Collectins and galectins in the abomasum of goats susceptible and resistant to gastrointestinal nematode infection.

Originally described in cattle, conglutinin belongs to the collectin family and is involved in innate immune defense. It is thought that conglutinin provides the first line of defense by maintaining a symbiotic relationship with the microbes in the rumen while inhibiting inflammatory reactions caused by antibodies leaking into the bloodstream. Due to the lack of information on the similar lectins and sequence detection in goats, we characterized the goat conglutinin gene using RACE and evaluated the differences in its gene expression profile, as well as in the gene expression profiles for surfactant protein A, galectins 14 and 11, interleukin 4 and interferon-gamma in goats. We used Saanen and Anglo Nubian F2 crossbred goats monitored over a period of four months and characterized them as resistant (R) or susceptible (S) based on the average values of EPG counts. Goat conglutinin was similar to bovine conglutinin, but its gene expression varied among different tissues. However, as with bovine conglutinin, it was most highly expressed in the liver. Variation in conglutinin (R=24.3±3.9; S=23.5±2.6, p=0.059), protein surfactant A (R=23.8±5.2, S=24.4±2.3, p=0.16), galectin 14 (R=15.9±3.5, S=14.7±6.2, p=0.49) and galectin l1 gene expression (R=25.4±2.6, S=25.8±3.7, p=0.53) was not significant between groups. However, there were weak correlations between interleukin 4 and the protein surfactant A gene (r=0.459, p=0.02) and between interleukin 4 and galectin 11 (r=0.498, p=0.01). Strong correlation between interferon-gamma and galectin 14 (r=0.744, p=0.00) was observed. Galectin 14 was negatively correlated with the number of nematodes in the goat (r=-0.416, p=0.04) as well as the EPG count (r=-0.408, p=0.04). This is the first study to date that identifies the gene expression of conglutinin, surfactant protein A and galectins 14 and 11 in the goat abomasum. In conclusion, we present evidence that lectin is involved in the immune response to gastrointestinal nematodes, which suggests that collectins and galectins are involved in the molecular recognition of helminths.

Evaluation of PCR and multiplex PCR in relation to nested PCR for diagnosing Theileria equi / Avaliação da PCR e PCR multiplex com relação a nested PCR no diagnóstico de Theileria equi

Conventional PCR (PCRTeq) for diagnosing Theileria equi and multiplex PCR (M/PCRTeq-Bc) for diagnosing T. equi and Babesia caballi were comparatively evaluated with nested PCR (N/PCR-Teq) for diagnosing equine piroplasmosis. In DNA sensitivity determinations, in multiple dilutions of equine blood that had tested positive for T. equi, PCR-Teq and N/PCR-Teq detected hemoparasite DNA in the larger dilutions (1:128), but did not differ significantly from the M/PCRTeq-Bc (1:64). In analyses on equine serum tested by ELISA, there was high agreement between this serological test and PCR-Teq (k = 0.780) and moderate agreement with N/PCR-Teq (k = 0.562) and M/PCRTeq-Bc (k = 0.488). PCR-Teq found a higher frequency of T. equi both in extensively and intensively reared horses, but this was not significant in relation to N/PCR-Teq (P>0.05), and both PCRs indicated that there was an endemic situation regarding T. equi in the population of horses of this sample. PCR-Teq was only significantly different from M/PCR-Teq-Bc (P<0.05). PCR-Teq presented high sensitivity and specificity, comparable to N/PCR-Teq, but with the advantage of higher speed in obtaining results and lower costs and risks of laboratory contamination. This accredits PCR-Teq for epidemiological studies and for determinations on affected horses.

Uma PCR convencional (PCRTeq) para diagnóstico de Theileria equi e PCR multiplex (M/PCRTeq-Bc) para diagnóstico T. equi e Babesia caballi foram avaliadas comparativamente a nested PCR (N/PCR-Teq) no diagnóstico de piroplasmose equina. Na determinação da sensibilidade com DNA em diluições múltiplas de sangue de equino positivo para T. equi, as PCR-Teq e N/PCR-Teq detectaram DNA do hemoparasito nas diluições maiores (1:128), mas não diferiu significativamente da M/PCRTeq-Bc (1:64). Na análise com soros de equinos testados por ELISA houve uma concordância elevada entre esse teste sorológico e a PCR-Teq (k =0,780) e moderada com N/PCR-Teq (k = 0.562) e M/PCRTeq-Bc (k = 0.488). A PCR-Teq determinou freqüência maior de T. equi tanto nos equinos de criação extensiva como na intensiva, entretanto essa não foi significativa com relação N/PCR-Teq (P>0.05), e ambas PCRs indicaram que há uma situação endêmica para T. equi na população de equinos que constaram da amostragem. A PCR-Teq diferiu significativamente apenas para a M/PCRTeq-Bc (P<0.05). A PCR-Teq apresentou alta sensibilidade e especificidade comparável a N/PCR-Teq, mas com a vantagem de maior rapidez na obtenção dos resultados e menor custo e risco de contaminação no laboratório. Isso credencia a PCR-Teq para estudos epidemiológicos e para determinação de equinos portadores.

