Infección de transmisión vertical por Toxoplasma gondii: seguimiento de los hijos de mujeres con primoinfección en una institución de asistencia médica colectiva; 2010-2015

La toxoplasmosis congénita (TC) afecta 1 a 2 niños cada 1.000 nacimientos al año. La mayoría de los recién nacidos infectados son asintomáticos pero la ausencia de tratamiento puede determinar secuelas oftalmológicas y neurológicas. Objetivo: describir el seguimiento de los hijos de mujeres con primoinfección por Toxoplasma gondii durante el embarazo derivados a una Policlínica de Infectología de la Médica Uruguaya entre diciembre de 2010 y mayo de 2015. Material y método: se incluyeron los hijos de mujeres con primoinfección por T.gondii durante el embarazo entre el 1 de diciembre de 2010 y el 31 de mayo de 2015. Se confirmó primoinfección mediante determinación inmunoenzimática de IgG e IgM específicas, complementada por IgM por inmunofluorescencia indirecta o test de avidez de IgG según el caso. El diagnóstico de infección congénita se realizó por la presencia de IgM o títulos de IgG estables o en aumento en los primeros 9 meses de seguimiento del niño. Resultados: se diagnosticó primoinfección en 34 mujeres. La mayoría controló adecuadamente el embarazo y ninguna presentó infección por VIH, sífilis o Chagas. Se confirmó TC en 3 niños nacidos a término, con peso adecuado, hijos de mujeres con primoinfección adquirida en el tercer trimestre y tratadas con espiramicina. Uno presentó coriorretinitis, los otros fueron asintomáticos. En todos la IgM fue negativa, el diagnóstico se confirmó con curva de IgG. Todos recibieron piremetamina, sulfadiazina y ácido folínico sin efectos adversos. A la fecha continúan en tratamiento y seguimiento dos de los tres niños. Discusión y conclusión: la captación temprana de la mujer embarazada, la indicación oportuna de medidas de prevención constituyen pilares fundamentales para reducir la TC. El tratamiento oportuno y adecuado puede prevenir las secuelas.

Determinación de la prevalencia serológica de brucella abortus, toxoplasma gondii, sarcocystis spp y los factores de riesgo asociados en estudiantes de medicina veterinaria de la Universidad de la Salle

Tesis (Médico Veterinario). -- Universidad de La Salle. Facultad de Ciencias Agropecuarias. Programa de Medicina Veterinaria, 2013

Pathogenicity and phenotypic sulfadiazine resistance of Toxoplasma gondii isolates obtained from livestock in northeastern Brazil

Toxoplasma gondii is the causative protozoan agent of toxoplasmosis, which is a common infection that is widely distributed worldwide. Studies revealed stronger clonal strains in North America and Europe and genetic diversity in South American strains. Our study aimed to differentiate the pathogenicity and sulfadiazine resistance of three T. gondii isolates obtained from livestock intended for human consumption. The cytopathic effects of the T. gondii isolates were evaluated. The pathogenicity was determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using a CS3 marker and in a rodent model in vivo. Phenotypic sulfadiazine resistance was measured using a kinetic curve of drug activity in Swiss mice. IgM and IgG were measured by ELISA, and the dihydropteroate synthase (DHPS) gene sequence was analysed. The cytopathic effects and the PCR-RFLP profiles from chickens indicated a different infection source. The Ck3 isolate displayed more cytopathic effects in vitro than the Ck2 and ME49 strains. Additionally, the Ck2 isolate induced a differential humoral immune response compared to ME49. The Ck3 and Pg1 isolates, but not the Ck2 isolate, showed sulfadiazine resistance in the sensitivity assay. We did not find any DHPS gene polymorphisms in the mouse samples. These atypical pathogenicity and sulfadiazine resistance profiles were not previously reported and served as a warning to local health authorities.

