Longitudinal seroepidemiological survey and risk factors for bluetongue virus infection in sheep in the state of Parana, Brazil, from 2014 to 2017.

The Bluetongue disease is an infectious and non-contagious viral disease mainly transmitted through hematophagous vector of the Culicoides genus, to domestic and wild ruminants. The aim of this study was to evaluate the antibodies occurrence, persistence and potential risk factors associated with bluetongue virus infection in sheep flocks in the state of Parana, Brazil. The competitive ELISA test was applied to evaluate 690 blood serum samples from 22 farms in eight mesoregions of Parana in 2014, and 270 sheep blood serum samples from 10 of the 22 previously studied farms in 2017. A questionnaire was applied to evaluate the risk factors associated with BTV infection. In 2014 and 2017, the numbers of seroreactive sheep were found to be 28.26% (195/690) and 41.11% (111/270), respectively, representing 95.45% (21/22) and 90% (9/10) of the flocks. The significant variables considered as risk factors were Culicoides presence (P < 0.0001; OR = 8.83 and 95% CI 4.28-18.22); genealogical record (P < 0.0001; OR = 0.23 and 95% CI 0.12-0.45) and use of sheepfold (P = 0.0208; OR = 0.36 and 95% CI 0.15-0 0.86). It was determined that BTV infection is endemic in Parana and persists in the mesoregions where the climate is favorable to vector proliferation.

Seroprevalence and Risk Factors Related to Bovine Brucellosis in Continental Ecuador.

Bovine brucellosis is a worldwide zoonotic contagious disease. According to World Animal Health Information System reports Ecuador has presented an increasing number of bovine brucellosis outbreaks in the continental territory over the past years (756 in 2018 versus 964 in 2021), generating economic losses for producers and causing a risk to public health. A cross-sectional study was conducted to investigate the seroprevalence of bovine brucellosis and associated risk or protective factors between May and June 2018. This stratified random study was implemented in 290 cattle herds located in the 23 provinces of continental Ecuador, which represents a total of 3737 cows aged 24 months or older. A competitive ELISA was used to detect Brucella antibodies. Simultaneously, an epidemiological survey was implemented to assess the brucellosis risk or protective factors. The apparent prevalence of bovine brucellosis at the herd level was 21.3% (95% CI: 16.8-26.6) and 6.2% (95% CI: 5.5-7) at the animal level. Univariate and multivariate logistic regression analyses were performed to determine the relationship between the potential factors associated with the presence of bovine brucellosis. The risk factors identified after multivariate analysis were a surface in ha per herd > 70 ha (OR = 2.73; 95% CI: 1.18-6.32) and the number of parturitions per animal (two or more with OR ≥ 1.8 and p-value ≤ 0.047). On the contrary, the protective factors were the region (farms located in the eastern region) and the absence of reported clinical signs. In addition, in herds where extensive production predominates, farmers have a low level of knowledge, and the farm biosecurity level is low. These results can guide the authorities in managing the risk factors identified, understanding the current epidemiological situation in Ecuador, improving the bovine brucellosis control program and food safety, as well as increase the one-health approach.

Comparison of serological techniques for the diagnosis of equine infectious Anemia in an endemic area of Argentina.

Equine Infectious Anemia is a transmissible viral disease present worldwide, caused by an RNA virus. Viral transmission is mainly mechanical through blood or its products most frequently by blood-sucking arthropods and iatrogenesis as well. OIE recommends Coggins Test as the diagnostic method of choice. Some ELISA tests detect antibodies earlier than the Coggins test, but may produce false-positive results. Currently, new techniques for EIA diagnosis are being developed, such as fluorescence polarization assay which is a simple method for measuring antigen-antibody interaction. The aim of this study was to assess cELISA and Fluorescence Polarization Assay performance for the serological diagnosis of EIA by comparing their results with those of the Coggins test. Tests were performed on 91 workhorses from an endemic zone in the northeast region of Argentina. From the total samples analyzed, 42 tested negative and 49 tested positive in the Coggins test. Same results were obtained using FPA. Using the cELISA, 41 negative results and 50 positive results were obtained. The Receiver Operating Characteristic analysis showed that FPA performance was excellent. Therefore FPA is proposed as an outstanding EIA diagnostic test to be validated in the near future by its simplicity, speed, and objective interpretation of results.

