Reproducibility of double agar gel immunodiffusion test using stored serum and plasma from paracoccidioidomycosis patients

Background: Serological evaluation performed by double agar gel immunodiffusion test (DID) is used for diagnosis, evaluation of severity, management of paracoccidioidomycosis patients, and development of new clinical studies. For these reasons, the Botucatu Medical School of UNESP maintains a serum bank at the Experimental Research Unit with patient clinical data. This study aimed to evaluate the influence of the freeze-thaw cycle and different blood matrices on the titration of circulating antibodies. Methods: The study included 207 patients with confirmed (etiology-demonstrated) or probable (serology-demonstrated) paracoccidioidomycosis, and DID was performed with culture filtrate from Paracoccidioides brasiliensis B339 as antigen. First experiment: the antibody levels were determined in serum samples from 160 patients with the chronic form and 20 with the acute/subacute form, stored at ­80o C for more than six months. Second experiment: titers of 81 samples of serum and plasma with ethylenediaminetetraacetic acid (EDTA) or heparin, from 27 patients, were compared according to matrix and effect of storage at ­20o C for up to six months. Differences of titers higher than one dilution were considered discordant. Results: First experiment: test and retest presented concordant results in serum stored for up to three years, and discordant titers in low incidence in storage for four to six years but high incidence when stored for more than six years, including conversion from reagent test to non-reagent retest. Second experiment: serum, plasma-EDTA and plasma-heparin samples showed concordant titers, presenting direct correlation, with no interference of storage for up to six months. Conclusions: Storage at ­80o C for up to six years has no or little influence on the serum titers determined by DID, permitting its safe use in studies depending on this parameter. The concordant titrations in different blood matrices demonstrated that the plasma can be used for immunodiffusion test in paracoccidioidomycosis, with stability for at least six months after storage at ­20o C.(AU)

First record of antibodies to the bluetongue virus in ewe (Ovis aries) in the state of Amazonas, Brazil / Primeiro registro de anticorpos para o vírus da língua azul em ovino (Ovis aries) no estado do Amazonas, Brasil

The objective of this work was to describe the first record of antibodies to the Bluetongue Virus (BTV) in ewe, in the state of Amazonas. The ewe, which was in twin pregnancy, gave birth on May 9, 2015, but a lamb died hours after delivery. Veterinary service was then requested by the owner, where emaciation, loss of wool, pyrexia, apathy, dyspnea, mucoid nasal secretion, facial, lingual and submandibular edema were observed. There was a visit by the Agricultural Defense Agency of the State of Amazonas to the property and blood samples were collected from the animal. The whole blood and serum were sent to the National Agricultural Laboratory, where it was possible to detect the presence of specific antibodies to BTV, through the Agar Gel Double Immunodiffusion. The ewe was submitted to a new blood collection, following the same protocols and the samples were sent to the Biological Institute of São Paulo, confirmed diagnosis. The animal in a serious clinical condition, could not resist and died in July 2015. The occurrence of an allochthonous case, in an area where vector insects occur, can trigger an endemic process in the Amazon region. With this, the epidemiological control of these occurrences is necessary, in order to avoid the spread of the disease in the country.

O objetivo do trabalho foi descrever o primeiro registro de anticorpos para o Vírus da Língua Azul (VLA) em ovino, no estado do Amazonas. A ovelha, que se encontrava em gestação gemelar, pariu no dia 9 de maio de 2015, porém um cordeiro faleceu horas após o parto. Foi então solicitado serviço veterinário por parte do proprietário, onde foi observado emaciação, perda de lã, pirexia, apatia, dispneia, secreção nasal mucoide, edema facial, lingual e submandibular. Houve visita da Agência de Defesa Agropecuária do Estado do Amazonas na propriedade e coletadas amostras de sangue do animal. O sangue total e soro foram enviados ao Laboratório Nacional Agropecuário, no qual foi possível detectar a presença de anticorpos específicos para VLA, através do teste de Imunodifusão Dupla em Gel de Ágar. A ovelha foi submetida a uma nova coleta de sangue, seguindo os mesmos protocolos e as amostras foram enviadas ao Instituto Biológico de São Paulo, confirmando diagnóstico. O animal em estado clínico grave, não resistiu e veio a óbito em julho de 2015. A ocorrência de um caso alóctone, em uma área de ocorrência de insetos vetores, pode desencadear um processo de endemia na região amazônica. Com isso, o controle epidemiológico destas ocorrências, se fazem necessários, afim de se evitar a disseminação da doença no país.

Blue tongue virus in dairy cattle in the Southern region of Minas Gerais, Brazil - serological survey

