PCR-RFLP Genotyping of Toxoplasma Gondii from Chickens from Espírito Santo State, Southeast Region, Brazil

Veterinary Parasitology 192 (2013) 111–117

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PCR-RFLP genotyping of Toxoplasma gondii from chickens from Espírito

Santo state, Southeast region, Brazil: New genotypes and a new SAG3

marker allele

a,∗ a b c a a

H.F.J. Pena , S.N. Vitaliano , M.A.V. Beltrame , F.E.L. Pereira , S.M. Gennari , R.M. Soares

Departamento de Medicina Veterinária Preventiva e Saúde Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade de São Paulo, São Paulo,

SP, Brazil

Curso de Medicina Veterinária, Universidade de Vila Velha, Rua Viana, s/n, Bairro Boa Vista, Vila Velha, CEP 29102-690, ES, Brazil

Núcleo de Doenc¸ as Infecciosas, Universidade Federal do Espírito Santo, Av. Marechal Campos, 1468, CEP 29040-091, Vitória, ES, Brazil

a r t i c l e i n f o

a b s t r a c t

Article history:

Brazil is one of the regions with the highest prevalences of Toxoplasma gondii in humans

Received 27 June 2012

and animals. Because free-range chickens become infected by feeding from ground con-

Received in revised form 3 October 2012

taminated with oocysts, the prevalence of T. gondii in this host has been widely used as

Accepted 4 October 2012

an indicator of the strains prevalent in the environment. The genetic variability among T.

gondii isolates from different healthy and sick hosts all over the world has been recently

Keywords:

studied. Three clonal genetic lineages (Types I, II and III) were initially recognised as pre-

Toxoplasma gondii

Chickens dominant in Western Europe and the United States. T. gondii strains are genetically diverse

Genotypes in South America. In Brazil, recombination plays an important role in strain diversiﬁca-

PCR-RFLP tion. The objective of this study was to genetically characterise T. gondii isolates from

Diversity free-range chickens from Espírito Santo state, Southeast region, Brazil, using polymerase

Brazil chain reaction-restriction fragment length polymorphism (PCR-RFLP). A total of 44 isolates

among 47 previously described isolates (TgCkBr234-281) from free-range chickens were

included in this study. Strain typing was performed using 12 PCR-RFLP markers: SAG1,

SAG2, alt. SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, Apico and CS3. Eleven geno-

types were identiﬁed. Ten isolates (23%) were grouped into four novel genotypes. Four

isolates, distributed in four counties, corresponded to the Type BrI lineage, the genotype

found most frequently in Brazil. No clonal Types I, II or III lineages were found. Two novel

genotypes were represented by single isolates. Unique alleles were identiﬁed for the mark-

ers SAG1, c22-8 and CS3, and for the ﬁrst time, a unique allele was found for the marker

SAG3. Although a large number of T. gondii genotypes have already been identiﬁed from a

variety of animal hosts in Brazil, new genotypes are continuously identiﬁed from different

animal species. This study conﬁrmed the diversity of T. gondii in Brazil and demonstrates

clonal Type I, II and III lineages are rare in this country.

1. Introduction Brazil is one of the regions with the highest prevalences of T.

gondii in humans and animals (Bahia-Oliveira et al., 2003;

Toxoplasma gondii can infect almost all homoeother- Sobral et al., 2005; Cavalcante et al., 2006). Because free-

mic animals, including humans (Dubey and Beattie, 1988). range chickens become infected by feeding from ground

contaminated with oocysts, the prevalence of T. gondii

in this host has been widely used as an indicator of the

∗ strains prevalent in the environment (Ruiz and Frenkel,

Corresponding author. Tel.: +55 11 3091 1403; fax: +55 11 3091 7928.

