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Archive: 1999 Session - Appendix 11

1999 Session of the Research Group of the Standing Technical Committee of EuFMD


Serosurveillance of FMD in the Transcaucasian Countries: role of the 3ABC-ELISA


Gusev A.A[1], Amadori M.[2], Berlinzani A.[2], Brocchi E.[2], Zakharov V.M.[1], Fomina T.A.[1], Dudnikov S.A.[1], Schekotova O.A.[1]
[1] All-Russian Research Institute for Animal Health, Vladimir, Russian Federation
[2] Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia, Brescia, Italy


The potential threat for Europe of FMD introduction from the Transcaucasian area has been highlighted by the Veterinary Services of the CIS Countries for a long time. This led to the concept of a FMD vaccine buffer zone, as first proposed by the CVOs of the CIS countries in a document signed on 29/10/1996. The document was discussed in the session of January 20-24, 1997, by the OIE European FMD Commission, which reviewed the proposal and declared its willingness to examine a detailed plan. The issue gained considerable priority following the appearance of a new A type FMDV strain in Iran and Turkey, significantly different from the A22 vaccine strains employed in that area. The further spread of this strain into Armenia was later announced during an ad hoc EuFMD mission to Moscow and Vladimir in August 1998; the results of this mission were then discussed at the OIE Commission for Europe meeting, held in Prague at the end of September 1998. The OIE Regional Commission recommended that an OIE/FAO/EC tripartite group should work together with the Veterinary Services of the CIS Countries to develop a common strategy for the control of FMD in the region, including a programme for a vaccination buffer zone. Following this recommendation, a meeting of the involved parties took place at ARRIAH on 24 November 1998. On the basis of these discussions, two OIE/EuFMD/EC missions to Transcaucasia and ARRIAH ensued in March 1999 to review the situation of FMD in that area and the capability of the National Veterinary Services to combat the disease under the co-ordination of ARRIAH. The conclusions and recommendations of the missions were accepted by the 33rd Session of EuFMD, held in Rome in April 1999. Following the results of the two expert mission and, in particular, the favourable report about the capability of ARRIAH to provide the necessary logistics, expertise and infrastructure, an agreement was signed by FAO and ARRIAH for a provision of funds to be used for epidemiological surveillance of FMD, an emergency FMD vaccination campaign in the Transcaucasian Countries and the preparation of a FMD vaccine stock. With respect to serosurveillance, a strict co-operation was achieved between ARRIAH and the Brescia Laboratory, in the framework of a three-year project funded by the Italian Ministry of Foreign Affairs. In particular, there was an exchange of reagents and expertise in the field of the antibody tests for FMDV non-structural proteins (NSPs). Thus, the 3ABC ELISA developed in Brescia (1 ) was used for the serosurveillance in the Transcaucasian Countries together with other antibody tests.



Materials and Methods

Sampling. 4 members of the ARRIAH staff worked in Georgia and Armenia in June and July 1999 to collect sera according to a sampling plan agreed upon with the local veterinary authorities; the relevant areas are shown in figures 1 and 2. Unfortunately, no detailed written documentation was available about the previous vaccination campaigns in Georgia and Armenia. According to the local veterinary authorities, the usual bivalent O and A type vaccine of ARRIAH was under use; in Georgia, also the bivalent vaccine of the local producer "Biokombinat Tabakhmelskii" was under use in the Garbadan district and in the autonomic territory of Adjarskii (Kobulety town, Khelvaciauri village).
675 cattle sera and 92 ovine sera were collected in 8 districts of Georgia (two of them located in the autonomous territory of Adjarskii) and 2 districts of Armenia within Arabat Province (Arabat and Artasciatski districts). The survey was carried out before the vaccination campaign with a bivalent O and A type vaccine, including the A Armenia Î98 variant strain.
Antibody tests for FMDV structural proteins. All the sera were tested by Liquid Phase Blocking Sandwich ELISA (2) for antibody to the O1 (O194) and A22 FMD vaccine strains at a single dilution. Ab-positive sera were also tested in dilution.
72 cattle and ovine sera from Georgia were also tested by microserumneutralization . In this test, two or four dilutions of the sera were reacted at the same time with 100 TCID50 of A22 virus and a suspension of IBRS-2 cells (106/ml).
Test for FMDV NSPs. The 3ABC-ELISA was performed in Brescia and in Vladimir as previously described (1), with a minor modification: sera were tested at a 1:100 dilution which is more suited for use with an anti-bovine IgG conjugate based on monoclonal antibodies. 139 Ab-positive sera were included into this latter assay on the basis of a balanced representation of all the inhabited areas of the survey, reported in Table 1.
Statistical analysis. The differences between the prevalences of antibody-positive animals were checked by the c 2 test. The threshold for significance was set at P<0.05.




Antibody tests for FMDV structural proteins
Results are shown in Tables 1 and 2. As can be inferred from Table 2, the percentages of O1 and A22 antibody-positive cattle and sheep were by far higher in the sampling areas of Armenia than in those of Georgia; the differences are significant for cattle (P<0.05). Concerning the microserumneutralization for A22 antibody, it must be stressed that some discrepancies were observed with respect to LPBE. Thus, for instance, 7 out 24 sera from Kobuleti (Georgia) and 17 out of 25 sera from farm 2 of Khelvaciauri (Georgia) were scored positive by microserumneutralization, only.


