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The production performance of a livestock herd can be compromised by various diseases. In sheep, maedi-visna (MV) infections, which have a chronic nature, are caused by a virus (maedi-visna virus (MVV)) belonging to the genus Lentivirus of the Retroviridae family. The infection can cause significant economic losses and has considerable health impacts on sheep breeding in production systems. Due to the importance of this disease in sheep flocks, the objective was to conduct a serosurvey of MVV in the states of Ceará (CE), Rio Grande do Norte (RN), Paraíba (PB), and Sergipe (SE). A total of, 3332 serum samples were collected in the four states, 1011 in CE, 931 in RN, 459 in PB, and 931 in SE, with the number of samples proportional to the actual herd size of each state. The samples were analyzed using the agar gel microimmunodiffusion test (AGID). Reproducers were revaluated using western blotting (WB). In addition to this serological survey, we administered an investigative questionnaire to identify possible risk factors that facilitate the introduction and spread of diseases (location, category, sex, breed type, creation system, production, herd size, and association with goats). After analysis of the sera using the AGID test, there was zero prevalence. Revaluating breeders by WB revealed a 5.5% prevalence of MV in the four states studied, with prevalences for the states of CE, RN, Paraiba, and SE of 2.3% (2/88), 10.4% (8/77), 3.6% (1/28), and 4.7% (2/42), respectively, corresponding to 13 breeders containing antibodies to the virus. These findings emphasized that the choice of diagnostic tests is extremely important for the early detection of seropositive animals and thus the prevention of the spread of the virus among herds in the region. [Estudo soroepidemiológico da maedi-visna em ovinos nos estados do Ceará, Rio Grande do Norte, Paraíba e Sergipe]. Resumo: O desempenho produtivo de um rebanho pode ser comprometido por diversas enfermidades. Em relação aos ovinos, à ocorrência da Maedi - Visna (MV) doença infecciosa, de caráter crônico, causada por um vírus (Maedi-Visna Vírus - MVV) pertencente ao gênero Lentivirus da família Retroviridae. A infecção pode causar importantes perdas econômicas e elevados impactos sanitários nos sistemas de produção da ovinocultura. Em virtude da importância desta enfermidade nos rebanhos ovinos, o objetivo do estudo foi realizar um levantamento soroepidemiológico do vírus da Maedi - Visna (MVV) nos estados do Ceará (CE), Rio Grande do Norte (RN), Paraíba (PB) e Sergipe (SE). Para tanto, foram coletadas 3332 amostras de sangue nos quatro estados, sendo 1011 do CE, 931 do RN, 459 da PB e 931 de SE, sendo este número de amostras proporcional ao rebanho efetivo de cada estado. As amostras foram analisadas utilizando o teste de microimunodifusão em gel de ágar (IDGA). Os reprodutores foram reavaliados pela técnica de Western Blot (WB). Associado a esse levantamento sorológico foi aplicado um questionário epidemiológico para identificar possíveis fatores de risco que podem facilitar a introdução e disseminação de enfermidades (localização, categoria, sexo, tipo racial, sistema de criação, produção, tamanho de rebanho e associação com caprinos). Após análise dos soros pelo teste de IDGA foi verificada uma soroprevalência nula nos rebanhos estudados. Os reprodutores reavaliados pelo WB apresentaram uma prevalência geral de 5,5% da MV nos quatro estados estudados, sendo que os estados do Ceará, Rio Grande do Norte, Paraíba e Sergipe apresentaram, 2,3% (2/88), 10,4% (8/77), 3,6% (1/28) e 4,7% (2/42) respectivamente, correspondendo a 13 reprodutores que apresentaram presença de anticorpos. Diante desses resultados, ressalta-se que a escolha dos testes diagnósticos é de extrema importância para que haja uma detecção precoce de animais soropositivos, e assim evitar a disseminação do vírus nos rebanhos da região. Made available in DSpace on 2018-09-26T00:42:27Z (GMT). No. of bitstreams: 1 CNPC2018Seroepidemiological.pdf: 312914 bytes, checksum: d6f6707fe9acafbcfa45385a89af2191 (MD5) Previous issue date: 2018-09-25

Small ruminant lentiviruses (SRLV) are widely distributed in the world and cause productive and economic losses. These pathogens have high genetic variability, which hinders diagnosis by molecular methods. Molecular characterization studies are good strategies for elaborating programs aimed to control or eradicate diseases. Hence, this study aimed to sequence, characterize and compare strains of SRLV, which were isolated from naturally infected goats from the states of Ceara, Rio Grande do Norte and Minas Gerais, Brazil, based on total nucleotide sequences of gag and pol genes. SRLV isolates from the microbiological collection of Sheep and Goat of Embrapa were cultured in goat synovial membrane. Supernatants containing cells were stored and viral particles were purified with sucrose density gradient centrifugation for RNA extraction. Then, sequencing in HiSeq 2500 platform (Illumina) was performed to obtain the total genome of the SRLV strains. The software MEGA version 7.0 was used for sequence editing and phylogeny analysis. A total of 17 isolates were multiplied in cellular culture and six were viable, which were two from Minas Gerais (MG), one from Rio Grande do Norte (RN) and three from Ceara (CE). Two sequences were obtained from the sequencing by synthesis, one from MG (BRMG CNPC) and one from RN (BRRN CNPC). From the total SRLV genome (9,200 bases), sample BRMG CNPC generated a consensus of 6,378 bases, approximately 69,34% of the viral genome and sample BRRN CNPC generated a consensus of 5,351 bases, which comprised 58.16% of the total genome. Samples in this study demonstrated closer phylogenetic relation with genotype B (B1 group). The remaining strains sequenced in Brazil are also present in the same group with the exception of three samples from genotype A. Immunodominant regions of gag gene and motifs of pol gene were similar to the standard CAEV Cork strain. For the first time, the complete gag and pol genes were sequenced in Brazil from the strain BRMG CNPC isolated in Minas Gerais state. In addition, 2490 bases from the total of 3316 bases were sequenced from the pol gene of the BRRN CNPC sample from Rio Grande do Norte.

This study aimed to evaluate by means of Nested Polymerase Chain Reaction (nPCR), co-cultivation and sequencing, with genetic comparison between strains (mother/newborn), the occurrence of vertical transmission of Small Ruminant Lentiviruses (SRLV) from naturally occurring nannies infected for their offspring. For the detection of SRLV seropositive progenitors, blood was collected from 42 nannies in the final third of gestation in tubes with and without anticoagulant. The diagnostic tests used were Western Blot (WB) and nPCR. During the period of birth, the same blood collection procedure was performed on 73 newborns at zero hours of birth, with the same diagnostic tests. Seventeen blood samples from seven-day-old kids, proven positive for SRLV by nPCR, chosen at random, were subjected to coculture in goat synovial membrane (GSM) cells for 105 days. The pro-viral DNA extracted from the cell supernatant from the coculture was subjected to nPCR. For DNA sequencing from the nPCR products, nine positive samples were chosen at random, four nannies with their respective offspring, also positive. Each sample was performed in triplicate, thus generating 27 nPCR products of which only 19 were suitable for analysis. Among the 42 pregnant goats, in 50% (21/42) pro-viral DNA was detected by nPCR, while in the WB, only 7.14% (3/42) presented antibodies against SRLV. Regarding neonates, of the 73 kids, 34 (46.57%) were positive for the virus, using the nPCR technique, while in the serological test (WB), three positive animals (4.10%) were observed. The coculture of the 17 samples with a positive result in the nPCR was confirmed in viral isolation by amplification of the SRLV pro-viral DNA. When aligned, the pro-viral DNA sequences (nannies and their respective offspring) presented homology in relation to the standard strain CAEV Co. It was concluded that the transmission of SRLV through intrauterine route was potentially the source of infection in the newborn goats.

