The Prevalence of Tick-Borne Encephalitis Virus in Wild Rodents Captured in Tick-Borne Encephalitis Foci in Highly Endemic Lithuania.

Wild rodents are considered to be one of the most important TBEV-amplifying reservoir hosts; therefore, they may be suitable for foci detection studies. To investigate the effectiveness of viral RNA detection in wild rodents for suspected TBEV foci confirmation, we trapped small rodents (n = 139) in various locations in Lithuania where TBEV was previously detected in questing ticks. Murine neuroblastoma Neuro-2a cells were inoculated with each rodent sample to maximize the chances of detecting viral RNA in rodent samples. TBEV RNA was detected in 74.8% (CI 95% 66.7-81.1) of the brain and/or internal organ mix suspensions, and the prevalence rate increased significantly following sample cultivation in Neuro-2a cells. Moreover, a strong correlation (r = 0.88; p < 0.05) was found between the average monthly air temperature of rodent trapping and the TBEV RNA prevalence rate in cell culture isolates of rodent suspensions, which were PCR-negative before cultivation in cell culture. This study shows that wild rodents are suitable sentinel animals to confirm TBEV foci. In addition, the study results demonstrate that sample cultivation in cell culture is a highly efficient method for increasing TBEV viral load to detectable quantities.

The Prevalence, Seroprevalence, and Risk Factors of Tick-Borne Encephalitis Virus in Dogs in Lithuania, a Highly Endemic State.

The rising awareness and increasing number of case reports of tick-borne encephalitis (TBE) in dogs indicate that the virus might be an important tick-borne pathogen in dogs, especially in endemic areas. Therefore, the aim of the present study was to investigate the prevalence rate of TBEV RNA and TBEV-specific antibodies in clinical samples of dogs living in a highly endemic region of Lithuania and to evaluate the main risk factors for severe disease course and death. The blood samples (n = 473) of dogs were collected in two veterinary clinics in central Lithuania. Tick-borne encephalitis virus (TBEV) RNA was detected in 18.6% (88/473; CI 95% 15.2-22.4) and TBEV-specific antibodies were found in 21.6% (102/473; CI 95% 17.9-25.6) of dog blood serum samples after confirmation with a virus neutralization test. The death/euthanasia rate was 18.2% (16/88; CI 95% 10.8-27.8) in PCR-positive dogs. Male dogs were more likely to develop neurological symptoms (p = 0.008). Older dogs (p = 0.003), dogs with the presence of neurological symptoms (p = 0.003), and dogs with the presence of TBEV-specific antibodies (p = 0.024) were more likely to experience worse outcomes of the disease. The results of the present study demonstrate that TBEV is a common and clinically important pathogen in dogs in such endemic countries as Lithuania.

Acute and Sub-Chronic Intraperitoneal Toxicity Studies of the Elsholtzia ciliata Herbal Extract in Balb/c Mice.

Elsholtzia ciliata essential oil (E. ciliata) has been reported to have an impact on the cardiovascular system. However, its toxicity remains unknown. Therefore, the objective of this investigation was to evaluate the toxicological aspects of the E. ciliata extract. Male Balb/c mice were subjected to either acute (a single dose administered for 24 h) or sub-chronic (daily dose for 60 days) intraperitoneal injections of the E. ciliata extract. The mice were assessed for blood hematological/biochemical profiles, mitochondrial functions, and histopathological changes. Additionally, in vitro cytotoxicity assessments of the E. ciliata extract were performed on immobilized primate kidney cells (MARC-145, Vero) and rat liver cells (WBF344) to evaluate cell viability. The control groups received an equivalent volume of olive oil or saline. Our results demonstrated no significant detrimental effects on hematological and biochemical parameters, mitochondrial functions, cellular cytotoxicity, or pathological alterations in vital organs following the intraperitoneal administration of the E. ciliata extract over the 60-day sub-chronic toxicity study. In general, E. ciliata displayed no indications of toxicity, suggesting that the E. ciliata extract is a safe natural product with a well-defined therapeutic and protective index (found to be 90 and 54, respectively) in Balb/c mice.

microRNAs Are Abundant and Stable in Platelet-Rich Fibrin and Other Autologous Blood Products of Canines.

