Outbreak of bovine herpetic meningoencephalomyelitis in Southern Brazil

Background: Herpetic meningoencephalitis is an infectious contagious disease worldwide distributed, most often caused by bovine alphaherpesvirus type 5 (BoHV-5), although bovine alphaherpesvirus type 1 (BoHV-1) may occasionally be the causative agent. The disease is characterized by subacute to acute clinical onset, often affecting animals submitted to stressful situations. Clinical signs are mainly neurologic due to meningoencephalitis and cortical necrosis. The involvement of the spinal cord has also been reported, however in BoHV-1 associated disease only. The aim of this report is to describe an outbreak of bovine meningoencephalomyelitis associated to BoHV-5.Case: In August 2017, nine 1-year-old calves died in a beef cattle farm with a flock of approximately 400 bovines. The animals presented neurological clinical signs characterized by excessive salivation, nasal and ocular discharges, incoordination, apathy, head tremors, head pressing, wide-based stance, recumbency followed by convulsions and paddling. According to the owner and referring veterinarian, affected animals displayed severe clinical signs with rapid progression and often leading to death in up to seven days. Four of these calves were submitted for necropsy, and gross lesions were present in the brain, characterized by mild to moderate multifocal hemorrhagic and soft areas. On cut surface, extensive areas of dark brown discoloration and malacia were observed. Histologically, lesions were characterized by extensive areas of liquefactive necrosis in the cerebral cortex grey matter, associated with inflammatory infiltrates composed of neutrophils, lymphocytes, plasma cells and foamy macrophages, as well as multifocal to coalescing areas of hemorrhage and fibrin deposition. Intranuclear eosinophilic inclusion bodies were rarely observed in neurons and astrocytes. On leptomeninges, there was diffuse inflammatory infiltrates of lymphocytes and plasma cells.[...]

Outbreak of bovine herpetic meningoencephalomyelitis in Southern Brazil

Background: Herpetic meningoencephalitis is an infectious contagious disease worldwide distributed, most often caused by bovine alphaherpesvirus type 5 (BoHV-5), although bovine alphaherpesvirus type 1 (BoHV-1) may occasionally be the causative agent. The disease is characterized by subacute to acute clinical onset, often affecting animals submitted to stressful situations. Clinical signs are mainly neurologic due to meningoencephalitis and cortical necrosis. The involvement of the spinal cord has also been reported, however in BoHV-1 associated disease only. The aim of this report is to describe an outbreak of bovine meningoencephalomyelitis associated to BoHV-5.Case: In August 2017, nine 1-year-old calves died in a beef cattle farm with a flock of approximately 400 bovines. The animals presented neurological clinical signs characterized by excessive salivation, nasal and ocular discharges, incoordination, apathy, head tremors, head pressing, wide-based stance, recumbency followed by convulsions and paddling. According to the owner and referring veterinarian, affected animals displayed severe clinical signs with rapid progression and often leading to death in up to seven days. Four of these calves were submitted for necropsy, and gross lesions were present in the brain, characterized by mild to moderate multifocal hemorrhagic and soft areas. On cut surface, extensive areas of dark brown discoloration and malacia were observed. Histologically, lesions were characterized by extensive areas of liquefactive necrosis in the cerebral cortex grey matter, associated with inflammatory infiltrates composed of neutrophils, lymphocytes, plasma cells and foamy macrophages, as well as multifocal to coalescing areas of hemorrhage and fibrin deposition. Intranuclear eosinophilic inclusion bodies were rarely observed in neurons and astrocytes. On leptomeninges, there was diffuse inflammatory infiltrates of lymphocytes and plasma cells.[...](AU)

Osteochondroma in a young cat infected by feline leukemia virus / Ostecondroma em um felino jovem infectado pelo vírus da leucemia felina

Osteochondromas are primary bone tumors characterized by cartilage-covered bone projections involving single or multiple masses (osteochondromatosis). This study reports the clinical and pathological findings from a young domestic cat with osteochondroma in the humerus. During the clinical evaluation, the animal had pronounced right forelimb musculature atrophy and an increased distal humeral volume. Histopathological examination of the neoplasm revealed a proliferative lesion characterized mostly by endochondral ossification and peripheral foci of proliferating cartilage tissue. Further testing using immunohistochemical staining and polymerase chain reaction revealed the presence of feline leukemia virus antigens in the hematopoietic cells of the bone marrow and FeLV proviral DNA in the peripheral blood lymphocytes. Clinical and pathological findings are consistent with osteochondroma. This neoplasm occurred in an eight-month-old feline with humeral enlargement that had been present since two months old.

O osteocondroma é um tumor ósseo primário que se caracteriza por apresentar uma projeção óssea recoberta por cartilagem que pode ser formado por uma única ou por múltiplas massas (osteocondromatose). O objetivo deste trabalho é descrever os achados clínicos e patológicos de um felino jovem com osteocondroma no úmero. Na avaliação clínica, o felino apresentava atrofia acentuada da musculatura do membro torácico direito além de um aumento de volume em região distal do úmero. No exame histopatológico, observou-se uma lesão proliferativa composta em sua maior parte por ossificação endocondral com focos periféricos de tecido cartilaginoso proliferado. Testes de imuno-histoquímica e reação em cadeia da polimerase revelaram a presença de antígenos de FeLV em células da linhagem hematopoiética da medula óssea e DNA proviral em linfócitos do sangue periférico, respectivamente. Os achados clínicos e patológicos foram consistentes com um osteocondroma em um felino jovem, com oito meses de idade, com lesão em úmero desde os dois meses de idade.

