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Descriptive and spatial epidemiology of bovine cysticercosis in North-Eastern Spain (Catalonia)
Veterinary Parasitology 159 (2009) 43–48
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Descriptive and spatial epidemiology of bovine cysticercosis in North-Eastern Spain (Catalonia) A. Allepuz a,b,*, S. Napp a, A. Picado a,d, A. Alba a, J. Panades c, M. Domingo a,b, J. Casal a,b a
Centre de Recerca en Sanitat Animal (CReSA), UAB-IRTA, Campus de la Universitat Auto`noma de Barcelona, 08193 Bellaterra, Barcelona, Spain Departament de Sanitat i Anatomia Animals, Universitat Auto`noma de Barcelona, 08193 Bellaterra, Barcelona, Spain c Departament d’Agricultura, Alimentacio´ i Accio´ Rural de la Generalitat de Catalunya, Gran Via de les Corts Catalanes, 612-614, 08007, Barcelona, Spain d Infectious and Tropical Diseases Department, London School of Hygiene and Tropical Medicine, London, UK b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 3 June 2008 Received in revised form 13 August 2008 Accepted 25 September 2008
From March 2005 to December 2007, 284 animals from 67 cattle farms (24 dairy and 43 beef) affected by bovine cysticercosis were detected in the region of Catalonia (NorthEastern Spain). Dairy farms were almost twice more likely to be affected than beef farms (OR = 1.79, 95% CI = 1.08–2.96, p < 0.05), and infected premises have a statistically significant (p < 0.05) larger number of animals when compared to uninfected farms in Catalonia. The geographical distribution of the infected farms was evaluated and two statistically significant clusters were identified. The most likely cluster was located in the western part of the study region, with 8 out of 10 farms infected. Epidemiological investigations revealed that the 8 farms belonged to the same company. The secondary cluster was located in Eastern Catalonia with 12 infected farms out of 167 cattle farms. No epidemiological links were found among the 12 infected premises. A questionnaire, based on the EFSA risk assessment, was used to assess the most likely route of introduction into each affected farm. Water supply for animals was the route with the highest score in 41.8% of the cases. ß 2008 Elsevier B.V. All rights reserved.
Keywords: Bovine cysticercosis Routes of infection Geographical distribution
1. Introduction Bovine cysticercosis is a disease caused by the larval infection of Taenia saginata in cattle (EFSA, 2004). T. saginata occurs in the small intestine of humans who are the final host of this tapeworm (Abuseir et al., 2007). Humans get infected by eating raw or undercooked meat containing viable cysticerci. The tapeworm develops in the small intestine and becomes sexually mature in about 3 months, producing gravid proglottids, which are mobile and either migrate from the host’s anus spontaneously or
* Corresponding author at: Centre de Recerca en Sanitat Animal (CReSA), UAB-IRTA, Campus de la Universitat Auto`noma de Barcelona, 08193 Bellaterra, Barcelona, Spain. Tel.: +34 93 581 10 47; fax: +34 93 581 32 97. E-mail address: [email protected] (A. Allepuz). 0304-4017/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2008.09.027
are shed in faeces. The presence of the tapeworm in the intestine can cause some abdominal discomfort, mild diarrhoea, weight loss and pruritus caused by migrating proglottids (Dorny and Praet, 2007). The prevalences in humans are highly variables within a country and between countries. This variability in prevalence is due to hygienic habits, quality of meat inspection and culinary habits. However, human taeniosis is not a notifiable disease and reported prevalences are only indicative (Cabaret et al., 2002). Transmission to animals occurs upon contamination of food or water by faeces of infected humans (Geysen et al., 2007). The contaminated material can derive directly from human faeces or via sewage plants after flooding or sewage sediment distributed on pastures (Abuseir et al., 2007). Direct transmission of eggs, resulting from hand rising of suckling calves by tapeworm carriers has been reported, but appears to be rare (Dorny and Praet, 2007).
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A. Allepuz et al. / Veterinary Parasitology 159 (2009) 43–48
European legislation (Anon., 2004) requires that a systematic investigation for bovine cysticercosis in all bovine animals over 6 weeks old must be carried out. The inspection is done by the Official Veterinarian at the slaughterhouse by visual examination and by cuts in masseter muscles, tongue, diaphragm and heart. If the infestation is generalised, the carcass and offal are rejected. However, if infestation is localised, the part of the carcass or offal affected must be rejected and the remainder can be kept in cold storage. Therefore, the disease causes economical losses to the cattle industry mainly due to condemnation, refrigeration and downgrading of infected carcasses. These losses have been estimated to amount to 30–45% of the value of the carcasses (Geerts, 1990). Epidemiological surveys on bovine cysticercosis are scarce in Europe (Boone et al., 2007). The prevalence of bovine cysticercosis in Europe is based on meat inspection reports, and ranges from 0.007% to 6.8% with a wide variation between countries, regions and abattoirs (Cabaret et al., 2002; Dorny and Praet, 2007). However, meat inspection has a low sensitivity, resulting in an underestimation of the prevalence of bovine cysticercosis (Kyvsgaard et al., 1990; Dorny et al., 2000; Dorny and Praet, 2007). Moreover, few EU countries report their data to the OIE and these data are rather fragmentary, so reliable conclusions are difficult to make (Dorny and Praet, 2007). In March 2005, the detection of a series of cases of bovine cysticercosis in Catalonian slaughterhouses resulted in an outbreak investigation on the farms initially affected, and the establishment of a monitoring programme targeting slaughterhouses and cattle farms. This paper presents the results of the first 3 years of monitoring programme. The aims of this study are to describe the demographic, geographical and temporal characteristics of infected cattle herds in Catalonia from 2005 to 2007, and to asses the most likely route of introduction of the eggs of T. saginata in the affected farms. 2. Materials and methods 2.1. Descriptive analyses The study was conducted in Catalonia. This region has an area of 32,105 km2 and there are approximately 626,400 cattle in 7496 farms, which represent the 10% of the total cattle population of Spain (Anon., 2007a). Data on the cattle farms in Catalonia: type of farm (beef or dairy), census and geographical coordinates were provided by the Department of Agriculture of the Autonomous Government of Catalonia. Data on the farms affected by bovine cysticercosis as well as the number of animals detected, date of detection and number of slaughterhouses that have reported cases of cysticercosis, were provided by the Department of Health of the Autonomous Government of Catalonia. Diagnostic of infestation was done by the Official Veterinarian at the slaughterhouse by visual examination and by cuts in masseter muscles, tongue, diaphragm and heart according to the European legislation (Anon., 2004). When visual diagnosis was not clear, samples were sent to the
