Epidemiologic significance of Toxoplasma gondii infections in chickens (Gallus domesticus): the past decade.

Toxoplasma gondii infections are common in humans and animals worldwide. Domestic free-range chickens (Gallus domesticus) are excellent sentinels of environmental contamination with T. gondii oocysts because they feed on the ground. Chickens can be easily infected with T. gondii; however, clinical toxoplasmosis is rare in these hosts. Chickens are comparatively inexpensive and thus are good sentinel animals for T. gondii infections on the farms. Here, the authors reviewed prevalence, the persistence of infection, clinical disease, epidemiology and genetic diversity of T. gondii strains isolated from chickens worldwide for the past decade. Data on phenotypic and molecular characteristics of 794 viable T. gondii strains from chickens are discussed, including new data on T. gondii isolates from chickens in Brazil. This paper will be of interest to biologists, epidemiologists, veterinarians and parasitologists.

First isolation and genotyping of Toxoplasma gondii from free-range chickens on São Luis island, Maranhão state, Brazil, with a new genotype described.

Toxoplasma gondii is a protozoan with worldwide distribution that infects warm-blooded vertebrates, including humans. Toxoplasma is considered to be monospecific, but three classical clonal lineages (Types I-III) have been described, especially for isolates from North America and Europe. However, recently, several studies have shown that many isolates, particularly from South America, are different from the classic types and highly diverse, with the presence of specific clonal lineages and non-archetypal genotypes. In order to isolate and genotypically characterize T. gondii, 60 free-range naturally infected chickens (Gallus domesticus) from four municipalities on São Luis island, state of Maranhão, Brazil, were first serologically screened for IgG anti-T. gondii antibodies by means of the immunofluorescent antibody test (IFAT≥1:16). Heart and brain samples from seropositive chickens were bioassayed in mice. The isolates obtained from bioassaying were genotyped by means of PCR-RFLP (restriction fragment length polymorphism) using 11 markers and by microsatellites (MS) using 15 markers. The frequency of anti-T. gondii antibodies in the chickens examined was 25.0% (15/60). Five isolates of T. gondii were obtained and named TgCkBrMA1 to 5. Four genotypes were described. One of them is reported for the first time. No classic clonal lineages of types I, II or III were found, but the Brazilian clonal lineage BrI was identified. MS analysis revealed five genotypes. The results corroborate studies already developed in other regions of Brazil, thus indicating that T. gondii has high genetic diversity in Brazil.

Phenotypic and genotypic characterization of two Toxoplasma gondii isolates in free-range chickens from Uberlândia, Brazil.

The aim of this study was to determine the seroprevalence of Toxoplasma gondii infection in free-range chickens from Uberlândia, Minas Gerais state, Brazil, and characterize the genotypic and phenotypic features of two isolates of this parasite, considering the importance of these hosts in the epidemiology of toxoplasmosis. Serum samples from 108 free-range chickens were obtained from ten different districts, and submitted to the modified agglutination test (MAT) for the presence of anti-T. gondii antibodies, and brain and heart tissue samples from infected chickens were processed for mouse bioassay. An overall seroprevalence of 71·3% was found and antibody titres ranged from 16 to 4096. After confirmation of seropositivity by mouse bioassay, the determination of the T. gondii genotypes of two isolates was performed by PCR-RFLP, using primers for the following markers: SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, new SAG2, Apico and CS3. These T. gondii isolates, designated TgChBrUD1and TgChBrUD2, were obtained from heart samples of free-range chickens. The TgChBrUD1 isolate belonged to ToxoDB PCR-RFLP genotype 11 and the TgChBrUD2 isolate belonged to ToxoDB PCR-RFLP genotype 6. Both isolates demonstrated high virulence in a rodent model, with the TgChBrUD1 isolate able to induce brain cysts, in accord with its pattern of multiplication rates in human fibroblast culture. Taken together, these results reveal high prevalence of T. gondii infection in free-range chickens throughout Uberlândia, indicating an important degree of oocyst environmental contamination and the existence of considerable risk for T. gondii transmission to humans by consumption of free-range chicken as a food source.