PREVALENCE, ISOLATION, AND GENETIC CHARACTERIZATION OF TOXOPLASMA GONDII IN CHICKEN

Toxoplasma gondii (T. gondii) is one of the most successful parasites in the world because of its capability of infecting all warm-blooded animals. It has been reported that up to one third of the world population is infected with this parasite. Chickens are recognized as good indicators of the environmental T. gondii oocysts contamination because they obtain food from the ground. Thus, the prevalence of T. gondii in chicken provides more insight related to public health concern from T. gondii. Previous studies have shown a high isolation rate from free-range chickens raised in the United States. The objectives of this study were to evaluate the microbial safety and infection of T. gondii in free-range chickens available at the grocery stores and farms for the consumers to purchase and genotype T. gondii isolates. Chicken hearts were obtained from the local markets and also from the farms raising free- range chickens. Heart juice was obtained from cavities of each heart. Modified agglutination test (MAT) for detection of IgG antibodies was conducted with those heart juice samples with titer of 1:5, 1:25, and 1: 100. Each seropositive heart was pepsin digested and bioassayed into a group of two mice. Six weeks post inoculation (p.i.) mice were bled and euthanized to examine the infection of T. gondii. In addition, multiplex multilocus nested PCR-RFLP was performed to genetically characterize T. gondii isolates with eleven PCR-RFLP markers including SAG1, SAG2, altSAT2, SAG3, BTUB, GRA6, c22-8, c29-a, L358, PK1, and Apico. One hundred fifty from a total of 997 samples (15.0%) were found seropositive for T. gondii. No viable T. gondii was isolated from chicken hearts that were sampled. A total of four genotypes were identified, including one new genotype and three previously identified genotypes. The results suggest that T. gondii oocysts could present in the environment and infect the food animals. T. gondii prevalence in chicken hearts could reflect the environmental contamination of T. gondii and prevalence information can be used to manage T. gondii infection risk.

Ocorrência da infecção por Toxoplasma gondii e avaliação da imunização em caprinos do sertão do cabugi, Rio Grande do Norte

Toxoplasmosis is one zoonosis caused by Toxoplasma gondii protozoan. Goats, amongst the production animals, are one of the species most susceptible to this parasite, being one them main involved agents in ovine and goat abortions, determining great economic losses and implications for public health, since the presence it parasite in the products of goat origin, consist in one of the main sources of infection for the man. In this study 244 blood samples in 8 farms situated in 4 cities from the Sertão do Cabugi region, Rio Grande do Norte State, northeast of Brazil and, tested by ELISA assay. The results had shown a prevalence of 47.13% for anti- T. gondii antibodies and a significant association between positivity and variable evaluated as age, locality and property. The IgG avidity assay evaluated in 115 positive samples was carried to discriminate acute and chronic infection. Twelve samples (10.4%) had presented antibodies of low avidity while 103 (89.6%) presented high avidity antibodies; indicating that most of the animals was precocious exposure to the parasite. Significant difference was verified only for the variable sex. We also evaluate the capacity of recombinant adenoviruses codifying SAG1, SAG2, SAG3 and CMV in inducing activation of specific immune response in goat. These 109 animals received 109 pfu of the AdSAG1, AdSAG2, AdSAG3, AdCMV or PBS in vaccine protocol with 3 immunizations. Serum samples of the each animal, before and after mmunization, had been submitted to the ELISA. The results demonstrate that the immunizations had induced the production of IgG antibodies specific against T. gondii proteins

Serological evidence of Toxoplasma gondii in hunted wild boar from Portugal

Toxoplasmosis is an important parasitic zoonosis with a worldwide distribution, being the parasitic disease with the highest occurrence in Europe. Wild boar has an important role in the epidemiological cycle of Toxoplasma gondii as an intermediate host, that can potentially infect humans when the meat is consumed raw or undercooked. The purpose of this work was to determine the presence of antibodies to T. gondii in serum of hunted wild boar. During the hunting season 2011/2012, sera samples were collected from 97 wild boar and tested for IgG antibodies to T. gondii, using the modified agglutination test. Twenty out of the 97 wild boar (20.6%) were seropositive for T. gondii IgG antibodies. Multivariate logistic regression analysis showed that males and older animals were associated with T. gondii seropositivity. These results show that T. gondii has an important presence in wild boar population from Portugal, suggesting a potential zoonotic risk for humans when wild boar meat or meat products are consumed raw or undercooked.

