Inactivation of the BSE agent by the heat and pressure process for manufacturing gelatine.

Dietary exposure to the bovine spongiform encephalopathy (BSE) agent is the probable cause of variant Creutzfeldt-Jakob disease in people. The industrial manufacturing process for the production of gelatine and colloidal protein by the heat and pressure process was downscaled accurately and its capacity to remove or inactivate bse infectivity was investigated. Gelatine was made from bones experimentally contaminated with mouse brain infected with the 301V strain of mouse-passaged bse agent in which the infective titre was 10(8.7) ID50/g. No infectivity was detected in the extracted protein (> or =10(0.45) ID50/g), and the calculated clearance factor was 10(6.5) ID50 or more.

Afghanistan and the development of alternative systems of animal health in the absence of effective government.

This case study describes the efforts by both non-governmental organisations and United Nations agencies to develop an alternative system for delivering animal health services in Afghanistan, during a period in which there was effectively no government. The authors examine the period from the mid-1980s to the year 2003. During this time, Afghanistan experienced war and severe civil unrest, resulting in the collapse of the veterinary infrastructure. As most trained animal health professionals had fled the country, an initial emphasis was placed on training intermediate and lower-level veterinary auxiliary personnel, as well as on the implementation of emergency treatment and vaccination campaigns. Gradually this programme has developed from an emergency-oriented approach to a more development-oriented process, resulting in a community-based system of animal health care in more than 250 districts (out of approximately 360). Some 500 paraveterinarians, trained for a period of five months, play a pivotal role in this programme, supported in outlying villages by trained vaccinators and basic veterinary workers. In this paper, the authors present an estimation of the impact of this programme. Essential elements of the programme are, as follows: the recruitment of trainees from areas where need has been identified; an emphasis on practical and problem-oriented training; the deployment of staff in so-called 'veterinary field units', supervised by more highly qualified staff and monitors; a guaranteed supply of veterinary medicines, anthelmintics and vaccines; a gradually increasing rate of cost recovery. The ultimate objective of the programme is to establish a self-sustaining system, based on the 'user-pays' principle. The paper concludes by describing the present-day problems of the animal health infrastructure in Afghanistan. Not only must the new government try to regain its central position, it must also assimilate two decades of development in the veterinary sector, which has occurred largely outside governmental control.

Bovine spongiform encephalopathy in sheep?

Bovine spongiform encephalopathy (BSE) in sheep has not been identified under natural conditions at the time of writing and remains a hypothetical issue. However, rumours about the possible finding of a BSE-like isolate in sheep have led to great unrest within the sheep industry, among the general public and within governmental and regulatory bodies. The difficulties of implementing a proper risk assessment and pre-emptive measures, in the absence of a confirmed case, are described. The authors attempt to list what is known about experimental BSE in sheep, the distribution of infectivity in the host, some aspects of risk assessment and management and the most promising methods for differentiating BSE from scrapie in the same host. As for the latter, new and promising methods are being developed and appear suitable for initial screening of isolates of transmissible spongiform encephalopathies, but in the absence of proper validation, use of the 'classical' strain-typing in a mouse panel is still indicated.

[Where does BSE in the Netherlands come from?]. / Waar komt BSE in Nederland vandaan?

It is only in the last 5 years that the Netherlands has been confronted with cases of bovine spongiform encephalopathy (BSE). The cases diagnosed to date have not been clearly linked to imports from the United Kingdom. This article describes the various possible explanations for the Dutch cases. The risk factors involved, have either a connection with imported BSE, local origin of BSE, or both. These factors can also be divided into introductory risk and propagation risk, terms that were also used in an EU risk assessment study. Research at ID-Lelystad since the early 1990s and at IKC-Ede has tried to assess the relative importance of the various risk factors, the results of which are discussed in this paper. The paper does not deal with the specifics of the cases diagnosed to date, because of the absence of an in-depth epidemiological investigation, but provides a general assessment of the risk factors that might have played a role. Important factors have been, in addition to the initial imports of cattle and meat and bone-meal from the UK, the continuing imports from other countries with covert BSE and the cross-contamination within the animal feed production lines. Emphasis is on the period of the early and mid-1990s, the period in which most calves with diagnosed BSE were born.

Diagnosis of bovine spongiform encephalopathy: a review.

