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Scrapie is a transmissible spongiform encephalopathy in sheep and an example of a disease that may be controlled through breeding for disease resistance. Member states of the European Union have introduced strategies for breeding against scrapie based on the selection of genetically resistant breeding rams. An ambitious strategy adopted in The Netherlands consisted of selecting resistant rams for breeding throughout both breeding and production sectors. Mathematical modelling of the effect of a breeding program on the spreading capacity of scrapie in a national flock is needed for making assessments on how long a breeding strategy needs to be maintained to achieve disease control. Here we describe such a model applied to the Dutch situation, with the use of data on the genetic content of the Dutch sheep population as well as on scrapie occurrence in this population. We show that the time needed for obtaining scrapie control depends crucially on two parameters measuring sheep population structure: the between-flock heterogeneity in genotype frequencies, and the heterogeneity of mixing (contact rates) between sheep flocks. Estimating the first parameter from Dutch genetic survey data and assuming scenario values for the second one, enables model prediction of the time needed to achieve scrapie control in The Netherlands.

TSE strains are routinely identified by their incubation period and vacuolation profile in the brain after intracerebral inoculation and serial passaging in inbred mouse lines. There are some major drawbacks to this method that are related to the variation in vacuolation that exists in the brains of mice infected with the same TSE strain and to variation between observers and laboratories in scoring vacuolation and determining the final incubation period. Aim: We investigated the potential of PrPSc immunohistochemistry and triplex Western blotting as possible alternative methods to differentiate between TSE strains. Methods: TSE reference strains ME7, 87A/87V, 22A/22C, 79A/79V and 301C/301V were intracerebrally inoculated in RIII or VM inbred mice that differ in their PrP genotype. Immunohistochemical PrPSc profiles were drawn up by scanning light microscopy both on coronal and sagittal sections. Results: On the basis of the localization of PrPSc in the cerebral cortex, hippocampus, and cerebellar cortex and the overall type of PrPSc staining, all TSE strains could be well differentiated from each other through their typical strain dependent characteristics. In addition, Western blot showed that the combination of glycosylation profile and 12B2 epitope content of PrPSc allowed to distinguish between all reference strains except for ME7 and 22A in VM mice. Conclusion: TSE strains in mice can be identified on the basis of their PrPSc profile alone. The potential to identify TSE strains in ruminants with these PrPSc profiles after a single primary passage in mice will be the topic of future studies.

International audience; Efforts to differentiate bovine spongiform encephalopathy (BSE) from scrapie in prion infected sheep have resulted in effective methods to decide about the absence of BSE. In rare instances uncertainties remain due to assumptions that BSE, classical scrapie and CH1641-a rare scrapie variant-could occur as mixtures. In field samples including those from fallen stock, triplex Western blotting analyses of variations in the molecular properties of the proteinase K resistant part of the disease-associated form of prion protein (PrPres) represents a powerful tool for quick discrimination purposes. In this study we examined 7 deviant ovine field cases of scrapie for some typical molecular aspects of PrPres found in CH1641-scrapie, classical scrapie and BSE. One case was most close to scrapie with respect to molecular mass of its non-glycosylated fraction and N-terminally located 12B2-epitope content. Two cases were unlike classical scrapie but too weak to differentiate between BSE or CH1641. The other 4 cases appeared intermediate between scrapie and CH1641 with a reduced molecular mass and 12B2-epitope content, together with the characteristic presence of a second PrPres population. The existence of these 2 PrPres populations was further confirmed through deglycosylation by PNGaseF. The findings indicate that discriminatory diagnosis between classical scrapie, CH1641 and BSE can remain inconclusive with current biochemical methods. Whether such intermediate cases represent mixtures of TSE strains should be further investigated e.g. in bioassays with rodent lines that are varying in their susceptibility or other techniques suitable for strain typing.

