Volume 2: Science
3. The nature and cause of BSE
The continuing epidemic and alternative routes of BSE transmission
Analysis of pedigree data and other genetic studies
Maternal cohort study
Embryo transfer studies
Studies of the infectivity of maternal tissues
Lateral transmission
Summary

3.101 Although MBM had been identified as the probable vector of transmission of BSE, it was important to consider whether there were alternative routes. The possibility that maternal transmission played a role in transmission was considered early in the epidemic. ¹ There was evidence of maternal transmission of scrapie in sheep but not in other TSEs such as kuru, non-familial CJD and transmissible mink encephalopathy (TME). ² It was therefore essential to determine if it occurred in cattle, as procedures put in hand for arresting the epidemic depended on maternal transmission not being an important factor. Lateral transmission was also considered since this, too, had been identified as a transmission route for scrapie (see Chapter 2).

3.102 Both these routes were considered by several independent scientists to be responsible for the propagation of the disease. Professor Richard Lacey proposed that the BSE epidemic had spread initially through the recycling of affected cattle in the MBM but had later become endemic through maternal and lateral transmission. ³ Other scientists, including Dr Helen Grant, raised concern about maternal transmission, highlighting the fact that if MAFF was relying on the scrapie origin theory, it should consider the likelihood of vertical transmission. ⁴ Professor Ivor Mills of Cambridge University recommended that calves born to BSE cows should not be used for breeding. ⁵

3.103 Epidemiological data suggested that the emergence from 1991 of BSE in animals born after the introduction of the ruminant feed ban in 1988 (so-called born-after-the-ban animals, or BABs) was most likely to have been due to exposure to ruminant protein in feed which had been manufactured before the feed ban and was still in the feed supply chain or on farms. There was, however, also the possibility that maternal and/or lateral transmission were taking place.

3.104 Several approaches were examined to address the issue:

1. analysis of pedigree data (1988);
2. long-term cohort study (1989);
3. embryo transfer study (1990); and
4. infectivity of placentae, intestine and milk (1989 and 1995).

Analysis of pedigree data and other genetic studies

3.105 As mentioned in paragraph 3.15, an attempt was made in 1987 to investigate herds with multiple cases to look for evidence of genetic inheritance, which might either be responsible for or confer susceptibility to the disease. The results, published in the Veterinary Record, ruled out single gene inheritance. ⁶ However, Wijeratne's analysis of the same data was not inconsistent with an autosomal recessive inheritance for BSE. ⁷ Neither analysis was sufficient to provide information on genetic susceptibility. The investigations into genetic susceptibility are discussed in detail later in this chapter (see paragraphs 3.151-3.158).

Maternal cohort study

3.106 The most important piece of work carried out to test maternal transmission was the cohort study (MAFF projects SE0202 and SE2101), which started in 1989 at the CVL. In brief, 300 calves from affected dams were compared with 300 control calves from unaffected dams for the subsequent development of BSE. The calves were purchased by MAFF from the owners and held on MAFF experimental farms. It was realised that some of the calves in both groups would have been exposed to infected cattle feed, but the size and design of the study, and the fact that both groups would have been similarly exposed, were to be taken into account in the analysis of the results.

3.107 Interim results of the cohort study were available in May 1996 ⁸ and the final results were published in September 1997. ⁹ BSE had occurred in 14 per cent of the offspring of affected dams and in 4.3 per cent of those born to unaffected dams. The difference was statistically significant but unfortunately did not distinguish between maternal transmission of the BSE agent and maternal transmission of a genetic susceptibility factor. None of the calves was tested for a bovine prion mutation, and a complex genetic association study using multiple polymorphic genetic markers would have been needed to determine if there were significant differences in genotype between subjects and controls. This was not possible in 1990 and would have been difficult to set up even in 1997 unless DNA samples had been banked for all 600 animals.

3.108 The results of the cohort study were analysed independently by several groups, all of whom published their results in 1997. ¹⁰ It seems from these analyses that maternal transmission might be responsible for up to 10 per cent of affected cattle, but that this level would not be sufficient to maintain the BSE epidemic. Indeed, to maintain the epidemic, each affected animal would have to have one affected offspring. There has been no new evidence since 1997 to confirm or refute this figure, and 10 per cent remains the commonly accepted value for the contribution of maternal transmission.

