Selenium concentrations from the polo ponies that died within hours of each in Florida were up to 20 times normal levels, the University of Florida has revealed.
The university's pathologists peformed tests on tissue samples from 15 of the 21 ponies that began dying shortly before a match in the United States Polo Open. The six other bodies were sent to the Florida Department of Agriculture and Consumer Services' diagnostic laboratory in Kissimmee.
The unversity said necropsies (autopsies) of all 21 horses yielded no answers, nor did subsequent microscopic examinations. Even the University of Florida Racing Laboratory, which routinely conducts toxicology screening tests of race horses in the state, revealed nothing initially that could explain the horses' sudden deaths.
But subsequent tests conducted by univesity toxicologist David Barber verified the presence of life-threatening concentrations of selenium in the horses' blood and liver samples.
The concentrations were found to be 10 to 15 times higher than normal in the blood and 15 to 20 times higher than normal in the liver.
Dr John Harvey, the college's executive associate dean and a board-certified clinical pathologist, said: "Our role in testing was key, not only because we were able to verify the toxic levels of selenium found to be present in the blood and liver of these horses, but also because through additional testing conducted at our Racing Lab, we were able to rule out the presence of common performance-enhancing drugs.
"This is significant, because in ruling out other drugs that could have killed these horses, we essentially were able to corroborate the assessment that, indeed, these deaths were likely caused by an accidental overdose rather than due to malicious or criminal intent."
The university received 15 horses for autopsies around 3am on Monday, April 20.
An eight-person pathology team led by Dr Lisa Farina and Dr Jeff Abbott, both board-certified anatomic pathologists, immediately set to work conducting autopsies on eight of the 15 animals.
The task was completed at about 5pm. Soon after, a request came to undertake an examination of the remaining seven. All of the examinations, including those performed at the university and those conducted at the state's diagnostic laboratory, were complete by day's end on Tuesday.
On Wednesday morning, Farina and her colleague, Dr Michael Dark, an assistant professor of anatomic pathology, began examining slides with tissue samples under the microscope while a CNN reporter videotaped them at work.
By this time, reports had surfaced that individuals associated with the polo team had admitted giving vitamin supplement shots to the horses shortly before they died.
Speculation intensified as to what exactly had been administered and whether any of the ingredients in the supplement injections could have caused the horses' deaths.
Meanwhile, blood samples taken from some horses before they died had arrived at the university and were being tested at the racing laboratory under the direction of Dr Richard Sams. Pathologists and toxicologists remained puzzled, as nothing conclusive had yet emerged.
On Thursday, April 22, the focus of the story took a dramatic shift when a spokesperson for a private pharmacy said that the horses had received an incorrect dose of one of the ingredients used in a vitamin compound with which the horses had been injected. Because of ongoing law-enforcement and other investigations, the pharmacy did not initially release the name of the specific ingredient.
On the basis of this information, Dr David Barber, an associate professor in the Centre for Environmental and Human Toxicology in the college's department of physiological sciences, worked until late Thursday night conducting analysis of inorganic components of the vitamin supplement.
Testing was performed on samples from affected and an unaffected horse.
Barber was not informed which horse was the "control" horse. He guessed the obvious after completing the testing.
The unaffected horse was the only horse among those tested that showed normal levels of selenium in its blood, compared with very elevated levels detected in the blood of other horses tested. A pharmacy spokesperson later confirmed that selenium was the ingredient in question.