High prevalence and abundant atypical genotypes of Toxoplasma gondii isolated from lambs destined for human consumption in the USA

Little information is available on the presence of viable Toxoplasma gondii in tissues of lambs worldwide. The prevalence of T. gondii was determined in 383 lambs (<1 year old) from Maryland, Virginia and West Virginia, USA. Hearts of 383 lambs were obtained from a slaughter house on the day of killing. Blood removed from each heart was tested for antibodies to T. gondii by using the modified agglutination test (MAT). Sera were first screened using 1:25, 1:50, 1: 100 and 1:200 dilutions, and hearts were selected for bioassay for T. gondii. Antibodies (MAT, 1:25 or higher) to T. gondii were found in 104 (27.1%) of 383 lambs. Hearts of 68 seropositive lambs were used for isolation of viable T. gondii by bioassay in cats, mice or both. For bioassays in cats, the entire myocardium or 500g was chopped and fed to cats, one cat per heart and faeces of the recipient cats were examined for shedding of T. gondii oocysts. For bioassays in mice, 50g of the myocardium was digested in an acid pepsin solution and the digest inoculated into mice; the recipient mice were examined for T. gondii infection. In total, 53 isolates of T. gondii were obtained from 68 seropositive lambs. Genotyping of the 53 T. gondii isolates using 10 PCR-restriction fragment length polymorphism markers (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico) revealed 57 strains with 15 genotypes. Four lambs had infections with two T. gondii genotypes. Twenty-six (45.6%) strains belong to the clonal Type II lineage (these strains can be further divided into two groups based on alleles at locus Apico). Eight (15.7%) strains belong to the Type III lineage. The remaining 22 strains were divided into 11 atypical genotypes. These results indicate high parasite prevalence and high genetic diversity of T. gondii in lambs, which has important implications in public health. We believe this is the first in-depth genetic analysis of T. gondii isolates from sheep in the USA.     

Transplacental toxoplasmosis in naturally-infected white-tailed deer: Isolation and genetic characterisation of Toxoplasma gondii from foetuses of different gestational ages

Clinical toxoplasmosis is most severe in congenitally-infected hosts. In humans, transmission of Toxoplasma gondii from the mother to the foetus is considered to be most efficient during the last trimester of pregnancy but clinical congenital toxoplasmosis is more severe if transmission occurs during the first trimester. However, there are no data on the rate of congenital transmission of T. gondii with respect to gestational age in any host during natural infection. In the present study, attempts were made to isolate T. gondii by bioassay in mice inoculated with tissues from foetuses of 88 naturally-exposed white-tailed deer from Iowa and Minnesota. Viable T. gondii was isolated from foetuses of six of 61 deer in early pregnancy (45-85 days of gestation) from Iowa and foetuses of nine of 27 deer from Minnesota in mid-gestation (130-150 days) of a gestational period of 7 months. The 15 T. gondii isolates obtained from foetal deer were PCR-restriction fragment length polymorphism genotyped using polymorphisms at 10 nuclear markers including SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and an apicoplast marker, Apico. Five genotypes were revealed, including the clonal Type II and III lineages, and three non-clonal genotypes. DNA sequencing analysis of representative isolates at loci SAG2, c22-8, L358 and PK1 revealed that the three non-clonal genotypes are closely related to the clonal Type I, II and III lineages. It is very likely that these non-clonal genotypes were derived from genetic crosses among the three clonal Type I, II and III lineages. The most common genotype was Type II, commonly found in humans in North America and Europe, suggesting the possible link of transmission from game animals to humans.     

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 S?o 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.     

