Comparison of three techniques for production goat lentivirus antigen used in the agar gel immunodifusion test

A Artrite Encefalite Caprina (AEC) é uma enfermidade que causa perdas econômicas consideráveis, incluindo perda na produção de leite e diminuição da vida útil do animal. No diagnóstico desta enfermidade o teste de imunodifusão em gel de ágar (IDGA) é utilizado mundialmente como o teste de triagem. Este trabalho teve como objetivo testar três diferentes concentrações de soro fetal bovino (SFB) na produção do antígeno (Ag) para o diagnóstico da AEC, verificar dentre três métodos o mais eficiente para efetuar a concentração e qual a concentração do antígeno produzido mais apropriada para o teste. Tanto o método do AMICON, como o da concentração do Ag por diálise são indicados, entretanto o sistema AMICON, apesar dos custos de implantação, promoveu menor perda de antígeno, maior rapidez e praticidade. Com relação à quantidade de soro fetal bovino (SFB) colocada após a inoculação viral observou-se que 5% de SFB foi a quantidade que apresentou melhores resultados. A concentração do antígeno mais indicada é de 100 vezes, pois permite a detecção de anticorpos contra o vírus da AEC (LVC) por duas proteínas (gp 135 e p28). A purificação do Ag por precipitação/ultracentrifugação, utilizada para provas imunoenzimáticas (ELISA e Dot-BLOT), não apresentou resultados satisfatórios para o IDGA.The Caprine Arthritis Encephalitis (CAE) is a disease who cause considerable economic losses, including loss in the milk production and reduction of the useful life of the animal. In the diagnosis of this disease the agar gel immunodifusion test (AGID) is used worldwide as the selection test. The objective of thid work was to test three different concentrations of bovine fetal serum (BFS) in the production of the antigen (Ag) for the diagnosis of the CAE virus (CAEV), to verify amongst the three methods the most efficient concentration and which the antigen concentration of the antigen produced is appropriate for the test. The method of the AMICON and the concentration of the Ag for dialysis was indicated, however the system AMICON, despite the implantation costs, promoted minor loss of antigen, little time expense in the processing and greater simplicity. With relation to the amount of BFS placed after the viral inoculation it was verified that 5% of BFS the amount that presented better resulted. The antigen concentration 100 times was more indicated, therefore it allowed the diagnosis of the CAEV for two proteins (gp 135 and p28). The concentration of the Ag for precipitation/ultracentrifugation, used for imunoenzimatic tests, did not present resulted satisfactory used in the AGID

Risk factors in caprine lentivirus transmission through semen

O objetivo deste trabalho foi avaliar a presença do DNA pró-viral do lentivírus caprino (LVC) em ejaculados de machos infectados naturalmente, e verificar a influência da lavagem do sêmen e da presença de inflamação testicular sobre a carga viral. Foram realizadas oito coletas de sêmen de sete reprodutores soropositivos para o LVC: quatro antes dos animais sofrerem dano testicular e quatro depois. Entre as coletas realizadas na mesma semana, em uma, o ejaculado era lavado, para retirada do plasma seminal, e na outra, não. O DNA pró-viral do LVC foi identificado pela reação em cadeia da polimerase Nested (PCR Nested), e pelo isolamento viral. O vírus foi isolado em 7,1% das amostras. A PCR identificou o DNA pró-viral em 35,7% do total das amostras: 17,9% nas amostras lavadas e 53,6% das amostras de sêmen integrais. O dano ao testículo permite maior fluxo do vírus para o sêmen, pois antes do dano, 21,4% das amostras foram positivas e pós-dano, 50%. A transmissão do LVC pelo sêmen de reprodutores caprinos é potencializada pela presença de inflamações testiculares e pelo fato de o sêmen criopreservado conter o LVC na forma infectante.The objective of this work was to evaluate the presence of the DNA provirus of the caprine lentivirus (LVC) in ejaculates of naturally infected males, and to verify the influence of the wash of the semen as well as the presence of testicle inflammation on the viral load. Eight semen collections of seven soropositive reproducers were accomplished, four before testicle injury and four after injury. Amongst the collections carried out at the same week, in one the ejaculate was washed, to withdraw the plasma seminal, and in the other it was not. The provirus DNA was identified both by Nested polymerase chain reaction technique (Nested PCR) and by the viral isolation. The virus was isolated in 7.1% of the samples. The PCR identified the provirus DNA in 35.7% of all samples, 17.9% in the washed samples and 53.6% of the integral semen samples. The injury of the testicle tends to greater flow of virus for the semen, therefore, before injury, 21.4% of the samples were positive and after-injury, 50%. Risk of transmission of the LVC by semen of goat reproducers is strengthened by the presence of testicle inflammations and the fact that the criopreserved semen contains the LVC in infecting form

