Overview of the risks of disease transmission cell cultures, which are routinely used Bielanski, Important characteristics of the TSEs which

296 A .E. Wrathall Livestock Production Science 62 2000 287 –316 have been reported too, and it is evident that these and often require substantial use of biological materi- technologies do carry risks as well as benefits. For als. Disease risks associated with oestrus synchroni- example, transgenic goats and pigs intended for sation and superovulation arise mainly because production of valuable proteins in milk for human donors and recipients may be treated with potentially therapy were found eventually to develop peculiar contaminated hormonal products. mammary lesions Ebert and Schindler, 1993. The IVP embryos are the foundation for most ad- foetal oversize problems, already mentioned, which vanced reproductive technologies, including cloning seem to be associated with in vitro culture are also and transgenics, but due to the properties of their impeding progress with genetic modification tech- zonae pellucidae, which seem to make them ‘sticky’, nologies. Another more hypothetical risk is that they are less amenable to pathogen removal by carriage of or susceptibility to infection, including washing than in vivo derived embryos Stringfellow infection with TSE agents, might permeate a narrow- and Wrathall, 1995; Marquant-Le Guienne et al., ly based, genetically modified population and remain 1998; Trachte et al., 1998; Booth et al., 1999; in it undetected, only to manifest itself many years Langston et al., 1999 The potential for pathogen later. exposure during oocyte collection, IVF and culture is further increased by batch production methods, and by the many substances of animal origin, including

3. Overview of the risks of disease transmission cell cultures, which are routinely used Bielanski,

by reproductive technologies 1998. Most laboratories collect oocytes weekly but culture to the morula blastocyst stage takes up to 9 3.1. General comments days, so inevitably there is some overlap, with attendant risks of introducing new infections into It must be emphasised at the outset that the risks ongoing batches. of transmitting infectious diseases by AI, embryo transfer and other reproductive technologies are extremely small, especially if established sanitary

4. Important characteristics of the TSEs which

protocols are followed. Broadly it can be said that influence transmission risks in-vivo-derived embryos seem to carry the lowest disease risks, with the risks of semen and IVP The TSEs are unique diseases, having many embryos somewhat higher, but the disease risks of characteristics which set them apart from those moving live animals are greater than any of these. caused by conventional infectious agents such as Surgical procedures create added disease risks if bacteria and viruses. For this reason they are often they are used in reproductive technologies. In small termed ‘unconventional infections’. Notable features ruminants, for example, it is normal for embryo of the TSEs include the following. transfer and sometimes AI to be done surgically, or at least by laporoscopy, so the risks are inevitably • The obscure nature of their causal agents which higher than for techniques without intentional pene- seem to contain no nucleic acid; thus, despite tration of the peritoneal cavity. Disease risks of major variations between agent strains, the ge- surgery, and also of ultrasound and manual interven- netic coding mechanism is a mystery. tions per rectum or per vagina in the larger rumin- • The extreme resistance of the agents to inactiva- ants, are largely those of mechanical transfer of tion by standard physical and chemical treatments infection from one animal to another by contami- such as dry heat and radiation, and many chemi- nated instruments, or by the operator’s hands Divers cal disinfectants. et al., 1995, and are not necessarily due to carriage • The epidemiology natural spread of TSEs be- of infection via the gametes or embryos per se. The tween animals is poorly understood, and there is advanced technologies such as semen and embryo scant information about threshold infective doses. sexing, cloning and genetic modification, all tend to • Their incubation periods are very long, i.e. years carry higher risks simply because they involve rather than days, weeks or months, but once prolonged culture and or complex instrumentation, clinical disease appears it is invariably fatal. A .E. Wrathall Livestock Production Science 62 2000 287 –316 297 • The absence of quick and effective tests for ticular infectious disease is never transmitted by presence of infection in living, preclinical and