Infection, Genetics and Evolution 113 (2023) 105473

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Infection, Genetics and Evolution

journal homepage: www.elsevier.com/locate/meegid

Genotyping of toxoplasma gondii isolates from México reveals

non-archetypal and potentially virulent strains for mice
Claudia Patricia Rico-Torres a, 1, Luis Fernando Valenzuela-Moreno a, 1, Héctor Luna-Pastén a,
Carlos Cedillo-Peláez a, Dolores Correa a, 2, Elizabeth Morales-Salinas b, José Juan Martínez-
Maya b, Bruna Farias Alves c, Hilda Fátima Jesus Pena c, **, Heriberto Caballero-Ortega a, *
a
Laboratorio de Inmunología Experimental, Instituto Nacional de Pediatría, Insurgentes Sur 3700C, Colonia Insurgentes Cuicuilco, Alcaldía Coyoacán, C.P. 04530
Ciudad de México, Mexico
b
Facultad de Medicina Veterinaria y Zootecnia, Universidad Nacional Autónoma de México, Av. Universidad 3000, Circuito Exterior s/n, Alcaldía Coyoacán, C.P.
04510, Ciudad Universitaria, Ciudad de México, Mexico
c
Laboratório de Doenças Parasitárias, Departamento de Medicina Veterinária Preventiva e Saúde Animal, Faculdade de Medicina Veterinária e Zootecnia, Universidade
de São Paulo - USP, São Paulo, SP, Brazil

A R T I C L E I N F O A B S T R A C T

Keywords: Genotyping and virulence studies of Toxoplasma gondii are essential to investigate the pathogenesis of strains
Toxoplasma gondii circulating worldwide. In this study, eight T. gondii isolates obtained from a congenitally infected newborn, a
Genotyping calf, two cats, three dogs, and a wallaby from five states of México were genotyped by Mn-PCR-RFLP with 11
PCR-RFLP
typing markers (SAG1, SAG2 5′3’, alt. SAG2, SAG3, BTUB, GRA6, c22–8, c29–2, L358, PK1 and Apico), five
Microsatellite analysis
virulence markers (CS3, ROP16, ROP17, ROP18 and ROP5), 15 microsatellite markers (TUB-2, W35, TgM-A, B18,
Sequencing
Virulence B17, M33, IV.1, XI.1, M48, M102, N60, N82, AA, N61, N83), and sequencing. A phylogenetic network was built to
México determine the relationship between Mexican isolates and those reported worldwide. Six different genotypes were
identified by polymerase chain reaction-restriction fragment length polymorphism (PCR–RFLP), ToxoDB #8,
#10, #28 (n = 3), #48, #116, and #282. Genotyping by microsatellite analysis differentiated the three PCR-
RFLP genotype #28 isolates into two strains, revealing a total of seven microsatellite genotypes. Three
different allele combinations of ROP18/ROP5 virulence markers were also found, 3/3, 1/1, and 4/1. The last two
combinations are predicted to be highly virulent in the murine model. According to the phylogenetic network,
the T. gondii strains studied here are related to archetypal strains I and III, but none are related to the strains
previously reported in México. The genotypes identified in this study in different species of animals demonstrate
the great genetic diversity of T. gondii in México. The ToxoDB-PCR-RFLP #28 genotype was found in three
isolates from different hosts and states. Additionally, four of the isolates are predicted to be highly virulent in
mice. The next step will be to perform in vitro and in vivo assays to determine the phenotype of these T. gondii
isolates in murine models.

1. Introduction 2022). At least 318 genotypes distributed across 16 haplogroups and 6

clades from around the world have been described (Lorenzi et al., 2016;
Toxoplasma gondii is one of the most successful pathogens due to its Meireles et al., 2022). A few intercontinental T. gondii lineages represent
worldwide distribution and ability to infect virtually any warm-blooded the majority of the isolates in the world (type I, II, III, BrI/Africa 1
animal (terrestrial and marine mammals and birds) (Delgado et al., -ToxoDB #6-), while in Central and South America, most of the isolated

* Correspondence to: H. Caballero-Ortega, Insurgentes Sur 3700C, Col. Insurgentes Cuicuilco, Ciudad de México C.P. 04530, Mexico.
** Correspondence to: H. Fátima Jesus Pena, Av. Prof. Dr. Orlando Marques de Paiva, 87, Cidade Universitária Armando de Salles Oliveira, São Paulo, SP CEP
05508-270, Brasil.
E-mail addresses: hfpena@usp.br (H.F.J. Pena), hcaballero_2000@yahoo.com.mx (H. Caballero-Ortega).
1
These authors have contributed equally to this work and share first authorship.
2
Present address: Dirección de Investigación. Centro de Investigación en Ciencias de la Salud. Fac. Ciencias de la Salud. Universidad Anáhuac México; Huxqui­
lucan, EdoMex, México.

