Volume 2: Science
2. The Spongiform Encephalopathies - knowledge existing in 1986
Transmission of TSEs
Maternal transmission
Lateral transmission
Iatrogenic transmission
Efficiency of routes of transmission
Species barrier effect
Summary

2.101 By 1986 research on TSEs had included work on the transmissible nature of the diseases, covering natural and experimental routes of transmission, in particular maternal, lateral and iatrogenic. Maternal transmission of disease includes transmission from dam to offspring in the uterus, and transmission via milk. Lateral transmission comprises passing on infection through contact with animals, or with their environment (eg, contaminated pasture), and iatrogenic involves transmission through medical intervention. Research on other TSEs gave a useful background on which to base expectations of likely transmission routes in BSE.

Maternal transmission

Scrapie

2.102 In 1986 scrapie was already known to be unusual among TSEs in that it was endemic in the flocks of many countries worldwide. In 1988, on the basis of a voluntary survey, Morgan estimated that a third of British flocks were affected, with infected flocks distributed throughout the UK. ¹ (A discussion of Morgan's results can be found at paragraphs 2.7-2.9.)

2.103 Although it had often been presumed that the disease was genetically determined, this could not explain its extent. ² Research demonstrating the importance of the contribution of maternal transmission had been carried out, and is described in the following paragraphs.

2.104 In 1966 Dickinson and co-workers implanted an embryo from the mating of a scrapie-free ewe and ram into a ewe that was the offspring of a scrapie-affected sire. ³ Inoculation of the recipient ewe with scrapie material resulted in scrapie in the ewe and in her lamb. A control - a natural-born full sibling of the lamb - did not develop the disease. This result was suggestive of maternal transmission, though the authors acknowledged that one case alone did not provide proof, and suggested that no firm conclusions could be drawn from this isolated experiment.

2.105 In another study in 1966, Dickinson inoculated pregnant ewes subcutaneously with scrapie-affected brain suspension. ⁴ Scrapie developed in 2 out of 4 offspring of these ewes, after 8 and 13 months. Because natural scrapie normally develops after 18 months of age, the authors considered that the scrapie in the two lambs was unlikely to be naturally occurring, and that maternal transmission was probably responsible.

2.106 Incidence of scrapie in commercial flocks had also demonstrated the role of maternal transmission in the spread of the disease. In 1974 Dickinson and co-workers carried out breeding experiments in flocks present at the Moredun Institute farm in Scotland, and found the overall incidence in offspring from affected ewes to be 62 per cent, and from unaffected ones 38 per cent. ⁵ These results suggested that maternal transmission had, indeed, an important role in establishing the endemicity of scrapie.

2.107 However, the maternal route did not fully explain transmission between animals. In the late 1960s Hourrigan had demonstrated that lateral transmission was also important. ⁶ Sheep and goats previously unexposed to scrapie were placed in direct contact with previously exposed animals. The animals were bred only within their respective groups. The incidence of scrapie in offspring whose sire and dam were both scrapie-free was 25 per cent. In those where only the sire was scrapie-affected the incidence was 39 per cent, and where only the dam was affected 42 per cent. When both sire and dam were scrapie-affected, 14 of the 18 offspring developed scrapie. These results confirmed that both lateral and maternal transmission were likely to have played a role in the spread of the disease. The infectivity status of the sire suggested that genetic susceptibility also influenced the incidence of scrapie in offspring.

2.108 The infectivity of foetal membranes as a possible means of lateral transmission was also considered, although an inconclusive picture had emerged by 1986. In 1972 Pattison and co-workers inoculated sheep and goats using material derived from foetal membranes of pregnant ewes affected with scrapie. ⁷ Inoculation was either intracerebral or by oral dosing. Scrapie occurred in 5 of the 12 sheep and 3 of the 18 goats dosed orally, and in 4 of the 12 sheep and 1 of the 18 goats inoculated intracerebrally. From further analysis of the results it was concluded that 4 out of 6 membranes contained scrapie infectivity. However, it was not possible to exclude naturally occurring disease.

2.109 Conflicting evidence had been obtained from work in 1982 using foetal membranes from two pregnant ewes incubating natural scrapie. ⁸ The membranes were used to prepare material that was then injected intracerebrally into ten mice. No transmission of infectivity was detected over an observation period of 24 months.

