Retrospective study of poxviruses diagnosed in cattle from Goiás State, Brazil (2010-2018) / Estudo retrospectivo de poxviroses diagnosticadas em bovinos no estado de Goiás (2010-2018)

A retrospective study of poxvirus infections diagnosed in cattle from Goiás state (GO), Brazil, from 2010 to 2018, was performed. All cases have been investigated by the GO Official Veterinary Service (Agrodefesa), from which technical forms and protocols of veterinary diagnosis laboratories were reviewed. In most cases, samples of oral or cutaneous tissues and/or swabs were submitted for virological diagnosis by polymerase chain reaction (PCR) and/or virus isolation. Thirty seven outbreaks/cases of vesicular disease were notified in cattle of 25 counties; in 33 cases the animals presented lesions clinically compatible with poxviruses. The etiology of 25 out of 33 outbreaks/cases was confirmed as poxviruses by PCR and/or viral isolation: 13 as bovine vaccinia virus (VACV), six as pseudocowpox virus (PCPV), five as bovine papular stomatitis virus (BPSV) and one coinfection (VACV and an Orf virus-like parapoxvirus). The laboratory confirmed that cases occurred mainly in dairy cattle (19/25) and during the dry season (22/25). In adult cattle, gross changes were observed mainly in the teats and udder and included vesicles, ulcers, crusts, papules and scars and varied of type, severity and affected region, depending on the poxvirus species. In calves, the main lesions were ulcers in the mouth and muzzle. Zoonotic lesions compatible with poxvirus infections were observed for all diagnosed poxviruses, affecting especially the hands of milkers and other farm workers. Our data demonstrate the sanitary and economic relevance of these diseases and the wide circulation of different poxviruses in cattle from GO.

Foi realizado um estudo retrospectivo das infecções por poxvírus diagnosticadas em bovinos do estado de Goiás (GO), entre 2010 e 2018. Todos os casos foram investigados pela Agência Goiana de Defesa Agropecuária (Agrodefesa). Foram revisados formulários técnicos e protocolos de laboratórios de diagnóstico veterinário. Na maioria dos casos, amostras de tecidos orais ou cutâneos e/ou swabs foram encaminhadas para diagnóstico virológico. Foram notificados 37 surtos/casos de doença vesicular em bovinos em 25 municípios; em 33 casos os animais apresentavam lesões clinicamente compatíveis com poxvírus. A etiologia de 25 de 33 surtos/casos foi confirmada como poxvírus por PCR e/ou isolamento viral: 13 como vírus vaccínia (VACV), seis como vírus pseudocowpox (PCPV), cinco como vírus da estomatite papular bovina (BPSV) e um caso de coinfecção (VACV e um parapoxvírus semelhante ao Orf vírus). Os casos confirmados laboratorialmente ocorreram principalmente em bovinos leiteiros (19/25) e durante a estação seca (22/25). Em bovinos adultos, alterações macroscópicas foram observadas principalmente nas tetas e úbere e incluíram vesículas, úlceras, crostas, pápulas e cicatrizes e variaram quanto ao tipo, gravidade e região afetada, dependendo da espécie do poxvírus. Em bezerros, as principais lesões foram úlceras na boca e focinho. Lesões zoonóticas compatíveis com infecção por poxvírus foram observadas em todas as poxviroses diagnosticadas, afetando principalmente as mãos dos ordenhadores e outros trabalhadores rurais. Nossos dados demonstram a relevância sanitária e econômica dessas doenças e a ampla circulação de diferentes poxvírus em bovinos de GO.