Coxiella burnetii is the causative agent of the zoonotic disease Q fever. Since its first recognition as a disease in the 1930s, the knowledge about the agent and the disease itself has increased. This review summarizes the current knowledge on C. burnetii and Q fever, its pathogenesis, diagnosis and control. C. burnetii is a bacterium which naturally replicates inside human or animal host cells. The clinical presentation of Q fever varies per host species. C. burnetii infection in animals is mainly asymptomatic except for pregnant ruminants in which abortions and stillbirth can occur. In humans, the disease is also mainly asymptomatic, but clinical presentations include acute and chronic Q fever and the post-Q fever fatigue syndrome. Knowledge of the pathogenesis of Q fever in animals and excretion of C. burnetii in infected animals is crucial in understanding the transmission routes and risks of human infection. Our studies indicated that infected pregnant animals only excrete C. burnetii during and after parturition, independent of abortion, and that C. burnetii phase specific serology can be a useful tool in the early detection of infection. Domestic ruminants are the main reservoir for human Q fever, which has a major public health impact when outbreaks occur. In outbreaks, epidemiological source identification can only be refined by genotypic analysis of the strains involved. To control outbreaks and Q fever in domestic ruminants, vaccination with a phase 1 vaccine is effective. Future challenges are to identify factors for virulence, host susceptibility and protection.

Q fever is a zoonosis caused by the bacterium Coxiella burnetii. One of the largest reported outbreaks of Q fever in humans occurred in the Netherlands starting in 2007; epidemiologic investigations identified small ruminants as the source. To determine the genetic background of C. burnetii in domestic ruminants responsible for the human Q fever outbreak, we genotyped 126 C. burnetii–positive samples from ruminants by using a 10-loci multilocus variable-number tandem-repeat analyses panel and compared them with internationally known genotypes. One unique genotype predominated in dairy goat herds and 1 sheep herd in the human Q fever outbreak area in the south of the Netherlands. On the basis of 4 loci, this genotype is similar to a human genotype from the Netherlands. This finding strengthens the probability that this genotype of C. burnetii is responsible for the human Q fever epidemic in the Netherlands.

Transmissible spongiform encephalopathies (TSEs) or prion diseases are fatal neurodegenerative disorders and include among others Creutzfeldt–Jakob disease in humans, bovine spongiform encephalopathy (BSE) in cattle, and scrapie in sheep. The central event in disease development in TSEs is the refolding of the normal host-encoded cellular prion protein (PrP) into abnormal and disease associated prion protein. The agent is thought to consist mainly or exclusively of these pathologically folded PrP molecules. The exact molecular mechanisms underlying this process and the role of normal PrP in the conversion to pathological isoforms of PrP are still poorly understood. The highly conserved PrP gene structure and organisation however, suggests that its function is important, even though PrP knockout mice appear to develop normally. Conversion of normal PrP is initiated by interaction with abnormal PrP (or “agent”) resulting in refolding of normal PrP into new pathological PrP (“agent replication”). Normal PrP was shown to interact/bind with many different molecules including metal ions, nucleic acids, several (receptor) proteins, and the prion protein itself. The processes underlying agent replication (normal to abnormal PrP conversion) are most likely initiated by selective interaction between PrP molecules and potentially influenced by chaperone molecules. Thus far no vaccine, disease reversing therapeutic compounds or strategies (cure) exists, although there are some compounds capable of slowing the progression of prion disease. Studies towards interference to date have primarily focussed on inference with the interaction between normal and pathological isoforms of PrP in order to develop therapeutic strategies or find compounds capable of inhibiting prion propagation. Most described strategies are either directed at depletion of normal PrP and thus preventing pathological PrP formation and accumulation, or are based on preventing interaction between normal and abnormal PrP. Other therapeutic strategies focus on selective (self-)interaction of normal PrP molecules. Increased understanding of these interactions and the processes in which normal PrP plays a (active) role, could potentially lead to new modes of inhibiting prion protein conversion in which the physiological function(s) of normal PrP is retained. Ultimately this may lead to therapeutic strategies that are effective not only as a prophylactic but also in later stages of prion disease development. Here we review the data underlying these PrP-based approaches.

It is generally accepted that the bovine spongiform encephalopathy (BSE) epidemic is transmitted by feed contamination with meat and bone meal (MBM). Whether in some cases substances other than MBM have caused the spread of this disease cannot be excluded at present. Detailed knowledge about country-specific transmission routes and relevant risk factors is important to perform accurate risk analyses and to control BSE. In the present study all possible information of Dutch BSE cases was collected. The general epidemiological data were first used for a comprehensive descriptive analysis of the Dutch BSE epidemic. A case-control study was performed to identify the risk factors associated with the occurrence of BSE cases in the Netherlands. Sixty-eight of the 76 BSE cases enrolled in the case-control study. With an oral questionnaire information was collected about cow and farm management. The same questionnaire was used in interviews at 128 randomly selected control farms. Detailed analysis of the case anamneses showed that all affected cows could have been exposed to MBM from pig or poultry feed as a result of cross-contamination. In the case-control study, only feed producer appeared to be a relevant risk factor. Differences in log odds between feed producers were related to the moment of separation of production lines and to the origin of the MBM. The results suggest that there were meaningful differences in the level of infectivity in MBM from different origins at the time that cross-contamination was still possible. No other risk factors, either farm or cow related, were significantly associated with the occurrence of BSE.

