High Prevalence of Enterocytozoon bieneusi Genotype BEB6 in Wild Boars in Lorestan Province, Iran: Potential Source of Zoonotic Transmission
-
Ehsan Javanmard
,
Hanieh Mohammad Rahimi
,
Ali Taghipour
,
Sara Nemati
,
Mehdi Mohebali
,
Mostafa Rezaeian
,
Ali Badrifar
,
Elham Kazemirad
,
Hamed Mirjalali
Abstract
Background: We aimed to investigate the prevalence of Enterocytozoon bieneusi and the circulating genotypes in wild boar in the western regions of Iran.
Methods: Fifty-two fecal samples were collected from wild boars in Lorestan province, Iran. After DNA extraction, the presence of E. bieneusi was evaluated by real-time PCR. A nested PCR targeting the internal transcribed region (ITS) was employed to characterize genotypes. The PCR products were sequenced, and the genetic diversity and relationships among the genotypes were identified using MEGA X and DnaSp (V5) software.
Results: Sixteen (30%) samples were positive for E. bieneusi using real-time PCR, and 11 (21%) were positive for nested PCR, which was sequenced. All 11 positive samples were identified as the BEB6 genotype (Group 2). The haplotype diversity was noted to be 0.182, and the nucleotide diversity, calculated using DnaSp, was 0. 00085.
Conclusion: The findings highlight the high prevalence of the genotype BEB6 in wild boars. The presence of this genotype suggests the circulation of E. bieneusi between domesticated animals and wild boars in Iran.
1. Han B, Pan G, Weiss LM. Microsporidiosis in humans. Clin Microbiol Rev. 2021;34(4): e0001020.
2. Weber R, Deplazes P, Mathis A. Microsporidia. Clin Microbiol Rev. 2015; 18(3):423-45.
3. Yildirim A, Okur M, Uslug S, et al. First report on the molecular prevalence of Enterocytozoon bieneusi in horses in Turkey: genotype distributions and zoonotic potential. Parasitol Res. 2020;119:2821-2828.
4. Sazmand A, Khordadmehr M, Önder Z, et al. Novel zoonotic Enterocytozoon and Encephalitozoon genotypes in domestic pigeons (Columba livia domestica) in Iran: Public health implications. Curr Res Parasitol Vector Borne Dis. 2024; 7:100232.
5. Li W, Xiao L. Ecological and public health significance of Enterocytozoon bieneusi. One Health. 2020;12:100209.
6. Stark D, Barratt J, Van Hal S, et al. Clinical significance of enteric protozoa in the immunosuppressed human population. Clin Microbiol Rev. 2009;22(4):634-50.
7. Wang Y, Li XM, Yang X, et al. Global prevalence and risk factors of Enterocytozoon bieneusi infection in humans: a systematic review and meta-analysis. Parasite. 2024;31:9.
8. Javanmard E, Nemati S, Sharifdini M, et al. The first report and molecular analysis of Enterocytozoon bieneusi from raccoon (Procyon lotor) in north of Iran. J Eukaryot Microbiol. 2020;67(3):359-68.
9. Yildirim Y, Al S, Duzlu O, et al. Enterocytozoon bieneusi in raw milk of cattle, sheep and water buffalo in Turkey: Genotype distributions and zoonotic concerns. Int J Food Microbiol. 2020;334:108828.
10. Ruan Y, Xu X, He Q, et al. The largest meta-analysis on the global prevalence of microsporidia in mammals, avian and water provides insights into the epidemic features of these ubiquitous pathogens. Parasit Vectors. 2021;14:186.
11. Li DF, Zhang Y, Jiang YX, et al. Genotyping and zoonotic potential of Enterocytozoon bieneusi in pigs in Xinjiang, China. Front Microbiol. 2019;10:2401.
12. Li W, Feng Y, Xiao L. Diagnosis and molecular typing of Enterocytozoon bieneusi: the significant role of domestic animals in transmission of human microsporidiosis. Res Vet Sci. 2020;133:251-61.
13. Bergmann H, Schulz K, Conraths FJ, et al. A review of environmental risk factors for African swine fever in European wild boar. Animals (Basel). 2021;11(9):2692.
14. Markov N, Economov A, Hjeljord O, et al. The wild boar Sus scrofa in northern Eurasia: a review of range expansion history, current distribution, factors affecting the northern distributional limit, and management strategies. Mamm Rev. 2022;52:519-37.
15. Taghipour A, Bahadory S, Khazaei S, et al. Global molecular epidemiology of microsporidia in pigs and wild boars with emphasis on Enterocytozoon bieneusi: A systematic review and meta‐analysis. Vet Med Sci. 2022;8(3):1126-36.
