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 serodiagnostic tests answer the question about which horses had an infection with Sarcocystis neurona in the central nervous tissues that is the disease, EPM.

Tools had to be developed that could define infection and disease, and I became part of that process in 1999. Wisdom came from one of my advisors. Dr. Ellis Greiner, a parasitologist at UF. He often said "science is self-correcting", he was right, it just may take a generation or two for EPM science to self-correct.

I found the best source of information in 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 would later correct the science about EPM.

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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 settled science 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. S. falcatula also infects other birds, (many of the intermediate hosts are described in the literature), the hosts include cowbirds and grackles.

S. fayeri sarcocyst in a horse muscle

Box showed that the oocysts release the infectious stage of the parasite in the cells of the birds intestine and in fifteen days or so cysts, called sarcocysts, develop in the birds muscles. The obligate intracellular parasites (the parasite must remain in a cell) are carried to the muscle in the bloodstream. Finally, feeding the infected muscle tissue to opossums infects 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. This is the way horses get equine muscular sarcocystosis (EMS).

Dr. Box showed that most intermediate hosts get brain inflammation and the infected animals show signs including depression. Her work showed that organisms that were in muscles did not establish new infections to neighboring muscles! Take note of this important point about these parasistes and what it means for cases of EPM. It means the parasites found in cysts are terminally committed, they can't go back and infect more cells in the intermediate host. To continue in the life cycle the cysts found in the muscle tissue must be eaten by the definitive host. This is the biology of Sarcocystis and true for all the species of Sarcocystis parasites. She determined this part of the Sarcocystis biology by looking at the size (the cysts get larger with age— new cysts are small and large cysts are older) of the sarcocyst.

There is still quite a lot of discussion at EPM meetings about horses having tiny, even microscopic, cysts that can cause persistent infections. We published data to refute that; it was based on work done by Dr. Box. If there are oocysts shed in the environment by an opossum there will be chronic exposure of new gut infections in the horse. Horses don't get muscle cysts from S. neurona, these parasites are eliminated by the immune system. It isn't persistant infection that keeps signs present— but repeated, low dose exposure to new oocysts and chronic stimulation of the immune system. There is much false information about EPM, EPM forums are rife with disinformation, we believe Dr. Greiner, this science will be self-correcting. It may take a new generation of scientists and bloggers.

The correctable science of S. neurona and EPM

In the 1990's the race was on to discover the intermediate host for S. neurona. Developing an experimental model of the disease was a top priority for the scientists. Dr. Clara Fenger succeeded in using molecular biological techniques to show the opossum was a definitive host for S. neurona. This was a big step. Now scientists could find the intermediate host, define the life-cycle and create an infection model, if they could get a good supply of infectious oocysts from the opossum. The beauty of lab infections is oocysts could be throughly characterized and the same oocysts could be used for multiple experiments. Unfortunately, opossums aren't good laboratory animals.

The next best source of infectious oocysts was road-kill opossums, even though everyone knew the opossum hosted more than one species of Sarcocystis. It was guaranteed that the oocysts obtained from road-kill animals were multiple species. It was impossible to ensure a homogeneous population of parasites from the dead animals for multiple experiments. The oocysts were gathered from feral opossum gut scrapings and administered, by the millions, to horses in an effort to make an equine model.

In 1997, Dr. Fenger claimed victory for an experimental model of EPM induced from road-kill opossum oocysts. Quiet a feat— if it had been true. Later, the Saville group added stress after oocyst administration (stress was induced by transporting horses). They also claimed 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 in the experiments but parasites were never recovered from the animals tissues.

Dr. Fenger used feral opossums to get oocysts for her infection challenge, and fed oocysts to budgies to prove her infection source was indeed S. falcatula. She completed the life-cycle by feeding the budgie muscles to opossums and recovering oocysts. She found the horses seroconverted on the S. neurona Western blot. She reported clinical signs but couldn't recover parasites in the central nervous system. She reported histopathological lesions "consistent with" EPM. The title of her publication 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. Had it been true there would be more confidence the budgie-infecting oocysts used by Fenger induced disease in horses. It wouldn't be for another fifteen years in a collaboration with Dr. Mike Grigg, that molecular data was gleaned from DNA Pathogenes provided and showed that the S. falcatula used by the UF team was a strain of S. falcatula that had a surface antigen identical to the SAG 6 strain of S. neurona. The important biological difference in SAG 6-S. falcactula and SAG 6-neurona is the ability to infect horses. Infection challenge experiments done at UF in 1999 with SAG 6 falcatula failed to cause signs of EPM or seroconversion.

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 reinforced Box's conclusions that 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 they could not be infected with S. 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 any signs of EPM. They had done this experiment multiple times to prove the falcatula-neurona connection, the answer was no, they were not the same organism. 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 oocysts from opossums that were fed muscle cysts from sparrows, but she didn't have enough so 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. But experiment was compromised by the use of feral opossum material.

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 was deceiving. Important to my future work was recognizing that to satisfy two Sarcocystis rules 1) the parasite is an obligate intracellular parasite and 2) it must travel through the blood to reach the muscles. The only logical conclusion is that blood cells must 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 as well! I believe that is why they couldn't ever find the parasite in the central nervous system of the horse, the 'coon filtered out the horse-infecting strain.

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 with S. neurona. There are other exciting clues in the literature, as well as solutions to why so many horses have S. neurona antibodies yet few horses have the disase EPM. And why serodiagnostic tests won't be useful to identify the horse with S. neurona in the central nervous system tissues of horses with neurological signs. There is more and we will lead you to the facts.

The Trojan Horse Model Experiments

Journeyman's Guide