Isolation and genetic characterization of Toxoplasma gondii from raccoons (Procyon lotor), cats (Felis domesticus), striped skunk (Mephitis mephitis), black bear (Ursus americanus), and cougar (Puma concolor) from Canada

Viable Toxoplasma gondii was isolated by bioassay in mice from tissues of 2 feral cats (Felis domesticus), 2 raccoons (Procyon lotor), a skunk (Mephitis mephitis) trapped in remote locations in Manitoba, Canada, and a black bear (Ursus americanus) from Kuujjuaq, northern Quebec, Canada. Genotyping of these T. gondii isolates using polymorphisms at 10 nuclear markers including SAGI, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and an apicoplast marker Apico revealed 4 genotypes. None of the isolates was clonal archetypal Types I, II, and III found in the United States. These results are in contrast with the Type II genotype that is widespread in domestic animals and humans throughout the United States and Europe. This is the first genotyping of T. gondii isolates from this part of North America.     

High prevalence and genotypes of Toxoplasma gondii isolated from goats, from a retail meat store, destined for human consumption in the USA

Little information is available concerning the presence of viable Toxoplasma gondii in tissues of goats worldwide. In the present study, hearts of 234 goats obtained from a local USA grocery store were examined for T. gondii infection. Blood clot or fluid removed from each heart was tested for antibodies to T. gondii by using the modified agglutination test (MAT). Antibodies to T. gondii were found in 125 (53.4%) of 234 goats, with titers of 1:5 in 20, 1:10 in 44, 1:20 in 16, 1:40 in five, 1:160 in five, 1:320 in five, and 1:640 or higher in 30 goats. Hearts of 112 goats (46 goats <1:5, and 66 goats 1:10 or higher) were used for isolation of viable T. gondii by bioassays in mice. For bioassays, 50 g of the myocardium were digested in an acid pepsin solution and the digest inoculated into mice; the recipient mice were examined for T. gondii infection. Toxoplasma gondii was isolated from 29 goats; from hearts of one of 46 with titers of <1:5, one of nine with titers of 1:10, one of three with titers of 1:40, and 26 of 40 with titers of 1:160 or higher. Two isolates were highly virulent to outbred Swiss Webster mice; all infected mice died of toxoplasmosis, irrespective of the dose. All T. gondii isolates were subsequently grown in cell cultures. Genotyping of the 29 T. gondii isolates using 10 PCR-restriction fragment length polymorphism markers (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico) from DNA obtained from cell culture grown tachyzoites revealed 12 genotypes. Nine isolates were clonal Type II lineage, four isolates had type II alleles at all loci except a type I allele at the Apico locus, and four isolates were clonal Type III. The remaining 12 strains were divided into nine atypical genotypes, including five new and four previously identified genotypes. DNA sequences of four introns (EF1, HP2, UPRT1 and UPRT7) and two genes (GRA6 and GRA7) were generated for the five new genotypes. Comparing these sequences with previously published data revealed no unique sequences in these goat strains. Taken together, these results indicate high parasite prevalence and moderate genetic diversity of T. gondii in goats, which have important implications in public health. We believe this is the first genetic analysis of T. gondii isolates from goats in the USA.     

First report of genotyping of Toxoplasma gondii isolates from wild birds in China

Toxoplasma gondii is an important cosmopolitan opportunistic protozoan parasite, which threatens the health of human beings and animals. Genetic characterization of isolates from South America has revealed high genetic diversity. In contrast, isolates from North America and Europe were highly clonal, with 3 major lineages known as the Types I, II, and III. However, limited information on T. gondii genotypes has been reported in The People's Republic of China. Here we conducted a survey to determine genetic diversity of this parasite in wild birds of China. In total, tissues from breast muscle of 178 wild birds, including 98 common pheasants ( Phasianus colchicus ), 35 tree sparrows ( Passer montanus ), 22 house sparrows ( Passer domesticus ), 20 saxaul sparrows ( Passer ammodendri ), and 1 cinnamon sparrow ( Passer rutilans ), were tested for T. gondii infection, 4 of which were found to be positive for the T. gondii B1 gene by PCR amplification. These positive DNA samples were typed at 10 genetic markers, including 9 nuclear loci, i.e., SAG1, 5'- and 3'-SAG2, alternative SAG2, SAG3, GRA6, L358, PK1, c22-8, c29-2, and an apicoplast locus Apico. Of these, 3 isolates were genotyped with complete data for all loci, and 2 genotypes (Type I and Type II variant) were identified. This is the first report of genetic typing of T. gondii isolates from wild birds from different regions in China. The results suggest that the Type I and II variant strains are circulating in wild birds in China, and these birds are potential reservoirs for T. gondii transmission.     