https://doi.org/10.1016/j.meegid.2023.105473
Received 19 April 2023; Received in revised form 14 June 2023; Accepted 20 June 2023
Available online 22 June 2023
1567-1348/© 2023 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
C.P. Rico-Torres et al. Infection, Genetics and Evolution 113 (2023) 105473

strains are hybrids from the intercontinental lineages and wild strains 2.3. Origin of the isolates
from the Americas (Galal et al., 2022). Epidemiological studies have
determined that the genotypes and clones of T. gondii circulating in Isolates of T. gondii were obtained from 2008 to 2016 (Table 1). One
different regions of the world are factors related to the pathogenesis of of them was from a congenitally infected newborn (TgHumMxCdMx1),
the agent (Gargaté et al., 2022). The virulence of a strain in murine and seven were from domestic and wild animals: three dogs (Canis
models can be defined by the outcome of the infection, where type I familiaris), two domestic cats (Felis catus), one calf (Bos taurus) and one
strains are highly virulent, type II strains are intermediately virulent and Bennet's wallaby (Macropus rufogriseus). TgCowMxHgo1 and
type III strains are avirulent, while non-archetypal strains have variable TgCatMxHgo1 were isolated from the brain of a calf and a cat, respec­
virulence (Su et al., 2002; Sanchez and Besteiro, 2021). Recently, 204 tively, both from Tizayuca, Hidalgo. TgDogMxCdMx1 was obtained
isolates worldwide were analyzed, and based on mortality in outbred from a pool of tissues (brain, heart, liver, lung, spleen and striated
murine models, a ratio of phenotypic diversity of 33%, 35%, and 32% of muscle) from a stray dog from México City. The other isolates had been
highly virulent, intermediately virulent and avirulent isolates, respec­ previously described (Table 1).
tively, was demonstrated. Of these, 57 isolates were obtained from
human clinical cases, and more than half were highly virulent in the 2.4. Bioassay in mice
murine model. These results could be biased because most of those
isolates were obtained from clinical cases, and thus far, there is no link The three new isolates were obtained following the methodology
between virulence in mice and humans (Calero-Bernal et al., 2022). described by Rico-Torres et al. (2015) and according to the Mexican
Some loci that play an important role in T. gondii virulence were guidelines for the handling and care of mice issued in NOM-062-ZOO-
identified when tested in a murine model. A complex formed by ROP17, 1999. Briefly, tissues were manually macerated in a mortar with ster­
ROP18 and ROP5 is involved in the phosphorylation and inactivation of ile 7.2 pH phosphate buffered solution (PBS) and 10,000 IU of penicillin
immunity-related GTPases (IRGs) and, together with ROP16, modulates G (PiSA, México); then, 500 μL of the homogenate was inoculated
the host immune response during the invasion of the parasite (Weil­ intraperitoneally into BALB/c mice, according to Table 1. The inocu­
hammer and Rasley, 2011; Zhang et al., 2019). However, some of the lated mice were monitored for 60 days by indirect ELISA and detection
ROP18/ROP5 allele combinations are highly predictive of virulence in a of clinical signs of T. gondii infection. Animals with severe disease were
murine model, but since humans lack the genes that encode for the IRGs, euthanized, and imprints of the lung and brain were examined under
they employ distinct immune mechanisms to control T. gondii infection optical microscopy to view tachyzoites or tissue cysts. Mice that sur­
(Gazzinelli et al., 2014; Shwab et al., 2016). vived for 60 days were also euthanized and were considered positive if
México is located in a geographic transition zone between North and tachyzoites or tissue cysts were found in their tissues. All isolates were
South America. Several genotypes have been reported throughout the further intraperitoneally propagated in mice, and the tachyzoites ob­
country. In the northern state of Durango, ToxoDB #9, #73, #74, #155, tained were cryopreserved in 90% fetal bovine serum (ATCC cat.
and #222 genotypes have been isolated from dogs, cats, pigeons and a 30–2020), and 10% dimethyl sulfoxide (DMSO, Sigma cat. D2650–100
puma, and most of their alleles were type II (Dubey et al., 2009, 2013). mL) and stored in liquid nitrogen at − 196 ◦ C until use (Döşkaya et al.,
In the center of the country, ToxoDB #2 and #10 genotypes and mixed 2013).
infections related to type I strains have been found in chickens and cases
of congenital toxoplasmosis in humans (Dubey et al., 2009; Rico-Torres 2.5. DNA extraction
et al., 2018). In southern México, high genetic diversity in T. gondii,
including ToxoDB #10, and mixed infections related to type I and III DNA was extracted from thawed tachyzoites of each isolate following
strains have been described in cats, pigs and wildlife (Cubas-Atienzar the manufacturer's instructions for the Qiagen Gentra® Puregene®
et al., 2018; Torres-Castro et al., 2016; Valenzuela-Moreno et al., 2019). Tissue kit (Hilden, Germany). The obtained DNA was quantified in a
In this study, we aimed to genotype isolates from diverse regions of spectrophotometer (Thermo Scientific Nanodrop™ 1000, MA, USA) and
México from different definitive and intermediate hosts using an stored at − 20 ◦ C until use. The isolates not previously genotyped were
expanded panel of virulence markers. We carried out three different subjected to genotyping by PCR-RFLP and/or MS (Table 1).
genotyping approaches, including PCR-RFLP, microsatellite analysis
(MS) and sequencing, to investigate the diversity of T. gondii in México 2.6. PCR for detection of T. gondii and Mn-PCR-RFLP genotyping
and to predict the virulence degree of the isolates as reported in murine
models. The three new isolates were verified to be T. gondii by amplification
of the B1 gene or the 529 bp noncoding repetitive region (SeqRep529)
2. Materials and methods by end-point PCR using the primers described by Homan and Vercam­
men (2000) and Robert-Gangneux et al. (2010).
2.1. Ethical considerations The genotypes of 5/8 isolates determined by PCR-RFLP have been
previously published (Table 1). The other three isolates were genotyped
This study was approved by the Institutional Review Board of the by multilocus nested-polymerase chain reaction-restriction fragment
Instituto Nacional de Pediatría of the Ministry of Health of México length polymorphism (Mn-PCR-RFLP) for 11 genetic markers: SAG1,
(Organization number IORG0011533), which includes the Research and SAG2 5′ 3′, alt. SAG2, SAG3, BTUB, GRA6, c22–8, c29–2, L358, PK1, and
Animal Care Committee (protocol number 2012/013 and 2020/039) Apico (Su et al., 2010). All PCR assays were performed with AmpliTaq
and human cases referred to INP due to clinical T. gondii infection. Gold™ polymerase (Thermo Fisher Scientific, cat. 4311806, MA, USA).
Appropriate controls were included in each assay, i.e., DNA samples of
2.2. Participating laboratories strains representative of the three archetypal lineages, RH, Me49 and
VEG (Toxoplasma gondii ATCC® 50861™) strains (types I, II and III,
All PCR-RFLP assays (typing and virulence markers) were performed respectively), were used as positive controls, and sterile water was used
at the Experimental Immunology Laboratory of the Instituto Nacional de as a negative control. All PCR products were visualized in 1.5% agarose-
Pediatría, México. Sequencing was carried out at the Institute of Biology TBE gels stained with ethidium bromide (EtBr). To ensure that there was
of UNAM, México. Microsatellite genotyping was carried out at the no cross contamination during the nested assays, positive and negative
Parasitic Diseases Laboratory, Department of Preventive Veterinary reamplification controls were included (Rico-Torres et al., 2022). After
Medicine and Animal Health, Faculdade de Medicina Veterinária e nested PCR, the resulting amplicons were digested with the specific
Zootecnia, Universidade de São Paulo, Brazil. restriction enzymes (New England, Biolabs®, USA) for each marker