Genetic characterization of Toxoplasma gondii from Brazilian wildlife revealed abundant new genotypes.

This study aimed to isolate and genotype T. gondii from Brazilian wildlife. For this purpose, 226 samples were submitted to mice bioassay and screened by PCR based on 18S rRNA sequences. A total of 15 T. gondii isolates were obtained, including samples from four armadillos (three Dasypus novemcinctus, one Euphractus sexcinctus), three collared anteaters (Tamandua tetradactyla), three whited-lipped peccaries (Tayassu pecari), one spotted paca (Cuniculus paca), one oncilla (Leopardus tigrinus), one hoary fox (Pseudalopex vetulus), one lineated woodpecker (Dryocopus lineatus) and one maned wolf (Chrysocyon brachyurus). DNA from the isolates, originated from mice bioassay, and from the tissues of the wild animal, designated as "primary samples", were genotyped by PCR-restriction fragment length polymorphism (PCR/RFLP), using 12 genetic markers (SAG1, SAG2, alt.SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L258, PK1, CS3 and Apico). A total of 17 genotypes were identified, with 13 identified for the first time and four already reported in published literature. Results herein obtained corroborate previous studies in Brazil, confirming high diversity and revealing unique genotypes in this region. Given most of genotypes here identified are different from previous studies in domestic animals, future studies on T. gondii from wildlife is of interest to understand population genetics and structure of this parasite.

Toxoplasma gondii and Neospora caninum in domestic cats from the Brazilian semi-arid: seroprevalence and risk factors / Toxoplasma gondii e Neospora canimum em gatos no semiárido nordestino: soroprevalência e fatores de risco

In the present study we aimed to establish the seroprevalence of T. gondii and N. caninum in stray and domiciled cats from the municipality of Patos, Paraíba state, Brazil. Blood samples were collected from 201 animals: 132 domiciled cats and 69 stray cats. An epidemiological questionnaire was conducted with all cat owners. Indirect immunofluorescent antibody test (IFAT) was performed at cut-offs of 1:16 and 1:50 for T. gondii and N. caninum, respectively. Overall prevalence of seroreagent cats for T. gondii was 43.8 percent. We found a prevalence of 47.7percent in domiciled cats and 36.2 percent in stray cats. Antibody titers ranged from 1:16 (cut-off) to 1:8192; 1:128 was the most frequent titer. No statistical difference was observed between domiciled cats and stray cats. Correlation was verified between seroreagent for T. gondii and age and hunting habit (P<0.05). No animals tested seroreagent for N. caninum. It was possible to conclude that there is high prevalence of cat seroreagent for T. gondii and that N. caninum is not present in cats from the area studied...

Este trabalho teve como objetivo determinar frequência de Toxoplasma gondii e Neospora caninum em gatos domiciliados e errantes do município de Patos, mesorregião do sertão paraibano. Foram coletadas amostras de sangue provenientes de 201 gatos, 132 domiciliados e 69 errantes. Nos domiciliados, foi aplicado um questionário epidemiológico aos proprietários. A reação de imunofluorescência indireta (RIFI) foi realizada levando-se em consideração os pontos de corte 1:16 e 1:50 para T. gondii e N. caninum, respectivamente. Foi obtida uma prevalência total de 43.8 por cento de gatos sororreagentes para T. gondii. Nos domiciliados, a prevalência foi de 47,7 por cento (63/132), e nos errantes de 36,2 por cento (25/69), com títulos variando de 1:16 a 1:8192, sendo a titulação 1:128 a mais frequente. Não se observou diferença estatística entre animais domiciliados e errantes. Verificou-se correlação entre sororreagentes a T. gondii e idade e hábito de caçar (P<0.05). Nenhum animal foi sororreagente para N. caninum. Concluiu-se que é alta a prevalência de gatos sororreagentes para T. gondii e que o N. caninum não está presente em gatos na área estudada...

Toxoplasma gondii in free-ranging wild small felids from Brazil: molecular detection and genotypic characterization.

