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Archive: 1999 Session - Appendix 19

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



Immunising value of foot-and-mouth disease virus antigens stored at 4oC for seven years


M. Danes, M. Gruia, Daniela Niƒ
Keywords: Foot-and-mouth disease; foot-and-mouth disease vaccine



The control of foot-and-mouth disease (FMD) called for large scale use of inactivated vaccines in the areas of high epidemiological risk, along with a limited application in disease free areas, highly exposed (seaports, airports, border lines) of the disease free areas. The discontinuance of the vaccine production and the ban on its use imposed the development of regional vaccines banks and laboratories authorised to keep stocks of inactivated concentrated FMD virus antigens ready for formulation as a vaccine. The emergency application, in outbreaks, of the newly formulated FMD vaccine requires the antigenic properties, the 145S particle concentration / vaccinal dose, the circulating serotypes and the immunologic adjuvant selection to be known in view of inducing - to the sensitive animals - a rapid, sound immune response protective against the natural infection.

The serological tests were used in many laboratories as an alternative to determining the efficacy of the FMD vaccines for cattle and pigs [1,2].

This paper aims at testing, by the liquid phase blocking-ELISA (LPB-ELISA), the efficiency of sev-eral FMD viral suspensions stored at 4oC for 7 years.

Material and method
FMD virus (strains A5, O1, and C) suspensions obtained on cell cultures (BHK21Cl13), inactivated with 0.01 M BEI, adsorbed on 32 g% dry weight bentonite, and concentrated 4 to 6 times were stored at 4oC for 7 years. The initial characteristics of the viral suspensions are presented in Table 1.



An amount of viral suspensions enough to ensure 8 to 16 µg of 146S particle / vaccinal dose was used to formulate a trivalent (A5, O1, C) vaccine containing 6 mg saponin / vaccinal dose.

The immunising value was tested by the LPB-ELISA [3] in 10 FMD antibodies-free catlle that were allotted into 3 groups depending on the subcutaneously administered dose (Table 2).

Blood was sampled 7, 14 and 21 days post vaccination, and the post vaccinal antibodies level test-ed by the LPB-ELISA [4].
21 days post vaccination the animals were inoculated with 1 vaccine dose (5 ml, by the subcu-taneous route).
The specific post vaccinal antibodies were tested on blood sampled 7, 14, 21, 28, 60, 90, 120 and 150 days post second vaccination.




The post vaccinal antibodies development after first vaccination was different. The results are pre-sented in Table 3.

The analysis of Table 3.3 results evidences the specific antibodies development at 14 days for serotype A5 in animals # 6 and 65 and for serotype C in animal # 6. Antibodies specific for sero-type 01 were present, 21 days post vaccination, in the blood of animal # 61.
A nonuniform immune response developed following first vaccination with the FMD vaccine prepared from antigens stored at 4oC for 7 years.
The immune response intensity was higher in the Group 3 animals inoculated with 2 doses admi-nistered at different sites.
With the same group, the occurrence of antibodies to 2 of the 3 antigens (A5 and C) composing the vaccine was noticed 14 days post vaccination in 2 out of the 3 vaccinated animals.The nonuniform development of the FMD antibodies was the argument in favour of the revaccination, with one vac-cinal dose, 21 days post first vaccination. The revaccination results ¿ recorded after 7, 14 and 21 days ¿ are presented in Table 4.

These results show that an immune response expressed as the 100% inhibition of the FMD virus antigens used in the LPB-ELISA developed 7 days post revaccination. The animals in all the three groups had antibodies to all the serotypes even if they had not been demonstrated at 21 days post first vaccination, by the LPB-ELISA.
The sera from animals # 1 , 7 and 61 ¿ collected 7 days post vaccination ¿ were used to de-termine the intensity of the immune response induced. The results are presented in Table 5.

The titration of the 3 animals sera reflects a particularly intense immune response to all the serotypes. Serum titres were detected at dilution 1:1024 for serotype A5, and at dilution 1:2048 for serotypes 01 and C.
The immunity length was monitored monthly for 5 months after the second vaccination. The results in the Figure illustrate the post vaccinal antibodies reduction in time.

Immunity duration in the animals vaccinated with the trivalent FMD vaccine prepared from antigens stored at 4oC for 7 years




In all the cases, the immunity duration remained high for 5 months post second vaccination. At 5ý months the sera from animals in Group 1 started turning negative; with the other two groups, the sera turned negative from 6 to 6ý months.
The inactivated, concentrated and bentonite-adsorbed FMD viral suspensions preserved their im-munologic properties for over 7 years when stored at 4oC.
Despite the drawback represented by the high amount of vaccine, the storing of semi-prepared antigens is an advantage on account of the reduced time necessary for formulation in the case of emergency vaccines. The antigenic diversity and the occurrence of new isolates seem, of course, to limit the usefulness of preformulating emergency FMD vaccines.
The results of this experiment demonstrated once more that the rapidity with which the specific FMD antibodies develop depends on the antigen concentration / vaccinal dose. Also, the antigen -
- immunologic adjuvant - body reactivity ratio is essential in obtaining a fast and sound immune response.



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