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Introduction
Dirofilaria immitis is a widely distributed filarial nematode parasite. The domestic dog (Canis familiaris) and some wild canids are the usual definitive hosts of D. immitis and thus serve as the main reservoirs of infection. Among the canids, the domestic dog was found to be more frequently affected by D. immitis, followed by coyotes then red foxes (Pappas & Lunzman, Reference Pappas and Lunzman1985). In areas where D. immitis is endemic, it can be the cause of morbidity and mortality in cats (Kramer & Genchi, Reference Kramer and Genchi2002; Blagburn et al., Reference Blagburn, Dillon, Tillson, Brawner, Welles, Johnson, Spencer, Kaltenboeck, Boothe and Rynders2007). The overall prevalence in cats in endemic areas is between 5 and 10% of that in dogs in any given area (Litster & Atwell, Reference Litster and Atwell2008). In both dogs and cats, mosquitoes act as intermediate hosts and must have fed on an infected microfilaraemic reservoir host prior to feeding on and infecting a cat (Dillon et al., Reference Dillon, Blagburn and Weinber2007).
Cats have a natural resistance to infection with D. immitis, resulting in relatively low worm burdens, slower growth and life span (2–3 years) compared to dogs, few reproductively active females, as well as rare and short microfilaraemia (Browne et al., Reference Browne, Carter, Levy, Snyder and Johnson2005). Although cats infected with D. immitis develop significant pathological changes in the respiratory system, the majority of the cats remain asymptomatic and only a small proportion of infected cats develop overt disease (Blagburn et al., Reference Blagburn, Dillon, Tillson, Brawner, Welles, Johnson, Spencer, Kaltenboeck, Boothe and Rynders2007). Newly arriving L5 larvae in the lungs and their subsequent death can result in an acute pulmonary inflammatory response and lung injury (Dillon et al., Reference Dillon, Blagburn and Weinber2007). This initial phase is often misdiagnosed as asthma or allergic bronchitis but, in reality, it is part of a syndrome now known as heartworm associated respiratory disease (HARD) (Blagburn et al., Reference Blagburn, Dillon, Tillson, Brawner, Welles, Johnson, Spencer, Kaltenboeck, Boothe and Rynders2007). It represents a spectrum of pathological and clinical presentations including coughing, dyspnoea, vomiting and sudden death (Blagburn et al., Reference Blagburn, Dillon, Tillson, Brawner, Welles, Johnson, Spencer, Kaltenboeck, Boothe and Rynders2007). Unlike in dogs, where the adult worm burden may be very large, as few as two adult heartworms can be associated with major pathological lesions in the cardiac and respiratory systems in cats, with resultant fatality (Borgarelli et al., Reference Borgarelli, Venco, Piga, Bonino and Ryan1997; Nogami & Sato, Reference Nogami and Sato1997).
The diagnosis of feline heartworm disease is elusive. Many cats are misdiagnosed with allergic bronchitis or feline asthma, while others experience acute death and a diagnosis is often never reached (Dillon et al., Reference Dillon, Blagburn and Weinber2007). Given the potential for serious cardiac and respiratory pathology and sudden death, it is important that prevalence estimates be established for cats in heartworm endemic areas. Serology for heartworm antigen and antibody has become an important diagnostic tool for detecting heartworm infection in cats. Combining results from antigen and antibody tests achieved greater sensitivity than using either test alone (Berdoulay et al., Reference Berdoulay, Levy, Snyder, Pegelow, Hooks, Tavares, Gibson and Salute2004) and served as the basis for conducting antigen and antibody tests in this study.
Heartworm infection reported in cats in various countries varies from a much lower level (one-tenth) to similar to that in dogs within the same area (Miller et al., Reference Miller, Atkins, Robertson, Soll and Knight1998). In the USA, prevalence of D. immitis in cats has been reported to be between 5 and 20% of the prevalence in the dog population (Carleton & Tolbert, Reference Carleton and Tolbert2004). In Japan the prevalence of feline heartworm infection was between 0.8 and 4.1% (Nogami & Sato, Reference Nogami and Sato1997) whereas, in Italy, Kramer & Genchi (Reference Kramer and Genchi2002) reported D. immitis prevalence in cats to be as high as 27%. Differences in prevalence and infection rates in these studies may be influenced by the different test methods applied, as well as the breeding habits and prevalence of mosquitos in these different topographical regions.
