Isolation of toxoplasma gondii from goats from Brazil

Goats are economically important in many countries, and little is known of caprine toxoplasmosis in Brazil. Antibodies to Toxoplasma gondii were assayed in the sera of 143 goats from 3 Brazilian states, using modified agglutination test (MAT titer ≥1:25); 46 (32.2%) tested positive. Samples of brain, heart, diaphragm, and masseter of seropositive animals were pooled, digested in pepsin, and bioassayed in mice. Viable T. gondii specimens were isolated from tissue homogenates of 12 goats; the isolates were designated TgGtBr1-12. Ten of the 12 isolates killed 100 of infected mice, indicating that goats can harbor mouse-virulent T. gondii and, hence, can serve as a source of infection for humans. © 2009 American Society of Parasitologists.

Study of Toxoplasma infection in Brazilian wild mammals: Serological evidence in Dasypus novemcinctus Linnaeus, 1758 and Euphractus sexcinctus Wagler, 1830

Toxoplasmosis is a worldwide distributed zoonosis that affects man and most warm-blooded animals, with a great economic impact in animal and public health. Serum samples from nine 9-banded armadillos, three 6-banded armadillos, three coatimundis, two opossums and one nutria were submitted for anti-Toxoplasma gondii antibody detection by means of a modified direct agglutination method. Encephalic tissue of three 6-banded armadillos, one 9-banded armadillo, one coatimundi and one nutria were digested in acid pepsin solution and inoculated into Swiss mice for parasite isolation. Only one serum sample from a nine-banded armadillo and two from six-banded armadillos reacted producing titers equal to 256, 512 and 512, respectively. T gondii was isolated in two 6-banded armadillos, one of which was not positive in the serological test. (c) 2005 Elsevier B.V. All rights reserved.

An enzyme-linked immunosorbent assay (elisa) for the detection of antibodies against babesia boris in cattle

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

The detection of Toxoplasma gondii by comparing cytology, histopathology, bioassay in mice, and the polymerase chain reaction (PCR)

The objective of this study was to compare the different methods of detecting Toxoplasma gondii in sheep tissue, tested serologically positive by the indirect immunofluorescent antibody test (IFAT). Brain, diaphragm, and blood samples were collected from 522 sheep slaughtered at the São Manuel abattoir, São Paulo State, Brazil. Brain and diaphragm samples from IFAT seropositive animals were digested by both trypsin and pepsin and then injected into mice. Part of the digested samples was used to prepare slides for Giemsa staining and in the polymerase chain reaction (PCR). Tissue fragments were fixed in formalin and examined using hematoxilin-eosin (HE). Forty of the sheep (7.7%) were IFAT positive. T. gondii was isolated in 23 (59.0%) of the 39 mice with pepsin-digested brain samples and in 27 (69.0%) of the 39 with trypsin-digested brain samples. Injection of diaphragm samples led to T. gondii isolation in 26 (66.7%) of the 39 pepsin-digested samples and 21 (53.8%) of the 39 trypsin-digested samples. Cytological and hystopathological examination of both brains and diaphragms was negative in all examined sheep. PCR was positive in 7 (17.9%) of the trypsin and 2 (5.1%) of the pepsin-digested samples, while 9 (23.1%) of the trypsin and 3 (7.7%) of the pepsin-digested samples showed T. gondii DNA. T. gondii isolation rate in mice (n = 34; 85.0%) was significantly higher than detection by PCR (n = 15; 37.5%). © 2001 Elsevier Science B.V.

Avaliação da eficiência dos métodos Direto, COPROTEST e Rugai no diagnóstico da estrongiloidíase

Strongyloidiasis, a relatively common parasitism in tropical and sub-tropical areas, is the result of the infection by the smaller nematode, the Strongyloides stercoralis. Humans can be infected by this parasite, which has in its vital cycle free-life forms of male and female individuals able to live in the ground, and with another step necessary parasitism in the intestinal wall. The diagnostic of the infection is routinely done by the microscopic observation of the larva in stool samples and the high sensibility of urn method over another one allows an trustable and efficient diagnostic. The efficiency of three methods (Direct, COPROTEST and Rugai) used in the Parasitology Sector of the NAC-LACAL in Araraquara (SP) to diagnosis the strongiloidiasis were evaluated. A number of 2346 samples of stool of patients from NAC-LACAL and Nestor Goulart Reis Hospital were analyzed in the period between August and December of 2002. The Rugai Method with an positivity index of 65 % was elected as the most efficient of thee ones.

