Bjesnoća u životinja i postekspozicijska zaštita ljudi u Sloveniji.

Over the last decade cases of rabies in animals in Central Europe have decreased. The objective of this study was to analyse the influence of the changing number of laboratory confirmed rabies in animals on post-exposure rabies treatment (PET) of humans in the Republic of Slovenia. This article presents data on the number of PET patients during the period 1992-2001. In the first five years of observation, the ratio between treated patients and laboratory confirmed rabid animals was 1.0 to 3.6 respectively. Over subsequent years this ratio gradually changed to 116.2, falling to 6.3 in 2001. The main carrier of rabies in Slovenia was the red fox, but people were treated for rabies mostly because of being bitten by a dog whose owner was unknown. There was an association between PET patients and animal rabies (correlation coefficient r = 0.77; r2 0.59; 95% confidence limits -0.07 < r2 <0.89). The average number of PET patients was 40.2 per 100,000 inhabitants/year (minimum 30.3, maximum 52.0, sd 6.3) and did not change as significantly as did the number of rabid animals in the same time period. Because of existence of a huge reservoir of rabies virus in animals almost all over the world, local focuses, migration of animals and travellers, bites from animals with unknown owners, and some possibility of importing rabies by pets and other animals, it is difficult to satisfy the need for post-exposure rabies treatment of humans in those regions where rabies in animals are in decline.U posljednjih 10 godina u Središnjoj Europi smanjuje se broj životinja oboljelih od bjesnoće. Istražen je utjecaj smanjenja broja bjesnoće u životinja na opseg postekspozicijske zaštite (PEZ) ljudi u Republici Sloveniji. Istraživanje je obavljeno za razdoblje od 1992. do 2001. godine. U prvoj polovici praćenog razdoblja omjer između vakciniranih osoba i laboratorijski potvrđene bjesnoće iznosio je 1,0 do 3,6. U narednim godinama taj se omjer postupno povećao na 116,2, a do 2001 godine se ponovno smanjio na 6,3. Utvrđena je povezanost izmedju PEZ ljudi i pojave bjesnoće u životinja (r = 0,77; r2 0,59; 95% CI -0,07 < r2 <0,89). Prosječan broj PEZ na godinu bio je 40,2 na 100.000 stanovnika (najmanji 30,3 najveći 52,0) i nije se tako dinamično mijenjao kao broj oboljelih životinja od bjesnoće. Potreban je daljnji nadzor bjesnoće u životinja te PEZ, budući da je rezervoar virusa bjesnoće diljem svijeta izrazito velik, te da postoje lokalna žarišta bjesnoće i u državama koje su bjesnoću gotovo u cijelosti iskorjenile, a i zbog međunarodnog prometa putnika i životinja

Sumnjivi i nepodudarni rezultati u dijagnostici bjesnoće testom imuno-fluorescencije.

A total of 7339 fox, dog, cat, cattle, sheep and other mammalian brains were tested to rabies virus antigen. Results of fluorescent antibody test (FAT) were evaluated by two microscopists. Doubtful and discordant results in FAT were analyzed again in virus isolation test (VIT) using mouse neuroblastoma (NA) cell line. Twenty-eight brain samples were determined as doubtful, while 9 brain samples were determined to be positive by one microscopist and negative by the other. Samples which were shown as doubtful and discordant in FAT were retested in VIT. Seventeen of these were positive in the VIT.Na prisutnost antigena virusa bjesnoće bilo je pretraženo ukupno 7339 uzoraka mozgova lisica, mačaka, goveda, ovaca i drugih vrsta životinja. Rezultate izravnog imunofluorescentnog testa vrednovala su dva stručnjaka. Uzorci koji su u testu imunofluorescencije davali sumnjive rezultate, ili se rezultati stručnjaka nisu slagali bili su pretraženi izdvajanjem virusa na kulturi stanica. Rabljena je stanična linija neuroblastoma miševa. Dvadeset osam uzoraka bilo je proglašeno sumnjivim na bjesnoću. Rezultat prvog mikroskopskog pregleda u devet uzoraka bio je pozitivan, a drugog negativan. Od ukupno 37 uzoraka, koji su bili ponovno pretraženi izdvajanjem virusa, 17 je bilo pozitivnih na bjesnoću

Vaccination against rabies and protective antibodies - comparison of elisa and fluorescent antibody virus neutralization (favn) assays

