BSE in France: epidemiological analysis and predictions

Attention throughout Europe continues to focus on bovine spongiform encephalopathy (BSE) with increasing evidence linking it to the new variant of Creutzfeldt-Jakob disease (vCJD) in humans. The age- and cohort-specific incidence of BSE in French cattle was modelled as a function of the survival distribution, the cohort-specific incidence of BSE infection, the underreporting rate of BSE cases, and the age-specific probability, conditional on survival, that an infected animal would experience clinical onset. The results reveal that thousands of French cattle were infected with BSE over the course of the epidemic. However, case incidence is predicted to decline in future years.     

Spatial dynamics of plant species in an agricultural landscape in the Netherlands

This study examined the changes in distribution patterns of 13 herbaceous plant species from 1998 to 2000 in ditch banks along the edges of arable fields in the Netherlands. The objective was to test if spatial dynamics could be related to spatial isolation and disturbance of habitat and to the dispersal and seed bank characteristics of the species. Knowledge of these relations should be used to increase the effectivity of agri-environmental schemes aiming at an increase of botanical diversity. All species frequently colonized empty patches and populations in occupied patches frequently went extinct. Most colonization events occurred within 50 m of conspecific source patches in the preceding year, but colonization events in patches at distances more than 200 m from conspecific source patches were also observed. The colonization probabilities decreased with isolation distance. For nine species this relation was statistically significant, after correction for year and habitat. The extinction probabilities increased with isolation. For only four species this relation was statistically significant. Both colonization and extinction probabilities were more often statistically significant related to isolation for species with transient seed banks than species with persistent seed banks. Implications for management options aiming at survival of plant species in fragmented landscapes are discussed.     

Estimating incidence of the French BSE infection using a joint analysis of both asymptomatic and clinical BSE surveillance data

Bovine Spongiform Encephalopathy (BSE) clinical surveillance data were the main source of information to perform back-calculation of BSE infection incidence. Since 2001, systematic BSE screening tests enhanced the clinical surveillance and allowed to detect some preclinical, i.e. asymptomatic, cases of BSE. We propose a method to incorporate additional information provided by screening tests. It was the first time that a back-calculation model was developed for a full BSE clinical surveillance. In the spirit, our approach resembles what it was done in the Acquired Immune Deficiency Syndrome (AIDS) epidemic to incorporate the Human Immunodeficiency Virus (HIV) diagnosis. Nevertheless, in the BSE epidemic, we had to consider different surveillance systems, their peculiarity, and the phenomenon of communicating vessels between these surveillance systems. In addition, both the preclinical sensitivity of tests and the status of BSE cases, asymptomatic or clinical, were not precisely known. We applied the model to the French BSE epidemic in order to obtain an updated estimate of the incidence of BSE infection. Our back-calculation model fitted very well the observed data of each surveillance system. We detected a lengthening of the incubation period and estimated that the number of infections was very small in the late 1990s and zero in July 2001.     

