[Titration of rabies antibodies with the rapid fluorescence focus inhibition test]. / Titrácia antirabických protilátok rýchlym fluorescencným fokus inhibicným testom (RFFIT).

Antirabies virus neutralization antibodies in sera and/or transudates modified RFFIT method by Smith et al. (1973). Sera were titrated on Lab-Tek 8 chamber TC slides. Sera and/or transudates (content of pleural cavity) as well as the challenge virus strain (vaccination strains of the rabies virus Vnukovo-32/107th passage and/or CVS 11/Paris) were incubated at 37 degrees C during 90 minutes subsequently BHK-21/C13 cell culture was added. The cultures were fixed after 24 to 48 hours and stained with antirabic fluorescent conjugate (Bioveta a.s., Ivanovice n. H., Czech Republic). The highest dilution of the virus was used as the challenge dose where 50 percent of the cells in the examined range of view were infected (fluorescent inclusions can be observed). The antirabic reference serum was used as a control in RFFIT in each examined serum. To ensure a good control, the serum was diluted to contain 0.5 IU/ml of antirabic virus neutralization antibodies. Sera and/or transudates which were sent to our Laboratory were examined in this way. We examined 40 sera or pleural transudates of orally vaccinated foxes by those methods. These sera were sent to National reference laboratories for rabies (NRPB) in Kosice. Samples were examined for the monitoring of efficiency of oral antirabic vaccination. The parallel quantification of antirabic antibodies by virus neutralization test (VNT) in vivo was applied to mice and indirect haemagglutination test (NHT). The results of these three tests are comparable or in correlation. RFFIT has many advantages. When using highly attenuated strain Vnukovo-32/107th passage as the challenge virus in RFFIT method the potential risk of laboratory exposition is absent.

Immunogenic and antigenic activity of an experimental oral rabies vaccine prepared from the strain Vnukovo-32/107.

The immunogenic and antigenic activity of an experimental live oral rabies vaccine prepared from the strain Vnukovo-32/107 was evaluated on the basis of results obtained in 3 sets of experiments. These were carried out as model experiments on white mice, then on target animals--red foxes (Vulpes vulpes) and a related species--farm-bred polar foxes (Alopex lagopus). For quantitative determination of the immunogenic activity of the orally or subcutaneously administered rabies vaccines in model experiments on mice a method was used that had been developed in our laboratory. Antibodies were detected and quantified by an ELISA kit that had also been developed in our lab. Tenacity of the experimental vaccine (infectious tissue culture medium after yolk addition) was verified at different temperatures; the effects of storage temperature upon virus titre and immunogenic activity were investigated. An important part of the experiments--evaluation of the antigenic and immunogenic activity of the live vaccine at oral vaccination (vaccination baits, conditions simulating field vaccination) was carried out in foxes. The immunogenic activity (challenge experiments with a street virus on day 180 and 360 after vaccination) was evaluated in common foxes (Vulpes vulpes). The results document a high immunogenic and antigenic activity of the experimental live oral rabies vaccine. The strain Vnukovo-32/107 is suitable for the industrial manufacturing of vaccination baits. In the target species--common foxes challenged on day 180 after primovaccination an 83% protection was observed. Challenge on day 180 after revaccination (or day 360 after primovaccination), the orally immunized foxes proved to be 100% protected. For parallel evaluation of the immunogenic activity of an oral vaccine and for antibody titration it is recommended to employ the quantitative mice test and an ELISA technique, respectively.

Quantification of residual virulence of the Vnukovo-32/107 rabies virus vaccination strain.

The present work summarizes the results of 11 groups of experiments carried out with the aim to complexly quantify the residual virulence of a cold mutant of the Vnukovo-32/107 rabies virus vaccination strain intended for the preparation of an oral rabies vaccine (Kamark) for the immunization of free-living carnivores. According to WHO prescriptions, residual virulence was quantified in experiments on carnivores, mainly red foxes (Vulpes vulpes)--the presumed target species, and farm-bred polar foxes (Alopex lagopus)--a related species. Further experiments were carried out in cats, dogs, non-target autochthonous micromammals, predatory birds (Microtus arvalis, Apodemus flavicollis, Falco tinnunculus) and in a large number of laboratory animals--white mice. At oral administration (including extremely high doses) the strain Vnukovo-32/107 proved to be apathogenic to the target carnivores--Vulpes vulpe and Alopex lagopus as well as cats, dogs and the autochthonous micromammals. For Falco tinnunculus the strain proved to be apathogenic even at intramuscular and intracerebral administration. The residual virulence of the Vnukovo-32/107 vaccination strain, also quantified by comprehensive model experiments on white mice of different weight categories that had been infected orally, subcutaneously, intramuscularly, intracerebrally, by contact, with ingestion of rabic material or by modelled immune suppression, proved to be extremely low-levelled. The strain under investigation revealed a high level of attenuation and a low level of residual virulence and proved to be suitable for the preparation of non-reactogenic oral vaccine intended for foxes, an extremely susceptible target species.

[Quantification of levels of serum antirabies antibodies in vaccinated individuals]. / Kvantifikácia hladiny antirabických protilátok v sére vakcínovaných l'udí.

