Historical analysis of epizootiological situation of rabies on the territory of the Slovak Republic.

Currently, Slovakia is a rabies-free country, but the epizootiological situation of rabies was not always favorable. The main reservoir species of rabies virus in the first half of the last century was the domestic dog. Since 1906, hundreds of cases were reported, of which approximately 90% were infected dogs. The disease had a typical urban character. Since 1929, the number of rabid domestic animals decreased due to the implementation of dog vaccination campaigns in particular parts of Slovakia. From the second half of 1950s, red foxes (Vulpes vulpes) have become an important reservoir of the RABV. In this time period urban rabies in Slovakia changed into sylvatic form. One effective method of prevention and control of wildlife rabies is an oral rabies vaccination of red foxes. It is carried out in Slovakia since 1993. A detailed development of the rabies epizootiological situation on the territory of the Slovak Republic until the application of oral antirabies immunisation of foxes and the current situation after its performance is the main object of this review. Keywords: rabies; Lyssavirus; red fox; incidence; oral vaccination.

[Correction of measurement errors in the ELISA test]. / Korekcia chýb merania v teste ELISA.

The authors followed the influence of the arrangement of measured and control samples on microtitration plate on the ELISA test results by quantitative evaluation of rabies antibodies titres in human blood serum. They performed the test under identical conditions in all microtitration plate wells. They calculated the result according to five calibration curves--four of them were obtained by different positioning of the control positive and negative sera and the fifth curve was obtained from average values. The results from the four various positional calibration curves were significantly statistically different from the average. However, the average calibration curve--obtained from four measurements--led to identical value of rabies antibodies in cases with different dilutions of sera. The authors propose to follow some principles of measured and control samples arrangement on microtitration plates in order to minimalize the errors, caused by their dishomogeneity. (Tab. 2, Fig. 3, Ref. 10.)

The effectiveness of inactivated, purified and concentrated experimental rabies vaccine for veterinary use: immunogenic activity.

The authors prepared an experimental, inactivated, concentrated and highly purified rabies vaccine from the strain Vnukovo-32/107. The purification and concentration (simultaneous--in one operation) was carried out by affinity chromatography. The content of rabies antigen in the vaccine was 4.256 UE/cm3 (determined by ELISA method). The authors used their own lipoid adjuvant--type oil in water--to resolve lyophilized vaccine to original volume. It is based on esters of fatty the acids. The effectiveness--immunogenic activity--of the experimental preparation was compared with that of commercial rabies vaccines and the reference vaccine by means of the NIH and NRLR methods. The results obtained (2.826 units/cm3 by the NIH and 3.085 units/cm3 by the NRLR method) confirmed that the method of affinity chromatography could be used to prepare a rabies vaccine, exhibiting effectiveness superior to commercial preparations available, and complying with the WHO criteria.

[Isolation and identification of Lyssavirus strains from an area of Slovakia where oral antirabies vaccine was administered]. / Izolácia a identifikácia lyssa vírusových kmenov z oblasti vykonania orálnej antirabickej vakcinácie na Slovensku.

The study was aimed at isolation and subsequent identification of strains of rabies virus by means of monoclonal antibodies from foxes killed in the vaccination zone within the complex preliminary monitoring of oral antirabies vaccination. The results obtained indicate that the vaccines for oral antirabies vaccination used in Slovakia did not contain any vaccination strain pathogenic to the extremely sensitive target species-the fox (Vulpes vulpes).

[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 fungal and yeast glucan and levamisole on the level of the cellular immune response in vivo and leukocyte phagocytic activity in mice]. / Vplyv fungálneho a kvasinkového glukánu a levamizolu na úroven celulárnej imunitnej odpovede in vivo a fagocytárnu aktivitu krvných leukocytov u mysí.

The level of cell-mediated immune response in vivo was investigated using the test of delayed hypersensitive reaction (DHR) to DNFB, along with the phagocytic activity (PA) of blood leucocytes in mice after subcutaneous implantation of fungal and yeast glucan and levamisol in dependence on the dose and administration schedule. The soluble form of fungal glucan (Pleurotus ostreatus) potentiated the DHR significantly at a dose of 10 mg/kg (but not at a dose of 50 mg/kg) while it was administered during DNFB sensitization (P < 0.05)-Tab. I and when its pre-medication effect was investigated (days -7 and -14; P < 0.05) with regard to the time of sensitization (Tab. II). The identical dose of glucan also had a positive effect (P < 0.05 or 0.01) on the percentual proportion of phagocytic cells (PC) reaching the maximum in the 2nd and 3rd week of investigation, as well as on the phagocytic activity index (P < 0.05; 3rd week) and percentage of neutrophil granulocytes (P < 0.05; 2nd week)-Tab. III. Yeast glucan (Saccharomyces cerevisiae) showed a potentiating effect on the DHR to DNFB only in the case of its pre-medication use; its soluble form was effective at both doses (10 mg and 50 mg/kg) in days -7 and -14 (P < 0.05), and its corpuscular form at a dose of 50 mg/kg on days -7, -14 and -21 (P < 0.05 or 0.01)-Tab. II. PA parameters of blood leucocytes displayed a stimulative effect only on the PC percentage. The most significant effects in this case were observed in the soluble form (both doses) in the 2nd and 3rd week (P < 0.01 and 0.05, resp.) and in the insoluble form (both doses) in the 3rd and 4th week of observation (P < 0.05 and 0.01, resp.). An increase in the number of neutrophil granulocytes was significant in the 2nd (P < 0.05 or 0.01; corpuscular form) and 3rd week of the experiment (P < 0.01; soluble form)-Tab. III. Levamisol affected both investigated parameters (DHR and PA) only at a dose 20 mg/kg (10 mg/kg-no effect). Its potentiating effect on the DHR level was observed both for its administration at the time of sensitization (P < 0.05) and for its administration on days 7 (P < 0.05) and 14 (P < 0.01) before DNFB sensitization (Tabs. I and II). A statistically significant increase in PC was recorded in weeks 2, 3 and 4 (P < 0.05 or 0.01), a statistically significant increase in the number of neutrophil granulocytes in the 3rd week of investigation (P < 0.05). The phagocytic activity index was not affected.

