1. Blood Sampling with Filter Paper Strips Developed under Poultry Management Technique Improvement Project and Its Availability in Haemagglutination Inhibition Test for Newcastle Disease Case Study Koike Ikuo 1 1 ex-JICA Expert, (D.V.M., M.A) Technical Consultant

2. particular for small-scale farming (vaccination in the farms) On the schedule Vaccine efficacy make money losing money ?

3. Vaccine efficacy Protective immune response Virus neutralization (VN) Haemagglutination Inhibition (HI) Blood sampling Enzyme-linked immunosorbent assay (ELISA), Agar gel precipitation test, etc.

4. Haemagglutination Inhibition (HI) Test for Assessment of Immune Response after Vaccination Blood sampling -Sample- blood/serum, -Bleeding tool- syringe & needle, filter paper & puncture needle, cotton soaked with Disinfectant (alcohol + povidon-iodine) -Materials for HI test- chicken red blood cell(CRBC) suspension, Phosphate buffer solution(PBS), Haemagglutination(HA) antigen suspension, serum -Instrument for HI test- dropper & diluter(25 ul & 50 ul ) microplate with 96 wells of V bottom multichannel micropipette (25 ul & 50 ul ) microplate with 96 wells of U bottom reservoirs for CRBC, PBS, HA -Other Instrument/ Tool- centrifuge, tubes, tips,pipette, cylinder for measuring,

5. -Case in Japan - Dr.Kenzo Nobuto (pioneer in Japan ) Available commercially 30 mm 18 mm 10 mm 5 mm blood absorbing area (strip type) 13mm 30 mm 20 mm (disc type) -thickness: 0.3mm - Newcastle Disease (HI) Toxoplasmosis (Indirect Agglutination) Canine Distemper (VN) Japanese Encephalitis (HI, VN) Mycoplasmosis(AG) Hog Cholera (VN), etc. Application to;

6. Cost Comparison Between FPS and Disposable Syringe in Bleeding for ND HI Test Item FPS (Filter Paper Strip) Materials Filter Paper of Whatman, Cost 10-12US$ for 100 papers of 32 cm in diameter Samples Available About 9,000. Disposable Syringe Plastic and attached Needle(2.5-3 ml) 6 US$ for 100 pieces / box 100 (6 x 90=540 US$) 9000 ( Plastic Waste ) Plastic pollution

7. Approach to Verify Applicability of the FPS Determination of FPS and Amount of PBS(-) for Elution of Serum from FPS Field Test Comparison of Eluate from FPS and Original Serum Statistical Analysis Variance Analysis, Check of Correlation

8. 1.Uniform material and quality in every batch of product. 2.Available at local dealers. Screening work in view of absorbing efficiency and matching with a puncher Maker: Whatman, Cat. No. :1113320 Final selection Puncher : for punching out disks of regular size, 6mm diameter About 40 mm 20 mm 5 disks punched out with the puncher 300 ul PBS Correspond to 1 :10 dilution of serum Cat. No. :1113320; 1113 as ID, 320 as diameter (mm)

9. Local Site Japan

10. One set in the local veterinary hospital,

11. Preparation for Bleeding with Filter Paper Strips １． Cut out the strip from the filter paper Strip About 90 strips are cut out from a filter paper of 32 cm in diameter

12. 2. Needle and Alcohol-Iodine cotton Alcohol: Ethanol (70-80%) for Disinfection Iodine:Povidone-Iodine is recommended Alcohol : Iodine=5 : 1 3. Fine wire or String with Nods (used to hang FPS for drying)

13. 1. Disinfection of the bleeding spot with (wing vein region) alcohol-iodine cotton 2. Puncture the wing vein with the needle 3.Soak the blood into the FPS 4. Leave the FPS dry with hanging, avoiding exposure to sun 6. Keep the FPS in a sunproof box etc. 5. Disinfect the needle with alcohol-iodine cotton for next bleeding

