10.06.2010

Fusszeile 1

Epidemiology and diagnosis of bovine

cysticercosis

Peter Deplazes

Rome 2010

Institute of Parasitology, University of Zurich, Switzerland

Taenia saginataCysticercus bovis

Div

isio

n of

Gas

troe

nter

olog

y, F

acul

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of M

edic

ine,

Dok

uz E

ylül

Uni

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ity,

Izm

ir, T

urke

y

10.06.2010 / 3

Prevalence

Ilsoe et al. (1990)

Mobius (1993)

Zimmermann (1985)

Van Knappen & Buys (1985)

Geerts et al. (1992)

Garate (1999)

Pawlowski (1999)

Battelli (1999)

Cysticercosis in cattle:Abattoir records in Spain

Occurrence of cysticercosis in cattle at slaughterh ouse in Northern Spain (Garcia-Castro, 2003)

Cysticercosis in cattle:Abattoir records in Italy

Occurrence of cysticercosis in cattle slaughtered i n Italy during the period 1999-2000 (Osservatorio epidemiologico veterinario reg ionale dellaLombardia)

CI 95%:

(0.16-0.21)

0.00-0.25)

0.00-0.20

Signifi-cance?

Cysticercosis in cattle:Abattoir records in Switzerland

EU approved abattoirs in Switzerland

years Heifer Bull/ Ox Cow

N Prevalence N Prevalence N Prevalence

# 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%

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Fusszeile 2

Distribution of bovine cysticercosis in Switzerland ( April 2005 –April 2006) N = 1‘377 (reports of 6 EU-approved abatto irs)

Known parasite transmission

Insufficient diagnostic tools

Doubtful strategy for control

Discussion

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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)

Taenia saginata prevalence in the human population of Switzerland?

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DateTotalamount

ResultMicroscope

Resultmultiplex 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 l2-3 taeniid

eggs267 bp395 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 eggs267 bp395 bp

Hymenolepis spp.n.d.

*Trachsel et al., 2007.

Analyses of effluent from a sewage purification plant

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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

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Cysticercosis in cattle:financial losses for farmers in Switzerland

Losses for farmers Cow Beef cattle

Total loss per animal (no insurance)

€ 465.- € 685.-

Total loss per animal (with insurance)

€ 238.- € 498.-

% deduction (from normal meat price per kilo)

45.3 % 51.0 %

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Risk analysis for cysticercosis in cattle: Inclusion criteria on farmlevel

119 positive farms66 negative farms

Animals at least 9 months on the farm

So far no records of cysticercosis on thefarm

Questioning during farm visit or by phone interview

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

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Fusszeile 3

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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

Stool examination for T. saginatainfections

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Definitions of factors included in the risk analysis for bovine cysticercosis

Variable on farm level Definition

Herd size cattle units (1 unit = 500 kg live weight)

Animal category Cow, beef cattle (each conventional or mother-cow-husbandry), heifer

Roughage area Area administered where cattle graze and where grass isharvested (ha)

Grazing time Hours per year animal spends on pasture

Transhumance Grazing on alpine pastures at least once

Streams Direct or indirect (flooded pastures or fields) access to waterfrom streams

Fresh grass Feeding grass in the stable without storage

Purchased roughage Hey or silage (grass/corn) also from external production

External manure Manure from other farms spread on land

Domestic sewage Waste water from households connected to effluent pond

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Definitions of factors included in the risk analysis for bovine cysticercosis

Variable on farm level Definition

Employees Employee(s) working on farm within past five years

Visitors Organised activities on farm (events, open days and otherhospitality activities), horse pension

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Definitions of factors included in the risk analysis for bovine cysticercosis

Variable for farm surrounding Definition

Leisure activities Sports activities, lido, playground, open-air party, fairgroundnear or on farmland

Picnic place/ viewpoint Frequently visited

Military Exercises on farm area

Car park Parking places (public or not)

Wild camping Observed camping activities (individual or camps) on unofficialcampground

Railway line Railway line along or through farm land

Swiss federal railways: Around 60% of wagons havean open toilet system

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Statistical analysis

• 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

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Risk factors in analysis based on 19 variables tested

herd sizeorganic or conventionalcontact streams feeding surfacegrazing timetranshumancepurchased roughagegrass feedingexternal manuredomestic sewage

employeesvisitorsleisure activitiesmilitary activitiesparking placeswild campingpicnic place/ viewpointrailway line(animal category)

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Fusszeile 4

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Significant results of the logisticregression analysis

VariablePresent (% of the farms)

Oddsratio

95% C.I.b p-value (LRT)

Controlfarms

Farms with cysticercosis

Railway line 7.58 22.69 3.72 1.38-11.91 0.008

Leisureactivities

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

Purchasedroughage

18.18 33.61 2.89 1.37-6.49 0.009

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 onewithout the relevant variable

b) 95% confidence intervals 10.06.2010 / 20

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 cysticercosis.