parents to their offspring during natural breeding, clinical cases, and also for infectivity in tissues then prima facie, it is unlikely that it will ever be and fluids. carried specifically by semen, oocytes or embryos. • The fact that host genetic factors can strongly Nevertheless, transmission could occur in artificial influence TSE susceptibility and incubation breeding due to contamination by other tissues, or if periods, particularly in sheep and goats and man contaminated equipment is used. Possible natural and mouse. disease transmission routes are: These and other features of TSEs are now consid- • horizontal or lateral transmission: the spread of ered in more detail. infection between unrelated animals via direct or indirect contact at any time, or to the offspring 4.1. Nature of TSE agents after parturition; • vertical transmission: the spread of infection from In all TSEs a characteristic proteinase-resistant parent male or female to offspring via germ- res Sc insoluble protein, referred to as PrP or PrP , plasm spermatozoa or oocytes at fertilisation, or accumulates in the central nervous system CNS. in utero during prenatal life; Many now believe TSE infections are caused by • maternal transmission: the spread of infection Sc particles consisting solely of PrP , or ‘prions’, and from the dam to her offspring either vertically that infectivity arises when the natural, soluble cell via female germplasm, or across the placenta, or c glycoprotein PrP is transformed by abnormal horizontally in the immediate post-parturient Sc folding at the molecular level into insoluble PrP or period via milk, saliva, faeces, etc.. prion protein Prusiner, 1995; Weber and Aguzzi, c 1997. However, in recent work, when PrP was Despite much study, natural routes of TSE trans- Sc transformed in vitro into protease resistant PrP , the mission are still poorly understood. Long incubation latter was found not to be infectious Hill et al., periods make it difficult to link clinical cases to their 1999a. original sources of infection, and genetic predisposi- TSE strains are subtypes of TSE infectious agents tions passed from parents to their offspring, especial- capable of maintaining their distinctive phenotypic ly in sheep scrapie, make epidemiological interpreta- characteristics such as disease incubation periods, tions even more complicated. Most TSEs can be CNS lesion profiles, and possibly tropisms for spe- transmitted experimentally by injecting or feeding cific cell types, when passaged within a host species, infected material e.g. brain from affected animals to or even within other species. A minority view is that others of the same species, and sometimes to differ- nucleic acid might exist as small, hitherto uniden- ent species, but the relevance of this to natural Sc, tified virus-like particles associated with the PrP transmission routes is uncertain. and that the agent strain variations are due to genetic Epidemiological studies have shown that most polymorphisms Farquhar et al., 1998; Hunter, cases of BSE in the UK arose from dietary exposure 1999. However, proponents of the protein-only to infected meat and bonemeal Wilesmith et al., hypothesis e.g. Collinge et al., 1996; Safar et al., 1988; Kimberlin and Wilesmith, 1994 and this, 1998 argue that strain-specific properties of TSE though unintentional, was a man-made transmission agents are encoded by conformational patterns of the route. The mean incubation period for BSE is about Sc PrP protein. 5 years, with a probable range of 2 to 7 years, and it is believed that cows giving birth in the latter stages 4.2. Natural transmission epidemiology of TSEs of incubation, or after clinical onset, are more likely in domesticated ruminants to transmit the disease to their offspring than those calving in early incubation Donnelly et al., 1997; Knowledge of natural disease transmission routes Wilesmith et al., 1997; Donnelly, 1998. Thus, while is basic to gauging the risks of transmission by no maternal transmission could be detected in off- reproductive technologies. For example, if a par- spring born more than 2 years before clinical onset in 298 A .E. Wrathall Livestock Production Science 62 2000 287 –316 the dam, the risk thereafter was enhanced, rising to explanation van Keulen et al., 1999. Some e.g. | 10 in the last 6 months of the maternal incuba- Ridley and Baker, 1998 have proposed that the tion period, and possibly more in those born after disease is entirely due to genetic causes; however, clinical onset. Some remain sceptical about these the fact that genetically susceptible sheep in scrapie- data, arguing that the apparent maternal transmission free countries do not succumb indicates