3.109 Most recently, retrospective interim analysis of the maternal cohort study has been carried out and set in historical context. It was shown that the enhanced risk for offspring of BSE-affected dams would have been apparent several years before completion of the study, had emerging results been monitored by a Data Monitoring Committee (DMC). ¹¹ The role of a DMC is to review accumulating data to see if the purposes for which the trial has been set up are being achieved, at which point it would release the study's findings. The establishment of a DMC had been suggested by the Department of Health, since it was common practice for such a committee to analyse data during comparative and case control studies. ¹² This suggestion was, however, rejected by MAFF scientists for reasons associated with the design of the study; interpretation of the period-of-birth effects at interim analyses would have been complicated by the incubation period. ¹³ The maternal cohort study also determined that a prohibitively large number of animals would have been required if it was to have distinguished between direct maternal transmission, a maternally associated risk or inherited susceptibility.

Embryo transfer studies

3.110 The embryo transfer study (SE1801) was carried out at the CVL, starting in April 1990. It aimed to determine whether the BSE agent was carried by the early embryo, but it was also thought that it might provide evidence for maternal transmission, should this be demonstrated. A further objective, of commercial interest, was to determine whether animals with genetically determined traits of economic importance could be rescued by embryo transfer from BSE-affected dams to uninfected recipients.

3.111 Two hundred suspect BSE cows were superovulated and artificially inseminated. Half of the cattle were inseminated with semen from BSE-positive bulls and half from BSE-negative bulls. After seven days, embryos were flushed from the uterus by uterine lavage, washed, and implanted in surrogate dams, which were guaranteed free of BSE. In effect, this meant that the surrogate dams had to be imported from New Zealand. The project was thus very expensive, and the need for it questioned by several experts. Neither a negative nor a positive control group was included in the project.

3.112 Interim results of the study in 1994 revealed no definite conclusions, but seemed to suggest lack of transmissibility by the embryo route. ¹⁴ Further results in 1995 came to a similar conclusion - at that time 2,388 embryos had been collected from BSE-positive dams, of which 587 had been transferred, resulting in 255 calves none of which had yet developed the disease. Further interim results are not available, though it has been shown that embryos and uterine flushings from BSE affected cattle are not infective to mice. ¹⁵ Final results are expected in March 2001.

Studies of the infectivity of maternal tissues

3.113 The infectivity of placental and uterine tissue was also a relevant area of investigation, and several studies were set up to address this issue. The first study, carried out at the NPU and the Royal Veterinary College (RVC), started in 1987 and was reported in 1996. It examined the infectivity of tissues including placenta, embryo tissue and uterine flushings by inoculating the tissues into mice (SE1401/1420), but no positive transmissions were observed. ¹⁶ Inoculation of infective material into experimental animals is the only way to measure the infectivity of TSE agents. This method, known as 'bioassay', is discussed in detail in paragraph 1.43.

3.114 Studies looking at the oral infectivity of foetal membranes in calves were carried out at the CVL, starting in November 1989 (SE1804/1818). Twelve calves, which had not been exposed to MBM, were dosed with homogenised foetal membranes from BSE-affected cattle in the last month of gestation, 90 ml as a drench (ie, introduced at the back of the throat over the tongue to enter the stomach directly) and 5 ml instilled into each nostril on three occasions. ¹⁷ Interim results from six subjects and three controls were reported in November 1994 and suggested no evidence of infectivity from mouse bioassay, post-mortem histopathology or clinical signs. ¹⁸ However, the final results are not due until mid-2000.

3.115 It is conceivable that maternal transmission might occur through milk from a BSE-infected dam. The mammary gland is a holocrine organ in which the secretory cells are included along with their secretory product: milk thus contains mammary cells, cellular organelles and also cells from the lymphoreticular system. It therefore has the potential to transmit TSE. However, evidence from the kuru epidemic shows that affected mothers do not transmit the disease to their offspring, which therefore excludes the possibility of transmission by milk, in kuru at least.

3.116 Infectivity in milk from affected cattle was tested in experimental mice both by intracerebral injection of 20 microlitre doses of unmodified milk (plus 0.1ml intraperitoneal injections) and by feeding mice with an average of 300 ml of milk per mouse. The results were reported in 1995 and no infectivity was found by either route. ¹⁹ Transmission has not been undertaken experimentally in calves, although this bioassay is known to be a thousandfold more sensitive than the mouse bioassay (see discussion in paragraph 3.218). ²⁰ However, results of the case control study examining maternal transmission (see paragraphs 3.121-3.122 below) do not suggest the occurrence of transmission through milk.

