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Appendix 20 ELISA results from Armenia, Georgia and Azerbaijan: Statistical evidence for recent FMD circulation? - Matthias Greiner
Appendix 20
ELISA results from Armenia, Georgia and Azerbaijan: statistical evidence for recent FMD circulation?
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Matthias Greiner, International EpiLab, Danish Institute for Food and Veterinary
Background EUFMD RG has requested an analysis of ELISA data collected in May to July 2005 in three selected study regions in Armenia, Georgia and Azerbaijan (see Carsten Pötzsch’s presentation of the study for further details) with the main objective to investigate the hypothesis that FMD circulated in the study regions in the last one or two years. This assessment should account for possible positive reactions due to the use of local non-pure vaccine. It can be assumed that older, multiple vaccinates will have more sero-responses than younger (e.g. 1-2 years) animals. Data The data sheet was cleaned (age expressed in years for all data from Georgia; The”>” sign for high ELISA titres was removed, “<10” was recoded “9”, “<=10” was recoded “9.9”; ELISA results labelled with “?“ were set to missing; one Asial result “0” recoded “9”; at three spelling versions of “BEYLIAGAN” were unified; region, county (rayon) and location (village or town) were recoded numerically. Three SP ELISAs (Manisa, A Iran, Asia 1) and one NSP ELISA (3abc) were included in the analysis; retest and Cedi results were not considered. For analyses, log-transformed variables were used for Age (logage) and the four tests (spi, sp2, sp3, nsp) (see Tab. 1 for a summary).
Association between age and seroresponse To our knowledge, some vaccination was used in all study areas. In the absence of more precise information about the vaccination history, we can use the age as a crude proxy for the number of vaccine doses an animal has received during its live. SP reactivity, and to a lesser extent NSP-reactivity due to the use of impure vaccines, should be positively correlated with age under the hypothesis of serological response to vaccination. High anti-SP serum titres in younger animals, on the other hand, could be an indication of recent exposure. Age is an important variable for this analysis. There is some indication for inconsistent age recordings among the three regions (Fig. 1). In Armenia, the age was given in somewhat more crude intervals than in the other regions. The age distribution for Georgia doesn’t seem plausible (maybe due to the colour coding to differentiate between month and age recordings in the same variable). A graphical analysis did not reveal any marked association between age and the continuous SP and NSP results for any of the tests or regions (Fig. 2). This is confirmed by linear regression models (outcome log ELISA results versus log age), which did not explain more the 2% of the variation in the serological variable (results not shown).
NSP positivity in relation to SP positivity While all SP tests are clearly correlated (Fig. 3) the association between NSP and Sp test results is less obvious. It seems that a subgroup of result exist for which higher SP titres are associated with higher NSP titres (Fig. 4). This effect is most obvious for the 0 Manisa and Asia 1 test. A hypothetical explanation could be that some heavily vaccinated animals (i.e. SP strong positive) reacted with the vaccine antigen. It is not possible to rule out an effect of circulating infection in these animals. This analysis used the mean of the SP ELISA results. The observed number of results for the 16 combinations of the test results was plotted against the expected number, which is generated under the assumption that all test results are independent (Fig. 5). The combination, where all tests except 3abc are positive and the combination of all tests negative occur more frequently than expected.
Distribution of responders The occurrence of SP and NSP positive test results should be homogeneously distributed over the locations and counties if the assumptions are true that all locations/counties have used the same vaccination regime in the past and no current or past clusters of infection exist. The distribution of the continuous test results is given for each county in the three regions (Fig. 9-11). Due to the high correlation among the SP tests, the mean of the log transformed results was used for this analysis. In some counties, a marked spread of results into the higher range of the measurement scale is found. This pattern is different among the counties in the three regions and may indicate current or recent FMDV infection or marked differences in the vaccination coverage or efficacy.
Discussion For Armenia the following vaccination scheme was reported. Armenian bivalent A/O types lapin, 4mg (1995-1998); Russian bivalent sorbent vaccine, 2mg (1999-2002); Russian trivalent A/O/Asia- 1 types vaccine, 3mg (2003-2005); revaccination was made in the given year (Keith Sumption). “Animals are vaccinated in the first year of life, if vaccine is available. They are not booster vaccinated in Georgia and only sometimes in Armenia and Azerbaijan. The vaccination history provided for Armenia does probably reflect the general vaccination policy. It can be assumed that in some years and areas other or no vaccines at all were used. Also, movements and exchange of animals with other areas have occurred” (Carsten Pötzsch). This information does not allow any differentiation of animals according to their vaccination status. More precise data vaccination history (on village level) would allow better interpretation of the data. The results are consistent with but do not provide a proof of current or recent infection in some counties in the three study regions. Under the strong assumption that the vaccination effect is identical in all study regions, the observed heterogeneity in the SP and NSP positivity rate may be interpreted as an indication of circulating infection in some counties. As age is a very important variable, the project coordinator may want to check the measurement procedure and outcomes for consistency and plausibility.
Figures and Tables
Table 1: Summary statistics of the data used for the analysis
Figure 1: Age distribution by region
Figure 2: Log ELISA results from top to bottom (O Manisa, A Iran, Asia 1, 3 abc) versus log age.
Figure 3: Scatter plots of the three SP tests
Figure 4: NSP results versus the mean of the results of the three SP tests
Figure:5 Empirical (obs) and expected (expert opinion under assumption of no circulating infection) probability of positive NSP results in relation to SP test results.
