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Published in final edited form as: Vet Parasitol. 2008 Aug 5;157(0):299–305. doi: 10.1016/j.vetpar.2008.07.036

Genetic diversity of Toxoplasma gondii isolates from chickens from Brazil

JP Dubey a,*, GV Velmurugan a, A Chockalingam b, HFJ Pena c, L Nunes de Oliveira c, CA Leifer b, SM Gennari c, LMG Bahia Oliveira d, C Su e
PMCID: PMC4448939  NIHMSID: NIHMS400212  PMID: 18804329

Abstract

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

Keywords: Toxoplasma gondii, Chickens, Genotype, PCR-RFLP, Brazil

1. Introduction

Toxoplasma gondii infections are widely prevalent in human beings and other animals worldwide (Dubey and Beattie, 1988). Humans become infected post-natally by ingesting tissue cysts from undercooked meat, consuming food or drink contaminated with oocysts, or by accidentally ingesting oocysts from the environment. However, only a small percentage of exposed adult humans or other animals develop clinical signs of disease. Whether the severity of toxoplasmosis in immunocompetent hosts is due to the parasite strain, host variability, or to other factors is largely unknown. Recently, attention has been focused on the genetic variability among T. gondii isolates from apparently healthy and sick hosts. In humans in French Guiana and Suriname, severe cases of toxoplasmosis in immuno-competent patients have been related to T. gondii strains with unusual genetic characteristics (Ajzenberg et al., 2004; Demar et al., 2007). An unusually high prevalence of clinical ocular toxoplasmosis in Erechim, Brazil is thought to be related to characteristics of prevailing T. gondii isolates (Glasner et al., 1992; Khan et al., 2006, 2007).

Most T. gondii isolates from human and animal sources have been grouped into one of three clonal lineages (Type I, II and III) by multilocus enzyme electrophoresis, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), and microsatellite typing (Dardé et al., 1992; Howe and Sibley, 1995; Ajzenberg et al., 2002a,b). We have started a worldwide evaluation of genetic diversity of T. gondii isolates based on DNA derived from live parasites. Our attention has been focused on South America, especially Brazil, because of the collaborative success in obtaining tissues from animals for the isolation of T. gondii. Initial results indicate that the isolates of T. gondii from Brazil are biologically and genetically different from those in North America and Europe (Dubey et al., 2002; Lehmann et al., 2006; Dubey et al., 2007a,b,c). T. gondii isolates from asymptomatic chickens from Brazil were more pathogenic to mice than isolates from Europe or North America, irrespective of the genotype (Dubey et al., 2006). Additionally, most isolates from chickens from Brazil were not clonal, and Type II lineage was absent (Dubey et al., 2007a,c).

Our initial studies were based on one marker (SAG2) (Dubey et al., 2002) and six microsatellites (Lehmann et al., 2006). In the present paper we genotyped 94 T. gondii isolates (previously analyzed with only SAG2) using 10 PCR-RFLP markers to achieve a high resolution in identification and evaluated the entire PCR-RFLP data set on chickens from Brazil (Table 1). Results indicate a very high genetic diversity among isolates of T. gondii, irrespective of the geographic location.

Table 1.