Pathology Department (Barcelona Veterinary College), where they were confirmed by histopathology. To estimate whether there were differences between the proportion of beef and dairy farms infected, we used Pearson’s chi-square test and calculated the odds ratio (OR) with its 95% confidence interval (CI). A non-parametric two-sample test (Mann–Whitney U-test) was used to compare the number of animals in infected and noninfected farms. 2.2. Spatial analysis To explore the spatial distribution of the infected farms in the region, we run a purely spatial analysis based on the Bernoulli model with SaTScan1 v6.1 (http://www.SaTScan.org) as described by Kulldorf (1997). With the Bernoulli model, farms were classified as either cases or non-cases. A farm was classified as a case if at least one infected animal was detected. The spatial scan statistic searches for clusters using a variable circular window size to detect spatial clusters in large areas, while controlling for the underlying population (Kulldorf, 1997). The circle is centred on each of the points. For each point, the size of the circle varies from zero to some upper limit specified by the user (50% of the population at risk). Under the null hypothesis, the expected number of cases in each area is proportional to its population size. For each circle a likelihood ratio statistic is computed based on the number of observed and expected cases within and outside the circle (Aamodt et al., 2006). The p-value is obtained through Monte Carlo hypothesis testing by comparing the rank of the maximum likelihood from the real data set with the maximum likelihood from the random data sets. We scanned only for clusters with high rates (more cases than expected). The number of Monte Carlo simulations was set at 999. Only statistically significant (p-value < 0.05) nonoverlapping clusters were reported (Kulldorf, 2005). The farms affected with cysticercosis and the spatial clusters detected in Catalonia were represented in a map using ArcMap1 v9.1 (ESRI, Redlands, CA). Detailed epidemiological investigations were carried out on clustered farms to determine possible links among them. 2.3. Most likely route of T. saginata introduction A questionnaire, based on the EFSA risk assessment (EFSA, 2004), was used to assess the most likely route of introduction on each affected farm. The questionnaire included questions on risk factors related to five potential routes of introduction: water supply for animals, feed, pastures, personnel and other routes. Each of these risk factors had an associated score which reflected the probability of introducing bovine cysticercosis (EFSA, 2004) (Table 1). This scoring system was used to evaluate the most likely route of introduction in each of the affected farms, as the risk factor with the highest score was considered the most likely route of introduction of T. saginata. The questionnaires were completed by official veterinarians from the Department of Agriculture by on-site personal interview with the farm owners. An 82% response
A. Allepuz et al. / Veterinary Parasitology 159 (2009) 43–48 Table 1 Routes of introduction, associated risk factors and corresponding risk scores according to EFSA’s opinion on Cysticercus Risk Assessment (EFSA, 2004). Route
Risk factors
Score
Water supply for animals
Use of untreated surface water (river/lake) Use of untreated local water (e.g. wells) Use of treated local water Use of municipal water Floods potentially spreading T. saginata eggs on the grazing and/or feed components
4 3
Regularlya Irregularlya Regular or irregularb
2 1 4 3 2
Feed
Roughage used multi – source Roughage used single – source Concentrate used multi – source Concentrate used single – source
2 1 2 1
Pastures
Animals kept mainly outdoor Animals kept combined indoor and outdoor Animals kept indoor only Use of untreated sewage as fertilisers Use of treated sewage as fertilisers
4 3
Visit endemic area in the last year
4
Personnel
Other routes
a b
Farm located near camping sites Farm located near public footpaths Waters known as receiving sewage. Waters not receiving sewage.
1 4 3
4 2
45
28.6% of the bovines slaughtered in Catalonia. By 2007 the number of slaughterhouses reporting bovine cysticercosis increased to 9 (responsible for the 73.1% of the bovines slaughtered in the region). The number of infected animals detected per farm varies from 1 to 70. The fact that there was a single farm responsible for 70 of the 284 cases reported in Catalonia resulted in an epidemiological investigation. This farm was a medium size fattening farm located in Western Catalonia. Given that the animals originated from different countries suggested that the animals were infected at the farm rather infected at the farm of origin. The epidemiological investigation was not able to determine with precision the source of infection, although water and feed were suggested as the most likely sources of contamination of the farm. The number of times infected animals were detected in a particular farm varies from 1 to 17, although in most of the farms (73%) infected animals were detected just 1 time (Table 3). A statistically significant association between type of farm (beef vs. dairy) and infection status has been found. Dairy farms were almost twice more likely to be affected than beef farms (OR = 1.79, 95% CI = 1.08–2.96, p < 0.05) In both dairy and beef farms the farm size (number of animals) seems to be related to the presence of bovine cysticercosis: infected farms have a statistically significant (p < 0.05) larger number of animals when compared to the uninfected farms in Catalonia. 3.2. Spatial analysis
rate was obtained, not being possible to complete the questionnaire in 12 of the affected farms. The data was double entered checked and corrected. 3. Results 3.1. Descriptive analysis From March 2005 to December 2007, 284 animals from 67 cattle farms (24 dairy and 43 beef herds) affected by bovine cysticercosis were detected. Infected farms were reported almost every month. The mean prevalence of infected animals is 1.8 per 10,000 slaughtered animals. The prevalence of infected animals increased throughout the study period from 0.015% in 2005 to 0.022% in 2007 (Table 2). The number of slaughterhouses which detected bovine cysticercosis also increased during the study period. In 2005 bovine cysticercosis was detected in 4 different slaughterhouses, which were responsible for the Table 2 Number of T. saginata bovines infected and number of cattle slaughtered in Catalonia from March 2005 to December 2007. Year
Number of animals detected
Cattle slaughtered
Prevalence (%)
2005 (from March) 2006 2007
75 97 112
499,846 567,024 498,351
0.015 0.017 0.022
The spatial analysis evidenced that the geographical distribution of the infected farms is clustered. The SaTScan identified two statistically significant clusters (Fig. 1). The most likely cluster had an area of 9 km2 and was located in the West part of the study region. The relative risk was 99.3, in this area there were 8 out of 10 farms infected. Epidemiological investigations revealed that the 8 farms belonged to the same company. Furthermore, 2 farms located outside the cluster, but belonging to the same company were also affected. The large number of animals infected and the fact that the animals originated from different regions in Spain and different countries in Europe practically discard the possibility of the animals being infected in origin. Tracing back of movements revealed that all the infected
Table 3 Number of times Cysticercus bovis infected animals were detected in a farm from March 2005 to December 2007 in Catalonia. Number of times infected animals were detected in a farm
Percentage
1 2 3 4 5 11 12 17
73.1 11.9 3.0 4.5 1.5 1.5 3.0 1.5
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A. Allepuz et al. / Veterinary Parasitology 159 (2009) 43–48
Fig. 1. Geographical distribution of the 67 T. saginata infected farms detected between 2005 and 2007 (triangles), and location of the most likely and the secondary cluster (circles) in Catalonia.