otherwise. might in fact be due to genetic variation in suscep- As for maternal transmission, this probably occurs tibility to BSE. However, there is little evidence that transplacentally or soon after birth. Moderate levels Sc the known PrP gene polymorphisms in cattle affect of infectivity and PrP have been found in placentae susceptibility, as they do in sheep. Further, as Pattison et al., 1974; Race et al., 1998 and this is pointed out by Donnelly et al. 1997, the fact that thought to contaminate and persist in pens and the maternal transmission risk is positively correlated pastures, leading also to horizontal transmission. It with incubation stage makes a genetic explanation has been found that scrapie tends to be common in unlikely. Maternal transmission in cattle is now sheep born to infected dams in infected environments generally accepted therefore, though how and when Hourrigan and Klingsporn, 1996. Whether maternal the calves are exposed, i.e. transplacentally during transmission occurs only in late incubation in sheep, gestation, at parturition, or in the early post-natal as in cattle, is not clear, although Hoinville 1996 period via maternal secretions excretions, is still mentions that the risk to lambs born the year before unknown. Experimental and epidemiological inves- onset of the dam’s clinical signs is similar to that in tigations have shown no clear evidence for horizontal lambs born in the year of disease onset. transmission of BSE in cattle. Scrapie cases in goats are uncommon and most Scrapie, which affects sheep and goats, is the can be traced to contacts with affected sheep. It is commonest of the natural TSEs, and at least in sheep also assumed from tissue infectivity studies that there is good evidence for both horizontal and natural transmission routes and pathogenic mecha- maternal transmission Dickinson et al., 1974; Hoin- nisms resemble those in sheep Andrews et al., 1992; ville, 1996; Wrathall, 1997; Woolhouse et al., 1998. Wood et al., 1992. Onset of natural scrapie usually However, because sheep carry a variety of different occurs between 2 to 4 years, but after experimental PrP gene polymorphisms which strongly influence inoculation the incubation can be as little as 20 susceptibility, the epidemiology of scrapie in differ- months. Maternal transmission in goats, although ent sheep breeds and populations is very complex. In probable, has not been documented. Until recently it infected flocks with a high proportion of genetically was believed that genetic factors had little effect on susceptible animals the disease tends to be common, scrapie incubation in goats, but new studies Gol- with clinical signs typically appearing at 2 to 4 years dmann et al., 1996, 1998 have revealed PrP gene of age, whereas in those with many resistant animals polymorphisms which do have a marked influence. clinical scrapie is rare and mainly occurs in older Chronic wasting disease CWD, a naturally oc- sheep. In some countries, such as Australia and New curring TSE in deer, was first reported in North Zealand, despite significant numbers of genetically America in 1967. Affected species include Rocky susceptible sheep Hunter and Cairns, 1998; Bossers Mountain elk or wapiti, mule deer Odocoileus et al., 1999, scrapie is not seen, which suggests an hemionus, white-tailed deer Odocoileus vir- absence of endemic infection and a need to avoid its ginianus and certain hybrids. Both captive and free importation. In countries with endemic scrapie, on ranging animals have been affected Williams and the other hand, selective breeding for resistance, Young, 1992; Spraker et al., 1997 and there are based on PrP genotyping blood tests, has good mounting concerns about spread into the expanding potential for control of the disease Dawson et al., deer farming industry in North America Zeman et 1998. al., 1998. Horizontal transmission of CWD is The mechanisms of horizontal transmission of thought to occur and maternal transmission is also scrapie between animals are still unknown, but probable. The minimum incubation period is about ingestion from contaminated pastures leading to 18 months but most cases occur at 3 to 4 years of infection of the alimentary tract is one possible age. Genetic effects on incubation in deer seem not A .E. Wrathall Livestock Production Science 62 2000 287 –316 299 Sc to have been studied. The infective agent responsible the presence of PrP does not necessarily equate for CWD differs from those of scrapie and BSE in with TSE infectivity, so bioassays, usually in mice, that it does not readily transmit experimentally to are also required. These are very time consuming mice or hamsters; transmissions to ferrets, mink and and