Transmissible spongiform encephalopathy strains can be differentiated by their behavior in bioassays and by molecular analyses of the disease-associated prion protein (PrP) in a posttranslationally transformed conformation (PrP Sc ). Until recently, isolates from cases of bovine spongiform encephalopathy (BSE) appeared to be very homogeneous. However, a limited number of atypical BSE isolates have recently been identified upon analyses of the disease-associated proteinase K (PK) resistance-associated moiety of PrP Sc (PrP res ), suggesting the existence of at least two additional BSE PrP res variants. These are defined here as the H type and the L type, according to the higher and lower positions of the nonglycosylated PrP res band in Western blots, respectively, compared to the position of the band in classical BSE (C-type) isolates. These molecular PrP res variants, which originated from six different European countries, were investigated together. In addition to the migration properties and glycosylation profiles (glycoprofiles), the H- and L-type isolates exhibited enhanced PK sensitivities at pH 8 compared to those of the C-type isolates. Moreover, H-type BSE isolates exhibited differences in the binding of antibodies specific for N- and more C-terminal PrP regions and principally contained two aglycosylated PrP res moieties which can both be glycosylated and which is thus indicative of the existence of two PrP res populations or intermediate cleavage sites. These properties appear to be consistent within each BSE type and independent of the geographical origin, suggesting the existence of different BSE strains in cattle. The choice of three antibodies and the application of two pHs during the digestion of brain homogenates provide practical and diverse tools for the discriminative detection of these three molecular BSE types and might assist with the recognition of other variants.

In intra-EU trade, the health status of animals is warranted by issuing a health certificate after clinical inspection in the exporting country. This certificate cannot provide guarantee of absence of infection, especially not for diseases with a long incubation period and no overt clinical signs such as bovine tuberculosis (bTB). The Netherlands are officially free from bTB since 1999. However, frequent reintroductions occurred in the past 15 years through importation of infected cattle. Additional testing (AT) of imported cattle could enhance the probability of detecting an imported bTB infection in an early stage. The goal of this study was to evaluate the effectiveness of risk-based AT for bTB in cattle imported into The Netherlands. A generic stochastic import risk model was developed that simulates introduction of infection into an importing country through importation of live animals. Main output parameters are the number of infected animals that is imported (Ninf), the number of infected animals that is detected by testing (Ndet), and the economic losses incurred by importing infected animals (loss). The model was parameterized for bTB. Model calculations were optimized to either maximize Ndet or to minimize loss. Model results indicate that the risk of bTB introduction into The Netherlands is very high. For the current situation in which Dutch health checks on imported cattle are limited to a clinical inspection of a random sample of 5–10% of imported animals, the calculated annual Ninf = 99 (median value). Random AT of 8% of all imported cattle results in Ndet = 7 (median value), while the median Ndet = 75 if the sampling strategy for AT is optimized to maximize Ndet. However, in the latter scenario, loss is more than twice as large as in the current situation, because only calves are tested for which cost of detection is higher than the expected gain of preventing a possible outbreak. When optimizing the sampling strategy for AT to minimize loss, only breeding and production cattle are selected for AT resulting in Ndet = 1 (median value). Loss is; however, reduced by 75% if compared to the current situation. We conclude that the effectiveness of AT can greatly be improved by risk-based sampling. The optimal sampling strategy for risk-based AT for bTB is highly dependent on the objective of AT. If economic losses are to be contained, AT should focus on breeding and production cattle originating from high-risk countries.

We here describe the identification and characterization of three novel secreted Mycobacterium avium subsp. paratuberculosis antigens of 9, 15 and 34 kDa (Map2609, Map2942c and Map0210c, respectively) by screening a genomic expression library with a serum of a naturally infected clinical cow. The 9, 15 and 34 kDa antigens display strong homology to previously described M. tuberculosis antigens, TB8.4, MPT53 and Erp, respectively. Furthermore, these antigens were shown to be recognized by antibodies from infected cattle, when tested with a limited number of sera from subclinical (n = 7) and clinical (n = 3) infected cattle.

