Infection with different Neospora caninum strains causes differences in the glycosylation pattern in the uteri and placentae of Neospora caninum-infected heifers.

Neospora caninum is an obligate intracellular parasite that causes abortion in ruminants. Different strains produce differences in the severity of disease outcomes. These differences may cause physiological or pathological changes in cells, modifying the intercellular interactions and intracellular transport pathways that could be evidenced by identifying the terminal sugars. This study aimed to characterize the oligosaccharide pattern in the bovine placenta and uterus after infection with tachyzoites of three different strains of N. caninum (Nc-1, Nc-6 Argentina and Nc Spain-7) during early gestation. Fourteen heifers were inoculated intravenously on day 70 of gestation with 2 × 108 N. caninum tachyzoites and samples of placentae and uteri were analysed by histology and lectin histochemistry. In the infected groups, severe placentitis was associated with changes in lectin binding in the vascular endothelium by Lens culinaris agglutinin (LCA), Pisum sativum agglutinin (PSA) and Ricinus communis I (RCA-I) lectins, in the epithelial cells of the endometrial glands by RCA-I, Dolichos biflorus agglutinin (DBA), succinylated wheat germ agglutinin, peanut agglutinin (PNA), concanavalin-A (CON-A), LCA, PSA and Phaseolus vulgaris erythroagglutinin (PHA-e), and in the trophoblast layer by PNA, CON-A, LCA, PSA, PHA-e, soybean agglutinin, RCA-I, DBA and Bandieraea simplicifolia agglutinin (BSA-I). The results suggest that N. caninum causes changes in the glycosylation pattern in the maternofetal interface tissues and might cause abortions in early gestation due to changes in the cellular structure of the placenta.

Evaluation of biological behavior of Toxoplasma gondii atypical isolates # 14 and # 163.

Toxoplasma gondii is an obligate intracellular protozoan parasite capable of infecting warm-blooded animals, including humans. A highly diverse genetic population has been reported in Central and South America, predominating mainly atypical genotypes. Different genotypes showed different biological behavior in mice. The aim of this study was to evaluate the biological behavior of T. gondii isolates obtained from Macropus rufogriseus (TgMr) and Saimiri boliviensis (TgSb) identified as atypical genotypes # 14 and # 163, respectively. Strains RH, ME49 and VEG were used as reference for clonal types I, II and III, respectively. In vitro invasion and replication capacity assays were analyzed at 6 and 18 hpi, respectively. In vivo assay was performed in Swiss mice (n = 30) using 1 × 102 and 1 × 103 parasites/mouse as infective doses (ME49, VEG, TgMr, TgSb and negative control). Morbi-mortality and tissues PCR were assessed. Lymphoproliferation assays were performed and gamma interferon was measured by ELISA. The ME49 strain showed the highest invasion, followed by TgSb and VEG, while RH and TgMr presented the lowest invasions. The RH strain and the TgSb isolate showed more endodyogeny events (fastest doubling times) than VEG and ME49 strains and the TgMr isolate. Both atypical isolates showed high virulence (100% morbi-mortality, at 8-10 dpi) and parasite DNA was detected in all tissue samples. Splenocytes from mice inoculated with TgMr and TgSb registered the highest values of gamma interferon. An in vitro invasion-replication index was established which correlates inversely with virulence in mice. In conclusion, T. gondii atypical isolates # 14 and # 163 showed a different in vitro behavior than clonal strains, with low invasion-replication indexes but being highly virulent in mouse model.

Toxoplasmosis congénita: Diagnóstico serológico, RPC, aislamiento y caracterización molecular de Toxoplasma gondii / Congenital toxoplasmosis: Serology, PCR, parasite isolation and molecular characterization of Toxoplasma gondii

Resumen Introducción El diagnóstico de toxoplasmosis congénita (TC) en el recién nacido es muy importante porque debe recibir tratamiento siempre, sintomático o no, para evitar o aminorar las secuelas de la enfermedad. Objetivo Evaluación comparativa de los métodos disponibles en la institución para el diagnóstico de TC. Materiales y Métodos Se evaluaron métodos diagnósticos en 67 niños cuyas madres cursaron toxoplasmosis aguda durante el embarazo. Se utilizó la técnica de Sabin Feldman para IgG al nacimiento y durante el seguimiento serológico hasta el año de vida. Para determinar IgM, IgA e IgE se utilizó la técnica immunosorbent agglutination assay (ISAGA). El diagnóstico directo se realizó por reacción de polimerasa en cadena (RPC), aislamiento y caracterización molecular del parásito. Resultados La sensibilidad (S) de ISAGA IgM fue 87%, ISAGA IgA 91% y la especificidad (E) fue 100% para ambas; cuando se realizaron en conjunto, la S aumentó a 98%. La detección de IgE contribuyó al diagnóstico cuando se la detectó sólo en la sangre del neonato y no en sangre materna. Se aisló el parásito en cuatro casos de TC, uno fue genotipo II y los otros tres, genotipos "atípicos". La S del aislamiento fue 80% y la E 100%. Conclusión Los métodos serológicos utilizados mostraron una buena eficacia diagnóstica. Un caso fue detectado sólo por el aislamiento y la caracterización molecular tiene gran valor epidemiológico.