Trypanosoma vivax infection dynamics in a cattle herd maintained in a transition area between Pantanal lowlands and highlands of Mato Grosso do Sul, Brazil

Trypanosoma vivax outbreaks in beef cattle in the Pantanal region of Mato Grosso do Sul state, Brazil, causes relevant economical impact due to weight loss, abortion and mortality. Cattle moved from the Pantanal to adjacent areas of this ecosystem for breeding and fattening is a common feature. Therefore an epidemiological study on breeding cows in the transition area between Pantanal lowland and adjacent highlands of Mato Grosso do Sul was performed to determine the T. vivax infection dynamics and outbreak risk. Three experimental groups were formed: Group 1 consisted of cows parasitologically negative by the Woo test and in the enzyme-linked immunosorbent assay for T. vivax antibody detection (Tv-ELISA-Ab); Group 2 parasitologically negative and positive in the Tv-ELISA-Ab; and in Group 3 cows were parasitologically positive and with positive reactions in the Tv-ELISA-Ab. During 24 months, the cows' dislodgment between the above established groups was monitored by Woo test and Tv-ELISA-Ab exams. The tabanid population was also monitored and the highest number occurred during the rainy season. Although parasitemias were detected only in the first four samplings of the experimental period, the cows could be considered as trypanotolerant, because no clinical signs were observed. Despite the higher T. vivax incidence during the dry season, no disease symptoms were seen. Even though T. vivax epidemiological situation in the herd was characterized as endemic with seasonal variation, the probability of outbreaks was null within the conditions of the study.

Surtos de Trypanosoma vivax em bovinos de corte do Pantanal foram responsáveis por relevante impacto econômico, devido a perda de peso, abortos e mortalidade. Um manejo comum é o deslocamento de bovinos do Pantanal baixo para áreas adjacentes desse ecosistema para reprodução e engorda. Por essa razão, foi efetuado um estudo epidemiológico em rebanho de vacas movidas para uma área de transição entre Pantanal baixo e planalto do Estado de Mato Grosso do Sul para determinar a dinâmica de infecção do T. vivax e o risco de surto. Três grupos experimentais foram formados: Grupo 1; composto por vacas parasitologicamente negativas no teste de Woo e no exame sorológico de imunoadsorção enzimática para detecção de anticorpos contra T. vivax (Tv-ELISA-Ab); Grupo 2, vacas negativas parasitológicamente e com reação positiva no Tv-ELISA-Ab; e no Grupo 3, positivas parasitologicamente e no Tv-ELISA-Ab. Durante 24 meses o deslocamento das vacas entre esses grupos experimentais foi determinado pelo monitoramento mensal realizado pelo teste de Woo e Tv-ELISA-Ab. Durante esse período a população de tabanídeos na área experimental foi determinada e as maiores populações ocorreram no período das chuvas. Parasitemias de T. vivax foram detectadas apenas nas quatro primeiras amostragens do período experimental, apesar da elevação de incidência determinada sorológicamente tenha ocorrido no período seco do ano. Portanto, T. vivax foi endêmico no rebanho e a ausência de manifestação clínica sugere que os bovinos sejam tripanotolerantes e o risco de surto seja nulo nas condições em que o experimento foi executado, pois a manifestação clínica da doença esta associada à presença de parasitemia.

[Transcription of genes of membrane proteins of Brazilians isolates of Anaplasma marginale]. / Transcrição de genes de proteínas de membrana de isolados brasileiros de Anaplasma marginale.

This work shows the transcription profile of membrane protein genes in three Brazilian isolates of Anaplasma marginale (Rio Grande do Norte, Pernambuco-Zona da Mata, and Pernambuco-Sertão). RNA was purified from cattle blood experimentally-infected with the three isolates of A. marginale. After reverse transcription, genes omp1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, and 14; opag1-3; virB3, 9, and 10; am097, 197, 254, 854, and 956 were amplified by PCR, with specific primers. Transcripts were detected for all genes, except omp2, 3 e opag3 in all isolates and for omp7 in one out of the three isolates analyzed. Absence of transcription for opag3 and omp7 diverge from the North American isolates of A. marginale. Reasons for such differences were discussed.

