New diagnostic approaches of testing for Infectious bovine rhinotracheitis (IBR)

Transcription

1 New diagnostic approaches of testing for Infectious bovine rhinotracheitis (IBR) Gaunitz C, Schroeder C, Buerger N, Engemann C, Gabert J LDL - Labor Diagnostik Leipzig, Germany

2 Agenda The disease The German IBR control program Performance of diagnostic tests for IBR New diagnostic approaches: CATTLETYPE BHV1 gb Ab CATTLETYPE Milk Prep Summary and Outlook

3 Disease Bovine Herpesvirus 1 (BHV1) is the causative agent of several diseases worldwide in cattle, including Infectious Bovine Rhinotracheitis, Infectious Pustular Vulvovaginitis, balanoposthitis and abortion. It is spread through aerosol transmission, sexual contact and artifical insemination. Like other herpesviruses, BHV1 causes a lifelong latent infection and shedding of the virus. Symptoms of infectious bovine rhinotracheitis include fever, discharge from the nose, cough, difficulty breathing, and loss of appetite. Ulcers commonly occur in the mouth and nose. Mortality may reach 10 percent.

4 Infectious Bovine Rhinotracheitis (respiratory form of BHV-1 infection) Photo courtesy of: Prof. Doll University of Giessen, Germany mouth breathing, dyspnea, fever, Nasal discharge, inflamed nares, salivation

5 German IBR control program BHV1 categories (herds) BHV1 categories (cattle) BHV1 free In eradication others FLI, Annual Animal Health Report 2008 German BHV1-regulation exists since ,1% of dairy- and suckler cow herds are BHV1- or BHV1 ge-free Strategies with DIVA vaccines (Differentiation Vaccinated from Infected Animals) with ge-deletion vaccines and testing of ge specific antibodies integrated IBR ge marker vaccinated herds are tested on individual blood samples IBR ge-elisa not recommended in Germany for bulk milk samples cost efficient bulk milk testing just on non-vaccinated herds

6 Performance of diagnotic tests for IBR gb specific ELISAs highest sensitivity, detection limit and repeatability (EU BHV1 ring trial 2004, Kramps) ge specific ELISA perform less well in serum and milk samples regarding sensitivity and specificity IBR gb and IBR ge-elisas (blocking ELISAs) having sensitivity problems testing milk and bulk milk samples

7 New diagnostic approaches CATTLETYPE BHV1 gb Ab Detection of antibodies to the glycoprotein B of Bovine Herpesvirus 1 (BHV1) in serum, plasma and milk samples CATTLETYPE Milk Prep Concentration and purification of antibodies from individual, pooled and bulk milk samples. Optimized and standardized from known methods* CATTLETYPE BHV1 gb Ab * Forschner, E., Bünger, I. (1986) Detection of IBR IPV, EBL and brucellosis antibodies in samples of bulk milk using a simple concentration method, Dtsch. tierärtztl. Wochenschr. 93, CATTLETYPE Milk Prep

8 CATTLETYPE BHV1 gb Ab sensitivity gb-specific blocking ELISA detects infection and vaccination For serum, plasma and milk samples coloured ready-to-use reagents protocol: or overnight Excellent sensitivity and specificity automatable Available as 5 plates (stripes) or 20 plates Testkit

9 CATTLETYPE BHV1 gb Ab sensitivity OIE-Reference-panel* sample Reference status PI result EU1 pos 90,7% pos EU2 weak pos 75,6% pos EU3 neg 0,3% neg Sample set Status n CATTLETYPE BHV1 gb Ab pos neg correct MW PI s B Infected % 89,9% 0,043 C Vaccinated % 97,5% 0,087 D Vacc. + challenged % 92,1% 0,004 G Non-infected % 8,7% 0,047 BHV % 85,8% - *Kramps et al. Vet Mic. 102, , 2004

10 CATTLETYPE BHV1 gb Ab sensitivity OIE-Reference-panel* BHV1-infection trial (sample set E) Serial dilution of a positive sample (sample set F) 100% 90% 80% 70% 60% PI 50% 40% 30% 20% 10% 0% 1:16 1:32 1:64 1:128 1:256 1:512 1:1024 *Kramps et al. Vet Mic. 102, , 2004