Background: Blue tongue (BT) is a noncontagious viral disease transmitted by hematophagous arthropods, especially of the genus Culicoides. The economic impact of the disease is related not only to deaths in sheep herds but also to the possible correlation of virus infection with the development of other diseases, such as pneumonia, abortion and movement problems. The economic losses caused by Blue Tongue are linked to restrictions on the import and export of animals and their genetic material and to the reproductive disorders associated with this disease. In addition, the fact that cattle take the role of reservoir, combined with the care by other countries with outbreaks of infection and biological contamination of their products, hinders trade in Mercosul, United States and Europe. Cattle are affected by Blue Tongue Virus in endemic areas and in some epidemic areas, but the development of clinical disease is rare. The clinical signs, when evident, range from reproductive losses, such as embryonic death, abortion, fetal malformation, temporary sterility, infertility in bulls, stillbirths and the birth of weak animals. The objective of this study was to determine the epidemiological aspects of Blue Tongue Virus (BTV) infection in dairy cattle in the Lavras region, state of Minas Gerais, Brazil. Materials, Methods & Results: A cross-sectional study was conducted to evaluate the frequency of cattle and herds seropositive for Blue Tongue in the southern region of Minas Gerais. In this study, 54 dairy farms were visited. A total of 586 serum samples were collected from cows of reproductive age. Sampling was random, and serum samples were collected from lactating cows over 24 months of age by puncture of the jugular vein and/or coccidian vein. The samples were transported and stored at the Setor de Patologia Veterinária, at the Universidade Federal de Lavras (SPV-UFLA), where they were centrifuged, and the serum aliquots were obtained, transferred to microtubes and kept at -20°C until the serological tests were performed. The samples were tested with the agarose gel immunodiffusion test (AGID) for anti-blue tongue virus antibodies. The AGID test is more practical and is the main method used to identify Blue Tongue Virus seroprevalence in different ruminant species. They are considered important tools for epidemiological surveillance of the disease. A prevalence of 83.28% was observed among animals that were seropositive for Blue Tongue Virus (488/586; IC 95% = 80.0 - 86.21). In addition, 100% (54/54; IC 95% = 93.4 - 100.0) of the farms had at least 1 positive animal, with rates ranging from 45.45% to 100% within the herds and where 22.22% of the farms had rates of 100% of the animals being positive. Discussion: Blue Tongue is a disease known to affect domestic and wild ruminants in Brazil. However, there is a lack of more precise information about its epidemiology and occurrence in the country and of joint efforts of researchers, producers and the government to understand in detail both the biology of vectors and the viral biology of Blue Tongue Virus in Brazil. This is the first record of detection of anti-blue tongue virus antibodies in cattle in the southern region of Minas Gerais. The results suggest that Blue Tongue Virus is present in cattle in the study area.

Multiparametric assay of screening for the diagnosis of mycoses of interest in Public Health: standardization of methodology / Ensaio multiparamétrico de triagem para diagnóstico de micoses de interesse em Saúde Pública: padronização da metodologia

The standardization and validation of a multiplex assay requires the combination of important parameters such as sensitivity and specificity, acceptable levels of performance, robustness, and reproducibility. We standardized a multiparametric Dot-blot aimed at the serological screening of paracoccidioidomycosis, histoplasmosis, and aspergillosis. A total of 148 serum were evaluated: 10 from healthy subjects, 36 from patients with paracoccidioidomycosis, 62 from patients with histoplasmosis, and 40 from patients with aspergillosis. It was found that the multiparametric Dot-blot showed a high percentage of cross-reactivity. However, when evaluated individually, in the serological screening of histoplasmosis, a good performance was observed when compared to the double immunodiffusion assay, considered the gold standard test, with 100% co-positivity and 83.3% co-negativity. The performance of serological screening for aspergillosis was not satisfactory when compared to double immunodiffusion, showing 71.4% co-positivity and 100% co-negativity. The evaluation of the stability of nitrocellulose membranes showed that membranes sensitized with H. capsulatum antigen remained stable for 90 days and those sensitized with A. fumigatus antigen for 30 days. We conclude that the use of crude antigens was not suitable for the standardization of the multiparametric Dot-blot assay, due to the high cross-reactivity, and that further tests should be performed with purified proteins (AU).

A padronização e validação de um ensaio multiplex requer a combinação de parâmetros importantes, como sensibilidade e especificidade, níveis aceitáveis de desempenho, robustez e reprodutibilidade. Este trabalho padronizou um Dot-blot multiparamétrico visando a triagem sorológica da paracoccidioidomicose, histoplasmose e aspergilose. Foram avaliadas 148 amostras de soro: 10 de indivíduos saudáveis, 36 de pacientes com paracoccidioidomicose, 62 de pacientes com histoplasmose e 40 de pacientes com aspergilose. Verificou-se que o Dot-blot multiparamétrico apresentou elevado percentual de reatividade cruzada. Entretanto, quando avaliado individualmente, na triagem sorológica da histoplasmose observou-se bom desempenho quando comparado ao ensaio de imunodifusão dupla, considerado o teste padrão ouro, com 100% de co-positividade e 83,3% de co-negatividade. O desempenho da triagem sorológica da aspergilose não foi satisfatório quando comparado a imunodifusão dupla, apresentando 71,4% de co-positividade e 100% de co-negatividade. A avaliação da estabilidade das membranas de nitrocelulose mostrou que membranas sensibilizadas com antígeno de H. capsulatum permaneceram estáveis por 90 dias e as sensibilizadas com antígeno de A. fumigatus, por 30 dias. Concluímos que o uso de antígenos brutos não foi adequado para a padronização do ensaio de Dot-blot multiparamétrico, devido ao alto índice de reatividade cruzada, e que novos testes devem ser realizados com proteínas purificadas (AU).

Risk factors and detection of brucella ovis in naturally infected rams and ewes / Fatores de risco e detecção de brucella ovis em carneiros e ovelhas naturalmente infectados

The objective of this study was to verify the occurrence of ovine brucellosis using Agar Gel Immunodiffusion (AGID) and Polymerase Chain Reaction (PCR) techniques, as well as to identify the main risk factors associated with infection in sheep flocks belonging to municipalities in the microregion from Teresina, PI, Brazil. A total of 100 urine and blood samples were collected from sheep aged 6 months or older. The urine samples were submitted to conventional PCR and the blood samples were examined by the AGID technique. Of the 100 blood samples, 17 (17%) were reactive to the AGID test. In conventional PCR of 100 urine samples, six (6%) were positive. Risk factors associated to infection by B. ovis included the rearing system (OR=0.19), feed management (OR=0.05), presence of dystotic births (OR=4.50), miscarriages (OR=3.75) and source of water offered to the animals (OR=0.19). Thus, it was concluded that it is possible to detect the occurrence of animals with ovine brucellosis since PCR is a reliable method to confirm infection. Furthermore, there are risk factors associated to infection by B. ovis in the municipalities studied.(AU)

