Dirofilaria immitis Infection in Dogs from Underserved, Native American Reservations in the United States

Introduction

Dirofilaria immitis is a mosquito-borne filarial worm that resides in the pulmonary arterial branches of canids that can be found in varying degrees throughout the United States.^1–6 The distribution of D. immitis infections is affected by climatic conditions (i.e., temperature and rainfall). These factors not only affect the availability of mosquito vectors, but also, where temperature is concerned, determine how long it takes for infective stage larvae (L₃) to develop in vector mosquitoes.^3,7–10 The domestic dog (Canis familaris) is the major vertebrate host infected in the United States. “Spill over” into other animal species is seen to the greatest degree in the regions of the United States where the D. immitis infection levels in dogs is the highest.^4,11–18

There is no organized surveillance program in the United States to monitor D. immitis infections in dogs. There are no incidence data available to show whether D. immitis infection in dogs is increasing, decreasing, or remaining static in any region of the United States. The veterinary profession has been prescribing prophylactic drugs for D. immitis for over 30 yr, but has yet to develop a national surveillance program to monitor changes in the prevalence and incidence in dogs with exposure risk of infection.

As climate changes progress, the L₃ development time in the vector mosquitoes will also change. As a result of an increase in ambient temperature, the period of transmission at various latitudes in the United States could be lengthened, resulting in an increased transmission success. Without baseline data and an ongoing surveillance program, recognizing such increases in the transmission success of the parasite will be delayed. It would eventually be recognized once there was an increase in disease, but this would be too late.

Dogs on Native American reservations throughout the United States offer a potential stationary, sentinel population to monitor D. immitis infections. These dogs are outdoors almost all of their lives, rarely (if ever) leave the reservation, are not administered prophylactic medication to prevent D. immitis infection, and are available for examination. Reservations are located at different latitudes and elevations across the United States and offer an opportunity to establish sentinel sites to monitor changes in infection levels of D. immitis in the domestic canine population. These reservations could also serve as locations for incidence studies as they are visited by the Rural Veterinary Service (RVS) of the Humane Society of the United States (HSUS) on a regular basis.

The purpose of this study was threefold. The first goal was to determine if the dog owners in these reservation communities would cooperate in filling out the questionnaire and permit their dog(s) to be tested. Next, the authors sought to determine the prevalence of D. immitis infection in dogs living on these reservations. The final purpose was to evaluate the influence ambient temperature in combination with rainfall has on the prevalence of D. immitis within the dog populations surveyed.

Materials and Methods

Blood samples were collected in 6 mL ethylenediaminetetraacetic acid tubes^a from dogs on Native American reservations in California, Washington, Idaho, Nevada, Arizona, New Mexico, North Dakota, South Dakota, Minnesota, and Wisconsin by the RVS of the HSUS between February 2004 and August 2007. A total of 1,822 dogs were surveyed for D. immitis infection using an enzyme-linked immunosorbent assay (ELISA) antigen capture test^b. The test was run according to the directions of the manufacturer with the exception that the wells were washed 10 times between reagents 1 and 2 instead of only 5 times to eliminate residual hemoglobin and red cell stroma in the plasma that can produce false positive results. Each dog owner provided answers to a questionnaire that requested information regarding each dog's gender, age, breed, outdoor-indoor management, travel history, use of heartworm prophylaxis, and previous results, if any, of any previously performed diagnostic tests for D. immitis. All blood samples that tested positive to the ELISA were subsequently examined for evidence of microfilariae following sample preparation with a nuclepore filter and acid phosphatase stain.¹⁹

Reservation communities were grouped based on the degrees of north latitude, 30 yr mean monthly temperatures, and 30 yr mean annual rainfall.²⁰ Using the daily temperature data, a mean monthly temperature was calculated for each grouped community. The number of months per year during which L₃ could develop in vectors within 30 days or less was also determined.^7–10 A reproductive index for D. immitis was calculated by multiplying the number of months of the year that L₃ could develop within vectors in 30 days or less (Table 1, column B) by the 30 yr mean annual rainfall for the same reservation community (Table 1, column C).

