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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.

cyst microscopic cropSarcocystosis is one of the most frequent infections of animals, the organisms are found in the muscles and the central nervous system! There are, perhaps, 196 species of Sarcocystis, yet the complete life cycle is only known for 26 of them. Sarcocystis require a prey-predator, 2 host, relationship to survive. Each host supports different stages of the life cycle. Hosts vary for each species of Sarcocystis.  Intermediate hosts support several species of Sarcocystis, each species use different definitive hosts.

People get sarcocystosis, two species use people as the definitive host.  Humans also serve as intermediate (or aberrant hosts) for several other species. Raw beef can infect you with S. hominis and raw pork can give you S. suihominis. Gastrointestinal symptoms are present when Sarcocystis cause disease in people  definitively hosting the parasite. Muscle pain is reported in people that serve Sarcocystis as an intermediate host. Most of the time people are asymptomatic with muscular sarcocystosis. People can get sick from an enterotoxin associated with Sarcocystis that infect horse muscles. if horse meat is uncooked.

Horses get sarcocystosis from dogs, the organism causing infections is S. equicanis (bertrami). In the US the horse-infecting organism is named S. fayeri. Donkeys harbor S. asinus, but experiments may show that this is actually S. fayeri. The visible difference in equicanis (bertrami) and fayeri is the thickness of cyst walls in muscles observed under the microscope. Our picture above is fayeri,  a thick-walled sarcocyst.  S. bertrami forms a thin-walled cyst. Dogs shed infective betrami organisms 8-10 days after eating infected muscle tissue. Horses eat the organisms from dog feces-contaminated feed.  It takes two to three weeks before signs appear in horses.  Signs include fever, neurological signs, apathy, and inappetence  a couple of months after infection. Muscle enzymes can be elevated in infected horses and increased enzymes can be measured in blood samples.

Sarcocystis fayeri cysts are found in skeletal muscles and the heart.  Ten and 25 days after infection (two waves of organisms are released from the gut) the developing parasites are found in arteries of the heart, brain, and kidney.  Muscle cysts (sarcocysts) are first seen at 55 days .  And by 77 days the muscle tissue can infect dogs to start the cycle again. We are taught that S. fayeri is only mildly pathogenic, horses develop anemia and fever after infections and some horses may have a stiff gait. More virulent isolates can cause inflammation of muscles and, in one case, the horse developed autoimmune anemia.  People that eat raw horse meat can get food poisoning, this is due to a toxin associated with the cysts.  A fetus can be infected by S. fayeri by crossing the placenta, so foals can be born with mature cysts in their muscles.

Malnourished horses show clinical muscle inflammation (myositis) and muscle atrophy that can be associated with S. fayeri. Muscle cysts are usually unassociated with clinical signs or muscle inflammation, leading to conclusions that fayeri infections are benign. Most clinicians agree S. fayeri isn’t an issue in horses.  However, there are some researchers that think S. fayeri should be considered in horses with neuromuscular disease. Scientists at UC Davis examined muscle tissues from horses with and without a history of neuromuscular disease.  They found an association, but not statistical significance, between disease and cysts.  More horses with neuromuscular disease had S. fayeri cysts when compared to the number of cysts found in horses that died from other causes.

We decided to look at the problem a different way.  While UC Davis looked at cysts, we chose to look for S. fayeri antitoxin in the serum of horses. An advantage to antitoxin analysis is that the test is performed in the live horse.  We found that 24% of normal horses had antitoxin in their serum. We also found that S. neurona antibodies were more often associated with neuromuscular disease in horses  than S. fayeri antitoxin (antibodies against the toxin released from cysts). That said, horses were more often infected with both species of Sarcocystis, not just one strain.

Horses infected with S. neurona and S. fayeri were more likely to show disease. We looked at inflammation using CRP (C-reactive protein) and found that CRP was detected in horses with and without apparent disease. We did find that significantly more horses with neuromuscular disease had an elevated CRP when compared to normal horses (p= .0135).  However, the data we needed to link the UC Davis study and ours was missing.  We needed to show  the relationship of antitoxin found in horses (our test) with sarcocysts found on histopathological slides (the gold-standard test for fayeri-sarcocystosis).

We examined three tissues (muscle tissue from the heart, esophagus, and skeletal muscle) from thirty-two horses (that’s a big number in horse studies) and measured the presence of antitoxin.  Our results confirmed that our serum test was almost the same as the results you would get if a full post-mortem exam was conducted. Also, in our study the infected horses were asymptomatic.

The conclusions thus far are that S. fayeri can be detected pre-mortem and should be considered in horses with signs of sarcocystosis or neuromuscular disease that has no other cause.  Also, horses with neuromuscular disease are more likely to have an elevated CRP.  In our S. fayeri study horses did have elevated CRP values but the inflammation was associated with other gastrointestinal parasites, not EMS (equine muscular sarcocystosis).

