Wolbachia and Heartworm: Why Doxycycline Is Needed in Heartworm Treatment

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

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.

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.

FIGURE 1. Pulmonary pathologic features associated with the death of heartworms in experimentally infected heartworm-positive dogs pretreated with ivermectin and doxycycline before receiving melarsomine injections. The top two panels are pictures of representative lungs obtained during necropsy. The bottom panels are photomicrographs of hematoxylin-eosin–stained lung sections. Photographs courtesy of John McCall, PhD, and Laura Kramer, DVM, PhD.

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

[ultimatetables 15 /]

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?
  

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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.