2
C.P. Rico-Torres et al. Infection, Genetics and Evolution 113 (2023) 105473

Table 1
Epidemiological data of Toxoplasma gondii isolates obtained from different states of México.
Isolation Host Origin Year Tissues used to Infected mice **Days of Genotyping by Genotyping by MS Predicted
attempt / Total mice euthanasia PCR-RFLP virulence in
isolations mouse

TgCowMxHgo1 Calf Hidalgo 2008 Brain 1/3 7 Unpublished Unpublished Unpublished

Mexico
TgDogMxCdMx1 Dog 2008 Pool of tissues^ 1/2 60 Unpublished Unpublished Unpublished
City
Rico-Torres et al.,
TgCatMxCol2 Cat Colima 2008 Pool of tissues^ 1/2 21 Unpublished Unpublished
2015
Brain 1/1
TgCatMxHgo1 Cat Hidalgo 2013 Heart 0/1 60 Unpublished Unpublished Unpublished
Diaphragm 0/1
Infected mice:
Lung 1/2 Valenzuela-
9–21
TgWbMxPue1 &Wallaby Puebla 2013 Liver 2/2 Moreno et al., Unpublished Unpublished
Not infected
Spleen 1/2 2022
mouse: 60
Mexico Rico-Torres et al.,
TgHumMxCdMx1* &Human 2014 Blood 1/2 60 Unpublished Unpublished
City 2018
Infected
Brain Valenzuela- Valenzuela-
0/1 mouse: 20
TgDogMxChp3 Dog Chiapas 2016 Heart + Moreno et al., Moreno et al., Unpublished
1/1 Not infected
diaphragm 2020 2020
mouse: 60
Infected
Brain Valenzuela- Valenzuela-
0/1 mouse: 13
TgDogMxChp11 Dog Chiapas 2016 Heart + Moreno et al., Moreno et al., Unpublished
1/1 Not infected
diaphragm 2020 2020
mouse: 60

*Congenital infection. ^Brain, heart, liver, lung, spleen and striated muscle. **Infected mice with serious clinical signs of illness were euthanized before 60 days.
&Isolates obtained from clinical cases, virulent for the host of origin.

