Isolation of Sarcocystis neurona from an opossum (Didelphis albiventris) in Argentina

Sarcocystis neurona is an Apicomplexan parasite which affects awide range of animal hosts. This protozoan is the main cause ofequine protozoal myeloecephalitis (EPM) inWestern Hemispherehorses. The parasite reproduces sexually in the intestine ofdefinitive hosts (DH) and asexually in tissues of intermediate andaberrant hosts. The geographical distribution of S. neurona isrelated with the distribution its definitive hosts, the opossumsDidelphis virginiana and D. albiventris. A recent serological studyconducted in Argentinean horses using S. neurona antigenrevealed an overall seroprevalence of 26.1%. However, the parasitehas not been isolated in Argentina. Tissues from an opossum (D.albiventris) hunted by dogs in a farm from the central region ofBuenos Aires province were collected. Horses raised in the farmshowed a 50% (10/20) S. neurona seroprevalence. One seropositivehorse developed neurological signs and evidenced clinicalimprovement after a 2 month treatment with Ponazuril. A completenecropsy of the opossum was conducted and the intestinalmucosal scraping was subjected to a parasitological study withsucrose solution. A high amount of Sarcocystis spp. oocysts/sporocystswere observed (Fig. 1). DNA was extracted from concentratedoocysts with a commercial kit (ZR Fecal DNA, ZymoResearch). The sample was identified as S. neurona by specificPCR-restriction fragment length polymorphism (RFLP) and bysequencing of a fragment of the 18S rRNA gene. Approximately 5 x105 oocysts were subjected to a pepsin-HCl digestion followed bya physical disruption. Released sporozoites were used to infectfresh BM cell cultures, maintained by 3 passages during 2 monthsand further preserved in liquid nitrogen. This study represents thefirst isolation of S. neurona in Argentina. Further studies will beconducted in order to identify antigen expression as well as tocompare genetic characteristics between the isolated strain andreference strains.Fil: Moré, Gastón Andrés. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Departamento de Epizootiología y Salud Pública. Laboratorio de Inmunoparasitología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; ArgentinaFil: Rambead, M.. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Departamento de Epizootiología y Salud Pública. Laboratorio de Inmunoparasitología; ArgentinaFil: Braun, F.. No especifíca;Fil: Campero, Lucía María. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Departamento de Epizootiología y Salud Pública. Laboratorio de Inmunoparasitología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; ArgentinaFil: Walkosksi, A.. No especifíca;Fil: Venturini, M. C.. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Departamento de Epizootiología y Salud Pública. Laboratorio de Inmunoparasitología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina10th International Equine Infectious Diseases ConferenceCiudad Autónoma de Buenos AiresArgentinathe Equine Infectious Disease Conferenc

An Ibero-American inter-laboratory trial to evaluate serological tests for the detection of anti-<i>Neospora caninum</i> antibodies in cattle

We carried out an inter-laboratory trial to compare the serological tests commonly used for the detection of specific Neospora caninum antibodies in cattle in Ibero- American countries. A total of eight laboratories participated from the following countries: Argentina (n = 4), Brazil (n = 1), Peru (n = 1), Mexico (n = 1), and Spain (n = 1). A blind panel of well-characterized cattle sera (n = 143) and sera representative of the target population (n = 351) was tested by seven in-house indirect fluorescent antibody tests (IFATs 1–7) and three enzyme-linked immunosorbent assays (ELISAs 1–3; two in-house and one commercial). Diagnostic performance of the serological tests was calculated and compared according to the following criteria: (1) the BPre-test information,^ which uses previous epidemiological and serological data; (2) the BMajority of tests,^ which classifies a serumas positive or negative according to the results obtained by most tests evaluated. Unexpectedly, six tests showed either sensitivity (Se) or specificity (Sp) values lower than 90%. In contrast, the best tests in terms of Se, Sp, and area under the ROC curve (AUC) values were IFAT 1 and optimized ELISA 1 and ELISA 2. We evaluated a high number of IFATs, which are the most widely used tests in Ibero-America. The significant discordances observed among the tests regardless of the criteria employed hinder control programs and urge the use of a common test or with similar performances to either the optimized IFAT 1 and ELISAs 1 and 2.Facultad de Ciencias Veterinaria

Detection of antibodies against to Brucella abortus, Leptospira spp. and Apicomplexa

