Argentinean agid test for diagnosis of equine infectious anemia: six years of history

Equine infectious anemia (EIA) is a disease of high economic impact on the equine industry worldwide. Since horses are frequent travelers, EIA falls under strict regulatory control programs in many countries. In Argentina the national animal health authority (SENASA) states that all horses imported, moving within the country, or congregating at public assemblies must have a negative EIA report conducted within the previous 2 months. The agent causing EIA is a RNA virus from the Retroviridae family and its major capsid protein named p26 is the most immunogenic protein in the viral particle. Thus, the detection of specific antibodies directed to p26 is the aim of most diagnosis tests available in the world. The agar gel immunodifusion (AGID) is the officially accepted method to certify the diagnosis of EIA in Argentina. Since 2009 IncuINTA was working on the scaling up and production of the KIT AIE IDGA RP26, an Argentinean AGID test entirely developed in the laboratory containing a recombinant p26 protein to detect EIA antibodies in horses’ serum. Until 2015 IncuINTA produced two pilot batches and six commercial batches (one per year) containing from 24000 determinations in 2011 to 39600 determinations in 2015. Since the product was launched in 2011, the sales were increased 109%. Up to date we have placed on the market 170640 determinations. As expected, the number of laboratories buying the KIT AIE IDGA RP26 was also increasing through time being 26 in 2011 and 36 in 2015. This number of clients represents 17% of the 207 laboratories authorized by SENASA to diagnose EIA in Argentina. These laboratories are located mostly in Buenos Aires, Santa Fe, Entre Ríos, Formosa, La Pampa, Rio Negro, Cordoba, Corrientes, Salta and Tucum an provinces. Until 2009 there was no Argentinean EIA test available in our market being the imported ones very expensive. IncuINTA, which is a R&D laboratory, could scale up, produce and sell the KIT AIE IDGA RP26 during six consecutive years. After this success, IncuINTA perspective is to increase the number of batches each year to be able to attend the demand of most diagnosis laboratories in the country.Fil: Bok, Marina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Asenzo, G.. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; ArgentinaFil: Vena, M. M.. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; ArgentinaFil: Parreño, V.. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; ArgentinaFil: Wigdorovitz, A.. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Virología; Argentina10th International Equine Infectious Diseases ConferenceBuenos AiresArgentinaUniversity of Kentuck

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

A novel neutralization sensitive and subdominant RAP-1-related antigen (RRA) is expressed by Babesia bovis merozoites

Objective. The Babesia bovis genome encodes a rap-1 related gene denominated RAP-1 related antigen (RRA). In this study, we analysed the pattern of expression, immunogenicity and functional relevance of RRA. Methods. Phylogenetic analysis was performed using the program Phylip. Expression of rra was analysed by Northern blots, RT-PCR, immunoprecipitation, Western blots and immunofluorescence. RRA antigenicity was tested by T-cell proliferation and Western blot analysis, and functional relevance was determined in an in vitro neutralization assay. Results. RRA is more closely related to RAP-1b of Babesia bigemina than to B. bovis RAP-1, and it is highly conserved among distinct strains. Transcriptional analysis suggests lower numbers of rra transcripts compared to rap-1. Immunoprecipitation of metabolically labelled B. bovis proteins with antibodies against synthetic peptides representing predicted antigenic regions of RRA confirmed the expression of a ∼43 kDa RRA in cultured merozoites. Antibodies present in B. bovis hyperimmune sera, but not in field-infected cattle sera, reacted weakly with recombinant RRA, and no significant stimulation was obtained using recombinant RRA as antigen in T-cell proliferation assays, indicating that RRA is a subdominant antigen. Antibodies against RRA synthetic peptides reacted with merozoites using immunofluorescence, and were able to significantly inhibit erythrocyte invasion in in vitro neutralization tests, suggesting functional relevance for parasite survival. Conclusion. B. bovis express a novel subdominant RAP-1-like molecule that may contribute to erythrocyte invasion and/or egression by the parasite.Fil: Suarez, Carlos E.. United States Department of Agriculture. Agriculture Research Service; Estados Unidos. Washington State University; Estados UnidosFil: Laughery, Jacob M.. Washington State University; Estados UnidosFil: Bastos, Reginaldo G.. Washington State University; Estados UnidosFil: Johnson, Wendell C.. United States Department of Agriculture. Agriculture Research Service; Estados UnidosFil: Norimine, Junzo. Washington State University; Estados UnidosFil: Asenzo, Gustavo Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas; ArgentinaFil: Brown, Wendy C.. Washington State University; Estados UnidosFil: Jacobsen, Monica Ofelia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas; ArgentinaFil: Goff, Will L.. United States Department of Agriculture. Agriculture Research Service; Estados Unido

Veterinary Parasitology

Texto completo: acesso restrito. p. 200–206This 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 0.1 fg/μl to 0.01 ng/μl 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