Association of Wolbachia with heartworm disease in cats and dogs

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Abstract

Although the presence of adult Dirofilaria immitis in the pulmonary arteries and its associated arteritis and thromboembolic disease can explain some of the manifestations of canine and feline heartworm disease, the cause of other findings remains unclear. Cats with D. immitis antibodies but lacking adult parasites in the pulmonary arteries frequently develop histological lesions of the airways, resulting in a condition termed Heartworm-Associated Respiratory Disease. All D. immitis parasites harbor Wolbachia pipientis bacteria and D. immitis-infected animals can have circulating Wolbachia antibodies and pro-inflammatory Wolbachia antigens (WSP) deposited in tissues. Little is known about the role that Wolbachia plays in lung disease of animals naturally infected with D. immitis. The purpose of this study was to determine the contribution of Wolbachia to the pathogenesis of natural heartworm disease in cats and dogs. We hypothesized that animals having sufficient Wolbachia burden to be detected in lung tissue by immunohistochemistry and/or polymerase chain reaction (PCR) would have more severe pulmonary disease than those with bacteria below the limits of detection. We further hypothesized that animals that were immunoreactive to pro-inflammatory WSP would have more severe pulmonary lesions than those that were seronegative for WSP antibodies. Blood and lung tissue samples were collected from cats and dogs representing three different D. immitis infection statuses: heartworm-free, heartworm-exposed, heartworm-infected. There was a positive but weak correlation between the magnitude of D. immitis antibody titers and WSP titers in cats (r = 0.57, p < 0.001) and in dogs (r = 0.39, p < 0.001). Pulmonary lesions were more common in HW-infected animals than in HW-free animals. Pulmonary arteriolar occlusion was more common in HW-infected cats (57%; p = 0.003) than in HW-infected dogs (17%). Although pulmonary lesions were most common in HW-infected animals, there was no clear additive effect when either Wolbachia DNA/WSP was detected in lung tissue or when circulating Wolbachia antibodies were detected. There were no significant differences in the magnitude of pulmonary lesion scores within each HW-infection status group regardless of whether Wolbachia DNA/WSP or antibodies were detected. The relationship between Wolbachia and lung pathology in heartworm-infected animals remains to be determined. The lack of clear evidence for a role of Wolbachia in heartworm disease creates a dilemma for veterinarians treating animals in D. immitis-endemic areas. Although the indiscriminant use of antibiotics should be avoided, many clinicians prescribe doxycycline based on the favorable responses observed in human filarial diseases and promising results from the first published studies of doxycycline use in D. immitis-infected dogs.

Introduction

In recent years, it has been recognized that a large array of helminths and arthropods are colonized by Wolbachia pipientis, a symbiotic Gram-negative intracellular bacterium closely related to the genus Rickettsia (Bandi et al., 1998, McCall et al., 2005). Human and animal filarial nematodes, including the agents of human river blindness (Onchocerca volvulus), human lymphatic filariasis (Brugia malayi), and feline and canine heartworm disease (Dirofilaria immitis), harbor Wolbachia in a mutually dependent manner (Kramer et al., 2003, Taylor, 2003, Kozek, 2005, McCall et al., 2005). Wolbachia organisms are maternally transmitted from one filarial generation to the next, and bacteria can be recovered from all life stages of the parasite. Filariae rendered free of Wolbachia by treatment with tetracycline antibiotics show inhibition of maturation, survival, and reproduction (Genchi et al., 1998, Bandi et al., 1999, Casiraghi et al., 2002, Makepeace et al., 2006, Pfarr and Hoerauf, 2006). Wolbachia are released in large numbers and at death of the parasite and during production and release of microfilariae (Bandi et al., 1999, Taylor, 2003, Taylor et al., 2005). Wolbachia-associated-molecules (WAMs) have recently been shown to be associated with inflammation in the parasitized mammalian hosts (Bazzocchi et al., 2000, Bazzocchi et al., 2003, Taylor, 2003, Morchon et al., 2004, Kozek, 2005, Kramer et al., 2005a, Kramer et al., 2005b, McCall et al., 2005). These inflammatory responses appear to be most profound when the parasite dies naturally or as a result of anti-filarial drug treatment. In contrast, the death of parasites rendered free of Wolbachia stimulates less intense inflammation in the host. This observation has led to treatment strategies for river blindness and lymphatic filariasis that include pretreatment with tetracycline antibiotics to reduce the Wolbachia numbers, followed by ivermectin to eliminate the parasites (Taylor, 2003, Makepeace et al., 2006).

Canids are the only significant natural reservoir for D. immitis, but other species, including humans, can be incidental hosts. Although cats are naturally more resistant to D. immitis, feline infection is likely to occur anywhere the parasite is found in dogs. The vast majority of L3 larvae transmitted to dogs is expected to mature into adult worms, whereas most L3 larvae entering cats undergo high mortality in the pulmonary arteries at the L5 stage approximately 90 days after infection. This results in a low prevalence of adult worms in cats (Snyder et al., 2000, Levy et al., 2003, Berdoulay et al., 2004).