Cows affected with bovine spongiform encephalopathy (BSE) display chronic neurological signs consisting of behavioural changes, abnormalities of posture and movement, and/or hyperaesthesia. At present, there are no laboratory test available to diagnose BSE in the live animal. In this article, we describe the post-mortem diagnostic examination of brains from BSE-suspected cattle as currently performed at ID-Lelystad. The routine laboratory diagnosis of BSE consists of histopathological examination of the brain and detection of the modified prion protein, PrP(BSE), in brain tissue. These tests, however, have the disadvantage of being laborious and time consuming, so that results are available only after several days. Recently, at ID-Lelystad a new post-mortem test has been developed that enables screening of larger volumes of brain samples for PrP(BSE) within 1 day. This BSE test is especially suited for slaughterline monitoring. A preliminary validation study has shown that both sensitivity and specificity are 100% compared to the gold diagnostic standard of histopathology.

Pathogenesis of natural scrapie in sheep.

Although scrapie has been known for a long time as a natural disease of sheep and goats, the pathogenesis in its natural host still remains unclear. To study the pathogenesis of natural scrapie, we used immunohistochemistry to monitor the deposition of PrP(Sc) in various tissues, collected during a natural scrapie infection from sheep with the PrP(VRQ)/PrP(VRQ) genotype which were purposely bred for their short incubation period for natural scrapie. PrP(Sc) was present in the lymphoid tissues of all animals from the age of 5 months onwards. At this age, PrP(Sc) was detected in the neural tissues only in the enteric nervous system (ENS) at the level of the duodenum and ileum. At the age of 10 months, PrP(Sc) was not only found in the ENS but also in the ganglion mesentericum cranialis/coeliacum, the dorsal motor nucleus of the vagus, and the intermediolateral column of the thoracic segments T8-T10. PrP(Sc) was detected for the first time in the nucleus tractus solitarius and ganglion nodosus at 17 months of age and in the ganglion trigeminale and several spinal ganglia at 21 months of age. Since the scrapie agent consists largely, if not entirely of PrP(Sc), these results indicate that the ENS acts as a portal of entry to the neural tissues for the scrapie agent followed by centripetal and retrograde spread through sympathetic and parasympathetic efferent fibers of the autonomic nervous system to the spinal cord and medulla oblongata respectively. PrP(Sc) accumulation in sensory ganglia occurs after infection of the CNS and is therefore probably due to centrifugal and anterograde spread of the scrapie agent from the CNS through afferent nerve fibers.

Scrapie-associated prion protein in the gastrointestinal tract of sheep with natural scrapie.

The scrapie-associated prion protein (PrPSc), which is closely associated with scrapie infectivity, accumulates in the brain and lymphoid tissues of sheep with natural scrapie. The most probable portal of entry of the scrapie agent in sheep is the alimentary tract; little attention, however, has been paid to the gastro-intestinal tract in scrapie research. In this study, we examined the presence and distribution of PrPSc within the gastro-intestinal tract of sheep with natural scrapie and scrapie-negative sheep. It was found that PrPSc accumulated in the enteric nervous system (ENS) of all scrapie-infected sheep but not in scrapie-negative sheep. The distribution of PrPSc within the ENS was then studied along the entire gastro-intestinal tract in seven scrapie-infected sheep carrying various PrP genotypes. In sheep with the highest genetically determined susceptibility to scrapie, PrPSc was detected in the ENS from the oesophagus to the rectum. In sheep with a lower genetic susceptibility to scrapie, PrPSc was present in the ENS of the forestomachs, small intestine and large intestine but not in the oesophagus. In a scrapie-negative sheep with a PrP genotype associated with scrapie resistance, no PrPSc was seen in the ENS at any site along the gastro-intestinal tract. The presence of PrPSc within the ENS of scrapie-infected sheep indicates a possible role of the ENS in the pathogenesis of natural scrapie as a portal of entry to the central nervous system.

[Epidemiologic aspects of scrapie and BSE including an analysis of risk factors]. / Epidemiologische aspecten van scrapie en BSE inclusief een analyse van risicofactoren.

After a discussion of the different hypotheses about the causative agent of prion diseases, various aspects of the two most important animal prion diseases, i.e. BSE and scrapie are described. This thesis focuses on the search for a preclinical diagnosis. A major breakthrough was the discovery of a new technique for detecting the disease-associated protein in tonsillar biopsies from scrapie-infected sheep long before clinical signs appeared. Another essential part of the studies described in this thesis concerned a risk analysis for BSE in a country like the Netherlands. Major risk factors were assessed, including an assessment of the efficacy of Dutch rendering procedures in the inactivation of the agents of scrapie and BSE.

Further observations on the impact of a veterinary programme in Afghanistan on seasonal livestock mortality.