following a previously described methodology (Su et al., 2010). agarose-TBE gels and photodocumented. The restriction products from
the ROP17 locus that had no similarity to the reference strains were
2.7. Microsatellite genotyping further resolved in 4 to 15% polyacrylamide gels stained with EtBr to
improve the resolution.
The genotypes of 2/8 isolates determined by MS had been previously
published (Table 1). Six of the eight isolates were subjected to MS 2.9. Sequencing
genotyping following the methodology published by Ajzenberg et al.
(2010). Briefly, 15 microsatellite markers were included in a single The TgDogMxChp3 isolate showed a non-archetypal ROP17 RFLP
multiplex assay: TUB2, W35, TgM-A, B18, B17, M33, MIV.1 and MXI.1 pattern; thus, it was again amplified using internal primers and
distinguished archetypal I, II and III strains from non-archetypal strains, sequenced for the sense and antisense strands at the Institute of Biology-
while M48, M102, N60, N82, AA, N61, and N83 were used to trace the UNAM, México. The electropherograms were analyzed using SnapGene
T. gondii fingerprint. Electrophoresis was conducted in an automatic viewer® v4.1.4, and the mean Phred quality was over 30 in both se­
sequencer (3500 Genetic Analyzer AB Hitachi, USA), and DNA from the quences. The consensus sequence obtained from both strands was
PTG strain (archetypal type II) was used as a positive control. All results aligned and compared to those of ROP17 sequences of reference strains
were analyzed with GeneMapper 4.1 software (Applied Biosystems, (GT1 -TGGT1_258580-, Me49 -TGME49_258580- and VEG -
USA). As described, the forward primer was labeled at the 5′ end with 6- TGVEG_258580- available at www.toxodb.org) and known strains that
FAM (TUB2, B18, M33, MXI.1, M48, N61, and N83), HEX (W35, TgM-A, bear alleles 3 and 4 (Cougar and MAS) available at https://toxodb.org/
B17, MIV.1, and N82) or NED (M102, N60, and AA). MS primers were toxo/app/search/organism/GenomeDataTypes/result). The consensus
synthetized at Applied Biosystems™. sequence was compared with virtual digestion products using the
Benchling® digestion tool (www.benchling.com) to confirm the RFLP
2.8. PCR-RFLP genotyping for virulence markers pattern observed in the polyacrylamide gel electrophoresis. The ob­
tained sequence was deposited in GenBank® (www.ncbi.nlm.nih.go
The genotyping of virulence markers in this study is unprecedented. v/genbank) with accession number OP850033.
The eight isolates were included in the Mn-PCR-RFLP to genotype CS3
(Pena et al., 2008), ROP16, ROP17, ROP5, and ROP18 (Shwab et al., 2.10. Phylogenetic network
2016) virulence markers. Since the internal and external primers of the
CS3 marker have the same melting temperature (tm) as the oligonu­ To determine the phylogenetic relationship between the Mexican
cleotides of the ROP markers, it was also included in the Mn-PCR-RFLP. isolates, including those reported by Alvarado-Esquivel et al. (2011) and
All PCR assays were performed using AmpliTaq Gold™ polymerase, and Dubey et al. (2004, 2009, 2013) and those representative of each hap­
DNA of the RH, Me49 and VEG (types I, II and III, respectively) strains logroup (RH, GT1, Me49, PTG, PRU, VEG, CTG, MAS, GUY-RUB, FOU,
were included as positive controls, while sterile water was used as a Wiktor, CAST, TgCtBr05, TgCatBr01, p89, GUY-VAND, Cougar, Type
negative control. The PCR products were resolved in 1.5% agarose-TBE 12, Type 12-X-A, Chinese1, GAB2–2007-GAL-DOM2, and TgCtCo05), a
gels. Additional positive and negative reamplification controls were also dataset of multilocus PCR-RFLP typing markers (Su et al., 2010) was
included to discard cross contamination during nested assays (Rico- composed and analyzed by SplitsTree4 (Huson, 1998; Huson and Bry­
Torres et al., 2022). Restriction assays were performed as previously ant, 2006).
described using recommended restriction enzymes (New England, Bio­
labs® USA, and Thermo Scientific® USA), and the results were
compared with the patterns obtained for reference strains (Pena et al.,
2008; Shwab et al., 2016). All restriction products were resolved in 3%

3
C.P. Rico-Torres et al. Infection, Genetics and Evolution 113 (2023) 105473

3. Results

Ajzenberg et al., 2010

Valenzuela-Moreno

Valenzuela-Moreno
Shwab et al., 2018
3.1. Bioassay in mice

Su et al., 2012
Su et al., 2012
Su et al., 2012

Su et al., 2012

Su et al., 2012

et al., 2020

et al., 2020
Eight isolates obtained via bioassay in mice from five different states

This study

This study

This study

This study
This study
This study
Reference
in México were included in the present study (three were new isolates
described here). A total of twenty-two mice were used to inoculate tissue
samples from the different hosts. Of these, eleven mice were infected: 8/
11 mice had serious clinical signs of disease and were euthanized or died

Non-archetypal §

Non-archetypal ^
Non-archetypal*
Non-archetypal*
Non-archetypal^

Non-archetypal
Non-archetypal

Non-archetypal
Type I variant
between 7 and 20 days postinoculation, and 3/11 mice survived to 60

MS genotype

archetypal*§
days, showing no clinical signs of infection but were serologically pos­

*Congenital infection. §Identical MS typing alleles as p89. ^Identical MS typing alleles as TgCkBr130. TgCatMxCol2 and TgDogMxCdMx1 have identical MS genotype (alleles in bold).
Type III
Type III

Type III
Type II
Type I
itive (Table 1).