Comparative analysis of Japanese and foreign L-type BSE prions

L-type bovine spongiform encephalopathy (BSE) is an atypical form of BSE. To characterize the Japanese L-type BSE prion, we conducted a comparative study of the Japanese and foreign L-type BSE isolates. The L-type BSE isolates of Japan, Germany, France and Canada were intracerebrally inoculated into bovinized prion protein-overexpressing transgenic mice (TgBoPrP). All the examined L-type BSE isolates were transmitted to TgBoPrP mice, and no clear differences were observed in their biological and biochemical properties. Here, we present evidence that the Japanese and Canadian L-type BSE prions are identical to those from the European cases.Key words: prion, atypical BSE, L-type BSEBovine spongiform encephalopathy (BSE) is one of the transmissible spongiform encephalopathies (TSEs), or prion diseases, in cattle. TSE is characterized by spongiform changes in the central nervous system (CNS) and the accumulation of an abnormal prion protein (PrP^Sc) in the CNS.¹ PrP^Sc has been regarded as the major component of TSE pathogens.²BSE was detected in the UK in 1986,³ and subsequently spread to the other European countries, Japan and North America.^4–6 BSE is thought to be caused by a single prion strain, based on the analyses of its biological and biochemical characteristics.⁷ From 2003, however, several atypical neuropathological and molecular phenotypes of BSE (atypical BSE) have been detected in Japan, several European countries and North America.^6,8–17 Currently, based on the molecular size of the proteinase-digested non-glycosylated form of PrP^Sc, atypical BSE is classified into two groups (L-type and H-type).¹⁴L-type BSE cases have been identified in the European countries, including Italy, France, Germany, Netherland, Poland and in Canada and Japan.^8–15 Two L-type BSE cases have been identified in Japan. One case was detected in a healthy 23-mo-old Holstein steer (BSE/JP8),⁸ and the other was detected in a 14-y-old black Japanese beef cattle (BSE/JP24).⁹ The latter case was successfully transmitted to bovinized transgenic mice and cattle, and the biological and biochemical properties differed from that of classical BSE (C-BSE).^18,19 However, it is unclear whether Japanese L-type BSE prion is identical to that of L-type BSE isolates from other countries. To characterize the Japanese L-type BSE isolate, we performed a comparative study of the Japanese and foreign L-type BSE isolates.A transmission study using experimental animals is a useful approach for prion characterization. Therefore, we performed a transmission study of the L-type BSE isolates in bovinized prion protein (PrP)-overexpressing transgenic mice (TgBoPrP).²⁰ Brain samples of L-type BSE-affected cattle from Japan (BSE/JP24),⁹ France,¹⁰ Germany¹¹ and Canada¹² were used in this study. The brain homogenates were intracerebrally inoculated into TgBoPrP using previously described methods in reference ¹⁸. All animal experiments were reviewed by the Committee of the Ethics on Animal Experiment of the National Institute of Animal Health.All the examined L-type BSE isolates were transmitted to TgBoPrP, and the affected mice developed progressive neurological diseases. Japanese L-type BSE isolate-affected TgBoPrP exhibited a unique clinical sign, the circling behavior. The same phenotype was observed when TgBoPrP were inoculated with German, French and Canadian L-type BSE isolates. On the other hand, in the first passage the incubation period for the Japanese L-type BSE isolate was significantly different from that of the other L-type BSE isolates (

Comparative analysis of Japanese and foreign L-type BSE prions

L-type bovine spongiform encephalopathy (BSE) is an atypical form of BSE. To characterize the Japanese L-type BSE prion, we conducted a comparative study of the Japanese and foreign L-type BSE isolates. The L-type BSE isolates of Japan, Germany, France and Canada were intracerebrally inoculated into bovinized prion protein-overexpressing transgenic mice (TgBoPrP). All the examined L-type BSE isolates were transmitted to TgBoPrP mice, and no clear differences were observed in their biological and biochemical properties. Here, we present evidence that the Japanese and Canadian L-type BSE prions are identical to those from the European cases.     

Inactivation of the BSE agent

In the studies carried out so far, the BSE agent has proved to be just as resistant as other TSE agents to inactivation by procedures such as autoclaving or exposure to sodium hydroxide that are effective with conventional microorganisms. However, in common with other TSE agents, the BSE agent appears to be effectively inactivated by exposure to sodium hypochlorite solutions containing high levels of available chlorine. Not surprisingly, the BSE agent has been found to survive at least some of the rendering processes that were used to process tissues discarded by abattoirs in the EU during the early 1980s. Despite the survival of BSE infectivity after autoclaving or exposure to sodium hydroxide, it is known that combining these procedures results in a very reliable degree of inactivation for TSE agents generally. The combination of heat and alkali has also been shown to be effective with a mouse-passaged strain of BSE agent, even at a temperature of only 100 degrees C for a minute. Also, in carrying out BSE-spiked validation studies relating to the safety of bone-derived gelatin, it has also been found that the exposure of acid-treated bone (which is free from any obvious remains of fatty or proteinaceous tissue) to 0.3 M sodium hydroxide for two hours knocks out any residual BSE infectivity.     

