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EPM testing: what does a test measure?


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There are no pre-mortem EPM tests! The lack of a test to diagnose EPM by detecting live S. neurona parasites in the central nervous system (CNS), the definition of EPM, is due to the disease process, the organisms are gone. The next best diagnostic evaluator of an EPM horse, because one can't detect the live organisms, is a comparison between known diseased horses and clinically normal horses, and then finding the measurable differences between them. That is what I tried to do during my PhD work, and twenty-two years ago everyone had similar ideas. The goal was to find the definitive marker for horses harboring live Sarcocystis neurona parasites in the CNS tissue. If you understand what a test measures, and what changes in an animal after infection and how infection differs from disease, you will understand the limits and differences between tests that all of us developed. None reached the Holy Grail of EPM testing! However, there are useful tools to help you, the greatest one is knowledge. Knowledge lets you select and interpret the most useful tests.

 

Organisms, infections, and disease


Organisms are composed of proteins and the genetic machinery that makes those proteins. Nature is lazy, evolutionary speaking, when Nature finds a good thing, it keeps it. Many examples of protein pathways that are present in ancient bacteria continue to be used by humans and horses. It is no surprise some proteins of S. neurona, the pathogenic protozoa that can cause EPM, are similar to all living organisms and some fungi. Closer to our topic of understanding tests, S. neurona shares proteins and pathways with Apicomplexan parasites. Apicomplexans are a taxonomic group, a phylum of diverse parasitic protozoans, that have a complex life cycle, usually involving both asexual and sexual generations, often in different hosts.


Protozoa infect hosts (horses) with several possible outcomes. An outcome can be that the host is unhospitable, and the organism dies, most often the case with S. neurona infecting horses. You won't find S. neurona in the muscle of a horse. An evolutionary truce may allow the parasite to infect the host, remaining quietly and unobtrusively conducting its parasite duties with little or no consequence to the host. This unobtrusive relationship is often the case with S. fayeri infections in horses, called equine muscular sarcocystosis or EMS. The often benign sarcocysts are found in the horses muscles. A more serious outcome is that the infection takes a hold and a battle with the host ensues. Horses are inhospitable to S. neurona, horses are aberrant hosts for this parasite and some of these infections result in EPM. And progressive consequences of infections with unknown organisms cause chronic inflammation and ultimately PNE, S. neurona can do that too.

horse is natural host

% horses in US with antibodies

produce sarcocyst in horse

can invade CNS

produces EPM-like signs

S. neurona

no

80%

no

yes

yes

S. fayeri

yes

>80%

yes

no

yes

S. falcatula

no

60%

no

no

yes

 

The host isn't without defenses, some defensive reactions are innate. These innate reactions are universal to all foreign comers. You could correctly guess a test that measures innate reactions won't be specific to Sarcocystis that infect horses. Keep in mind S. neurona and S. fayeri both infect horses and cause different diseases, but the diseases can look clinically similar.


The second line of host defense mechanisms is called adaptive immunity. Adaptive immunity takes days to weeks after infection and respond with cells primed to specifically attack the invader. The response is both organism-specific and also against molecules that are common to all parasitic protozoa. The response to non-organism specific molecules are less useful in diagnosis of disease, non-specific adaptive responses to S. neurona and S. fayeri and S. falcatula will be similar. Adaptive responses develop only to the the parts of the protozoa that the immune system detects.


Let me clarify this for you. Adaptive immunity will detect foreign molecules that are shared among Apicomplexans and also those that are unique to a species of protozoa, and even a "serotype". Serotypes are the different strains of Sarcocystis sp. that the hosts immune system can detect. You are probably suspecting that if you wanted to diagnose S. neurona sarcocystosis (an infection) one would select the the most specific test possible. The non-organism specific responses, that are shared between neurona and fayeri, won't distinguish between these pathogens. You will need organism specific responses to do that. Also, hold the thought that infections with S. neurona, most of the time, don't cause EPM (EPM means the parasites had to travel from the gut to the central nervous system). Most of the time the S. neurona organism is eliminated by the adaptive immune system. Sarcocystis fayeri can produce a disease-causing toxin and there is an adaptive response to the toxin. Because no one has demonstrated a S. neurona toxin a cleaver way to distinguish the horse-infecting sarcocystis sp. is by looking for that specific toxin.


Give yourself a gold star if you recognize that once a horse is infected with the parasite the horse makes antibodies, one arm of adaptive immune response. Those antibodies won't distinguish where the parasite goes, the gut or the CNS. Ah, you say, what if I find antibodies in the cerebrospinal fluid, CSF, isn't that proof the organism is in the CNS? Not true! A nice experiment conducted by Dr. Martin Furr showed that giving a large protein to an animal, a protein that is too big to enter the CNS (he used bovine serum albumin) resulted in antibodies produced by and detected in the CNS. We'll have to find another way to diagnose EPM disease other than CSF testing.

 

If an organism, such as S. neurona, comes under attack from the hosts innate and adaptive immunity, it can respond and change using several strategies. There are many immune evasion tricks that parasites have learned as they evolved along with horses. A sneaky strategy is invading the hosts own cells, hiding inside a cell such as a white blood cell, and thereby becoming undetectable to the host. Sarcocystis do that, not just neurona. That is a useful method a parasite uses to uber from the gut to the muscle (the intended target of all Sarcocystis) without coming under attack by the host. And a useful method to create an animal model if you are a scientist!