The aim of the study was monitoring the efficacy of primary vaccination against rabies and the need for booster doses. These studies validate at the same time recent technological improvements in laboratory diagnostics of the level of rabies protection in human sera. Research was carried out into the level of antibodies, considering that an antibody titer ≥0.5 IU/mL is protective. We used Platelia rabies ELISA kit (BIO-RAD Laboratories) for the detection of rabies virus anti-glycoprotein antibodies in 41 human sera of previously healthy veterinarian students. Neutralisation rabies virus antibodies were also measured simultaneously by fluorescent antibody virus neutralization (FAVN) test. Two to eight years prior to entering the study subjects had received rabies treatment with human diploid cell vaccine (HDCV, Rabivac, Chiron Germany) according to the schedule: one vaccine on days 0, 7, 21 and 365. Mean level of rabies antibody detected by ELISA was 19.6 (SD 18.8 minimum 1 maximum 56). Results were higher in the groups vaccinated recently. No subject had titer ≤0.5 IU/mL in ELISA, as well as in FAVN. In FAVN test the average titer was higher, reaching 54.4 (SD 44.3 minimum 0.7 maximum 152.5). An immune-complex-like reaction occurring after administration of the booster doses of rabies vaccine is the reason for reconsideration of the needs for administration of booster rabies vaccines. At the same time, the need for mass protection of professionals exposed subjects to rabies virus is real everywhere. Results of these studies indicate that HDCV is highly protective in both FAVN and ELISA tests. A high level of protection lasts at least 8 years in human sera. Average levels of detected rabies antibodies were lower in ELISA in comparison with FAVN test. A correlation between the two tests was found

Vaccination against rabies and protective antibodies - comparison of elisa and fluorescent antibody virus neutralization (favn) assays

The aim of the study was monitoring the efficacy of primary vaccination against rabies and the need for booster doses. These studies validate at the same time recent technological improvements in laboratory diagnostics of the level of rabies protection in human sera. Research was carried out into the level of antibodies, considering that an antibody titer ≥0.5 IU/mL is protective. We used Platelia rabies ELISA kit (BIO-RAD Laboratories) for the detection of rabies virus anti-glycoprotein antibodies in 41 human sera of previously healthy veterinarian students. Neutralisation rabies virus antibodies were also measured simultaneously by fluorescent antibody virus neutralization (FAVN) test. Two to eight years prior to entering the study subjects had received rabies treatment with human diploid cell vaccine (HDCV, Rabivac, Chiron Germany) according to the schedule: one vaccine on days 0, 7, 21 and 365. Mean level of rabies antibody detected by ELISA was 19.6 (SD 18.8 minimum 1 maximum 56). Results were higher in the groups vaccinated recently. No subject had titer ≤0.5 IU/mL in ELISA, as well as in FAVN. In FAVN test the average titer was higher, reaching 54.4 (SD 44.3 minimum 0.7 maximum 152.5). An immune-complex-like reaction occurring after administration of the booster doses of rabies vaccine is the reason for reconsideration of the needs for administration of booster rabies vaccines. At the same time, the need for mass protection of professionals exposed subjects to rabies virus is real everywhere. Results of these studies indicate that HDCV is highly protective in both FAVN and ELISA tests. A high level of protection lasts at least 8 years in human sera. Average levels of detected rabies antibodies were lower in ELISA in comparison with FAVN test. A correlation between the two tests was found

Kontrola arteritisa konja na jednoj ergeli.

An epidemiology of infection with equine arteritis virus (EAV) on one stud farm with approximately 350 horses in the period from 1995 to 2008 was studied. Infection was detected by virological methods, using a virus neutralisation test (VNT) for EAV antibody detection in serum samples, and virus isolation and RT-PCR test for virus detection in semen. No clinical picture of the disease was observed. The highest seroprevalence (nearly 100%) was among stallions and old mares, while seroprevalence among young fi llies, before mating, was lower than 9%. A high incidence for seroconversion was detected among fi llies after mating. Virus was detected by RT-PCR and by a virus isolation test in the semen of 40.7% of 76 seropositive stallions. The 8 stallions, which were shedding EAV, were infected within the period of the first three years after birth, but the other 12 seropositive stallions, which were negative for EAV in semen samples, became firstly seropositive 5 years after birth. In this study we confirmed that the major transmission of EAV on the stud farm occurred from shedding stallions to fillies during the mating time, but an important role of virus transmission to other horses is also played by contact between different groups of animals. Virus positive stallions were castrated and a new breeding unit for young foals was established. EAV negative foals were vaccinated and were bred outside the farm up to 3 years of age.Prikazana je epizootiologija arteritisa konja na jednoj ergeli s 350 konja u razdoblju od 1995. do 2008. godine. Zaraza je bila dokazana na osnovi serološke pretrage virus neutralizacijskim testom (VNT), izdvajanja i identifikacije virusa te RT-PCR-om u sjemenu pastuha. Klinički znakovi bolesti nisu bili primijećeni. Najveća seroprevalencija (gotovo 100%) bila je dokazana u pastuha i starih kobila dok je seroprevalencija u ždrjebica prije pripusta bila manja od 9%. Visoka incidencija serokonverzije bila je dokazana u ždrjebica nakon pripusta. Virus je bio dokazan RT-PCR-om i izdvojen iz sjemena 40,7% od 76 serološki pozitivnih pastuha. Osam pastuha koji su izlučivali virus arteritisa bilo je zaraženo u prvim trima godinama života, a ostalih 12 serološki pozitivnih u kojih virus nije bio izdvojen iz sjemena postali su prvi put serološki pozitivni pet godina nakon ždrijebljenja. Potvrđeno je da se virus u najvećoj mjeri prenosio s pastuha koji su izlučivali virus na ždrjebice za vrijeme pripusta. Za prijenos virusa važan je bio i izravan dodir među različitim skupinama životinja. Pastusi pozitivni na virus bili su kastrirani te je osnovana nova uzgojna jedinica za ždrebad. Ždrebad negativna na virus arteritisa bila je cijepljena, a do treće godine držana izvan ergele