Water buffalo industry has become a profitable activity worldwide, including the Northeast of Argentina (NEA). However, research on diseases affecting this species is scarce. The aim of the present study was to detect antibodies against Brucella abortus, Leptospira spp., Neospora caninum, Toxoplasma gondii, and Sarcocystis spp. in 500 water buffalo cows from five ranches (100 animals each) in the NEA. Serum samples were tested for B. abortus by fluorescence polarization assay, Leptospira spp. By microagglutination test, and N. caninum, T. gondii, and Sarcocystis spp. by indirect fluorescent antibody tests. Overall, the proportion of seropositive animals was 6.4, 22.2, 42.2, 25.4, and 50.8 % for brucellosis, leptospirosis, neosporosis, toxoplasmosis, and sarcocystosis, respectively. The proportion of seropositive animals for all diseases was statistically different among herds (p<0.05). Statistical differences were also detected among age groups for brucelosis and neosporosis (p<0.05). The detection of specific antibodies to B. abortus, Leptospira spp., and several Apicomplexa protozoans in water buffaloes in the NEA is reported in this study.Fil: Konrad, José Luis. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional del Nordeste. Facultad de Cs.veterinarias; ArgentinaFil: Campero, Lucía María. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Caspe, Gastón S.. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Corrientes. Estación Experimental Agropecuaria Mercedes; ArgentinaFil: Brihuega, Bibiana. Instituto Nacional de Tecnología Agropecuaria; ArgentinaFil: Draghi, Graciela. Instituto Nacional de Tecnología Agropecuaria; ArgentinaFil: Moore, Dadin Prando. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Crudeli, Gustavo A.. Universidad Nacional del Nordeste. Facultad de Cs.veterinarias; ArgentinaFil: Venturini, María C.. Universidad Nacional de la Plata. Facultad de Ciencias Veterinarias; ArgentinaFil: Campero, Carlos M.. Instituto Nacional de Tecnología Agropecuaria; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias; Argentin

Anti-Neospora caninum and anti-Sarcocystis spp. specific antibodies cross-react with Besnoitia besnoiti and influence the serological diagnosis of bovine besnoitiosis

Bovine besnoitiosis control remains a challenge because the disease continues to spread and control relies solely on accurate diagnosis coupled to management measures. However, recent studies have reported that routinely used ELISAs may raise a high number of false-positive results. Herein, cross-reactions between Besnoitia besnoiti antigens and anti-Neospora caninum and/or anti-Sarcocystis spp.-specific antibodies were studied in an in house ELISA since N. caninum and Sarcocystis spp. are closely related parasites, and both infections are highly prevalent in cattle worldwide. The serum panel was composed of the following categories: sera from B. besnoiti-seronegative (n=75) and -seropositive cattle (n=66), B. besnoiti-based-ELISA false-positive reactors (n=96) together with N. caninum (n=36) and Sarcocystis spp. (n=42) -seropositive reference cattle sera. B. besnoiti tachyzoite based western blot (WB) results classified animals as seropositive or seronegative. Sera were analyzed for the detection of anti-N. caninum by WB and ELISA and anti-Sarcocystis spp.-specific antibodies by WB and IFAT. Those samples recognizing a Sarcocystis spp. 18-20kDa antigenic region and N. caninum 17-18kDa immunodominant antigen were considered to be Sarcocystis spp. and N. caninum seropositive, respectively. The category of B. besnoiti based-ELISA false-positive reactors showed the highest number of sera with specific anti-Sarcocystis spp. and anti-N. caninum antibodies (74%; 71/96), followed by the N. caninum-seropositive cattle category (52.8%; 19/36). In contrast, few B. besnoiti-seronegative and -seropositive cattle showed antibodies against Sarcocystis spp. and N. caninum (10.7%; 8/75 and 1.5%; 1/66), respectively). This study revealed that B. besnoiti false-positive ELISA results were associated not only with the presence of anti-N. caninum and anti-Sarcocystis spp. antibodies (χ2: 78.36; p<0.0001; OR: 34.6; CI: 14-88) but also with high antibody levels against them using ELISA and IFAT tests, respectively (p<0.05; t-test). These results may explain why only some animals seropositive to Sarcocystis spp. and/or N. caninum are Besnoitia false-positive reactors. Therefore, sera meeting these requirements should be included in future validations of serological tests for bovine besnoitiosis.Fil: García Lunar, P.. Universidad Complutense de Madrid; EspañaFil: Moré, Gastón Andrés. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Departamento de Epizootiología y Salud Pública. Laboratorio de Inmunoparasitología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Campero, Lucía María. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Departamento de Epizootiología y Salud Pública. Laboratorio de Inmunoparasitología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Ortega Mora, L. M.. Universidad Complutense de Madrid; EspañaFil: Álvarez García, G.. Universidad Complutense de Madrid; Españ

Changes in the carbohydrate expression in cattle and buffaloes infected with the abortigenic protozoan Neospora caninum