2.110 The same study had also considered the possibility that suckling lambs might become infected from their dam's milk. Mammary gland tissue from two scrapie-affected ewes, and colostrum at parturition from six high-risk ewes (from families with incidence of disease in at least two of the last three generations in the pedigree) had also been used to prepare material for intracerebral injection into ten mice. No transmission of infectivity was detected.

2.111 Although maternal transmission of scrapie had been seen in sheep and goats, it was not observed in mice infected with experimental scrapie. Investigation into this finding in 1973 suggested that the maturity of the immune system might be important in this respect. ⁹ Researchers found that certain cells of the lymphoreticular system (LRS) ¹⁰ might be necessary for early replication of scrapie agent. Since neonatal mice are relatively immature immunologically, they were assumed to lack the cells necessary for the initiation of pathogenesis. Sheep, on the other hand, are born relatively mature immunologically.

Other TSEs

2.112 The transmission of Chronic Wasting Disease (CWD) or natural North American Transmissible Mink Encephalopathy (TME) had not been linked to a maternal route. However, one report in 1983 had suggested that experimental Russian TME might be maternally transmitted. ¹¹ This is not supported by any other findings.

2.113 In studying kuru, epidemiological analysis had been conducted in the late 1950s on children born after the cessation of mortuary rituals, including cannibalism. Children of kuru-affected mothers were not found to develop kuru, ¹² suggesting that maternal transmission was not involved.

2.114 During a long-term study of monkeys and chimpanzees inoculated with the infective agents of kuru, CJD and scrapie, ten offspring were born to affected animals. No evidence for maternal transmission was found as none of these offspring went on to develop a clinical disease characteristic of a spongiform encephalopathy. ¹³

2.115 Both Gerstmann-Sträussler Syndrome (GSS) and Familial Fatal Insomnia (FFI) are known to be inherited disorders in certain families, passing from one generation to the next. There has been no evidence to suggest that maternal transmission, from mother to child in the uterus or transmission via milk, is involved in either. ¹⁴

Lateral transmission

Scrapie

2.116 Lateral transmission of scrapie has been demonstrated by many scientists. A long-term study in the 1940s and 1950s involved grazing a pasture alternately with affected and healthy sheep. ¹⁵ Seven of 26 originally healthy sheep developed scrapie. The diet of these sheep was not recorded.

2.117 Rida, a strain of scrapie, has been prevalent in northern areas of Iceland for many years. Some evidence indicates that the disease was first observed there in 1878. ¹⁶ Observations reported in 1979 indicated that a single or a few infected sheep transmitted rida to a new flock when housed together with the unaffected flock during the winter. ¹⁷ In addition, previously healthy sheep became affected when they were moved into the northern areas to replace the culled sheep population. ¹⁸ Possible vectors for this lateral transmission were thought to be pasture contamination and/or exposure to feed and drinking water contaminated by the previous affected flock. Field mice were also considered possible vectors in that they often lived in sheep houses during the winter. Experimental studies did not, however, offer conclusive results to support this possibility. ¹⁹ Evidence obtained in 1999 suggests that fly larvae and pupae may act as vectors and/or reservoirs for scrapie. More recent evidence suggests that material from hay mites from farms with a history of scrapie, transmitted scrapie when injected into mice; mites may act as a reservoir for scrapie. This is discussed later in the context of lateral transmission (paragraphs 3.118ff). ²⁰

2.118 In 1968 goats and Scottish Blackface sheep, a breed that had never developed scrapie naturally, were kept for long periods in the same pen as scrapie-affected sheep. Results suggested that lateral transmission did occur, since 10 out of 17 goats and 3 out of 11 sheep developed scrapie. ²¹

2.119 In 1974 Dickinson and co-workers kept Scottish Blackface sheep under normal commercial conditions in lifetime contact with a line of sheep that went on to develop scrapie. ²² Scrapie occurred in 28 per cent of the purebred Scottish Blackface contact group, compared with an absence of scrapie in the isolated source flock.

