And goats AND mice

THINGS CHANGED IN THE EARLY 1950s. By then scrapie had turned up in Canada, the United States, and Australia in the wake of importation of sheep from the United Kingdom. Those countries, along with New Zealand, imposed an embargo on such sheep unless they could be guaranteed scrapie-free. Now, finding the source of the disease became an economic issue. Motivated and well financed, British veterinarians resumed large-scale experimentation at two major research centers: the Agricultural Research Council's institute in Compton, Berkshire, and the Moredun Research Institute in Edinburgh. Although they were unable to identify the mysterious scrapie virus, they made many important discoveries, three of which I shall discuss here.

The first related to the distribution of the infectious agent in the various organs of a sick animal. It was known to be found in the brain and the spinal cord, but what about other organs? To find out, it was necessary to prepare macerates (crushed tissue preparations) of the various organs of sick animals and see if these could transmit scrapie to healthy animals. Two of the researchers, Iain Pattison and Geoffrey C. Millson, used goats in their experiments. These, in fact, turned out to be far more susceptible than sheep to inoculation; attempts at goat-to-goat transmission were generally 100 percent successful, while the figure was around 25 percent in sheep. Their experiments showed that large quantities of the infectious agent were present in the brain and in the nearby pituitary gland, and somewhat smaller quantities in the cerebrospinal fluid, the sciatic nerve, and the adrenal and salivary glands. Very small quantities were found in muscle tissue, but it could not be detected in the blood or the urine. The infectious agent, therefore, was not confined to the nervous system—the only tissues in which lesions had been observed.

The second discovery also resulted from Pattison and Millson's experiments with goats. Sheep-to-goat transmission by intracerebral inoculation—injection into the brain—resulted in two clinically distinct types of scrapie: "drowsy," which was mainly manifested in neurological symptoms from the outset, and "scratching," whose initial symptoms involved itching before progressing to the neurological variety. If brain tissue from a goat with the "drowsy" clinical type was used to inoculate another goat, this would result several months later in a case of "drowsy" scrapie. Similarly, brain tissue from a goat with the "scratching" type would cause "scratching" scrapie. There appeared to be two strains of the scrapie agent, causing somewhat different diseases. Patti-son and Millson offered the theory that these two viral strains were also found among sheep, which could explain the diversity of clinical symptoms that had been observed, as well as the fact that the names for the disease varied by locality—the French had called the disease tremblante because their sheep were affected mainly by the "drowsy" strain, while the British had called it scrapie because their sheep harbored the "scratching" virus. Here was yet another example of the many ways in which The Disease could disguise itself. And the question of different strains would arise repeatedly in the course of the hunt. It remains at issue today.

The third discovery I want to highlight was made by a close colleague of Pattison and Millson, Richard Chandler, whose discovery was to expedite research considerably. In 1961, Chandler succeeded in transmitting scrapie to mice. This completely new outcome was published in the British medical journal The Lancet, whereas almost all previous work in the field had been published in veterinary journals. As we shall soon see, this was when the hunt for The Disease began to interest non-veterinary physicians.

Chandler was not deterred by the failure of his predecessors in transmitting scrapie to mice; he was determined to try. He was encouraged by two factors. The first was the work of Pattison, Millson, and a few other researchers, who had perfected a method for effectively and re-producibly transmitting the infectious agent in goats. Successive transmissions in goats had, so to speak, stabilized the agent, which now gave rise to relatively consistent symptoms in a time frame that was itself comparatively consistent and quite short: from three to seven months. The second factor had its roots in his own work on the susceptibility of mice to bacterial infections. Now, bacteriologists do not work with mice simply trapped in the wild. For many, many years they have bred a number of different lines of mice originating with wild "founders." The stability of each line is maintained by inbreeding. Thus, the mice of a given inbred line are genetically very similar, while mice of different lines display genetic differences that reflect those between the founders of the respective lines.

Chandler worked with three different inbred lines, and observed a varying susceptibility to bacterial infection. He theorized that they might also have differing degrees of susceptibility to the causative agent of scrapie. He inoculated the brains of mice from each inbred line with extracts of brain tissue from goats suffering from scrapie. He carried out two parallel experiments using brain tissue from goats with the "drowsy" and the "scratching" strains of scrapie, respectively. After an incubation period of seven and a half to nine months, several mice manifested typically neurological symptoms. He wrote:

The symptoms suggested disorder of function of the motor nerves, especially those associated with the hindquarters and tail. The mice stood with their hindquarters lowered close to the ground, and they were reluctant to move. The hind legs were occasionally dragged, the mice eventually walking with a stiff and rolling gait. The tail was held in an unnatural manner—stiffly and often to one side. If curled over a finger, the tail often retained a circular form for several minutes. When held up by their tails affected mice usually brought their hind feet together, whereas normal mice usually splay their hind legs. Some of the affected mice had ruffled coats and arched backs.1

When the mice were sacrificed, examination of their nervous systems showed lesions that were characteristic of scrapie.

