The pathogenesis of filarial disease is characterised by both acute and chronic inflammation. Filarial nematodes harbour intracellular bacteria belonging to the genus Wolbachia. These bacteria play an important role in the biology of the parasite and appears to have an essential role in the embryogenesis of these nematodes (antibiotic treatment inhibits production of microfilariae). Wolbachia are released into the blood of the host following the death of filarial worms, microfilarial turnover and pharmacological treatment. Massive release of Wolbachia into the blood can determine severe systemic inflammatory reactions. Indeed, the release of bacteria has been shown to be associated with production of pro-inflammatory cytokine, neutrophil recruitment and an increase in specific immunoglobulins. We have identified a Wolbachia molecule (the Wolbachia surface protein, WSP) that is able to: 1) evoke cellular immune responses in onchocerciosis patients including a release of TNF-alpha, interleukin (IL)-12, IL-8, and interferon (IFN)-gamma; 2) stimulate canine neutrophil chemokinesis and IL-8 production. We have also observed that animals and humans infected with filarial nematodes mount a specific humoral response to WSP and that the predominance of IgG2 antibodies is indicative of a Th1-type, cell mediated response. Therefore the role of Wolbachia in the host response to filarial infection may be due to the inflammatory/immunomodulatory activity of Wolbachia associated proteins.
Filariasis is still a major health problem in humans and animals. More than 140 million people are infected with the filarial nematodes Brugia malayi, Onchocerca volvulus and Wuchereria bancrofti which are responsible for the majority of human cases of filarial disease (Ottesen 1992, 1995). The heartworm disease of dogs and cats is caused by another filarial nematode, Dirofilaria immitis (Boreham and Atwell, 1988). The pathogenesis of filarial disease is characterised by acute and chronic inflammation. One of the main goals of experimental studies on Filariasis has been the identification of filarial molecules responsible of immunological and pathological responses (e.g., Rao et al., 1999).
Wolbachia, a gram negative bacterial endosymbiont belonging to the Rickettiales, has been described in the body of various species of filarial nematodes (Sironi et al. 1995; Bandi et al., 1998, Casiraghi et al., 2001). These bacteria are present in the lateral chords of both males and females, in the reproductive apparatus of females and also in the larvae present in the vector (Bandi et al., 2001). Wolbachia has also been shown to be transovarially transmitted from female worms to the offspring. In addition, a 100% prevalence of infection in the filarial species positive for Wolbachia is suggestive of an obligatory symbiosis between bacteria and its host (Bandi et al., 2001). Furthermore, drugs like tetracycline, known to be effective against Rickettsia-like bacteria, have been shown to cause detrimental effects on filarial nematodes which harbour Wolbachia, and no effects on filarial nematodes which do not harbour these bacteria (Bandi et al., 1999, Hoerauf et al., 1999; Langworthy et al., 2000). In fact, tetracycline treatments reduce the population of Wolbachia in filarial worms and interferes with the L4-L5 moult of the parasite (Casiraghi et al., 2002). Furthermore, antibiotic treatments of human affected by O. volvulus have been shown an inhibition of the production of microfilariae (Hoerauf 2000, 2001). The effective reduction of Wolbachia in the body of filarial nematodes after antibiotic treatment has been monitored using both Real time PCR (Casiraghi et al., 2002) and immunohistochemical/immunogold detection (Kramer et al., 2003). However, since a chemically modified tetracycline which does not have anti-microbial activity, has been shown to block the filarial moulting, the casual effect of tetracycline treatment, Wolbachia clearance and filarial attrition is still to be evaluated (Rajan, 2004).
The presence of Wolbachia in the body of filarial nematodes and the study of the biological mechanisms involved in the interaction between bacteria, the filaria and the human and animal hosts, permit a re-examination and re-interpretation of immunological and immunopathological investigation on filariasis.
Wolbachia has been shown to be implicated in the immunology of filariasis. Indeed, antibodies against a surface protein of Wolbachia are present in sera of animals (cats: Bazzocchi et al., 2000, Morchon R et al., 2004; dogs: Simon et al. in preparation) and humans (Simon et al. 2003; Punkosdy et al., 2003) infected by filariae. Dogs naturally infected with D. immitis mount a specific humoral response to WSP and the predominance of IgG2 antibodies is indicative of a TH1-type cell mediated response (Simon et al., in preparation). This has been previously observed in murine models (Marcos-Atxutegi et al., 2003) and in humans (Brattig et al., 2004) to indicate an involvement of Wolbachia in immune polarisation during infection.
Wolbachia are released following the death of filarial worms, microfilarial turnover and pharmacological treatment (Taylor et al., 2001). A massive release of bacteria into the blood of the host coincides with the development of severe systemic inflammatory reactions (Cross et al., 2001).