2.120 In 1964 sheep and goats previously unexposed to scrapie were placed in direct contact with previously exposed animals. The animals were bred only within their respective groups. Scrapie developed in 5 of the 140 previously unexposed sheep, whereas it did not develop in any of the previously unexposed goats. Dairy goats born in scrapie-affected flocks, and removed from scrapie exposure at 6 months or older developed scrapie, while those removed at birth did not. Sheep born in scrapie-affected flocks and removed from exposure at birth or at 4, 9 or 20 months developed scrapie. There was a progressive increase in scrapie incidence among those removed at older ages. It was also found that lambs exposed to a contaminated environment from birth to weaning at about 4 months of age were more readily infected and succumbed to scrapie earlier than lambs (from scrapie-free flocks) first exposed after weaning. ²³

2.121 Possible sources of infection were considered. Foetal membranes were tested by inoculation into mice, but were not found to be infective. Other materials including faeces, urine, saliva, milk, semen, and colostrum were used for intracerebral or subcutaneous injection, or oral dosing of mice. ²⁴ However, the mice did not develop scrapie when treated by any of these routes.

2.122 Mice were also exposed to grass from pastures and manure from sheds believed to be contaminated with scrapie. ²⁵ Preliminary results indicated that mice did not develop scrapie through lateral transmission from these contaminated sources, but a species barrier effect may have complicated the study.

2.123 In addition, Dickinson, in the early 1970s, considered a novel route of lateral transmission of scrapie in sheep, through feed containing meat and bone meal (MBM) derived from scrapie-infected carcasses. These speculations derived from the difficulty he faced in trying to establish a scrapie-free flock. He had tried to eliminate all known routes of infection, using farms that had experienced no cases of scrapie, and which derived their flocks from older rams that would, if infected with scrapie, already have been showing signs of infection. However, the flocks did not remain scrapie-free. This inevitably leads one to speculate that some animals may have eaten infected MBM, a possibility not ruled out by recent evidence to the Inquiry from Dr Dickinson. ²⁶ The possibility of infection through MBM was not investigated and it is not known to what extent this factor compromised studies on lateral and maternal transmission.

Other TSEs

2.124 By 1986 available evidence for lateral transmission of TSEs was inconclusive. There was evidence of lateral transmission of scrapie and CWD, but little evidence to support the occurrence of lateral transmission of kuru and CJD. The transmission vector in TME had not been established, but transmission of the disease by subcutaneous inoculation had led Marsh and Hanson in 1979 to propose that natural transmission might be initiated via wounds. These wounds might have been caused by fighting between littermates at feeding time, resulting in penetration of the skin by teeth contaminated with infected feed. ²⁷

2.125 CWD was reported in Rocky Mountain elk two years after the first occurrence in mule deer. ²⁸ Elk had fence-line contact with clinically normal and CWD-affected deer and were occasionally housed in the same pens. This suggested a lateral route of transmission, and the safety implications of reusing pens or buildings that had housed scrapie-affected sheep were then considered. ²⁹

2.126 No case of kuru had ever occurred among immigrants from other parts of Papua New Guinea living in the area inhabited by the Fore people. ³⁰ In addition, no cases of the disease were seen in natives from elsewhere in Papua New Guinea eating and living together with Fore people in whom kuru developed while they were away from home.

2.127 There had been two reports of conjugal disease in which husband and wife died of CJD within a few years of each other, which suggested the possibility of lateral transmission. However, they were later thought to be coincidental. ³¹ Since 1986 there has been a further report of CJD in a husband and wife. ³² The occurrence of TSE in a cat and its owner, with onset within weeks of one another, has also been described. ³³

Iatrogenic transmission

Scrapie

2.128 By 1986 iatrogenic transmission of TSEs - ie, by medical interventions - had been demonstrated by several groups. The first reported case in 1937 concerned the louping-ill vaccination programme for sheep. The vaccine was prepared as a saline solution of brain, spinal cord and spleen from sheep which, five days earlier, had been inoculated intracerebrally with formalin-inactivated louping-ill virus. ³⁴ The vaccine was then applied by subcutaneous injection. The first reports of problems with this vaccine were received in June 1937, when sheep vaccinated two years previously started developing scrapie. The disease was eventually identified in over 1,000 sheep, even in breeds in which scrapie seldom occurred. ³⁵

2.129 It was apparent that the louping-ill vaccine of 1935 had become contaminated with the causal agent of scrapie. It was deduced that one batch of the vaccine was contaminated, and this was traced back to the original source of sheep material. It was discovered that tissues from lambs that had previously been part of experiments in lateral transmission of scrapie had been used by mistake. Although the lambs had seemed healthy and clinical signs of scrapie in their fellow experimental group did not appear until February 1936, they had apparently been harbouring infection which was then transmitted through vaccination. This mistake provided an early recognition of the danger of transmission from subclinically infected animals.

