Next Article in Journal
The Isolation and Identification of Pseudoalteromonas sp. H27, a Bacterial Strain Pathogenic to Crassostrea gigas
Next Article in Special Issue
Treatment Protocols for Gestational and Congenital Toxoplasmosis: A Systematic Review and Meta-Analysis
Previous Article in Journal
Taxane-Producing Fungi Isolated from Taxus globosa Tree Bark
Previous Article in Special Issue
Antiproliferative and Morphological Analysis Triggered by Drugs Contained in the Medicines for Malaria Venture COVID-Box Against Toxoplasma gondii Tachyzoites
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Microorganisms
Volume 13
Issue 2
10.3390/microorganisms13020301
microorganisms-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Communication

Investigation of Virulence-Related Markers in Atypical Strains of Toxoplasma gondii from Brazil

by
Júlia Gatti Ladeia Costa
1,†,
Érica Santos Martins-Duarte
2,†,
Lorena Velozo Pinto
2,
Ramon Araujo de Castro Baraviera
2,
Wagner Martins Fontes do Rego
2 and
Ricardo Wagner de Almeida Vitor
2,*
1
Departamento de Ciências Biológicas, Universidade do Estado de Minas Gerais, Ibirité 32412-190, Brazil
2
Departamento de Parasitologia, Universidade Federal de Minas Gerais, Belo Horizonte 31270-901, Brazil
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Microorganisms 2025, 13(2), 301; https://doi.org/10.3390/microorganisms13020301
Submission received: 20 December 2024 / Revised: 17 January 2025 / Accepted: 27 January 2025 / Published: 29 January 2025
(This article belongs to the Special Issue Parasites and Infection: Strategies to Control, Diagnose, and Treat Parasitic Diseases)
Browse Figures
Review Reports Versions Notes

Abstract

:
Toxoplasma gondii is an obligate intracellular protozoan parasite distributed worldwide that infects a wide range of warm-blooded animals, including humans. Recent studies sought to clarify the relationship between the alleles GRA15, ROP5, ROP16, ROP17, and ROP18 and the virulence of T. gondii isolates in mice. This work aims to analyze the variability of genes that express T. gondii virulence proteins of 103 strains. Most strains were virulent for mice (76/103–73.79%); within these, 30 were 100% lethal, and 46 caused a cumulative mortality range from 20% to 93%. For the GRA15 marker, most strains presenting allele 2 were non-lethal. For the ROP17 marker, allele 4 was associated with mortality, compared to allele 1. For the ROP18 marker, alleles 1 and 4 were associated with mortality, compared to alleles 2 and 3. A combined analysis of alleles showed low cumulative mortality when the strains presented alleles 3 and 1 for ROP18 and ROP16, respectively. On the other hand, allele 4 of ROP17 was a determinant for virulence when associated with ROP18 allele 3 and ROP16 allele 1. Our analysis shows that ROP18 is the primary determinant of the virulence of atypical strains in mice. Additionally, ROP17 genotyping should not be overlooked, as it has proven critical to enhance this prediction.

1. Introduction

Toxoplasmosis is a worldwide zoonosis that presents various clinical conditions, from asymptomatic infection to severe systemic manifestations. In immunocompetent individuals, the disease, in general, is asymptomatic. However, latent toxoplasmosis may be associated with neurological disorders such as schizophrenia [1]. In immunocompromised individuals, sequelae caused by Toxoplasma gondii have been observed in individuals with acquired immunodeficiency syndrome (AIDS), in treatment with immunosuppressive drugs, or in those who have received an organ or bone marrow transplant. In congenital toxoplasmosis, the parasite can cause abortion or the birth of children with ocular and neurological disorders [2,3].
Studies carried out with strains of T. gondii isolated from patients or animals from South America have shown a high rate of genetic polymorphism [4]. Recent studies sought to clarify the relationship between the alleles identified by polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) of the protein virulence markers, such as dense granule protein 15 (GRA15), rhoptry protein 5 (ROP5), ROP16, ROP17, and ROP18, with the virulence of T. gondii isolates in mice [5]. Among these proteins, ROP16 and ROP17 were associated with the modulation of the host’s immune response by the parasite, and ROP18 and ROP5 were highlighted as the main determinants of T. gondii virulence in mice [5]. Rêgo et al. [6] demonstrated that in Brazilian atypical strains of T. gondii, the ROP18 protein, analyzed alone or in combination with ROP5, effectively determined the parasite’s virulence for mice.
Different mouse strains, life cycle stages of the parasite, and routes of inoculation are used in T. gondii virulence assays. These limit comparisons considering genotype and virulence in mice and the integration of data [7]. Here, we analyze a large number of atypical isolates obtained in Brazil, including those isolated from humans, with a standardized methodology to evaluate the variability of genes that express T. gondii virulence proteins.

2. Materials and Methods

2.1. Toxoplasma gondii Strains

All 103 strains used in this work were previously isolated by our group (Supplementary Table S1) and have been maintained cryopreserved in N2 banks at the Laboratory of Toxoplasmosis at the Universidade Federal de Minas Gerais—UFMG, Brazil. According to Brazilian law, access to Brazilian genetic heritage was approved by SisGen protocols A3F9195, AD3C00F, AA14CC9, AB9C3CF, A375FCF, AC52DBB, and AD3745E.