E-mail address: [email protected] (H.F.J. Pena). 1980; Dubey et al., 2007a). The genetic variability among

112 H.F.J. Pena et al. / Veterinary Parasitology 192 (2013) 111–117

◦ ◦ ◦

T. gondii isolates from different healthy and sick hosts all 94 C for 30 s, 60 C for 1 min and 72 C for 2 min. The nested

over the world has been recently studied. Three clonal PCR products were treated with restriction enzymes and

genetic lineages (Types I, II and III) were initially recognised resolved in agarose gels by electrophoresis to reveal the

as predominant in Western Europe and the United States RFLP patterns of the isolates.

(Howe and Sibley, 1995; Ajzenberg et al., 2002); however, Mouse virulence data for these T. gondii isolates pre-

a re-evaluation of the population of T. gondii in wildlife in viously reported by Beltrame et al. (2012) were used to

North America revealed a new clonal lineage referred to determine if the CS3 marker can predict parasite virulence

haplogroup 12 (Khan et al., 2011). in mice.

In Brazil, a highly reticulated phylogenetic relationship

was identiﬁed when several genetic markers were used 2.2. SAG3 marker sequencing and genetic diversity of T.

to analyse Brazilian isolates, suggesting that recombina- gondii

tion plays an important role in strain diversiﬁcation in

this country (Pena et al., 2008; Soares et al., 2011). The The nested PCR products for the isolate TgCkBr274,

objective of this study was to genetically characterise T. with a unique allele for the marker SAG3 revealed by PCR-

gondii isolates from free-range chickens from Espírito Santo RFLP, were sequenced along with the nested products for

(ES) state, Southeast region, Brazil, using polymerase chain the strains used as positive controls (Type I, Type II and

reaction-restriction fragment length polymorphism (PCR- Type III). To purify these PCR products, a clean-up sys-

® ®

RFLP) to provide new information on the distribution of T. tem (COSTAR Spin-X , Corning, NY, USA) was used. The

gondii genotypes and on T. gondii diversity in this country. puriﬁed PCR products were sequenced in both directions

using the internal primers (P43S2 and P43AS2) and the

2. Materials and methods ABI PRISM Big Dye Terminator v3.1 sequencing kit (Applied

Biosystems, USA). The sequencing products were analysed

2.1. Multilocus PCR-RFLP strain typing on an ABI377 automated sequencer. Derived sequences

from TgckBr274, RH, PTG and CTG were deposited in Gen-

A total of 44 isolates from 47 previously described T. Bank (accession numbers JX218224, JX218225, JX218226,

gondii isolates (TgCkBr234-281) (Beltrame et al., 2012) JX218227, respectively) and were aligned against each

from free-range chickens in six counties in ES state other using ClustalX (Thompson et al., 1997).

(Colatina, Guarapari, Linhares, Marechal Floriano, Serra and The evolutionary history was inferred using a reticu-

Vila Velha), Southeast region, Brazil, were included in this lated network (Su et al., 2006; Dubey et al., 2008; Soares

study. These counties were located 50–110 km apart from et al., 2011), combining the RFLP genotype results from

each other. For each isolate, peritoneum exudates posi- this paper and the representative RFLP genotypes results

tive for tachyzoites from two infected mice were used to described to date in Brazil. Using Simpson’s Diversity Index

extract DNA using a standard phenol–chloroform protocol, (D) (Simpson, 1949), the genetic diversity of T. gondii

as described in detail by Pena et al. (2006). was calculated and compared with values obtained in

Strain typing was performed using 12 PCR-restriction other studies. This index represents the probability that

fragment length polymorphism (PCR-RFLP) markers: SAG1, two individuals randomly selected from a sample will

SAG2, alt. SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, belong to different species. This value is calculated as

PK1, Apico (Su et al., 2006; Dubey et al., 2007a) and CS3 follows:

(Pena et al., 2008). Reference strains, including Type I (RH), s

Type II (PTG), Type III (CTG) and other strains (TgCgCa1,

D = 1 − nj(nj − 1)

N N −

MAS, TgCatBr5), were used in the genotyping assays as pos- ( 1)

n=1

itive controls. Brieﬂy, the target DNA sequences were ﬁrst

ampliﬁed by two rounds of multiplex PCR using external

primers, followed by nested PCR using internal primers for 3. Results

individual markers as described previously (Su et al., 2006;

