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EPM Clues from Sarcocystis falcatula

Did you ever wonder why diagnosing EPM due to Sarcocystis neurona is difficult? That was an initial conundrum when I began my PhD project. Why didn't the available diagnostic tests answer the question about which horses had an infection with Sarcocystis neurona and the disease, EPM? Tools had to be developed to define infection and disease, and I became part of that process in 1999. Valuable guidance came from one of my advisors. Ellis Greiner, a parasitologist at UF often said "science is self-correcting", he was right, it just may take a generation for EPM.


I found sage advice from the literature, especially the infection studies done by Dr. Edith Box. She was retired by the time I read her work. Her three papers detailing infection of the definitive and intermediate hosts of Sarcocystis falcatula could later correct the science about EPM.


 

What Dr. Box taught about S. falcatula


Edith Box re-described Sarcocystis falcatula in 1984. The original description was in 1893, and it was described again in 1929. It was known then that the opossum is a definitive host for falcatula. Dr. Box knew that she could infect birds using the infective oocysts found in the feces of the opossum. She showed that S. falcatula could infect the budgie, the sparrow, and the canary, but not ducks. Falcatula also infects other birds, (many of the intermediate hosts are described in the literature), the hosts include cowbirds and grackles.

parasite cyst in the muscle of a horse
S. fayeri sarcocyst in a horse muscle

She showed that the oocysts release the parasites in the gut of the bird and in fifteen days or so cysts, called sarcocysts, develop in the birds muscles. The obligate intracellular parasites are carried in the bloodstream. Finally, feeding the infected muscle tissue to opossums will infect the opossum. That completes the life cycle of the parasite. By the way, the natural intermediate host for S. fayeri is the horse and the definitive host is the dog.


Dr. Box showed that most intermediate hosts get brain inflammation and these animals show signs including depression. She surmised that organisms from muscles did not establish new infections locally. That means the parasites in the cyst are terminally committed, they can't go back and infect more cells in the intermediate host and must be ingested by the definitive host. This is true for all the Sarcocystis. She determined that by looking at the age of the sarcocyst. There is still quite a lot of discussion at EPM meetings about horses having tiny cysts that can cause persistent infections. We published data to refute that; it was based on work done by Dr. Box. There are other false starts about EPM that linger with incorrect information.


The correctable science of S. neurona and EPM


In the 1990's the press was on to discover the intermediate host for S. neurona. Developing an experimental model of the disease was a top priority. Dr. Clara Fenger used molecular biological techniques to show the opossum was a definitive host for S. neurona. This was a big step. Now scientists could define the life-cycle and create an infection model if they could get a good supply of infectious oocysts.


The next best source of infectious oocysts was road-kill opossums, even though everyone knew the opossum hosted more than one species. These oocysts were gathered from feral opossum gut scrapings and administered, by the millions, to horses. In 1997 Dr. Fenger claimed victory for an experimental model and later, the Saville group added stress after oocyst administration, also claiming that the elusive equine model of EPM had been achieved. Alas, these scientists couldn't demonstrate the organism in the brain of the infected animals, a requirement for true model. Many horses were used but parasites weren't found.


Dr. Fenger used feral opossums for her infection challenge, and fed them to budgies to prove her infection source was indeed S. falcatula. She completed the life-cycle by feeding the muscles to opossums and recovering oocysts. She found the horses seroconverted on the S. neurona Western blot. She saw clinical signs but couldn't recover parasites in the central nervous system. She reported histopathological lesions "consistent with" EPM. Her title claims to have experimentally induced EPM and that the opossum was a true definitive host for S. neurona.


Team UF led by John Dame and Rob MacKay were collecting muscle cysts from cowbirds and grackles to show that S. neurona and S. falcatula were one in the same and thus the first to describe the true life-cycle for neurona. They published their hypothesis in 1995.


In 1997 Antoinette Marsh showed that S. neurona and S. falcatula were biologically different, bird infection studies proved that they were not the same organism. Tanhauser, a member of the Dame team, used molecular techniques developing molecular markers that could differentiate neurona oocysts from falcatula oocysts, and therefore the opossum carried more than one Sarcocystis species. (Some laboratories took these markers to say what was neurona, and that isn't what these markers could do. Differentiating two organisms with random markers isn't the same as an identifying marker for an organism).


Dr. Marsh showed budgies could be infected with S. falcatula but not neurona. The experiments wrapped up when Dame's team challenged horses with S. falcatula in 1999, the horses failed to show seroconversion after challenge, much less, show signs of EPM. They had done this experiment multiple times to prove the falcatula-neurona connection, the answer was they were not the same. This was not to be the last of the "S. neurona is S. falcatula" controversy. One clue why Fenger may have another Sarcocystis sp. in her study is found in the source of the oocysts used for infections. She used opossums fed the muscle cysts from sparrows, but she supplemented the sporocysts from two additional feral opossums. The difference between the Fenger and Dame studies, in addition to clinical signs, was seroconversion after challenge. Dame's challenged horses were negative, yet Fenger's challenged horses were positive.


Edith Box set the foundation for EPM research


Dr. Box showed that the opossum was promiscuous in the range of sarcocystis it hosted, adding oocysts from feral animals could be deceiving. Important to my future work was recognizing that to satisfy two rules that are: the parasite is an obligate intracellular parasite and must travel through the blood to reach the muscles, required blood cells to be infected by intermediate stage merozoites. That is the point of many laboratory hours and summarized in my first publication.


Another clue from Box was that the intermediate host is far more selective and can be used to filter out desired species, like the budgie did for falcatula. That played a part in Saville's studies because they used the raccoon as a host. The raccoon harbors multiple, different, and confounding species of Sarcocystis! I believe that is why they couldn't ever find the parasite in the central nervous system of the horse.


I hope you can see the importance of reading the literature from early biological studies carefully, and you should have enough information to follow my path to developing the Trojan Horse model of EPM. There are other exciting clues in the literature, as well as the solution. Yes, Dr. Greiner, you'll be glad to know that the EPM literature is being corrected! If you find a clue, please contact me and let's see if you are a good EPM detective.



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