The objective of this study was to demonstrate iatrogenic transmission of small ruminant lentivirus (SRLV) from goats to sheep and horizontal transmission between sheep. The study was conducted on a farm with separate goat and sheep rearing, and animals were monitored for lentivirus occurrence by clinical examination and testing by immunoblotting (IB), agar gel immunodiffusion (AGID), and nested polymerase chain reaction (nPCR). Positive results had not been observed in the sheep flock until this study. Conversely, virus positive dairy goats were known. For this reason, the farm performed the caprine arthritis-encephalitis (CAE) control program. The study was designed with a sheep group that presented positive animals for SRLV by nPCR. It was verified that three newborn animals in this group were rejected by their mothers and consequently received milk from the goat herd. These three animals remained with another 20 sheep of the same age, totaling 23 animals. After one year, during monitoring, 11 of the 23 animals in the group presented positive results in the nPCR and three demonstrated seroconversion by IB. Of the animals that had received goat milk, two had positive results in the nPCR and IB. The 11 animals positive in the nPCR were followed and it was verified that five animals did not present further positive results in the nPCR, nor seroconversion; two continued presenting positive results in the nPCR but were negative in the IB and AGID and four were positive in the nPCR, IB, and AGID. Thus, it was possible to demonstrate iatrogenic interspecific infection and the occurrence of horizontal caprine lentivirus transmission among sheep. [Infecção de ovinos pelo lentivírus caprino]. Resumo: O objetivo foi demonstrar a transmissão iatrogênica de lentivírus de pequenos ruminantes (LVPR) de caprinos para ovinos e a transmissão horizontal entre ovinos. O estudo foi conduzido em propriedade com criação não consorciada de caprinos e ovinos, monitorada para ocorrência de lentiviroses, a partir de acompanhamento clínico e testes de immunoblotting (IB), imunodifusão em gel de agarose (IDGA) e reação em cadeia da polimerase do tipo nested (nPCR). Resultados positivos no rebanho ovino não haviam sido observados até então. Por outro lado, sabia-se que o rebanho caprino leiteiro era positivo. Por este motivo, a propriedade realizava programa de controle da artrite-encefalite caprina (CAE). O estudo foi delineado em um lote de ovinos que apresentou animais positivos para LVPR na nPCR. Verificou-se que três animais neonatos deste lote foram rejeitados por suas mães e por isso receberam leite do rebanho caprino. Estes três animais permaneceram com outros 20 ovinos de mesma faixa etária, totalizando 23 animais. Após um ano, durante a realização do monitoramento, dos 23 animais do lote, 11 apresentaram resultados positivos na nPCR e três demonstraram soroconversão por IB. Dos animais que haviam recebido leite do rebanho caprino, dois tiveram resultados positivos na nPCR e no IB. Os 11 animais positivos na nPCR foram acompanhados, verificando-se que cinco animais não apresentaram mais resultados positivos na nPCR, nem soroconverteram; dois continuaram apresentando resultados positivos na nPCR, mas negativos no IB e IDGA e quatro apresentaram-se positivos na nPCR, no IB e no IDGA. Dessa forma, foi possível demonstrar a infecção interespecífica de forma iatrogênica e a ocorrência de transmissão horizontal do lentivírus caprino entre ovinos. Made available in DSpace on 2018-10-06T00:41:36Z (GMT). No. of bitstreams: 1 CNPC2018Sheep.pdf: 169811 bytes, checksum: 20856e1b8518b49b8c3f85f3c43af5bc (MD5) Previous issue date: 2018-10-05

The objective of this study was to verify the presence of small ruminant lentivirus in the amniotic fluid of goats using molecular tests and viral isolation by cocultivation in the amniotic fluid of naturally infected goats. The study analyzed eight goats: seven were small ruminant lentivirus-positive and one was negative. The amniotic fluid was collected from each of the eight animals during cesarean section at 147 days of pregnancy. Cocultivation was undertaken using secondary goat nictitating membrane cell cultures obtained by explant from a small ruminant lentivirus-negative calf followed by trypsinization and sub-cultivation of the cells for 63 days. During this period, five supernatant collections were performed for DNA extraction and subsequent nested polymerase chain reaction. DNA was extracted from the amniotic fluid after 3 h of cellular sedimentation, from which a sample of 600 μL was taken from the sediment and another 600 μL sample from the supernatant. After DNA extraction, nested polymerase chain reaction was performed. Of the eight goats, 62.5 % (05/08) were small ruminant lentivirus-positive, with 43.75 % (07/16) of the total samples positive when considering the two repetitions (supernatant and cell sediment). Moreover, positivity was confirmed by small ruminant lentivirus pro-viral DNA amplification in the cell supernatant throughout the cocultivation period. Small ruminant lentivirus were present in the amniotic fluid samples from the naturally infected goats indicating an intrauterine transmission route. Moreover, this biological fluid can be adopted for the diagnosis of these lentiviruse because it is an important risk factor related to intrauterine transmission.

Background: Caprine Arthritis Encephalitis (CAE) is a disease that causes productive losses in dairy goat flocks due to the reduction in milk production, followed by lesions in joints and mammary glands. An early diagnosis is essential, considering that there is frequent occurrence of asymptomatic animals. Hence, this study aimed to perform a comparison of immunological and molecular based diagnostic tests, represented by Agar Gel Immunodiffusion (AGID), Western Blot (WB) and nested Polymerase Chain Reaction (nPCR). In addition, the mammary glands (MG) of dairy goats were clinically evaluated. Material, Methods & Results: Blood collection and clinical examination were performed in 1191 dairy goats of 12 farms located in Northeastern and Southeastern regions of Brazil. Serological (AGID, WB) and molecular (nPCR) test results were compared and the data, along with MG alterations, were analyzed using Epi-info 7 and WinEpiscope 2.0. Seroprevalence in AGID test was 41.14% (490/1191). In WB, 51.47% (613/1191) of animals were seropositive and nPCR detected 69.44% (827/1191) positive animals. Hence, WB was more sensitive (P < 0.001) than AGID. However, nPCR detected more positive animals than AGID (P < 0.001) and WB (P < 0.001). The analysis of mammary glands revealed that 105 out of 1096 nanny goats presented alterations, of which 101 presented altered consistency, 16 presented elevated temperatures and 60 had enlarged retromammary lymph nodes. There was significant statistic difference (P < 0.05) only when comparing the results of serological tests with MG alterations.Discussion: In general, AGID technique is most frequently used when screening flocks for the disease due to the practicality and low cost this test presents. However, the results demonstrated that AGID detected the lowest number of positive animals. This low sensitivity that the test presented may be attributed to its antigen-antibody interaction mechanism, considering that agar gel precipitation requires multiple interactions. In addition, WB was more effective than AGID in detecting antibodies. On the other hand, nPCR was important for the detection of infected animals that serological tests failed to detect. The intermittence of immunological response observed in the serological tests may be explained by the variation of antibodies levels that may occur during life. Likewise, viral compartmentalization would justify the intermittent detection of proviral DNA. Hence, the results can be influenced by the viral intermittence, test sensitivity, late seroconversion and statistic values that can be calculated (sensitivity, specificity, positive predictive value, negative predictive level and kappa). Crossing the results of the diagnostic tests with the different mammary gland alterations, it was shown that there was a statistically significant difference (P

