Isolation and characterization of Toxoplasma gondii strains from stray cats revealed a single genotype in Beijing, China

Cats are essential in the epidemiology of Toxoplasma gondii because they are the only hosts that can excrete the environmentally resistant oocysts in nature. This study was aimed to determine the seropositivity, distribution of genotypes and mouse virulence of T. gondii from stray cats in Beijing, China. A total of 64 serum samples, 23 feces and tissue samples were collected from stray cats in Beijing. Antibodies to T. gondii were assayed by the modified agglutination test (MAT). 57.8% (37/64) of these stray cats had titers of 1:20 or higher and were considered positive with infection. T. gondii oocysts were not found in feces of the 23 cats. Tissues of 23 cats were bioassayed in mice and 11 T. gondii isolates were obtained. The genotype of these isolates were identified by 11 PCR-RFLP markers, including SAG1, (3'+5')SAG2, alt.SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and an apicoplast marker, Apico. Only one genotype was identified. This genotype, designated as ToxoDB genotype #9, was previously reported in cats, pigs and human from Guangdong and Gansu provinces in China and animals from a few other countries. To determine mouse virulence of this lineage of parasites, one isolate was randomly selected and inoculated into BABL/c mice, the result showed that it is intermediately virulent to mice. These results indicated that an atypical, intermediately virulent T. gondii lineage is widespread in China. The high seropositivity of T. gondii in stray cats posts potential risk of transmission of the parasite to human population in the region.     

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 Irm?os, Pernambuco State, Northeastern Brazil, for 1 year and 8 years, respectively. The wild black-eared opossum was captured in S?o 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 characterization of Toxoplasma gondii isolates from pigs intended for human consumption in Brazil

This study genetically Toxoplasma gondii isolates obtained from pigs intended for human consumption in northeastern Brazil; multilocus PCR-RFLP and sequencing techniques were utilized. Bioassays were conducted using the brain and tongue of 20 pig heads purchased at butcher shops in the city of Ilheus, Bahia, Brazil. Overall, 11 T. gondii isolates designated TgPgBr06-16 were identified. Application of multilocus PCR-RFLP with seven molecular markers (SAG1, SAG2, SAG3, BTUB, C22-8, PK1 and Apico) identified six different genotypes. Isolates TgPgBr 06, 08, 11, 12, 14 and 15 were indistinguishable by this technique, forming a single genotype; the remaining isolates were characterized as distinct genotypes. However, when five genetic markers (SAG1, SAG2, SAG3, BTUB and c22-8) were employed in multilocus PCR-sequencing, all eleven strains of T. gondii were shown to be different. All isolates differed from Type I, II and III clonal genotypes using both genotyping techniques. These results demonstrate that the multilocus PCR-RFLP assay underestimated the true diversity of the T. gondii population in this study. Thus, DNA sequencing is the preferred technique to infer the genetic diversity and population structure of T. gondii strains from Brazil. Moreover, it is necessary to develop new molecular markers to group and characterize atypical T. gondii isolates from South America.     

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, Maranh?o, Paraná, Pernambuco, Rio de Janeiro, Rio Grande do Norte, S?o 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.     

Biologic and genetic comparison of Toxoplasma gondii isolates in free-range chickens from the northern Pará state and the southern state Rio Grande do Sul, Brazil revealed highly diverse and distinct parasite populations

The prevalence of Toxoplasma gondii in 84 free-range chickens (34 from the northern Pará state, and 50 from Rio Grande do Sul, the southern state) from Brazil, South America was determined. Antibodies to T. gondii were assayed by the modified agglutination test (MAT), and found in 39 (46.4%) of 84 chickens with titers of 1:10 in one, 1:20 in two, 1:40 in four, 1:80 in seven, 1:160 in five, 1:320 in six, 1:640 in eight and > or =1:1280 in six. Hearts and brains of 45 chickens with titers of 1:20 or less were pooled and fed to two T. gondii-free cats. Hearts and brains of 39 chickens with titers of 1:10 or higher were bioassayed in mice. Feces of cats were examined for oocysts. One cat fed tissues from 31 chickens with titers of less than 1:10 from Rio Grande do Sul shed T. gondii oocysts. T. gondii was isolated by bioassay in mice from 33 chickens with MAT titers of 1:20 or higher. All infected mice from 10 isolates died of toxoplasmosis. All 34 isolates (15 from Pará, 19 from Rio Grande do Sul) were genotyped using 11 genetic markers including SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, a new SAG2 and Apico. Eleven genotypes were revealed for Pará isolates and seven genotypes for Rio Grande do Sul. No genotype was shared between the two geographical locations. These data suggest that T. gondii isolates are highly diverse and genetically distinct between the two different regions in Brazil that are 3500 km apart.     

