Evaluation of IFA, MAT, ELISAs and immunoblotting for the detection of anti-Toxoplasma gondii antibodies in paired serum and aqueous humour samples from experimentally infected pigs

The study evaluated the efficiency of diagnostic laboratory methods to detect anti-Toxoplasma gondii antibodies in paired serum and aqueous humour samples from experimentally infected pigs. 18-mixed breed pigs were used during the experiment; these were divided into two groups, G I (infected group, n = 10) and G2 (uninfected group, n = 8). Infection was performed with 4 x 10(4) VEG strain oocysts at day 0 by the oral route in G1 animals. All pigs were euthanized at day 60, when retina, aqueous humour, and blood samples were collected. Anti-T gondii antibody levels were assessed in serum (s) and aqueous humour (ah) by indirect immunofluorescence assay (IFA), modified agglutination test (MAT), m-ELISA (using crude membranes from T gondii tachyzoites as antigen) and r-ELISA (using rhoptries from T gondii tachyzoites as antigen). Polymerase chain reactions (PCR) of samples from the retina were performed by using Tox4 and Tox5 primers. Antibody titers of G1 animals ranged from 128 to 1024 and from 16 to 256 in serum and aqueous humour, respectively. There were differences in the correlation coefficients between IFA(s) x IFA (ah) (r = 0.62, P = 0.05), MAT(s) x MAT (ah) (r = 0.97, P < 0.0001); however, there was no significant difference between r-ELISA(s) x r-ELISA (ah) (r = 0. 14, P = 0.7). Antibodies present in serum and aqueous humour recognized similar antigens. Samples of retina were positive by PCR in 30% (3/10) of infected pigs. G2 animals remained without antibody levels and were PCR negative throughout the experiment. These results suggest that the use of a combination of tests and immunoblotting for paired aqueous humour and serum samples could improve the sensitivity and specificity for the diagnosis of ocular toxoplasmosis. (c) 2007 Elsevier Ltd. All rights reserved.

Toxoplasma gondii in the peripheral blood of patients with acute and chronic toxoplasmosis

Background and aims Toxoplasmic retinochoroiditis may recur months or years after the primary infection. Rupture of dormant cysts in the retina is the accepted hypothesis to explain recurrence. Here, the authors present evidence supporting the presence of Toxoplasma gondii in the peripheral blood of immunocompetent patients. Methods Direct observation by light microscopy and by immunofluorescence assay was performed, and results were confirmed by PCR amplification of parasite DNA. Results The authors studied 20 patients from Erechim, Brazil, including acute infected patients, patients with recurrent active toxoplasmic retinochoroiditis, patients with old toxoplasmic retinal scars, and patients with circulating IgG antibodies against T gondii and absence of ocular lesions. Blood samples were analysed, and T gondii was found in the blood of acutely and chronically infected patients regardless of toxoplasmic retinochoroiditis. Conclusions The results indicate that the parasite may circulate in the blood of immunocompetent individuals and that parasitaemia could be associated with the reactivation of the ocular disease.

Detection of anti-Toxoplasma gondii antibodies in experimentally and naturally infected non-human primates by Indirect Fluorescence Assay (IFA) and indirect ELISA

The Indirect Fluorescence Assay (IFA) and the indirect ELISA were comparatively used to detect IgG and IgM antibodies for Toxoplasma gondii in experimentally and naturally infected primates. In the experimentally infected group, antibodies of diagnostic value were detected at day 9 post-infection (PI) with the IFA (IgG and IgM) and with IgG-ELISA. IgM-ELISA detected antibodies for T. gondii starting at day 3 PI until the end of the experiment (102 days PI). Of the 209 naturally infected sera tested, from many zoos of State of Sao Paulo, 64.59 and 67.94% were positive in the IgG-IFA test and IgG-ELISA respectively. IgM-ELISA test detected seropositivity in 52.63% of the sera although IgM-IFA test detected it in only in 0.96% of the samples. The differential toxoplasmosis diagnosis was accomplished with Neospora caninum by IFA, observing 61 (29.2%) seropositive animals for this parasite and 149 (70.8%) negative. Sixty animals were positive for both T. gondii and N. caninum. Pneumonia, splenomegaly, and intestinal ulcers were macroscopically observed. Unremarkable interstitial pneumonia, enteritis, colitis, splenitis, and glomerulitis were microscopically observed. The immunohistochemical stain could not detect the presence of T. gondii in the tissues of the animals infected experimentally.

