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Various microRNAs (miRNAs) present in autologous blood products of canines have not been studied recently. We aimed to elucidate the existence of miRNAs in platelet-rich fibrin (PRF) and the stability of canine autologous blood products under various storage conditions. Total RNAs were isolated from PRF and other autologous blood products following newly adapted protocols used in commercial kits for plasma and tissue samples. Quantitative real-time polymerase chain reaction analysis (qPCR) was used to detect miRNAs in autologous blood products. The miR-16, miR-21, miR-155, and miR-146a were abundant in PRF and other autologous blood products of canines. Furthermore, we found they could maintain stability under protracted freezing temperatures of −30 °C for at least one month. Our findings revealed that PRF might be a stable resource for various canine miRNAs.

This study evaluated the influence of fermentation with Lactobacillus plantarum LUHS135 and Lactobacillus paracasei LUHS244, ultrasonication, and different methods of dehydration on the content of IgG, IgA, and IgM in bovine colostrum (BC), as well as the antimicrobial activity of the treated and fresh BC samples [fresh = BC; freeze dried = BClyoph; vacuum dried (+45°C) = BCvacdried; BC fermented with LUHS135 = BCLUHS135; BC fermented with LUHS244 = BCLUHS244; BC fermented with LUHS135 and freeze dried = BCLUHS135lyoph; BC fermented with LUHS244 and freeze dried = BCLUHS244 lyoph; BC fermented with LUHS135 and vacuum dried = BCLUHS135 vacdried; BC fermented with LUHS244 and vacuum dried = BCLUHS244 vacdried; BC ultrasonicated and freeze dried = BCultr lyoph; BC ultrasonicated and vacuum dried = BCultr vacdried]. The antimicrobial activity was assessed against Klebsiella pneumoniae, Salmonella enterica, Pseudomonas aeruginosa, Acinetobacter baumanni, Proteus mirabilis, methicillin-resistant Staphylococcus aureus, Enterococcus faecalis, Enterococcus faecium, Bacillus cereus, Streptococcus mutans, Enterobacter cloacae, Citrobacter freundii, Staphylococcus epidermis, Staphylococcus haemolyticus, and Pasteurella multocida using the agar well diffusion method, as well as in liquid medium. In liquid medium analysis showed that the fermented BC samples had the broadest antimicrobial spectrum (of 15 tested pathogenic strains, BCLUHS135 vacdried and BCLUHS135lyoph inhibited 13; BCLUHS244 vacdried inhibited 12; and BCLUHS135, BCLUHS244, and BCLUHS244 lyoph inhibited 11). Based on the inhibition zones, BCLUHS135lyoph samples exhibited the broadest inhibition spectrum, inhibiting the growth of 12 of the 15 tested pathogenic strains). According to the lactic acid bacteria strain selected for BC fermentation, different properties of the BC will be obtained. To ensure a broad antimicrobial spectrum and high IgG content, fermentation with LUHS135 can be recommended (IgG concentration in BCLUHS135 was retained), whereas fermentation with LUHS244 will provide a high IgM concentration (IgM concentration increased by 48.8 and 21.6% in BCLUHS244 and BCLUHS244lyoph samples, respectively). However, IgA is very sensitive for fermentation, and further studies are needed to increase IgA stability in BC. Finally, fermented BC can be recommended as a food/beverage ingredient, providing safety, as well as improved functionality through displaying a broad spectrum of antimicrobial activities.

Dietary components, such as lactic acid bacteria (LAB), bovine colostrum, apple production by-products, and essential oils, can favorably alter the host immune system and gut microbiota, however, their cumulative effect as multicomponent nutraceutical supplement has not been investigated. Therefore, the present study is the first one to evaluate a combination of LAB, bovine colostrum, dehydrated apple pomace, and essential oils for their immunomodulatory and prebiotic properties in the swine model. This study shows that supplementary feeding of pigs using multicomponent nutraceutical resulted in a statistically significant decrease in proportions of T cytotoxic and double-positive (CD4+CD8+low) cells within the CD3+ cell population at 28 DPI, compared to the beginning of the experiment (0DPI). Conversely, a statistically significant increase in proportions of B cells (accompanied by an increase in IgG concentration) and macrophage/monocyte cells within viable cell population at 28 DPI, compared to the beginning of the experiments, was observed. Furthermore, changes in the bacterial composition of gut microbiota in pigs fed with multicomponent nutraceutical changed significantly, with a 1.78 times higher number of probiotic strains (Bifidobacterium, Streptococcus, Faecilbacterium) at the end of the experiment, compared to control group animals. This study shows a positive effect of the nutraceutical formula used on the changes of gut microbiota by facilitating an increase in probiotic bacteria strains and possible anti-inflammatory properties.

