Genetic characterization of Toxoplasma gondii revealed highly diverse genotypes. from human congenital toxoplasmosis in southeastern Brazil

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1 JCM Accepts, published online ahead of print on 2 January 2013 J. Clin. Microbiol. doi: /jcm Copyright 2013, American Society for Microbiology. All Rights Reserved. 1 2 Genetic characterization of Toxoplasma gondii revealed highly diverse genotypes from human congenital toxoplasmosis in southeastern Brazil 3 4 Genotyping of Toxoplasma gondii from newborns Ana Carolina de Aguiar Vasconcelos Carneiro a,f, Gláucia Manzan Queriroz de Andrade b,f, Júlia Gatti Ladeia Costa a, Breno Veloso Pinheiro a, Daniel Vitor Vasconcelos-Santos d,f, Adriana de Melo Ferreira a, Chunlei Su e, José Nélio Januário c,f, Ricardo Wagner de Almeida Vitor a,f Departamento de Parasitologia a, Departamento de Pediatria b, Núcleo de Ações e Pesquisa em Apoio Diagnóstico c and Departamento de Oftalmologia d, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil; Department of Microbiology, University of Tennessee, Knoxville, TN e ; UFMG Congenital Toxopasmosis Brazilian Group f Address correspondence to R. W. A. Vitor, ricardovitor@icb.ufmg.br 1

2 Recent studies of Toxoplasma gondii isolates from animals in Brazil have revealed high genetic diversity. Many of these isolates are virulent to mice. It is speculated that these isolates may also be virulent to humans. However, there is very limited data regarding T. gondii strains from human infection. Therefore it is not clear whether there is any association between parasite genotypes and disease phenotypes. In this study, a total of 27 T. gondii strains were isolated from congenital toxoplasmosis in Minas Gerais state, Brazil. The genetic variability was assessed by Restricted Fragment Length Polymorphism in 11 loci (SAG1, 5 +3 SAG2, SAG2 alt, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, Apico). Genetic analysis of 24 strains revealed 14 different genotypes, including 7 previously identified from animals and 7 new types. The widespread genotype BrII, accounted for 29% (7/24) of the isolates and was the dominant genotype involved in this study. This is the first report of genotyping of T. gondii isolates obtained from blood of newborns with congenital toxoplasmosis. Genotypic characterization of these isolates suggests high genetic diversity of T. gondii in this human population in Brazil. Future studies are needed to determine the source of contamination of this human population. Keywords. Toxoplasma gondii, Congenital Toxoplasmosis, Genotyping, PCR-RFLP, Isolates, Newborn patients

3 Toxoplasma gondii is a widely distributed Apicomplexan parasite of great medical importance. T. gondii infection in humans may lead to congenital, ocular or encephalic toxoplasmosis. Congenital toxoplasmosis has been associated with abortion and stillbirth, as well as neonatal mortality and morbidity. Prevalence of congenital toxoplasmosis varies according to geographic location and is determined by social, cultural and other factors, such as type of biological sample and diagnostic assay used (1). In Brazil, prevalence of congenital toxoplasmosis ranges from 0.3/1,000 newborns in Ribeirão Preto - SP (2) to 5.0/1,000 in Uberlândia - MG (3). In Belo Horizonte - MG, the prevalence of T. gondii infection in newborns is one in 1,540 live births (4). A more recent population-based study involving the entire state of Minas Gerais revealed one case of congenital toxoplasmosis in every 770 live births (1.3/1,000). 79.8% of the newborns with congenital toxoplasmosis had retinochoroidal lesions in at least one eye (5). Several factors can be related to the severity of congenital toxoplasmosis, including parasite strain and host genetic variability and immune response. Genetic diversity of T. gondii strains has been an interesting and important research topic. T. gondii was considered to have a clonal population genetic structure with three major lineages (Types I, II and III) in Europe and North America (6). In these regions, congenital toxoplasmosis was mainly associated with strains classified as Type II (7, 8). However, recent studies using multilocus markers showed high genetic diversity in South America, which is absent in strains from North America and Europe (9, 10, 11). More recently, analysis of isolates from domestic animals in Brazil revealed 48 RFLP genotypes, with four of these isolates being considered to be common clonal lineages, designated as types BrI BrII, BrIII and BrIV (12). 3

