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Madariaga virus (MADV) is a member of the eastern equine encephalitis virus (EEEV) complex that circulates in Central and South America. It is a zoonotic, mosquito-borne pathogen, belonging to the family Togaviridae. Disturbances in the natural transmission cycle of this virus result in outbreaks in equines and humans, leading to high case fatality in the former and acute febrile illness or neurological disease in the latter. Although a considerable amount of knowledge exists on the eco-epidemiology of North American EEEV strains, little is known about MADV. In Brazil, the most recent isolations of MADV occurred in 2009 in the States of Paraíba and Ceará, northeast Brazil. Because of that, health authorities have recommended vaccination of animals in these regions. However, in 2019 an equine encephalitis outbreak was reported in a municipality in Ceará. Here, we present the isolation of MADV from two horses that died in this outbreak. The full-length genome of these viruses was sequenced, and phylogenetic analyses performed. Pathological findings from postmortem examination are also discussed. We conclude that MADV is actively circulating in northeast Brazil despite vaccination programs, and call attention to this arbovirus that likely represents an emerging pathogen in Latin America.

As enfermidades do sistema nervoso central (SNC) são frequentemente relatadas em bovinos no Brasil. Apesar de Minas Gerais ter o segundo maior rebanho bovino do país, há escassez de informações referentes às doenças neurológicas que acometem esses animais. O Laboratório de Saúde Animal do Instituto Mineiro de Agropecuária (LSA/IMA) é o responsável pelo diagnóstico das enfermidades neurológicas dos animais de produção no Estado, com ênfase para a raiva e as encefalopatias espongiformes transmissíveis. Foi realizado um estudo retrospectivo dos dados referentes às amostras de SNC de bovinos com síndrome neurológica avaliadas pelo LSA/IMA de janeiro/2003 a junho/2010, com o objetivo de determinar o perfil das amostras encaminhadas para análise no serviço de defesa sanitária animal, com ênfase no diagnóstico da raiva bovina. Foram consideradas características do animal (sexo, idade, raça e tipo de morte) e da amostra (método de conservação e responsável pela coleta), sendo nas positivas para raiva, avaliada sua composição, assim como as alterações histopatológicas encontradas. Os dados relacionados à frequência de positividade nas diferentes categorias foram submetidos à análise pelo Teste Exato de Fisher. Durante o período avaliado, foram analisadas 3.731 amostras de bovinos com doença neurológica, havendo predomínio de fêmeas e mestiços, o que reflete a composição do rebanho do Estado. O método de conservação foi o principal problema encontrado, sendo apenas 25,89% das amostras encaminhadas em gelo e formol a 10%. Verificou-se uma diminuição gradativa no envio de material para análise. Quanto a raiva bovina diagnosticada no Estado, foram avaliadas 3.703 amostras pela imunofluorescência direta (IFD) e prova biológica (PB), com 41,58% de positividade, sendo dessas 282 submetidas a histopatologia. A frequência de positividade foi influenciada pela raça, idade e tipo de morte do animal. A composição da amostra alterou significativamente o resultado das análises, havendo maior frequência de positividade naquelas compostas por três ou mais fragmentos de SNC, tanto na IFD/PB, quanto na histopatologia. O bulbo, fragmento de eleição para o diagnóstico da EEB, tem sido erroneamente enviado refrigerado e não em formol a 10%. Cerebelo, tálamo, tronco encefálico e medula apresentaram maior frequência de corpúsculos de Negri que cérebro e gânglio trigeminal. O infiltrado inflamatório não supurado foi menos frequente no cérebro, que nos demais fragmentos avaliados. Conclui-se que as amostras de bovinos com síndrome neurológica enviadas ao serviço de defesa sanitária animal de Minas Gerais apresentam características distintas, sendo o método de conservação o principal problema encontrado. Além disso, a raiva bovina diagnosticada na população estudada é influenciada pelas características do animal e da amostra, sendo indicado o envio de diferentes fragmentos do SNC para análise, conservados adequadamente, o que contribui para um diagnóstico mais preciso. Diseases of the central nervous system (CNS) are often reported in cattle in Brazil. Although the State of Minas Gerais has the second largest cattle herd in the country, there is little information from this state concerning neurological diseases that affect cattle. The Laboratório de Saúde Animal of the Instituto Mineiro de Agropecuária (LSA/IMA) is