(New)
Non-
3.2. PCR-RFLP genotyping

N83

306
306
306
310
312
312
314
314
314

312

306

314

314

314

314
Of the eight isolates included in this study, six different genotypes

N61

103

107
were found. One was ToxoDB #8 - type BrIII (TgDogMxChp3), one was

93
93
87
91
89
89
87
89

89

87

89

87

89
archetypal type I ToxoDB #10 (TgCowMxHgo1), three were ToxoDB

263
263
265
265
267
269
261
265
265

263

263

279

261

263

277
#28 (TgCatMxCol2, TgDogMxCdMx1 and TgDogMxChp11), one was

AA
ToxoDB #48 (TgCatMxHgo1), one was ToxoDB #116 (TgWbMxPue1)
and one was ToxoDB #282 (TgHumMxCdMx1) (Table 2).

N82

123
123
119
111
111
111
111
111
111

111

119

111

111

113

119
Fingerprinting Markers
3.3. Microsatellite genotypes

N60

138
138
145
142
153
147
142
140
142

147

145

142

145

142

147
From the eight isolates, seven genotypes were identified:

M102

168
168
168
174
188
190
190
174
192

190

166

192

190

190

168
TgCowMxHgo1 was a type I variant (type II TgM-A allele), making it a
new genotype; TgCatMxHgo1 was an archetypal type III strain and was
M48

211
211
almost identical to the CTG strain but with a 263 AA allele; and five

209
215
213
215
213
237
213

215

209

213

213

213

211
isolates were non-archetypal genotypes: TgHumMxCdMx1 and
TgDogMxChp3 shared the same typing marker alleles with p89 (ToxoDB
MXI.1

358
358
358
356
356
356
356
356
356

356

358

356

356

356

358
#08), although they were independent strains with differences among
the fingerprinting markers. Additionally, the TgWbMxPue1 isolate
MIV.1

shared the same typing alleles with the Brazilian strain TgCkBr130, but

276
276
274
274
278
278
278
278
278

278

274

278

278

278

276
they had differences in four fingerprinting markers (M102, N82, AA and
N61). Finally, TgCatMxCol2, TgDogMxCdMx1 and TgDogMxChp11
M33

165
165
169
169
165
165
165
165
169

165

169

165

165

169

165
shared the same typing alleles, but the latter had differences at the N60,
N82, AA, N61, and N83 markers. Isolates TgCatMxCol2 and

342
342
B17

342
336
336
336
348
362
348

336

342

348

348

348

342
TgDogMxCdMx1 were identical in all 15 MS markers (Table 2).

158
158
B18

160
158
160
160
160
160
160

160

160

160

160

160

158
3.4. Prediction of virulence degree in mice by specific genetic markers
PCR-RFLP and Microsatellite genotyping of Toxoplasma gondii isolates from México.

Five different combinations were found with a predominance of al­

TgM-

205
205
209
207
205
205
205
205
205

205

207

205

205

205

205
leles related to the RH and VEG strains, and four strains displayed non-
A

archetypal alleles as well (Table 3). Four strains had a type I allele, and
Typing Markers

W35

248
248
248
242
242
242
242
242
242

242

248

242

242

242

248
four had a type III allele at the CS3 marker. The ROP16 marker revealed
that all strains had the type 1 allele, while ROP17 identified six type 1
TUB2

strains, one type 2, and one type 4, which were found to be identical to
291
291
291
289
289
289
291
291
289

289

291

291

291

289

291

the MAS strain (Fig. 1; Supplementary Fig. 1). At the ROP18 locus, we
found three different alleles, one type 1, four type 3, and three type 4
ToxoDB PCR-

(Fig. 2). Finally, four isolates were type 1 and four type 3 at the ROP5
locus.
#116

#282

#116
RFLP

#10

#19

#48

#10

#28
#28

#28
#1
#2
#2
#8

#8

3.5. Phylogenetic network

Country

México

México

México

México

México
México
México

México
Brazil
Brazil

The isolates included in this study formed three groups. The

USA
USA
USA
USA
USA

TgCowMxHgo1 isolate was grouped in the branch of the type I strains

with GT1. The TgDogMxChp3 isolate was grouped with the p89 (type
Wallaby
Chicken
Human

