FRANK J. MILNE STATE-OF-THE-ART LECTURE


ing a metacercariae containing the rickettsia agent Neorickettsia helminthotheca and then develop fever and severe gastroenteritis. The fish get the infec- tion from a fluke stage originating in a freshwater stream snail. We formed the hypothesis that the agent of PHF had something to do with snails. The immediate response from colleagues was not positive, and indeed I was ridiculed at meetings and in the hallways when other PHF researchers asked me how many snails horses had to eat to come down with PHF, knowing of course that horses don’t eat snails. Undeterred, we tested 400 freshwater snails and got the first DNA evidence of E. risticii in nature in about 15% of snails in the Shasta River adjacent to the pastures and barns where horses had developed Shasta River Crud. We attempted transmission by stomach tubing research horses with infected snails but got no clinical disease. So our team of Gerhart Reubel, Nicola Pusterla, Christian Leutennegger, Jeff Barlough, Joon Seok Chae, and Elfriede DeRock went to the river, brought the snails back, and put them in an aquar- ium in the lab. There, the snails one day released “white stuff” which, when examined under a micro- scope, contained swimming cercariae. We DNA tested the cercariae and they were positive for E. risticii. We attempted transmission via stomach tube and, again, there was no infection in horses given the infected cercariae. We next thought that maybe the cercariae penetrate and burrow into the skin when horses cross the river. So we taught horses how to stand in buckets of water and we added the infected cercariae from the aquariums but again no transmission. We tried putting the cer- cariae in drinking water; no transmission. We knew we needed an isolate and not just DNA evi- dence so we did a bold experiment involving trans- mission by dissecting the infected snails and injecting the infected tissue into 3 research horses, who were given penicillin and gentamicin which have no killing spectrum against E. risticii.We had done it! We got disease transmission with fe- ver, diarrhea, and mild colic. We isolated the agent from the blood of that horse on cell culture. We inoculated the cell culture and the next horse got the same disease. We had therefore achieved the first isolation of the agent from nature. The next question was how the agent gets from


the river to the horse. We used gumshoe sleuthing to answer this question interviewing several older wranglers at the ranch who remembered cases of Shasta River Crud. We asked if all the cases had direct access to the river and the answer was no. Several were stall confined and lived over 100 yards from the river. So we pursued this lead. The killer (E. risticii) had to walk or be transported by a carrier on ground or flight to the barn. We then began to collect, with the direction of a UC Davis freshwater entomologist, all insect larvae on the rocks next to the snails that released the cercariae. The cercariae would rapidly float downstream fol-


lowing release so they had to go to the next stage somewhere close to form metacercariae. We deter- mined over 17 species of insects were infected with the agent. We brought caddisfly larvae back to the lab and put them in our aquarium culture. Our lab looked like the Petco section for fish. One after- noon in the cold room on the second floor of Tupper Hall, the caddisfly larvae hatched and adult caddis- flies were flying around the room. Pusterla caught 8 of them, which we fed to a research horse. This horse came down with severe disease; fever, diar- rhea, mild colic followed by laminitis. No other IV transmission studies with E. risticii had produced laminitis, as developed in naturally occurring cases. Our next step was determining how snails became infected. We knew something had to eat the flying insects and return the agent to the river to complete the fluke cycle. We saw lots of bats on the river in the evening and began a study, which determined that the bats had a fluke that was infected with E. risticii. The eggs of the fluke contained the agent, which were released by the bats while flying over the river allowing transmission to the snails. The modus operandi of the killer was revealed and


is this: The killer struck its victims by emerging from the creek within the body of a small insect, mayfly, stonefly, caddisfly, etc., and flew to pastures, or perhaps where lights were on in the barn, dropped into feed buckets or grass or hay and the horse ingested them and came down with the infec- tion. The horse is a dead end host and not contagious. Copyright use authorized by Elsevier: Pusterla N, Madigan JE. Neorickettsia risticii in- fection. In: Sellon D, Long M, eds., Equine infec- tious diseases. 2nd ed. Amsterdam: Elsevier, 2013.


Etiology


Neorickettsia risticii (formerly Ehrlichia risticii)is the etiologic agent of EN, also called Potomac Horse Fever, equine monocytic ehrlichiosis,or equine ehrli- chial colitis. Neorickettsia risticii has recently been classified based on genetic analysis in the genera Neorickettsia among three other species: Neorick- ettsia sennetsu (human agent of Sennetsu fever), Neorickettsia helminthoeca (agent of salmon poison- ing in the dog), and an ehrlichia-like bacterium pres- ent in the metacercarial stage of the fluke Stellantchasmus falcatus (SF agent).1 Based on se- quence analysis of the 16S rRNA gene, N. risticii shares 98.9% homology with N. sennetsu, 94.8% with N. helminthoeca, and 99.1% with the SF agent. Strain variance has been determined among 11 N. risticii strains, with a maximum divergence of 0.7%. Neorickettsia risticii is a Gram-negative coccus


and stains dark blue to purple with Giemsa stain and Romanowsky stain, red with Macchiavello stain, and pale blue with hematoxylin and eosin (H&E). The organism tends to occupy one side of the cytoplasm rather than being symmetric and is


AAEP PROCEEDINGS  Vol. 60  2014 107


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