Dubey et al., 2007a; Pena et al., 2008). Each multiplex PCR 3.1. Multilocus PCR-RFLP strain typing

reaction was carried out in 25 ␮l containing 1× PCR buffer,

2 mM MgCl2, 200 ␮M each of the dNTPs, 0.10 ␮M each of The genotyping results for the 44 chicken T. gondii iso-

the forward and reverse primers, 0.5 units of Platinum Taq lates for all PCR-RFLP markers are shown in Table 1. Eleven

␮

DNA Polymerase (Invitrogen) and 1.5 l of DNA extract. PCR-RFLP genotypes were detected. No clonal Types I, II

◦

The reaction mixture was incubated at 95 C for 4 min, fol- or III lineages were found. Chicken isolates from Marechal

◦ ◦ ◦

lowed by 25 cycles of 94 C for 30 s, 55 C for 30 s and 72 C Floriano and Colatina counties accounted for four geno-

for 1.5 min. The multiplex products were 1:1 diluted in types each; Vila Velha and Linhares accounted for three,

water and then used for a second round of ampliﬁcation Guarapari for two, and Serra for one. Most of the analysed

(nested PCR) with internal primers for each marker sepa- isolates from chickens from ES state had genotypes previ-

rately. The nested PCR was carried out in 25 ␮l containing ously described in other states (São Paulo, Rio de Janeiro

1× PCR buffer, 2 mM MgCl2, 200 ␮M each of the dNTPs, and Paraná) in the same region of Brazil.

␮

0.30 M each of the forward and reverse primers, 0.5 units The Type BrI genotype (ToxoDB RFLP genotype #6) was

of Platinum Taq DNA Polymerase (Invitrogen) and 1.5 ␮l found in four isolates, each from a different county, indi-

of diluted multiplex PCR products. The reaction mixture cating that this genotype is well distributed in the state

◦

was treated at 95 C for 4 min, followed by 35 cycles of of ES. This genotype has already been found in ﬁve other

H.F.J. Pena et al. / Veterinary Parasitology 192 (2013) 111–117 113 and

BrI

Sergipe

ToxoDB RFLP-genotype #6 Type #162 #109 #14 #75 #65 #108 #206 #213 #214 #215 Ceará, Brazil.

from

(state) Bahia,

(SP)

–

11 Brazil

Norte, isolates

71,

10,

from 9,

(PR) 62,

(PR)

other –

–

(MS)

(RJ) 55,

– 30

–

(MS).

104

Grande

with

54, isolates

(RO) 87

21, (SP)

207 TgShBr8,

Sul

j –

Rio

– 102, 53,

(RJ) (RJ)

86,

17, 9

(PA) (RS) (SP) do

(CE) – –

other

203, (SP) (SP) (SP) (SP) (SP) 124 47,

– – – (SP) 4,

–

(RJ)

identity

79, 101,

12, – – – – –

(SP) 90 88 7

–

with

and

Grosso

Rondônia, genotype genotype genotype genotype

TgCkBr201, TgCatBr42, TgCkBr144 TgCkBr153 TgDgBr3, TgCatBr2, TgCkBr10 TgCkBr55, TgCkBr98, TgCkBr123, TgCkBr177 TgGtBr2, TgCpBr14 Mato

TgCkBr82, Identity c d e f f f f g h i j TgCpBr1 f TgCatBr15 g TgCkBr48, TgCatBr82 TgCkBr89 TgCatBr57 New New New New TgDgBr19 analysis

Paraná, k

from

CS3 I II II II I II I II I I u-2

Janeiro, PCR-RFLP

chickens de

Apico I I III III III I I I III I I

Rio

and PK1 I III III III III III III III III I III Brazil,

of .

Paulo, (SP)

L358 I I III III I I I I I I I São

region

Paulo

(2007a)

al.

