Kit performance
When the kit was stored at 37°C, the values for the control monoclonal antibody (Mab) declined over time. They remained above the accepted limit (0.5 OD) for three weeks, after which, the percentage inhibition (PI) value obtained for the laboratory internal positive reference serum decreased slightly, from a mean of 61PI to 54PI, but clearly remained within acceptable limits.
Once the kit had been formatted and a preliminary batch had been produced in an industrial production process by IDEXX-Montpellier SAS, the specificity of the test was assessed on 478 serum samples of French origin of known infection status. The distribution of results followed a Gaussian curve, with a mean PI of 25.5. There was no correlation between mean cELISA titer and mycoplasma infection status. Indeed, Mycoplasma mycoides subsp. capri (Mmc) was the most frequently isolated mycoplasma species (six of the seven herds for which infection was detected on the basis of tests on milk), whereas Mycoplasma capricolum subsp. capricolum (Mcc) was detected only once. The 40 serum samples with the highest titers, including two individual serum samples with PI values of 56 and 57, were retested twice, to determine the titer more precisely. The highest mean PI value for a negative serum was 54. A cutoff point of 55PI was applied, so the test was strictly specific.
An internal reference serum, with a titer close to the cutoff point, was tested in 392 replicates, on four different dates, by three technicians. All the results were pooled and the distribution was evaluated. The curve obtained was strictly Gaussian, with a mean PI value of 56. The standard deviation (SD) of this mean was 3.5. Further testing over a one-year period showed the uncertainty of measurement around the mean to be 8PI for serum samples with titers around the cutoff point. This uncertainty was estimated taking into account all the reagents, equipment and parameters critical for a cELISA test, as specified by the ISO-17025 standard. The most important factors are the kit itself, the precision of the pipettes, the training and experience of the technicians, and the linearity and repeatability of the ELISA reader. This uncertainty of measurement has two main components: the performance of the kit itself and the way the test is implemented in the laboratory. The same kit may therefore yield different uncertainties in different laboratories.
We present here only the data generated at CIRAD. However, the test was also performed locally elsewhere, notably in Ethiopia, Kenya, Mali, Mauritania, Pakistan, Senegal and Tajikistan. At all these sites, correct results were obtained for the controls, demonstrating the robustness of the kit.
Five sites were selected for the evaluation of CCPP seroprevalence with this cELISA (Figure 1-A). France was selected as a representative site at which CCPP is absent but other mycoplasmas of the mycoides cluster are isolated frequently. The other sites selected were in countries in which CCPP is known to occur.
Seroprevalence in Central Asia
A preliminary test was performed on seven serum samples collected during a CCPP outbreak confirmed by PCR, in South-West Tajikistan. These serum samples were collected from goats that had shown respiratory signs in the previous weeks or months. Six of the seven samples tested positive and titers were very high in some cases, with PI values of up to 90.
The 359 serum samples collected from the Wakhan District in Afghanistan and the 199 serum samples retrieved from the contiguous Ishkoshim and Murghob districts in Tajikistan tested negative. These results are highly consistent with the absence of clinical signs of CCPP in this part of the Central Asian highlands (Figure 1-B).
In the province of Gilgit-Baltistan in northern Pakistan, serological results confirmed the clinical observations suggestive of CCPP in this area, but the results obtained differed between sampling sites. In the Gilgit District only four of the 150 serum samples tested were positive, giving a prevalence of 2.7% (95% CI: 0.7%-6.7%). By contrast, in the Diamer District, 115 of the 260 serum samples tested were positive, giving a prevalence of 44.2% (95% CI: 38.1%-50.5%), a value significantly higher than that obtained for Gilgit. The reasons for this difference are unknown, but further sampling, with the testing of a larger sample spread over a broader geographical area should increase our understanding of the variation of CCPP seroprevalence in Gilgit-Baltistan.
In the Shuro-Obod District and the contiguous Darvoz District in southern Tajikistan, 20 of the 197 serum samples analyzed tested positive, giving a prevalence of 10.1% (95% CI: 6.3%-15.2%), confirming the clinical observations suggestive of CCPP in this region bordering Afghanistan [20]. More precise results should become available in the future, as a nationwide serological survey is currently being carried out as part of a technical cooperation project of the Food and Agriculture Organization (FAO) of the United Nations. This project should provide precise estimates of CCPP prevalence in the Tajik goat stock.
Seroprevalence in infected African countries and Mauritius
We tested 1000 goat serum samples from the Afar region of Ethiopia, from sites at which vaccination had never been carried out; 146 of these samples tested positive (Figure 1-C). The individual seroprevalence in this region was therefore 14.6% (95% CI: 13%-17%). Prevalence varied between sites, with values of 10 and 12% obtained for the Dubti district and of 5.3 and 23% for the Hadar district.
In the Narok district in Kenya (Figure 1-D), serum samples were collected from each of the goats in 10 herds (46 to 180 serum samples per herd), the owners of which agreed to participate in the CCPP vaccination campaign. Positive results were obtained for every herd tested. Seroprevalence differed considerably between herds, with between 6 and more than 90% of the serum samples from a given herd testing positive. Herd prevalence may therefore be highly heterogeneous in enzootic zones and may depend, in particular, on the time elapsed since the start of the CCPP outbreak in the herd concerned.
In Mauritius, only nine of the 62 herds tested positive, with at least one serum sample having a PI value greater than 63 (the cutoff point plus the uncertainty of measurement) (Figure 1-E). Herd prevalence was therefore estimated at about 15%, with an individual prevalence of 8%. Within the positive herds, the percentage of serum samples testing positive was high, from 26 to 80%. In Mauritius, it was observed that positive herds followed animal husbandry practices favoring contact with goats from other farms.
Detection of antibodies following vaccination
Three goats vaccinated at CIRAD with a batch of the reference vaccine displayed rapid marked seroconversion, which was detectable after as little as one week. PI values peaked three weeks after vaccination (74, 87 and 92 respectively) and remained stable at for at least five weeks thereafter. The same animals were retested five months after vaccination: two continued to test positive (PI values of 62 and 76, respectively), whereas the third tested negative.
At AU-PANVAC, groups of two to three goats were vaccinated with various proportions of antigen and adjuvant. Goats vaccinated with the reference vaccine displayed seroconversion of a similar intensity to that seen at CIRAD (Figure 2-A). By contrast, the use of lower concentrations of antigen (Figure 2-C and D) and/or adjuvant (Figure 2-B and D) resulted in seroconversion that was either less pronounced or lasted for a shorter period. The negative controls, which received an injection of saponin alone, displayed no seroconversion (Figure 2-E). Only one of the animals receiving antigen alone (Figure 2-F) displayed transient seroconversion. No seroconversion was observed in the other two. These results confirm that the cELISA was able to detect the antibodies induced by CCPP vaccination. They also show that vaccine quality directly affected the intensity and duration of seroconversion. Lower antigen content and the use of smaller amounts of adjuvant resulted in weaker responses.