Background Rabies vaccination of wildlife carnivores is a powerful tool to prevent, control and eliminate rabies. The presence of neutralizing rabies antibodies in blood is considered a reliable indicator of adequate vaccination. The main purpose of the present study was to analyze the seroprevalence of specific antibodies in target populations of Lithuanian red fox (RF) and raccoon dog (RD) during the oral rabies vaccination (ORV) campaigns during the 2010–2019 period. Results Over the ten-year period, 7,261 RF and 2,146 RD sera samples were collected post-mortem in field conditions and tested using a commercial standardized enzyme-linked immunosorbent assay (ELISA) kit in Lithuania. In the ORV spring and autumn vaccination periods, 31.8% (20.3–43.4 95% CI – 95% confidence interval) and 31.7% (21.2–42.1 95% CI) of RF, and 34.1% (22.5–45.7 95% CI) and 34.7% (22.7–46.7 95% CI) of RD sera samples, respectively, were identified as ELISA-positive (seroconversion ≥ 0.5 EU/mL—Equivalent Units per Millilitre). The seroprevalence analysis in adult/ juvenile animal subpopulations indicated that 34.9% (27.2–42.5 95% CI) and 29.2% (20.3–37.9 95% CI) of RF, and 35.6% (25.2–46.0 95% CI) and 30.6% (20.2–40.9 95% CI) of RD sera samples, respectively, were identified as ELISA-positive (seroconversion ≥ 0.5 EU/mL). Statistically strong determinate correlations (r) between the serological results (pos.%) in RF adult/juvenile animal subpopulations (r = 0.937) and between RF and RD positive seroconvert (pos.%) sera samples during the spring vaccinations (r = 0.864) were demonstrated. In different ORV periods, 14–29% of RF and 7–25% of RD sera samples were identified as ELISA-negative (seroconversion Conclusions The 2010–2019 ORV programme has been an effective tool in both RF and RD populations in Lithuania. The rabies-free status of Lithuania was self-declared in 2015 with only three rabies cases identified in buffer zones since then. The percentage of ELISA-positive serum samples (seroconversion ≥ 0.5 EU/mL) during the different periods of vaccination was similar in RF and RD populations—32% and 34% respectively. The identified seroconversion average of 21.5% in RF and 16% in RD sera samples were officially identified as ELISA-negative (seronversion

Direct laser writing (DLW) based on the femtosecond (fs) pulse-induced light-matter interaction expanded considerably during the last decades. The key advantage of using fs lasers for DLW is the possibility to exploit various nonlinear light-matter interaction regimes as well as control the thermal aspect of the process. This work is dedicated to exploring the capabilities of expanding DLW in several possible biomedical application areas where fs lasers could yield a very attractive, high throughput solution. Namely, we will be discussing how hybrid additive-subtractive DLW can be exploited for the high-throughput fabrication of integrated microfluidic systems. Furthermore, a mechanical flexible scaffold will be presented. Finally, a possibility to produce very high precision metalized 3D structures by using pre-existing high-throughput multi-photon polymerization capabilities will be shown. In all cases, attention will be placed on the unique capabilities of fs-lasers in DLW as well as practical considerations of the processes and their up-scaling.

Various animal species have been evaluated in depth for their potential as Tick-borne encephalitis virus (TBEV) sentinel species, although evidence for equine capacity is incomplete. Therefore, a comprehensive cross-sectional stratified serosurvey and PCR analysis of selected horses (n = 301) were performed in TBEV endemic localities in Lithuania. Attached and moving ticks (n = 241) have been collected from aforementioned hosts to evaluate natural infectivity of TBEV vectors (Ixodes spp.) in the recreational environments surrounding equestrian centers. All samples were screened for TBEV IgG and positive samples were confirmed by virus neutralization test (VNT). 113 (37.5%) horses from all counties of Lithuania tested positive for TBEV IgG, revealing age and sex indifferent results of equine seroprevalence that were significantly dependent on pedigree: horses of mixed breed were more susceptible to infection possibly due to their management practices. TBEV prevalence in equine species corresponded to TBEV-confirmed human cases in the precedent year. As much as 3.9% of horses were viraemic with TBEV-RNA with subsequent confirmation of TBEV European subtype. 4/38 of tested tick pools were positive for TBEV-RNA (Minimal infectious rate 1.2%). Several unknown microfoci were revealed during the study indicating areas of extreme risk close to popular human entertainment sites. The study provides important evidence in favor of horses&rsquo, usage as sentinel species, as equines could provide more detailed epidemiological mapping of TBEV, as well as more efficient collection of ticks for surveillance studies.