Osteochondroma in a young cat infected by feline leukemia virus / Ostecondroma em um felino jovem infectado pelo vírus da leucemia felina

Osteochondromas are primary bone tumors characterized by cartilage-covered bone projections involving single or multiple masses (osteochondromatosis). This study reports the clinical and pathological findings from a young domestic cat with osteochondroma in the humerus. During the clinical evaluation, the animal had pronounced right forelimb musculature atrophy and an increased distal humeral volume. Histopathological examination of the neoplasm revealed a proliferative lesion characterized mostly by endochondral ossification and peripheral foci of proliferating cartilage tissue. Further testing using immunohistochemical staining and polymerase chain reaction revealed the presence of feline leukemia virus antigens in the hematopoietic cells of the bone marrow and FeLV proviral DNA in the peripheral blood lymphocytes. Clinical and pathological findings are consistent with osteochondroma. This neoplasm occurred in an eight-month-old feline with humeral enlargement that had been present since two months old.(AU)

O osteocondroma é um tumor ósseo primário que se caracteriza por apresentar uma projeção óssea recoberta por cartilagem que pode ser formado por uma única ou por múltiplas massas (osteocondromatose). O objetivo deste trabalho é descrever os achados clínicos e patológicos de um felino jovem com osteocondroma no úmero. Na avaliação clínica, o felino apresentava atrofia acentuada da musculatura do membro torácico direito além de um aumento de volume em região distal do úmero. No exame histopatológico, observou-se uma lesão proliferativa composta em sua maior parte por ossificação endocondral com focos periféricos de tecido cartilaginoso proliferado. Testes de imuno-histoquímica e reação em cadeia da polimerase revelaram a presença de antígenos de FeLV em células da linhagem hematopoiética da medula óssea e DNA proviral em linfócitos do sangue periférico, respectivamente. Os achados clínicos e patológicos foram consistentes com um osteocondroma em um felino jovem, com oito meses de idade, com lesão em úmero desde os dois meses de idade.(AU)

Bovine Herpesvirus 4 in Parana State, Brazil: case report, viral isolation, and molecular identification

Bovine Herpesvirus 4 (BoHV-4) is a member of Gammaherpesvirinae sub-family and belongs to genus Rhadinovirus. This virus has been associated with different clinical manifestations and research activity has put forward a strong correlation among virus infection, postpartum metritis, and abortion. The goal of this work was to characterize a virus strain isolate from a cows uterine outflow. From swabs drawn of uterine secretion, a virus strain was isolated and characterized by its cytopathology, morphology, and molecular biology approaches. In culture there was CPE development, characterized mainly by long strands with several small balloons along them, radiated from infected cells. Electron microscopy analysis revealed virus particles that had icosahedrical capsid symmetry surrounded by a loose envelope, typical of a herpesvirus. A 2,571 bp PCR product after HindIII digestion generated four fragments, whose base pair composition were 403, 420, 535, and 1,125 bp. Restriction enzymes HindIII and BamHI generated the expected diagnostic bands as well as a 2,350 bp hypermolar fragment as a result of BamHI treatment to demonstrate that agent was a bovine herpesvirus 4, appertaining to DN-599 group.(AU)

Detection of human adenovirus, rotavirus and enterovirus in water samples collected on dairy farms from Tenente Portela, Northwest of Rio Grande do Sul, Brazil

Viral gastroenteritis and other waterborne diseases are a major concern for health in Brazil. A number of studies were conducted about the presence of viruses on water samples from Brazilian areas. However, the knowledge about the occurrence of viral contamination of drinking water sources in rural settings of the country is insufficient. On the present work, 15 samples from 5 dairy farms located at the municipality of Tenente Portela were collected and analysed for the presence of human adenoviruses (HAdV), as well as human enteroviruses (EV) and rotaviruses (RV). HAdV was present on 66.66% of the water samples, and have been found in all samples from artesian wells and springs, which are used as sources of drinking water for the individuals inhabiting those farms. EV and RV found only in one sample each. The detection rates of HAdV on the water from these dairy farms are alarming and point towards a situation of elevated environmental contamination by fecal microorganisms of human origin and poor basic sanitation conditions.(AU)

Bovine herpesviruses do not play a major role in the differential diagnosis of rabies in cattle in southern Brazil

