Immune response dynamics of recent and chronic small ruminant lentivirus infection in the male reproductive system / Dinâmica da resposta imunológica da infecção recente e crônica por lentivírus de pequenos ruminantes no sistema reprodutor de machos

The objective of this study was to analyze the immune responses of bucks to small ruminant lentivirus (SRLV) with a focus on the reproductive system of males with recent and chronic infection. A total of 12 bucks were selected, six seronegative and six seropositive with chronic natural infection for more than 18 months (chronic infection group). After selecting the animals, the six seronegative males were intravenously inoculated with caprine arthritis-encephalitis virus (CAEV)-Co viral strain at a titer of 10-5,6 TCID50/mL. After viral inoculation, this group was called the recent infection group and was monitored weekly with the chronically infected group for 180 days with blood serum and seminal plasma Western Blot (WB) analysis. Of the animals with chronic SRLV infection, 18.94% (50/264) showed anti-SRLV antibodies in at least one of the samples, and 81.06% (214/264) were negative. Anti-SRLV antibodies were detected in 27.27% (36/132) of the blood serum samples from this group, while 10.60% (14/132) were reactive in the seminal plasma WB test. The animals inoculated with CAEV-Co became seropositive after the third week of viral inoculation. In this group, 31.06% (41/132) of seminal plasma samples had anti-SRLV antibodies, and of these, 70.73% (29/41) coincided with blood serum results. Of the remaining 29.27% (12/41), the seminal plasma sample of only three animals (RIA2, RIA3, and RIA5) had anti-SRLV antibodies. One of the animals with a recent infection presented anti-SRLV antibodies only in seminal plasma samples, possibly due to virus compartmentalization. Intermittent viral shedding was observed in both biological samples, regardless of the infection stage. The immune response in bucks with recent SRLV infection is more significant than in chronically infected animals. Regardless of the stage of infection, there is a fluctuation in antibody levels, therefore, this creates a risk of false-negative samples when performing the diagnosis.

O objetivo desse estudo foi analisar a resposta imunológica aos lentivírus de pequenos ruminantes (LVPR) com enfoque no sistema reprodutor de machos com infecção recente e crônica. Para isso, foram selecionados 12 reprodutores caprinos, sendo seis soronegativos e seis soropositivos com infecção natural crônica há mais de 18 meses (grupo com infecção crônica). Após seleção dos animais, os seis machos soronegativos foram inoculados com a cepa viral do vírus da artrite encefalite caprina (CAEV)-Co, título 10-5,6 TCID50/mL, por via intravenosa. A partir da inoculação viral este agrupamento passou a ser denominado de grupo com infecção recente e juntamente com o grupo com infecção crônica foram acompanhados, semanalmente por 180 dias, com análise dos testes de Western Blot (WB) no soro sanguíneo e plasma seminal. Nos animais com infecção crônica para LVPR, 18,94% (50/264) apresentaram anticorpos anti-LVPR em pelo menos uma das distintas amostras, e 81,06% (214/264) tiveram resultados negativos. Das amostras de soro sanguíneo do referido grupo, em 27,27% (36/132) detectou-se anticorpos anti-LVPR, enquanto que no plasma seminal 10,60% (14/132) foram reagentes no teste de WB. Nos animais inoculados com o CAEV-Co, ocorreu a soroconversão após a terceira semana da inoculação viral. Nesse grupo, 31,06% (41/132) das amostras de plasma seminal tiveram anticorpos anti-LVPR, sendo que dessas 41, 70,73% (29/41) coincidiram com resultado das amostras de soro sanguíneo. Nos 29,27% (12/41) restante, houve a detecção somente no plasma seminal e eram amostras provenientes de três animais (AIR2, AIR3 e AIR5). Em um dos animais com infecção recente, só foi identificado anticorpos anti-LVPR em amostras de plasma seminal, possivelmente em função da compartimentalização do vírus. Intermitência viral foi observada em ambas as amostras biológicas, independentemente do estágio de infecção. Conclui-se que a resposta imunológica em reprodutores com infecção recente LVPR é mais acentuada do que em animais cronicamente infectados. E, independentemente do estágio da infecção há uma flutuação nos níveis de anticorpos, sendo, portanto, um fator de risco, em virtude da existência de amostras falso-negativo ao realizar o diagnóstico.