Objetivou-se verificar a ocorrência da brucelose ovina através das técnicas de Imunodifusão em Gel de Ágar (IDGA) e Reação em Cadeia da Polimerase (PCR), bem como identificar os principais fatores de risco associados à infecção nos rebanhos ovinos pertencentes a municípios da microrregião de Teresina, PI, Brasil. Foram colhidas 100 amostras de urina e de sangue de ovinos com idade superior ou igual a seis meses. As amostras de urina foram submetidas a PCR convencional e as amostras de sangue à técnica de IDGA. Das 100 amostras de sangue 17 (17%) foram reagentes ao teste de IDGA. Já na PCR convencional das 100 amostras de urina, seis (6%) foram positivas. Ressalta-se que três animais foram positivos em ambos os testes. Como fatores associados à infecção por B. ovis, observou-se o tipo de sistema de criação (OR=0,19), o manejo alimentar (OR=0,05), presença de partos distócicos (OR=4,50), abortamentos (OR=3,75) e a fonte de água fornecida aos animais (OR=0,19). Assim, conclui-se que foi possível detectar a ocorrência de animais com brucelose ovina, uma vez que a PCR é um método confirmatório. Além disso, há fatores de risco associados à infecção por B. ovis nos municípios estudados.(AU)

Risk factors and detection of brucella ovis in naturally infected rams and ewes / Fatores de risco e detecção de brucella ovis em carneiros e ovelhas naturalmente infectados

The objective of this study was to verify the occurrence of ovine brucellosis using Agar Gel Immunodiffusion (AGID) and Polymerase Chain Reaction (PCR) techniques, as well as to identify the main risk factors associated with infection in sheep flocks belonging to municipalities in the microregion from Teresina, PI, Brazil. A total of 100 urine and blood samples were collected from sheep aged 6 months or older. The urine samples were submitted to conventional PCR and the blood samples were examined by the AGID technique. Of the 100 blood samples, 17 (17%) were reactive to the AGID test. In conventional PCR of 100 urine samples, six (6%) were positive. Risk factors associated to infection by B. ovis included the rearing system (OR=0.19), feed management (OR=0.05), presence of dystotic births (OR=4.50), miscarriages (OR=3.75) and source of water offered to the animals (OR=0.19). Thus, it was concluded that it is possible to detect the occurrence of animals with ovine brucellosis since PCR is a reliable method to confirm infection. Furthermore, there are risk factors associated to infection by B. ovis in the municipalities studied.

Objetivou-se verificar a ocorrência da brucelose ovina através das técnicas de Imunodifusão em Gel de Ágar (IDGA) e Reação em Cadeia da Polimerase (PCR), bem como identificar os principais fatores de risco associados à infecção nos rebanhos ovinos pertencentes a municípios da microrregião de Teresina, PI, Brasil. Foram colhidas 100 amostras de urina e de sangue de ovinos com idade superior ou igual a seis meses. As amostras de urina foram submetidas a PCR convencional e as amostras de sangue à técnica de IDGA. Das 100 amostras de sangue 17 (17%) foram reagentes ao teste de IDGA. Já na PCR convencional das 100 amostras de urina, seis (6%) foram positivas. Ressalta-se que três animais foram positivos em ambos os testes. Como fatores associados à infecção por B. ovis, observou-se o tipo de sistema de criação (OR=0,19), o manejo alimentar (OR=0,05), presença de partos distócicos (OR=4,50), abortamentos (OR=3,75) e a fonte de água fornecida aos animais (OR=0,19). Assim, conclui-se que foi possível detectar a ocorrência de animais com brucelose ovina, uma vez que a PCR é um método confirmatório. Além disso, há fatores de risco associados à infecção por B. ovis nos municípios estudados.

First study of Brucella ovis antibodies in purebred sheep flocks in the State of Parana, Brazil / [Primeiro estudo de anticorpos para Brucella ovis em rebanhos de ovinos puros de Origem, no estado do Paraná, Brasil]

Brucella ovis, a non-zoonotic species, is the etiological agent of ovine brucellosis, an infectious disease of clinical or subclinical occurrence in sheep flocks. Until then, there is no serological study of anti-Brucella ovis antibodies in purebred sheep herds. This study aimed to determine the presence of anti-Brucella ovis antibodies in purebred sheep flocks with breeding purposes from Parana State. Blood samples from 728 animals, of which 563 were females and 165 males, between 8 and 56 months of age from the six major sheep producing mesoregions of Parana, were submitted to detection of anti-Brucella ovis antibodies by the Agar Gel Immunodiffusion technique using an antigen from the bacteria Brucella ovis (Reo 198). The results indicate the presence of this disease in purebred sheep from Parana State in a low occurrence of 0.27% (2/728). The only two positive animals were rams, Santa Inês breed, from the same flock in the East Center region of Parana, without clinical disease. In conclusion, Brucella ovis is present in purebred sheep in Parana State, Brazil, and this low occurrence may have occurred due to rigorous breeding systems that may contribute to reduce the transmission of this disease.(AU)

Brucella ovis, espécie não zoonótica, é o agente etiológico da brucelose ovina, doença infecciosa de ocorrência clínica ou subclínica. Atualmente, não existe estudo sorológico de anticorpos anti-Brucella ovis em rebanhos de ovinos puros de origem. Este estudo teve como objetivo determinar a presença de anticorpos anti-Brucella ovis em rebanhos ovinos de raça pura de origem, com fins reprodutivos do estado do Paraná. Amostras de sangue de 728 animais, sendo 563 fêmeas e 165 machos, entre oito e 56 meses de idade, pertencentes a seis principais mesorregiões produtoras de ovinos no Paraná, foram submetidas à detecção de anticorpos anti-Brucella ovis pela técnica de imunodifusão em ágar gel usando-se um antígeno da bactéria Brucella ovis (Reo 198). Os resultados indicam a presença da doença em ovinos puros de origem do estado do Paraná em baixa ocorrência de 0,27% (2/728). Os dois únicos animais positivos foram reprodutores da raça Santa Inês, do mesmo rebanho da região Centro Leste do Paraná, sem manifestação clínica. Em conclusão, Brucella ovis está presente em ovinos puros de origem no estado do Paraná, e essa baixa ocorrência pode ter ocorrido devido a sistemas rigorosos de criação, que podem contribuir para a redução da transmissão dessa doença.(AU)