The authors developed a kit for the purpose of assessment of anti-rabies antibodies by the ELISA immunoenzymatic method in human immunized sera. The results of the detection and quantification of anti-rabies antibodies acquired by the ELISA method were compared with those originating from classical procedures (virusneutralizing test on mice, indirect hemagglutination test), and a sufficient correlation and sensitivity of the immunoenzymatic method were detected. By means of the developed test it is possible to detect the particular level of anti-rabies virusneutralizing IgG antibodies. (Tab. 2, Fig. 1, Ref. 25).

[Improving the laboratory diagnosis of rabies and titration of rabies viruses]. / Zdokonalenie laboratórnej diagnostiky besnoty a titrácie vírusu besnoty.

For primary isolation and titration of street strains of the rabies virus from brains of suspected animals, an assay prepared on the cell culture BHK-21/C 13 (rabies infection test - RTCIT) was used. The above assay proved to be reliable and its sensitivity proved to be comparable to the standard mouse inoculation test. Through this test, the results were obtained within 24 to 48 hrs on Lab-Tek tissue culture chamber/slides. It was found out that DEAE-dextran added to the cell culture only slightly increased the invasiveness of the virus in the samples tested. The method described herein is able to substitute the mouse inoculation test (MIT). In our laboratory, 20 vaccination strains of the rabies virus Vnukovo-32/107 and 25 street strains of the rabies virus (delivered from the field) - original fox brain suspensions. And 10 brain suspensions were negative when tested in laboratory conditions (by PMIF, RTCIT as well as by MIT methods).

[The effect of adjuvant substances on the antigenic activity of cellular antirabies vaccine in experiments on cattle]. / Vplyv adjuvantných látok na antigénnu aktivitu bunkovej antirabickej vakcíny v pokusoch na hovädzom dobytku.

Trials were conducted with young cattle to study the effect of adjuvants, applied subcutaneously and intramuscularly, upon the antigenic activity of live and inactivated cell rabies vaccine prepared from the Vnukovo -32 strain at the level of the 107th series cell passage. Cerebral vaccine of Fermi type was also used in the trials for comparison. The antibodies were parallelly titrated by four methods, three of which were conducted in vitro. The levels of antirabies antibodies indicate a possibility of fortifying the antigenic activities of inactivated vaccine by means of the Bioveta Nitra oil adjuvant and the activities of the live vaccine by means of adjuvant prepared after Buchnev . The antigenic activity of the Czechoslovak-produced cerebral rabies vaccine for veterinary use is extraordinarily low.

[Distribution of rabies antigen in the central nervous system of rats and mice experimentally infected with rabies strains isolated from hamsters (Cricetus cricetus)]. / Distribúcia rabického antigénu v CNS potkanov a mysí experimentálne infikovaných kmenmi vírusu besnoty izolovanými z chrckov rol'ných (Cricetus cricetus).

The comparative experiments were carried out to study the distribution of the rabies antigen in the central nervous system (CNS) of sewer-rats and mice experimentally infected with three "hamster" strains (for comparison also with "fox" strain 1151); it was found out that with microscopical observation of preparations stained by the method of direct immunofluorescence the "hamster" strains produced a blended picture of fluorescing particles characteristic of strains with a reduced virulence and virulent strains. As for mice infected with strains 3 O and 7 E the rabies antigen was detected in all parts of CNS as early as 24 hours after infection. In this period the rabies antigen strain 9 E was not detected in lumbar spinal cord and that with strain 1151 was detected only in the Ammonian horns. After 48 hours the rabies antigen in mice was detected in all parts of CNS with all four strains used. In sewer-rats, with regard to their lower susceptibility to the rabies virus, the first detection of rabies antigen in CNS was recorded on the 6th day after infection with strains 3 O, 7 E and 1151, whereas with strain 9 E as late as the 9th day in lumbar spinal cord and not in all animals.

[Pathogenesis of experimental rabies infection in foxes and cats infected with viruses isolated from hamsters (Cricetus cricetus)]. / Patogenéza experimentálnej rabickej infekcie lísok obycajných a maciek domácich infikovaných vírusom izolovaným z chrckov rol'ných (Cricetus cricetus).

All the infected foxes (9) contracted the disease and died from rabies the 20th-21st day from infection with the virus isolated from hamster. Out of the total number of 9 cats experimentally infected by intramuscular infection, seven showed symptoms of clinical disease on the 18th-34th day from infection. The infected dogs, wolves and rabbits did not show clinical disease. In the post mortem examination of eight foxes the rabies virus or rabies antigen was detected in all parts of the CNS and in n. ischiadicus. Of the extraneural organs, the virus was present, in all animals, in the pharyngeal salivary glands, and in one fox also in the tongue. The bioassay on the eye was positive in all cases. The rabies antigen was detected in 4 foxes in the thigh muscle, in liver and spleen, and in all 8 foxes in lungs and cornea, in 5 cases in kidney, and in 3 cases in heart and tongue. Seven cats were examined post mortem by the inoculation test on mice and the PMIF-staining test; it was found that the rabies virus, or rabies antigen, was distributed in the same manner as in foxes.