2.2. PCR-RFLP Genotyping of T. gondii

PCR-RFLP protocols for GRA15, ROP16, and ROP18 were performed according to Dubey et al., 2014 [8]. PCR-RFLP protocols for ROP5 and ROP17 were performed according to Shwab et al. [5].
Genomic DNA samples from T. gondii strains were obtained according to Carneiro et al. [9] and provided by the Laboratory of Toxoplasmosis (UFMG). Briefly, DNA extraction of the tachyzoites was performed using the Promega Wizard genomic DNA purification kit following the manufacturer’s instructions. Genomic DNA stocks were stored at −20 °C to 4 °C before analysis. The RH88 (type I), ME49 (type II), PTG (II), VEG (type III), and MAS (atypical) strains were used as controls and references. The negative control was distilled water in the presence of primers. For each locus, PCR was performed on parasite genomic DNA using external primers, followed by a second round of PCR with nested primers using external primer products as templates. Primer sequences and restriction enzymes for PCR-RFLP genotyping of rhoptry gene loci ROP18, ROP5, ROP16, and ROP17 were the same as previously described [5,8]. The amplified products were digested using restriction endonucleases (New England BioLabs, Ipswich, MA, USA) specific for each marker according to the manufacturer’s instructions. The DNA of the digested products was purified by extraction with an equal volume of phenol/chloroform (1:1), subjected to polyacrylamide gel (5%) electrophoresis, stained with silver nitrate, and photographed.

2.3. Parasite Virulence Determination

Strain virulence information (Supplementary Table S1) was obtained from previously published data from bioassays in mice [6,9,10,11,12,13,14,15]. Five female BALB/c mice, six to eight weeks old, were inoculated intraperitoneally (i.p.) with 10, 100, or 1000 tachyzoites of each strain in 0.2 mL of PBS (pH 7.2). A total of 15 mice per strain were used. Five animals inoculated i.p. with PBS were maintained as negative controls. All efforts were made to minimize animal suffering during the study. Mouse mortality was observed for a period of 30 days. ELISA (anti-T. gondii IgG) was performed on all surviving mice to confirm infection. The survivor mice that did not seroconvert were excluded from the experiment. RH (virulent) and ME49 (nonvirulent) strains were used as references for comparison. According to Saraf et al. [7], cumulative mortality was calculated based on the number of mice that died divided by the number of mice infected, analyzing three sequential inoculation dosages, with the lowest dose resulting in only partial infection. The Animal Ethics Committee (CEUA) of the Universidade Federal de Minas Gerais (UFMG), Minas Gerais, Brazil, approved all experiments and procedures, including euthanasia and blood collection (CEUA Protocols: 013/2007, 128/2010, 266/2012, 067/2016, and 048/2018).

2.4. Statistical Analyses

The analysis of the correlation between allele types (GRA15, ROP5, ROP16, ROP17, and ROP18) and the median mouse cumulative mortality was performed using the non-parametric Mann–Whitney U test (individual comparisons) and the Kruskal–Wallis test (group analysis) followed by Dunn’s test for multiple comparisons. Statistical correlation between lethality and genotype was analyzed using Pearson chi2. Analyses were performed using GraphPad Prism version 5.0 (GraphPad Software, La Jolla, CA, USA), and p < 0.05 was considered significant.

3. Results

In total, we studied 103 atypical strains isolated from four different Brazilian states: Espírito Santo state (ES) and Minas Gerais state (MG), both located in Southeast Brazil, with a predominantly tropical climate, and Piauí state (PI) and Rio Grande do Norte state (RN), both located in the Northeast region of Brazil with a predominantly semi-arid climate (Figure 1A). Considering the hosts, 29 strains were obtained from humans (28.16%), 29 (28.16%) from chickens, 21 (20.39%) from pigs, 10 (9.71%) from goats, 8 (7.77%) from dogs, and 6 (5.83%) from wild birds (Supplementary Table S1). Most strains showed some virulence level for mice (76/103–73.79%); within these, 30 were 100% lethal, and 46 caused a cumulative mortality ranging from 20% to 93%. Within the 103 strains, previous studies showed that 39 different genotypes were identified, with 26 strains of unique genotypes, 17 identified as ToxoDB #163, 14 as #11(BrII), 7 as #108, 6 as #146, 6 as #206, 5 as #8 (BrIII), 5 as #109, 4 as #13, 4 as #24, 3 as #57, 2 as #6 (BrI), 2 as #41, and 2 as #19 (Supplementary Table S1).
The atypical strains presented diverse combinations of virulence factors. Representative gel images of PCR-RFLP genotyping are presented in Supplementary Figure S1. There was no difference between the accumulated mortality from strains obtained in different geographic regions (p = 0.2083). The diversity of alleles found for the studied markers GRA15, ROP5, ROP16, ROP17, and ROP18 in strains obtained from different geographic regions is shown in Figure 1B. To validate the correlation of the allele types for GRA15, ROP5, ROP16, ROP17, ROP18, and combinations of ROP18/ROP5 with the virulence of T. gondii strains in mice, we plotted allele types against the cumulative mortality (Figure 2). The cumulative mortality was calculated from published data (Supplementary Table S1). Despite a large variation in the percentage of cumulative mortality within allele types, the differences between the medians were significant for some virulence factor types. For the GRA15 marker, most strains presenting allele 2 showed cumulative mortality of 0%, and the median cumulative mortality in isolates that presented alleles 1 or 3 was around 90% (Figure 2A). For the ROP17 marker, we observed that the median cumulative mortality of the isolates presenting allele 4 was significantly higher than that of allele 1 (Figure 2B). For the ROP16 and ROP5 markers, there were no differences in the cumulative mortality within the different alleles (Figure 2C,E).
The isolates that presented allele 1 of the ROP18 marker had 100% cumulative mortality, a significantly different value from those that had the 2 or 3 (p < 0.001) allele (Figure 2D). The median cumulative mortality for the isolates with alleles 1 or 4 of the ROP18 marker was higher than that of isolates with alleles 2 or 3. Equally, analysis of the combined alleles of ROP18/ROP5 with mouse virulence indicated that the combination had an overall stronger correlation. Six different combinations were identified (Figure 2F). Some combination segregation with high or low mouse virulence was statistically significant.
The relation of alleles 4 and 3 of ROP18 with high or low cumulative mortality, respectively, was evident when a combined analysis of alleles of ROP18 and ROP16 was performed. While no significant statistical difference was seen between the combinations of ROP18 allele 4 with ROP16 alleles 1 or 2 (Figure 3B), a significant difference was seen when the strains presented alleles 3 and 1 for ROP18 and ROP16, respectively, compared to those with ROP18 allele 4 (Figure 3B). The relation of allele 4 of ROP18 with cumulative mortality in strains presenting ROP16 allele 2 was irrespective of the ROP17 allele, as no significant difference was seen between the presence of ROP17 alleles 4 and 3 (Figure 3E). However, allele 4 of ROP17 was a determinant for the virulence of strains with ROP18 allele 3 and ROP16 allele 1 (Figure 3D). In the strains harboring alleles 3 and 1 of ROP18 and ROP17, respectively, the presence of alleles 1/3 or 2 of GRA15 did not influence virulence (Figure 3C).
To identify alleles that could be possible molecular markers of lethal strains, we also analyzed the frequency of alleles in two groups of strains according to their lethality (Table 1). Only 2 isolates from 50 that presented ROP18 allele 4 were non-lethal. ROP18 allele 1 and allele 2 were only found in lethal and non-lethal strains, respectively. Allele 4 of ROP5 and allele 3 of ROP17 were identified only in lethal strains, but all of them also harbored ROP18 allele 4. Strains harboring ROP17 allele 4 also showed a higher frequency of lethal phenotype. A higher frequency of non-lethal phenotype was observed in strains presenting GRA15 allele 2, ROP18 allele 3, and ROP5 allele 1 (Table 1). However, all strains with ROP5 allele 1 also harbored ROP18 allele 3, and the non-lethal phenotype is possibly associated with ROP18 alleles. Equally, most strains with ROP16 allele 2 presented a lethal phenotype; however, those also harbored ROP18 allele 4. In conclusion, our analysis shows that ROP18 is the major determinant of the virulence of atypical strains in mice.