Risk factors and detection of brucella ovis in naturally infected rams and ewes / Fatores de risco e detecção de brucella ovis em carneiros e ovelhas naturalmente infectados

The objective of this study was to verify the occurrence of ovine brucellosis using Agar Gel Immunodiffusion (AGID) and Polymerase Chain Reaction (PCR) techniques, as well as to identify the main risk factors associated with infection in sheep flocks belonging to municipalities in the microregion from Teresina, PI, Brazil. A total of 100 urine and blood samples were collected from sheep aged 6 months or older. The urine samples were submitted to conventional PCR and the blood samples were examined by the AGID technique. Of the 100 blood samples, 17 (17%) were reactive to the AGID test. In conventional PCR of 100 urine samples, six (6%) were positive. Risk factors associated to infection by B. ovis included the rearing system (OR=0.19), feed management (OR=0.05), presence of dystotic births (OR=4.50), miscarriages (OR=3.75) and source of water offered to the animals (OR=0.19). Thus, it was concluded that it is possible to detect the occurrence of animals with ovine brucellosis since PCR is a reliable method to confirm infection. Furthermore, there are risk factors associated to infection by B. ovis in the municipalities studied.

Objetivou-se verificar a ocorrência da brucelose ovina através das técnicas de Imunodifusão em Gel de Ágar (IDGA) e Reação em Cadeia da Polimerase (PCR), bem como identificar os principais fatores de risco associados à infecção nos rebanhos ovinos pertencentes a municípios da microrregião de Teresina, PI, Brasil. Foram colhidas 100 amostras de urina e de sangue de ovinos com idade superior ou igual a seis meses. As amostras de urina foram submetidas a PCR convencional e as amostras de sangue à técnica de IDGA. Das 100 amostras de sangue 17 (17%) foram reagentes ao teste de IDGA. Já na PCR convencional das 100 amostras de urina, seis (6%) foram positivas. Ressalta-se que três animais foram positivos em ambos os testes. Como fatores associados à infecção por B. ovis, observou-se o tipo de sistema de criação (OR=0,19), o manejo alimentar (OR=0,05), presença de partos distócicos (OR=4,50), abortamentos (OR=3,75) e a fonte de água fornecida aos animais (OR=0,19). Assim, conclui-se que foi possível detectar a ocorrência de animais com brucelose ovina, uma vez que a PCR é um método confirmatório. Além disso, há fatores de risco associados à infecção por B. ovis nos municípios estudados.

Mycobacterium avium subspecies paratuberculosis and bovine leukemia virus seroprevalence and associated risk factors in dairy herds in Minas Gerais state, Brazil / [Soroprevalência do Mycobacterium avium subespécie paratuberculosis e vírus da leucemia bovina e fatores de risco associados em rebanhos leiteiros de Minas Gerais, Brasil]

Mycobacterium avium subesp. paratuberculosis (MAP) e o vírus da leucemia bovina (BLV) são agentes que causam grandes perdas econômicas nos rebanhos. O objetivo deste estudo foi avaliar a situação epidemiológica da paratuberculose bovina (PTB) e leucose enzoótica bovina (EBL) em rebanhos leiteiros de Lagoa Formosa, Minas Gerais, Brasil. Foram coletadas 236 amostras de sangue de vacas, as quais foram submetidas aos testes ELISA e imunodifusão em gel de ágar para detecção de anticorpos contra MAP e BLV. A soroprevalência de anticorpos contra MAP e BVL foi de 20% para os rebanhos e 6% para os animais e de 85% para os rebanhos e 50,42% para os animais, respectivamente. A presença dessas enfermidades deve servir como um alerta para os produtores e veterinários, para que concentrem maior atenção na implementação de medidas higiênico-sanitárias, incorporando elementos de vigilância com base nos riscos identificados no estudo.(AU)

Intercurrence between bovine leukosis virus infection and mastitis in dairy cattle / Intercorrência entre a infecção pelo vírus da leucose bovina e a mastite em bovinos leiteiros

There are several factors affecting global milk production leading to severe economic losses. Mastitis and leukosis are highly prevalent diseases especially in Brazilian cattle herds. The present study aimed to evaluate the correlation between mastitis and the bovine leukemia virus (BLV) infection in naturally infected cows belonging to dairy herds in Minas Gerais State. Additionally, the prevalence of both diseases, pathogens causing mastitis, and their association with BLV infection and persistent lymphocytosis were evaluated. The prevalence of BLV was determined using the agar gel immunodiffusion test (AGID) in 1,392 lactating cows from ten dairy herds. From the AGID results, 240 cows were selected and divided into two experimental groups: A case group composed of 160 BLV-positive cows (BLV-P) and the control group of 80 BLV-negative cows (BLV-N). Based on serological and leucocytic evaluations, the animals were classified into three subgroups: BLV-N, BLV-P without persistent lymphocytosis (BLV-PWPL), and BLV-positive with persistent lymphocytosis (BLV-PPL). The California Mastitis Test (CMT) was performed on days 0 (D0) and 60 (D60) in the subgroups and the milk from CMT-positive glands were analyzed. BLV infection was present in 100% of herds, with an overall prevalence of 80.9% in cows older than 24 months. Among BLV-P animals, 74% were classified as BLV-PWPL and 26% as BLV-PPL. Coagulase-negative Staphylococcus (73.30%), Corynebacterium spp. (12.04%), Streptococcus agalactiae (8.90%), and Staphylococcus aureus (5.76%) were predominant (61.41%). There were no statistical differences among the scores of clinical or subclinical mastitis with the pathogen type (contagious or environmental) in the BLV-PWPL or BLV-PPL subgroups. However, BLV-P animals showed higher CMT scores than BLV-N animals, suggesting a higher predisposition to subclinical mastitis in cows affected by leukosis.(AU)

