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Journal of Clinical Microbiology, February 2005, p. 862-869, Vol. 43, No. 2
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.2.862-869.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Population-Level Retrospective Study of Neurologically Expressed Disorders in Ruminants before the Onset of Bovine Spongiform Encephalopathy (BSE) in Belgium, a BSE Risk III Country

C. Saegerman,¹ D. Berkvens,² L. Claes,³ A. Dewaele,⁴ F. Coignoul,⁴ R. Ducatelle,⁵ D. Cassart,⁴ B. Brochier,⁶ F. Costy,⁷ S. Roels,⁸ H. Deluyker,^9, E. Vanopdenbosch,⁸ and E. Thiry^10*

Federal Agency for the Safety of the Food Chain, Administration of Control Policy, Secretariat of the Scientific Committee,¹ Department of Microbiology, Section of Virology, Scientific Institute of Public Health,⁶ Institut Pasteur, Rabies Service,⁷ Veterinary and Agrochemical Research Center (CODA/CERVA), National Reference Laboratory for Veterinary TSE, Brussels,⁸ Prince Leopold Institute of Tropical Medicine, Department of Animal Health, Antwerp,² Dierengezondheidszorg Vlaanderen, Leefdaal,³ Department of Pathology,⁴ Department of Infectious and Parasitic Diseases, Virology, Epidemiology, and Viral Diseases, Faculty of Veterinary Medicine, University of Liège, Liège,¹⁰ Vakgroep Pathologie, Bacteriologie en Pluimveeziekten,⁵ Vakgroep Verloskunde, Voortplantig en Bedrijfsdiergeneeskunde, Faculteit Diergeneeskunde, Universiteit Ghent, Merelbeke, Belgium⁹

Received 25 June 2004/ Returned for modification 12 September 2004/ Accepted 17 October 2004

Top Abstract Introduction Materials and Methods Results Discussion References

 
A retrospective epidemiological study (n = 7,875) of neurologically expressed disorders (NED) in ruminants before the onset of the bovine spongiform encephalopathy epidemic (years studied, 1980 to 1997) was carried out in Belgium. The archives of all veterinary laboratories and rabies and transmissible spongiform encephalopathy (TSE) epidemiosurveillance networks were consulted. For all species, a significantly higher number of NED with virological causes (rabies) was reported south of the Sambre-Meuse Valley. During the period 1992 to 1997, for which the data were complete, (i) the predicted annual incidence of NED varied significantly as a function of species and area (higher numbers in areas where rabies was present) but was always above 100 cases per million, and (ii) the mean incidence of suspected TSE cases and, among them, those investigated by histopathological examination varied significantly as a function of species and area. The positive predictive value of a presumptive clinical diagnosis of NED ranged from 0.13 (game) to 0.63 (sheep). Knowledge of the positive predictive value permits the definition of a reference point before certain actions (e.g., awareness and training campaigns) are undertaken. It also shows the usefulness of a systematic necropsy or complementary laboratory tests to establish an etiological diagnosis. TSE analysis of a small, targeted historical sampling (n = 48) permitted the confirmation of one case and uncovered another case of scrapie. The results of the present study help to develop and maintain the quality of the worldwide clinical epidemiological networks for TSE, especially in countries that in the past imported live animals, animal products, and feedstuffs from countries with TSE cases.

Top Abstract Introduction Materials and Methods Results Discussion References

 
In affected ruminants, transmissible spongiform encephalopathies (TSE) cause neurological signs that can be classified into three categories: disturbances in behavior, sensitivity, and locomotion. In addition, some general clinical signs are also observed (4, 8, 44, 71, 78, 80). The course of the disease is progressive and always fatal. The lesions are restricted to the central nervous system, although the pathogenesis of infection implies a primary replication step of TSE agents in the lymphoid organs, followed by a neuroinvasive phase (32, 45, 64, 68).

Scrapie occurs in sheep, goats (49), and mouflons (80). Scrapie in sheep has been an endemic disease for more than 250 years (18, 49). Scrapie affects adult animals, with a peak age at onset of 2 to 3 years (8, 15). Not a single clinical case of scrapie has been diagnosed in animals younger than 6 months (52). The first description of the natural disease in goats dates back to 1942 (12). Subsequently, only a few cases of scrapie have been reported for this species (8, 18). In France, a clinically suspected case of spongiform encephalopathy in a cow was described in 1883, but no brain block was conserved (57). This case is still an enigma. Currently, scrapie is considered an infectious disease with maternal and horizontal contagious transmission, where host genetic factors play a central role (1, 19, 62).