Retrospective study of poxviruses diagnosed in cattle from Goiás State, Brazil (2010-2018) / Estudo retrospectivo de poxviroses diagnosticadas em bovinos no estado de Goiás (2010-2018)

A retrospective study of poxvirus infections diagnosed in cattle from Goiás state (GO), Brazil, from 2010 to 2018, was performed. All cases have been investigated by the GO Official Veterinary Service (Agrodefesa), from which technical forms and protocols of veterinary diagnosis laboratories were reviewed. In most cases, samples of oral or cutaneous tissues and/or swabs were submitted for virological diagnosis by polymerase chain reaction (PCR) and/or virus isolation. Thirty seven outbreaks/cases of vesicular disease were notified in cattle of 25 counties; in 33 cases the animals presented lesions clinically compatible with poxviruses. The etiology of 25 out of 33 outbreaks/cases was confirmed as poxviruses by PCR and/or viral isolation: 13 as bovine vaccinia virus (VACV), six as pseudocowpox virus (PCPV), five as bovine papular stomatitis virus (BPSV) and one coinfection (VACV and an Orf virus-like parapoxvirus). The laboratory confirmed that cases occurred mainly in dairy cattle (19/25) and during the dry season (22/25). In adult cattle, gross changes were observed mainly in the teats and udder and included vesicles, ulcers, crusts, papules and scars and varied of type, severity and affected region, depending on the poxvirus species. In calves, the main lesions were ulcers in the mouth and muzzle. Zoonotic lesions compatible with poxvirus infections were observed for all diagnosed poxviruses, affecting especially the hands of milkers and other farm workers. Our data demonstrate the sanitary and economic relevance of these diseases and the wide circulation of different poxviruses in cattle from GO.(AU)

Foi realizado um estudo retrospectivo das infecções por poxvírus diagnosticadas em bovinos do estado de Goiás (GO), entre 2010 e 2018. Todos os casos foram investigados pela Agência Goiana de Defesa Agropecuária (Agrodefesa). Foram revisados formulários técnicos e protocolos de laboratórios de diagnóstico veterinário. Na maioria dos casos, amostras de tecidos orais ou cutâneos e/ou swabs foram encaminhadas para diagnóstico virológico. Foram notificados 37 surtos/casos de doença vesicular em bovinos em 25 municípios; em 33 casos os animais apresentavam lesões clinicamente compatíveis com poxvírus. A etiologia de 25 de 33 surtos/casos foi confirmada como poxvírus por PCR e/ou isolamento viral: 13 como vírus vaccínia (VACV), seis como vírus pseudocowpox (PCPV), cinco como vírus da estomatite papular bovina (BPSV) e um caso de coinfecção (VACV e um parapoxvírus semelhante ao Orf vírus). Os casos confirmados laboratorialmente ocorreram principalmente em bovinos leiteiros (19/25) e durante a estação seca (22/25). Em bovinos adultos, alterações macroscópicas foram observadas principalmente nas tetas e úbere e incluíram vesículas, úlceras, crostas, pápulas e cicatrizes e variaram quanto ao tipo, gravidade e região afetada, dependendo da espécie do poxvírus. Em bezerros, as principais lesões foram úlceras na boca e focinho. Lesões zoonóticas compatíveis com infecção por poxvírus foram observadas em todas as poxviroses diagnosticadas, afetando principalmente as mãos dos ordenhadores e outros trabalhadores rurais. Nossos dados demonstram a relevância sanitária e econômica dessas doenças e a ampla circulação de diferentes poxvírus em bovinos de GO.(AU)

Poxviruses diagnosed in cattle from Distrito Federal, Brazil (2015-2018).

