Institute of Parasitology, University of Zurich, Switzerland Taenia saginata Cysticercus bovis Epidemiology and diagnosis of bovine Division of Gastroenterology, Faculty of Medicine, Dokuz Eylül University, Izmir, Turkey Peter Deplazes Rome 2010 Prevalence Abattoir records in Spain Occurrence of in cattle at slaughterhouse in Northern Spain (Garcia-Castro, 2003) Ilsoe et al. (1990) Mobius (1993) Zimmermann (1985) Van Knappen & Buys (1985) Geerts et al. (1992) Garate (1999) Pawlowski (1999) Battelli (1999) 10.06.2010 / 3 Abattoir records in Italy Occurrence of in cattle slaughtered in Italy during the period 1999-2000 (Osservatorio epidemiologico veterinario regionale della Lombardia) Abattoir records in Switzerland EU approved abattoirs in Switzerland years Heifer Bull/ Ox Cow N Prevalence N Prevalence N Prevalence Significance? CI 95%: (0.16-0.21) 0.00-0.25) # 1 8 40 335 0.63% 66 215 0.24% 142 839 1.48% # 2 4 32 368 0.22% 40 046 0.13% 57 447 1.17% # 3 5.5 18 554 0.2% 36 126 0.01% 71 996 0.49% # 4 10 6 397 0.39% 23 065 0.14% 17 189 0.45% # 5 2 11 025 1.05% 44 402 0.18% 48 494 0.99% 0.00-0.20 Fusszeile 1
Discussion Taenia saginata prevalence in the human population of Switzerland? Known parasite transmission Insufficient diagnostic tools Doubtful strategy for control Distribution of bovine in Switzerland (April 2005 April 2006) N = 1 377 (reports of 6 EU-approved abattoirs) Taeniid egg in effluent from sewage purification plant: isolated by a sieving method Taeniid egg identification by PCR and sequencing: (Trachsel et al., Parasitology, 2007) 10.06.2010 / 8 Analyses of effluent from a sewage purification plant Date Total amount Result Microscope Result multiplex PCR* Sequencing results 03.10.06 200 l - 267 bp T. saginata 09.10.06 300 l - negativ - 11.10.06 200 l - 267 bp T. saginata 13.10.06 200 l 2-3 taeniid eggs 267 bp 395 bp n.d. E. multilocularis 18.10.06 250 l - 267 bp Diphyllobothrium spp. 23.10.06 200 l - 267 bp T. saginata 25.10.06 200 l eggs 267 bp 395 bp Hymenolepis spp. n.d. Transmission in Switzerland: Most farms are family-run Average roughage area per farm: 22.3 ha Herd size per farm: 36.2 cattle units 60% without employees, 40% with an average of 3.2 employees *Trachsel et al., 2007. 10.06.2010 / 9 10.06.2010 / 10 financial losses for farmers in Switzerland Losses for farmers Cow Beef cattle Risk analysis for in cattle: Inclusion criteria on farm level 119 positive farms 66 negative farms Total loss per animal (no insurance) Total loss per animal (with insurance) 465.- 685.- 238.- 498.- Animals at least 9 months on the farm So far no records of on the farm % deduction (from normal meat price per kilo) 45.3 % 51.0 % confirmed infection: visual/ PCR (PCR as described: Gonzalez et al. 2000) homemade infection: animals on the same farm for at least 9 months with viable cysticerci 24 months with calcified lesions Questioning during farm visit or by phone interview 10.06.2010 / 11 10.06.2010 / 12 Fusszeile 2
Stool examination for T. saginata infections 317 samples from 49 farms (41% of the positive farms) Coproscopy: all negative Coproantigen-ELISA (Deplazes et al. 1991): 308 samples analysed 266 negative (86.3%) 35 not interpretable (11.4%) 7 positive (?) (2.3%) Egg isolation by filtration from coproantigen-positive samples (Mathis et al. 1996) - all negative 10.06.2010 / 13 Definitions of factors included in the risk analysis for bovine Variable on farm level Herd size Definition cattle units (1 unit = 500 kg live weight) Animal category Cow, beef cattle (each conventional or mother-cowhusbandry), heifer Roughage area Grazing time Transhumance Streams Fresh grass Purchased roughage External manure Domestic sewage Area administered where cattle graze and where