16 p.-3 fig.-5 tab., Seroprevalence rates and molecular characterization of hepatitis E virus (HEV) prevalent in the Lithuanian human population has not yet been evaluated. Immunosuppressed individuals have been recognized as a risk group for chronic hepatitis due to HEV genotype 3 (HEV-3) infections. The objectives of the present study were to determine prevalence rates of anti-HEV antibodies among inflammatory bowel disease (IBD) patients and solid organ transplant (SOT) recipients, to isolate and characterize HEV strain present in the Lithuanian human population, and to investigate its capacity to infect non-human primate (MARC-145 and Vero), swine (PK-15) and murine (Neuro-2a) cells in vitro. In the present study, the significant difference of anti-HEV IgG prevalence between healthy (3.0% (95% CI 0–6.3)) and immunosuppressed individuals (12.0% [95% CI 8.1–15.9]) was described. Moreover, our findings showed that anti-HEV IgG seropositivity can be significantly predicted by increasing age (OR = 1.032, p < 0.01), diagnosis of IBD (OR = 4.541, p < 0.01) and reception of SOT (OR = 4.042, This research was partly funded by the Lithuanian University of Health Sciences Science Foundation (LSMUSF), grant number 119-05 and COST Action CA15116 ASF-STOP, supported by COST (European Cooperation in Science and Technology).

The first cases of African swine fever (ASF) were detected in the Lithuanian wild boar population in 2014. Since then, the disease spread slowly through the whole country, affecting both, wild boar and domestic pigs. In the other Baltic states, which both are also affected by ASF since 2014, the recent course of ASF prevalence suggests that the countries might be well under way of disease elimination. In contrast, in Lithuania the epidemic seems to be still in full progress. In the present study, we aimed to extend a previous prevalence study in Lithuania. Looking at ASF virus (ASFV) and seroprevalence estimates of wild boar in all months of 2018 and in all affected municipalities in Lithuania, the course of ASF was evaluated on a temporal and spatial scale. A non-spatial beta-binomial model was used to correct for under- or overestimation of the average prevalence estimates. Within 2018 no big differences between the prevalence estimates were seen over time. Despite of the lower sample size, highest ASFV prevalence estimates were found in dead wild boar, suggesting higher detection rates through passive surveillance than through active surveillance. Accordingly, with the maximum prevalence of 87.5% in May 2018, the ASFV prevalence estimates were very high in wild boar found dead. The number of samples originating from hunted animals (active surveillance) predominated clearly. However, the ASFV prevalence in those animals was lower with a maximum value of 2.1%, emphasizing the high value of passive surveillance. A slight increase of the seroprevalence in hunted wild boar could be seen over time. In the center of Lithuania, a cluster of municipalities with high ASFV and seroprevalence estimates was found. The results of the study indicate that ASFV is still circulating within the Lithuanian wild boar population, constituting a permanent risk of disease transmission into domestic pig holdings. However, additional, more recent data analyses are necessary to re-evaluate the course of ASF in Lithuania and thus, to be able to make a statement about the stage of the ASF epidemic in the country. This is of huge importance for Lithuania for evaluating control measures and their efficacy, but also for neighbouring countries to assess the risk of disease spread from Lithuania.