4 Genetic analysis of T. gondii infecting humans is important to understand epidemiology, transmission patterns and mechanisms of the disease. However, difficulty to obtain T. gondii strains from humans is a limiting factor (13, 14, 11). In this study, we described the genetic and biological characteristics of T. gondii isolates from newborns with congenital toxoplasmosis in the state of Minas Gerais, Brazil METHODS Study Population. This study was part of an investigation on neonatal screening for congenital toxoplasmosis conducted by a multidisciplinary research group (UFMG- Brazilian Congenital Toxoplasmosis Group) in the Minas Gerais state, southeastern Brazil. From November 1st, 2006 to May 31st, 2007, a total of 146,307 children were tested for anti-t. gondii IgM antibodies in dried blood samples on filter paper using the Toxo IgM kit (Q-Preven, Symbiosis, Leme, Brazil). Confirmative serologic tests were carried out for 220 infants with positive or undetermined screening results. These infants were tested for anti-t. gondii IgG, IgA and IgM by enzyme-linked fluorometric assay - ELFA-VIDAS (biomérrieux SA, Lyon, France). Out of these 220 cases, 178 infants tested positive persistence of anti-t. gondii IgG antibodies in serum at the age of 12 months. IgM tests (Q-Preven and ELFA-VIDAS ) showed a moderate level of discordance. However this was expected, since the collection of blood on filter paper for the initial screening (Q-Preven ) was made in children aged 7 to 10 days after birth. Confirmatory tests (ELFA-VIDAS ) were made after 31 to 86 days after birth (mean: 55.6 days). It is likely that, at this moment, the levels of IgM previously initially detected by Q-Preven had decreased in some children and were no longer detected by ELFA-VIDAS. Ophthalmologic examinations were performed for all 220 children 4

5 according to the method described previously (5). The protocols used in this study were approved by the local Human Research Ethics Committee (COEP-Federal University of Minas Gerais, protocol 298/06) Toxoplasma gondii isolates. Peripheral blood samples from children ranging between 31 and 86 days (average age of \ days) was collected. Heparinized peripheral blood samples (0.5 ml) were centrifuged at 2,000g for 15 minutes and the blood cell sediments containing erythrocytes and leukocytes were resuspended in 0.2 ml of phosphate buffered saline solution (PBS) ph 7.2. For parasite isolation, 0.1 ml of this cell suspension was inoculated intraperitoneally (i.p.) in each of two, six to eightweek-old female Swiss mice. Thirty days after inoculation, each surviving mouse was bled via retro-orbital plexus. The blood (0.1mL) was centrifuged and the plasma was used to perform ELISA for anti-t. gondii IgG antibodies (15). Animals that died before 30 days post inoculation were examined for the presence of tachyzoites in the peritoneum or cysts in the brain. All surviving mice were sacrificed by cervical dislocation. The brains of ELISA seropositive mice were removed, macerated, and 1.0 ml PBS ph 7.2 was added. Ten µl of brain lysates were used to search for the presence of tissue cysts under light microscopy. To determine mouse virulence of T. gondii isolates, tachyzoites were first produced from the peritoneal cavities of five Swiss mice i.p. inoculated with brain cysts, as described previously (16). Five to seven days after inoculation, the parasites were collected and washed from the peritoneal cavity with PBS ph 7.2 and used for virulence assay (fresh tachyzoites) or stored as frozen pellets at 20 o C, until genomic DNA extraction. In the case of virulent strains, the infected Swiss reservoirs were treated with sulfadiazine for 10 days after infection to obtain cysts (17). The protocols conducted in this study were approved by the local 5

6 Animal Ethics Committee (CETEA-Federal University of Minas Gerais, protocol 013/2007) Parasite virulence. The same criteria adopted previously (18) were applied to determine the virulence of the isolates of T. gondii. Five female BALB/c mice were i.p. inoculated with 10 0, 10 1, 10 2 or 10 3 tachyzoites of each isolate in 0.2mL of PBS (ph7.2). Five animals inoculated i.p. with PBS were maintained as negative controls. Mice mortality was observed over a 30-day period. The survivors were bled via retroorbital plexus and the sera were tested by ELISA. The mice which did not seroconvert were excluded from the experiment. All the surviving mice were sacrificed by cervical dislocation to search for tissue cysts in the brain. For comparison, RH (virulent) and ME49 (non-virulent) strains were used as references. Isolates killing 100% of the infected mice were classified as virulent. Isolates with LD 100 greater than 10 3 tachyzoites were classified as non-virulent, and those with an intermediate pattern between the two extremes were classified as of intermediate virulence (6). DNA extracts and multilocus PCR-RFLP genotyping of T. gondii. DNA extraction of the tachyzoites was performed using the Promega Wizard genomic DNA purification kit following the manufacturer s instructions. The genotyping of isolates obtained from newborns with congenital toxoplasmosis was determined by PCR-RFLP patterns of eleven DNA segments including SAG1, 3 SAG2 + 5'SAG2, alt. SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico, as described previously (19). However, we performed a PCR in a single stage using only the internal markers, 6