Seroprevalence studies of D. immitis in dogs have been undertaken in several Caribbean countries, including several localities in Cuba (range 7–19%), several localities in Curacao (range 9–11%), Grand Bahamas (53%), several areas in Puerto Rico (range 3.1–20.4%) and the Dominican Republic (18%) (Kozek et al., Reference Kozek, Vazquez, Gonzalez, Iguina, Sanchez, de Jesus, Cardona, Gomez, Seneriz, Diaz-Umpierre, Soll and Knight1995). In Grenada, antigen testing and necropsy studies for D. immitis in dogs revealed a prevalence of 14.8% and 21%, respectively (Bhaiyat, pers. comm. 2005). To the authors' knowledge, there are no documented reports on the prevalence of D. immitis in cats in the Caribbean. The above findings on D. immitis in dogs in the Caribbean in general, and Grenada in particular, served as the basis for undertaking this study. Our main objective was to determine the seroprevalence of D. immitis in feral cats in Grenada by employing both antibody and antigen tests.
Materials and methods
From 2004 to 2008, feral cats were collected from various parishes of Grenada. Every Thursday evening, humane cat traps containing food and water were set in shaded areas and checked the following morning. Trapped cats were transported to the Small Animal Hospital at St. George's University. They were anaesthetized with xylazine (2 mg/kg) and ketamine (11 mg/kg) given intramuscularly, based on measurement of body weight in kilograms for each cat. Approximately 3 ml of blood were drawn from each cat. Blood was centrifuged at 3000 g for 5 min and sera were stored at − 20°C in airtight vials until tested. The age of cats was subjectively estimated by dentition using dentition eruption tables and noting markers for periodontal disease and subsequent wear on teeth (Lobprise & Wiggs, Reference Lobprise and Wiggs2000). Information on gender and area of the island where the cat was collected were recorded upon sample collection. Additional information regarding the general health status of the cats was also recorded. Healthy cats were spayed or castrated and released the next day in the same area where they were trapped.
One hundred and thirty-seven serum samples (from 74 male and 63 female cats) were collected. Sera were tested for D. immitis antigen using an enzyme-linked immunosorbent assay (Snap Feline Heartworm Antigen Test; IDEXX Laboratories, Inc., Westbrook, Maine, USA) according to the manufacturer's instructions. Sera were also tested for antibodies (IgG) using lateral-flow immunoassay (Heska's Solo Step® FH; Heska, Loveland, Colorado, USA) as per manufacturer's instructions.
Results and discussion
Of the 137 cats tested, 11 were antibody positive (8%, 95% confidence interval (CI) 3.5–12.5%) of which seven were antigen positive (5.1%, 95% CI 1.4–8.8%). Cats testing both antibody and antigen positive are presumed to be actively infected with heartworm disease at the time of blood collection. All of the cats that tested antigen positive also tested antibody positive. Therefore, the prevalence of heartworm infection in Grenada is 5.1% (95% CI 1.4–8.8%). None of the cats that tested antibody negative yielded a positive result at antigen testing, ruling out the presence of heartworm infection at the time of blood collection. Four cats were antibody positive but antigen negative. The interpretation for this could be that the four cats had cleared the active heartworm infection, thus signifying exposure as opposed to infection. Of the 11 antibody-positive cats, seven were male (9.4%) and four were female (6.3%). Three male cats (4.0%) and four female cats (6.3%) were antigen positive. Age ranged from less than 1 year to 4 years. One male cat less than 1 year of age tested positive to antibody, and one female cat less than 1 year of age reacted positively to the antigen test. These two relatively younger cats were likely exposed to heartworm very early in life.
While the use of serological testing alone can limit a study like this, it has been shown in a previous study that combining results from antigen and antibody tests achieves greater sensitivity than using either test alone (Berdoulay et al., Reference Berdoulay, Levy, Snyder, Pegelow, Hooks, Tavares, Gibson and Salute2004). Although the feline heartworm antibody test is useful for screening for exposure to heartworms, it does not necessarily indicate active infection. A positive antibody test indicates exposure to migrating larvae as well as adults, not the presence of adult heartworms specifically, and should be combined with antigen testing to confirm infection versus previous exposure (Nelson & Cuoto, Reference Nelson and Cuoto2003). IDEXX laboratories report a sensitivity value of 87% (95% CI 78.68–94.4%) and specificity value of 100% (95% CI 97.4–100%) for their Feline Heartworm Antigen test kit (IDEXX Laboratories test kit insert, 2002). Exact data on sensitivity and specificity of Heska's Solo Step Feline heartworm antibody test are unavailable, although the company does report the test to be both highly sensitive and specific. Berdoulay et al. (Reference Berdoulay, Levy, Snyder, Pegelow, Hooks, Tavares, Gibson and Salute2004) did find that the heartworm antibody test in cats detected 62.1–72.4% of heartworm infections and had a wider range of false-positive results.