3.117 The above analyses suggest that the role of maternal transmission in the spread of BSE remains uncertain.

Lateral transmission

3.118 The infectivity of placenta was also important in relation to lateral transmission of BSE. This route of transmission had long been accepted as being an important factor underlying the endemic nature of scrapie, ²¹ and although no such evidence existed for the human diseases kuru and sporadic CJD, the lateral route was thought to be important in other animal TSEs. Chronic Wasting Disease (CWD), for example, was thought to have been spread by lateral transmission from neighbouring elk. ²² The outbreak of disease in kudu in London Zoo in 1993 ²³ was also indicative of lateral transmission, since none (except possibly the first case) had been exposed to foods containing ruminant-derived protein. In mink, early transmission of TME by subcutaneous inoculation had led to the proposal that natural transmission might be initiated via wounds. ²⁴

3.119 Lateral transmission of scrapie was thought to occur orally through pasture contamination (see paragraphs 2.116-2.123). One possible source of pasture contamination was foetal membranes from scrapie-affected ewes, which had been shown to be infective experimentally. ²⁵ Other suggested sources include infected faeces, urine and oronasal secretions, but there is no documented experimental evidence to support these. ²⁶ Recent evidence suggests that fly larvae and pupae may act as vectors for scrapie, since the internal contents of larvae fed with scrapie-infected brain material were capable of transmitting disease to hamsters following oral inoculation. ²⁷

3.120 The fact that infectivity has not been detected in cattle placentae by mouse bioassay ²⁸ suggests that, if lateral transmission were to occur in BSE, it is not through pastures contaminated with placental tissues, but by some other route.

3.121 The appearance of BSE from 1991 in cattle born after the introduction of the feed ban (BABs) (see paragraphs 3.135-3.140) made investigation of all possible routes of infection even more urgent. An investigation of risk factors was thus initiated at the CVL, as part of the programme of epidemiology work (project SE0201), starting in 1993. ²⁹ It was designed as a case control study and sought evidence of direct transmission by maternal and lateral routes. The study in particular sought data about the management of calving in the herds in which the selected BAB case was born. The exposure to the infective agent was considered in terms of the BSE status of the BAB's mother, and the number of subsequently infected cattle that calved within a week of the selected BAB case, as well as the month of birth of the subject.

3.122 The results, published in 1995, reported no significant increase in incidence of BSE in herds where calves were born to BSE-affected dams, compared with controls. There was a statistically significant risk for animals born between one and three days after an affected dam had given birth, though this result may not indicate a causal association. ³⁰ It was concluded that direct transmission between cattle (for example, via contaminated pasture) could not account for the majority of BABs, and that no conclusive evidence thus existed for such transmission in cattle.

Summary

3.123 The decline in the BSE epidemic which followed the introduction of the ruminant feed ban, and which occurred after a lag period due to the four-year average incubation time, confirmed conclusively that the epidemic had spread via the inclusion of MBM in commercial cattle feed. The disappointment and concern about the occurrence of disease in a substantial number of cattle born more than a year after the introduction of the ban raised the possibility that alternative routes of transmission were responsible for the slow tailing off of the epidemic. The earlier BABs could be accounted for by 'carry-over', ie, the continued use of previously stored feed containing MBM either on the farm or distributed from existing stocks. It later emerged that cattle feed had almost certainly continued to be contaminated with MBM from feed containing MBM prepared exclusively for pigs and poultry. The incidence of BABs was found to be higher in regions where pigs and poultry were farmed as well as cattle. The contamination could have occurred either in the feedmills or on the farms. It was not clear what proportion of BABs were due to feed contamination or to alternative routes of transmission, particularly maternal and lateral transmission. MBM was banned from all animal feed in August 1996. At the time this volume went to press, just one animal born after that date has been affected.

3.124 The results of the maternal cohort study suggest that up to 10 per cent of BSE cases early in the epidemic were the result of maternal transmission. As indicated in paragraph 3.107, the study was flawed in that maternal transmission could not be distinguished from maternal susceptibility. A case control study was unable to confirm this level of maternal transmission, although it did suggest that lateral transmission might have occurred in the herd from affected animals within a few days of calving. The possible mechanism of infection via lateral transmission remains unsolved. Foetal membranes from affected dams have not transmitted infection experimentally in calves or mice; neither have uterine flushings or milk. Preliminary results from the transfer of early embryos from affected dams to uninfected surrogate dams likewise indicate that the disease is not transmissible in this way. However, controlled experiments have not been done in which unaffected animals are introduced into BSE-affected herds. This type of experiment in sheep first demonstrated the lateral transmission of scrapie (paragraph 2.120).

3.125 Among untested mechanisms for lateral transmission are faecal and other contamination of pasture, and the occurrence of an intermediate host. It is known that TSE infectivity can persist after being buried in the soil for at least three years. ³¹ There is evidence that field mice and maggots can carry the TSE agent following feeding on infected cattle waste, and it is possible that either can act to contaminate the feed or pasture provided for cattle.