Transcrição de genes de proteínas de membrana de isolados brasileiros de Anaplasma marginale / Transcription of genes of membrane proteins of Brazilians isolates of Anaplasma marginale

Este trabalho demonstra o padrão de transcrição de genes de proteínas de membrana em três isolados brasileiros de A. marginale (Rio Grande do Norte, Pernambuco-Zona da Mata e Pernambuco-Sertão). O RNA foi purificado a partir de sangue de bovinos infectados experimentalmente com os três isolados de A. marginale. Após transcrição reversa, os genes omp1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13 e 14; opag1-3; virB3, 9, 10; am097, 197, 254, 854 e 956 foram amplificados por PCR, com oligonucleotídeos iniciadores específicos. Detectaram-se transcritos para todos os genes analisados, exceto omp2, 3 e opag3 em todos os isolados e do gene omp7 em um dos isolados estudados. A ausência de transcrito para os genes opag3 e omp7 diverge do observado em isolados americanos da riquétsia. Possíveis razões para essas diferenças são discutidas.

This work shows the transcription profile of membrane protein genes in three Brazilian isolates of Anaplasma marginale (Rio Grande do Norte, Pernambuco-Zona da Mata, and Pernambuco-Sertão). RNA was purified from cattle blood experimentally-infected with the three isolates of A. marginale. After reverse transcription, genes omp1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, and 14; opag1-3; virB3, 9, and 10; am097, 197, 254, 854, and 956 were amplified by PCR, with specific primers. Transcripts were detected for all genes, except omp2, 3 e opag3 in all isolates and for omp7 in one out of the three isolates analyzed. Absence of transcription for opag3 and omp7 diverge from the North American isolates of A. marginale. Reasons for such differences were discussed.

ELISA com MSP5 recombinante truncada para detecção de anticorpos contra Anaplasma marginale em bovinos / ELISA based on recombinant truncated MSP5 for detection of antibodies against Anaplasma marginale in cattle

Os objetivos deste estudo foram produzir e solubilizar a proteína MSP5 recombinante truncada de Anaplasma marginale, e avaliar seu desempenho em um ensaio de imunoadsorção enzimática indireto (ELISA) para detecção de anticorpos contra a riquétsia. O gene msp5, exceto a região N-terminal hidrofóbica, foi amplificado por PCR, clonado em plasmídeo pTrcHis-TOPO e expresso em Escherichia coli. A solubilização da proteína recombinante foi avaliada em diferentes pHs e concentrações de uréia. A sensibilidade e a especificidade do ensaio foram avaliados testando-se 66 soros de animais infectados experimentalmente com A. marginale e 96 soros negativos, com o estado de infecção destes animais confirmado por PCR. Um total de 1.666 amostras de soros bovino, provenientes do Brasil - Rio Grande do Sul (73), Mato Grosso do Sul (91), Pernambuco (86), Bahia (314) e Minas Gerais (267)-, Uruguai (32) e Costa Rica (803) foram testadas nos ELISAs com MSP5 truncada e com MSP1a recombinantes e a concordância entre os dois testes foi avaliada. O ELISA indireto com MSP5 truncada foi capaz de detectar animais infectados com 96,97 por cento de sensibilidade e 100 por cento de especificidade. Nos animais infectados experimentalmente, o ELISA detectou anticorpos do 12° até o último dia de observação (37° dia). Os ELISAs para MSP5 e MSP1a apresentaram concordância de 95,67 por cento, com índice kappa de 0,81. Os resultados discordantes apresentaram uma diferença significativa (p <0,001). Anticorpos contra A. marginale foram detectados em animais de todas as regiões estudadas. O ELISA com MSP5 recombinante truncada apresentou bom desempenho na detecção de anticorpos contra A. marginale, com alta sensibilidade e especificidade, representando uma importante ferramenta para o diagnóstico da anaplasmose bovina em estudos epidemiológicos.