animals had stayed at some point (although not during the same period) in a single farm belonging to the company, where calves were milk-fed for 2 months before being transferred to the other farms (fattening farms). Therefore the possibility of the animals getting infected at that farm could not be ruled out. In this farm the water was obtained from a channel. However, the infection of the animals at the fattening farms was also possible as the source of feed and straw was also common for all the farms. On the other hand, all animals from the farms located in the cluster were kept indoors, and roughage did not come from pastures fertilised with sewage sludge. The secondary spatial cluster, located in Eastern Catalonia has an area of 255 km2 and includes 167 cattle farms. Bovine cysticercosis was detected in 12 of them, presenting an increased risk of 9.4 compared to farms outside the cluster. No epidemiological links were found among the 12 infected premises. 3.3. Most likely route of infection The scoring system used shows that water supply for animals was the route with the highest score in 41.8% of the cases (23 farms), followed by feed and other routes in 9.1% (5 farms) and 12.7% (7 farms) respectively. Personnel and pastures seemed less frequent as they appeared to be responsible for the infection of 9.1% (5 farms) and 5.5% (3 farms) respectively. In 12.7% (7 farms), the highest score was shared by 2 risk factors, whereas in 7.3% (4 of the farms), the highest score was shared by 3 risk factors. For one farm, the route of infection was considered as unknown, as none of the risk factors scored over 1.
4. Discussion Epidemiological data on bovine cysticercosis are scarce in Europe, and therefore prevalence data in order to compare the situation between countries is difficult to find. Moreover, the reporting in the different countries is not standardised, and prevalence data are difficult to collate and compare. Bovine cysticercosis is present at a low level in Western European countries, and the prevalences vary between 0.007% and 2.4% (SCVMPH, 2000). In Northern Spain, Garcia Castro (2003), based on detailed meat inspection, reported prevalences of 0.54% in animals kept outdoors at pastures. More recently, a study carried out in Belgium reports prevalences of bovine cysticercosis, based on official meat inspection, of 0.22% in 2001, 0.44% in 2003, 0.34% in 2004 and 0.28% in 2005 (Boone et al., 2007). This paper provides information on bovine cysticercosis prevalence in Catalonia (North-Eastern Spain) that was not previously reported. The observed mean prevalence (0.018%) is low compared to the figures reported in Belgium by Boone et al. (2007) and Northern Spain by Garcia Castro (2003). A possible explanation to this lower prevalence is that in Catalonia most of the animals are kept indoors. Grazing on pastures potentially contaminated with T. saginata eggs derived from human faeces directly or via sewage sediment distributed in pastures (Abuseir et al., 2007), free access of cattle to surface water (rivers, lakes, canals) and flooding of pastures have been described as important environmental risk factors for the detection of bovine cysticercosis in a herd (Boone et al., 2007). Therefore, differences in the production systems may, to some extent, explain the differences in prevalences. On the other hand, mean prevalence of infected animals was calculated from data given by the authorities. The
A. Allepuz et al. / Veterinary Parasitology 159 (2009) 43–48
validity of these data depends on the quality of the visual meat inspection. Findings by the current palpation and incision techniques at routine meat inspection cannot be considered as an accurate indicator of the true occurrence of bovine cysticercosis (EFSA, 2004). The classical meat inspection techniques detect only a minor fraction of the carcasses infected with cysticerci (Dorny et al., 2000). The fact that the probability of cyst detection at routine post mortem meat inspection depends on the number of cysts present in the animal (Walter and Koske, 1980) and that most animals in Europe are lightly infected and that only a proportion of cysts are located at the predilection sites (Dorny and Praet, 2007), contribute to the low sensitivity of meat inspection. Moreover, there may be differences in cysticercosis detection efficacy due to differences in motivation and skills of inspectors or meat inspection facilities (EFSA, 2004). In our study, bovine cysticercosis prevalence was estimated from routine meat inspection at slaughterhouses and, therefore, is likely to be an underestimation of the real prevalence. However, the extent to what prevalence was underestimated is difficult to quantify. Dorny et al. (2000) found that the prevalence obtained by means of an ELISA was at least 10 times higher than the prevalence obtained by routine meat inspection. In Belgium, the increase in the number of cases from 2001 to 2003 was attributed to a more careful inspection at abattoirs, rather than to a real increase in the prevalence (Boone et al., 2007). Something similar is likely to have happened in Catalonia. As neither the diagnostic approach, nor the staff involved, have changed, the increase in the number of cases detected is more likely to be related to the efforts made by the Veterinary Authorities (courses, seminars and meetings) in order to improve the detection at slaughterhouses, than to a real increase of prevalence. The increase in the number of slaughterhouses detecting cysticercosis (from 3 in 2005 to 9 in 2007) may be considered as an indication of the improvements made in the detection. Besides, the number of T. saginata carrierships in Spain does not seem to have increased. The number of cases reported to the authorities was 43 in 2005, 42 in 2006 and 17 in the first half of 2007 (Anon., 2007b), although these data should be taken with caution as it is not a notifiable disease. On the other hand, in most of the farms infected animals have been detected only one time, a result which is consistent with what happens in other European countries (Dorny and Praet, 2007). This could be due to the fact that most infections are the result of the accidental ingestion of eggs which are found disseminated in the environment, and therefore light infections are more common than heavy infections (Dorny and Praet, 2007). We found that the proportion of dairy farms affected was higher than the proportion of beef farms. This could be attributable to the fact that in dairy cattle the age of the animals at slaughter is normally higher than in beef cattle. Dorny et al. (2000) found that sero-prevalence was positively correlated with increasing age. As the infection is accidental, the risk of exposure increases with the age of the animals (Dorny and Praet, 2007). Unfortunately, data on the age of the animals were not recorded.
