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.

Immune response to Neospora caninum live tachyzoites in prepubertal female calves.

The aim of the present study was to characterize the specific immune response in prepubertal female calves inoculated with Neospora caninum. Forty-eight N. caninum-seronegative 6-month-old Angus female calves were randomly allocated into two groups: group A calves were inoculated subcutaneously (sc) with 1 × 106 tachyzoites of the low virulence NC-Argentina LP1 isolate in sterile phosphate-buffered saline (PBS); group B calves were mock inoculated sc with sterile PBS. Calves from group A developed a specific immune response characterized by the production of IgG antibodies and the expression of IFN-γ and TNF-α cytokines. Animals did not present any febrile reaction or reactions at the site of inoculation. Although chronic N. caninum infection was developed in 50% of calves of group A after inoculation, according to the presence of antibodies against rNc-SAG4, antigen characteristic of bradyzoites, N. caninum antibodies dropped below the cut-off of ELISA from day 210 post-inoculation onwards. Future trials using the same group of inoculated animals will allow the characterization of the evolution of the immune response during pregnancy and to determine whether the immunization with the local isolate is able to prevent congenital transmission and to protect against heterologous challenges.

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.

Toxoplasma gondii isolates from chickens in an area with human toxoplasmic retinochoroiditis.

The aim of this study was to detect, isolate and genetically characterize Toxoplasma gondii from tissues obtained from free range chickens which were breed in farms from patients with toxoplasmic retinochoroiditis in Misiones, Argentina. Thirty three samples of head (refrigerated = 18 and frozen = 15) from free range chickens were processed. Refrigerated (n = 18) chicken central nervous systems (CNS) were bioassay in mice. DNA was obtained from all samples (n = 33) and PCR was performed using TOX5-TOX8 T. gondii specific primers. Positive PCR samples were characterized by nested-PCR and restriction fragment length polymorphism using the markers SAG2, BTUB, GRA6, SAG3, PK1, L358, C22-8, C29-2 and Apico. T. gondii DNA was amplified in 30.3% (10/33) of CNS samples. Isolates were obtained in 27.7% (5/18) of inoculated CNS samples (TgCk11-9Arg, TgCk13-5Arg, TgCk14-5Arg, TgCk14-6Arg and TgCk14-7Arg). Seven samples showed a restriction pattern to all markers and were identified as atypical with several alleles type III. Genotyping of T. gondii from samples of patients with retinochoroiditis in the same area could improve the understanding of the epidemiology of toxoplasmosis in the region.

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 38kDa 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.

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.

First report of seroprevalence of Toxoplasma gondii and Neospora caninum in dairy sheep from Humid Pampa, Argentina.

The aim of this study was to describe the occurrence of antibodies to Toxoplasma gondii and Neospora caninum in dairy sheep from the Humid Pampa region, Argentina. Blood samples from 704 dairy sheep belonging to six flocks were collected. Using a cut off titer of 1:50, an indirect fluorescence antibody test was used. Antibodies to T. gondii or N. caninum were detected in 17.3 % (n = 122) and 3 % (n = 21), respectively. All the flocks had at least one seropositive animal to T. gondii but two of them had no seropositive sheep to N. caninum. Fifty-two of 122 (42.6 %) positive samples to T. gondii had antibody titers higher than 1:400. There was a significantly higher proportion of T. gondii seropositive animals in females and older sheep (p < 0.05). Ten of 21 (52.3 %) positive samples to N. caninum had antibody titers higher than 1:400. This is the first report of seroprevalence of T. gondii and N. caninum in dairy sheep from Humid Pampa, Argentina. Further research is required for a better understanding of the role of toxoplasmosis and neosporosis in dairy sheep in Argentina.

Detection of antibodies against Brucella abortus, Leptospira spp., and Apicomplexa protozoa in water buffaloes in the Northeast of Argentina.

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 brucellosis 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.

[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.

Serologic profiles for Sarcocystis sp. and Neospora caninum and productive performance in naturally infected beef calves.