The objective of this study was the production and solubilization of recombinant truncated MSP5 of Anaplasma marginale and the evaluation of its performance in an enzyme-linked immunosorbent assay (ELISA), to detect antibodies against the rickettsia in cattle. The fragment of msp5 gene, except the hydrophobic N-terminal region, was amplified by PCR, cloned in pTrcHis-TOPO plasmid and expressed in Escherichia coli. Solubilization of the recombinant protein was evaluated in different pHs and concentrations of urea. The sensibility and specificity of the assay were evaluated with 66 sera from cattle experimentally-infected and 96 sera from cattle free of A. marginale defined by polymerase chain reaction for msp5 gene. Serum samples from 1,666 cattle from Brazil - states of Rio Grande do Sul (73), Mato Grosso do Sul (91), Pernambuco (86), Bahia (314) and Minas Gerais (267), Uruguay (32) and Costa Rica (803), were tested by ELISAs with recombinant truncated MSP5 and with recombinant MSP1a, and the agreement between both ELISAs was calculated. ELISA with recombinant truncated MSP5 protein detected infected animals with sensibility of 96.97 percent and specificity of 100 percent. In cattle experimentally-infected, the ELISA detected antibodies from the 12th day post-infection (DPI) to the end of the experiment, at the 37th DPI. The agreement between the ELISAs with truncated MSP5 and MSP1a antigens was 95.67 percent, with a kappa index of 0.81. Disagreement results showed significative difference (p <0.001). Antibodies for A. marginale were detected in animals of the all the region analyzed. The ELISA with recombinant truncated MSP5 showed a good performance in ELISA for detention of antibodies against A. marginale, with high sensitivity and specificity, representing an important tool for the diagnosis of anaplasmose bovine in epidemiological studies.

The development of an enzyme-linked immunosorbent assay for Trypanosoma vivax antibodies and its use in epidemiological surveys.

There are data indicating that the distribution of Trypanosoma vivax in the Brazilian territory is expanding with potential to reach other areas, where the vectors are present. The detection of anti-trypanosomal antibodies in serum provides important information of the trypanosomal status in cattle herds. For this reason, an enzyme-linked immunosorbent assay (Tv-ELISA-Ab) with crude antigen from one Brazilian isolate of T. vivax was developed and evaluated. The sensitivity and specificity were respectively 97.6 and 96.9%. In the evaluation of cross-reactions, three calves inoculated with T. evansi trypimastigotes blood forms showed optical densities (OD) under the cut-off during the whole experimental period, except one at 45 days post-inoculation. With relation to Babesia bovis, B. bigemina, and Anaplasma marginale, which are endemic hemoparasites in the studied area, the cross-reactions were shown to be 5.7, 5.3, and 1.1%, respectively. The first serological survey of Pantanal and state of Pará showed that T. vivax is widespread, although regions within both areas had significantly different prevalences. Therefore, this Tv-ELISA-Ab may be a more appropriate test for epidemiological studies in developing countries because the diagnostic laboratories in most countries may be able to perform an ELISA, which is not true for polymerase chain reaction.

Resposta imune específica de bovinos experimentalmente sensibilizados com inóculos inativados de Mycobacterium bovis e Mycobacterium avium / Specific immune response of cattle to experimental sensibilization by inactivated Mycobacterium bovis and Mycobacterium avium

O diagnóstico presuntivo da tuberculose bovina é baseado na análise da resposta imune celular a antígenos micobacterianos. Procedeu-se à simulação experimental de sensibilização por Mycobacterium bovis e Mycobacterium avium inativados em bovinos a fim de acompanhar a resposta imune a partir do teste cervical comparativo e da evolução da produção específica de interferon-gama, além de identificar a interferência de reações inespecíficas por M. avium nos resultados dos testes. Verificou-se que os animais desencadearam resposta de hipersensibilidade tardia contra os bacilos inativados, e que ambos os testes diagnósticos da tuberculose bovina foram eficientes na identificação dos animais sensibilizados com M. bovis e na discriminação das reações geradas pela inoculação dos bovinos com M. avium.

The presumptive diagnosis of bovine tuberculosis is based on analysis of the immune response to micobacterial antigens. This experimental simulation of sensibilization by Mycobacterium bovis and Mycobacterium avium in cattle aimed to verify the immune response by both the cervical comparative test and the evolution of the specific production of gamma-interferon, and also to identify interference of unspecified reactions by M. avium on the test results. The results support that the experimental animals started a response of delayed hypersensitivity to the inactivated bacilli, and that both diagnostic tests for bovine tuberculosis were efficient for the identification of animals sensitized with M. bovis and for discrimination of reactions generated by inoculation of cattle with M. avium.