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.

Peripheral mononuclear cells composition in platelet-rich fibrin in canines with chronic conditions.

Platelet-rich fibrin (PRF) is a hot research topic because of its regenerative effect in humans. However, data reporting about its application in companion animals is lacking. The study aimed to supplement currently available data on PRF cell composition in canine patients by isolating peripheral blood mononuclear cells (PBMC), namely T cells, matured B cells, monocytes and macrophages, and adapting current protocols of cell flow cytometry for PRF analysis. The canine patient population was divided into three subgroups: animals with periodontitis only, animals with neoplasia and periodontitis, and healthy controls. Individual clinical parameters of the patients and evaluation of the wound healing quality were included in the research. In the present study, canine PRF cell composition was analyzed for the first-time using cell flow cytometry protocol. A higher proportion of PBMC cells related to wound healing (CD3+, CD3+ CD4+ CD8-, CD14+) were found in the PRF of control, periodontitis and neoplasia groups compared to the respective blood samples, which implies a positive outcome associated with clinical PRF usage in canine patients. Proportions of monocytes and macrophages were higher in PRF samples compared to the blood of healthy patients and periodontitis-affected patients. However, inflammatory and neoplastic processes do not affect the distribution of PBMC.

Bulk Milk Tank Samples Are Suitable to Assess Circulation of Tick-Borne Encephalitis Virus in High Endemic Areas.

A reliable surveillance strategy of tick-borne encephalitis virus (TBEV) is necessary to ensure adequate disease control measures. However, current approaches assessing geographical TBEV circulation are ineffective or have significant limitations. In this study we investigated a total of 1363 goat and 312 sheep bulk tank milk samples for the presence of TBEV. Samples were collected from systematically selected farms in Lithuania every 4-5 days from April to November in 2018 and 2019. To validate results, we additionally tested 2685 questing ticks collected in the vicinity of milk collection sites. We found 4.25% (95% CI 3.25-5.47) and 4.48% (95% CI 2.47-7.41) goat and sheep milk samples to be positive for TBEV, respectively. Furthermore, geographical distribution of TBEV in milk samples coincided with the known TBE endemic zone and was correlated with incidence of TBE in humans in 2019. When sampling time coincides, TBEV detection in milk samples is as good a method as via flagged ticks, however bulk milk samples can be easier to obtain more frequently and regularly than tick samples. The minimal infectious rate (MIR) in ticks was 0.34% (CI 95% 0.15-0.64). Therefore, our results confirm that testing milk serves as a valuable tool to investigate the spatial distribution of TBEV at higher resolution and lower cost.

Investigation of Immunomodulatory and Gut Microbiota-Altering Properties of Multicomponent Nutraceutical Prepared from Lactic Acid Bacteria, Bovine Colostrum, Apple Production By-Products and Essential Oils.

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.

Molecular Characterization and Seroprevalence of Hepatitis E Virus in Inflammatory Bowel Disease Patients and Solid Organ Transplant Recipients.

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, <0.05). Locally isolated HEV strain clustered within genotype 3i subtype of genotype 3 and was capable of infecting MARC-145 cells. This study demonstrates higher HEV seroprevalence in the risk group compared to healthy control individuals without confidence interval overlap. The high level of genetic homology between human and animal strains in Lithuania and the capacity of locally isolated strains to infect cells of non-human origin suggests its potential for zoonotic transmission.

Analysis of seroprevalence in target wildlife during the oral rabies vaccination programme in Lithuania.

