Original Article

Phylogenetic Analysis and Genetics Polymorphisms Evaluation of ROP8 and B1 Genes of Toxoplasma gondii in Livestock and Poultry Hosts of Yazd, Qom and Golestan Provinces of Iran


Background: A high correlation is observed between specific clonal lineages and host types in toxoplasmosis. The main objectives of this study were comparing polymorphism and evolutionary analysis of the B1 and ROP8 genes, as well as the evaluation of phylogenic and Toxoplasma gondii isolates obtained from different hosts and regions.

Methods: Overall 96 brain/ diaphragm tissue samples of livestock and poultry from three provinces of Iran (cows: 9 from Yazd, 9 from Qom; sheep: 19 from Yazd, 7 from Qom; goats: 7 from Yazd, 4 from Qom; one camel from Yazd and 37 chickens, 2 roosters and one duck from Golestan) were tested during 2018-19. A nested PCR and PCR-PCR methods were developed with the B1 and ROP8 genes. Evaluation of genetic proximity, genetic diversity and evolutionary analysis were done using MEGA-X and DnaSP5 software. Thirty samples of both genes were sequenced (18 B1 and 12 ROP8 genes), and submitted to the GenBank (MN275903-MN275932).

Results: Tajima's D index analyses showed that both genes were in the negative direction of evolution. The B1 gene was more sensitive than the ROP8 gene. The ROP8 gene showed better and more acceptable results in terms of the relationship between the host and the genotyping of the samples.

Conclusion: The B1 gene was only an attractive target for rapid detection of T. gondii parasites, whereas the ROP8 gene due to a high level of polymorphism was able to isolate the three clonal lineages (type I, II and III), inter-types and even atypical strains from different isolates of T. gondii.