This paper reports further results of a two year livestock mortality survey in Afghanistan, where a war of more than a decade had completely disrupted the veterinary field services. A questionnaire-based survey to measure the impact of a veterinary field programme indicated that average annual mortality in cattle, sheep, and goats was substantially lower in districts that received veterinary services (covered districts) than in districts without any veterinary services (control districts). The impact of the programme varies according to the season and the age group of the animals involved. The programme lacked impact in winter, in particular in adult small ruminants. The highest impact was noted in the spring and autumn for adult goats, and summer and autumn for adult sheep. The impact of the programme was also limited (in small ruminants even completely absent) during the suckling period in young animals. The highest impact was noted in the post-weaning period in small ruminants, when approximately four times more lambs and kids died in the control districts than in the covered districts. It was concluded that the major impact of the veterinary programme was achieved when animals are in contact with other flocks during grazing seasons, when parasitic and infectious diseases are present and against which the veterinary programme is directed. Additional inputs-including extension activities-are therefore required to improve the effect of the programme in the winter and in the neonatal period.

Studies on the efficacy of hyperbaric rendering procedures in inactivating bovine spongiform encephalopathy (BSE) and scrapie agents.

The efficacy of the procedures in use at the two rendering plants in the Netherlands was assessed on a laboratory-scale using procedures that simulated the pressure cooking part of the rendering process. A pool of bovine spongiform encephalopathy (BSE)-infected brainstem from the United Kingdom and a pool of scrapie-infected brainstem from Dutch sheep were used to spike the rendering materials. The mixtures were subjected to various time-temperature combinations of hyperbaric heat treatment related to the conditions used in Dutch rendering plants in the early 1990s, and to the combination of 20 minutes at 133 degrees C required by the EU Directive on rendering of 1996. The efficacy of the procedures in inactivating BSE or scrapie infectivity was measured by titrating the materials before and after heat treatment in inbred mice, by combined intracerebral and intraperitoneal inoculations at limiting dilutions. Two independent series of experiments were carried out. The design of the study allowed for minimum inactivations of up to 2.2 log (2.0 in the second series) to be measured in the diluted infective material and 3.1 log in the undiluted material. After 20 minutes at 133 degrees C there was a reduction of BSE infectivity of about 2.2 log in the first series (with some residual infectivity detected), and in the second series more than 2.0 log (with no residual infectivity detected). With undiluted brain material there was an inactivation of about 3.0 log (with some residual infectivity detected). With the same procedure, scrapie infectivity was reduced by more than 1.7 log in the first series and by more than 2.2 log in the second series. With undiluted brain material there was an inactivation of more than 3.1 log. In each case no residual scrapie infectivity was detected. The BSE agent consistently appeared to be more resistant to heat inactivation procedures than the scrapie agent, particularly at lower temperatures and shorter times.

Tonsillar biopsy and PrPSc detection in the preclinical diagnosis of scrapie.

Preliminary findings have indicated that in naturally infected sheep, fully susceptible to scrapie (VRQ-homozygous), PrPSc can be detected in the tonsils approximately one year before the expected onset of clinical disease, whereas no immunostaining can be detected in animals with a semi-resistant genotype. This paper describes the technique for taking tonsillar biopsies from sheep and gives the results of the completed experiment. In another experiment PrPSc was detected even earlier in comparable VRQ-homozygous sheep born and raised in different surroundings. At three-and-a-half months of age no PrPSc could be detected in three homozygous susceptible sheep (VRQ/VRQ), but PrPSc was detected at four months in one similar sheep. At eight months of age all seven sampled VRQ/VRQ sheep showed positive immunostaining in the biopsies, but none of the biopsies from three VRQ/ARQ heterozygotes showed any immunostaining; they were positive when sampled at 14 to 15 months of age. Biopsies from VRQ/ARR sheep were negative throughout this period. On the basis of the established or expected incubation period, PrPSc could thus be detected in the tonsils of live susceptible animals at between one-third and a half of the incubation period, more than one-and-a-half years before clinical signs normally appear in both these genotypes.

Prion protein and scrapie susceptibility.