7 without pre amplification by multiplex PCR using external primers. The amplification reactions were carried out in a final volume of 10 µl, containing 2 µl of green 5X 135 buffer (Promega), 25 mm of MgCl 2, 2.5 mm of each deoxynucleotide (datp/dttp/dgtp/dctp, Invitrogen), 5u/μL of Taq DNA polymerase (Promega), 5 pmol of each primer and 1 μl of DNA. A negative control, without DNA, was included in each reaction. The strains RH88 (type I), ME49 (type II) and VEG (type III) were used as controls. The first amplification step consisted of four minutes of denaturation at 95 C, followed by 35 cycles of denaturation at 94 C for 30 seconds, annealing at 60 C for 60 seconds, and extension at 72 C for 90 seconds. The extension step at the final cycle was extended to five minutes. The PCR products were visualized in 5% polyacrylamide gel stained with silver nitrate. The amplified products were digested using the appropriate restriction endonucleases, according to previously published method (19). The digestions were conducted at a final volume of 10 μl, containing 3 μl of the PCR product, 1 μl of the corresponding buffer and 2.5U (0.25μL) of the enzyme, at 37 C for 3 hrs, according to the manufacturer's instruction. The DNA of the digested products was purified by extraction with an equal volume of phenol/chloroform (1:1), submitted to polyacrylamide gel (5%) electrophoresis, stained with silver nitrate and photographed. Data analysis. The profiles found after digestion with restriction endonucleases were compared with the profiles of the reference strains in a virtual database ToxoDB ( The study database included serological results, demographical data (age, gender, place of birth) of each child, as well as bioassay, virulence, genotyping results and clinical signs. To reveal the genetic relationship of all the parasite isolates, 7

8 the composite dataset of multilocus PCR-RFLP genotyping was analyzed by SplitsTree4 (20, 21). The composite dataset was based on most recent genotyping results from Brazil (22). The result is presented as a reticulated network to describe complex relationships of these T. gondii strains RESULTS Strain isolation and virulence determination of the T. gondii isolates. One hundred seventy eight infants with persistence of anti-t. gondii IgG antibodies at the age of 12 months were selected for this study. We tested one blood sample from each child by bioassay. Twenty seven isolates of T. gondii were obtained from 27 newborns with congenital toxoplasmosis, demonstrating parasitemia in 15.2% (27/178). The isolates were designated as TgCTBr1 to TgCTBr27 (TgCTBr: Tg = T. gondii; CT = Congenital Toxoplasmosis, Br = Brazil), following the chronological order in which isolation was performed. Only one of two mice inoculated with the blood of newborn infants was infected with T. gondii, except TgCTBr9. In this case the two mice became infected. The isolate TgCTBr6 was lost before the production of tachyzoites to DNA extraction and virulence experiment. Table 1 presents data of 27 newborns from which T. gondii was isolated. It shows the age of the children at the time blood was collected for the bioassay, ranging between 31 and 86 days (average age of \ days), gender, major clinical signs and confirmative serologic results. The newborns from which T. gondii was isolated came from different regions of the state of Minas Gerais (Fig. 1), with isolates being obtained from 10 out of the 12 regions. The isolates were divided into three groups according to 8

