Interpreting S. neurona antibody tests

This is Legend, he's not feeling well. Michelle has owned Legend for about 11 years. She says “He is basically my 1200 pound baby”. She moved to Arizona, Illinois, and back to Arizona over several years and then to Texas for 4 months. He never had any serious health problems until his EPM nightmare. And then Legend became Michelle’s full time occupation. She felt she had to move him back home despite worrying about stressing him with a trailer ride. She mentally made the decision that she’d never let him suffer.

Antibodies 101…antigens and epitopes

Antibodies are made by a host in response to an infection, and antibody responses are specific to an organism. Organisms display proteins that are available to the hosts immune system, these proteins are called antigens. Antigens are like fingerprints. An antibody is made by the body and binds individual amino acids of a protein antigen, the recognized sequence is called an “epitope”. Epitopes can be linear or conformational. Conformational epitopes are a huge issue in recognizing organisms in diagnostic tests, re-read the blog Western Blot, do’s and don’ts if you forgot why! The bottom line was that purifying antigens (immunoprecipitation) was a powerful tool to identify S. neurona in infected horses.

Western Blot do's and don's

Sidebar: The host makes antibodies to any foreign protein that is not recognized as “self”. Antibodies that are made against “self” result in autoimmune disease. The basis of the Sidewinder test is measuring antibodies against myelin protein 2 in horses. It is relevant to EPM diagnosis because an autoimmune disease won’t be resolved with anti-parasitic agents.

Sarcocystis neurona displays antigens on its surface that directs the horses reaction to the invasion. Did you know that there are different genotypes of S. neurona? The horse can’t differentiate the organism at the DNA level. However, there are three serotypes of this parasite, this is the level the horse can distinguish. The serotypes represent the mutually exclusive, immunodominant surface antigens displayed by the three different strains of S. neurona. Our tests measure that specific reaction to the surface antigen.

S. neurona is called the ‘master of disguise’ because as the infection progresses in the horse, neurona changes the antigens it displays on its surface! S. neurona displays different antigens at different stages of development. Stage related antigen expression has important implications in detecting disease-causing strains. That deception, antigen switching, is an immune invasion tactic often employed by protozoa.

Sidebar: Also, organisms differ in their virulence. Virulence is related to genotype, and perhaps serotype. Another factor of infection versus disease is dose, how many organisms the host contacts. If you read much of the EPM animal model literature, you are aware that dose and route of infection are important in producing disease. The pandemic certainly made us all aware of infection (production of antibodies and an immune response) and disease (a morbid result of infection). Both situations produce antibodies. Only disease should be treated.

Our animal infection model studies taught us that the titer increases with duration of infection, not the degree of clinical signs displayed when the animal is diseased. The clinical signs are related to cytokine responses, remember cytokines are part of the innate immune response and occur earlier than antibody production. Innate responses are not specific but highly important in inflammation.

Antibodies were shown in an experiment to protect against challenge in the Trojan Horse model. We did show in a serum neutralization assay that a rSAG1 (recombinant) vaccine, and resulting immunity, did not neutralize SAG 5 organisms. However, following a natural infection with SAG 1, neutralizing antibodies were produced against a SAG 5 strain. Here’s why. The horse makes antibodies against the whole organism, proteins that are shared between serotypes. Antibodies to non-specific, shared antigens can be partially protective.

Protective antibody paper

We developed three ELISA tests to monitor S. neurona infections in your horse. We use recombinant proteins representing SAG 1, SAG 5, and SAG 6 antigens. The tests measure antibodies against these immunodominant antigens of S. neurona in any body fluid. The three individual tests results distinguish the serotype of the infecting organism, or organisms, infecting a horse from one sample of serum. A titer is a number that indicates the level of antibody response to an infection at one point in time. The SAG ELISA tests give you three titers that give the antibody profile of infection with S. neurona.

To demonstrate the interpretation of the test, check out the graph. It shows the antibody level stacked up for each of 9 horses. You get a titer, a number, reported by a test result. This is illustrated as each square represents the serotype, SAG 1, 5 or 6, and the level of the titer. Other tests measure only the height of the stack given as one number while our tests tell you what makes up the stack.

For example, most tests that analyzed horse 1 and 5 samples would have the same titer.

We report three titers, each test represents a serotype specific to S. neurona. The serotype analysis tells you horse 1 has a SAG 6 infection. There is no antibody against SAG 1 or SAG 5, S. neurona. That means that horse 1 is not diseased and may get better without treatment. SAG 6 S. neurona infects horses, may cause inflammation, but isn’t EPM-associated. That isn’t true for horse 5, he may need specific intervention because the majority of his infection is due to SAG 1 organisms. SAG 1 and 5 are EPM-disease associated.

What do no or low antibody titers mean…

If there are no antibodies detected in the sample, the result is reported as 2 (negative). If there is some antibody detected, but isn’t it significant (as measured by challenge studies) we report a 4 (negative). Horse 7 in the graph is negative. The horse should be retested in four weeks, if there are clinical signs, because antibody levels could change. It takes 17 days for a horse to produce antibodies to infection. That is why if the sample is negative, we like to wait a few weeks and test again if the horse has clinical signs. Absence of antibody is due to no infection or very early infection. If there is a four-fold rise in titer (reported as a number) in a second assay, that means the infection is active. What is crucial here, to show active infection, is a change in the titer. A single value isn’t useful.

