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Apicomplexans (Sarcocystis and Toxoplasma) are common infections in horses but these infections generally don’t cause clinical signs.  When infection doesn’t elicit clinical signs it is called a sub-clinical infection. However, even these non-progressive infections stimulate inflammatory cytokines. Cytokines are tightly regulated by the host to control infections, cytokine release results in both positive and negative effects. An imbalance or dysregulation of cytokines can play a part in tissue  damage and can be associated with protozoal infections. Pathogenic protozoa initially cause gut infections and in some cases they encyst in muscle tissue. Other parasites can encyst in horse tissues, small strongyle and tape worm larvae are examples.

C-reactive protein (CRP) is a non-specific marker for inflammation.  CRP is not specific (to one cytokine or infection) because it regulates several cytokines that are stimulated by infection.

A 2005 multi-center study found statistically significant gene signatures in horses with acute S. neurona sarcocystosis (EPM).  The cytokine signature was not found in chronically infected EPM horses.  Chronic infections were defined as horses that were clinically abnormal one month after infection by parasites. The disease-gene profiles were related to induced inflammatory cytokines and their regulatory molecules. Cytokine profiles in neural tissues in horses with EPM and herpes virus infection showed inflammatory cytokines were present in diseased, but not clinically normal horses. Again, these cytokine profiles were not specific to the organism but were present in inflammatory disease.

Some cytokines cause host tissue damage.  Host tissues are damaged by unregulated or dysregulated cytokines. The regulatory molecule CRP is useful to detect sub-clinical inflammation in horses that may have dysregulated pathways that cause tissue damage. As cytokine damage progresses the animals will show clinical signs.

We have some anecdotal information using CRP to detect inflammatory disease. An anecdote is an interesting story often proposed to support or demonstrate a point. Because anecdotes are singular events, they hold no scientific weight.  The person offering the anecdote is convinced of the outcome and not much will sway them from their opinion. Our anecdotal evidence leads us to think that 10% of horses tested for CRP will show up as a ‘0’ on the test result and these horses will continue to be ‘0’ on future testing.  We based our anecdotal opinion on 1000 tests that returned a 0-test result from over one hundred horses. The results were comprised of data that was compared to data from 15,000 horses. It seems like enough data.  We often hear clinicians confidently report anecdotal stories from several cases they have treated. Is our hypothesis true?

We ran a study using good laboratory practices (GLP) to test our hypothesis: the test will not be valid on 10% of horses. (We surmised that there is a genetic disposition in 10% of horses that preclude them from testing positive.)  Studies performed according to GLP are assigned the top rank of 1 (reliable without restriction) and are preferred by agencies. These GLP studies require many hundreds of man-hours for planning, performing, monitoring, recording, archiving and reporting. The studies have a lot of quality assurance (QA) built in.  And then there is QA on the QA. These studies are not inexpensive, they run into the mid-six figures by the time the final report is issued.

Based on GLP study results we can tell you our hypothesis is false!  Horses with a CRP of 0 will not always be tested as 0.  Further, horses with an elevated CRP can return to a CRP of 0 with proper treatment. The most likely cause of the elevated CRP is inflammation associated with encysted intestinal parasites. It may be important, but not proven, that treating the parasites and the dysregulated inflammatory cytokine responses are necessary.

We suggest, based on our GLP study results, that an elevated CRP should be taken seriously and treated by using an objective approach. The CRP serum concentration does remain elevated a bit longer than the days the literature suggests. The acute phase protein rises within days and our data indicated that 14 days is a reasonable time to expect a response (a decrease in CRP) after appropriate therapy. If you got a CRP result as ‘0’ it is not an indication that CRP testing has no value. 


Toxoplasmosis is a disease caused by Toxoplasma gondii , Toxo infects most mammals and birds. Cats (the definitive host) shed the infective material making cat litter a risk factor for people and other animals! In horses, toxoplasmosis is usually asymptomatic, thus the disease is considered subclinical. Cysts form in horse tissues and antibodies are found in the serum from this chronic, subclinical infection.  The seroprevalence in horses in the United States varies widely and is reported to be between 0 and 90%!  Risk factors for horses include water contaminated with cat litter or feces.