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 S?o 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.     

Biologic and genetic characteristics of Toxoplasma gondii isolates in free-range chickens from Costa Rica, Central America

The prevalence of Toxoplasma gondii in free-ranging chickens is a good indicator of the prevalence of T. gondii oocysts in the soil because chickens feed from the ground. The prevalence of T. gondii in 144 free-range chickens (Gallus domesticus) from Costa Rica was determined. Antibodies to T. gondii were assayed by the modified agglutination test (MAT), and found in 60 (40.1%) of 144 chickens with titers of 1:5 in 16, 1:10 in 5, 1:20 in 2, 1:40 in 3, 1:80 in 5, and 1:160 or higher in 29. Tissues of all chickens were bioassayed for T. gondii in mice or cats. Hearts and brains of 52 chickens with titers of 1:5 or higher and 16 chickens with doubtful titers were pooled and bioassayed in mice. Tissues from 76 chickens with MAT titers of 1:10 or less were pooled and fed to three T. gondii-free cats. Fecal floats of cats were bioassayed orally in mice but were negative for T. gondii oocysts. T. gondii was isolated by bioassay in mice from 32 chickens with MAT titers of 1:10 or higher. All infected mice from 4 of the 32 isolates died of toxoplasmosis. Genotyping of these 32 isolates using polymorphisms at the loci SAG1, SAG2, SAG3, BTUB and GRA6 revealed five genotypes. Five isolates had type I alleles and one isolate had type III alleles at all loci. The rest 26 isolates contained the combination of type I and II or I and III alleles and were divided into three genotypes. None was found to have genotype II alleles at all five loci. This is the first report of genetic characterization of T. gondii isolates from Costa Rica, Central America.     

Genotyping studies of Toxoplasma gondii isolates from Africa revealed that the archetypal clonal lineages predominate as in North America and Europe

Until recently, Toxoplasma gondii was considered to be 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. However, little is known of the genetics of T. gondii strains from Africa. In this study, we genotyped 19 T. gondii isolates from chickens from six African countries (Egypt, Kenya, Nigeria, Congo, Mali, and Burkina Fasco) using 10 PCR-RFLP markers (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico). The results revealed four genotypes. Thirteen isolates belong to the Type III lineage, five isolates have Type II alleles at all loci except apico and they belong to the Type II lineage. One isolate from Nigeria had atypical genotype. In general, these isolates were mostly clonal Type III and II strains that predominate in North American and European. DNA sequencing at several loci for representative isolates confirmed the results of PCR-RFLP genotyping. Taken together with recent studies of T. gondii isolates from Africa, it is clear that the three clonal lineages (Types I, II and III) predominate not only in North America and Europe, but also in Africa.     

Prevalence of Toxoplasma gondii in dogs from Sri Lanka and genetic characterization of the parasite isolates

The prevalence of Toxoplasma gondii in 86 street dogs from Sri Lanka was determined. Antibodies to T. gondii were assayed by the modified agglutination test (MAT) and found in 58 (67.4%) of 86 dogs with titers of 1:20 in eight, 1:40 in four, 1:80 in 10, 1:160 in 22, 1:320 in six, 1:640 in five, and 1:1280 or higher in three. Hearts, tongues, and brains (either separately or pooled) of 50 dogs with MAT titers of 1:40 were selected for isolation of T. gondii by bioassays in mice. For bioassays, canine tissues were digested in pepsin and homogenates were inoculated subcutaneously into mice; the mice receiving canine tissues were examined for T. gondii infection. In all, T. gondii was isolated from 23 dogs. Interestingly, dog organs varied in their capacity to induce T. gondii infection in mice, muscles producing more positive results than the brain. The T. gondii isolates obtained from 23 seropositive dogs were PCR-RFLP genotyped using polymorphisms at 10 nuclear markers including SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, a new SAG2, and an apicoplast marker Apico. Mixed infection with two genotypes was observed in one dog. Four genotypes were revealed, including three unique genotypes in addition to one belonging to the predominant Type III lineage. The 24 isolates were designated as TgDgSl 1-24.     