expensive to perform. In peripheral tissues, as Sc a goat have been reported however Bartz et al., distinct from the CNS tissues, neither PrP nor TSE 1998. Little is known about susceptibility of other infectivity seem to be associated with pathological domesticated ruminants to TSEs, though one case of lesions. BSE in a bison has occurred in a British zoo MAFF, A guide to potential levels of TSE infectivity in 1997. various tissues is shown in Table 1 which is based mainly on bioassay results from naturally and ex- 4.3. TSE infectivity in different body tissues perimentally affected sheep, goats and cattle in many different studies, most of which are covered in detail Understanding the risks of TSE transmission by elsewhere e.g. Hoinville, 1996; Wrathall, 1997; reproductive technologies requires a knowledge of MAFF, 1998. Despite all the studies, allocation of TSE infectivity in different types of tissue, and how tissues to infectivity categories is fraught with diffi- Sc it is detected. Presence of PrP , the characteristic culty, and it is unlikely that Table 1 is wholly proteinase-resistant protein of the TSEs, can be accurate. Doubts arise for various reasons, not least demonstrated in the CNS and some other tissues of because some of the published work lacks detail on affected animals by immunohistochemical and im- methodology. Results also vary according to stage of munoblotting tests which, if applied to biopsies of infection, PrP genotype, breed and species. For accessible tissues such as lymph node, tonsil or example, in cattle with BSE as compared to sheep nictitating membrane, can also be useful for ante- and goats with scrapie infectivity has been detected mortem diagnosis of TSEs O’Rourke et al., 1998; by mouse bioassay in relatively few tissues, i.e. Race et al., 1998; Schreuder et al., 1998. However, CNS, trigeminal ganglion, retina, distal ileum and Table 1 a,b,c Guide to potential levels of TSE infectivity in tissues of affected ruminants Category Tissue type 1. No detected infectivity Skeletal muscle, heart, kidney, urine, thyroid, mammary gland d milk, blood clotted and serum , saliva, connective tissue, skin, cartilage, faeces, testis, epididymis, prostate, seminal vesicle, e e semen, ovary , uterus , uterine flushings, embryos in-vivo-derived e ones at 7-days post-fertilisation, foetus f 2. Low infectivity Thymus, liver, lung , kidney, adrenal gland, pancreas, nasal g mucosa, salivary gland , bone marrow 3. Medium infectivity Lymphoreticular tissues, especially those associated with the pharynx e.g. tonsil, alimentary tract e.g. Peyer’s patches and eye e.g. nictitating membrane; also spleen, intestines small and large, placenta, peripheral nerve 4. High infectivity Central nervous system e.g. brain, spinal cord, eye, dorsal h h h root ganglia, pituitary gland , pineal gland , dura mater a Information condensed from many original reports see text. b Infectivity critically dependent on incubation stage, assay sensitivity and many other factors see text. c In cattle few non-CNS tissues have been shown to harbour infectivity; i.e. distal ileum, bone marrow. d Inconclusive evidence exists that certain white blood cells might carry infectivity see text. e Detected by Hourrigan 1988, 1990 in sheep by mouse bioassay but not confirmed by other workers. f If killed by stunning pithing, infected brain emboli may lodge in lung and possibly other tissues. g Detected by Pattison and Millson 1962 in goat salivary gland by bioassay in goats, but not confirmed. h High risk due to close association with CNS, and evidence of iatrogenic CJD transmission in man. 300 A .E. Wrathall Livestock Production Science 62 2000 287 –316 possibly bone marrow, and other tissues have tested appear to be proportionately much lower than in negative. Whereas bioassays, if positive, are valuable sheep and goats with scrapie. for categorising the risk of particular tissues, confi- dence in negative results obtained when heterologous 4.4. TSE spread within the body of an infected species e.g. mice are used for the tests may be animal misplaced, particularly if only a few samples are tested, or the tissue dilution factor is high. Testing At this juncture it is important to consider how for BSE infectivity in bovine tissues by inoculating TSE infection spreads to the CNS from its portal of mice could be as much as 1000-fold less sensitive entry into the body. The traditional view, based on than testing in cattle, the natural host species Wells experimental work with scrapie in mice Kimberlin et al., 1998. Thus, even if a particular ruminant and Walker, 1988; Fraser et al., 1992 is that after tissue tests negative in mice, analogies with other initial replication