Transmissibilidade de Lentivírus de Pequenos Ruminantes para cabritos e cabras adultas por meio de sêmen infectado experimentalmente / Transmissibility of Small Ruminants Lentivirus in kids by experimentally infected semen

A Artrite Encefalite Caprina se caracteriza por ser multissistêmica e infecciosa, causada por um lentivírus. O estudo teve como objetivo avaliar a transmissibilidade do Lentivírus Caprino, para fêmeas e sua prole, por meio de sêmen infectado experimentalmente. Para tanto, onze fêmeas livres de CAEV foram inseminadas artificialmente com sêmen de bode livre de CAEV ao qual foi adicionado CAEV-Cork para obter título infectante com carga viral em 105 TCID50/ml. (grupo experimental 1). Destas, seis obtiverem prenhez confirmada, e a sua prole (n=6) constituiu o grupo experimental 2. Duas cabras livres de CAEV foram inseminadas artificialmente com sêmen do mesmo bode, sem o inócuo viral, constituindo-se o grupo controle. O diagnóstico da infecção pelo Lentivírus Caprino, foi realizado por IDGA, cELISA e nested-PCR. As fêmeas foram monitoradas durante 210 dias pós inseminação artificial. Já as proles foram imediatamente separadas das mães após o nascimento, e monitoradas nos momentos hora zero, aos quinze dias de idade e mensalmente, até doze meses de idade. Em relação às cabras, 56,96%(9/158) apresentaram positividade para cELISA, 24,05% (38/158) foram positivas a IDGA e nenhuma para nested-PCR. Em relação aos cabritos, 11,28% (15/133) amostras positivas para nested-PCR, 5,26% (7/133) amostras positivas para IDGA e nenhum para cELISA. As proles do grupo controle apresentaram resultados negativos para as três técnicas. A positividade encontrada em nested-PCR pode indicar grande importância para identificação de animais infectados, porém soronegativos, em situações de soroconversão tardia. De acordo com os resultados, concluiu-se que há a transmissão do Lentivírus caprino para a prole e para as mães pelo sêmen infectado.(AU)

Caprine Arthritis Encephalitis is a multisystemic infectious disease, caused by a lentivirus. The objective of this study was to evaluate the transmissibility of caprine lentivirus to goats and their offspring, through experimentally infected semen. Therefore, eleven free-CAEV goats were artificially inseminated using semen from a free-CAEV buck experimentally infected with CAEV-Cork strain (experimental group one). Pregnancy was confirmed in only six goats and their offspring (n=6) constituted the experimental group two. Two free-CAEV females were artificially inseminated with semen from the same seronegative buck, without viral inoculum to constitute the control group. The diagnosis of caprine lentivirus infection was performed using AGID, cELISA and nested-PCR. All females were monitored for 210 days after artificial insemination. Kids were immediately separated from their mothers after birth, and monitored at zero time, 15 days old and monthly until 12 months old. Regarding goat samples, 56.96% (9/159) were positive in cELISA, 24.05% (38/158) were positive in IDGA and none was positive in nested-PCR. Regarding to the offspring samples, 11.28% (15/133) and 5.26% (7/133) were positive in nested-PCR and IDGA, respectively, while no sample was positive in cELISA. The control group showed no positives in the three techniques. The positivity observed to nested-PCR may show its importance to identify infected, but seronegative animals, in late seroconversion situations. According to results, the transmission of caprine lentivirus to offspring and their mothers through infected semen is possible.(AU)

Transmissibilidade de Lentivírus de Pequenos Ruminantes para cabritos e cabras adultas por meio de sêmen infectado experimentalmente / Transmissibility of Small Ruminants Lentivirus in kids by experimentally infected semen

A Artrite Encefalite Caprina se caracteriza por ser multissistêmica e infecciosa, causada por um lentivírus. O estudo teve como objetivo avaliar a transmissibilidade do Lentivírus Caprino, para fêmeas e sua prole, por meio de sêmen infectado experimentalmente. Para tanto, onze fêmeas livres de CAEV foram inseminadas artificialmente com sêmen de bode livre de CAEV ao qual foi adicionado CAEV-Cork para obter título infectante com carga viral em 105 TCID50/ml. (grupo experimental 1). Destas, seis obtiverem prenhez confirmada, e a sua prole (n=6) constituiu o grupo experimental 2. Duas cabras livres de CAEV foram inseminadas artificialmente com sêmen do mesmo bode, sem o inócuo viral, constituindo-se o grupo controle. O diagnóstico da infecção pelo Lentivírus Caprino, foi realizado por IDGA, cELISA e nested-PCR. As fêmeas foram monitoradas durante 210 dias pós inseminação artificial. Já as proles foram imediatamente separadas das mães após o nascimento, e monitoradas nos momentos hora zero, aos quinze dias de idade e mensalmente, até doze meses de idade. Em relação às cabras, 56,96%(9/158) apresentaram positividade para cELISA, 24,05% (38/158) foram positivas a IDGA e nenhuma para nested-PCR. Em relação aos cabritos, 11,28% (15/133) amostras positivas para nested-PCR, 5,26% (7/133) amostras positivas para IDGA e nenhum para cELISA. As proles do grupo controle apresentaram resultados negativos para as três técnicas. A positividade encontrada em nested-PCR pode indicar grande importância para identificação de animais infectados, porém soronegativos, em situações de soroconversão tardia. De acordo com os resultados, concluiu-se que há a transmissão do Lentivírus caprino para a prole e para as mães pelo sêmen infectado.(AU)