Toxoplasma gondii Infection in Hunted Wild Boars (Sus scrofa): Heart Meat Juice as an Alternative Sample to Serum for the Detection of Antibodies

Toxoplasmosis is a global zoonosis caused by the protozoan parasite Toxoplasma gondii. Detection of antibodies to T. gondii in serum samples from hunted animals may represent a key step for public health protection. It is also important to assess the circulation of this parasite in wild boar population. However, in hunted animals, collection of blood is not feasible and meat juice may represent an alternative sample. The purpose of the present study was to evaluate heart meat juice of hunted wild boars as an alternative sample for post-mortem detection of antibodies to T. gondii by modified agglutination test (MAT). The agreement beyond chance between results from meat juice assessed with Cohen’s kappa coefficient revealed that the 1:20 meat juice dilution provided the highest agreement. McNemars’s test further revealed 1:10 as the most suitable meat juice dilution, as the proportion of positive paired samples (serum and meat juice from the same animal) did not differ at this dilution. All together, these results suggest a reasonable accuracy of heart meat juice to detect antibodies to T. gondii by MAT and support it as an alternative sample in post-mortem analysis in hunted wild boars.

Toxoplasma-SPECIFIC IgG SUBCLASS ANTIBODY RESPONSE IN CEREBROSPINAL FLUID SAMPLES FROM PATIENTS WITH CEREBRAL TOXOPLASMOSIS

SUMMARY Cerebral toxoplasmosis can be highly debilitating and occasionally fatal in persons with immune system deficiencies. In this study, we evaluated the Toxoplasma gondii-specific IgG subclass antibody response in 19 cerebrospinal fluid (CSF) samples from patients with cerebral toxoplasmosis who had a positive IgG anti-T. gondii ELISA standardized with a cyst antigen preparation. There were no significant differences between the rates of positivity and the antibody concentrations (arithmetic means of the ELISA absorbances, MEA) for IgG1 and IgG2, but the rates of positivity and MEA values for these two IgG subclasses were significantly higher than those for IgG3 and IgG4. The marked IgG2 response in CSF from patients with cerebral toxoplasmosis merits further investigation.

Toxoplasma antibody and stool parasites in public school children, Rolândia, Paraná, Brazil

The occurrence of toxoplasmosis and enteroparasitosis was studied in 434 children from elementary schools in the rural and urban areas of Rolândia, Paraná State, Brazil. Sera and fecal samples from all the students were submitted to IFA for Toxoplasma gondii and coproparasitological tests, respectively. The children were tested by Amsler grid and 72 of them were examined for the presence of lesions compatible with ocular toxoplasmosis. Some variables were tested but none showed increased risk for toxoplasmosis. The distribution according to sex and age and also same other variables are presented and discussed. Correlations between Amsler's grid test, toxoplama RIFI, occurrence of eyes lesions and enteroparasitosis are also considered.

Humoral responses and immune protection in mice immunized with irradiated T. gondii tachyzoites and challenged with three genetically distinct strains of T. gondii

Toxoplasma gondii is an obligate intracellular parasite that infects a variety of mammals and birds. T. gondii also causes human toxoplasmosis; although toxoplasmosis is generally a benign disease, ocular, congenital or reactivated disease is associated with high numbers of disabled people. Infection occurs orally through the ingestion of meat containing cysts or by the intake of food or water contaminated with oocysts. Although the immune system responds to acute infection and mediates the clearance of tachyzoites, parasite cysts persist for the lifetime of the host in tissues such as the eye, muscle, and CNS. However, T. gondii RH strain tachyzoites irradiated with 255 Gy do not cause residual infection and induce the same immunity as a natural infection. To assess the humoral response in BALB/c and C57BL/6J mice immunized with irradiated tachyzoites either by oral gavage (p.o.) or intraperitoneal (i.p.) injection, we analyzed total and high-affinity IgG and IgA antibodies in the serum. High levels of antigen-specific IgG were detected in the serum of parenterally immunized mice, with lower levels in mice immunized via the oral route. However, most serum antibodies exhibited low affinity for antigen in both mice strain. We also found antigen specific IgA antibodies in the stools of the mice, especially in orally immunized BALB/c mice. Examination of bone marrow and spleen cells demonstrated that both groups of immunized mice clearly produced specific lgG, at levels comparable to chronic infection, suggesting the generation of IgG specific memory. Next, we challenged i.p. or p.o. immunized mice with cysts from ME49. VEG or P strains of T. gondii. Oral immunization resulted in partial protection as compared to challenged naive mice: these findings were more evident in highly pathogenic ME49 strain challenge. Additionally, we found that while mucosal IgA was important for protection against infection, antigen-specific IgG antibodies were involved with protection against disease and disease pathogenesis. Most antigen responsive cells in culture produced specific high-affinity IgG after immunization, diverse of the findings in serum IgG or from cells after infection, which produced low proportion of high-avidity IgG. (C) 2011 Elsevier B.V. All rights reserved.