The biology of Toxo makes the tests that are used in studies that detect antibodies against Toxo important.  Some strains of Toxo are more infectious than others (virulent strains), and some strains don't cause disease at all. Toxo is a protozoa that can be lethal in some animals (a mouse for example) but the same strain isn’t damaging to another species--that makes the selection of strains used in experiments and diagnostics important.

It is generally accepted that horses are less sensitive to the pathological effects of Toxo infections. In the United States, the best guess for the number of horses in the general population that are subclinically infected with Toxo is between 12-14%. That number was much higher in a group of horses studied in California-- about 25% of the healthy horses in that study were seropositive. The California study examined neurologic horses, those with clinical disease, and showed that they were more likely to be seropositive to Toxo when the sick group was compared to non-neurologic, or healthy horses. These results beg the question, are co-infections a risk factor for EPM in horses?

Marine mammals infected with both S. neurona (the agent that causes EPM in horses) and Toxo were associated with an increased severity of infection.  Co-infections were bad for these marine animals. When horses in the Eastern United States were evaluated for subclinical infections of Toxo and protozoal myeloencephalitis over a six-year period, co-infections were not found. These data indicate that horses seropositive T. gondii antibodies didn’t have an increase in clinical EPM.  The study made these conclusions looking at the organisms S. neurona and N. hughesi, the protozoans associated with EPM. Subclinical S. neurona sarcocystosis (seropositive, healthy horses) is most common--antibodies against S. neurona are found in more than 80% of horses, yet less than 1% of the horse population is diagnosed with EPM. Neospora hughesi is less common, 34% of healthy horses have Neospora antibodies and of those perhaps 2% have EPM.

There could be a relationship between other protozoa that infect horses; the first on our list is S. fayeriSarcocystis fayeri was more common in horses with neuromuscular disease (when compared to normal horses) as shown in two studies.  These studies didn’t show that subclinical S. fayeri facilitates EPM (caused by S. neurona) but, EPM was more prevalent in horses that had equine muscular sarcocystosis. Another interesting finding was that statistically, horses with sarcocystosis were more likely to have an elevated C-reactive protein (CRP). Interestingly, it was reported that horses seropositive for Toxo have a higher incidence of pro-inflammatory cytokines and CRP values.

When you ask us what can sustain an elevated CRP in a healthy horse we will give you a list that includes encysted small strongyle larvae, gastric and hind-gut ulcer disease, equine muscular sarcocystosis, and subclinical Toxo. Protozoa can keep pro-inflammatory cytokines active and that makes subclinical inflammation chronic in horses and chronic inflammation can produce disease.

What will it take to develop a useful diagnostic test for Toxo in horses?  First, a model of disease.  Koch’s postulates must be completed.  Linking disease to the infection is important, not just detecting antibodies that were made in response to subclinical infections that didn't  progress to disease. Experimentally inducing toxoplasmosis hasn’t been accomplished in horses.  It is more complicated that it appears on the surface because Toxo shows strain-related virulence differences in hosts.

It would be easiest to find a horse with acute, clinical toxoplasmosis and isolate the organism—that has not been done yet.  As prevalent as Toxo is, one wonders why. An organism isolated from a clinically ill horse could be used to infect horses, validate the model, and answer questions about species susceptibility. The available virulent mice strains or strains isolated from humans may not be useful strains to infect horses or used for diagnostics.  We wonder if there is a need for detecting Toxo in horses because, as mentioned above, in a 6-year retrospective study no Toxo infections were related to disease in horses.

However, determining that chronic inflammation is due to Toxo may be important in polyneuritis equi.  There are breadcrumbs that lead us in that direction, but as yet no hard evidence.  We’ll get back to you after we develop a model for toxoplasmosis in horses, followed by investigation of the statistical significance in various equine populations, diseased and healthy.

cyst microscopic cropSarcocystosis is one of the most frequent infections of animals, the organisms are found in the muscles and the central nervous system! There are, perhaps, 196 species of Sarcocystis, yet the complete life cycle is only known for 26 of them. Sarcocystis require a prey-predator, 2 host, relationship to survive. Each host supports different stages of the life cycle. Hosts vary for each species of Sarcocystis.  Intermediate hosts support several species of Sarcocystis, each species use different definitive hosts.

