Effects of doxycycline on early infections of Dirofilaria immitis in dogs
Introduction
Earlier studies with rodent filarial models have demonstrated that tetracycline drugs are highly effective against 3rd- and 4th-stage larvae and juvenile worms of filarial parasites that harbor Wolbachia endosymbionts (Bosshardt et al., 1993, Hoerauf et al., 1999, McCall et al., 1999, Townson et al., 2000, Casiraghi et al., 2002) but are not effective against those that do not harbor the bacteria (Hoerauf et al., 1999, McCall et al., 1999). These drugs have also been shown to block embryogenesis and gradually eliminate microfilariae and adult worms in species that harbor them but months to years are often required for these stages to die (Bandi et al., 1999, Hoerauf et al., 1999, Hoerauf et al., 2000a, Hoerauf et al., 2001, Hoerauf et al., 2003a, Hoerauf et al., 2003b, McCall et al., 1999, McCall et al., 2008a, McCall et al., 2008b, Langworthy et al., 2000, Casiraghi et al., 2002, Chirgwin et al., 2003, Volkmann et al., 2003, Gilbert et al., 2005, Taylor et al., 2005a, Taylor et al., 2005b). Recent studies also showed that Dirofilaria immitis microfilariae in blood from dogs treated with doxycycline that were fed to mosquitoes developed to 3rd-stage larvae that appeared to be normal in motility and appearance but did not complete their development in the vertebrate host (McCall et al., 2008a, McCall et al., 2008b). A similar observation has been made on Litomosoides sigmodontis, which is transmitted by mites (Arumugam et al., 2008) and Brugia pahangi, which is transmitted by mosquitoes (McCall, unpublished data). Tetracycline drugs, particularly doxycycline, are becoming more widely used by veterinarians prior to adulticidal therapy to reduce the inflammatory response associated with dead heartworms in dogs (AHS, 2010, Kramer et al., 2008, Kramer et al., 2011). Many of the heartworm-positive dogs undergoing arsenical (melarsamine dihydrochloride) therapy harbor larval and/or juvenile stages of the parasite as well as adult worms and microfilariae. Melarsamine has been shown to be highly effective against 2- and 4-month-old heartworms as well as adult worms, but it is not considered to be microfilaricidal, and its effects on young worms of other ages have not been reported (Dzimianski et al., 1989, Keister et al., 1992, McCall et al., 2010). A protocol supported by the AHS (2010) includes pretreatment for 1 month with doxycycline and for 2–3 months with prophylactic doses of macrocyclic lactone preventives before administering melarsomine (Nelson, 2010) which should kill heartworm larvae that are 1–3 months old (Atkins and Miller, 2003). However, the recent concern about the possibility of the existence of populations of heartworms that are resistant to macrocyclic lactone preventives encourages the search for alternative drugs that could be used to kill any resistant strains (AHS, 2010).
The objective of this study was to assess the effects of a widely used adulticidal dosage and treatment schedule of doxycycline (10 mg/kg body weight administered orally twice daily for 1 month) on the early larval stages and juvenile worms of D. immitis in dogs with dual infections of heartworms and B. pahangi, a lymphatic-dwelling filarial parasite. This paper is a report of part of a complex study that is still ongoing and includes some of the completed data sets for the D. immitis portion of the study.
Section snippets
Animals
A total of 20 purpose-bred male (12) and female (8) beagles were used. Just before the day of infection (Day 0), the dogs were 4.2–5.3 months of age and weighed 5.9–11.4 kg. All dogs were negative for heartworm microfilariae and antigen prior to infection with infective stages of D. immitis. The dogs were housed in mosquito-proof indoor pens in a purpose-built building, with controlled temperature and ventilation systems. The dogs were fed an appropriate quantity of commercially available
Worm counts
Treatment during the first month of infection (Group 1) was 100% effective in preventing the establishment of heartworm infection (Table 1). Treatment on Days 40–69 (Group 2) was 98.4% effective (P < 0.0001 vs. control), with only two of the dogs in this group harboring worms, and these surviving female worms were stunted (i.e., about 20% shorter in length than worms from control dogs; P < 0.004). One dog had two female worms and the other dog had one male and one female worm. The male worm was 14.2
Discussion
Although individual filarial worms appear to vary widely in their Wolbachia load, this bacteria, which is found in all individuals of the filarial species known to harbor them, are considered endosymbionts and thus essential for worm survival (Sironi et al., 1995, Taylor et al., 2001, Taylor et al., 2005a, Taylor et al., 2005b). Wolbachia-derived proteins have been shown to be directly associated with the inflammatory response, innate and adaptive immune responses, and tolerized immunological
Conflict of interest
J.W. McCall, L. Kramer, C. Genchi, J. Guerrero, M.T. Dzimianski, P. Supakorndej, A. Mansour, S.D. McCall, N. Supakorndej, G. Grandi, and B. Carson have no personal or financial relationship with other persons or organizations that could inappropriately influence or bias this study.