Caprine Arthritis Encephalitis is a multisystemic infectious disease, caused by a lentivirus. The objective of this study was to evaluate the transmissibility of caprine lentivirus to goats and their offspring, through experimentally infected semen. Therefore, eleven free-CAEV goats were artificially inseminated using semen from a free-CAEV buck experimentally infected with CAEV-Cork strain (experimental group one). Pregnancy was confirmed in only six goats and their offspring (n=6) constituted the experimental group two. Two free-CAEV females were artificially inseminated with semen from the same seronegative buck, without viral inoculum to constitute the control group. The diagnosis of caprine lentivirus infection was performed using AGID, cELISA and nested-PCR. All females were monitored for 210 days after artificial insemination. Kids were immediately separated from their mothers after birth, and monitored at zero time, 15 days old and monthly until 12 months old. Regarding goat samples, 56.96% (9/159) were positive in cELISA, 24.05% (38/158) were positive in IDGA and none was positive in nested-PCR. Regarding to the offspring samples, 11.28% (15/133) and 5.26% (7/133) were positive in nested-PCR and IDGA, respectively, while no sample was positive in cELISA. The control group showed no positives in the three techniques. The positivity observed to nested-PCR may show its importance to identify infected, but seronegative animals, in late seroconversion situations. According to results, the transmission of caprine lentivirus to offspring and their mothers through infected semen is possible.(AU)

Transmissibilidade de Lentivírus de Pequenos Ruminantes para cabritos e cabras adultas por meio de sêmen infectado experimentalmente

ABSTRACT: Caprine Arthritis Encephalitis is a multisystemic infectious disease, caused by a lentivirus. The objective of this study was to evaluate the transmissibility of caprine lentivirus to goats and their offspring, through experimentally infected semen. Therefore, eleven free-CAEV goats were artificially inseminated using semen from a free-CAEV buck experimentally infected with CAEV-Cork strain (experimental group one). Pregnancy was confirmed in only six goats and their offspring (n=6) constituted the experimental group two. Two free-CAEV females were artificially inseminated with semen from the same seronegative buck, without viral inoculum to constitute the control group. The diagnosis of caprine lentivirus infection was performed using AGID, cELISA and nested-PCR. All females were monitored for 210 days after artificial insemination. Kids were immediately separated from their mothers after birth, and monitored at zero time, 15 days old and monthly until 12 months old. Regarding goat samples, 56.96% (9/159) were positive in cELISA, 24.05% (38/158) were positive in IDGA and none was positive in nested-PCR. Regarding to the offspring samples, 11.28% (15/133) and 5.26% (7/133) were positive in nested-PCR and IDGA, respectively, while no sample was positive in cELISA. The control group showed no positives in the three techniques. The positivity observed to nested-PCR may show its importance to identify infected, but seronegative animals, in late seroconversion situations. According to results, the transmission of caprine lentivirus to offspring and their mothers through infected semen is possible.

RESUMO: A Artrite Encefalite Caprina se caracteriza por ser multissistêmica e infecciosa, causada por um lentivírus. O estudo teve como objetivo avaliar a transmissibilidade do Lentivírus Caprino, para fêmeas e sua prole, por meio de sêmen infectado experimentalmente. Para tanto, onze fêmeas livres de CAEV foram inseminadas artificialmente com sêmen de bode livre de CAEV ao qual foi adicionado CAEV-Cork para obter título infectante com carga viral em 105 TCID50/ml. (grupo experimental 1). Destas, seis obtiverem prenhez confirmada, e a sua prole (n=6) constituiu o grupo experimental 2. Duas cabras livres de CAEV foram inseminadas artificialmente com sêmen do mesmo bode, sem o inócuo viral, constituindo-se o grupo controle. O diagnóstico da infecção pelo Lentivírus Caprino, foi realizado por IDGA, cELISA e nested-PCR. As fêmeas foram monitoradas durante 210 dias pós inseminação artificial. Já as proles foram imediatamente separadas das mães após o nascimento, e monitoradas nos momentos hora zero, aos quinze dias de idade e mensalmente, até doze meses de idade. Em relação às cabras, 56,96%(9/158) apresentaram positividade para cELISA, 24,05% (38/158) foram positivas a IDGA e nenhuma para nested-PCR. Em relação aos cabritos, 11,28% (15/133) amostras positivas para nested-PCR, 5,26% (7/133) amostras positivas para IDGA e nenhum para cELISA. As proles do grupo controle apresentaram resultados negativos para as três técnicas. A positividade encontrada em nested-PCR pode indicar grande importância para identificação de animais infectados, porém soronegativos, em situações de soroconversão tardia. De acordo com os resultados, concluiu-se que há a transmissão do Lentivírus caprino para a prole e para as mães pelo sêmen infectado.

Seroepidemiological survey of Theileria equi and Babesia caballi in horses from a rural and from urban areas of Paraná State, southern Brazil.