Detection of Maedi-Visna virus from sheep bronchoalveolar lavage by Nested PCR evaluation of different primers pairs / Detecção do vírus Maedi-Visna em amostras de lavado bronco-alveolar de ovinos através da técnica de Nested PCR utilizando diferentes pares de primers

Background: Small ruminant lentiviruses (SRLV) are characterized by a high degree of genetic variability related to your replication process, resulting in several strains in different geographic regions. The Polymerase Chain Reaction (PCR) is very successful in the detection of proviral DNA of SRLV, however, due to the high variability of the lentivirus genome, the efficiency and sensibility of PCR depends mainly on the specificity of the primers designed and the choice of the amplified target viral region. The aim of this study was to compare detection of Maedi Visna Virus (MVV) from bronco alveolar lavage samples of sheep by Nested PCR using primers for the gag and LTR genes. Materials, Methods & Results: Samples of sheep bronchoalveolar lavage (n = 58) from slaughterhouse in the Metropolitan Region of Fortaleza were previously tested by nested PCR using primers for region gag. Thereafter, these samples were tested by nested PCR with primers designed for the LTR region. Both tests were conducted using thermocycler (Biocycler®) under the following conditions: initial denaturation at 94C for 5 min, followed by 35 cycles of denaturation at 94C for 1 min, annealing of primers at 56C for 1 min and extension of DNA at 72C for 45 s with a final extension at 72 for 7 min. The first and second round were performed under the same conditions. Every amplification was performed [...]

Detection of Maedi-Visna virus from sheep bronchoalveolar lavage by Nested PCR evaluation of different primers pairs / Detecção do vírus Maedi-Visna em amostras de lavado bronco-alveolar de ovinos através da técnica de Nested PCR utilizando diferentes pares de primers

Background: Small ruminant lentiviruses (SRLV) are characterized by a high degree of genetic variability related to your replication process, resulting in several strains in different geographic regions. The Polymerase Chain Reaction (PCR) is very successful in the detection of proviral DNA of SRLV, however, due to the high variability of the lentivirus genome, the efficiency and sensibility of PCR depends mainly on the specificity of the primers designed and the choice of the amplified target viral region. The aim of this study was to compare detection of Maedi Visna Virus (MVV) from bronco alveolar lavage samples of sheep by Nested PCR using primers for the gag and LTR genes. Materials, Methods & Results: Samples of sheep bronchoalveolar lavage (n = 58) from slaughterhouse in the Metropolitan Region of Fortaleza were previously tested by nested PCR using primers for region gag. Thereafter, these samples were tested by nested PCR with primers designed for the LTR region. Both tests were conducted using thermocycler (Biocycler®) under the following conditions: initial denaturation at 94C for 5 min, followed by 35 cycles of denaturation at 94C for 1 min, annealing of primers at 56C for 1 min and extension of DNA at 72C for 45 s with a final extension at 72 for 7 min. The first and second round were performed under the same conditions. Every amplification was performed [...](AU)

Detection of proviral sequences of the caprine arthritis encephalitis virus in saliva / Detecção de sequências do DNA proviral do vírus da artrite encefalite caprina em saliva

Background: Goats can be infected by a lentivirus called the caprine arthritis encephalitis virus (CAEV) that causes an infectious disease characterized by a chronic. For replication, the CAEV, integrate as a provirus in the DNA of the host cell genome. By consequence, infection of cells persistent life-long infection of the animal. The CAEV can be found in most body fluids, It has been demonstrated its presence in blood, milk, semen. However, the search for the CAEV in other body fluids besides blood is important to assess possible viral transmission. The aim of this study was to determine whether the presence of proviral DNA sequences in saliva of animals infected.Materials, Methods & Results: The study was carried out on the farm belonging to the Embrapa Goat and Sheep Research Center, located in the municipality of Sobral, Ceará, Brazil. To assess the oral fluid for the presence of CAEV, samples of saliva from eight infected breeders were collected by suctioning saliva from the oral cavity on the side region of the breeders lower molar teeth using a probe coupled to a plastic 5 mL syringe. And pro-viral DNA was extracted from the samples using NaCl and proteinase K. Two rounds of polymerase chain reaction (nested PCR) were carried out to amplify the final 187 pb fragment of the pro-viral DNA. All the oligonucleotide primers were determined from the gag region...(AU)