The development of an enzyme-linked immunosorbent assay for Trypanosoma vivax antibodies and its use in epidemiological surveys

There are data indicating that the distribution of Trypanosoma vivax in the Brazilian territory is expanding with potential to reach other areas, where the vectors are present. The detection of anti-trypanosomal antibodies in serum provides important information of the trypanosomal status in cattle herds. For this reason, an enzyme-linked immunosorbent assay (Tv-ELISA-Ab) with crude antigen from one Brazilian isolate of T. vivax was developed and evaluated. The sensitivity and specificity were respectively 97.6 and 96.9 percent. In the evaluation of cross-reactions, three calves inoculated with T. evansi trypimastigotes blood forms showed optical densities (OD) under the cut-off during the whole experimental period, except one at 45 days post-inoculation. With relation to Babesia bovis, B. bigemina, and Anaplasma marginale, which are endemic hemoparasites in the studied area, the cross-reactions were shown to be 5.7, 5.3, and 1.1 percent, respectively. The first serological survey of Pantanal and state of Pará showed that T. vivax is widespread, although regions within both areas had significantly different prevalences. Therefore, this Tv-ELISA-Ab may be a more appropriate test for epidemiological studies in developing countries because the diagnostic laboratories in most countries may be able to perform an ELISA, which is not true for polymerase chain reaction.

Comparison between indirect enzyme-linked immunosorbent assays for Anaplasma marginale antibodies with recombinant major surface protein 5 and initial body antigens.

Indirect enzyme-linked immunosorbent assays (ELISAs) based on recombinant major surface protein 5 (rMSP5) and initial body (IB) antigens from a Brazilian isolate of Anaplasma marginale were developed to detect antibodies against this rickettsia in cattle. Both tests showed the same sensitivity (98.2%) and specificities (100% for rMSP5 and 93.8% for IB ELISA) which did not differ statistically. No cross-reactions were detected with Babesia bigemina antibodies, but 5 (rMSP5 ELISA) to 15% (IB ELISA) of cross-reactions were detected with B. bovis antibodies. However, such difference was not statistically significant. Prevalences of seropositive crossbred beef cattle raised extensively in Miranda county, state of Mato Grosso do Sul, Brazil, were 78.1% by rMSP5 ELISA and 79.7% by IB ELISA. In the analysis of sera from dairy calves naturally-infected with A. marginale, the dynamics of antibody production was very similar between both tests, with maternal antibodies reaching the lowest levels at 15-30 days, followed by an increase in the mean optical densities in both ELISAs, suggesting the development of active immunity against A. marginale. Results showed that all calves were seropositive by one-year old, characterizing a situation of enzootic stability. The similar performances of the ELISAs suggest that both tests can be used in epidemiological surveys for detection of antibodies to A. marginale in cattle.

Comparison between indirect enzyme-linked immunosorbent assays for Anaplasma marginale antibodies with recombinant major surface protein 5 and initial body antigens

Indirect enzyme-linked immunosorbent assays (ELISAs) based on recombinant major surface protein 5 (rMSP5) and initial body (IB) antigens from a Brazilian isolate of Anaplasma marginale were developed to detect antibodies against this rickettsia in cattle. Both tests showed the same sensitivity (98.2 percent) and specificities (100 percent for rMSP5 and 93.8 percent for IB ELISA) which did not differ statistically. No cross-reactions were detected with Babesia bigemina antibodies, but 5 (rMSP5 ELISA) to 15 percent (IB ELISA) of cross-reactions were detected with B. bovis antibodies. However, such difference was not statistically significant. Prevalences of seropositive crossbred beef cattle raised extensively in Miranda county, state of Mato Grosso do Sul, Brazil, were 78.1 percent by rMSP5 ELISA and 79.7 percent by IB ELISA. In the analysis of sera from dairy calves naturally-infected with A. marginale, the dynamics of antibody production was very similar between both tests, with maternal antibodies reaching the lowest levels at 15-30 days, followed by an increase in the mean optical densities in both ELISAs, suggesting the development of active immunity against A. marginale. Results showed that all calves were seropositive by one-year old, characterizing a situation of enzootic stability. The similar performances of the ELISAs suggest that both tests can be used in epidemiological surveys for detection of antibodies to A. marginale in cattle.