Chronic wasting disease (CWD) has emerged as an important wildlife disease in North America since the 1970s (79), and over the past 5 years, the known distribution has expanded to free-ranging cervids and to game-farming industries (77). CWD is horizontally transmitted, and environmental contamination may play an important role in local maintenance of the disease (78).

Bovine spongiform encephalopathy (BSE) was first recognized and defined as a pathological entity in the United Kingdom in November 1986 (70). Initial epidemiological investigations and examination of archived brains indicated that the first clinical cases occurred around April 1985 (44). The disease affects adult animals, with a peak age at onset of 4 to 5 years (2, 30, 35, 71). The age range of clinically confirmed cases is very wide (from 20 months to almost 20 years) (16), although BSE is rarely confirmed in animals younger than 30 months (54). The specific origin is not clear, but the marker of the disease is the prion protein PrP^Sc (PrP scrapie) (50). In the late 1970s, a reduction in the use of hydrocarbon solvents in the production of meat and bone meal coincided with the accepted start of exposure of the cattle population in Great Britain (2, 3, 72, 73, 74). The first cattle with confirmed BSE cases were born at the time of this change (17). Most BSE cases resulted from the recycling within the cattle population of infected cattle tissues via meat and bone meal (71, 74). The duration of clinical signs is 1 to 2 months on average, but it can be less than 2 weeks (44, 48, 56, 75) or as long as 1 year (44). Currently BSE can be confirmed only postmortem, by pathological examination of brain tissue. The histological changes are typical: microscopic lesions in the central nervous system consist of bilaterally symmetrical, noninflammatory vacuolization of neuronal perikarya and grey-matter neuropil (71). The new variant of Creutzfeldt-Jakob disease was first identified in the United Kingdom in 1996 (76). Subsequently, several investigations have indicated a link with BSE (5, 37, 61).

Before the onset of BSE during the second half of the 1980s, there were no specific surveillance programs for TSE in ruminants in most countries. In Belgium, notification of rabies has been compulsory since 1967 and notification of ruminant TSE has been compulsory since 1990. The first scrapie case in Belgium was recognized in 1963, i.e., before compulsory notification. It appeared in a Suffolk ram, which was imported from the United Kingdom (40). The first BSE case was diagnosed in October 1997 (66). No case of CWD has ever been reported in Europe.

In the literature little information has been available on the definition of reported neurologically expressed disorder (NED) and on those NED cases that were suspected of TSE. Almost no information on the incidence of NED in wild ruminants has been found. In the United States, the National Animal Health Monitoring System (NAHMS) has registered an incidence of 4,000 per million sheep above the age of 1 year that were culled or died with "behavioral faults" in 1995 (10). For cattle, some information is available but comparison of the different studies is not feasible. According to Heim et al. (36), neurological pathologies have a preferential distribution in bovines under the age of 1 year (29.3%) and in those aged 4 to 5 years (18.9%). The VIALINE network (Vigilance, Alerte, Intervention, et Évaluation) in Haute Normandie (France) mentions a total mortality rate of 5.1% in 1993, and 13.5% of these animals were above the age of 2 years. Of these, 8% had neurological diseases (23). Combining these figures yields a mortality rate from neurological diseases of 550 per million cattle above the age of 2 years. Between 1992 and 1999, an annual average of 90 clinically suspected BSE cases per million cattle above the age of 2 years was reported to the veterinary authorities in Switzerland (passive surveillance); 43% of these cases were confirmed (20). In the United States, NAHMS recorded a mortality rate of 1,000 dairy cows per million due to "lack of coordination or severe depression" in 1995 and a morbidity rate in beef herds of 1,000 breeding females with "neurological problems" per million in 1996 (9, 11). In beef herds, this rate, expressed as affected bovines per thousand, doubles when the herd size is below 100 head and is nil when the herd size is more than 300 head (9). Finally, a limited retrospective study in New Zealand, based on histopathological diagnoses made for 28 cattle suspected of BSE, is available (38).

Information on ruminant neurological cases is very scarce. However, it is extremely important to get epidemiological estimates of these cases, because most of the neurological disorders enter into the differential diagnosis of TSE in ruminants.