Sarcocystis sp. and Neospora caninum infections affect cattle worldwide causing important economic losses. The objective of the present study was to trace serologic profiles for Sarcocystis sp. and N. caninum in naturally infected beef calves and analyze their relationship with transmission routes and productive performance. Samples were collected in two cow-calf operations located in Buenos Aires province, Argentina. In farm 1, 43 calves were bled and weighed three times. In farm 2, 69 calves were bled and weighed six times. Sarcocystis sp. and N. caninum immunofluorescence antibody test (IFAT) titers were averaged for each sampling point in order to trace serologic profiles for each infection. Categories were created to evaluate differences in daily weight gain. For S. cruzi antigen, animals were separated in a low-titer (< or = 200) and high-titer group (>200); for N. caninum, animals were grouped as infected and uninfected. Sarcocystis sp. antibody titer as well as the number of infected animals increased gradually over time in both farms. In farm 2 the low-titer group had significantly higher daily weight gain than the high-titer group. For N. caninum 44% (farm 1) and 65% (farm 2) of calves were considered infected, and the serological profile was horizontal or decreasing over time. However, seroprevalence increased in both farms and vertical and horizontal transmission frequency were estimated between 18.5%-29% and 22-25.5%, respectively. No differences were detected in daily weight gain between N. caninum groups from both farms. This is the first report of serological profiles for Sarcocystis sp. and N. caninum by IFAT in naturally infected beef calves and their relationship to different transmission routes and productive performance.

Molecular characterization of Cryptosporidium spp. from domestic pigs in Argentina.

Cryptosporidiosis in pigs is caused by different Cryptosporidium species or genotypes, with C. suis and C. scrofarum considered porcine specific species. There is scarce information on Cryptosporidium infection in pigs in South America. A total of 520 individual faecal samples were obtained from 1, 2, 3 and 4 week old piglets (n = 130 from each age group), from 13 Argentinean intensive pig farms. The diagnosis of species of Cryptosporidium combined microscopy and molecular techniques. Genotyping from samples with Cryptosporidium oocysts at microscopy was performed by genus-specific and species-specific nested PCR targeting 18S rRNA gene fragments, and sequencing. Microscopic analysis detected Cryptosporidium oocysts in 47/520 (9%) faecal samples from 11/13 (85%) farms, with farm infection rates between 0 and 17.5%. Presence of Cryptosporidium oocysts was associated with diarrhea. The proportion of microscopically positive samples was not associated with piglet age. A total of 15/47 (32% of samples with oocyst compatible structures) were positive by genus and species-specific nested PCR. Species-specific PCR and sequencing showed presence of C. suis, C. scrofarum, and both species in 3, 8 and 4 samples, respectively. The proportion of positive samples on each specific PCR was similar between age groups, being C. suis proportion slightly higher in 4 week old piglets. The use of molecular tools allowed the confirmation of C. suis and C. scrofarum infection in Argentinean pigs. Cryptosporidiosis was widely distributed in the main pig husbandry area from Argentina, with a low to moderate intra farm infection rate.

Congenital human toxoplasmosis caused by non-clonal Toxoplasma gondii genotypes in Argentina.

Toxoplasmosis, a worldwide distributed zoonosis, can be transmitted congenitally affecting fetuses and developing variable clinical signs. Different Toxoplasma gondii genotypes and infective dose are related factors with different clinical manifestations. Several studies indicate that atypical strains could produce more severe clinical manifestations compared to typical strains. Umbilical cord blood (n = 37) and placenta (n = 19) were collected at birth from women with acute T. gondii infection and processed for isolation by mice bioassay. Six isolates were obtained and identified as TgHm14-4Arg, TgHm15-02Arg, TgHm16-01Arg, TgHm16-02Arg, TgHm17-01Arg and TgHm17-02Arg. Three genotypes described previously on Toxo-DB were identified: #138 identified in chickens from Brazil, #182 isolated from eared doves from Brazil, #14 from wallaby kangaroos and chickens from Argentina, chickens from Brazil, Colombia, Chile and Venezuela, cats and dogs from Brazil and Colombia and also coyotes from USA indicating worldwide distribution of these genotypes. Two new allele combinations were obtained showing high genotypes diversity in Argentina. Four of the isolates (TgHm14-4Arg, TgHm15-02Arg, TgHm16-01Arg, TgHm16-02Arg) and two of them (TgHm17-01Arg, TgHm17-02Arg) produced chronic and acute infections in mice, respectively. Until now, seven T. gondii isolates have been obtained from humans in Argentina, and all were atypical or non-clonal genotypes. The identification of atypical strains causing congenital toxoplasmosis and circulating in our region, make important to perform the serological screenings according Argentine Consensus of Toxoplasmosis and to apply and monitoring treatments earlier in pregnancy. To achieve this aim, it is necessary to inform general population about T. gondii infection, diagnostics and control measures. These results should serve to generate awareness about congenital toxoplasmosis in South America.