TSE strains are routinely identified by their incubation period and vacuolation profile in the brain after intracerebral inoculation and serial passaging in inbred mouse lines. There are some major drawbacks to this method that are related to the variation in vacuolation that exists in the brains of mice infected with the same TSE strain and to variation between observers and laboratories in scoring vacuolation and determining the final incubation period.We investigated the potential of PrP(Sc) immunohistochemistry and triplex Western blotting as possible alternative methods to differentiate between TSE strains.TSE reference strains ME7, 87A/87V, 22A/22C, 79A/79V and 301C/301V were intracerebrally inoculated in RIII or VM inbred mice that differ in their PrP genotype. Immunohistochemical PrP(Sc) profiles were drawn up by scanning light microscopy both on coronal and sagittal sections.On the basis of the localization of PrP(Sc) in the cerebral cortex, hippocampus, and cerebellar cortex and the overall type of PrP(Sc) staining, all TSE strains could be well differentiated from each other through their typical strain dependent characteristics. In addition, Western blot showed that the combination of glycosylation profile and 12B2 epitope content of PrP(Sc) allowed to distinguish between all reference strains except for ME7 and 22A in VM mice.TSE strains in mice can be identified on the basis of their PrP(Sc) profile alone. The potential to identify TSE strains in ruminants with these PrP(Sc) profiles after a single primary passage in mice will be the topic of future studies.

To the Editor: Q fever is a zoonosis caused by the bacterium Coxiella burnetii. The Q fever outbreak in the Netherlands affected ≈4,000 humans during 2007–2010 (1). In this outbreak, 1 genotype of C. burnetii appeared to be responsible for abortions in small ruminants and for clinical disease in humans (2,3). However, little is known about the outbreak genotype and the prevalence of C. burnetii in possible additional reservoirs for human Q fever (i.e., cats, dogs, horses, sheep, and cattle) in the Netherlands. We aimed to search for possible additional reservoirs for human Q fever in the Netherlands. Placentas from 15 cats, 54 dogs, and 31 horses were collected in 2011 at 5 veterinary practices. Placentas were collected by targeted sampling at breeding facilities and during parturition with veterinary assistance. In addition, 27 ovine, 11 caprine, 16 porcine, 8 equine, and 139 bovine placentas (originating from aborting animals from throughout the Netherlands that were submitted in 2011 to investigate the abortion cause) were included in the study. Samples were stored at −20°C before testing. DNA was extracted from the allantochorion of the placenta and analyzed as described (2). Samples with sufficient DNA load (cycle threshold [Ct] value

Q fever is a zoonosis caused by the intracellular bacterium Coxiella burnetii. Both humoral and cellular immunity are important in the host defence against intracellular bacteria. Little is known about the immune response to C. burnetii infections in domestic ruminants even though these species are the major source of Q fever in humans. To investigate the goat’s immune response we inoculated groups of pregnant goats via inhalation with a Dutch outbreak isolate of C. burnetii. All animals were successfully infected. Phase 1 and Phase 2 IgM- and IgG-specific antibodies were measured. Cellular immune responses were investigated by interferon-gamma, enzyme-linked immunosorbent spot test (IFN-¿ Elispot), lymphocyte proliferation test (LPT) and systemic cytokines. After two weeks post inoculation (wpi), a strong anti-C. burnetii Phase 2 IgM and IgG antibody response was observed while the increase in IgM anti-Phase 1 antibodies was less pronounced. IgG anti-Phase 1 antibodies started to rise at 6 wpi. Cellular immune responses were observed after parturition. Our results demonstrated humoral and cellular immune responses to C. burnetii infection in pregnant goats. Cell-mediated immune responses did not differ enough to distinguish between Coxiella-infected and non-infected pregnant animals, whereas a strong-phase specific antibody response is detected after 2 wpi. This humoral immune response may be useful in the early detection of C. burnetii-infected pregnant goats.

