AHS Heartworm HotlineWolbachia and Heartworm: Why Doxycycline Is Needed in Heartworm Treatment
Andy Moorhead, DVM, MS, PhD
University of Georgia College of Veterinary Medicine
The Heartworm Hotline column is presented in partnership between Today’s Veterinary Practice and the American Heartworm Society (heartwormsociety.org). The goal of the column is to communicate practical and timely information on prevention, diagnosis, and treatment of heartworm disease, as well as highlight current topics related to heartworm research and findings in veterinary medicine.
The treatment of adult heartworm (Dirofilaria immitis) infection in dogs has changed significantly since the days when veterinarians’ only option was injectable thiacetarsamide sodium. First, the introduction of melarsomine dihydrochloride in the mid-1990s revolutionized the treatment of adult heartworm infection by providing superior efficacy via intramuscular administration.1 The next great leap forward in heartworm treatment came with the discovery that some filarial worms harbor a type of bacteria, Wolbachia, and that elimination of these bacteria proved beneficial to the animal and reduced the complications of disease.2
This article explains why Wolbachia are important in the pathogenesis of heartworm disease and why eliminating them through the use of doxycycline is so important to the treatment of the heartworm-infected animal.
WHAT ARE WOLBACHIA AND WHY ARE THEY IMPORTANT TO THE HEARTWORM?
Wolbachia bacteria are related to Rickettsia, which are intracellular bacterial parasites that live within certain invertebrates, including some flies and nematodes.3 Wolbachia are bacterial symbionts. As endosymbionts, Wolbachia bacteria require the host organism, the heartworm, to survive; equally, the heartworm requires Wolbachia for its survival.4
The exact function of Wolbachia in heartworms is unknown.5 It has been hypothesized that the presence of these bacteria may aid in energy metabolism.6 Populations of Wolbachia, which are present in all heartworm life stages, expand between the third-stage infectious larvae (L3) and fourth-stage larvae (L4). For this reason, it is hypothesized that preventing the expansion of the Wolbachia population will prevent heartworm development.7
Wolbachia possess surface proteins (WSPs)8 that are responsible in part for the pathogenesis of heartworm disease. When adult heartworms die naturally or as a result of melarsomine administration, the bacteria and their components, including WSPs, are released. The WSPs recruit neutrophils and other immune cells; as these cells are recruited on the microscopic level in the blood vessels, partial blockage of vessels can result, which can impede blood flow. Furthermore, inflammation of the dog’s lungs can manifest in clinical signs, such as coughing and dyspnea.2,9
Although we do not understand the complete role of Wolbachia in the pathogenesis of heartworm disease, experimental and clinical trials have shown that elimination of the bacteria using doxycycline decreases both macroscopic and microscopic pathology, as well as clinical signs.2,7,10
WHY IS DOXYCYCLINE IMPORTANT IN HEARTWORM TREATMENT?
The American Heartworm Society (AHS) recommends that doxycycline be incorporated into an adulticidal treatment regimen11 to eliminate Wolbachia. Elimination of Wolbachia has 2 clinical benefits: (1) reducing the WSPs released into the bloodstream, thus decreasing inflammation in the canine host; and (2) reducing worm mass, thereby decreasing the severity of pulmonary thromboembolisms (PTEs) associated with adulticidal treatments.2
The AHS recommendation is based on findings from important clinical studies:
- McCall et al7 demonstrated that in animals administered prophylactic doses of ivermectin and doxycycline before melarsomine treatment, there were fewer inflammatory infiltrates in the lungs and a lower possibility of development of PTEs (Figure 1). The dose used in this study was 10 mg/kg q24h for an extended period (weeks 1 to 6, 10 to 11, 16 to 17, 22 to 25, and 28 to 33) before melarsomine treatment.
- Kramer et al12 administered doxycycline to dogs at 20 mg/kg q24h for 30 days, with or without a prophylactic dose of ivermectin. Two months after the end of the doxycycline regimen, animals were administered melarsomine using the standard 3-dose protocol. In this study, the groups receiving doxycycline had almost no thrombi in their lungs when compared with melarsomine-only controls. This study further supports the use of doxycycline for reducing the pathology associated with Wolbachia released from dying heartworms.
