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United States Department of Agriculture

Agricultural Research Service

Research Project: DETERMINANTS OF ANAPLASMA MARGINALE TRANSMISSION AT THE VECTOR/PATHOGEN INTERFACE

Location: Animal Diseases Research

2008 Annual Report


1a.Objectives (from AD-416)
Our objective in this project is to investigate the factors influencing transmission of Anaplasma marginale by Dermacentor andersoni. We hypothesize that there are interactions between the vector and the pathogen that are determinants of transmission. Our first objective is to set up a field study to examine the relationship between tick vector competence and transmissibility of Anaplasma marginale strains at field sites selected for differences in vector abundance and pathogen strain composition. We will collect ticks annually and determine the susceptibility phenotype of the population at each site by determining the proportion of ticks that are susceptible to midgut infection with A. marginale; midgut susceptibility is a surrogate marker for vector competence. Using a longitudinal survey of a cohort of cattle at each site we will test the hypothesis that some strains of A. marginale are more highly transmissible than others. The first objective will also provide ticks and A. marginale isolates for study in the subsequent objectives. Our next 2 objectives target vector competence of the tick population. First we will attempt to establish if vector competence is a stable genetic characteristic of the tick populations at our field sites by testing the hypotheses that:.
1)the proportion of ticks that are susceptible to midgut infection with A. marginale within each population (i.e. the population susceptibility phenotype) is stable characteristic of the population from one year to the next, and.
2)that there is limited gene flow between populations of D. andersoni. Secondly, we will determine if tick innate immune responses regulate vector competence by testing the hypotheses that.
1)there are differences between tick populations in sequence or expression of tick defensins, and.
2)that these differences correlate with phenotypes which are associated with vector competence for A. marginale. Our final two objectives target A. marginale strain transmissibility. First, we will identify common genetic markers of highly transmissible A. marginale strains collected in our field study, and test the hypothesis that these strains share genetic determinants that are associated with, and are predictive of, more efficient transmission by ticks. We will then identify the outer membrane protein (OMP) structure of these highly transmissible A. marginale strains and test the hypotheses that.
1)highly transmissible A. marginale strains share conserved OMPs, and.
2)that immunization with conserved cross-linked OMPs will induce protection against challenge by heterologous A. marginale strains. By simultaneously approaching studies of the determinates of transmission of A. marginale from the prospective of tick vector competence and from the prospective of strain transmissibility we can begin to define the parameters that influence transmission, including parameters relating to the vector, the pathogen, and to their interaction.


1b.Approach (from AD-416)
Our objective in this project is to investigate the factors influencing transmission of Anaplasma marginale by Dermacentor andersoni. Our first approach is to set up a field study to examine the relationship between tick vector competence and transmissibility of Anaplasma marginale strains at field sites selected for differences in vector abundance and pathogen strain composition. We will collect ticks annually and determine the susceptibility phenotype of the population at each site by determining the proportion of ticks that are susceptible to midgut infection with A. marginale; midgut susceptibility is a surrogate marker for vector competence. Our next approach is to target vector competence of the tick population. We will establish if vector competence is a stable genetic characteristic of the tick populations at our field sites. And secondly, we will determine if tick innate immune responses regulate vector competence by testing if there are differences between tick populations in sequence or expression of tick defensins, and whether these differences correlate with phenotypes which are associated with vector competence for A. marginale. Our final approachs target A. marginale strain transmissibility. First, we will identify common genetic markers of highly transmissible A. marginale strains collected in our field study, and test the hypothesis that these strains share genetic determinants that are associated with, and are predictive of, more efficient transmission by ticks. We will then identify the outer membrane protein (OMP) structure of these highly transmissible A. marginale strains and test the hypotheses that.
1)highly transmissible A. marginale strains share conserved OMPs, and.
2)that immunization with conserved cross-linked OMPs will induce protection against challenge by heterologous A. marginale strains. Formerly 5348-32000-023-00D (12/06).


3.Progress Report
This project is part of NP #103, Animal Health program component #7, Prevention and Control of Parasitic Diseases, Problem Statement 7B: Hemoparasitic diseases result in significant export and production problems for the U.S. cattle and equine industries. We have continued to collect ticks and test cattle for A. marginale infection at our ranch study sites in four distinctly different geographic locations (north central Washington, south eastern Washington, east central Oregon and west central Montana). Anaplasma strain typing of the infected cattle in the study herds has been done but the infection prevalence at these sites has been lower than expected. Consequently analysis of relative transmissibility of field strains has not yet been possible. To increase the number of field strains available for analysis we have added a 5th site in central Oregon (Burns) that has a higher Anaplasma infection prevalence. We have also proceeded with analysis of genetic factors related to strain transmissibility using previously collected highly transmissible and non-transmissible lab strains. Four A. marginale field strains have been collected from 2 of the ranch sites; these strains have been strain typed, amplified in splenectomized cattle, and preserved as frozen stabilates for future study. Ticks from the four ranch sites have been tested for susceptibility to A. margianle; ticks from all four of the populations were susceptible to gut infection after 2 days of feeding and all were competent to transmit the St. Maries strain of A. marginale from a persistently infected to an uninfected host after 7 days of feeding. Defensins are peptides produced in response to bacterial infection and we have hypothesized that these peptides may play a role in vector competence for A. margianle. We have sequenced the defensin gene from a sample of D. andersoni ticks from each of the populations and shown that it is highly conserved between populations. A quantitative PCR analysis has shown there to be a relatively high level of constitutive expression of this gene in blood fed ticks. Work to examine the relationship between tick defensin and vector competence for A. margianle is ongoing. Population genetic analysis of the ticks from the different populations has proceeded along two fronts. In collaboration with colleagues at Northern Arizona University we have developed a set of Amplified Fragment Length Polymorphism (AFLP) markers that can be used in a whole genome screen for genetic variation between populations and we are continuing to use Single Nucleotide Polymorphisms (SNPs) in the 16s mitochondrial RNA gene to examine genetic differences between ticks at the population level. To date our studies have shown that there is limited gene flow between populations. Whether this will lead to differences between populations in vector competence for A. marginale remains to be seen. The composition of the surface of Anaplasma marginale was characterized and used in a vaccine study which showed that use of cross-linked surface complex induced protection against high-level parasite challenge.


4.Accomplishments
1. Validate A. marginale MSP5 cELISA test for detection of A. ovis infection in sheep and Anaplasma sp. in wildlife.

The role of wildlife and sheep in maintaining and transmitting Anaplasma ovis is unknown and this knowledge is important in deriving control methods. The commercially available cELISA kit for detecting A. marginale infections in cattle that was developed by the Animal Disease Research Unit of ARS in collaboration with Washington State University was validated for detection of A. ovis infection of sheep and Anaplasma sp. infections in wildlife. The extension of this assay is allowing the derivation of infection percentages in wildlife and sheep and derivation of the role of infection in disease transmission. This accomplishment addresses NP #103, Animal Health, and Problem Statement #7B: Hemoparasitic Diseases.

2. The antibacterial peptide defensin is constitutively expressed in blood fed Dermacentor andersoni at very high levels.

Defensins are antibacterial peptides that are produced by organisms in response to bacterial infection; and we have hypothesized that tick defensins (components of ticks which protect them from pathogens) play a role in vector competence for A. marginale. Scientists with the Animal Disease Research Unit of ARS have shown that D. andersoni has a defensin gene that is homologous to one that has been described from D. variabilis. Analysis of the level of expression of defensin shows that it is expressed at relatively high levels in the guts of blood fed ticks. This research provides an additional avenue to block transmission of the economically important pathogen. This accomplishment addresses NP #103, Animal Health, and Problem Statement #7B: Hemoparasitic Diseases.

3. Risk assessment of vector borne transmission of Anaplasma marginale by ticks and biting flies

Control of vector borne diseases is dependent on knowledge concerning the relative efficiencies of vector transmission. Scientists with the Animal Disease Research Unit of ARS in collaboration with Washington State University demonstrated that tick-borne transmission of Anaplasma marginale is at least two orders of magnitude more efficient that mechanical transmission by biting flies. The identification of isolates of Anaplasma marginale which are not transmissible by ticks is an issue in the control of this important cattle disease, since we have not yet discovered the method(s) by which they are maintained in nature. This work indicates that non-tick transmissible isolates of Anaplasma marginale are unlikely to be maintained in nature through biting fly transmission and provides further guidance as to transmission control points. This accomplishment addresses NP #103, Animal Health, and Problem Statement #7B: Hemoparasitic Diseases.

4. Successful vaccination against Anaplasma marginale with a complex of surface proteins.

Eventual control of economic losses due to Anaplasma marginale is needed to provide a safe and efficacious vaccine. Previous work has shown that components of the organism capable of inducing protective immunity are, at least in part located on the surface of the organism. Scientists with the Animal Disease Research Unit of ARS in collaboration with Washington State University showed protection using cross-linked surface complex from Anaplasma marginale against high-level bacteriemia and anemia upon challenge of cattle. This accomplishment provides clear guidance in vaccine design for this important pathogen. This accomplishment addresses NP #103, Animal Health, and Problem Statement #7B: Hemoparasitic Diseases.


5.Significant Activities that Support Special Target Populations
None


6.Technology Transfer

None

Review Publications
Scoles, G.A., Goff, W.L., Lysyk, T.J., Lewis, G.S., Knowles Jr, D.P. 2008. Validation of an Anaplasma marginale cELISA for use in the diagnosis of A. ovis infections in domestic sheep and Anaplasma spp. in wild ungulates. Veterinary Microbiology. 130:184-190.

Scoles, G.A., Miller, J.A., Foil, L.D. 2008. Comparison of the Efficiency of Biological Transmission of Anaplasma marginale (Rickettsiales: Anaplasmataceae) by Dermacentor andersoni Stiles (Acari: Ixodidae) with Mechanical Transmission by the Horse Fly, Tabanus fuscicostatus Hine (Diptera: Muscidae). Journal of Medical Entomology. 45(1):109-114.

Futse, J.E., Brayton, K.A., Dark, M.J., Knowles Jr, D.P., Palmer, G.H. 2008. Superinfection as a driver for genomic diversification in antigenically variant pathogens. Proceedings of the National Academy of Sciences. 105(6):2123-2127.

Noh, S.M., Brayton, K.A., Brown, W.C., Norimine, J., Munske, G.R., Davitt, C.M., Palmer, G.H. 2008. Composition of the Surface Proteome of Anaplasma marginale and Its Role in Protective Immunity Induced by Outer Membrane Immunization. Infection and Immunity. 76(5):2219-2226.

Last Modified: 9/10/2014
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