Coxiella burnetii is an intracellular bacterial pathogen that causes Q fever. Infected pregnant goats are a major source of human infection. However, the tissue dissemination and excretion pathway of the pathogen in goats are still poorly understood. To better understand Q fever pathogenesis, we inoculated groups of pregnant goats via the intranasal route with a recent Dutch outbreak C. burnetii isolate. Tissue dissemination and excretion of the pathogen were followed for up to 95 days after parturition. Goats were successfully infected via the intranasal route. PCR and immunohistochemistry showed strong tropism of C. burnetii towards the placenta at two to four weeks after inoculation. Bacterial replication seemed to occur predominantly in the trophoblasts of the placenta and not in other organs of goats and kids. The amount of C. burnetii DNA in the organs of goats and kids increased towards parturition. After parturition it decreased to undetectable levels: after 81 days post-parturition in goats and after 28 days post-parturition in kids. Infected goats gave birth to live or dead kids. High numbers of C. burnetii were excreted during abortion, but also during parturition of liveborn kids. C. burnetii was not detected in faeces or vaginal mucus before parturition. Our results are the first to demonstrate that pregnant goats can be infected via the intranasal route. C. burnetii has a strong tropism for the trophoblasts of the placenta and is not excreted before parturition; pathogen excretion occurs during birth of dead as well as healthy animals. Besides abortions, normal deliveries in C. burnetii-infected goats should be considered as a major zoonotic risk for Q fever in humans.

Background Susceptibility of sheep to scrapie infection is known to be modulated by the PrP genotype of the animal. In the Netherlands an ambitious scrapie control programme was started in 1998, based on genetic selection of animals for breeding. From 2002 onwards EU regulations required intensive active scrapie surveillance as well as certain control measures in affected flocks. Here we analyze the data on genotype frequencies and scrapie prevalence in the Dutch sheep population obtained from both surveillance and affected flocks, to identify temporal trends. We also estimate the genotype-specific relative risks to become a detected scrapie case. Results We find that the breeding programme has produced a steady increase in the level of genetic scrapie resistance in the Dutch sheep population. We also find that a significant decline in the prevalence of scrapie in tested animals has occurred a number of years after the start of the breeding programme. Most importantly, the estimated scrapie prevalence level per head of susceptible genotype is also declining significantly, indicating that selective breeding causes a population effect. Conclusions The Dutch scrapie control programme has produced a steady rise in genetic resistance levels in recent years. A recent decline in the scrapie prevalence per tested sheep of susceptible prion protein genotype indicates that selective breeding causes the desired population effect.

Background Following EU decision 2003/100/EC Member States have recently implemented sheep breeding programmes to reduce the prevalence of sheep with TSE susceptible prion genotypes. The present paper investigates the progress of the breeding programme in the Netherlands. The PrP genotype frequencies were monitored through time using two sets of random samples: one set covers the years 2005 to 2008 and is taken from national surveillance programme; the other is taken from 168 random sheep farms in 2007. The data reveal that although the level of compliance to the breeding programme has been high, the frequency of susceptible genotypes varies substantially between farms. The 168 sheep farms are a subset of 689 farms participating in a postal survey inquiring about management and breeding strategies. This survey aimed to identify how much these strategies varied between farms, in order to inform assessment of the expected future progress towards eradication of classical scrapie. Results On the one hand, we found that compliance to the national breeding program has been high, and the frequency of resistant genotypes is expected to increase further in the next few years. On the other hand, we observed a large variation in prevalence of the scrapie resistant PrP genotype ARR between farms, implicating a large variation of genetic resistance between farms. Substantial between-flock differences in management and breeding strategies were found in the postal survey, suggesting considerable variation in risk of scrapie transmission between farms. Conclusions Our results show that although there has been a good progress in the breeding for scrapie resistance and the average farm-level scrapie susceptibility in the Netherlands has been significantly reduced, still a considerable proportion of farms contain high frequencies of susceptible genotypes in their sheep population. Since 2007 the breeding for genetic resistance is voluntarily again, and participation to selective breeding can decrease as a result of this. This, together with the patterns of direct and indirect contact between sheep farms, might present a challenge of the aim of scrapie eradication. Communication to sheep owners of the effect of the breeding programme thus far, and of the prospects for classical scrapie eradication in The Netherlands might be essential for obtaining useful levels of participation to the voluntary continuation of the breeding programme.