Background: Rabies has long been recognized as the major cause of encephalitis in cattle in Latin American countries. It has been estimated that nearly 50.000 cattle heads per year are lost due to encephalitis in that subcontinent, with a significant economic impact on cattle productive chains. In Brazil only, 2.500 to 3.000 cattle heads are estimated to be lost every year due to rabies. However, it is believed that rabies incidence in cattle is much larger, since usually only a few samples from affected animals in disease outbreaks are submitted to diagnostic laboratories. Rabies encephalitis is promptly and accurately diagnosed; however, particularly when rabies is excluded as causa mortis, the agent responsible for neurological disease of infectious origin often remains undetermined. Two bovine herpesviruses (BoHVs), bovine herpesvirus type 1 (BoHV-1) and bovine herpesvirus type 5 (BoHV-5) are major pathogens of cattle which are widely disseminated in Brazil. As usual in herpesvirus' biology, these tend to infect a large number of hosts and establish lifelong latent infections which may occasionally be reactivated. Both viruses, particularly BoHV-5, are often recovered from cases of neurological disease in cattle. The participation of BoHVs in the differential diagnosis of rabies must be evaluated. Besides, there might be associations between the occurrence of rabies and BoHV infections that deserve investigation. The aim of this study was to investigate whether bovine herpesvirus 1 and 5 would play a significant role in cases of neurological disease where rabies was the presumptive clinical diagnosis. In addition, associations between the occurrence of rabies and BoHV infections were searched for. The approach adopted for conducting such investigations was based on the search for viral nucleic acids as well as classical virus isolation on tissues of cattle submitted to rabies diagnosis over a two-year period, including rabies-positive and rabies-negative specimens. Materials, Methods & Results: Brain tissue samples of 101 cattle originally submitted to rabies diagnosis were collected over a two year period (2009-2010) from various municipalities within the state of Rio Grande do Sul, Brazil. Thirty nine of these samples had the diagnosis of rabies confirmed by standard laboratory diagnostic methods. Aliquots of tissues were submitted to DNA extraction and examined in search for genomes of bovine herpesviruses (BoHV) types 1 (BoHV-1) and 5 (BoHV-5) by as well as for infectious virus. Bovine herpesvirus genomes were detected in 78/101 (77.2%) samples, in which BoHV-1 genomes were detected in 26/78 (25.7%), BoHV-5 genomes in 22/78 (21.8%) and mixed BoHV infections (BoHV-1 and BoHV-5 genomes) were detected in 30/101 (29.7%) samples. In the 39 samples with confirmed rabies diagnosis, BoHV-1 DNA was detected in 9/39 (23%), BoHV-5 DNA in 6/39 (15.4%) and mixed infections with both BoHV types in 16/39 (41%) samples. However, no infectious herpesvirus was recovered from any of the specimens examined. Discussion: The high prevalence of BoHV1 and BoHV-5 infections was evidenced in the sampled population, but the absence of infectious BoHVs indicate that these were not associated to the occurrence of the cases of encephalitis where rabies was the primary suspicion. In addition, no association was detected between occurrence of rabies and detection of BoHVs, since the frequency of detection of herpesvirus genomes did not significantly differ between rabies-positive and rabies-negative samples. The detection of BoHV DNA in scattered areas of the brain with no infectious virus suggests that latency may take place in different regions of the brain.

Cortical necrosis and cerebral atrophy in calves experimentally infected with bovine herpesvirus type 5

Bovine encephalitis herpesvirus, or bovine herpesvirus type 5 (BoHV-5), a member of the family Herpesviridae, subfamily Alphaherpesvirinae, is long recognized as the causative agent of bovine herpesvirus encephalitis. The disease caused by BoHV-5 is characterized by signs of nervous impairment, consequent to non-suppurative meningoencephalitis. Although bovine herpetic encephalitis is a rare event in herds from the Northern Hemisphere, BoHV-5 infections are an important cause of central nervous system disease in cattle in Brazil and Argentina. Recovery of animals from clinical illness has been documented before, both in naturally infected animals and experimentally infected individuals.[...]

Cortical necrosis and cerebral atrophy in calves experimentally infected with bovine herpesvirus type 5

Bovine encephalitis herpesvirus, or bovine herpesvirus type 5 (BoHV-5), a member of the family Herpesviridae, subfamily Alphaherpesvirinae, is long recognized as the causative agent of bovine herpesvirus encephalitis. The disease caused by BoHV-5 is characterized by signs of nervous impairment, consequent to non-suppurative meningoencephalitis. Although bovine herpetic encephalitis is a rare event in herds from the Northern Hemisphere, BoHV-5 infections are an important cause of central nervous system disease in cattle in Brazil and Argentina. Recovery of animals from clinical illness has been documented before, both in naturally infected animals and experimentally infected individuals.[...](AU)

Blood or serum collected on filter paper for detection of antibodies to bovine herpesvirus type 1 (BoHV-1)

The method of collection as well as the packaging conditions in which samples are submitted to laboratories play a critical role on the acquisition of reliable results on diagnostic tests. Alternative methods however have been proposed, as the adsorption of blood or serum in filter paper. In this work, it was evaluated the viability of using serum or whole blood samples from bovines collected in filter paper for serological testing against bovine herpesvirus type 1 (BoHV-1).[...]

Blood or serum collected on filter paper for detection of antibodies to bovine herpesvirus type 1 (BoHV-1)

The method of collection as well as the packaging conditions in which samples are submitted to laboratories play a critical role on the acquisition of reliable results on diagnostic tests. Alternative methods however have been proposed, as the adsorption of blood or serum in filter paper. In this work, it was evaluated the viability of using serum or whole blood samples from bovines collected in filter paper for serological testing against bovine herpesvirus type 1 (BoHV-1).[...](AU)

Transfecção de DNA de BoHV-5 com fosfato de cálcio em diferentes tipos de cultivos celulares / Transfection of BoHV-5 DNA with calcium phosphate in different types of cell culture