4. Discussion

Toxoplasma gondii is a successful protozoa parasite due to its ability to manipulate host cell behavior [16]. Rhoptry proteins (ROPs) function at the parasitophorous vacuole to inhibit the recruitment of immunity-related GTPases (IRGs) and guanylate-binding proteins (GBPs), playing a critical role in host–parasite interactions [17,18]. ROP5 and ROP18 work synergistically to block innate immune mechanisms triggered by IFN-γ in murine hosts and serve as predictors of strain virulence in mice [5,19]. Consistent with our findings, previous studies have shown that the combined analysis of ROP18 and ROP5 can predict high virulence in mice from Brazilian strains isolated from pigs, goats, and even cases of human congenital toxoplasmosis [6,20]. However, most strains analyzed in this study exhibited ROP5 allele 3, which made it difficult to assess the contribution of this allele to the observed virulence.
In this work, we found ROP18 allele 4 in 49 isolates; in contrast, only 9 isolates had allele 1. Additionally, the combination of ROP18 allele 2/ROP5 allele 3 had not been previously reported, and strains carrying these alleles showed low cumulative mortality. The ROP18 allele 4/ROP5 allele 4 combination was first identified in T. gondii samples from Misiones province, Argentina, leading the authors to propose a possible regionalization of the ROP18/ROP5 profile [21]. However, in our study, we analyzed many strains and detected the same ROP18 allele 4/ROP5 allele 4 combination in four strains from Minas Gerais, Brazil. This finding underscores the importance of analyzing isolates from different regions to validate the predictive value of the ROP18/ROP5 profile for T. gondii virulence in mice, especially considering that standardized bioassays in mice are time-consuming, costly, and not feasible in all laboratories [7,22].
ROP5 forms complexes with the ROP18 and ROP17 kinases, working together to regulate acute virulence in mice [23]. ROP16 allele 1/2 can phosphorylate STAT3 and STAT6, leading to the alternative activation of macrophages (M2), which promotes parasite replication within host cells [19]. When analyzed together, our results indicate that ROP18 alleles 1 and 4, irrespective of the ROP5 or ROP16 background, are strongly associated with the virulence of Brazilian strains, showing a high predictive value for cumulative mortality in mice.
In contrast, ROP18 allele 3 is associated with lower virulence and may be influenced by the ROP17 background. Etheridge et al. [23] showed that while ROP17 is not directly responsible for mortality in mice, it contributes to the enhanced virulence associated with the ROP18/ROP5 combination. Hamilton et al. [24] suggested that ROP17 may play a role in determining virulence, which could explain cases where strains predicted to be non-lethal based on the ROP18/ROP5 allele combination are found to be virulent in mouse bioassays. This makes ROP17 a priority gene for genotyping analysis [25]. Thus, determining virulence may be more complex when examining only a few alleles.
GRA15 allele 2 emerged as an important marker in the non-lethal strains identified in this study. In clonal type II strains, it facilitates NF-kB nuclear translocation, induces macrophages toward a classically activated (M1) phenotype, and enhances the host’s innate immunity against T. gondii infection [26]. Notably, the only lethal strain with GRA15 allele 2 also harbored ROP18 allele 4 and ROP17 allele 4, both key virulence markers, despite the presence of ROP16 allele 2.
Like the current study, most research has focused on the mechanisms by which ROP proteins confer virulence in mice. However, how those virulence factors would impact the outcome of T. gondii infection in humans still needs to be discovered [27]. However, type I and atypical strains are frequently associated with symptomatic and severe forms of toxoplasmosis [28,29,30,31,32], and some studies shown the possible association of ROP18 with congenital and ocular diseases in humans [33,34,35].
To date, strain virulence cannot be reliably assessed using PCR-RFLP genotyping alone, except for the three archetypal strains [36]. Virulence protein polymorphisms were accessed in our study using the PCR-RFLP technique. This methodology limits the detection of single nuclear polymorphisms (SNPs) that are recognized by restriction enzymes and may not be sufficient to detect all polymorphisms in key genes that contribute to phenotypic and pathogenic differences in T. gondii strains [16]. So, a future perspective is to sequence the virulence proteins of isolates that presented alleles related to virulence but were non-lethal to mice or vice versa. However, a growing body of research, including our study, indicates that the combination of ROP18 and ROP5 loci offers a strong predictive capacity for virulence in mice, as proposed by Shwab et al. [5].
Our analysis shows that ROP18 is the major determinant of the virulence of Brazilian atypical strains in mice, and highlights the higher prevalence of strains with allele 4. However, how that allele would modulate the immune system in mice or humans has yet to be found. Additionally, ROP17 genotyping should not be overlooked, as it has proven to be a key factor in enhancing virulence prediction. Establishing the fundamental roles of these genetic markers in identifying the virulence of T. gondii will allow, in the future, the identification of virulent strains circulating in the environment, especially in livestock. Consequently, more appropriate measures to control the parasite will be adopted.
A limitation of our study is that we evaluated 103 isolates of T. gondii of only 39 genotypes. Thus, it is necessary to study more strains, with different genotypes obtained from both animals and humans, from other Brazilian regions and worldwide, to confirm our findings. We expect this work to stimulate other research groups to include similar or other analyses with more strains of identical or different genotypes from different world regions. Thus, over time, this analysis will gain strength and will support whether ROP18 is the major virulence determinant of T. gondii in mice and, potentially, in humans.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/microorganisms13020301/s1: Table S1: GRA15, ROP18, ROP16, ROP5, and ROP17 genotypes of Toxoplasma gondii strains, according to lethality and cumulative mortality for mice. Figure S1: Representative gel images of PCR-RFLP genotyping (markers GRA15, ROP5, ROP16, ROP17, and ROP18). Sample IDs are at the top of the gel images; genotype results are at the bottom. MK is the DNA size marker.