Diverse and atypical genotypes identified in Toxoplasma gondii from dogs in São Paulo, Brazil

The prevalence of Toxoplasma gondii in 118 unwanted dogs from S?o Paulo City, S?o Paulo State, Brazil, was determined. Antibodies to T. gondii were assayed by the modified agglutination test and found in 42 (35.8%) dogs, with titers of 1:20 in 10, 1:40 in 6, 1:80 in 5, 1:160 in 5, 1:320 in 6, 1:640 in 7, and 1:1,280 or higher in 3. Hearts and brains of 36 seropositive dogs were bioassayed in mice, or cats, or both. Tissues from 20 seropositive dogs were fed to 20 T. gondii-free cats. Feces of cats were examined for oocysts. Toxoplasma gondii was isolated from 15 dogs by a bioassay in mice, from the brain alone of 1, from the heart alone of 4, and from both brains and hearts of 10. All infected mice from 5 of 15 isolates died of toxoplasmosis during primary infection. Four additional isolates were obtained by bioassay in cats. Genotyping of these 19 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 (an apicoplast marker Apico) revealed 12 genotypes. One isolate had Type III alleles at all 11 loci, and the remaining 18 isolates contained a combination of different alleles and were divided into 11 genotypes. The absence of Type II in Brazil was confirmed. The result supports previous findings that T. gondii population genetics is highly diverse in Brazil.     

Endemic toxoplasmosis in pigs on a farm in Maryland: isolation and genetic characterization of Toxoplasma gondii

The prevalence of Toxoplasma gondii was investigated on a poorly managed pig farm in Maryland. Serum and tissue samples from 48 of the 100 pigs on the farm were available for T. gondii evaluation. Serological testing was performed using both ELISA and the modified agglutination test (MAT). Antibodies to T. gondii were detected by ELISA in 12 of 48 animals, while antibodies were detected in 34 of 48 pigs by MAT with titers of 1:10 in 1, 1:20 in 4, 1:40 in 7, 1:80 in 3, 1:160 in 8, 1:320 in 3, 1:640 in 4, and 1:1,280 in 4. Hearts of 16 pigs with MAT titers of 1:10 or higher were bioassayed for T. gondii in cats; 11 cats shed T. gondii oocysts. Hearts of 22 pigs were autolyzed and bioassayed only in mice; T. gondii was isolated from 3 of these 22 pigs. Genetic typing of the 14 T. gondii isolates using the SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico loci revealed 4 genotypes; 10 isolates belonged to type II lineage (genotypes 1 and 2), 3 belonged to genotype 3, and 1 belonged to genotype 4. Genotype 1 and 2 have type II alleles at all genetic loci, except the former has type II allele and the latter has a type I allele at locus Apico. Both genotypes 1 and 2 are considered to belong to the clonal type II lineages. Genotype 3 and 4 are nonclonal isolates. Results document high prevalence of T. gondii in pigs on a farm in Maryland.     

Isolation of Toxoplasma gondii from animals in Durango, Mexico

Little is known concerning the epidemiology of Toxoplasma gondii infection in people and animals in rural Mexico. Serum samples and tissues from 150 dogs (Canis familaris), 150 cats (Felis catus), 65 opossums (Didelphis virginianus), 249 rats (Rattus spp.), 127 mice (Mus musculus), and 69 squirrels (Spermophilus variegatus) from the Durango area were evaluated for T. gondii infection. Using a modified agglutination test and a serum dilution of 1:25, antibodies to this parasite were found in 68 (45.3%) of 150 dogs, 14 (9.3%) of 150 cats, 11 (16.6%) of 66 opossums, 2 (0.8%) of 249 rats, 4 (3.1%) of 127 mice, and 0 of 69 squirrels. Tissues (brain and heart) of dogs, cats, opossums, rats, mice, and squirrels were bioassayed in mice for the presence of T. gondii. Viable T. gondii was isolated in tissues from 3 of 28 seropositive dogs and 5 of 8 seropositive cats, but not from the other animals. The DNA obtained from the 3 T. gondii isolates from dogs, 6 isolates from 5 cats, and 4 isolates from free-range chickens from Mexico, previously isolated, were genotyped. The PCR-RFLP typing, which used 11 markers (B 1, SAGI, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico), identified 5 genotypes. One genotype (the 4 chicken isolates) belongs to the clonal Type III lineage, three genotypes were reported in previous reports, and 1 genotype is unique.     