International audience; Many studies have been undertaken in rodents to study the pathogenesis of transmissible spongiform encephalopathies (TSE). Only a few studies have focused on the pathogenesis of bovine spongiform encephalopathy (BSE) and scrapie in their natural hosts. In this review, we summarize the most recent insights into the pathogenesis of BSE and scrapie starting from the initial uptake of TSE agents and crossing of the gut epithelium. Following replication in the gut-associated lymphoid tissues (GALT), TSE agents spread to the enteric nervous system (ENS) of the gut. Infection is then carried through the efferent fibers of the post-ganglionic neurons of the parasympathetic and sympathetic nervous system to the pre-ganglionic neurons in the medulla oblongata of the brain and the thoracic segments of the spinal cord. The differences between the pathogenesis of BSE in cattle and scrapie in sheep are discussed as well as the possible existence of additional pathogenetic routes.

It is generally accepted that the bovine spongiform encephalopathy (BSE) epidemic is transmitted by feed contamination with meat and bone meal (MBM). Whether in some cases substances other than MBM have caused the spread of this disease cannot be excluded at present. Detailed knowledge about country-specific transmission routes and relevant risk factors is important to perform accurate risk analyses and to control BSE. In the present study all possible information of Dutch BSE cases was collected. The general epidemiological data were first used for a comprehensive descriptive analysis of the Dutch BSE epidemic. A case-control study was performed to identify the risk factors associated with the occurrence of BSE cases in the Netherlands. Sixty-eight of the 76 BSE cases enrolled in the case-control study. With an oral questionnaire information was collected about cow and farm management. The same questionnaire was used in interviews at 128 randomly selected control farms. Detailed analysis of the case anamneses showed that all affected cows could have been exposed to MBM from pig or poultry feed as a result of cross-contamination. In the case-control study, only feed producer appeared to be a relevant risk factor. Differences in log odds between feed producers were related to the moment of separation of production lines and to the origin of the MBM. The results suggest that there were meaningful differences in the level of infectivity in MBM from different origins at the time that cross-contamination was still possible. No other risk factors, either farm or cow related, were significantly associated with the occurrence of BSE.

Chronic wasting disease (CWD) is an emerging transmissible spongiform encephalopathy (prion disease) of North American cervids, i.e., mule deer, white-tailed deer, and elk (wapiti). To facilitate in vitro studies of CWD, we have developed a transformed deer cell line that is persistently infected with CWD. Primary cultures derived from uninfected mule deer brain tissue were transformed by transfection with a plasmid containing the simian virus 40 genome. A transformed cell line (MDB) was exposed to microsomes prepared from the brainstem of a CWD-affected mule deer. CWD-associated, protease-resistant prion protein (PrP CWD ) was used as an indicator of CWD infection. Although no PrP CWD was detected in any of these cultures after two passes, dilution cloning of cells yielded one PrP CWD -positive clone out of 51. This clone, designated MDB CWD , has maintained stable PrP CWD production through 32 serial passes thus far. A second round of dilution cloning yielded 20 PrP CWD -positive subclones out of 30, one of which was designated MDB CWD2 . The MDB CWD2 cell line was positive for fibronectin and negative for microtubule-associated protein 2 (a neuronal marker) and glial fibrillary acidic protein (an activated astrocyte marker), consistent with derivation from brain fibroblasts (e.g., meningeal fibroblasts). Two inhibitors of rodent scrapie protease-resistant PrP accumulation, pentosan polysulfate and a porphyrin compound, indium (III) meso-tetra(4-sulfonatophenyl)porphine chloride, potently blocked PrP CWD accumulation in MDB CWD cells. This demonstrates the utility of these cells in a rapid in vitro screening assay for PrP CWD inhibitors and suggests that these compounds have potential to be active against CWD in vivo.

Molecular typing of the abnormal form of the prion protein (PrP(Sc)) has come to be regarded as a powerful tool in the investigation of the prion diseases. All evidence thus far presented indicates a single PrP(Sc) molecular type in variant Creutzfeldt-Jakob disease (termed type 2B), presumably resulting from infection with a single strain of the agent (bovine spongiform encephalopathy). Here we show for the first time that the PrP(Sc) that accumulates in the brain in variant Creutzfeldt-Jakob disease also contains a minority type 1 component. This minority type 1 PrP(Sc) was found in all 21 cases of variant Creutzfeldt-Jakob disease tested, irrespective of brain region examined, and was also present in the variant Creutzfeldt-Jakob disease tonsil. The quantitative balance between PrP(Sc) types was maintained when variant Creutzfeldt-Jakob disease was transmitted to wild-type mice and was also found in bovine spongiform encephalopathy cattle brain, indicating that the agent rather than the host specifies their relative representation. These results indicate that PrP(Sc) molecular typing is based on quantitative rather than qualitative phenomena and point to a complex relationship between prion protein biochemistry, disease phenotype and agent strain.

