ssBioMed CentFilaria Journal
Open AcceResearchEvidence against Wolbachia symbiosis in Loa loaHelen F McGarry1, Ken Pfarr2, Gill Egerton1, Achim Hoerauf2, Jean-Paul Akue3, Peter Enyong4, Samuel Wanji5, Sabine L Klger6, Albert E Bianco7, Nick J Beeching8 and Mark J Taylor*1
Address: 1Molecular and Biochemical Parasitology, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, UK, 2Department of Helminthology, Bernhard Nocht Institute for Tropical Medicine, 20359 Hamburg, Germany, 3Department of Medical Parasitology, International Center for Medical Research of Franceville, BP 769, Franceville, Gabon, 4Tropical Medicine Research Station, P.O. BOX 55, Kumba, Cameroon, 5Research Foundation in Tropical diseases and Environment, P.O. Box 474, Buea, Cameroon, 6Department of Haematology, Box 234, Addenbrookes NHS Trust, Hills Road, Cambridge CB2 2QQ, UK, 7Wellcome Trust, 183 Euston Road, London, UK and 8Clinical Research Group, Liverpool School of Tropical Medicine, Liverpool, L3 5QA, UK
Email: Helen F McGarry - firstname.lastname@example.org; Ken Pfarr - email@example.com; Gill Egerton - firstname.lastname@example.org; Achim Hoerauf - email@example.com; Jean-Paul Akue - firstname.lastname@example.org; Peter Enyong - email@example.com; Samuel Wanji - firstname.lastname@example.org; Sabine L Klger - Sabineklager@aol.com; Albert E Bianco - email@example.com; Nick J Beeching - firstname.lastname@example.org; Mark J Taylor* - email@example.com
* Corresponding author Equal contributors
AbstractBackground: The majority of filarial nematode species are host to Wolbachia bacterialendosymbionts, although a few including Acanthocheilonema viteae, Onchocerca flexuosa and Setariaequina have been shown to be free of infection. Comparisons of species with and without symbiontscan provide important information on the role of Wolbachia symbiosis in the biology of thenematode hosts and the contribution of the bacteria to the development of disease. Previousstudies by electron microscopy and PCR have failed to detect intracellular bacterial infection in Loaloa. Here we use molecular and immunohistological techniques to confirm this finding.
Methods: We have used a combination of PCR amplification of bacterial genes (16S ribosomalDNA [rDNA], ftsZ and Wolbachia surface protein [WSP]) on samples of L. loa adults, third-stagelarvae (L3) and microfilariae (mf) and immunohistology on L. loa adults and mf derived from humanvolunteers to determine the presence or absence of Wolbachia endosymbionts. Samples used in thePCR analysis included 5 adult female worms, 4 adult male worms, 5 mf samples and 2 samples ofL3. The quality and purity of nematode DNA was tested by PCR amplification of nematode 5SrDNA and with diagnostic primers from the target species and used to confirm the absence ofcontamination 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 andsections 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, Litomosoidessigmodontis 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 WSPwere negative for all DNA samples from L. loa. Positive PCR reactions were obtained from DNAsamples derived from species known to be infected with Wolbachia, which confirmed the suitability
Published: 2 May 2003
Filaria Journal 2003, 2:9
Received: 13 March 2003Accepted: 2 May 2003
This article is available from: http://www.filariajournal.com/content/2/1/9
2003 McGarry et al; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.Page 1 of 7(page number not for citation purposes)
of the primers and PCR conditions. The quality and purity of nematode DNA samples was verifiedby PCR amplification of 5S rDNA and with nematode diagnostic primers. Additional analysis by
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'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 inL. loa.
BackgroundThe majority of filarial nematodes are infected with Wol-bachia endosymbionts, including the major pathogenicspecies in humans, Wuchereria bancrofti, Brugia malayi andOnchocerca volvulus [1,2]. Research on the symbiosis ofWolbachia bacteria and filarial nematodes has highlightedthe contribution of bacteria to inflammatory diseasepathogenesis and the use of antibiotic therapy as a novelmethod of treatment . A few filarial nematode spe-cies, including Acanthocheilonema viteae, Onchocerca flexu-osa and Setaria equina, are free of Wolbachia infection . Studies using these species have helped define the con-tribution of Wolbachia to inflammatory pathogenesis  and the effects of antibiotic depletion on develop-ment and fertility [14,15]. Determining the extent of Wol-bachia infection in filarial nematodes could also shed lighton the evolutionary history of the symbiosis and giveinsight into the nature of the mutualistic association.
The association of Wolbachia with severe inflammatoryreactions post-treatment of B. malayi and O. volvulus withivermectin or diethylcarbamazine [10,16,17] promptedus to examine whether L. loa was infected with Wolbachiaand thus could potentially contribute to the rare but seri-ous severe adverse neurological events (SAE) followingivermectin treatment . Previous electron microscopystudies have failed to find intracellular bacteria in L. loamicrofilariae [6,19] and adults [20,21] and PCR analysisof microfilariae from two patients also failed to detectWolbachia . Here we have used molecular and immu-nohistochemical analysis to confirm this finding in alarger number of samples derived from different endemicareas.