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The cluster analysis evidenced that the risk of infection was not homogeneously distributed over the entire region. There are some areas where the risk of infection is higher as compared to the entire region. In the most likely cluster the risk of infection is almost 100 times higher as compared to the rest of Catalonia. Besides, in contrast to what usually happens in Catalonia, several animals from these farms were affected. Despite the presence of common factors, among the affected farms in the cluster, the origin of the outbreak could not be clearly established. When cysticercosis outbreaks occur, the source of contamination is seldom determined (Scandrett and Gajadhar, 2004). In the secondary cluster the risk of infection is more than 9 times higher as compared to the rest of Catalonia. The epidemiological investigation was not able to find any link (common source) which could explain the increased risk. Demographic pressure has been suggested to be a risk factor, as higher population density can increase the risk of bovine cysticercosis (Boone et al., 2007). Unlike what happens in Western Catalonia, in the eastern area (Barcelona province), the high population density could be an explanation for the increased risk. In contrast to our findings, Kyvsgaard et al. (1991) reported no tendency towards the geographical concentration of the affected farms in Denmark. Boone et al. (2007) inspected visually the geographical distribution of the infected farms in Belgium by means of a geographic information system, and found that the infected farms were distributed all over the study area. However, they did not apply any statistical technique in order to test whether there was any pattern in the spatial distribution of the infected farms. Furthermore, some spatial pattern on the infected farms might be present, as a herd located in the province of Nimburg was 4 times more likely to be infected with T. saginata cysticercus than farms in other provinces. Water supply for animals was the most frequent risk factor with the highest score (23 farms). This is consistent with a recent study which found 3 environmental factors related to water (access of animals to surface water, flooding of pastures and proximity with a source of waste water), associated with an increased risk of bovine cysticercosis (Boone et al., 2007). The role of water in the transmission of the disease had been previously proposed by other authors (Guilhon, 1975; Kyvsgaard et al., 1991). Besides, current wastewater management practices seem to contribute to the dissemination of T. saginata eggs, which contaminate water streams and surface water (Dorny and Praet, 2007). The results of the most likely route of infection should be taken with care as it is a semi-quantitative approach and the scores given are based on the opinion of experts rather than in real quantitative data. Therefore, the change in the weighting given to the different risks may affect the results obtained. However, we regard this system as a simple method to get an estimation of what may be the most likely route of infection at farm level and the relative importance of the different risk factors in Catalonia.
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5. Conclusion Bovine cysticercosis is present in North-Eastern Spain. The number of infected animals has increased between 2005 and 2007, parallel to the raise in the number of slaughterhouses which detect infected animals. This increase seems more likely to be related to the efforts made by the Veterinary Authorities to improve the detection of bovine cysticercosis in slaughterhouses, rather than to the increase of taenia carrierships. Dairy and larger farms seem to be at higher risk of bovine cysticercus infection, and water supply for animals appears to be the most frequent route of infection. The location of the farm may also have an influence on the risk of bovine cysticercosis. Acknowledgements This study has been possible thanks to the Official Veterinarians from the Department of Agriculture of the Catalonian Autonomous Government, who were responsible of completing the questionnaires, and to Ariadna Garcia, who dealt with the questionnaires. References Aamodt, G., Samuelsen, S.O., Skrondal, A., 2006. A simulation study of three methods for detecting disease clusters. Int. J. Health Geogr. 12, 5–15. Abuseir, S., Ku¨hne, M., Schneider, T., Klein, G., Epe, C., 2007. Evaluation of a serological method for the detection of Taenia saginata cysticercosis using serum and meat juice samples. Parasitol. Res. 101, 131–137. Anon., 2004. Regulation (EC) no 854/2004 of the European parliament and of the council of 29 April 2004 laying down specific rules for the organisation of official controls on products of animal origin intended for human consumption. Anon., 2007a. Estadı´sticas ganaderas. Available from:.
Boone, I., Thys, E., Marcotty, T., Borchgrave, J., Ducheyne, E., Dorny, P., 2007. Distribution and risk factors of bovine cysticercosis in Belgian dairy and mixed herds. Prev. Vet. Med. 82, 1–11. Cabaret, J., Geerts, S., Madeleine, M., Ballandonne, C., Barbier, D., 2002. The use of urban sewage sludge on pastures: the cysticercosis threat. Vet. Res. 33, 575–597. Dorny, P., Vercammen, F., Brandt, J., Vansteenkiste, W., Berkvens, D., Geerts, S., 2000. Sero-epidemiological study of Taenia saginata cysticercosis in Belgian cattle. Vet. Parasitol. 88, 43–49. Dorny, P., Praet, N., 2007. Taenia saginata in Europe. Vet. Parasitol. 149, 22–24. EFSA, 2004. Opinion of the Scientific Panel on Biological Hazards on ‘‘Risk assessment of a revised inspection of slaughter animals in areas with low prevalence of Cysticercus’’. EFSA J. 176, 1–24. Garcia Castro, M.C., 2003. Higiene e Inspeccio´n de carnes – vol. II, bases cientificas y legales de los dictamenes de matadero. Benito Moreno, Edit. Diaz de Santos, Madrid, 269 pp. Geerts, S., 1990. Taenia saginata: een eeuwig probleem? Verhand. Kon. Acad. Geneesk. Belg. 52, 537–564. Geysen, D., Kanobana, K., Victor, B., Rodriguez-Hidalgo, R., Borchgrave, J., Brandt, J., Dorny, P., 2007. Validation of meat inspection results for Taenia saginata cysticercosis by PCR-restriction fragment length polymorphism. J. Food Protect. 70 (1), 236–240. Guilhon, J., 1975. Le roˆle de la pollution hydrique dans l’e´tiologie et l’e´pide´miologie de la cysticercose bovine et du te´niasis humain. Rec. Med. Vet. 151, 39–45. Kulldorf, M., 1997. A spatial scan statistic. Commun. Stat. Theor. M. 26, 1481–1496. Kulldorf, M., 2005. SaTScan User Guide for version 6.0. Available form:. Kyvsgaard, N.C., Ilsoe, B., Henriksen, S.A., Nansen, P., 1990. Distribution of Taenia saginata cysts in carcases of experimentally infected calves and its significance for routine meat inspection. Res. Vet. Sci. 49, 29– 33. Kyvsgaard, N.C., Ilsøe, B., Willeberg, P., Nansen, P., Henriksen, S.A., 1991. A case-control study of risk factors in light Taenia saginata cysticercosis in Danish cattle. Acta Vet. Scand. 32, 243–252. Scandrett, W.B., Gajadhar, A.A., 2004. Recovery of putative taeniid eggs from silt in water associated with an outbreak of bovine cysticercosis. Can. Vet. J. 45, 758–760. SCVMPH, 2000. Opinion of the Scientific Committee on veterinary measures relating to public health on the control of taeniosis/cysticercosis in man and animals. Available from: . Walter, M., Koske, J.K., 1980. Taenia saginata cysticercosis: a comparison of routine meat inspection and carcase dissection results in calves. Vet. Rec. 106, 401–402.
