The current method for Babesia spp. serodiagnosis based on a crude merozoite antigen is a complex and time-consuming procedure. An indirect enzyme-linked immunosorbent assay (iELISA) based on a recombinant multi-antigen of Babesia bovis (rMABbO) was developed for detection of antibodies in bovines suspected of infection with this parasite. The multi-antigen comprises gene fragments of three previously characterized B. bovis antigens: MSA-2c, RAP-1 and the Heat Shock protein 20 that are well-conserved among geographically distant strains. The cutoff value for the new rMABbo-iELISA was determined using 75 known—positive and 300 known—negative bovine sera previously tested for antibodies to B. bovis by the gold-standard ELISA which uses a merozoite lysate. A cutoff value of ≥35% was determined in these samples by receiver operator characteristic (ROC) curve analysis, showing a sensitivity of 95.9% and a specificity of 94.3%. The rMABbo-iELISA was further tested in a blind trial using an additional set of 263 field bovine sera from enzootic and tick-free regions of Argentina. Results showed a good agreement with the gold standard test with a Cohen’s kappa value of 0.76. Finally, the prevalence of bovine babesiosis in different tick enzootic regions of Argentina was analyzed where seropositivity values among 68–80% were obtained. A certain level of cross reaction was observed when samples from B. bigemina infected cattle were analyzed with the new test, which can be attributed to shared epitopes between 2 of the 3 antigens. This new rMABbo-iELISA could be considered a simpler alternative to detect anti Babesia spp. antibodies and appears to be well suited to perform epidemiological surveys at the herd level in regions where ticks are present.
Anaplasma marginale is an obligate intraerythrocytic bacterium of bovines, responsible for large economic losses worldwide. It is mainly transmitted by Rhipicephalus (Boophilus) microplus ticks and, despite mounting evidence suggesting transovarial transmission, the occurrence of this phenomenon remains controversial. We evaluated the vector competence of R. microplus larvae vertically infected with A. marginale to transmit the bacterium to a naïve bovine. A subgroup of engorged female ticks collected from an A. marginale-positive animal was dissected and the presence of the pathogen in its tissues was confirmed. A second subgroup of ticks was placed under controlled conditions for oviposition. After confirming the presence of A. marginale in the hatched larvae, an experimental infestation assay was conducted. Larvae were placed on an A. marginale-free splenectomized calf. The bacterium was detected in the experimentally infested bovine 22 days post-infestation. We analyzed the A. marginale diversity throughout the transmission cycle using the molecular marker MSP1a. Different genotypes were detected in the mammalian and arthropod hosts showing a reduction of strain diversity along the transmission process. Our results demonstrate the vertical transmission of A. marginale from R. microplus females to its larvae, their vector competence to transmit the pathogen, and a bottleneck in A. marginale strain diversity.
Background
Anaplasma marginale, a gram-negative obligate intracellular microorganism, infects erythrocytes of ruminants and other host species from tropical and subtropical world regions. This pathogen of importance for cattle production, is known to be transmitted mechanically and through hematophagous parasites, mainly by Rhipicephalus microplus ticks. Even though transovarial transmission of A. marginale has been previously reported in R. microplus ticks, there is no evidence of A. marginale transmission from the resulting unfed larvae to a susceptible host. The aim of the present study was to evaluate the transovarial transmission of A. marginale in the R. microplus tick under natural conditions and to study the vector competence of the infected hatched larvae.
Methods
Engorged female R. microplus ticks were sampled from A. marginale positive bovines from an enzootic area in Argentina, and larvae were obtained from a subgroup of these females. After identifying A. marginale in the females and their larvae, selected ticks collected from a single bovine were used. After spawn, hatched larvae were seeded on a susceptible healthy bovine and another subgroup of engorged R. microplus ticks were subjected to dissection to recover the salivary glands and ovaries for A. marginale identification. Anaplasma marginale was detected by PCR and molecularly characterized by means of cloning and sequence all msp1α fragments while genotyping.
Results
A. marginale was detected in naturally infected bovines, in different organs of engorged R. microplus females that were parasitizing them, and in the larvae obtained from these females. A. marginale infection of the susceptible bovine was corroborated both from DNA detection and from compatible structures in a blood smear. The characterization of A. marginale msp1α genotypes involved throughout the infection process provided relevant information about the dynamics of the transmission process.
Conclusions
The results demonstrate the transovarial transmission of A. marginale in R. microplus ticks under natural conditions and, through an experimental infection assay, the vector competence of the infected R. microplus larvae to transmit A. marginale to a susceptible bovine. The high genetic diversity of A. marginale in natural transmission settings seems to be one of the key factors for the transovarial transmission to occur.
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