Although the presence of adult D. immitis in the pulmonary arteries and its associated arteritis and thromboembolic disease can explain some of the manifestations of canine and feline heartworm disease, the cause of other findings remains unclear. This is particularly true for cats, which frequently develop generalized severe bronchointerstitial disease and extrapulmonary signs in response to very low parasite intensities (Atkins et al., 2000). In cats, even a single worm can result in fatal disease. We recently evaluated pulmonary pathology in cats with natural D. immitis infection. Lung lesions characterized by pulmonary arterial occlusive hypertrophy were common in cats with adult worms and in cats that were free of adult worms but having circulating D. immitis antibodies, suggesting that even transient infection leaves cats with long-lasting pulmonary pathology (Browne et al., 2005).

All D. immitis parasites harbor Wolbachia. The bacteria are present in all developmental stages and are located in the hypodermal cells of the lateral chords and in the female reproductive organs (Kramer et al., 2003, Kozek, 2005). D. immitis-infected animals can have circulating Wolbachia antibodies and Wolbachia antigens deposited in tissues (Bazzocchi et al., 2000, Morchon et al., 2004, Kozek, 2005, Kramer et al., 2005b, Kramer et al., 2008). Macrophages containing Wolbachia have been identified in the lung, liver, and kidneys of dogs with natural D. immitis infection (Kramer et al., 2005b). Wolbachia surface protein (WSP) stimulates canine neutrophil chemotaxis and IL-8 production (Bazzocchi et al., 2003). Stimulation of canine vascular endothelial cells with WSP resulted in production of cyclooxygenase-2, 5-lipooxygenase, leukotriene B4, intracellular adhesion molecules, E-cadherin, and vascular endothelial growth factor (Simon et al., 2008). In cats, experimental D. immitis infection resulted in production of antibodies against both D. immitis and Wolbachia within 2 months of exposure to infective larvae (Morchon et al., 2004). Treatment with ivermectin 30 days later to abort D. immitis infection was associated with disappearance of D. immitis antibodies, but persistence of Wolbachia antibodies. Dogs experimentally infected with D. immitis and treated with doxycycline in combination with ivermectin and melarsomine had less pulmonary pathology than dogs treated with melarsomine alone (Kramer et al., 2008). More interestingly, a recent study has shown that ivermectin/doxycycline combination is also adulticidal (Bazzocchi et al., 2008). Taken together, these studies suggest a role for Wolbachia in the pathogenesis of heartworm disease.

If recently reported experiences with antibiotic treatment of human filarial diseases prove to be predictive of responses in naturally infected animals, then treatment with doxycycline prior to adulticide therapy in dogs may reduce the Wolbachia numbers in D. immitis, leading to fewer inflammatory side-effects when the larvae and adults die. Doxycycline therapy may be especially helpful for cats, which suffer from substantial and life-threatening effects of heartworm disease, but for which adulticide therapy is associated with high complication rates and is generally contraindicated. Elimination of inflammatory Wolbachia organisms may allow cats to coexist more comfortably with their parasites, even if an actual cure is not feasible. However, it is not currently known what role Wolbachia plays in the pathogenesis of natural heartworm disease.

The purpose of this study was to determine the contribution of Wolbachia to the pathogenesis of natural heartworm disease in cats and dogs. Since all D. immitis parasites harbor Wolbachia, it is difficult to differentiate the effects of the parasite from those of its endosymbiont bacteria. We hypothesized that animals having sufficient Wolbachia numbers to be detected in lung tissue by immunohistochemistry and/or by PCR would have more severe pulmonary disease than those with bacteria below the limits of detection. We further hypothesized that animals that were immunoreactive to pro-inflammatory WSP would have more severe pulmonary lesions than those that were seronegative for WSP antibodies.

Section snippets

Materials and methods

For this study, samples were collected from cats and dogs representing three different D. immitis infection statuses. The heartworm-free (HW-free) groups were composed of 30 cats and 30 dogs that were free of any evidence of current or previous D. immitis infection (seronegative for D. immitis antigen and antibodies and negative for parasites at necropsy). The heartworm-exposed (HW-exposed) groups were composed of 30 cats and 30 dogs with evidence of either larval-stage infection or past

Serology

There was a positive but weak correlation between the magnitude of D. immitis antibody titers and WSP titers in cats (r = 0.57, p < 0.001) and in dogs (r = 0.39, p < 0.001). Antibodies against WSP were detected in a majority of HW-infected cats and dogs and in variable proportions of animals in other HW-status groups (Table 4). Of the 90 cats in the study, 10 had FIV antibodies (five from the HW-infected group and five from the HW-exposed group) and one was infected with FeLV (from the HW-exposed

Discussion

In this study, we identified the presence of Wolbachia in HW-defined groups of cats and dogs to determine if there was an association between the bacterium and the severity of pulmonary lesions characteristic of heartworm disease. Although pulmonary lesions were most severe in animals with adult D. immitis there was no clear additive effect when Wolbachia was present in sufficient amounts for detection in the lungs or when immunoreactivity against Wolbachia was detected. These findings suggest

Acknowledgements

This work was supported by a grant from the Morris Animal Foundation. We thank Aundria West, Jennifer Hawley, Melissa Brewer, Cassie Quest, and Abigail Carreno for their technical assistance, and Dr. Cynda Crawford for her careful review of this manuscript.

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