Hilda Fátima de Jesus Pena

Geographical patterns of Toxoplasma gondii genetic diversity revealed by multilocus PCR-RFLP genotyping

In recent years, an extensive collection of Toxoplasma gondii samples have been typed using a set of 10 PCR-RFLP genetic markers. Here we summarize the data reported until the end of 2012. A total of 1457 samples were typed into 189 genotypes. Overall, only a few genotypes dominate in the northern hemisphere, which is in stark contrast to the southern hemisphere where hundreds of genotypes coexist with none being notably dominant. PCR-RFLP genotype #1 (Type II clonal), #2 (Type III), #3 (Type II variant) and #10 (Type I) are identified globally. Genotypes #2 and #3 dominate in Africa, genotypes #9 (Chinese 1) and #10 are prevalent in Asia, genotypes #1, #2 and #3 are prevalent in Europe, genotypes #1, #2, #3, #4 and #5 dominate in North America (#4 and #5 are collectively known as Type 12). In Central and South America, there is no clear dominance of any genotype even though a few have relatively higher frequencies. Statistical analysis indicates significant differences among populations in Africa, Asia, Europe, North America, and Central and South America, with only Europe and North America exhibiting similar diversity. Collectively, the results revealed distinct population structures and geographical patterns of diversity in T. gondii.

Genetic diversity of Toxoplasma gondii isolates from chickens from Brazil

Until recently, Toxoplasma gondii was considered clonal with very little genetic variability. Recent studies indicate that T. gondii isolates from Brazil are genetically and biologically different from T. gondii isolates from USA and Europe. In the present study, we retyped 151 free range chicken isolates from Brazil including 117 newly isolated samples from 11 geographically areas (Alagoas, Bahia, Ceará, Maranhão, Paraná, Pernambuco, Rio de Janeiro, Rio Grande do Norte, São Paulo, Sergipe, and Rondonia) and 34 previously reported isolates from the very north (Pará) and the very south (Rio Grande do Sul). Ten PCR-RFLP markers including SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico were used to genotype all isolates. Overall analysis of 151 T. gondii isolates revealed 58 genotypes. Half (29/58) of these genotypes had single isolate and the other half of the genotypes were characterized with two or more isolates. Only 1 of 151 isolates was clonal Type I strain and 5 were clonal Type III strains. Two isolates had mixed infections. Clonal Type II strain was absent. One strain was Type II at all loci, except BTUB. The results confirm high genetic diversity of T. gondii isolates from Brazil.

First isolation of Sarcocystis neurona from the South American opossum, Didelphis albiventris, from Brazil.

Sarcocystis neurona was isolated from sporocysts from two of eight South American opossums, Didelphis albiventris, from Brazil. Interferon gamma gene knock out (KO) mice fed sporocysts from two opossums developed neurologic sarcocystosis. S. neurona was demonstrated in the brains of infected KO mice by immunohistochemical staining with anti-S. neurona antibody. The parasite was cultivated in cell culture and S. neurona DNA was isolated from cultured merozoites. This is the first report of isolation of S. neurona from Brazil and the first report from its new host, D. albiventris.

Prevalence of anti-Toxoplasma gondii and anti-Neospora caninum antibodies in sheep from Mossoró, 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 Mossoró, 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 agents 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.

OCCURRENCE OF CATTLE SARCOCYSTIS SPECIES IN RAW KIBBE FROM ARABIAN FOOD ESTABLISHMENTS IN THE CITY OF SAO PAULO, BRAZIL, AND EXPERIMENTAL TRANSMISSION TO HUMANS

Fifty samples of raw kibbe from 25 Arabian restaurants in the city of São Paulo, Brazil, were examined for the presence of bovine Sarcocystis species, using light and electron microscopy, and for infectivity to humans. Sarcocysts were found in all 50 samples. Based on cyst wall structure, S. hominis (94%), S. hirsuta (70%), and S. cruzi (92%) were identified (mostly as mixed infections). Different raw kibbe samples, positive for S. hominis in fresh preparations, were offered as a meal for 7 human volunteers. Six volunteers (85.7%), 2 of whom developed diarrhea, excreted sporocysts in feces. The prepatent period lasted 10–14 (12 ± 1.8) days and the patent period lasted 5–12 (8.8 ± 1.1) days.