Contents lists available at ScienceDirect
Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar
Descriptive and spatial epidemiology of bovine cysticercosis in North-Eastern Spain (Catalonia) A. Allepuz a,b,*, S. Napp a, A. Picado a,d, A. Alba a, J. Panades c, M. Domingo a,b, J. Casal a,b a
Centre de Recerca en Sanitat Animal (CReSA), UAB-IRTA, Campus de la Universitat Auto`noma de Barcelona, 08193 Bellaterra, Barcelona, Spain Departament de Sanitat i Anatomia Animals, Universitat Auto`noma de Barcelona, 08193 Bellaterra, Barcelona, Spain c Departament d’Agricultura, Alimentacio´ i Accio´ Rural de la Generalitat de Catalunya, Gran Via de les Corts Catalanes, 612-614, 08007, Barcelona, Spain d Infectious and Tropical Diseases Department, London School of Hygiene and Tropical Medicine, London, UK b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 3 June 2008 Received in revised form 13 August 2008 Accepted 25 September 2008
From March 2005 to December 2007, 284 animals from 67 cattle farms (24 dairy and 43 beef) affected by bovine cysticercosis were detected in the region of Catalonia (NorthEastern Spain). Dairy farms were almost twice more likely to be affected than beef farms (OR = 1.79, 95% CI = 1.08–2.96, p < 0.05), and infected premises have a statistically significant (p < 0.05) larger number of animals when compared to uninfected farms in Catalonia. The geographical distribution of the infected farms was evaluated and two statistically significant clusters were identified. The most likely cluster was located in the western part of the study region, with 8 out of 10 farms infected. Epidemiological investigations revealed that the 8 farms belonged to the same company. The secondary cluster was located in Eastern Catalonia with 12 infected farms out of 167 cattle farms. No epidemiological links were found among the 12 infected premises. A questionnaire, based on the EFSA risk assessment, was used to assess the most likely route of introduction into each affected farm. Water supply for animals was the route with the highest score in 41.8% of the cases. ß 2008 Elsevier B.V. All rights reserved.
Keywords: Bovine cysticercosis Routes of infection Geographical distribution
1. Introduction Bovine cysticercosis is a disease caused by the larval infection of Taenia saginata in cattle (EFSA, 2004). T. saginata occurs in the small intestine of humans who are the final host of this tapeworm (Abuseir et al., 2007). Humans get infected by eating raw or undercooked meat containing viable cysticerci. The tapeworm develops in the small intestine and becomes sexually mature in about 3 months, producing gravid proglottids, which are mobile and either migrate from the host’s anus spontaneously or
* Corresponding author at: Centre de Recerca en Sanitat Animal (CReSA), UAB-IRTA, Campus de la Universitat Auto`noma de Barcelona, 08193 Bellaterra, Barcelona, Spain. Tel.: +34 93 581 10 47; fax: +34 93 581 32 97. E-mail address: [email protected] (A. Allepuz). 0304-4017/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.vetpar.2008.09.027
are shed in faeces. The presence of the tapeworm in the intestine can cause some abdominal discomfort, mild diarrhoea, weight loss and pruritus caused by migrating proglottids (Dorny and Praet, 2007). The prevalences in humans are highly variables within a country and between countries. This variability in prevalence is due to hygienic habits, quality of meat inspection and culinary habits. However, human taeniosis is not a notifiable disease and reported prevalences are only indicative (Cabaret et al., 2002). Transmission to animals occurs upon contamination of food or water by faeces of infected humans (Geysen et al., 2007). The contaminated material can derive directly from human faeces or via sewage plants after flooding or sewage sediment distributed on pastures (Abuseir et al., 2007). Direct transmission of eggs, resulting from hand rising of suckling calves by tapeworm carriers has been reported, but appears to be rare (Dorny and Praet, 2007).
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European legislation (Anon., 2004) requires that a systematic investigation for bovine cysticercosis in all bovine animals over 6 weeks old must be carried out. The inspection is done by the Official Veterinarian at the slaughterhouse by visual examination and by cuts in masseter muscles, tongue, diaphragm and heart. If the infestation is generalised, the carcass and offal are rejected. However, if infestation is localised, the part of the carcass or offal affected must be rejected and the remainder can be kept in cold storage. Therefore, the disease causes economical losses to the cattle industry mainly due to condemnation, refrigeration and downgrading of infected carcasses. These losses have been estimated to amount to 30–45% of the value of the carcasses (Geerts, 1990). Epidemiological surveys on bovine cysticercosis are scarce in Europe (Boone et al., 2007). The prevalence of bovine cysticercosis in Europe is based on meat inspection reports, and ranges from 0.007% to 6.8% with a wide variation between countries, regions and abattoirs (Cabaret et al., 2002; Dorny and Praet, 2007). However, meat inspection has a low sensitivity, resulting in an underestimation of the prevalence of bovine cysticercosis (Kyvsgaard et al., 1990; Dorny et al., 2000; Dorny and Praet, 2007). Moreover, few EU countries report their data to the OIE and these data are rather fragmentary, so reliable conclusions are difficult to make (Dorny and Praet, 2007). In March 2005, the detection of a series of cases of bovine cysticercosis in Catalonian slaughterhouses resulted in an outbreak investigation on the farms initially affected, and the establishment of a monitoring programme targeting slaughterhouses and cattle farms. This paper presents the results of the first 3 years of monitoring programme. The aims of this study are to describe the demographic, geographical and temporal characteristics of infected cattle herds in Catalonia from 2005 to 2007, and to asses the most likely route of introduction of the eggs of T. saginata in the affected farms. 2. Materials and methods 2.1. Descriptive analyses The study was conducted in Catalonia. This region has an area of 32,105 km2 and there are approximately 626,400 cattle in 7496 farms, which represent the 10% of the total cattle population of Spain (Anon., 2007a). Data on the cattle farms in Catalonia: type of farm (beef or dairy), census and geographical coordinates were provided by the Department of Agriculture of the Autonomous Government of Catalonia. Data on the farms affected by bovine cysticercosis as well as the number of animals detected, date of detection and number of slaughterhouses that have reported cases of cysticercosis, were provided by the Department of Health of the Autonomous Government of Catalonia. Diagnostic of infestation was done by the Official Veterinarian at the slaughterhouse by visual examination and by cuts in masseter muscles, tongue, diaphragm and heart according to the European legislation (Anon., 2004). When visual diagnosis was not clear, samples were sent to the