Doxycycline not only has effects on adult worms but also decreases numbers of microfilariae and prevents development of heartworm in subsequently infected dogs.
- Decrease in microfilariae In microfilaremic heartworm-infected dogs administered doxycycline for 30 days at a dose of 10 mg/kg q12h, microfilariae levels slowly declined by 12 to 13 months after administration of doxycycline.
- In McCall et al,7 elimination of microfilariae was noted in 9 weeks with an increased amount of doxycycline (10 mg/kg q24 h for weeks 1 to 6 and then weeks 10 to 11, 16 to 17, 22 to 25, and 28 to 33).
- Prevention of adult heartworm development in subsequently infected dogs13
- Microfilaremic heartworm-positive dogs were administered doxycycline for 30 days at a dose of 10 mg/kg q12h. At different time points, including 161 days after doxycycline administration, microfilaremic blood was fed to laboratory-reared mosquitoes.
- Sixteen days after feeding, infective L3s were harvested from the mosquitoes and dogs were experimentally infected.
- When the dogs were necropsied at least 7 months after infection, no adults were found in the experimental animals.
It is hypothesized that doxycycline had eliminated Wolbachia from microfilariae. Therefore, Wolbachia expansion after the L3 to L4 molt could not occur and heartworms could not fully develop. In essence, the use of doxycycline in heartworm-infected dogs prevented heartworm infection in subsequent animals. The fact that doxycycline can essentially prevent heartworm transmission makes its use mandatory when macrocyclic lactone resistance is suspected.
HOW SHOULD PRACTITIONERS TIME DOXYCYCLINE ADMINISTRATION?
Under the current AHS guidelines (Table 1), a 4-week dose regimen of oral doxycycline should be initiated immediately after diagnosis and concurrent with the first dose of a macrocyclic lactone heartworm preventive. A second dose of heartworm preventive is given at the end of the doxycycline regimen.11 A month later, the first dose of melarsomine is administered.
However, if there are extenuating circumstances, melarsomine may be administered immediately after the last dose of doxycycline instead of waiting an extra month because at least some of the Wolbachia population will be eliminated by this time.
It is unknown whether the extra month between the end of the last doxycycline dose and the beginning of melarsomine is necessary for the observed reduction in lung pathology associated with Wolbachia elimination. In Kramer et al,2 a lack of arterial lesions was demonstrated after waiting for 2 months after the completion of a 30-day doxycycline regimen. On the basis of this study, the following questions should be asked:
- Are the Wolbachia completely eliminated after 1 month of doxycycline, or is further time needed?
- Even if Wolbachia are killed during this 1-month time period, have the WSPs, a cause of host inflammation, been completely eliminated?
- Are the clinical benefits of doxycycline dependent on the amount of time (ie, 0, 1, or 2 months) between the end of the doxycycline regimen and the first melarsomine injection?
Further research is needed to answer these questions and to determine whether different doxycycline protocols are effective.