Why are we still interested in S. fayeri? Certainly our results support the strain of S. fayeri  infecting our 32 horses was non-virulent and support the view of most clinicians that S. fayeri may not be an issue in most horses, just some of them.

For us, it’s about the toxin.  The toxin is an actin-depolymerizing factor (ADF).  Another Apicomplexan, Toxoplasma gondii, has a very similar ADF. The TG-ADF can protect lab animals against T. gondii infections. Interestingly, some animals with lethal S. neurona infections also were infected with T. gondii. The difference between protection (mice) versus no-ADF protection (sea otters) could be stage of infection (with TG), ,strain-virulence, or something we didn't think of yet.

Can S. fayeri-ADF protect against S. neurona infections in horses? Is ADF species specific? Is ADF a virulence factor or even a survival factor for the organism? Are there cyst-producing S. fayeri organisms that don't produce ADF? Does S. neurona produce an ADF? And, can a S. neurona be incorporated into a fayeri sarcocyst? These are all questions to be answered by experiment.

Here is our opinion. It is worth monitoring S. fayeri infection in horses. It is worth considering S. fayeri infection as a cause of neuromuscular disease in horses showing weakness, muscle atrophy, and inflammation for which there is no other explanation. The disease EMS should be considered in old and debilitated horses.  Sarcocystis fayeri should be considered in horses with chronic inflammation. When  horses showing weakness do not have antibodies against S. neurona, EMS should be considered. Horses that provide meat that is fed to dogs should be monitored for S. fayeri.

If you have questions about S. fayeri, give us a call.

It is a common thought that infection by more than one disease-causing protozoa is linked to increased severity of disease.  This was shown in marine mammals with protozoan encephalitis, is it similar in horses?

One study sought to understand if horses with presumptive EPM had antibody evidence of infection with other protozoal infections. Documenting more than one protozoal infection is possible because horses get infections with Neospora and/or Toxoplasma.  The criteria for EPM in dead animals rested on the presence of lesions (not parasites) in central nervous tissues.  They included cases from live horses that had a presumptive diagnosis of EPM.  EPM was presumed when clinical signs were present, excluding other disease based on ancillary testing, and SAG ELISA positive serum and CSF for Sarcocystis neurona (in this study the ratio was less than or equal to 50).  The scientists compared the presence of Neospora or Toxoplasma antibodies in their selected cases to a group that were not expected to have protozoal infections.  The expected-negative group was comprised of horses with a diagnosis of cervical vertebral malformation (CVM) and negative for SAG ELISA of serum and CSF (in this study the ratio was less than or equal to 50).

Overall, they found 12.9% of horses were antibody positive for Neospora.  The proportion of EPM cases that tested positive did not differ from the proportion of CVM cases that tested positive.  A similar finding with  Toxoplasma, antibodies were found in 14.9% of suspect EPM cases and CVM horses.  A qualifier in the study was that these horses were from the eastern US.  It is possible that Neospora is more common in the west, however one study does not support that.

They found no horses with co-infections! Another interesting finding was that they didn’t find sub-clinical infection’s either. A sub-clinical infection was a case in which antibody was detected in the serum but not enough antibody in the CSF to be considered EPM.  They concluded that their data did not support protozoal co-infection as a common finding in horses with neurologic disease from the eastern US, and further co-infection with a protozoal species is unlikely to play an important role in development of clinical disease caused by S. neurona infection.

You had us nodding our heads in agreement until they said “protozoal species”.  We assumed that an implied qualifier might be “the data did not support protozoal co-infection with Toxoplasma or Neospora”…yes.

We disagree that co-infections aren’t present in horses with S. neurona! We presented evidence to the attendees at the 2nd EPM Society Workshop in Tahoe City, CA on October 26, 2017 to show that Sarcocystis fayeri and Sarcocystis neurona antibodies were present in horses with clinical neuromuscular disease.  And in some horses with sub-clinical disease, antibodies present and not ataxic. And in normal horses!  We showed that 87% of normal horses have co-infections with Sarcocystis and is in direct opposition to the findings in the above experiment. Two protozoal infections in one animal.  We might speculate that S.fayeri is protective against developing EPM in S. neurona exposed horses.  We can find some data to support this speculation because the protein we use to measure S. fayeri is a protein that protects animals against Toxoplasma infections. Our data would not argue against their findings, we just disagree that horses can’t harbor two species of protozoa.