Author Contributions

Conceptualization, J.G.L.C. and R.W.d.A.V.; methodology, J.G.L.C.; validation, W.M.F.d.R.; formal analysis, J.G.L.C., W.M.F.d.R. and É.S.M.-D.; investigation, J.G.L.C., L.V.P. and R.A.d.C.B.; resources, R.W.d.A.V.; data curation, J.G.L.C. and É.S.M.-D.; writing—original draft preparation, J.G.L.C.; writing—review and editing, R.W.d.A.V. and É.S.M.-D.; visualization, J.G.L.C. and R.W.d.A.V.; supervision, R.W.d.A.V.; project administration, J.G.L.C. and R.W.d.A.V.; funding acquisition, R.W.d.A.V. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Conselho Nacional de Desenvolvimento e Pesquisa (CNPq) (grants 305574/2021-3 and 308082/2023-0), and Fundação de Amparo à Pesquisa de Minas Gerais (FAPEMIG) (grant APQ-00537-17). J.G.L.C. was supported by a post-doctoral fellowship from CNPq (grant 152652/2016-7). R.W.d.A.V and É.S.M.-D. are Research Fellows at CNPq.

Institutional Review Board Statement

The animal study protocol was approved by the Animal Ethics Committee (CEUA) of the Universidade Federal de Minas Gerais (UFMG), Minas Gerais, Brazil, Protocols: 013/2007, 128/2010, 266/2012, 067/2016, and 048/2018.

Informed Consent Statement

Not applicable.

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors on request.