Tendo em vista a relevância econômica e social da produção de leite e a ampla ocorrência da leucose e mastite em rebanhos brasileiros, foi proposto o presente estudo com o objetivo de avaliar a correlação entre a mastite e a infecção pelo vírus da leucose bovina em vacas leiteiras naturalmente infectadas de 10 rebanhos leiteiros da região sul do estado de Minas Gerais. Além disso, foi avaliada a prevalência de ambas as doenças e dos patógenos causadores de mastite e sua associação com infecção por BLV e linfocitose persistente. A prevalência do VLB foi avaliada pelo teste de imunodifusão em gel de ágar (IDGA) em 1.392 vacas em lactação nos rebanhos estudados. De acordo com os resultados do IDGA, 240 vacas foram selecionadas para compor dois grupos experimentais: 1-caso: composto por 160 vacas BLV positivas (BLV-P) e 2-controle: composto por 80 vacas BLV negativas (BLV-N). De acordo com as avaliações sorológicas e leucocitárias, os animais foram classificados em três subgrupos: BLV-N; BLV-P sem linfocitose persistente (BLV-PWPL) e BLV positivos com linfocitose persistente (BLV-PPL). O California Mastitis Test (CMT) e a cultura do leite das glândulas CMT positivas foram realizados nos dias 0 (D0) e 60 (D60). A leucose foi detectada em 100% dos rebanhos com prevalência média de 80,9%. Entre os animais BLV-P, 74% foram classificados como BLV-PWPL e 26% como BLV-PPL. Staphylococcus coagulase negativos (73,30%), seguido por Corynebacterium spp. (12,04%), Streptococcus agalactiae (8,90%) e Staphylococcus aureus (5,76%) foram os agentes predominantes. Não houve diferença estatística entre os escores de mastite clínica ou subclínica e nem entre o tipo de patógeno (contagioso ou ambiental) nos subgrupos de animais BLV-PWPL ou BLV-PPL relacionados ao BLV-N. No entanto, animais BLV-P apresentaram escores mais elevados no CMT em relação aos animais BLV-N, sugerindo maior predisposição à mastite subclínica em vacas com leucose.(AU)

Western Blot no imunodiagnóstico de lentivírus de pequenos ruminantes / Western Blot in immunodiagnosis of small ruminant lentivirus

Background: Small ruminant lentivirus (SRLV) belong to genus Lentivirus, family Retroviridae. These viruses causecaprine arthritis encephalitis (CAE) and maedi visna (MV), infectious diseases that cause economic, production, and reproductive losses. There are no effective treatments or vaccines for these diseases. Thus, early detection via serology hasgreat importance for control of SRLV. Therefore, the objective of this review is to demonstrate the potential of the westernblot (WB) test as an immunodiagnostic test for SRLV.Review: In general, immunodiagnosis of SRLV is performed via agar gel immunodiffusion (AGID) and indirect enzymelinked immunosorbent assay (ELISA), which can detect antibodies in several different biological samples but is used preferably with serum and blood plasma. However, WB has demonstrated efficacy in the early diagnosis of immunoglobulinsagainst SRLV, presenting higher sensitivity and specificity than the serological tests usually used, because this techniquecan detect antibodies at a dilution as much as 256 times greater than that of AGID and 32 times greater than that of ELISA.SRLV infection and consequent immunological activation result in the induction of cellular and humoral responses. Additionally, around the third week, production of antibodies directed mainly toward viral capsid proteins (p25 and p28)occurs. After the fifth week, production of immunoglobulins directed toward other viral proteins occurs. Because of thepersistence of SRLV infection, serology is considered to be the most practical means to diagnosis. Each serological testhas a percentage specificity and distinct sensitivity, as well as advantages and disadvantages in its applicability. It shouldbe noted that there is no gold standard test for diagnosis of SRLV infection. Moreover, SRLV are characterized by escapemechanisms such as genetic diversity, mutagenic potential, viral intermittence...

Is bluetongue virus a risk factor for reproductive failure in tropical hair sheep in Brazil?

Background: Bluetongue is a vector-borne viral disease transmitted by midges from the genus Culicoides. The disease caninfect most of the ruminant and camelid species, but the severe disease is most often seen in european wool and muttonsheep breeds. In this sense, there is a gap in the knowledge on BTV infection in hair sheep breeds from tropical zones.Thus, this study aimed at establishing whether exposure to BTV is a risk factor for reproductive failure in Santa Inês ewes,a hair sheep breed, reared under tropical conditions in Brazil.Materials, Methods & Results: A retrospective cross-sectional study was carried out in sheep farms in São Paulo state,Brazil, after the rainy season. Serum samples from 110 Santa Inês ewes with a history of reproductive disorders, in the last6 months, which were included: abortion, premature birth, stillbirth, retention of placenta, infertility, estrus repetition, fetalmalformation, weak lamb birth and neonatal death were collected. The presence of antibodies against BTV was assessedby agar gel immunodiffusion method (AGID). Serology to the infectious agents Brucela ovis, Lepstopira spp., Toxoplasmagondii, Neospora caninum and Campylobacter sp. were also assessed. Bivariate associations between the outcome andindividual explanatory variables were assessed using the Fisher’s exact test. Abortion was the most common reproductive disorder (53%; 74/139) observed, followed by estrus repetition (12%; 17/139) and infertility (11%; 15/139). Otherdisorders related to the conceptus totaled nearly one fourth of the reported disorders. A total of 20% (22/110) of the eweswere seropositive to BTV. A higher frequency of BTV seropositive than BTV seronegative ewes with a history of abortionwas found. Also, abortion with seroreactivity to BTV was tested for prevalence ratio that showed 1.38 [95% CI 1.10-1.74;P = 0.030]. With regards to the abortion involvement of other infectious diseases associated with the seropositive ewes to...