Carbohidrate pattern in the placenta was studied in different species showing a great variation between species. The sacharydes of the surface glycoconjugates are important to the adhesion between conceptus and uterus. Besides, reproductive diseases as tritrichomonosis and campylobacteriosis generate changes in this pattern in cattle. The aim of this study was to characterize the saccharide in placenta of cattle and buffaloes after experimental infection with the abortigenic protozoan Neospora caninum (N. caninum) at early gestation by lectinhistochemistry.Fil: Caspe, S. G.. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Corrientes; ArgentinaFil: Campero, C.. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce. Área de Investigación en Producción y Sanidad Animal; ArgentinaFil: Moore, Dadin Prando. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce. Área de Investigación en Producción y Sanidad Animal; ArgentinaFil: Campero, Lucía María. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Departamento de Epizootiología y Salud Pública. Laboratorio de Inmunoparasitología; ArgentinaFil: Ortega Mora, L. M.. Universidad Complutense de Madrid; EspañaFil: Bacigalupe, Diana. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Departamento de Epizootiología y Salud Pública. Laboratorio de Inmunoparasitología; ArgentinaFil: Lischinsky, L. H.. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Sala, J. M.. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Corrientes; ArgentinaFil: Venturini, C.. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Departamento de Epizootiología y Salud Pública. Laboratorio de Inmunoparasitología; ArgentinaFil: Konrad, José Luis. Universidad Nacional del Nordeste. Facultad de Ciencias Veterinarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste; ArgentinaFil: Barbeito, Claudio Gustavo. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Departamento de Epizootiología y Salud Pública. Laboratorio de Inmunoparasitología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Caracterización biológica y genética de la cepa de <i>Neospora caninum</i> NC-6 Argentina y aplicación práctica de la tipificación de microsatélites en infecciones experimentales en bovinos

La infección por Neospora caninum es una de las principales causas de abortos bovinos. Los objetivos de este trabajo fueron caracterizar genéticamente el aislamiento de N.caninum NC-6 Argentina utilizando el análisis de microsatélites y estudiar su comportamiento biológico mediante inoculaciones experimentales en bovinos preñados, evaluando la respuesta inmune producida y la ocurrencia de transmisión transplacentaria. Se inocularon vacas preñadas de 65 días de gestación, seropositivas y seronegativas a N. caninum, con 5 x 107 taquizoítos de la cepa NC-6 y se sacrificaron a los 108 +/- 2 días de gestación. Se tomaron muestras de sueros periódicamente y se les realizó inmunofluorescencia indirecta para anticuerpos. Se obtuvieron muestras de sangre los días 30 y 37, se estimularon in vitro con N. caninum y se analizó la producción de interferón gamma (IFNγ). Se tomaron muestras de órganos de las madres, las placentas y los fetos que fueron procesadas por histopatología, inmunohistoquímica y PCR para ADN de N. caninum Las muestras positivas se analizaron para la tipificación de los microsatélites. Los animales inoculados incrementaron significativamente los títulos de anticuerpos anti-N.caninum y la producción de IFNγ respecto a los controles. Una vaca seropositiva inoculada abortó, un feto del grupo seronegativo no fue viable y el resto de los fetos fueron viables pero presentaron lesiones. La PCR fue positiva en los fetos de las vacas seronegativas y en 2/3 fetos de las seropositivas. El análisis de microsatélites demostró que el ADN presente tenía un patrón idéntico a NC-6 Argentina. Éste es el primer reporte de una infección experimental de bovinos con la cepa de N. caninum aislada en Argentina. Esta cepa demostró su patogenicidad en animales seropositivos y seronegativos, fue capaz de atravesar la placenta y fue patógena para los fetos, el análisis de microsatélites demostró que la cepa hallada en las placentas era NC-6 Argentina.El presente trabajo ha sido galardonado con el Premio Mayor AAPAVET (Asociación Argentina de Parasitología Veterinaria) - Premio Anual Rioplatense 2010/2011 “Congreso Mundial de Parasitología Veterinaria” en la categoría Mejor trabajo original de investigación.Facultad de Ciencias Veterinaria

An Ibero-American inter-laboratory trial to evaluate serological tests for the detection of anti-Neospora caninum antibodies in cattle

