Real time polymerase chain reaction to diagnose Anaplasma marginale in cattle and deer (Ozotoceros bezoarticus leucogaster) of the Brazilian Pantanal / Reação da polimerase em cadeia tempo real para diagnóstico de Anaplasma marginale em bovino e veado campeiro do Pantanal brasileiro Ozotoceros bezoarticus leucogaster

Epizootiological study of Anaplasma marginale in regions that contain various reservoir hosts, co-existence of rickettsiapathogens, and common vectors is a complicated task. To achieve diagnosis of this rickettsia in cattle and campeiro deer ofBrazilian Pantanal, a comparison was made between a real time polymerase chain reaction (RT-PCR) with intercalating SybrGreen fluorochrome and primers based on msp5 gene of A. marginale; a conventional PCR (C-PCR); and parasitologicalexamination using thin blood smear stained with Giemsa-MayGrunwald. Both PCRs showed good performance in thediagnosis of A. marginale in cattle, and were superior to the parasitological exam. The RT-PCR detected seven positive campeirodeer (16.3%). This rate was significantly higher compared to C-PCR, which identified one animal as positive (2.3%), andalso compared to parasitological diagnosis, which did not find any positive animals. The dissociation temperature average ofpositive reactions in cattle (81.72 °C ± 0.20) was identical to dissociation temperature found in the cervids (81.72 °C ± 0.12),suggesting that both animal species were infected with A. marginale. We concluded that RT-PCR can be used for A. marginalediagnosis and in epizootiological studies of cattle and cervids; in spite of the small number of campeiro deer samples, theresults indicated that this wildlife species has importance in the Anaplasma epizootiology in the Brazilian Pantanal.(AU)

O estudo epizootiológico de Anaplasma marginale em regiões que existem vários reservatórios, co-existência de espéciesde riquétsias patógenas e vetores comuns é uma tarefa complicada. Com o objetivo de obter o diagnóstico dessa riquétsia embovinos e veado campeiro do Pantanal brasileiro foi avaliada uma reação da polimerase em cadeia em tempo real (PCR‑TR)com o fluoróforo intercalante de fita dupla de DNA Sybr Green e iniciadores baseados na seqüência do gene msp5 deA. marginale comparando-a a uma PCR convencional (PCR-C) e ao exame parasitológico de esfregaço fino de sangue coradocom Giemsa-MayGrunwald. Ambas PCRs apresentaram bom desempenho no diagnóstico de A. marginale nos bovinos,o qual foi superior ao exame parasitológico. O PCR-TR detectou sete veados campeiros positivos (16,3%), o que foisignificativamente maior comparado ao PCR-C identificando um animal como positivo (2,3%), e ao exame parasitológiconão encontrou nenhum animal positivo. A média da temperatura de dissociação das reações positivas para amostras debovinos (81,72 °C ± 0,20) foi idêntica àquelas dos cervídeos ( 81,72 °C ± 0,12), o que sugere que ambas espécies animaisforam infectadas por A. marginale. Concluímos que PCR-TR pode ser utilizada para diagnóstico e estudos epizootiológicosde A. marginale em bovinos e cervídeos. Apesar da pequena amostragem de veado campeiro os resultados indicam que essaespécie de animal selvagem tem importância na epizootiologia do Anaplasma no Pantanal brasileiro.(AU)

Evaluation of an enzyme-linked immunosorbent assay to detect antibodies against Anaplasma marginale

A rapid indirect enzyme-linked immunosorbent assay (ELISA) was developed for measuring antibodies against Anaplasma marginale using a partially soluble antigen prepared from semi-purified initial bodies from erythrocytes with 80.0% of rickettsiaemia. This technique utilized alkaline phosphatase and p-nitrophenyl phosphate as reaction indicators. The high sensitivity (100.0%) was confirmed with sera from 100 calves experimentally-infected with A. marginale. All of these animals showed seroconversion before or at the same time of the first rickettsiaemia or even when it was not detected. Also the elevated specificity (94.0%) was confirmed by the low percentage of cross-reactions with sera from animals experimentally-infected with Babesia bigemina and Babesia bovis (1.4 and 6.6%, respectively). Performances of ELISA and indirect fluorescent antibody test (IFAT) with 324 sera from enzootically stable area did not show statistical difference (P>0.05), since the former showed 96.9% and the latter 97.2% of positive reactions. The advantage of this ELISA is a shorter execution time than others developed until now, allowing more samples to be analyzed. (AU)

Evaluation of an ELISA for detection of antibodies to Babesia bigemina in cattle and it's application in an epidemiological survey in Brazil

An indirect enzyme-linked immunosorbent assay (ELISA) using a crude antigen was evaluated for its performance to detect Babesia bigemina antibodies. The sensitivity and specificity were 98.0 per cent and 99.0 per cent, respectively. In agreement with the high specificity, no cross-reactions were verified with sera from calves inoculated three times with 10 7(subscribe) Babesia bovis organisms. With regard to the comparison of ELISA and indirect fluorescent antibody test (IFAT) in detecting antibodies against B. bigemina in calves experimentally infected with five Brazilian geographical isolates of this hemoparasite, IFAT was able to detect antibodies one day earlier in most of the calves' sera. There was a good agreement between results shown by ELISA and IFAT with sera from an enzootically stable area (k=0.61). However, there was no agreement between these serological tests with sera from an enzootically unstable area (k=0.33). The ELISA was employed in an epidemiological survey using with 1,367 sera from four counties in the Pantanal of Mato Grosso do Sul and characterized this region as an enzootically stable area, since the prevalence ranged from 87.7 to 98.9 per cent. Therefore, this ELISA with high sensitivity, specificity and performance similar to IFAT can be employed in serological diagnosis of B. bigemina (AU)