Is bluetongue virus a risk factor for reproductive failure in tropical hair sheep in Brazil?

Background: Bluetongue is a vector-borne viral disease transmitted by midges from the genus Culicoides. The disease caninfect most of the ruminant and camelid species, but the severe disease is most often seen in european wool and muttonsheep breeds. In this sense, there is a gap in the knowledge on BTV infection in hair sheep breeds from tropical zones.Thus, this study aimed at establishing whether exposure to BTV is a risk factor for reproductive failure in Santa Inês ewes,a hair sheep breed, reared under tropical conditions in Brazil.Materials, Methods & Results: A retrospective cross-sectional study was carried out in sheep farms in São Paulo state,Brazil, after the rainy season. Serum samples from 110 Santa Inês ewes with a history of reproductive disorders, in the last6 months, which were included: abortion, premature birth, stillbirth, retention of placenta, infertility, estrus repetition, fetalmalformation, weak lamb birth and neonatal death were collected. The presence of antibodies against BTV was assessedby agar gel immunodiffusion method (AGID). Serology to the infectious agents Brucela ovis, Lepstopira spp., Toxoplasmagondii, Neospora caninum and Campylobacter sp. were also assessed. Bivariate associations between the outcome andindividual explanatory variables were assessed using the Fishers exact test. Abortion was the most common reproductive disorder (53%; 74/139) observed, followed by estrus repetition (12%; 17/139) and infertility (11%; 15/139). Otherdisorders related to the conceptus totaled nearly one fourth of the reported disorders. A total of 20% (22/110) of the eweswere seropositive to BTV. A higher frequency of BTV seropositive than BTV seronegative ewes with a history of abortionwas found. Also, abortion with seroreactivity to BTV was tested for prevalence ratio that showed 1.38 [95% CI 1.10-1.74;P = 0.030]. With regards to the abortion involvement of other infectious diseases associated with the seropositive ewes to...(AU)

Western Blot no imunodiagnóstico de lentivírus de pequenos ruminantes / Western Blot in immunodiagnosis of small ruminant lentivirus

Background: Small ruminant lentivirus (SRLV) belong to genus Lentivirus, family Retroviridae. These viruses causecaprine arthritis encephalitis (CAE) and maedi visna (MV), infectious diseases that cause economic, production, and reproductive losses. There are no effective treatments or vaccines for these diseases. Thus, early detection via serology hasgreat importance for control of SRLV. Therefore, the objective of this review is to demonstrate the potential of the westernblot (WB) test as an immunodiagnostic test for SRLV.Review: In general, immunodiagnosis of SRLV is performed via agar gel immunodiffusion (AGID) and indirect enzymelinked immunosorbent assay (ELISA), which can detect antibodies in several different biological samples but is used preferably with serum and blood plasma. However, WB has demonstrated efficacy in the early diagnosis of immunoglobulinsagainst SRLV, presenting higher sensitivity and specificity than the serological tests usually used, because this techniquecan detect antibodies at a dilution as much as 256 times greater than that of AGID and 32 times greater than that of ELISA.SRLV infection and consequent immunological activation result in the induction of cellular and humoral responses. Additionally, around the third week, production of antibodies directed mainly toward viral capsid proteins (p25 and p28)occurs. After the fifth week, production of immunoglobulins directed toward other viral proteins occurs. Because of thepersistence of SRLV infection, serology is considered to be the most practical means to diagnosis. Each serological testhas a percentage specificity and distinct sensitivity, as well as advantages and disadvantages in its applicability. It shouldbe noted that there is no gold standard test for diagnosis of SRLV infection. Moreover, SRLV are characterized by escapemechanisms such as genetic diversity, mutagenic potential, viral intermittence...(AU)

True prevalence and spatial distribution of equine infectious anemia virus (EIAV) in horses from northeast region of Brazil

Equine infectious anemia (EIA) is a viral infection, caused by a lentivirus of the Retroviridae family, Orthoretrovirinael subfamily and its occurrence generates significant economic losses due to culling of positive animals as a measure of infection control. The objective of this work was to determine the prevalence of horses positive for equine infectious anemia virus (EIAV) and to identify the occurrence of areas with higher densities of cases in the states of Paraíba (PB), Pernambuco (PE), Rio Grande do Norte (RN) and Ceará (CE), Northeast region of Brazil, during the rainy (May and June) and dry (October and November) periods of 2017 and 2018. Serum samples from 6,566 horses from the states of PB, PE, RN and CE, Brazil, provided by the Laboratório Veterinária Diagnóstico - Ltda., were used. Serological diagnosis of EIA was performed using indirect enzyme-linked immunosorbent assay (ELISA) as a screening test and agar gel immunodiffusion test (AGID) as a confirmatory test. The apparent prevalence was obtained by dividing the number of seroreactive animals by the total number of animals, while the true prevalence was estimated by adjusting the apparent prevalence, considering the sensitivity (100%) and specificity (98.6%) of the diagnostic protocol used. For the construction of Kernel estimates, the Quartic function was used. In the dry season, of the 1,564 animals sampled, 28 were serologically positive, of which 19 belonged to the state of Ceará, 7 to Paraíba and 2 to Rio Grande do Norte. In 2018, it was observed that, during the rainy season, 26 of the 1,635 horses were seroreactive, with 19 cases resulting from Ceará, 4 from Paraíba and 3 from Pernambuco. In the dry season, 32 of the 1,526 animals were seroreactive to EIAV, of which 26 were from Ceará, 3 from Paraíba, 1 from Rio Grande do Norte and 2 from Pernambuco. In the dry period of 2017, the CE had a real prevalence of 1.22% (95% CI = 0.05 - 2.99%). In 2018, during the rainy season, prevalences of 0.03% (95% CI = 0 - 1.18%) were identified in CE and 1.69% (95% CI = 0 - 8.38%) in PE. Regarding the 2018 dry period, a prevalence of 1.32% (95% CI = 0.26 - 2.84%) was found in the state of CE. In both dry and rainy periods of 2017, the presence of spatial clusters of animals positive for EIA was observed, mainly in the border areas among the states of CE, PE, PB and RN. In 2018, there was a variation in the distribution of areas with higher densities of cases between the rainy and dry periods. The state of CE had the highest prevalence of positive animals and the presence of areas with higher densities of EIA cases in both climatic periods, in the years 2017 and 2018. In some municipalities of the CE, important sporting events of agglomeration of animals take place, which can favor the transmission of EIAV by facilitating the contact of infected and susceptible animals. Population density may be a factor associated with the higher prevalence observed in this region, as it has the second largest herd among the states studied. Higher densities indirectly contribute to the occurrence of infectious diseases, as they favor the contact of infected and susceptible animals. The occurrence of higher densities of cases in the border areas of the states of PE, RN, CE, and PB may be related to the greater movement of animals in these regions, favoring the indirect contact of infected horses with susceptible ones. The observed results demonstrate the circulation of the EIAV in four states in the Northeast region of Brazil.(AU)

