Transovarial Transmission of Anaplasma marginale in Rhipicephalus (Boophilus) microplus Ticks Results in a Bottleneck for Strain Diversity.

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.

Development of highly sensitive one step-PCR tests for improved detection of B. bigemina and B. bovis.

Bovine babesiosis caused by Babesia bigemina and B. bovis is an economically relevant tick-borne disease distributed over tropical and subtropical world regions. Animals that recover from the clinical disease can remain persistently infected, and those carriers are epidemiologically relevant since they can act as a source of infection to other animals through the tick bite. According to the manual of the World Organisation for Animal Health (OIE), the recommended molecular diagnosis test for both parasites is a nested polymerase chain reaction (nPCR) based on an amplification of a fragment of the rap-1 gene. Since nPCRs are time consuming, have a higher cost and risk of contamination, we propose a single step PCR for B. bigemina (BbiVESA) and B. bovis (BboVESA) based on the amplification of the multi-copy ves-1α gene. We developed these methods and we achieved a detection limit of 1 × 10-12 % parasitemia for B. bigemina and of 1 × 10-6 % for B. bovis using reference strains, which compared to the reference OIE tests, results in an improvement in sensitivity of six orders for B. bigemina. Finally, we tested 48 field samples from a babesiosis enzootic region where we were able to detect a higher proportion of positive animals with both VESA methods than with the reference rap-1 nPCRs. This difference was statistically significant for each Babesia species. Concordance between both diagnostic schemes based on Cohen's kappa coefficient showed minimal to non-agreement (κ = 0.32) for B. bigemina and non-agreement (κ = 0.16) for B. bovis since BbiVESA and BboVESA PCR tests showed a significantly higher detection capacity. In conclusion, the high sensitivity of the assay, together with the lower demand of time and reagents make the VESA PCR methods developed here a valuable diagnostic tool for the molecular detection and epidemiological survey of both Babesia pathogens.

Molecular diagnosis of Leishmania spp. in dogs of a subtropical locality of Argentina.

Leishmaniosis is a tropical and subtropical vector-borne disease caused by hemoparasites of the genus Leishmania. The disease can infect humans, as well as domestic and wildlife animals. Dogs are the main reservoir for L. infantum, the aetiological agent of visceral leishmaniosis (VL) in America, and a domestic source of L. braziliensis, the most widespread aetiological agent of American tegumentary leishmaniosis. Infected dogs can develop a clinical syndrome called canine leishmaniosis (CanL), which presents with skin lesions, mild fever; additionally hepatomegaly and splenomegaly can be observed, although asymptomatic infections are frequent. Direct microscopic observation of the parasite in bone marrow, blood, skin scrapings and conjunctival swab samples is the gold standard of diagnosis and is usually complemented with serological tests, and to a lesser extent, molecular detection of the parasite. In Argentina, leishmaniosis is an emerging disease, with a growing number of human and canine clinical cases since 2006. Our study was carried out in Mercedes, a town located in the subtropical north-eastern area of Argentina, where dogs with positive parasitological test results for Leishmania spp. must be euthanized according to local regulations. We evaluated the presence of Leishmania spp. DNA in the blood of dogs (n = 166) from urban and peri-urban zones. Genomic DNA was extracted from whole blood using Chelex 100 resin and a conserved 116 bp region of the kinetoplastid DNA was amplified by conventional PCR. Clinical signs, age and gender were recorded. Our results showed that 120 out of 166 surveyed dogs (72%) were positive for Leishmania spp. DNA of which only seven were positive by parasitological and serological tests. No significant correlation between positive cases and gender or age groups was found. This report shows the high prevalence of this disease in Argentina and contributes to improve public health policy with regard to diagnosis, prevention and treatment of infected dogs.

Development of an Indirect ELISA Based on a Recombinant Chimeric Protein for the Detection of Antibodies against Bovine Babesiosis.

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.

Epidemiology of Babesia, Anaplasma and Trypanosoma species using a new expanded reverse line blot hybridization assay.

Vector-borne hemoparasitic infections are a major problem that affects livestock industries worldwide, particularly in tropical and subtropical regions. In this work, a reverse line blot (RLB) hybridization assay was developed for the simultaneous detection and identification of Anaplasma, Babesia and bovine trypanosomes, encompassing in this way the most relevant hemoparasites that affect cattle. A total of 186 bovine blood samples collected from two different ecoepidemiological regions of northeast Argentina, with and without tick control, were analyzed with this new RLB. High diversity of parasites, such as Babesia bovis, B. bigemina, Anaplasma marginale and three different Trypanosoma species, was found. High rates of coinfections were also detected, and significant differences were observed not only in the prevalence of parasites but also in the level of coinfections between the two analyzed areas. Regarding the Trypanosoma genus, we provide molecular evidence of the presence of T. vivax and T. theileri for the first time in Argentina. Besides, since the RLB is a prospective tool, it allowed the identification of a yet unknown bovine trypanosome which could not be assigned to any of the bovine species known so far. In the present study we provide new insights on the prevalence of several pathogens that directly impact on livestock production in Argentina. The RLB assay developed here allows to identify simultaneously numerous pathogenic species which can also be easily expanded to detect other blood borne pathogens. These characteristics make the RLB hybridization assay an essential tool for epidemiological survey of all vector-borne pathogens.