A study on the analytical sensitivity of 6 BSE tests used by the Canadian BSE reference laboratory

Bovine spongiform encephalopathy (BSE) surveillance programs have been employed in numerous countries to monitor BSE prevalence and to protect animal and human health. Since 1999, the European Commission (EC) authorized the evaluation and approval of 20 molecular based tests for the rapid detection of the pathological prion protein (PrP^sc) in BSE infection. The diagnostic sensitivity, convenience, and speed of these tests have made molecular diagnostics the preferred method for BSE surveillance. The aim of this study was to determine the analytical sensitivity of 4 commercially available BSE rapid-test kits, including the Prionics®-Check WESTERN, the Prionics® Check-PrioSTRIP™, the BioRad® TeSeE™ ELISA, and the IDEXX® HerdChek™ EIA. Performances of these tests were then compared to 2 confirmatory tests, including the BioRad® TeSeE™ Western Blot and the modified Scrapie Associated Fibrils (SAF)/OIE Immunoblot. One 50% w/v homogenate was made from experimentally generated C-type BSE brain tissues in ddH₂O. Homogenates were diluted through a background of BSE-negative brainstem homogenate. Masses of both positive and negative tissues in each dilution were calculated to maintain the appropriate tissue amounts for each test platform. Specific concentrated homogenization buffer was added accordingly to maintain the correct buffer condition for each test. ELISA-based tests were evaluated using their respective software/detection platforms. Blot-protocols were evaluated by manual measurements of blot signal density. Detection limitations were determined by fitted curves intersecting the manufacturers'' positive/negative criteria. The confirmatory SAF Immunoblot displayed the highest analytical sensitivity, followed by the IDEXX® HerdChek™ EIA, Bio-Rad® TeSeE™ Western Blot, the Bio-Rad® TeSeE™ ELISA, Prionics®-Check PrioSTRIP™, and Prionics®-Check WESTERN™, respectively. Although the tests performed at different levels of sensitivity, the most sensitive and least sensitive of the rapid tests were separated by 2 logs in analytical sensitivity, meeting European performance requirements. All rapid tests appear suitable for targeted BSE surveillance programs, as implemented in Canada.     

Zooplankton in the Schelde estuary, Belgium and The Netherlands. Spatial and temporal patterns

The zooplankton fauna of the Zeeschelde estuary (Belgium) wasinvestigated over 10 months by means of monthly sampling. CanonicalCorrespondence Analysis (CCA) was used to relate the speciesdistribution to environmental factors. The variation in thespecies data was significantly (P < 0.05) related to a setof 10 environmental variables (chlorinity, NH₄⁺, temperature,PO₄-P^-, DW, Chl a and Chl b, NO₂-N, NO₃-N and pH). The mainspatial and seasonal gradients were associated with chlorinityand temperature respectively. The brackish water zone was dominatedby the calanoid Eurytemora affinis in spring, succeeded by Acartiatonsa and mysid species during summer. In the freshwater transect,cyclopoids dominated, together with several cladoceran species.Thermophilic cyclopoid species (Thermocyclops oithonoides, Th.crassus and Mesocyclops leuckarti) occurred during periods ofmaximal temperature. The cyclopoids Acanthocyclops robustus,Paracyclops poppei and Cyclops vicinus, the cladocerans Daphnialongispina, Chydorus sphaericus and Bosmina longirostris togetherwith the numerically dominant rotifers, oligochaetes, nematodesand juvenile copepods seemed little affected by environmentalgradients.     

