Natural history of zoonotic Ehrlichia species in the United States and discovery of a novel ehrlichial pathogen

Abstract

Ehrlichia are obligate intracellular pathogens, transmitted by ixodid ticks, of both animals and humans. Ehrlichiae are emerging diseases in the USA, and the discovery of new species proceeds more rapidly than the development of models to study these agents. Laboratory animals were evaluated as models for tick transmission with E. chaffeensis, using a quantitative real-time PCR assay we developed to follow the kinetics of infection. Five strains of mice were evaluated: the wild-type strain and four immune-compromised strains. Wild-type C57BL/6 mice developed transient infections and transmitted ehrlichiae to some ticks, but ticks did not maintain the pathogen transstadially. MHCI, MHCII, and iNOS deletion mutants were more susceptible, with MHCII mutants displaying significantly delayed clearance of infection, but no ticks acquired the pathogen. Rag1 deletion mutants developed fulminant, fatal infections; some ticks acquired ehrlichiae but failed to maintain the pathogen transstadially. Guinea pigs and gerbils were resistant to infection, with no evidence of tick transmission. Laboratory goats were infested with A. americanum ticks from a field site endemic for E. chaffeensis and E. ewingii. Goats became infected with E. ewingii and with a novel ehrlichiosis, PME, but not with E. chaffeensis. Goats were competent reservoirs for both ehrlichiae and transmitted them to feeding ticks. PME was shown to be genetically and antigenically similar to E. ruminantium but not identical. All goats infected with E. ewingii and PME displayed acute febrile syndromes, followed by months of asymptomatic chronic infection. This was the first report of clinical disease in goats caused by Ehrlichia from the USA. A person who visited the PME-endemic field site found a feeding tick on his arm and then developed persistent neck pain; molecular testing identified PME, but serological testing using the only available antigen (E. chaffeensis) was inconclusive. Clinical signs rapidly responded to doxycycline; the laboratory confirmation of infection, combined with this clinical response, suggests that PME is zoonotic. The natural transmission cycle of PME was examined. White-tailed deer (Odocoileus virginianus), the reservoir for E. chaffeensis and E. ewingii, were shown to be naturally infected with PME and to transmit the pathogen to ticks. A sensitive and specific PCR assay for PME was developed and applied to a large sample of ticks collected throughout the range of A. americanum. Ticks containing PME were detected throughout the vector’s range, both from vegetation and from people. The conserved gltA gene and the variable map1 gene were sequenced from the positive ticks, revealing complete identity of gltA and two distinct clades of map1. The work described in this thesis adds significantly to the understanding of zoonotic ehrlichioses from the USA. A goat model for E. ewingii was established. PME, a new ehrlichiosis that infects goats and people, was discovered, and its natural transmission cycle and range were established. Prior to this work, ehrlichioses endemic to the USA were not recognized as agents of disease in goats, and PME was still undiscovered; it is unknown how many clinical infections with these zoonotic agents may have gone undiagnosed and untreated.