Mouse-virulent Toxoplasma gondii isolated from feral cats on Mona Island, Puerto Rico

Cats are essential in the life cycle of Toxoplasma gondii because they are the only hosts that can excrete the environmentally resistant oocysts. Samples of serum, feces, and tissues from cats from Mona, a remote island off the coast of Puerto Rico, were examined for T. gondii infection. Antibodies to T. gondii were assayed by the modified agglutination test and found in 16 of 19 (84.2%) of cats, with titers of 1:10 in 2, 1:80 in 1, 1:160 in 4, 1:320 in 3, and 1:1,280 or higher in 6. Tissues of 19 of the 20 cats were bioassayed in mice for T. gondii infection. Toxoplasma gondii was isolated from tissues of 12 cats: from the hearts of 9, skeletal muscle of 10, and brain of 1 cat. All infected mice from 10 of 12 isolates died of acute toxoplasmosis during primary infection. Genotyping of these 12 T. gondii isolates (designated (TgCatPr 1-12) by 10 multilocus PCR-RFLP markers, i.e., SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and an apicoplast marker Apico, and the 6 multilocus microsatellite markers TUB2, W35, TgM-A, B18, B17, and M33, revealed 7 genotypes; 5 isolates had Type I alleles at all loci except at 1 microsatellite locus, and the remainder were atypical. The latter isolates of T. gondii were different biologically and phenotypically from the feline isolates from the rest of the Americas. One isolate (TgCatPr 12) was a mixed infection with 2 genotypes.     

Isolation of Toxoplasma gondii from bottlenose dolphins (Tursiops truncatus)

Toxoplasma gondii infection in marine mammals is intriguing and indicative of contamination of the ocean environment and coastal waters with oocysts. In previous serological surveys, >90% of bottlenose dolphins (Tursiops truncatus) from the coasts of Florida, South Carolina, and California had antibodies to T. gondii by the modified agglutination test (MAT). In the present study, attempts were made to isolate T. gondii from dead T. truncatus. During 2005, 2006, and 2007, serum or blood clot, and tissues (brain, heart, skeletal muscle) of 52 T. truncatus stranded on the coasts of South Carolina were tested for T. gondii. Antibodies to T. gondii (MAT 1:25 or higher) were found in 26 (53%) of 49 dolphins; serum was not available from 3 animals. Tissues (heart, muscle, and sometimes brain) of 32 dolphins (26 seropositive, 3 seronegative, and 3 without accompanying sera) were bioassayed for T. gondii in mice, or cats, or both. Tissues of the recipient mice were examined for T. gondii stages. Feces of recipient cats were examined for shedding of T. gondii oocysts, but none excreted oocysts. Toxoplasma gondii was isolated from hearts of the 3 dolphins (2 with MAT titers of 1:200, and 1 without accompanied serum) by bioassay in mice. Genotyping of these 3 T. gondii isolates (designated TgDoUs1-3) with the use of 10 PCR-RFLP markers (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico) revealed 2 genotypes. Two of the 3 isolates have Type II alleles at all loci and belong to the clonal Type II lineage. One isolate has a unique genotype. This is the first report of isolation of viable T. gondii from T. truncatus.     