1. Dubey JP. Toxoplasmosis of animals and humans. 2nd ed. Beltsville. Maryland: U.S.A; CRC press 2016.
2. Sharif M, Sarvi Sh, Shokri A, et al. Toxoplasma gondii infection among sheep and goats in Iran: a systematic review and meta-analysis. Parasitol Res. 2015; 114(1):1-16.
3. Tenter AM, Heckeroth AR, Weiss LM. Toxoplasma gondii: from animals to humans. Int J Parasitol. 2000; 30(12-13):1217-1258.
4. Guo M, Dubey JP, Hill D, et al. Prevalence and risk factors for Toxoplasma gondii infection in meat animals and meat products destined for human consumption. J Food Prot. 2015; 78(2):457-476.
5. Habibi GR, Imani AR, Gholami MR, et al. Detection and identification of Toxoplasma gondii type one infection in sheep aborted fetuses in Qazvin Province of Iran. Iran J Parasitol. 2012; 7(3):64–72.
6. Howe DK, Sibley LD. Toxoplasma gondii comprises three clonal lineages: correlation of parasite genotype with human disease. J Infect Dis. 1995; 172(6):1561-1566.
7. Galal L, Ajzenberg D, Hamidović A, et al. Toxoplasma and Africa: one parasite, two opposite population structures. Trends Parasitol. 2018; 34(2):140-154.
8. Sharif M, Amouei A, Sarvi S, et al., Genetic diversity of Toxoplasma gondii isolates from ruminants: a systematic review. Int J Food Microbiol. 2017; 258:38-49.
9. Ajzenberg D, Banuls AL, Su C, et al. Genetic diversity, clonality and sexuality in Toxoplasma gondii. Int J Parasitol. 2004; 34(10):1185-1196.
10. Daryani A, Sarvi Sh, Aarabi M, et al. Seroprevalence of Toxoplasma gondii in the Iranian general population: a systematic review and meta-analysis. Acta Trop. 2014; 137:185-194.
11. Rahimi MT, Daryani A, Sarvi Sh, et al. Cats and Toxoplasma gondii: A systematic review and meta-analysis in Iran. Onderstepoort J Vet Res. 2015; 82(1):01-10.
12. Liu Q, Wang ZD, Huang SY, et al. Diagnosis of toxoplasmosis and typing of Toxoplasma gondii. Parasit Vectors. 2015; 8(1):292-306.
13. Asgari Q, Mohammadpour I, Pirzad R, et al., Molecular and Serological Detection of Toxoplasma gondii in Stray Cats in Shiraz, South-central, Iran. Iran J Parasitol. 2018. 13(3):p. 430–439.
14. Hamilton CM, Robins R, Thomas R, Oura C, et al. Prevalence and genetic diversity of Toxoplasma gondii in free-ranging chickens from the Caribbean. Acta parasitologica. 2019; 64(4):738-44.
15. Foroutan M, Ghaffarifar F, Sharifi Z, et al. Vaccination with a novel multi-epitope ROP8 DNA vaccine against acute Toxoplasma gondii infection induces strong B and T cell responses in mice. Comp Immunol Microbiol Infect Dis. 2020; 69:101413.
16. Wang JL, Li TT, Li ZY, et al. Rhoptry protein 47 gene sequence: a potential novel genetic marker for population genetic studies of Toxoplasma gondii. Exp Parasitol. 2015; 154:1-4.
17. Li ZY, Chen J, Lu J, et al. Sequence variation in ROP8 gene among Toxoplasma gondii isolates from different hosts and geographical localities. Genet Mol Res. 2015; 14(3): 11403-11409.
18. Dubey JP. Refinement of pepsin digestion method for isolation of Toxoplasma gondii from infected tissues. Vet Parasitol. 1998; 74(1):75-77.
19. Grigg ME, Boothroyd JC. Rapid Identification of Virulent Type I Strains of the Protozoan Pathogen Toxoplasma gondii by PCR-Restriction Fragment Length Polymorphism Analysis at theB1 Gene. J Clin Microbiol. 2001; 39(1):398-400.
20. Altschul SF, Gish W, Miller W, et al. Basic local alignment search tool. J Mol Biol. 1990; 215(3):403-410.
21. Librado P, Rozas J. DnaSP v5: a software for comprehensive analysis of DNA polymorphism data. Bioinformatics. 2009; 25(11):1451-1452.
22. Tajima F. Evolutionary relationship of DNA sequences in finite populations. Genetics. 1983; 105(2):437-460.
23. Ivović V, Vujanić M, Živković T, Klun I, et al. Molecular detection and genotyping of Toxoplasma gondii from clinical samples. In: Djaković OD, editor. Toxoplasmosis. Rijeka: Croatia; 2012. P. 5:1-18.
24. Rozas J. DNA sequence polymorphism analysis using DnaSP. Methods Mol Biol. 2009; 537:337-350.
25. Nei M, Gojobori T. Simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions. Mol Biol Evol. 1986; 3(5):418-426.
26. Guéguen L, Duret L. Unbiased estimate of synonymous and nonsynonymous substitution rates with nonstationary base composition. Mol Biol Evol. 2018; 35(3):734-42.
27. Watterson GA. On the number of segregating sites in genetical models without recombination. Theor Popul Biol. 1975; 7(2):256-276.
28. Fotouhi-Ardakani R, Dabiri S, Ajdari S, et al. Assessment of nuclear and mitochondrial genes in precise identification and analysis of genetic polymorphisms for the evaluation of Leishmania parasites. Infect Genet Evol. 2016; 46:33-41.
29. Kalambhe D, Gill JPS, Singh BB. Molecular detection of Toxoplasma gondii in the slaughter sheep and goats from North India. Vet Parasitol. 2017; 241:35-38.
30. Khan A, Su C, German M, et al. Genotyping of Toxoplasma gondii strains from immunocompromised patients reveals high prevalence of type I strains. J Clin Microbiol. 2005; 43(12):5881-5887.
31. Sibley LD, Khan A, Ajioka JW, et al. Genetic diversity of Toxoplasma gondii in animals and humans. Philos Trans R Soc Lond B Biol Sci. 2009; 364(1530):2749-2761.
32. Saeij JPJ, Boyle JP, Boothroyd JC. Differences among the three major strains of Toxoplasma gondii and their specific interactions with the infected host. Trends Parasitol. 2005; 21(10):476-481.
33. Taniguchi Y, Appiah-Kwarteng C, Murakami M, et al. Atypical virulence in a type III Toxoplasma gondii strain isolated in Japan. Parasitol Int. 2018; 67(5):587-592.
34. Sroka J, Bilska-Zajac E, Wojcik-Fatla A, et al. Detection and Molecular Characteristics of Toxoplasma gondii DNA in Retail Raw Meat Products in Poland. Foodborne Pathog Dis. 2019; 16(3):195-204.
35. Carrillo C, Lu Z, Borca MV, et al. Genetic and phenotypic variation of foot-and-mouth disease virus during serial passages in a natural host. J Virol. 2007; 81(20):11341-11351.
36. Kumar S, Miller LK. Effects of serial passage of Autographa californica nuclear polyhedrosis virus in cell culture. Virus Res. 1987; 7(4):335-349.
37. Waldeland H, Pfefferkorn ER, Frenkel JK. Temperature-sensitive mutants of Toxoplasma gondii: pathogenicity and persistence in mice. J Parasitol. 1983; 69:171-175.
IssueVol 16 No 4 (2021) QRcode
SectionOriginal Article(s)
DOI https://doi.org/10.18502/ijpa.v16i4.7870
Toxoplasma gondii Phylogenic analysis Genetics polymorphisms evaluation

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How to Cite
Azimpour-Ardakan T, Fotouhi-Ardakani R, Hoghooghi-Rad N, Rokni N, Motallebi A. Phylogenetic Analysis and Genetics Polymorphisms Evaluation of ROP8 and B1 Genes of Toxoplasma gondii in Livestock and Poultry Hosts of Yazd, Qom and Golestan Provinces of Iran. Iran J Parasitol. 2021;16(4):576-586.