The ELISA antigen capture test used in this study has been evaluated for sensitivity and specificity in both low and high D. immitis prevalence canine populations. Antigen positive and antigen negative dogs were either subsequently necropsied to check for adult D. immitis or had a blood examination performed to check for microfilariae.^4,21–23 The specificity of the test has been consistently high, ranging from 98% to 100% in dogs that have had proven infections by necropsy. The sensitivity has improved over the years from 90% in early reports, to 98–100% in later evaluations using improved reagents.^5,21–23 For the purposes of calculating true prevalence, the specificity of the test has been considered to be 100% and the sensitivity of the test to be 98%. In the current study, calculations of true prevalence from observed prevalence were performed using the method reported in Greiner and Gardner (2000).²⁴

Over the 30 mo study period, 11,418 dogs were spayed or neutered by the RVS of the HSUS. Approximately 16% of those also participated in the D. immitis study.

Results

Table 1 shows the data collected on each of the reservation communities studied. χ² analysis showed that there was no significant difference in the distribution of infection with D. immitis between genders (P>0.25). Because there was no significant difference in infection between male and female dogs, numbers for each gender were combined for further analysis. There was an exponential relationship between the POR of heartworm infection and the reproductive index. For every 100 unit increase in reproductive index, the POR of heartworm infection more than doubled (POR =2.10; 95% CI, 1.46–3.00).

Table 1 Physical Characteristics of Communities Studied and Biometric and D. immitis Prevalence Data of Dogs Examined

F, female; M, male; NA, not applicable

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Table 1 (continued)

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Reservation communities at different Northern (°N) latitudes had true prevalences of D. immitis that were not significantly different from those at much lower latitudes. For example, North Dakota reservation communities at 46--49° N latitude had true prevalence of 0.017 and South Dakota reservations, at 43--45° N latitude, had true prevalence of 0.013. These were not significantly different from the Arizona reservation communities' true prevalence of 0.012. The Arizona communities were at 34–39° N latitude and had nearly twice the number of months for development of L₃ infective larvae in 30 days or less than either North Dakota or South Dakota (5.50 mo in Arizona versus 2.75 mo and 2.80 mo in North and South Dakota, respectively). The reproductive index of the Minnesota reservation communities at 47°30′–48° N latitude was a little over two times that of the New Mexico reservation communities (124.50 versus 59.48) even though the latter communities were between 35–36°30′ N latitude. The prevalence of D. immitis in the dogs in the Minnesota communities was four times that in the New Mexico communities, even though the ambient temperature in the New Mexico communities allowed a longer time in which development of L₃ could take place in 30 days or less (Table 1).

The median age of all the dogs examined was 2.03 yr with a range of 1.58–3.20 yr. In general, the age class examined was biased in that these dogs were brought to the RVS clinics for neutering and hence were not likely to represent the median age of all dogs on the reservation communities. Nonetheless, the age of the dogs from most of the reservation communities included in this study indicated about 2 yr of exposure to infection on their respective reservations.

Finally, 74% of all the ELISA-positive dogs (Table 1) had microfilariae of D. immitis in their blood sample.

Discussion

Sentinel populations are of value in assessing changes in the dynamics of parasite populations provided the factors that affect exposure risk are controlled for between the host populations being used as sentinels. Over a 30 mo period, this study drew samples from 36 Native American reservation communities located in 10 states (Table 1).

The reservation communities were characterized by °N latitude, average 30 yr mean annual rainfall in cm, and the total mo/yr development of L₃ could take place within the vector in 30 days or less.²⁰ A reproductive index for D. immitis for each community was calculated based upon two major factors influencing the reproductive success of this filarial worm: water availability(which affects vector abundance and seasonal activity); and developmental time to L₃ in the vector (which influences the number of infective mosquitoes available to transmit the parasite at any given time).²⁵ Multiplying these two independent variables (Table 1, (B) x (C) column) provided a relative evaluation of each reservation's climatic factors as they affect D. immitis transmission.