IgM and IgA Antibody Responses in 12 Cases of Human Acquired Toxoplasmosis

The persistence, in some subjects, of specific IgM antibodies to Toxoplasma gondii for several months after the acute phase of infection has complicated the interpretation of serological test results for toxoplasmosis. Several reports have emphasized the value of the detection of Toxoplasma-specific IgA antibodies for the diagnosis of acute toxoplasmosis. In this article, we report the follow-up profiles of Toxoplasma-specific IgM and IgA antibodies in serum samples obtained from 12 patients at various intervals after the onset of the clinical manifestations of infection. IgM antibodies were detected by the indirect immunofluorescence (IIF) test, antibody capture enzyme-linked immunosorbent assay (cELISA) and enzyme-mediated chemilluminescent technique (CmL). IgA antibodies were quantified by the direct ELISA (dELISA) and cELISA procedures. As defined by the manufacturer of the cELISA test for IgA used, most patients with acute toxoplasmosis have antibody levels > 40 arbritary units per ml (AU/ml). At values > 40 AU/ml, the cELISA for IgA detected significant antibody levels for a shorter time than the other techniques used for IgM and IgA detection. However, IgA levels £ 40 AU/ml do not exclude the possibility of acute toxoplasmosis since such levels can be reached very soon after infection with T. gondii. The results obtained in the present study show that the serological diagnosis of acute toxoplasmosis may not be such an easy task. Our data suggest that use of the IgA-cELISA concomitantly with IgM antibody screening could permit, in some circumstances, a more efficient diagnosis of acute acquired toxoplasmosis

IgG Antibody responses in mice coinfected with Toxocara canis and other helminths or protozoan parasites

The immune response expressed by IgG antibodies in BALB/c mice experimentally infected with Toxocara canis, was studied with the aim of verifying the possible in vivo cross-reactivity between antigens of T. canis and other parasites (Ascaris suum, Taenia crassiceps, Schistosoma mansoni, Strongyloides venezuelensis and Toxoplasma gondii). Experiments included three groups of mice: one infected only by T. canis, another with one of the other species of parasites and a third concomitantly infected with T. canis and the other species in question. Animals were bled by orbital plexus at 23, 38 and 70 days post infection (p.i.). Sera were analyzed for anti-Toxocara antibodies by ELISA and Immunoblotting, using excretion-secretion antigens (ES), obtained from culture of third-stage larvae of T. canis. For all experiments a control group comprised by ten non-infected mice was used. Only in the case of A. suum infection, in these experimental conditions, the occurrence of cross-reactivity with T. canis was observed. However, in the case of co-infection of T. canis - S. mansoni, T. canis - S. venezuelensis and T. canis - T. crassiceps the production of anti-Toxocara antibodies was found at levels significantly lower than those found in mice infected with T. canis only. Co-infection with S. mansoni or S. venezuelensis showed lower mortality rates compared to what occurred in the animals with single infections. Results obtained in mice infected with T. canis and T. gondii showed significant differences between the mean levels of the optical densities of animals infected with T. canis and concomitantly infected with the protozoan only in the 23rd day p.i.

IgG Antibody responses in mice coinfected with Toxocara canis and other helminths or protozoan parasites

The immune response expressed by IgG antibodies in BALB/c mice experimentally infected with Toxocara canis, was studied with the aim of verifying the possible in vivo cross-reactivity between antigens of T. canis and other parasites (Ascaris suum, Taenia crassiceps, Schistosoma mansoni, Strongyloides venezuelensis and Toxoplasma gondii). Experiments included three groups of mice: one infected only by T. canis, another with one of the other species of parasites and a third concomitantly infected with T. canis and the other species in question. Animals were bled by orbital plexus at 23, 38 and 70 days post infection (p.i.). Sera were analyzed for anti-Toxocara antibodies by ELISA and Immunoblotting, using excretion-secretion antigens (ES), obtained from culture of third-stage larvae of T. canis. For all experiments a control group comprised by ten non-infected mice was used. Only in the case of A. suum infection, in these experimental conditions, the occurrence of cross-reactivity with T. canis was observed. However, in the case of co-infection of T. canis - S. mansoni, T. canis - S. venezuelensis and T. canis - T. crassiceps the production of anti-Toxocara antibodies was found at levels significantly lower than those found in mice infected with T. canis only. Co-infection with S. mansoni or S. venezuelensis showed lower mortality rates compared to what occurred in the animals with single infections. Results obtained in mice infected with T. canis and T. gondii showed significant differences between the mean levels of the optical densities of animals infected with T. canis and concomitantly infected with the protozoan only in the 23rd day p.i.