The present study had three goals: first, to determine the annual incidence of NED in Belgium, a country classified as level III for geographical BSE risk; second, to determine whether TSE were present in a country before the first report; and third, once TSE had been detected, to monitor the evolution of incidence in space and time.

The present epidemiological retrospective study constitutes the first validation, at a population level, of the requirements of the Office International des Epizooties (OIE), which are based on expert opinion (81).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Population at risk. The inclusion criteria depended on the species: cattle older than 12 months, small ruminants older than 6 months, and game of all age. Ruminant population size data in Belgium were extracted from the annual registration for the period of 1980 to 1997 (available from the National Institute of Statistics), more specifically called the "15 May census."

Evolution in time. The period studied started on 1 January 1980 and ended on 31 December 1997 (the year of the first indigenous BSE case in Belgium). The starting date was selected on the basis of the trade of live animals, animal products, and meat and bone meal from the United Kingdom, where BSE occurs, to other European countries (41, 58, 59, 67) and to countries outside Europe (33, 34) and on the basis of an average BSE incubation time of 4 to 5 years (71).

Spatial distribution. Belgium has been free of rabies since July 2001 (83). Rabies was enzootic during the period under investigation. Belgium can be divided into three regions according to previous rabies prevalence. One region, the Sambre-Meuse Valley (SMV), forms a natural border that separates the other two: the area north of the SMV, where rabies was absent, and the area south of the SMV, where rabies was present. The data from the SMV itself are of limited importance and are not presented here.

Definitions. For the purpose of this study, a reported NED case is defined as a case for which either nervous clinical signs were reported to the veterinary laboratory, or a diagnosis of neurological disorder was made at autopsy or through another laboratory test, or, in the absence of the above information, analyses were performed on the central nervous system. A reported NED case with suspected TSE is defined as a reported NED case for which TSE could not be excluded (which could not be explained by any other cause) or a reported NED case, whatever the nature, for which either the survival time was 7 days or more, or the animal was euthanized before 7 days. To meet the objectives of the present study, this definition was deliberately made broader than the definitions of suspected cases proposed by the European Parliament and Council (28) and by the OIE (81, 82). The annual incidence rate is the ratio of the number of reported NED cases, or of the number of reported NED cases suspected of TSE, to the population at risk.

Database. The rabies and TSE epidemiosurveillance networks, as well the veterinary diagnostic network, report cases of NED. In the first two networks, game of all ages, all cattle above the age of 20 months, and all small ruminants above the age of 12 months, suspected of rabies but testing negative, have been examined for TSE since 1990. The veterinary diagnostic network consists of the provincial veterinary laboratories, the National Reference Laboratory for Veterinary TSE (CODA/CERVA), and the Veterinary Medicine Faculties at the University of Liège (UL) and the University of Ghent (UG).

A database with detailed information about all these cases was established. It contained the following information: laboratory, reference number, date of entry, locality of origin, race, sex, age, clinical signs, results of laboratory tests and/or autopsy, date of result, and indication of whether biological material was forwarded to other laboratories (in order to avoid double entries). The archive search began with those for 1 January 1980. The archives were complete for the period 1992 to 1997. An exhaustive list of NED cases per species was constructed, with the goal of standardizing the data independently of the source (54).

Pathological examination. (i) Targeted samples. Brain blocks have been produced by the TSE networks from 1990 on and have been available in the Department of Pathology of the Faculty of Veterinary Medicine of UL since 1980 and in the corresponding department of UG since 1990. Thirty animals were analyzed at UL, and 18 animals were analyzed at UG. The samples were taken between 1983 and 1997 and came from 17 bovines (1 below, and 16 above, the age of 24 months), 24 sheep (2 below, and 22 above, the age of 12 months), and 7 goats (1 below, and 6 above, the age of 12 months).

(ii) Histology. For histological examination, all tissues were fixed in 4% phosphate-buffered formalin, routinely processed, embedded in paraffin wax, and sectioned at a thickness of 5 µm. Sections were stained with hematoxylin and eosin stains.

