Mosquito Transmission Of Dog Heartworm (Dirofilaria Immitis), From The Laboratory To The Field: Dna Barcoding Blood Meal Analysis For Vector Incrimination, Refining Degree-Day Development Models For Diurnal Temperature Fluctuation, And Assessing Knowledge And Socio-Behavioral Risk Factors In Two Endemic Residential Areas.

Abstract

Dog heartworm is a mosquito-borne filarial disease found globally that can be potentially fatal to dogs and cats if left untreated. Current control measures rely solely on preventive drugs (macrocyclic lactones) to kill immature larvae in susceptible hosts, or immiticide to eliminate adult heartworms from infected animals; however there is evidence of resistance in wild D. immitis populations. Little is known about dog heartworm transmission ecology and how its wide range of potential mosquito vectors contribute to disease maintenance across differing habitats and domestic dog populations. Incriminating key mosquito vectors and identifying socio-behavioral risk factors for transmission would help create informed vector control approaches to manage or eradicate dog heartworm disease. In my research, I have employed and developed effective methods for vector incrimination and surveillance, investigated the effects of temperature fluctuation on D. immitis development in mosquito vectors, and conducted door-to-door questionnaires and entomological surveys in endemic communities to determine community knowledge and practices regarding dog heartworm and mosquitoes. DNA barcoding can be a useful tool for mosquito blood meal analysis, and can reveal vector-host links that are crucial to understanding the potential disease risk from mosquito vector species in an area. To determine blood feeding patterns of mosquito species in Ithaca, NY, I deployed various trapping and collection methods in high human and wildlife traffic areas and performed blood meal analysis on wildcaught specimens. Many DNA barcoding primers and methods are available for this purpose, but success rates and host identification standards are very rarely reported. To determine the limits of my method I designed a series of quality control analyses using blood samples taken from local wildlife, which were extracted and analyzed individually and as mixed blood samples. I discovered inherent biases of each primer set, and describe for the first time the identification of both hosts in a mixed blood sample from a single DNA barcode query of sequence databases. Recommendations for blood meal analysis projects of different scales are provided as well as implications of the method for disease surveillance and vector incrimination. D. immitis extrinsic incubation period is predicted by a standard heartworm development unit (HDU) degree-day model that is used to define time periods and geographical areas at risk of dog heartworm transmission. The HDU model is based on average daily temperature above the accepted 14°C threshold for D. immitis development in the mosquito vector, but this ignores temperature fluctuation above and below the baseline average temperature. To see if D. immitis extrinsic incubation period differs between fluctuating and constant temperature conditions, and to determine the validity of the HDU model, I infected cohorts of Liverpool strain Ae. aegypti with D. immitis microfilariae and monitored larval development upon dissection. Larval developmental stage was compared between constant and fluctuating treatments of the same average daily temperature, and HDU predictions were assessed against hourly calculations of development unit accumulation. Temperature fluctuation was more accurately modeled by hourly computations of development units than daily calculations, and larvae in fluctuating treatments that spanned the developmental threshold were detected in mosquito heads eight days sooner than larvae at the constant temperature, and they developed sooner than predicted by the standard HDU model. The implication for HDU models predicting transmission period or geographical range is that hourly calculations of heartworm development units should be used for data regarding fluctuating temperature or temperatures approaching the developmental threshold, otherwise, standard HDU models are likely to underestimate heartworm transmission risk at the beginning and end of the transmission season and in colder regions. Dog heartworm is primarily a disease of domestic dogs and can cause serious complications in domestic cats, therefore, these animals' well-being relies on their owners' knowledge and practices regarding heartworm prevention and mosquito avoidance. To determine community knowledge regarding dog heartworm, mosquito-borne, disease, and mosquito reduction/avoidance practices, I designed and administered a knowledge, attitudes, and practices questionnaire to residents in two dog heartworm endemic communities. Entomological surveys and continued adult mosquito collections were continued in both neighborhoods after the questionnaires, and the data were analyzed to determine residents' knowledge level, disease concern, and prevention practices. Most residents were not aware that mosquitoes transmit dog heartworm, but it was of high concern to them. Most pet owners had their animals on preventive medication, and owning a dog was associated with correct mosquito knowledge regarding important breeding sites and daily activity. The two most common reasons for pet owners not administering preventive medication to their animals were 1) being unaware of the risk to their pet, and 2) consciously deciding that the risk to their particular pet was low. Recommendations for public health messaging by veterinarians and health officials are to emphasize the risks and cost of heartworm disease treatment in comparison to prevention, highlight the importance of mosquito avoidance and reduction, and to provide detailed information regarding mosquito activity and common garden items that can serve as container-breeding sites.