2.130 More recently, another possible case of iatrogenic transmission of scrapie has been reported, in sheep vaccinated against contagious agalactia. ³⁶ The vaccine used consisted of homogenised, filtered organs (central nervous system, mammary gland and mammary lymph nodes) from germ-free sheep, but the scrapie status of these animals was unknown.

CJD

2.131 The first recognised case of iatrogenic CJD had occurred in 1974. A 55-year-old woman who presented with CJD-like symptoms had received a corneal transplant 18 months earlier from a donor who was subsequently found to have died of CJD. ³⁷ CJD was confirmed post-mortem in the recipient and by transmission of the disease to a chimpanzee.

2.132 One study from 1960 had described seven patients who had undergone neurosurgical procedures, who later developed a 'subacute spongiform encephalopathy' which was fundamentally different from CJD. ³⁸ An examination of the case notes of these patients in 1982 suggested that iatrogenic transmission had occurred on three occasions. ³⁹ A close temporal relationship of neurosurgical procedures on two CJD-affected patients and these three patients, unaffected at the time, was detected.

2.133 Transmission of CJD by contaminated neurosurgical instruments (silver electrodes used for brain examination) was first recorded in detail in 1977. ⁴⁰ The electrodes, which had previously been implanted in the brain of a CJD patient, were disinfected using alcohol and sterilised in formaldehyde vapour for at least 48 hours. They were then used in surgical procedures on two patients suffering from severe forms of epilepsy. CJD developed in both patients two years after surgery. The ages of the patients (17 and 23 years), the rarity of the disease, the known possibility of transmission, and the inadequacy of the sterilisation procedures all suggested that iatrogenic transmission had occurred from the first patient.

2.134 Transmission of CJD in guinea pigs by contaminated blood products was demonstrated in 1978. ⁴¹ Transmission was shown to be successful via intracerebral, subcutaneous, intramuscular and intraperitoneal inoculation of buffy coat (white blood cells). Since 1986 this result has been confirmed in mice. ⁴²

2.135 Pituitary hormones derived from human cadavers had been used from 1959 onwards to treat short stature and infertility. ⁴³ Treatment with human growth hormone (hGH) was prolonged, so that a young patient might have received more than 2,000 injections over a period of 10-15 years. Between 1959 and 1985 almost 2,000 children were treated with hGH in the UK. The first case of CJD in a young adult treated with the hormone was identified in 1985. ⁴⁴ By 1986, 16 recipients of hGH had died and a further 3 recipients were believed to be suffering terminally from CJD. ⁴⁵

2.136 It was estimated in 1985 that 30,000 pituitary glands from human cadavers were required annually to meet the need of the 800 UK recipients of hGH. ⁴⁶ Therefore, although CJD was rare, there was a reasonable probability of a CJD-contaminated pituitary being included in the batch process, leading to the infection of many vials of hormone. ⁴⁷

2.137 In 1985 the safety of the hGH extraction procedure was tested on a small experimental scale. ⁴⁸ Material from scrapie-affected mouse brain (as an analogue for CJD-affected tissue) was mixed with human pituitary tissue. No infectivity was detected by tests (using mice). It was concluded that the final product had a very low risk of containing any infective agent, but only if the handling and preparative procedures were adequate to prevent contamination from earlier processing stages. However, it was not possible to guarantee that contamination could not occur during normal production, and so pituitary hGH was withdrawn from patient use in the UK in 1985 and replaced with synthetic recombinant hormone. ⁴⁹

2.138 In order to complete the picture, we mention that more recently, iatrogenic transmission of CJD has been demonstrated in over 60 patients following neurosurgical operations involving cadaveric dura mater grafts. ⁵⁰ It appears likely that batch processing of samples allowed cross-contamination of tissue from healthy and infected donors. Dura mater grafts have largely been replaced by synthetic materials or by grafts of fibrous tissue derived from the individual undergoing the graft. The emergence of disease in these 60 patients demonstrates the continual risk of accidental transmission from human-derived tissues.