grass is harvested (ha) Hours per year animal spends on pasture Grazing on alpine pastures at least once Direct or indirect (flooded pastures or fields) access to water from streams Feeding grass in the stable without storage Hey or silage (grass/corn) also from external production Manure from other farms spread on land Waste water from households connected to effluent pond 10.06.2010 / 14 Definitions of factors included in the risk analysis for bovine Variable on farm level Definition Definitions of factors included in the risk analysis for bovine Variable for farm surrounding Definition Employees Visitors Employee(s) working on farm within past five years Organised activities on farm (events, open days and other hospitality activities), horse pension Leisure activities Picnic place/ viewpoint Military Car park Wild camping Railway line Sports activities, lido, playground, open-air party, fairground near or on farmland Frequently visited Exercises on farm area Parking places (public or not) Observed camping activities (individual or camps) on unofficial campground Railway line along or through farm land Swiss federal railways: Around 60% of wagons have an open toilet system 10.06.2010 / 15 10.06.2010 / 16 Statistical analysis Risk factors in analysis based on 19 variables tested Logistic regression analysis Explanatory variables = risk factors + biologically comprehensible interactions 1. Pre-selection of factors Stepwise backwards selection with Aikaike information criterion (AIC; Software R) 2. Final selection (as AIC is very conservative) Likelihood-ratio test herd size organic or conventional contact streams feeding surface grazing time transhumance purchased roughage grass feeding external manure domestic sewage employees visitors leisure activities military activities parking places wild camping picnic place/ viewpoint railway line (animal category) 10.06.2010 / 17 10.06.2010 / 18 Fusszeile 3
Significant results of the logistic regression analysis Variable Present (% of the farms) Control farms Farms with Odds ratio 95% C.I. b p-value (LRT) Railway line 7.58 22.69 3.72 1.38-11.91 0.008 Leisure activities 4.55 13.45 3.58 1.05-16.59 0.039 Car park 9.09 26.05 3.05 1.20-8.91 0.020 Purchased roughage 18.18 33.61 2.89 1.37-6.49 0.009 Discussion Direct transmission on farms could not be demonstrated in this study! Attempts to diversify the income in the Swiss agriculture, including tourist activities and farm events, might contribute to new risks for. Old risks such as railway lines crossing farm grounds should in a long term be of minor importance. New strategies for control have to be considered Visitors 7.58 21.01 2.87 1.06-9.22 0.013 a) LRT: Likelihood-ratio test based on the final model compared to the one without the relevant variable b) 95% confidence intervals 10.06.2010 / 19 10.06.2010 / 20 Epidemiology EU Epidemiology EU Allepuz et al., (2008). Describtive and spatial epidemiology of bovine in North-Eastern Spain (Catalonia) : Most likely route of T. saginata introduction: - Questionnaire (based on the EFSA risk assessment) - on each affected farm (N = 67) - 5 risk factors included (water supply for animals, feed, pastures, personnel and other routes) - 82% response - Water supply for animals was the route with the highest score in 41.8% of the cases (23 farms) - Feed in 9.1% (5 farms) - other routes in 12.7% (7 farms) 10.06.2010 / 21 Boone et al., (2007). Distribution and risk factors of bovine in Belgian dairy and mixed herds : - Questionnaire to 1500 farms (750 positive and 750 negative) - 56.1% response - 28 factors included - province - number of cattle slaughtered in 2003 - Flooding of pastures - free access of cattle to surface water - Proximity to