In addition to the strong indication that it was possible to transmit scrapie to mice, this experiment yielded another important result: Transmission was successful only with material from the brains of goats that had the "drowsy" strain, and only in one of the three inbred lines of mice. The second point recalled the varying susceptibility to scrapie of different breeds of sheep, as often reported by farmers.

In a 1963 article, Chandler confirmed that he had transmitted scrapie to mice. He indicated that scrapie could in fact be transmitted from goats to any of his three inbred lines of mice, but that this was far more difficult in two lines, where only a tiny fraction of the animals developed the disease, and only after far longer incubation periods (thirteen to fifteen months, as compared with seven to nine months). Chandler then set about effecting mouse-to-mouse transmission, which he did without difficulty. But here, the infectious agent acquired two new properties. First, it had somehow adapted to its new host, bringing on the disease more quickly, in only four to five months. And second, it developed with equal speed and effectiveness in all three inbred lines of mice. The strain of scrapie that had adapted to mice was in some way different from the original strain that had adapted to goats. The changes were seen too when Chandler successfully retransmitted the disease from mice to goats after several mouse-to-mouse transmissions. That success was proof positive that the disease observed in mice was indeed scrapie. As noted, however, only the "drowsy" type could be trans mitted to mice. But with transmission in the opposite direction, goats inoculated with the infectious agent from mice with "drowsy" scrapie sometimes came down with the "drowsy" type and other times with the "scratching" type—and sometimes with a mixture of the two. Such a change had never been observed in goat-to-goat transmission.

It was thus learned that scrapie could be transmitted between species as different as goats and mice; that transmission could be more or less difficult depending on the genetic traits of the animals concerned; and that once transmission had occurred, the agent adapted to its new host by acquiring new properties.

By using mice, Chandler was able to do a series of basic experiments whose cost and duration would have been prohibitive using goats or sheep. For example, he carried out measurements of the infectious agent; previously, scientists had simply used undiluted extracts of various tissues for inoculation, and the result was basically all or nothing— either the inoculated animal developed scrapie or it did not. Of course, in those previous experiments the incubation period and the percentage of animals that contracted the disease provided some idea of the quantity of infectious agent in the inoculation, but this was very imprecise. Chandler, who was able to use a virtually unlimited number of animals, prepared a series of different concentrations of tissue extracts and used them to inoculate a great number of mice. Using an extract of mouse brain, he observed that the more dilute the extract, the longer the incubation period. But even with a 1 : 100,000 extract (equivalent to one tablespoon of extract in more than 390 gallons of water), he succeeded in causing the disease in less than six months in all inoculated mice. And some mice contracted scrapie eight to nine months after inoculation with a 1:100,000,000 extract (one tablespoon per 390,000 gallons—the equivalent of a typical Olympic-sized swimming pool). Clearly, there is a considerable quantity of infectious agent in the brains of sick mice. Considering that a mouse can be inoculated with about one one-hundredth of a cubic centimeter (0.00034 fluid ounce), it is clear that extracts from the brain of a single infected mouse would suffice to con taminate millions or even billions of other mice.

Chandler also studied the effect of different methods of inoculation: injection into the brain (intracerebrally) and into the spinal cord, through the peritoneum (intraperitoneally), subcutaneously, and orally, using a gastric tube. In all cases it proved possible to transmit the disease, although with only partial success using oral inoculation; in those cases only about half the animals had sickened after nine months, while all others had shown symptoms in less than seven months.

Mice would now supplant sheep and goats in scrapie research. The fact is that any scientist seeking to purify the causative agent (that is, to separate it from the numerous particles and molecules present in any cell extract or bodily fluid) needed to inoculate hundreds or thousands of animals in order to measure the infectious potency of various samples. This was possible using mice, but less so with sheep or goats. So we will soon be able to shift our attention away from those friendly farm animals. But first, we have to return to the thorny question of how scrapie is transmitted in nature.

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