de Negri (CNs) ou imuno-marcação em ao menos uma das áreas analisadas. Para o estudo da intensidade das lesões histológicas, as lesões foram classificadas em graus 0, 1, 2 e 3 e a positividade do teste na presença ou ausência de CN em uma das três áreas analisadas. Para verificar a influência da evolução clínica da doença foram criados 4 grupos de análise conforme o estado clínico do animal no momento da eutanásia, sendo: M1 = animal eutanasiado em estação, M2 = eutanasiado em decúbito esternal, M3 = eutanasiado em decúbito lateral, M4 = animal com morte natural. Dos 34 encéfalos avaliados a IHQ foi positiva em 100% dos casos, a IFD foi positiva em 97,05%, sendo que na amostra negativa a presença de RABV foi confirmada por IVCC. A histologia com HE, através da visualização das CNs, foi positiva em 88,23 % dos casos, e o teste de EDS, foi positivo em 82,35%. Todas as técnicas de diagnóstico apresentaram casos positivos em todos os grupos analisados. Cada caso foi positivo em, pelo menos, dois métodos de diagnóstico. Todos os casos que continham CN foram positivos para raiva nos demais testes. Nesse estudo observou-se que as variáveis analisadas intensidade de lesão e evolução clínica no momento da eutanásia tiveram influência somente nas técnicas de HE e EDS, que se baseiam na pesquisa do CN para formação do diagnóstico, mas não interferiram na eficácia do diagnóstico realizado através da detecção do antígeno viral realizado por IFD e IHQ. Todas as amostras RABV isoladas incluídas no presente estudo apresentam linhagem genética característica de morcegos hematófagos Desmodus rotundus. A avaliação de qRT-PCR demostrou que a quantidade de vírus não interferiu na positividade dos testes. Esse trabalho mostra que a IHQ e a IFD são técnicas seguras de diagnóstico e que mesmo em animais eutanasiados em estágios iniciais de evolução clínica com intensidades leve de lesões histológicas, são capazes de detectar o RABV.

The equine influenza virus (EIV) H3N8 subtype is responsible for all EIV outbreaks worldwide while the H7N7 subtype is less pathogenic and is considered extinct as it has not been confirmed in outbreaks since 1980. Although EIV is enzootic in Brazil, few reports describe the actual EIV antibody status in the country. The aims of this study were: - to evaluate the efficiency of different serum treatments described by the World Organisation for Animal Health (OIE) and the World Health Organization (WHO) to remove non-specific haemagglutination inhibitors for the haemagglutination inhibition (HI) assay for EIV - to evaluate the presence of EIV antibodies by HI, enzyme-linked immunosorbent assay and agar gel immunodiffusion in 83 non-vaccinated equines from Sao Paulo State - to evaluate a strategy to better analyse equine sera for EIV antibodies. Although there was no statistical difference among treatments, receptor-destroying enzyme treatment followed by chicken erythrocyte adsorption showed more consistent results, which corroborate the OIE and WHO recommendation to use this treatment preferentially. The HI results suggest equine H3N8 virus circulation among the animals tested from Sao Paulo State. The algorithm suggested here could be used to guide antibody detection against equine influenza virus in equines, improving the test specificity by aiming to avoid false positive results.

Bovine papillomavirus (BPV) infection is endemic in Brazilian herds. Papillomaviruses are oncogenic, with a trophic response in squamous epithelial and mucosal tissues, and are associated with asymptomatic infections, proliferative benign skin lesions (papillomas), and malignant epithelial lesions (carcinomas). The presence and expression of BPV in the blood of healthy and papillomatosis-affected cattle has been demonstrated. Experimental inoculation of Bovine papillomavirus (BPV) into calf meninges can result in meningiomas and papillomatosis, but it´s not known if its natural infection causes neoplasia and neurological syndrome in cattle. We assessed the frequency of BPV in 300 Central Nervous System (CNS) samples from cattle with neurological syndrome from several Brazilian regions obtained from surveillance of neurological syndrome. Samples were negative for rabies, Neospora caninum, BoHV-1 and BoHV-5, bovine leukemia virus, and catarrhal malignant fever (PCR). Samples were fixed in 10% buffered formalin and submitted to macroscopic examination. For histological analysis, slides were submitted to a staining protocol using hematoxylin and eosin. PCR for BPV detection was applied in CNS frozen samples using generic primers FAP59 and FAP64 (L1 gene). Thirteen (4.3%) samples were positive for BPV by PCR, with 11 of these showing no pathological changes in microscopy, and two exhibiting nonspecific non-purulent meningoencephalitis. No CNS samples showed neoplasia. Nine of the 13 BPV positive samples (69.2%) came from females and four (30.8%) from males. The 13 positive animals were age 5 to 168 months with seven over 36 months (53.8%). Five were dairy cattle, four crossbred, and three beef cattle. Only one of the 13 positive samples provided sufficient BPV DNA for sequencing, which emonstrated 99% identity to samples of BPV-1 obtained from cutaneous papillomas in cattle in Brazil. The small quantity of BPV DNA in the CNS and the low number of PCR-positive samples may be associated with low neurotropism, unspecific inflammation, or BPV-infected lymphocytes in CNS tissues or bloodstream. Natural BPV-1 infection was not associated with cerebral neoplasia or neurological syndrome.

Among the diseases that affect equines, bacterial diseases play an important role from a health and economic point of view, especially leptospirosis and brucellosis. The study aimed to provide information on the occurrence of anti-Leptospira spp. and anti-Brucella abortus antibodies in donkeys of São Paulo state. We found a frequency of 62.4% (53/85) antibodies against Leptospira spp. The donkeys were not seropositive for Brucella spp.

The etiology of diseases that affect the central nervous system (CNS) of equids was investigated. Samples (n = 218) collected from equids showing clinical signs of nervous or behavioral changes were analyzed, of which 37 (17.0%) were positive for rabies, 13 (6.0%) for the presence of protozoans (one Sarcocystis neurona, 12 Toxoplasma gondii), three (1.4%) for equine herpesvirus type 1 myeloencephalopathy, and 24 (11%) for bacterial encephalitis. Histopathology of the CNS revealed one (0.4%) case of cryptococcal myelomeningoencephalitis and 20 (9.2%) cases of equine leukoencephalomalacia. Central nervous system samples were positive for Sarcocystis neurona and Toxoplasma gondii by nested PCR-ITS1 followed by nucleotide sequencing. Diagnosis of equine herpesvirus 1 was confirmed by cell isolation and polymerase chain reaction followed by sequencing of the GD and polymerase (ORF 30) genes in three samples. No case of equine encephalomyelitis was diagnosed in samples analyzed by isolation in mice, VERO cell cultures, and RT-PCR for the nsP1 gene. Bacterial agents (Staphylococcus spp., Streptococcus spp., Bacillus spp., Enterobacteriaceae spp., Corynebacterium spp., and nonfermenting gram-negative bacillus) were detected in pure or preponderant cultures. Diagnosis was conclusive in 45% of samples, indicating that other infectious and noninfectious etiologies of encephalitis and encephalopathy should be considered for investigation.