in the lymphoreticular tissues, species including man, plus a basic knowledge of especially those of the alimentary tract and spleen, TSE pathogenesis, may indicate that it would be infection spreads along autonomic nerve fibres to the prudent to allocate it to a potentially non-negative spinal cord, and thereafter to the brain. Replication in risk category until proved otherwise. Possible exam- the CNS occurs first in those parts of the spinal cord ples include the spleen and placenta in cattle. or brain from which nerves connect to the sites of In Table 1, in contrast to similar tables in some primary infection, which implies spread of the agent other publications e.g. WHO, 1997; Advisory Com- via the peripheral nervous system. An alternative or mittee, 1998, potentially high infectivity tissues e.g. additional route about which there is currently much CNS are placed in the high numerical category, i.e. speculation is that infectivity might be carried in the category 4, whilst those without detectable infectivi- blood by mobile cells from the lymphoreticular ty are mainly in category 1. This is to facilitate a system. quantitative approach to risk. Apart from CNS and some contiguous tissues 4.4.1. Possibility of haematogenous carriage of which can carry high levels of infectivity it is evident TSE infectivity from Table 1 that several peripheral tissues can also Follicular dendritic cells FDCs are known to support at least some TSE replication. Of particular play a key role in TSE replication in the importance in this respect are lymphoreticular tis- lymphoreticular tissue McBride et al., 1992; van sues, especially those e.g. tonsil and Peyer’s pat- Keulen et al., 1996; Hill et al., 1997, 1999b but, ches associated with the alimentary tract. The spleen being non-mobile cells, they are unlikely to carry is another potential source of infectivity in sheep and infection into the blood stream themselves. Recent goats, although none has been found in spleen or work Blattler et al., 1997; Klein et al., 1997, 1998; lymph nodes of cattle with BSE. Nevertheless, BSE Collinge and Hawke, 1998 suggests that B lympho- infectivity has been detected to a limited extent in cytes, which are not only mobile and circulate in the bone marrow of affected cattle Wells et al., 1998, blood part of the ‘buffy coat’ fraction, but also 1999b, and, surprisingly, it has also been found in interact closely with FDCs, might act as ‘carriers’ of the spleen of sheep challenged with BSE Foster et infectivity. If correct this has important implications al., 1996a. for the reproductive technologies. Occurrence of scrapie and BSE infectivity in Direct evidence for TSE infectivity in blood has spleen of sheep, and possible BSE infectivity in the come mainly from studies with hamsters, mice and bone marrow but not spleen of cattle, might mean humans. For example, Casaccia et al. 1989 demon- that TSE agents infect different types of strated low levels in concentrated blood from lymphoreticular cell in these two species. Alter- scrapie-infected hamsters infected intraperitoneally natively their affinity might be for a type of cell using other hamsters challenged intracerebrally for residing in different sites. Whatever the case, infec- the bioassays, thereby avoiding the problem of non- tivity levels in peripheral tissues of BSE cattle homologous testing. Brown et al. 1998 reported A .E. Wrathall Livestock Production Science 62 2000 287 –316 301 presence of infectivity in samples of buffy coat, sions or use of blood products in any species, plasma, and Cohn plasma fractions I-plus-II-plus III, including humans. Thus the risks, if they exist, must but not in the red blood cell component or in Cohn be very small, and should be balanced against the plasma fractions IV or V the albumen fraction from established benefits of using blood products. clinically ill mice that had been inoculated with a human CJD strain. Brown also claims to have 4.4.2. Iatrogenic transmissions detected CJD infectivity in plasma, buffy coat and The term ‘iatrogenic transmission’ means inadver- whole blood of human patients Brown, 1998. So tent and preventable induction of disease by medical far as ruminants are concerned, apart from an early veterinary treatments or procedures Webster’s report of transmission of a scrapie-like illness to Medical Dictionary, 1986 so it includes disease mice by inoculation of serum from an affected ram induced by reproductive technologies. Iatrogenic Gibbs et al., 1965, infectivity does not appear to transmissions of CJD in the medical field have had have been detected in blood or serum from scrapie- much publicity, with at least 80 known cases arising affected sheep and