Acknowledgments

The authors would like to thank Rosalida Estevan Nazar Lopes for the valuable technical assistance and Pró-Reitoria de Pesquisa of the Universidade Federal de Minas Gerais for support.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Flegr, J. How and Why Toxoplasma Makes Us Crazy. Trends Parasitol. 2013, 29, 156–163. [Google Scholar] [CrossRef]
  2. Deganich, M.; Boudreaux, C.; Benmerzouga, I. Toxoplasmosis Infection during Pregnancy. Trop. Med. Infect. Dis. 2022, 8, 3. [Google Scholar] [CrossRef]
  3. Layton, J.; Theiopoulou, D.-C.; Rutenberg, D.; Elshereye, A.; Zhang, Y.; Sinnott, J.; Kim, K.; Montoya, J.G.; Contopoulos-Ioannidis, D.G. Clinical Spectrum, Radiological Findings, and Outcomes of Severe Toxoplasmosis in Immunocompetent Hosts: A Systematic Review. Pathogens 2023, 12, 543. [Google Scholar] [CrossRef] [PubMed]
  4. Pardini, L.; Bernstein, M.; Carral, L.A.; Kaufer, F.J.; Dellarupe, A.; Gos, M.L.; Campero, L.M.; Moré, G.; Messina, M.T.; Schneider, M.V.; et al. Congenital Human Toxoplasmosis Caused by Non-Clonal Toxoplasma gondii Genotypes in Argentina. Parasitol. Int. 2019, 68, 48–52. [Google Scholar] [CrossRef]
  5. Shwab, E.K.; Jiang, T.; Pena, H.F.J.; Gennari, S.M.; Dubey, J.P.; Su, C. The ROP18 and ROP5 Gene Allele Types Are Highly Predictive of Virulence in Mice across Globally Distributed Strains of Toxoplasma gondii. Int. J. Parasitol. 2016, 46, 141–146. [Google Scholar] [CrossRef]
  6. Rêgo, W.M.F.; Costa, J.G.L.; Baraviera, R.C.A.; Pinto, L.V.; Bessa, G.L.; Lopes, R.E.N.; Vitor, R.W.A. Association of ROP18 and ROP5 Was Efficient as a Marker of Virulence in Atypical Isolates of Toxoplasma gondii Obtained from Pigs and Goats in Piauí, Brazil. Vet. Parasitol. 2017, 247, 19–25. [Google Scholar] [CrossRef] [PubMed]
  7. Saraf, P.; Shwab, E.K.; Dubey, J.P.; Su, C. On the Determination of Toxoplasma gondii Virulence in Mice. Exp. Parasitol. 2017, 174, 25–30. [Google Scholar] [CrossRef] [PubMed]
  8. Dubey, J.P.; Van Why, K.; Verma, S.K.; Choudhary, S.; Kwok, O.C.H.; Khan, A.; Behinke, M.S.; Sibley, L.D.; Ferreira, L.R.; Oliveira, S.; et al. Genotyping Toxoplasma gondii from Wildlife in Pennsylvania and Identification of Natural Recombinants Virulent to Mice. Vet. Parasitol. 2014, 200, 74–84. [Google Scholar] [CrossRef] [PubMed]
  9. Carneiro, A.C.A.V.; Andrade, G.M.; Costa, J.G.L.; Pinheiro, B.V.; Vasconcelos-Santos, D.V.; Ferreira, A.M.; Su, C.; Januário, J.N.; Vitor, R.W.A. Genetic Characterization of Toxoplasma gondii Revealed Highly Diverse Genotypes for Isolates from Newborns with Congenital Toxoplasmosis in Southeastern Brazil. J. Clin. Microbiol. 2013, 51, 901–907. [Google Scholar] [CrossRef]
  10. Brandão, G.P.; Ferreira, A.M.; Melo, M.N.; Vitor, R.W.A. Characterization of Toxoplasma gondii from Domestic Animals from Minas Gerais, Brazil. Parasite 2006, 13, 143–149. [Google Scholar] [CrossRef]
  11. Ferreira, A.d.M.; Vitor, R.W.A.; Gazzinelli, R.T.; Melo, M.N. Genetic Analysis of Natural Recombinant Brazilian Toxoplasma gondii Strains by Multilocus PCR-RFLP. Infect. Genet. Evol. 2006, 6, 22–31. [Google Scholar] [CrossRef] [PubMed]
  12. Clementino Andrade, M.M.; Pinheiro, B.V.; Cunha, M.M.; Carneiro, A.C.a.V.; Andrade Neto, V.F.; Vitor, R.W.A. New Gentotypes of Toxoplasma gondii Obtained from Farm Animals in Northeast Brazil. Res. Vet. Sci. 2013, 94, 587–589. [Google Scholar] [CrossRef]
  13. Ferreira, T.C.R.; Buery, J.C.; Moreira, N.I.B.; Santos, C.B.; Costa, J.G.L.; Pinto, L.V.; Baraviera, R.C.d.A.; Vitor, R.W.A.; Fux, B. Toxoplasma gondii: Isolation, Biological and Molecular Characterisation of Samples from Free-Range Gallus gallus Domesticus from Countryside Southeast Brazil. Rev. Bras. Parasitol. Vet. 2018, 27, 384–389. [Google Scholar] [CrossRef] [PubMed]
  14. Rêgo, W.M.F.; Costa, J.G.L.; Baraviera, R.C.A.; Pinto, L.V.; Bessa, G.L.; Lopes, R.E.N.; Silveira, J.a.G.; Vitor, R.W.A. Genetic Diversity of Toxoplasma gondii Isolates Obtained from Free-Living Wild Birds Rescued in Southeastern Brazil. Int. J. Parasitol. Parasites Wildl. 2018, 7, 432–438. [Google Scholar] [CrossRef] [PubMed]
  15. Pinheiro, B.V.; Noviello, M.d.L.M.; Cunha, M.M.; Tavares, A.T.; Carneiro, A.C.A.V.; Arantes, R.M.E.; Vitor, R.W.A. Pathological Changes in Acute Experimental Toxoplasmosis with Toxoplasma gondii Strains Obtained from Human Cases of Congenital Disease. Exp. Parasitol. 2015, 156, 87–94. [Google Scholar] [CrossRef]
  16. Cheng, W.; Wang, C.; Xu, T.; Liu, F.; Pappoe, F.; Luo, Q.; Xu, Y.; Lu, F.; Shen, J. Genotyping of Polymorphic Effectors of Toxoplasma gondii Isolates from China. Parasit. Vectors 2017, 10, 580. [Google Scholar] [CrossRef] [PubMed]