As condições para uma transfecção eficaz pelo método de fosfato de cálcio podem variar substancialmente. Neste estudo foi testado o tipo de cultivo celular mais apropriado para transfecção de DNA de herpesvírus bovino tipo 5 (BoHV-5) por esta técnica. Primeiramente foi avaliada a capacidade de multiplicação do BoHV-5 em três diferentes tipos de células. Para isto, a concentração viral foi calculada pelo método de dose infectante para cultivo celular / 50% (TCID50/mL). O BoHV-5 foi titulado em células de linhagem de rim de macaco verde Africano (VERO), células de linhagem de rim de suíno (PKsC3), células primárias de testículos de terneiro (TT) e células de linhagem de rim de bovino (MDBK). Em células VERO e PKsC3 a concentração viral máxima obtida foi 103,3 TCID50/mL, enquanto que em células MDBK o título foi de 106,8 TCID50/mL, similar ao encontrado para as células TT. Para avaliar a eficácia da transfecção nessas células, foi utilizado DNA plasmideal expressando um gene repórter (EGFP). Os resultados mostraram um valor de 4,5% de células fluorescentes para células TT, de 2,9% para PKsC3, de 0,5% para VERO e de 0,05% para MDBK. Quando células PKsC3 e TT foram transfectadas com 0,5 μg de DNA purificado de BoHV-5, somente as células TT apresentaram resultado positivo, com duas placas virais por poço. Para investigar a influência da concentração de DNA viral, células TT foram transfectadas com 0,5 μg e 1 μg de DNA purificado de BoHV-5, produzindo duas e quatro placas, respectivamente. Nas condições experimentais testadas neste estudo os resultados indicam que as células TT poderiam ser boas candidatas para serem utilizadas para a transfecção de DNA de BoHV-5.

The conditions for an efficacious transfection by calcium phosphate method can vary substantially. In this study the most appropriate cell type for transfection of bovine herpesvirus type 5 (BoHV-5) DNA using this technique was tested. It was first evaluated the capacity of BoHV-5 multiplication in three different cell types. For this purpose virus concentration was calculated by tissue culture infectious dose 50% assay (TCID50/mL). BoHV-5 was titrated in kidney cells from African green monkey (VERO), cell clone of pig kidney (PKsC3), primary cell culture of calf testis (TT) and bovine kidney cells (MDBK). In Vero and PK15 cells maximum virus concentration reached 103,3 TCID50/mL, whereas in MDBK cells the titre was 106,8 TCID50/mL, similar to that of TT cells. In order to evaluate the transfection efficacy in these cells, plasmid DNA expressing a reporter gene (EGFP) was used. Results showed 4.5% fluorescent cells for TT, 2.9% for PKsC3, 0.5% for VERO and 0.05% for MDBK cells. When PKsC3 and TT cells were transfected with 0.5 mg of BoHV-5 purified DNA, only TT cells gave positive result with two plaques per well. To investigate the influence of viral DNA concentration, TT cells were then transfected with 0.5 mg and 1.0 mg of purified BoHV-5 DNA producing two and four plaques, respectively. Under experimental conditions tested in this study the results indicate that TT cells could be good candidates to be used for transfection of BoHV-5 DNA.

Transfecção de DNA de BoHV-5 com fosfato de cálcio em diferentes tipos de cultivos celulares / Transfection of BoHV-5 DNA with calcium phosphate in different types of cell culture

As condições para uma transfecção eficaz pelo método de fosfato de cálcio podem variar substancialmente. Neste estudo foi testado o tipo de cultivo celular mais apropriado para transfecção de DNA de herpesvírus bovino tipo 5 (BoHV-5) por esta técnica. Primeiramente foi avaliada a capacidade de multiplicação do BoHV-5 em três diferentes tipos de células. Para isto, a concentração viral foi calculada pelo método de dose infectante para cultivo celular / 50% (TCID50/mL). O BoHV-5 foi titulado em células de linhagem de rim de macaco verde Africano (VERO), células de linhagem de rim de suíno (PKsC3), células primárias de testículos de terneiro (TT) e células de linhagem de rim de bovino (MDBK). Em células VERO e PKsC3 a concentração viral máxima obtida foi 103,3 TCID50/mL, enquanto que em células MDBK o título foi de 106,8 TCID50/mL, similar ao encontrado para as células TT. Para avaliar a eficácia da transfecção nessas células, foi utilizado DNA plasmideal expressando um gene repórter (EGFP). Os resultados mostraram um valor de 4,5% de células fluorescentes para células TT, de 2,9% para PKsC3, de 0,5% para VERO e de 0,05% para MDBK. Quando células PKsC3 e TT foram transfectadas com 0,5 μg de DNA purificado de BoHV-5, somente as células TT apresentaram resultado positivo, com duas placas virais por poço. Para investigar a influência da concentração de DNA viral, células TT foram transfectadas com 0,5 μg e 1 μg de DNA purificado de BoHV-5, produzindo duas e quatro placas, respectivamente. Nas condições experimentais testadas neste estudo os resultados indicam que as células TT poderiam ser boas candidatas para serem utilizadas para a transfecção de DNA de BoHV-5.(AU)

The conditions for an efficacious transfection by calcium phosphate method can vary substantially. In this study the most appropriate cell type for transfection of bovine herpesvirus type 5 (BoHV-5) DNA using this technique was tested. It was first evaluated the capacity of BoHV-5 multiplication in three different cell types. For this purpose virus concentration was calculated by tissue culture infectious dose 50% assay (TCID50/mL). BoHV-5 was titrated in kidney cells from African green monkey (VERO), cell clone of pig kidney (PKsC3), primary cell culture of calf testis (TT) and bovine kidney cells (MDBK). In Vero and PK15 cells maximum virus concentration reached 103,3 TCID50/mL, whereas in MDBK cells the titre was 106,8 TCID50/mL, similar to that of TT cells. In order to evaluate the transfection efficacy in these cells, plasmid DNA expressing a reporter gene (EGFP) was used. Results showed 4.5% fluorescent cells for TT, 2.9% for PKsC3, 0.5% for VERO and 0.05% for MDBK cells. When PKsC3 and TT cells were transfected with 0.5 mg of BoHV-5 purified DNA, only TT cells gave positive result with two plaques per well. To investigate the influence of viral DNA concentration, TT cells were then transfected with 0.5 mg and 1.0 mg of purified BoHV-5 DNA producing two and four plaques, respectively. Under experimental conditions tested in this study the results indicate that TT cells could be good candidates to be used for transfection of BoHV-5 DNA.(AU)