Genetic characterisation of Toxoplasma gondii in wildlife from North America revealed widespread and high prevalence of the fourth clonal type

Little is known of the genetic diversity of Toxoplasma gondii circulating in wildlife. In the present study wild animals, from the USA were examined for T. gondii infection. Tissues of naturally exposed animals were bioassayed in mice for isolation of viable parasites. Viable T. gondii was isolated from 31 animals including, to our knowledge for the first time, from a bald eagle (Haliaeetus leucocephalus), five gray wolves (Canis lupus), a woodrat (Neotoma micropus), and five Arctic foxes (Alopex lagopus). Additionally, 66 T. gondii isolates obtained previously, but not genetically characterised, were revived in mice. Toxoplasma gondii DNA isolated from these 97 samples (31 + 66) was characterised using 11 PCR-restriction fragment length polymorphism (RFLP) markers (SAG1, 5′- and 3′-SAG2, alt.SAG2, SAG3, BTUB, GRA6, c22–8, c29–2, L358, PK1 and Apico). A total of 95 isolates were successfully genotyped. In addition to clonal Types II, and III, 12 different genotypes were found. These genotype data were combined with 74 T. gondii isolates previously characterised from wildlife from North America and a composite data set of 169 isolates comprised 22 genotypes, including clonal Types II, III and 20 atypical genotypes. Phylogenetic network analysis showed limited diversity with dominance of a recently designated fourth clonal type (Type 12) in North America, followed by the Type II and III lineages. These three major lineages together accounted for 85% of strains in North America. The Type 12 lineage includes previously identified Type A and X strains from sea otters. This study revealed that the Type 12 lineage accounts for 46.7% (79/169) of isolates and is dominant in wildlife of North America. No clonal Type I strain was identified among these wildlife isolates. These results suggest that T. gondii strains in wildlife from North America have limited diversity, with the occurrence of only a few major clonal types.     

Genetic characterization of Toxoplasma gondii isolates from China

Toxoplasma gondii infections are prevalent in humans and animals worldwide. In North America and Europe, T. gondii is highly clonal, consisting of three distinct lineages (Types I, II and III), whereas in South America, T. gondii is highly diverse with a few lineages expanded in the population. However, there is limited data on the diversity of T. gondii in Asia. Here we report the genetic characterization of T. gondii isolates from different hosts and geographical locations in China using the multilocus PCR–RFLP. A total of 17 T. gondii isolates from humans (3 strains), sheep (1 strain), pigs (5 strains) and cats (8 strains) were typed at 10 genetic markers including 9 nuclear loci SAG1, SAG2, SAG3, BTUB, GRA6, L358, PK1, c22-8, c29-2 and an apicoplast locus Apico. Four genotypes were revealed, including three previously reported and one new genotype. Three isolates belong to the clonal Type I lineage, one isolate belongs to the clonal Type II lineage, and the rest 13 isolates are grouped into two genotypes. This is the first report of genetic typing of T. gondii isolates from different hosts and geographical locations in China using a number of genetic markers, which has implications for the studies of population genetic structures of T. gondii, as well as for the prevention and control of T. gondii infections in humans and animals in China.     

Isolation of viable Toxoplasma gondii from feral guinea fowl (Numida meleagris) and domestic rabbits (Oryctolagus cuniculus) from Brazil

Toxoplasma gondii was isolated from a feral guinea fowl (Numida meleagris) and domestic rabbits (Oryctologus cuniculus) from Brazil for the first time. Serum and brains from 10 guinea fowl and 21 rabbits from Brazil were examined for T. gondii infection. Antibodies to T. gondii were found in 2 of 10 fowl and 2 of 21 rabbits by the modified agglutination test (titer 1∶25 or higher). Viable T. gondii (designated TgNmBr1) was isolated from 1 of the 2 seropositive fowl by bioassay in mice but not from the 8 seronegative fowl by bioassay in cat. Viable T. gondii was isolated from both seropositive rabbits (designated TgRabbitBr1, TgRabbitBr2) by bioassay in mice from 1 and by bioassay in cat from the other. The TgRabbitBr1 strain was highly virulent for out-bred mice; mice fed 1 infective oocyst died of acute toxoplasmosis. The remaining 2 isolates were relatively avirulent for mice; lethal dose for mice was 10,000 oocysts. All 3 isolates were grown in cell culture, and tachyzoite-derived DNA were genotyped using 10 PCR-restriction fragment length polymorphism markers (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico). The TgNmBr1 was found to be clonal Type II, a rare finding in Brazil in any host. The rabbit isolates were atypical, similar to isolates from cats from Brazil (TgRabbitBr1 was identical to TgCatBr5, and TgRabbitBr2 was identical to TgCatBr1, a common genotype in Brazil denoted type BrII). This is the first genetic characterization of T. gondii isolates from the rabbits and guinea fowl in Brazil and the first host record for T. gondii in the guinea fowl.     