Monoclonal antibody directed against Neospora caninum tachyzoite carbohydrate epitope reacts specifically with apical complex-associated sialylated beta tubulin

Monoclonal antibodies (mabs) were generated against whole sonicated Neospora caninum tachyzoites as immunogen. Initial ELISA screening of the reactivity of hybridoma culture supernatants using the same antigen and antigen treated with sodium periodate prior to antibody binding resulted in the identification of 8 supernatants with reactivity against putative carbohydrate epitopes. Following immunoblotting, mab6D12 (IgG1), binding a 52/48-kDa doublet, and mab6C6 (IgM), binding a 190/180-kDa doublet, were selected for further studies. Immunofluorescence of tachyzoite-infected cultures localized the corresponding epitopes not to the surface, but to interior epitopes at the apical part of N. caninum tachyzoites. During in vitro tachyzoite to bradyzoite stage conversion, mab6C6 labeling translocated toward the cyst periphery, while for mab6D12 no changes in localization were noted. Upon extraction of tachyzoites with the nonionic detergent Triton-X-100, the 52-kDa band recognized by mab6D12 was present exclusively in the insoluble, cytoskeletal fraction of both N. caninum and Toxoplasma gondii tachyzoites. Tandem mass spectrometry analysis identified this protein as N. caninum beta tubulin. The 48-kDa band labeled by mab6D12 was a Vero cell protein contamination. The protein(s) reacting with mab6C6 could not be conclusively identified by mass spectrometry. Immunofluorescence consistently failed to label T. gondii tachyzoites, indicating that beta tubulin in T. gondii and N. caninum could be differentially modified or that the reactive epitope in T. gondii is masked. Immunogold TEM of isolated apical cytoskeletal preparations and dual immunofluorescence with antibody to tubulin confirmed that mab6D12 binds to the anterior part of apical complex-associated microtubules. The sodium periodate sensitivity of the beta tubulin associated epitope was confirmed by immunoblotting and ELISA, and treatment of N. caninum cytoskeletal proteins with sialidase prior to mab6D12 labeling resulted in a profound loss of antibody binding, suggesting that mab6D12 reacts with sialylated beta tubulin.

The antibody response in experimental ocular toxoplasmosis

BACKGROUND: The dynamics of the humoral immune response in ocular toxoplasmosis (OT) are poorly understood. We therefore investigated this process in a rabbit model of the disease. MATERIALS AND METHODS: Of 24 infection-naïve adult rabbits, 12 were left untreated and 12 were systematically infected with 5,000 tachyzoites of the non-cyst-forming BK strain of Toxoplasma gondii. Three months later, all rabbits were inoculated transvitreally with 5,000 tachyzoites of Toxoplasma gondii. Paired samples of aqueous humor and serum were analyzed temporally for their total and specific IgG contents. RESULTS: In infection-naïve rabbits with primary OT, specific IgG reached detectable levels in the inoculated eyes between 5 and 15 days after inoculation. In infection-immunized rabbits with secondary OT, a significant increase in specific IgG was regularly detected after 5 days. The antibody ratio C was diagnostic (>/=3) from day 15 onward in primary OT and from day 21 onward in secondary OT. In the uninfected partner eyes, the antibody ratio C was found sporadically diagnostic from day 15 onward in primary OT, but at no time in secondary OT. Specific IgG persisted both locally and in the serum until the end of the monitoring period (100 days). CONCLUSION: Our findings relating to the rabbit model of OT reveal three features of clinical relevance: a diagnostic window precedes the establishment of a humoral immune response; specific antibodies persist long after the cessation of disease activity; and in primary OT, the antibody ratio C may also increase in the uninfected partner eye.