9 the virulence phenotype for BALB/c mice. The time between inoculation of the blood of newborn in two Swiss mice and isolation of T. gondii are in Table S1 in the supplemental material. Fourteen isolates (54%) were characterized as of intermediate virulence, ten (38%) as virulent and only two (8%) as non virulent (Table 1) Genotyping analysis of T.gondii. The complete genotype was obtained in 25/27 (92%) isolates (Table 2). It was not possible to carry out the complete genotyping in one sample (TgCTBr16) due to the non-amplification of some markers as well as due to the occurrence of extremely polymorphic digestion products. Isolate TgCTBr6 was lost before DNA was obtained. Representative result of PCR RFLP analysis (BTUB marker) is shown in Figure S2 in the supplemental material. Tables 1 and 2 present the isolates grouped according to the genotypes identified. From the 25 isolates typed, 14 different genotypes were identified, and one isolate (TgCTBr19) had mixed infection, in which a combination of two alleles was observed in four of the 11 loci analyzed, i.e., SAG2, alt.sag2, BTUB and c29-2. Of the 14 genotypes found, 7 are considered new types that were not reported previously, and seven were previously identified including ToxoDB PCR-RFLP genotype #8, #11, #36, #41, #67, #108 and #162 (Table 1, 2). The 7 new genotypes are designated following the scheme of ToxoDB PCR-RFLP genotype numbers. The new types are #206 (TgCTBr01, 03 and 25), #207 (TgCTBr10), #208 (TgCTBr13), #209 (TgCTBr21), #210 (TgCTBr22), #211 (TgCTBr24), and #212 (TgCTBr26). Seven isolates (TgCTBr02, 08, 09, 11, 14, 20 and 27) matched to genotype #11 (also known as Type BrII). This genotype was previously identified from domestic animals in Southeastern Brazil (12, 23, 24, 25, 26, 27). Geographical distribution of genotype #11 in Minas Gerais was 9

10 clearly widespread among 6 of 10 regions in which positive isolation of T. gondii from congenital toxoplasmosis patients was achieved (Figure 1). Isolate TgCTBr05 matched, to genotype #8 (a.k.a Type BrIII), was previously identified from domestic animals in another areas of Brazil (12, 23, 25, 26, 28, 29). Isolate TgCTBr18 matched to genotype #36, which was previously identified from chickens in Rio de Janeiro, (23). Isolates TgCTBr15 and TgCTBr23 matched to genotype #41, which was previously identified from chickens in Amazon, Brazil (23) and capybaras in Sao Paulo state (26). Isolate TgCTBr07 matched to genotype #67, which was previously identified from dogs in Sao Paulo city (25), and cats in Sao Paulo state (12). Isolates TgCTBr04 and TgCTBr17 matched to genotype #108, which was previously identified from cat in Sao Paulo state (12). Isolate TgCTBr12 matched to genotype #162, which was previously identified from capybara in Sao Paulo state (26). The genetic relationship of the 24 isolates of T. gondii obtained from newborns (excluding isolate TgCTBr19, due to its mixed infection origin), together with previously published data were compared using SplitsTree4 software (20, 21). The results are presented in Figure 2. The 14 genotypes identified from the 24 congenital cases showed high diversity and are scattered in the network. Regarding virulence in mice, isolates with identical genotypes presented similar phenotypes. A descriptive analysis did not show any association between the genotype of the isolates and retinochoroiditis in the newborns. Likewise, no association between the genotypes and the sites of origin of the isolates was observed DISCUSSION 10

11 Most of the isolates obtained and genotyped in Brazil are from domestic animals including free-range chickens, cats, dogs, sheep, and goat; little is known about the genetics of T. gondii isolates from humans (22). To understand the epidemiology of toxoplasmosis, it is important to determine how and from which sources the parasite is transmitted from animals to humans. In the present study, we isolated and genotyped T. gondii from peripheral blood of newborns. The high positivity observed in the bioassay experiments confirms the presence of viable tachizoites in the blood circulation of these transplacentally infected infants, even after a period of two to three months of life. Isolation of T. gondii in infants up to 86 days old can be explained by the immunoimmaturity of the newborns, which allows parasitemia to occur longer than in immunocompetent adults (30). The 27 isolates were obtained from children from different regions in Minas Gerais, showing that T. gondii is widely distributed in the state. We observed variability in the mouse virulence of the T. gondii isolates from cases of congenital toxoplasmosis. Ninety two percent (24/26) of the isolates are intermediately or highly virulent to mice. A previous study analyzed the virulence of isolates of T. gondii obtained from domestic animals in the state of Minas Gerais, showing a higher prevalence of samples with high/intermediate virulence phenotype (31). Several factors interfere in the virulence of T. gondii, including parasite stage, infection route, infective dose, inoculation mode, mouse lineage and intrinsic characteristics of the isolate (32). Although few studies have been carried out with isolates of T. gondii derived from cases of congenital toxoplasmosis (33), it is known that, in general, the isolates obtained in Brazil are virulent (10). Of the 24 isolates completely genotyped in this study, 20 originated from newborns with retinochoroiditis. 11

