Serologic Investigation on Tick-Borne Encephalitis Virus, Kemerovo Virus and Tribec Virus Infections in Wild Birds.

The present study reports on serosurvey on the tick-borne encephalitis virus European subtype (TBEV; genus Flavivirus), and the tick-borne Kemerovo (KEMV) and Tribec (TRBV) orbivirus (genus Orbivirus) infections in tick-infested and non-infested birds. No virus RNA was detected in the blood clots. Birds were infested mostly by Ixodes ricinus, but Haemaphysalis concinna and I. frontalis were observed too. TBEV, KEMV and TRBV neutralising antibodies (NAb) were detected in the screening microtitration neutralisation test (µVNT). Seropositive samples were further examined in simultaneous µVNT to distinguish TBEV infection from WNV and USUV. KEMV and TRBV infections were also further examined by µVNT against each other. The demonstrated results point to increased TBEV and TRBV seroprevalence in birds over the past several years. This is the first study on KEMV infection in the Slovak bird population, and seropositive juvenile birds suggest its occurrence in a new geographic area. The results indicate the significance of tick infestation rates, seropositivity and specific NAb titre. The reservoir role of birds for TBEV, KEMV and TRBV remains unclear. However, targeted monitoring of birds and vectors is an effective measure of surveillance of arbovirus introduction into new geographic areas.

Opportunities and Limitations of Molecular Methods for Studying Bat-Associated Pathogens.

Bats have been identified as reservoirs of zoonotic and potentially zoonotic pathogens. Significant progress was made in the field of molecular biology with regard to infectious diseases, especially those that infect more than one species. Molecular methods, sequencing and bioinformatics have recently become irreplaceable tools in emerging infectious diseases research and even outbreak prediction. Modern methods in the molecular biology field have shed more light on the unique relationship between bats and viruses. Here we provide readers with a concise summary of the potential and limitations of molecular methods for studying the ecology of bats and bat-related pathogens and microorganisms.

Serological survey of lyssaviruses in synanthropic bats and human exposure to bats in Slovakia.

INTRODUCTION: Bats are considered natural reservoirs for lyssaviruses. A total of 17 out of 19 known lyssaviruses circulate in bat populations. Lyssaviruses cause rabies in animals and humans. The transmission of lyssaviruses from European bats to terrestrial animals and humans is rare, but the risk of infection still exists even in developed countries. Slovakia is currently a rabies-free country. OBJECTIVE: The aim of the study was to assess the potential circulation of EBLV-1 in synanthropic bats present in human inhabited buildings, and to give an overview of human exposure to bats. MATERIAL AND METHODS: A passive serological survey targeted the prevalence of antibodies to bat lyssaviruses in synanthropic bats between 2009 - 2019. A total of 598 bats of the species Pipistrellus pipistrellus, Pipistrellus pygmaeus, Eptesicus serotinus, Nyctalus noctula and Vespertilio murinus were captured in buildings mainly in Eastern Slovakia, and examined by the rapid fluorescent focus inhibition test (RFFIT). RESULTS: Lyssavirus-specific antibodies were detected in 2 (0.3%) of the 598 examined bats. Additionally, brain tissues of bats found dead were examined using the standard fluorescent antibody test (FAT) with negative results. An overview of available data on human exposure to bats recorded in Slovakia from 2007 - 2019 is also included. CONCLUSIONS: The study confirmed the presence of lyssavirus antibodies in synanthropic bats in Slovakia, suggesting the active circulation of bat lyssaviruses in bat populations exploiting human buildings. Although the seroprevalence was found to be extremely low, the results show that any case of human exposure to bats must be treated with caution in order to protect public health.

The first serological evidence of Anaplasma phagocytophilum in horses in Slovakia.

Anaplasma phagocytophilum is the causative agent of granulocytic anaplasmosis. It affects humans and several wild and domesticated mammals, including horses. The aim of our study was a preliminary survey of the occurrence of these re-emerging pathogens in horses in Slovakia. The sera from 200 animals of different ages and both sexes were tested for the presence of A. phagocytophilum antibodies by indirect immunofluorescence assay. Subsequently, detection of the 16S rRNA gene fragment of A. phagocytophilum was attempted by polymerase chain reaction (PCR) in each blood sample. Our results confirmed the presence of specific antibodies in 85 out of 200 individuals (42.5%), but no significant changes were found between the animals of different ages and sexes. However, the PCR analysis did not detect any positive animals. Our data represent one of the highest values of seropositivity to A. phagocytophilum in horses in Central Europe. These results may contribute to a better understanding of the circulation of A. phagocytophilum in this region, thus indicating a potential risk to other susceptible species.