Efficiency of routes of transmission

2.139 By the 1960s experimental transmission of scrapie to sheep and goats had been demonstrated by intracerebral, intraspinal, intraocular and subcutaneous inoculation of scrapie-affected material. In 1961 scrapie was experimentally transmitted to sheep and to goats by the oral route. ⁵¹ The number of cases of scrapie produced in sheep by the oral route was comparable with the number of cases expected following intracerebral or subcutaneous inoculation of a similar group of sheep. The incubation periods ranged from 5.5 to 10.5 months, and these were again thought to be similar to those observed following direct inoculation.

2.140 In 1977 the repeated passage of a scrapie strain in golden hamsters had been studied, including the effects of route of inoculation on incubation period. ⁵² Material for inoculation was prepared from brains taken from clinically affected hamsters. Animals were examined for up to 300 days after inoculation. Infectivity was more effective by intracerebral than by intraperitoneal inoculation. The authors suggested that this difference was due to degradation or removal of the infectivity when delivered via the intraperitoneal route.

2.141 Similar results were obtained by scientists during 1975 and 1978, but with mice as the test species. ⁵³ Infectivity was again more effective by intracerebral than by intraperitoneal inoculation. In addition, these workers found that the subcutaneous route was even less effective than the intraperitoneal route. However, the intravenous route produced levels of scrapie infectivity which were almost as high as intracerebral inoculation. Kimberlin and Walker considered that this could be due to the immediate dispersion of the infection through the bloodstream by intravenous injection, compared with a more localised infection from inoculation by the subcutaneous route. ⁵⁴

2.142 The authors also suggested that intraperitoneal and subcutaneous routes of injection might allow the agent to persist at the injection site, favouring the active destruction of much of the agent by the host's phagocytic cells (ie, cells whose function is to ingest and destroy micro-organisms and other particles). This theory of a non-specific host-defence mechanism had been supported by evidence from Kimberlin and other scientists in 1975 (see paragraph 2.145).

Species barrier effect

2.143 By 1986 there was substantial evidence from studies of experimental and natural disease (usually scrapie), suggesting a species barrier effect, ie, natural barriers which resisted the transmission of TSEs from one species to another. The 'barrier' became evident through observation of the incubation time. Indeed, studies had shown that when scrapie was inoculated into a new species, the incubation period at first passage was significantly longer than those seen at later passages in the same species. ⁵⁵

2.144 Although most studies had used scrapie, in 1986 Kimberlin and co-workers also demonstrated the species barrier with TME. They transmitted TME from mink to hamsters by intracerebral inoculation. ⁵⁶ An incubation period of over 600 days was observed across this species barrier (mink to hamsters), compared with the 130-230 days seen within the first species (mink).

2.145 The mechanism behind this effect was not fully understood, although studies to investigate it had been undertaken. In 1975 Kimberlin and co-workers suggested that an immune response to the scrapie agent might be responsible for the effect, leading to passive removal of much of the injected agent. This was demonstrated by repeated intraperitoneal injections of normal hamster brain material into mice before inoculation with scrapie-affected hamster brain extract. ⁵⁷ The authors postulated that an immune response was induced in the mice as a result of sensitising with normal hamster brain material; and that this immune response inhibited the scrapie infection.

2.146 Natural isolates of scrapie were known to contain mixtures of different agent strains. The balance of these strains in the host species altered when the agent was passaged through a different species. Competition between strains of agent was known, and there was evidence to suggest that agents could block one another when injected at the same time. ⁵⁸

2.147 It had also been suggested that the extended incubation period observed when an infection passed the species barrier could reflect the low pathogenicity for the new host of the main strain in the original mixture. The reduced incubation period at later passages could be due to the selection of a strain of greater pathogenicity for the new host. Alternatively, the adaptation of scrapie to a new species might involve an alteration in the properties of a single agent. Indeed, Kimberlin and Walker produced experimental evidence to support this possibility in 1978. ⁵⁹ Experiments with hamsters and mice showed that different strains affected the two species differently, and that the proportions of the different strains changed during sequential passage through each species. They concluded that at least four factors were at play with respect to different agent strains and the species barrier effect:

1. the relative amounts of each agent strain originally present;
2. modification of pathogenesis due to association of agents with alien tissue;
3. the relative pathogenicities of the strains for the new host; and
4. interactions between the strains.