Identification of H-type BSE in Portugal

ABSTRACT

During the bovine spongiform encephalopathy (BSE) epidemic, Portugal was the third most affected country. As a result of a successful national eradication plan, the number of BSE affected animals has been progressively declining in Portugal with no cases identified in 2013. However, within the scope of this active surveillance scheme, we have identified the first H-type BSE case born after the introduction of the reinforced ban in fallen stock. Here, we report the phenotypic features of this case and the analysis of the protein coding sequence of prnp as well as the prnp promoter and intron 1 insertion-deletions.     

Analysis of dam-calf pairs of BSE cases: confirmation of a maternal risk enhancement.

We investigate whether a calf born to a dam that develops bovine spongiform encephalopathy (BSE) (prior or subsequent to the birth) is itself at an enhanced risk of developing BSE. Analyses utilize the main database on reported BSE cases in the British cattle herd maintained by the Central Veterinary Laboratory in Weybridge to trace the dams of BSE-affected animals born following the ruminant feed ban in July 1988. The data reveal a significantly enhanced risk of disease in calves born to BSE-affected dams, with the risk being greatest when birth occurs after the onset of clinical signs of disease in the dam. The dependence of the maternally enhanced risk on the maternal incubation stage at birth argues for a significant component of direct maternal transmission of the aetiological agent of BSE, and offers little support for the hypothesis of genetic predisposition. Using a statistical likelihood model, we obtain estimates of the rate of direct maternal transmission by maternal incubation stage; however, biases in the available data make these values minimum estimates.     

Implications of BSE infection screening data for the scale of the British BSE epidemic and current European infection levels

The incidence of confirmed clinical cases of bovine spongiform encephalopathy (BSE) in Great Britain continues to decline, but the recent discovery of cases in previously unaffected countries (including Israel, Japan, Poland, Slovenia and Spain) has heightened concerns that BSE transmission was more intense and widespread than previously thought. We use back-calculation methods to undertake an integrated analysis of data on infection prevalence in apparently healthy cattle and the incidence of confirmed clinical disease. The results indicate substantial underascertainment of clinical cases over the course of the British epidemic, and consequently that two- to fourfold more animals were infected than previously estimated. Upper bounds on the predicted size of the new variant Creutzfeldt-Jakob Disease (vCJD) epidemic are unaffected, as the prediction methods employed fit to observed vCJD mortality data, and are not sensitive to estimates of the absolute magnitude of past human exposure to BSE-infected cattle, only to relative changes in exposure through time. We also estimate the per-head incidence of infection in cattle born between 1993 and 1997 in other European Union countries, using data on the testing of apparently healthy cattle slaughtered for consumption. Infection incidence for cattle born after mid-1996 was highest in Greece, Italy and Belgium, with Spain and The Netherlands having intermediate levels, and estimates for Great Britain, Germany and France being comparably low.     

Sheep and goat BSE propagate more efficiently than cattle BSE in human PrP transgenic mice

A new variant of Creutzfeldt Jacob Disease (vCJD) was identified in humans and linked to the consumption of Bovine Spongiform Encephalopathy (BSE)-infected meat products. Recycling of ruminant tissue in meat and bone meal (MBM) has been proposed as origin of the BSE epidemic. During this epidemic, sheep and goats have been exposed to BSE-contaminated MBM. It is well known that sheep can be experimentally infected with BSE and two field BSE-like cases have been reported in goats. In this work we evaluated the human susceptibility to small ruminants-passaged BSE prions by inoculating two different transgenic mouse lines expressing the methionine (Met) allele of human PrP at codon 129 (tg650 and tg340) with several sheep and goat BSE isolates and compared their transmission characteristics with those of cattle BSE. While the molecular and neuropathological transmission features were undistinguishable and similar to those obtained after transmission of vCJD in both transgenic mouse lines, sheep and goat BSE isolates showed higher transmission efficiency on serial passaging compared to cattle BSE. We found that this higher transmission efficiency was strongly influenced by the ovine PrP sequence, rather than by other host species-specific factors. Although extrapolation of results from prion transmission studies by using transgenic mice has to be done very carefully, especially when human susceptibility to prions is analyzed, our results clearly indicate that Met129 homozygous individuals might be susceptible to a sheep or goat BSE agent at a higher degree than to cattle BSE, and that these agents might transmit with molecular and neuropathological properties indistinguishable from those of vCJD. Our results suggest that the possibility of a small ruminant BSE prion as vCJD causal agent could not be ruled out, and that the risk for humans of a potential goat and/or sheep BSE agent should not be underestimated.     