RESUMEN. En la presente revision se presentan algunos aspectos de la biologia y comportamiento de las especies de quiropteros hematofagos Diphylla ecaudata y Diaemus youngi . Y se analizan las principales caracteristicas anatomicas que permiten hacer la diferenciacion de estas especies con Desmodus rotundus . Es notable la falta de informacion sobre las dos primeras especies y la necesidad de capacitacion para la correcta identificacion de las mismas, lo que redundara en un direccionamiento mas especifico para controlar las poblaciones de Desmodus rotundus . ABSTRACT. This review presents some aspects about the biology and behavior of the hematophagous bats Diphylla ecaudata and Diaemus youngi . It is notable the lack of information regarding both species, and the need of training in order to identify them properly from Desmodus rotundus. We analyze the main anatomical features that allow differentiation of these species. This will result in a more specific control of Desmodus rotundus populations.

The direct-fluorescent antibody test (dFAT) is considered the "gold standard" assay to diagnose rabies. However, it is crucial to develop molecular techniques, such as RT-PCR and RT-qPCR, since many laboratories lack the needed supplies for performing complementary methods (viral isolation, for example). For this purpose, diagnostic techniques must be specific and sensitive to guarantee accuracy. This present investigation aimed to detect rabies virus (RABV) in 126 clinically suspected cattle in Brazil using different diagnostic tests [dFAT, mouse inoculation test (MIT), immunohistochemistry (IHC), RT-PCR and RT-qPCR] and to compare those results obtained under routine laboratory conditions. The results of the present investigation demonstrate that the molecular techniques are more sensitive and may detect low viral load, even though the non-homogeneous viral distribution caused a false-negative result in dFAT. We also observed a usual alteration in antigens distribution among regions of the central nervous system (CNS). By both dFAT and IHC assays, the most reliable CNS structures were thalamus and midbrain. Although this investigation demonstrated diagnostic sensitivity and specificity close to 100 % in all laboratory techniques employed, a dFAT auxiliary test is required for bovine specimens, such as molecular techniques, when there are poor sampling conditions (low viral load combined with unavailability of brainstem structures).

Equine herpesvirus type 1 (EHV-1) is an important pathogen that causes abortion, neonatal disease, respiratory disorders, and neurological syndrome in equine populations worldwide. To evaluate EHV-1 as a cause of abortion and perinatal mortality in Brazil, tissue samples from 105 aborted equine fetuses, stillbirths, and foals up to one month of age were examined using virus isolation, immunohistochemistry (IHC), histopathology, and nested polymerase chain reaction (PCR). Two fetuses were positive for EHV-1 by PCR, one of which showed syncytia and eosinophilic intranuclear inclusion bodies in bronchial epithelia, but it was negative by virus isolation. The other showed no characteristic histological lesions, but it was positive by viral isolation. No sample was positive by IHC. The results presented low occurrence of EHV-1 in the studied population and suggested that the use of a combination of techniques increases the likelihood of an accurate diagnosis of EHV-1., O herpes-vírus equino tipo 1 (HVE-1) é um importante agente patogênico causador de aborto, doença neonatal, distúrbios respiratórios e síndrome neurológica em populações de equinos em todo o mundo. Para avaliar a ocorrência do HVE-1 como agente causal de abortamento e mortalidade perinatal no Brasil, foram examinadas amostras de 105 fetos equinos abortados, natimortos e potros de até 1 mês de idade, utilizando as técnicas de isolamento viral, imuno-histoquímica (IHQ), histopatologia e reação em cadeia da polimerase aninhada (nested-PCR). Dois fetos foram positivos na análise de PCR, e um deles apresentou corpúsculos de inclusão viral eosinofílicos e sincícios no epitélio brônquico, porém foi negativo na análise de isolamento viral. O outro feto não apresentou lesões histológicas características de infecção herpética, mas foi positivo na análise de isolamento viral. Nenhuma amostra apresentou resultado positivo pela análise de IHQ. Os resultados demonstraram baixa ocorrência de HVE-1 na população estudada e que o uso de diferentes técnicas diagnósticas aumenta a probabilidade de um diagnóstico preciso para o HVE-1., Facultad de Ciencias Veterinarias

The genetic lineage of rabies virus (RABV) associated with dogs has not been found in the state of Sao Paulo since 1998, and all cases of rabies in domestic animals reported since then have involved the RABV lineage that circulates in bats. As there has been a change in the rabies transmission cycle in cats and dogs, we decided to analyze the tests used to diagnose rabies in these animals in the 15-year period from 2002 to 2016 in the state of Sao Paulo. During this period, 85,508 central nervous system (CNS) samples from dogs and cats were submitted to the Rabies Diagnosis Section at the Pasteur Institute of Sao Paulo for testing. All of the samples were tested by the fluorescent antibody test (FAT) and at least one of the following three tests: mouse inoculation test (MIT), rabies tissue culture infection test (RTCIT) and reverse transcription polymerase chain reaction (RT-PCR). Of all the samples tested, twenty were positive in at least one of these assays. Four other positive samples were identified at other institutions in the state of Sao Paulo. Of the twenty samples that tested positive at the Pasteur Institute of Sao Paulo, nine were tested by FAT, and the results were subsequently confirmed by other techniques; five gave inconclusive results, and therefore, other techniques had to be used as soon as possible in case the samples were positive; and six were negative by FAT and positive by one or more of the following tests: RTCIT, MIT and RT-PCR. Genetic typing of isolates from eighteen samples identified them as the lineage circulating in bats. In light of this finding, which indicates that genetic lineages associated with bats are circulating in domestic animals in the state of Sao Paulo, when the results of FAT carried out with samples from aggressive cats and dogs are inconclusive, complementary tests should be used. Decomposing samples and samples for which FAT was inconclusive should be tested using molecular techniques so that a definitive result can be obtained quickly and timely post-exposure prophylaxis can be administered to exposed individuals.