waste water effluent within a range of 200m around the farm 10.06.2010 / 22 Diagnosis at meat inspection Prevention / Meat inspection: EU-Legislation: Directive 64/433/EEC (OJ 121, 29.7.1964, p. 2012/64) amended by Directive 95/23/EC (OJ L 243, 11.10.1995, p.7) 25. The official veterinarian must examine, in particular: (d) the pericardium and the heart, the latter being incised lengthwise so as to open the ventricles 26. In addition, the official veterinarian must systematically carry out: A. An investigation for : (a) in bovine animals over six weeks old, at the level of: - the tongue, of which the musculature must be incised lengthwise on the lower surface, without damaging the organ excessively; - the oesophagus, which must be freed from the trachea; - the heart, which, in addition to the incision provided for in paragraph 25 (d), must be split from two opposite points from the auricles to the apex of the external and internal masseters, which shall be incised along two planes parallel to the mandible from its lower edge to its upper muscular insertion; - the diaphragm, the muscular part of which must be freed from the serous part; - the muscular surfaces of the carcass which are directly visible; 10.06.2010 / 23 Cutting musculature: - inner & exterior cheek muscles - heart Visual examination: 1 cm 10.06.2010 / 24 Fusszeile 4
Diagnosis at meat inspection: an old story Sensitivity of routine meat inspection: Dewhirst et al. (1967). Comparison of prescribed US inspection procedures for bovine with more extensive examinations of the predilection sites showed that the prescribed procedures did not detect infection in at least 26% of the carcasses.. Geerts et al., (1980). Hearts of 100 slaughtered cattle, approved fit for human consumption at meat inspection, [ ]. In 25 per cent of the animals one or more caseous or calcified cysts were recovered. Walther & Koske, (1980). [ ] At dissection 75.9% were found infected. However, only 38.3% of the infected carcasses were detected at meat inspection. Diagnosis at meat inspection Sensitivity of routine meat inspection: Hörchner, (1983). [ ] Exact examination of wholesome recognised slaughtered animals performed at different slaughterhouses in Europe showed changes ranging between 11.4 and 25% particularly in the muscles of heart. [ ] Dorny et al., (2000). [ ], the prevalence of bovine was more than 10 times higher with the antigen detection ELISA than by classical meat inspection. Underestimation of the prevalence by a factor 3 to 10 10.06.2010 / 25 10.06.2010 / 26 Diagnosis at meat inspection Sensitivity of routine meat inspection: Estimation by Walther & Koske (1980) of the sensititiviy of detecting cysticerci at routine post mortem meat inspection: Predilection sites Lopes et al., (2010): Preferential infection sites of Cysticercus bovis in cattle experimentally infected with Taenia saginata eggs 10.06.2010 / 27 10.06.2010 / 28 Diagnosis at meat inspection: specificity High as confirmed by PCR even in calcified lesions: Methods: 6 additional muscle cuts (heart) 3 EU-approved abattoirs in Switzerland (N=1 088) routine diagnosisperformed at meat inspection microscopical and molecular confirmation of positive abattoir result Taenia-specific primers Cest3/5 (Trachsel et al., 2007) 10.06.2010 / 29 Fusszeile 5