A retrospective study of officially diagnosed poxvirus infections in cattle in Distrito Federal (DF), Brazil, between 2015 and 2018 was performed. All cases were investigated by the DF Official Veterinary Service. In the most cases, samples of oral, cutaneous (teats, udder) or foot lesions were submitted to molecular diagnosis by PCR. In approximately 70% of the cases, additional samples were also submitted for histopathology. Ninety-three out of 2,467 clinically examined cattle (from 385 farms) presented suggestive and/or compatible lesions with poxviruses. Fifty-two out of these 93 cases were confirmed as poxviruses: 27 vaccinia virus (VACV), 9 pseudocowpox virus (PCPV), 8 bovine papular stomatitis virus (BPSV), 5 coinfection by PCPV and BPSV and 3 unidentified parapoxvirus. The clinical cases were observed in farms with different exploration (beef, dairy or mixed) from 9 out of 30 administrative regions of DF. Gross findings consisted of papules, vesicles, ulcers, scabs and scars and varied of type, severity and affected tissue, according to the detected virus. A single human case was observed associated with a BPSV infection. Histologically, the lesions were very similar, independently of the detected poxvirus, and included mild to moderate, superficial, multifocal inflammatory infiltrate of lymphocytes, plasma cells, macrophages and/or neutrophils, with acanthosis and parakeratotic hyperkeratosis, usually associated with serous content, cellular debris and spongiosis. In the ulcerated lesions, there were focally extensive areas of necrosis with severe infiltrate of neutrophils in the adjacent connective tissue. Few to moderate amount of 4- to 8-µm eosinophilic inclusion bodies were observed in the cytoplasm of keratinocytes in 6 cases (2 of VACV, 2 of PCPV and 2 of PCPV/BPSV coinfection). Data of the current study demonstrate the wide circulation of different poxviruses in cattle from DF.

Short communication: Parapoxvirus and Orthopoxvirus coinfection in milk of naturally infected cows.

Several studies have shown the occurrence of poxvirus infections associated with exanthematic lesions in cattle from many Brazilian states. Coinfection between viruses belonging to 2 genera, Orthopoxvirus (OPXV) and Parapoxvirus (PPV), was already identified from the lesions of affected cows and humans. The DNA and infectious viral particles of Vaccinia virus, an OPXV, have been detected in milk of naturally and experimentally infected cows. However, to date no reports have described the detection of Pseudocowpox virus, a PPV, in milk. Thus, we investigated the presence of PPV and OPXV in milk samples obtained from dairy cows from a Brazilian region with exanthematic disease outbreaks. From 2011 to 2014, 6 dairy farms with exanthematic disease outbreaks involving dairy cows, calves, and humans were visited. Twelve crusts of cows' teat lesions and 60 milk samples were collected. The crusts and milk samples were analyzed by PCR to detect OPXV or PPV DNA. According to the analyzed crusts, we detected PPV infection in 4 of the 6 visited farms, from which we investigated the PPV contamination in milk. From the 40 milk samples tested, PPV DNA was detected in 12 samples. Of these milk samples, 8 were positive for both PPV and OPXV. This is the first report of PPV DNA detection in milk samples from affected cows, indicating that the virus may be present in milk and potentially contaminating dairy products associated or not with OPXV. In addition to the lesions caused by direct contact, the presence of 2 or more poxvirus species in milk showed that the effect of zoonotic exanthematic diseases on public health and animal husbandry is relevant and cannot be overlooked.

A multiplex PCR for viruses associated with exanthematic and vesicular disease in cattle.

Exanthematic and papulo-vesicular lesions in the udder and teats of milking cows are fairly common in some Brazilian dairies, especially those with poor sanitary conditions and hand milking. The orthopoxvirus Vaccinia virus (VACV) and the parapoxviruses Pseudocowpox virus (PCPV) and Bovine popular stomatitis virus (BPSV) have been frequently associated with such conditions. Elsewhere, Bovine herpesvirus 2 (BoHV-2) has also been associated with similar clinical signs. Thus, we herein describe a conventional multiplex PCR designed to detect the genome of these viruses in clinical samples while differentiating among them by amplicon size. For this, primer sets targeting the orthopoxvirus vascular growth factor (amplicon size 292bp), PCPV (374bp) and BSPV (607bp) B2L genes, and the BoHV-2 DNA polymerase gene (138bp) were selected. The chosen primers anneal within the same temperature range and do not interfere with each other during the PCR amplification. PCR conditions were initially standardized for each agent in individual PCR reactions firstly using the target virus as positive control followed by using a mixture of all four virues. Lastly, a multiplex PCR containing the four sets of primers was set up to amplify all four targeted viruses in one reaction. The multiplex PCR was able to detect DNA extracted from cell culture supernatants containing 20 TCID50 of BoHV-2 and 50 TCID50 of VACV. Further, the test could detect the viral genomes in 1:10, 1:50 and 1:1000 dilutions of total DNA extracted from clinical specimens (e.g. scabs, crusts) of natural cases (PCPV, VACV and BPSV) and 1:10 dilutions of DNA extracted from scabs collected from BoHV-2 experimentally infected cattle. A possible amplification of other orthopoxviruses, predicted by in silico analysis, was considered to not represent an important pitfall since these are exotic in Brazil, very rare, or viruses not associated with cattle. For definitive agent identification amplicon sequencing needs to be conducted. Thus, this multiplex PCR seems suitable for initial detection and identification of the agents involved in exanthematic and vesicular disease, providing a sensitive and specific diagnosis for such conditions in dairy cows.