People get sarcocystosis, two species use people as the definitive host.  Humans also serve as intermediate (or aberrant hosts) for several other species. Raw beef can infect you with S. hominis and raw pork can give you S. suihominis. Gastrointestinal symptoms are present when Sarcocystis cause disease in people  definitively hosting the parasite. Muscle pain is reported in people that serve Sarcocystis as an intermediate host. Most of the time people are asymptomatic with muscular sarcocystosis. People can get sick from an enterotoxin associated with Sarcocystis that infect horse muscles. if horse meat is uncooked.

Horses get sarcocystosis from dogs, the organism causing infections is S. equicanis (bertrami). In the US the horse-infecting organism is named S. fayeri. Donkeys harbor S. asinus, but experiments may show that this is actually S. fayeri. The visible difference in equicanis (bertrami) and fayeri is the thickness of cyst walls in muscles observed under the microscope. Our picture above is fayeri,  a thick-walled sarcocyst.  S. bertrami forms a thin-walled cyst. Dogs shed infective betrami organisms 8-10 days after eating infected muscle tissue. Horses eat the organisms from dog feces-contaminated feed.  It takes two to three weeks before signs appear in horses.  Signs include fever, neurological signs, apathy, and inappetence  a couple of months after infection. Muscle enzymes can be elevated in infected horses and increased enzymes can be measured in blood samples.

Sarcocystis fayeri cysts are found in skeletal muscles and the heart.  Ten and 25 days after infection (two waves of organisms are released from the gut) the developing parasites are found in arteries of the heart, brain, and kidney.  Muscle cysts (sarcocysts) are first seen at 55 days .  And by 77 days the muscle tissue can infect dogs to start the cycle again. We are taught that S. fayeri is only mildly pathogenic, horses develop anemia and fever after infections and some horses may have a stiff gait. More virulent isolates can cause inflammation of muscles and, in one case, the horse developed autoimmune anemia.  People that eat raw horse meat can get food poisoning, this is due to a toxin associated with the cysts.  A fetus can be infected by S. fayeri by crossing the placenta, so foals can be born with mature cysts in their muscles.

Malnourished horses show clinical muscle inflammation (myositis) and muscle atrophy that can be associated with S. fayeri. Muscle cysts are usually unassociated with clinical signs or muscle inflammation, leading to conclusions that fayeri infections are benign. Most clinicians agree S. fayeri isn’t an issue in horses.  However, there are some researchers that think S. fayeri should be considered in horses with neuromuscular disease. Scientists at UC Davis examined muscle tissues from horses with and without a history of neuromuscular disease.  They found an association, but not statistical significance, between disease and cysts.  More horses with neuromuscular disease had S. fayeri cysts when compared to the number of cysts found in horses that died from other causes.

We decided to look at the problem a different way.  While UC Davis looked at cysts, we chose to look for S. fayeri antitoxin in the serum of horses. An advantage to antitoxin analysis is that the test is performed in the live horse.  We found that 24% of normal horses had antitoxin in their serum. We also found that S. neurona antibodies were more often associated with neuromuscular disease in horses  than S. fayeri antitoxin (antibodies against the toxin released from cysts). That said, horses were more often infected with both species of Sarcocystis, not just one strain.

Horses infected with S. neurona and S. fayeri were more likely to show disease. We looked at inflammation using CRP (C-reactive protein) and found that CRP was detected in horses with and without apparent disease. We did find that significantly more horses with neuromuscular disease had an elevated CRP when compared to normal horses (p= .0135).  However, the data we needed to link the UC Davis study and ours was missing.  We needed to show  the relationship of antitoxin found in horses (our test) with sarcocysts found on histopathological slides (the gold-standard test for fayeri-sarcocystosis).

We examined three tissues (muscle tissue from the heart, esophagus, and skeletal muscle) from thirty-two horses (that’s a big number in horse studies) and measured the presence of antitoxin.  Our results confirmed that our serum test was almost the same as the results you would get if a full post-mortem exam was conducted. Also, in our study the infected horses were asymptomatic.

The conclusions thus far are that S. fayeri can be detected pre-mortem and should be considered in horses with signs of sarcocystosis or neuromuscular disease that has no other cause.  Also, horses with neuromuscular disease are more likely to have an elevated CRP.  In our S. fayeri study horses did have elevated CRP values but the inflammation was associated with other gastrointestinal parasites, not EMS (equine muscular sarcocystosis).