Introducao e objetivos: O herpesvirus equino tipo 1 (EHV-1) e um patogeno de suma importância, responsavel por perdas significativas nos planteis, fato que, sob o ponto de vista economico, o torna uma ameaca potencial a criacao mundial de cavalos uma vez que a sua distribuicao e cosmopolita. Esse virus tem sido identificado como a causa de abortamentos, mortalidade neonatal, doenca respiratoria e manifestacoes neurologicas em cavalos [1,2]. Modelos experimentais de infeccao pelo EHV-1 utilizando roedores, como camundongos e hamsters, sao uteis para o estudo da resposta do hospedeiro ao virus, pois muitos dos aspectos da etiopatogenia da doenca nessas especies se assemelham aos observados no hospedeiro natural [3]. O camundongo e um excelente modelo para estudo da etiopatogenia da encefalite causada por estirpes neuropatogenicas do herpesvirus equino tipo 1 (EHV-1) [4]. No entanto, pesquisas recentes evidenciaram que a infeccao estabelecida pela via intranasal em hamsters resultou em sintomas mais agudos e severos do que nos camundongos, sugerindo uma maior susceptibilidade da especie ao agente [5,6]. Com base nesses dados, o presente trabalho foi delineado para avaliar as alteracoes respiratorias e neurologicas decorrentes da infeccao pelo EHV-1 em hamsters, comparando a susceptibilidade desse modelo com estudos realizados em camundongos e equinos. Materiais e metodos: Hamsters Sirios machos, com tres semanas de idade, foram infectados pela via intranasal com estirpes do EHV-1, obtidas de fetos abortados e potros infectados pelo virus (A4/72, A9/92, A3/97 e ISO/72). Os hamsters foram pesados e examinados diariamente observando-se o aparecimento de manifestacoes neurologicas e/ou respiratorias da doenca. De acordo com o aparecimento dos sintomas, grupos de cinco hamsters foram submetidos a eutanasia por overdose de isoflurano e deles foram coletados os materiais: SNC, pulmoes, timo, figado e baco que foram encaminhados tanto para a realizacao de isolamento viral em cultura de celulas E-dermal como para exame histopatologico. Em uma segunda etapa que tambem utilizou cinco hamsters por grupo, inoculados com as mesmas estirpes virais, apos eutanasia por aplicacao intraperitoneal de overdose de cetamina e xilazina, foi realizada para a obtencao do lavado bronco-alveolar (LBA). Foi determinada a contagem total e diferencial de globulos brancos a partir do LBA dos hamsters. Resultados e discussao: De forma similar aos experimentos realizados com camundongos, os hamsters desafiados com as estirpes A4/72 e A9/92 apresentaram manifestacoes clinicas severas no 3o dia pos-inoculacao (dpi) tais como a perda de peso representada no grafico 1, apatia, dispneia, desidratacao, decubito e morte. Tambem foram observados sinais neurologicos como hiperexcitabilidade, paralisia espastica, perda de propriocepcao, andar em circulos e convulsoes. Ao contrario do modelo murino, em que nao foi desenvolvida a doenca, os hamsters inoculados com as estirpes A3/97 e ISO/72 apresentaram sintomas clinicos e neurologicos no 4o dpi, onde as alteracoes respiratorias foram as mais evidentes, com destaque para a epistaxe. O isolamento do virus do SNC foi positivo em todos os animais; no entanto, os pulmoes foram positivos apenas nos grupos infectados pelas estirpes A9/92 e A4/72. Nos demais orgaos houve variacao de resultado entre os grupos como visualizado na tabela 1. O LBA mostrou que a contagem total de leucocitos apresentou maior numero de celulas brancas nos hamsters infectados por A4/72 quando comparado com A9/92, A3/97 e ISO/72 e grupo controle. Entretanto, o aumento de leucocitos total encontrado nos grupos de hamsters inoculados nao foi significativo (p>0,05) quando comparado ao valor de leucocitos total do grupo controle. Macrofagos ativados com citoplasma bastante vacuolizado, alguns deles contendo grânulos intracitoplasmaticos e uma grande quantidade de eritrocitos foi observada em esfregacos da maioria dos animais inoculados ao contrario dos esfregacos do grupo controle, que em sua maior parte apresentou apenas celulas macrofagicas de citoplasma vacuolizado e raros eritrocitos. Os valores apresentados na tabela 2 revelam que a contagem total e diferencial de leucocitos variou tanto entre as estirpes como tambem entre os individuos de um mesmo grupo. De modo semelhante ao descrito em cavalos em experimentos de inoculacao com EHV-1 [7], na contagem diferencial no LBA notou-se nos hamsters infectados apresentaram um aumento no numero de neutrofilos acompanhado de um decrescimo de macrofagos, enquanto no grupo controle a celula predominante foi o macrofago variando de 90 a 100% da contagem total dos leucocitos como observado na tabela 2. Os grupos de hamsters infectados por A3/97 e A4/72 apresentaram um aumento significativo (p 0,05) de neutrofilos e uma diminuicao nao significativa de macrofagos. A diferenca encontrada nos numeros de linfocitos e monocitos entre os grupos inoculados e o grupo controle nao foi significativa. Conclusao: Os sintomas e os sinais clinicos observados durante o experimento indicaram que o hamster e a especie mais susceptivel frente a infeccao causada pelo EHV-1, quando comparada ao modelo murino. Alem disso, notou-se que a resposta dos macrofagos alveolares do hamster frente a infeccao pelo EHV-1 foi similar a do equino, o que torna a especie um atrativo modelo experimental para posteriores estudos com o agente viral.

Rabies is one of the most important zoonotic diseases and is caused by several rabies virus (RABV) variants. These variants can exhibit differences in neurovirulence, and few studies have attempted to evaluate the neuroinvasiveness of variants derived from vampire bats and wild carnivores. The aim of this study was to evaluate the neuropathogenesis of infection with two Brazilian RABV street variants (variant 3 and crab-eating fox) in mice. BALB/c mice were inoculated with RABV through the footpad, with the 50% mouse lethal dose (LD50) determined by intracranial inoculation. The morbidity of rabies in mice infected with variant 3 and the crab-eating fox strain was 100% and 50%, respectively, with an incubation period of 7 and 6 days post-inoculation (dpi), respectively. The clinical disease in mice was similar with both strains, and it was characterized initially by weight loss, ruffled fur, hunched posture, and hind limb paralysis progressing to quadriplegia and recumbency at 9 to 12 dpi. Histological lesions within the central nervous system (CNS) characterized by nonsuppurative encephalomyelitis with neuronal degeneration and necrosis were observed in mice infected with variant 3 and those infected with the crab-eating fox variant. However, lesions and the presence of RABV antigen, were more widespread within the CNS of variant-3-infected mice, whereas in crab-eating fox-variant-infected mice, RABV antigens were more restricted to caudal areas of the CNS, such as the spinal cord and brainstem. In conclusion, the results shown here demonstrate that the RABV vampire bat strain (variant 3) has a higher potential for neuroinvasiveness than the carnivore variant.

Bovine herpesvirus type 5 (BoHV-5) is an important pathogen that causes meningoencephalitis in cattle. Few studies have used the mouse as a model for BoHV-5 infection. Despite the fact that BoHV-5 can infect mice with immune deficiencies, little is known about viral replication, immune response, and the course of infection in the central nervous system (CNS) of wild-type mice. Therefore, the aim of this study was to evaluate the response in the CNS of BALB/c mice acutely infected with BoHV-5 at different days post-inoculation (dpi). BoHV-5, when inoculated intracranially, was able to infect and replicate within the CNS of BALB/c mice. Until 15 dpi, the mice were able to survive without showing prominent neurological signs. The infection was accompanied by a Th1 immune response, with a significant expression of the cytokines IFN-γ and TNF-α and chemokine CCL-2. The expression of these cytokines and chemokines was most significant in the early course of infection (3 and 4 dpi), and it was followed by meningoencephalitis with perivascular cuffing and periventriculitis, composed mainly of macrophages and lymphocytes. After the expression of cytokines and chemokine, the mice were able to curb BoHV-5 acute infection in the brain, since there was a decrease in the number of BoHV-5 DNA copies after 3 dpi and viable viral particles were not detected after 6 dpi. Importantly, BoHV-5 was able to infect the trigeminal ganglia during acute infection, since a large number of BoHV-5 DNA copies were detected on 1 and 2 dpi.