TABLE 1. American Heartworm Society Treatment Protocol for Dogs
|Day 0||Dog diagnosed and verified as heartworm positive: ¥ Positive antigen (Ag) test verified with microfilaria (MF) test ¥ If no microfilariae are detected, confirm with 2nd Ag test from a different manufacturer Begin exercise restriction. ¥ The more pronounced the signs, the stricter the exercise restriction If the dog is symptomatic: ¥ Stabilize with appropriate therapy and nursing care Prednisone prescribed at 0.5 mg/kg BID 1st week, 0.5 mg/kg SID 2nd week, 0.5 mg/kg EOD 3rd and 4th weeks =|
|Day 1||Administer heartworm preventive. ¥ If microfilariae are detected, pretreat with antihistamine and glucocorticosteroid, if not already on prednisone, to reduce risk of anaphylaxis Observe for at least 8 hours for signs of reaction|
|Days 1–28||Administer doxycycline 10 mg/kg BID for 4 weeks. ¥ Reduces pathology associated with dead heartworms Disrupts heartworm transmission|
|Day 30||Administer heartworm preventive.|
|Day 60||Administer heartworm preventive. First melarsomine injection 2.5 mg/kg intramuscularly (IM) Prescribe prednisone 0.5 mg/kg BID 1st week, 0.5 mg/kg SID 2nd week, 0.5 mg/kg EOD 3rd and 4th weeks. Decrease activity level even further. Cage restriction/on leash when using yard|
|Day 90||Administer heartworm preventive. Second melarsomine injection 2.5 mg/kg IM|
|Day 91||Third melarsomine injection 2.5 mg/kg IM Prescribe prednisone 0.5 mg/kg BID 1st week, 0.5 mg/kg SID 2nd week, 0.5 mg/kg EOD 3rd and 4th weeks. Continue exercise restriction for 6 to 8 weeks following last melarsomine injections.|
|Day 120||Test for presence of microfilariae. ¥ If positive treat with a microfilaricide and retest in 4 weeks Establish year-round heartworm prevention.|
|Day 271||Antigen test 6 months after completion; screen for microfilariae.|
|BID, twice daily; EOD, every other day; SID, once daily. Reprinted by permission from the American Heartworm Society|
CAN DOXYCYCLINE BE USED IN ALTERNATIVE TREATMENT REGIMENS?
The AHS does not recommend the use of multiple doses of macrocyclic lactone heartworm preventives for adulticidal treatment of heartworm infection.11 Nonetheless, in clinical situations where treatment with melarsomine is not feasible, doxycycline and macrocyclic lactones have been used as an alternative treatment. Only studies combining prophylactic doses of ivermectin or topical moxidectin with doxycycline have been reported in the literature.
- In naturally infected dogs, administration of ivermectin (6 mcg/kg q15d for 180 days) plus doxycycline (10 mg/kg q24h) resulted in 8 of 11 (72.7%) dogs becoming antigen negative after 300 days.10
- In an experimental study using 16 dogs (8 treated and 8 controls) that received transplanted adult heartworms, the treated dogs received topical moxidectin (2.5%) plus imidacloprid (10%) for 10 months. Doxycycline (10 mg/kg q12h) was administered concurrently during the first month of moxidectin/imidacloprid treatment. At the end of the study, dogs were necropsied. An average of 10.6 heartworms were found in control dogs compared with 0.6 heartworms in experimental dogs. Furthermore, 5 of the 8 experimental dogs had no heartworms.14
Although these regimens have shown some efficacy, the AHS recommends melarsomine in heartworm treatment except when melarsomine administration is not feasible. These alternative treatment studies do, however, reinforce that doxycycline is extremely important in the treatment of adult heartworm infection.
CONCLUSIONS AND LINGERING QUESTIONS
Several important questions remain regarding the use of doxycycline in heartworm treatment:
- Is minocycline a viable alternative to doxycycline (Box 1)?
- Is a full month of doxycycline needed for it to have an effect during heartworm treatment? In other words, what is the minimum dose needed for elimination of Wolbachia from heartworms?
- Would a different regimen of doxycycline be more effective in the adulticidal regimen?
- Does a regimen of doxycycline increase the efficacy of melarsomine administration?
BOX 1. Minocycline Versus Doxycycline in Heartworm Treatment
Anecdotally, minocycline has been used in the place of doxycycline for treatment of heartworm, typically at the standard dose of 10 mg/kg q12h for 30 days. Although no formal studies have been published on the effect of minocycline on heartworm, efficacy has been demonstrated in vitro against a related filarial parasite.15 Recent modeling work also suggests that minocycline will be effective as a substitute for doxycycline in the AHS treatment protocol.
Papich16 used pharmacokinetic/ pharmacodynamic analysis to determine potentially effective doses of minocycline for use in heartworm treatment. Results of this analysis suggest that a q12h dose of 5 mg/kg minocycline in dogs will have an effect on Wolbachia in heartworms. Furthermore, minocycline is more lipophilic and binds less easily to proteins, suggesting that it could have superior tissue penetration compared with doxycycline.