Human
Sheep

BrIII) strain and was very close to the isolates TgCatMxHgo1 and
Goat
Host

Dog

Dog

Dog
Calf
Cat

Cat

Cat

Cat
Pig

TgWbMxPue1, both sharing genetic relationships with type III strains,

and thus were placed in the same branch as the VEG strain. Other sub­
TgHumMxCdMx1

TgDogMxCdMx1

TgDogMxChp11

group (TgCatMxCol2, TgDogMxCdMx1 and TgDogMxChp11) shared a

TgCowMxHgo1

TgDogMxChp3
Present Study
TgCatMxHgo1

TgWbMxPue1
TgCatMxCol2

branch with the CAST strain, close to type I strains. Interestingly, the
TgCkBr130
TgCatBr5

TgHumMxCdMx1 isolate did not share a branch with any Mexican or

Table 2

haplogroup representative strain and was between the type III and
VEG
CTG
PTG
GT1

p89
ID

GAB2–2007-GAL-DOM2 branches (Fig. 3).

4
C.P. Rico-Torres et al. Infection, Genetics and Evolution 113 (2023) 105473

Fig. 1. Representative RFLP patterns for Toxoplasma gondii ROP17 marker alleles. (A) A type 4 allele was found in the TgDogMxChp3 (D3) isolate. RH, Me49 and
VEG are type I, II and III reference strains, respectively. (B) In silico digestion from the ROP17 sequences downloaded from www.toxodb.org (GT1, Me49, VEG,
COUGAR and MAS) and the sequence obtained from D3. Virtual digestion performed at www.benchling.com. The ROP17 genotype of each strain is indicated in
Arabic numerals below. RFLP products were resolved in 4–15% polyacrylamide gels and stained with EtBr. MW: Molecular Weight marker of 50 bp.

Fig. 2. Representative RFLP patterns for Toxoplasma gondii ROP18 marker alleles. (A) Restriction fragments of four Mexican isolates are shown: Lanes 1)
TgDogMxChp11; 2) TgCatMxCol2; 3) TgCowMxHgo1; and 4) TgDogMxCdMx1. Reference strains RH and Me49 for ROP18 are included. (B) Additionally, GT1, MAS,
and TgCatBr5 strains are included in the virtual digestion. *Oligonucleotides used for the amplification of the ROP18 type 1 and type 2 alleles do not amplify ROP18
type 3 of the VEG strain. The ROP18 genotype of each strain is indicated in Arabic numerals below the fig. MW: Molecular Weight marker of 50 bp.

Table 3
Virulence multilocus PCR-RFLP typing for Toxoplasma gondii Mexican isolates.
Isolate ID ToxoDB PCR-RFLP Virulence genetic markers Predicted virulence in mouse+

CS3 ROP16§ ROP17^ ROP18 ROP5

RH #10 I 1 1 1 1 High virulence

Me49 #1 II 2 2 2 2 Low virulence
VEG #2 III 1 2 3 3 Low virulence
p89 #8 (BrIII) NA 1 4 3 3 Low virulence
TgCatBr3 #8 (BrIII) NA 1 1 3 3 Low virulence
Present study
TgDogMxChp3 #8 (BrIII) III 1 4 3 3 Low virulence
TgCowMxHgo1 #10 I 1 1 1 1 High virulence
TgCatMxCol2 #28 I 1 1 4 1 High virulence
TgDogMxChp11 #28 I 1 1 4 1 High virulence
TgDogMxCdMx1 #28 I 1 1 4 1 High virulence
TgCatMxHgo1 #48 III 1 2 3 3 Low virulence
TgWbMxPue1 #116 III 1 1 3 3 Low virulence
TgHumMxCdMx1* #282 III 1 1 3 3 Low virulence

*Congenital infection. §Strains type I and III have allele 1. ^Strains type II and III have allele 2. +Based on Shwab et al. (2016). NA: not available.

4. Discussion able to describe four different genotypes, ToxoDB #8 (type BrIII), #28,
#116 and #282 (Rico-Torres et al., 2015, 2018; Valenzuela-Moreno
The genetic characterization of T. gondii is of vital importance for et al., 2020, 2022). In the present study, we describe genotype #28
epidemiological and clinical studies to determine the genotypes present in México City, archetypal type I #10 and genotype #48, both
involved in toxoplasmosis cases and how these genotypes are related isolated from samples of Hidalgo, a state near México City. The type I
and distributed in different regions of the world (Fernández-Escobar genotype (ToxoDB #10) had already been described from DNA-positive
et al., 2022). Previously, using 11 PCR-RFLP typing markers, we were blood of a severe case of congenital toxoplasmosis of the central region

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C.P. Rico-Torres et al. Infection, Genetics and Evolution 113 (2023) 105473

