This beautiful horse is Nizhoni, or Zhoni as her friends call her. The word Nizhoni is Navajo for beautiful. And her spirit is just that.
We think of Zhoni as an old friend because we have known her since she was 17 years old, and at 28, she is going strong. She is a cross between a Tennessee Walker and a paint horse, that is what makes her such a great trail companion. She was one of the horses that gave us clues us about horses diagnosed with relapsing EPM.
Relapsing EPM
In the "old days" we listened to the experts at the EPM society meeting talk about relapsing cases of EPM. We heard that all horses had antibodies (more than 80%), but gosh darn it, where were the parasites hanging out? It was a common idea that only immuno-compromised horses could get EPM. That dogma didn't really make a lot of sense to us and that's when we did a deep dive into cases and developed new tests to find out what was going on. A few studies helped us parse the nuances of infection and disease. A review here may be useful to you as well.
Life stages of Sarcocystis in the intermediate host
Clue number 1: Rule #1 Sarcocystis enter the blood stream from the gut
Keep in mind what Edith Box taught us, she said that all Sarcocystis are ingested, the zoites (and merozoites) are released into the gut and then gain access the blood stream of the intermediate host. That is called a parasitemia. From the blood they travel to the muscles, that is what sarcocysts are, the end stage of a Sarcocystis infection that came via the blood.
Clue number 2: Rule #2 Sarcocystis are obligate intracellular parasites
There is a another rule, and rules must be followed, and that is these parasites are obligate intracellular parasites. They can not live outside a cell. Researchers unsuccessfully injected the merozoites into the CNS of horses trying to create a model. This is the rule that prevented that pathway to infection, the merozoites were not in any cell.
In 2004 we published our Trojan Horse model, you can read about it using the button. Bayer and Schering used our model prior to publication to learn more about their EPM medications. Our model proves that immunocompetent horses can be experimentally infected if you follow rule #2.
There was another experiment that is worth understanding. A paper published in 2004 showed that infecting immunodeficient horses with Sarcocystis neurona does not result in neurologic disease! The Arabian foals used in the experiment suffered from severe combined immune deficiency (SCID). The experiment included some horses that were immunocompetent (this cohort was called the IC group). Surprisingly, the immunocompetent horses reacted much differently to S. neurona challenge.
Arabian foals with SCID
Arabian foals with SCID lack specific B and T cell responses because they have a genetic mutation . It is a devastating genetic disorder and an opportunity to investigate the role of adaptive immune responses in the pathogenesis of some equine diseases. These foals generally succumb to bacterial pneumonia or adenovirus early in life.
In the experiment, two 2-4 week old SCID foals were infected orally with infective S. neurona from the feces or intestines of wild, or previously infected, opossums. Another SCID foal was infected by intravenous (IV) administration of merozoites (braking Rule #2). The three SCID animals developed a prolonged parasitemia and persistent infection of muscle, lung, liver, and spleen tissues, but did not develop neurologic signs. Sarcocystis neurona was not detected in the neuronal tissues.
SCID horses were infected, but not diseased. The SCID foals don't develop inflammation. Demonstrated here is the double edge sword of inflammation. Inflammatory cells are a response to infection and induce clinical signs. The responding inflammatory cells eliminate the parasites. Functional inflammatory cells are absent in these foals, resulting in no signs and a persistent parasitemia.
The immunocompetent cohort of the experiment
Two orally infected, immunocompetent (2-4 week old) foals used in this experiment didn't develop a detectable parasitemia. And when the parasites were given IV to four IC foals, the induced parasitemia was short lived. In contrast to the SCID animals, some of the IC horses developed neurologic signs. One horse infected orally showed signs at day 42 but improved starting at day 48. This horse wasn't euthanized until day 471. Another foal infected orally never showed clinical signs of disease. Neither of these horses had parasites isolated from the CNS.
The four immunocompetent animals that were infected IV had different outcomes from the orally infected IC foals. One never showed signs of disease and parasites were not isolated from the CNS. The three remaining IV challenged animals had evidence of parasites, two by PCR and one by culture. The parasite was not detected in the viscera (muscle, lung, liver, and spleen) in these IC foals.
The moral of this story: inflammation is important
Infected SCID foals are unable to clear S. neurona from visceral tissues and the infection does not result in neurologic signs; in contrast, IC foals rapidly control the parasitemia as well as infections of visceral tissues, but can experience neuroinvasion and exhibit clinical signs of neurologic disease.
When taken together with our model, the results of the SCID experiment can be understood. Our model uses the leukocytes from the blood to transport the parasite to the neural tissue. It is the leukocytes that elicit an inflammatory reaction seen as neurologic signs. The SCID foals lack the functional leukocytes to transport the parasites to the CNS and lack the inflammatory reaction associated with clinical signs. The immunocompetent horse quickly eliminates the infection. The inflammatory reaction is evident in these horses. Occasionally the parasites can be engulfed by leukocytes and rather than elimination, a parasite can be transported (in a cell!) to the CNS.
Important points
Before you go off the deep end about the occasional parasite that can get into the CNS consider these facts. In the Trojan Horse model we infect cells in vitro, using 6000 parasites. We do that for several consecutive days. The oral infection in the SCID experiment uses 500,000 oocysts (each carries four sporozoites). Oral infection required 100 times more oocysts than the Trojan model and none of the infections resulted in disease. Opossums don't shed 500,000 in their scat. To get enough material, researchers have to scrape the intestines and pool the oocysts.
The SCID experiment used 500,000,000 organisms injected IV. Five hundred million. That is 100,000 times more than we needed because we followed rule #1. That should indicate to you that it is very difficult for an organism to get from the gut and into to the blood and then into a leukocyte (that won't kill it) and then get into the CNS. It is extremely common for the innate immune system to start an inflammatory reaction upon infection. The inflammation can look like parasitic disease.
Inflammatory cells are exploited by the parasites. Cells engulf parasites and Uber them to the CNS, but this is rare. The parasites want to end the ride at a muscle cell. Inflammatory cells are always stimulated when parasites enter the gut tissues and the inflammatory cycle begins. Parasites to paresis goes through inflammation.
Zhoni didn't have EPM. Zhoni didn't have antibodies to S. neurona. She suffered from dysregulated inflammation. The only clue was the timing of the signs. And that's where Blue Eyed Playboy helped us five years earlier.
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