Characterization of Toxoplasma gondii isolates from free range chickens from Paraná, Brazil

The prevalence of Toxoplasma gondii in free range chickens is a good indicator of the prevalence of T. gondii oocysts in the environment because chickens feed from the ground. In the present study, prevalence of T. gondii in 40 free range chickens (Gallus domesticus) from a rural area surrounding Paraná, Brazil was assessed. Blood, heart, and brain from each chicken were examined for T. gondii infection. Antibodies to T. gondii, assayed with the modified agglutination test (MAT> or =1:5) were found in 16 chickens. Hearts and brains of seropositive (MAT> or =1:5) chickens were bioassayed in mice. Additionally, hearts and brains of seronegative (MAT<1:5) chickens were bioassayed in two T. gondii-free cats (12 chickens per cat). T. gondii was isolated from 13 of 16 (81%) seropositive chickens. Of the two cats fed tissues pooled form seronegative chickens, one shed T. gondii oocysts. Nine of the 13 T. gondii isolates killed 100% of infected mice. The T. gondii isolate from the cat was also virulent for mice. Genotyping of 13 chicken isolates of T. gondii using the SAG2 locus indicated that seven isolates were type I and six were type III; three of these type III isolates killed all infected mice suggesting that all strains virulent for mice are not type I. The isolate from the feces of the cat fed chicken tissues was type I.     

First isolation and molecular characterization of Toxoplasma gondii from finishing pigs from São Paulo State, Brazil

Toxoplasma gondii infection is widely prevalent in humans in Brazil. Among the food animals, pigs are considered the most important meat source of T. gondii for infection in humans. In the present study, we report the first isolation of viable T. gondii from finishing pigs in Brazil. Antibodies to T. gondii were found in 49 (17%) of 286 pigs prior slaughter using the modified agglutination test (MAT) at a serum dilution of 1:25. Attempts were made to isolate T. gondii from 28 seropositive pigs. Samples of heart, brain, and tongue from each pig were pooled, digested in acid pepsin, and bioassayed in five mice per pig. Viable T. gondii was isolated from seven pigs; all isolates were lethal for mice. Restriction fragment length polymorphism on products of SAG2 locus amplified by PCR revealed that two isolates were Type I and five were Type III. The results indicate that phenotypically and genetically T. gondii isolates from pigs from Brazil are distinct from isolates of T. gondii from pigs in the USA.     

Prevalence of Toxoplasma gondii in dogs from Colombia, South America and genetic characterization of T. gondii isolates

The prevalence of Toxoplasma gondii in 309 unwanted dogs from Bogotá, Colombia, South America was determined. Antibodies to T. gondii were assayed by the modified agglutination test (MAT) and found in 52 (16.8%) of 309 dogs with titers of 1:20 in 20, 1:40 in six, 1:80 in 17, 1:160 in three, 1:320 in three, 1:1280 or higher in three. Some organs obtained after necropsy of dogs (hearts, tongues and brains, either separately or pooled) were used in bioassays carried out in mice (37 samples, of which 20 were assayed with separate organs and 17 were assayed with pooled organs), cats (pooled organs from six) and pooled organs of two dogs both in mice and cat. Mice receiving dog tissues were examined for T. gondii infection. Feces of cats that received dog tissues were examined for oocyst shedding. In total, T. gondii was isolated from tissues of 20 dogs (16 by bioassays in mice, 3 by bioassay in cats and 1 by bioassay in mice and cat). All infected mice from 7 of 17 isolates bioassayed in this host died of toxoplasmosis during primary infection. Only 10 of the 20 dogs whose tissues were bioassayed separately induced infections in mice. Interestingly, dog organs varied in their capacity to induce T. gondii infection in mice, hearts and tongues producing more positive results than the brain. The 20 T. gondii isolates obtained from seropositive dogs were PCR-RFLP genotyped using polymorphisms at 10 nuclear markers including SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, a new SAG2 and an apicoplast marker Apico. Ten genotypes were revealed. These genotypes are different from the three predominant Types I, II and III lineages that are widely spread in North America and Europe. A new allele denoted u-3 at PK1 locus was identified in three isolates. This result supports previous findings that T. gondii population is highly diverse in Colombia.     