Detection of multiple viral infections in cattle and buffalo with suspected vesicular disease in Brazil.

Vesicular diseases are of high importance for livestock, primarily because of foot-and-mouth disease (FMD), which is a high-morbidity disease that generates direct losses caused by low milk production, weight loss, and indirect losses because of the need for sanitary barriers. Other vesicular diseases are also of importance for livestock because of direct impacts or because their clinical signs may be confused with those of FMD. We report herein the detection of multiple infections in cattle with suspected vesicular disease in the Brazilian states of Amazonas (AM), Mato Grosso (MT), and Roraima. Thirty-seven epithelial samples from cattle and 1 sample from a buffalo were sent to the laboratory for testing for FMDV and similar disease agents. All samples from MT were positive for parapoxvirus (Pseudocowpox virus and Bovine papular stomatitis virus). In addition, 3 samples were positive for Bluetongue virus, and 5 samples were positive for Bovine herpesvirus 1 Among these samples, 1 was positive for all of these 3 agents. Only 2 samples from AM were negative for parapoxvirus. The molecular tests conducted in this study detected multiple infections, with a high prevalence of parapoxvirus.

Coinfection by Vaccinia virus and an Orf virus-like parapoxvirus in an outbreak of vesicular disease in dairy cows in midwestern Brazil.

The current report describes an outbreak of vesicular disease affecting dairy cows in midwestern Brazil in which a coinfection with 2 poxviruses-Vaccinia virus (VACV) and a parapoxvirus-was demonstrated. Milking cows presented vesicles, painful reddish or whitish papules, and scabby proliferative lesions in the teats and udder, in a clinical course of approximately 10-21 days. Histologically, multifocal areas of moderate to severe acanthosis, spongiosis, hypergranulosis, and parakeratotic or orthokeratotic hyperkeratosis with adjacent focally extensive ulcers were observed in the epidermis. Rounded eosinophilic inclusion bodies were observed in the cytoplasm of epithelial cells of areas with acanthosis or necrosis. Moderate inflammatory infiltrate of lymphocytes, plasma cells, neutrophils, and macrophages were observed in some dermal areas. Two people milking the affected cows developed lesions on the hands, painful papules which progressed to ulcerative and scabby lesions in 4-7 days. Electron microscopy of scabs from 1 cow revealed the concomitant presence of orthopoxvirus and parapoxvirus particles. Scabs from 2 cows were positive by polymerase chain reaction for the parapoxvirus B2L gene; 1 of the scabs was also positive for the VACV vgf gene. Nucleotide sequencing of the B2L amplicon revealed a similarity of 96-99% with Orf virus (ORFV) and lower identity with Pseudocowpox virus (92-95%) and Bovine papular stomatitis virus (85-86%). Nucleotide sequencing of a region of parapoxvirus DNA polymerase gene revealed a high similarity (98-100%) with ORFV sequences. Thus, an unusual coinfection with VACV and a parapoxvirus, likely ORFV, was demonstrated in the outbreak.

Bovine papular stomatitis affecting dairy cows and milkers in midwestern Brazil.