(iii) Immunohistochemistry. All immunohistochemical staining was carried out on 5-µm-thick dewaxed sections. Rehydrated sections were placed in a bath of 98 or 100% formic acid at room temperature (RT) for 30 min. After being rinsed with dematerialized water, sections were sterilized at 125°C for 30 min. Endogenous peroxidase was inactivated by covering the sections with a bath of 0.3% hydrogen peroxide in methanol for 30 min at RT. Incubations with primary antibodies were performed at room temperature for 1 h. The antibody used was R524-7/IDDLOMH-AA7 (ID-Lelystad, Lelystad, The Netherlands), a polyclonal rabbit anti-PrP peptide serum diluted 1/1,500. As a secondary antibody, a biotinylated goat anti-rabbit antibody (EO432; DAKO, Glostrup, Denmark) was applied for 10 min at RT, followed by a 5-min incubation in peroxidase-conjugated streptavidin (PO397; DAKO), both diluted 1/500 at RT. 3,3'-Diaminobenzidine tetrahydrochloride (DAB) (Sigma, St Louis, Mo.) was used as a chromogen in the presence of hydrogen peroxide. Sections were counterstained with hematoxylin (Gill 3; Prosan N.V., Merelbeke, Belgium) for 30 s, mounted with DPX mountant (BDH Laboratory Supplies, Poole, England), and covered with a coverglass. This immunohistochemistry procedure has been performed in Belgium since 1996.

(iv) Transmission electron microscopy. For examination of the fibers associated with TSE (scrapie-associated fibers [SAF]), the fresh brain stem material was dissected and discarded, and 1-g aliquots were homogenized in 10% (wt/vol) N-lauroylsarcosine and processed by the Hilmert and Diringer technique (39). After proteinase K digestion, the final pellet was resuspended in 50 µl of distilled water and negatively stained for electron microscopy as described by Scott et al. (60). Examinations were carried out with a Philips EM 208S transmission electron microscope at a magnification of x22,000.

Statistical analysis. Statistical analyses were carried in STATA/SE 8 (63). Unless otherwise indicated, negative binomial regression was used for analysis of the annual incidence rates of reported NED cases, suspected rabies cases, and suspected TSE cases, by considering three independent variables for the first two rates (species, originating area, and year) and four for the suspected TSE cases (species, originating area, year, and number of suspected rabies cases). Predicted values were used for the annual incidence rates of reported NED cases and suspected TSE cases in order to better highlight the results of the statistical analysis in the respective table. This was not done for the incidence rate of suspected or histologically examined TSE cases, in order to allow comparison of the results with the OIE requirements.

The cattle population south of the SMV was taken as the reference population. The following modification of the formula of Cannon and Roe was used to estimate the number of brains to be examined in order to detect BSE, if present in at least 1% of NED cases, with a 99% probability (7, 22, 46), as recommended by the OIE (81):

where n is the sample size needed to detect one or more BSE-affected animals in the sample, a is the confidence level of observing at least one affected animal in the sample (in our case, the confidence level is 99%), D is the number of TSE-affected animals in a population with the selected minimum annual incidence, and N is the parent population size.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Etiological classification of the reported NED cases. The numbers of reported NED cases in Belgium between 1980 and 1997 were 5,261 for cattle, 1,642 for sheep, 528 for goats, and 446 for game. Table 1 shows the distribution by type of etiology according to Saegerman et al. (54). Laboratory diagnosis was not always performed because of the cost of investigation. Among the reported cases with determination of the cause, 80% or more (depending on species) were included on the basis of a laboratory diagnosis, 7 to 19% (depending on species) were based on necropsy diagnosis, and 5% or less (depending on species) were based on clinical diagnosis. In all species, the biological-cause group was the most common during the period studied. Among cattle, a virological cause was most frequently encountered, followed by a bacteriological cause. This order was reversed for the other species, especially for goats and game. A parasitological cause was rarely put in evidence. Among nonbiological causes, metabolic and nutritional causes were more frequently found for cattle, sheep, and goats, and a chemical cause was most commonly recorded for game. An unconventional transmissible agent was diagnosed in sheep (nine cases of scrapie), goats (one case of scrapie), and cattle (one case of BSE). Except for cattle (² = 5.51; df = 2; P = 0.064), the proportions of cause groups in the areas north and south of the SMV were significantly different (² = 78; df = 2; P < 0.001). Significantly different proportions of etiological causes were found north and south of the SMV for cattle (² = 1,111; df = 7; P < 0.001), sheep (² = 739; df = 7; P < 0.001), goats (² = 155; df = 6; P < 0.001), and game (² = 193; df = 3; P < 0.001). A significantly higher number of cases with virological causes (rabies) was reported south of the SMV (P < 0.003 by Fisher's exact test). No etiological cause was found in 41.5% (cattle), 39% (sheep), 40.5% (goats), and 77% (game) of the cases.

