As we burn 20 year old files, as is our custom, we incinerated the early challenge experiment raw data. The signed exam forms, the CBC results, the CSF taps. In 2000 there were a lot of unknowns about equine protozoal myeloencephalitis. Then the thinking was a challenge model would answer some questions. One group was trying to create disease from road-kill opossum-gut-scrapings and another group gave oocysts to horses and then shipped the animals, to stress them. These models were proven ineffective— the challenged horses didn’t get parasites that could be recovered from the central nervous system. Marquis was under development and there were rumors about a vaccine.
A new recruit to the cause, I left UF with my newly printed PhD and started Pathogenes Inc. The most significant project was developing an animal model for EPM. Bayer listened to my pitch and funded a 4-horse feasibility study that was a success. All three of the challenged animals developed EPM: that included antibodies detected in the CSF, clinical signs and isolation of the organism from the CNS. The control animal remained healthy. Bayer was impressed, they funded a 20 horse challenge experiment.
In the proposed 20-horse trial, ten horses were given Ponazuril (10 mg/kg) and ten horses were given placebo. Due to a delay in starting the study the horses were treated much longer than the planned 14 days before the start of the challenge. The animals were selected and observed by two independent observers (board certified veterinarians, prominent in the EPM field). Keep in mind the treatment responses are decided prior the initiation of the study—that prevents wiffle waffling the data after the study. The response to treatment was scored as a complete success-prevention of clinical signs of EPM and a negative CSF or no clinical signs; a partial success was scored when there were no clinical signs but the CSF was positive; and a failure was when there were clinical signs of disease and the CSF was positive.
Eighteen of the horses converted from a negative to positive CSF test result after challenge, 10 placebo-treated and eight Ponazuril-treated horses. Clinical signs were observed in 8 of the 10 placebo-treated and none of the Ponazuril-treated horses. As expected, the placebo-treated horses were complete failures. Two Ponazuril treated horses were successes, and eight ponazuril-treated horses were partial successes.
Bayer’s take aways from the two feasibility experiments was that EPM was induced in 13 of 14 placebo-treated animals (93%) in two studies and clearly showed the efficacy of the model and that horses given Ponazuril prior to challenge did not get clinical signs of EPM while maintained on the drug, but they did develop thecal antibody.
There was a lot more shown in these experiments. A cellular immunity assay showed that interferon gamma (IFNϒ) was down regulated in chronic infection and measuring this cytokine indicates active infections. We could answer the oft asked question is this an active infection? The biggest “miss” was documenting the acute signs of EPM. The animal care takers saw these profound challenge signs, the study evaluators examined the horses at 30-day intervals thereby missing the opportunity to see these signs. It was important to get this information out so we published the acute signs paper-13 years later.
There had been considerable discussion in previous studies using other “EPM models” that immunoconversion in the CNS was indicative of disase and could be considered the parameter for infection. If true, one could only conclude that all the horses, Ponazuril-treated or not, got disease based on CSF immunoconversion. This didn’t bring smiles to the Company Team.
Another novel observation was that treated horses contaminated the communal water troughs resulting in drug levels in all horses. Horses in the next study would need to be isolated by treatment groups. Surprisingly, a trend was observed that was not previously known: after continued high doses in horses the drug levels begin to decline. The study made it possible to find a level for which horses were protected. And serum could be used rather than CSF. This was not observed in prior pharmacokinetic data because the experiments were not carried out for extended times. In discussions with manufacturing pharmacological scientists in Germany their leading hypothesis was that horses start making an enzyme (an induced enzyme) that more efficiently eliminates the drug when given for extended periods in high doses. No smiles on that thought and one that would not get funded to prove true.
Another study was conducted. Twenty four horses were divided into 4 treatment groups. A placebo-controlled group, a low dose/middle dose/ and maximum dose treatment. Again, the model induced disease in these horses and there was no dose in which the majority of the animals were without clinical signs. In this experiment intrathecal antibodies measurably declined leading to the conclusion the organism was killed at the lower doses and the pathophysiological CNS clearance of antibody was 2-3 months-“never shown so clearly in any species” said the Team. Exciting as these novel observations were, no one on the Company Team was smiling.
This experiment did not make the company happy, again they did not like the results. The horses got CNS infections and developed EPM, whether they were treated with Ponazuril or not. The Company Team got the message that EPM is a syndrome and not synonymous with sarcocystosis. It isn’t just a semantic issue, the infection in the CNS results in mild inflammation. A misconception persisted, they felt in natural disease ataxia was permanent while in the study it was not permanent, meaning they modified the disease with successful outcome. [22 years later and we understand the inflammatory component of the natural disease can be treated! Too bad all those folks are retired now, I’d sure like to explain it to them. Set the record straight. The dogma persists that with natural disease, damage is permanent. Hard to change that paradigm!] It would have helped all these years to have the Company Team stand shoulder to shoulder with us.
The conclusion was disease was prevented at a high dose and modified at a moderate dose. At even lower doses parasites were compromised, CSF intrathecal antibodies dropped to undetected, and inflammatory signs were prevented. Disease was not prevented, at some doses the EPM syndrome was modulated.
They concluded that in natural disease inflammation continues after the parasites are gone. How I wish that concept was publicized by them all those years ago! Contracts kept us mute for 8 years-and then we were free to bring new studies to the equine community. Our earnest work started in 2010. They didn’t feel it was worth the label change to “81% effective in acute cases” from “treats 60% of chronic cases get 1-2 grades better”. The big thing they acknowledged, in silence, was “until practitioners recognize acute signs we can’t fight the perception that EPM is a chronic disease!” They promised the perception would change, saying Ohio was hot on it. We are still waiting for the Buckeyes to chime in.
Another big point was these experiments showed that SAG1 was a potent stimulator of interferon gamma and that interferon gamma is down regulated in S. neurona infections. If, they said, interferon gamma could be stimulated in the horse prior to challenge the model could work, most likely it would work without drug. That made a vaccine to stimulate a protective immune response cheaper than it was to produce drug. We followed that thought and published data in 2009.
As we watch the flames and the papers curl into ash, we sadly reflect on how different it could have been. We swore to do things differently, transparently. Twenty-two years and millions of dollars later, we question if practitioners appreciated the effort.
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