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 < 0.5 EU/mL), but with low (0.125 < 0.49 EU/mL) antibody (Abs) titres. 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 < 0.5 EU/mL), but with low 0.125 < 0.49 EU/mL Abs titres. That low, but positive seroconversion participated in the formation of populations overall immune status and can influence the interpretation of oral vaccination efficacy.

Fabrication of Polydimethysiloxane (PDMS) Dense Layer on Polyetherimide (PEI) Hollow Fiber Support for the Efficient CO2/N2 Separation Membranes.

The development of thin layer on hollow-fiber substrate has drawn great attention in the gas-separation process. In this work, polydimethysiloxane (PDMS)/polyetherimide (PEI) hollow-fiber membranes were prepared by using the dip-coating method. The prepared membranes were characterized by Scanning Electron Microscope (SEM), energy-dispersive X-ray spectroscopy (EDX), and gas permeance measurements. The concentration of PDMS solution and coating time revealed an important influence on the gas permeance and the thickness of the PDMS layer. It was confirmed from the SEM and EDX results that the PDMS layer's thickness and the atomic content of silicon in the selective layer increased with the growth in coating time and the concentration of PDMS solution. The composite hollow-fiber membrane prepared from 15 wt% PDMS solution at 10 min coating time showed the best gas-separation performance with CO2 permeance of 51 GPU and CO2/N2 ideal selectivity of 21.

Cross-Sectional Study on the Prevalence and Factors Influencing Occurrence of Tick-Borne Encephalitis in Horses in Lithuania.

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' 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.

African swine fever in the Lithuanian wild boar population in 2018: a snapshot.

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.

The Influence of Essential Oils on Gut Microbial Profiles in Pigs.

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 "liquid/solid" 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.

Preparation and Characterization of Polyphenylsulfone (PPSU) Membranes for Biogas Upgrading.

Asymmetric polyphenylsulfone (PPSU) membranes were fabricated by a non-solvent induced phase inversion method. Glycerin and silica nanoparticles were added into the polymer solution to investigate their effects on the material properties and gas separation performance of prepared membranes. The morphology and structure of PPSU membranes were analyzed by scanning electron microscopy (SEM), the surface roughness of the selective layer was analyzed by atomic force microscopy (AFM), and the surface free energy was calculated based on the contact angle measurements by using various solvents. The gas separation performance of PPSU membranes was estimated by measuring the permeability of CO2 and CH4. The addition of glycerin as a nonsolvent into the polymer solution changed the cross-section structure from finger-like structure into sponge-like structure due to the delayed liquid-liquid demixing process, which was confirmed by SEM analysis. The incorporation of silica nanoparticles into PPSU membranes slightly increased the hydrophilicity, which was confirmed by water contact angle results. PPSU membrane fabricated from the polymer solution containing 10 wt.% glycerin showed the best CO2/CH4 selectivity of 3.86 and the CO2 permeability of 1044.01 Barrer. Mixed matrix PPSU membrane containing 0.1 wt.% silica nanoparticles showed the CO2/CH4 selectivity of 3.16 and the CO2 permeability of 1202.77 Barrer.

Hepatitis E genotype 3 virus isolate from wild boar is capable of replication in non-human primate and swine kidney cells and mouse neuroblastoma cells.

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.

Fermented, ultrasonicated, and dehydrated bovine colostrum: Changes in antimicrobial properties and immunoglobulin content.

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.

Correction to: Prevalence and phylogenetic analysis of hepatitis E virus in pigs, wild boars, roe deer, red deer and moose in Lithuania.

Following publication of the original article [1], we have been notified of a typing error in the "Discussion" section.

Prevalence and spatiotemporal distribution of African swine fever in Lithuania, 2014-2017.

