Progress 11/15/99 to 11/30/03
Outputs
This is the final progress report of the above research grant. We have been very successful for this research project and made more progress than we originally proposed in the grant proposal. We published three papers in a high quality peer-reviewed scientific journals. 1. As shown in Manuscript 1 and 2 we identified N. risticii DNA in xiphidiocercariae from freshwater snails in the Elimia (Goniobasis) species, also of the Pleuroceridae family, including Elimia livescens in Ohio (Kanter et al., 2000) and Elimia virginica in Pennsylvania (Mott et al., 2002). Xiphidiocercariae that were identified in Ohio and Pennsylvania, but not those found in California, possess a stylet that is used to bore through chitinous exoskeletons. These xiphidiocercariae come from digenetic trematodes that utilize insects as their second intermediate hosts. This fact suggests that, unlike N. helminthoeca or the SF agent, insects rather than the fish were the second intermediate hosts of N.
risticii-infected trematodes. 2. In manuscript 2, therefore, we tested larvae and adult aquatic insects (mayflies, caddisflies) carrying metacercariae for N. risticii DNA by PCR analysis and we found that trematodes in these insects are infected with N. risticii (Mott et al., 2002). This suggests that the bacteria are transstadially transmitted from the cercaria to the metacercaria stages of the trematode life cycle. 3. In manuscript 2 we tested horizontal transmission of N. risticii from trematodes in the metacercaria stage to horses through Koch's postulate. When horses were fed caddisflies from the PHF endemic region, these horses developed clinical signs of PHF and live N. risticii were isolated from the horses' blood (Mott et al., 2002). Comparison of the DNA sequences revealed an exact match between the original N. risticii from the caddisflies that were fed to the horses and the N. risticii found in the horses' blood or in cultures isolated from the blood of sick horses
(Mott et al., 2002). 4. Although considerable progress had been made in recent years, the species of this infected trematode and the definitive host where it sexually reproduces remained unknown. In manuscript 3, we isolated a gravid trematode from the intestines of Eptesicus fuscus big brown bats from the PHF endemic region of Pennsylvania and tested it for N. risticii infection by PCR analysis and indirect immunofluorescence staining. We found this trematode is infected with N. risticii and identified it as Acanthatrium oregonense. Pusterla et al (2003) recently reported that pools of digenetic trematodes, Acanthatrium sp. and Lecithodendrium sp. (species were not identified) recovered from two Myotis yumanensis bats in California, tested positive for the 5' segment of the N. risticii 16S rRNA gene by PCR analysis. Our manuscript 3 describes our study, conducted in Pennsylvania between 2001 and 2003, that identifies a trematode species infected by N. risticii and its definitive
host. Our results provide new information on the role of A. oregonense as natural reservoir and vector of N. risticii.
Impacts
Potomac horse fever (PHF) is a life-threatening disease of horses characterized by fever, loss of appetite, depression, and watery diarrhea. PHF is caused by a small bacterium called, Neorickettsia risticii, which infects blood leukocytes and cells lining the gut wall of the horse. The long-term goal of this research project is to identify where and how N. risticii exists in nature, and how the horse gets infected. Previous studies indicate that the disease is not contagious and vectorborne. Recent studies suggest that flukes parasitizing the fresh-water snail harbor N. risticii. Ehrlichial genes were identified by using a very sensitive and specific laboratory method called polymerase chin reaction (PCR) in a specific type of flukes infesting a specific type of snails collected from stream waters, in central Ohio and northern California where PHF frequently occurs. We proposed to (1) isolate live N. risticii from these flukes by mouse inoculation and cell culture;
(2) clone fluke isolates and sequence 3 different genes of E risticii isolates and compare these sequences with those of local horse isolates; and (3) inoculate selected fluke isolates of N. risticii into horses to examine their infectivity and pathogenicity. These approaches permit us to prove the existence of live N. risticii in flukes, which are infectious and potentially pathogenic to the horse. Therefore, the proposed research provides much needed information to develop preventive strategies for limiting exposure, thus limiting use of ineffective vaccines and dependence on antibiotic therapy.
Publications
- Kanter, M., J. Mott, N. Ohashi, B. Fried, S. Reed, Y. Lin, and Y. Rikihisa (2000) Analysis of 16S rRNA and 51-kDa antigen gene and transmission in mice of Ehrlichia risticii in virgulate trematodes from Elimia livescens snails in Ohio. J. Clin. Microbiol. 38: 3349-3358.
- Mott, J, Y. Muramatsu, E. Seaton, C. Martin, S. Reed, and Y. Rikihisa. (2002) Molecular Analysis of Ehrlichia risticii in Adult Aquatic Insects in Pennsylvania, in Horses Infected by Ingestion of Insects, and Isolated in Cell Culture. J. Clin. Microbiol. 40: 690-693.
- Rikihisa, Y., C. Zhang, M. Kanter, Z. Cheng, N. Ohashi, and T. Fukuda (2004) Analysis of p51, groESL, and the major antigen P51 in various species of Neorickettsia, an obligatory intracellular bacterium of trematodes and mammals. J. Clin. Microbiol. 42: 3823-3826.
- Gibson, K. E. Y. Rikihisa, C. Zhang, and C. Martin (2005) Neorickettsia risticii is vertically transmitted in the trematode Acanthatrium oregonense and horizontally transmitted to bats. Environ. Microbiol. 7: In press. Feb., 2005
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