Close this window to return to IVIS

www.ivis.org

Proceedings of the
World Small Animal Veterinary Association
Mexico City, Mexico 2005

Hosted by:

Reprinted in the IVIS website with the permission of the WSAVA

Published in IVIS with the permission of the WSAVA Close window to return to IVIS
www.ivis.org

Infectious Diseases
DIROFILARIA/WOLBACHIA SYMBIOSIS: A FRIEND OR A FOE

DIROFILARIA/WOLBACHIA: AMIGO O ENEMIGO?

Genchi Claudio1, Bazzocchi Chiara1, Kramer Laura2, Genchi Marco1, Bandi Claudio1

Abstract:
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.

Resumen:
La patogenia de la filarais se caracteriza por presentar inflamacin aguda y crnica. Los nematodos filar
ideos alojan bacterias intracelulares que pertenecen al genero Wolbachia. Estas bacterias juegan un papel
importante en la biologa del parsito y parecen tambin tener un rol esencial en la embriogenesis de estos
nematodos (tratamiento con antibiticos inhibe la produccin de microfilarias). Esta Wolbachias son
liberadas en la circulacin del hospedero cuando se produce la muerte de los parsitos, cuando hay
liberacin de microfilarias y a consecuencia de un tratamiento farmacolgico. La liberacin masiva de
Wolbachia a la sangre puede llevar a producir reacciones inflamatorias severas de tipo sistmico. Esta
liberacin de bacterias ha sido demostrado estar asociada a liberacin de citoquinas pro inflamatorias,
atraccin de neutrofilos y en un incremento en produccin de inmunoglobulinas especificas. Hemos
identificado una molcula proveniente de la superficie de la Wolbachia ( WSP) que es capaz de ; 1) evocar
respuestas celulares en pacientes humanos con oncocercosis que incluyen una liberacin del factor de
necrosis tumoral alfa , interleucina (IL)-12. IL-8 e interferon gama; 2) estimula la quimiocinesis de
neutrofilos y la produccin de IL-8. Tambin hemos observado que tanto animales como humanos
infectados con filarias montan una respuesta humoral especfica al WSP con predominancia de anticuerpos
IgG2 indicativos de una respuesta celular Th1. Concluimos que el rol de la Wolbachia sobre la respuesta del
hospedero a la infeccin con filarias puede ser debida a la actividad inflamatoria inmunomoduladora de las
protenas provenientes de la Wolbachia.

1 DIPAV, Sezione di Patologia Generale e Parassitologia, Facolt di Medicina Veterinaria, Universit degli
Studi di Milano, Via Caloria 10, 20133 Milano Italy
2Dipartimento di Produzioni Animali, Biotecnologie Veterinarie, Qualit e Sicurezza degli Alimenti,
Facolt di Medicina Veterinaria, Via del Taglio 4, 43100 Parma, Italy

Filariosis 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 filariosis 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

Proceedings of the WSAVA Congress, Mexico City, Mexico 2005

Published in IVIS with the permission of the WSAVA Close window to return to IVIS
www.ivis.org

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 filariosis.

Wolbachia has been shown to be implicated in the immunology of filariosis. 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 Andr 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.

References

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.

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.

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.

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.

Proceedings of the WSAVA Congress, Mexico City, Mexico 2005

Published in IVIS with the permission of the WSAVA Close window to return to IVIS
www.ivis.org

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.

Boreham, P. F. L. & Atwell, R. B. 1988 Dirofilariasis. Boca Raton, FL: CRC Press.

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, 439446.

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.

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.

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.

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.

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.

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.

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.

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, 381386.

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.

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.

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.

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

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.

Ottesen, E. A. 1992 Infection and disease in lymphatic filariasis: an immunological perspective.

Parasitology 104, S71-S79.

Ottesen, E. A. 1995 Immune responsiveness and the pathogenesis of human onchocerciasis. J. Infect. Dis.
171, 659-671.

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.

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.

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.

Proceedings of the WSAVA Congress, Mexico City, Mexico 2005

Published in IVIS with the permission of the WSAVA Close window to return to IVIS
www.ivis.org

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.

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, 180181.

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.

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.

Proceedings of the WSAVA Congress, Mexico City, Mexico 2005