Brazil harbors the largest number of wild Neotropical felid species, with ten of the twelve species recorded in the American continent. Although these animals are considered to be definitive hosts for Toxoplasma gondii, there are few descriptions of the parasite in these species. Here, we performed a molecular detection of T. gondii by amplification of the marker ITS-1 from tissue samples obtained from 90 free-ranging wild small Neotropical felids from Rio Grande do Sul - Brazil. Of the sampled animals, 34.4% (n=31) were positive including the species Puma yagouaroundi - jaguarondi (9/22), Leopardus geoffroyi - Geoffroy's cat (6/22), Leopardus tigrinus - oncilla (8/28), Leopardus wiedii - margay (6/10), Leopardus pardalis - ocelot (1/1) and Leopardus colocolo - Pampas cat (1/7). Toxoplasma DNA was detected with a frequency of 14.6% (63/433) in primary samples of tongue (16/56), brain (8/43), skeletal muscle (15/83), heart (7/63), diaphragm (3/56), vitreous humor (2/44), eye muscle (6/44) and eyeball (6/44). Multilocus PCR-RFLP genotyping of eleven small Neotropical felids using the molecular markers SAG1, 5'3'SAG2, alt. SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, Apico and CS3 allowed the partial characterization of eight genotypes. We fully characterized two new genotypes that have not been described previously in Brazil (Lw#31Tn from L. wiedii and Py#21Sm from P. yagouaroundi) and one genotype Py#56Br from P. yagouaroundi that has been described previously in isolates from cats, dogs and capybaras from São Paulo state. This study constitutes the first detection and genotypic characterization of T. gondii in free-ranging felids in Brazil, demonstrating the occurrence of the parasite in wild populations and suggesting its potential transmissibility to humans and other domestic and wild animals.

Seroprevalence of Toxoplasma gondii in wild marsupials and rodents from the Atlantic forest of Pernambuco state, northeastern region, Brazil.

Felids are important in the epidemiology of Toxoplasma gondii because they are the only hosts that can excrete the environmentally resistant oocysts in their feces. Cats acquire T. gondii infection in nature by ingesting tissues of small mammals and birds. Serum samples of 223 feral marsupials and 174 feral rodents captured in 7 segments of the Atlantic Forest of the State of Pernambuco, northeastern region of Brazil, and in urban areas of the municipality of Recife were examined for antibodies to T. gondii by the modified agglutination test (MAT). Antibodies (MAT ≥ 25) were found in 6.7% (15 of 223) of the marsupials and 5.7% (10 of 174) of the rodents. No association was observed between seropositivity in marsupials or rodents and sex, age, or different areas of collection (P > 0.05). This is the first study on the seroprevalence of T. gondii in marsupials and rodents performed in the Atlantic Forest of the northeastern region of Brazil. The presence of antibodies to T. gondii are reported for the first time in long-furred woolly mouse opossum ( Micoureus demerarae ), murine mouse opossum ( Marmosa murina ), brown four-eyed opossum ( Metachirus nudicaudatus ), and gray short-tailed opossum ( Monodelphis domestica ).

First isolation and genotyping of Toxoplasma gondii from bats (Mammalia: Chiroptera).

There are currently no reports on the isolation and molecular examination of Toxoplasma gondii from bats. Here, we report the isolation and genotypic characterisation of two T. gondii isolates from bats. A total of 369 bats from different municipalities in São Paulo state, southeastern Brazil, were captured and euthanised, and collected tissues (heart and pectoral muscle) were processed for each bat or in pools of two or three bats and bioassayed in mice (a total of 283 bioassays). Eleven PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) markers were used to genotype positive samples: SAG1, SAG2 (5'-3'SAG2 and alt. SAG2), SAG3, BTUB, GRA6, L358, c22-8, c29-2, PK1, CS3 and Apico. The parasite was isolated from two bats from São Paulo city: an insectivorous bat, the velvety free-tailed bat Molossus molossus, and a hematophagous bat, the common vampire bat Desmodus rotundus. Isolates were designated TgBatBr1 and TgBatBr2, respectively. The genotype of the isolate from M. molossus (TgBatBr1) has been previously described in an isolate from a capybara from São Paulo state, and the genotype from the D. rotundus isolate (TgBatBr2) has already been identified in isolates from cats, chickens, capybaras, sheep, a rodent and a common rabbit from different Brazilian states, suggesting that this may be a common T. gondii lineage circulating in some Brazilian regions. Isolation of T. gondii from a hematophagous species is striking. This study reveals that bats can share the same isolates that are found in domesticated and wild terrestrial animals. This is the first report of the isolation and genotyping of T. gondii in chiropterans.

