Pathogenes Inc.

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Equine protozoal myeloencephalitis (EPM) is primarily caused by the apicomplexan parasite Sarcocystis neurona (click to see the parasite). In the United States, EPM is the most commonly diagnosed neurologic disease of the horse  Click this link to see an active case of EPM.  A NAHMS study found that EPM was the most important infectious disease facing the equine industry. Opossums are definitive hosts for S. neurona in the US and shed infective S. neurona sporocysts.  Intermediate hosts of S. neurona harbor sarcocysts in muscle tissue and have been identified in the muscles of cats, raccoons, armadillos, sea otters and skunks. Studies from Michigan and Florida have reported S. neurona antibodies in 7% of domestic cats tested.  Domestic cats as well as raccoons and armadillos are reported to be natural carriers of this parasite.  These natural intermediate hosts occasionally exhibit neurological disease that is associated with parasites in the central nervous system.  For the last 10 years we have examined the disease progression from the day of infection until the clinical course was clear.  Understanding some of the pathophysiology of disease and the host response to infection is interesting and complex.  We collaborate with scientists across the United States and in Canada, Germany, and Australia.  Our collaborators include individual scientists, PhD candidates and published authors.  Groups of scientists at pharmaceutical, diagnostic, and vaccine companies also contribute to our knowledge base. You can catch up with what we are doing by visiting our blog. 

 Initial Infection

     After an animal ingests sporocysts of Sarcocystis the parasite readily invades and multiplies in intestinal tissues.  This gut phase of infection is followed by a parasitemia (parasites in the blood) in the natural intermediate host for many Sarcocystis species.  The parasites can move from the blood to other organs, usually the muscle thereby setting up the natural progression of disease in a normal host.  This results in muscle cysts. 

Equine Infections

    Although the horse is a natural host for some Sarcocystis  (S. fayeri, S. equicanis) the horse is an unnatural host for S. neurona.  The horse ingests virulent S. neurona sporocysts shed by the opossum (its complicated) leading to infection in the gut.  The gut infection allows parasites to enter cells (leukocytes, white blood cells) that migrate to lymph tissues and the blood stream, called a parasitemia.  The contact with lymph tissues and entry into the blood stream result in production of serum antibodies that can be detected by tests such as ELISA.  We believe that these infected cells (white blood cells) are the carriers of the parasite to the brain causing encephalitis. 

Diseases resulting from infections

     The signs that horses have after infection are recognized as dose related.  The number of sporocyts that the horse ingests can determine the outcome of the infections.  In other animals, sarcocystosis (infections) produce different signs that are recognized as different diseases--multi-organ infection with parasites or neurological disease in which the parasite is no longer present in the central nervous system.  

    The horse can recover from mild infections.  In these infections the animal produces antibodies (IgM or IgG) that can be measured by ELISA tests.  Generally in these infections the antibody levels decline rapidly and mild signs, if present, abate quickly.

    Some animals exhibit a neurologic stage, encephalitis, with tissue damage directly from parasites (septic disease) or tissue damage from inflammation-- the  immune systems fighting the infection that are associated with the parasites.  This results in clinical EPM recognized by early signs, ataxia, or lameness.  In fatal cases sometimes the organisms are recovered from the nervous tissues.  The antibodies that are measured by ELISA increase with time, the serum Peptide ELISA can detect this increase in antibodies in 2 to 4 weeks after initial infections.  Measuring the antibody levels several times can be valuable in evaluating the horse.

     In some neurologic disease that follows infections the parasite cannot be recovered from the neural tissues, aseptic disease.  Aseptic disease is recognized in sarcocystis infections in several animal species and has been demonstrated in the transport stress model of EPM.  The recognition of this form of the disease is important because anti-protozoal therapy is not effective.  These animals will be recognized as chronic relapsing or unresponsive to treatment.  Anti-inflammatory drugs or specific immune modulators (vaccination) are appropriate therapies.

Responses to disease   

    Disease also results in measurable responses such as (specific) antibodies or (non-specific) inflammatory biomarkers.  Certainly IgM and IgG are increased, usually appearing from 15 days after infection has progressed to the blood stage.  Specific serum tests, SAG 1 ELISA and the Peptide ELISA can determine the presence of parasites that cause disease.  It was shown that there was no advantage to test cerebral spinal fluid (CSF), diagnosis could be made from the serum alone.