Genetic diversity of T. gondii isolates from different regions of Brazil

T. gondii isolate No. of isolates States in Brazil Reference for isolates Genotype
TgCkBr 117, 119, 120, 122, 123, 124, 126, 127, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140 20 Rond?nia Dubey et al. (2006) 7 genotypes – this study
TgCkBr 7, 8, 10, 11, 13, 16, 17, 19, 23, 24 10 São Paulo Dubey et al. (2002, 2006) 6 genotypes – this study
TgCkBr 26, 27, 28, 30, 31, 32, 33, 34, 36, 37, 38, 40, 41, 42, 44, 45, 46, 47, 48, 49, 50, 51, 52, 54, 55, 56, 57, 58, 59, 60, 61, 62, 64, 65, 66, 67, 69, 74, 75, 76, 78, 79, 80, 81, 82, 84, 85, 86, 87, 88, 89, 90, 92. 53 Rio de Janeiro Dubey et al. (2003a,b, 2006) 29 genotypes – this study
TgCkBr 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 104 11 Paraná Dubey et al. (2003a,b, 2006) 7 genotypes – this study
TgCkBr 107–116, 141–145 15 Pará Dubey et al. (2007a) 11 genotypes – Dubey et al. (2007a)
TgCkBr 146–164 19 Rio Grande do Sul Dubey et al. (2007a) 9 genotypes – Dubey et al. (2007a)
TgCkBr 165, 166 2 Pernambuco de Oliveira et al. (in press) 2 genotypes – this study
TgCkBr 167–170 4 Rio Grande do Norte de Oliveira et al. (in press) 3 genotypes – this study
TgCkBr 171, 172 2 Maranhão de Oliveira et al. (in press) 1 genotype – this study
TgCkBr 173–176 4 Bahia de Oliveira et al. (in press) 3 genotypes – this study
TgCkBr 177–182 6 Ceará de Oliveira et al. (in press) 5 genotypes – this study
TgCkBr 183 1 Sergipe de Oliveira et al. (in press) 1 genotype – this study
TgCkBr 184–187 4 Alagoas de Oliveira et al. (in press) 3 genotypes – this study
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2. Materials and methods

For the present study, in total 151 T. gondii isolates from 11 different regions of Brazil (Fig. 1) were genotyped (Table 1). Of these SAG2 data on 94 isolates were previously reported (Dubey et al., 2002, 2003a,b, 2006). For the present study, these cryopreserved 94 T. gondii isolates were revived in mice at the Animal Parasitic Diseases Laboratory, Beltsville, MD (Dubey et al., 2002). Viable T. gondii parasites were collected from lung tissue of mice that died, and from the brains of mice that survived for more than 30 days after inoculation with cryopreserved material were processed for the present study, and were cryopreserved in liquid nitrogen with DMSO (Dubey and Beattie, 1988) for future studies. Additionally, 23 isolates were recently obtained at the Universidade de São Paulo, São Paulo, SP, Brazil (de Oliveira et al., in press); these isolates were genetically characterized for the first time. Genetic data on the remaining 34 isolates had been published previously (Dubey et al., 2007a) and incorporated in the present study. Details of T. gondii isolation in mice have been published in detail in the papers listed in Table 1 and not repeated here.

Fig. 1.

Fig. 1

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Map of Brazil with sources of chickens sampled.

T. gondii DNA was extracted from tissues of positive mice using DNeasy kit (Qiagen) and genotyped using the genetic markers SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico (Dubey et al., 2006; Su et al., 2006). Data on 34 T. gondii isolates recently published (Dubey et al., 2007a) from chickens from Pará and Rio Grande do Sul (Table 1) were combined with data on newly genotyped 117 isolates and analyzed by SplitsTree4 (Huson, 1998; Huson and Bryant, 2006) in the present study.

3. Results

Multilocus PCR-RFLP genotyping of a total of 151 T. gondii isolates from free range chickens in 13 geographically areas including the states of Alagoas, Bahia, Ceará, Maranhão, Paraná, Pernambuco, Rio de Janeiro, Rio Grande do Norte, São Paulo, Sergipe, Rondönia, Pará and Rio Grande do Sul identified 58 genotype groups (Genotype #1–58) and two isolates with mixed infection (Table 2). Twenty-nine of the 58 multilocus genotypes identified are characterized with two or more isolates, while the other 29 genotypes have single isolate each. Only 1 (TgCkBr 146, now lost) of 151 isolates was clonal Type I strain, and 5 were clonal Type III strains. No clonal Type II lineage was found. One strain (TgCkBr 168) was Type II at all loci, except BTUB. The four major T. gondii lineages (Type BrI, BrII, BrIII and BrIV) previously identified from different animal hosts in Brazil (Pena et al., 2008) were also identified from chickens from much wider geographical regions in this study. In addition, the genotype group #21 was also identified in several states in Brazil (Table 2). Phylogenetic relationships of these 149 chicken isolates (exclude the two with mixed infection) were analyzed by SplitsTree4 (Huson, 1998; Huson and Bryant, 2006). The result is presented as reticulated network in Fig. 2. This results show that there is no clear clustering of genotypes with different geographical regions, and there is lack of type II alleles in the parasite population in Brazil. In addition, most genotypes are found only in one of the 13 states analyzed in this study, with exception of the lineages Type III, BrI, BrII, BrIII, BrIVand Genotype groups #21 (with 10 isolates from six different states in Brazil (Table 1, Table 2 and Fig. 2).