Genetic and biologic characteristics of Toxoplasma gondii infections in free-range chickens from Austria

The prevalence of Toxoplasma gondii in free-ranging chickens is a good indicator of the prevalence of T. gondii oocysts in the soil because chickens feed from the ground. The prevalence of T. gondii in free-range chickens (Gallus domesticus) from 11 Bio-farms in Austria was determined. Antibodies to T. gondii assayed by the modified agglutination test (MAT) were found in 302 of 830 (36.3%) chickens with titers of 1:10 in 50, 1:20 in 69, 1:40 in 53, 1:80 in 40, 1:160 or higher in 90. Hearts of 218 chickens with MAT titers of 10 or higher were bioassayed individually in mice. Tissues from 1183 chickens were pooled and fed to 15, T. gondii-free cats. Feces of the cats were examined for oocysts; 11 cats shed T. gondii oocysts. T. gondii was isolated from 56 chickens by bioassay in mice. Thus, there were 67 isolates of T. gondii from these chickens. Genotyping of these 67 isolates using the SAG2 locus indicated that all 33 were Type II. Phenotypically and genetically these isolates were different from T. gondii isolates from Brazil. None of the isolates was virulent for mice. This is the first report of isolation of T. gondii from chickens from Austria.     

Occurrence of Toxoplasma gondii and Hammondia hammondi oocysts in the faeces of cats from Germany and other European countries

Faecal samples of 24,106 cats from Germany and other European countries were examined microscopically in a veterinary laboratory in Germany between October 2004 and November 2006 to estimate the prevalence of animals shedding Toxoplasma gondii or Hammondia hammondi oocysts. Oocysts of 9-15 microm size with a morphology similar to that of H. hammondi and T. gondii were found in 74 samples (0.31%). A total of 54 samples were further characterised to achieve a species diagnosis and to determine the genotype of T. gondii isolates by PCR and PCR-RFLP. From these samples, 48 isolates were obtained: 26 (0.11%) were finally identified as T. gondii and 22 (0.09%) as H. hammondi. T. gondii-positive samples came from Germany, Austria, France and Switzerland while H. hammondi was detected in samples from Germany, Austria and Italy. In two samples (one T. gondii and one H. hammondi), PCR indicated the presence of Hammondia heydorni DNA. No Neospora caninum DNA was detected in any of the feline faecal samples. Twenty-two of the 26 T. gondii isolates could be genotyped. A PCR-RFLP analysis for the SAG2, SAG3, GRA6 and BTUB genes revealed T. gondii genotype II in all cases. Morphologically, H. hammondi oocysts exhibited a statistically significantly smaller Length-Width-Ratio than T. gondii oocysts.     

Genetic and biologic characterization of Toxoplasma gondii isolates of cats from China

Cats are important in the epidemiology of Toxoplasma gondii infection because they are the only hosts that can excrete the environmentally resistant oocysts. In the present study, prevalence of T. gondii was determined in serum, feces, and tissues of 34 cats from People's Republic of China. Antibodies to T. gondii were assayed by the modified agglutination test and found in 27 of 34 (79.4%) cats with titers of 1:40 in one, 1:80 in one, 1:160 in three, 1:320 in three, 1:640 in eight, and 1:1280 or higher in 11 cats. T. gondii oocysts were not found in feces of any cat as ascertained by bioassay in mice. Tissues (brain, heart, and tongue) of 27 seropositive cats were pooled and bioassayed in mice (8 cats) or cats (19 cats). T. gondii was isolated from tissues of 17 of 27 seropositive cats. Genotyping of these 17 T. gondii isolates using polymorphisms at 10 nuclear markers including SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and a new SAG2, and an apicoplast marker Apico revealed two genotypes. This is the first report of genetic typing of T. gondii isolates from cats from China.     