Background. Congenital toxoplasmosis diagnosis in the newborn is a very important issue due to the need for early treatment to prevent future sequels. Aim To compare available methods at the institution for the diagnosis of congenital toxoplasmosis. Material and Methods In this study we have evaluated the different diagnostic tests used in 67 congenital exposed newborns, including serological tests, PCR, parasite isolation and molecular characterization. Results The ISAGA IgM and IgA tests showed sensitivity (Se) of 87 and 91%, respectively, and specificity (Sp) of 100%. When ISAGA IgM and IgA were performed simultaneously, the Se increased to 98% and the Sp was 100%. The presence of IgE contributed to the diagnosis when it was detected in the child's serum but not in maternal blood. In four congenital infected children the parasite was isolated and genotyped: one was genotype II and the other three were "atypical" genotypes. No parasite was isolated in children without congenital toxoplasmosis. Discussion Overall, serological tests showed a good diagnostic performance although in one case they were all negative and isolation was the only tool to identify the infection. We conclude that it is essential to use all diagnostic tests in every single exposed child, including if possible, molecular characterization due to its epidemiological implication.

Evaluación y comparación de pruebas serológicas para la detección de la neosporosis bovina en Argentina / Evaluation and comparison of serological methods for the detection of bovine neosporosis in Argentina

Neospora caninum is a protozoan parasite that causes abortion and important economic losses in cattle worldwide. The accurate diagnosis of neosporosis is essential for management and control measures. The aims of this study were: i) to evaluate the performance of an in-house enzyme-linked immunosorbent assay based on the 38 kDa native antigen (p38-ELISA) to diagnose bovine neosporosis in Argentina using a well- characterized local sera panel from experimentally infected and naturally exposed cattle and ii) to compare the diagnostic performance and agreement of three N. caninum serological tests: p38-ELISA, indirect fluorescence antibody test (IFAT) and immunoblotting (IB) using the same sera panel. Serum samples testing either positive or negative by IFAT and IB were considered "Relative Standards of Comparison" (RSC) and used for p38-ELISA evaluation. Receiver operating characteristics analysis revealed that p38-ELISA was highly accurate (area under the curve= 0.982) according to RSC with a cut-off index of 0.0905. Relative sensitivity and specificity of p38-ELISA were 97.8 % and 99.5 %, respectively and agreement between RSC and p38-ELISA was almost perfect (k= 0.97). The evaluation and performance comparison of serological tests were performed according to the definition of gold standard based on the decision of the "majority of tests". All tests displayed high sensitivity and specificity values (greater than 95 %); and excellent agreement. This study describes the accurate performance of p38-ELISA evaluated locally and the highly accurate diagnostic performance of the studied tests for the detection of anti-N. caninum antibodies in cattle from Argentina.

Neospora caninum es un parásito protozoo responsable de abortos y pérdidas económicas en bovinos. La realización de un diagnóstico serológico preciso y con resultados comparables obtenidos por diferentes pruebas contribuye al manejo de este problema y a encarar medidas de control. Los objetivos del presente trabajo fueron los siguientes: 1) evaluar en Argentina una prueba de enzimoinmunoensayo in-house con el antígeno nativo de 38 kDa de N. caninum (ELISA-p38) para el diagnóstico de la neosporosis bovina, utilizando un panel de sueros locales bien caracterizados, procedentes de bovinos infectados de modo experimental o naturalmente expuestos; 2) comparar el desempeño y establecer el nivel de concordancia de tres pruebas serológicas para la detección de N. caninum, ELISA-p38, inmunofluorescencia indirecta (IFI) e inmunoblot (IB), con el mismo panel de sueros. Los sueros que resultaron positivos o negativos a IFI e IB fueron considerados como estándares relativos de comparación (ERC) para evaluar la prueba de ELISA-p38. El análisis de característica operativa del receptor determinó que la prueba de ELISA-p38 fue altamente precisa (área bajo la curva= 0,982) usando el punto de corte 0,0905. La sensibilidad y especificidad relativa del ELISA-p38 fue 97,8 % y 99,5 %, respectivamente, con una concordancia casi perfecta (k= 0,97) respecto del ERC. La comparación del desempeño de las pruebas se realizó usando como gold standard el criterio de la decisión de la "mayoría de las pruebas". Las pruebas exhibieron altos valores de sensibilidad y especificidad (mayores del 95 %) y excelente concordancia. Este trabajo describe un buen desempeño de la prueba de ELISA-p38 evaluada localmente y adecuada performance diagnóstica de las pruebas serológicas analizadas para la detección de anticuerpos anti N. caninum en bovinos de Argentina.

First isolation and molecular characterization of Toxoplasma gondii from a human placenta in Argentina.

Blood sample and placenta were taken from a 37-week pregnant woman; serologic results indicated acute toxoplasmosis. Placenta was inoculated into mice. Seropositive mice were sacrificed and tissue cysts from brain were inoculated into new mice. Specific DNA was detected by PCR, and the isolate was characterized as Type II by nPCR-RFLP for nSAG2, SAG3, BTUB, GRA6, c29-2, c22-8, L358, PK1 and Apico markers. This is the first isolation and molecular characterization of Toxoplasma gondii from humans in Argentina.