  17. Jensen, K.D.C.; Camejo, A.; Melo, M.B.; Cordeiro, C.; Julien, L.; Grotenbreg, G.M.; Frickel, E.-M.; Ploegh, H.L.; Young, L.; Saeij, J.P.J. Toxoplasma gondii Superinfection and Virulence during Secondary Infection Correlate with the Exact ROP5/ROP18 Allelic Combination. mBio 2015, 6, e02280. [Google Scholar] [CrossRef]
  18. Kim, E.W.; Nadipuram, S.M.; Tetlow, A.L.; Barshop, W.D.; Liu, P.T.; Wohlschlegel, J.A.; Bradley, P.J. The Rhoptry Pseudokinase ROP54 Modulates Toxoplasma gondii Virulence and Host GBP2 Loading. mSphere 2016, 1, e00045-16. [Google Scholar] [CrossRef]
  19. Saeij, J.P.J.; Boyle, J.P.; Coller, S.; Taylor, S.; Sibley, L.D.; Brooke-Powell, E.T.; Ajioka, J.W.; Boothroyd, J.C. Polymorphic Secreted Kinases Are Key Virulence Factors in Toxoplasmosis. Science 2006, 314, 1780–1783. [Google Scholar] [CrossRef]
  20. de Melo, R.P.B.; Wanderley, F.S.; Porto, W.J.N.; Pedrosa, C.d.M.; Hamilton, C.M.; de Oliveira, M.H.G.S.; Ribeiro-Andrade, M.; Rêgo, R.C.d.S.; Katzer, F.; Mota, R.A. Description of an Atypical Toxoplasma gondii Isolate from a Case of Congenital Toxoplasmosis in Northeastern Brazil. Parasitol. Res. 2020, 119, 2727–2731. [Google Scholar] [CrossRef] [PubMed]
  21. Bernstein, M.; Rudzinski, M.; Schneider, V.; Messina, M.; Gos, M.L.; Helman, E.; Dellarupe, A.; Unzaga, J.M.; Venturini, M.C.; Moré, G.; et al. Genetic Characterization of Toxoplasma gondii from Human and Chicken Isolates from Argentina. Parasitol. Res. 2024, 123, 129. [Google Scholar] [CrossRef]
  22. Calero-Bernal, R.; Fernández-Escobar, M.; Katzer, F.; Su, C.; Ortega-Mora, L.M. Unifying Virulence Evaluation in Toxoplasma gondii: A Timely Task. Front. Cell. Infect. Microbiol. 2022, 12, 868727. [Google Scholar] [CrossRef]
  23. Etheridge, R.D.; Alaganan, A.; Tang, K.; Lou, H.J.; Turk, B.E.; Sibley, L.D. The Toxoplasma Pseudokinase ROP5 Forms Complexes with ROP18 and ROP17 Kinases That Synergize to Control Acute Virulence in Mice. Cell Host Microbe 2014, 15, 537–550. [Google Scholar] [CrossRef] [PubMed]
  24. Hamilton, C.M.; Black, L.; Oliveira, S.; Burrells, A.; Bartley, P.M.; Melo, R.P.B.; Chianini, F.; Palarea-Albaladejo, J.; Innes, E.A.; Kelly, P.J.; et al. Comparative Virulence of Caribbean, Brazilian and European Isolates of Toxoplasma gondii. Parasit. Vectors 2019, 12, 104. [Google Scholar] [CrossRef] [PubMed]
  25. Bernstein, M.; Pardini, L.; Bello Pede Castro, B.; Unzaga, J.M.; Venturini, M.C.; Moré, G. ROP18 and ROP5 Alleles Combinations Are Related with Virulence of T. gondii Isolates from Argentina. Parasitol. Int. 2021, 83, 102328. [Google Scholar] [CrossRef]
  26. Rosowski, E.E.; Lu, D.; Julien, L.; Rodda, L.; Gaiser, R.A.; Jensen, K.D.C.; Saeij, J.P.J. Strain-Specific Activation of the NF-kappaB Pathway by GRA15, a Novel Toxoplasma gondii Dense Granule Protein. J. Exp. Med. 2011, 208, 195–212. [Google Scholar] [CrossRef] [PubMed]
  27. Niedelman, W.; Gold, D.A.; Rosowski, E.E.; Sprokholt, J.K.; Lim, D.; Farid Arenas, A.; Melo, M.B.; Spooner, E.; Yaffe, M.B.; Saeij, J.P.J. The Rhoptry Proteins ROP18 and ROP5 Mediate Toxoplasma gondii Evasion of the Murine, but Not the Human, Interferon-Gamma Response. PLoS Pathog. 2012, 8, e1002784. [Google Scholar] [CrossRef] [PubMed]
  28. Gilbert, R.E.; Freeman, K.; Lago, E.G.; Bahia-Oliveira, L.M.G.; Tan, H.K.; Wallon, M.; Buffolano, W.; Stanford, M.R.; Petersen, E. European Multicentre Study on Congenital Toxoplasmosis (EMSCOT) Ocular Sequelae of Congenital Toxoplasmosis in Brazil Compared with Europe. PLoS Negl. Trop. Dis. 2008, 2, e277. [Google Scholar] [CrossRef]
  29. Khan, A.; Jordan, C.; Muccioli, C.; Vallochi, A.L.; Rizzo, L.V.; Belfort, R.; Vitor, R.W.A.; Silveira, C.; Sibley, L.D. Genetic Divergence of Toxoplasma gondii Strains Associated with Ocular Toxoplasmosis, Brazil. Emerg. Infect. Dis. 2006, 12, 942–949. [Google Scholar] [CrossRef] [PubMed]
  30. de Lima Bessa, G.; de Almeida Vitor, R.W.; Dos Santos Martins-Duarte, E. Toxoplasma gondii in South America: A Differentiated Pattern of Spread, Population Structure and Clinical Manifestations. Parasitol. Res. 2021, 120, 3065–3076. [Google Scholar] [CrossRef]
  31. Switaj, K.; Master, A.; Borkowski, P.K.; Skrzypczak, M.; Wojciechowicz, J.; Zaborowski, P. Association of Ocular Toxoplasmosis with Type I Toxoplasma gondii Strains: Direct Genotyping from Peripheral Blood Samples. J. Clin. Microbiol. 2006, 44, 4262–4264. [Google Scholar] [CrossRef]
  32. Vallochi, A.L.; Muccioli, C.; Martins, M.C.; Silveira, C.; Belfort, R.; Rizzo, L.V. The Genotype of Toxoplasma gondii Strains Causing Ocular Toxoplasmosis in Humans in Brazil. Am. J. Ophthalmol. 2005, 139, 350–351. [Google Scholar] [CrossRef] [PubMed]