Acknowledgements
Funding for this study was provided by TRS Labs Inc., Athens, Georgia. The authors gratefully acknowledge Sheila Gross for performing the statistical analyses and Kathy Newcomb for her assistance in editing the manuscript.
References (61)
- et al.
Infection of the intermediate host with Wolbachia-depleted Litomosoides sigmodontis microfilariae: Impaired L1 to L3 development and subsequent sex-ratio distortion in adult worms
Int. J. Parasitol.
(2008) - et al.
Effects of tetracycline on the filarial worms Brugia pahangi and Dirofilaria immitis and their bacterial endosymbiont Wolbachia
Int. J. Parasitol.
(1999) - et al.
Immunological role of the endosymbionts of Dirofilaria immitis: the Wolbachia surface protein activates canine neutrophils with production of IL-8
Vet. Parasitol.
(2003) - et al.
Combined ivermectin-doxycycline treatment has microfilaricidal and adulticide activity against D. immitis in experimentally infected dogs
Int. J. Parasitol.
(2008) - et al.
Neutrophil accumulation around Onchocerca worms and chemotaxis of neutrophils are dependent on Wolbachia endobacteria
Microbes Infect.
(2001) - et al.
Tetracycline treatment and sex-ratio distortion; a role for Wolbachia in the moulting of filarial nematodes?
Int. J. Parasitol.
(2002) - et al.
Brugia pahangi and Wolbachia: the kinetics of bacteria elimination, worm viability, and host responses following tetracycline treatment
Exp. Parasitol.
(2003) - et al.
Severe reactions to filarial chemotherapy and release of Wolbachia endosymbionts into blood
Lancet
(2001) - et al.
Quantification of Wolbachia bacteria in Brugia malayi through the nematode lifecycle
Mol. Biochem. Parasitol.
(2004) - et al.
Endosymbiotic bacteria in worms as targets for a novel chemotherapy in filariasis
Lancet
(2000)
Depletion of Wolbachia endobacteria in Onchocerca volvulus by doxycycline and microfilaridermia after ivermectin treatment
Lancet
Doxycycline in the treatment of human onchocerciasis: kinetics of wolbachia endobacteria reduction and inhibition of embryogenesis in female Onchocerca worms
Microb. Infect.
What is new in the Wolbachia/Dirofilaria interactions?
Vet. Parasitol.
Immune response to and tissue localization of the Wolbachia surface protein (WSP) in dogs with natural heartworm (Dirofilaria immitis) infection
Vet. Immunol. Immunopathol.
Wolbachia and its influence on the pathology and immunology of Dirofilaria immitis infection
Vet. Parasitol.
Heartworm and Wolbachia: therapeutic implications
Vet. Parasitol.
Heartworm disease in animals and humans
Adv. Parasitol.
Population dynamics of Wolbachia bacterial endosymbionts in Brugia malayi
Mol. Biochem. Parasitol.
Specific IgG antibody response against antigens of Dirofilaria immitis and its Wolbachia endosymbiont bacterium in cats with natural and experimental infections
Vet. Parasitol.
Dogs with patent Dirofilaria immitis infection have higher expression of circulating IL-4, IL-10 and iNOS mRNA than those with occult infection
Vet. Immunol. Immunopathol.
iNOs expression is stimulated by the major surface protein (rWSP) from Wolbachia bacterial endosymbiont of Dirofilaria immitis following subcutaneous injection in mice
Parasitol. Int.
A close relative of the arthropods endosymbiont Wolbachia in a filarial worm
Mol. Biochem. Parasitol.
Macrofilaricidal activity after doxycycline treatment of Wuchereria bancrofti: a double-blind, randomised placebo-controlled trial
Lancet
Is there a better way to administer heartworm adulticidal therapy?
Vet. Med.
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
Wolbachia surface protein (WSP) inhibits apoptosis in human neutrophils
Par. Immunol.
Prophylactic activity of tetracycline against Brugia pahangi in jirds (Meriones unguiculatus)
J. Parasitol.
The major surface protein of Wolbachia endosymbionts in filarial nematodes elicits immune responses through TLR2 and TLR4
J. Immunol.
Macrofilaricidal effect of 4 weeks of treatment with doxycycline on Wuchereria bancrofti
Trop. Med. Int. Health.
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