Kontrola arteritisa konja na jednoj ergeli.

An epidemiology of infection with equine arteritis virus (EAV) on one stud farm with approximately 350 horses in the period from 1995 to 2008 was studied. Infection was detected by virological methods, using a virus neutralisation test (VNT) for EAV antibody detection in serum samples, and virus isolation and RT-PCR test for virus detection in semen. No clinical picture of the disease was observed. The highest seroprevalence (nearly 100%) was among stallions and old mares, while seroprevalence among young fi llies, before mating, was lower than 9%. A high incidence for seroconversion was detected among fi llies after mating. Virus was detected by RT-PCR and by a virus isolation test in the semen of 40.7% of 76 seropositive stallions. The 8 stallions, which were shedding EAV, were infected within the period of the first three years after birth, but the other 12 seropositive stallions, which were negative for EAV in semen samples, became firstly seropositive 5 years after birth. In this study we confirmed that the major transmission of EAV on the stud farm occurred from shedding stallions to fillies during the mating time, but an important role of virus transmission to other horses is also played by contact between different groups of animals. Virus positive stallions were castrated and a new breeding unit for young foals was established. EAV negative foals were vaccinated and were bred outside the farm up to 3 years of age.Prikazana je epizootiologija arteritisa konja na jednoj ergeli s 350 konja u razdoblju od 1995. do 2008. godine. Zaraza je bila dokazana na osnovi serološke pretrage virus neutralizacijskim testom (VNT), izdvajanja i identifikacije virusa te RT-PCR-om u sjemenu pastuha. Klinički znakovi bolesti nisu bili primijećeni. Najveća seroprevalencija (gotovo 100%) bila je dokazana u pastuha i starih kobila dok je seroprevalencija u ždrjebica prije pripusta bila manja od 9%. Visoka incidencija serokonverzije bila je dokazana u ždrjebica nakon pripusta. Virus je bio dokazan RT-PCR-om i izdvojen iz sjemena 40,7% od 76 serološki pozitivnih pastuha. Osam pastuha koji su izlučivali virus arteritisa bilo je zaraženo u prvim trima godinama života, a ostalih 12 serološki pozitivnih u kojih virus nije bio izdvojen iz sjemena postali su prvi put serološki pozitivni pet godina nakon ždrijebljenja. Potvrđeno je da se virus u najvećoj mjeri prenosio s pastuha koji su izlučivali virus na ždrjebice za vrijeme pripusta. Za prijenos virusa važan je bio i izravan dodir među različitim skupinama životinja. Pastusi pozitivni na virus bili su kastrirani te je osnovana nova uzgojna jedinica za ždrebad. Ždrebad negativna na virus arteritisa bila je cijepljena, a do treće godine držana izvan ergele

Sumnjivi i nepodudarni rezultati u dijagnostici bjesnoće testom imuno-fluorescencije.