Prevalence of anti-Toxoplasma gondii and anti-Neospora caninum antibodies in swine from Northeastern Brazil

A serologic survey was conducted among 130 swine slaughtered in the public slaughterhouse of the city of Patos, Paraíba State, Northeastern Brazil, to determine the prevalence of anti-Toxoplasma gondii and anti-Neospora caninum antibodies, and to verify possible associations between sex of the animals and antibody prevalence. The sera were analyzed by indirect antibody tests, considering 1:64 (T. gondii) and 1:50 (N. caninum) dilutions as cut-off points. The prevalence of anti-T. gondii antibodies was 36.2% (47/130) (95% CI = 27.9 - 45.0%) with reciprocal titers ranging from 64 to 2,048, and of anti-N. caninum antibodies was 3.1% (4/130) (95% CI = 0.8 - 7.7%) with reciprocal titers ranging from 50 to 6,400. Three of the four N. caninum-positive samples were also positive for T. gondii antibodies. All Neospora and Toxoplasma IFAT-positive animals were also positive for confirmatory immunoblotting techniques using total and purified N. caninum and T. gondii tachyzoite antigens, i.e., p38 (NcSRS2) and p30 (TgSAG1). There was no association between sex of animals and prevalence of anti-T. gondii and anti-N. caninum antibodies. This is the first indication of N. caninum natural infection in pigs from Brazil.

Occurrence of anti-Neospora caninum and anti-Toxoplasma gondii antibodies in dogs with visceral leishmaniasis

Uninfected dogs and those naturally infected with Leishmania chagasi exhibiting different clinical forms of disease were evaluated for the presence of anti-Neospora caninum and anti-Toxoplasma gondii antibodies. Blood samples were collected from 110 mongrel dogs. Sera were tested using the indirect fluorescent antibody test (IFAT), and the animals with visceral leishmaniasis (VL) (n=60) were classified clinically. Out of the 110 sera investigated, 5 (4.5%) were positive for N. caninum (IFAT≥50) and 36 (32.7%) for T. gondii (IFAT≥16). Anti-L. chagasi antibody titers in asymptomatic dogs (n=10) were found to be significantly lower (P<0.05) than those in oligosymptomatic ones (n=22), which were in turn significantly lower (P<0.05) than those in symptomatic ones (n=28). No association between Leishmania and N. caninum infections was observed. Among dogs infected with L. chagasi, a tendency (P=0.053) towards an association between the infection with T. gondii and the appearance of VL symptoms was observed, suggesting that the clinical manifestation of VL in dogs may enhance their susceptibility to T. gondii. The possible influence of the immunosuppressive status of canine leishmaniasis in the different clinical forms of the disease is discussed.

Presence of antibodies against Toxoplasma gondii, Neospora caninum and Leishmania infantum in dogs from Piauí

This study aimed to evaluate the presence of antibodies against Neospora caninum, Toxoplasma gondii and Leishmania infantum in dogs attended at the Veterinary Hospital of the Federal University of Piauí, Northeastern Brazil, where there are no reports of the occurrence of N. caninum and T. gondii in dogs. Serum samples from 530 dogs of genders, different ages and breeds from the municipality of Teresina and nearby towns were analyzed using three indirect fluorescent antibody tests, each one targeting one of the three agents. The associations between the parasites and gender, breed and age of the dogs were assessed by the chi-square test (p > 0.05). The occurrence of antibodies to N. caninum, T. gondii and L. infantum was 3.2, 18.0 and 78.1%, respectively. Toxoplasma gondii was more frequently found in older dogs (p < 0.05) whereas L. infantum was more common in animals aged between 1 to 3 years (p < 0.05). In order to evaluate potential associations between the presence of anti-N. caninum and anti-T. gondii antibodies and Leishmania infection, 240 dogs were selected (120 positive and 120 negative for Leishmania spp.), based on serological and parasitological diagnoses. No association was found between Leishmania spp. and the coccidian parasites (p > 0.05). The results confirm the exposure of dogs to these parasites in the State of Piauí.