The list of animal viruses has been frequently added of new members raising permanent concerns to virologists and veterinarians. The pathogenic potential and association with disease have been clearly demonstrated for some, but not for all of these emerging viruses. This review describes recent discoveries of animal viruses and their potential relevance for veterinary practice. Dogs were considered refractory to influenza viruses until 2004, when an influenza A virus subtype H3N8 was transmitted from horses and produced severe respiratory disease in racing greyhounds in Florida/USA. The novel virus, named canine influenza virus (CIV), is considered now a separate virus lineage and has spread among urban canine population in the USA. A new pestivirus (Flaviviridae), tentatively called HoBi-like pestivirus, was identified in 2004 in commercial fetal bovine serum from Brazil. Hobi-like viruses are genetically and antigenically related to bovine viral diarrhea virus (BVDV) and induce similar clinical manifestations. These novel viruses seem to be widespread in Brazilian herds and have also been detected in Southeast Asia and Europe. In 2011, a novel mosquito-borne orthobunyavirus, named Schmallenberg virus (SBV), was associated with fever, drop in milk production, abortion and newborn malformation in cattle and sheep in Germany. Subsequently, the virus disseminated over several European countries and currently represents a real treat for animal health. The origin of SBV is still a matter of debate but it may be a reassortant from previous known bunyaviruses Shamonda and Satuperi. Hepatitis E virus (HEV, family Hepeviridae) is a long known agent of human acute hepatitis and in 1997 was first identified in pigs. Current data indicates that swine HEV is spread worldwide, mainly associated with subclinical infection. Two of the four HEV genotypes are zoonotic and may be transmitted between swine and human by contaminated water and undercooked pork meat. The current distribution and impact of HEV infection in swine production are largely unknown. Avian gyrovirus type 2 (AGV2) is a newly described Gyrovirus, family Circoviridae, which was unexpectedly found in sera of poultry suspected to be infected with chicken anemia virus (CAV). AGV2 is closely related to CAV but displays sufficient genomic differences to be classified as a distinct species. AGV2 seems to be distributed in Brazil and also in other countries but its pathogenic role for chickens is still under investigation. Finally, the long time and intensive search for animal relatives of human hepatitis C virus (HCV) has led to the identification of novel hepaciviruses in dogs (canine hepacivirus [CHV]), horses (non-primate hepaciviruses [NPHV] or Theiler's disease associated virus [TDAV]) and rodents. For these, a clear and definitive association with disease is still lacking and only time and investigation will tell whether they are real disease agents or simple spectators.

The equine herpesvirus type 1 (EHV-1) is an important cause of myeloencephalopathy and respiratory disease in horses. Animal models for EHV-1 infection have been specially developed using mice and Syrian hamsters (Mesocricetus auratus). However, few studies have attempted to evaluate the pathogenesis of EHV-1 infection in the central nervous system (CNS) and respiratory system of hamsters. Therefore, the aim of this study was to evaluate the pathogenesis of four Brazilian EHV-1 strains within the CNS and lungs of Syrian hamsters. Hamsters intranasally infected with A4/72, A9/92, A3/97, and Iso/72 EHV-1 strains developed severe neurological and respiratory signs and died during acute EHV-1 infection within 3 to 5days post-inoculation. However, neurological signs were more severe in A4/72 and A9/92-infected hamsters, whereas respiratory signs were more prominent in A3/97 and Iso/72-infected hamsters. In the latter, lesions in the CNS were predominantly inflammatory, whereas in A4/72 and A9/92-infected hamsters, neuronal and liquefactive necrosis were the predominant lesions. EHV-1 infected hamsters also developed an interstitial pneumonia with infiltration of alveolar septa by macrophages, neutrophils, and lymphocytes, with the exception of A9/92-infected hamsters, which developed severe hemorrhages within the airways. EHV-1 antigens were detected along with CNS and pulmonary lesions. EHV-1 was also recovered from CNS of all infected hamsters, whereas the virus was recovered from the lungs of A4/72, A9/92, and Iso/72-infected hamsters. Brazilian EHV-1 strains caused both severe CNS and respiratory disease in hamsters, thus making this species an interesting model for EHV-1 infection in the CNS and respiratory system.

Background Bats have been implicated as the main reservoir of coronavirus (CoV). Thus the role of these hosts on the evolution and spread of CoVs currently deserve the attention of emerging diseases surveillance programs. On the view of the interest on and importance of CoVs in bats the occurrence and molecular characterization of CoV were conducted in bats from Brazil. Findings Three hundred five enteric contents of 29 bat species were tested using a panCoV nested RT-PCR. Nine specimens were positive and eight was suitable for RdRp gene sequencing. RdRp gene phylogeny showed that all CoVs strains from this study cluster in Alphacoronavirus genus, with one Molossidae and one Phlyllostomidae-CoV specific groups. Phylogenetic analyses of two S gene sequences showed a large diversity within the Alphacoronavirus genus. Conclusions This study indicated a CoV-to-host specificity and draws attention for CoV detection in Cynomops sp, a potential new reservoir. The phylogenetic analyses indicate that diversity of CoV in bats is higher than previously known.

Intranasal inoculation of equid herpesvirus type-1 (EHV-1) Brazilian strains A4/72 and A9/92 induced an acute and lethal infection in four different inbred mouse strains. Clinical and neurological signs appeared between the 2nd and 3rd day post inoculation (dpi) and included weight loss, ruffled fur, a hunched posture, crouching in corners, nasal and ocular discharges, dyspnoea, dehydration and increased salivation. These signs were followed by increased reactivity to external stimulation, seizures, recumbency and death. The virus was recovered consistently from the brain and viscera of all mice with neurological signs. Histopathological changes consisted of leptomeningitis, focal haemorrhage, ventriculitis, neuronal degeneration and necrosis, neuronophagia, non-suppurative inflammation, multifocal gliosis and perivascular infiltration of polymorphonuclear and mononuclear cells. Immunohistochemical examination demonstrated that EHV-1 strains A4/72 and A9/92 replicated in neurons of the olfactory bulb, the cortex and the hippocampus. In contrast, mice inoculated with the EHV-1 Brazilian strain A3/97 showed neither weight loss nor apparent clinical or neurological signs; however, the virus was recovered consistently from their lungs at 3 dpi. These three EHV-1 strains showed distinct degrees of virulence and tissue tropism in mice. EHV-1 strains A4/72 and A9/92 exhibited a high degree of central nervous system tropism with neuroinvasion and neurovirulence. EHV-1 strain A3/97 was not neurovirulent despite being detected in the brains of infected BALB/c nude mice. These findings indicate that several inbred mouse strains are susceptible to neuropathogenic EHV-1 strains and should be useful models for studying the pathogenesis and mechanisms contributing to EHV-induced myeloencephalopathy in horses.

This report describes the first detection of an equine herpesvirus 1 (EHV-1) neuropathogenic variant (G2254/D752) in Brazil from a case of fatal equine herpesvirus myeloencephalopathy (EHM) in a mare. The results of nucleotide sequencing of the EHV-1 ORF30 gene showed that two other Brazilian EHV-1 isolates from EHM cases are representatives of the non-neuropathogenic variant (A2254/N752), suggesting that other unidentified factors are probably also involved in the neuropathogenicity of EHV-1 in horses. These findings will contribute to the epidemiological knowledge of EHV-1 infection in Brazil.

Bovine papillomaviruses (BPV) are the causal agents of benign and malignant lesions; they can cause dramatic economic losses in cattle. Although 10 virus types have been described, three types are most common in tumors, namely BPV-1, -2 and -4. Previous studies have reported BPV in blood cells and the possibility of blood acting as a latent virus site and/or transmission agent of virus dissemination. We studied a Holstein dairy herd in Pernambuco, Brazil, in which several animals showed severe cutaneous papillomatosis, without previous determination of BPV types. Blood samples and short-term lymphocyte cultures were collected from 54 cows. We compared the BPV types detected in peripheral blood to those identified in the respective lymphocyte cultures: BPV-1 was detected in 74% and BPV-2 in 87% of the whole blood samples. Simultaneous virus presence (BPV-1 and BPV-2) was found in 65% of the blood samples. BPV-1 or BPV-2 were detected in the lymphocyte cultures in 93% of the samples, and both in 89%. The detection of viral DNA in whole blood and in lymphocyte cultures is evidence that this virus is carried by lymphocytes.

