at individual animal level

strain homologous to that of the vaccine. Use an SP test employing a virus homologous to the vaccine and use a threshold determined by either a homologous potency test or an estimate derived from calibration sera or based on past experience of the test.

c) Demonstrating that vaccination has elicited sufficient immunity to protect animals against challenge by a virus strain circulating in the region and likely to be a threat. Use an SP test employing a locally circulating virus (or equivalent strain) and use a threshold determined by either a heterologous potency test or an estimate derived from calibration sera or based on past experience of the test.

3.5.1 Post-vaccination monitoring to assess population immunity at individual animal level

a) Suggested values for the expected proportion of animals with a specific level of antibodies in the following age groups: – age 0–6 months – expected proportion 60%; – age 6–12 months – expected proportion 70%; – age 12–24 months – expected proportion 80%; – age > 24 months – expected proportion 90%.

b) Allowable standard error – 10%.

c) Level of confidence – 95%. – Based on the above values, the following number of epiunits for each of the age groups will be required: - age 0–6 months – 26 epi-units, collect 10 samples per unit (total 260 samples); - age 6–12 months – 26 epi-units, collect 7 samples per unit (total 182 samples); - age 12–24 months – 26 epi-units, collect 4 samples per unit (total 104 samples); - age > 24 months – 26 epi-units, collect 2 samples per unit (total 52 samples).

– A total of at least 598 blood samples will then be required for this study (which will reduce to 338 if the age-group six months and under is not sampled).

The sample size should be increased by one additional epi-unit per group, in order to compensate for possible withdrawals.

– Selection of epi-units depends on the available sampling frame:

- Should a reliable list of epi-units and the estimated number of animals for each epi-unit exist (and their distribution in the four age groups), then epi-units may be selected with probability proportional to size (PPS) taking into account that PPS may be different for each of the four age groups considered for sampling.

If this procedure for selecting primary sampling units (PSUs) has been utilised, then analysis of samples can be conducted using the procedure described in

Annex 2 (example II.a – option 1). - Should only a reliable list of epi-units be available, then epi-units may be selected by simple random sampling (SRS). If this procedure for selecting

PSUs has been utilised, then analysis of samples can be conducted using the procedure described in

Annex 2 (example II.a – option 2).

– In each selected epi-unit, animals from each age-group may be selected by SRS or systematic random sampling.

– Collect blood samples according to the procedure established (at the time of vaccination and/or at any point in time).

– Analyse the samples: - Determine the proportion of animals with detectable levels of SP against the homologous vaccine strains and NSP antibodies. It is also advisable to measure SP antibody titres against field strain(s) to measure protection against circulating virus.

– Determine the mean titres of SP antibodies.

– Calculate the level of SP antibodies and the confidence interval for each age group: - If epi-units were selected with PPS, then use equations 13, 14, and 15 (in Annex 2). - If epi-units were selected by SRS, then use equations 16, 14, and 17 (in Annex 2). The proposed procedure is based on the assumption that the minimum age for being eligible for vaccination is six months and that campaigns will be carried out every six months. The age-cohort of animals six months and under will contribute to the overall estimate of immunity and will possibly allow also the assessment of the presence of maternal antibodies and how passive immunity contributes to the overall population immunity.

The sample size proposed has been estimated for different expected proportions (from 60% to 90%), assuming that, when regular vaccination is carried out, the proportion of SP-positive animals will progressively increase with age. It should also be noted that the number of the primary sampling units sampled should be always above 25 epiunits, to conform to statistical theory regarding unbiased parameter estimates (13).