UDC 619:616.98:578.835.2:616-079.4

doi: 10.15389/agrobiology.2014.6.73eng


D.N. Afonina, N.Ye. Kamalova, A.V. Kan'hina, A.M. Timina

Federal Centre for Animal Health, FGBU «VNIIZZh», mkr. Yur’evets, Vadimir, 600901 Russia, e-mail afonina@arriah.ru, kamalova@arriah.ru, kanshina@arriah.ru, timina@arriah.ru

Received March 31, 2014

In Russia the preventive vaccination dominates over other protective and preventive measures against foot and mouth disease (FMD). However, the development of the subclinical infection in immune animals should not be ruled out as these animals become virus carriers and pose a potential threat for susceptible animal population. Therefore, the design and reduction to practice of tests for identification of virus-carrier animals continues to be relevant. Secretory immunoglobulins A (sIgA) provide the first line of protection against many infectious agents, they are capable of inhibiting virus intracellular replication and serve as a transmitter of virus neutralization. As reported by foreign authors there is a possibility to identify virus-carrier animals using a rapid ELISA-based test detecting secretory IgA (sIgA-ELISA). Previously we determined sIgA-ELISA optimal conditions for detection virus-specific sIgA in saliva samples in one dilution. In the given paper results of validation and evaluation of the sIgA-ELISA test-system («sandwich» ELISA) are shown as compared with other laboratory methods. The paper presents data on testing bovine biological samples (96 animals with body weight of 200-300 kg at the age of 18-24 months) from FMD-free agricultural enterprises (Vladimir Oblast) collected before and after vaccination from animals, immunized with different batches of FMD vaccine and subject to experimental infection. Samples of saliva, blood sera and esopharyngeal fluid collected with an interval of 3-4 days and up to 94 days after infection were tested using sIgA-ELISA, NP-ELISA (detection of antibodies to virus nonstructural proteins), microneutralization test and Real-Time PCR. Besides, relative sensitivity, specificity and accuracy of the test was evaluated using formulae recommended by the World Organisation for Animal Health (OIE) and compared with results of a reference test conducted by Satya Parida (World Reference Laboratory for Foot and Mouth Disease — WRL, Pirbright Institute, Great Britain) according to a validated method. Out of five preliminary immunized and then infected animals without clinical signs one animal (№ 8895) demonstrated the presence of sIgA in saliva in sIgA-ELISA on day 18 after infection at PP (positivity percent) = 53.00±0.05 % and at other dates (from 21 to 94 days) — at PP from 41.00±0.11 to 115.00±0.41 %. The obtained results were confirmed using the microneutralization test (sIgA titer on day 18 was 1.20±0.05 lg, during a period from day 21 to day 46 the titer was 1.58±0.08-1.90±0.13 lg). At that, the microneutralization test showed significant variations in values and it is undesirable for an identification test. Results of detecting antibibodies to FMD virus nonstructural proteins on day 18-94 were positive. FMD virus RNA was detected using PCR in esopharyngeal fluid samples during a period from day 28 to day 60 after experimental infection, but sIgA-ELISA showed more unambiguous results as compared with PCR. A latent infection was also confirmed in the other animal (№ 8898). Thus, it was demonstrated that the suggested test-system on the basis of indirect «sandwich» ELISA for detection of sIgA to FMD virus in bovine saliva samples made it possible to identify virus-carrier animals according to diagnostic characteristics. After considering results of testing 91 saliva samples the sensitivity of the developed test-system as compared with the reference one (WRL) was 100 %, relative specificity — 97.7 %, accuracy — 99.0 %. The high reproducibility of two test-systems was confirmed on the basis of κ-statistics (κ = 0.99).

Keywords: foot and mouth disease, secretory immunoglobulin A, solid phase immunosorbent assay, virus carrier state, post-vaccination control.


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