Serum neutralization with different types and subtypes of bovine herpesvirus 1 and 5 / Soroneutralização com diferentes tipos e subtipos de herpesvírus bovinos 1 e 5

The serum neutralization (SN) test is the gold standard method to measure neutralizing antibodies to bovine herpesviruses. However, in view of the further subdivisions of bovine herpesviruses in types/subtypes, defining which virus to use at challenge in SN tests may be difficult. In view of that, this study was carried out to re-evaluate (SN) sensitivity with different types/subtypes of bovine herpesviruses types 1 (BoHV-1) and 5 (BoHV-5) as challenge viruses. Bovine sera (n=810) were collected from two distinct geographic regions and tested by SN with three type 1 viruses (BoHV-1.1 strains "Los Angeles" and "EVI123/98"; BoHV-1.2a strain "SV265/96") and three type 5 viruses (BoHV-5a strain "EVI88/95"; BoHV-5b strain "A663" and BoHV-5c "ISO97/95"). SN tests were performed with a 1 hour incubation of the serum-virus mixtures at 37ºC against 100 TCID50 of each of the viruses. SN sensitivity varied greatly depending on the challenge virus used in the test. The highest sensitivity (327 positive/810 total sera tested; 40.37 percent) was attained when the positive results to the six viruses were added together. No association could be found between any particular type or subtype of virus and the sensitivity of the test. When positive results to each single strain were considered, SN sensitivity varied from 41.7 percent to 81.7 percent, depending on the virus and the geographic region of origin of the sera. Variation was detected even when challenge viruses belonged to the same subtype, where disagreement between positive results reached 41 percent. These results indicate that one hour incubation SN tests against single viruses, as performed here, may display a significantly low sensitivity (p=0.05); performing SN tests against a number of different viruses may increase considerably SN sensitivity. Furthermore, the choice of virus used for challenge is critical in SN tests. In addition, sera from different geographic regions may give rise to disagreeing results with different strains of BoHV-1 and BoHV-5. This might be particularly relevant for control programs and in international trade, were maximum sensitivity should be targeted.(AU)

O teste de soroneutralização (SN) é o método padrão para a mensuração de anticorpos neutralizantes para herpesvírus bovinos. Entretanto, com as subdivisões propostas destes agentes em tipos e subtipos, a definição de qual amostra utilizar como virus de desafio à SN pode ser difícil. Em vista disso, este estudo foi realizado para re-avaliar a sensibilidade de testes de SN utilizando diferentes tipos e subtipos de herpesvírus bovinos tipos 1 (BoHV-1) e 5 (BoHV-5) como amostras de desafio. Soros bovinos (n=810) foram coletados de duas regiões geográficas distintas e testados frente a amostras do tipo 1 (BoHV-1.1: amostras "Los Angeles" e "EVI123/98", BoHV-1.2a: amostra "SV265/96") e três amostras do tipo 5 (BoHV-5a: "EVI88/95"; BoHV-5b: "A663" e BoHV-5c "ISO97/95"). Os testes de SN foram realizados com incubação de 1 hora a 37ºC da mistura soro-vírus, frente a 100 doses infectantes para 50 por cento dos cultivos celulares (DICC50) de cada um dos vírus. A sensibilidade da SN variou grandemente em função do vírus utilizado no teste. A maior sensibilidade (327 soros positivos/810 soros testados; 40.37 por cento) foi alcançada quando os resultados positivos frente aos seis diferentes vírus foram somados. Nenhuma associação foi detectada entre determinado tipo/subtipo de vírus e a sensibilidade do teste. Quando resultados positivos frente a cada vírus foram considerados isoladamente, a sensibilidade da SN variou entre 41,7 por cento a 81,7 por cento, dependendo do vírus de desafio e da região geográfica de origem das amostras de soro. Variação foi detectada mesmo quando as amostras de desafio pertenciam a um mesmo subtipo; a discrepância entre os resultados positivos atingiu até 41 por cento. Estes resultados indicam que testes de SN contra amostras isoladas de vírus podem apresentar uma sensibilidade notadamente baixa; o emprego de diferentes amostras de vírus de desafio pode aumentar consideravelmente a sensibilidade da prova. Além disso, a escolha da amostra de vírus para a realização do teste é crítica. Outro achado importante é que sorors de diferentes regiões geográficas podem dar resultados discordantes frente a diferentes amostras de BoHV-1 e BoHV-5. Estes achados são particularmente relevantes para programas de controle destas infecções e para o comércio internacional, onde a sensibilidade deve ser maximizada.(AU)

Experimental infection of rabbits with a recombinant bovine herpesvirus type 5 (BoHV-5) gI, gE and US9-negative / Infecção experimental de coelhos com um recombinante do herpesvírus bovino tipo 5 defectivo na gI, gE e US9