A total of 7339 fox, dog, cat, cattle, sheep and other mammalian brains were tested to rabies virus antigen. Results of fluorescent antibody test (FAT) were evaluated by two microscopists. Doubtful and discordant results in FAT were analyzed again in virus isolation test (VIT) using mouse neuroblastoma (NA) cell line. Twenty-eight brain samples were determined as doubtful, while 9 brain samples were determined to be positive by one microscopist and negative by the other. Samples which were shown as doubtful and discordant in FAT were retested in VIT. Seventeen of these were positive in the VIT.Na prisutnost antigena virusa bjesnoće bilo je pretraženo ukupno 7339 uzoraka mozgova lisica, mačaka, goveda, ovaca i drugih vrsta životinja. Rezultate izravnog imunofluorescentnog testa vrednovala su dva stručnjaka. Uzorci koji su u testu imunofluorescencije davali sumnjive rezultate, ili se rezultati stručnjaka nisu slagali bili su pretraženi izdvajanjem virusa na kulturi stanica. Rabljena je stanična linija neuroblastoma miševa. Dvadeset osam uzoraka bilo je proglašeno sumnjivim na bjesnoću. Rezultat prvog mikroskopskog pregleda u devet uzoraka bio je pozitivan, a drugog negativan. Od ukupno 37 uzoraka, koji su bili ponovno pretraženi izdvajanjem virusa, 17 je bilo pozitivnih na bjesnoću

A Step Forward in Molecular Diagnostics of Lyssaviruses – Results of a Ring Trial among European Laboratories

Rabies is a lethal and notifiable zoonotic disease for which diagnostics have to meet the highest standards. In recent years, an evolution was especially seen in molecular diagnostics with a wide variety of different detection methods published. Therefore, a first international ring trial specifically designed on the use of reverse transcription polymerase chain reaction (RT-PCR) for detection of lyssavirus genomic RNA was organized. The trial focussed on assessment and comparison of the performance of conventional and real-time assays. In total, 16 European laboratories participated. All participants were asked to investigate a panel of defined lyssavirus RNAs, consisting of Rabies virus (RABV) and European bat lyssavirus 1 and 2 (EBLV-1 and -2) RNA samples, with systems available in their laboratory. The ring trial allowed the important conclusion that conventional RT-PCR assays were really robust assays tested with a high concordance between different laboratories and assays. The real-time RT-PCR system by Wakeley et al. (2005) in combination with an intercalating dye, and the combined version by Hoffmann and co-workers (2010) showed good sensitivity for the detection of all RABV samples included in this test panel. Furthermore, all used EBLV-specific assays, real-time RT-PCRs as well as conventional RT-PCR systems, were shown to be suitable for a reliable detection of EBLVs. It has to be mentioned that differences were seen in the performance between both the individual RT-PCR systems and the laboratories. Laboratories which used more than one molecular assay for testing the sample panel always concluded a correct sample result. Due to the markedly high genetic diversity of lyssaviruses, the application of different assays in diagnostics is needed to achieve a maximum of diagnostic accuracy. To improve the knowledge about the diagnostic performance proficiency testing at an international level is recommended before using lyssavirus molecular diagnostics e.g. for confirmatory testing

The successful elimination of sylvatic rabies using oral vaccination of foxes in Slovenia

Sylvatic rabies was present in Slovenia between 1973 and 2013, with the red fox as the main reservoir of the rabies virus. The first oral rabies vaccination (ORV) control program in foxes started in 1988, using the manual distribution of baits. Significant improvement of fox vaccination was achieved with the aerial distribution of baits, starting in 1995 and successfully finished with the final, fifty-ninth vaccination campaign in 2019. Between 1979 and 2019, a total of 86,471 samples were tested, and 10,975 (12.69%) rabies-positive animals were identified. Within the ORV, two different vaccines were used, containing modified live virus strain Street Alabama Dufferin (SAD) B19 and SAD Bern, while the last ORV campaigns were completed in 2019, with a vaccine containing a genetically modified strain of SPBN GASGAS. Molecular epidemiological studies of 95 rabies-positive samples, originating from red foxes, badgers, cattle, dogs, martens, cats, and horses, revealed a low genetic diversity of circulating strains and high similarity to strains from neighboring countries. During the elimination program, few vaccine-induced rabies cases were detected: three in red foxes and one case in a marten, with no epidemiological relevance. Slovenia has been officially declared a country free of rabies since 2016

Diagnostics procedures in rabies

Rabies is a major zoonosis for which diagnostic techniques can only be performed in the laboratory. Laboratory techniques are preferably oriented on tissue removed from the cranium: hippocampus (Ammon's horn), cerebellum and the medulla oblongata or tissue liquids. Clinical observation may only lead to a suspicion of rabies. The only way to perform a reliable diagnosis of the disease is to identify the virus or some of its specific components using laboratory tests such as histological identification of characteristic cell lesions, immunochemical identification of rabies virus antigen and virus isolation. Serological tests are rarely used in epidemiological surveys but much more frequently in control of the vaccination programs (e.g. oral vaccination). Most commonly used serological tests are the virus neutralization test on cell culture (FAVN), virus neutralization in mice and ELISA