Why are we still interested in S. fayeri? Certainly our results support the strain of S. fayeri  infecting our 32 horses was non-virulent and support the view of most clinicians that S. fayeri may not be an issue in most horses, just some of them.

For us, it’s about the toxin.  The toxin is an actin-depolymerizing factor (ADF).  Another Apicomplexan, Toxoplasma gondii, has a very similar ADF. The TG-ADF can protect lab animals against T. gondii infections. Interestingly, some animals with lethal S. neurona infections also were infected with T. gondii. The difference between protection (mice) versus no-ADF protection (sea otters) could be stage of infection (with TG), ,strain-virulence, or something we didn't think of yet.

Can S. fayeri-ADF protect against S. neurona infections in horses? Is ADF species specific? Is ADF a virulence factor or even a survival factor for the organism? Are there cyst-producing S. fayeri organisms that don't produce ADF? Does S. neurona produce an ADF? And, can a S. neurona be incorporated into a fayeri sarcocyst? These are all questions to be answered by experiment.

Here is our opinion. It is worth monitoring S. fayeri infection in horses. It is worth considering S. fayeri infection as a cause of neuromuscular disease in horses showing weakness, muscle atrophy, and inflammation for which there is no other explanation. The disease EMS should be considered in old and debilitated horses.  Sarcocystis fayeri should be considered in horses with chronic inflammation. When  horses showing weakness do not have antibodies against S. neurona, EMS should be considered. Horses that provide meat that is fed to dogs should be monitored for S. fayeri.

If you have questions about S. fayeri, give us a call.

Acetylcholine_binding_proteinOur 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.


funny rat We predict that in the year 2050 there will be no animal experimentation.  That includes animal testing for drug licensing.  Today, 100 million animals are used in experiments, about 13% of those are involved in studies that are required to license drugs.

Happily, a large step toward the goal of providing alternatives to terminal animal studies was published recently.  The FDA commissioner Scott Gottlieb, MD, stated that the FDA is trying to reduce, replace, or refine the use of animals in research. This is heartening news. We hate the current standards for new drug development that require euthanizing animals in studies. Even if a drug is commonly used in other species—if a new indication is under investigation, terminal studies are required. We’ve repeatedly argued against this position.  We lost the debates. But FDA is not insensitive to the issue.

On the horizon are simulation models that include in vitro (non-animal) dissolution tests and computer modeling that will replace animals in some studies.   A first step to animal-alternative testing is the validation of simulation methods designed to show the equivalence of a proposed generic formulation to an approved drug. We can predict a future that allows these same models for new drug development.  Currently, FDA requires data to understand how a drug performs in a live animal.  In our case, mountains of data obtained from goats and dogs and cats won’t substitute for horses.

A part of the generic licensing process involves an animal drug developer to perform “bioequivalence studies”.  These live animal studies compare the originally approved product and a proposed generic version to see if they are similar enough to link them in terms of safety and effectiveness. These are expensive studies and these studies are ignored by compounders.  Another subject for another day.

Back to the good news. There are studies afoot at FDA that will create a physical model to simulate properties of a specific drug when tested in live animals. The proposed studies use dogs. The live animal data will validate the simulations for anti-parasite formulations that are currently used in dogs.  If the studies are successful, animal drug sponsors may use these data to aid in designing in vitro studies.  And that will save the lives of animals.  The initial studies used to equate the simulation to the live animal are designed to use a small number of anima.  The animals are subjected to minimally invasive procedures, perhaps as simple as a blood draw. And more good news, the lab-dogs will be adopted as pets.

The FDA is encouraging drug companies to develop and validate alternatives to animal testing, including in vitro dissolution tests and computer modeling. For now some drugs may still require live animal studies, and these studies may require euthanizing the test subjects to support a new animal drug. We’d like to get back to the future.  We developed a simulation method that is designed to test the equivalence of a proposed generic anti-protozoal medication against approved drugs.  As licensed EPM drugs age out of patents there will be an opportunity for licensed generics. We are refining an animal model that can prevent excessive horse use and will design and test the validation parameters.  I suspect that it will take a year or so to get the rubber stamp on a validated model.  We would like to be instrumental in the movement to decrease animal experimentation.