PCR-RFLP genotyping of Toxoplasma gondii from chickens from Espírito Santo state, Southeast region, Brazil: new genotypes and a new SAG3 marker allele.

Brazil is one of the regions with the highest prevalences of Toxoplasma gondii in humans and animals. Because free-range chickens become infected by feeding from ground contaminated with oocysts, the prevalence of T. gondii in this host has been widely used as an indicator of the strains prevalent in the environment. The genetic variability among T. gondii isolates from different healthy and sick hosts all over the world has been recently studied. Three clonal genetic lineages (Types I, II and III) were initially recognised as predominant in Western Europe and the United States. T. gondii strains are genetically diverse in South America. In Brazil, recombination plays an important role in strain diversification. The objective of this study was to genetically characterise T. gondii isolates from free-range chickens from Espírito Santo state, Southeast region, Brazil, using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). A total of 44 isolates among 47 previously described isolates (TgCkBr234-281) from free-range chickens were included in this study. Strain typing was performed using 12 PCR-RFLP markers: SAG1, SAG2, alt. SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, Apico and CS3. Eleven genotypes were identified. Ten isolates (23%) were grouped into four novel genotypes. Four isolates, distributed in four counties, corresponded to the Type BrI lineage, the genotype found most frequently in Brazil. No clonal Types I, II or III lineages were found. Two novel genotypes were represented by single isolates. Unique alleles were identified for the markers SAG1, c22-8 and CS3, and for the first time, a unique allele was found for the marker SAG3. Although a large number of T. gondii genotypes have already been identified from a variety of animal hosts in Brazil, new genotypes are continuously identified from different animal species. This study confirmed the diversity of T. gondii in Brazil and demonstrates clonal Type I, II and III lineages are rare in this country.

Seroprevalence and isolation of Toxoplasma gondii from free-range chickens from Espírito Santo state, southeastern Brazil.

Prevalence of Toxoplasma gondii infection in 510 free-range (FR) chickens (380 from 33 small farms, and 130 from a slaughter house for FR chickens) from Espírito Santo state, southeastern Brazil, was investigated. Antibodies to T. gondii were sought using commercial indirect haemagglutination (IHAT, Imuno-HAI Toxo(®), Wama Diagnóstica, São Paulo, Brazil, cut-off 1:16) and the modified agglutination test (MAT, cut-off 1:25) tests. Attempts were made to isolate viable T. gondii from seropositive chickens by bioassay in mice. Pooled samples of brain, heart and quadriceps muscle of one thigh (total 40 g) from 64 chickens with IHAT titers of ≥ 1:16 were minced, digested in pepsin and bioassayed in mice. Antibodies to T. gondii were found in 40.4% (206/510) FR chickens by IHAT (titer ≥ 1:16) and 38.8% (198/510) by MAT (titer ≥ 1:25); concordance between IHAT and MAT was 81.6% (kappa index=0.614). Viable T. gondii was isolated (designated TgCkBr234-281) from 48 of 64 (75%) seropositive (IHAT titers ≥ 1:32) FR chickens. Most isolates of T. gondii were virulent for mice; 100% of mice inoculated with 44 of 48 isolates died of toxoplasmosis within 30 days post inoculation (p.i). An epidemiological investigation revealed that people living in rural areas have little knowledge about the parasite and about the risk of acquiring it from raw meat. Results indicated that the locally available IHAT was useful for screening of chicken sera for T. gondii antibodies.

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 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.

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 São Paulo state, Brazil were genetically characterised using 10 PCR restriction fragment length polymorphism markers including SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 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.