11 CATTLETYPE BHV1 gb Ab specificity Distribution of negative serum sample population (n = 746) Neg. Cut-Off Pos. Cut-Off 746 serum samples of negative herds frequency doubtful result no false positive Diagnostic specificity: 99,9 % % Inhibition

12 CATTLETYPE BHV1 gb Ab milk samples Distribution of negative (n = 265) and positive (n = 190) milk samples frequency sensitivity: 100 % specificity: 99,9 % % Inhibition

13 CATTLETYPE BHV1 gb Ab conclusion New blocking ELISA for detektion of gb-specific antibodies Suitable for serum, plasma and milk samples Excellent sensitivity and specificity Very good alternative to other commercial gb test kit User friendly

14 CATTLETYPE BHV1 gb Ab ELISA for detection of antibodies to glycoprotein B of Bovine Herpesvirus 1 CATTLETYPE Milk Prep Reagent pack for concentration and purification of antibodies from individual, pooled and bulk milk samples

15 CATTLETYPE BHV1 Milk Prep Goals Method for concentration and purification of antibodies from individual, pooled and bulk milk samples Use of bulk milk samples in less sensitive ELISAs e.g. BHV1 monitoring (ge) in bulk milk Easy method and protocol suitable for routine diagnostic Controls integrated

16 CATTLETYPE BHV1 Milk Prep Casein precipitation centrifugation 15 min neutralisation adding matrix Incubation 2h centrifugation 5 min sample/matrix transfer to MicroSpin 2x Washing centrifugation 3 s Elution neutralisation ELISA

17 CATTLETYPE BHV1 Milk Prep protocol Casein precipitation Time need: 2-4 samples ca. 3h 12 samples ca. 3,5h Incubation 2h therefrom 2h incubation time Equipment needed: centrifuge (50ml devices) rotating platform, shaker centrifuge (2ml) water-jet vaccuum pump 2x Washing Elution ELISA

18 CATTLETYPE BHV1 Milk Prep protocol Casein precipitation centrifugation 15 min neutralisation adding matrix Incubation 2h centrifugation 5 min sample/matrix transfer to MicroSpin 2x Washing centrifugation 3 s Elution neutralisation ELISA

19 CATTLETYPE BHV1 Milk Prep protocol Casein precipitation centrifugation 15 min neutralisation adding matrix Incubation 2h Bindung 2h centrifugation 5 min sample/matrix transfer to MicroSpin 2x Washing centrifugation 3 s Elution neutralisation ELISA

20 CATTLETYPE BHV1 Milk Prep protocol Casein precipitation centrifugation 15 min neutralisation Adding matrix Incubation 2h centrifugation 5 min sample/matrix transfer to MicroSpin 2x Washing centrifugation 3 s Elution neutralisation ELISA

21 CATTLETYPE BHV1 Milk Prep protocol Casein precipitation centrifugation 15 min neutralisation Incubation 2h centrifugation 5 min sample/matrix transfer to MicroSpin 2x Washing centrifugation 3 s Elution neutralisation ELISA

22 CATTLETYPE BHV1 Milk Prep protocol Casein precipitation centrifugation 15 min neutralisation Incubation 2h centrifugation 5 min sample/matrix transfer to MicroSpin 2x Washing centrifugation 3 s Elution neutralisation ELISA

23 CATTLETYPE BHV1 Milk Prep protocol Casein precipitation centrifugation 15 min neutralisation Incubation 2h centrifugation 5 min sample/matrix transfer to MicroSpin 2x Washing centrifugation 3 s Elution neutralisation ELISA

24 CATTLETYPE BHV1 Milk Prep Milk Prep in combination with CATTLETYPE BHV1 gb Ab using pooled milk samples Pools of 25 Pools of 50 Pools of 75 Pools of 100 PI result PI result PI result PI result WT ,7% pos Pool 55,1% pos 25,6% neg 14,1% neg 9,1% neg Eluat 77,7% pos 89,0% pos 76,3% pos 71,6% pos B43/07 90,8% pos Pool 23,0% neg 1,3% neg -4,8% neg -5,3% neg Eluat 78,7% pos 67,5% pos 55,1% pos 51,1% suspect PI < 45% = negative PI 45% bis 55% = suspect PI > 55% = positive