Condição imunológica de bovinos das raças Holandesa e Nelore frente a Babesia bovis e B. bigemina em duas regiões do Estado de São Paulo / Immunological condition of cattle in Holstein and Nelore breed in regard to Babesia bovis and B. bigemina in two regions of the State of São Paulo

A resposta imunológica de uma população frente a um agente infeccioso pode variar entre as raças e o manejo dessa população. Dessa maneira, torna-se relevante a pesquisa regional, visando o conhecimento da inter-relação do agente com seu hospedeiro. Partindo desses pressupostos, investigou-se a ocorrência de imunoglobulinas da classe IgG, anti-Babesia bovis e anti-B. bigemina nas raças Nelore (Bos indicus) e Holandesa (Bos taurus), em duas regiões do Estado de São Paulo, distantes a 300 km. Pelo método de ELISA indireto, foram testadas 1.161 amostras de soro de bovinos. As freqüências médias de anticorpos mostraram que ambas as regiões se encontram em situação de estabilidade enzoótica para B. bovis para ambas as raças estudadas, embora haja tendência para área marginal na região de Presidente Prudente para raça Nelore. No referente a B. bigemina ambas as regiões são de estabilidade enzoótica para a raça Holandesa e de instabilidade enzoótica para a raça Nelore. Essa constatação é um alerta sanitário, pois casos agudos da doença ou surtos específicos de B. bigemina na raça Nelore podem ocorrer nessas regiões.

The immunological reply of a population to an infectious agent can vary between races and handling of this population. Regional research becomes important, in order to know the interrelation between the agent and its host. In this way, the occurrence of immunoglobulins of class G, anti-Babesia bovis and anti-Babesia bigemina in the Nelore (Bos indicus) and Hostein breed (Bos taurus), was investigated in two regions of the State of São Paulo, 300 km distant from each other. For the indirect method of ELISA, 1,161 bovine serum samples were tested. The medium frequencies of antibodies showed that in the two regions exists an enzootic stability for B. bovis in both breeds studied; even so there was a tendency of marginal area for the Nelore breed in one of the regions. Regarding B. bigemina, in both regions exists enzootic stability for the Hostein and enzootic instability for the Nelore breed. Therefore, acute cases of the disease or specific outbreaks by B. bigemina infection in the Nelore breed may occur in these regions.

Serological survey of Babesia bovis, Babesia bigemina, and Anaplasma marginale antibodies in cattle from the semi-arid region of the state of Bahia, Brazil, by enzyme-linked immunosorbent assays.

The objectives of this work were to determine the prevalence of Babesia bovis, Babesia bigemina, and Anaplasma marginale detecting antibodies in cattle raised in the semi-arid region of the state of Bahia, Brazil, through indirect enzyme linked immunosorbent assays (ELISA) and to compare the performances of indirect enzyme-linked immunosorbent assays with crude (I-ELISA-CrAnaAg) and recombinant major surface protein-5 (I-ELISA-MSP-5Ag), as antigens to detect antibodies against A. marginale. An stable enzootic area was found in Senhor do Bonfim and Euclides da Cunha for B. bovis that showed 86 and 95.5% of prevalence, respectively, and for B. bigemina with 90.8 and 91.3%. On the other hand, Uauá and Juazeiro, were characterized as enzootically unstable areas, since prevalences were: B. bovis--63.7 and 56.4% and B. bigemina--53 and 54.8%, respectively. The prevalence of A. marginale in the four municipalities was above 97% with I-ELISA-CrAnaAg and 94.8% with I-ELISA-MSP-5Ag which is an indication of stable enzootic condition for the rickettsia. The I-ELISA-CrAnaAg and I-ELISA-MSP-5Ag showed a highly significant association (r = 0.977), which means that both ELISA tests are suitable for epidemiological studies of A. marginale.

Development of enzyme-linked immunosorbent assays based on recombinant MSP1a and MSP2 of Anaplasma marginale

Indirect enzyme-linked immunosorbent assays (ELISAs) based on recombinant MSP1a and MSP2 from a Brazilian isolate of Anaplasma marginale were developed to detect antibodies against this rickettsia in cattle. The high sensitivities (99 percent for both tests) and specificities (100 percent for both tests) were confirmed with sera from cattle positive or negative for A. marginale antibodies, respectively, by immunofluorescent antibody test. By the analysis of 583 sera from cattle of three regions of the state of Pernambuco, Brazil, the agreement between both tests was high, with a kappa index of 0.89. The similar performances of the ELISAs suggest that both tests can be used in epidemiological surveys for detection of antibodies to A. marginale in cattle.