False-negative antibody testing can occur in an estimated 3–14% of cases, and is usually associated with a single worm infection (Nelson & Cuoto, Reference Nelson and Cuoto2003). This is a concern as the worm burden in cats usually tends to be low. In cases where antibody testing is negative but clinical suspicion for disease is present, it is suggested that serological testing be repeated using a different antibody test and combining results with an antigen test. Radiography of the thoracic cavity, echocardiography, complete blood count and broncho-alveolar lavage are additional diagnostic tests that may be helpful in supporting a diagnosis of feline heartworm disease when clinical suspicion is present (Nelson & Cuoto, Reference Nelson and Cuoto2003). Detection of microfilaria was not considered in this study due to the rare and transient nature of microfilaraemia in cats (Nelson & Cuoto, Reference Nelson and Cuoto2003). A molecular survey using polymerase chain reaction (PCR) analysis could be considered as an additional diagnostic test in future studies in this region.
A previous study showed a heartworm disease seroprevalence of 14.8% in dogs in Grenada (Bhaiyat, pers. comm.). It has been shown that prevalence of heartworm varies from one-tenth to the same rate as dogs in heartworm endemic areas (Miller et al., Reference Miller, Atkins, Robertson, Soll and Knight1998; Litster & Atwell, Reference Litster and Atwell2008). In this study 8% (95% CI 3.5–12.5%) of cats showed antibody seropositivity and 5.1% (95% CI 1.4–8.8%) of cats were antigen positive.
Some previous studies have shown that older age is associated with a higher prevalence of D. immitis infection in cats (Liu et al., Reference Liu, Song, Lee, Lee, Lee, Hayasaki, You and Kim2005). In contrast, in this study there was no significant association between older cats (>1 to 4 years) and younger cats less than 1 year of age (P>0.05, χ2). Other studies have also shown no significant association between age and prevalence of infection (Kramer & Genchi, Reference Kramer and Genchi2002).
Kramer & Genchi (Reference Kramer and Genchi2002) showed a statistically significant higher prevalence in male versus female cats. Our study did not show a significant difference (P>0.05, χ2) between male and female cats with respect to D. immitis exposure (i.e. antibody positivity) or infection (i.e. antigen positivity). Our results are in agreement with Miller et al. (Reference Miller, Atkins, Robertson, Soll and Knight1998), Robertson (Reference Robertson, Soll and Knight1998), Atkins et al. (Reference Atkins, Defrancesco, Coats, Sidley and Keene2000) and Liu et al. (Reference Liu, Song, Lee, Lee, Lee, Hayasaki, You and Kim2005), who indicated that male and female cats are at similar risk for exposure to heartworm. The fact that the sample size in this study was relatively smaller than that in the Kramer and Genchi study, coupled with a lower seroprevalence of feline heartworm disease in Grenada, could explain the lack of association between gender and heartworm exposure or infection rates of cats.
Although data on the gastrointestinal parasite status of the cats in this study is lacking, feline heartworm antibody tests have been shown to have minimal to no cross-reactivity to gastrointestinal parasitic infections in cats (McCall et al., Reference McCall, Guerrero and Supakorndej1998; Nelson & Cuoto, Reference Nelson and Cuoto2003).
Adulticidal therapies traditionally used in dogs are considered risky in felines and can be fatal. Therefore, supportive care is more often indicated for cats exhibiting clinical signs. Heartworm prophylaxis is usually recommended for cats in areas where heartworm disease is endemic in dogs (Levy et al., Reference Levy, Snyder, Taveres, Hooks, Pegelow, Slater, Hughes and Salute2003).
To our knowledge, this study is the first published report detailing serological evidence of heartworm exposure and infection in cats in Grenada. Further studies are required to investigate associated clinical and laboratory findings in cats presenting with clinical signs that could be related to heartworm disease. Because heartworm infection is endemic in local dogs and has a relatively high prevalence in Grenadian cats, we recommend that Grenadian cats are protected using heartworm prophylactic drugs.
Acknowledgements
This study was funded by a small research initiative grant from St. George's University, to which we are grateful.