Seroprevalence and influence of bovine leukemia virus on the incidence of mastitis in dairy herds

Background: The enzootic bovine leukosis (EBL) is a viral disease with a chronic aspect and its etiological agent is anoncogenic deltaretrovirus called bovine leukemia virus (VLB). It has a wide distribution in dairy cattle herds, and it maytake several years before the first clinical manifestations occur. Animals seropositive for VLB are more susceptible todeveloping infectious diseases such as mastitis. The objective of this study was to evaluate the seroprevalence of EBL, theprevalence of mastitis, the possible association between leukosis and mastitis, and the risk factors related to leukosis inmunicipalities of the Caparaó region of Espírito Santo, Brazil.Materials, Methods & Results: The prevalence of enzootic bovine leukosis and its association with mastitis were evaluatedin an 854 clinically healthy crossbred (Bos tauros tauros x Bos taurus indicus) dairy cows were used, in different lactationphases, from farms located in the 12 municipalities that compose the Caparaó Capixaba micro-region, in the southernpart of Espírito Santo, Brazil, in the period from February to December 2015. The seroprevalence of EBL was determinedby the agar gel immunodiffusion test, the presence of clinical mastitis was ascertained by the black background mug testand of subclinical mastitis by the CMT. The reuse of needles, type of milking, reproductive management, calf mortality,presence of reproductive problems and milk yield were the risk factors assessed. The association between the variableswas estimated by the odds ratio (95% confidence interval). The significance of the associations was determined by theChi-square test. The prevalence of EBL, clinical and subclinical mastitis was 56.79%, 5.50% and 43.55%, respectively.There was a positive association between the EBL virus and the presence of...

Seroprevalence and influence of bovine leukemia virus on the incidence of mastitis in dairy herds

Background: The enzootic bovine leukosis (EBL) is a viral disease with a chronic aspect and its etiological agent is anoncogenic deltaretrovirus called bovine leukemia virus (VLB). It has a wide distribution in dairy cattle herds, and it maytake several years before the first clinical manifestations occur. Animals seropositive for VLB are more susceptible todeveloping infectious diseases such as mastitis. The objective of this study was to evaluate the seroprevalence of EBL, theprevalence of mastitis, the possible association between leukosis and mastitis, and the risk factors related to leukosis inmunicipalities of the Caparaó region of Espírito Santo, Brazil.Materials, Methods & Results: The prevalence of enzootic bovine leukosis and its association with mastitis were evaluatedin an 854 clinically healthy crossbred (Bos tauros tauros x Bos taurus indicus) dairy cows were used, in different lactationphases, from farms located in the 12 municipalities that compose the Caparaó Capixaba micro-region, in the southernpart of Espírito Santo, Brazil, in the period from February to December 2015. The seroprevalence of EBL was determinedby the agar gel immunodiffusion test, the presence of clinical mastitis was ascertained by the black background mug testand of subclinical mastitis by the CMT. The reuse of needles, type of milking, reproductive management, calf mortality,presence of reproductive problems and milk yield were the risk factors assessed. The association between the variableswas estimated by the odds ratio (95% confidence interval). The significance of the associations was determined by theChi-square test. The prevalence of EBL, clinical and subclinical mastitis was 56.79%, 5.50% and 43.55%, respectively.There was a positive association between the EBL virus and the presence of...(AU)

Non-specific reactions caused by vaccination in agar gel immunodiffusion for diagnosis of equine infectious anemia / Reações inespecíficas provocadas por vacinação na imunodifusão em gel de ágar para diagnósticodaanemia infecciosa equina

Equine infectious anemia (EIA) is one of the most important diseases from the health and economic point of view for equidae breeding, as it does not have treatment and vaccines. The Brazilian Ministry of Agriculture, Livestock and Food Supply (MAPA) instituted mandatory sanitary measures that include the official diagnosis by the agar gel immunodiffusion (AGID) test and sacrifice of seropositive animals to control this disease. Seventy-two seronegative equines, challenged with different vaccines, were used to verify the occurrence of non-specific reactions in the AGID techniques. Five serological controls were performed one week after vaccination, at seven-day intervals. The results indicated that the use of vaccines in equines in a period that precedes the performance of laboratory tests for the diagnosis of EIA does not induce seroconversion. However, 11.11% of the equines vaccinated against influenza, encephalomyelitis, equine rhinopneumonitis, and tetanus, and 15.38% of those vaccinated against leptospirosis had non-specific negative reactions to AGID. In this study, there was a non-specific line in the AGID for EIA described by Ordinance No. 84/1992 by MAPA but already mentioned in the Normative Instruction 55 of 26 November 2018.

