In comparative experiments the diagnostic effectiveness of four methods of laboratory diagnostics of rabies – the mouse intracerebral inoculation test (MICIT, MIT), the rabies tissue culture infection test (RTCIT), the rapid rabies enzyme immune diagnosis test (RREID) and a molecular-genetic method, the nested reverse transcription polymerase chain reaction (nRT-PCR) – was quantified by the titration of serial dilutions of brain viral suspensions. The threshold value of the tests, i.e., the highest dilution of a specimen, which the method used is able to detect as a positive one, was determined. Further advantages and disadvantages of the tested methods were compared as well. Experimental optimization of procedures for RNA extraction was carried out and the optimum primer for RNA transcription to cDNA was selected. The RREID method was carried out in two variants: detection of the rabies antigen in a clarified (centrifugated) as well as in a non-clarified (noncentrifugated) brain suspension. In the experiments three autochthonous street isolates of rabies virus (in the form of primary isolates) were used; they had been isolated from naturally infected red foxes (Vulpes vulpes) and a lynx (Lynx lynx). The results of comparative experiments revealed a relative correlation of the diagnostic effectiveness of standard methods (MICIT and RTCIT), with standard MICIT being the more sensitive one, RTCIT however having several other advantages (among others the speed of performance) and thus being preferred. For quantitative comparison of diagnostic effectiveness two other methods (RREID and nRT-PCR) were examined in that street isolates of rabies virus, which revealed the highest titer after titration by MICIT and RTCIT. The sensitivity of the RREID method proved to be rather low. If used with noncentrifugated brain suspensions this method may yield nonspecific reactions. If compared particularly with RREID the nRT-PCR is characterized by a considerably higher diagnostic effectiveness. The sensitivity of nRT-PCR is not affected by preliminary clarification of the brain suspension. The reverse primer N12 seems to be more suitable for transcription of the extracted RNA to cDNA than random hexamers.

218Detection and quantification of rabies antibodies is intended in the first place for checking the immunity to rabies or effectiveness of rabies vaccines. Detection and quantification of virus neutralisation rabies antibodies in the serum is based on inhibition of rabies infection in vivo in animals or in vitro in cell cultures (Atanasiu, 1973;Bourhy and Sureau, 1991). Several suitable procedures have been recommended for determination of titres of virus neutralisation antibodies. e methods most frequently used for quantification of immune response in vaccinated animals after rabies vaccination challenge are serum neutralisation methods carried out on mice and in cell cultures (Smith et al., 1996). WHO recommends in vivo virus neutralisation test on mice (VNT) and in vitro rapid fluorescence focus inhibition test (RFFIT). e VNT on mice is time demanding and too expensive for routine use in virological laboratories. Recently it has been replaced by sensitive, less expensive and more rapid in vitro tests. e RFFIT is highly sensitive and advantageous because of its low time demand. e application of the RFFIT for detection and quantification of rabies antibodies also requires an OIE standard (WHO, 1992). e FAVN (fluorescent antibody virus neutralisation) test was first described by Zalan et al. (1979), and later reworked and modified in the AFSSA laboratory, Malzéville, Nancy, France. It is simple, rapid, safe and economically sustainable, it allows conduct of many serological examinations needed to check animals, particularly dogs, exported to other countries but also checking the immune status of vaccinated animals (Cliquet et al., 1998(Cliquet et al., , 2000. MATERIAL AND METHODS Examined seraEighty-three sera were chosen for comparison of individual methods for rabies antibodies detection from both non-vaccinated (12 sera) and vaccinated (71 sera) pet dogs. Before examination, they were inactivated by exposure to 56°C for 30 min and stored at -20°C. For comparison of reliability, sensitivity and reproducibility of rabies antibodies detection methods, if the sera were taken from vaccinated dogs we selected those that exhibited titres of rabies antibodies lower than 1.0 IU/cm 3 (International Units/cm 3 ), or near the level 0.5 IU per cm 3 , respectively, as detected by the RFFIT. e tests also included titration of reference serum (Copenhagen, Denmark, 30 IU in an ampoule, the dilution of serum containing 0.5 IU/cm 3 antibodies was used for the titration), and the control titration . e results of rabies antibodies levels in non-vaccinated dogs obtained by all three methods were in correlation. e comparison of rabies antibody titres determined in vaccinated dogs using VNT and FAVN methods showed 86.6% correspondence, while those obtained by RFFIT and FAVN methods corresponded in almost 95% of cases.

The authors verified the possibility of antioxidative protection of squalene adjuvant emulsions by the antioxidants α-tocopherol and β-carotene. They determined the influence of β-carotene on the stability and antigenic effectiveness of adjuvant emulsion in combination with rabies vaccine. The composition of the adjuvant emulsions or vaccines was: 2.5% squalene; 6% detergents; 0.5% antioxidant; 91% water phase. The oxidative injury after UV-irradiation was followed by the detection of the peroxide value of the emulsions. The stability of the emulsions was evaluated by the determination of the emulsion's particle size. The level of rabies antibodies (RAB) in mice sera until day 90 after vaccination, was determined by the rapid fluorescent focus inhibition test. In the in vitro system of squalene adjuvant, α-tocopherol acted as a prooxidant, while β-carotene effectively reduced the oxidative injury. The homogenization of the squalene adjuvant during a prolonged period from 8 to 10 min did not change the particle size. The oxidation processes were efficiently reduced by β-carotene during the preparation process and also during the 70-d storage. The vaccine with β-carotene induced a gradual increase in the RAB levels with the highest value on day 28. While the inactivated rabies vaccine with adjuvant without β-carotene developed a rapid formation of RAB, the application of the vaccine with β-carotene induced a slower but more uniform production of RAB. The level of RAB was significantly higher after the application of the vaccine with β-carotene and reached the protective value of 0.5 IU/mL, in contrast to the vaccine without β-carotene.

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In the presented work, we compared the results of determination of rabies antibodies using three in vitro methods: rapid fluorescent focus inhibition test (RFFIT), fluorescent antibody virus neutralisation test (FAVNT) and the immunoenzymatic assay (ELISA). 196 dog sera samples were examined with FAVNT, RFFIT methods and the ELISA test. Sera with low and sufficiently high titre of antibodies had a similar result in determining by all methods. A critical level of rabies antibodies close to the required protection level (0.5 IU/cm 3 ) was seen in sera of 18 dogs (9.18%); these were the sera obtained after primary vaccination of dogs. At this level, even small differences can cause a change in the assessment of the patient's serum seronegativity or seropositivity. Therefore, it is important to choose the appropriate method that has sufficiently strict criteria while having a good reproducibility.