In recent years, the intake of ultra-processed foods has increased dramatically worldwide. Missing natural foods in the diet raise the need of biologically active food components that could compensate for this deficiency and help maintain proper immune status. This study used pigs as an animal model for the assessment of the impact of consumption of Origanum vulgare plant extract combined with Mentha piperita and Thymus vulgaris essential oils on microbial profile in intestines. A single group of weaned pigs received basal diet, while the other group basal diet supplemented with plant extract and two essential oils in the form of bilayer tablets prepared using &ldquo, liquid/solid&rdquo, phase technology. Metagenomic sequencing was performed with the aim to investigate changes of microbial communities in ileum, caecum, and colon. The results demonstrated that the combination of essential oils was non cytotoxic, and had a positive effect on the microbial composition in the large intestine of pigs due to significant increase in the number of probiotic bacteria. The amount of Lactobacillus was 2.5 times and Bifidobacterium 1.9 times higher in the animal group fed with supplement. The combination, however, had some negative impact on the variety of minor species in the distal part of the ileum. Additional studies need to be performed to obtain knowledge on how combinations of essential oils can change bacterial variety in the proximal part of the gastrointestinal tract.

Background Wild boar-derived hepatitis E (HEV) genotype 3 virus has been successfully isolated in cell lines of human origin only. Considering the zoonotic potential and possible extrahepatic localisation of genotype 3 strain, it is important to investigate the viability of cell lines of different animal and tissue origins. Therefore, the objective of the present study was to determine the permissiveness of non-human primate (MARC-145 and Vero) and swine (PK-15) cell lines of kidney origin, and a mouse neuroblastoma (Neuro-2a) cell line for isolation of wild boar-derived HEV genotype 3. Results This study showed that MARC-145, PK-15, Neuro-2a and Vero cell lines were permissive to wild boar-derived HEV genotype 3 subtype 3i harbouring viral genome equivalents of 1.12 × 107 copies/ml, 2.38 × 105 copies/ml, 2.97 × 107 copies/ml and 4.01 × 107 copies/ml after five serial passages respectively. In all permissive cell lines, HEV was continuously recovered from growth medium between five and at least 28 days post-infection. Peak loads of HEV genome equivalents were observed on days 7, 12, 19 and 30 in MARC-145 (2.88 × 107 copies/ml), Vero (4.23 × 106 copies/ml), Neuro-2a (3.15 × 106 copies/ml) and PK-15 (2.24 × 107 copies/ml) cell lines respectively. In addition, successful virus isolation was confirmed by immunofluorescence assay targeting HEV capsid protein and sequencing of HEV isolate retrieved from cell cultures. Conclusions This study showed that wild boar-derived HEV genotype 3 subtype 3i strain was capable of infecting cell lines of animal origin, including primate and porcine kidney cells (MARC-145, PK-15 and Vero), and mouse neuroblastoma cells (Neuro-2a), supporting the notion of the capacity of HEV genotype 3 to cross the species barrier and extra-hepatic localisation of the virus. These findings warrant further studies of tested cell lines to investigate their capacity as an efficient system for HEV propagation. HEV isolates from other wild animal hosts should be isolated on tested cell lines in order to generate more data on HEV transmission between wild animal populations and their role as sources of human infections.

Background Hepatitis E virus (HEV) is one of the major causes of acute viral hepatitis worldwide. In Europe, food-borne zoonotic transmission of HEV genotype 3 has been associated with domestic pigs and wild boar. Controversial data are available on the circulation of the virus in animals that are used for human consumption, and to date, no gold standard has yet been defined for the diagnosis of HEV-associated hepatitis. To investigate the current HEV infection status in Lithuanian pigs and wild ungulates, the presence of viral RNA was analyzed by nested reverse transcription polymerase chain reaction (RT-nPCR) in randomly selected samples, and the viral RNA was subsequently genotyped. Results In total, 32.98 and 22.55% of the domestic pig samples were HEV-positive using RT-nPCR targeting the ORF1 and ORF2 fragments, respectively. Among ungulates, 25.94% of the wild boar samples, 22.58% of the roe deer samples, 6.67% of the red deer samples and 7.69% of the moose samples were positive for HEV RNA using primers targeting the ORF1 fragment. Using primers targeting the ORF2 fragment of the HEV genome, viral RNA was only detected in 17.03% of the wild boar samples and 12.90% of the roe deer samples. Phylogenetic analysis based on a 348-nucleotide-long region of the HEV ORF2 showed that all obtained sequences detected in Lithuanian domestic pigs and wildlife belonged to genotype 3. In this study, the sequences identified from pigs, wild boars and roe deer clustered within the 3i subtype reference sequences from the GenBank database. The sequences obtained from pig farms located in two different counties of Lithuania were of the HEV 3f subtype. The wild boar sequences clustered within subtypes 3i and 3h, clearly indicating that wild boars can harbor additional subtypes of HEV. For the first time, the ORF2 nucleotide sequences obtained from roe deer proved that HEV subtype 3i can be found in a novel host. Conclusion The results of the viral prevalence and phylogenetic analyses clearly demonstrated viral infection in Lithuanian pigs and wild ungulates, thus highlighting a significant concern for zoonotic virus transmission through both the food chain and direct contact with animals. Unexpected HEV genotype 3 subtype diversity in Lithuania and neighboring countries revealed that further studies are necessary to understand the mode of HEV transmission between animals and humans in the Baltic States region.