Development of a multiplex real time PCR to differentiate Sarcocystis spp. affecting cattle.

Cattle are intermediate hosts of Sarcocystis cruzi, Sarcocystis hirsuta and Sarcocystis hominis which use canids, felids or primates as definitive hosts (DH), respectively, and in addition of Sarcocystis sinensis from which the DH is unknown. The aims of the present study were to develop and optimize a multiplex real time PCR for a sensitive and specific differentiation of Sarcocystis spp. affecting cattle and to estimate the prevalence of Sarcocystis spp. in Argentinean cattle. The 18S rRNA genes from individual sarcocysts were amplified and cloned to serve as controls. For the amplification of bovine Sarcocystis spp. a total of 3 primers were used in combination with specific individual probes. Each assay was evaluated and optimized individually and subsequently combined in a multiplex assay (BovSarcoMultiplex real time PCR). The analytical specificity of the multiplex assay was assessed using 5 ng of DNA of heterologous Sarcocystis spp. and other apicomplexan parasites, and no positive reactions were observed other than for the species the PCR targeted. The analytical sensitivity ranged between 0.0125 and 0.125 fg of plasmid DNA (equivalent to the DNA of 2-20 plasmid DNA copies) or resembling DNA of 0.1-0.3 bradyzoites. A total of 380 DNA loin samples from Argentina were tested and 313, 29, 14 and 2 were positive for S. cruzi, S. sinensis, S. hirsuta and S. hominis, respectively. S. sinensis was the most prevalent species among thick walled Sarcocystis spp. in Argentinean cattle. Mixed infections were detected in 8.9% of all samples. Diagnostic sensitivity and specificity for the BovSarcoMultiplex real time PCR relative to previous microscopic examination for thin and thick-walled cyst were 91.5% and 41.7%, 36.3% and 95.9% respectively. Improved DNA extraction methods may allow to further increase the specific and sensitive detection of Sarcocystis spp. in meat samples.

[Advances in the development of vaccines for bovine neosporosis]. / Avances en el desarrollo de vacunas contra la neosporosis bovina.

Neosporosis, a disease caused by the obligate intracellular protozoan Neospora caninum, produces abortions in cattle. The severe economic losses in cattle industry justify the need to develop control measures for preventing bovine abortion. Apicomplexan parasitic resistance is associated with T helper 1 immune response mediated by CD4 cytotoxic T lymphocytes, the production of interferon-gamma, interleukin-12, tumor necrosis factor and immunoglobulin G2. The reduction of vertical transmission in subsequent pregnancies and the low levels of abortion repetition suggests the existence of protective immune mechanisms. Inoculation with live tachyzoites before mating protects against infection and abortion. Antecedents of the development of live vaccines against other protozoa stimulate research to develop a live vaccine against N. caninum. On the other hand, an inactivated vaccine with low efficacy against neosporosis is useful in the prevention of abortion in farms with epizootic disease. A neosporosis vaccine should avoid abortion, transplacental transmission and infection persistence. In the present work, advances in vaccine development including lysate of tachyzoites, live parasites, recombinant antigens and vaccine vectors are reviewed.

Coinfección con toxoplasma gondii y virus de la inmunodeficiencia felina (FIV) / Toxoplasma gondii and feline immunodeficiency virus (FIV) coinfection

Se utilizaron 100 sueros de gatos provenientes de la ciudad de La Plata y alrededores con el fin de determinar la relación entre la infección con FIV y la infección con T. gondii. Los sueros se analizaron para determinar anticuerpos anti-FIV y anti-T. gondii por las pruebas de inmunobloting e inmunofluorescencia indirecta, respectivamente. Para el análisis de los resultados los sueros se clasificaron en cuatro grupos según presentaran o no anticuerpos anti-FIV y anti-T. gondii (Tox): Grupo 1) FIV+ Tox+; Grupo 2) FIV+ Tox-; Grupo 3) FIV- Tox+; Grupo 4) FIV- Tox-. Se establecieron tres categorías por grupo con respecto a los signos clínicos de los gatos estudiados. El porcentaje de sueros positivos a FIV y a T. gondii (34,5 por ciento, 19/55) fue significativamente mayor que el porcentaje de sueros positivos a FIV y negativos a T. gondii (17,7 por ciento, 8/45; p < 0,05, prueba exacta de Fisher). Los títulos de anticuerpos anti T. gondii fueron significativamente mayores en el Grupo 1 con respecto al Grupo 3. La distribución de los signos clínicos fue significativamente diferente entre los 4 grupos. El porcentaje de animales con signos clínicos potencialmente compatibles con FIV y/o toxoplasmosis fue: Grupo 1: 57,9 por ciento, Grupo 2: 75 por ciento; Grupo 3: 48,6 por ciento y Grupo 4: 27 por ciento. Los resultados del presente trabajo indican la asociación entre la infección con el FIV y la infección con T. gondii