SEROPREVALENCE AND ISOLATION OF TOXOPLASMA GONDII FROM SHEEP FROM SAO PAULO STATE, BRAZIL

Sheep are important in the epidemiology of Toxoplasma gondii infection, but little is known of ovine toxoplasmosis in Brazil. Antibodies to T. gondii were assayed in sera of 495 sheep from 36 countries of Sao Paulo state. Brazil, using the modified agglutination test (MAT titer >= 1:25); 120 (24.2%) sheep tested positive. Samples of brain, heart, and diaphragm of 85 seropositive sheep were pooled, digested in pepsin, and bioassayed in mice. Toxoplasma gondii was isolated from tissue homogenated of 16 sheep, and the isolated were designated TgShBr 1-16. Six of the 16 T. gondii isolated killed 100% of infected mice. Results indicate that asymptomatic sheep can harbour mouse-virulent T. gondii; hence, they can serve as a source of infection for humans.

Galectin-3 plays a modulatory role in the life span and activation of murine neutrophils during early Toxoplasma gondii infection

Galectins are beta-galactoside-binding lectins involved in several biological processes and galectin-3 (Gal-3) is related to modulation of immune and inflammatory responses. This study aimed to evaluate the role of Gal-3 in the life span and biological functions of murine neutrophils during in vitro infection by virulent Toxoplasma gondii RH strain. Inflammatory peritoneal neutrophils (N phi) from C57BL/6 wildtype (WT) and Gal-3 knockout (KO) mice were cultured in the presence or absence of parasites and analyzed for phosphatidylserine (PS) exposure and cell death using Annexin-V and propidium iodide staining, and cell viability by MU assay. Cell toxicities determined by lactate dehydrogenase (LDH), degranulation by lysozyme release, and cytokine production were measured in NO culture supernatants. Phorbol myristate acetate (PMA)- or zymosan-dependent reactive oxygen species (ROS) were measured in N phi cultures. Our results demonstrated that Gal-3 is involved in the increase of the viable Not. number and the decrease of PS exposure and cell death following T. gondii infection. We also observed that Gal-3 downmodulates gondii-induced N phi toxicity as well as N phi degranulation regardless of infection. Furthermore, Gal-3 expression by N phi was associated with increased levels of IL-10 in the beginning and decreased levels of TNF-alpha later on, regardless of parasite infection, as well as with decreased levels of IL-6 and increased IL-12 levels, following early parasite infection. Our results also showed that Gal-3 suppresses PMA- but not zymosan-induced ROS generation in N phi following T. gondii infection. In conclusion, Gal-3 plays an important modulatory role by interfering in N phi life span and activation during early T gondii infection. (C) 2009 Elsevier GmbH. All rights reserved.

Quality control of Toxoplasma gondii in meat packages: Standardization of an ELISA test and its use for detection in rabbit meat cuts

Toxoplasma gondii causes severe disease both to man and livestock and its detection in meat after slaughtering requires PCR or biological tests. Meat packages contain retained exudate that could be used for serology due to its blood content. Similar studies reported false negative assays in those tests. We standardized an anti-T. gondii IgG ELISA in muscle juices from experimentally infected rabbits, with blood content determination by cyanhemoglobin spectrophotometry. IgG titers and immunoblotting profiles were similar in blood, serum or meat juice, after blood content correction. These assays were adequate regardless of the storage time up to 120 days or freeze-thaw cycles, without false negative results. We also found 1.35% (1/74) positive sample in commercial Brazilian rabbit meat cuts, by this assay. The blood content determination shows ELISA of meat juice may be useful for quality control for toxoplasmosis monitoring. (C) 2011 Elsevier Ltd. All rights reserved.