  33. Hernández-de-Los-Ríos, A.; Murillo-Leon, M.; Mantilla-Muriel, L.E.; Arenas, A.F.; Vargas-Montes, M.; Cardona, N.; de-la-Torre, A.; Sepúlveda-Arias, J.C.; Gómez-Marín, J.E. Influence of Two Major Toxoplasma gondii Virulence Factors (ROP16 and ROP18) on the Immune Response of Peripheral Blood Mononuclear Cells to Human Toxoplasmosis Infection. Front. Cell. Infect. Microbiol. 2019, 9, 413. [Google Scholar] [CrossRef]
  34. Torres-Morales, E.; Taborda, L.; Cardona, N.; De-la-Torre, A.; Sepulveda-Arias, J.C.; Patarroyo, M.A.; Gomez-Marin, J.E. Th1 and Th2 Immune Response to P30 and ROP18 Peptides in Human Toxoplasmosis. Med. Microbiol. Immunol. 2014, 203, 315–322. [Google Scholar] [CrossRef] [PubMed]
  35. Sánchez, V.; de-la-Torre, A.; Gómez-Marín, J.E. Characterization of ROP18 Alleles in Human Toxoplasmosis. Parasitol. Int. 2014, 63, 463–469. [Google Scholar] [CrossRef] [PubMed]
  36. Rico-Torres, C.P.; Valenzuela-Moreno, L.F.; Luna-Pastén, H.; Cedillo-Peláez, C.; Correa, D.; Morales-Salinas, E.; Martínez-Maya, J.J.; Alves, B.F.; Pena, H.F.J.; Caballero-Ortega, H. Genotyping of Toxoplasma gondii Isolates from México Reveals Non-Archetypal and Potentially Virulent Strains for Mice. Infect. Genet. Evol. 2023, 113, 105473. [Google Scholar] [CrossRef] [PubMed]
Microorganisms 13 00301 g001
Figure 1. Distribution of Toxoplasma gondii strains according to the Brazilian regions from which they were isolated. (A) Map of Brazil showing the four states where the 103 Toxoplasma gondii strains were isolated: ES (Espírito Santo state), MG (Minas Gerais state), PI (Piauí state), and RN (Rio Grande do Norte state). In total, 5 strains were obtained from chickens from ES; 54 strains were obtained from MG (29 from humans—25 from peripheral blood of newborns with congenital toxoplasmosis and 4 from amniotic fluid; 8 from dogs; 11 from chickens; and 6 from free-living wild birds); 25 strains were obtained from PI (16 from pigs and 9 from goats); and 19 strains were obtained from RN (13 from chickens, 5 from pigs, and 1 from goat). (B) Distribution of alleles of the virulence protein genes GRA15, ROP5, ROP16, ROP17, and ROP18 identified by PCR-RFLP in 103 strains isolated in the Northeast and Southeast regions of Brazil.
Figure 1. Distribution of Toxoplasma gondii strains according to the Brazilian regions from which they were isolated. (A) Map of Brazil showing the four states where the 103 Toxoplasma gondii strains were isolated: ES (Espírito Santo state), MG (Minas Gerais state), PI (Piauí state), and RN (Rio Grande do Norte state). In total, 5 strains were obtained from chickens from ES; 54 strains were obtained from MG (29 from humans—25 from peripheral blood of newborns with congenital toxoplasmosis and 4 from amniotic fluid; 8 from dogs; 11 from chickens; and 6 from free-living wild birds); 25 strains were obtained from PI (16 from pigs and 9 from goats); and 19 strains were obtained from RN (13 from chickens, 5 from pigs, and 1 from goat). (B) Distribution of alleles of the virulence protein genes GRA15, ROP5, ROP16, ROP17, and ROP18 identified by PCR-RFLP in 103 strains isolated in the Northeast and Southeast regions of Brazil.
Microorganisms 13 00301 g001
Microorganisms 13 00301 g002
Figure 2. Virulence of T. gondii strains in mice, categorized by dense granule GRA15 gene and rhoptry genes ROP5, ROP16, ROP17, and ROP18 allele types. Cumulative mouse mortality for 103 Toxoplasma gondii strains with published bioassay results was categorized for alleles of GRA15 (A), ROP17 (B), ROP16 (C), ROP18 (D), ROP5 (E), and the ROP18/ROP5 (F) combinations. The percentage of mouse cumulative mortality was plotted per allele type, with each dot representing the percent mortality reported for an individual parasite strain. The gray bars indicate the median cumulative mortality for each allele type. The number (n) of strains representing each allele type is in parentheses below allele identification. Different letters above allele data (a or b) indicate statistical differences as determined by Mann–Whitney U tests. Differences between data are significant at p < 0.001 for GRA15 and ROP18, p = 0.003 for ROP17, and p < 0.05 for the ROP18/ROP5 allele combinations.
Figure 2. Virulence of T. gondii strains in mice, categorized by dense granule GRA15 gene and rhoptry genes ROP5, ROP16, ROP17, and ROP18 allele types. Cumulative mouse mortality for 103 Toxoplasma gondii strains with published bioassay results was categorized for alleles of GRA15 (A), ROP17 (B), ROP16 (C), ROP18 (D), ROP5 (E), and the ROP18/ROP5 (F) combinations. The percentage of mouse cumulative mortality was plotted per allele type, with each dot representing the percent mortality reported for an individual parasite strain. The gray bars indicate the median cumulative mortality for each allele type. The number (n) of strains representing each allele type is in parentheses below allele identification. Different letters above allele data (a or b) indicate statistical differences as determined by Mann–Whitney U tests. Differences between data are significant at p < 0.001 for GRA15 and ROP18, p = 0.003 for ROP17, and p < 0.05 for the ROP18/ROP5 allele combinations.