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TABLE 1. Estimation of the most frequently reported NEDsa by species of ruminants in Belgium, 1980 to 1997

Predicted annual incidence rate. (i) Predicted annual incidence rate of reported NED. Table 2 shows the predicted incidence rate for reported NED (PIR-NED) for the period 1992 to 1997. No difference could be demonstrated between different years, and the final model included only the variables "species" and "area of origin." PIR-NED in cattle was significantly higher south of the SMV, and PIR-NED was significantly higher in sheep and goats than in cattle. Game had a higher PIR-NED than cattle in the area north of the SMV. South of the divide, the situation was reversed.

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TABLE 2. Predicted mean incidence rates of reported NEDs in Belgium during the period 1992 to 1997

(ii) Predicted annual incidence rate of suspected TSE cases. The predicted annual incidence rates of suspected TSE cases (PIR-TSE) are given in Table 3. PIR-TSE was lower north of the SMV, irrespective of the species. This difference was constant over time. PIR-TSE was lowest in cattle and game, higher in sheep, and significantly higher in goats. There was a negative trend over time in all species, except for cattle, where there was a significant increase. PIR-TSE was furthermore positively correlated with the predicted annual incidence rate of suspected rabies cases (PIR-rabies), again in all species. PIR-rabies for the period 1992 to 1997 is shown in Table 4. The following trends were observed: there was no significant effect of time, except for cattle, for which there was a significant increase in the number of suspected rabies cases over time both north and south of the SMV; PIR-rabies was always higher south of the SMV, irrespective of species; and PIR-rabies was higher for sheep and goats than for cattle and game, which had similar rates.

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TABLE 3. Predicted mean incidence rates of reported NED cases with suspected TSE in Belgium during the period between 1992 and 1997a

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TABLE 4. Predicted mean incidence rates of suspected rabies cases in Belgium during the period 1992 to 1997

(iii) Incidence rate of suspected and histologically examined TSE cases. Table 5 presents the observed annual incidence rate of histologically examined TSE cases (IR-TSEHE). The statistical analysis revealed that significantly more records were obtained for goats, both north and south of the SMV. The incidence decreased significantly over time in all species, and there was a correlation between IR-TSEHE and PIR-NED in both regions, although the relationship was stronger in the north.

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TABLE 5. Mean incidence rates of reported NED cases with suspected TSE and with complete histological examination in Belgium during the period between 1992 and 1997

Positive predictive value of presumptive clinical diagnosis. The level of agreement between the presumptive clinical diagnosis (actually made or written in the anamnesis) and the necropsy findings was calculated for the Faculties of Veterinary Medicine of UL and UG for 224 cattle older than 12 months, 112 sheep and 49 goats older than 6 months, and 8 game animals of all ages. The obtained positive predictive values of presumptive clinical diagnosis versus necropsy were as follows: 0.50 for cattle (95% confidence interval [95% CI], 0.43 to 0.56), 0.63 for sheep (95% CI, 0.53 to 0.71), 0.49 for goats (95% CI, 0.34 to 0.64), and 0.13 for game (95% CI, 0.003 to 0.53).

Pathological examination of targeted samples. On the basis of the present study, a supplementary targeting of the available brain blocks from suspected TSE cases (e.g., presence of suspected clinical signs and/or presence of vacuoles in reports) was carried out.