The objective of this study was to evaluate the seroepidemiological data of Babesia caballi and Theileria equi in horses from a rural settlement and carthorses from urban areas of Paraná State, southern Brazil. A total of 198 horses, including 32 from the rural settlement and 166 carthorses from Colombo (n=48), Pinhais (n=76), Londrina (n=24), and Curitiba city (n=18) was sampled and tested using a commercial competitive inhibition ELISA (cELISA) test. Out of the 198 horses, 193 (97.5%) were seropositive for at least one piroplasm species. Antibodies to T. equi were detected in 155/198 horses (78.3%), antibodies to B. caballi were detected in 137/198 horses (69.2%), and antibodies to both were detected in 99/198 (50.0%) horses. Horses living in the rural settlement and Colombo were more likely to be seropositive to T. equi than those in Curitiba (p<0.05). Horses older than 5 years were more likely to be seropositive for T. equi than those younger than 5 years (p<0.05). No significant association was found between gender or the presence of ticks and seropositivity to T. equi (p>0.05). In conclusion, the high seroprevalences to B. caballi and T. equi observed in this study emphasize that active surveillance programs are critical for monitoring animal health status, particularly because carthorses may act as urban disseminators of these piroplasms.

Detection of Theileria equi and Babesia caballi infections in Venezuelan horses using Competitive-Inhibition ELISA and PCR.

The focus of this study was the detection of equine piroplasmosis in Distrito Capital, Miranda, Aragua, Guárico and Apure States from Venezuela, using two methods: Competitive-Inhibition ELISA and multiplex PCR and the analysis of the possible differences in occurrence in relation to the primary purpose of the horses, which is related to varied degrees of exposure to tick. Antibody levels to Babesia caballi and Theileria equi were assessed in 694 equine serum samples using Competitive-Inhibition ELISA, while PCR assays were performed in 136 horses, using two sets of oligonucleotides to establish the presence of T. equi, B. caballi or both. The overall seroprevalence of equine piroplasmosis was 50.2%, antibodies to B. caballi were found in 161 horses (23.2%), whereas 97 (14.0%) were seropositive to T. equi and 90 (13.0%) were positives to both parasites (mixed infections). PCR determinations (n=136) showed a prevalence of 66.2%, distributed in 84 (61.8% positives) for T. equi and, 6 (4.4%) were positive to both parasites. The cELISA showed higher levels of prevalence of B. caballi and mixed infections, as compared to the PCR method. This discrepancy can be explained by the different parameters that are evaluated by each technique, PCR detect the parasite itself, while cELISA detects antibodies to the parasite. By PCR, the highest prevalence was found in Apure state, where 92.3% of the samples were positive to T. equi infections. In this locality, free grazing animals are used for livestock management. This high prevalence may be linked to the tick species present in that area. More epidemiological studies will be necessary to assess the epidemiological status of equine piroplasmosis in Venezuela.

Soroprevalência de Anaplasma marginale em bovinos da região Centro-Sul do estado do Paraná, Brasil, por um teste imunoenzimático competitivo utilizando proteína recombinante MSP5-PR1 / Seroprevalence of Anaplasma marginale in cattle from Center-South Region of Paraná State, Brazil by a competitive ELISA test with recombinant MSP5-PR1 protein

Foi determinada a prevalência de Anaplasma marginale em 223 soros de bovinos com idade maior ou igual a dois anos, das regiões de Ponta Grossa, Guarapuava e Laranjeiras do Sul, região Centro-Sul do estado do Paraná. O teste imunoenzimático de competição PR1 (cELISA-PR1) foi utilizado para determinar a presença ou ausência de anticorpos anti-A. marginale. Dos 223 soros analisados, 130 (58,74 por cento) foram positivos pelo cELISA-PR1, sugerindo ser essa região de instabilidade de enzoótica, com uma porcentagem significativa de animais susceptíveis à infecção por A. marginale, com risco potencial para desenvolver a anaplasmose. As características de tipo de exploração da propriedade, sistema de criação, manejo e forma de comercialização dos animais foram avaliadas. A análise estatística não demonstrou haver diferença significativa entre as variáveis estudadas e a positividade dos animais.

Anaplasma marginale prevalence was determined in 223 sera samples in 2-year old or older cattle, from the Center- Southern Region of the Paraná State, including Ponta Grossa, Guarapuava and Laranjeiras do Sul municipalities. A survey of antibodies IgG class against Anaplasma marginale was performed through a competitive immune absorbent assay (cELISA-PR1). From the 223 sera examined, 130 (58.74 percent) reacted to cELISA-PR1 test, suggesting an region of enzootic instability, with a significant percentage of animals susceptible to infection by A. marginale and potentially in risk to develop anaplasmosis. The kind of exploration in the property, the breeding and handling system, the presence of other animals (ovine/caprine, horses, wild animals), and means of commercialization of animals were analyzed. The statistical analysis showed that there were no significant differences among the analyzed variables.

Soroprevalência de Anaplasma marginale em bovinos da região Centro-Sul do estado do Paraná, Brasil, por um teste imunoenzimático competitivo utilizando proteína recombinante MSP5-PR1

Anaplasma marginale prevalence was determined in 223 sera samples in 2-year old or older cattle, from the Center- Southern Region of the Paraná State, including Ponta Grossa, Guarapuava and Laranjeiras do Sul municipalities. A survey of antibodies IgG class against Anaplasma marginale was performed through a competitive immune absorbent assay (cELISA-PR1). From the 223 sera examined, 130 (58.74%) reacted to cELISA-PR1 test, suggesting an region of enzootic instability, with a significant percentage of animals susceptible to infection by A. marginale and potentially in risk to develop anaplasmosis. The kind of exploration in the property, the breeding and handling system, the presence of other animals (ovine/caprine, horses, wild animals), and means of commercialization of animals were analyzed. The statistical analysis showed that there were no significant differences among the analyzed variables.