Freqüência de parasitos gastrintestinais em cães e gatos atendidos em hospital-escola veterinário da cidade de São Paulo / Frequency of gastrointestinal parasites in dogs and cats referred to a veterinary school hospital in the city of São Paulo

Fecal samples from 1755 dogs and 327 cats were examined for the presence of helminths and protozoan forms. From the total samples, 486 (27.7 percent) dogs and 103 (31.5 percent) cats presented at least one parasite. The main genus of parasite in dogs were Ancylostoma (12.7 percent), Giardia (8.5 percent), Cystoisosopora (4.4 percent), Toxocara (2.6 percent), and Cryptosporidium (2.4 percent). The ocurrence of Ancylostoma was associated to male dogs, older than one year, while Giardia, Cryptosporidium, Cystoisospora and Toxocara were associated to dogs younger than one year (P<0.05). Among cats, the most frequent parasites were Cryptosporidium (11.3 percent), Giardia (8.3 percent), Cystoisosopora (8.3 percent), Toxocara (6.1 percent), and Ancylostoma (2.1 percent). Cryptosporidium and Cystoisosopora were more prevalent in cats younger than one year (P < 0.05)

Molecular phylogenetic analysis in Hammondia-like organisms based on partial Hsp70 coding sequences.

The 70 kDa heat-shock protein (Hsp70) sequences are considered one of the most conserved proteins in all domains of life from Archaea to eukaryotes. Hammondia heydorni, H. hammondi, Toxoplasma gondii, Neospora hughesi and N. caninum (Hammondia-like organisms) are closely related tissue cyst-forming coccidians that belong to the subfamily Toxoplasmatinae. The phylogenetic reconstruction using cytoplasmic Hsp70 coding genes of Hammondia-like organisms revealed the genetic sequences of T. gondii, Neospora spp. and H. heydorni to possess similar levels of evolutionary distance. In addition, at least 2 distinct genetic groups could be recognized among the H. heydorni isolates. Such results are in agreement with those obtained with internal transcribed spacer-1 rDNA (ITS-1) sequences. In order to compare the nucleotide diversity among different taxonomic levels within Apicomplexa, Hsp70 coding sequences of the following apicomplexan organisms were included in this study: Cryptosporidium, Theileria, Babesia, Plasmodium and Cyclospora. Such analysis revealed the Hammondia-like organism to be the lowest divergent group when compared to other groups within the phylum Apicomplexa. In conclusion, the Hsp70 coding sequences proved to be a valuable genetic marker for phylogenetic reconstruction and may constitute a good candidate to be used with other genes for species phylogeny within this group of organisms.

Isolation and molecular detection of Neospora caninum from naturally infected sheep from Brazil.

Neospora caninum was isolated from a naturally infected sheep from Brazil by bioassay in dogs. Approximately 70g of brain from each of two 4-month-old sheep with indirect fluorescent antibodies (>or=1:50) to N. caninum was offered to a different IFAT negative dog (Sheep n. 302, IFAT 1:400-Dog 1 and Sheep n. 342, IFAT 1:50-Dog 2). Parasite DNA was detected in both sheep brains using a PCR targeting the Nc-5 gene of N. caninum. Shedding of Neospora-like oocysts was noticed only in Dog 1, from 10 days post-inoculation (PI) to 25 days PI (a total of approximately 27,600 oocysts). Seventy days after infection, Dog 1 was euthanized and brain/cerebellum and medulla were collected and submitted to molecular methods, as were the oocysts, to confirm the identity of the isolate. Serum samples collected weekly from both dogs from the infection to the end of the experimental period had no antibodies anti-N. caninum by IFAT (<1:50). Oocysts, brain/cerebellum and medulla specimens of Dog 1 proved positive by a PCR assay targeting the Nc-5 gene of N. caninum. In addition, the oocysts have the DNA amplified by a PCR based on primers directed to the common toxoplasmatiid ITS1 sequence. The PCR products of ITS1 were sequenced, confirming again the isolate as N. caninum. Oocysts were also orally inoculated in two Swiss white mice two Mongolian gerbils (Meriones ungulatus) and two large vesper mice (Calomys callosus) (10(3)oocysts/animal). The rodents were sacrificed 2 months PI, and fresh preparations of brains showed Neospora thick-walled cysts in gerbil brains, but molecular detection using the Nc-5 PCR assay revealed DNA parasite in gerbil and also C. callosus brains. This is the first report of isolation and sequencing of N. caninum from a Brazilian sheep and the first report of molecular detection of N. caninum from C. callosus.