in charge of the diagnosis of neurological diseases of livestock in the State, with emphasis on rabies and transmissible spongiform encephalopathies. A retrospective study was conducted on data from cattle with neurologic diseases evaluated by the LSA/IMA from January/2003 to June/2010, aiming to determine the profile of CNS samples sent for analysis, with emphasis on the diagnosis of bovine rabies. Issues related to the animal (sex, age, breed and type of death) as well as to the sample (method of conservation, person in charge the collection, and region of the CNS sampled) were evaluated. Data on frequency of rabies positive samples were analyzed by Fisher's exact test. During the period studied, 3,731 samples from cattle with neurological signs were analyzed, with a predominance of females and crossbred cattle. The method of preservation was the main problem encountered with only 25.89% of samples sent both refrigerated and fixed in 10% formaldehyde. There was a gradual decrease in submission of samples during the course of this study. All 3,703 samples were evaluated by direct fluorescent antibody test (DFA) and biological test (BT) for rabies, 41.58% being positivity for rabies, and 282 of those samples being subjected to histopathology examination. The frequency of positivity was influenced by breed, age, and type of death. Composition of the sample significantly influenced the results, with higher frequency of positivity in samples containing three or more CNS fragments by DFA, BT, or histopathology. The medulla, which is the fragment of choice for diagnosis of BSE, has often been mistakenly submitted under refrigeration, but not in 10% formalin. Cerebellum, thalamus, brain stem, and spinal cord had higher frequency of Negri bodies than the cerebral cortex and trigeminal ganglia. The nonsuppurative inflammatory infiltrate was less frequent in the cerebral cortex than in other CNS fragments. In conclusion, CNS samples from cattle with neurological syndrome sent to the animal health protection service of Minas Gerais are heterogeneous, and the preservation method was the major problem hindering and adequate diagnosis. In addition, diagnosis of rabies was influenced by parameters of the animal as well as the CNS sample. Submission of properly preserved fragments from various segments of the CNS contributes to a more accurate diagnosis of rabies in cattle.

To the Editor: Malignant catarrhal fever (MCF) is an acute, generalized, and usually fatal disease previously thought to be restricted to mammals of the order Artiodactyla, often members of the subfamilies Bovinae, Cervidae, and Suidae (1). Although animals of the order Perissodactyla, family Equidae, have previously been considered not susceptible to ovine herpesvirus 2 (OvHV-2), we show that horses may be infected by this virus. In July 2006, in the state of Minas Gerais, Brazil, neurologic signs developed acutely in a 6-month-old foal; signs included muscle spasms, rigidity of the neck and limbs, difficulty standing, and hind-limb paralysis. The foal also had severe dyspnea and profuse sweating and died 1 day after the onset of clinical signs. Histopathologic findings included a marked lympho-histiocytic fibrinoid necrotizing vasculitis affecting small blood vessels and arterioles in the kidney and liver (Figure) and associated with lymphocytic interstitial nephritis and mild multifocal granulomatous hepatitis. The vasculitis lesions were strikingly similar to those observed in cattle with MCF caused by OvHV-2 or alcelaphine herpesvirus 1. Both forms of the disease have a wide spectrum of clinical manifestations, but histopathologic findings for the 2 forms are similar (2,3). In addition, the foal had severe and diffuse interstitial pneumonia characterized by thickening of the alveolar walls and interstitial accumulation of macrophages, proliferation of type II pneumocytes, and accumulation of cell debri in the alveolar lumen. Granulomatous inflammation, characterized by mild multifocal to coalescent accumulation of epithelioid macrophages, was also observed in the spleen and lymph nodes. Surprisingly, the brain showed only moderate congestion and mild, multifocal, perivascular hemorrhage. Figure Liver of foal with vasculitis associated with intralesional ovine herpesvirus 2 DNA, showing segmental, severe, fibrinoid, necrotizing arteriolitis. Hematoxylin and eosin stain. Scale bar = 100 μm. Although horses are not considered susceptible to OvHV-2, histopathologic findings in this case were consistent with MCF-like lesions. Thus, we looked for any history of direct or indirect contact between the affected foal and goats or sheep. Indeed, on this