Foi determinada a prevalência de Anaplasma marginale em 223 soros de bovinos com idade maior ou igual a dois anos, das regiões de Ponta Grossa, Guarapuava e Laranjeiras do Sul, região Centro-Sul do estado do Paraná. O teste imunoenzimático de competição PR1 (cELISA-PR1) foi utilizado para determinar a presença ou ausência de anticorpos anti-A. marginale. Dos 223 soros analisados, 130 (58,74%) foram positivos pelo cELISA-PR1, sugerindo ser essa região de instabilidade de enzoótica, com uma porcentagem significativa de animais susceptíveis à infecção por A. marginale, com risco potencial para desenvolver a anaplasmose. As características de tipo de exploração da propriedade, sistema de criação, manejo e forma de comercialização dos animais foram avaliadas. A análise estatística não demonstrou haver diferença significativa entre as variáveis estudadas e a positividade dos animais.

Evaluation of experimental conditions for quantification of LT produced by human derived enterotoxigenic Escherichia coli strains

The heat-labile toxin (LT) is a key virulence-associated factor associated with the non-invasive secretory diarrhea caused by enterotoxigenic Escherichia coli (ETEC) strains either in humans or domestic animals. Several LT detection methods have been reported but quantification of the toxin produced by wild-type ETEC strains is usually performed by the GM1 ganglyoside enzyme-linked immunosorbent assay (GM1 ELISA). In this study we conducted the optimization of an alternative LT-quantification method, the antibody-capture ELISA (cELISA). Detailed analysis of the appropriate dilutions of capture and detecting LT-specific antibodies significantly improved the sensitivity of the method. Additionally, testing of different LT extraction techniques indicated that sonic disruption of the bacterial cells enhanced LT recovery yields, in contrast to the usual procedure based on addition of polymyxin B to the culture medium as well as extraction methods based on chloroform or Triton X-100. Moreover, the present data indicate that performance of the LT extraction method based on polymyxin B treatment can vary among wild ETEC strains.

A toxina termo-lábil (LT) é um fator de virulência associado à diarréia secretora não invasiva causada por linhagens de Escherichia coli enterotoxigênica (ETEC) em humanos ou animais domésticos. Diversos métodos de detecção de LT foram descritos na literatura, no entanto, a quantificação da toxina produzida por linhagens selvagens de ETEC é geralmente realizada por ensaio imunoenzimático com o gangliosídeo GM-1 (GM-1 ELISA). Neste estudo, conduzimos uma otimização experimental de um método alternativo de quantificação de LT, o ELISA de captura (cELISA). Análise detalhada de diluições apropriadas dos anticorpos LT específicos de captura e detecção melhorou significantemente a sensibilidade do método. Em adição, testes com diferentes técnicas de extração de LT indicaram que a ruptura das células por ultra-som, mas não o tratamento com polimixina B, clorofórmio ou Triton X-100, aumentou o rendimento da recuperação de LT. Além disto, os dados apresentados demonstram que o desempenho do método de extração de LT baseado no tratamento com polimixina B pode variar entre linhagens selvagens de ETEC.

Detection of bovine viral diarrhea virus by amplification on polycation-treated cells followed by enzyme immunoassay / Detección del virus de la diarrea viral bovina por amplificación sobre células tratadas con policationes seguida de enzimoinmunoensayo

A bovine viral diarrhea virus (BVDV) amplification method combined with an enzyme immunoassay was developed to detect BVDV antigens in seropositive cattle. Reconstitution assays conducted by adding decreasing amounts of BVDV (Singer strain) to Madin-Darby bovine kidney (MDBK) cells showed that the sensitivity threshold of the combined assay was 10-7 TCID50. BVDV amplification was carried out in polycation (DEAE-Dextran and polybrene)- treated MDBK cells. Treated cells were able to replicate both ether-treated virus and neutralizing antibody-coated virus. Ammonium chloride decreased virus replication in polycation-treated cells, suggesting viral penetration by endocytosis. BVDV detection was tested in leukocytes from 104 seropositive cattle from 2 unvaccinated commercial closed dairy herds with high seroprevalence. Lysates and co-cultures of peripheral blood leukocytes (PBL) were tested, directly or after up to 6 blind passages in normal or polycation-treated cells. BVDV was detected in 10/104 cattle after only one co-culture of PBL in treated cells. No virus was detected in whole blood or plasma samples. BVDV positive and negative cattle were retested three times, achieving consistent results. The finding of immune carriers supports the possibility that these animals may constitute an epidemiological risk.