Papillomaviruses have been reported to be very difficult to grow in cell culture. Also, there are no descriptions of cell cultures from lesions of bovine cutaneous papillomatosis, with identification of different bovine papilloma virus (BPV) DNA sequences. In the present report, we describe primary cell cultures from samples of cutaneous lesions (warts). We investigated the simultaneous presence of different BPV DNA sequences, comparing the original lesion to different passages of the cell cultures and to peripheral blood. BPV 1, 2 and 4 DNA sequences were found in lesion samples, and respective cell cultures and peripheral blood, supporting our previous hypothesis of the possible activity of these sequences in different samples and now also showing how they can be maintained in different passages of cell cultures.

RESUMO Foi realizado um estudo retrospectivo de 26 cavalos portadores de obstrucao aerea recorrente (OAR), examinados no Hospital Veterinario da Faculdade de Medicina Veterinaria e Zootecnia da Universidade de Sao Paulo, entre dezembro de 1997 e novembro de 2004. Durante esse periodo foram atendidos 3101 animais (equinos, asininos e muares), dos quais 182 apresentavam afeccoes respiratorias (5,9%) e os casos de OAR representaram 0,8% do total de equideos atendidos. A maioria dos cavalos acometidos pela OAR (96,1%) apresentava idade superior a cinco anos. Todos os animais eram mantidos estabulados e 76,9% deles apresentaram duracao da enfermidade superior a tres meses. A ocorrencia de OAR nao teve influencia sazonal. A queixa comum relatada em 100% dos cavalos acometidos pela OAR foi a presenca de tosse cronica. Dados referentes a dispneia, intolerância ao exercicio e corrimento nasal foram frequentes em 88,4%, 61,5% e 60,5% dos casos de OAR, respectivamente. Ruidos anormais na auscultacao toracica e taquipneia foram dados frequentemente detectados no exame fisico em 88,4% e 69,2% dos cavalos portadores dessa enfermidade, respectivamente. A porcentagem de 78,5 dos 14 casos de OAR examinados por endoscopia tinha excesso de secrecoes respiratorias na traqueia e a diferenca maxima da pressao intrapleural estava aumentada em 71,4% desses animais. A citologia do lavado traqueobronquico foi o exame complementar com maior sensibilidade no auxilio diagnostico dessa afeccao, pois 85,7% dos 21 casos de OAR examinados apresentaram elevacao no numero de neutrofilos; portanto, foi um importante indicador clinico de inflamacao pulmonar. Palavras-chave: Obstrucao aerea recorrente, doenca pulmonar obstrutiva cronica, equino. RETROSPECTIVE STUDY OF RECURRENT AIRWAY OBSTRUCTION (RAO) IN HORSES: 26 CASES (1997-2004) ABSTRACT A retrospective study was done from 26 horses with recurrent airway obstruction (RAO), which were clinically examined at Veterinary Hospital of Faculdade de Medicina Veterinaria e Zootecnia da Universidade de Sao Paulo, from December of 1997 to November of 2004. During this period, it was attended 3101 animals (horses, donkeys, mules and burros); in which 182 of them showed respiratory disorders (5.9%) and the occurrences of equine RAO were 0.8% of total equid cases. The majority of RAO horses (96.1%) were older than five years. All horses were exposed to indoors, which 76.9% of cases had the duration of disease longer than three months. The occurrence of RAO in horses did not have any season influence. Chronic coughing was the most common historical complaint in horses with RAO, which was reported in 100% of them. Dyspnea, exercise intolerance and nasal discharge were also found as indicators of RAO cases in 88.4%, 61.5% and 60.5% of the horses, respectively. Abnormal lung sounds and increased respiratory rates were the frequently detected findings on physical examination in 88.4% and 69.2% RAO horses, respectively. A percentage of 78.5 of 14 horses examined by endoscopy with RAO had excess volumes of respiratory secretions in their trachea and the maximal intrapleural pressure changes were raised in 71.4% of these horses. Most of 21 RAO cases (85.7%) examined with tracheobronchial wash cytology had elevated neutrophils counts and it was the most sensitive clinical indicator of pulmonary inflammation. Key words: Recurrent airway obstruction, chronic obstructive pulmonary disease, equine. INVESTIGACION RETROSPECTIVA DE 26 CASOS CLINICOS DE OBSTRUCCION AEREA RECURRENTE (OAR) EN CABALLOS (1997-2004) R ESUMEN Una investigacion retrospectiva fue hecha a partir de 26 caballos con obstruccion aerea recurrente (OAR), que fueron examinados en el Hospital Veterinario de la Faculdade de Medicina Veterinaria e Zootecnia da Universidade de Sao Paulo, en el periodo de diciembre de 1997 y noviembre de 2004. Durante este periodo, fueron atendidos 3101 animales (equinos, asininos y muares), de los cuales 182 presentaban los desordenes respiratorios (5,9%) y las ocurrencias de OAR eran 0,8% del total de los equidos. La mayoria de los caballos con OAR (96,1%) presentaban edad superior a los cinco anos. Todos los animales fueron expuestos al establo, y 76,9% de los cuales habian tenido duracion de la enfermedad superior a los tres meses. La ocurrencia de OAR no tiene influencia de las estaciones del ano. La queja comun de 100% de los caballos que le padecen era la presencia de tos cronica. Los datos que se refieren a la respiracion dificultosa, a la intolerancia al ejercicio y a la descarga nasal fueron frecuentes en los 88,4%, 61,5% y 60,5% de los animales afectados, respectivamente. Los ruidos anormales en la auscultacion toracica y la frecuencia respiratoria aumentada fueron signos clinicos detectados en el examen fisico en los 88,4% y 69,2% de los caballos afectados, respectivamente. El porcentaje de 78,5 de los 14 casos de OAR examinados por endoscopia tenia exceso de secrecion en la traquea y la diferencia maxima de presion intrapleural estaba aumentada en 71,4% de estos caballos. La mayoria de los 21 casos de OAR (85,7%) examinados con citologia de los lavajes traqueales presentaba elevado numero de neutrofilos y fue el indicador clinico mas sensible de inflamacion pulmonar. Palabras-clave: Obstruccion aerea recurrente, enfermedad pulmonar obstructiva cronica, equino

Summary Seventeen serum biochemical variables were determined in 40 donkeys of the Brazilian breed (34 females and 6 males) aged from 3 to 19 years. Mean ± standard deviation (SD) (minimum–maximum) values, obtained by automated analysis, were as follows: glucose, 58.35 ± 10.40 (44.00–90.00) mg/dL; cholesterol, 88.41 ± 9.86 (73.58–124.26) mg/dL; serum protein, 6.82 ± 0.40 (6.00–7.52) g/dL; albumin, 3.13 ± 0.21 (2.65–3.69) g/dL; creatinine, 1.80 ± 0.14 (1.51–2.19) mg/dL; urea, 24.25 ± 5.37 (14.12–34.39) mg/dL; lactate, 20.10 ± 4.58 (12.99–33.47) mg/dL; aspartate aminotransferase (AST), 295.81 ± 62.79 (173.71–466.07) IU/L; creatine kinase (CK), 158.00 ± 76.94 (51.69–440.33) IU/L; γ-glutamil-transferase (GGT), 45.82 ± 13.34 (26.17–86.38) IU/L; lactate dehydrogenase (LDH), 576.02 ± 156.32 (213.53–1162.81) IU/L; alkaline phosphatase (AP), 345.36 ± 65.90 (227.25–490.16) IU/L; calcium (Ca), 8.54 ± 0.18 (8.19–8.90) mg/dL; phosphorus (P), 2.76 ± 0.38 (1.99–3.97) mg/dL; chloride (Cl), 106.05 ± 3.20 (99.00–112.00) mEq/L; sodium (Na), 121.50 ± 4.14 (116.00–132.00) mEq/L; and potassium (K), 3.70 ± 0.42 (2.80–4.40) mEq/L. Comparisons of biochemical ranges obtained for the Brazilian donkey breed with reference ranges for other donkey breeds suggested that most values were similar. Biochemical values determined in the present study serve as reference ranges for donkey populations and can be used for health control and diagnosis of diseases.