Soluble extracts of filarial nematodes harbouring Wolbachia were injected into the corneal stroma of a murine model of river blindness inducing a pronounced corneal inflammation characterized by neutrophil infiltration and development of corneal haze and thickness. Extract of parasite that do not harbour bacteria or depleted of Wolbachia organisms by antibiotics do not induce keratitis (Saint Andrè et al., 2002). Using electron microscopy, neutrophils were found to completely surround the injected microfilariae harbouring Wolbachia into the corneal stroma (Gilette-Ferguson et al., 2004). Moreover neutrophils have been shown to ingest Wolbachia, as shown by immunostaining with antibodies against the Wolbachia surface protein (Gilette-Ferguson er al., 2004). Worm nodules from untreated onchocerciasis patients displayed a strong neutrophil infiltrate adjacent to the live adult worms. In contrast, in onchocerciasis patients treated with antibiotics to eliminate the endobacteria from O. volvulus and to render the worms sterile, the neutrophil accumulation around live adult filariae was drastically reduced. Other studies showed a neutrophil chemotaxis and activation induced directly by endobacterial products (Brattig et al., 2001). Moreover in dogs infected by D. immitis,neutrophils accumulate in kidneys and in the wall of pulmonary arteries. Wolbachia is thought to contribute to these inflammatory phenomena through its surface protein WSP. Indeed, starting from neutrophil purified from dogs infected by D. immitis, it has been demonstrated that WSP is able to stimulate neutrophil chemokinesis and IL-8 production (Bazzocchi et al., 2003). We have recently reported the specific immune response to Wolbachia in heartworm affected dogs (Marcos-Atxutegi et al, 2004) and positive staining for the Wolbachia surface protein in lungs and kidneys from D. immitis-infected dogs (Kramer et al, 2004). Furthermore, preliminary data from our laboratory indicates that antibiotic treatment before adulticide therapy in dogs with heartworm disease leads to a decrease in pro-inflammatory cytokines, particularly IL-8. Recently WSP has been demonstrated to act also as an inducer of the innate immune system through TLR2 and TLR4. In fact, WSP elicited the release of TNF-alpha, IL-12 and IL-8 from cultured blood cells of both Onchocerca volvulus-infected and uninfected people. Blood cells of onchocerciosis patients exposed to WSP also generated down regulating mediators (Brattig et al., 2004). In conclusion, there is an overall consistency of experimental data indicating that Wolbachia plays a central role in the immunology and pathogenesis of filarial disease.
1. Bandi, C., Anderson, T. J. C., Genchi, C. & Blaxter, M. L. 1998 Phylogeny of Wolbachia in filarial nematodes. Proc. R. Soc. Lond. B 265, 2407-2413.
2. Bandi, C., McCall, J.W., Genchi, C., Corona, S., Venco, L. and Sacchi, L. 1999 Effects of tetracycline on the filarial worms Brugia pahangi and Dirofilaria immitis and their bacterial endosymbionts Wolbachia. International Journal for Parasitology 29, 357-364.
3. Bandi C, Trees AJ, Brattig NW. 2001 Wolbachia in filarial nematodes: evolutionary aspects and implications for the pathogenesis and treatment of filarial diseases. Vet Parasitol. 98, 215-238.
4. Bazzocchi, C., Ceciliani, F., Mc Call, J.W., Ricci, I., Genchi, C. & Bandi, C. 2000 Antigenic role of the endosymbionts of filarial nematodes: IgG response against the Wolbachia surface protein in cats infected with Dirofilaria immitis. Proc. R. Soc. Lond. B. 267, 2511-2516.
5. Bazzocchi C, Genchi C, Paltrinieri S, Lecchi C, Mortarino M, Bandi C. 2003 Immunological role of the endosymbionts of Dirofilaria immitis: the Wolbachia surface protein activates canine neutrophils with production of IL-8. Vet Parasitol. 117, 73-83.
6. Boreham, P. F. L. & Atwell, R. B. 1988 Dirofilariasis. Boca Raton, FL: CRC Press.
7. Brattig NW, Buttner DW and Hoerauf A 2001 Neutrophil accumulation around Onchocerca worms and chemotaxis of neutrophils are dependent on Wolbachia endobacteria. Microb. Infect. 36, 439-446.
8. Brattig NW, Bazzocchi C, Kirschning CJ, Reiling N, Buttner DW, Ceciliani F, Geisinger F, Hochrein H, Ernst M, Wagner H, Bandi C, Hoerauf A. 2004 The major surface protein of Wolbachia endosymbionts in filarial nematodes elicits immune responses through TLR2 and TLR4. J Immunol. 173, 437-445.
9. Casiraghi M, Anderson TJC, Bandi C, Bazzocchi C, Genchi C. 2001 A phylogenetic analysis of filarial nematodes: comparison with the phylogeny of Wolbachia endosymbionts. Parasitology 122, 93-103.
10. Cross HF, Haarbrink M, Egerton G, Yazdanbakhsh M, Taylor MJ. 2001 Severe reactions to filarial chemotherapy and release of Wolbachia endosymbionts into blood. Lancet. 358, 1873-1875.
11. Gillette-Ferguson I, Hise AG, McGarry HF, Turner J, Esposito A, Sun Y, Diaconu E, Taylor MJ, Pearlman E. 2004 Wolbachia-induced neutrophil activation in a mouse model of ocular onchocerciasis (river blindness). Infect Immun. 72, 5687-5692.
