Elsevier

Veterinary Parasitology

Volume 176, Issue 4, 22 March 2011, Pages 361-367
Veterinary Parasitology

Effects of doxycycline on early infections of Dirofilaria immitis in dogs

https://doi.org/10.1016/j.vetpar.2011.01.022Get rights and content

Abstract

The antifilarial effects of tetracycline drugs were first demonstrated when they were found to be highly effective against L3 and L4 of Brugia pahangi and Litomosoides sigmodontis in rodent models. Tetracyclines are also now known to have activity against microfilariae and adult Dirofilaria immitis, but assessment of their activity against larval and juvenile heartworms has not been reported previously. This study assessed the effects of doxycycline administered orally at 10 mg/kg twice daily for 30-day periods at selected times during the early part of the life cycle of D. immitis in dogs with dual infections of D. immitis and B. pahangi. Twenty beagles were randomly allocated by weight to four groups of five dogs each. On Day 0, each dog was given 50 D. immitis L3 and 200 B. pahangi L3 by SC injection. Dogs received doxycycline on Days 0–29 (Group 1); Days 40–69 (Group 2); or Days 65–94 (Group 3). Group 4 served as untreated controls. Blood samples were collected for microfilariae counting and antigen testing. Necropsy for collection of adult heartworms and selected tissues were performed Days 218–222. Heartworms recovered were examined by immunohistology, conventional microscopy/transmission electron microscopy, and molecular biology techniques. No live heartworms were recovered from dogs in Group 1; dogs in Group 2 had 0 to 2 live worms (98.4% efficacy), and dogs in Group 3 had 0–36 live worms (69.6% efficacy). All control dogs had live adult heartworms (25–41). The live worms recovered from dogs in Groups 2 and 3 were less developed and smaller that worms from control dogs. Microfilariae were not detected in any dogs in Groups 1 and 2; one dog in Group 3 had 1 microfilariae/ml at necropsy. All control dogs had microfilariae at necropsy. One dog in Group 1 was antigen positive at one sampling (Day 166). One dog in Group 2 was antigen positive Days 196 and 218–222 and three dogs in Group 3 were antigen positive at one or more samplings All five control dogs were antigen positive at all three sampling times. These findings suggest that doxycycline at 10 mg/kg orally twice daily for 30 days has efficacy against migrating tissue-phase larvae and juvenile worms and will delay or restrict microfilarial production.

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.

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