Testing Questions

The tests we run at Pathogenes answer specific questions. They do not diagnose disease. A diagnosis is made by clinical exam that is supported by testing. In most cases, the tests are helpful to rule out some etiologies. Because exposure to some microbes and antibodies are developed against these organisms are common, a horse may be seropositive and not be diseased. For example, most horses have antibodies against S. neurona but they do not have EPM. EPM is rare and sarcocystosis is common.

The SAG 1, 5, 6 ELISA’s answer the following questions:

“Has the horse been exposed to S. neurona in the environment?”

“What serotypes of S. neurona has the horse been infected with?”

“What is the duration of the exposure to S. neurona in the horse?”

“A negative test will rule out the possibility of EPM.”

History: The SAG1 ELISA was developed during my PHD program (1999-2001) with the intent to develop an assay to diagnose EPM in the horse. Later on the SAG5 and SAG6 ELISA's were developed at Pathogenes Inc to investigate strain virulence. It became evident that EPM is a syndrome with two disease phases, acute and chronic. Animal challenge studies proved that chronic sarcocystosis was due to inflammation. The parasites were eliminated from the central nervous system (CNS) by the time disease was chronic but clinical signs persisted. The clinical signs were a response to innate immunity. Chronic sarcocystosis is not influenced by anti-parasitic agents.

Acute sarcocystosis is initiated with infection of the gut cells and the release of protozoa from the intestinal cells into the blood stream a few days later. Parasites invade monocytes in the gut and, if they are carried into the CNS through the blood stream, the disease is called EPM.  Intestinal cells are involved in a coordinated effort to increase inflammation and accelerate gut cell turnover through innate immunity. Parasites are maintained in white blood cells in the bloodstream where they are exposed to the immune system allowing the horse to develop long term immunity. Antibodies can be found in the serum and the central nervous system. The initial gut inflammatory responses induce clinical signs. When the horse has repeated exposure in the environment the protective antibody responses are maintained, antibody titers don't go down. Chronic sarcocystosis is maintained because new infections start in the gut from the contaminated environment, however after the first “insult” (an initial infection) an antibody response is stronger and can more efficiently remove the parasites before they can get into the circulation.

The SAG ELISA’s don’t diagnose EPM, no antibody test can do that because antibodies from the peripheral circulatory system are found in the CNS. The SAG1, SAG5, and SAG6 are antibody tests and values take months to decline, repeat testing should be after the antibodies are expected to decrease.

The Sarcocystis fayeri ELISA answers the question:

            “Does my horse have an anti-toxin produced by S. fayeri?”

History: Sarcocystis fayeri produces an enterotoxin that induces the production of antibodies, the anti-toxin is detected by the S. fayeri ELISA test. Horses we tested in an FDA clinical study had S. fayeri sarcocystosis identified by histology and the S. fayeri ELISA test. The disease caused by the enterotoxin is dose dependent and it only induces clinical signs when enough toxin is released from the parasite, presumably in heavy infections. Most horses are infected with S. fayeri but do not have detectable anti-toxin. In a survey Pathogenes conducted, six percent of horses with neuromuscular disease had S. fayeri anti-endotoxin. The pathogenesis of infection is similar to S. neurona infection except the parasite doesn’t cross into the CNS but forms sarcocysts in muscle tissues. It hasn’t been determined if the toxin is released when the parasite is in the parasitemic phase or if it is released when the sarcocysts are removed naturally. Some parasitologists believe that the sarcocyst is walled off and are never removed and other prominant parasitologists believe the cysts are broken down and removed after several months. Sarcocystosis fayeri infections are only susceptible to anti-protozoal treatment when they are in the intestine or the parasitemic stage of infection. If heavy S. fayeri iintestinal infections are treated it is possible for the parasites to initiate enteritis, induce a “leaky” hindgut, and clinical signs of enterotoxemia.

The S. fayeri ELISA is an antibody test and values take months to decline, repeat testing should be after the antibodies are expected to decrease.

The C-reactive protein ELISA answers the following question:

“Does the horse have inflammation causing CRP to be elevated?”

History: Although it is not specific for a disease, CRP is produced by the liver in response to inflammation. Causes of an elevated CRP are infections (bacterial, viral, protozoal) and autoimmune diseases. An elevated CRP can be a sign of an underlying medical condition that requires attention. In our lab an elevated CRP in horses is associated with sarcocystosis and was statistically correlated to polyneuritis. We found the higher the CRP value the more likely there will be a  demyelinating polyneuropathy. A CRP test will not differentiate between S. neurona and S. fayeri but will detect inflammation associated with both parasites.