Toxoplasma gondii: The severity of toxoplasmic encephalitis in C57BL/6 mice is associated with increased ALCAM and VCAM-1 expression in the central nervous system and higher blood-brain barrier permeability

In order to investigate the differential ALCAM, ICAM-1 and VCAM-1 adhesion molecules mRNA expression and the blood-brain barrier (BBB) permeability in C57BL/6 and BALB/c mice in Toxoplasma gondii infection, animals were infected with ME-49 strain. It was observed higher ALCAM on day 9 and VCAM-1 expression on days 9 and 14 of infection in the central nervous system (CNS) of C57BL/6 compared to BALB/c mice. The expression of ICAM-1 was high and similar in the CNS of both lineages of infected mice. In addition, C57BL/6 presented higher BBB permeability and higher IFN-gamma and iNOS expression in the CNS compared to BALB/c mice. The CNS of C578L/6 mice presented elevated tissue pathology and parasitism. In conclusion, our data suggest that the higher adhesion molecules expression and higher BBB permeability contributed to the major inflammatory cell infiltration into the CNS of C57BL/6 mice that was not efficient to control the parasite. (C) 2010 Elsevier Inc. All rights reserved.

Prevalence of Toxoplasma gondii and Neospora caninum infections in sheep from Federal District, central region of Brazil

Serum samples from 1028 sheep were collected from 32 herds within Federal District, in the central region of Brazil. The samples were examined by indirect fluorescent antibody test (IFAT) using sera diluted 1:64 and 1:50 as cut-off values for the detection of antibodies against Toxoplasma gondii and Neospora caninum, respectively. The observed prevalence for T. gondii infection was 38.22% (26.81%< CI 0.95 < 49.62%), and the titers ranged from 64 to 65536. The observed prevalence for N. caninum infection was 8.81% (7.08%< CI 0.95 < 10.53%). The titers ranged from 50 to 51200. The reactant sera to both pathogens corresponded to 4.67% of the samples. The risk factors were not determined because of the absence of negative herds for T. gondii and the high proportion of positive herds for N. caninum (87.50%). The prevalence for T. gondii infection was significantly higher among males than in females. The present work is the first report on seroprevalence of T. gondii and N. caninum in sheep from Federal District and shows that infection by both parasites is widespread in the ovine population from this region.

Prevalence of anti-Toxoplasma gondii and anti-Neospora caninum antibodies in sheep from Mossoro, Rio Grande do Norte, Brazil

Toxoplasma gondii is an obligate intracellular parasite with a variety of hosts, responsible for reproductive problems and economic losses in sheep flocks. Neospora caninum was recently identified and its clinical presentation in sheep is similar to that of toxoplasmosis, which can cause repeated abortions, though less frequently in this species. In order to confirm the prevalence of these agents in the city of Mossoro, Rio Grande do Norte, Brazil, 409 serum samples from adult sheep (364 females and 45 males) were tested by the indirect immunofluorescence antibody test, using cut-off point at a dilution of 1:64 and 1:50 for T. gondii and N. caninum, respectively. From the 35 properties examined, 23 (65.7%)had at least one seropositive animal for T gondii and six (17.1%) for N. caninum. The prevalence of seropositive animals for T. gondii was 20.7% and for N. caninum 1.8%. There was no association between the presence of the agent`s antibody and gender, reports of reproductive problems and presence of dogs and/or cats in the properties. T. gondii is well distributed and N. caninum has low prevalence in sheep and in the properties of the studied region. (C) 2008 Elsevier B.V. All rights reserved.