Bovine papular stomatitis virus (BPSV) is a parapoxvirus associated with papular and erosive lesions on the muzzle, lips, and oral mucosa of cattle. Teats of milking cows are occasionally affected, and the infection is frequently transmitted to human beings. The present report describes an outbreak of BPSV infection affecting cows in midwestern Brazil, with human involvement. The disease was observed in neighboring small hand-milking farms, affecting 20 milking cows. The signs included painful reddish papules, ulcers, and scabby proliferative lesions on the teats, with a clinical course of 7-12 days. Affected cows presented severe local pain, not allowing the completion of milking. Histologically, acanthosis, spongiosis, and parakeratotic hyperkeratosis with adjacent focally extensive ulcers and multifocal inflammatory infiltrate were observed in the epidermis. Eosinophilic inclusion bodies were noted in the cytoplasm of epithelial cells. Personnel milking the affected cows developed lesions on the hands, painful papules that progressed to ulcerative and scabby lesions in 4-7 days. A polymerase chain reaction using a set of pan-parapoxvirus primers for the B2L gene performed on DNA extracted from scabs amplified a 590-bp product, which when sequenced, revealed similarities of 99%, 85%, and 84% with BPSV, Pseudocowpox virus, and Orf virus, respectively. A phylogenetic tree based on the B2L sequence was constructed, showing that the virus clustered with BPSV isolates. Although clinical cases compatible with BSPV infection have been frequently described in Brazil, the present report identifies the agent associated with cattle and human disease in the country.

Nested-multiplex PCR detection of Orthopoxvirus and Parapoxvirus directly from exanthematic clinical samples.

BACKGROUND: Orthopoxvirus (OPV) and Parapoxvirus (PPV) have been associated with worldwide exanthematic outbreaks. Some species of these genera are able to infect humans and domestic animals, causing serious economic losses and public health impact. Rapid, useful and highly specific methods are required to detect and epidemiologically monitor such poxviruses. In the present paper, we describe the development of a nested-multiplex PCR method for the simultaneous detection of OPV and PPV species directly from exanthematic lesions, with no previous viral isolation or DNA extraction. METHODS AND RESULTS: The OPV/PPV nested-multiplex PCR was developed based on the evaluation and combination of published primer sets, and was applied to the detection of the target pathogens. The method showed high sensitivity, and the specificity was confirmed by amplicon sequencing. Exanthematic lesion samples collected during bovine vaccinia or contagious ecthyma outbreaks were submitted to OPV/PPV nested-multiplex PCR and confirmed its applicability. CONCLUSION: These results suggest that the presented multiplex PCR provides a highly robust and sensitive method to detect OPV and PPV directly from clinical samples. The method can be used for viral identification and monitoring, especially in areas where OPV and PPV co-circulate.

Epidemiologia da poxvirose bovina no Estado do Espírito Santo, Brasil / Epidemiology bovine poxviruse in the Espírito Santo State, Brazil

O trabalho consistiu no estudo epidemiológico do surto de poxvirose bovina no Estado do Espírito Santo no período de 2002 a 2005. A coleta dos dados deu-se em 28 propriedades de gado bovino leiteiro de 08 municípios do Sul do Estado do Espírito Santo com casos de doenças pústulo-vesicular em bovinos, no período de agosto de 2002 a maio de 2005. Observou-se que a prevalência da doença no gado bovino leiteiro variou de 10,2% a 100% das propriedades com média de 52,7%. Através de isolamento viral e PCR do gene Timidina Quinase (TK), identificou-se o vírus do gênero Orthopoxvirus como o agente etiológico da poxvirose bovina. Em todas as propriedades constatou-se uma perda econômica, principalmente relacionada à queda na produção leiteira. A poxvirose bovina assumiu um caráter epidêmico no Sul do Estado do Espírito Santo, com grande impacto econômico e importância em saúde pública, como uma zoonose ocupacional entre os ordenhadores. Baseadas nos dados obtidos, medidas de cunho fiscal-sanitário e de orientação ao produtor agropecuário foram implementadas, visando conter a disseminação da doença entres as propriedades, e os outros municípios do Estado.(AU)