This article presents briefly current views on the role of prion protein (PrP) in Transmissible Spongiform Encephalopathies or prion diseases and the effect of PrP polymoryhisms on the susceptibility to these diseases, with special emphasis on sheep scrapie. The PrP genotype of sheep appears to be a major risk factor for scrapie, and polymorphisms at codons 136, 154, and 171 modulate the susceptibility of sheep for scrapie. Nevertheless, scrapie is not a spontaneous genetic disease alone. We describe an in vitro system in which sheep PrP variants show characteristics which reflect their linkage with in vivo scrapie susceptibility. Studies with this in vitro system not only confirm that scrapie susceptibility is determined by the PrP genotype of the target animal, but also suggest that the PrP genotype of the animal that is the source of the infectious agent plays an important role in determining scrapie susceptibility. The behaviour of PrP variants in this in vitro system may be an indicator for the transmissibility of prion diseases.

Control of scrapie eventually possible?

After a brief description of the scrapie situation in the Netherlands, the technical progress made in aspects of scrapie diagnosis is reported. Emphasis is placed on the use of immuno-histochemistry (IHC) in the post-mortem histological diagnosis and on the recently published preclinical test for scrapie, in which IHC is applied to tonsillar biopsies. These two approaches use the same IHC technique and enable us to confirm suspected scrapie in individual animals, and for certain genotypes even in the preclinical phase. The tonsillar biopsy method could eventually lead to an infection- or PrPSc-detection method at flock level. Further work is required, including validation of the assay for various breeds, genotypes, and strains of the agent, and the conversion of the test into a more practical assay. The article continues with a discussion of several scrapie control strategies, describing briefly schemes tried in various countries, and elaborates on a proposed scrapie control scheme that could be suitable for the Netherlands. This scheme is essentially based on breeding for resistance, based on PrP genotyping.

Risk of BSE from the import of cattle from the United Kingdom into countries of the European Union.

This study assesses quantitatively the risk that other countries, in particular those within the European Union, have incurred by importing cattle from the United Kingdom during the period before or shortly after the ban on the import of live breeding stock was introduced in 1989. It does this by assessing the probability that animals imported from the UK in a certain year would have become a detected BSE case, had they not been exported. Using the annual incidence rates available for separate birth cohorts and a given culling rate, a cumulative incidence for each birth cohort was calculated. These figures were then combined with the numbers of live breeding cattle imported from the UK into the other countries of the EU, to give an import-related risk index for each country, assuming that their culling rates were similar to that in Great Britain. The countries could thus be categorised in terms of the number of cases of BSE they might have expected.

PrP genotype contributes to determining survival times of sheep with natural scrapie.

Several allelic variants of the sheep PrP gene are associated with scrapie susceptibility. However, it is not known whether, and to what extent, the PrP genotype contributes to determining survival times of scrapie sheep. We therefore determined the PrP genotype and life spans of over 50 Flemish and Swifter sheep within a single scrapie-affected flock. Eighty-three per cent of the scrapie sheep were homozygous for the PrP(VQ) allele (polymorphic amino acids at codons 136 and 171 are indicated) and these sheep died from scrapie at a mean age of 25 months. In sheep heterozygous for PrP(VQ), development of scrapie was delayed or did not occur. Sheep with at least one PrP(AR) allele, including PrP(VQ)/PrP(AR) sheep, did not develop scrapie. No scrapie sheep were found without a PrP(VQ) allele. We conclude that the PrP genotype contributes to determining survival times of sheep with natural scrapie. Additionally, we describe two novel sheep PrP allelic variants.

Livestock mortality in Afghanistan in districts with and without a veterinary programme.

This paper reports on livestock mortality in Afghanistan where a war has been going on for more than a decade, with complete disruption of the veterinary field services. The study attempted to measure the impact of a veterinary field programme carried out mainly by paravets. The study also provides valuable data on the impact of diseases in the absence of any veterinary intervention. The veterinary programme, implemented by a Dutch non-governmental organisation (NGO) for approximately 3 years, consisted essentially of vaccination against major infectious diseases and administration of anthelmintic drugs for nematodes and liver flukes. Veterinary personnel were also involved in curative treatments. Livestock mortality figures were collected by questionnaire over a period of 2 years from more than 700 farmers in randomly selected villages in 4 covered districts with a veterinary programme and 4 control districts without a veterinary programme. The average annual mortality rates for calves, lambs and kids respectively were 16.2%, 17.3% and 19.1% in the covered areas, against 21.5%, 25.2% and 24.6% in the control areas. Adult mortality figures were 3.8%, 7.4% and 5.4% in the covered areas, against 5.3%, 13.6% and 15.6% in the control areas for cattle, sheep and goats respectively. The survey indicated significant differences (P < 0.01 in cattle, P < 0.001 in small ruminants) in livestock mortality between the control and covered areas and it is concluded that these differences were attributable to the presence or absence of the animal health programme.