The aim of this study was to evaluate in vitro and in vivo the effect of sodium dodecyl sulfate (SDS) on the caprine lentivirus (CLV) in colostrum and milk. This was performed to develop a practical and efficient method of blocking the lactogenic transmission of the virus. In the in vitro experiment, colostrum and milk were treated with 0.25%; 0.50% and 1% SDS. Then, somatic cells of colostrum and milk were submitted to co-culture with caprine synovial membrane cells (CSM). In the in vivo test, goats were fed with colostrum and milk provided from CLV-positive goats treated with SDS in the same concentrations used in the in vitro experiment. Animals were tested by nested polymerase chain reaction (nPCR) and Western blot (WB) assays. In the in vitro experiment, inhibitory activity against CLV without inactivation occurred in colostrum with all SDS concentrations. However, concentrations of 0.25 and 0.5% SDS presented only inhibitory activity against CLV in milk cells, and 1% concentration provided inactivation of the virus. In the in vivo tests, none of the three concentrations of SDS was effective in inactivating LVC in colostrum or goat milk, which was confirmed by seroconversion and presence of proviral DNA in animals afterwards. [Avaliação in vitro e in vivo do dodecil sulfato de sódio (SDS) como inativador do lentivírus caprino (LVC) em colostro e leite]. Resumo: O objetivo da pesquisa foi avaliar in vitro e in vivo o efeito do dodecil sulfato de sódio (SDS) sobre o lentivírus caprino (LVC) no colostro e no leite, a fim de desenvolver um método prático e eficiente no bloqueio da via de transmissão lactogênica do vírus. No experimento in vitro, o colostro e o leite de cabras positivas foram tratados com SDS a 0,25%, 0,50% e 1,0%. Em seguida, as células somáticas do colostro e do leite foram obtidas e direcionadas ao cocultivo com células de membrana sinovial caprina (MSC). No teste in vivo, os cabritos foram alimentados com colostro e leite providos de cabras positivas para LVC, tratados com SDS nas mesmas concentrações usadas no teste in vitro. Os animais foram acompanhados pelos testes de reação em cadeia da polimerase nested (nPCR) e western blot (WB). Nos resultados in vitro, no colostro, observou-se que, em todas as concentrações de SDS, ocorreu uma atividade inibitória contra o LVC, sem a inativação. Em relação às células do leite, o SDS apresentou, nas concentrações de 0,25 e 0,5%, atividade inibitória contra o LVC, e na concentração de 1%, houve inativação viral. Nos testes in vivo, as três concentrações de SDS testadas não foram efetivas na inativação do LVC no colostro e no leite caprino, o que se comprovou pela soroconversão e pela presença de DNA proviral nos animais. Made available in DSpace on 2018-09-12T00:39:29Z (GMT). No. of bitstreams: 1 CNPC2018Invitro.pdf: 784450 bytes, checksum: a259846f11ab6746f0c67f0f432de2de (MD5) Previous issue date: 2018-09-11

This study was conducted to evaluate caprine arthritis encephalitis virus (CAEV) transmission among sheep using 15 lambs that were distributed in 2 experimental groups. The exposed group consisted of 10 lambs that remained with their mothers, who were experimentally infected with CAEV. The non-exposed group was characterized as the control group and was comprised of 5 lambs that remained with their CAEV-negative mothers. Blood samples were collected monthly from birth until 1 year of life. To evaluate the transmission, an agar gel immunodiffusion test (AGID), enzyme immunoassay (ELISA), immunoblotting (IB), and nested polymerase chain reaction (nPCR) techniques were used. The non-exposed group was negative in all of the tests throughout the whole experiment. In the exposed group, 2 individuals had positive nPCR results. Positive nPCR samples were sequenced for comparison with the original goat strains and were shown to be similar to the CAEV-Cork strain. Seroconversion was not detected, and clinical manifestations were not observed. Thus, after 1 year of observation, it was verified that CAEV transmission among sheep is possible; however, with discreet frequency. This was an initial study, and other experiments are needed to analyze the adaptive capacity of the CAEV to remain in an infected sheep flock and cause the disease. [Transmissão do vírus da artrite encefalite caprina entre ovinos]. Resumo: O estudo foi conduzido para avaliar a transmissão do vírus da artrite encefalite caprina (CAEV) entre ovinos, utilizando 15 cordeiros, distribuídos em dois grupos experimentais. O grupo exposto foi constituído por 10 cordeiros, mantidos com suas mães, que foram infectadas, experimentalmente, com CAEV. O grupo não exposto caracterizou-se como grupo controle e foi formado por cinco cordeiros, mantidos com suas matrizes, negativas para CAEV. Foram colhidas amostras de sangue mensalmente, do periodo que compreende o nascimento até um ano de vida. Para avaliar a transmissão, foram utilizadas as técnicas de imunodifusão em gel de agarose (IDGA), ensaio imunoenzimático (ELISA), immunoblotting (IB) e reação em cadeia da polimerase do tipo nested (nPCR). O grupo não exposto se manteve negativo aos testes durante todo o experimento. Já no grupo exposto, dois indivíduos apresentaram resultados positivos na nPCR. As amostras positivas na nPCR foram sequenciadas para serem comparadas com as cepas originais de caprinos, comprovando se tratar de lentivírus semelhante à cepa CAEV-Cork. A soroconversão não foi detectada e a manifestação clínica não foi observada. Sendo assim, após um ano de observação, verificou-se que a transmissão do CAEV entre ovinos é possível, entretanto, com discreta frequência. Este foi um estudo inicial, e outros experimentos são necessários para analisar a capacidade adaptativa do CAEV de permanecer em rebanho ovino infectado e, com isso, causar doença. Made available in DSpace on 2018-09-21T00:46:25Z (GMT). No. of bitstreams: 1 CNPC2018Transmission.pdf: 1669152 bytes, checksum: 57ddeb25c242586e1b38691f197f4dae (MD5) Previous issue date: 2018-09-20