16. Feng S, Jia T, Huang J, et al. Identification of Enterocytozoon bieneusi and Cryptosporidium spp. in farmed wild boars (Sus scrofa) in Beijing, China. Infect Genet Evol. 2020;80:104231.
17. Lee H, Seo MG, Lee SH, et al. Distribution and genotypic analysis of Enterocytozoon bieneusi from wild boars in Korea. Med Mycol. 2021;59(9):934-8.
18. Verweij JJ, Ten Hove R, Brienen EA, et al. Multiplex detection of Enterocytozoon bieneusi and Encephalitozoon spp. in fecal samples using real-time PCR. Diagn Microbiol Infect Dis. 2007;57(2):163-7.
19. Javanmard E, Mirjalali H, Niyyati M, et al. Molecular and phylogenetic evidences of dispersion of human-infecting microsporidia to vegetable farms via irrigation with treated wastewater: one-year follow up. Int J Hyg Environ Health. 2018;221(4):642-51.
20. Kumar S, Stecher G, Li M, et al. MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol. 2018;35(6):1547-9.
21. Rozas J, Sánchez-DelBarrio JC, Messeguer X, et al. DnaSP, DNA polymorphism analyses by the coalescent and other methods. Bioinformatics. 2003;19(18):2496-7.
22. Tajima F. Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics. 1989;123(3):585-95.
23. Zhang K, Zheng S, Wang Y, et al. Occurrence and molecular characterization of Cryptosporidium spp., Giardia duodenalis, Enterocytozoon bieneusi, and Blastocystis sp. in captive wild animals in zoos in Henan, China. BMC Vet Res. 2021;17(1):332.
24. Masuda A, Wada M, Saho H, et al. Prevalence and molecular characterization of the zoonotic enteric protozoans Cryptosporidium spp., Enterocytozoon bieneusi, and Blastocystis from Pallas's Squirrels (Callosciurus erythraeus) in Kanagawa Prefecture, Japan. Microbiol Spectr. 2021;9(3): e0099021.
25. Didier ES. Microsporidiosis: an emerging and opportunistic infection in humans and animals. Acta Trop. 2005;94(1):61-76.
26. Rojas A, Germitsch N, Oren S, et al. Wildlife parasitology: sample collection and processing, diagnostic constraints, and methodological challenges in terrestrial carnivores. Parasit Vectors. 2024;17(1):127.
27. Weiss LM, Vossbrinck CR. Molecular biology, molecular phylogeny, and molecular diagnostic approaches to the microsporidia. The Microsporidia And Microsporidiosis. 1999:129-71.
28. Subrungruang I, Mungthin M, Chavalitshewinkoon-Petmitr P, et al. Evaluation of DNA extraction and PCR methods for detection of Enterocytozoon bienuesi in stool specimens. J Clin Microbiol. 2004;42(8):3490-4.
29. Polley SD, Boadi S, Watson J, et al. Detection and species identification of microsporidial infections using SYBR Green real-time PCR. J Med Microbiol. 2011;60(Pt 4):459-66.
30. Stensvold CR, Jirků-Pomajbíková K, Tams KW, et al. Parasitic intestinal protists of zoonotic relevance detected in pigs by metabarcoding and real-time PCR. Microorganisms. 2021;9(6):1189.
31. Rubio J, Lanza M, Fuentes I, et al. A novel nested multiplex PCR for the simultaneous detection and differentiation of Cryptosporidium spp., Enterocytozoon bieneusi and Encephalitozoon intestinalis. Parasitol Int. 2014;63(5):664-9.
32. Perec-Matysiak A, Leśniańska K, Buńkowska-Gawlik K, et al. Zoonotic genotypes of Enterocytozoon bieneusi in wild living invasive and native carnivores in Poland. Pathogens. 2021;10(11):1478.
33. Wang H, Zhang Y, Wu Y, et al. Occurrence, molecular characterization, and assessment of zoonotic risk of Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi in pigs in Henan, Central China. J Eukaryot Microbiol. 2018;65(6):893-901.
34. Li W, Diao R, Yang J, et al. High diversity of human-pathogenic Enterocytozoon bieneusi genotypes in swine in northeast China. Parasitol Res. 2014;113:1147-53.
35. Sak B, Kváč M, Hanzlíková D, Cama V. First report of Enterocytozoon bieneusi infection on a pig farm in the Czech Republic. Vet Parasitol. 2008;153(3-4):220-4.
36. Reetz J, Nöckler K, Reckinger S, et al. Identification of Encephalitozoon cuniculi genotype III and two novel genotypes of Enterocytozoon bieneusi in swine. Parasitol Int. 2009;58(3):285-92.
37. Dashti A, Rivero‐Juarez A, Santín M, et al. Enterocytozoon bieneusi (Microsporidia): Identification of novel genotypes and evidence of transmission between sympatric wild boars (Sus scrofa ferus) and Iberian pigs (Sus scrofa domesticus) in Southern Spain. Transbound Emerg Dis. 2020;67(6):2869-80.
38. Němejc K, Sak B, Květoňová D, et al. Prevalence and diversity of Encephalitozoon spp. and Enterocytozoon bieneusi in wild boars (Sus scrofa) in Central Europe. Parasitol Res. 2014;113:761-7.
39. Mirjalali H, Mirhendi H, Meamar AR, et al. Genotyping and molecular analysis of Enterocytozoon bieneusi isolated from immunocompromised patients in Iran. Infect Genet Evol. 2015;36:244-249.
40. Tavalla M, Mardani-Kateki M, Abdizadeh R, et al. Molecular diagnosis of potentially human pathogenic Enterocytozoon bieneusi and Encephalitozoon species in exotic birds in Southwestern Iran. J Infect Public Health. 2018;11(2):192-6.
41. Delrobaei M, Jamshidi S, Shayan P, et al. Molecular detection and genotyping of intestinal microsporidia from stray dogs in Iran. Iran J Parasitol. 2019;14(1):159-166.
42. Muadica AS, Messa Jr AE, Dashti A, et al. First identification of genotypes of Enterocytozoon bieneusi (Microsporidia) among symptomatic and asymptomatic children in Mozambique. PLoS Negl Trop Dis. 2020;14(6): e0008419.
43. Feng Y, Xiao L, Liu D. Population Genetics of Enterocytozoon bieneusi. Food Microbiol. CRC Press; 2021:309-16.
44. Prasertbun R, Mori H, Pintong A-r, Sanyanusin S, et al. Zoonotic potential of Enterocytozoon genotypes in humans and pigs in Thailand. Vet Parasitol. 2017;233:73-9.
45. Mohammad Rahimi H, Mirjalali H, Zali MR. Molecular epidemiology and genotype/subtype distribution of Blastocystis sp., Enterocytozoon bieneusi, and Encephalitozoon spp. in livestock: concern for emerging zoonotic infections. Sci Rep. 2021;11(1):17467.
46. Zhao W, Zhou H, Yang L, et al. Prevalence, genetic diversity and implications for public health of Enterocytozoon bieneusi in various rodents from Hainan Province, China. Parasit Vectors. 2020;13:438.
47. Henriques-Gil N, Haro M, Izquierdo F, et al. Phylogenetic approach to the variability of the microsporidian Enterocytozoon bieneusi and its implications for inter-and intrahost transmission. Appl Environ Microbiol. 2010;76(10):3333-42.
2. Weber R, Deplazes P, Mathis A. Microsporidia. Clin Microbiol Rev. 2015; 18(3):423-45.
3. Yildirim A, Okur M, Uslug S, et al. First report on the molecular prevalence of Enterocytozoon bieneusi in horses in Turkey: genotype distributions and zoonotic potential. Parasitol Res. 2020;119:2821-2828.
4. Sazmand A, Khordadmehr M, Önder Z, et al. Novel zoonotic Enterocytozoon and Encephalitozoon genotypes in domestic pigeons (Columba livia domestica) in Iran: Public health implications. Curr Res Parasitol Vector Borne Dis. 2024; 7:100232.
5. Li W, Xiao L. Ecological and public health significance of Enterocytozoon bieneusi. One Health. 2020;12:100209.
6. Stark D, Barratt J, Van Hal S, et al. Clinical significance of enteric protozoa in the immunosuppressed human population. Clin Microbiol Rev. 2009;22(4):634-50.
7. Wang Y, Li XM, Yang X, et al. Global prevalence and risk factors of Enterocytozoon bieneusi infection in humans: a systematic review and meta-analysis. Parasite. 2024;31:9.
8. Javanmard E, Nemati S, Sharifdini M, et al. The first report and molecular analysis of Enterocytozoon bieneusi from raccoon (Procyon lotor) in north of Iran. J Eukaryot Microbiol. 2020;67(3):359-68.
9. Yildirim Y, Al S, Duzlu O, et al. Enterocytozoon bieneusi in raw milk of cattle, sheep and water buffalo in Turkey: Genotype distributions and zoonotic concerns. Int J Food Microbiol. 2020;334:108828.
10. Ruan Y, Xu X, He Q, et al. The largest meta-analysis on the global prevalence of microsporidia in mammals, avian and water provides insights into the epidemic features of these ubiquitous pathogens. Parasit Vectors. 2021;14:186.
11. Li DF, Zhang Y, Jiang YX, et al. Genotyping and zoonotic potential of Enterocytozoon bieneusi in pigs in Xinjiang, China. Front Microbiol. 2019;10:2401.
12. Li W, Feng Y, Xiao L. Diagnosis and molecular typing of Enterocytozoon bieneusi: the significant role of domestic animals in transmission of human microsporidiosis. Res Vet Sci. 2020;133:251-61.
13. Bergmann H, Schulz K, Conraths FJ, et al. A review of environmental risk factors for African swine fever in European wild boar. Animals (Basel). 2021;11(9):2692.
14. Markov N, Economov A, Hjeljord O, et al. The wild boar Sus scrofa in northern Eurasia: a review of range expansion history, current distribution, factors affecting the northern distributional limit, and management strategies. Mamm Rev. 2022;52:519-37.
15. Taghipour A, Bahadory S, Khazaei S, et al. Global molecular epidemiology of microsporidia in pigs and wild boars with emphasis on Enterocytozoon bieneusi: A systematic review and meta‐analysis. Vet Med Sci. 2022;8(3):1126-36.
16. Feng S, Jia T, Huang J, et al. Identification of Enterocytozoon bieneusi and Cryptosporidium spp. in farmed wild boars (Sus scrofa) in Beijing, China. Infect Genet Evol. 2020;80:104231.
17. Lee H, Seo MG, Lee SH, et al. Distribution and genotypic analysis of Enterocytozoon bieneusi from wild boars in Korea. Med Mycol. 2021;59(9):934-8.
18. Verweij JJ, Ten Hove R, Brienen EA, et al. Multiplex detection of Enterocytozoon bieneusi and Encephalitozoon spp. in fecal samples using real-time PCR. Diagn Microbiol Infect Dis. 2007;57(2):163-7.
19. Javanmard E, Mirjalali H, Niyyati M, et al. Molecular and phylogenetic evidences of dispersion of human-infecting microsporidia to vegetable farms via irrigation with treated wastewater: one-year follow up. Int J Hyg Environ Health. 2018;221(4):642-51.
20. Kumar S, Stecher G, Li M, et al. MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol. 2018;35(6):1547-9.
21. Rozas J, Sánchez-DelBarrio JC, Messeguer X, et al. DnaSP, DNA polymorphism analyses by the coalescent and other methods. Bioinformatics. 2003;19(18):2496-7.
22. Tajima F. Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics. 1989;123(3):585-95.
23. Zhang K, Zheng S, Wang Y, et al. Occurrence and molecular characterization of Cryptosporidium spp., Giardia duodenalis, Enterocytozoon bieneusi, and Blastocystis sp. in captive wild animals in zoos in Henan, China. BMC Vet Res. 2021;17(1):332.
24. Masuda A, Wada M, Saho H, et al. Prevalence and molecular characterization of the zoonotic enteric protozoans Cryptosporidium spp., Enterocytozoon bieneusi, and Blastocystis from Pallas's Squirrels (Callosciurus erythraeus) in Kanagawa Prefecture, Japan. Microbiol Spectr. 2021;9(3): e0099021.
25. Didier ES. Microsporidiosis: an emerging and opportunistic infection in humans and animals. Acta Trop. 2005;94(1):61-76.
26. Rojas A, Germitsch N, Oren S, et al. Wildlife parasitology: sample collection and processing, diagnostic constraints, and methodological challenges in terrestrial carnivores. Parasit Vectors. 2024;17(1):127.
27. Weiss LM, Vossbrinck CR. Molecular biology, molecular phylogeny, and molecular diagnostic approaches to the microsporidia. The Microsporidia And Microsporidiosis. 1999:129-71.
28. Subrungruang I, Mungthin M, Chavalitshewinkoon-Petmitr P, et al. Evaluation of DNA extraction and PCR methods for detection of Enterocytozoon bienuesi in stool specimens. J Clin Microbiol. 2004;42(8):3490-4.
29. Polley SD, Boadi S, Watson J, et al. Detection and species identification of microsporidial infections using SYBR Green real-time PCR. J Med Microbiol. 2011;60(Pt 4):459-66.
30. Stensvold CR, Jirků-Pomajbíková K, Tams KW, et al. Parasitic intestinal protists of zoonotic relevance detected in pigs by metabarcoding and real-time PCR. Microorganisms. 2021;9(6):1189.
