ROTADIAL: The first nanobody-based immunoassay to detect Group A Rotavirus.

ROTADIAL is a rapid nanobody (Nb)-based ELISA assay able to identify Rotavirus group A (RVA) in feces from pediatric patients. The assay is based on a sandwich of two patented llama-derived Nbs directed to the inner capsid viral protein VP6 from RVA. Nbs are directed to conformational epitopes of VP6 and recognized all human RVA strains tested, representing ideal reagents for their use in immunodiagnostic tests for RVA detection. All the steps are carried out at room temperature, bringing results in less than two hours. This assay, named ROTADIAL, was validated with a reference panel of feces from pediatric patients from Argentina. The overall sensitivity and specificity of the ROTADIAL test, when compared to a commercial test, was 100 % (100/100) and 99 % (99/100) respectively. ROTADIAL presented optimal analytical performance, being capable of detecting RVA regardless of the presence of other common human enteric infectious agents and is the first RVA-diagnostic assay developed using Nbs, worldwide.

Development of fluorogenic probe-based PCR assays for the detection and quantification of bovine piroplasmids.

This paper reports two new quantitative PCR (qPCR) assays, developed in an attempt to improve the detection of bovine piroplasmids. The first of these techniques is a duplex TaqMan assay for the simultaneous diagnosis of Babesia bovis and B. bigemina. This technique is ideal for use in South America where bovids harbour no theilerids. The second technique, which is suitable for the diagnosis of both babesiosis and theileriosis worldwide, involves fluorescence resonance energy transfer (FRET) probes. In FRET assays, Babesia bovis, B. divergens, Babesia sp. (B. major or B. bigemina), Theileria annae and Theileria sp. were all identifiable based on the melting temperatures of their amplified fragments. Both techniques provided linear calibration curves over the 0.1fg/microl to 0.01ng/microl DNA range. The assays showed good sensitivity and specificity. To assess their performance, both procedures were compared in two separate studies: the first was intended to monitor the experimental infection of calves with B. bovis and the second was a survey where 200 bovid/equine DNA samples from different countries were screened for piroplasmids. Comparative studies showed that duplex TaqMan qPCR was more sensitive than FRET qPCR in the detection of babesids.

Development of an indirect ELISA for the diagnosis of equine piroplasmosis.

An indirect ELISA (iELISA) for the detection of specific anti-Theileria equi antibodies in horse serum was developed. Its performance showed good concordance (K= 0.79) when compared with a competitive ELISA recommended by the World Organisation for Animal Health. Horse serum samples from two provinces located in the north and east of Argentina (Formosa and Entre Rios, respectively) were analyzed by this iELISA. A high percentage of positive horses were found in Formosa, consistent with the climatic conditions of the region that are apt for the development of tick vectors. Surprisingly, seropositive animals were also detected, although in a lower proportion, in Entre Rios, which has a temperate weather and is presumably tick free. Breeding of thoroughbred racing horses is an important economic asset of Argentina. Since equine piroplasmosis is a constraint for horse export, the epidemiological situation in different regions of the country needs to be assessed for the implementation of control measures. The iELISA developed in this work provides a convenient tool for this type of study.

Search for Babesia bovis vaccine candidates.

Babesia bovis is a tick-borne apicomplexan pathogen that remains an important constrain for the development of cattle industries worldwide. Effective control can be achieved by vaccination with live attenuated forms of the parasite, but they have several drawbacks and thus the development of alternative subunit vaccines, either based in recombinant versions of full size proteins or in recombinant or synthetic peptides containing combinations of protective B-cell and T-cell epitopes is needed. Our current strategies for the identification of vaccine candidate antigens include the identification of functionally relevant antigens, bioinformatics, and comparative genomics using the recently sequenced B. bovis genome. These led us to the functional and immunological characterization of members of the VMSA gene family, a group of well conserved putative cysteine and serine proteases, and to the definition of a surface exposed B-cell epitope present in the Merozoite Surface Antigen-2c. Work in progress is focused in defining additional epitopes, and to determine whether they are neutralization-sensitive. These approaches might unravel useful vaccine candidates for B. bovis, and will increase our understanding of the pathogenicity mechanisms of these and related hemoparasites.

A quantitative PCR assay for the detection and quantification of Babesia bovis and B. bigemina.

The haemoparasites Babesia bovis and Babesia bigemina affect cattle over vast areas of the tropics and temperate parts of the world. Microscopic examination of blood smears allows the detection of clinical cases of babesiosis, but this procedure lacks sensitivity when parasitaemia levels are low. In addition, differentiating between similar haemoparasites can be very difficult. Molecular diagnostic procedures can, however, overcome these problems. This paper reports a quantitative PCR (qPCR) assay involving the use of SYBR Green. Based on the amplification of a small fragment of the cytochrome b gene, this method shows both high sensitivity and specificity, and allows quantification of parasite DNA. In tests, reproducible quantitative results were obtained over the range of 0.1 ng to 0.1 fg of parasite DNA. Melting curve analysis differentiated between B. bovis and B. bigemina. To assess the performance of the new qPCR procedure it was used to screen for babesiosis in 40 cows and 80 horses. B. bigemina was detected in five cows (three of these were also found to be positive by standard PCR techniques targeting the 18S rRNA gene). In addition, B. bovis was detected in one horse and B. bigemina in two horses using the proposed method, while none was found positive by ribosomal standard PCR. The sequences of the B. bigemina cytochrome b and 18S rRNA genes were completely conserved in isolates from Spain and Argentina, while those of B. bovis showed moderate polymorphism.