Serological survey of Babesia bovis, Babesia bigemina, and Anaplasma marginale antibodies in cattle from the semi-arid region of the state of Bahia, Brazil, by enzyme-linked immunosorbent assays

The objectives of this work were to determine the prevalence of Babesia bovis, Babesia bigemina, and Anaplasma marginale detecting antibodies in cattle raised in the semi-arid region of the state of Bahia, Brazil, through indirect enzyme linked immunosorbent assays (ELISA) and to compare the performances of indirect enzyme-linked immunosorbent assays with crude (I-ELISA-CrAnaAg) and recombinant major surface protein-5 (I-ELISA-MSP-5Ag), as antigens to detect antibodies against A. marginale. An stable enzootic area was found in Senhor do Bonfim and Euclides da Cunha for B. bovis that showed 86 and 95.5 percent of prevalence, respectively, and for B. bigemina with 90.8 and 91.3 percent. On the other hand, Uauá and Juazeiro, were characterized as enzootically unstable areas, since prevalences were: B. bovis - 63.7 and 56.4 percent and B. bigemina - 53 and 54.8 percent, respectively. The prevalence of A. marginale in the four municipalities was above 97 percent with I-ELISA-CrAnaAg and 94.8 percent with I-ELISA-MSP-5Ag which is an indication of stable enzootic condition for the rickettsia. The I-ELISA-CrAnaAg and I-ELISA-MSP-5Ag showed a highly significant association (r = 0.977), which means that both ELISA tests are suitable for epidemiological studies of A. marginale.

Development of enzyme-linked immunosorbent assays based on recombinant MSP1a and MSP2 of Anaplasma marginale.

Indirect enzyme-linked immunosorbent assays (ELISAs) based on recombinant MSP1a and MSP2 from a Brazilian isolate of Anaplasma marginale were developed to detect antibodies against this rickettsia in cattle. The high sensitivities (99% for both tests) and specificities (100% for both tests) were confirmed with sera from cattle positive or negative for A. marginale antibodies, respectively, by immunofluorescent antibody test. By the analysis of 583 sera from cattle of three regions of the state of Pernambuco, Brazil, the agreement between both tests was high, with a kappa index of 0.89. The similar performances of the ELISAs suggest that both tests can be used in epidemiological surveys for detection of antibodies to A. marginale in cattle.

Progressos na imunização contra Anaplasma marginale / Progresses in immunization against Anaplasma marginale

Até o presente momento, as imunizações contra anaplasmose em rebanhos bovinos utilizam organismos vivos ou mortos. No entanto, esforços têm sido realizados nos últimos anos com o objetivo de desenvolver uma nova geração de vacinas. A membrana externa de Anaplasma marginale é capaz de induzir reposta imune protetora contra desafio homólogo e parcialmente protetora contra desafio heterólo-go. Nela foram identificadas seis proteínas principais de superfície (MSPs), as quais têm sido alvo de estudos para o desenvolvimento de imunógenos contra a anaplasmose. Destas proteínas, MSP1a e MSP2 têm demonstrado maior potencial como imunógenos, protegendo os animais contra desafio com isolados virulentos homólogos e heterólogos de A. marginale, apesar do polimorfismo de tamanho da primeira proteína e variabilidade do gene que codifica a segunda. Uma outra alternativa para a imunização contra A. marginale é o cultivo in vitro dessa riquétsia. Organismos inativados provenientes de cultivo em células IDE8 de Dermacentor variabilis foram testados como imunógeno. Os animais apresentaram uma significativa diferença na redução do volume globular após desafio e não apresentaram sinais clínicos de anaplasmose. Além da proteção conferida por este tipo de imunógeno, os organismos provenientes de cultura de células de carrapato são livres de células e patógenos de bovinos, o que é uma vantagem significativa quando comparado aos processos tradicionais de imunização

Antigenic characterization of Brazilian isolates of Anaplasma marginale.

Antigenic characterization of Anaplasma marginale isolates, by identifying conserved and variable epitopes of major surface proteins (MSP), is an important tool for vaccine development against this rickettsia. The B cell epitopes of A. marginale isolates from three microregions of the State of Pernambuco and one from the State of Mato Grosso do Sul, Brazil, were characterized by indirect fluorescent antibody technique (IFAT) and Western blot (WB) with 15 monoclonal antibodies (MAbs). The epitope recognized by MAb ANA22B1 (MSP-1a) was conserved by IFAT and WB (73-81 kDa). MSP-2 epitopes recognized by MAbs ANAO58A2 and ANAO70A2 were conserved by IFAT, while ANAO50A2 and ANA66A2 epitopes were polymorphic; in the WB, the MAbs ANAO50A2 and ANAO70A2 identified bands of 45 kDa only in the Pernambuco-Mata isolate. None of the isolates reacted with MAb ANAR75C2 (MSP-3). The MSP-4 epitope recognized by MAb ANAR76A1 was conserved by IFAT, as well as the MSP-5 epitope recognized by MAb ANAF16C1 by IFAT and WB (16 kDa). The MAbs ANAR17A6, ANAR83B3, ANAR94C1, ANAO24D5 and ANAR19A6 identified conserved epitopes by IFAT. MSP-1, MSP-2 and MSP-4, which previously showed partial protection in experimental trials, are also potential immunogens to be employed in Brazil, due to the B cell epitope conservation.