This study aimed to carry out a serum epidemiological survey of goat arthritis encephalitis in the sisal region of Bahia, Brazil, and to evaluate risk factors. We evaluated 831 samples of goat blood serum among males and females older than six months, from 49 farms distributed among the municipalities of Araci, Cansanção, Conceição do Coité, Itiúba, Monte Santo, Nordestina, Queimadas, Santa Luz, São Domingos and Valente. An epidemiological questionnaire for the analysis of possible risk factors was applied. All sera were tested for immune-agar gel. The seroprevalence obtained in surveyed herds was 1.56% (13/831). There was significant difference (p

Background: Contagious agalactia is an infectious disease caused by Mycoplasma agalactiae (M. agalactiae) that occurs in small ruminants leading to productive and economic losses. Due to the similarity of clinical signs presented in Caprine Arthritis Encepha­litis (CAE), which is a viral disease, a differential diagnosis is important. Therefore, this study aimed to investigate the presence of anti-Mycoplasma agalactiae antibodies in dairy goat flocks in Ceará State and possible correlation with CAE.Materials, Methods & Results: The research was performed in four mesoregions in Ceará State (Metropolitan Region of Fortaleza- MRF; Northeast Ceará - NeC; North Ceará - NC; Sertões in Ceará - SC), from which 16 productions located in 10 cities with the highest representativeness for goat milk production within the State or mesoregion were sampled. A total of 417 females and 69 males (486 animals) of breeds with dairy production aptitude, pure or crossbreed, maintained in semi-intensive or intensive systems, were tested. Blood serum was obtained by venipuncture of the jugular vein with vacuum pressure syringe followed by centrifugation at 1,500 g for 10min. Antibodies against the caprine arthritis encephalitis virus (CAEV) were detected with micro technique of agarose gel immunodiffusion (AGID) and Western Blot (WB). The anti-Mycoplasma agalactiae antibodies were detected with commercial kit of enzymatic immunoassay (IDEXX Laboratories™). Seroprevalence of M. agalactiae in dairy goat flocks in Ceará State was 0.62% (3/486). From the total of 16 visited productions, 18.75% (3/16) had seropositive animals for M. agalactiae located in MRF, NC and SC mesoregions. CAE was diagnosed in 56.25% (9/16) of productions with AGID and in 81.25% (13/16) with WB. In addition, 5.2% (25/486) of animals were seropositive for CAE with AGID and 16.6% (80/486) with WB. Animals that reacted positive for M. agalactiae were all females of pure breed with milk production aptitude in distinct mesoregions submitted to intensive rearing system. None of these animals was positive in neither test (AGID or WB) for CAE. Therefore, no correlation of results obtained in diagnosis of M. agalactiae by ELISA and CAEV by AGID or WB (P < 0.05) was identified. However, two out of three productions that were positive for M. agalactiae presented positive results for CAEV with frequencies of 10% and 20%.Discussion: Seroprevalence of M. agalactiae in Ceará State was low in comparison with other Brazilian states and even other countries. However, the presence of the pathogen in more than one mesoregion indicates that the disease occurs in different loca­tions within the State. Therefore, flocks in Ceará are susceptible to the infection, which may be favored by uncontrolled commerce that occurs with deficient surveillance, associated with the importation of animals to improve flock genetic quality. The presence of the pathogen in dairy goats may contribute to significant losses in the local production. On the other hand, CAE was diagnosed in nearly all productions proving the dissemination of this lentivirus infection among dairy goat flocks in Ceará State. Although an association between these diseases was not identified, the presence of a retrovirus in the organism may favor co-infection with another micro-organism, promoting the deficiency in the immune system of the host. In conclusion, M. agalactiae is present in different mesoregions of the Ceará State and control measures should be adopted in short term to prevent pathogen dissemination and, consequently reduce economic and productive losses in the local dairy goat production. No correlation was identified between the prevalence of infection by CAEV and M. agalactiae in this study.Keywords: correlation, diagnosis, caprine lentivirus, mycoplasmosis.

Resumo: A transmissão da artrite encefalite caprina pelo sêmen inviabiliza o uso de reprodutores soropositivos de alto valor genético. Como o vírus da artrite encefalite caprina (CAEV), é encontrado no sêmen na forma de RNA viral (vírus livre) ou DNA pró-viral dentro das células não espermáticas, possibilita que pesquisas com plantas com potencial antiviral sejam algo promissor para viabilizar o sêmen de reprodutores caprinos de grande valor zootécnico infectados. Diante disso, com essa revisão objetivou-se elucidar o potencial de fitocompostos, com comprovada ação antiviral, que poderiam vir a ser alvo de estudo para inativar o lentivírus caprino no sêmen. Inúmeras plantas de muitas famílias botânicas já foram estudadas para validação de efeito antiviral contra diversos vírus que acometem animais e humanos. Folhas, raízes, flores, e sementes demonstraram ao longo do tempo em sua constituição fitoquímica substâncias antivirais promissoras. De acordo com o tipo de planta, não apenas algumas partes dela, mas regiões inteiras da mesma são alvos das pesquisas por fitocompostos a ser usado como alternativa no combate as doenças virais. A base dos estudos nessa vertente são os metabólitos secundários, pois nas pesquisas têm apresentado compostos bioativos antivirais de alto potencial atuando nas diferentes fases do ciclo de desenvolvimento viral. Dessa forma, a biodiversidade da flora brasileira aliada à tendência de formar fitocompostos poderiam contribuir no avanço das pesquisas, a fim de encontrar uma alternativa de base natural, potencialmente eficaz, para eliminar os riscos de transmissão do CAEV pelo sêmen. [[Prospectives of herbal remedies in the treatment of caprine semen infected by Caprine Arthritis Encephalitis virus]. Abstract: The transmission of Caprine Arthritis Encephalitis in semen limits the use of seropositive bucks that have high genetic value. The Caprine Arthritis Encephalitis virus (CAEV) may be found in the form of RNA virus (free virus) or pro-viral DNA within non-spermatic cells in semen. Thus, researches involving plants with potential antiviral activity are promising for developing techniques that allow the use of semen from high-quality production goats that are infected by this virus. Therefore, this review aimed to elucidate the potential of phytocompounds that have known antiviral activity and could be used in studies to assess inactivation of the caprine lentivirus in semen. Several plants from many botanical families have been tested for validation of antiviral effect against viruses that infect animals and humans. Leaves, roots, flowers and seeds have demonstrated promising antiviral substances in their phytochemical composition. According to the type of plant, entire regions of the specimens are used for researching phytocompounds that can serve as alternatives in the treatment of viral diseases. Most of the studies involving this subject focus on secondary metabolites and several have identified high potential compounds with bioactive antiviral activity in different stages of the viral development cycle. Hence, the biodiversity of Brazilian flora, which has a tendency for phytocompound formation, provides adequate material for research aimed at finding a natural alternative to eliminate the risk of CAEV transmission through semen. Made available in DSpace on 2019-09-27T00:41:18Z (GMT). No. of bitstreams: 1 cnpc2019Perspectivas.pdf: 231992 bytes, checksum: 140c812e42b31d726cd363880d1d4f76 (MD5) Previous issue date: 2018