Evaluation of Solvent Toxicity of Plant Extract with Antiviral Action in Refrigerated Goat Semen

Background: Caprine Arthritis Encephalitis Virus have been detected in sperm of breeding goats causing economic losses. In order to control the virus, researches aiming to identify natural extracts with potential antiviral effects are performed. However, aqueous or ethanolic extracts must be diluted in dimethyl sulfoxide (DMSO), which is a substance with unknown effects in sperm quality when present in diluting media. Therefore, this study aimed to evaluate sperm viability of refrigerated caprine semen diluted in media containing DMSO. This was performed to provide data that aid in researches involving the use of this component with natural extracts that may inactivate the caprine lentivirus in sperm.Materials, Methods & Results: The experiment was performed at the Laboratory of Seminal Technology in Embrapa Goats and Sheep in the city of Sobral, Brazil. Sperm viability was assessed in caprine semen refrigerated in two dilution media with crescent concentrations of DMSO. Sperm samples of five goats seronegative for the caprine lentivirus were pooled and diluted in minimal essential medium (MEM) enriched with glucose at 0.01 M added of crescent concentrations of DMSO (0%, 1.5%, 1.75%, 2.0%, 2.25% and 2.5%). The same breeders provided the pool of sperm to test Tris added 2.5% of egg yolk and the same concentrations of DMSO previously mentioned. Treatments were refrigerated at 7°C and evaluated up until four h after DMSO addition. Individual progressive motility (MIP), sperm vigor (V), percentage of spermatozoa reactive to hypoosmotic test (HO) and morphologically normal (NOR) were evaluated. IPM, vigor and NOR remained within normal standards for the caprine species in all treatments test. Percentage results of spermatozoa reactive to hypoosmotic was higher in Tris yolk with values ranging between 34.66% to 46.33%. Sperm vigor was positively correlated (r = 0.85) with IPM in the MEM diluted pool of sperm. In Tris yolk, vigor and hypoosmotic test correlated moderately (r = 0.63, r = 0.54, respectively) with IPM. Tris yolk medium added DMSO presented the highest percentage of reactivity to hypoosmotic test in all treatments when compared to MEM added DMSO.Discussion: The fact that DMSO is easily homogenized in water, ethylic alcohol and most organic solvents favors its use in diluting natural extracts. These components are a possible source of products that inactivate caprine arthritis encephalitis virus in sperm, which is the key to promoting the safe use of genetic material of infected breeders, in addition to commercial use of germplasm. In this study, there was no interference of DMSO in the analyzed parameters when added in a maximum concentration of 2.5% to MEM and Tris yolk, which is in accordance with standard values for goats. In addition, Tris yolk may promote greater protection to the membrane of sperm cells, which was demonstrated by hypoosmotic test. This medium could be ideal to be used in new methodologies that incorporate DMSO. In conclusion, DMSO added to dilution media Tris yolk and MEM did not interfere with the quality of refrigerated caprine sperm, which maintained viability. These results indicate that this substance did not present harmful effects to the genetic material, promoting the use as solvent of extracts from plant compounds with potential anti-viral effect. The information in this study may aid new research performed in this area

Seroprevalence and Risk Factors of Chlamydophila abortus Infection in Goats of the State of Rio Grande do Norte, Brazil