Bovine herpesvirus type 5 (BoHV-5) is a major cause of viral meningoencephalitis in cattle. The expression of different viral proteins has been associated with BoHV-5 neuropathogenesis. Among these, gI, gE and US9 have been considered essential for the production of neurological disease in infected animals. To evaluate the role of gI, gE and US9 in neurovirulence, a recombinant from which the respective genes were deleted (BoHV-5 gI-/gE-/US9-) was constructed and inoculated in rabbits of two age groups (four and eight weeks-old). When the recombinant virus was inoculated through the paranasal sinuses of four weeks-old rabbits, neurological disease was observed and death was the outcome in 4 out of 13 (30.7 percent) animals, whereas clinical signs and death were observed in 11/13 (84.6 percent) of rabbits infected with the parental virus. In eight weeks-old rabbits, the BoHV-5 gI-/gE-/US9- did not induce clinically apparent disease and could not be reactivated after dexamethasone administration, whereas wild type BoHV-5 caused disease in 55.5 percent of the animals and was reactivated. These findings reveal that the simultaneous deletion of gI, gE and US9 genes did reduce but did not completely abolish the neurovirulence of BoHV-5 in rabbits, indicating that other viral genes may also play a role in the induction of neurological disease.(AU)

O herpesvírus bovino tipo 5 é uma das principais causas de meningoencefalite viral em bovinos. A expressão de diferentes proteínas virais tem sido associada à neuropatogenia do BoHV-5. Entre estas, a gI, gE e US9 têm sido consideradas essenciais para a indução de sinais neurológicos nos animais infectados. Para avaliar o papel das proteínas gI, gE e US9 na neurovirulência, construiu-se um recombinante no qual os genes que codificam estas proteínas foram deletados, denominado BoHV-5 gI-/gE-/US9-. Este vírus foi inoculado em coelhos de idades diferentes (quatro e oito semanas de idade). Quando o vírus recombinante foi inoculado nos seios paranasais de coelhos de quatro semanas de idade, doença neurológica e morte foram observadas em 4 dos 13 (30,7 por cento) animais, enquanto que sinais clínicos e morte foram observados em 11/13 (84,6 por cento) dos coelhos infectados com o vírus parental. Em coelhos de oito semanas de idade, o BoHV-5 gI-/gE-/US9- não induziu sinais clínicos aparentes e, após tentativa de reativação viral por tratamento com dexametasona, o vírus não foi re-excretado. Por outro lado, o vírus selvagem causou doença clínica em 55,5 por cento dos coelhos e foi re-excretado após tratamento com dexametasona. Estes achados revelam que a deleção simultânea dos genes gI, gE e US9 reduziu mas não aboliu completamente a neurovirulência do BoHV-5 em coelhos, indicando que outros genes virais possam ter papel na indução da doença neurológica.(AU)

Neuropatogênese experimental da infecção pelo herpesvírus bovino tipo 5 em coelhos: [revisão] / Experimental neuropathogenesis of bovine herpesvirus 5 infection in rabbits: [revision]

Vários aspectos da biologia do herpesvírus bovino tipo 5 (BoHV-5) têm sido estudados em coelhos, que desenvolvem infecção aguda e doença neurológica após inoculação experimental. A infecção aguda é seguida pelo estabelecimento de infecção latente, que pode ser reativada natural ou artificialmente. Os primeiros experimentos nesta espécie estabeleceram um protocolo de inoculação e monitoramento da infecção, e caracterizaram os principais aspectos virológicos, clínicos e patológicos da infecção aguda. A patogenia da infecção aguda, desde a replicação viral nos sítios de inoculação, vias e cinética de transporte viral até o encéfalo, distribuição e replicação viral no sistema nervoso central (SNC), tropismo celular e tecidual, manifestações clínicas e patologia no SNC foram detalhadamente estudados nestes animais. Posteriormente, vários aspectos biológicos e moleculares da infecção latente também foram elucidados a partir de inoculações de coelhos. Os coelhos também têm sido utilizados para estudar o fenótipo (neuroinvasividade, neurovirulência) de isolados de campo e de cepas vacinais recombinantes, proteção por imunidade passiva, proteção vacinal, eficácia de drogas anti-virais e terapêuticas de suporte da infecção neurológica. Este modelo experimental também foi utilizado para o estudo da origem e distribuição dos estímulos elétricos produzidos durante as convulsões - uma característica da infecção neurológica pelo BoHV-5 -, e para testes de medicamentos anti-convulsivantes. Ressalvadas as diferenças que certamente existem entre bovinos - os hospedeiros naturais - e coelhos, as observações oriundas deste modelo experimental tem contribuído sobremaneira para o conhecimento da biologia do BoHV-5. O presente trabalho apresenta uma coletânea de resultados e observações, publicadas ou não pelo grupo, ao longo de mais de uma década, envolvendo inoculações de coelhos para estudar diversos aspectos da infecção pelo BoHV-5.(AU)

Several aspects of the biology of bovine herpesvirus 5 (BoHV-5) have been studied in rabbits, which develop acute infection and neurological disease upon experimental inoculation. The acute infection is followed by the establishment of latent infection, which can be naturally or artificially reactivated. The first experiments in rabbits established a protocol for virus inoculation and monitoring the infection, and characterized the main virological, clinical and pathological aspects of the acute infection. The pathogenesis of acute infection, from the initial viral replication at site of inoculation, pathways and kinetics of viral transport to the brain, distribution and virus replication in the central nervous system (CNS), cellular and tissue tropism, clinical signs and CNS pathology have been extensively studied using this animal model. Subsequently, several biological and molecular aspects of latent BoHV-5 infection have also been elucidated upon inoculation of rabbits. Rabbits have also been used to investigate the phenotype (neuroinvasiveness, neurogrowth) of field isolates and recombinant vaccine candidates, protection by passive immunity, vaccine protection, the efficacy of anti-viral drugs and support therapies for neurological disease. This animal model was also used to investigate the origin and distribution of electric impulses involved in seizures - a hallmark of BoHV-5 induced neurological infection - and also to test the efficacy of anti-convulsivants. In spite of the possible differences between rabbits and cattle - the natural host of the virus - the observations taken from this experimental model have greatly contributed to the knowledge of the biology of BoHV-5 infection. The present article presents a review of the main published and unpublished results and observations by our group, comprising more than a decade of studies on the pathogenesis of BoHV-5 infection in the rabbit model.(AU)