12 Comparing Brazilian and European infants with congenital toxoplasmosis, it was observed that T. gondii causes more severe ocular disease in the former than in the latter (34). These authors concluded that the differences in frequency, size, and multiplicity of the retinochoroidal lesions between the two populations may have been due to infections with more virulent strains of the parasite that predominate in Brazil and are rarely found in Europe. In this study, we completely genotyped 24 T. gondii isolates from peripheral blood of newborns with congenital toxoplasmosis. Analysis of the multilocus PCR-RFLP revealed 14 genotypes, suggesting high diversity of the parasite isolated from the human population in Minas Gerais, similar to results reported by other authors in studies of parasites isolated from other host species (12, 23, 28). Of the 14 genotypes identified, four were identified in two or more isolates (#11, #41, #108 and #206), while the other ten genotypes are represented by only one isolate. Among the 14 genotypes, 7 were previously reported in animals and 7 were described for the first time in the literature (Table 2), with six presenting only one isolate. This result reconfirmed the high diversity of T. gondii strains in Brazil (23, 29, 28). Although the T. gondii population is highly diversified in Brazil, some clonal genotypes circulate in the hosts. The previously identified clonal isolates BrII (#11) and BrIII (#8) were found in this study. These genotypes had already been described in several hosts such as sheep, chicken, and cats (12, 23, 29), but this is the first report of these two genotypes in humans. The isolation of strains with identical genotypes in distinct geographic regions (genotypes #11 and #8) suggests the widespread distribution of clonal genotypes of T. gondii in Minas Gerais, Brazil. 12

13 In this study, the isolate TgCTBr19 presented mixed profiles in four of the eleven markers used. This mixed infection may have occurred due to simultaneous and sequential infections (reinfections), with parasites of different genotypes, acquired from oocysts directly from the environment or through ingestion of tissue cysts in the intermediate hosts infected with two distinct isolates of the parasite, possibilities already described in the literature (35, 36). Studies show evidences of mixed infections mainly associated to congenital cases (36). The occurrence of mixed genotypes in Brazil corroborates the hypothesis that several genotypes of T. gondii must be widely disseminated and circulating simultaneously (23, 12, 26, 29, 28). In Brazil, overall, isolates are recombinant and virulent (10, 12, 37). Isolate TgCTBr05, the only one classified as of type BrIII, was shown to be non-virulent to mice, corroborating previous study (12). However, the seven isolates identified as genotype #11 (BrII), showed variable virulence. Biological differences between the isolates of the same genotype must not be neglected and it may be possible that the genetic markers used in this study are incapable of reflecting possible phenotypic differences between the isolates under question (28). Thus, there is no clear correlation between the genotypes of the isolates of T. gondii obtained from newborns in the state of Minas Gerais and virulence to mice. A recent study in Brazil has not shown any concrete evidence, either, of correlation between virulence of T. gondii for mice and different genotypes (38). No association between the parasite genotype and retinochoroiditis in the newborns was observed, with further studies being necessary to define the role played by phenotypic and genotypic characteristics of T. gondii in the development of ocular lesions. 13

14 The results of this study indicate that a significant proportion of cases of congenital toxoplasmosis (62.5%, 15/24) were caused by T. gondii genotypes previously reported from animals (Table 3). In particular, 29% (7/24) cases were caused by the common T. gondii genotype #11 (BrII) that was previously reported from a variety of animals in Brazil (23, 24, 25, 12, 26, 27). Nine T. gondii isolates were grouped into 7 new genotypes that were not reported before, indicating high diversity of the parasite in the population. Further studies of sampling of T. gondii in animals from Minas Gerais are needed to better understand the population structure of T. gondii and transmission of the parasites among animals and humans. This is the first report of genotyping of isolates obtained from the blood of newborns, providing important technical and scientific information as to epidemiology of congenital toxoplasmosis in Brazil. ACKNOWLEDGEMENTS This work was supported by Conselho Nacional de Desenvolvimento e Pesquisa - CNPq (Grants /2009-3, /2010-8); Fundação de Amparo à Pesquisa de Minas Gerais - FAPEMIG (Grant ); Secretaria de Saúde de Minas Gerais (SES-MG) and Núcleo de Ações e Pesquisa em Apoio Diagnóstico (NUPAD-UFMG). Ana Carolina de Aguiar Vasconcelos Carneiro is the recipient of a scholarship from the CNPq. We thank Rosalida Estevan Nazar Lopes for technical assistance. Technical support from the UFMG - Congenital Toxoplasmosis Brazilian Group (UFMG-CTBG) is greatly appreciated: Ana Carolina de Aguiar Vasconcelos Carneiro, Daniel Vitor Vasconcelos-Santos, Danuza O. Machado Azevedo, Ericka V. Machado Carellos, Fernando Oréfice, Gláucia M. Queiroz-Andrade, José Nélio Januário, Luciana 14