São c29-2 I I I I I I I III III I I

states:

et .

from Southeast

c22-8 u-1 II III III II u-1 II II u-1 II II (2011) Dubey

following

state, al. by

(2010)

isolates

the

al. .

GRA6 II III III III III III III III III II III . Santo et

. from

Soares

(2009)

genotyped

BTUB I III III III III III III III III I III cabyparas (2007b) by .

al.

Ragozo (2008)

Espírito

al. ).

and ). by al.

states et chickens

(2006) SAG3 III III III III III III III III III III u-1 Yai et from

1997

Sul

2006 al.

goats by

from genotyped al.,

et Dubey

Pena

al.,

Su by et by SAG2 I III II III II II II II II I II

genotyped

chickens sheep,

states by

a ( isolates

Grande

Howe Sul genotyped (

cats,

Rio

SAG2 do of (AL):

genotype

genotyped state genotyped

free-range 5 I III I III I I I I I I I and

respectively, sequence and genotyped

sequence Grosso

state

state studies

Paulo Pará from

(SE) goat, gene

state

PCR-RFLP SAG1 I I I u-1 I I u-1 I u-1 u-1 I gene (2008) São

Mato Paulo

the Paulo from

and

(CE), al.

the of

isolates

et São Paraná Vila from São

of from

Velha genotyping

(BA), sheep

-end

from chickens from gondii

from 5 the M.

Linhares Vila Serra,

Dubey -ends

from in (RN), 3

the cats by

chickens cats

dogs

capybaras from Velha

Floriano Floriano Floriano on

and

(RO), -

Velha County Linhares, M. Colatina Colatina M. Vila Colatina, Colatina, Guarapari Guarapari M. Floriano, from from from

from

from 5 isolates

Toxoplasma included

based isolates on

(PR),

genotyped

(SP):

240, 279 271 only

isolates – isolates isolates

isolates

(RJ), isolates (RS):

248,

based

marker was 281 270, 239, 278,

(PR): (SP): (SP), (SP):

261–264 (SP): (PA), (MS): 247,

– – TgGtBr –

– – – –

277, 269, 241 238, 246, 260 252

genotyping

SAG2

marker

respectively, 243,

marker

273, 268, 250, 237, 235, 245, 259, 1

new

253–256, 242, SAG2 A TgCatBr TgCatBr TgDgBr TgCkBr TgCkBr TgCkBr TgCpBr TgShBr, CS3 i j f c a e 265, 267, 249, 236, 272 TgCkBr 280 234, 244, 258, 257 TgCkBr 274 TgCkBr TgCkBr TgCkBr TgCkBr TgCkBr TgCkBr TgCkBr Isolate TgCkBr TgCkBr g k b d h Table Multilocus Alagoas,

114 H.F.J. Pena et al. / Veterinary Parasitology 192 (2013) 111–117

Fig. 1. Nested polymerase chain reaction using P43S2 and P43AS2 primers (A) and restriction patterns after digestion with NciI restriction enzyme (B)

for SAG3 marker from DNA of Brazilian T. gondii isolate TgCkBr274 (genotype #215). RH (Type I), PTG (Type II) and CTG (Type III) were used as reference

strains. Fragments were resolved in 2.5% agarose gel. M, molecular marker: Gene Ruler 50 bp DNA ladders (Fermentas, Hanover, MD, USA). (C) negative

control.

hosts (dogs, cats, capybaras, sheep and goats) and in chick- No difference was observed between alleles I and II of

ens from Brazil. Type BrI has been described not only in the marker CS3 when mouse mortality was compared. Both

isolates from Brazilian states in the same region as ES (São alleles were strongly associated with virulence in mice, as

Paulo, Rio de Janeiro and Paraná states) but also in isolates 100% and 98.7% of mice harbouring strains with alleles I

from the Northeast region (Pará and Rondônia states) and and II died within 4 weeks post inoculation, respectively,

Central-West region (Mato Grosso do Sul state). according to Beltrame et al. (2012). No allele III at CS3 was

Chicken isolates TgCkBr267-271 from Marechal Flo- found among the genotypes.