Se desarrolló un método de detección de antígenos del virus de la diarrea viral bovina (BVDV) combinando amplificación viral con enzimoinmunoensayo. El método combinado presentó una sensibilidad de 10-7 TCID50 en ensayos con diluciones decrecientes de BVDV cepa Singer sobre la línea celular MDBK. La amplificación del título viral se efectuó sobre células MDBK tratadas con policationes Estas células replicaron tanto el BVDV tratado con éter como el unido a anticuerpos. La replicación viral en las células tratadas disminuyó ante la presencia de cloruro de amonio, lo que sugiere la penetración viral por endocitosis. El BVDV se determinó en leucocitos de 104 bovinos seropositivos de dos rodeos en producción, cerrados y con alta seroprevalencia. Los leucocitos de sangre periférica (LSP) fueron lisados y analizados directamente o luego de hasta 6 pasajes ciegos sobre células normales o tratadas con policationes. El BVDV se detectó en 10 de los 104 animales después de solamente un cultivo de LSP en células tratadas. No se pudo detectar presencia viral en las muestras de sangre o plasma. Los estudios se repitieron tres veces en animales BVDV positivos y negativos, con resultados consistentes. El hallazgo de bovinos seropositivos portadores del virus indica la posibilidad de que estos animales puedan significar un riesgo epidemiológico.

Comparação dos testes sorológicos de Imunofluorescência Indireta, Conglutinação Rápida, ELISA indireto e ELISA por competição para a detecção de anticorpos contra o Anaplasma marginale em soros de bovinos de diferentes áreas enzoóticas / Comparison of the serologic tests of Indirect Immunofluorescence,Rapid Conglutination, Indirect ELISA and Competition ELISA fordetection of antibodies against Anaplasma marginale in cattle sera fromdifferent enzootic areas

Os Testes Sorológicos de Conglutinação Rápida (TCR) Imunofluorescência Indireta (IFI) e Imunoenzimáticos Indireto (iELISA) utilizando ELISA por competição (cELISA), como padrão ouro, foram avaliados comparativamente para a detecção de anticorpos contra o Anaplasma marginale. Foram utilizadas 453 amostras de soros sangüíneos de bovinos vacinados e não-vacinados e de áreas de estabilidade e instabilidade enzoótica. O iELISA, IFI e TCR apresentaram respectivamente, índice kappa=0,77 (substancial), 0,57 e 0,49 (moderado), sensibilidade de 90,6, 90,2 e 73,7 e especificidade de 86,6, 62,8, e 79,3. O iELISA apresentou o melhor desempenho e maior especificidade, podendo ser indicado na avaliação do perfil sorológico de rebanhos, na detecção de animais persistentemente infectados e de animais submetidos a programas de vacinação. As técnicas de IFI e TCR, mesmo apresentando desempenho inferior, podem ser recomendadas para a realização de inquéritos epidemiológicos e para o monitoramento de animais em trânsito entre as diferentes regiões geográficas

The serological techniques Rapid Conglutination Test (RCT), Indirect ELISA (iELISA) and IndirectImmunofluorescent Assay (IFA), using the competition ELISA (cELISA) as gold test, were comparativelyevaluated to detect antibodies against Anaplasma marginale. A total of 453 sera from vaccinated andnon vaccinated cattle and, collected from enzootic stability and instability areas were tested. iELISA, IFAand TCR presented kappa index = 0.77 (substantial); 0.57 and 0.49 (moderate), sensibility of 90.6%;90.2% and 73.7% and specificity of 86.6%; 62.8%, and 79.3%, respectively. Therefore, iELISA presentedbetter specificity than IFA and RCT, and can be indicated for more detailed serological investigations,detection of persistently infected animals in cattle herds and for monitorating of vaccination programs.IFA and TCR can be used in prevalence studies and to monitor cattle movement between differentgeographical regions

Comparison of the serologic tests of Indirect Immunofluorescence, Rapid Conglutination, Indirect ELISA and Competition ELISA for detection of antibodies against Anaplasma marginale in cattle sera from different enzootic areas / Comparação dos testes sorológicos de Imunofluorescência Indireta, Conglutinação Rápida, ELISA indireto e ELISA por competição para a detecção de anticorpos contra o Anaplasma marginale em soros de bovinos de diferentes áreas enzoóticas

The serological techniques Rapid Conglutination Test (RCT), Indirect ELISA (iELISA) and Indirect Immunofluorescent Assay (IFA), using the competition ELISA (cELISA) as gold test, were comparatively evaluated to detect antibodies against Anaplasma marginale. A total of 453 sera from vaccinated and non vaccinated cattle and, collected from enzootic stability and instability areas were tested. iELISA, IFA and TCR presented kappa index = 0.77 (substantial); 0.57 and 0.49 (moderate), sensibility of 90.6%; 90.2% and 73.7% and specificity of 86.6%; 62.8%, and 79.3%, respectively. Therefore, iELISA presented better specificity than IFA and RCT, and can be indicated for more detailed serological investigations, detection of persistently infected animals in cattle herds and for monitorating of vaccination programs. IFA and TCR can be used in prevalence studies and to monitor cattle movement between different geographical regions.