O presente estudo teve por objetivo avaliar funcionalmente os macrófagos lácteos "nonelicited" presentes por meio de testes de fagocitose, espraiamento e liberação de peróxido de hidrogênio. Foram colhidas 56 amostras de leite de 15 búfalas hígidas e mensuradas a contagem de células somáticas total e diferencial, a viabilidade celular, os testes de fagocitose, de espraiamento e a liberação de peróxido de hidrogênio. Dessas variáveis obteve-se respectivamente média de 14.500 cél/mL de leite; com mediana de 4,33% de linfócitos e médias e desvios padrão de 50,77% + 18,28 de células da série monócito/macrófago e 32,13% + 19,27 de polimorfonucleares. A viabilidade das células na suspensão foi 66,8% +15,8 e os índices de fagocitose e espraiamento foram 30,1% + 16,9 e 58,5% +13,3. Não houve diferença entre a liberação de H2O2 espontânea e induzida por PMA. Concluiu-se que os macrófagos presentes no leite de búfalas hígidas e espraiaram significativamente, além de apresentarem correlação com outro marcador de ativação celular, no caso, a liberação de peróxido de hidrogênio; mais da metade dos macrófagos aderidos fagocitaram partículas de zymosan; os fagócitos mantêm sua capacidade de liberar peróxido de hidrogênio, espontaneamente ou não, em grau máximo, com uma significativa variação entre amostras.

The role of the pulmonary alveolar macrophages (PAM) in the lung defense mechanism was evaluated in horses infected with equine hespesvirus-1 (EHV-1). Five adult horses were exposed to 10(6.6) TCID50 EHV-1 by intranasal instillation. Cytology of bronchoalveolar lavage (BAL) was performed using cytocentrifugation of samples and slides stained by Rosenfeld. Cell concentration was adjusted to 2´10(6) cells/ml, for the measurement of macrophage activity - spreading, phagocytosis of zymosan particles and release of hydrogen peroxide (H2O2). All animals were positive in virus isolation on the second, third and fifth days post-inoculation (DPI). Seroconversion was observed on the 14th DPI. Lymphocytosis was observed by BAL cytology on the 16th DPI. Measurement of macrophage activity demonstrated a marked increase in the spreading rate, on the 23rd and 30th DPI. Phagocytosis was decreased on the second DPI, and returned to levels similar to those observed before inoculation on the 23rd DPI. The amount of H2O2 released by PAM declined on day 2, but, by day 16, they returned to values similar to those observed before inoculation. The decline in PAM activity in the acute phase of disease is indirect evidence that these cells have an important role in lung defense mechanisms against this agent. O papel dos macrófagos alveolares (MA) nos mecanismos de defesa pulmonar foi estudado em cavalos infectados pelo herpesvírus eqüino tipo 1 (EHV-1). Cinco cavalos adultos foram inoculados com 10(6,6) TCID50 do EHV-1, por instilação intranasal. A citologia do lavado broncoalveolar (LBA) foi feita usando-se citocentrifugação das amostras e confecção de lâminas coradas por Rosenfeld. A concentração celular foi ajustada para 2´10(6) células/ml, para mensuração da atividade macrofágica - espraiamento, fagocitose de partículas de zymosan e liberação de peróxido de hidrogênio (H2O2). Observou-se soroconversão no 14º dia pós-inoculação (DPI) e isolamento viral positivo no segundo, terceiro e quinto DPI. Na citologia do LBA observou-se linfocitose no 16º DPI. Os testes de mensuração da atividade macrofágica demonstraram aumento significativo nos índices de espraiamento no 23º e 30º DPI e diminuição no índice de fagocitose no segundo DPI, retornando a valores semelhantes aos observados antes da inoculação a partir do 23º DPI. Houve diminuição na liberação de H2O2 no segundo DPI, e no 16º DPI esses valores retornaram aos observados antes da inoculação. O declínio da atividade fagocítica dos MA, na fase aguda da infecção, indicou que essas células atuam de forma importante nos mecanismos de defesa pulmonar contra esse agente.

Os flavivírus são considerados uma séria ameaça à saúde pública em diversas partes do mundo, pois muitos são agentes altamente patogênicos a seres humanos e animais, tais como os vírus da febre amarela, vírus do Oeste do Nilo, vírus da encefalite japonesa e vírus da dengue, capazes de causar encefalites ou febres hemorrágicas em seus hospedeiros. Muitos deles têm avançado a diferentes regiões geográficas onde sua circulação não havia sido detectada previamente, causando novos surtos. O diagnóstico clínico destas infecções é, muitas vezes, difícil, devido ao grande número de sintomas apresentados, que podem se confundir com outras enfermidades de diferentes causas etiológicas. Os principais métodos diretos utilizados atualmente no Brasil para detecção destes vírus são a inoculação intracerebral em camundongos neonatos, inoculação em culturas de células e RTPCR específica. O presente trabalho tem como objetivos avaliar a sensibilidade e validar a detecção dos vírus pertencentes ao gênero Flavivirus circulantes no Brasil por meio de uma reação single de RT-PCR em tempo real e implementá-la, tanto na rotina diagnóstica de casos com suspeita de arbovirose como na pesquisa de amostras de campo para monitoramento viral. Amostras dos flavivírus padrões da Febre Amarela, Bussuquara, Iguape, Ilheus, Encefalite de Saint Louis, Cacipacore e Zika foram quantificados por titulação em unidades formadoras de placa (UFP) ou TCID50 para se avaliar os limites de detecção para cada um deles por RT-qPCR que detecta o gênero Flavivirus. Os limites encontrados variaram de 0,01 UFP, para o vírus Ilheus, a 1 UFP, para os vírus da Febre Amarela e Iguape, e 1x101,6 TCID50/100µL para o vírus Bussuquara. Além disso, o presente trabalho foi capaz de identificar, após sequenciamento de cDNA gerado, os vírus Zika, isolado de um paciente febril, e os vírus Ilheus e Iguape, isolados a partir de diferentes espécies de Culicídeos, após uma única reação de RT-qPCR, e um possível novo flavivírus específico de insetos, isolado de mosquitos Aedes coletados em Guapiaçu, São Paulo. Não houve sinal de amplificação para os Alphavirus Mayaro e Chikungunya. O presente protocolo mostrou-se com alta sensibilidade e especificidade, podendo dessa forma ser utilizado para o diagnóstico diferencial dos diferentes flavivírus que ocorrem no Brasil, bem como para estudos de monitoramento viral em animais sentinelas e vetores, colaborando dessa forma com a saúde pública. Pode-se, ainda, detectar possíveis novos vírus específicos de artrópodes Flaviviruses are considered a serious threat to public health in many parts of the world, as many are highly pathogenic to humans and animals, such as Yellow Fever virus, West Nile virus, Japanese encephalitis virus and dengue virus, which are capable of causing encephalitis or hemorrhagic fever in their hosts. Many of them have spread to different geographic regions where their circulation had not been detected previously, causing new outbreaks. Diagnosis of these infections is often difficult, due to the large number of symptoms presented, which can be confused with other diseases of different etiological causes. The main direct methods currently used in Brazil for detecting these viruses are intracerebral inoculation in neonatal mice, inoculation in cell cultures and specific RT-PCR. The present work aims to evaluate the sensitivity and validate the detection of viruses belonging to the genus Flavivirus circulating in Brazil through a single real-time RT-PCR reaction and to implement it, both in the diagnostic routine of cases with arbovirus suspicions and in field samples for viral monitoring. Samples of the standard flaviviruses Yellow Fever, Bussuquara, Iguape, Ilheus, Saint Louis Encephalitis, Cacipacore and Zika were quantified by titration by plaque forming units (UFP) or TCID50 to evaluate the detection limits for each of them by RT- qPCR that detects genus Flavivirus. The limits found ranged from 0.01 PFU for Ilheus virus to 1 PFU for Yellow Fever and Iguape viruses and 1x101.6 TCID50 / 100L for the Bussuquara virus. In addition, the present work was able to identify, after cDNA sequencing Zika virus, isolated from a febrile patient, and both Ilheus and Iguape viruses, isolated from different species of Culicidae, and a possible new insect-specific flavivirus, isolated from Aedes mosquitoes collected in Guapiaçu, São Paulo. The Alphaviruses Mayaro and Chikungunya were not amplified. The present protocol shoed high sensitivity and specificity, and therefore it may may be used for the differential diagnosis of the different flaviviruses that occur in Brazil, as well as for viral monitoring studies in sentinel animals and vectors, thus collaborating with public health. It is also possible to detect new flavivirus that are arthopode-specific.