The pathogenesis of CRP elevation due to S. fayeri enterotoxin is indirect, enterotoxin induces host monocytes to increase CRP and this can lead to hindgut enteritis. In contrast to the indirect pathogenesis of increased CRP due to enterotoxin, endotoxins directly increase CRP production.

The C-reactive protein ELISA is not an antibody test and CRP values change quickly with successful treatment.

 The LPS ELISA answers the following question:

            “Does the horse have an endotoxemia?”

History: Endotoxins are produced by gram negative bacteria that release lipopolysaccharides (LPS) into the bloodstream. Enteritis may be due to compromise of the intestinal barrier (leaky hind gut) and not necessarily hind gut ulcers. Endotoxins directly increase CRP increasing inflammation. Causes of endotoxemia include a high-starch diet, high intensity exercise, stress, inflammatory bowel disease, hind-gut ulcers, and/or a disturbed microbiota. There is some evidence that anti-protozoal drugs in horses are associated with endotoxemia, probably due to release of parasite antigens from host cells or iatrogenic induction of a parasitemia.

The LPS ELISA is not an antibody test and conversion from a seropositive-to a seronegative value would indicate successful treatment.

The Equine Myelin Protein ELISA’s (MP2; MPP) answer the following question:

“Does the horse have antibody against myelin protein P2?”

History: Myelin protein P2 antibodies were detected by ELISA and associated with polyneuritis equi (PNE) in 1987. The work followed scientific inquiry into Guillian-Barre syndrome in people. Improvements in the tests for use in equine medicine were made possible by creating recombinant proteins as antigens rather than using myelin protein P2 homogenates. In a clinical study Pathogenes Inc. detected antimyelin protein 2 antibodies in horses with experimentally induced chronic S. neurona sarcocystosis and in natural histologically confirmed EPM cases. Horses have a polyneuropathy prior to developing demyelination and they test negative but generally have a high CRP value. The progression of autoimmune disease is followed clinically by measuring the population of antibodies detected against MP2. The progression of antibody production is 1) anti-myelin protein 2 antibodies (MP2) followed by 2) antibodies against an IL6 receptor on MP2 (MPP). The third pashe is a decrease in antibody to MP2 (negative) with antiodies against MPP, and finally 4) negative on both tests. Once the disease progresses to calcification or fibrosis of the MP2 the tests will be negative.

The driver of the autoimmune disease is CRP's enzyme action on MP2 and is over stimulation of innate immunity, irrespective of viral, bacterial, protozoal, or iatrogenic etiologies. Like the disease EPM, the etiologic agent is no longer present but chronic inflammation persists.

Converting from a seropositive-to-seronegative status would indicate successful treatment of the disease, however it will take antibodies months to resolve. Repeat testing should be after antibodies are expected to decline. Using CRP analysis in conjunction with Equine Myelin Protein ELLISA's is useful.

The Neurofilament light ELISA (NfL) measures the level of NfL and answers the question:

            “Does the horse have circulating NfL in the serum/plasma?”

History: The presence of NfL in the serum/plasma is an indicator of axon damage. Axon damage is related to equine degenerative myeloencephalopathy (EDM) in the horse, a disease similar to human Amyotrophic Lateral Sclerosis (ALS). The NfL test is not specific for EDM, NfL can be detected in horses with PNE and other axon damaging diseases.

NfL ELISA is not an antibody test, successful treatment is indicated by a seropositive-to- seronegative status.

The Neospora ELISA and the Lyme ELISA answer the following questions:

“Does the horse have exposure to Neospora hughesi?”

“Does the horse have antibody against the OspC antigen of Borrelia burgdorferi?”

History: Neospora and Borrelia both are associated with equine encephalomyelitis. The pathogenesis of Neospora in horses is not fully understood but infections are expected to mimic those of S. neurona. The Neospora ELISA tests for antibodies against recombinant NhSAG1 and is used to rule out exposure for inclusion into our Clinical Trials. The OspC antigen is a major outer surface protein of the Lyme disease bacterium, it is highly immunogenic in the early stages of infection and is used to rule out Lyme disease for inclusion into our Clinical Trials.

Neospora ELISA and Lyme ELISA are antibody tests and antibodies will decline slowly in the recovering horse. We use antibody levels to guide our decision for study inclusion.