Os testes sorológicos de Conglutinação Rápida (TCR) Imunofluorescência Indireta (IFI) e Imunoenzimáticos Indireto (iELISA) utilizando ELISA por competição (cELISA), como padrão ouro, foram avaliados comparativamente para a detecção de anticorpos contra o Anaplasma marginale. Foram utilizadas 453 amostras de soros sangüíneos de bovinos vacinados e não-vacinados e de áreas de estabilidade e instabilidade enzoótica. O iELISA, IFI e TCR apresentaram respectivamente, índice kappa=0,77 (substancial), 0,57 e 0,49 (moderado), sensibilidade de 90,6%, 90,2% e 73,7% e especificidade de 86,6%, 62,8%, e 79,3%. O iELISA apresentou o melhor desempenho e maior especificidade, podendo ser indicado na avaliação do perfil sorológico de rebanhos, na detecção de animais persistentemente infectados e de animais submetidos a programas de vacinação. As técnicas de IFI e TCR, mesmo apresentando desempenho inferior, podem ser recomendadas para a realização de inquéritos epidemiológicos e para o monitoramento de animais em trânsito entre as diferentes regiões geográficas.

Dynamic of the natural infection by Anaplasma marginale in Holstein cows and calves in the Londrina region, North of Paraná State, Brazil / Dinâmica da infecção natural pelo Anaplasma marginale em vacas e bezerros da raça Holandesa, na região de Londrina, Estado do Paraná, Brasil

The dynamic of the infection by Anaplasma marginale in Holstein cows and calves was studied in two dairy farms (A and B) in the Londrina region, North of Paraná. In the farm A the cows were maintained in the tie-stall system and, the calves in collective stall; in the farm B, the cows stayed in freestall system and the calves in individual cages. Every 15 days, blood samples were collected from the dams 45 days before parturition until 60 days post partum, and from their calves at birth until 240 days of age. Tick burden counting was also performed on dams and calves twice a month. Percentage of infected erythrocytes was established by Giemsa-stained smears. Blood and sera samples were examined by Polimerase Chain Reaction (PCR) and competitive Enzime-Linked Immunosorbent Assay (cELISA), respectively. In the cows, the anti-A. marginale antibody levels decreased close to the parturition, showing an adverse behavior among the farms. In the farm A the levels of antibodies increased between 30 and 60 days after the parturition and, in farm B the antibodies stayed in low levels during the accompaniment period of the cows. The anti-A. marginale antibody levels of the calves of the farm A, that were not high in the day of the birth, decreased more until 45 days post parturition and it increased again starting from 60 days, with maximum pick to the 105 days. In the farm B, where the ca

A dinâmica da infecção por Anaplasma marginale em vacas e bezerros da raça Holandesa foi estudada em duas propriedades leiteiras (A e B) com manejos distintos da região Norte do Paraná. Na propriedade A as vacas eram mantidas no sistema tie-stall e, os bezerros em bezerreiros coletivos; na propriedade B, as vacas em sistema free-stall e os bezerros em gaiolas individuais. A cada 15 dias efetuou-se a contagem de carrapatos e coletas de amostras de sangue das vacas e seus respectivos bezerros. As vacas foram monitoradas dos 45 dias antes até 60 dias após o parto, e seus bezerros, do nascimento até 240 dias de idade. A parasitemia foi determinada em esfregaços sangüíneos corados pelo Giemsa. O sangue total e as amostras de soro obtidas foram submetidos, respectivamente, as técnicas da PCR e cELISA. Nas vacas os níveis de imunoglobulinas diminuíram próximo ao parto, além de apresentarem uma dinâmica de anticorpos diferentes entre as duas propriedades. Na propriedade A os níveis de anticorpos aumentou entre 30 e 60 dias após o parto e na B os anticorpos permaneceram em níveis baixos no período de monitoramento das vacas. Os níveis de anticorpos dos bezerros da propriedade A que não eram altos no dia do nascimento, diminuíram ainda mais até 45 dias pós-parto e voltou a crescer a partir de 60 dias, com pico máximo aos 105 dias. Na propriedade B, onde os bezerros apresentavam nívei

Dynamic of the natural infection by Anaplasma marginale in Holstein cows and calves in the Londrina region, North of Paraná State, Brazil / Dinâmica da infecção natural pelo Anaplasma marginale em vacas e bezerros da raça Holandesa, na região de Londrina, Estado do Paraná, Brasil

The dynamic of the infection by Anaplasma marginale in Holstein cows and calves was studied in two dairy farms (A and B) in the Londrina region, North of Paraná. In the farm A the cows were maintained in the tie-stall system and, the calves in collective stall; in the farm B, the cows stayed in freestall system and the calves in individual cages. Every 15 days, blood samples were collected from the dams 45 days before parturition until 60 days post partum, and from their calves at birth until 240 days of age. Tick burden counting was also performed on dams and calves twice a month. Percentage of infected erythrocytes was established by Giemsa-stained smears. Blood and sera samples were examined by Polimerase Chain Reaction (PCR) and competitive Enzime-Linked Immunosorbent Assay (cELISA), respectively. In the cows, the anti-A. marginale antibody levels decreased close to the parturition, showing an adverse behavior among the farms. In the farm A the levels of antibodies increased between 30 and 60 days after the parturition and, in farm B the antibodies stayed in low levels during the accompaniment period of the cows. The anti-A. marginale antibody levels of the calves of the farm A, that were not high in the day of the birth, decreased more until 45 days post parturition and it increased again starting from 60 days, with maximum pick to the 105 days. In the farm B, where the ca