Evaluation of matrix metalloproteinases in blood from buck naturally infected with Caprine Arthritis Encephalitis in Semiarid region of Brazil / Avaliação das metaloproteinases de matriz no sangue de reprodutores caprinos naturalmente infectados com Artrite Encefalite Caprina na Região Semiárida do Brasil

Background: Studies have attempted to understand the importance of metalloproteinase (MMPs) in the pathogenesis ofdiseases caused by lentiviruses, being the human immunodeficiency virus (HIV) the most investigated. Despite advancesin studies with MMPs in others diseases, research correlating the presence and activity of gelatinases in animals affectedby caprine arthritis encephalitis virus (CAEV), a lentiviruses, are incipient and there is a need for research aiming to understand the dynamic of these enzymes in animals infected and its relation to pathological condition. The aim of this studywas to evaluate the presence and activity of the MMPs in blood serum of chronically infected bucks by CAEV.Materials, Methods & Results: The experiment was constituted by two groups (n = 5 each group). The first one wascomposed of five naturally infected bucks (4-5 years) and second group constituted of five seronegative bucks (3-4 years)for CAE. Serology was performed using the Western Blotting (WB) and confirmed by Polymerase Chain Reaction (PCR).These bucks belong to the experimental flock at Embrapa Goats and Sheep and the seropositive bucks were confirmed forCAE in the first two years. Blood samples were collected by puncturing the jugular vein from animals and evaluated byzymography (SDS-PAGE) using gelatin as substratum. Clinical examination was performed by evaluating the temperature (T), cardiac frequency (FC), respiratory frequency (FR) and clinical articular index (IAC). The IAC was calculatedby measuring the circumference of carpal joint and metacarpal bone height (difference between greater extent carpal andmetacarpal lesser extent). In infected and control groups were found molecular mass bands of 66 kDa (MMP-2), 72 kDa(pro-MMP-2), 86 kDa (MMP-9) and 92 kDa (pro-MMP-9). The correlation...(AU)

Detection of proviral sequences of the caprine arthritis encephalitis virus in saliva / Detecção de sequências do DNA proviral do vírus da artrite encefalite caprina em saliva

Background: Goats can be infected by a lentivirus called the caprine arthritis encephalitis virus (CAEV) that causes an infectious disease characterized by a chronic. For replication, the CAEV, integrate as a provirus in the DNA of the host cell genome. By consequence, infection of cells persistent life-long infection of the animal. The CAEV can be found in most body fluids, It has been demonstrated its presence in blood, milk, semen. However, the search for the CAEV in other body fluids besides blood is important to assess possible viral transmission. The aim of this study was to determine whether the presence of proviral DNA sequences in saliva of animals infected.Materials, Methods & Results: The study was carried out on the farm belonging to the Embrapa Goat and Sheep Research Center, located in the municipality of Sobral, Ceará, Brazil. To assess the oral fluid for the presence of CAEV, samples of saliva from eight infected breeders were collected by suctioning saliva from the oral cavity on the side region of the breeders lower molar teeth using a probe coupled to a plastic 5 mL syringe. And pro-viral DNA was extracted from the samples using NaCl and proteinase K. Two rounds of polymerase chain reaction (nested PCR) were carried out to amplify the final 187 pb fragment of the pro-viral DNA. All the oligonucleotide primers were determined from the gag region...

Evaluation of matrix metalloproteinases in blood from buck naturally infected with Caprine Arthritis Encephalitis in Semiarid region of Brazil / Avaliação das metaloproteinases de matriz no sangue de reprodutores caprinos naturalmente infectados com Artrite Encefalite Caprina na Região Semiárida do Brasil