Several studies have shown an accumulation of approximately 130°C days above 14°C (57.2°F) was necessary for the development of L₃ infective larvae in mosquito vectors to take place.^3,7–10 The lower the ambient temperature, the longer the mosquito must remain alive if L₃ are to develop. Estimations of daily survivorship of female mosquitoes have been achieved by capture, mark, release, and recapture studies. These studies have reported daily survivorship ranges between 0.63 and 0.82, depending upon the habitat, mosquito species involved and strain of mosquito.²⁶ In this study, 0.90 was selected as the daily survivorship value because it provided a liberal estimate and would not underestimate the transmission potential of the many different species of mosquitoes that vector D. immitis on the reservations studied. With a 0.90 daily survivorship, 10% of the mosquitoes would die each day. If there were 100 infected mosquitoes alive on day 0, then by day 30 there would be approximately 4 mosquitoes out of the original 100 still alive and carrying infective L₃. Beyond 30 days, the small number of surviving infective mosquitoes would be unlikely, by themselves, to sustain transmission, so the numbers of months out of the year in which development of L₃ can take place in 30 days or less are the most important transmission months.³ It must also be remembered that female mosquitoes are not host-specific. Blood meal studies have shown that many vectors of D. immitis readily feed on other mammals that do not support D. immitis development.^27,28 If other wild or domestic animals are present they can attract these infective mosquitoes, the infective mosquitoes will then not feed on dogs.

Some studies have suggested that the use of chemoprophylaxis to prevent D. immitis infection could be based upon the number of months in which development of L₃ infective larvae could occur within the ecological life expectancy of the mosquito vector.^7,8 The data reported here suggest that the amount and duration of water available for vector development in determining the transmission period of this filarial nematode should also be considered.

All of the dogs examined on the reservation communities in the 10 states surveyed resided outdoors all the time during the period of potential transmission, were not on prophylaxis, had no travel off of the reservation, and did not significantly differ in age across reservations. These criteria are the major elements that characterize a dog's exposure risk to D. immitis infection^1,2 All dogs in this study were equal in these criteria; therefore, all of the dogs were at equal management risk of exposure to infective mosquitoes in their respective communities. Differences in D. immitis prevalence in dogs of nearly the same age and exposure risk could logically be due to differences in transmission intensity from one area to another. The transmission intensity of a vector-borne agent is a function of infective vector abundance both in time and space. From the standpoint of the filarial worm, a large number of available vectors do not improve the transmission success if the ambient temperature is too low to allow development of infective larvae within the life expectancy of the vectors.² Likewise, the presence of microfilaremic dogs year round is not conducive to long transmission periods if, due to the lack of water availability for vector development and survival, the abundance of vectors is limited in space and time.²⁹

Western Washington is an example of the first of the two scenarios postulated above. A much larger sample size study conducted in Washington State showed nearly the same prevalence in dogs with no travel history out of Western Washington as the study reported herein.² Arizona and Southern California reservation communities are examples of the second scenario presented above. These reservations have ambient temperatures which would permit development of L₃ infective larvae in mosquito vectors for 5.5–6 mo out of the year in 30 days or less, but have limited rainfall. The prevalence of D. immitis infection in the dogs from these reservations is 4–10 times lower than that in both Wisconsin and Minnesota where the ambient temperature will allow L₃ to develop in mosquito vectors in 30 days or less for only 2–3 mo out of the year, but whose spring and summer rainfall is 2–3 times that of Arizona and Southern California (Table 1).

The results of this study indicate that hot, arid climates and cold, wet climates are not favorable for the development and transmission of D. immitis. Areas with rainfall at appropriate times of the year may provide ample breeding sites for mosquito vectors. Some of these vectors can develop at very low temperature. If the average ambient temperature for development of L₃ in the vectors is so low that it takes more than 30 days, the vectors are of little use to the reproductive success of D. immitis because the number of vectors still alive after 30 days is too few to maintain the transmission cycle.

Exact logistic regression was used to evaluate prevalence as a function of the arithmetic variable called the reproductive index for each reservation community. This value accounts for the fact that both rainfall and ambient temperature are operating independently to affect a transmission window for this filarial nematode in any particular geographic area. Both the 30 yr average annual rainfall and the 30 yr average ambient temperature for many communities are online. These data could be used to predict the PORs for different communities. Serological studies could then be done to test the value of such a predictive methodology. The Native American reservation communities located in many different climatic regions of the United States offer an ideal dog population to study. This study also suggests that calculating prophylactic use based solely on the ambient temperature necessary for L₃ development could result in recommendations for prophylactic use that are excessive in the hot, arid parts of the United States.