A positive titer is 8 or greater

A positive titer, 8 or above, shows up on the illustration and is reported as a number interpreted as a positive result. A positive titer indicates in the last 6-10 months the horse had an infection that stimulated an immune response. One value can’t tell you if it was in the past, or if the infection is active. The clinical exam, antibody titer to specific serotypes, and change in titer over 4 weeks are all useful information to determine current infections. A rise in titer in 2 to 4 weeks is good evidence of current infection. A decline in titer indicates the infection is gone and there isn’t re-infection. If the organism is present in the environment there will be daily exposure and the titer will remain elevated. In a clean environment it will take 10 months for the horse to become seronegative.

Horses can, and often do, have mixed infections. The samples 2, 3, 4, 5, 6, and 8 in the illustration are seropositive for multiple serotypes of S. neurona. Horse 1 is seropositive for SAG 6. Horse 7 is negative for all serotypes. Horse 9 is seropositive for the SAG 1 strain. Remember, we said that titer increases with duration of infection. One might recheck horse 9 in a few weeks for a four-fold increase in titer if the horse has clinical signs consistent with sarcocystosis.

S. neurona SAG 6 antibodies and what it means

Horses have mild clinical signs with infections to the SnSAG 6 phenotype strain. We don’t believe this is due to parasites that have invaded the neurological tissues by crossing the blood brain barrier. We suspect that there are some common parasite antigens, maybe the SAG's 2, 3, 4, that produce mild, transient signs stimulating inflammation.

It’s also possible that SAG 6 antibodies may offer some protection against more virulent S. neurona infections. Infections with this serotype may be a complicated interplay between parasite growth rate, competition between organisms, or just plain adaptive immunity. As yet, we don’t know. We haven’t found a pattern in SAG titers to hang our hat on yet and we will have to work it out with more studies. Our data suggests that letting these infections run their course may result in protective immunity.

S. neurona SAG 5 antibodies and what it means

This serotype is made up of at least 3 genotypes. The SAG 5 serotypes can invade neural tissues, but they do so rarely. It was shown experimentally that infections with at least one SAG 5 strain infections are mild and self-limiting. Inflammation is a hallmark of this type of infection. The presence of SAG 6 antibodies may offer some protection against SAG 5 infections.

It was also shown that some strains are resistant to Marquis and diclazuril at recommended levels when used in experiments. Treating a resistant strain could result in relapsing clinical signs. There is no evidence that there is synergy between toltrazuril and diclazuril and we have no data that combining these treatments would increase efficacy.

The antibody profile in the graph indicates a SAG 5-predominant serotype in horse 6. The horses 2, 3, 4, and 8 have SAG 5 antibodies, these horses have duel infections with EPM-associated disease causing strains.

S. neurona SAG 1

We have no doubt that SAG 1 serotypes of S. neurona are more virulent and more common in equine S. neurona infections. Some of the strains are sensitive to ponazuril and diclazuril, some of them are quite resistant (data from Dr. David Lindsay).

Clinical Evaluation

Clinical evaluation and identifying the serotype that caused the infection/s with changes in antibody levels over time are valuable tools in managing S. neurona infections in horses. Testing multiple times over the course of disease are important to fully evaluate infection versus disease and monitor re-exposure. A single, screening assay isn't useful.

Take home messages…

you can’t compare test results between formats

Different tests measure different immune responses. Tests that measure common antigens, SAG's 2, 3, and 4 do not detect serotype specific antigens of S. neurona. You get a total amount of antibody, not an analysis of infecting strain.

Detecting serotype specific antigens avoid cross reactions, such as antibodies against S. fayeri. We don’t need to dilute sera to avoid cross-reactivity that is needed for tests using 2, 3, and 4.

Conformational epitopes are important! We detect the epitopes the horse recognizes, not a linear sequence of amino acids. A synthetic chimeric protein, 2, 4/3 doesn’t detect antibodies to conformational epitopes. Serotype specific IFA and Western Blots are possible but aren’t standards for commercial testing.

Sporozoites infect the horses gut cells, the infection progresses to merozoite stages that circulate in the blood. Both gut and blood infections elicit antibody production. Analysis of S. neurona by immunoblot of SAG antigens demonstrated that SAG’s 1, 2, 3, 4, and 6 are present in the sporozoite and merozoite stages. SAG 5 is present in the merozoite stage and greatly reduced in the sporozoite stage. Re-read the the 29 kDa paper and Western Blot Blog and see how experimental conditions can change the level of detection in an experiment.

29 kDa Antigen of S. neurona

The Do's and Don'ts of Western Blot Testing

Antibodies are evidence of infection but not disease. Antibodies can be protective against disease. Don't treat antibodies...treat clinical signs.

Michelle says “Right now, after treatment, he seems really good. So hopefully were on the road to recovery, whatever that may mean. I don't care if I can ever ride him. I just want him happy and healthy.” And so do we, Michelle.

Before treatment Legend's profile shows a mixed infection. After treatment the SAG 1 and 6 went down. A year later he has antibodies to SAG 5 and 6. The profile reveals the serotypes that are in his environment and helps his veterinarian plan his care.