A raiva é uma doença aguda, progressiva e infecciosa do sistema nervoso central de mamíferos, causada pelo vírus da raiva (RABV). Embora possa ser prevenida por vacina, continua sendo um grave problema de saúde pública, além de ser responsável pela morte de seres humanos e muitos outros animais, incluindo os de interesse econômico. Este estudo teve como objetivo avaliar a relação entre polimorfismos dos genes que codificam as proteínas P e L de amostras de RABV pertencentes a variantes antigênicas 2 e 3 e períodos de incubação e títulos em camundongos. Para isso, foram selecionadas amostras isoladas de diferentes reservatórios de raiva de mamíferos das Ordens Carnivora e Chiroptera e amostras de bovinos, de áreas endêmicas para o vírus da raiva. As sequências obtidas foram utilizadas para a construção de árvores filogenéticas para procurar os padrões de segregação de linhagens. Os resultados mostraram que não houve marcadores ou polimorfismos que explicam as variações nos períodos de incubação e de letalidade entre cepas pertencentes a variantes antigênicas 2 e 3. Esta informação pode ser usada para discussões sobre a importância de reservatórios de raiva, a dinâmica do vírus da manutenção e evolução das diferentes formas desta zoonose entre os animais infectados, contribuindo para um estudo mais aprofundado sobre a busca de marcadores moleculares para patogênese. Rabies is an acute, progressive and infectious disease of the central nervous system of mammals, caused by Rabies virus (RABV). Although preventable by vaccine, it remains a serious public health problem, and is responsible for the death of humans and many other animals, including those of economic interest. This study aimed to assess the relationship between polymorphisms in genes encoding the P and L proteins of RABV samples belonging to antigenic variants 2 and 3 and incubation periods and titers in mice. For this, samples isolated from different mammalian rabies reservoirs of the Orders Carnivora and Chiroptera and samples of cattle from endemic areas for rabies virus were selected. The sequences obtained were used to construct phylogenetic trees to search for the segregation patterns of strains. The results showed that there were no markers or polymorphisms that explain variations in incubation periods and lethality amongst strains belonging to antigenic variants 2 and 3. This information might be used for discussions about the importance of rabies reservoirs, the dynamics of the virus maintenance and evolution of the different forms of this zoonotic disease among infected animals, contributing to further study about the search for molecular markers for pathogenesis.

C. perfringens é uma bactéria anaeróbia presente no intestino delgado do homem e animais em equilíbrio e, sob a ação de alguns fatores predisponentes como mudança brusca de alimentação ou super alimentação, stress no manejo ou alto parasitismo intestinal, há a proliferação do microrganismo com a consequente produção de potentes toxinas que provocam a morte do animal. Dentre as toxinas principais destaca-se a toxina alfa, importante fator de virulência, produzida por todos os tipos de C. perfringens, sendo os pertencentes ao tipo A os maiores produtores. A fim de caracterizar o microrganismo em suspeitas de enterotoxemia em ruminantes, trabalhamos com 61 amostras de intestino delgado de bovinos e 12 de ovinos como grupo estudo e no grupo controle composto de animais hígidos levados ao abate, 73 amostras de intestino delgado de bovinos e 24 de ovinos. Foram realizados procedimentos de isolamento e tipagem molecular de C. perfringens e quantificação celular, detecção molecular da toxina β2, além de avaliações moleculares qualitativa (PCR convencional) e quantitativa (PCR em tempo real) do gene da toxina alfa dos diferentes isolados. Em 29 amostras do grupo estudo bovino (47,54%) e em 4 (33,33%) do grupo estudo ovino isolou-se o microrganismo, em contrapartida no grupo controle bovino não houve isolamento do bacilo e 5 amostras do grupo controle ovino (20,83%) foram positivas. Houve diferença estatisticamente significante somente entre os grupos de bovinos (p