The purpose of this study was to investigate the epidemiological data of bovine poxviruses in the State of Espírito Santo during the years 2002 to 2005. The sample consisted of 28 properties that produce cow's milk in 08 cities from the South of the State, in wich were present cases of bonive pustulo-vesicular disease between 2002 august to 2005 may. The bovine disease was found in 10,2% to 100% of the properties with a mean of 52,7%. Throughout virus isolation techniques and PCR of the Timidin Kinase gene, an Orthopoxvirus was identified as the etiological agent of the bovine yiruses. All the properties reported economic losts related to a decrease in milk production. The bovine poxviruses assumed an epidemic feature in the South of the State of Espírito Santo promoting a big economic impact and representing an important public health worry as an occupational zoonosis mainly for the milkers. Thus, based on the obtained data, policy and sanitary statements, and educational strategies, were applied in the properties in order to stop the disease dissemination over the neighbor cities.(AU)

Epidemiologia da poxvirose bovina no Estado do Espírito Santo, Brasil / Epidemiology bovine poxviruses in the Espírito Santo State, Brazil

O trabalho consistiu no estudo epidemiológico do surto de poxvirose bovina no Estado do Espírito Santo no período de 2002 a 2005. A coleta dos dados deu-se em 28 propriedades de gado bovino leiteiro de 08 municípios do Sul do Estado do Espírito Santo com casos de doenças pústulo-vesicular em bovinos, no período de agosto de 2002 a maio de 2005. Observou-se que a prevalência da doença no gado bovino leiteiro variou de 10,2% a 100% das propriedades com média de 52,7%. Através de isolamento viral e PCR do gene Timidina Quinase (TK), identificou-se o vírus do gênero Orthopoxvirus como o agente etiológico da poxvirose bovina. Em todas as propriedades constatou-se uma perda econômica, principalmente relacionada à queda na produção leiteira. A poxvirose bovina assumiu um caráter epidêmico no Sul do Estado do Espírito Santo, com grande impacto econômico e importância em saúde pública, como uma zoonose ocupacional entre os ordenhadores. Baseadas nos dados obtidos, medidas de cunho fiscal-sanitário e de orientação ao produtor agropecuário foram implementadas, visando conter a disseminação da doença entres as propriedades, e os outros municípios do Estado.

The purpose of this study was to investigate the epidemiological data of bovine poxviruses in the State of Espírito Santo during the years 2002 to 2005. The sample consisted of 28 properties that produce cow's milk in 08 cities from the South of the State, in wich were present cases of bonive pustulo-vesicular disease between 2002 august to 2005 may. The bovine disease was found in 10,2% to 100% of the properties with a mean of 52,7%. Throughout virus isolation techniques and PCR of the Timidin Kinase gene, an Orthopoxvirus was identified as the etiological agent of the bovine yiruses. All the properties reported economic losts related to a decrease in milk production. The bovine poxviruses assumed an epidemic feature in the South of the State of Espírito Santo promoting a big economic impact and representing an important public health worry as an occupational zoonosis mainly for the milkers. Thus, based on the obtained data, policy and sanitary statements, and educational strategies, were applied in the properties in order to stop the disease dissemination over the neighbor cities.

Detection of poxvirus in cattle associated with human cases in the State of Rio de Janeiro: preliminary report

This preliminary report describes human and cow cases of poxvirus that recently ocurred in the State of Rio de Janeiro. The electron microscopic findings were consistent with parapoxviral and orthopoxviral infection. Orthopoxvirus strains were isolated from human and cow cases. Detailed viral characterization by means of genetical techniques is under investigation. Based on these informations, poxviral diseases should be also considered an emerging viral zoonosis that can affect human beings.

Detection of poxvirus in cattle associated with human cases in the State of Rio de Janeiro: preliminary report.

This preliminary report describes human and cow cases of poxvirus that recently occurred in the State of Rio de Janeiro. The electron microscopic findings were consistent with parapoxviral and orthopoxviral infection. Orthopoxvirus strains were isolated from human and cow cases. Detailed viral characterization by means of genetical techniques is under investigation. Based on these informations, poxviral diseases should be also considered an emerging viral zoonosis that can affect human beings.