A dinâmica da infecção por Anaplasma marginale em vacas e bezerros da raça Holandesa foi estudada em duas propriedades leiteiras (A e B) com manejos distintos da região Norte do Paraná. Na propriedade A as vacas eram mantidas no sistema tie-stall e, os bezerros em bezerreiros coletivos; na propriedade B, as vacas em sistema free-stall e os bezerros em gaiolas individuais. A cada 15 dias efetuou-se a contagem de carrapatos e coletas de amostras de sangue das vacas e seus respectivos bezerros. As vacas foram monitoradas dos 45 dias antes até 60 dias após o parto, e seus bezerros, do nascimento até 240 dias de idade. A parasitemia foi determinada em esfregaços sangüíneos corados pelo Giemsa. O sangue total e as amostras de soro obtidas foram submetidos, respectivamente, as técnicas da PCR e cELISA. Nas vacas os níveis de imunoglobulinas diminuíram próximo ao parto, além de apresentarem uma dinâmica de anticorpos diferentes entre as duas propriedades. Na propriedade A os níveis de anticorpos aumentou entre 30 e 60 dias após o parto e na B os anticorpos permaneceram em níveis baixos no período de monitoramento das vacas. Os níveis de anticorpos dos bezerros da propriedade A que não eram altos no dia do nascimento, diminuíram ainda mais até 45 dias pós-parto e voltou a crescer a partir de 60 dias, com pico máximo aos 105 dias. Na propriedade B, onde os bezerros apresentavam nívei

Seroprevalence fo Anaplasma marginale in dairy cattle and, studies on the dynamics of natural infection of Holstein calves in Southern Brazil / Soroprevalência de Anaplasma marginale em bovinos leiteiros e estudos sobre a dinâmica da infecção natural em bezerros holandeses no Sul do Brasil

Sera of 708 animals (cows, heifers and calves) from 13 dairy herds in the Londrina region of Paraná, Brazil, were tested for antibodies to Anaplasma marginale by a competitive ELISA assay (cELISA). Ten 2 to 20 days old Holstein calves, from one of the 13 herds studied, were monitored during one year. Blood samples from each calf were collected monthly and tick burden counting was performed every fortnight. Percentage of infected erythrocytes was established by Giemsa-stained smears, and sera samples were examined by cELISA to detect antibodies against A. marginale. In the 13 herds, 92.94% of the animals were seropositive to A. marginale, which indicates that Londrina is an area of enzootic stability. Among the three animal categories (cows, heifers and calves), the rates were 98.29%, 96.64% and 81.25%, respectively. Passive transfer of maternal antibodies to calves was demonstrated by cELISA. From ten calves, nine (90%) were seropositive at the first sampling, revealing colostral antibodies anti-A . marginale. These antibodies remained in calves for 2 to 3 months. After this period the calves were infected with ticks, and then all of them were seropositive to Anaplasma. Five 4 to 7 months old calves showed rickettsemia ranging from 0.1% to 3.8%. Two of them were treated with tetracycline. The rickettsemia and clinical signs of anaplasmosis of these calves were coincident with t

Soros de 708 animais (vacas, novilhas e bezerros) oriundos de 13 propriedades leiteiras da região de Londrina, Estado do Paraná, Brasil, foram testados para a presença de anticorpos contra Anaplasma marginale pelo teste ELISA competitivo (cELISA). Dez bezerros recém nascidos, pertencentes a um dos 13 rebanhos utilizados no levantamento sorológico, foram monitorados durante um ano. De cada bezerro foram colhidas amostras de sangue mensalmente e a cada quinze dias foi realizada a contagem de carrapatos. A porcentagem de eritrócitos parasitados foi determinada pela coloração de Giemsa em esfregaços sanguíneos e as amostras de soros foram submetidas ao cELISA para a detecção de anticorpos contra A. marginale. Dos animais dos rebanhos, 92,94% foram soropositivos para A. marginale indicando que a região de Londrina é uma área de estabilidade enzoótica. Nas três categorias de animais estudadas (vacas, novilhas e bezerros) as taxas de soropositivos foram de 98,29%, 96,64% e 81,25%, respectivamente. Nove dos 10 bezerros (90%) foram positivos pelo cELISA logo após o nascimento, o que demonstra a transferência de anticorpos colostrais. Os níveis de anticorpos colostrais foram detectados até o segundo e o terceiro mês de idade com um decréscimo neste período. Os níveis de anticorpos contra A. marginale voltaram a crescer nos meses seguintes, após os animais se infestarem com o Boophilus mi