12. Hoerauf, A., Nissen-Pahle, K., Schmetz, C., Henkle-Duhrsen, K., Blaxter, M.L., Buttner, D.W., Gallin, M., Al-Qaoud, K.M., Lucius, M. and Fleischer, B. 1999 Tetracycline therapy targets Intracellular bacteria in the filarial nematode Litomosoides sigmodontis and results in filarial infertility. Journal of Clinical Investigation 103, 11-18.
13. Hoerauf, A., Volkmann, L., Hamelmann, C., Adjei, O., Autenrieth, I.B., Fleischer, B., Buttner, D.W. 2000 Endosymbiotic bacteria in worms as targets for a novel chemotherapy in filariasis [letter]. Lancet. 355, 1242-1243.
14. Hoerauf A, Mand S, Adjei O, Fleischer B, Buttner DW. 2001 Depletion of Wolbachia endobacteria in Onchocerca volvulus by doxycycline and microfilaridermia after ivermectin treatment. Lancet. 357, 1415-1416.
15. Kramer L.H., Passeri B., Corona S.. Simoncini L., Casiraghi M. 2003 Immunohistochemical/immunogold detection and distribution of the endosymbiont Wolbachia of Dirofilaria immitis and Brugia pahangi using a polyclonal antiserum raised against WSP ( Wolbachia surface protein) Parasitol Res 89, 381-386.
16. Kramer L.H., Sacchi L., Corona S., Passeri B., Calvi L.E. 2004 The intracellular bacteria of filarial nematodes and their implications in the pathogenesis of filariasis: histological, immunhistochemical and ultrastructural studies. Parassitologia, 46, 135.
17. Langworthy, N.G., Renz, A., Mackenstedt, U., Henkle-Duhrsen, K., De Bronsvoort, M.B., Tanya, V.N., Donnelly, M.J., Trees, A.J. 2000 Macrofilaricidal activity of tetracycline against the filarial nematode Onchocerca ochengi: elimination of Wolbachia precedes worm death and suggests a dependent relationship. Proc. R. Soc. Lond. B Biol. Sci. 267, 1063-1069.
18. Marcos-Atxutegi C, Kramer LH, Fernandez I, Simoncini L, Genchi M, Prieto G, Simon F. 2003 Th1 response in BALB/c mice immunized with Dirofilaria immitis soluble antigens: a possible role for Wolbachia? Vet Parasitol. 28;117-130.
19. Marcos-Atxutegi C, Gabrielli S., Kramer LH, Cancrini G., Simon F. 2004 Antibody response against Dirofilaria immitis antigens and the Wolbachia endosymbiont in naturally infected canine and human hosts. Parassitologia, 46, 116
20. Morchon R, Ferreira AC, Martin-Pacho JR, Montoya A, Mortarino M, Genchi C, Simon F. 2004 Specific IgG antibody response against antigens of Dirofilaria immitis and its Wolbachia endosymbiont bacterium in cats with natural and experimental infections. Vet Parasitol. 2004 125, 313-321.
21. Ottesen, E. A. 1992 Infection and disease in lymphatic filariasis: an immunological perspective. Parasitology 104, S71-S79.
22. Ottesen, E. A. 1995 Immune responsiveness and the pathogenesis of human onchocerciasis. J. Infect. Dis. 171, 659-671.
23. Punkosdy, G. A., D. G. Addiss, P. J. Lammie . 2003 Characterization of antibody responses to Wolbachia surface protein in humans with lymphatic filariasis. Infect. Immun. 71, 5104-5114.
24. Rajan TV. 2004 Relationship of anti-microbial activity of tetracyclines to their ability to block the l3 to L4 molt of the human filarial parasite Brugia malayi. Am J Trop Med Hyg. 71,24-28.
25. Rao, U. R., Nasarre, C., Coleman, S. U., Horohov, D. W. & Klei, T. R. 1999 Granulomatous inflammatory response to recombinant filarial proteins of Brugia species. Am. J. Trop. Med. Hyg. 60, 251-254.
26. Saint Andre A., Blackwell N.M., Hall L.R., Hoerauf A., Brattig N.W., Volkmann L., Taylor M.J., Ford L., Hise A.G., Lass J.H., Diaconu E., Pearlman E., 2002 The role of endosymbiotic Wolbachia bacteria in the pathogenesis of river blindness. Science. 295, 1892-1895.
27. Simon F., Prieto G., Morchon R., Bazzocchi C., Bandi C., Genchi C. 2003 Immunoglobulin G Antibodies against the Endosymbionts of Filarial Nematodes (Wolbachia) in Patients with Pulmonary Dirofilariasis Clinical An Diagnostic Laboratory Immunology, 10, 180-181.
28. Sironi, M., Bandi, C., Sacchi, L., Di Sacco, B., Damiani, G., Genchi, C. 1995 A close relative of the arthropod endosymbiont Wolbachia in a filarial worm. Molec. Biochem. Parasitol. 74, 223-227.
29. Taylor M.J., Cross H.F., Ford L., Makunde W.H., Prasad G.B.K.S. and Bilo K. 2001 Wolbachia bacteria in filarial immunity and disease. P. Immunol. 23, 401-409.