   It is important to distinguish between pathogenic sarcocystis that cause septic or aseptic disease and those organisms that do not.  Non-specific markers (SAG 2, 3, 4) can determine the presence of both parasites that do and do not cause septic disease or neurological disease in the horse.  But they cannot distinguish between pathogens and non-pathogens.  Past comparisons of the antibody response in the cerebral spinal fluid (CSF) and serum didn't increase the value of testing when using these non-specific markers.  The Western Blot antigens have been favorably compared to the SAG 2, 3, and 4 ELISA antigens.  It remains to be seen if differential testing can improve the use of these antigens.

Specific immune responses   

The horses produce antibodies (humoral immune responses) and cellular immune responses that play important roles in  infections.   It is thought that many horses are exposed to S. neurona by ingesting sporocysts but only a small percent of the population get EPM.  This indicates that the horse, recognized as an unnatural or aberrant host, can fight the infection successfully.  The immune system can be stimulated to fight the infection by specific immune stimulators (rSAG1).  The specific stimulation of the humoral immune system protected horses from septic and aseptic disease.

Strains of S. neurona important in disease    

   The majority of sarcocystis that have been isolated from diseased horses have the SAG1 protein and this is the only antigen type that has been recovered from the CNS following experimental infections.  All commercial tests use material derived from the SAG 1 strain.  Organisms have been isolated from normal horses that had structurally similar but antigenically distinct surface proteins and were not associated with inflammation.  These organisms could be S. betrami (S. equicanis) or S. fayeri that use the horse as a natural intermediate host and do not cause disease.  In a few horses with neurological disease another antigen type of S. neurona was recovered, but so far this antigen type only causes aseptic disease in experimental infections.  This antigen type, as well as a third, cause septic disease in sea otters.  Cats get rare septic and aseptic sarcocystosis and more commonly have muscle cysts.  The strain used in the determination of the presence of antibodies in cats was not the SAG 1 strain, more studies comparing the three antigen types in cats are ongoing.

More research on strains of S. neurona

     Recently it was shown that all the sarcocystis that were tested displayed proteins SAG 2, 3, and 4 (Wendte, 2010) including S. falcatula.  Sarcocystis falcatula has confounded research on EPM and was once thought to be the species causing disease in horses.  

     The opossum is  the source of oocysts, oocysts from opossums passed to raccoons led researchers to believe they had biologically purified S. neurona from S. falcatula. Extensive infection challenge studies with a raccoon strain Sn37R, while producing antibodies that are detected by SAG2 protein do not cause septic disease.  The early results led some to believe that SAG 1 and SAG 5 were immunologically cross-reactive. The Sn37R isolate was actually composed of two strains of S. neurona Sn138 and Sn744, were genetically different (the former has SAG5 while the latter has SAG1), and possibly undergo biological selection in the laboratory raccoon to favor Sn138.  The elimination of a SAG1 strain from the challenge organisms is one explanation why the organism was not found in the CNS of those horses (the SAG 1 strain produces septic disease).  Repeated experiments using higher doses did not lead to septic disease.  An alternative interpretation is that strain Sn138 only produces aseptic disease.   

S. neurona, common or not?   

    The Western Blot has been the standard for determining the serological prevalence of S. neurona in animal populations.  Using the SAG2 protein detected on the Western Blot is the reason so many "normal" horses are seropositive.  The SAG1 specific ELISA test detects about 6% of the horse population as infected with S. neurona.  While low dose infections seem to recover and antibodies decline, this estimate is closer to the level of recognized disease in horses.

Remaining questions 

     As researchers know more about Sarcocystis neurona infections in the horse the question of the antigen types contacting horses (see its complicated above) and the pathogenicity of these strains will be elucidated.  Without doubt, mixed populations of oocysts contact horses and have been used in many experiments.  An observation by researchers that study S. neurona oocyst infections is that the ability of strains used to experimentally infect some animals (budgies, immunodeficient mice, horses) and not others is an indication that oocysts from opossums are not S. neurona but another species (Dame 1995, Marsh 1998, Marsh 2003, Rosanno 2005, Ellison unpublished data).  Oocysts representing several species from opossums have complicated studies-- including those that use sporocysts from the opossum.  In our sporocyst model we identified lung lesions in challenged horses, however we too used mixed oocysts from an opossum.  We will continue to do research using molecular tools as well as biological assays to provide answers to questions surrounding infections, disease, and treatment.