Among the samples examined by immunohistochemistry and histopathology, three were positive for TSE. A first brain block from a sheep that was presented at UL in March 1983 was confirmed as a case of scrapie. For this case, the immunohistochemistry confirmed the original histopathological diagnosis (neuron with pathognomonic cytoplasmic vacuolization). A second brain block from the TSE network was classified as negative for scrapie in November 1992, because the result of histopathology was not conclusive and the SAF result was negative. For this sample also, the result of immunohistochemistry was positive (multiple sites with brownish positive staining). This sample originated from a 5-year-old Hampshire ewe imported from the United Kingdom. The third brain block from UG, from a 3-year-old goat, was histologically diagnosed as scrapie in August 1992(neuron with pathognomonic multiple cytoplasmic vacuolization), but this diagnosis was not confirmed by immunohistochemistry in our study. All other samples tested remained negative.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present study had three goals: (i) to determine the annual incidence of NED; (ii) to determine whether TSE were present in a country before the first report; and (iii) once TSE had been detected, to monitor the evolution of incidence in space and time. Our study allowed us (i) to identify the strengths and weaknesses of the organization of the network, (ii) to choose the appropriate awareness and training campaigns for the participants in the epidemiological network, and (iii) to devise appropriate measures, implement them, and monitor their effectiveness. The geographical TSE risk assessment (25, 26, 27) and TSE risk mapping methodologies would enable identification of the countries and areas where TSE emerge and of the animal groups at risk (21, 24, 33, 65). Targeted passive and active surveillance might be performed in these geographical areas and for these animal groups at risk.

Etiological classification of the reported NEDs. Even though the laboratory archives were incomplete before 1992, all laboratories processed a sufficient number of samples to make them representative of the bovine population in their vicinity. Moreover, the same types of causes were found in every region before and after 1992. It is therefore assumed that the percentages of the morbidity causes calculated for every area during the period 1992 to 1997 were indeed representative for the population at risk. The fairly high percentage of cases for which no etiological cause could be established (around 40% of reported NED cases for domestic ruminants to 77% for game) is in line with previous observations elsewhere: a similar percentage (39%) was found in a Swiss study of cattle with nervous signs and suspected of rabies (29). This result could be due partly to a metabolic or toxicological pathology producing no lesion (47). Improvements in examination techniques and acquisition of more experience could probably reduce this percentage by half (36).

During the period of this study, the role of the SMV as a dividing line for the distribution of causes of NED in Belgium was pivotal, essentially due to the rabies prevalence in the south. Differences in causes between countries had already been noted previously (42, 43, 53, 69). Knowledge about the distribution of NED causes and their associated risk factors should be improved, because the development of decision support tools is based on such knowledge (see, e.g., references 13, 14, and 53).

Predicted annual incidence rates of NED and suspected TSE cases. The annual incidence rates of NED and suspected TSE cases are higher south of the SMV than north of the SMV, and the PIR-TSE is correlated with the annual incidence rate of suspected rabies. Thus, the main hypothesis to explain this observation is that south of the SMV, where rabies is prevalent, farmers and veterinarians have historically been more aware (56). Moreover, PIR-NED is underestimated because many NED cases are not reported if the cause and/or therapy is known. In addition, the IR-TSEHE is directly correlated with the PIR-NED. Any measure resulting in an increase in NED reporting has a nonspecific knock-on effect on reporting of suspected and histologically examined TSE cases. Progressive increases in numbers reported through the specific TSE network were noted toward the end of the study period, particularly for cattle (data not shown). Overall, PIR-NED is higher, and there are more suspected TSE cases, in small ruminants. Several hypotheses can be put forward to explain this observation. In Belgium, a considerable number of livestock holders have a few small ruminants, and research into the cause of morbidity is more frequent with hobby farmers, because they consider their animals to be pets. The "true" number of registered small ruminants has increased in Europe following the episode of food-and-mouth disease in Great Britain (31). Even when these facts are taken into account, the rate in small ruminants remains higher. In the United States, the NAHMS network has registered a NED incidence rate around fourfold higher for sheep than for cattle.

According to the OIE requirement, based on expert opinion, the annual incidence rate of NED for cattle in all countries is 100 cases per million animals, irrespective of their BSE status (81, 82). The present retrospective study provides the first external validation of this requirement at the population level. Because no clinical sign is pathognomonic for BSE (54, 82), laboratory examination of brains is essential for an efficient BSE epidemiological network. OIE (81) calculates the minimum number of samples that must be examined in order to have a probability of 99% to detect at least one case, if the disease is present in 1% of the cattle with NED. According to this OIE requirement and the number of suspected BSE cases for which a complete histopathological examination has been carried out, the power of the epidemiosurveillance effort for the period 1992 to 1997 was on average 59, 28, and 33%, respectively, for the area south of the SMV, the area north of the SMV, and Belgium as a whole. If all suspected BSE cases were analyzed (by using a definition deliberately broader than the definition of suspected cases proposed by the OIE or the European Commission [28, 82]), the power would become 99, 28, and 52%, respectively. This suggests the necessity of organizing awareness and training campaigns to improve presumptive BSE clinical sign detection for all participants in the epidemiosurveillance network, especially in countries that in the past imported live animals, animal products, and feedstuffs from countries where TSE occurs. This need particularly is addressed by the methodology for geographical BSE risk (GBR) assessment, developed by the Scientific Steering Committee of the European Commission to classify countries according to their BSE risks (25, 26, 27). Thus, in Belgium, with the onset of the BSE epidemic and one information campaign, the power of the BSE epidemiosurveillance network increased to 99% (for Belgium and the area south of the SMV) and to around 70% (north of the SMV) in 1998 and 1999 (55, 56).