We carried out an inter-laboratory trial to compare the serological tests commonly used for the detection of specific Neospora caninum antibodies in cattle in Ibero-American countries. A total of eight laboratories participated from the following countries: Argentina (n = 4), Brazil (n = 1), Peru (n = 1), Mexico (n = 1), and Spain (n = 1). A blind panel of well-characterized cattle sera (n = 143) and sera representative of the target population (n = 351) was tested by seven in-house indirect fluorescent antibody tests (IFATs 1–7) and three enzyme-linked immunosorbent assays (ELISAs 1–3; two in-house and one commercial). Diagnostic performance of the serological tests was calculated and compared according to the following criteria: (1) the “Pre-test information,” which uses previous epidemiological and serological data; (2) the “Majority of tests,” which classifies a serum as positive or negative according to the results obtained by most tests evaluated. Unexpectedly, six tests showed either sensitivity (Se) or specificity (Sp) values lower than 90%. In contrast, the best tests in terms of Se, Sp, and area under the ROC curve (AUC) values were IFAT 1 and optimized ELISA 1 and ELISA 2. We evaluated a high number of IFATs, which are the most widely used tests in Ibero-America. The significant discordances observed among the tests regardless of the criteria employed hinder control programs and urge the use of a common test or with similar performances to either the optimized IFAT 1 and ELISAs 1 and 2.Fil: Campero, Lucía María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Departamento de Epizootiología y Salud Pública. Laboratorio de Inmunoparasitología; ArgentinaFil: Moreno Gonzalo, Javier. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Departamento de Epizootiología y Salud Pública. Laboratorio de Inmunoparasitología; ArgentinaFil: Venturini, María Cecilia. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Departamento de Epizootiología y Salud Pública. Laboratorio de Inmunoparasitología; ArgentinaFil: Moré, Gastón Andrés. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Departamento de Epizootiología y Salud Pública. Laboratorio de Inmunoparasitología; ArgentinaFil: Dellarupe, Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Departamento de Epizootiología y Salud Pública. Laboratorio de Inmunoparasitología; ArgentinaFil: Rambeaud, Magdalena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Departamento de Epizootiología y Salud Pública. Laboratorio de Inmunoparasitología; ArgentinaFil: Echaide, Ignacio Eduardo. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria Rafaela; ArgentinaFil: Valentini, Aurora Beatriz. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria Rafaela; ArgentinaFil: Campero, Lucía María. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Moore, Dadin Prando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Departamento de Epizootiología y Salud Pública. Laboratorio de Inmunoparasitología; ArgentinaFil: Cano, Dora B.. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires Sur. Estación Experimental Agropecuaria Balcarce; ArgentinaFil: Fort, Marcelo. Instituto Nacional de Tecnología Agropecuaria. Centro Regional La Pampa-San Luis. Estación Experimental Agropecuaria Anguil; ArgentinaFil: Mota, Rinaldo A.. Universidade Federal Rural Pernambuco; BrasilFil: Serrano Martínez, Marcos E.. Universidad Peruana Cayetano Heredia; PerúFil: Cruz-Vázquez, Carlos. Instituto Tecnológico el Llano Aguascalientes; MéxicoFil: Ortega Mora, Luis M.. Universidad Complutense de Madrid; EspañaFil: Álvarez García, Gema. Universidad Complutense de Madrid; Españ

Association of bovine viral diarrhea virus, bovine herpesvirus 1, and Neospora caninum with late embryonic losses in highly supplemented grazing dairy cows

The objectives of this study were: 1- to evaluate the association of Bovine Viral Diarrhea Virus (BVDV), Bovine Herpes Virus 1 (BoHV-1), and Neospora caninum (N. caninum) with the risk for Late Embryonic Loss (LEL) in grazing dairy cows, 2- to evaluate blood progesterone concentration at the time of LEL occurrence, and 3- to describe a novel ultrasound-guided technique for conceptus sampling. We run a prospective cohort study involving 92 cows (46 LEL and 46 NLEL). An LEL cow was that having an embryo with no heartbeat, detached membranes, or floating structures, including embryo remnants detected at pregnancy check by ultrasonography (US) 28-42 days post-AI, whereas an NLEL cow was that with embryo heartbeats detectable by US at pregnancy check 28-42 d post-IA. We took two blood samples from every cow at pregnancy check by US (the day of LEL detection) and 28 d later to perform serological diagnosis of BVDV, BoHV-1, and N. caninum; and to measure blood progesterone concentration at pregnancy check (28-42 d post-AI). We also sampled the conceptus from all the LEL cows. We performed PCR to detect BVDV, BoHV-1, and N. caninum in sampled conceptuses from LEL cows. Finally, we evaluated the associations of risk factors (serological titers, seroconversion, and progesterone) with LEL odds with logistic models. The risk for LEL was associated with serological titers to BVDV (P = 0.03) and tended to be associated with seroconversion to BVDV, given that 19.6% (9/46) in LEL and 6.5% (3/46) in NLEL cows seroconverted to BVDV (P = 0.09). In addition, BVDV was detected in conceptuses from LEL cows that seroconverted to BVDV but not in LEL cows that did not seroconvert. Conversely, the risk for LEL was not associated with the titers or seroconversion to BoHV-1 and N. caninum. BoHV-1 and N. caninum were not identified in any of the conceptuses. Finally, blood progesterone concentration was similar in LEL and NLEL cows, and it was not associated with the risk for LEL (P = 0.54). In conclusion, BVDV infection is a risk factor for LEL in dairy cows