Prevalence of Toxoplasma gondii in cats from Colombia, South America and genetic characterization of T. gondii isolates

Cats are important in the epidemiology of Toxoplasma gondii infection because they are the only hosts that can excrete the environmentally-resistant oocysts. In the present study, prevalence of T. gondii was determined in serum, feces, and tissues of 170 unwanted cats from Colombia, South America. Antibodies to T. gondii were assayed by the modified agglutination test and found in 77 of 170 (45.2%) cats with titers of <1:5 in 93, 1:5 in eight, 1:10 in 17, 1:20 in 10, 1:40 in seven, 1:80 in four, 1:160 in eight, 1:320 in six, and 1:640 or higher in 17 cats. T. gondii oocysts were not found in feces of any cat as ascertained by bioassay in mice. Tissues (brain, heart, tongue) of 116 cats were bioassayed in mice or cats. T. gondii was isolated from tissues of 15 of the 42 cats with titers of 1:40 or higher and not from any of the 90 cats titers of 1:20 or lower. Of the 29 cats whose tissues were bioassayed individually, T. gondii was isolated from the tongues of nine, hearts of eight, and brains of five. Mice inoculated with tissues of 12 of 15 infected cats died of toxoplasmosis; with nine T. gondii isolates all infected mice died. Overall, 65 of 92 (70%) of T. gondii-infected mice died of toxoplasmosis. Genotyping of these 15 isolates using polymorphisms at the SAG1, SAG2, SAG3, BTUB, and GRA6 loci revealed that three isolates (TgCtCo1, 2, and 7) had Type I alleles and one isolate (TgCtCo8) had Type II allele at all five loci. Eleven isolates contained the combination of Type I and III alleles and were divided into three genotypes, with TgCtCo3,5,6,9,12,13 and 15 had alleles I, I, III, I and III, TgCtCo4,10,11 had alleles I, III, III, I and I, and TgCtCo14 had alleles I, III, III, III, and III, at loci SAG1, SAG2, SAG3, BTUB and GRA6, respectively. All infected mice from each group had identical genotype except one mouse infected with TgCtCo5 had a Type III allele at locus BTUB and a unique allele (u-1) at locus SAG1 indicating mixed infection for TgCtCo5, whereas the rest seven mice had a Type I alleles at both loci.     

Biologic and genetic characteristics of Toxoplasma gondii isolates in free-range chickens from Nicaragua, Central America

The prevalence of Toxoplasma gondii in free-ranging chickens is a good indicator of the prevalence of T. gondii oocysts in the soil because chickens feed from the ground. The prevalence of T. gondii in 98 free-range chickens (Gallus domesticus) from Nicragua was determined. Antibodies to T. gondii were assayed by the modified agglutination test (MAT), and found in 84 (85.7%) of 98 chickens with titers of 1:5 in 10, 1:10 in eight, 1:20 in seven, 1:40 in nine, 1:80 in 11, 1:160 in one, 1:200 in 27, 1:400 in six, 1:800 four, and 1:3200 in one bird. Hearts and brains of 32 chickens with titers of 1:10 or less were pooled and fed to three T. gondii-free cats. Hearts and brains of 66 chickens with titers of 1:20 or higher were bioassayed in mice. Feces of cats were examined for oocysts. The cat fed tissues from eight chickens with titers of 1:10 shed T. gondii oocysts. The two cats fed tissues of 24 chickens with titers of 1:5 or less did not shed oocysts. T. gondii was isolated by bioassay in mice from 47 chickens with MAT titers of 1:20 or higher. All infected mice from six isolates died of toxoplasmosis. Overall, 41 of 170 (24.1%) mice that became infected after inoculation with chicken tissues died of toxoplasmosis. Genotyping of these 48 isolates (47 from mice and 1 from pooled tissues) using polymorphisms at the loci SAG1, SAG2, SAG3, BTUB and GRA6 revealed eight genotypes. Six isolates had Type I alleles, three isolate had Type II alleles and six isolates had Type III alleles at all loci. Four isolates had mixed infections. Two isolates have a unique allele at SAG1 locus and combination of I and III alleles at other loci. The rest 27 isolates contained the combination of Type I and III alleles and were divided into four genotypes. More than one genotypes were often isolated in chickens from the same household, indicating multiple genotypes were circulating in the same environment. This may explain the high frequency of mixed infections observed. High rate of mixed infection in intermediate hosts such as chickens may facilitate genetic exchange between different parasite lineages in definitive feline hosts. This is the first report of genetic characterization of T. gondii isolates from Nicragua, Central America.     