riano county had RFLP genotype #162, which has been

previously described only in a capybara from São Paulo 3.2. SAG3 marker sequencing and genetic diversity of T.

state (same region). Chicken isolates TgCkBr249, 250 and gondii

252 from Colatina county had RFLP genotype #109, which

has been previously found in a chicken isolate, but this After sequence analysis, the nested PCR product for

chicken isolate was from Ceará state, located in the North- the marker SAG3 from TgCkBr274 was shown to have an

east region. Chicken isolates 236, 237 and 241, from insertion of 21 nucleotides between positions 1003 and

Colatina county, had RFLP genotype #14, which has been 1004 from the start of the gene (numbering based on a

previously reported in a cat, a dog and two chickens also homologous sequence of T. gondii registered in GenBank

in the Southeast region and in a chicken in the South with the accession number JF312642). This insertion cor-

region (Rio Grande do Sul state). RFLP genotype #108 corresponds to the insertion of seven amino acids in the

responded to 17 chicken isolates from two counties in this putative protein. In addition to the 21 bp insertion, other

study and has been reported before only in a cat from São single-nucleotide polymorphisms were detected in the

Paulo (TgCatBr57). This genotype is most likely a common new sequence. This sequence differed from RH at three

genotype circulating in the state of ES. nucleotide positions (1027, 1053 and 1061), from CTG at

In the present study, ten isolates (23%) distributed in three nucleotide positions (1027, 1037 and 1046), and from

four counties were grouped into four novel RFLP genotypes PTG at six nucleotide positions (981, 1001, 1005 1027, 1044

based on the 12 markers analysed. The data were uploaded and 1076), as shown in Fig. 2.

at ToxoDB.org and designated as genotypes #206, #213, The evolutionary reconstruction revealed a population

#214 and #215. with high genetic diversity, with taxa positioned in the typ-

The new genotypes identiﬁed had unique alleles for the ical star-like network (Fig. 3). The value of D for the whole

markers SAG1 (genotypes #206, #214 and #215), c22-8 population surveyed in this study was 0.86.

(genotype #213) and CS3 (genotype #215), and for the ﬁrst

time, a unique allele, denoted u-1, was found for the marker 4. Discussion

SAG3 (genotype #215).

The enzyme NciI cut the SAG3 fragment from RH into Free-range chickens from different regions have been

three smaller segments of 65, 62 and 99 bp, respectively, extensively surveyed for T. gondii isolation and genotyping

whereas the homologous PCR products from CTG were cut in Brazil (Dubey et al., 2008).

into two fragments of 65 and 161 bp. The SAG3 fragment In the present study, only non-archetypal genotypes

from PTG was not cleaved by NciI. The RFLP analysis of the were found, supporting other ﬁndings that archetypal

SAG3 fragment from TgCkBr274 (genotype #215) revealed lineages are rare in Brazil. Seven atypical genotypes, cor-

a pattern different from those of the three archetypes. In responding to 34 chicken isolates from ﬁve counties, had

this case, the SAG3 fragment from TgCkBr274 was cleaved already been reported for isolates from different hosts,

into three segments of 65, 83 and 99 bp (Fig. 1). mostly from the Southeast region of Brazil. The presence

H.F.J. Pena et al. / Veterinary Parasitology 192 (2013) 111–117 115

Fig. 2. Alignment of sequences of nested PCR products from SAG3 locus from T. gondii Types I, II, and III and genotype # 215. Shaded boxes at the ends of

the sequences correspond to hybridisation sites of primers. The shaded box in the middle of the alignment corresponds to insertion sequence found in the

genotype #215. Unshaded boxes correspond to the cleavage sites for the enzyme NciI.

Fig. 3. Neighbour-Net phylogenetic network with representatives of T. gondii genotypes available in ToxoDB. Genotypes in ES state are marked in red, and

clonal genotypes are marked in blue.

116 H.F.J. Pena et al. / Veterinary Parasitology 192 (2013) 111–117

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