Pathology Department (Barcelona Veterinary College), where they were confirmed by histopathology. To estimate whether there were differences between the proportion of beef and dairy farms infected, we used Pearson’s chi-square test and calculated the odds ratio (OR) with its 95% confidence interval (CI). A non-parametric two-sample test (Mann–Whitney U-test) was used to compare the number of animals in infected and noninfected farms. 2.2. Spatial analysis To explore the spatial distribution of the infected farms in the region, we run a purely spatial analysis based on the Bernoulli model with SaTScan1 v6.1 (http://www.SaTScan.org) as described by Kulldorf (1997). With the Bernoulli model, farms were classified as either cases or non-cases. A farm was classified as a case if at least one infected animal was detected. The spatial scan statistic searches for clusters using a variable circular window size to detect spatial clusters in large areas, while controlling for the underlying population (Kulldorf, 1997). The circle is centred on each of the points. For each point, the size of the circle varies from zero to some upper limit specified by the user (50% of the population at risk). Under the null hypothesis, the expected number of cases in each area is proportional to its population size. For each circle a likelihood ratio statistic is computed based on the number of observed and expected cases within and outside the circle (Aamodt et al., 2006). The p-value is obtained through Monte Carlo hypothesis testing by comparing the rank of the maximum likelihood from the real data set with the maximum likelihood from the random data sets. We scanned only for clusters with high rates (more cases than expected). The number of Monte Carlo simulations was set at 999. Only statistically significant (p-value < 0.05) nonoverlapping clusters were reported (Kulldorf, 2005). The farms affected with cysticercosis and the spatial clusters detected in Catalonia were represented in a map using ArcMap1 v9.1 (ESRI, Redlands, CA). Detailed epidemiological investigations were carried out on clustered farms to determine possible links among them. 2.3. Most likely route of T. saginata introduction A questionnaire, based on the EFSA risk assessment (EFSA, 2004), was used to assess the most likely route of introduction on each affected farm. The questionnaire included questions on risk factors related to five potential routes of introduction: water supply for animals, feed, pastures, personnel and other routes. Each of these risk factors had an associated score which reflected the probability of introducing bovine cysticercosis (EFSA, 2004) (Table 1). This scoring system was used to evaluate the most likely route of introduction in each of the affected farms, as the risk factor with the highest score was considered the most likely route of introduction of T. saginata. The questionnaires were completed by official veterinarians from the Department of Agriculture by on-site personal interview with the farm owners. An 82% response
A. Allepuz et al. / Veterinary Parasitology 159 (2009) 43–48 Table 1 Routes of introduction, associated risk factors and corresponding risk scores according to EFSA’s opinion on Cysticercus Risk Assessment (EFSA, 2004). Route
Risk factors
Score
Water supply for animals
Use of untreated surface water (river/lake) Use of untreated local water (e.g. wells) Use of treated local water Use of municipal water Floods potentially spreading T. saginata eggs on the grazing and/or feed components
4 3
Regularlya Irregularlya Regular or irregularb
2 1 4 3 2
Feed
Roughage used multi – source Roughage used single – source Concentrate used multi – source Concentrate used single – source
2 1 2 1
Pastures
Animals kept mainly outdoor Animals kept combined indoor and outdoor Animals kept indoor only Use of untreated sewage as fertilisers Use of treated sewage as fertilisers
4 3
Visit endemic area in the last year
4
Personnel
Other routes
a b
Farm located near camping sites Farm located near public footpaths Waters known as receiving sewage. Waters not receiving sewage.
1 4 3
4 2
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28.6% of the bovines slaughtered in Catalonia. By 2007 the number of slaughterhouses reporting bovine cysticercosis increased to 9 (responsible for the 73.1% of the bovines slaughtered in the region). The number of infected animals detected per farm varies from 1 to 70. The fact that there was a single farm responsible for 70 of the 284 cases reported in Catalonia resulted in an epidemiological investigation. This farm was a medium size fattening farm located in Western Catalonia. Given that the animals originated from different countries suggested that the animals were infected at the farm rather infected at the farm of origin. The epidemiological investigation was not able to determine with precision the source of infection, although water and feed were suggested as the most likely sources of contamination of the farm. The number of times infected animals were detected in a particular farm varies from 1 to 17, although in most of the farms (73%) infected animals were detected just 1 time (Table 3). A statistically significant association between type of farm (beef vs. dairy) and infection status has been found. Dairy farms were almost twice more likely to be affected than beef farms (OR = 1.79, 95% CI = 1.08–2.96, p < 0.05) In both dairy and beef farms the farm size (number of animals) seems to be related to the presence of bovine cysticercosis: infected farms have a statistically significant (p < 0.05) larger number of animals when compared to the uninfected farms in Catalonia. 3.2. Spatial analysis
rate was obtained, not being possible to complete the questionnaire in 12 of the affected farms. The data was double entered checked and corrected. 3. Results 3.1. Descriptive analysis From March 2005 to December 2007, 284 animals from 67 cattle farms (24 dairy and 43 beef herds) affected by bovine cysticercosis were detected. Infected farms were reported almost every month. The mean prevalence of infected animals is 1.8 per 10,000 slaughtered animals. The prevalence of infected animals increased throughout the study period from 0.015% in 2005 to 0.022% in 2007 (Table 2). The number of slaughterhouses which detected bovine cysticercosis also increased during the study period. In 2005 bovine cysticercosis was detected in 4 different slaughterhouses, which were responsible for the Table 2 Number of T. saginata bovines infected and number of cattle slaughtered in Catalonia from March 2005 to December 2007. Year
Number of animals detected
Cattle slaughtered
Prevalence (%)
2005 (from March) 2006 2007
75 97 112
499,846 567,024 498,351
0.015 0.017 0.022
The spatial analysis evidenced that the geographical distribution of the infected farms is clustered. The SaTScan identified two statistically significant clusters (Fig. 1). The most likely cluster had an area of 9 km2 and was located in the West part of the study region. The relative risk was 99.3, in this area there were 8 out of 10 farms infected. Epidemiological investigations revealed that the 8 farms belonged to the same company. Furthermore, 2 farms located outside the cluster, but belonging to the same company were also affected. The large number of animals infected and the fact that the animals originated from different regions in Spain and different countries in Europe practically discard the possibility of the animals being infected in origin. Tracing back of movements revealed that all the infected
Table 3 Number of times Cysticercus bovis infected animals were detected in a farm from March 2005 to December 2007 in Catalonia. Number of times infected animals were detected in a farm
Percentage
1 2 3 4 5 11 12 17
73.1 11.9 3.0 4.5 1.5 1.5 3.0 1.5
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Fig. 1. Geographical distribution of the 67 T. saginata infected farms detected between 2005 and 2007 (triangles), and location of the most likely and the secondary cluster (circles) in Catalonia.