Anemia Infecciosa Equina é uma das enfermidades mais importantes sob o ponto de vista sanitário e econômico para a equideocultura, por não possuir tratamento e vacinas. Para controle desta doença, o Ministério da Agricultura, Pecuária e Abastecimento (MAPA) instituiu medidas sanitárias obrigatórias em todo território nacional que incluem o diagnóstico e sacrifício dos animais soropositivos. Para verificar a ocorrência de reações inespecíficas na técnica de IDGA utilizou-se 72 equinos soronegativos, desafiados com diferentes vacinas. Uma semana após a vacinação, realizou-se cinco controles sorológicos, em intervalos de sete dias. Os resultados indicaram que o uso de vacinas em equinos em período que antecede a realização de exames laboratoriais para diagnóstico de AIE, não induz a soroconversão. Entretanto, 11,11% dos equinos vacinados contra influenza, encefalomielite, rinopneumonite equina e tétano, e 15,38% dos que foram vacinados contra leptospirose apresentaram reações negativas inespecíficas ao IDGA. Neste estudo, verificou-se uma linha inespecífica no IDGA para AIE.

Non-specific reactions caused by vaccination in agar gel immunodiffusion for diagnosis of equine infectious anemia / Reações inespecíficas provocadas por vacinação na imunodifusão em gel de ágar para diagnósticodaanemia infecciosa equina

Equine infectious anemia (EIA) is one of the most important diseases from the health and economic point of view for equidae breeding, as it does not have treatment and vaccines. The Brazilian Ministry of Agriculture, Livestock and Food Supply (MAPA) instituted mandatory sanitary measures that include the official diagnosis by the agar gel immunodiffusion (AGID) test and sacrifice of seropositive animals to control this disease. Seventy-two seronegative equines, challenged with different vaccines, were used to verify the occurrence of non-specific reactions in the AGID techniques. Five serological controls were performed one week after vaccination, at seven-day intervals. The results indicated that the use of vaccines in equines in a period that precedes the performance of laboratory tests for the diagnosis of EIA does not induce seroconversion. However, 11.11% of the equines vaccinated against influenza, encephalomyelitis, equine rhinopneumonitis, and tetanus, and 15.38% of those vaccinated against leptospirosis had non-specific negative reactions to AGID. In this study, there was a non-specific line in the AGID for EIA described by Ordinance No. 84/1992 by MAPA but already mentioned in the Normative Instruction 55 of 26 November 2018.(AU)

Anemia Infecciosa Equina é uma das enfermidades mais importantes sob o ponto de vista sanitário e econômico para a equideocultura, por não possuir tratamento e vacinas. Para controle desta doença, o Ministério da Agricultura, Pecuária e Abastecimento (MAPA) instituiu medidas sanitárias obrigatórias em todo território nacional que incluem o diagnóstico e sacrifício dos animais soropositivos. Para verificar a ocorrência de reações inespecíficas na técnica de IDGA utilizou-se 72 equinos soronegativos, desafiados com diferentes vacinas. Uma semana após a vacinação, realizou-se cinco controles sorológicos, em intervalos de sete dias. Os resultados indicaram que o uso de vacinas em equinos em período que antecede a realização de exames laboratoriais para diagnóstico de AIE, não induz a soroconversão. Entretanto, 11,11% dos equinos vacinados contra influenza, encefalomielite, rinopneumonite equina e tétano, e 15,38% dos que foram vacinados contra leptospirose apresentaram reações negativas inespecíficas ao IDGA. Neste estudo, verificou-se uma linha inespecífica no IDGA para AIE.(AU)

Seropositivity for Maedi-Visna virus in sheep in Porto Acre city - Western Amazon, Brazil / Soropositividade para o vírus Maedi-Visna em ovinos da cidade de Porto Acre - Amazônia Ocidental, Brasil

Lentivirosis of small ruminants (LVPR) are chronic and degenerative infectious diseases, caused by Lentivirus, associated with numerous losses such as: drop in meat and milk production, predisposition to secondary infections, expenses with veterinary assistance and, even, early disposal of animals. In the northern region of Brazil, the epidemiological situation is poorly understood. Thus, this study aimed to determine the seropositivity of sheep for Lentivirus in Porto Acre city, Western Amazon, Brazil. 122 blood samples from sheep were collected and as a diagnostic method, agarose gel immunodiffusion was used, using the p28 protein of the capsid as antigen. The seropositivity of the sheep to the test was 8.2% (10/122). In 80% (4/5) of the investigated properties, the presence of seropositive animals was detected. It is worth noting that the acquisition of small ruminants from other states likely represented a risk to sheep health in the municipality of Porto Acre, Western Amazon, Brazil. It is concluded that there is a need for more systematic investigations on the prevalence of LVPR in the state of Acre.

As lentiviroses de pequenos ruminantes (LVPR) são enfermidades infecciosas crônicas e degenerativas, causadas por Lentivírus, associadas a inúmeros prejuízos como: queda na produção de carne e leite, predisposição a infecções secundárias, gastos com assistência veterinária e, até mesmo, descarte precoce dos animais. Na região norte do Brasil, a situação epidemiológica é pouco elucidada. Objetivou-se, assim, por meio deste estudo, determinar a soropositividade de ovinos para Lentivírus no município de Porto Acre, Amazônia Ocidental, Brasil. Foram coletadas 122 amostras de sangue de ovinos e como método diagnóstico foi empregada a imunodifusão em gel de agarose, utilizando a proteína p28 do capsídeo como antígeno. A soropositividade dos ovinos ao teste foi de 8,2% (10/122). Em 80% (4/5) das propriedades investigadas, detectou-se a presença de animais soropositivos. É válido ressaltar ainda que a aquisição de pequenos ruminantes advindos de outros estados provavelmente representou um risco à sanidade ovina no município de Porto Acre, Amazônia Ocidental, Brasil. Conclui-se que existe a necessidade de mais investigações sistemáticas sobre a prevalência de LVPR no estado do Acre.