15 Macedo Resende, Olindo Assis Martins-Filho, Ricardo Wagner de Almeida Vitor, Roberta M. Castro Romanelli, Waleska Teixeira Caiaffa, Wesley R. Campos REFERENCES Tenter AM, Heckeroth AR, Weiis ML Toxoplasma gondii: from animals to humans. Int. J. Parasitol. 30: Carvalheiro CG, Mussi-Pinhata MM, Yamamoto AY, De Souza CB, Maciel LM Incidence of congenital toxoplasmosis estimated by neonatal screening: relevance of diagnostic confirmation in asymptomatic newborn infants. Epidemiol. Infect. 133: Segundo GRS, Silva DAO, Mineo JR, Ferreira MS A comparative study of congenital toxoplasmosis between public and private hospitals from Uberlândia, MG, Brazil. Mem. Inst. Oswaldo Cruz 99: Queiroz GM, Resende LM, Goulart EMA, Siqueira AL, Vitor RWA, Januário JN Hearing loss in congenital toxoplasmosis detected by newborn screening. Rev. Bras. Otorrinolaringol. 74: Vasconcelos-Santos DV, Machado DO, Campos WR, Oréfice F, Andrade GMQ, Carellos EVM, Romanelli RM, Januario JN, Resende LM, Martins- Filho O, Carneiro ACAV, Vitor RWA, Caiaffa WT Congenital toxoplasmosis in southeastern Brazil: results of early ophthalmologic examination of a large cohort of neonates. Ophthalmol. 116: Howe DK, Sibley LD Toxoplasma gondii comprises three clonal lineages, correlation of parasite genotype with human disease. J. Infect. Dis. 172:

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21 FIG 1 Origin of the T. gondii isolates from newborns in the twelve regions of Minas Gerais state, Brazil. #PCR-RFLP genotype; *nd: genotyping not done; **Mix: mixed genotype

22 FIG 2 NeighborNet phylogenetic network of Toxoplasma gondii isolates from Brazil. TgCTBr1 to TgCTBr27: newborns isolates from Minas Gerais

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24 TABLE 1 Toxoplasma gondii isolates from Minas Gerais, Brazil, by location, patient gender, age, occurrence of clinical signs and mouse-virulence. Patient Age PCR-RFLP Patient Location Clinical signs IgM screening a ELFA-IgA b ELFA-IgM b ELFA-IgG b Isolates Mouse-virulence gender (days) Genotype 1 Norte de Minas Male 62 ACRL TgCTBr02 Intermediate #11 (Br II) 2 Triângulo Mineiro / Male 41 ACRL TgCTBr08 Virulent #11 (Br II) Alto do Paranaíba 3 Vale do Rio Doce Female 51 ACRL, IC, * TgCTBr09 Virulent #11 (Br II) 4 Metropolitana de Belo Male 78 ARL, ** + nd nd + TgCTBr11 Virulent #11 (Br II) Horizonte 5 Noroeste de Minas Male 63 ACRL TgCTBr14 Intermediate #11 (Br II) 6 Sul/ Sudoeste de Minas Female 73 CRL TgCTBr20 Intermediate #11 (Br II) 7 Triângulo Mineiro / Male 80 no TgCTBr27 Intermediate #11 (Br II) Alto do Paranaíba 8 Oeste de Minas Female 31 no TgCTBr05 Avirulent #8 (BrIII) 9 Zona da Mata Male 45 CRL TgCTBr01 Virulent #206 (New) 10 Vale do Jequitinhonha Male 52 no TgCTBr03 Virulent #206 (New) 11 Zona da Mata Male 64 ACRL TgCTBr25 Virulent #206 (New) 12 Oeste de Minas Male 86 CRL TgCTBr15 Intermediate #41 13 Norte de Minas Male 65 ACRL TgCTBr23 Virulent #41 14 Central Mineira Male 31 ACRL TgCTBr04 Virulent # Zona da Mata Male 73 ACRL TgCTBr17 Virulent # Triângulo Mineiro / Female 43 ACRL TgCTBr07 Intermediate #67 Alto do Paranaíba 17 Vale do Rio Doce Male 44 ARL TgCTBr10 Avirulent #207 (New)