Field evaluation of safety during gestation and horizontal spread of a recombinant differential bovine Herpesvirus 1 (BoHV-1) vaccine / Avaliação a campo da segurança para vacas prenhes e capacidade de disseminação horizontal de uma vacina diferencial recombinante contra o Herpes-vírus Bovino tipo 1 (BoHV-1)

Infecções pelo herpesvírus bovino tipo 1 (BoHV-1) são importantes causas de doença respiratória, reprodutiva e abortos em bovinos. A vacinação é freqüentemente empregada para minimizar as perdas produzidas pela infecção. Todavia, a imunização de vacas durante a prenhez com algumas vacinas contendo vírus vivo modificado (MLV) pode ocasionalmente causar abortos. Em trabalho prévio, nosso grupo desenvolveu uma vacina recombinante de BoHV-1 construída a partir de um isolado brasileiro de BoHV-1 (Franco et al., 2002a) do qual o gene que codifica para a glicoproteína E (gE) foi artificialmente deletado. Tal recombinante (gE-) vem sendo avaliado como vacina diferencial, isto é, capaz de permitir a diferenciação entre animais vacinados e infectados. No presente estudo, o potencial de disseminação do vírus recombinante foi avaliado em um rebanho de gado de corte, em condições de campo. Para tanto, a segurança da vacina gE- quando aplicada durante a prenhez foi avaliada pela inoculação intramuscular de 107,4 doses infectantes para 50% dos cultivos celulares (DICC50) do vírus em 22 fêmeas prenhes (14 previamente soronegativas e 8 previamente soropositivas para BoHV-1) em diferentes fases da gestação. Outras 15 vacas prenhes foram mantidas como controles não-vacinados. Não ocorreram abortos, natimortos ou anormalidades fetais em nenhum dos grupos. Soroconversão foi observada nas fêmeas vacinadas previamente soronegativas. Em um segundo experimento, 4 novilhas foram inoculadas pela via intranasal com 107,6 DICC50 do vírus recombinante, sendo mantidos em contato com 16 novilhas em uma área de campo, a uma densidade de 1 animal por hectare. Os animais foram monitorados quanto à presença de sinais clínicos; amostras de soro foram coletadas nos dias 0, 30, 60 e 180 após a vacinação. Soroconversão foi observada apenas nos animais vacinados e não nos contatos...(AU)

Bovine herpesvirus type 1 (BoHV-1) is recognized as a major cause of respiratory, reproductive disease and abortion in cattle. Vaccination is widely applied to minimize losses induced by BoHV-1 infections; however, vaccination of dams during pregnancy with modified live virus (MLV) vaccines has been occasionally associated to abortions. We have previously reported the development of a BoHV-1 recombinant virus, constructed with basis on a Brazilian BoHV-1 (Franco et al. 2002a) from which the gene coding for glycoprotein E (gE) was deleted (gE-) by genetic manipulation. Such recombinant has been previously evaluated in its potential as a differential vaccine (gE- vaccine) that allows differentiation between vaccinated and infected animals. Here, in the first part of the present study, the safety of the gE- vaccine during pregnancy was evaluated by the intramuscular inoculation of 107.4 tissue culture 50 % infective doses (TCID50) of the virus into 22 pregnant dams (14 BoHV-1 seronegative; 8 seropositive), at different stages of gestation. Other 15 pregnant dams were kept as non-vaccinated controls. No abortions, stillbirths or fetal abnormalities were seen after vaccination. Seroconversion was observed in both groups of previously seronegative vaccinated animals. In the second part of the study, the potential of the gE- vaccine virus to spread among beef cattle under field conditions was examined. Four heifers were inoculated intranasally with a larger amount (107,6 TCID50) of the gE- vaccine (to increase chances of transmission) and mixed with other sixteen animals at the same age and body condition, in the same grazing area, at a population density equal to the average cattle farming density within the region (one cattle head per 10,000 m2), for 180 days. All animals were monitored daily for clinical signs. Serum samples were collected on days 0, 30, 60 and 180 post-vaccination. Seroconversion was observed only in vaccinated heifers... (AU)

Comparative pathogenicity of bovine herpesvirus 1 (BHV-1) subtypes 1 (BHV-1.1) and 2a (BHV-1.2a) / Avaliação comparativa da patogenicidade de herpesvírus bovinos tipo 1 (BHV-1) subtipos 1 (BHV-1.1) e 2a (BHV-1.2a)