animals had stayed at some point (although not during the same period) in a single farm belonging to the company, where calves were milk-fed for 2 months before being transferred to the other farms (fattening farms). Therefore the possibility of the animals getting infected at that farm could not be ruled out. In this farm the water was obtained from a channel. However, the infection of the animals at the fattening farms was also possible as the source of feed and straw was also common for all the farms. On the other hand, all animals from the farms located in the cluster were kept indoors, and roughage did not come from pastures fertilised with sewage sludge. The secondary spatial cluster, located in Eastern Catalonia has an area of 255 km2 and includes 167 cattle farms. Bovine cysticercosis was detected in 12 of them, presenting an increased risk of 9.4 compared to farms outside the cluster. No epidemiological links were found among the 12 infected premises. 3.3. Most likely route of infection The scoring system used shows that water supply for animals was the route with the highest score in 41.8% of the cases (23 farms), followed by feed and other routes in 9.1% (5 farms) and 12.7% (7 farms) respectively. Personnel and pastures seemed less frequent as they appeared to be responsible for the infection of 9.1% (5 farms) and 5.5% (3 farms) respectively. In 12.7% (7 farms), the highest score was shared by 2 risk factors, whereas in 7.3% (4 of the farms), the highest score was shared by 3 risk factors. For one farm, the route of infection was considered as unknown, as none of the risk factors scored over 1.
4. Discussion Epidemiological data on bovine cysticercosis are scarce in Europe, and therefore prevalence data in order to compare the situation between countries is difficult to find. Moreover, the reporting in the different countries is not standardised, and prevalence data are difficult to collate and compare. Bovine cysticercosis is present at a low level in Western European countries, and the prevalences vary between 0.007% and 2.4% (SCVMPH, 2000). In Northern Spain, Garcia Castro (2003), based on detailed meat inspection, reported prevalences of 0.54% in animals kept outdoors at pastures. More recently, a study carried out in Belgium reports prevalences of bovine cysticercosis, based on official meat inspection, of 0.22% in 2001, 0.44% in 2003, 0.34% in 2004 and 0.28% in 2005 (Boone et al., 2007). This paper provides information on bovine cysticercosis prevalence in Catalonia (North-Eastern Spain) that was not previously reported. The observed mean prevalence (0.018%) is low compared to the figures reported in Belgium by Boone et al. (2007) and Northern Spain by Garcia Castro (2003). A possible explanation to this lower prevalence is that in Catalonia most of the animals are kept indoors. Grazing on pastures potentially contaminated with T. saginata eggs derived from human faeces directly or via sewage sediment distributed in pastures (Abuseir et al., 2007), free access of cattle to surface water (rivers, lakes, canals) and flooding of pastures have been described as important environmental risk factors for the detection of bovine cysticercosis in a herd (Boone et al., 2007). Therefore, differences in the production systems may, to some extent, explain the differences in prevalences. On the other hand, mean prevalence of infected animals was calculated from data given by the authorities. The
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validity of these data depends on the quality of the visual meat inspection. Findings by the current palpation and incision techniques at routine meat inspection cannot be considered as an accurate indicator of the true occurrence of bovine cysticercosis (EFSA, 2004). The classical meat inspection techniques detect only a minor fraction of the carcasses infected with cysticerci (Dorny et al., 2000). The fact that the probability of cyst detection at routine post mortem meat inspection depends on the number of cysts present in the animal (Walter and Koske, 1980) and that most animals in Europe are lightly infected and that only a proportion of cysts are located at the predilection sites (Dorny and Praet, 2007), contribute to the low sensitivity of meat inspection. Moreover, there may be differences in cysticercosis detection efficacy due to differences in motivation and skills of inspectors or meat inspection facilities (EFSA, 2004). In our study, bovine cysticercosis prevalence was estimated from routine meat inspection at slaughterhouses and, therefore, is likely to be an underestimation of the real prevalence. However, the extent to what prevalence was underestimated is difficult to quantify. Dorny et al. (2000) found that the prevalence obtained by means of an ELISA was at least 10 times higher than the prevalence obtained by routine meat inspection. In Belgium, the increase in the number of cases from 2001 to 2003 was attributed to a more careful inspection at abattoirs, rather than to a real increase in the prevalence (Boone et al., 2007). Something similar is likely to have happened in Catalonia. As neither the diagnostic approach, nor the staff involved, have changed, the increase in the number of cases detected is more likely to be related to the efforts made by the Veterinary Authorities (courses, seminars and meetings) in order to improve the detection at slaughterhouses, than to a real increase of prevalence. The increase in the number of slaughterhouses detecting cysticercosis (from 3 in 2005 to 9 in 2007) may be considered as an indication of the improvements made in the detection. Besides, the number of T. saginata carrierships in Spain does not seem to have increased. The number of cases reported to the authorities was 43 in 2005, 42 in 2006 and 17 in the first half of 2007 (Anon., 2007b), although these data should be taken with caution as it is not a notifiable disease. On the other hand, in most of the farms infected animals have been detected only one time, a result which is consistent with what happens in other European countries (Dorny and Praet, 2007). This could be due to the fact that most infections are the result of the accidental ingestion of eggs which are found disseminated in the environment, and therefore light infections are more common than heavy infections (Dorny and Praet, 2007). We found that the proportion of dairy farms affected was higher than the proportion of beef farms. This could be attributable to the fact that in dairy cattle the age of the animals at slaughter is normally higher than in beef cattle. Dorny et al. (2000) found that sero-prevalence was positively correlated with increasing age. As the infection is accidental, the risk of exposure increases with the age of the animals (Dorny and Praet, 2007). Unfortunately, data on the age of the animals were not recorded.