Fig. 3. Phylogenetic network of Toxoplasma gondii isolates from several animal species, including one human, from different Nearctic and Neotropical regions of
México, using PCR-RFLP data. The reference genotypes included are in bold: clonal types I, II, and III; Brazilian clonal types BrI, BrII, BrIII, BrIV, and representative
T. gondii strains of the haplogroups are included (circled in black), including some Mexican isolates reported in the literature (green). The genotype IDs of the
respective strains of this study are in red and listed for each taxonomic branch. (For interpretation of the references to colour in this figure legend, the reader is
referred to the web version of this article.)

of México and in a feral cat from Quintana Roo (Rico-Torres et al., 2018; background of the strain. Isolate TgWbMxPue1 shares the same MS
Valenzuela-Moreno et al., 2019). The ToxoDB #48 genotype has been typing alleles as strain TgCkBr130 (isolated from a chicken from Brazil),
previously reported in a rabbit, a rat and chickens from Argentina, and both are ToxoDB #116 but have different fingerprinting profiles,
Brazil, Guiana and Venezuela (Bernstein et al., 2018; Costa et al., 2021; probably because they are independent strains (Shwab et al., 2018). The
Feitosa et al., 2017; Shwab et al., 2014). We had already found the TgHumMxCdMx1 isolate has the same MS typing alleles as
ToxoDB #28 genotype in two states of México (TgCatMxCol2 and TgDogMxChp3 and strain p89 (isolated from a pig in the USA), even
TgDogMxChp11). To date, this is the more frequent genotype present in though they have different PCR-RFLP genotypes (#282 and #8,
the Western and Southeast regions, and it could be present throughout respectively); their PCR-RFLP profiles are almost identical, except for
the tropical region of México (Rico-Torres et al., 2015; Valenzuela- the alt. SAG2 and PK1 markers both have a strong type III background
Moreno et al., 2020). This genotype has also been described in Brazil, and a type II c22–8 allele. Finally, the three ToxoDB #28 isolates had the
Colombia and the USA (Shwab et al., 2014). Of the eight isolates same MS typing alleles, and TgCatMxCol2 and TgDogMxCdMx1 are
described herein, most of the typed alleles are types I and III, with scarce closely related, sharing the same 15 MS allele profile, suggesting that
representation of type II alleles. This differs from the isolates reported in they could be clones. These two isolates were obtained from different
northern México, where ToxoDB #9, #74 and #222 genotypes have a geographic regions of México (approximately 500 km of distance be­
majority of type II alleles (Dubey et al., 2009, 2013). This phenomenon tween Colima City and México City), but both isolates were obtained in
could be because Durango State is located in the Nearctic region and is the same year and, currently, globalization could make it possible that in
separated from the Neotropical region by the Sierra Madre Oriental and a very small time window, the same strain of T. gondii could contaminate
Sierra Madre Occidental mountain ranges, which function as natural meat products or infect domestic animals in different regions. Another
barriers and as climate transition regions, limiting the transit of wildlife hypothesis is homoplasy, in which two different unrelated strains may
and preventing the dispersion of T. gondii (Morrone, 2019). have the same allele combination, but this would be quite unlikely
In this study, we used MS to determine whether isolates with the because all 15 markers are located on 11 different chromosomes with
same ToxoDB genotype could be clones (as seen in outbreaks) or are different mutation rates (Dardé et al., 2020; Deiró et al., 2021).
different strains that have independently diversified. Seven different For the ROP gene analysis, we compared the results obtained to those
genotypes were identified, two of which had already been previously reported by Shwab et al. (2016) to determine if any of these isolates bear
published (Valenzuela-Moreno et al., 2020), and here, we describe five a new combination of alleles. The TgCowMxHgo1 isolate remained a
additional genotypes. The TgCowMxHgo1 isolate is a rare type I variant clonal type I strain, and all five markers were type I. TgDogMxChp3
that has a type II allele at TgM-A. The TgCatMxHgo1 isolate is an (ToxoDB #8) had a different virulence genotype than Brazilian strains
archetypal type III MS genotype almost identical to the reference type III (TgCatBr3, 4, 58, 59, 60, 73, and 74) but had the same pattern as the p89
strain CTG (14/15 markers) (Ajzenberg et al., 2010); however, its PCR- strain isolated from a pig in the USA, which makes sense since these
RFLP genotype is almost a type III strain except for the SAG1 locus (type isolates share the same MS typing alleles. It also suggests that although
I). Thus, the MS genotype is due to the strong type III genetic ToxoDB #8 has been found all over the American continent, there are