Made available in DSpace on 2014-09-30T18:18:25Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-04-01Bitstream added on 2014-10-01T14:03:35Z : No. of bitstreams: 1 S0102-09352014000200014.pdf: 1430988 bytes, checksum: ab5146a04db176f51f6cb1cef5ee68aa (MD5) A artrite-encefalite caprina (CAE) é diagnosticada rotineiramente pela técnica de imunodifusão em gel de agarose (IDGA), que é considerada pouco sensível. Objetivou-se com este estudo padronizar testes de Elisa-i e Western Blot (WB) para diagnóstico precoce de anticorpos em caprinos contra CAEV e comparar os resultados obtidos nesses testes com a prova de IDGA. Para a padronização dos testes Elisa-i e WB, utilizaram-se diferentes concentrações e diluições de antígeno, soros e conjugado. No Elisa-i, adotaram-se microplacas rígidas com 96 poços, sendo a combinação de concentração de 0,5µg/poço de antígeno e diluições de 1:100 de soro e 1:1500 de conjugado a que apresentou melhor resultado. No WB foram utilizadas membranas de nitrocelulose, definindo-se as diluições de 1:50 de soro e 1:15000 de conjugado. Para avaliar o desempenho das técnicas, 222 amostras de soro caprino foram testadas e os dados obtidos foram comparados com o IDGA. A sensibilidade e a especificidade do Elisa-i/IDGA, WB/IDGA e WB/Elisa-i foram de 70% e 91%, 100% e 72,6%, 84,6% e 76,5%, concomitantemente. O índice Kappa desses testes foi de 0,35, 0,2 e 0,36, respectivamente. As técnicas de Elisa-i e WB apresentaram-se mais sensíveis que a IDGA, podendo ser utilizadas como ferramentas para o diagnóstico precoce da CAE. Caprine arthritis-encephalitis (CAE) is routinely diagnosed with the Agarose Gel Immunodiffusion (AGID) technique, which is considered to have low sensitivity. The objective of this study was to standardize testing i-Elisa and Western Blot for early detection of antibodies against CAEV in goats and compare the results obtained in these tests with proof of AGID. For standardization of i-Elisa and WB, different concentrations and dilutions of antigen, sera and conjugate were used. In the i-Elisa, rigid microplate with 96 wells was adopted, and the combination that showed the best result was a concentration of 0.5µg/ well of antigen and dilutions of the serum of 1:100 and conjugate of 1:1500. In the WB nitrocellulose membranes were used, and the dilutions of the serum were defined at 1:50 and conjugate at 1:15000. To evaluate the performance of the techniques, 222 goat serum samples were tested and the data were compared with the AGID. The sensitivity and specificity of Elisa-i/IDGA, WB/AGID and WB/Elisa-i were 70% and 91%, 100% and 72.6%, 84.6% and 76.5%, concomitantly. The Kappa index of these tests was 0.35, 0.2 and 0.36, respectively. The i-Elisa and WB techniques were more sensitive than the AGID and can be used as tools for early diagnosis of CAE. Universidade Estadual do Ceará Faculdade de Veterinária Universidade Estadual Paulista Empresa Brasileira de Pesquisa Agropecuária Embrapa Caprinos e Ovinos Universidade Estadual Vale do Acaraú Universidade Federal da Bahia Escola de Medicina Veterinária e Zootecnia Agência de Defesa Agropecuária do Estado do Ceará Universidade Estadual Paulista

The mammalian Wharton's jelly of umbilical cord (WJUC) is a promising source of multipotent cells, providing advantages due to ethical implications, ease of collection and the absence of teratomas in pre-clinical trials. Ovine multipotent cells have already been isolated from various tissues, however there are no reports using umbilical cords in this species. This study aimed to investigate the best medium to transport the umbilical cord, to isolate and maintain ovine WJUC cells and to compare in vitro growth and mesodermal differentiation potential. Eight ovine umbilical cords were obtained during parturition, sectioned and transported in six different media: MEM, low glucose DMEM, M199, RPMI 1640, PBS and saline. For each transportation medium, four culture media were used and the tissue was explanted in 24-well plates and cultured in MEM, low glucose DMEM, M199 and RPMI 1640, all with 10% FBS. Every experiment was conducted with low-passage (P2), investigating MTT viability during four days and adipogenic, chondrogenic and osteogenesis differentiation was induced in vitro. The most effective transport medium (p

Azevedo D.A.A., Araújo J.F., Sousa A.L.M. de, Dias R.P., Souza T.S. de, Andrioli A. & Pinheiro R.R. [Caprine nictitating membrane cell culture for production of small ruminants lentivirus antigen.] Produção de antí- geno de lentivírus de pequenos ruminantes através da cultura celular de membrana nictitante caprina. Revista Brasileira de Medicina Veterinária, 37(4):316-320, 2015. Empresa Brasileira de Pesquisa Agropecuária (Embrapa Caprinos e Ovinos), Fazenda Três Lagoas, Estrada Sobral/Jordão Km 4, Sobral, CE 62010-970, Brasil. E-mail: dalvaazevedo@outlook.com Small Ruminants lentivirus (SRLV) belong to the Retroviridae family and is the etiologic agent of caprine arthritis encephalitis in goats and maedi-visna in sheep. Cells of the monocytic-phagocytic lineage are the main viral targets and infected hosts cannot develop a curative immune response. We evaluated whether nictitating membrane (NM) cells can be cultivated in vitro and also whether it is a feasible system for SRLV antigen production for immunodiagnosis purposes. MN cells were collected from a SRLV negative animal and cultivated in minimal essential media supplemented with bovine fetal serum. Subcultures were inoculated with caprine arthritis encephalitis virus (CAEV) Cork strain, the supernatant was collected, clarified by centrifugation (3.400 g for 20 minutes), concentrated using the AMICON® system. Samples were subjected to agar gel immunodiffusion (AGID) tests. MN cells presented satisfactory performance, were amenable to several subcultivation., Os Lentivírus de Pequenos Ruminantes (LVPR) pertencem à família Retroviridae e causam enfermidades crônicas e progressivas, conhecidas como artrite-encefalite caprina em caprinos e maedi-visna em ovinos. As células da linhagem monocítico fagocitária são os principais alvos do vírus no organismo animal, que não consegue desenvolver resposta imune curativa. Baseando-se no fato de que a membrana nictitante (MN) sofre naturalmente infecção, no organismo do animal, por estes vírus, objetivou-se avaliar se essas são passiveis para o cultivo e se possuem bom crescimento em cultura, visando à produção de antígeno de lentivírus caprino (LVC) para técnica de imunodiagnóstico. Sendo assim, realizou-se explant de células de MN de um animal negativo para LVPR. Essas células foram sub-cultivadas por tripsinização e então inoculadas com a cepa padrão CAEV Cork. Os sobrenadantes foram coletados, clarificados por centrifugação a 3.300g e concentrados em sistema AMICON®. Após realizou-se o teste de Imunodifusão em Gel de Agarose (IDGA) do antígeno produzido. As células de MN demonstraram bom crescimento em cultura, foram permissíveis ao LVC e com isso pôde-se produzir antígeno, o qual apresentou uma evidente linha de precipitação no teste.