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

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.

Population structure of Toxoplasma gondii in Argentina.

The protozoan Toxoplasma gondii is worldwide distributed showing a particular population structure that may differ among continents and countries. The aim of this study was to analyze the T. gondii population structure in Argentina and compare it with genotyping information from other South American countries. For the analysis, 39 samples from Argentina (isolates from the provinces of Buenos Aires, Misiones, Entre Ríos and San Luis) were genotyped using 10 multilocus PCR-RFLP markers including SAG1, SAG2 (5'-3'SAG2, alt. SAG2), SAG3, BTUB, GRA6, C22-8, C29-2, L358, PK1, and Apico. The T. gondii DNA samples were obtained from domestics animals (chickens n = 20; cats n = 3; pigs n = 2; goat n = 1; rabbit n = 1), humans (n = 6), zoo animals (n = 5) and a rat (n = 1). Phylogenetic relationship of these Argentinean isolates together with representative reference genotypes was determined by phylogenetic network analysis. Thirty-seven Argentinean samples belonged to 21 genotypes and two samples were genotyped at 8 of the 10 loci and considered incomplete characterized. Among these 37 typed samples, five genotypes were not previously reported. The majority of the samples grouped with the Type III (ToxoDB PCR-RFLP genotype #2) lineage. The clonal Type II (ToxoDB genotypes #1 and #3) was also identified. Our results suggest a unique population structure with combination of unique genotypes and the common Type II and Type III lineages in Argentina. Nevertheless, different regions showed distinctive pattern of genotypes, revealing a higher variability in Northern provinces.

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.

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.

Assessment of diagnostic accuracy of a commercial ELISA for the detection of Toxoplasma gondii infection in pigs compared with IFAT, TgSAG1-ELISA and Western blot, using a Bayesian latent class approach.

Serological methods are the most commonly used diagnostic tools to detect Toxoplasma gondii infections in pigs. In the absence of a readily available 'gold standard', an estimation of diagnostic accuracy is difficult to assess. A commercial ELISA (PrioCHECK® Toxoplasma Ab porcine ELISA, Prionics, Schlieren, Switzerland) for the diagnosis of T. gondii infection in pigs was evaluated in naturally infected animals from two distinct populations; indoor and outdoor living animals. An assessment of diagnostic accuracy, using a Bayesian latent class approach with adjustment for within indoor and outdoor farm clustering using random effects, was performed. Tests used for comparison were: IFAT; ELISA using native affinity-purified P30 (SAG1) T. gondii tachyzoite surface antigen (TgSAG1-ELISA); and Western blot with T. gondii tachyzoites lysate. The data set comprised 297 pig serum samples across outdoor (n=149) and indoor (n=148) farms in Argentina. The estimated sensitivity and specificity for the commercial ELISA were 98.9% (95% credible interval: 96.2; 100) and 92.7% (95% credible interval: 87.7; 96.6), respectively. The analysis of sera and plasma from pigs (n=6) experimentally inoculated with 5,000 T. gondii oocysts revealed a pronounced antibody response beginning 2 weeks p.i. until the end of the observation period (11 weeks p.i.) in all animals. Meat juice obtained from inoculated animals after euthanasia also tested positive. These results suggest that the PrioCHECK® Toxoplasma Ab porcine ELISA may be a useful tool to perform serological diagnosis of T. gondii infections in pigs to control Toxoplasma infection in pigs and humans.

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.

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