A significant increase in the incidence of caseous lesions in the lymph nodes of slaughter pigs prompted a large-scale investigation in five slaughterhouses in The Netherlands. In total, 158,763 pigs from 2,899 groups underwent gross examination. At least one pig with caseous lesions in the submaxillary and/or mesenteric lymph nodes was observed in each of 154 of the 2,899 groups examined (5%). In total, 856 pigs (0.5%) were affected. As many as five pigs in each of 141 of the 154 positive groups (91.5%) had lymph node lesions. Greater numbers of pigs with affected lymph nodes were found in 13 groups (8.5%). Four pigs had lesions in the kidneys, liver, or spleen. Acid-fast bacteria were detected by microscopic examination of 121 of 292 Ziehl-Neelsen-stained smears of caseous lesions (41%). In a follow-up study, Mycobacterium avium complex (MAC) bacteria were isolated from 219 of 402 affected lymph nodes (54.2%). Ninety-one of the isolated strains were analyzed by restriction fragment length polymorphism (RFLP) typing with insertion sequence IS 1245 as a probe. All but 1 of these 91 strains contained IS 1245 DNA, indicating that pigs in The Netherlands carried almost exclusively M. avium bacteria and no other bacteria of MAC. Only one pig isolate exhibited the bird-type RFLP pattern. MAC isolates from 191 human patients in The Netherlands in 1996 were also typed by RFLP analysis. Computer-assisted analysis showed that the RFLP patterns of 61% of the human isolates and 59% of the porcine isolates were at least 75% similar to the RFLP patterns of the other group of strains. This indicates that pigs may be an important vehicle for M. avium infections in humans or that pigs and humans share common sources of infection.

Background Diagnosis based on prion detection in lymph nodes of sheep and goats can improve active surveillance for scrapie and, if it were circulating, for bovine spongiform encephalopathy (BSE). With sizes that allow repetitive testing and a location that is easily accessible at slaughter, retropharyngeal lymph nodes (RLN) are considered suitable organs for testing. Western blotting (WB) of brain homogenates is, in principle, a technique well suited to both detect and discriminate between scrapie and BSE. In this report, WB is developed for rapid diagnosis in RLN and to study biochemical characteristics of PrPres.

Background Molecular mechanisms underlying prion agent replication, converting host-encoded cellular prion protein (PrPC) into the scrapie associated isoform (PrPSc), are poorly understood. Selective self-interaction between PrP molecules forms a basis underlying the observed differences of the PrPC into PrPSc conversion process (agent replication). The importance of previously peptide-scanning mapped ovine PrP self-interaction domains on this conversion was investigated by studying the ability of six of these ovine PrP based peptides to modulate two processes; PrP self-interaction and conversion. Results Three peptides (octarepeat, binding domain 2 -and C-terminal) were capable of inhibiting self-interaction of PrP in a solid-phase PrP peptide array. Three peptides (N-terminal, binding domain 2, and amyloidogenic motif) modulated prion conversion when added before or after initiation of the prion protein misfolding cyclic amplification (PMCA) reaction using brain homogenates. The C-terminal peptides (core region and C-terminal) only affected conversion (increased PrPres formation) when added before mixing PrPC and PrPSc, whereas the octarepeat peptide only affected conversion when added after this mixing. Conclusion This study identified the putative PrP core binding domain that facilitates the PrPC-PrPSc interaction (not conversion), corroborating evidence that the region of PrP containing this domain is important in the species-barrier and/or scrapie susceptibility. The octarepeats can be involved in PrPC-PrPSc stabilization, whereas the N-terminal glycosaminoglycan binding motif and the amyloidogenic motif indirectly affected conversion. Binding domain 2 and the C-terminal domain are directly implicated in PrPC self-interaction during the conversion process and may prove to be prime targets in new therapeutic strategy development, potentially retaining PrPC function. These results emphasize the importance of probable PrPC-PrPC and required PrPC-PrPSc interactions during PrP conversion. All interactions are probably part of the complex process in which polymorphisms and species barriers affect TSE transmission and susceptibility.