Microorganisms 13 00301 g002
Microorganisms 13 00301 g003
Figure 3. Cumulative mouse mortality for 103 Toxoplasma gondii strains categorized for ROP18/ROP17/ROP16 (A,D,E) ROP18/ROP16 (B), and GRA15/ROP18/ROP17 (C) allele combinations. Percentage of mouse cumulative mortality is plotted against allele type, with each individual dot representing the percentage of mortality reported for an individual parasite strain. The gray bars indicate the median cumulative mortality for each allele type. The number (n) of strains representing each allele type is given in parentheses below allele identification. Different letters above allele data (a, b, or c) indicate statistical differences as determined by Mann–Whitney U tests. Differences between data are significant at p < 0.0001 for the ROP18/ROP16 allele combinations (B) and at p = 0.0004 for the ROP18/ROP16/ROP17 allele combinations (D).
Figure 3. Cumulative mouse mortality for 103 Toxoplasma gondii strains categorized for ROP18/ROP17/ROP16 (A,D,E) ROP18/ROP16 (B), and GRA15/ROP18/ROP17 (C) allele combinations. Percentage of mouse cumulative mortality is plotted against allele type, with each individual dot representing the percentage of mortality reported for an individual parasite strain. The gray bars indicate the median cumulative mortality for each allele type. The number (n) of strains representing each allele type is given in parentheses below allele identification. Different letters above allele data (a, b, or c) indicate statistical differences as determined by Mann–Whitney U tests. Differences between data are significant at p < 0.0001 for the ROP18/ROP16 allele combinations (B) and at p = 0.0004 for the ROP18/ROP16/ROP17 allele combinations (D).
Microorganisms 13 00301 g003
Table 1. Distribution of alleles of the virulence protein genes GRA15, ROP5, ROP16, ROP17, and ROP18 identified by PCR-RFLP in Brazilian atypical strains according to lethality in mice.
Table 1. Distribution of alleles of the virulence protein genes GRA15, ROP5, ROP16, ROP17, and ROP18 identified by PCR-RFLP in Brazilian atypical strains according to lethality in mice.
Lethal StrainsNon-Lethal Strains
GRA15n (f)n (f)Totalp value *
1 or 375 (98.7%)14 (51.9%)89 (86.4%)<0.001
21 (1.3%)13 (48.1%)14 (13.6%)
Total7627103
ROP5
11 (1.3%)3 (11.1%)4 (3.9%)0.04
371 (93.4%)24 (88.9%)95 (92.2%)
44 (5.3%)0 (0.0%)4 (3.9%)
Total7627103
ROP16
146 (60.5%)23 (88.5%)69 (67.6%)0.007
230 (39.5%)3 (11.5%)33 (32.4%)
Total7626102 a
ROP17
111 (15.1%)17 (63.0%)28 (28.0%)<0.001
22 (2.7%)3 (11.1%)5 (5.0%)
34 (5.5%)0 (0.0%)4 (4.0%)
456 (76.7%)7 (25.9%)63 (63.0%)
Total7327100 b
ROP18
110 (13.3%)0 (0.0%)10 (9.8%)<0.001
20 (0.0%)3 (11.1%)3 (2.9%)
317 (22.7%)22 (81.5%)39 (38.2%)
448 (64.0%)2 (7.4%)50 (49.0%)
Total7527102 a
a incomplete ROP16 and ROP18 genotyping for one strain. b incomplete ROP17 genotyping for three strains. * Pearson’s chi2.
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Costa, J.G.L.; Martins-Duarte, É.S.; Pinto, L.V.; Baraviera, R.A.d.C.; do Rego, W.M.F.; Vitor, R.W.d.A. Investigation of Virulence-Related Markers in Atypical Strains of Toxoplasma gondii from Brazil. Microorganisms 2025, 13, 301. https://doi.org/10.3390/microorganisms13020301

AMA Style

Costa JGL, Martins-Duarte ÉS, Pinto LV, Baraviera RAdC, do Rego WMF, Vitor RWdA. Investigation of Virulence-Related Markers in Atypical Strains of Toxoplasma gondii from Brazil. Microorganisms. 2025; 13(2):301. https://doi.org/10.3390/microorganisms13020301

Chicago/Turabian Style

Costa, Júlia Gatti Ladeia, Érica Santos Martins-Duarte, Lorena Velozo Pinto, Ramon Araujo de Castro Baraviera, Wagner Martins Fontes do Rego, and Ricardo Wagner de Almeida Vitor. 2025. "Investigation of Virulence-Related Markers in Atypical Strains of Toxoplasma gondii from Brazil" Microorganisms 13, no. 2: 301. https://doi.org/10.3390/microorganisms13020301

APA Style

Costa, J. G. L., Martins-Duarte, É. S., Pinto, L. V., Baraviera, R. A. d. C., do Rego, W. M. F., & Vitor, R. W. d. A. (2025). Investigation of Virulence-Related Markers in Atypical Strains of Toxoplasma gondii from Brazil. Microorganisms, 13(2), 301. https://doi.org/10.3390/microorganisms13020301

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop