Research

Evidence against Wolbachia symbiosis in Loa loa

Helen F McGarry^1* , Ken Pfarr^2* , Gill Egerton¹, Achim Hoerauf², Jean-Paul Akue³, Peter Enyong⁴, Samuel Wanji⁵, Sabine L Kläger⁶, Albert E Bianco⁷, Nick J Beeching⁸ and Mark J Taylor¹

¹  Molecular and Biochemical Parasitology, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, UK

²  Department of Helminthology, Bernhard Nocht Institute for Tropical Medicine, 20359 Hamburg, Germany

³  Department of Medical Parasitology, International Center for Medical Research of Franceville, BP 769, Franceville, Gabon

⁴  Tropical Medicine Research Station, P.O. BOX 55, Kumba, Cameroon

⁵  Research Foundation in Tropical diseases and Environment, P.O. Box 474, Buea, Cameroon

⁶  Department of Haematology, Box 234, Addenbrookes NHS Trust, Hills Road, Cambridge CB2 2QQ, UK

⁷  Wellcome Trust, 183 Euston Road, London, UK

⁸  Clinical Research Group, Liverpool School of Tropical Medicine, Liverpool, L3 5QA, UK

Filaria Journal 2003, 2:9doi:10.1186/1475-2883-2-9

Published:	2 May 2003

Abstract

Background

The majority of filarial nematode species are host to Wolbachia bacterial endosymbionts, although a few including Acanthocheilonema viteae, Onchocerca flexuosa and Setaria equina have been shown to be free of infection. Comparisons of species with and without symbionts can provide important information on the role of Wolbachia symbiosis in the biology of the nematode hosts and the contribution of the bacteria to the development of disease. Previous studies by electron microscopy and PCR have failed to detect intracellular bacterial infection in Loa loa. Here we use molecular and immunohistological techniques to confirm this finding.

Methods

We have used a combination of PCR amplification of bacterial genes (16S ribosomal DNA [rDNA], ftsZ and Wolbachia surface protein [WSP]) on samples of L. loa adults, third-stage larvae (L3) and microfilariae (mf) and immunohistology on L. loa adults and mf derived from human volunteers to determine the presence or absence of Wolbachia endosymbionts. Samples used in the PCR analysis included 5 adult female worms, 4 adult male worms, 5 mf samples and 2 samples of L3. The quality and purity of nematode DNA was tested by PCR amplification of nematode 5S rDNA and with diagnostic primers from the target species and used to confirm the absence of contamination from Onchocerca sp., Mansonella perstans, M. streptocerca and Wuchereria bancrofti. Immunohistology was carried out by light and electron microscopy on L. loa adults and mf and sections were probed with rabbit antibodies raised to recombinant Brugia malayi Wolbachia WSP. Samples from nematodes known to be infected with Wolbachia (O. volvulus, O. ochengi, Litomosoides sigmodontis and B. malayi) were used as positive controls and A. viteae as a negative control.

Results

Single PCR analysis using primer sets for the bacterial genes 16S rDNA, ftsZ, and WSP were negative for all DNA samples from L. loa. Positive PCR reactions were obtained from DNA samples derived from species known to be infected with Wolbachia, which confirmed the suitability of the primers and PCR conditions. The quality and purity of nematode DNA samples was verified by PCR amplification of 5S rDNA and with nematode diagnostic primers. Additional analysis by 'long PCR' failed to produce any further evidence for Wolbachia symbiosis. Immunohistology of L. loa adults and mf confirmed the results of the PCR with no evidence for Wolbachia symbiosis.

Conclusion

DNA analysis and immunohistology provided no evidence for Wolbachia symbiosis in L. loa.