Toxoplasma gondii: Detection by mouse bioassay, histopathology, and polymerase chain reaction in tissues from experimentally infected pigs

In the present study, we evaluated three techniques, mouse bioassay, histopathology, and polymerase chain reaction (PCR) to detect Toxoplasma gondii infection in tissues from experimentally infected pigs. Twelve mixed breed pigs, seronegative for T. gondii using an indirect immunofluorescent antibody test (IFAT), were used. Ten pigs were infected with 4 × 104 VEG strain oocysts, and two were maintained as uninfected controls. Animals were killed 60 days pos infection. Muscle (heart, tongue, diaphragm, and masseter) and brain samples were collected to investigate the presence of T. gondii tissue cysts by the different assay methods. For the bioassay, samples of brain (50 g) and pool of muscle samples (12.5 g of tongue, masseter, diaphragm, and heart) were used. PCR was performed using Tox4 and Tox5 primers which amplified a 529 bp fragment. The DNA extraction and PCR were performed three times, and all tissue samples were tested individually (brain, tongue, masseter, diaphragm, and heart). For histopathology, fragments of tissues were fixed in 10% of buffered formal saline and stained with HE. Histopathological results were all negative. PCR showed 25/150 (16.6%) positive samples, being 17/120 (14.1%) and 8/30 (26.6%) from muscle, and brain tissues, respectively. Tissue cysts of T. gondii were identified by mouse bioassay in 54/98 (55.1%) samples, being 31/48 (64.6%) from muscle samples, and 23/50 (46.0%) from brain samples. Toxoplasma gondii isolation in muscle samples by mouse bioassay was higher than in PCR (P < 0.01). Results indicate that DNA from pig tissues interfered with 529-bp-PCR sensitivity, and mouse bioassay was better than PCR in detecting T. gondii in tissues from pigs. © 2006 Elsevier Inc. All rights reserved.

The taxonomic and phylogenetic relationships of Trypanosoma vivax from South America and Africa

The taxonomic and phylogenetic relationships of Trypanosoma vivax are controversial. It is generally suggested that South American, and East and West African isolates could be classified as subspecies or species allied to T. vivax. This is the first phylogenetic study to compare South American isolates (Brazil and Venezuela) with West/East African T. vivax isolates. Phylogeny using ribosomal sequences positioned all T. vivax isolates tightly together on the periphery of the clade containing all Salivarian trypanosomes. The same branching of isolates within T. vivax clade was observed in all inferred phylogenies using different data sets of sequences (SSU, SSU plus 5.8S or whole ITS rDNA). T. vivax from Brazil, Venezuela and West Africa (Nigeria) were closely related corroborating the West African origin of South American T. vivax, whereas a large genetic distance separated these isolates from the East African isolate (Kenya) analysed. Brazilian isolates from cattle asymptomatic or showing distinct pathology were highly homogeneous. This study did not disclose significant polymorphism to separate West African and South American isolates into different species/subspecies and indicate that the complexity of T. vivax in Africa and of the whole subgenus Trypanosoma (Duttonella) might be higher than previously believed. © 2006 Cambridge University Press.

Interclonal Variations in the Molecular Karyotype of Trypanosoma cruzi: Chromosome Rearrangements in a Single Cell-Derived Clone of the G Strain

Trypanosoma cruzi comprises a pool of populations which are genetically diverse in terms of DNA content, growth and infectivity. Inter- and intra-strain karyotype heterogeneities have been reported, suggesting that chromosomal rearrangements occurred during the evolution of this parasite. Clone D11 is a single-cell-derived clone of the T. cruzi G strain selected by the minimal dilution method and by infecting Vero cells with metacyclic trypomastigotes. Here we report that the karyotype of clone D11 differs from that of the G strain in both number and size of chromosomal bands. Large chromosomal rearrangement was observed in the chromosomes carrying the tubulin loci. However, most of the chromosome length polymorphisms were of small amplitude, and the absence of one band in clone D11 in relation to its reference position in the G strain could be correlated to the presence of a novel band migrating above or below this position. Despite the presence of chromosomal polymorphism, large syntenic groups were conserved between the isolates. The appearance of new chromosomal bands in clone D11 could be explained by chromosome fusion followed by a chromosome break or interchromosomal exchange of large DNA segments. Our results also suggest that telomeric regions are involved in this process. The variant represented by clone D11 could have been induced by the stress of the cloning procedure or could, as has been suggested for Leishmania infantum, have emerged from a multiclonal, mosaic parasite population submitted to frequent DNA amplification/deletion events, leading to a 'mosaic' structure with different individuals having differently sized versions of the same chromosomes. If this is the case, the variant represented by clone D11 would be better adapted to survive the stress induced by cloning, which includes intracellular development in the mammalian cell. Karyotype polymorphism could be part of the T. cruzi arsenal for responding to environmental pressure. © 2013 Lima et al.

Sexual transmission of Toxoplasma gondii in sheep

Male sheep of reproductive age were distributed into three groups: GI, a sheep inoculated (oral) with 2.0×105 oocysts of the P strain of Toxoplasma gondii; GII, a sheep infected (subcutaneous) with 1.0×106 tachyzoites of the RH strain of T. gondii; and GIII, a sheep kept as a control (not infected). After the inoculation of the males, 12 breeding ewes, which were not pregnant and which were serologically negative for reproductive diseases (particularly toxoplasmosis), were distributed into three groups, synchronized, and subsequently exposed to natural mating with previously inoculated males. The distribution was as follows: five ewes that underwent natural mating with the GI male, five ewes that were exposed to natural mating with the GII male, and two ewes that were mated with the non-infected male (control). Serum samples of all the ewes were collected on days -30, -14, -7, -1, and 0 (days before natural mating) and on days 1, 3, 5, 7, 11, 14, and weekly until birth; the presence of serum antibodies against T. gondii was assessed by IFAT. Using a bioassay and PCR, T. gondii was isolated from the semen of the infected reproducing sheep before mating. Following natural mating, 5 of the 12 females displayed antibodies specific for T. gondii; of these animals, two of the ewes underwent natural mating with the male inoculated with oocysts (GI) and three with the male infected with tachyzoites (GII). One of the females that displayed antibodies specific to this coccidian and that underwent natural mating with the GII sheep had a macerated fetus on the 70th day following coverage. Using a bioassay after the birth, it was possible to isolate T. gondii from samples of the pool of tissues from the five females that seroconverted after natural mating and from their respective lambs. Using PCR, the DNA of T. gondii was isolated from the pool of tissues from one and two females exposed to natural mating with the reproductive males infected with the oocysts and tachyzoites, respectively. Using this technique, it was also possible to diagnose the presence of the parasite in the pool of tissues from the lambs of one female that underwent natural mating with the male sheep infected with oocysts. These results demonstrated the sexual transmission of T. gondii in the sheep species with consequent vertical transmission to their lambs. © 2013 Elsevier B.V.

Estudo epidemiológico da amebíase no Estado do Pará utilizando diferentes metodologias para diagnóstico

A epidemiologia da amebíase está sendo reavaliada desde que a E. histolytica (patogênica) foi considerada espécie distinta de E. dispar (não patogênica). Neste estudo, investigou-se a freqüência da amebíase em uma amostra de residentes do Pará por diferentes técnicas de diagnóstico e avaliou-se a patogenia do parasito. Os participantes (n = 845) forneceram material fecal e destes, 191 foram entrevistados quanto aos sintomas de diarréia, cólicas intestinais, constipação, náuseas e vômito. Foram também analisados 8 exsudatos de pacientes com suspeita de amebíase hepática. As amostras foram observadas sob microscopia de luz e a confirmação de E. histolytica feita a partir da pesquisa de antígenos. Um total de 98 amostras fecais e todos os exsudatos foram semeados em meio Pavlova para isolamento e posterior caracterização bioquímica e molecular (identificação de espécie e genotipagem). Isolados de outras regiões do Brasil foram também genotipados. A positividade obtida foi de 29,35% (248/845) e não houve correlação com a faixa etária. A microscopia revelou baixa sensibilidade (45,26%; 74/334), porém elevada especificidade (87,03%; 260/334) quando comparada ao ELISA. Houve relação significativa (OR 4,4026) entre a presença de sintomas e a positividade no ELISA, sendo a diarréia (58,82%) e a cólica intestinal (58,82%) os sintomas mais relatados. Nenhum exsudato foi positivo no exame a fresco, porém 7 foram positivos no ELISA. Obteve-se 22 isolados de material fecal e a caracterização da HE foi possível em 13, dos quais 7 E. histolytica e 6 E. dispar. O DNA de 22 isolados e dos exsudatos foram testados para identificação molecular de espécie e genotipagem. Do total, 16 cultivos (9 cepas mistas, 4 E. dispar e 3 E. histolytica) e 5 exsudatos (todos E. histolytica) amplificaram na PCR. A genotipagem identificou adicional positividade para E. histolytica em um exsudato e revelou diferentes polimorfismos de comprimento para o locus 1-2 de E. histolytica e E. dispar do Pará e de outras regiões do Brasil e um caso de co-infecção por diferentes genótipos de E. dispar. Nossos resultados revelam que a amebíase invasiva é um importante problema de saúde pública em nossa população e grande variedade de genótipos de E. histolytica contribuem para a doença no Brasil.

Influence of soil texture in the recovery of Toxocara canis eggs by a flotation method

In epidemiological surveys, the evaluation of soil contamination by Toxocara canis eggs requires a quick and easy method for the isolation of parasite eggs from soil samples. The efficiency of flotation methods is influenced by sample size, soil texture, degree of soil contamination, pretreatment, flotation solutions and time of flotation. This investigation was designed to evaluate the influence of soil texture in the recovery of T. canis eggs with the centrifugal flotation technique of Dada (Dada, B.J.O., 1979. A new technique for the recovery of Toxocara eggs from soil. J. Helminthol., 53: 141-144). Four types of soil (clay silt, sandy, silty clay and sand) were artificially contaminated with T. canis eggs (200 eggs per gram). Zinc sulphate (specific gravity 1.20) and sodium dichromate (specific gravity 1.35) were used as flotation solutions. Twenty replicated examinations were performed for each type of soil and flotation solution. There was a statistically significant difference in the results depending on soil type. The highest recovery percentages were observed in soils rich in sand (62.5% for sand and 38.0% for sandy soil). Differences were also observed with different flotation solutions. Sodium dichromate solution was more efficient for recovering T. canis eggs, regardless of the soil texture. © 1994.

A Social Parasite Evolved Reproductive Isolation from Its Fungus-Growing Ant Host in Sympatry

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Parasite diversity, patterns of MHC II variation and olfactory based mate choice in diverging three-spined stickleback ecotypes

Ecological speciation has been the subject of intense research in evolutionary biology but the genetic basis of the actual mechanism driving reproductive isolation has rarely been identified. The extreme polymorphism of the major histocompatibility complex (MHC), probably maintained by parasite-mediated selection, has been proposed as a potential driver of population divergence. We performed an integrative field and experimental study using three-spined stickleback river and lake ecotypes. We characterized their parasite load and variation at MHC class II loci. Fish from lakes and rivers harbor contrasting parasite communities and populations possess different MHC allele pools that could be the result of a combined action of genetic drift and parasite-mediated selection. We show that individual MHC class II diversity varies among populations and is lower in river ecotypes. Our results suggest the action of homogenizing selection within habitat type and diverging selection between habitat types. Finally, reproductive isolation was suggested by experimental evidence: in a flow channel design females preferred assortatively the odor of their sympatric male. This demonstrates the role of olfactory cues in maintaining reproductive isolation between diverging fish ecotypes.