Avaliação clínica e hematológica em bezerros Nelore infectados experimentalmente com isolados de Babesia bigemina das regiões Sudeste, Nordeste e Norte do Brasil / Clinical and hematological evaluation of Nelore calves experimentally infected with isolates of Babesia bigemina from the Southeastern, Northeastern and Northern regions of Brazil

O presente trabalho teve por objetivo estudar comparativamente as alteraçöes clínicas e hematológicas desencadeadas por isolados de Babesia bigemina das regiöes Sudeste, Nordeste e Norte do Brasil em bezerros Nelore infectados experimentalmente. Foram utilizados 18 bezerros com idade entre sete e nove meses, isentos de anticorpos contra Babesia sp. e criados livres de carrapatos. Três animais foram previamente inoculados com 2,0x10(9) eritrócitos parasitados (EP) para cada isolado. Os outros 15 bezerros foram subdivididos em três grupos de cinco animais, que foram subinoculados com 1,0x10(10) EP dos respectivos isolados. Foram avaliadas as alteraçöes clínicas e hematológicas por meio da determinação da parasitemia, do hemograma, do fibrinogênio plasmático, da contagem de reticulócitos, da análise descritiva da medula óssea e da fragilidade osmótica eritrocitária, no decorrer de 30 dias, perfazendo um total de sete momentos de observaçäo. O acompanhamento da resposta imunológica pelo teste de imunofluorescência indireta foi realizado diariamente até o 10º dia pós-inoculaçäo (DPI) e posteriormente no 15º, 20º, 25º e 30º DPI. Clinicamente, observou-se uma manifestaçäo muito branda da doença. Os achados laboratoriais revelaram baixos níveis de parasitemia; decréscimo nos valores do eritrograma; ausência de reticulócitos; diminuiçäo inicial na contagem total dos leucócitos, neutrófilos e linfócitos com posterior elevaçäo do número destas células; hipercelularidade da série eritrocítica e decréscimo da relaçäo mielóide:eritróide mais acentuada entre o 8º e 12º DPI e um aumento da fragilidade osmótica eritrocitária nos grupos inoculados com os isolados sudeste e nordeste. Nenhum dos três isolados de B. bigemina desencadeou a forma clínica característica da enfermidade, apesar de induzirem uma resposta imune humoral

Antigenic characterization of Brazilian isolates of Anaplasma marginale

Antigenic characterization of Anaplasma marginale isolates, by identifying conserved and variable epitopes of major surface proteins (MSP), is an important tool for vaccine development against this rickettsia. The B cell epitopes of A. marginale isolates from three microregions of the State of Pernambuco and one from the State of Mato Grosso do Sul, Brazil, were characterized by indirect fluorescent antibody technique (IFAT) and Western blot (WB) with 15 monoclonal antibodies (MAbs). The epitope recognized by MAb ANA22B1 (MSP-1a) was conserved by IFAT and WB (73-81 kDa). MSP-2 epitopes recognized by MAbs ANAO58A2 and ANAO70A2 were conserved by IFAT, while ANAO50A2 and ANA66A2 epitopes were polymorphic; in the WB, the MAbs ANAO50A2 and ANAO70A2 identified bands of 45 kDa only in the Pernambuco-Mata isolate. None of the isolates reacted with MAb ANAR75C2 (MSP-3). The MSP-4 epitope recognized by MAb ANAR76A1 was conserved by IFAT, as well as the MSP-5 epitope recognized by MAb ANAF16C1 by IFAT and WB (16 kDa). The MAbs ANAR17A6, ANAR83B3, ANAR94C1, ANAO24D5 and ANAR19A6 identified conserved epitopes by IFAT. MSP-1, MSP-2 and MSP-4, which previously showed partial protection in experimental trials, are also potential immunogens to be employed in Brazil, due to the B cell epitope conservation