Population structure and mouse-virulence of Toxoplasma gondii in Brazil

Recent studies found that isolates of Toxoplasma gondii from Brazil were biologically and genetically different from those in North America and Europe. However, to date only a small number of isolates have been analysed from different animal hosts in Brazil. In the present study DNA samples of 46 T. gondii isolates from cats in 11 counties in Sao Paulo state, Brazil were genetically characterised using 10 PCR restriction fragment length polymorphism markers including SAG1, SAG2, SAG3, STUB, GRA6, c22-8, c29-2, L358, PKI and Apico. An additional marker, CS3, that locates on chromosome VIIa and has previously been shown to be linked to acute virulence of T. gondii was also used to determine its association to virulence in mice. Genotyping of these 46 isolates revealed a high genetic diversity with 20 genotypes but no clonal Type I, II or III lineage was found. Two of the 46 isolates showed mixed infections. Combining genotyping data in this study with recent reported results from chickens, dogs and cats in Brazil (total 125 isolates) identified 48 genotypes and 26 of these genotypes had single isolates. Four of the 48 genotypes with multiple isolates identified from different hosts and locations are considered the common clonal lineages in Brazil. These lineages are designated as Types BrI, BrII, BrIII and BrIV. These results indicate that the T. gondii population in Brazil is highly diverse with a few successful clonal lineages expanded into wide geographical areas. In contrast to North America and Europe, where the Type II clonal lineage is overwhelmingly predominant, no Type II strain was identified from the 125 Brazil isolates. Analysis of mortality rates in infected mice indicates that Type BrI is highly virulent, Type BrIII is non-virulent, whilst Type BrII and BrIV lineages are intermediately virulent. In addition, allele types at the CS3 locus are strongly linked to mouse-virulence of the parasite. Thus, T. gondii has an epidemic population structure in Brazil and the major lineages have different biological traits. (C) 2007 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Isolation and genetic characterisation of Toxoplasma gondii from a red-handed howler monkey (Alouatta belzebul), a jaguarundi (Puma yagouaroundi), and a black-eared opossum (Didelphis aurita) from Brazil

Toxoplasma gondii isolates are highly diverse in domestic animals from Brazil. However, little is known about the genetics of this parasite from wild mammals in the same region. Reveal genetic similarity or difference of T. gondii among different animal populations is necessary for us to understand transmission of this parasite. Here we reported isolation and genetic characterisation of three T. gondii isolates from wild animals in Brazil. The parasite was isolated by bioassay in mice from tissues of a young male red handed howler monkey (Alouatta belzebul), an adult male jaguarundi (Puma yagouaroundi), and an adult female black-eared opossum (Didelphis aurita). The monkey and the jaguarundi had inhabited the Zoo of Parque Estadual Dois Irmaos, Pernambuco State, Northeastern Brazil, for 1 year and 8 years, respectively. The wild black-eared opossum was captured in Sao Paulo State, Southeastern Brazil, and euthanised for this study because it was seropositive for T. gondii (titre 1:100 by the modified agglutination test, MAT). Ten PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) markers, SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico, were used to genotype the isolates. T. gondii was isolated from the brain and heart homogenate of the monkey, the muscle homogenate of the jaguarundi, and the heart homogenate of the black-eared opossum. This was the first isolation of T. gondii from a neotropical fetid from Brazil. The isolate from the monkey (TgRhHmBr1) was not virulent in mice, whereas the isolates from the jaguarundi (TgJagBr1) and the black-eared opossum (TgOpBr1) were virulent in mice. The genotype of the isolate from the monkey has been identified in isolates from a goat and ten chickens in the same region of Brazil, suggesting that it may be a common lineage circulating in this region. The genotypes of the isolates from the jaguarundi and the black-eared opossum have not been previously reported. Although there are already 88 genotypes identified from a variety of animal hosts in Brazil, new genotypes are continuously being identified from different animal species, indicating an extremely high diversity of T. gondii in the population. (C) 2010 Elsevier B.V. All rights reserved.

SEROPREVALENCE OF TOXOPLASMA GONDII ANTIBODIES IN CAPTIVE WILD MAMMALS AND BIRDS IN BRAZIL

In this study, serum samples of 203 animals from different locations, from zoos and breeding facilities from the north and northeast regions of Brazil, were analyzed for the presence of anti-Toxoplasma gondii antibodies by the modified agglutination test (MAT) with a cutoff of 1:25. Of the sampled animals, 184 were adult mammals of both sexes and 19 were birds. Antibodies were found in 61 of 184 mammals, and no association between sex and age of the animals and the presence of T. gondii antibodies was observed (P < 0.05). Anti-T gondii antibodies were not found in birds. Toxoplasma gondii was detected in Brazilian tapir (Tapirus terrestris) for the first time.