Abstrract: This study was conducted in order to evaluate the transmission of caprine lentivirus to sheep using different experimental groups. The first one (colostrum group) was formed by nine lambs receiving colostrum from goats positive for small ruminant lentiviruses (SRLV). The second group (milk group) was established by nine lambs that received milk of these goats. Third was a control group, consisting of lambs that suckled colostrum and milk of negative mothers. Another experimental group (contact group) was formed by eight adult sheep, confined with two naturally infected goats. The groups were monitored by immunoblotting (IB), enzyme-linked immunosorbent assay (ELISA), agar gel immunodiffusion (AGID) and nested polymerase chain reaction (nPCR). All lambs that suckled colostrum and milk of infected goats and six sheep of the contact group had positive results in the nPCR, although seroconversion was detected only in three of the exposed animals, with no clinical lentiviruses manifestation, in 720 days of observation. There was a close relationship between viral sequences obtained from infected animals and the prototype CAEV-Cork. Thus, it was concluded that SRLV can be transmitted from goats to sheep, however, the degree of adaptation of the virus strain to the host species probably interferes with the infection persistence and seroconversion rate.

This study aimed to isolate cells from the Wharton's jelly of umbilical cord (WJUC) of sheep collected during natural parturition using different culture media, in addition to reporting for the first time the permissiveness of these cells to in vitro infection by small ruminant lentiviruses. Ten umbilical cords were collected from healthy sheep. Each cord explants were grown in different media consisting of MEM, low glucose DMEM, M199, and RPMI-1640. The permissiveness of infection of sheep cells from WJUC was tested with CAEV-Cork and MVV-K1514 strains, inoculating 0.1 MOI of each viral strain. Four supernatants from each strain were obtained from WJUC sheep cell cultures infected in different media. The results demonstrated the presence of cytopathic effect after the in vitro infection by CAEV-Cork and MVV-K1514 with all of the tested culture media. Nested-PCR detected proviral DNA in all supernatants. Supernatants containing CAEV-Cork viruses had TCID50/ml titres of 105.5 in MEM, 104.0 in low glucose DMEM, 105.0 in M199, and 105.7 in RPMI-1640. Supernatants containing the MVV-K1514 virus had TCID50/ml titres of 104.3 in MEM, 103.5 in low-glucose DMEM, 104.7 in M199, and 103.5 in RPMI-1640. Sheep cells from WJUC are permissive to in vitro infection by small ruminant lentivirus. [Células da geleia de Wharton do cordão umbilical ovino mantidas em diferentes meios de cultivo são permissivas à infecção in vitro por lentivírus de pequenos ruminantes]. Resumo: O objetivo deste estudo foi isolar células da geleia de Wharton do cordão umbilical (GWCU) ovino coletado por ocasião do parto natural, utilizando-se diferentes meios de cultivo, além de relatar, pela primeira vez, sua permissividade à infecção in vitro por lentivírus de pequenos ruminantes (LVPRs). Dez cordões umbilicais foram coletados de ovelhas hígidas e soronegativas para LVPRs pelo teste de imunodifusão em gel de agarose (IDGA). De cada cordão, explantes foram cultivados em quatro meios distintos que consistiram em MEM, DMEM baixa glicose, meio 199 e RPMI-1640, todos acrescidos de 10% de soro fetal bovino em estufa com atmosfera úmida e 5% de CO2 a 37ºC. A permissividade de infecção das células GWCU ovino foi testada frente às cepas CAEV-Cork e MVV-K1514, inoculando-se 0,1 MOI de cada cepa viral e corando as monocamadas com May Grunwald Giemsa para visualização do efeito citopático. Foram obtidos quatro sobrenadantes CAEV-Cork e quatro MVV-K1514, provenientes do cultivo de células GWCU ovino infectadas por 21 dias em meios distintos, dos quais foram realizadas titulação em membrana sinovial caprina e extração do DNA pró-viral para realização de nested-PCR e eletroforese em gel de agarose a 2%. Os resultados demonstraram a presença de efeito citopático na infecção in vitro tanto por CAEV-Cork como por MVV-K1514 em todos os meios de cultivo, sendo visualizados sincícios e lise celular em microscópio invertido. A nested-PCR detectou o DNA pró-viral tanto do CAEV-Cork como do MVV-K1514 em todos os sobrenadantes. Os sobrenadantes contendo o vírus CAEV-Cork apresentaram títulos em TCID50/mL de 105,5 em MEM, 104,0 em DMEM baixa glicose, 105,0 em meio 199 e 105,7 em RPMI-1640. Os sobrenadantes contendo o vírus MVV-K1514 apresentaram título em TCID50/mL de 104,3 em MEM, 103,5 em DMEM baixa glicose, 104,7 em meio 199 e 103,5 em RPMI-1640. Células GWCU ovino são permissivas à infecção in vitro pelos lentivírus de pequenos ruminantes CAEV-Cork e MVV-K1514. [ALICE ANDRIOLI PINHEIRO].

With the objective of detecting the presence of caprine lentivirus (CLV) in ewe milk and in ram semen, ten matrixes and four reproducers experimentally infected with CLV were used. Samples of ewe milk were collected during the four months of lactation, five collections per animal, totaling 50 samples. Regarding the rams, eight semen collections were made per animal, during one year of experimentation, totaling 32 samples. The milk and semen samples were submitted to DNA extraction and the nested polymerase chain reaction test (nPCR) to detect CLV proviral DNA. Eight (16%) of the milk samples were positive in nPCR originating from two ewes. Only one (3.12%) semen sample was positive. The amplification products were sequenced, and were confirmed to be a CLV genomic sequence. Thus, the presence of CLV proviral DNA in sheep milk and semen was demonstrated, confirming the feasibility of infection between species, and alerting to the risk of spreading infections. [Lentivírus caprino em leite e sêmen de ovinos]. Resumo: Com o objetivo de detectar a presença do lentivírus caprino (LVC) no leite de ovelhas e no sêmen de carneiros, utilizaram-se 10 matrizes e quatro reprodutores infectados experimentalmente com o LVC. Foram coletadas amostras de leite das ovelhas durante os quatro meses de lactação, ocorrendo cinco coletas por animal, totalizando 50 amostras. Quanto aos carneiros, realizaram-se oito coletas de sêmen por animal, durante um ano de experimentação, totalizando 32 amostras. As amostras de leite e de sêmen foram submetidas à extração de DNA e à prova de reação em cadeia da polimerase do tipo nested (nPCR) visando à detecção de DNA proviral do LVC. Oito (16%) amostras de leite foram positivas na nPCR oriundas de duas ovelhas. Apenas uma (3,12%) amostra de sêmen apresentou positividade. Produtos da amplificação foram sequenciados, confirmando-se tratar de sequência genômica do LVC. Dessa forma, demonstrou-se a presença do DNA proviral do LVC em leite e sêmen de ovinos, confirmando a viabilidade da infecção entre espécies e, assim, alertando sobre o risco de que a infecção seja disseminada. Made available in DSpace on 2017-09-24T00:12:27Z (GMT). No. of bitstreams: 1 CNPC2017Caprine.pdf: 376528 bytes, checksum: 3ae3a9af4fdd105cae76695fa5801913 (MD5) Previous issue date: 2017-09-22 [ALICE ANDRIOLI PINHEIRO].

