Simultaneous Detection of Antigen and Antibodies of African Swine Fever in a Novel Combo Lateral Flow Assay.

African swine fever (ASF) is a contagious disease of wild boar and domestic pigs notifiable to the World Organisation for Animal Health due to its high socio-economic impact. ASF is caused by the complex ASF virus (ASFV), and it can present different clinical manifestations that can be confused with other diseases; for this reason, laboratory testing is necessary for the proper diagnosis of clinically suspected animals. Despite the efforts put into it over decades, no treatment or safe vaccine is globally available, and disease control is based on early diagnosis and the implementation of strict biosecurity measures. In this context, rapid tests have the potential to accelerate and facilitate the identification of infected animals by giving fast on-site results. In this work, we improved the available point-of-care assays for the diagnosis of the disease by the development of a more specific antigen test and a more sensitive antibody test. This antibody detection test allowed for the earlier detection of infected animals than two commercial indirect ELISAs (statistically significant). Moreover, we developed a combined dual rapid test, unifying, in the same cassette, an antigen detection strip and an antibody detection strip. In this study, we confirmed that this combo approach is a useful tool for implementing rapid tests in the field since it increases the percentage of positive samples detected, even when PCR turns negative, while maintaining a good specificity.

Evaluation of SARS-CoV-2 passive surveillance in Lithuanian mink farms, 2020-2021.

The newly emerged SARS-CoV-2, causing COVID-19 in humans, is also infecting American mink (Neovison vison), used in fur production. Since 2020, passive surveillance of SARS-CoV-2 in mink farms was implemented in Lithuania. Here, we describe data from a survey of all 57 active Lithuanian mink farms carried out during November-December 2021 to complement passive surveillance in the country. In all 57 mink farms, nasopharyngeal swab samples were collected from dead or live mink and tested by real-time RT-PCR. Dead mink samples were tested in pools of 5, while live mink samples were tested individually. In 19 mink farms, blood serum was collected and tested for antibodies to determine previous exposure to the virus. Environmental samples from 55 farms were also collected and tested in pooled samples by real-time RT-PCR. The present survey has detected 22.81% viral RNA-positive mink farms and a high number of mink farms that were exposed (84.21, 95% CI 67.81-100%) to the virus. The increasing exposure of mink farms to the virus due to growing human COVID-19 cases and limitations of passive surveillance could explain the observed epidemiological situation of SARS-CoV-2 in Lithuanian mink farms, compared to the few positive farms previously detected by passive surveillance. The unexpected widespread exposure of mink farms to SARS-CoV-2 suggests that passive surveillance is ineffective for early detection of SARS-CoV-2 in mink. Further studies are needed to reveal the present status in previously infected mink farms.

Do Seropositive Wild Boars Pose a Risk for the Spread of African Swine Fever? Analysis of Field Data from Latvia and Lithuania.

In 2020, ASF occurred in wild boars throughout Latvia and Lithuania, and more than 21,500 animals were hunted and tested for the presence of the virus genome and antibodies in the framework of routine disease surveillance. The aim of our study was to re-examine hunted wild boars that tested positive for the antibodies and negative for the virus genome in the blood (n = 244) and to see if the virus genome can still be found in the bone marrow, as an indicator of virus persistence in the animal. Via this approach, we intended to answer the question of whether seropositive animals play a role in the spread of the disease. In total, 2 seropositive animals out of 244 were found to be positive for the ASF virus genome in the bone marrow. The results indicate that seropositive animals, which theoretically could also be virus shedders, can hardly be found in the field and thus do not play an epidemiological role regarding virus perpetuation, at least not in the wild boar populations we studied.

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 African Swine Fever Epidemic in Wild Boar (Sus scrofa) in Lithuania (2014-2018).

In January 2014 the first case of African swine fever (ASF) in wild boar of the Baltic States was reported from Lithuania. It has been the first occurrence of the disease in Eastern EU member states. Since then, the disease spread further affecting not only the Baltic States and Poland but also south-eastern Europe, the Czech Republic and Belgium. The spreading pattern of ASF with its long-distance spread of several hundreds of kilometers on the one hand and the endemic situation in wild boar on the other is far from being understood. By analyzing data of ASF cases in wild boar along with implemented control measures in Lithuania from 2014-2018 this study aims to contribute to a better understanding of the disease. In brief, despite huge efforts to eradicate ASF, the disease is now endemic in the Lithuanian wild boar population. About 86% of Lithuanian's territory is affected and over 3225 ASF cases in wild boar have been notified since 2014. The ASF epidemic led to a considerable decline in wild boar hunting bags. Intensified hunting might have reduced the wild boar population but this effect cannot be differentiated from the population decline caused by the disease itself. However, for ASF detection sampling of wild boar found dead supported by financial incentives turned out to be one of the most effective tools.

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.