Inter-laboratory validation of the 3ABC-ELISA
In June 1999 a colleague from ARRIAH carried out in Brescia the test on the following samples: 5 cattle sera collected 21 days after A type infection, 5 cattle sera after FMDV O type infection , 1 cattle serum after FMDV C type infection, 4 sera of cattle vaccinated with O/A bivalent vaccine, one serum of a naive steer. These samples were tested in parallel with cattle sera after vaccination and/or infection collected in Italy. All the sera were classified correctly and there was no false positive reaction. In July 1999, 83 cattle and sheep sera of the above serosurvey in Georgia were divided and an aliquot of each was shipped to Brescia for an evaluation of 3ABC-specific antibody, meant as an internal control of test reproducibility among laboratories; sera were collected in Garbadani (33 sera) and Mzkheta districts (50 sera); all of these sera were scored negative in both laboratories.


Test for FMDV NSPs on sera from the survey
42 out of 139 sera were 3ABC-positive; interestingly, all positive sera were clustered in two areas of Georgia (Borzhomi and Akhalzikhe districts) and seven of Armenia (Berdik, Arabat, Uruadsor, Armash, Gorovan, Noiakert, Urualandzh) (see Table 1; 3ABC-positive sites are marked with an asterisk). With regard to the cattle sera from Armenia, the 3ABC-positive sites were characterised also by the highest prevalence of antibody-positive head by LPBE (see Table 1). It must be stressed that all the 139 sera of this survey were also re-tested in Brescia: 17 sera (12.2 %) were scored differently, the differences being mainly related to border line, threshold reactions; only one district (Borzhomi) was differently allocated after re-testing.




The Transcaucasian Countries are particularly at risk for FMD. An analysis of historical records shows that FMD has often spread from Central Asia into the Transcaucasian Countries and then further north into the European part of the Russian Federation. During the Soviet period, effective control measures had been applied over many years, which led to the practical disappearance of FMD in most districts of the former USSR. A favourable situation had been achieved also in the Transcaucasian Countries thanks to the prophylactic vaccination and the policy of strict border controls. Since the collapse of the Soviet Union, there has been a substantial relaxation of the above measures; in particular, the vaccination coverage has been very poor. Owing to the above, FMD has repeatedly occurred in the area, whereas a further spread to north has been prevented until now by a very effective vaccine buffer zone in the southern part of the Russian Federation. Due to the extensive husbandry conditions, transhumance of cattle and sheep may be responsible for wide-range dissemination of FMD due to free intermingling during the summer pasture period of animals of different districts, which then return to their farms scattered over a large territory; in addition, there is evidence that sheep are transported over long distances across the national borders. The control of the disease is therefore extremely difficult; it must be stressed that in case of FMD outbreaks the adopted measures are usually limited to isolation of the affected animals and, sometimes, ring vaccination. This may provide ample opportunity for virus survival and re-circulation under the field conditions, keeping in mind that both Georgia and Armenia were affected by repeated FMD epizootics from 1992 to date; the most serious of these (sustained by an O type virus) was present in 30 districts of Georgia in 1997. In this respect, it is interesting to note that the 3ABC-positive Akhalzikhe district was affected by FMD in 1998, together with Akhalkalaki, Ninozmind, Zalk and Aspinds districts. Unfortunately, no detailed information is available about FMD occurrence in Armenia in the past years. The wide differences of antibody prevalence among different districts and, interestingly, between Georgia and Armenia, lead to the reasonable conclusion that they are not due to a sampling bias, based on a simple difference of vaccination coverage. This inference is further confirmed by the detection of clusters of 3ABC-positive sera in defined areas of Armenia where the prevalence of LPBE-positive bovine sera was highest.

The analysis of the data confirms that a combination of antibody tests for structural and non-structural proteins of FMDV is most suited to depict the sanitary status of the animal population in areas affected by FMD outbreaks, where vaccination has been performed before and after the disease cases. On the basis of the available information, it is worth mentioning that the 3ABC-ELISA was able to detect convalescent animals about one year after the last reported outbreak.

Although the extent of this serosurvey does not allow for precise estimates of Ab prevalence in the population, it does confirm previous indications concerning the poor and irregular vaccination coverage in the Transcaucasian Countries and the patchy re-circulation of FMDV in some areas. Based on these findings, these Countries should not be considered endemic areas; they should be regarded instead as areas where infectious virus may be costantly introduced from the sorrounding endemic areas of Central Asia and persist for some time in certain sites; in turn, following virus persistance in ruminants, further flare-ups of the disease may occur due to the low vaccination coverage and the peculiar husbandry conditions.

The results of this antibody survey in Georgia and Armenia prompt to a long-term surveillance in the area; this may be possible if the Veterinary Services effectively liaise with each other and with the OIE reference laboratory, in the framework of a network aimed at a timely exchange of information. Further antibody surveys in the future should provide fundamental data about the efficacy of the future vaccination campaigns in terms of a stepwise, dramatic reduction of virus persistence in the field.




1) De Diego M. et al. (1997), Arch. Virol. 142, 2021-2033

2) Hamblin C. et al. (1986), J. Immunol. Meth. 93, 115-122.