BACKGROUND: The emergence in 2014 and persistence of African Swine Fever (ASF) in Lithuania has been linked to infected wild boar movement and close contact with the carcasses of other infected wild boars. Over time the number of reported cases of ASF in wild boars gradually increased, but no detailed epidemiological data has been available. Therefore, the objective of the present study was to determine ASF virus prevalence in wild boars and domestic pigs during the 2014-2017 period and further explore the current geographical distribution of the virus. RESULTS: Our study results show that ASF virus prevalence in hunted wild boars using PCR analysis increased from 0.83% (95% CI 0.69-0.98) to 2.27% (95% CI 2.05-2.48) from 2014 to 2016 respectively. However, there was a dramatic jump in the number of ASF positive wild boars cases in 2017 resulting in prevalence of 12.39% (95% CI 11.91-12.86) (p < 0.05). The average prevalence of ASF-specific antibodies in wild boar population during years 2014-2017 was 0.45% (95% CI 0.39-0.51) based on ELISA test results. Prevalence of ASF virus in domestic pigs ranged from 0.24% (95% CI 0.17% - 0.32) in 2015 to 2.74% (95% CI 2.33% - 3.15) in 2017. The average seasonal prevalence of ASF virus in pigs was statistically significant (p < 0.05) and ranged from 0% in spring to 3.68% (95% CI 3.32-4.05) in summer. Correlation between the pig density and number of recorded pig ASF cases in affected regions was only found in 2017 (R = 0.78, p < 0.05). No correlation was detected between the wild boar density and number of recorded pig or wild boar ASF - positive cases. CONCLUSIONS: This study provides the first results of ASF virus prevalence changes in Lithuania during the 2014-2017. The overall results confirm the relatively high prevalence of ASF virus in wild boar that was gradually increasing from 2014 to 2017. In the last year of study, the number of ASF positive cases in both domestic pigs and wild boars had unexpectedly increased several times. A better understanding of current status of the disease will enable better control and prevent further spread of ASF virus in Western Europe.

Prevalence and phylogenetic analysis of hepatitis E virus in pigs, wild boars, roe deer, red deer and moose in Lithuania.

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.

Detection and molecular characterization of porcine reproductive and respiratory syndrome virus in Lithuanian wild boar populations.

BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) is recognized worldwide as an important and economically devastating pathogen in pig production. Although PRRSV is widespread in domestic swine, there is a lack of information regarding PRRSV infection in European wild boars (Sus scrofa). Currently available information does not provide conclusive evidence that wild boars are a reservoir of PRRSV. Nevertheless, wild boars may be likely to become infected by domestic swine through occasional direct or indirect contact. Furthermore, wild boars can act as a reservoir for infectious diseases of domestic pigs. Therefore, the objectives of the present study were to determine the virus prevalence and further explore the epidemiology and diversity of PRRSV strains present in Lithuanian wild boars over a 5-year period. A total of 1597 tissue and serum samples from wild boars inhabiting 44 districts and ten counties in Lithuania were analysed using conventional nested reverse transcription polymerase chain reaction (RT-PCR) and real-time Taqman RT-PCR for the detection of PRRSV-specific open reading frame (ORF) 1 and 6 sequences. RESULTS: PRRSV was highly prevalent in Lithuanian wild boar populations, with an average rate of 18.66 % using conventional RT-PCR and 19.54 % using real-time RT-PCR. PRRSV was detected in 36.71 and 41.77 % of 237 hunting grounds tested by conventional RT-nPCR and real-time RT-PCR, respectively. No statistically significant differences in PRRSV prevalence were observed by geographic area in the ten Lithuanian counties. Animals infected with PRRSV were identified in all age groups; however, significantly higher prevalence rates were identified in subadult and adult wild boars than in juveniles up to 12 months old. No positive results were obtained using conventional PCR with Type 2 specific primers. Phylogenetic analysis of the partial ORF5 region revealed that ten wild boars harboured virus sequences belonging to genetic subtypes 3 and 4 and may therefore pose a serious threat to Lithuanian pig farms in which only subtype two strains are circulating. CONCLUSIONS: The results of virus prevalence and phylogenetic analyses strongly support the role of wild boars as a possible natural reservoir for PRRSV in Lithuania.