Background: Chlamydophilosis is a disease caused by the bacterium Chlamydophila abortus, responsible for reproductive damage in small ruminants, and economic losses to animal production. In addition, it is classified as a zoonosis once it presents symptoms similar to numerous bacterial and viral infections of the reproductive system, making clinical diagnosis difficult. The objective of this work was to estimate the seroprevalence of anti-C. abortus antibodies in goats and to identify the risk factors for the disease in two mesoregions of Rio Grande do Norte, Brazil.Material, Methods & Results: This research was carried out in two mesoregions of the State of Rio Grande do Norte (Central Potiguar - CP and West Potiguar - WP) located in the Northeast of Brazil. These mesoregions together represent 83% of the regional herd. A total of 54 properties were studied. Those are distributed in seven municipalities: Afonso Bezerra, Angicos, Lages and Pedro Avelino, in the CP mesoregion; and Apodi, Caraúbas, and Mossoró, in the OP mesoregion. We used 540 animals from herds characterized as non-defined breed (NDB) and native breed, regardless of their productive functions, being frequent the consortium of caprine and ovine species. Blood samples were collected through jugular venipuncture by vacuum system with 5 mL tubes without anticoagulant. After collection, the tubes were centrifuged at 1500 g for 15 min. Detection of anti-Chlamydophila sp. antibodies was performed by the Complement Fixation Test. In parallel with the collection, a questionnaire was applied addressing general property and producer characteristics. Seroprevalence of chlamydophilosis in the CP mesoregion was 3.04% (7/230) for the animals and 26.1% (6/23) for the properties, whereas, in the WP mesoregion, the values were 4.2% (13/310) and 35.5% (11/31), respectively. In the final logistic regression model, no variable analyzed was considered to be associated with a higher probability of C. abortus positive properties. There was no significant difference (P = 0.76) in the infection among the studied mesoregions. All the seven municipalities evaluated, had at least one positive property. No serology was found with titers greater than 1:32. The results of the suspects with titers of 1:16 were 3.9% (9/230) in the CP and 3.5% (11/310) in the WP mesoregion. None of the 82 breeding animals tested were C. abortus reagent, but three were suspected. The highest prevalence for the infection was seen in the matrices with 5.6% (18/321), followed by young females with 2.9% (1/35), young males with 1.2% (1/82) and breeding animals with 0% (0/82).Discussion: The seroprevalence to Chlamydophila abortus found in the state of Rio Grande do Norte was lower than that found in most Brazilian states, and even in other countries. The presence of properties with positive animals displays a risk of dissemination of the disease in the region. In addition, the presence of suspect animals may indicate a much larger number of positive animals. The existence of false-negative animals in the herd can lead to difficulties in controlling and eradicating this disease. Although none of the breeding animals were reagent to C. abortus in the CF test, the possibility of venereal transmission should not be discarded. As some animals remained suspects, it may indicate an initial infection phase and seroconversion process, possibly characterizing future transmitters of the bacterium via semen. However, even though seroprevalence was numerically low, this disease should be a cause for concern due to its severity and spread capacity among herds, and especially because it is a zoonosis. We conclude that chlamydophilosis is present in goat breeding in the state of Rio Grande do Norte, Brazil, and female animals are more susceptible to Chlamydophila abortus infection

Perspectivas do uso de fitoterápicos no tratamento do sêmen caprino infectado pelo vírus da Artrite Encefalite Caprina

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

Mycoplasma agalactiae in Dairy Goat Flocks Bred in State of Ceará in Association with Caprine Arthritis Encephalitis Virus

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 diagnosedin 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

Comparison of Serological and Molecular Tests for Diagnosis of Caprine Arthritis Encephalitis and Clinical Evaluation of Mammary Glands of Infected Dairy Goats

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 <0.05) only in the comparison of the results of the serological tests with GM alterations. Everything indicates that the humoral or cellular immune system being on stimulus is more propitious to find these changes. In conclusion, WB was more sensitive than AGID and, considering that nPCR can detect a larger number of animals infected with the goat lentivirus, it must be associated with a sensible serological test, such as Western Blot. In addition, infected animals have alterations in MG, which is more frequent in cases with positive serological results

Enfermidades Infeciosas de Pequenos Ruminantes: Epidemiologia, Impactos Econômicos, Prevenção e Controle: Uma Revisão

Nesta revisão bibliográfica, pretende-se apresentar de forma simples e sistemática as enfermidades possíveis de transmissão através da carne, leite e derivados de caprinos e ovinos. Em caprinos e ovinos as enfermidades possíveis de transmissão pela carne, leite e derivados são divididas neste trabalho em dois grupos: as ocasionadas pela ingestão de alimentos denominadas intoxicações alimentares e as zoonoses que causam risco para a saúde humana e pública. As enfermidades de origem microbiana transmitidas através da ingestão de alimentos contaminados por bactérias do tipo, Escherichia coli, Salmonella sp, Campylobacter sp, Clostridium sp, Staphylococcus sp.. No grupo das zoonoses citam-se a Listeriose, a Brucelose, a Tuberculose, a Toxoplasmose, a Raiva, a Leptospirose e a Febre Aftosa.In this bibliographic review, it is intended to show, on a simple and systematic form, the possible diseases transmitted through meat, milk and other derivates of “caprinos” and “ovinos”. In “caprinos” and “ovinos”, the possible diseases transmitted through meat, milk and other derivates are divided in this present work by two different groups: those occurred by ingesting food named food intoxications, and the “zoonoses”, which bring human and public health risks. The diseases that have a “microbiana” origin, are transmitted through the ingestion of food infected by bacterias such as Escherichia coli, Salmonella sp, Campylobacter sp, Clostridium sp, Staphylococcus sp. On the zoonoses group, it’s possible to talk about “Listeriose, a Brucelose, a Tuberculose, a Toxoplasmose, a Raiva, a Leptospirose e a Febre Aftosa