DNA barcoding of medically important mosquitoes and molecular detection of Rickettsia felis in the mosquitoes and the blood of domestic dogs and cats

Abstract

Rickettsia felis, an obligate intracellular bacterium, is the causative agent of human flea-borne spotted fever. The recent surge of reports of this disease has raised the concern of a near-future outbreak as the list of vectors, reservoirs, and hosts of this pathogen are increasing with recent studies. Due to the close inhabitation of humans and the mammalian hosts, cats and dogs, and the ubiquitous distribution of the main vector, Ctenocephalides felis, it is not uncommon for companion animals and humans to get infected with R. felis. Mosquitoes have also been found to carry this pathogen in different countries. As a result, this pathogen retains a potential to disseminate among the human population. Mosquito species identification was required to study field-level mosquitoes for R. felis. This thesis first establishes and validates a highly sensitive DNA barcoding method for medically important mosquitoes (Chapter 2). Then, the presence of R. felis DNA in mosquitoes and in the blood of domestic cats and dogs in the USA were investigated (Chapters 3 and 4). Field level mosquitoes were trapped in Auburn, Alabama, with mosquito trappers. A SYBR-qPCR system targeting the mitochondrial Cytochrome Oxidase C Subunit 1 (COI) was developed, optimized, and validated for the accurate determination of mosquito species. Furthermore, mosquitoes were tested for the presence of R. felis DNA using a gltA-based FRET-qPCR, a nested-qPCR targeting the gltA of Rickettsia, and a R. felis species-specific BioB-based qPCR system. Domestic cat blood samples from 752 cats from 43 states and domestic dog blood samples from 777 dogs from 45 states of the USA were tested for the presence of R. felis DNA with the same PCR systems. Following Sanger sequencing, the sequences were analyzed and were submitted to the GenBank. After assigning the accession numbers, phylogenetic trees were constructed. The AU-COI PCR system was validated which show sensitivity of a single copy of DNA/10 µL (Ae. japonicas, An. quadrimaculatus, Cx. nigripalpus, Cx. usquatissimus, Or. Alba, Ps. ferox, and Ur. sapphirina) and 10 copies/10 µL (An. punctipennis). Eight mosquito species were successfully identified by morphology, published PCR from Folmer et al. 1994, and the AU-COI PCR system. In addition, seven mosquito species were correctly identified by both of two PCR systems, but not by the morphology. Furthermore, nine mosquito species were only accurately identified by the AU-COI PCR, not by the published one or by the morphology. In this study, the AU-COI DNA barcoding method detected 24 mosquito species out of 128 individual mosquitoes, which included two new Culex and three new Psorophora species in Alabama. Anopheles punctipennis, Aedes vexans and Uranotaenia sapphirina were found positive for R. felis. Molecular techniques detected R. felis in 9% of the mosquito pools. Nine percent (5/57) of the mosquito pools, including An. punctipennis (3/6), Ae. vexans (1/4) and Ur. sapphirina (1/3), were positive by PCR, in each case with all three Rickettsia specific PCRs. One of the positive An. punctipennis, and the positive Ur. sapphirina pool contained only male mosquitoes. The 120-bp nucleotide sequences of the five mosquito pools positive in R. felis-specific BioB-based PCRs were identical to one another, and to that of R. felis URRWXCal2. There was only a single base pair difference amongst the 446-bp nucleotide sequences of the positive gltA-based PCRs which were 99.7–100% identical to recognized R. felis strains in GenBank. Four R. felis positive cats were from different states: Kansas, California, New York, and Texas, whereas three R. felis positive dogs were from Texas and Georgia. Low copy numbers of R. felis DNA (around 100 copies/ml whole blood) were found in four cats (4/752, 0.53%) and three dogs (3/777, 0.39%). The DNA barcoding of mosquitoes is unaffected by morphological variation between different stages of the life cycle. This technology precisely identifies even the cryptic mosquitoes which is vital for the accuracy of a surveillance program as it greatly reduces the burden on experienced taxonomists. Results indicated the presence of R. felis in mosquitoes and in domestic cats and dogs in the USA. There is still much to be understood about the vector and reservoir role of the wide range of arthropods that harbor R. felis. The growing reports of R. felis occurring in mosquito species around the world and the known role of mosquitoes in transmitting a wide range of important human and animal pathogens indicate the necessity of further studies to determine the role mosquitoes play in the epidemiology of infectious diseases. The low levels of infection in clinically ill animals are consistent with R. felis being an unlikely cause of disease in naturally infected dogs and cats.