Toxoplasma gondii infection in cats from São Paulo state, Brazil: seroprevalence, oocyst shedding, isolation in mice, and biologic and molecular characterization.

Cats are the most important hosts in the epidemiology of Toxoplasma gondii infections in humans and animals. Serologic and parasitological prevalence of T. gondii were determined in 237 cats from 15 counties in São Paulo state, Brazil. Antibodies to T. gondii were found in a 1:25 dilution of serum of 84 (35.4%) out of 237 cats by the modified agglutination test (MAT). Samples of brain, heart, tongue, and limb muscles (total 50 g) of 71 of the seropositive cats were pooled for each cat, digested in pepsin and bioassayed in mice. Faeces (1 g) from the rectum of each cat were examined microscopically for T. gondii-like oocysts and verified by bioassay in mice; T. gondii oocysts were found in the faeces of three (1.3%) of 237 cats. T. gondii was isolated from tissue homogenates of 47 cats. The DNA obtained from these 47 tissue isolates was characterized using the SAG2 locus: 34 (72.4%) isolates were type I, 12 (25.5%) were type III and one (2.1%) was mixed with types I and III. No type II isolates were detected. Most (23/34) of the type I isolates killed all infected mice and 7 of 12 type III isolates did not kill infected mice. Characterization of the SAG2 locus directly from tissue homogenates from 37 of 46 cats was successful. Genotypes obtained from these primary samples were the same as those from the corresponding isolates obtained in mice. Genotyping of the three oocyst isolates revealed that two were type I and one was type III. Molecular and biologic characteristics of T. gondii isolates from animals from Brazil are different from those from other parts of the world.

Ocorrência de protozoários e helmintos em amostras de fezes de cäes e gatos da cidade de Säo Paulo / Occurrence of protozoa and helminths in faecal samples of dogs and cats from Säo Paulo city

Foram examinadas, pelo Laboratório de Doenças Parasitárias do Departamento de Medicina Veterinária Preventiva e Saúde Animal, da Faculdade de Medicina Veterinária e Zootecnia da Universidade de Säo Paulo, no período de janeiro de 1991 a janeiro de 1995, amostras de 353 cäes e de 187 gatos, com idades variadas e de diferentes áreas da cidade de Säo Paulo. Os métodos de diagnóstico empregados foram: centrífugo-flutuaçäo em soluçäo de sacarose e centrífugo-sedimentaçäo em água-éter. Aproximadamente 45,0 por cento das amostras de cäes examinadas (160 amostras) apresentaram-se positivas e o percentual de ocorrência de parasitos, em relaçäo ao total de amostras foi: Ancylostoma spp. 20,40; Toxocara canis 8,49; Giardia sp. 7,65; Cryptosporidium parvum 2,83; Cystoisospora spp. 2,55; Sarcocystis spp. 1,70; Hammondia heydorni 0,85; e Spirocerca lupi, Trichuris vulpis e Dipylidium caninum 0,28. Das 187 amostras recebidas, provenientes de gatos, somente oito eram negativas e observou-se a ocorrência (por cento) de Cystoisospora spp. 38,5; Toxocara sp. 34,22; Giardia sp. 16,04; Cryptosporidium parvum 14,44; Ancylostoma spp. 13,37; Dipylidium caninum 10,69; Sarcocystis spp. 8,56; Physaloptera spp. 4,81; oocistos tipo "Hammondia-Toxoplasma" 1,60; Strongyloides stercoralis 1,60; e Platynosomum concinum 1,07. Em cäes, as associaçöes mais freqüentemente observadas foram: Ancylostoma spp. + T. canis em oito amostras e Ancylostoma spp. + Giardia sp. e Ancylostoma spp. + T. vulpis em cinco amostras. Em gatos, Toxocara sp. e Cystoisospora spp., encontrados em 24 amostras, e Toxocara sp. + Ancylostoma spp. em cinco amostras, foram as associaçöes mais comumente observadas