25 Increasing sensitivity of BHV1 gb ELISA for bulk milk by using CATTLETYPE Milk Prep Testing pooled milk samples in CATTLETYPE BHV1 gb Ab after milk concentration with CATTLETYPE Milk Prep 100% 80% PI im gb-elisa 60% 40% 20% 0% Positive (n = 116): 1 pos. in 49 neg. milk samples Negative (n = 73): Bulk milk of 50 from German IBR neg. herds -20% -40% positiv negativ

26 Increasing sensitivity of IBR ge ELISA for bulk milk by using CATTLETYPE Milk Prep IBRgE positive individual milk samples in pools with negative samples Milk concentration performed with CATTLETYPE Milk Prep Kit IBR ge positive milk samples can be detected in milk pools after milk concentration Maximum pool size depends on signal of positive sample in the pool S/N 0,8 = positive S/N > 0,8 = negative Milk concentrate Milk Sample S/N Pool Size S/N Result 25 0,185 pos WT 426 0, ,317 pos 75 0,201 pos 100 0,249 pos 25 0,319 pos WT 63 0, ,588 pos 75 0,631 pos 100 1,106 neg 25 0,498 pos WT 177 0, ,675 pos 75 0,948 neg 100 1,068 neg 25 0,628 pos WT 418 0, ,952 neg 75 1,067 neg 100 1,089 neg

27 Increasing sensitivity of IBR ge ELISA for bulk milk by using CATTLETYPE Milk Prep Herd A: 1000 cattle head IBR positive dairy herd in Saxonia-Anhalt Poor Dairy herd in Saxonia-Anhalt with a long year history of IBR ge positive and no progression of IBR eradication IBR marker vaccination 84 cows tested IBR ge positive from serum in the local state lab Individual milk samples testet Individual milk samples pooled in 49 negative milk samples Milk pools concentrated with CATTLETYPE Milk Prep 3 of 84 milk samples positive in pool of 50 (3,6%) 61 of 84 milk samples positive in pool after concentration (72,6%) S/N 1,2 1 0,8 0,6 I 0,4 0,2 0 Einzelmilch individual 50er-Eluat conc. 50 pool 50er-Pool sample HerdChek IBRgE S/N 0,8 = positive S/N > 0,8 = negative

28 leipzig.de Increasing sensitivity of IBR ge ELISA for bulk milk by using CATTLETYPE Milk Prep Herd B: 250 cattle head dairy herd in Lower-Saxony with recent IBR outbreak IBR outbreak 2009, still ongoing Individual milk samples of IBR ge positive cows testet Individual milk samples pooled in 49 negative milk samples Milk pools concentrated with CATTLETYPE Milk Prep 2 of 37 milk samples positive in pool of 50 (5,4%) 28 of 37 milk samples positive in pool after concentration (75,7%) Pools with strong positive milk samples are more likely to be detected 1,60 1,40 1,20 1,00 0,80 0,60 0,40 0,20 0,00 Einzelmilch individual 50er Pool pool 50er Eluat conc S/N HerdChek IBRgE S/N 0,8: positive; S/N > 0,8: negative

29 CATTLETYPE BHV1 Milk Prep summary IBR gb and IBR ge ELISA demonstrated poor sensitivity on pooled milk. Concentration of pool and bulk milk samples using CATTLETYPE Milk Prep does increase sensitivity of IBR gb and IBR ge-elisa. ge-positive cows with strong and medium signal in milk will be detected in pool of 50 after concentration No false positive reactions in concentrated milk samples CATTLETYPE Milk Prep is a simple method and practical for routine diagnostic

30 Outlook CATTLETYPE BHV1 gb Ab is approved by FLI for bulk milk of 50 in combination with CATTLETYPE Milk Prep. Pilot study in State of Thuringia about moving towards bulk milk testing for IBR ge using CATTLETYPE Milk Prep. Testing milk and bulk milk samples is an ecomonic way of surveillance of freedom of disease in cattle herds currently used for BLV, Brucella, BVDV, IBR. Possible applications for other disease control tests from bulk milk.