O presente estudo teve como objetivo examinar a capacidade de duas amostras de herpesvírus bovino tipo 1 (BHV-1) de diferentes subtipos (amostra EVI 123/96: BHV-1.1; amostra SV265/98: BHV-1.2a) de induzir doença respiratória em bovinos. Estas duas amostras são representativas de subtipos de BHV-1 prevalentes no Brasil. Os subtipos das amostras foram confirmados por análises com anticorpos monoclonais e com enzimas de restrição. As amostras foram inoculadas por via intranasal em sete bezerros de três meses de idade (quatro com BHV-1.1, três com BHV-1.2a), soronegativos para BHV-1, sendo outros três animais mantidos como controles não infectados. Nos dois grupos de animais inoculados, os sinais clínicos observados foram consistentes com o quadro de rinotraqueíte infecciosa bovina (IBR), incluindo febre, apatia, anorexia, descargas mucopurulentas nasais e oculares, conjuntivite, erosões e hiperemia na mucosa nasal, dispnéia, tosse, estridor traqueal e aumento dos linfonodos retrofaríngeos, submandibulares e cervicais. Não foram observadas diferenças significativas entre os escores clínicos atribuídos aos animais nos dois grupos. Igualmente, foram similares as quantidades de vírus re-isoladas dos animais infectados, à exceção de uma diferença significativa na disseminação de vírus pelas secreções nasais, a qual foi maior nos animais infectados com BHV-1.1 nos dias 1 a 3 pós-inoculação. Após reativação induzida por corticosteróides, foi observado recrudescimento dos sinais clínicos, os quais foram também similares em ambos os grupos. Em conclusão, as amostras de BHV-1 dos subtipos 1 e 2a não apresentaram diferenças significativas em sua patogenicidade sobre o trato respiratório nos animais inoculados, tanto após a infecção primária como após a reativação. (AU)

The study aimed to examine the capacity of two bovine herpesvirus type 1 (BHV-1) isolates of different subtypes (EVI 123/96, BHV-1.1; SV265/98, BHV-1.2a) to induce respiratory disease in calves. These two isolates are representative of the BHV-1 subtypes prevalent in Brazil. Viral subtypes were confirmed by monoclonal antibody analysis and by restriction enzyme digestion of viral genomes. The viruses were inoculated intranasally into seven 3 months old calves (four with BHV-1.1, three with BHV-1.2a). Three other calves of identical age and condition were kept as uninfected controls. In both groups of infected calves, the clinical signs observed were consistent with typical infectious bovine rhinothracheitis (IBR), including pyrexia, apathy, anorexia, nasal and ocular mucopurulent discharges, erosions on the nasal mucosa, conjunctivitis, lachrymation, redness of nasal mucosa, dyspnoea, coughing, tracheal stridor and enlargement of retropharingeal, submandibular and cervical lymphnodes. No significant differences were observed between the clinical scores attributed to both groups. Virus shedding in nasal and ocular secretions were also similar, apart from a significant difference in nasal virus shedding on day 1 to 3 post-inoculation, which was higher for BHV-1.1 than for BHV-1.2a. Following corticosteroid induced reactivation of the latent infection, recrudescence of clinical signs was also observed, with no significant differences on both groups. It was concluded that both subtypes BHV-1.1 and BHV-1.2a were able to induce clinically undistinguishable respiratory disease in calves, either subsequent to a primary infection or following reactivation.

A Brazilian glycoprotein E-negative bovine herpesvirus type 1.2a (BHV-1.2a) mutant is attenuated for cattle and induces protection against wild-type virus challenge

The authors previously reported the construction of a glycoprotein E-deleted (gE-) mutant of bovine herpesvirus type 1.2a (BHV-1.2a). This mutant, 265gE-, was designed as a vaccinal strain for differential vaccines, allowing the distinction between vaccinated and naturally infected cattle. In order to determine the safety and efficacy of this candidate vaccine virus, a group of calves was inoculated with 265gE-. The virus was detected in secretions of inoculated calves to lower titres and for a shorter period than the parental virus inoculated in control calves. Twenty one days after inoculation, the calves were challenged with the wild type parental virus. Only mild signs of infection were detected on vaccinated calves, whereas non-vaccinated controls displayed intense rhinotracheitis and shed virus for longer and to higher titres than vaccinated calves. Six months after vaccination, both vaccinated and control groups were subjected to reactivation of potentially latent virus. The mutant 265gE- could not be reactivated from vaccinated calves. The clinical signs observed, following the reactivation of the parental virus, were again much milder on vaccinated than on non-vaccinated calves. Moreover, parental virus shedding was considerably reduced on vaccinated calves at reactivation. In view of its attenuation, immunogenicity and protective effect upon challenge and reactivation with a virulent BHV-1, the mutant 265gE- was shown to be suitable for use as a BHV-1 differential vaccine virus (AU)

Development and standardization of an indirect ELISA for the serological dianosis of classical swine fever

Um ensaio imunoenzimático do tipo ELISA indireto (ELISA-I) foi desenvolvido e padronizado para o diagnóstico sorológico de peste suína clássica. Na comparação foram utilizadas novecentas e trinta e sete amostras de soros suínos, as quais foram testadas pelo teste de soroneutralização seguido de revelação por imunoperoxidase (NPLA), tomado como padrão, resultando em 223 amostras positivas e 714 negativas. Em relação ao NPLA, o ELISA-I apresentou sensibilidade de 98,21 por cento, especificidade de 92,86 por cento, valor preditivo positivo de 81,11 por cento, valor preditivo negativo de 99,4 por cento e precisão de 94,1 por cento. A análise estatística dos resultados revelou uma correlação muito forte (r=0,94) entre os dois testes. Quando comparado com um "kit" de ELISA disponível comercialmente, a performance de ambos em relação ao NPLA foi similar. Concluiu-se que o ELISA-I é um teste apropriado para triagem em larga escala de soros para a detecção de anticorpos contra o Vírus da Peste Suína Clássica (VPSC), embora não seja capaz de diferenciar entre anticorpos induzidos pelo VPSC ou outros pestivírus (AU)