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The cluster analysis evidenced that the risk of infection was not homogeneously distributed over the entire region. There are some areas where the risk of infection is higher as compared to the entire region. In the most likely cluster the risk of infection is almost 100 times higher as compared to the rest of Catalonia. Besides, in contrast to what usually happens in Catalonia, several animals from these farms were affected. Despite the presence of common factors, among the affected farms in the cluster, the origin of the outbreak could not be clearly established. When cysticercosis outbreaks occur, the source of contamination is seldom determined (Scandrett and Gajadhar, 2004). In the secondary cluster the risk of infection is more than 9 times higher as compared to the rest of Catalonia. The epidemiological investigation was not able to find any link (common source) which could explain the increased risk. Demographic pressure has been suggested to be a risk factor, as higher population density can increase the risk of bovine cysticercosis (Boone et al., 2007). Unlike what happens in Western Catalonia, in the eastern area (Barcelona province), the high population density could be an explanation for the increased risk. In contrast to our findings, Kyvsgaard et al. (1991) reported no tendency towards the geographical concentration of the affected farms in Denmark. Boone et al. (2007) inspected visually the geographical distribution of the infected farms in Belgium by means of a geographic information system, and found that the infected farms were distributed all over the study area. However, they did not apply any statistical technique in order to test whether there was any pattern in the spatial distribution of the infected farms. Furthermore, some spatial pattern on the infected farms might be present, as a herd located in the province of Nimburg was 4 times more likely to be infected with T. saginata cysticercus than farms in other provinces. Water supply for animals was the most frequent risk factor with the highest score (23 farms). This is consistent with a recent study which found 3 environmental factors related to water (access of animals to surface water, flooding of pastures and proximity with a source of waste water), associated with an increased risk of bovine cysticercosis (Boone et al., 2007). The role of water in the transmission of the disease had been previously proposed by other authors (Guilhon, 1975; Kyvsgaard et al., 1991). Besides, current wastewater management practices seem to contribute to the dissemination of T. saginata eggs, which contaminate water streams and surface water (Dorny and Praet, 2007). The results of the most likely route of infection should be taken with care as it is a semi-quantitative approach and the scores given are based on the opinion of experts rather than in real quantitative data. Therefore, the change in the weighting given to the different risks may affect the results obtained. However, we regard this system as a simple method to get an estimation of what may be the most likely route of infection at farm level and the relative importance of the different risk factors in Catalonia.
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5. Conclusion Bovine cysticercosis is present in North-Eastern Spain. The number of infected animals has increased between 2005 and 2007, parallel to the raise in the number of slaughterhouses which detect infected animals. This increase seems more likely to be related to the efforts made by the Veterinary Authorities to improve the detection of bovine cysticercosis in slaughterhouses, rather than to the increase of taenia carrierships. Dairy and larger farms seem to be at higher risk of bovine cysticercus infection, and water supply for animals appears to be the most frequent route of infection. The location of the farm may also have an influence on the risk of bovine cysticercosis. Acknowledgements This study has been possible thanks to the Official Veterinarians from the Department of Agriculture of the Catalonian Autonomous Government, who were responsible of completing the questionnaires, and to Ariadna Garcia, who dealt with the questionnaires. References Aamodt, G., Samuelsen, S.O., Skrondal, A., 2006. A simulation study of three methods for detecting disease clusters. Int. J. Health Geogr. 12, 5–15. Abuseir, S., Ku¨hne, M., Schneider, T., Klein, G., Epe, C., 2007. Evaluation of a serological method for the detection of Taenia saginata cysticercosis using serum and meat juice samples. Parasitol. Res. 101, 131–137. Anon., 2004. Regulation (EC) no 854/2004 of the European parliament and of the council of 29 April 2004 laying down specific rules for the organisation of official controls on products of animal origin intended for human consumption. Anon., 2007a. Estadı´sticas ganaderas. Available from:
Boone, I., Thys, E., Marcotty, T., Borchgrave, J., Ducheyne, E., Dorny, P., 2007. Distribution and risk factors of bovine cysticercosis in Belgian dairy and mixed herds. Prev. Vet. Med. 82, 1–11. Cabaret, J., Geerts, S., Madeleine, M., Ballandonne, C., Barbier, D., 2002. The use of urban sewage sludge on pastures: the cysticercosis threat. Vet. Res. 33, 575–597. Dorny, P., Vercammen, F., Brandt, J., Vansteenkiste, W., Berkvens, D., Geerts, S., 2000. Sero-epidemiological study of Taenia saginata cysticercosis in Belgian cattle. Vet. Parasitol. 88, 43–49. Dorny, P., Praet, N., 2007. Taenia saginata in Europe. Vet. Parasitol. 149, 22–24. EFSA, 2004. Opinion of the Scientific Panel on Biological Hazards on ‘‘Risk assessment of a revised inspection of slaughter animals in areas with low prevalence of Cysticercus’’. EFSA J. 176, 1–24. Garcia Castro, M.C., 2003. Higiene e Inspeccio´n de carnes – vol. II, bases cientificas y legales de los dictamenes de matadero. Benito Moreno, Edit. Diaz de Santos, Madrid, 269 pp. Geerts, S., 1990. Taenia saginata: een eeuwig probleem? Verhand. Kon. Acad. Geneesk. Belg. 52, 537–564. Geysen, D., Kanobana, K., Victor, B., Rodriguez-Hidalgo, R., Borchgrave, J., Brandt, J., Dorny, P., 2007. Validation of meat inspection results for Taenia saginata cysticercosis by PCR-restriction fragment length polymorphism. J. Food Protect. 70 (1), 236–240. Guilhon, J., 1975. Le roˆle de la pollution hydrique dans l’e´tiologie et l’e´pide´miologie de la cysticercose bovine et du te´niasis humain. Rec. Med. Vet. 151, 39–45. Kulldorf, M., 1997. A spatial scan statistic. Commun. Stat. Theor. M. 26, 1481–1496. Kulldorf, M., 2005. SaTScan User Guide for version 6.0. Available form: