Pattern recognition is an important process that emphasizes the the identification of data regularities in a given scenario. People are natural pattern-seekers. How many times have you heard things happen in three’s? Humans are hard wired to recognize sets of three events, even if science proves the events are unrelated!
Students are trained in veterinary school to become observers. The trained veterinary-observer develops into a diagnostician after years of clinical practice. The art of practice is a combination of science and observation enhanced by continued questioning of the objective outcomes one effects with treatments. A clinician adds tools to his/her toolbox over time.
Bioassays are tools that are available in the clinicians toolbox. Our passion is neurodegenerative diseases in horses and people. We bioassay a lot of samples from laboratory experiments and clinical submissions. Sometimes, we compare our results to other laboratories by running tests that evaluate similar disease conditions, using different testing platforms, in order understand the differences in case interpretation. Our head-to-head tests (duplicate samples that are run on different platforms) figure into our interpretations.
After results are obtained, recognized patterns are passed along to the field veterinarian. We are not immune to event-bias and overcome that tendency by using algorithms that are coupled with statistical analysis. The systems analysis procedure produces, in a finite number of steps, the answer to questions we pose. For example, is this horse likely to relapse?
Our algorithms sift through the data we get from tests and red flag results to which we should pay attention. A clinician may evaluate a case a year, or perhaps several cases over several years. Each case presents an individual interpretation of disease that makes field medicine enigmatic. We enter tens of thousands of results into our algorithm and give the succinct final analysis to the veterinarian. The data to feed the algorithm come from serum bioassays important to the diagnostician. At the very least, test results offer objective parameters to veterinarians on which to base their treatment decisions.
There are some new things to consider. And these topics were discussed in our recent Zoom meetings. We hope you joined in! To assist with clinical analysis of horses with neuromuscular disease, several bioassays are used. The term for the assayed molecule is “analyte”. Some of the assays detect antibody against foreign proteins as the analyte and include surface proteins of Sarcocystis neurona, (remember, these are unique to S. neurona and mutually exclusive to serotypes of each neurona species), or Neospora hughesii. Anti-toxin against Sarcocystis fayeri is an analyte and if disease is stimulated an autoimmune reaction follows. Two areas of the myelin protein P2 are analytes in our “Sidewinder” panel. There are bioassays for antigens that include C-reactive protein (CRP) and neurofilaments (NfL) molecules.
The principle difference one should consider between detecting antibodies versus detecting antigens as analytes is time frame for a change in the levels found in the serum. Some antibodies won’t decrease for months after they are produced against an antigen (foreign agent). Another consideration is that a naïve animal will show a reduction in antibodies much sooner than an animal that is “experienced” with the infection. That means prior exposure is important information that should be taken into consideration when examining a case.
An animal that is chronically exposed to an organism in the environment will maintain antibodies due to new gut infections and it can be tough to interpret these test results in the face of acute disease. That means prevalence of disease is an important consideration in analysis of these cases. The life-cycle of the organism is an important consideration. Does the organism complete the life-cycle in the host, as in S. fayeri or is it unable to mature, as in S. neurona infections in horses? Does the organism change it’s repertoire of antigens presented during infection, as does S. neurona? Did you consider that one infection, with a particular serotype of S. neurona may not protect against another serotype? Are antibodies produced against a serotype of S. neurona protective against infection but stimulatory to the inflammation that can become dysregulated?
Antigen molecules, such as CRP, an acute phase protein, are useful. CRP is elevated in inflammation when it is associated with the cytokine IL6. There are several innate plasma buffer systems that regulate IL6—>CRP, but occasionally the system becomes dysregulated. Chronic dysregulation can lead to an inflammatory condition and chronic inflammation can lead to an autoimmune disease. The presence of CRP indicates inflammation due to an infective process, however it isn’t specific to one particular organism. CRP is quick to be produced but in our analysis, it doesn’t decline in days. It can take weeks. Horses have several conditions that can keep the CRP value elevated; they include encysted parasites or hind gut ulcer disease.
One very dynamic marker in neurodegenerative disease is neurofilament light (NfL). Neurofilaments are cytoplasmic neuronal proteins highly expressed in large myelinated axons. The levels of NfL expressed in body fluids are in proportion to the degree of axonal damage (inflammatory, neurodegenerative, traumatic, and cerebrovascular diseases). The utility of NfL is based on the rapid decline of levels, within days, of effective therapy! The difficulty with another measurable neurofilament antigen, heavy chain, is that heavy chains can clump in some cells and clumps aren’t detected in live bioassays.
Our algorithm for suspect cases of EPM first evaluates levels of surface antigens from S. neurona, 1, 5, and 6, as well as CRP. If there is supporting history from bioassay, we can determine if the horse is experienced or naïve. Further analysis can determine if there is chronic exposure to the parasite in the environment. Our algorithm gives less attention to N. hughesii and Borrelia infections unless there is a high prevalence of disease (the algorithm uses zip code for the regional association). There is an association with S. fayeri and anti-myelin protein P2 antibody. For example, 786 horses with circulating S. fayeri anti-toxin and of those, 610 also had circulating anti-myelin protein P2 antibody. This is important to evaluate on a case-by-case basis, but points out that some equine muscular sarcocystosis can result in a demyelinating polyneuropathy. There are treatment implications to these data.
It may be useful for a clinician to distinguish between demyelinating, (has antibody against MP2), and non-demyelinating polyneuropathy because treatment and prognosis will vary between these presentations. While NfL responds quickly to successful treatment, this marker can be present in both demyelinating and non-demyelinating polyneuropathies. A panel of assays are useful to determine the pathogenesis of disease. Take advantage of our neurodegenerative disease panel by downloading our submission form. The data will be submitted into our algorithm and our interpretation returned to the veterinarian.