25 Metropolitana de Belo 18 Male 61 ACRL, IC TgCTBr12 Intermediate #162 Horizonte 19 Sul/ Sudoeste de Minas Female 64 no TgCTBr13 Virulent #208 (New) 20 Vale do Jequitinhonha Male 61 no TgCTBr18 Intermediate #36 21 Metropolitana de Belo Male 63 ACRL, IC TgCTBr21 Intermediate #209 (New) Horizonte 22 Vale do Jequitinhonha Female 69 ACRL TgCTBr22 Intermediate #210 (New) 23 Oeste de Minas Male 73 CRL TgCTBr24 Intermediate #211 (New) 24 Sul/ Sudoeste de Minas Male 67 ACRL TgCTBr26 Intermediate #212 (New) 25 Metropolitana de Belo Female 56 no TgCTBr19 Intermediate Mixed Horizonte 26 Norte de Minas Male 60 CRL TgCTBr16 Intermediate No typing data 27 Noroeste de Minas Female 40 no TgCTBr06 Nd No typing data a anti-t. gondii IgM antibodies in blood on filter paper using the TOXO IgM kit (Q-Preven, Symbiosis, Leme, Brazil); b anti-t. gondii IgA, IgM and IgG by the enzyme-linked fluorometric assay ELFA-VIDAS, biomérrieux SA, Lyon, France; ARL (unilateral or bilateral active retinochoroidal lesions); CRL (unilateral or bilateral cicatricial retinochoroidal lesions); ACRL (active and cicatricial retinochoroidal lesions ); IC (intracranial calcifications); * dehydration, hypoactivity, malnutrition, child died at 9 months of age; ** hepatomegaly, splenomegaly, adenomegaly, microphthalmia, child died at 4 months of age. Nd no data.

26 Table 2. Multilocus genotyping of Toxoplasma gondii isolates from newborns from Minas Gerais state, Brazil Genetic markers PCR-RFLP Isolates SAG1 5'-3' SAG2 alt.sag2 SAG3 BTUB GRA6 c22-8 c29-2 L358 PK1 Apico Genotype References RH88 I I I I I I I I I I I #10 [10] ME49 II or III II II II II II II II II II II #1 [10] VEG II or III III III III III III III III III III III #2 [10] TgCTBr01, 03, 25 (n=3) u-1 I II III III III II III I III I #206 (New) This study TgCTBr02, 08, 09, 11, 14, 20, 27 (n=7) I I II III III III I III I II III #11 (BrII) [12] TgCTBr04, 17 (n=2) I I II III III III II I I III I #108 [12] TgCTBr15, 23 (n=2) I I I III I II I I I I I #41 [23] TgCTBr05 (n=1) I III III III III III II III III III III #8 (BrIII) [12] TgCTBr07 (n=1) I III III III I III I III III u-1 III #67 [12] TgCTBr10 (n=1) I I u-1 III I II II I I I I #207 (New) This study TgCTBr12 (n=1) I III III III III III II I I III I #162 [26] TgCTBr13 (n=1) I I II III III III III I I III I #208 (New) This study TgCTBr16 (n=1) I Nd III Nd III III II Nd Nd Nd III Nd - TgCTBr18 (n=1) I I I III I III II I III I III #36 [23] TgCTBr19 (n=1) I I/III I/III III I/III III II I/III III I III Mixed - TgCTBr21 (n=1) u-1 I II I III II I I I I III #209 (New) This study TgCTBr22 (n=1) u-1 I II III III III II III I III III #210 (New) This study TgCTBr24 (n=1) I I I III I III I III III I III #211 (New) This study TgCTBr26 (n=1) I I I III III III I III I II III #212 (New) This study

27 Nd no data. u-1 is new allele that is different from the clonal type I, II and III alleles. Downloaded from on July 19, 2018 by guest

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