Biologic and molecular characteristics of Toxoplasma gondii isolates from striped skunk (Mephitis mephitis), Canada goose (Branta canadensis), black-winged lory (Eos cyanogenia), and cats (Felis catus)

Toxoplasma gondii isolates can be grouped into 3 genetic lineages. Type I isolates are considered virulent to outbred mice, whereas Type II and III isolates are not. In the present report, viable T. gondii was isolated for the first time from striped skunk (Mephitis mephitis), Canada goose (Branta canadensis), and black-winged lory (Eos cyanogenia). For the isolation of T. gondii, tissues were bioassayed in mice, and genotyping was based on the SAG2 locus. Toxoplasma gondii was isolated from 3 of 6 skunks, 1 of 4 Canada geese, and 2 of 2 feral cats (Felis catus) from Mississippi. All donor animals were asymptomatic. Viable T. gondii was also isolated from 5 of 5 lories that had died of acute toxoplasmosis in an aviary in South Carolina. Genotypes of T. gondii isolates were Type III (all skunks, lories, and the goose) and Type II (both cats). All 5 Type III isolates from birds and 2 of the 3 isolates from skunks were mouse virulent.     

Genetic characterization of Toxoplasma gondii isolates from pigs in southwestern China

The genetic diversity of Toxoplasma gondii varies in different geographical regions. Isolates of T. gondii in South America, for example, are genetically and biologically divergent from those in North America and Europe, where the population structure is highly clonal and composed mainly of 3 distinct lineages, i.e., Types I, II, and III. However, little is known of the T. gondii genotypes in the People's Republic of China. Toxoplasma gondii infection in pigs causes significant economic loss and presents a risk for human infection. We conducted a survey to determine the genetic diversity of this parasite in slaughtered pigs from Yunnan Province, southwestern China. In total, 412 DNA samples were extracted from hilar lymph nodes and livers of pigs from slaughterhouses in Yunnan Province in southwest China, 56 of which were found to be positive for the T. gondii SAG3 gene. These positive DNA samples were typed at 10 genetic markers, including 9 nuclear loci, i.e., SAG1, SAG2, SAG3, BTUB, GRA6, L358, PK1, c22-8, c29-2, and an apicoplast locus Apico. Of these, 5 isolates were genotyped with complete data for all loci. Only 1 genotype (ToxoDB 9) was identified, previously reported as a widespread lineage from pigs, cats, and human patients in China. The results indicate that this genotype may be the major T. gondii lineage in China and possibly all of eastern Asia. This is the first report of genetic typing of T. gondii isolates from pigs in China's southwestern Yunnan Province, the results of which have implications for the prevention and control of T. gondii infections in humans and other animals.     