Genetic and biologic characteristics of Toxoplasma gondii isolates in free-range chickens from Colombia, South America

The prevalence of Toxoplasma gondii in free-ranging chickens is a good indicator of the prevalence of T. gondii oocysts in the soil because chickens feed from the ground. The prevalence of T. gondii in 77 free-range chickens (Gallus domesticus) from Colombia, South America was determined. Antibodies to T. gondii were assayed by the modified agglutination test (MAT), and found in 32 (44.4%) of 72 chickens with titers of 1:5 in 4, 1:10 in 3, 1:20 in 1, 1:40 in 1, 1:80 in 8, 1:160 in 8, 1:320 in 3, and 1:640 or higher in 4. Hearts and brains of 31 seropositive chickens were pooled and bioassayed in mice. Tissues from 32 (16+16) seronegative chickens were pooled and fed to two, T. gondii-free cats, and tissues from nine chickens without matching sera were fed to one T. gondii-free cat. Feces of cats were examined for oocysts. T. gondii oocysts were excreted by a cat that was fed tissues of 16 seronegative chickens. T. gondii was isolated by bioassay in mice from 23 chickens with MAT titers of 1:20 or higher. All infected mice from 16 of the 23 isolates died of toxoplasmosis. Overall, 82 (81.1%) of 101 mice that became infected after inoculation with chicken tissues died of toxoplasmosis. Genotyping of these 24 isolates using polymorphisms at the SAG2 locus indicated that seven T. gondii isolates were Type I, 17 were Type III, and none was Type II. Phenotypically, T. gondii isolates from chickens from Colombia were similar to isolates from Brazil but different from the isolates from North America; most isolates from chickens from Brazil and Colombia were lethal for mice whereas isolates from North America did not kill inoculated mice. Genetically, none of the T. gondii isolates from Colombia and Brazil was SAG2 Type II, whereas most isolates from chickens from North America were Type II. This is the first report of genetic characterization of T. gondii isolates from Colombia, South America.     

Genetic diversity among sea otter isolates of Toxoplasma gondii

Sea otters (Enhydra lutris) have been reported to become infected with Toxoplasma gondii and at times succumb to clinical disease. Here, we determined genotypes of 39 T. gondii isolates from 37 sea otters in two geographically distant locations (25 from California and 12 from Washington). Six genotypes were identified using 10 PCR-RFLP genetic markers including SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico, and by DNA sequencing of loci SAG1 and GRA6 in 13 isolates. Of these 39 isolates, 13 (33%) were clonal Type II which can be further divided into two groups at the locus Apico. Two of the 39 isolates had Type II alleles at all loci except a Type I allele at locus L358. One isolate had Type II alleles at all loci except the Type I alleles at loci L358 and Apico. One isolate had Type III alleles at all loci except Type II alleles at SAG2 and Apico. Two sea otter isolates had a mixed infection. Twenty-one (54%) isolates had an unique allele at SAG1 locus. Further genotyping or DNA sequence analysis for 18 of these 21 isolates at loci SAG1 and GRA6 revealed that there were two different genotypes, including the previously identified Type X (four isolates) and a new genotype named Type A (14 isolates). The results from this study suggest that the sea otter isolates are genetically diverse.     

Characterization of Toxoplasma gondii isolates in free-range chickens from Chile, South America

The prevalence of Toxoplasma gondii in free-ranging chickens is a good indicator of the prevalence of T. gondii oocysts in the soil because chickens feed from the ground. The prevalence of T. gondii in 85 free-range chickens (Gallus domesticus) from Chile was determined. Antibodies to T. gondii were assayed by the modified agglutination test (MAT), and found in 47 of 85 (55.3.9%) chickens with titers of 1:5 in six, 1:10 in four, 1:20 in four 1: 40 in three, 1: 80 in nine, 1: 160 in four 1:320 in nine, and 1: 640 or higher in eight. Hearts and brains of 47 chickens with titers of 1:5 or higher were pooled for each chicken and bioassayed in mice. Tissues from 16 seronegative (MAT<1:5) chickens were pooled and fed to one T. gondii-free cat. Feces of the cat were examined for oocysts but none was found based on bioassay of fecal floats in mice. Hearts and brains from seven seronegative (<1:5) were pooled and bioassayed in mice; T. gondii was not isolated. T. gondii was isolated by bioassay in mice from 22 chickens with MAT titers of 1:20 or higher. Genotyping of these 22 isolates using polymorphisms at the loci SAG1, SAG2, SAG3, BTUB and GRA6 revealed three genotypes. Seventeen isolates had type II alleles and four isolates had type III alleles at all loci. One isolate contained the combination of type I and III alleles. This is the first report of genetic characterization of T. gondii isolates from Chile, South America.