Infections by small ruminant lentiviruses (SRLVs) affect goats and sheep causing chronic multisystemic diseases that generate great economic losses. The caprine lentivirus (CLV) and the ovine lentivirus (OLV) present tropism for cells of the monocyte/macrophage lineage, which are directly associated with the main route of transmission through the ingestion of milk and colostrum from infected animals. In this manner, controlling this route is of paramount importance. Currently, researches have investigated the use of chemical additives in milk that can preserve colostrum or milk and inactivate microbiological agents. Among the compounds, sodium dodecyl sulfate (SDS) has been shown to be satisfactory in the chemical inactivation of HIV and CLV in milk, and also as a biocide in goat colostrum. [Emprego do dodecil sulfato de sódio como inativador viral e suas perspectivas no controle de lentivírus de pequenos ruminantes]. Resumo: As lentiviroses de pequenos ruminantes (LVPRs) são infecções que afetam caprinos e ovinos, causando doenças multissistêmicas crônicas, ocasionando grandes perdas econômicas. Os agentes causadores, lentivírus caprino (LVC) e o lentivírus ovino (LVO), apresentam tropismo por células da linhagem monocítico-fagocitária, as quais estão diretamente associadas à principal via de transmissão, por meio da ingestão de leite e colostro provindos de animais infectados. Desse modo, o controle por esta via é de suma importância. Atualmente, pesquisas vêm sendo desenvolvidas para o uso de aditivos químicos no leite, que possam conservar o colostro ou leite, e inativar agentes microbiológicos presentes. Dentre estes, o dodecil sulfato de sódio (SDS) vem apresentando resultados satisfatórios na inativação química do HIV e LVC em leite, e ainda como biocida em colostro caprino. Made available in DSpace on 2019-11-18T18:06:07Z (GMT). No. of bitstreams: 1 cnpc2019Sodium.pdf: 498207 bytes, checksum: 0395f8468452fc5d662d9c754b53f271 (MD5) Previous issue date: 2019 [ALICE ANDRIOLI PINHEIRO]

Background: Caprine Arthritis Encephalitis (CAE) is a disease that causes productive losses in dairy goat flocks due to the reduction in milk production, followed by lesions in joints and mammary glands. An early diagnosis is essential, considering that there is frequent occurrence of asymptomatic animals. Hence, this study aimed to perform a comparison of immunological and molecular based diagnostic tests, represented by Agar Gel Immunodiffusion (AGID), Western Blot (WB) and nested Polymerase Chain Reaction (nPCR). In addition, the mammary glands (MG) of dairy goats were clinically evaluated. Materials, Methods & Results: Blood collection and clinical examination were performed in 1191 dairy goats of 12 farms located in Northeastern and Southeastern regions of Brazil. Serological (AGID, WB) and molecular (nPCR) test results were compared and the data, along with MG alterations, were analyzed using Epi-info 7 and WinEpiscope 2.0. Seroprevalence in AGID test was 41.14% (490/1191). In WB, 51.47% (613/1191) of animals were seropositive and nPCR detected 69.44% (827/1191) positive animals. Hence, WB was more sensitive (P < 0.001) than AGID. However, nPCR detected more positive animals than AGID (P < 0.001) and WB (P < 0.001). The analysis of mammary glands revealed that 105 out of 1096 nanny goats presented alterations, of which 101 presented altered consistency, 16 presented elevated temperatures and 60 had enlarged retromammary lymph nodes. There was significant statistic difference (P < 0.05) only when comparing the results of serological tests with MG alterations. Discussion: In general, AGID technique is most frequently used when screening flocks for the disease due to the practicality and low cost this test presents. However, the results demonstrated that AGID detected the lowest number of positive animals. This low sensitivity that the test presented may be attributed to its antigen-antibody interaction mechanism, considering that agar gel precipitation requires multiple interactions. In addition, WB was more effective than AGID in detecting antibodies. On the other hand, nPCR was important for the detection of infected animals that serological tests failed to detect. The intermittence of immunological response observed in the serological tests may be explained by the variation of antibodies levels that may occur during life. Likewise, viral compartmentalization would justify the intermittent detection of proviral DNA. Hence, the results can be influenced by the viral intermittence, test sensitivity, late seroconversion and statistic values that can be calculated (sensitivity, specificity, positive predictive value, negative predictive level and kappa). Crossing the results of the diagnostic tests with the different mammary gland alterations, it was shown that there was a statistically significant difference (P

Background: Contagious agalactia is an infectious disease caused by Mycoplasma agalactiae (M. agalactiae) that occurs in small ruminants leading to productive and economic losses. Due to the similarity of clinical signs presented in Caprine Arthritis Encephalitis (CAE), which is a viral disease, a differential diagnosis is important. Therefore, this study aimed to investigate the presence of anti-Mycoplasma agalactiae antibodies in dairy goat flocks in Ceará State and possible correlation with CAE. Materials, Methods & Results: The research was performed in four mesoregions in Ceará State (Metropolitan Region of FortalezaMRF; Northeast Ceará - NeC; North Ceará - NC; Sertões in Ceará - SC), from which 16 productions located in 10 cities with the highest representativeness for goat milk production within the State or mesoregion were sampled. A total of 417 females and 69 males (486 animals) of breeds with dairy production aptitude, pure or crossbreed, maintained in semi-intensive or intensive systems, were tested. Blood serum was obtained by venipuncture of the jugular vein with vacuum pressure syringe followed by centrifugation at 1,500 g for 10min. Antibodies against the caprine arthritis encephalitis virus (CAEV) were detected with micro technique of agarose gel immunodiffusion (AGID) and Western Blot (WB). The anti-Mycoplasma agalactiae antibodies were detected with commercial kit of enzymatic immunoassay (IDEXX Laboratories). Seroprevalence of M. agalactiae in dairy goat flocks in Ceará State was 0.62% (3/486). From the total of 16 visited productions, 18.75% (3/16) had seropositive animals for M. agalactiae located in MRF, NC and SC mesoregions. CAE was diagnosed in 56.25% (9/16) of productions with AGID and in 81.25% (13/16) with WB. In addition, 5.2% (25/486) of animals were seropositive for CAE with AGID and 16.6% (80/486) with WB. Animals that reacted positive for M. agalactiae were all females of pure breed with milk production aptitude in distinct mesoregions submitted to intensive rearing system. None of these animals was positive in neither test (AGID or WB) for CAE. Therefore, no correlation of results obtained in diagnosis of M. agalactiae by ELISA and CAEV by AGID or WB (P < 0.05) was identified. However, two out of three productions that were positive for M. agalactiae presented positive results for CAEV with frequencies of 10% and 20%. Discussion: Seroprevalence of M. agalactiae in Ceará State was low in comparison with other Brazilian states and even other countries. However, the presence of the pathogen in more than one mesoregion indicates that the disease occurs in different locations within the State. Therefore, flocks in Ceará are susceptible to the infection, which may be favored by uncontrolled commerce that occurs with deficient surveillance, associated with the importation of animals to improve flock genetic quality. The presence of the pathogen in dairy goats may contribute to significant losses in the local production. On the other hand, CAE was diagnosed in nearly all productions proving the dissemination of this lentivirus infection among dairy goat flocks in Ceará State. Although an association between these diseases was not identified, the presence of a retrovirus in the organism may favor co-infection with another micro-organism, promoting the deficiency in the immune system of the host. In conclusion, M. agalactiae is present in different mesoregions of the Ceará State and control measures should be adopted in short term to prevent pathogen dissemination and, consequently reduce economic and productive losses in the local dairy goat production. No correlation was identified between the prevalence of infection by CAEV and M. agalactiae in this study. [Mycoplasma agalactiae in dairy goat flocks bred in Ceará State of Ceará in association with Caprine Arthritis Encephalitis Virus]. Made available in DSpace on 2018-02-27T00:42:41Z (GMT). No. of bitstreams: 1 CNPC2018Mycoplasma.pdf: 384799 bytes, checksum: 41ca4219fc4691d2ffc562158295282c (MD5) Previous issue date: 2018-02-26 [ALICE ANDRIOLI PINHEIRO].