[Use of the skin test in the evaluation of true levels of cellular immunity and levels after experimental administration of glucan and levamisole in feed-lot bulls]. / Vyuzitie kozného testu pre hodnotenie aktuálnej úrovne celulárnej imunity a jej úrovne po experimentálnej aplikácii glukánu a levamizolu u výkrmových býckov.

Applicability of a skin test induced by dinitrofluorobenzene (DNFB) to quantification of the actual level of cellular immunity (CI) in vivo and its level after an experimental immunomodulation intervention were evaluated in two breeds (40 animals in each) of fattening bulls (10-11 months old). At the selected methodical procedure of intensity determination of the delayed type of hypersensitivity (DTH), its average value reached 4.5 +/- 1.5 mm in 80 animals, while in 77.5% of bulls its level ranged from 3.6 to 9.6 mm, in 18.7% from 2.0 to 3.5 mm and in 3.8% remained less than 2.0 mm. Evident expression of the reaction points to the possibility of application of the used methodical procedure of the skin test using DNFB to quantify the level of CI response in vivo in cattle. Percentual representation of animals according to the intensity of skin reaction (Tab. I) and concentration of total serum immunoglobulins (Cs-Ig) and serum IgG (Tab. II) indicates the different cellular and humoral state of animals in investigated breeds. This is also confirmed by the recorded average values of mentioned parameters which were significantly lower (P less than 0.01; or 0.05) in animals of the first breed (4.0 +/- 1.3 mm; 28.3 +/- 4.4 U ZST, 18.4 +/- 3.5 g.l-1) than in breed 2 (4.9 +/- 1.6 mm; 32.5 +/- 3.8 U ZST; 20.3 +/- 3.5 g.l-1). The animals of each breed were divided into four experimental groups with the approximately equal actual levels of DTH (Tab. III).(ABSTRACT TRUNCATED AT 250 WORDS)

[Rabies in the common hamster (Cricetus cricetus) in Slovakia]. / Besnota chrckov rol'ných (Cricetus cricetus) na Slovensku.

The trials were conducted within the full-scale research on the ecology of lyssa virus. In a period of the mass outbreak of common hamster population in the East Slovakian region, 283 hamsters were examined for rabies. Using the direct immunofluorescence method (DIFM), the rabies antigen was detected in the brain of five hamsters. Three virus strains (denoted as 3 O, 7 E, 9 E) were isolated by means of the inoculation test on sucking mice. On the basis of the detection of the nucleo-protein antigen by DIFM, or its inhibition, detection of the Babes-Negri bodies, determination of the neutralization index, titration on mice and determination of incubation time, the isolated strains were identified as the street strains of rabies virus. As determined by further detailed studies on biological characteristics (determination of the invasiveness index on animals with different susceptibility to rabies virus, determination of pathogenicity for different species of laboratory animals, different weight categories, with different methods of administration, invasiveness index), the "hamster" strains are included among those of intermediate virulence or reduced virulence. At intramuscular administration, the most virulent of the three "hamster" strains studied (3 O) induces a fatal course of rabies in common fox and cat; for wolves, dogs and rabbits it is apathogenic. This strain is also contained in the salivary glands of foxes and cats. In immunofluorescent detection of the rabies nucleoprotein antigen, the "hamster" strains formed a mixed picture of fluorescing particles, characteristic of street strains.

[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.

[Neuroallergic activity of anti-rabies vaccines]. / Neuroalergická aktivita antirabických vakcín.

The safety and neuroallergic activity of current commercial and experimental rabies vaccine were studied by detecting the patho-histological changes in the central nervous system of laboratory animals (guinea-pigs) according to the method recommended by the World Health Organization (Gispen, 1975). Six rabies vaccines were tested in the experiments. The vaccines are as follows: lyophilized rabies vaccine - human; lyophilized rabies vaccine - veterinary; rabies vaccine U. S. P. duck embryo - human; avianized rabies vaccine - veterinary; inactivated rabies vaccine from strain Vnukov-32 - human; live cell rabies vaccine from strain Vnukovo-32 - veterinary. Patho-histological changes indicating the neuroallergic activity of the vaccines were observed in laboratory animals (varying range and intensity of these activities, to which the following vaccines were applied: lyophilized vaccine - veterinary and lyophilized rabies vaccine - human. The cell rabies vaccines from strain Vnukovo-32 were found to be safe; they can be recommended for their merits (including nonreactogenicity) to be used in veterinary practice in rabies immunoprophylaxis.

[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.