Hepatitis E is an important public health concern disease that causes acute hepatitis in humans with different hepatitis E virus strains reported among domestic and wild animals. In Lithuania, seroprevalence studies on animals considered to be possible virus reservoirs, particularly in wildlife species, had never been investigated before. In order to assess the actual distribution of the virus in the domestic pig and wildlife species, serum samples originating from different geographic areas of Lithuania were tested for the presence of antibodies to the virus using enzyme-linked immunosorbent assay. The study results proved that hepatitis E virus infection is prevalent among domestic pigs, wild boar, moose, and roe deer in Lithuania, with the overall prevalence of IgG antibodies against hepatitis E virus being 43.75%, 57.05%, 11.76%, and 1.20%, respectively. Significantly higher (P< 0.01) seroprevalence values of 53.66% and 80% were observed in weaned pigs and adult wild boar. Herds with 15 001–30 000 pigs were 2.4 times more likely (P< 0.01) to test positive for hepatitis E virus antibodies (70.18%), than herds with ≤ 500 pigs (21.11%). The differences in seroprevalence rates between domestic pigs (from 25 to 66.67%) and wild boar (from 42.86 to 73.33% ) in all 10 counties were not significant (P> 0.05). Estimated low seroprevalence rate in moose, roe deer and absence of antibodies in red deer suggest that such wildlife species are accidental hosts of hepatitis E virus or at least hepatitis E virus is not present in Lithuanian cervides.

Hepatitis E virus (HEV) is one of the major causes of acute viral hepatitis worldwide. In Europe, food-borne zoonotic transmission of HEV genotype 3 has been associated with domestic pigs and wild boar. Controversial data are available on the circulation of the virus in animals that are used for human consumption, and to date, no gold standard has yet been defined for the diagnosis of HEV-associated hepatitis. To investigate the current HEV infection status in Lithuanian pigs and wild ungulates, the presence of viral RNA was analyzed by nested reverse transcription polymerase chain reaction (RT-nPCR) in randomly selected samples, and the viral RNA was subsequently genotyped.In total, 32.98 and 22.55% of the domestic pig samples were HEV-positive using RT-nPCR targeting the ORF1 and ORF2 fragments, respectively. Among ungulates, 25.94% of the wild boar samples, 22.58% of the roe deer samples, 6.67% of the red deer samples and 7.69% of the moose samples were positive for HEV RNA using primers targeting the ORF1 fragment. Using primers targeting the ORF2 fragment of the HEV genome, viral RNA was only detected in 17.03% of the wild boar samples and 12.90% of the roe deer samples. Phylogenetic analysis based on a 348-nucleotide-long region of the HEV ORF2 showed that all obtained sequences detected in Lithuanian domestic pigs and wildlife belonged to genotype 3. In this study, the sequences identified from pigs, wild boars and roe deer clustered within the 3i subtype reference sequences from the GenBank database. The sequences obtained from pig farms located in two different counties of Lithuania were of the HEV 3f subtype. The wild boar sequences clustered within subtypes 3i and 3h, clearly indicating that wild boars can harbor additional subtypes of HEV. For the first time, the ORF2 nucleotide sequences obtained from roe deer proved that HEV subtype 3i can be found in a novel host.The results of the viral prevalence and phylogenetic analyses clearly demonstrated viral infection in Lithuanian pigs and wild ungulates, thus highlighting a significant concern for zoonotic virus transmission through both the food chain and direct contact with animals. Unexpected HEV genotype 3 subtype diversity in Lithuania and neighboring countries revealed that further studies are necessary to understand the mode of HEV transmission between animals and humans in the Baltic States region.