Evidence against Wolbachia symbiosis in Loa loa

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

Abstracts from the NIHR INVOLVE Conference 2017

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Comparative Analysis of Glycosylated and Nonglycosylated Filarial Homologues of the 20-Kilodalton Retinol Binding Protein from Onchocerca volvulus (Ov20)

Ov20 is a structurally novel 20-kDa retinol binding protein secreted by Onchocerca volvulus. Immunological and biological investigation of this protein has been hampered by the inability to maintain O. volvulus in a laboratory setting. In an effort to find a system more amenable to laboratory investigation, we have cloned, sequenced, and expressed cDNA encoding homologues of Ov20 from two closely related filarial species, Brugia malayi (Bm20) and Acanthocheilonema viteae (Av20). Sequence comparisons have highlighted differences in glycosylation of the homologues. We present here an analysis of mouse immune responses to Ov20, Bm20, and Av20. The results suggest a strong genetic restriction in response to native Bm20 that is overcome when recombinant, nonnative material is used. Reactivity of human filarial sera to the three recombinant proteins confirmed previous specificity studies with Ov20 but highlighted important differences in the reactivity patterns of the O. volvulus and B. malayi homologues that may be due to differences in glycosylation patterns. Ov20 is a dominant antigen in infected individuals, while Bm20 is not. The availability of the B. malayi homologue enabled us to use defined murine reagents and inbred strains for genetic analysis of responsiveness in a way that is not possible for Ov20. However, the close sequence similarity between Ov20 and Av20 suggests that the A. viteae model may be more suited to the investigation of the biological functions of Ov20