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.

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.

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.

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.

Prevalence of methicillin-resistant Staphylococcus haemolyticus in companion animals: a cross-sectional study.

BACKGROUND: Among coagulase-negative staphylococci, Staphylococcus haemolyticus is the second most frequently isolated species from human blood cultures and has the highest level of antimicrobial resistance. This species has zoonotic character and is prevalent both in humans and animals. Recent studies have indicated that methicillin-resistant S. haemolyticus (MRSH) is one of the most frequent isolated Staphylococcus species among neonates in intensive care units. The aim of this study was to determine the presence of MRSH in different groups of companion animals and to characterize isolates according their antimicrobial resistance. METHODS: Samples (n = 754) were collected from healthy and diseased dogs and cats, female dogs in pure-breed kennels, healthy horses, and kennel owners. Classical microbiological tests along with molecular testing including PCR and 16S rRNA sequencing were performed to identify MRSH. Clonality of the isolates was assessed by Pulsed Field Gel Electrophoresis using the SmaI restriction enzyme. Antimicrobial susceptibility testing was performed using the broth micro-dilution method. Detection of genes encoding antimicrobial resistance was performed by PCR. Statistical analysis was performed using the R Project of Statistical Computing, "R 1.8.1" package. RESULTS: From a total of 754 samples tested, 12 MRSH isolates were obtained. No MRSH were found in horses and cats. Eleven isolates were obtained from dogs and one from a kennel owner. Ten of the dog isolates were detected in pure-breed kennels. The isolates demonstrated the same clonality only within separate kennels.The most frequent resistances of MRSH isolates was demonstrated to benzylpenicillin (91.7%), erythromycin (91.7%), gentamicin (75.0%), tetracycline (66.7%), fluoroquinolones (41.7%) and co-trimoxazole (41.7%). One isolate was resistant to streptogramins. All isolates were susceptible to daptomycin, rifampin, linezolid and vancomycin. The clone isolated from the kennel owner and one of the dogs was resistant to beta-lactams, macrolides, gentamicin and tetracycline. CONCLUSIONS: Pure-breed kennels keeping 6 or more females were determined to be a risk factor for the presence of MRSH strains. MRSH isolated from companion animals were frequently resistant to some classes of critically important antimicrobials, although they remain susceptible to antibiotics used exclusively in human medicine.

Raccoon dog rabies surveillance and post-vaccination monitoring in Lithuania 2006 to 2010.

BACKGROUND: Oral rabies vaccination (ORV) in rabies infected regions should target the primary rabies vector species, which in Lithuania includes raccoon dogs as well as red foxes. Specific investigations on ORV in raccoon dogs are needed e.g. evaluation of vaccine effectiveness under field conditions. The objective of the current study was to investigate the efficacy of the ORV programme 2006-2010 in Lithuania by examining the number of rabies cases and estimating the prevalences of a tetracycline biomarker (TTC) and rabies virus antibodies in raccoon dogs. METHODS: From 2006 to 2010, 12.5 million rabies vaccine-baits were distributed by aircraft. Baiting occurred twice per year (spring and autumn), targeting raccoon dogs and red foxes in a 63,000 km2 area of Lithuania. The mandibles of raccoon dogs found dead or killed in the vaccination area were analyzed by fluorescence microscopy for the presence of the TTC. Rabies virus sera neutralizing anti-glycoprotein antibody titres were determined using an indirect ELISA method and seroconversion (> 0.5 EU/ml) rates were estimated. RESULTS: During the study period, 51.5% of raccoon dog mandibles were positive for TTC. 1688 of 3260 tested adults and 69 of 175 tested cubs were TTC positive. Forty-seven percent of raccoon dog serum samples were positive for rabies virus antibodies. 302 of 621 investigated adults and 33 of 95 investigated cubs were seropositive. In the same time 302 of 684 and 43 of 124 tested samples were TTC and ELISA positive in spring; whereas 1455 of 2751 and 292 of 592 tested samples were TTC and ELISA positive in autumn. There was a positive correlation between the number of TTC and antibody positive animals for both adult and cub groups. CONCLUSIONS: ORV was effective in reducing the prevalence of rabies in the raccoon dog population in Lithuania. The prevalence of rabies cases in raccoon dogs in Lithuania decreased from 60.7% in 2006-2007 to 6.5% in 2009-2010.