Table 2.

Summary of T. gondii PCR-RFLP genotypes from chickens from Brazil

Genotypes (clonal types) T. gondii isolate Genetic markers
SAG1* (5′ + 3′) SAG2a SAG2b SAG3 BTUB GRA6 c22-8 c29-2 L358 PK1 Apico
Reference (I) RH88 I I I I I I I I I I I
Reference (II) PTG II or III II II II II II II II II II II
Reference (III) CTG II or III III III III III III III III III III III
Reference COUGAR I II II III II II II u-1 I u-2 I
Reference MAS u-1 I II III III III u-1 I I III I
Reference TgCatBr5 I III III III III III I I I u-1 I
#1 7 (TgCkBr 119, 120, 122, 129, 135, 137, 140) u-1 I II III III III III I I III I
#2 2 (TgCkBr 48, 88) u-1 I II III III III II I I III III
#3 1 (TgCkBr 96) u-1 I II III III III II I I III I
#4 3 (TgCkBr 75, 76, 92) u-1 I II III III III III III I III I
#5 (BrIV) 8 (TgCkBr 81, 147, 148, 151, 154, 160, 162, 163) u-1 I II III III III u-1 I I III I
#6 5 (TgCkBr 41, 42, 49, 60, 62) u-1 I II III I III u-1 I I I I
#7 1 (TgCkBr 46) u-1 I II III I III II I I I I
#8 1 (TgCkBr 178) u-1 I II III I III II III III I III
#9 8 (TgCkBr 38, 27, 44, 51, 65, 66, 78, 80) u-1 I II III III III u-1 I III III III
#10 1 (TgCkBr 45) u-1 I II III III III II I III III I
#11 1 (TgCkBr 177) I I II III III III III I III III III
#12 1 (TgCkBr 114) I I II III I III II I III III I
#13 2 (TgCkBr 171, 172) I I I III I II u-1 I III II III
#14 1 (TgCkBr 173) I I I III I II u-1 I I III III
#15 3 (TgCkBr 136, 138, 139) I I I III I II I I I I I
#16 (BrI) 12 (TgCkBr 123, 124, 55, 79, 86, 87, 10, 98, 101, 102, 104, 144) I I I III I II u-1 I I I I
#17 2 (TgCkBr 40, 47) I I I III III II u-1 I I I I
#18 1 (TgCkBr 146) I I I I I I I I I I I
#19 1 (TgCkBr 141) I I I I I I u-1 I I III III
#20 1 (TgCkBr 54) I I I III I III II I I I III
#21 10 (TgCkBr 165, 167, 170, 174, 176, 179, 180, 183, 184, 185 I I I I I III II III III I III
#22 1 (TgCkBr 169) I I I I I III II I III I III
#23 3 (TgCkBr 115, 142, 145) I I I I I I II I III I III
#24 4 (TgCkBr 59, 30, 34, 67) I I I III I III II I III I III
#25 1 (TgCkBr 186) I I I III III III II I III I III
#26 1 (TgCkBr 156) I I I III III III I I III I III
#27 1 (TgCkBr 74) u-1 III III III III III III I III III III
#28 3 (TgCkBr 82, 90, 153) I III III III III III III I III III III
#29 1 (TgCkBr 130) I III III III I III II III III III III
#30 (BrIII) 7 (TgCkBr 11, 7, 17, 131, 132, 133, 134) I III III III III III II III III III III
#31 1 (TgCkBr 8) I III III III III III II III III u-2 III
#32 3 (TgCkBr 111, 112, 182) I III III III III III III III III III I
#33 1 (TgCkBr 181) I III III III III III III III III III III
#34 5 (TgCkBr 31, 56, 158, 161, 164) II or III III III III III III III III III III III
#35 4 (TgCkBr 149, 150, 152, 157) II or III III III III III III I III III I III
#36 1 (TgCkBr 113) I III III I III III III III III I III
#37 1 (TgCkBr 110) I III III I III III III III III I I
#38 1 (TgCkBr 166) I III III I III III III I III I I
#39 2 (TgCkBr 26, 69) I III III III III III II I III III I
#40 3 (TgCkBr 126, 127, 117) I III III III I II II III I I III
#41 2 (TgCkBr 107, 108) I III III III III II u-1 I I I III
#42 2 (TgCkBr 99, 100) I III III III III II u-1 I I II I
#43 2 (TgCkBr 93, 94) I III III III III II I III I II I
#44 5 (TgCkBr 28, 33, 50, 52, 58) I III III III III III I I I u-1 I
#45 1 (TgCkBr 95) I III III III III III I I I III III
#46 2 (TgCkBr 155, 159) u-1 III III III III III u-1 I I III I
#47 1 (TgCkBr 109) I I II I III III II III I III III
#48 1 (TgCkBr 16) I I II I III III I I I II I
#49 2 (TgCkBr 13, 23) I I II III III III I III I II I
#50 (BrII) 3 (TgCkBr 57, 64, 97) I I II III III III I III I II III
#51 1 (TgCkBr 116) u-1 II II III III II II nd II II I
#52 1 (TgCkBr 168) II or III II II II III II II II II II II
#53 1 (TgCkBr 143) I I II III III II u-1 III III III I
#54 1 (TgCkBr 37) I I II III III II u-1 I I III I
#55 1 (TgCkBr 61) I I II I III II u-1 I I III I
#56 2 (TgCkBr 19, 24) I I II III III III u-1 I I u-2 I
#57 4 (TgCkBr 36, 32, 84, 85) I II II III III III u-1 I I III I
#58 1 (TgCkBr 89) I I II III III III u-1 I I III I
Mixed infections 1 (TgCkBr 175) I I + III I + III I + III I II + III II I + III I + III I I + III
1 (TgCkBr 187) I I I I + III I + III III II I + III III I III
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u-1 and u-2 are new alleles that are different from the clonal Type I, II and III alleles.