2.148 These were the main mechanisms thought in 1986 to underlie the species barrier effect. The effect and the assumptions behind it were crucially important to certain actions taken in response to BSE. These are discussed in Chapter 3.

2.149 Since 1986 progress in research in this area has been aided by the development of transgenic mice expressing foreign or mutant PrP genes. Mice expressing both hamster and mouse PrP were used to test the hypothesis that the species barrier for transmission of disease is due to differences in the nucleotide sequence of PrP. ⁶⁰ When these transgenic mice were inoculated with scrapie-infected hamster brain material, the mice developed disease. Normally, scrapie infectivity propagated in hamsters cannot be transmitted to non-transgenic mice. The normal barrier to transmission is removed by expression of the PrP gene of the same sequence as that of the inoculating species.

2.150 This ability to overcome the species barrier to transmission has been used in work with human TSEs. Experimental transmission studies of the human TSEs have until recently mainly involved transmission to primates, since transmission to rodents was not always successful and often produced disease only after long incubation times. ⁶¹ However, primate studies are hampered by both cost and ethical concerns. The development of transgenic mice expressing human PrP, but not mouse PrP, has overcome those problems, producing mice highly susceptible to human TSEs. ⁶²

Summary

2.151 Research before 1986 on the mechanisms by which scrapie and other TSEs were spread within species generated much information, and later we shall see how this helped to determine policy and assess risks when the BSE epidemic emerged. It was generally accepted that scrapie was endemic as a result of both maternal and lateral transmission. Maternal transmission of scrapie was evident in the higher proportion of affected offspring of affected ewes compared with the offspring of unaffected ewes. This was confirmed experimentally by transplanting an embryo from an unaffected ewe onto an affected ewe; the resultant lamb was affected. Also, experimental inoculation of scrapie into pregnant ewes resulted in affected offspring earlier than would have been expected if infection had occurred after birth. Lateral transmission was demonstrated when sheep previously unexposed to scrapie were placed within affected flocks and subsequently became affected. Lateral transmission was thought to occur by direct contact with affected animals, by contaminated pasture (possibly through ingestion of infected placentae), or by vectors such as field mice and seagulls. It is not known to what extent transmission of the scrapie agent via contaminated MBM in compound feed has compromised these studies, as little account seems to have been taken of this possibility, and there is nothing published on the matter.

2.152 Maternal transmission has not been found in other species. In CJD and in kuru affected mothers do not seem to pass the condition to their children. Maternal transmission does not occur in experimental mice inoculated with TSEs. Nor does it occur in Chronic Wasting Disease in mule deer or elk, and likewise probably not in TME.

2.153 On the other hand, there was evidence that lateral transmission was responsible for the spread of CWD and TME. In the latter case it might have occurred as a result of wounds inflicted by affected littermates during the fight for food.

2.154 In 1986 the occurrence of iatrogenic infection of CJD was in the forefront of the minds of scientists studying TSEs. It had just then been shown that children treated with cadaveric growth hormone for short stature were at risk of developing CJD some 10-15 years later.

2.155 Similar anecdotal evidence had previously been reported of infection following corneal and dura mater transplants. As long ago as 1937, sheep were reported as having contracted scrapie from a vaccine against louping-ill; brain tissue from lambs used previously in lateral transmission experiments of scrapie had been re-used mistakenly to source material for preparing the vaccine. This was probably the first indication that tissues from animals in the pre-clinical phase of scrapie infection could transmit disease.

2.156 A particular question of importance at that time was the question whether TSEs could be transmitted naturally between species. Transmission between sheep and goats was known. The possibility that the scrapie agent could cause CJD had been raised, but the incidence of CJD was no higher among occupational groups exposed to sheep than among other groups, or among those whose diets included sheepmeat and brains. Likewise scrapie had not passed to cattle herds which were farmed in close proximity to sheep, although this potential avenue had existed for more than 250 years. On the other hand, extensive research had shown that scrapie, CJD and other TSEs could be transmitted to certain experimental animals either orally or by inoculation.

2.157 Inoculation directly into the brain was the most certain route, but other routes of parenteral injection were effective, usually more so than the oral route. Once infection had been established across the species barrier, transmission within the new species was much faster, ie, incubation times were shorter, because the TSE agent had become 'species adapted'.