Some of the diseased horses we examined had two Sarcocystis infections and met the bar of serum/CSF ratio (<100), confirming EPM.  In diseased horses, we found 74% were Sarcocystis positive!  In diseased horses dual infections were more common than single species infections. When we only looked at single species infections we found that neurona was more common in diseased horses. These data still support the finding that a single infection is more pathogenic or sometimes S. fayeri is present but not protective. We found that some S. fayeri strains may not produce the protective protein under some as yet undefined conditions. The EPM-Neospora-Toxo study discussed above may have been stronger if they considered S. fayeri as a factor or stated that there are other protozoa that co-exist in normal and diseased horses. The relationship between Sarcocystis that infect horses remains undefined.

We, and UC Davis, have reported finding S. fayeri in horses with neuromuscular disease, although we look for the disease in different ways. We ran a study to compare our way (serum ELISA) with the UC Davis method (post-mortem tissue exam) finding a pretty good correlation between the methods.  Good enough to suggest pre-mortem serum testing is more palatable to the owner. The point is that multiple protozoa infect horses, multiple protozoa are in diseased horses and it takes a good (diagnostic eye) to discern the cause of clinical signs.

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Not all species of Sarcocystis harm the intermediate host! Species passed by dogs are usually more damaging to the host than those transmissible by other definitive hosts and generally severity of signs depends on the dose of parasites. Horses don’t show any outward signs with initial S fayeri infections transmitted by dogs. Only a few horses show signs when they are infected with S neurona. S neurona infection is passed to the horse through opossums.

When a horse succumbs to S fayeri disease it takes a large dose of oocysts and usually in a debilitated animal. Infection can cause fever, anemia, and hair loss. Infected horses can have a stiff gait and muscle soreness. Experimentally it takes millions of oocysts to get clinical signs with S neurona. Microscopically, inflammatory cells are increased but not associated with parasites or the cysts found in muscles, inflammation is associated with host tissue cell death. New infections are most likely spread from the gut, re-exposure probably plays a large part in chronic inflammation for both Sarcocystis.

Little is known about chronic sarcocystosis (in the natural intermediate host) to explain the cause of central nervous system signs often seen in intermediate host infections. Some cysts may rupture from time to time releasing toxins. It has been known for 100 years that an extract of parasites (the bradyzoites) release a toxin. S fayeri infected horsemeat is toxic to humans, interestingly this is how the toxin was discovered and identified in Japan. A toxin wouldn’t be expected from S neurona infections in horses because this parasite doesn’t make muscle cysts. If it is discovered that neurona does make cysts in the horse, then the test for the fayeri toxin would be useful. Detection of neurona toxin using the fayeri assay is expected because the toxin is conserved in Sarcocystis and other cyst-forming pathogenic protozoa. Scientists haven’t identified S neurona cysts in horses, but they are looking again at tissues, this time with a fine-tooth comb. Our work shows no link (statistics used here) between horses with clinical signs and antibodies against S neurona and fayeri anti-toxin. One would expect a link if S neurona made a toxin.

Sarcocystis induce antibody production and the level of antibody (titer) increases with the duration of infection. Antibody may play a part in protection. It is cellular immunity that recruits white blood cells from tissues and the blood stream to fight and protect against parasites, including protozoa. Cellular immunity is evident 2 weeks after initial infection from oocysts. In certain animals Sarcocystis can depress immunity. Some proteins that are released during infection are cross-reactive with other Sarcocystis. When non-specific tests are used any cross-reactive antibodies will need to be diluted out (the reason the SAG 2, 4/3 starts at 250). In our lab we look for antibody made against the fayeri toxin released from cysts because we associate toxin with disease in susceptible horses. When we look for antibody against S neurona we use neurona specific proteins to identify serotypes (SAG 1, 5, 6).

Protective immunity is most likely associated with a sporozoite (found in the oocyst) or first-generation schizont. In some species sporocysts induce protective immunity that persisted for 80 days to 280 days. Protection can result from as little as 100 sporocysts. Protective immunity has only been shown with homologous (the same) species. We are working on the protocol to induce protective immunity in field exposure to Sarcocystis. It has been shown in mice and horses that ponazuril will decrease antibody production (detected by antibody assays) but not prevent signs of EPM.  In a mouse experiment mice were given drugs to prevent infections.  When the animals were removed from treatment and challenged, they succumbed to the disease.  Some protozoal vaccines have been attempted in pigs and mice.  Live, killed, or fractions of bradyzoites induced antibodies but provided no protection in these species.  The persistence of live sarcocysts is probably not crucial for maintaining protective immunity.  Goats are protected from challenge infection after initial infection from sporocysts.  We are working on it, stay tuned!

Sarcocystis require 2 hosts to survive, a definitive host and an intermediate host.  The sexual cycle occurs in the definitive host and the asexual cycle occurs in the intermediate host.  Definitive hosts that are important in understanding EPM are the dog and the opossum.  The dog transmits Sarcocystis fayeri to horses, this results in Equine Muscle Sarcocystosis, EMS. The opossum transmits Sarcocystis neurona to horses, but horses are aberrant hosts, S neurona isn’t believed to make cysts in horses. Opossums can also harbor S falcatula, these organisms can look like S neurona to horses. The opossum can have other Sarcocystis species but they aren’t related to equine disease.