a

SAG2 marker based on 5′- and 3′-ends of the gene sequence (Howe et al., 1997).

b

A new SAG2 marker based on the 5′-end of the gene sequence (Su et al., 2006).

Fig. 2.

Fig. 2

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NeighborNet phylogenetic network of T. gondii isolates in chickens from Brazil. Isolates from different states of Brazil are color-coded for clarity. RH88, PTG, CTG, TgCgCa1 (a.k.a. Cougar1, COUGAR), MAS and TgCatBr5 are used as reference strains. Numeric number in parenthesis following each isolate’s identification number is the number of strains with the identical genotype from the same states.

4. Discussion

Multilocus genotyping data on these 151 chicken isolates (including two mixed infections) showed that half (29/58) of the identified genotype groups have a single isolate, indicating high diversity of T. gondii isolates in Brazil. This is in supporting of recent findings from cat and dog isolates in Brazil (Pena et al., 2008). The current study of chicken isolates confirms the previous finding in that the Type I strain is rare and there is lack of the clonal Type II strain in Brazil, which is in sharp contrast in that these two lineages are highly prevalent in North America and Europe (Dardé et al., 1992; Howe and Sibley, 1995; Ajzenberg et al., 2002a,b). Identification of the Type III, BrI, BrII, BrIII, BrIV and the genotype group #21 strains from different states of Brazil do suggest that these lineages are wide spread in different regions. The high proportion of genotypes with single isolate indicates high diversity of parasite strains in Brazil. This would suggest there could be many more unique genotypes circulating in the environment. To better understand molecular epidemiology and population structure of T. gondii in Brazil, a much deeper sampling of a variety of animal hosts is necessary.

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