Toxoplasma gondii: genotyping of strains from Brazilian AIDS patients with cerebral toxoplasmosis by multilocus PCR-RFLP markers

This study investigated the genetic characteristics of the Toxoplasma gondii strains isolated from 87 patients with cerebral toxoplasmosis and AIDS, treated in Sao Paulo State, Brazil. The laboratorial diagnosis of cerebral toxoplasmosis was based on positive serological exams and PCR of blood and/or cerebrospinal fluid. Four markers (5'-SAG2, 3'-SAG2, SAG3 and GRA6) were chosen to analyze the samples. Each having clear resolution to distinguish the three clonal lineages after PCR amplified targets were treated with restriction enzyme digestion (PCR-RFLP). The genotyping provided the following results: 40 patients (46%) were infected with strains classified as type I; 4 (4%), as type III; 13 (15%) were infected with polymorphic strains (unusual genotype); 6 patients with type I or II alleles; and 15 (17%) patients had strains not classified for any marker. PCR-RFLP, also classified 9 (11%) clinical isolates as type II, which is uncommon in South America. However, the sequencing of the nested-PCR products (of SAG3 marker) of type II and polymorphic isolates (of 5'-SAG2, SAG3 and GRA6 markers) showed a nucleotide polymorphism compared with the archetypal clonal genotypes (types I, II and III) and these isolates were considered as polymorphic strains. The markers used here were inappropriate to distinguish the most isolates considered as polymorphic strains. These data confirm other studies showing the high rate of genetic polymorphism in T. gondii strains isolated in Brazil.     

Genotypes and Mouse Virulence of Toxoplasma gondii Isolates from Animals and Humans in China

Background

Recent population structure studies of T. gondii revealed that a few major clonal lineages predominated in different geographical regions. T. gondii in South America is genetically and biologically divergent, whereas this parasite is remarkably clonal in North America and Europe with a few major lineages including Types I, II and III. Information on genotypes and mouse virulence of T. gondii isolates from China is scarce and insufficient to investigate its population structure, evolution, and transmission.

Methodology/Principal Findings

Genotyping of 23 T. gondii isolates from different hosts using 10 markers for PCR-restriction fragment length polymorphism analyses (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico) revealed five genotypes; among them three genotypes were atypical and two were archetypal. Fifteen strains belong to the Chinese 1 lineage, which has been previously reported as a widespread lineage from swine, cats, and humans in China. Two human isolates fall into the type I and II lineages and the remaining isolates belong to two new atypical genotypes (ToxoDB#204 and #205) which has never been reported in China. Our results show that these genotypes of T. gondii isolates are intermediately or highly virulent in mice except for the strain TgCtwh6, which maintained parasitemia in mice for 35 days post infection although it possesses the uniform genotype of Chinese 1. Additionally, phylogenetic network analyses of all isolates of genotype Chinese 1 are identical, and there is no variation based on the sequence data generated for four introns (EF1, HP2, UPRT1 and UPRT7) and two dense granule proteins (GRA6 and GRA7).

Conclusion/Significance

A limited genetic diversity was found and genotype Chinese 1 (ToxoDB#9) is dominantly circulating in mainland China. The results will provide a useful profile for deep insight to the population structure, epidemiology and biological characteristics of T. gondii in China.     

Seroprevalence and isolation of Toxoplasma gondii from free-range chickens in Ghana, Indonesia, Italy, Poland, and Vietnam

The prevalence of Toxoplasma gondii in free-ranging chickens (Gallus domesticus) is a good indicator of the prevalence of the parasite's oocysts in soil because chicken feed from the ground. The prevalence of T. gondii in free-range chickens from Ghana, Indonesia, Italy, Poland, and Vietnam was determined using the modified agglutination test (MAT). Antibodies to T. gondii were found in 41 (64%) of 64 chickens from Ghana, 24 (24.4%) of 98 chickens from Indonesia, 10 (12.5%) of 80 chickens from Italy, 6 (30%) of 20 chickens from Poland, and 81 (24.2%) of 330 chickens from Vietnam. Hearts and brains of chickens were bioassayed for T. gondii. Viable T. gondii was isolated from 2 chickens from Ghana, 1 chicken from Indonesia, 3 chickens from Italy, 2 chickens from Poland, and 1 chicken from Vietnam. Toxoplasma gondii isolates from 9 chickens were genotyped using 10 PCR-RFLP markers including SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico. A total of 7 genotypes was identified; the 3 isolates from chickens from Italy were clonal type II, and the others were nonclonal. This is the first report of genetic characterization of T. gondii isolates from animals from these countries.