Recipient Organization
UNIVERSITY OF MISSOURI
(N/A)
COLUMBIA,MO 65211
Performing Department
Veterinary Pathobiology
Non Technical Summary
Ehrlichia chaffeensis is the agent of human monocytic ehrlichiosis (HME), one of the most compeeling emerging tick -borne diseases of recent decades. Although Missouri has a high prevalence of HME, and dogs in other areas were shown to harbor infections, the prevalence of this pathogen among dogs in Missouri is unknown. Furthermore, the impact of this agent on dogs remains unknown, largely because we are yet to isolate E. chaffeensis from naturally infected dogs.
Animal Health Component
20%
Research Effort Categories
Basic
80%
Applied
20%
Developmental
(N/A)
Goals / Objectives
A long-term goal of this program is to understand the impact of E. chaffeensis on dogs. The central hypothesis proposed in the current application, which is an important step in reaching this goal, is that E. chaffeensis is prevalent among dogs in Missouri. This central hypothesis will be tested with the following specific aims. (1) Molecular survey for E. chaffeensis infections among dogs from six geographic regions of Missouri. The approach to this aim is to use E. chaffeensis-specific and tick-borne Anaplasmataceae-universal real time PCR assays to survey 390 blood samples (65 from each of 6 regions of Missouri) submitted to VMDL during the late spring through late summer of 2008. (2) Isolation of E. chaffeensis from naturally infected dogs. The objective for this aim is to culture E. chaffeensis from PCR-positive canine blood samples submitted to the VMDL, which will allow confirmation of canine infections. The approach to this aim is to refrigerate blood samples during PCR screening, and to culture PCR-positive samples. The rationale for this work is that, once we know the prevalence of E. chaffeensis among dogs in different regions of Missouri, we can use this data to focus on more detailed studies to determine the effects of natural infection of dogs with this pathogen. Furthermore, we intend to for the first time isolate E. chaffeensis from naturally infected dog samples that will be identified in this study. These isolated strains will then be available for in depth analysis to confirm that they are indeed E. chaffeensis, which is another important step in attaining the long-term goal of this work.
Project Methods
The prevalence of E. chaffeensis in a canine population would be instrumental in understanding the epizootiology of canine ehrlichioses, and will be important in determining risk factors associated with natural transmission of E. chaffeensis to dogs. The approach to this aim will be to use three PCR-based assays to survey blood samples submitted to VMDL. The first assay will help determine whether dogs in Missouri are naturally infected with E. chaffeensis, the second assay will serve as a test for E. canis, and the third assay is a 'catch-all' that will suggest whether other anaplasmal agents (e.g., A. phagocytophilum and E. ewingii) could be present among these dogs. In order to avoid geographic clustering of the data, the samples will be divided according to the six statistical areas described for the state of Missouri by the MU Economic and Policy Analysis Center. These areas were chosen due to their approximately equal size, but, for analysis purposes, we will also compare results for dogs from the major physiographic regions of Missouri (e.g., Glacial Till and Osage Plains, Ozark Plateau and Southeastern Lowlands). Only blood samples with anticoagulant and sufficient information (e.g., breed and location) will be processed for PCR. Remaining aliquots of whole blood samples assayed in aim 1 will be stored at 4 degree C until buffy coats of those samples can be tested by PCR. Blood from PCR-positive samples will be used to inoculate DH82 cells Each blood sample will be assayed for both E. canis and E. chaffeensis, and only those that test positive for E. chaffeensis alone will be cultured. We can isolate DNA and view PCR assay results within a single day and cultivate E. canis from canine carrier blood 24 hours after collection, and we will work further to identify a more precise window for cultivation of monocytotropic Ehrlichia after refrigeration of infected blood, by attempting to cultivate E. canis from carrier blood between 24-96 hours of refrigeration. In the event that we are limited to a 24-hour window, selection of PCR-positive samples for culture is still feasible. However, in the event that this approach does not work (i.e., we cannot gain access to samples soon enough to culture E. chaffeensis) we can attempt to have PCR-positive dogs bled again for more rapid turnaround preceding culture. Rapid access to blood from patients diagnosed with HME, prior to administration of antibiotics, will be key for isolation of these E. chaffeensis strains. Blood from patients diagnosed with HME will be immediately placed in DH82 cell culture as the samples are simultaneously screened for E. chaffeensis.