6
C.P. Rico-Torres et al. Infection, Genetics and Evolution 113 (2023) 105473

genetic differences between Northern and Southern American strains Funding

belonging to the same genotype that could have an impact on their
virulence. The three ToxoDB #28 strains (TgCatMxCol2, This work was partially supported by the Consejo Nacional de
TgDogMxCdMx1 and TgDogMxChp11) had the same allele combination Ciencia y Tecnología, México (CONACyT, grant number A1-S-21955)
as CAST and TgCtCo1 (with the type 4 allele at the ROP18 locus asso­ and by grants 2012/013 and 2020/039 from Instituto Nacional de
ciated with high virulence) but were different from TgCkBr142, which Pediatría and by Fundação de Amparo à Pesquisa do Estado de São Paulo
has type III alleles at the ROP17 and ROP18 loci. For ToxoDB #48, there (FAPESP, grant number 2018/26071-5).
was only one strain (TgCkGy22) that had been genotyped for these
markers, and it had a type 4 allele at the ROP17 locus that differed from CRediT authorship contribution statement
the TgCatMxHgo1 type 2 allele, implying that it was a new virulence
profile (Supplementary Table 1). Finally, ToxoDB #116 and #282 Claudia Patricia Rico-Torres: Investigation, Methodology, Valida­
(TgWbMxPue1 and TgHmMxCdMx1, respectively) showed a new viru­ tion, Formal analysis, Writing – original draft, Writing – review &
lence profile, since no strains with these genotypes have been previously editing. Luis Fernando Valenzuela-Moreno: Investigation, Methodol­
characterized by this panel. ogy, Validation, Formal analysis, Writing – original draft, Writing – re­
To date, the assessment of the virulence of a strain cannot be view & editing. Héctor Luna-Pastén: Investigation, Methodology,
determined using PCR-RFLP genotyping (except for the three archetypal Writing – review & editing. Carlos Cedillo-Peláez: Investigation,
strains). Standardized bioassays in outbred mice and cell culture assays Methodology, Writing – review & editing. Dolores Correa: Funding
are required to determine the virulence of each isolate, which could be acquisition, Investigation, Writing – review & editing. Elizabeth Mo­
time-consuming and expensive and cannot be performed in every lab­ rales-Salinas: Investigation, Writing – review & editing. José Juan
oratory (Saraf et al., 2017; Calero-Bernal et al., 2022; Gargaté et al., Martínez-Maya: Investigation, Writing – review & editing. Bruna
2022). A prediction of virulence in mice can be made based on the Farias Alves: Methodology, Writing – review & editing. Hilda Fátima
combination of the ROP18/ROP5 loci (Shwab et al., 2016). Of the eight Jesus Pena: Investigation, Formal analysis, Resources, Supervision,
isolates analyzed, we obtained three different ROP18/ROP5 combina­ Funding acquisition, Writing – original draft, Writing – review & editing.
tions, one of which (type 3/3) is expected to have low virulence and two Heriberto Caballero-Ortega: Conceptualization, Investigation, Formal
(1/1 and 4/1) to have high mortality. Even the 4/1 combination can analysis, Project administration, Resources, Supervision, Funding
reach up to 100% mortality, which means that half of the Mexican acquisition, Writing – original draft, Writing – review & editing.
isolates might be virulent in mouse bioassays. One aspect to be high­
lighted is that the three ToxoDB #28 isolates bear the ROP18 type 4
Declaration of Competing Interest
allele, which in all combinations would cause mortality between 80 and
100% in outbred mice, resulting along with the combination with ROP5,
The authors declare that they have no competing interests.
in a higher virulence genotype (even with the low virulence ROP5 type 5
allele). For the ROP5 marker, all the strains have type 1 or 3 alleles,
Data availability
which express proteins capable of blocking IRG oligomerization and
GTPase activity (Behnke et al., 2011; Reese et al., 2014). ROP17 works
TgDogMxChp3 OP850033 available at https://www.ncbi.nlm.nih.
collectively with ROP18 and ROP5 to block the IRG pathway by phos­
gov/genbank/
phorylating and contributing to the disassembly of IRG oligomers,
making them available for ROP5 and ROP18. Therefore, ROP17 is not
Acknowledgments
directly responsible for causing mortality in laboratory mice, but it may
contribute to the increase in virulence predicted for the ROP18/5
We are grateful to Dr. Rafael Saavedra from the Instituto de Inves­
combination, making it a priority gene for genotyping analysis (Ether­
tigaciones Biomédicas-UNAM (México) for generously providing RH
idge et al., 2014). The ROP16 marker showed that all isolates were type
and Me49 T. gondii strains. The authors thank Dr. Héctor Quiroz-Romero
1. This allele codes for the functional ROP16 protein that phosphorylates
from Facultad de Medicina Veterinaria y Zootecnia-UNAM for kindly
and activates STAT3 and STAT6, which serve as suppressors of pro-
helping us with an isolate of T. gondii from central México. We also thank
inflammatory cytokine production and result in the modulation of the
Rafael López-Reboseño and students Diana Ofelia Chávez Crisóstomo,
host immune response, preventing its death due to the exacerbated
Lizbeth Ramírez Pérez, and Noé Pacheco Coronel for their excellent
immune response (Saeij et al., 2007; Ihara and Nishikawa, 2021). Ac­
technical support and collection of biological samples from several an­
cording to Pena et al. (2008), alleles I and II of CS3 are related to
imal species and Dr. Fernando García Lacy for his English language
virulence in mice, while allele III has been associated with nonvirulence
review.
in this murine model. Therefore, in the case of the Mexican strains re­
ported here, four would be avirulent (allele III), and four would be
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