A bronquite infecciosa é uma doença viral, aguda e altamente contagiosa que afeta as aves da espécie Gallus gallus, de todas as idades, causando grandes perdas econômicas devido à mortalidade, queda na produção e qualidade dos ovos. Variações na sequência de aminoácidos na região hipervariável da subunidadade S1 da proteína de espícula (S) levam à emergência de novos sorotipos do vírus da bronquite infecciosa das galinhas (IBV). Este estudo teve como objetivo seqüenciar parcialmente o gene S1 para determinar a relação filogenética entre amostras vacinais e de campo com o intuito de investigar os genótipos circulantes. Cento e sessenta amostras foram coletadas, na forma de pools, de plantéis sintomáticos durante 2009/2010. Cada pool continha tecidos de 3 a 5 aves e foram compostos por diferentes órgãos (traquéia, pulmões, rins, conteúdo entérico, trato reprodutivo e swabs traqueais). As amostras foram triadas para a presença de IBV utilizando-se uma nested RT-PCR, direcionada a região 3UTR. As amostras positivas, (n=89) foram submetidas a nova nested RT-PCR dirigida para o gene S, resultando em 10 amplicons de 390pb que foram então submetidos a seqüenciamento de DNA e análise filogenética. As amostras brasileiras segregaram-se em dois grupos filogenéticos: Massachusetts (Mass) e tipos marcadamente brasileiros. Seis amostras agruparam-se no grupo Mass, com vacinas vivas atenuadas utilizadas no Brasil. Quatro amostras agruparam com cepas de campo brasileiras previamente descritas (acesso no GeneBank: FJ791257 to FJ791273). A identidade de aminoácidos no grupo Mass variou de 98% a 100%, sugerindo a detecção de vírus vacinal. Enquanto que entre as amostras brasileiras e o grupo Mass, a variabilidade de aminoácidos foi de 79% a 81%. Esses resultados mostram uma variação importante em genótipos virais circulantes nos plantéis industriais avícolas brasileiros. Portanto, o presente estudo reforça a contínua emergência e disseminação de novas linhagens de IBV no Brasil e a importância de constantes pesquisas e monitoramento, para que se compreendam aos fenômenos de emergência de novas linhagens e suas consequências, bem como para otimizar as medidas de controle do vírus. Infectious bronchitis is a viral disease, acute and highly contagious that affects birds of the Gallus gallus species, of all ages, causing huge economic losses due to mortality, drop in production and quality eggs. Differences between serotypes of IBV are due to variations in the amino acids sequence in S1 hypervariable region subunit of spike protein (S). This study aimed to partially sequence the S1 gene to determine the phylogenetic relationship between field and vaccine viral types, and to investigate circulating genotypes in Brazil. One hundred and sixty pool samples were collected from symptomatic flocks during 2009/2010. Each pool contained tissues from 3 to 5 birds and was composed by different organs (tracheas, lungs, kidneys, enteric contents, tract reproductive and tracheal swabs). Samples were screened for the presence of IBV using a nested RT-PCR targeted to the 3UTR. Positive samples (n=56) were submitted to a nested RT-PCR target to S gene resulting in 10 amplicons of 390pb that were then submitted to DNA sequencing and analysis. Brazilian IBV genotypes segregated in two phylogenetic groups: Massachusetts (Mass) and Brazilian types. Six samples clustered in the Mass group, with live attenuated vaccines used in Brazil. Four samples grouped with field Brazilian strains previously described (GeneBank accession: FJ791257 to FJ791273). In Mass cluster aminoacids identity range from 98% to 100%, suggesting vaccine virus detection, in this last case. Moreover, the verified identity between samples from this study and Mass group ranged 79% to 81%. Therefore the present study reinforces the emergence and circulation of news lineages of IBV in commercial Brazilian flocks, as well as the importance of consistent research and monitoring to better understand this phenomen and its consequences, aiming to improved control measures against the virus.

A diarreia é apontada mundialmente como uma das principais enfermidades que acometem bovinos. Estudos em diferentes países tem reconhecido a participação do Torovírus Bovino (BToV) na etiologia de enterites em bezerros e bovinos adultos. Devido à inexistência de dados a respeito da sua ocorrência no Brasil, o presente estudo foi conduzido buscando detectar a presença deste vírus em amostras fecais diarreicas. Através da técnica de nested-RT-PCR dirigida para o gene que codifica a proteína do nucleocapsídeo (N), analisou-se 80 amostras fecais de animais com diarreia, jovens e adultos, dos estados de Mato Grosso, Rio Grande do Sul e São Paulo. Obteve-se amplificação de fragmento do gene N em 5 das amostras estudadas. Após clonagem em vetor plasmidial de 3 das amostras, confirmou-se a especificidade dos fragmentos obtidos. Através de estudo de diversidade molecular, demonstrou-se a presença de duas linhagens brasileiras, ambas relacionadas com as sequências reportadas na Europa e Japão. Diarrhea is pointed worldwide as a major disease affecting cattle. Studies in different countries have recognized the role of Bovine Torovirus (BToV) in the etiology in enteritis in calves and adult cattle. Due to lack of data about its occurrence in Brazil, this study was conducted to detect the presence of this virus in diarrheic stool samples. Using the nested-RT-PCR technique directed for the gene encoding the nucleocapside protein (N gene), we analyzed 80 stool samples from animal with diarrhea, calves and adults, from states of Mato Grosso, Rio Grande do Sul and São Paulo. We obtained amplification of gene N fragments in 5 samples. Three positive for BToV samples were clones in a plasmidial vector and then submitted to sequencing for confirmation of specificity. Studies of molecular diversity were showed the presence of 2 strains of BToV in Brazil, both related with sequences reported in Europe and Japan.

Scrapie ou paraplexia enzoótica dos ovinos é uma doença neurodegenerativa fatal que acomete ovinos e raramente caprinos. A doença é influenciada por polimorfismos nos códons 136, 154 e 171 do gene prnp que codifica a proteína priônica. Os animais podem ser susceptíveis ou resistentes, de acordo com as sequências alélicas observadas nos referidos códons. No Brasil ocorreram apenas casos de animais que foram importados, sendo o país considerado livre da doença. Neste trabalho foi realizada a genotipagem dos diferentes polimorfismos associados ao desenvolvimento do scrapie e a categorização em animais susceptíveis e resistentes. Foram sequenciadas 118 amostras provenientes de ovinos da raça Santa Inês criados em propriedades localizadas no Estado de São Paulo. Destas amostras foram identificados 6 alelos e 11 genótipos (ARQ/ARQ, ARR/ARQ, ARQ/AHQ, ARQ/VRQ, AHQ/AHQ, ARR/ARR, ARR/AHQ, VRQ/VRQ, ARQ/TRQ, TRR/TRR, TRQ/TRQ), dentre os quais o genótipo ARQ/ARQ teve ocorrência de 56,7%. Em nosso estudo foi detectada a presença da tirosina no códon 136, observação rara na medida em quenão existem relatos nacionais e relatos envolvendo a raça Santa Inês descrevendo este polimorfismo. Com os resultados obtidos, foi possível determinar a existência de grande variabilidade genética relacionada à raça Santa Inês no Estado de São Paulo, apesar da variabilidade, apenas 1,69% dos genótipos observados são extremamente resistentes ao scrapie. Estes dados demonstram que a raça nativa Santa Inês pode ser considerada potencialmente susceptível ao scrapie. Enzootic paraplexia or scrapie is a fatal neurodegenerative disease affecting mainly sheep and rarely goats. The disease is influenced by polymorphisms at codons 136, 154 and 171 of prnp gene that encodes the prion protein. The animals may be susceptible or resistant to the development of the disease according to the allelic sequences observed in these codons. In Brazil there were only cases of scrapie in imported animals, therefore the country is considered free of the disease. This study performed the genotyping of different polymorphisms associated to the development of scrapie. Then, based on these findings the animals were categorized in resistant and susceptible. A total of 118 samples were sequenced from the Santa Ines sheep raised on properties located in the State of Sao Paulo. From these samples, 6 alleles and 11 genotypes were identified (ARQ / ARQ, ARR / ARQ, ARQ / AHQ, ARQ / VRQ, AHQ / AHQ, ARR / ARR, ARR / AHQ, VRQ / VRQ, ARQ / TRQ, TRR / TRR, TRQ / TRQ), the genotype ARQ / ARQ presented a frequency of 56.7%. It was also detected the presence of tyrosine at codon 136, which may be considered a rare observation, since there is no report regarding Santa Ines breeding presenting this polymorphism. These results showed the great genetic variability in Santa Ines in Sao Paulo and only 1,69% of the genotypes observed are extremely resistant to scrapie. These data demonstrate that the Santa Ines sheep can be considered potentially susceptible to scrapie.