Os lentivírus de pequenos ruminantes, vírus da artrite encefalite caprina e vírus maedi visna, pertencem a família Retroviridae. São relacionados genética e antigenicamente, causam doenças crônicas e progressivas, levando a perdas produtivas e econômicas. A transmissão interespécie desses vírus pode ser preocupante devido a recombinação genética desses lentívirus. O estudo da proteômica desses vírus torna-se necessário para o conhecimento da função dos genes. A análise proteômica sorológica (SERPA) associada a espectrometria de massas (EM), dentre muitas possibilidades, permite o conhecimento de proteínas de valor diagnóstico. Com isso objetivou-se delinear o perfil proteíco-imunogênico bidimensional de lentivírus de pequenos ruminantes caracterizando proteínas detentoras de alto valor diagnóstico do antígeno dos lentivírus de pequenos ruminantes. Primeiramente realizou-se explant de células de membrana sinovial caprina e posteriormente, com o subcultivo das células, a produção do antígeno de três cepas virais MVV K1514, CAEV Cork e LVC-CE. Essas amostras foram submetidas à eletroforese bidimensional e western blot. Proteínas imunorreativas foram escolhidas para o sequenciamento através de espectrometria de massas. Como o resultado elaborou-se o perfil proteíco-imunogênico bidimensional das cepas de LVPR, inclusive da cepa viral do estado do Ceará (LVC-CE), dados inéditos para o país. Seis proteínas tiveram sequencias homologas compatíveis com proteínas de CAEV e MVV, referentes às proteínas p28, p19 e p15. Demonstrou-se a similaridade da resposta imune entre as cepas estudadas. A técnica SERPA associada à EM permitiu a identificação e caracterização peptídica das proteínas imunogênicas das cepas padrão CAEV Cork e MVV K1514, e da amostra de LVPR nativa do estado do Ceará. Abstract: The small ruminant lentiviruses (SRLV), the caprine arthritis encephalitis virus and maedi visna virus belong to Retroviridae family. They are related genetically and antigenically, and induce chronic and progressive disease which leads to productive and economic losses. The interspecific transmission is worrying due to the genetic recombination of these viruses. The proteomic study is necessary in order to know the genes function. Serological proteome analysis (SERPA) in association with mass spectrometry, besides others possibilities, allow knowing high-value proteins for diagnosis. The aim of this study was to determine the two-dimensional proteinimmunogenic profile and characterize the high-value proteins for diagnostic of different strains of SRLV. Initially, goat synovial membrane cells were explanted and subculture for antigen production of three viral strains CAEV Cork, MVV K1514 and LVC-CE. These samples were submitted to two-dimensional electrophoresis and western blot. Immunoreactive proteins were chosen in order to be sequenced by mass spectrometry. The results showed two-dimensional protein-immunogenic profile of the SRLV strains. Six proteins had homologous sequences compatible to protein CAEV and MVV, related to protein p28 and p15. The SERPA in association with mass spectrometry allowed the immunogenic protein identification and peptide characterization of the two standard strains, CAEV Cork and MVV K1514, and also the national one isolated in Ceará state. Dissertação (Mestrado em Zootecnia) - Centro de Ciências Agrárias e Biológicas, Universidade Estadual Vale do Acaraú, Sobral. Orientador: Raymundo Rizaldo Pinheiro; Coorientadora: Alice Andrioli Pinheiro.

Resumo: Os Lentivírus de Pequenos Ruminantes (LVPR) pertencem à família Retroviridae e causam enfermidades crônicas e progressivas, conhecidas como artrite-encefalite caprina em caprinos e maedi-visna em ovinos. As células da linhagem monocítico fagocitária são os principais alvos do vírus no organismo animal, que não consegue desenvolver resposta imune curativa. Baseando-se no fato de que a membrana nictitante (MN) sofre naturalmente infecção, no organismo do animal, por estes vírus, objetivou-se avaliar se essas são passiveis para o cultivo e se possuem bom crescimento em cultura, visando à produção de antígeno de lentivírus caprino (LVC) para técnica de imunodiagnóstico. Sendo assim, realizou-se explant de células de MN de um animal negativo para LVPR. Essas células foram sub-cultivadas por tripsinização e então inoculadas com a cepa padrão CAEV Cork. Os sobrenadantes foram coletados, clarificados por centrifugação a 3.300g e concentrados em sistema AMICON®. Após realizou-se o teste de Imunodifusão em Gel de Agarose (IDGA) do antígeno produzido. As células de MN demonstraram bom crescimento em cultura, foram permissíveis ao LVC e com isso pôde-se produzir antígeno, o qual apresentou uma evidente linha de precipitação no teste. [Caprine nictitating membrane cell culture for production of small ruminants lentivirus antigen.]. Abstract: Small Ruminants lentivirus (SRLV) belong to the Retroviridae family and is the etiologic agent of caprine arthritis encephalitis in goats and maedi-visna in sheep. Cells of the monocytic-phagocytic lineage are the main viral targets and infected hosts cannot develop a curative immune response. We evaluated whether nictitating membrane (NM) cells can be cultivated in vitro and also whether it is a feasible system for SRLV antigen production for immunodiagnosis purposes. MN cells were collected from a SRLV negative animal and cultivated in minimal essential media supplemented with bovine fetal serum. Subcultures were inoculated with caprine arthritis encephalitis virus (CAEV) Cork strain, the supernatant was collected, clarified by centrifugation (3.400 g for 20 minutes), concentrated using the AMICON® system. Samples were subjected to agar gel immunodiffusion (AGID) tests. MN cells presented satisfactory performance, were amenable to several subcultivation.