The mean numbers of histologically investigated cases of NED during the period 1992 to 1997 (51 and 30 per million cattle south and north of the SMV, respectively) compare well with those for other countries at the same GBR level, level III: 10 per million in France (6) and 90 per million in Switzerland (20) during the same period.

Positive predictive value of presumptive clinical diagnosis. Determination of the positive predictive value of presumptive clinical diagnosis permits us to obtain a reference point before undertaking actions (e.g., awareness and training campaigns). In fact, it offers the possibility of following up and evaluating these actions continuously. It also shows the added benefits of systematically turning to necropsy examinations or complementary tests to establish an etiological diagnosis and above all to dispel uncertainty over the identification of suspected TSE cases (54, 82). The main purpose of a clinical TSE epidemiosurveillance network is to attain as high a sensitivity as possible (in order to identify every BSE case). This goal has to be promoted by permanent awareness and training campaigns for veterinarians, farmers, and other actors so that a higher number of nervous-disorder cases are reported and analyzed (28, 81). The low predictive value for game (0.13) can probably be explained by the low level of clinical observation and anamnesis possible for wildlife.

Pathological examination of targeted samples. The present study confirms that neuronal vacuolization can occasionally be observed in cattle in the absence of BSE (2). The additional importance of immunohistochemical staining in the control of TSE in ruminants is also proved. In fact, immunohistochemical staining revealed one extra TSE case and confirmed one suspected TSE case. The negative result upon histopathological examination is possibly an indication of a (sub)clinical case without clear signs. However, due to the lack of tonsillar tissue, confirmation could not be established (51). The absence of SAF in electron microscopy can be explained by the difficulty this technique encounters at such a large magnification. The fact that no BSE case was detected before October 1997 may be due to the low sample size.

The present retrospective study constitutes the first validation, at a population level, of the OIE requirements. The predicted mean incidence rate of NED in a GBR level III country is sufficiently high to detect confirmed BSE cases only when a minimal number of samples has been examined. The results of the present study help to develop and maintain the quality of the clinical epidemiological TSE networks worldwide, especially in countries that in the past imported live animals, animal products, and feedstuffs from countries where TSE occurs.

Finally, our study also revealed the two main limiting factors for this type of study: (i) standardization of the definitions and list of NEDs and (ii) archiving of data and brain blocks over time. The use of an identification and registration system and a laboratory information management system would undoubtedly make it easier to analyze data.

 
We thank the members of the Belgian TSE Advisory Group for their support. This group was founded by the Federal Agency for the Safety of the Food Chain in Belgium (FASFC). We also thank all our colleagues from the private and provincial veterinary investigation centers, the Pasteur Institute, the National Reference Laboratory for Veterinary TSE (CODA/CERVA), and the Faculties of Veterinary Medicine at the Universities of Liège and Ghent, as well as all the veterinarians in practice who have provided the necessary information. We particularly thank P. Dechamps (FASFC) for facilitating access to data. The calculations of population size were made possible by the help of R. Hellemans, then of the Agriculture Economics Centre, and of administrative staff for different regions of Belgium (for the size of the game population).

 
* Corresponding author. Mailing address: Department of Infectious and Parasitic Diseases, Virology, Epidemiology, and Viral Diseases, Faculty of Veterinary Medicine, University of Liège, Boulevard de Colonster, 20, B43b, B-4000 Liège, Belgium. Phone: 32/4 366-42-50. Fax: 32/4 366-42-61. E-mail: etienne.thiry{at}ulg.ac.be.

Present address: European Food Safety Authority, B-1140 Brussels, Belgium.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Clinical Microbiology, February 2005, p. 862-869, Vol. 43, No. 2
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.2.862-869.2005
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