MethodsParasitesNematode samples from infected humans and animalswere obtained with the approval of the ethics committeesand regulatory authorities of all institutions and countriesinvolved in this study.
Loa loaMicrofilariaeMicrofilariae samples were obtained from venous bloodsamples from individuals diagnosed with Loa loa fromCameroon (3), Gabon (2) and Benin (1). Whole blood
microfilariae, which were either frozen, fixed in 80% eth-anol or used directly for the extraction of DNA.
Third-stage larvae (L3)L3 larvae were collected from Chrysops fed on human vol-unteers from Cameroon. Engorged flies were maintainedin insectaries for 12 days at 2328C and 7780%humidity. Heads of infected flies were dissected in RPMImedium and the recovered L3s washed three times. Larvaewere either frozen in liquid N2 or used to inoculate a drill,Mandrillus leucophaeus, for the recovery of adult worms.
Adult wormsTwo adult female worms were obtained following surgicalremoval from infected individuals in Gabon and fixed in80% ethanol. Adult worms (three female and four maleworms) were recovered from subcutaneous tissues of atwo-year old drill born in captivity, seven months aftersubcutaneous inoculation with 200 L3 in the inguinalregion and fixed with 4% formaldehyde in phosphatebuffered saline.
PCRPCR analyses were conducted in two separate laborato-ries, in the Liverpool School of Tropical Medicine and theBernhard Nocht Institute for Tropical Medicine, Ham-burg, and are therefore described for each laboratory.
LiverpoolDNA was extracted from the parasites by the phenol/chlo-roform method, as follows. Worms were placed in 500 lof TEN (20 mM Tris pH 7.5; 50 mM EDTA; 100 mM NaCl)with 0.5% SDS, 0.1 mg/ml proteinase K and 1 l -mer-captoethanol, and incubated in a 55C water bath untilthe parasites were digested. Phenol: chloroform: isoamylalcohol (25:24:1, Sigma, UK) was added to the lysate, gen-tly mixed, and after a 2 minute centrifugation, the aque-ous phase was removed to a clean tube. The organic phasewas re-extracted with 200 l TEN and the aqueous phasescombined. To precipitate the DNA, 1.2 ml of room tem-perature ethanol was added and the DNA pelleted by cen-trifugation, followed by washing with ice cold 70%ethanol, centrifugation, and drying of the pellet; the pelletwas then resuspended in 200 l of sterile distilled water.DNA concentration was determined by absorbance at 260nm (Adult female, 226, 157 g/ml; microfilariae 73, 102Page 2 of 7(page number not for citation purposes)
samples were either frozen directly or filtered to collect g/ml; L3, 2 g/ml). By PCR, L. loa samples were con-firmed to be positive for L. loa DNA  and negative for
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Onchocerca species , M. perstans and M. streptocerca and Wuchereria bancrofti .
16s rDNAFor amplification of bacterial 16s rDNA, 5 l of DNA wasamplified with the eubacterial primers 27f (5'-GAG AGTTTG ATC CTG GCT CAG-3') and 1495r (5'-CTA CGG CTACCT TGT TAC GA-3') as previously described .
ftsZTo increase the sensitivity of the reaction , ftsZ prim-ers (ftsZ UNIF 5'-GG [CT] AA [AG] GGT GC [AG] GCAGAA GA-3' and ftsZ UNIFR 5'-ATC [AG]AT [AG]CC AGTTGC AAG-3')  were used with a proof-reading DNApolymerase enzyme (Bio-X-Act, Bioline, U.K.). Onemicrolitre of DNA was amplified with 0.4 M of eachprimer, 1 X buffer, 350 M dNTPs, 2.5 U DNA polymeraseand between 1.5 mM and 2.5 mM MgCl2. After an initialdenaturation at 95C for 2 minutes, samples were heatedat 94C for 10 seconds, 65C for 30 seconds, and 68Cfor 1.5 mins for a total of ten cycles, after which the sam-ples were amplified for an additional 20 cycles with anannealing temperature of 55C and an extension time of68C for 1.5 mins plus an extra 20 seconds each cycle.
WSPWSP primers were based on the sequence of Brugia malayiWolbachia WSP (WSP-FILF 5'-CGC TTG CAG TAC AATAGT GAG-3' and WSP-FILR 5'-GCT TCT GCA CCA ATAGTG CT-3'). One microlitre of adult or 5 l of microfilar-ial/L3 DNA was amplified with 0.2 M of each primer, 1Xbuffer that contained 1.5 mM MgCl2, 0.1 mM of eachdNTP, 2.5 U of HotStarTaq DNA polymerase and water to50 l. Following activation of the DNA polymerase at95C for 15 minutes, the mixes were heated at 94C for45 seconds, 60C for 45 seconds with a decrease of 1Cper cycle for 5 cycles, then at 55C for 35 cycles, with anextension step at 72C for 90 seconds and a final exten-sion step of 8 minutes.