Background: Studies have attempted to understand the importance of metalloproteinase (MMPs) in the pathogenesis ofdiseases caused by lentiviruses, being the human immunodeficiency virus (HIV) the most investigated. Despite advancesin studies with MMPs in others diseases, research correlating the presence and activity of gelatinases in animals affectedby caprine arthritis encephalitis virus (CAEV), a lentiviruses, are incipient and there is a need for research aiming to understand the dynamic of these enzymes in animals infected and its relation to pathological condition. The aim of this studywas to evaluate the presence and activity of the MMPs in blood serum of chronically infected bucks by CAEV.Materials, Methods & Results: The experiment was constituted by two groups (n = 5 each group). The first one wascomposed of five naturally infected bucks (4-5 years) and second group constituted of five seronegative bucks (3-4 years)for CAE. Serology was performed using the Western Blotting (WB) and confirmed by Polymerase Chain Reaction (PCR).These bucks belong to the experimental flock at Embrapa Goats and Sheep and the seropositive bucks were confirmed forCAE in the first two years. Blood samples were collected by puncturing the jugular vein from animals and evaluated byzymography (SDS-PAGE) using gelatin as substratum. Clinical examination was performed by evaluating the temperature (T), cardiac frequency (FC), respiratory frequency (FR) and clinical articular index (IAC). The IAC was calculatedby measuring the circumference of carpal joint and metacarpal bone height (difference between greater extent carpal andmetacarpal lesser extent). In infected and control groups were found molecular mass bands of 66 kDa (MMP-2), 72 kDa(pro-MMP-2), 86 kDa (MMP-9) and 92 kDa (pro-MMP-9). The correlation...

Detecção do vírus da Artrite Encefalite Caprina por nested PCR e nested RT-PCR em ovócitos e fluido uterino / Caprine Arthritis Encephalitis virus is detected in oocytes and uterine fluid by nested PCR and nested RT-PCR

A Artrite Encefalite Caprina (CAE) é uma enfermidade infectocontagiosa causada por um vírus pertencente ao gênero lentivírus, denominado de vírus da Artrite Encefalite Caprina (CAEV). O CAEV é encontrado em vários tecidos, como o nervoso, o pulmonar, o da glândula mamária e do trato genital masculino e feminino. Desta forma, objetivou-se com este trabalho identificar a presença do CAEV, pelas técnicas de diagnóstico moleculares, em ovócitos e fluido uterino, visando avaliar a possibilidade de transmissão do CAEV pela reprodução. Foram selecionadas 13 cabras comprovadamente infectadas, as quais foram submetidas à eutanásia para coleta do aparelho reprodutor, aspiração do fluido uterino e dissecção dos ovários para coleta de ovócitos. Para identificação do CAEV nas amostras coletadas, na forma de provírus e na forma livre, foram realizadas as técnicas de PCRn e RT-PCRn, respectivamente. Observaram-se que 53,8% dos ovócitos foram positivos à técnica de RT-PCRn, enquanto apenas 9,1% foram positivos à PCRn. A técnica de RT-PCRn também identificou o vírus no fluido uterino de 46,1% das fêmeas testadas. Embora as 13 cabras em experimento fossem portadoras do CAEV, 30,8% apresentaram resultados negativos na PCRn e RT-PCRn em todas as amostras analisadas (ovócito e fluido uterino). Conclui-se que a PCRn e a RT-PCRn podem ser utilizadas no diagnóstico da CAE tendo os ovócitos e o fluido uterino como materiais de análise, e que a presença do CAEV nestes materiais aponta para o risco da transmissão do CAEV através das tecnologias reprodutivas aplicadas às fêmeas.

Caprine arthritis-encephalitis (CAE) is an infectious disease caused by the caprine arthritis-encephalitis virus (CAEV), belonging to the lentivirus genus. The presence of the virus has been observed in the nervous system, respiratory tract and mammary gland, and also in the male and female genital tract. The objective of this study is to identify the virus in oocyte and uterine fluid of infected goats by molecular diagnostic techniques, in order to assess the possibility of CAEV transmission with reproduction. Thirteen infected goats were selected and submitted to euthanasia for the collection of the reproductive system, aspiration of the uterine fluid and dissection of ovaries for oocyte collection. In order to identify the CAEV in the collected material, in the protovirus and free forms, it was submitted to the nRT-PCR and nPCR techniques, respectively. As a result, it was observed that 53.8% of oocytes were positive to nRT-PCR, while only 9.1% were positive to nPCR. The nRT-PCR also identified the virus in the uterine fluid of 46.1% of the tested females. Even though the 13 goats had CAEV, 30.8% presented negative results in nPCR and nRTPCR in all of the analyzed samples (oocyte and uterine fluid). This work concludes that nRT-PCR and nPCR can be used in the diagnosis of CAE for the analysis with oocytes and uterine fluid, and that the presence of CAEV in these materials points out to the risk of CAEV transmission through reproductive technologies used in females