Assessing the presence of BSE and scrapie in slaughterhouse wastewater

Aims: This paper describes a procedure for evaluating the presence and the stability of the proteinase K-resistant form of the prion protein (PrP^res) in slaughterhouse wastewater. Methods and Results: Wastewater samples were spiked with either scrapie or bovine spongiform encephalopathy agents and PrP^res was concentrated and detected by western blotting. The detection limit was estimated to be 2–4 μg of either scrapie or BSE-infected brain tissue in 15 ml of sewage. Wastewater samples from three abattoirs were analysed, two of which had processed BSE-infected animals. No PrP^res was detected. The effect of sewage on the inoculum and the persistence of transmissible spongiform encephalopathy agents in wastewater were also considered. Conclusions: The results of the assay suggest that wastewaters from abattoirs where one positive BSE case has been identified would contain titres lower than 0·6–26 × 10⁻⁴ cattle oral ID₅₀ per litre resulting from specified risk material tissue contamination. Moreover, the effect of abattoir wastewaters is to reduce the persistence of PrP^res. Significance and Impact of the Study: The assay may be a useful tool for risk assessment studies and for reducing the potential risk of contamination with BSE via sewage sludge fertilizer procedures.     

The mobile epibenthic fauna of soft bottoms in the Dutch Delta (south-west Netherlands): Spatial structure

During 1989 monthly beam trawl samples were collected at 48 stations in the Dutch Delta. The annual mean densities of the demersal fishes and mobile epibenthic invertebrates at these stations were calculated. These data were then subjected to multivariate statistical techniques for an analysis of the spatial structure of the communities found and to study the relationship between these communities and their environment. The present study confirms the results of HENDERSON (1989) that, given a fairly limited number of environmental variables, mainly salinity and exposure/substratum type, quite accurate predictions of the type of community expected at a certain site can be made. The explicit inclusion of epibenthic invertebrates, a dominant group in most assemblages, in studies mainly targeted at demersal fishes, is strongly recommended. In the Dutch Delta rich and varied communities exist in the Voordelta. A number of groups, such as salmonids, anadromous species and starfish, expected in the westerschelde are absent or extremely rare probably as a consequence of pollution stress. The Oosterschelde is relatively poor in density terms but has a highly diverse epibenthic fauna dominated by fishes.     

PET-blot analysis contributes to BSE strain recognition in C57Bl/6 mice.

Identification of the strain of agent responsible for bovine spongiform encephalopathy (BSE) can be made histologically through the analysis of both distribution and intensity of brain vacuolar lesions after BSE transmission to mouse. Another useful way to distinguish the BSE agent from other prion strains is the study of the distribution of the abnormal prion protein (PrP(res)). For that purpose, paraffin-embedded tissue blot (PET-blot) method was applied on brains from C57Bl/6 mice infected with cattle BSE, experimental sheep BSE, or feline spongiform encephalopathy (FSE) from a cheetah. PrP(res) distribution was comparable, whichever of the three BSE agent sources was considered and was distinct from the PrP(res) distribution in C57Bl/6 mice inoculated with a French scrapie isolate or with a mouse-adapted scrapie strain (C506M3). These data confirm a common origin of infectious agent responsible for the British and French cattle BSE. They also indicate that PET-blot method appears as a precise complementary tool in prion strain studies because it offers easy and quick assessment of the PrP(res) mapping. Advantages and limits of the PET-blot method are discussed and compared with other established and validated methods of strain typing.