Investigated carcasses N = 1 088 Positive by routine meat inspection 20 (1.8 %) Positive by additional cuts 29 (2.7 %) Total positive cases 49 (4.5%) (N = 1 068) Additional findings (parasitic lesions) Heart surface 4 0.37 % Missed at inspection 1 0.09 % 1 additional cut 1 0.09 % 103 cysticerci were located 91.8% of the cases had a single cyst by total heart dissection, 8 animals also had viable cysts next to degenerated ones. routinely performed meat inspection of the heart: 60% (3/5) cysticerci of viable infection status additional examination of the heart: 25.8% (8/31) cysticerci of viable infection status 2 additional cuts 11 1.03 % 3 additional cuts 7 0.66 % 4 additional cuts 3 0.28 % 5 additional cuts 1 0.09 % 6 additional cuts 1 0.09 % Total 29 2.7 % Cysts per animal at routinely performed meat inspection with additional heart cuts: 50 45 44 Alternative tools for the diagnosis of bovine Detection of specific antigens in serum Animals 40 35 30 25 20 Detection of specific antibodies in serum or meat juice 15 10 5 0 3 2 1 2 3 Cysts Evaluation of diagnostic tools for bovine Detection of circulating Ag: MoAb B158C 11 A 10 / B60H 8 A 4 (anti-e/s) Evaluation of diagnostic tools for bovine Peptides for detection of specific Ab: (Characterized by Bonay et al., (2001) HP6-2 (major secreted antigen of activated oncospheres) Van Kerckhoven et al. (1998) Sensitivity: 92%, Specificity: 98.7% > 50 cysts per infected animal Ts45S-10 (T. ovis 45S homologue protective antigen) Abuseir et al. (2007): HP6-2 Se: 100% Sp: 98% Ts45S-10 Se: 100% Sp: 97% Dorny et al. (2000) CO-group: Positive animals: 100 young calves reared under hygienic conditions 9 experimentally infected animals Sensitivity: 12.8%, Specificity: 98.7% < 50 cysts per infected animal Ts45S-10 HP6-2 Fusszeile 6
Evaluation of serological tools for bovine : Antigens Evaluation of serological tools for bovine : Samples Antibody-detecting tests: ELISA Metacestodes Pestle / homogenization Ultrasonic mixer Freezing / defrosting Ultrasonic desintegration Centrifugation Crude antigen Isoelectrofocusing IEF F1 to F5 IEF Cca extract Affinity chromatography Crude cysticercus antigen extract (Cca) In vitro- Cultivation Concentration Larval E/S Antigen-detecting tests: ELISA In preparation, collaboration with P. Dorny Synthetic peptides: HP6-2 Ts45S-10 10.06.2010 / 37 Serum and meatjuice samples: Cut-off group: - 49 healthy cattle (serum and meat-juice) Positive samples: - 51 naturally-infected samples - 9 experimentally infected serum samples Negative samples: - 61 cattle without liver-fluke - 47 cattle with Fasciola hepatica infection - 168 cattle with Dicrocoelium dendriticum infection Samples collected at abattoir Evaluation of serological tools for bovine : results Test Serum of naturally infected cattle (N=51) % Sensitivity Meat juiceof naturally infected cattle (N=51) Peptide HP6-2 61.8 9.8 100 Peptide Ts45S-10 73.5 49.0 100 Crude cysticercus antigen extract (Cca) 82.4 70.6 100 IEF Cca extract 52.9 35.3 100 Larval E/S 88.2 76.5 100 Test % Spezifity (Serum) Serum of experimentally infected cattle Healthy (N=61) F. hepatica (N=47) D.dendriticum Peptide HP6-2 98.4 83.0 88.7 Peptide Ts45S-10 95.1 83.0 85.1 Crude cysticercus antigen extract (Cca) 90.2 42.6 90.5 IEF Cca extract 88.5 97.9 92.9 Larval E/S 96.7 91.5 95.8 * Abuseir et al. Parasitol Res., 2007 ** Ogunremi and Benjamin, Vet. Parasitol., 2009 Literature Sensitivity 100 %?* 100 %?* nd 92.9* Specificity 98 %?* 97 %?* nd 90.6 %* Factors influencing transmission and control Asymptomatic infection: few definitive hosts can contaminate large areas Survival in the environment Meet inspection with low sensitivity Vaccination commercially not available Chemotherapy expensive, will not eliminate calcified lesions Risk factors for bovine infection: variable Canadian control system Thank you for your attention! The premises remains under quarantine until such time as the source has been eliminated and there is evidence that the herd is free of the parasite. The CFIA may pay producers compensation up to established limits for carcasses condemned or treated for. 10.06.2010 / 41 10.06.2010 / 42 Fusszeile 7