Genetic diversity among capybara (Hydrochaeris hydrochaeris) isolates of Toxoplasma gondii from Brazil

Recent studies indicate that Toxoplasma gondii isolates of many domestic hosts from Brazil are genetically and biologically different from T. gondii isolates from USA and Europe. However, little is known about genetics of T. gondii isolates from wild mammals in Brazil. In this study, genotypes of 36 T. gondii isolates from capybaras (Hydrochaeris hydrochaeris) from six counties in São Paulo state, Brazil, were determined. Sixteen genotypes were identified using 11 genetic markers including SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, Apico and CS3. No classical clonal Type I and Type II isolates were found, confirming other findings that these lineages are rare in Brazil. Eight of these 36 isolates were grouped into the common clonal lineages in Brazil, previously designed as Types BrI, BrII and BrIII. Seven of the 16 genotypes were reported for the first time in this study. Three of the 36 isolates showed mixed infections. Analysis of mortality rates in infected mice indicated that Type BrI is highly virulent, Type BrII is intermediately virulent and Type BrIII is non-virulent, which is in agreement with previous report. The allele types at the CS3 locus are strongly linked to mouse-virulence of the parasite. These genotyping results support previous findings that the T. gondii population is highly diverse in Brazil.

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 Irmãos, Pernambuco State, Northeastern Brazil, for 1 year and 8 years, respectively. The wild black-eared opossum was captured in São 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 felid 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.

Genetic diversity among Toxoplasma gondii isolates of small ruminants from Brazil: Novel genotypes revealed

Recent studies indicated that Toxoplasma gondii isolates of many domestic animal hosts from Brazil are genetically and biologically different from those in USA and Europe. Despite of high pathogenicity of this parasite to small ruminants, the epidemiology and genetic diversity of T. gondii in these animals are not well understood in Brazil. In this study, a total of 28 T. gondii samples (16 isolates from sheep in São Paulo state, and 12 isolates from goats in the states of São Paulo and Rio Grande do Norte) were genotyped using genetic markers SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, Apico and CS3. Eleven genotypes were identified from these T. gondii isolates. Eight isolates (4 from sheep and 4 from goats) were grouped into the common clonal type BrI lineage. One sheep isolate was grouped to the type BrIII lineage. Five isolates grouped to three previously identified genotypes in Brazil, and 13 isolates grouped to six novel genotypes. Mixed genotype was found in one isolate from goat in São Paulo. No classical clonal Type I, II or III isolates were found, confirming previous reports that these clonal lineages are rare in Brazil. The allele types at the CS3 locus are strongly linked to mouse virulence of the parasite. The results of this study indicate that even though a large number of T. gondii genotypes have been identified from a variety of animal hosts in Brazil, high percentage of new genotypes are continuously identified from different animal species, suggesting extremely high diversity of T. gondii in the population.

Prevalence and risk factors for anti-Toxoplasma gondii antibodies in goats of the Seridó Oriental microregion, Rio Grande do Norte state, Northeast region of Brazil

The prevalence and risk factors for anti-Toxoplasma gondii antibodies were investigated in goats of the Seridó Oriental microregion, Rio Grande do Norte state, Northeast region of Brazil. Three hundred and sixty-six blood samples from goats collected by jugular venopuncture were used. For the serologic diagnosis of Toxoplasma gondii infection, the indirect fluorescent-antibody test (IFAT) with cut-off value 1:64 was carried out. The prevalence of anti-T. gondii antibodies was 30.6% [95% CI=25.9-35.6%] with titers ranging from 1:64 to 1:16,384. The multivariate logistic regression analysis showed that the risk factors associated to anti-T. gondii antibodies were presence of cats in the herd, extensive/semi-intensive management systems and lack of mineral supplementation.

Analysis of marine bivalve shellfish from the fish market in Santos city, Sao Paulo state, Brazil, for Toxoplasma gondii

The aim of this study was to determine if Toxoplasma gondii are present in oysters (Crassostrea rhizophorae) and mussels (Mytella guyanensis) under natural conditions using a bioassay in mice and molecular detection methods. We first compared two standard protocols for DNA extraction, phenol-chloroform (PC) and guanidine-thiocyanate (GT), for both molluscs. A total of 300 oysters and 300 mussels were then acquired from the fish market in Santos city, São Paulo state, Brazil, between March and August of 2008 and divided into 60 groups of 5 oysters and 20 groups of 15 mussels. To isolate the parasite, five mice were orally inoculated with sieved tissue homogenates from each group of oysters or mussels. For molecular detection of T. gondii, DNA from mussels was extracted using the PC method and DNA from oysters was extracted using the GT method. A nested-PCR (Polymerase Chain Reaction) based on the amplification of a 155 bp fragment from the B1 gene of T. gondii was then performed. Eleven PCR-RFLP (Restriction Fragment Length Polymorphism) markers, SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, CS3 and Apico, were used to genotype positive samples. There was no isolation of the parasite by bioassay in mice. T. gondii was not detected in any of the groups of mussels by nested-PCR. DNA of T. gondii was apparently detected by nested-PCR in 2 groups of oysters (3.3%). Genotyping of these two positive samples was not successful. The results suggest that oysters of the species C. rhizophorae, the most common species from the coast of São Paulo, can filter and retain T. gondii oocysts from the marine environment. Ingestion of raw oysters as a potential transmission source of T. gondii to humans and marine mammals should be further investigated.