by Siobhan P. Ellison
My business is consulting. That means we dispense advice. We don’t look into a crystal ball or read tea leaves. We base our opinions on an analysis of your case and immunodiagnostic testing. Analyzing a case involves looking at laboratory values, the case history, and physical examinations done by licensed veterinarians. The underpinning of our opinion is immunodiagnostic testing associated with neurological diseases and understanding the parameters and limits of each diagnostic test we use.
We are not on the sidelines when it comes to the literature on sarcocystosis. Our works are published so other experts, and you, can critically review our approach. We rely heavily on works of others to develop new theories that are tested by direct observations. Our hypotheses’ are scrutinized by our Statistician, he has a PhD in statistics, years in the pharmaceutical industry, and is considered one of the best in the industry. So when we are asked a question, we answer with our objective opinion with statistical significance.
Recently we were asked, “Can you clarify your comment that the “titer” is an indication of duration of infection (vs the degree of disease) and explain the difference between a negative IFAT test and the SAG titer of 8?” It seems to be a simple enough question. To get to the answer, one has to A) know how the organism behaves in the horse, B) correlate antibody levels with clinical disease in horses, C) understand the critical differences in quantitating antibody between test formats.
How does a Sarcocystis behave in an animals that it infects? The authority would be Dr. Edith Box. Throughout the 1980’s Dr. Box infected budgies with S. falcatula. It is historically important that budgies were lethally infected by S. falcatula. An aside to the question posed above is the 1995 hypothesis by researchers from the University of Florida. They presented molecular evidence that S. falcatula and S. neurona were the same organism! Their hypothesis was refuted by demonstrating that S. neurona does not infect budgies at all. Horses are infected by S. neurona, budgies not. Budgies, but not horses, are infected by S. falcatula. Until proven otherwise, another study conducted at UF is the law of the land. Their horse-infection study indicates that horses are not susceptible to S. falcatula infections.
How does a Sarcocystis behave in a natural host? S. falcatula in budgies is the model. Box showed that the organism is distributed throughout the organs from the gut (the end result is a muscle cyst). New flushes of organisms are not lingering in organs outside the gut. New infections didn’t spring up from muscle cysts either. Box showed that some birds did get infections that went to the brain. (We always suspected S. neurona wasn’t special in a proclivity for brain tissue).
Wait, you say. What about organisms that are not in the natural host? The horse is not a natural host for S. neurona. Usually Sarcocystis are highly host specific. Host specificity is how UF used biological studies to correct and retract their claims that neurona and falcatula were the same. When based on molecular data they were pretty darn similar. When tested in animals, not so. Other studies support that horses with an immune system quickly remove organisms just as the bird model shows for falcatula. Interestingly, horses with abnormal white blood cells do not clear the organisms at all…and paradoxically the immune-deficient horses do not show clinical signs. Heads up! The immune response to the organism caused the clinical signs, not the organism.
And now back to the basis for answering the original question. We did it by animal experiment, first developing an infection model in the horse. We infected 75 horses with S. neurona using white blood cells to mimic a “natural” infection. We didn’t evaluate the horses ourselves, we brought in EPM-experts from across the country (at least 2 for each study) to examine the horses as the disease progressed. They were unaware of which horses were infected and which ones were not so we “blinded” the experiment. Experts saw the horses before infection and then every month until the experiment was terminated four months later. They scored the disease using a meticulous neurological exam. Yes, they documented the horses got worse over time.
The horses’ blood and CSF fluid was taken every month at the same time the exams were conducted. We used a SAG 1 ELISA test and also sent duplicate samples to a Kentucky laboratory. Both laboratories tested the samples for the presence of antibodies. That way there was no bias in testing. We used only one test because a SAG 1 strain of S. neurona was used for the infections. In natural infections, there are three serotypes of S. neurona, SAG 1, SAG 5, and SAG 6. Each SAG is mutually exclusive-only one SAG is present in an organism. Multiple serotype infections are possible in the field because an opossum can be infected by all three serotypes. Three different infections. Our experiments were limited to a single serotype. We sent the KY lab a sample of SAG 1 to run in their test making sure they had the same marker that we had on our test. Thus, any difference between the tests would be a consequence of ability to detect and interpret a single protein.
Our Statistician crunched the numbers. He concluded that as time progressed, the clinical signs got worse and that was statistically significant. Also, as time progressed, antibodies increased, but the antibody level did not statistically correlate with the degree of clinical signs observed by the clinicians.