goats, or in blood clots, serum or from transplants of dura mater from cadavers which the buffy coat from BSE-affected cattle that were were subsequently shown or suspected of having had inoculated into mice. It is important to reiterate, CJD Brown, 1998. Smaller numbers have resulted however, that failure to detect infectivity does not from transplantation of eye tissues cornea and necessarily mean it is absent. Samples of concen- sclera Duffy et al., 1974 and also from use of trated blood or of specific types of blood cells from contaminated neurosurgical instruments or in- preclinically and clinically affected ruminants have tracerebral electrodes. In a recent case-control study seldom been tested, and bioassays, particularly in of risk factors for sporadic CJD in humans in mice, may be incapable of reliably detecting very Australia a range of surgical procedures were found low and intermittent levels of haematogenous infec- to be significantly associated with development of tivity Bolton, 1998. the disease Collins et al., 1999. The largest number The possibility of haematogenous carriage of TSE over 100 of known iatrogenic CJD cases, however, infectivity not only raises concerns about the use of has arisen from the use of pituitary hormones blood and blood products but, a priori, would imply growth hormone and gonadotrophins which were that most tissues and some secretions and excretions extracted from what were presumably infected from TSE-affected animals could also be potentially human cadavers Brown, 1998. Gonadotrophins infected. Concerns about haematogenous infectivity were mainly used for treatment of infertility and in have been particularly acute in the medical field IVF programmes. CJD incubation periods after CNS where, because certain individuals who died of CJD surgery or ocular exposure were often short 1 or 2 had been blood donors during their pre-clinical years whereas incubations following parenteral in- phase, the risks of transmission via blood or blood jection of pituitary hormones tended to be longer 5 components are being taken very seriously Will and to 35 years. Kimberlin, 1998. Among blood components per- Iatrogenic TSE transmissions have occurred in the ceived as a risk is serum albumen, which is used in veterinary field too, the best known example being human IVF, and, to the dismay of those practitioners an incident in the UK over 60 years ago in which and their patients who had already used them, some tissues brain, spinal cord and spleen from young batches of this and other blood products have had to sheep which must have been incubating scrapie were be withdrawn. In that the haematogenous TSE treated with formalin and used to make a vaccine hypothesis has already had an impact on the human against the tick-borne disease, louping-ill. Three reproductive technologies, its potential implications batches were made and several thousands of sheep for the ruminant technologies should not be lightly were vaccinated. Scrapie appeared 2.5 years later dismissed. Nevertheless, it is important to emphasise amongst sheep vaccinated with one of the batches, that no proven or probable instances of accidental and over 35 were affected on some farms Gordon, TSE transmission have arisen from blood transfu- 1946. Another outbreak of what appears to have 302 A .E. Wrathall Livestock Production Science 62 2000 287 –316 been iatrogenic scrapie occurred recently in Italy in IETS Stringfellow and Seidel, 1998. Quality con- sheep and goats vaccinated against the mycoplasmal trols should be based on these. disease, contagious agalactia, with a vaccine consist- Procedures involving surgery or laparoscopy used ing of homogenised, filtered ovine brain, mammary for AI and embryo collection and transfer in small gland and lymph nodes Capucchio et al., 1998. Of ruminants generally carry higher risks than the non- a total of over 1000 goats and 1000 sheep on three surgical procedures used to collect and transfer farms, 18.5 of the goats and 1.15 of the sheep embryos in cattle and other large ruminants. The developed scrapie. same applies to TVOR from live donors of whatever These incidents of iatrogenic TSE are salutary species, since this also involves invasion of the warnings of the hazards of using contaminated abdominal cavity. instruments or infected biological materials for medi- cal and veterinary purposes, including the reproduc- 5.2. Risks of transmission by instruments and tive technologies. Vigilance is essential to minimise equipment the risks. Although the risks of TSE transmission by instru- ments and equipment in reproductive technologies

5. Special risks of TSE transmission by are small, they are important. Disinfection proce-