Antioxidative Protection of Squalene Adjuvant and Rabies Vaccine with Adjuvant.

The authors verified the possibility of antioxidative protection of squalene adjuvant emulsions by the antioxidants α-tocopherol and ß-carotene. They determined the influence of ß-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 ß-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 ß-carotene during the preparation process and also during the 70-d storage. The vaccine with ß-carotene induced a gradual increase in the RAB levels with the highest value on day 28. While the inactivated rabies vaccine with adjuvant without ß-carotene developed a rapid formation of RAB, the application of the vaccine with ß-carotene induced a slower but more uniform production of RAB. The level of RAB was significantly higher after the application of the vaccine with ß-carotene and reached the protective value of 0.5 IU/mL, in contrast to the vaccine without ß-carotene.

Immunomodulatory effect of glucan on specific and nonspecific immunity after vaccination in puppies.

The objective of the study was to determine the immunostimulatory effect of ß-(1,3/1,6)-D-glucan in puppies. The effect exerted on the efficacy of vaccination, especially against canine parvovirus and rabies infection, was studied. The application of vaccine and glucan leads to significant increases in the nonspecific immunological parameters (phagocytic ability of leukocytes, blastogenic response of lymphocytes, metabolic and chemotactic activity of polymorphonuclear cells). The level of antibodies against canine parvovirus (Ab CPV) and rabies infection reached the most statistically significant values on the 28th day after the application of vaccine and a syrup containing ß-(1,3/1,6)-D-glucan (Group GV) as compared to the control group (Group V, puppies receiving only vaccine). Dogs without glucan supplementation did not produce such significant levels of antibodies. We can conclude that glucan has relevant immunostimulatory effects in dogs with altered immunity. The glucan product tested in this study (PleraSAN V, PLEURAN, Bratislava, Slovakia) could be used in the small animal clinical practice.

Experimental squalene adjuvant. II. Harmlessness and local reactogenity.

Model experiments on laboratory animals (guinea pigs) were carried out to test the possible allergic reaction (possibility of sensitisation) to the repeated administration of an experimental lipoid adjuvant prepared on the basis of squalene (experimental squalene adjuvant--ESA). No significant differences were observed between the animals sensitised-provoked with ESA and control animals. In order to evaluate the local tissue reactivity (local reactogenity), also with regard to the process dynamics to the administration of ESA, comparative patho-anatomical and patho-histological examinations of tissues were carried out in the location of adjuvant administration. The examinations indicated very low local reactogenity of the experimental lipoid adjuvant prepared in our laboratory. The test of pyrogenicity also confirmed the safety of ESA, the labelled lysate sensitivity lambda was under 0.25 IU/cm3.

Experimental squalene adjuvant. I. Preparation and testing of its effectiveness.

The authors prepared experimental lipoid adjuvant of the oil-in-water (O/W) type, based on squalene (ESA: experimental squalene adjuvant), intended for liquid inactivated veterinary vaccines. The final concentration of squalene in the vaccine was 5% (w/v). Detergents Poloxamer 105 in the concentration of 4% (w/v) and Abil-Care in the concentration of 2% were used as emulsifiers. The water component was inactivated virus suspension. Emulsification at decreased temperatures (up to 37 degrees C) fails to denaturate the immunization antigen. The effectiveness of experimental squalene adjuvant was tested using a liquid inactivated adjuvant rabies vaccine and a liquid porcine parvovirus vaccine in comparison with adjuvant-free vaccines or vaccines potentiated with aluminium hydroxide. The squalene adjuvant increased the immunogenic activity of non-potentiated rabies vaccine approximately 1.8-fold, while aluminium hydroxide increased the effectiveness of rabies vaccine only 1.08-fold. The antigenic activity, i.e. the level of specific antibodies after vaccination was significantly increased by using both adjuvants; ESA was more efficient than aluminium hydroxide.