Isolation and characterization of the female sex pheromone of the sugar beet cyst nematode, heterodera schachtii, and an analysis of male precopulatory behaviour /

The sugar beet cyst nematode, Heterodera schachtii, is a major agricultural pest. The disruption of the mating behaviour of this plant parasite in the field may provide a means of biological control, and a subsequent increase in crop yield. The H. schachtii female sex pheromone, which attracts homospecific males, was collected in an aqueous medium and isolated using high performance liquid chromatography. Characterization of the male-attractive material revealed that it was heat stable and water soluble. The aqueous medium conditioned by female H. schachtii was found to be biologically active and stimulated male behaviour in a concentration dependent manner. The activity of the crude pheromone was specific to males of H. schachtii and did not attract second stage juveniles. Results indicated that vanillic acid, a putative nematode pheromone, is not an active component of the H. schachtii sex pheromone. Male H. schachtii exhibited stylet thrusting, a poorly understood behaviour of the male, upon exposure to the female sex pheromone. This behaviour appeared to be associated with mate-finding and was used as a novel indicator of biological activity in bioassays. Serotonin, thought to be involved in the neural control of copulatory behaviour in nematodes, stimulated stylet thrusting. However, the relationship between stylet thrusting induced by the sex pheromone and stylet thrusting induced by serotonin is not clear. Extracellular electrical activity was recorded fi-om the anterior region of H. schachtii males during stylet thrusting, and appeared to be associated with this behaviour. The isolation of the female sex pheromone of H. schachtii may, ultimately, lead to the structural identification and synthesis of the active substance for use in a novel biological control strategy.

Isolation and characterization of a chitinase-secreting mutant of mortierella pusilla with altered interaction with the mycoparasite piptocephalis virginiana

Mortierella pusilla is a susceptible host and supports good growth of the mycoparasite, Piptocephalis virginiana. Uninucleate spores of M. pusilla were sUbjected to N-methyl-N'-nitro-nitrosoguanidine (MNNG). To attain a high mutation frequency , a 1o-minute exposure to 10 mg/ml MNNG was used and lead to the survival of about 10 % of the spores. The exposed spores then were plated on chitin or milk plates. Approximately 30,000 colonies were examined after mutagenesis on the screening media. A strain, MUT23 , with abnormal slow growth morphology was found to delay parasitism by £. virginiana. The particular morphology was not due to auxotrophy, because this strain displayed normal hyphae when glucose was used as the sole carbon source. One interesting phenomenon was that MUT23 showed an extensive clearing zone around the colony on colloidal chitin agar after 20-25 d. On the same conditions, wild type strain did not show this phenotype. In addition, the MUT23 strain produced the same normal hypha as the wild type strain when it was grown on colloidal chitin agar. The MUT23 was also able to produce more spores on colloidal chitin agar than on malt-yeast extract and minimal media. The parasite germ tubes formed appressoria at the point of contact on the cell surface of wild type and MUT23 grown for 6 days cell surface but not on the cel surface of MUT23 grown for 2 days. Thus, interaction between MUT23 strain and the mycoparasite was dependent on MUT23 age. The effect of MUT23 filtrate on germination of the parasite was tested. Lysis of germinated spores of the parasite were observed in concentrated MUT23 filtered solution. MUT23 was compared to the wild type strain for their chitinase production in sUbmerged culture. The chitinase isozymes of both wild type and MUT23 were shown by immunoblotting. Eight distinct chitinase molecules were detected. MUT23 showed markedly higher chitinase activity than the wild type cultured in chitin-containing medium. Maximum chitinase activities of MUT23 were 13.5 fold higher at 20 day of the culture then that of wild type.