Equine protozoal myeloencephalitis (EPM) refers to neuromuscular disease in horses, most often associated with S neurona.  Other protozoa, less often associated with EPM, include Neospora. The reaction to the protozoal infections include antibody production (we test for antibodies in serum and CSF) and inflammation.  That means disease is a syndrome. Understanding the biology, the life cycle of the parasite in it’s hosts, facilitates understanding, diagnosing, and treating EPM.

 

THE DEFINITIVE STAGES OF THE SARCOCYSTIS LIFE CYCLE

Definitive Host S fayeri: DOG              Definitive Host S neurona: OPOSSUM                       Definitive Host S falcatula: OPOSSUM

How does a horse get EPM? They ingest parasite “eggs” (oocysts) shed in feces from a definitive host and the horse immune responses set up an inflammatory cycle that benefits parasitic infection. Definitive hosts are infected by ingesting mature parasites found in muscle cysts (sarcocysts). Slow growing parasite stages, bradyzoites, are released from the digested tissues where they penetrate cells in the small intestine . The sexual stage (gametogony) of the parasite life cycle occurs here when microgamonts fuse with macrogamonts. After fertilization a protective wall develops around the newly formed zygote-it’s now an oocyst . This occurs within 24 hours of cyst ingestion. These processes don’t happen at the same time, oocyst development is asynchronous. This is important when treating animals. If a drug doesn’t kill all stages the drug is static, when the drug is removed the life cycle continues. The parasite in the oocyst divide forming 4 sporocysts in each oocyst. They are now “sporulated” and infectious to the intermediate host.

 

sporocysts

 

THE INTERMEDIATE STAGES OF THE SARCOCYSTIS LIFE CYCLE

Intermediate host S fayeri: HORSE     Intermediate Host S neurona: ARMADILLO (experimentally: Cat, Raccoon)   Intermediate Host S falcatula: GRACKLE, COW BIRD

Sarcocystis enter the asexual stage of the life cycle in the intermediate host (horse) ultimately forming muscle cysts (sarcocysts), this is equine muscular sarcocystosis or EMS. Toxins are sometimes released from degenerating cysts in horses, these toxins produce signs that look just like EPM!! Horses ingest infectious parasite “eggs” (oocysts that contain 4 sporozoites) that were deposited in feed or water by the definitive host, the dog. Sporozoites show up in 4-7 days in lymph nodes. Precystic replication, “schizogony”, occurs virtually throughout the body and these stages disappear before cysts form. (This is important because repeat exposure from the environment causes “new” infections). The next stage of the asexual parasite life cycle is called a “merozoite”. Merozoites are committed to make a cysts in muscle tissue (shown below). Merozoites that are not in muscle cells die because supportive nutrients are lacking. In muscle cells, the parasite is surrounded by a vesicle where it happily transforms into a metrocyte. A metrocyte divides into many daughter cells filling the muscle cyst (sarcocyst) with dormant, slow metabolizing bradyzoites, shown in a microscopic photo below. After 70 days or so, the sarcocyst is infectious to the definitive host and the cycle can begin again, after the definitive host dines on infected muscle tissues. Most muscle cysts begin to disappear after 3 months. Ruptured cysts can cause inflammation, but bradyzoites do not make new cysts-THERE IS NO REACTIVATION OF CHRONIC INFECTION, IRRESPCETIVE OF THE IMMUNE STATUS OF THE HOST. Sarcocystis fayeri causes cysts in horses, see infected muscle tissue below.

 

s fayeri muscle

cyst microscopic crop

 

 

Sarcocystis neurona schizonts (or merozoites) are found in horse neural tissues.  Horses are considered aberrant hosts because muscle cysts aren’t formed by the asexual merozoites.  A very interesting experiment is intentionally infecting horses with S neurona oocysts from the opossum.  If the horse is given a HUGE dose of oocysts, and the horse has a normal immune system, the merozoites are quickly removed-- because they can’t form cysts.  These horses show neurological disease within days, antibodies are produced against S neurona as well. When the same organisms are used to infect immunodeficient Arabian foals, they are called SCID foals and have abnormal white blood cells, the foals show no clinical signs!!  Very convincing evidence, to me, that disease and clinical signs are due to how the horse handles the infection. Horses make detectable antibodies against S falcatula, another protozoa harbored by opossums.  Our experiments show that blocking some receptors on white blood cells in normal horses, and challenge them with S neurona infection, they don’t show signs, just like an Arabian SCID foal.