31. Rubio J, Lanza M, Fuentes I, et al. A novel nested multiplex PCR for the simultaneous detection and differentiation of Cryptosporidium spp., Enterocytozoon bieneusi and Encephalitozoon intestinalis. Parasitol Int. 2014;63(5):664-9.
32. Perec-Matysiak A, Leśniańska K, Buńkowska-Gawlik K, et al. Zoonotic genotypes of Enterocytozoon bieneusi in wild living invasive and native carnivores in Poland. Pathogens. 2021;10(11):1478.
33. Wang H, Zhang Y, Wu Y, et al. Occurrence, molecular characterization, and assessment of zoonotic risk of Cryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi in pigs in Henan, Central China. J Eukaryot Microbiol. 2018;65(6):893-901.
34. Li W, Diao R, Yang J, et al. High diversity of human-pathogenic Enterocytozoon bieneusi genotypes in swine in northeast China. Parasitol Res. 2014;113:1147-53.
35. Sak B, Kváč M, Hanzlíková D, Cama V. First report of Enterocytozoon bieneusi infection on a pig farm in the Czech Republic. Vet Parasitol. 2008;153(3-4):220-4.
36. Reetz J, Nöckler K, Reckinger S, et al. Identification of Encephalitozoon cuniculi genotype III and two novel genotypes of Enterocytozoon bieneusi in swine. Parasitol Int. 2009;58(3):285-92.
37. Dashti A, Rivero‐Juarez A, Santín M, et al. Enterocytozoon bieneusi (Microsporidia): Identification of novel genotypes and evidence of transmission between sympatric wild boars (Sus scrofa ferus) and Iberian pigs (Sus scrofa domesticus) in Southern Spain. Transbound Emerg Dis. 2020;67(6):2869-80.
38. Němejc K, Sak B, Květoňová D, et al. Prevalence and diversity of Encephalitozoon spp. and Enterocytozoon bieneusi in wild boars (Sus scrofa) in Central Europe. Parasitol Res. 2014;113:761-7.
39. Mirjalali H, Mirhendi H, Meamar AR, et al. Genotyping and molecular analysis of Enterocytozoon bieneusi isolated from immunocompromised patients in Iran. Infect Genet Evol. 2015;36:244-249.
40. Tavalla M, Mardani-Kateki M, Abdizadeh R, et al. Molecular diagnosis of potentially human pathogenic Enterocytozoon bieneusi and Encephalitozoon species in exotic birds in Southwestern Iran. J Infect Public Health. 2018;11(2):192-6.
41. Delrobaei M, Jamshidi S, Shayan P, et al. Molecular detection and genotyping of intestinal microsporidia from stray dogs in Iran. Iran J Parasitol. 2019;14(1):159-166.
42. Muadica AS, Messa Jr AE, Dashti A, et al. First identification of genotypes of Enterocytozoon bieneusi (Microsporidia) among symptomatic and asymptomatic children in Mozambique. PLoS Negl Trop Dis. 2020;14(6): e0008419.
43. Feng Y, Xiao L, Liu D. Population Genetics of Enterocytozoon bieneusi. Food Microbiol. CRC Press; 2021:309-16.
44. Prasertbun R, Mori H, Pintong A-r, Sanyanusin S, et al. Zoonotic potential of Enterocytozoon genotypes in humans and pigs in Thailand. Vet Parasitol. 2017;233:73-9.
45. Mohammad Rahimi H, Mirjalali H, Zali MR. Molecular epidemiology and genotype/subtype distribution of Blastocystis sp., Enterocytozoon bieneusi, and Encephalitozoon spp. in livestock: concern for emerging zoonotic infections. Sci Rep. 2021;11(1):17467.
46. Zhao W, Zhou H, Yang L, et al. Prevalence, genetic diversity and implications for public health of Enterocytozoon bieneusi in various rodents from Hainan Province, China. Parasit Vectors. 2020;13:438.
47. Henriques-Gil N, Haro M, Izquierdo F, et al. Phylogenetic approach to the variability of the microsporidian Enterocytozoon bieneusi and its implications for inter-and intrahost transmission. Appl Environ Microbiol. 2010;76(10):3333-42.
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| Issue | Vol 20 No 1 (2025) | |
| Section | Original Article(s) | |
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| Enterocytozoon bieneusi Wild boars Iran Molecular analysis | ||
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How to Cite
1.
Javanmard E, Mohammad Rahimi H, Taghipour A, Nemati S, Mohebali M, Rezaeian M, Badrifar A, Kazemirad E, Mirjalali H. High Prevalence of Enterocytozoon bieneusi Genotype BEB6 in Wild Boars in Lorestan Province, Iran: Potential Source of Zoonotic Transmission. Iran J Parasitol. 2025;20(1):130-139.