14. NaCl ----------------------------- 7.4 g NaH 2 PO 4 · H 2 O---------------- 0.45 g Na 2 HPO 4 · 12 H 2 O-------------2.4 g Distilled water----------------- 1,000 ml pH 7.0 C 6 H 5 O 7 Na 3 (Sodium citrate) ----- 8.0 g NaCl --------------------------------- 4.2 g Glucose------------------------------20.5 g C 6 H 8 O 7 H 2 (Citric acid)-------------0.55 g Distilled water---------------------1,000 ml pH 6.1

15. 1. Data record 2. Punch out 5disks from each FPS (soaking in PBS of small tubes) ( soaking in PBS of the microtube ) PBS:300 ul/ tube, Soaking time:60 minutes or more,

17. Statistical Analysis Purpose: Verification of No Significant Difference Statistically Between Eluate of Filter Paper Strip and Original Sera Variance Analysis of Two Groups Verification of No Significant Difference between Two Means in the Distribution of Variance Ratio at Probability of 5 % (0.05)

18. HI titer of original serum (1:10 fold dilution) HI Antibody Titer PBS(ul) for elution 100200300400 : sample FPS soaked with blood In above case, 300 ul shall apply as provisory volume of PBS for elution of serum to be equivalent to no more than 1:10 fold dilution of its original serum. Test for Optimum Volume of PBS for Elution of Serum from FPS

19. Distribution of ND HI Antibody Titers in Sera Recovered from Filter Papers and Original One Separated from Whole Blood in Field Survey ND HI Titer (Reciprocal of Serum Dilution ) 0 * (5) ** 1 (10) 2 (20) 3 (40) 456 7891011 7 1151062221 Sera from F.P.S 61 16863312 * : indicates exponent x in a series of serum dilution of 1/5 x 2 -x, ** : indicates reciprocal of dilution fold of serum, (5) means antibody titer 5, F.P.S: Filter Paper Strip, Mean indicates the mean of HI titer expressed with exponent x of serum dilution

20. 1. 1. 1. Variance ( s 2 ) n= number of samples 2. Standard deviation ( s ) SD = = s 3. Standard error ( SE ) 4. Variance ratio (degree of freedom: f 1 =n 1 – 1, f 2 =n 2 – 1, ) 5. Mean of two variances 6. Significant difference between two means in the distribution of the variance ratio (degree of freedom: f 1 = 1, f 2 = n 1 + n 2 – 1 )

21. GroupNo. of samples (n) Mean ( y ) Variance ( S 2 1,S 2 2 ) Standard Deviation (S) Standard Error (SE) Original sera375.3789.5753.0940.508 Sera from FPS 375.6219.9083.1470.517 = 1.034 < 1.726 < F 36, 36 (0.05) < 1.776 F 0 < F 36, 36 (0.05) = 0.112 <3.960 < F 1, 72 (0.05) <4.001 F 0 < F 1, 72 (0.05) ( f 1 = 1, f 2 =37 +37–2=72 ) The variance ratio between two groups Significant difference between two groups’ means No significant difference between two groups’ means at a probability of 0.05 Analysis with statistical method (0.05: probability 5%)

23. Analogous Distribution of HI Antibody Titers in Eluate of Filter Paper and Original Sera

24. Check of Correlation Between Sera of FPS and Original One Coefficient of Correlation: r t o = r n - 2 1 – r 2 ( Degree of Freedom= n – 2 ) r = 0.981 t o = 29.91 29.91 > t 35(0.001) = 3.591 Correlation:0.98 Error rate: 0.1 %

25. 1.Drying the FPS soaked with blood should be made in the shade out of the direct sunbeam. 2.The FPS soaked with blood should not be exposed to volatile chemicals or volatilizing gas. 3.After completion of drying, the FPS should be kept into envelopes (plastic bags) or other appropriate storage box(container) for transportation. Cooling condition is preferable. 4.The FPS may be available at 4 ℃ for a few weeks and should be examined within the period, but if mold frequently grows under some environment, it should be used under a week. For longer storage, minus 20 ℃ or lower temperature is available. Notes for Handling of Filter Paper Strips Soaked with Blood