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Journal of Parasitology Research
Volume 2011 (2011), Article ID 129542, 2 pages
http://dx.doi.org/10.1155/2011/129542
Research Article

Molecular Identification of Enterocytozoon bieneusi Isolates from Nigerian Children

1Department of Veterinary Microbiology and Parasitology, Faculty of Veterinary Medicine, University of Ibadan, Ibadan, Nigeria
2Division of Foodborne, Waterborne, and Environmental Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA
3Department of Zoology, Lagos State University, Ojo, Lagos, Nigeria

Received 21 July 2011; Accepted 14 September 2011

Academic Editor: Takeshi Agatsuma

Copyright © 2011 Adekunle Bamidele Ayinmode et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

A study was conducted to detect and identify enteric microsporidian species in 43 children from Oyo state, Nigeria. Using nested polymerase chain reaction, 9.3% of the children were identified as positive for Enterocytozoon bieneusi. DNA sequencing of the PCR products showed the presence of three known genotypes (two isolates of genotype D and one of genotype K) and one new genotype. This study suggests that either human or animal (or both) could be the infection source for the children, since identified genotypes D and K have been previously detected in both immunocompromised and immunocompetent patients and domestic animals. The identification of high diversity also suggests intensive transmission of microsporidiosis in the studied area.

1. Introduction

Microsporidia are enteric protozoan parasites that are increasingly recognized as important pathogens in immunocompromised persons and malnourished children. Some of the species of this organism infecting humans have been found in animals and water, thereby raising concerns for zoonotic, foodborne, and waterborne transmission [13]. Enterocytozoon bieneusi is the most common microsporidian species in humans, infecting enterocytes and other epithelial cells of human [1]. Although given lesser attention, the parasite is emerging as an important etiology of diarrhoea in immunocompromised and immunocompetent persons [1]. Enterocytozoon bieneusi causes chronic diarrhea and weight loss in human-immunodeficiency-virus- (HIV-) infected patients, and the infection is increasingly observed in organ transplant recipients [46].

While there are a lot of studies on opportunistic organisms like Cryptosporidium and Giardia, microsporidiosis is rarely studied. This preliminary study was conducted to identify the species- and genotypes-involved microsporidiosis in children from Oyo state of Nigeria.

2. Materials and Methods

Fecal samples were collected randomly from 43 (33 (76.7%) diarrhoeic and 10 (23.3%) nondiarrhoeic) children attending an outpatient department of a hospital in Oyo state, southwestern Nigeria. These samples were processed for DNA extraction with FastDNA Spin Kit for soil (MP Biologicals, Solon, Ohio, USA). For the detection of E. bieneusi, a nested PCR was used to amplify a ~400-bp fragment of the internal transcribed spacer (ITS) region of the rRNA gene [7]. The PCR products were visualized on 1% agarose gel after ethidium bromide staining [7]. Enterocytozoon bieneusi was genotyped by sequence analysis of the ITS PCR products.

DNA sequencing of E. bieneusi ITS product was done using an ABI Prism 3130 Genetic Analyzer and the BigDye Terminator Sequencing Kit (Applied Biosystems, Foster City, Calif, USA). The nucleotide sequences obtained were aligned with each other and reference sequences downloaded from the GenBank using ClustalX (http://www.clustal.org/). E. bieneusi genotypes were determined by their similarity to the sequences from known genotypes and subtypes. The established terminology was used in naming the E. bieneusi genotypes [1].

3. Results and Discussion

PCR analysis of the ITS gene identified E. bieneusi in four samples (Table 1). DNA sequencing of the PCR products identified the presence of three genotypes. Two of the genotypes were known ones, D (in two isolates) and K (in one isolate), and one had two nucleotide substitutions comparing to genotype K (in one isolate). Among the four E. bieneusi-positive children, three had diarrhoea and one did not have diarrhoea (Table 1).

Table 1: Showing the prevalence of Enterocytozoon bieneusi isolates from Nigerian children.

While in this pilot study, only a small number of children were found positive for E. bieneusi, the genotype and subtype diversity of the parasite was high, suggesting that there was intensive transmission of microsporidiosis in this area. The source of E. bieneusi in the studied children is not known. However, both genotypes D and K identified in the study have been previously detected in both immunocompromised and immunocompetent patients and domestic animals. [1] While, these genotypes could be of animal origin, a data from Cryptosporidium investigation conducted on the same group of patient [8] suggests a strong possibility of anthroponotic transmission of these two E. bieneusi genotypes in the study area. More systematic collection of epidemiological data and sampling of animals, drinking water, and fresh produce are needed to clarify the source of E. bieneusi in Nigerian children.

Geographically isolated E. bieneusi genotypes/subtypes are probably circulating in Nigerian children, and even one of the E. bieneusi genotypes represents a new genotype.

In conclusion, our study reports high genotype and subtype diversity of E. bieneusi and also suggests that anthroponotic transmissions may play a significant role in the epidemiology of microsporidiosis. More studies are needed on the source and transmission of microsporidiosis in Nigeria and in other developing countries.

Acknowledgments

This research was supported by the John D. and Catherine T. MacArthur Foundation Overseas Training Grant from University of Ibadan (Nigeria) and the Center for Diseases Control, and Prevention, Atlanta, GA, USA. The authors thank Theresa Dearen for technical assistance. This study is part of a larger study with approval from the University of Ibadan/University College Hospital Ethics Committee with the assigned number: UI/EC/10/0129. The findings and conclusions in this paper are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention. This work was carried out at the Division of Foodborne, Waterborne, and Environmental Diseases, Centers for Disease Control and Prevention, Atlanta, Ga 30333, USA.

References

  1. R. J. Ten Hove, L. Van Lieshout, M. B. J. Beadsworth et al., “Characterization of genotypes of Enterocytozoon bieneusi in immunosuppressed and immunocompetent patient groups,” Journal of Eukaryotic Microbiology, vol. 56, no. 4, pp. 388–393, 2009. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  2. B. Dengjel, M. Zahler, W. Hermanns et al., “Zoonotic potential of Enterocytozoon bieneusi,” Journal of Clinical Microbiology, vol. 39, no. 12, pp. 4495–4499, 2001. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  3. I. M. Sulaiman, R. Fayer, A. A. Lal, J. M. Trout, F. W. Schaefer, and L. Xiao, “Molecular characterization of microsporidia indicates that wild mammals harbor host-adapted Enterocytozoon spp. as well as human-pathogenic Enterocytozoon bieneusi,” Applied and Environmental Microbiology, vol. 69, no. 8, pp. 4495–4501, 2003. View at Publisher · View at Google Scholar · View at Scopus
  4. R. T. Bryan and D. A. Schwartz, “Epidemiology of microsporidiosis,” in The Microsporidia and Microsporidiosis, M. Wittner, Ed., pp. 502–516, ASM Press, Washington, DC, USA, 1999.
  5. S. Fournier, O. Liguory, V. Garrait et al., “Microsporidiosis due to Enterocytozoon bieneusi infection as a possible cause of traveller's diarrhea,” European Journal of Clinical Microbiology and Infectious Diseases, vol. 17, no. 10, pp. 743–744, 1998. View at Publisher · View at Google Scholar · View at Scopus
  6. A. Guerard, M. Rabodonirina, L. Cotte et al., “Intestinal microsporidiosis occurring in two renal transplant recipients treated with mycophenolate mofetil,” Transplantation, vol. 68, no. 5, pp. 699–707, 1999. View at Publisher · View at Google Scholar · View at Scopus
  7. V. A. Cama, J. Pearson, L. Cabrera et al., “Transmission of Enterocytozoon bieneusi between a child and guinea pigs,” Journal of Clinical Microbiology, vol. 45, no. 8, pp. 2708–2710, 2007. View at Publisher · View at Google Scholar · View at PubMed · View at Scopus
  8. A. B. Ayinmode, B. O. Fagbemi, and L. Xiao, “Molecular characterization of Cryptosporidium in children in Oyo State, Nigeria: implication for infection sources,” Parasitology Research. In press.