And there was individual variation in the antibody response. Some really sick horses had lower antibodies when compared to not-so-affected horses that had high antibodies. The Kentucky test was less likely to identify infected horses. Twenty-five percent of the test results differed. That means that had we conducted the experiments only using the KY lab, the interpretation of the study would be different. That is why different scientists have different views of immunodiagnostics. We base our opinion on study results.
Subsequent experiments showed that horses that had never experienced an infection produced less antibody and dropped the antibodies quickly when compared to “experienced” horses. Experienced horses got a measurable antibody response quickly (the scientific word is amnestic response) and retain those antibodies for up to 10 months.
The experiments in 75 experimental infections allow us to say that in horses the antibody response to SAG 1 will increase over time and linger longer in an “experienced” horse and antibodies are more a reflection of duration of infection, not degree of disease.
A couple of things help explain part C of the question quantitating the difference between two tests. It is important to recognize that testing formats are different. That is to say what is measured (antibody against a protein) and how the protein is prepared for testing are different. It has been known for a long time that reducing conditions change how antibodies recognize a protein. Without going into the fascinating field of protein chemistry, suffice it to say, a protein is a string of amino acids. The amino acid sequence is the primary structure of the protein and that is determined by the DNA that codes for the protein.
The secondary structure of a protein is determined by folds in the backbone of the protein. This means side groups don’t interact in determining the secondary structure. The overall tertiary structure of the protein, its 3D structure, is determined by the reactions and bonding between side groups. The charge of individual amino acids, the affinity or aversion to water, and special bonds are important in tertiary folding of a protein. The amino acid cystine has very strong disulfide bonds to contribute to 3D structure. I digress to point out that Sarcocystis surface proteins characteristically have a lot of cystines and it can be expected these amino acids give important tertiary structure to the SAG’s. One would speculate, without any other knowledge about the protein, that the cystines give the protein its functionality.
A horses’ immune response is to the tertiary structure of the SAG proteins making antibodies to the 3D molecule, not the primary structure.
Think of a protein as a rope, twisted it up upon itself into its 3D structure. Now mentally dip that twisted up protein into a vat of dye-lets imagine red. You have tie-dyed your protein! When it is unfolded into its primary (linear) structure you will see where areas of the protein, that were far apart, touched when folded. Red marks are distributed seemingly randomly along the molecule and there are long areas between the marks that have no dye. The scientific term for the areas that touched are “conformational epitopes” and the areas that are side by side are called “linear epitopes”. An “epitope” is a section of amino acids that induce an antibody reaction in a host. You can correctly guess the amino acids do not have to be side by side to make the host react, in fact you expect the 3D structure to be more important in inducing a response. Conformational epitopes are important in immunodiagnostics.
Some testing formats use chemicals to stretch out the proteins into linear molecules. Other formats retain the 3D conformation of the protein. You can correctly guess that each test would measure antibodies that recognize the same protein, but by different markers. We have always favored conformational epitopes and run our tests without protein-straightening chemicals. Our thoughts are not original. It is long known that Sarcocystis infections are more recognizable when diagnostic tests that use conformational epitopes are selected.
We evaluated SAG 1 and recognized a relationship between breaking tertiary bonds and the concentration of chemicals used to break those bonds. It was dose dependent. That means that the KY lab that tested samples from the experimentally infected animals looked for linear epitopes. and depleted the reactions to SAG 1. We keep the SAG 1 for detection folded in its happy state.
You correctly point out that an IFAT test uses the whole organism. Are the surface proteins altered in the IFAT test? It depends. It matters how the organisms are prepared for the test. More important to the argument is that commercial IFAT tests are run on SAG 1 strains. Great for experiments using SAG 1 organisms to cause infections, but not so great in field infections that are caused by SAG 5 and SAG 6 strains, as well as the more common SAG 1 strain.
There are other complicating issues: Sarcocystis are known to selectively stop displaying some proteins during infection, different testing labs use different dilutions of the serum, some tests are measured by machine and some are subjectively evaluated by lab technicians. All things considered, an IFAT reported as negative at a 1:40 dilution tested on a SAG 1 strain is not comparable to a 1:8 dilution (titer) on a SAG 5 surface protein.
We take these things into consideration for you as we consult on your case.