More research is needed, but the hope is that minocycline can be used for heartworm treatment when doxycycline use is not possible for cost or availability reasons.
Answering these questions will advance our knowledge of the practical applications of doxycycline in the heartworm treatment protocol. Even without these answers, we know that Wolbachia are a significant contributor to pathology in dogs infected with heartworm. Because doxycycline can eliminate Wolbachia and reduce inflammation and PTEs, it should be used whenever possible in the treatment of heartworm-infected animals. Don’t let a heartworm-positive dog leave the clinic without it.
- McTier TL, McCall JW, Dzimianski MT, et al. Use of melarsomine dihydrochloride (RM 340) for adulticidal treatment of dogs with naturally acquired infections of Dirofilaria immitis and for clinical prophylaxis during reexposure for 1 year. Vet Parasitol 1994;55(3):221-233.
- Kramer L, Grandi G, Leoni M, et al. Wolbachia and its influence on the pathology and immunology of Dirofilaria immitis Vet Parasitol 2008;158(3):191-195.
- Bandi C, Anderson TJ, Genchi C, et al. Phylogeny of Wolbachia in filarial nematodes. Proc Biol Sci 1998;265(1413):2407-2413.
- Bandi C, Trees AJ, Brattig NW. Wolbachia in filarial nematodes: evolutionary aspects and implications for the pathogenesis and treatment of filarial diseases. Vet Parasitol 2001;98(1-3):215-238.
- Kozek WJ. What is new in the Wolbachia/Dirofilaria interaction? Vet Parasitol 2005;133(2-3):127-132.
- Voronin D, Bachu S, Shlossman M, et al. Glucose and glycogen metabolism in Brugia malayi is associated with Wolbachia symbiont fitness. PLoS One 2016;11(4):e0153812.
- McCall JW, Genchi C, Kramer L, et al. Heartworm and Wolbachia: therapeutic implications. Vet Parasitol 2008;158(3):204-214.
- Kramer LH, Tamarozzi F, Morchon R, et al. Immune response to and tissue localization of the Wolbachia surface protein (WSP) in dogs with natural heartworm (Dirofilaria immitis) infection. Vet Immunol Immunopathol 2005;106(3-4):303-308.
- Kramer L, Simon F, Tamarozzi F, et al. Is Wolbachia complicating the pathological effects of Dirofilaria immitis infections? Vet Parasitol 2005;133(2- 3):133-136.
- Grandi G, Quintavalla C, Mavropoulou A, et al. A combination of doxycycline and ivermectin is adulticidal in dogs with naturally acquired heartworm disease (Dirofilaria immitis). Vet Parasitol 2010;169(3-4):347-351.
- American Heartworm Society. Current canine guidelines for the diagnosis, prevention, and management of heartworm (Dirofilaria immitis) infection in dogs. 2014. heartwormsociety.org/veterinary-resources/american-heartworm-society-guidelines. Accessed May 2017.
- Kramer L, Grandi G, Passeri B, et al. Evaluation of lung pathology in Dirofilaria immitis-experimentally infected dogs treated with doxycycline or a combination of doxycycline and ivermectin before administration of melarsomine dihydrochloride. Vet Parasitol 2011;176(4):357-360.
- McCall JW, Kramer L, Genchi C, et al. Effects of doxycycline on heartworm embryogenesis, transmission, circulating microfilaria, and adult worms in microfilaremic dogs. Vet Parasitol 2014;206(1-2):5-13.
- Savadelis MD, Ohmes CM, Hostetler JA, et al. Assessment of parasitological findings in heartworm-infected beagles treated with Advantage Multi(R) for dogs (10% imidacloprid + 2.5% moxidectin) and doxycycline. Parasites Vectors 2017;10(1):245.
- Townson S, Tagboto S, McGarry HF, et al. Onchocerca parasites and Wolbachia endosymbionts: evaluation of a spectrum of antibiotic types for activity against Onchocerca gutturosa in vitro. Filaria J 2006;5:4.
- Papich MG. Considerations for using minocycline vs doxycycline for treatment of canine heartworm disease. Parasit Vectors [In press, 2017].