“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

Discussion

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Epidemiology EU

Allepuz et al., (2008). “Describtive and spatial epidemiology of bovine cysticercosis 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

Results:- Water supply for animals was the route with the hig hest score in 41.8% of the cases (23 farms)- Feed in 9.1% (5 farms)- other routes in 12.7% (7 farms)

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Epidemiology EU

Boone et al., (2007). “Distribution and risk factors of bovine cysticercosis in Belgian dairy and mixed herds”:

- Questionnaire to 1500 farms (750 positive and 750 negative)- 56.1% response- 28 factors included

Results:- province- number of cattle slaughtered in2003

- Flooding of pastures- free access of cattle to surfacewater

- Proximity to waste water effluentwithin a range of 200m around the farm

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Diagnosis at 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 cysticercosis: (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 / 24

Prevention / Meat inspection:

Cutting musculature:- inner & exterior cheek muscles- heartVisual examination:

1 cm

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Fusszeile 5

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Diagnosis at meat inspection: an old story

Sensitivity of routine meat inspection:Dewhirst et al. (1967). “Comparison of prescribed US inspection procedures for bovine cysticercosis 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.”

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Diagnosis at meat inspection

Sensitivity of routine meat inspection:

Hörchner, (1983). “[…] Exact examination of wholesome recognisedslaughtered 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 cysticercosis 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

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Diagnosis at meat inspection

Sensitivity of routine meat inspection:

Estimation by Walther & Koske (1980) of the sensititiviy ofdetecting cysticerci at routine post mortem meat inspection:

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Predilection sites

Lopes et al., (2010): “Preferential infection sites of Cysticercus bovis in cattle experimentally infected with Taenia saginata eggs”

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High as confirmed by PCR even in calcified lesions:

Diagnosis at meat inspection: specificity

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

„Increased sensitivity of routine meat inspection“

Taenia-specific primers Cest3/5 (Trachsel et al., 2007)

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Fusszeile 6

Results:� 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%)

„Increased sensitivity of routine meat inspection“

(N = 1‘068) Additional findings (parasitic lesions)

Heart surface 4 0.37 %

Missed at inspection 1 0.09 %

1 additional cut 1 0.09 %

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 %

Results:� 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

„Increased sensitivity of routine meat inspection“

Results:Cysts per animal at routinely performed meat inspection with additional heart cuts:

„Increased sensitivity of routine meat inspection“

44

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1 2 3

Ani

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Cysts

“Alternative tools for the diagnosis of bovine cysticercosis”

• Detection of specific antigens in serum

• Detection of specific antibodies in serum or meatjuice

“Evaluation of diagnostic tools for bovine cysticercosis”

Detection of circulating Ag: MoAb B158C11A10/ B60H8A4 (anti-E/S)

Van Kerckhoven et al. (1998)

Sensitivity: 92%, Specificity: 98.7% > 50 cysts per infected animal

Dorny et al. (2000)

Sensitivity: 12.8%, Specificity: 98.7% < 50 cysts per infected animal

Ts45S-10 HP6-2

“Evaluation of diagnostic tools for bovine cysticercosis”

Peptides for detection of specific Ab: (Characterized by Bonay et al., (2001)

HP6-2 (major secreted antigen of activated oncospheres)

Ts45S-10 (T. ovis 45S homologue protective antigen)

Abuseir et al. (2007): HP6-2 Se: 100% Sp: 98%

Ts45S-10 Se: 100% Sp: 97%

CO-group: 100 young calves reared under hygienic conditions

Positive animals: 9 experimentally infected animals

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Fusszeile 7

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Evaluation of serological tools for bovine cysticercosis: Antigens

Antibody-detecting tests: ELISA Antigen-detecting test s: ELISA

Metacestodes

Crude antigen

Pestle / homogenizationUltrasonic mixerFreezing / defrostingUltrasonic desintegrationCentrifugation

Crudecysticercus

antigen extract(Cca)

IEFF1 to F5

Affinity chromatography

Isoelectro-focusing

IEFCca

extract

Synthetic peptides:

HP6-2

Ts45S-10

Larval E/S

In vitro-CultivationConcentration

In preparation, collaboration with P. Dorny

Evaluation of serological tools for bovine cysticercosis: Samples

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

LiteratureSensitivity

100 % ?*

100 % ?*

nd

92.9*

Specificity

98 % ?*

97 % ?*

nd

90.6 %*

Evaluation of serological tools for bovine cysticercosis: results

Test

% Sensitivity

Serum of naturallyinfected cattle(N=51)

Meat juice ofnaturally infectedcattle (N=51)

Serum ofexperimentallyinfected cattle

Peptide HP6-2 61.8 9.8 100

Peptide Ts45S-10 73.5 49.0 100

Crude cysticercus antigenextract (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)

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 antigenextract (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 ** Ogunremiand Ben jamin, Vet. Parasitol., 2009

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 notavailableChemotherapy expensive, will not eliminate calcified lesionsRisk factors for bovineinfection: variable

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Canadian control system

„The premises remains underquarantine until such time asthe source has been eliminatedand there is evidence that theherd is free of the parasite.The CFIA may pay producerscompensation up to establishedlimits for carcasses condemnedor treated for cysticercosis“.

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Thank you for your attention !