Infection with Sarcocystis seems inevitable. Sarcocystis are niche organisms, they’ve adapted to infect the intestine of limited hosts and complete their destiny as a muscle cyst. Generally, the host is unharmed. Occasionally, things run amuck and the host becomes diseased. It is estimated that 80% of horses in the US get infected with Sarcocystis . Most of the time the horse-adapted fayeri infects muscles where it makes a sarcocyst, this disease is equine muscular sarcocystosis or EMS. Less than 10% of horses with EMS show signs of disease, but some infected horses do get sick.
Most horses in the United States also encounter S. neurona and most infected horses are none the worse for it. Horses don’t get sick because the immune system eliminates the organism. Sarcocystis neurona-infected horses develop antibodies and bountiful cytokines that are effective in killing the protozoa. A most important cytokine is interferon-gamma. But occasionally, the most recent estimate is 14 of 10,000 horses, suffer the devastating disease EPM.
It is believed that the organism invades the central nervous system (CNS) and causes physical damage. In nearly all cases of experimental (natural challenge) S. neurona infections in horses, no organisms were found in spite of producing clinical signs. Histologists noted plenty of inflammation present in CNS tissues of the infected horses. Were the organisms there and removed quickly? Could the organisms hide out in other tissues waiting for the right moment to manifest? Were the samples taken at an inopportune time? Was inflammation the culprit and no organisms ever got into the CNS? Those are unanswered questions that are being investigated.
There is circumstantial evidence that some horses don’t develop the right kind of immune response to eliminate the parasite, maybe these are the horses that get organisms in CNS tissues. It was surprising when horses with deficient immune systems, Arabian foals that show severe combined immune deficiency syndrome (SCID), were infected with S. neurona and surprisingly, they got plenty of organisms in their blood stream, none in the CNS, and no clinical signs. It looks like an inflammatory response (the SCID foal can’t produce) is responsible for transporting organisms to the CNS and producing clinical signs. When a population of normal horses were likewise challenged they got clinical signs, but no organisms were found.
Some expect that horses will only improve by 1 grade (on a scale of 0-5) with treatment. Also, 10-25% of those horses that do respond to treatment are expected to relapse after treatment begging the questions: Are horses relapsing because they are re-exposed and have a new infection or is the initial infection latent-ready to manifest at any opportunity? How long can a protozoa hide before re-emerging? Can the horse make protective immunity?
As scientists ponder the best way to prove what is happening (and don’t forget it could be more than one mechanism!) you need to know some things to effectively evaluate new information.
It is important to understand how protozoal parasites mature. I will use the term synchronous to mean they all mature at the same time. Protozoa progress through their life-cycle stages on a one-way path to complete their destiny as a sarcocyst. Parasites (merozoites) go through a couple of replications, move to the next stage, and finally when they reach the muscle they encyst as slow metabolizing bradyzoites. Bradyzoites aren’t expected to move from a muscle fiber to another muscle to make new cysts. Cysts degenerate, unless they are passed to the definitive host where the parasite can complete the sexual part of the life-cycle and begin a new generation of infectious organisms. After the initial gut infection (sporozoite) and a few rounds of replication the resulting merozoites can’t go back to an earlier stage.
In 2001 we showed that S. neurona could be released from some cells, but not others, using an ionophore. The optimum time of release was 10-20 days after infection and this could be repeated once in another 30 days. When we examined the stages of the parasites by electron microscopy it was apparent that the parasites were not synchronized because there were multiple stages in the cells. We didn’t find a method that satisfactorily synchronized the cultures. Even cloning a culture from one cell resulted in asynchronous maturation of the colony.
In one particular cell line parasites grew very slowly, it took 120 days for them to establish a colony. The slow growing culture was infectious when transferred to a more traditionally used cell line and when transferred, they matured quickly. The colony was still asynchronous. In horses we expect the gut infection is asynchronous.
Sarcocystis are generally restricted to the hosts they infect. For example, S. muris can infect mice but not horses. S. canis infects dogs but not horses. One point of interest is that opossums can be a definitive host for many sarcocystis-ones that infect skunks, cats, birds, mice, and horses. True hosts are ones that support the life-cycle. Aberrant hosts are hosts that don’t support the completion of the life-cycle.
Dr. David Lindsay is a master of growing Sarcocystis and published papers on chemicals that delay, but don’t kill specific protozoa. He published an interesting experiment that treated Toxoplasma-susceptible mice and then infected them with Toxoplasma. The take-home-message from his work was that mice that were allowed to produce an immune response faired better with later challenge when they were compared to animals that were treated during the infection process. When mice were treated they didn’t produce a protective immune response and subsequently succumbed to toxoplasmosis. He also published work that showed diclazuril fails to eliminate S. neurona from laboratory cultures and showed the ability of decoquinate to render the cultures sterile.
In a recent experiment it was shown that the interferon gamma-defective mouse could be infected with a mouse-opossum strain of S. neurona. Untreated mice were diseased and the organisms could be recovered from CNS tissues. The experiment further showed that diclazuril could inhibit S. neurona activity, but not eliminate the parasite, providing evidence that recurrent disease could be a result of persistent infection and treatment failure rather than simple reinfection in this mouse model. The take home message was that S. neurona can resume its activity after cessation of diclazuril in a live interferon-gamma deficient mouse.
One must be careful when interpreting study data from one animal to another. In the mouse experiment, the mice were injected with cultured organisms that did not allow stimulation of a natural immune response in the gut. A similar experiment in 2001 used mice that ingested sporocysts of the same Sarcocystis strain as the above experiment (a natural infection) and also administered diclazuril in the diet. After discontinuing treatment the mice did not have organisms in the CNS. The discordant results may be the method of administration of the protozoa or even when tissues were examined. after the discontinuation of therapy Obviously, there is more work to be done.
All the above considered, we have an issue with diclazuril used for our non-inferiority study. It isn’t a fear of persistent infection and relapse after treatment because that has not been shown in the horse. We did test several hundred horses with clinical EPM for a lack of interferon-gamma and didn’t find one. Our insurmountable task is showing that in our study a comparison drug, diclazuril, is as effective as it was when licensed. We have the daunting task of showing that diclazuril is 67% effective in treating EPM. If the statistics don’t support 67% of diclazuril-treated horses clinically improve when diagnosed with EPM (the horses must have CSF tap confirming disease before treatment) the study is not acceptable. When diclazuril was licensed to treat EPM clinical improvement was seen 59% when based on clinical signs. Because diclazuril was considered successful when antibody declined the CSF when there was no clinical change. the reported stats are 67% effectiveness. That didn’t fit our criteria of success and we won’t be asking for a post-treatment CSF sample. Other factors that render the data unacceptable are concomitant drugs with diclazuril, like DMSO, levamisole, steroids, phenylbutazone, flunixin , or firocoxib. If the attending clinician administers these treatments while waiting for CSF analysis, the case is not useable.
While we put on our thinking-cap, please fill out our survey if you treated your horse with diclazuril for 28 days (no other treatments within 6 months of treatment) and let us know the outcome of treatment. We can use the data to know if 67% effectiveness is an attainable goal.