PCR products were visualised on an agarose gel stainedwith ethidium bromide.
HamburgIndividual L. loa worms (4 male, 3 female) or microfilar-iae were homogenised in lysis buffer (50 mM Tris-HCl,pH 8; 20 mM EDTA; 2% SDS), then incubated for 30 min-utes at 37C with 0.1 volume of 10 mg/ml Proteinase K(Qiagen, Hilden, Germany). The DNA was extracted twicein phenol:chloroform, ethanol precipitated, and the pelletwas resuspended in 200 l water. The DNA concentrationas determined by absorbance at 260 nm had a range of15145 g/ml with an average of 53 g/ml. PCR of the
The following primer sets and annealing temperatureswere used to amplify the eubacterial 16S rDNA and ftsZsequences: 16S rDNA forward: AGA GTT TGA TCC TGGCTC AG, reverse: AAG AGG TGA TCC AGC C ; ftsZforward: CTT GGT GCT GGT GCT TTG CCT, reverse: TACCAA TCA TTG CTT TAC CCA. PCR was performed on 2 lof genomic DNA in a 50 l reaction in 1X Hotstar Taqbuffer (Qiagen, Hilden, Germany) with 1.5 mM MgCl2,0.2 M dNTPs, and 20 M of each primer. The cycle con-ditions were an initial step of 95C for 15 minutes, fol-lowed by 35 cycles of 94C for 30 seconds, 55C for 2minutes, 72C for 1 minute, and a final extension at 72Cfor 10 minutes. Products were separated on agarose gels in1X TBE and visualised with ethidium bromide. FtsZ prim-ers were also used with the Elongase taq polymerase mix(Invitrogen, Paisley, United Kingdom) with 2 mM Mg2+ asper the manufacturer's protocol.
ImmunohistologyAntisera to recombinant Brugia malayi Wolbachia WSPA rabbit was immunised and boosted with purifiedrecombinant Brugia malayi Wolbachia WSP protein andthe serum tested in a Western blot. A single band of 28kDa was detected in B. malayi protein extract, whereasthere was no recognition of a Wolbachia-free A. viteaeextract or when pre-immunisation serum was used (notshown). Likewise, when used in immunohistology, thisantibody specifically labelled Wolbachia from 14 speciesof filarial nematodes tested but did not cross react withany nematode tissue (D. W. Bttner, pers. comm.; ourunpublished observation).
Immuno-electron microscopyL. loa microfilariae were fixed and embedded for immu-noelectron microscopy as described previously . Sec-tions cut at 90 nm and mounted on nickle grids wereblocked with 1% bovine serum albumin in PBS with0.01% Tween 20 and then reacted with rabbit anti-WSPserum (dilutions of 1 in 20 to 1 in 100), washed and incu-bated with goat anti-rabbit colloidal gold conjugate (20nm diameter, British Biocell, UK). Sections were counter-stained with 2% aqueous uranyl acetate solution andexamined on a Phillips CM10 transmission electronmicroscope.
Light immunohistologyL. loa adult worms fixed with 4% formaldehyde in phos-phate buffered saline were embedded in paraffin. Sectionswere probed with rabbit anti-WSP serum (1:250) and vis-ualised using the alkaline phosphatase anti-alkaline phos-phatase (APAAP) method according to the manufacturer'srecommendations (Dako Diagnostika, Hamburg, Ger-many). Anti-rabbit mouse immunoglobulin was used as aPage 3 of 7(page number not for citation purposes)
nematode 5S rDNA was performed as previouslydescribed  to confirm the quality of the DNA.
secondary antibody (clone MR12/53, Dako Diagnostika)and Fast Red TR salt (Sigma) as the chromogen with
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haematoxylin (Merck) as the counterstain. Brugia malayiadult female worms were used as a positive control.
ResultsPCRTo determine the presence of Wolbachia in L. loa at themolecular level, PCR was performed on genomic DNAwith primers for the eubacterial 16S rDNA, ftsZ and WSPsequences. No PCR product was obtained with any of theprimer sets with L. loa and A. viteae DNA (Figure 1),although all DNA samples produced a nematode 5S rDNAsignal, indicating that there was DNA at sufficient concen-tration for detection in one round of PCR. The 16S rDNA,ftsZ and WSP primers were functional as all primer sets
ysis by 'long PCR', which has been reported to increase thesensitivity of the identification of Wolbachia in arthropods, was used; however, neither the Elongase polymer-ase mix nor the Bio-X-Act polymerase used with the ftsZprimer sets produced a signal from L. loa of the expectedmolecular weight (Figure 1).