Another useful parasite defense is by changing the detectable proteins that initially alerted the hosts immune system it was under attack, S. neurona can do that. It is like changing ones coat, if the immune system is looking for a red-coated parasite, stripping down to a blue sweater is effective. S. neurona does that, it stops expressing specific proteins on its surface, especially when anti-parasitic drugs are given. Parasites can use parts of the hosts own system to do their bidding. Parasites can mimic host proteins, mimicry is a strategy. There are more strategies, immune evasion is a fascinating topic.

 

A part of the disease process becomes the hosts reaction to the parasite. You have heard of auto-immune diseases, when the immune responses to a pathogen attack the host tissues. It can be due to molecular mimicry. Autoimmunity can be stimulated when host molecules are broken down and adaptive immunity forms in the debris removing reactions. Several human neurodegenerative diseases are good examples, Muscular Sclerosis, MS, is one example in which the adaptive immune system will attack the host tissue myelin.


Innate molecules called cytokines stimulate reactions and in turn stimulate more protective cytokines in an ever larger cascade of pro-inflammatory reactions. Inflammation in the CNS causes clinical signs that look like parasitic neuromuscular diseases. Inflammation causes tissue damage, sometimes nerves axons are broken down, releasing neurofilaments that are swept up and can found in the diseased horses blood stream. Sometimes myelin protein is shredded and degraded allowing adaptive immunity against these host tissues. There are regulatory cytokines that turn off pro-inflammatory reactions when appropriate, when the threat has passed. Cytokines and their regulators are short lived, some just for days. An important cytokine regulator is c-reactive protein, CRP. Clinical signs are present when CRP is dysregulated.


Adaptive immunity, in the form of antibodies, form and refine themselves into ever more specific fighters. Once the threat is neutralized, the antibodies linger, just to be sure the threat is gone. When no new or persistent threat is perceived, the antibody population wanes, usually over many months or even years. The antibody response can maintain itself if there is continued infection from the environment. If there is no continued stimulus, the primed cells are put into storage with a few are tasked with surveillance. The immune system is primed to fight again if needed, it is experienced and will jump into action with a stronger response when challenged.


EPM is a syndrome


A syndrome is a condition characterized by a set of associated signs. A part of the "EPM syndrome" is the inflammatory response that parasites set off in the horse. In fact, if you read the literature, in the majority of reported cases, the histopathology says "inflammatory lesions consistent with EPM with no parasites found". Disease just may be the inflammatory effects of some infections, in some horses. Inflammation in the tissues is a constant finding. Inflammation is a consequence of the pro-inflammatory cytokines and primed cells that are deployed to remove parasites like S. neurona. After these responses have finished their task, sometimes they don't turn off appropriately. Cytokines can initiate inflammation in the CNS without parasites and toxins can initiate inflammation in the CNS,

 

There is a disease process, called the by-stander mechanism, when the infecting threat is eliminated, but the immune systems are still engaged in battle. It is called 'by-stander' because the inciting cause won't be found, it was eliminated. Proposed by-stander mechanisms can be a result of virus, chronic disease (gastric ulcer disease), encysted small strongyles, encysted tape worms, bacteria, or even fungus. Diseases that linger due to a by-stander mechanism that you have heard of are Lyme's Disease and polyneuritis equi, PNE. Check out what the Centers for Disease Control, CDC, has to say about post-Lyme disease syndrome in people.


An antibody test won't differentiate syndromes but sometimes they point you in the right direction. Ultimately, antibody tests may give you the probable cause that started the process. Cytokine tests are useful, but as you now realize they are innate responses and short-lived. Anti-protozoal drugs won't stop the by-stander inflammatory process. Sometimes drugs just add to the cytokine storm, one reason not to use continual anti-protozoal agents in some cases.


When there is enough inflammation, in the right place, an autoimmune disease can manifest. That is what the disease MS is, inflammation and a host's reaction, attacking the covering of nerves (myelin) in the central nervous system. There are diseases that mimic MS, just like there are diseases that mimic neurodegenerative diseases in horses. It was once proposed that human MS was a result of Sarcocystis infections. Steroids are sometimes useful, but they don't turn off the cytokine reactions responsible for cytokines, they subdue the antibody producing cells. It is an interesting story that led MS researchers to horse spinal tissues for their research. Discovering reactive peptides on equine myelin proteins that were linked to PNE were important to us. We developed antibody tests to measure anti-myelin protein antibodies. Because these are antibody tests the level of antibody will lag behind clinical improvement.


Are you ready to understand diagnostic testing?


Now you might have enough knowledge to understand what tests could be useful, when they are useful, and what information about the different disease processes they provide. The Testing Tab on this web site can provide additional useful information. Your veterinarian is a valuable resource, discussion about the above topics and the relevance to your case should not be overlooked. In fact, the most valuable test is the neurological exam, repeated often and especially before and after a treatment is initiated. We ask for the neurological exam score on our submission form because it helps us in evaluating your case.


Be sure and email us with any questions you have about our testing. Ask us why we targeted the topics that were discussed here because we think testing strategy is a roadmap to understanding equine neurological diseases. Read our published literature, use the Publications tab, to understand why we selected and interrogated the molecules we did. These studies led to our understanding EPM, EMS, and PNE. You will also find out how we validated our markers of disease on the Publications section of this site. You should also ask the same questions for all tests you use.



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