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1 Classificatie: intern

2 Animal Health Service Deventer Jet Mars part 1: Paratuberculosis

3 ParaTB approach In the NL: control program, not an eradication program Quality of dairy products as starting point National program for dairy herds: all herds participate

4 ParaTB approach main goal: all herds obtain a status: herd infected herd probably uninfected ELISA monitoring instrument: Testing scheme Test quality

5 ParaTB approach Important for support of farmers: Herd examinations by ELISA Easy: Individual milk samples of all lactating cattle collected at milk production registration infected herds test every year ; seronegative herds every two years Repeatable test results High specificity: cut off important

6 Cut off ELISA faeces negative S/P value ELISA culture negative

7 Cut off ELISA S/P value ELISA Culture negative Low shedder

8 Cut off ELISA S/P value ELISA Culture negative Low shedder Middle or high shedder

9 ParaTB approach Important for support of farmers: High specificity 99.6% ELISA GD Cut off important - False-positives very rare. Seropositive cattle confirmation PCR faeces High sensitivity for detection high shedders Shedders must be removed Low shedders, or intermittend shedders can be missed in detection, and will be detected next year.

10 ParaTB approach Herd examinations by ELISA infected herds test every year ; probably uninfected herds every two years ELISA results correlate with stadium infection

11 ELISA vs shedding faeces % of infected cattle Sensitivity ELISA Clinical symptoms 1-2% % Heavy shedders 20-30% 60-90% Low shedders 60-70% 10-20%

12 >99 % Kweek positief ELISA confirmation faeces culture/pcr 1 Bevestigen met mestkweek >20% >50% >90% Melk ELISA (S/P w aarde)

13 Interpretation animal level Is this cow infected? Seronegative: probably uninfected Seropositive: probably infected If farmer chooses : confirmation faeces culture/pcr Faeces negative: the animal does not shed bacteria at this moment, but might start shedding in the near future Faeces positive: the animal is infectious en might be contaminating the bulk milk.

14 Interpretation herd level Is the herd infected? All animals seronegative: status A probably herd uninfected next monitoring 2 years later One or two seropositives: confirmation faeces Status A (faeces negative) or B/C (faeces positive) Multiple cattle seropositive: herd infected Status B/C remove heavy shedders. ELISA all cattle every year management infected herds

15 Paratbc-ELISA quality Indirect absorbed ELISA ELISA robot system Very important that test quality is excellent standard procedure ISO17025

16 Batch control procedure Kit controls Batch control sample panels Well-defined samples (24 milk and 24 sera) Acceptance criteria based on historic data of these samples in several batches (mean S/P% +/- 2 sd values) Internal control samples (n=2) Tested on every plate Criteria: mean S/P% +/-2 sd value in several batches Field samples comparison (results current batch vs. new batch)

17 Batch control BATCHNR nat DATUM Monsters OD OD OD OD PC (ext netto) NC (ext netto) IC1 (ext netto) IC1 SP ratio IC2 (ext netto) Gem. GW per berekend IC2 SP ratio sp ratio - 3 SD - 2SD +2SD + 3SD criteria NR SP Ratio SP Ratio SP Ratio SP Ratio N N N N N N P P P P P P P N N N N P P N N N N N

18 quality control Internal control samples Tested on every plate (n = 2) Criteria: mean S/P% +/-2D in several batches Proficiency tests (n=2: USDA and GD)

19 Trend analysis: ELISA control samples

20 Trend analysis: ELISA control samples

21 Trend analysis: PCR faeces in ELISA positive cattle

22 ParaTB Main issues herd management Closed herds Preventive management: hygiene; calf rearing, colostrum ELISA most practical monitoring instrument: high throughput testing reproducable results high specificity and a sufficient sensitivity on herd level cost effective Quality assurance important

23 End of part 1 Questions Paratbc?

24 Part 2: Brucella suis Animal Health Service Deventer

25 Brucellosis in swine Source: WAHID January 2017

26 Epidemiology Brucella suis Biovar 2 endemic in wild boar. Biovar 2 only reported in Europe In 2012 and 2013 a few cases in wild boar in Belgium and Limburg (NL) EFSA risk assessment at the request EC in 2009 zoonotic risk is very low Risk CVI 2013: same conclusion However, monitoring is important. NL is free of Brucellosis in commercially held pigs since late sixties

27 Brucella tests: Factors which may cause false-positive' results include: Exposure to antigenically related organisms, e.g., Salmonella, Yersinia, Pasteurella Laboratory test artifacts Factors which may cause false-negative reaction in serological tests include: Recent infection No sero-conversion - due to localization of infection or immunological incompetence

28 Test characteristics variation Parameter Average estimate [95% CrI] RBT Se [0.835; 0.913] Sp [0.939; 0.959] FPA (fluorescence Se [0.890; 0.970] polarization assay) Sp [0.917; 0.941] I-ELISA Se [0.607; 0.710] Sp [0.958; 0.976] C-ELISA1 Se [0.906; 0.989] Sp [0.942; 0.966] C-ELISA2 Se [0.907; 0.994] Sp [0.982; 1.0] Prevalence (Metropolitan France) [0.105; 0.134] Source: Praud A, Gimenez O, Zanella G, Dufour B, Pozzi N, Antras V, Meyer L, Garin-Bastuji B. Estimation of sensitivity and specificity of five serological tests for the diagnosis of porcine brucellosis. Preventive veterinary medicine Apr 1;104(1):

29 Brucella test GD Rose Bengal simple, rapid slide-type agglutination assay performed with a stained Brucella abortus suspension (ph ) and plain serum Limitation : Sensitivity, particularly in chronic cases Positive samples should be checked by: the Complement fixation test (CFT) or ELISA.

30 Testing for Brucella suis at GD In 2016: samples were tested by the Rose Bengal method >80% from the samples are from boars at insemination stations 1-3% of these samples are positive in RB All of these were negative at CVI using indirect Brucella ELISA (ID Vet)

31 Testing for Brucella suis at GD RB test: procedure for this test is simple But need to be followed exactly, especially the incubation time False positive reactions can be reduced by inactivation of the sera 56 C, 30 minutes

32 quality control standard procedure ISO17025 control sample delivered by National Reference Lab 1:4 and 1:8 dilution: 1:4 should be positive; 1:8 should be negative antigen batch (Pourquier/Idexx or ID.Vet) certificate National Reference Lab CVI Internal control samples(n = 2) Tested every run Proficiency test CVI every 6 months Interpretation check: all technicians perform the PTS and we compare their interpretation

33 Thank you for your attention Questions?

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