particular farm, horses shared food with 65 goats. On the basis of indirect contact with potential reservoirs of infection, the disease in the foal was suspected of being associated with a member of the MCF virus (MCFV) group (4). To determine whether the disease in the foal was associated with an MCFV, we obtained tissue samples from the foal at necropsy and collected blood from all 3 adult horses, including the dam, and 10 randomly selected goats on the farm. DNA was extracted from these samples, and PCR was performed to detect members of the MCFV group, including OvHV-2 (5), caprine herpesvirus 2 (6), and alcelaphine herpesvirus 1 (7). The tissues from the foal as well as peripheral blood mononuclear cells from all 3 adult horses and 8 tested goats were positive for OvHV-2 only. The adult horses had no clinical signs of infection for at least 8 months after the outbreak. To confirm the PCR detection of OvHV-2, we purified amplicons obtained from the dam of the affected foal, the affected foal, and 1 goat and processed them for automated sequencing. These nucleotide and deduced amino acid sequences were identical, and we deposited them in GenBank under accession nos. {"type":"entrez-nucleotide","attrs":{"text":"EU244694","term_id":"160347526"}}EU244694, {"type":"entrez-nucleotide","attrs":{"text":"EU718486","term_id":"189419074"}}EU718486, {"type":"entrez-nucleotide","attrs":{"text":"EU718487","term_id":"189419076"}}EU718487. PCR was conducted on tissue samples of the foal to test for differential diagnosis agents equine herpesvirus 1 (8), equine herpesvirus 4 (8), and equine arteritis virus (9). No amplification was observed. Considering that sheep and goats are the most important natural reservoirs of OvHV-2 (6), these results support the notion that infected goats were the most likely source of infection for horses in this outbreak. To further support this diagnosis of OvHV-2 infection, we attempted to detect viral DNA in the vascular wall of the foal’s hepatic arterioles that contained fibrinoid necrotizing lympho-histiocytic vasculitis by using laser capture microdissection on sections stained with hematoxylin and eosin (10). DNA extracted from these areas was PCR positive for OvHV-2, which confirmed the co-localization of OvHV-2 DNA sequences in the site of MCF-like lesions. Taken together, these findings confirm an emergent infectious disease associated with OvHV-2 infection in a horse, a species previously considered not susceptible to OvHV-2. The finding of vasculitis associated with intralesional OvHV-2 DNA sequences unequivocally demonstrates the pathogenic potential of this virus in foals. However, a cause-and-effect relationship between OvHV-2 infection and interstitial pneumonia as well as the granulomatous inflammation in the liver and spleen could not be established in this case. This report supports the notion that either equine infection is extremely rare or that this strain of OvHV-2 underwent recent modifications that expanded the host range.

Durante o período de Outubro de 2004 a Fevereiro de 2006, 75 amostras de sistema nervoso central (SNC) oriundas de eqüinos que morreram com sinais neurológicos no estado de Minas Gerais foram enviadas ao Laboratório de Virologia Comparada no ICB/UFMG para o diagnóstico de herpesvírus eqüino. Essas amostras foram previamente diagnosticadas negativas para o vírus da raiva através dos testes de imunofluorescência direta e inoculação em camundongos, no Laboratório de Saúde Animal (LSA) do Instituto Mineiro de Agropecuária (IMA). Dentre as amostras analisadas, 39 (52%) foram positivas para o herpesvírus eqüino 1 (EHV-1) através da técnica de reação em cadeia pela polimerase (PCR). Na maioria dos casos, o exame histopatológico do SNC revelou uma discreta vasculite com infiltrado perivascular de células mononucleares, congestão, trombose arterial e degeneração do tecido nervoso central. As amostras de SNC positivas para o EHV-1 foram coletadas de eqüinos oriundos de 30 municípios de Minas Gerais. Os casos de EHV-1 ocorreram de forma isolada não apresentando caráter sazonal. Na maioria dos casos (71,8%), a evolução dos sinais clínicos foi aguda, sendo que os sinais clínicos observados com mais freqüência foram ataxia, instabilidade dos membros posteriores, paralisia dos membros posteriores e decúbito. De acordo com informações relatadas pelo IMA, as infecções causadas pelo EHV-1 foram tão freqüentes como as infecções causadas pelo vírus da raiva em eqüinos no estado de Minas Gerais durante o período estudado. Portanto, torna-se importante a inclusão da encefalite pelo EHV-1 no diagnóstico diferencial de outras doenças do SNC de eqüinos no estado de Minas Gerais.