Retrospective analysis of sero-prevalence and bait uptake estimations in foxes after oral rabies vaccination programmes at European level: Lessons learned and paths forward.

Rabies caused by the Classical Rabies Virus (Lyssavirus rabies abbreviated RABV) in the European Union has been close to elimination mainly thanks to Oral Rabies Vaccination (ORV) campaigns targeting wildlife (primarily red foxes). ORV programmes co-financed by the European Commission include a monitoring-component to assess the effectiveness of the ORV campaigns at national level. This assessment is performed by a random collection of red foxes in the vaccinated areas with control of antibodies presence by serological analysis and control of bait uptake by detection of biomarkers (tetracycline incorporated into the baits) in the bones and teeth. ORV programmes aim to a vaccine coverage high enough to immunize (ideally) 70 % of the reservoir population to control the spread of the disease. European Union (EU) programmes that led to almost elimination of rabies on the territory have been traditionally found to have a bait uptake average of 70 % (EU countries; 2010-2020 period) while the seroconversion data showed an average level of 40 % (EU countries; 2010-2020 period). To better understand variations of these indicators, a study was been set up to evaluate the impact of several variables (linked to the vaccination programme itself and linked to environmental conditions) on the bait uptake and the seroconversion rate. Thus, pooling data from several countries provides more powerful statistics and the highest probability of detecting trends. Results of this study advocate the use of a single serological test across the EU since data variation due to the type of test used was higher than variations due to field factors, making the interpretation of monitoring results at EU level challenging. In addition, the results indicates a negative correlation between bait uptake and maximum temperatures reached during ORV campaigns questioning the potential impact of climatic change and associated increase of temperatures on the ORV programmes efficiency. Several hypotheses requesting additional investigation are drawn and discussed in this paper.

Molecular diversity of Paenibacillus larvae strains isolated from Lithuanian apiaries.

Paenibacillus larvae bacterium is known to be the causative agent of American foulbrood (AFB), a widespread, highly contagious and fatal disease in honey bees (Apis mellifera). There are four genotypes of Paenibacillus larvae that are named after their enterobacterial repetitive consensus (ERIC), and a fifth ERIC genotype has recently been found. In this study, a total of 108 independent P. larvae isolates from different geographical regions in Lithuania collected between 2011 and 2021 were investigated by molecular methods. The aims of this study were to detect which enterobacterial repetitive intergenic consensus (ERIC) genotype is the most common in Lithuania apiaries, identify and differentiate subtypes of the defined genotype by using multiple-locus variable number of tandem-repeat analysis (MLVA), and review how bacterial molecular diversity has changed over time in different parts of Lithuania. The obtained molecular analysis results showed that 100% of P. larvae bacterial isolates from Lithuania belong to the ERIC I genotype and can be differentiated to nine different subtypes by using the MLVA and capillary electrophoresis methods.

Zearalenone and Its Metabolites in Blood Serum, Urine, and Milk of Dairy Cows.

After oral contamination, zearalenone (ZEN) is rapidly absorbed in organisms and can be detected in biological fluids. In this study, we investigated the metabolites of ZEN in the biological fluids of cows (blood, urine, milk). The study was divided into three stages: preparation (the first stage), investigation (the second stage), and final stage (the third stage). Samples of biological fluids were taken 7, 21, and 35 days after the beginning of the study. At the first stage and at the second stage, the cows were fed a total mixed ration (TMR) with naturally contaminated mycotoxin-zearalenone (500 ± 75 µg/kg). In the third stage, the cows were fed a TMR without mycotoxins. This study established that at the second stage, the alpha-zearalenol concentrations in the serum increased by 92% and the beta-zearalanol in the urine decreased by 48% compared to the first stage (p < 0.05). The beta-zearalenol and zearalanone concentrations in the urine were higher compared to that of the alpha-zearalenol. The zearalenone concentration in the milk at the second stage was 35% higher than at the first stage (p < 0.05). A significant negative correlation (r = −0.540) was determined between the beta-zearalenol and beta-zearalanol concentrations in the urine and the positive significant correlation (r = 0.826) between the beta-zearalanol and alpha-zearalenol concentrations in the serum (p < 0.05). During the study, it was determined that feeding cows for two weeks with a TMR without mycotoxins can reduce concentrations of alpha-zearalanol, beta-zearalenol, and beta-zearalanol in the biological fluids and can reduce the concentrations of ZEN in the milk, but does not reduce the concentration of zearalanone.

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.

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.

Rabies in the Baltic States: Decoding a Process of Control and Elimination.

Rabies is a fatal zoonosis that still causes nearly 70, 000 human deaths every year. In Europe, the oral rabies vaccination (ORV) of red foxes (Vulpes vulpes) was developed in the late 1970s and has demonstrated its effectiveness in the eradication of the disease in Western and some Central European countries. Following the accession of the three Baltic countries--Estonia, Latvia and Lithuania--to the European Union in 2004, subsequent financial support has allowed the implementation of regular ORV campaigns since 2005-2006. This paper reviews ten years of surveillance efforts and ORV campaigns in these countries resulting in the near eradication of the disease. The various factors that may have influenced the results of vaccination monitoring were assessed using generalized linear models (GLMs) on bait uptake and on herd immunity. As shown in previous studies, juveniles had lower bait uptake level than adults. For the first time, raccoon dogs (Nyctereutes procyonoides) were shown to have significantly lower bait uptake proportion compared with red foxes. This result suggests potentially altered ORV effectiveness in this invasive species compared to the red foxes. An extensive phylogenetic analysis demonstrated that the North-East European (NEE) rabies phylogroup is endemic in all three Baltic countries. Although successive oral vaccination campaigns have substantially reduced the number of detected rabies cases, sporadic detection of the C lineage (European part of Russian phylogroup) underlines the risk of reintroduction via westward spread from bordering countries. Vaccine induced cases were also reported for the first time in non-target species (Martes martes and Meles meles).

In-Depth Characterization of Live Vaccines Used in Europe for Oral Rabies Vaccination of Wildlife.

Although rabies incidence has fallen sharply over the past decades in Europe, the disease is still present in Eastern Europe. Oral rabies immunization of wild animal rabies has been shown to be the most effective method for the control and elimination of rabies. All rabies vaccines used in Europe are modified live virus vaccines based on the Street Alabama Dufferin (SAD) strain isolated from a naturally-infected dog in 1935. Because of the potential safety risk of a live virus which could revert to virulence, the genetic composition of three commercial attenuated live rabies vaccines was investigated in two independent laboratories using next genome sequencing. This study is the first one reporting on the diversity of variants in oral rabies vaccines as well as the presence of a mix of at least two different variants in all tested batches. The results demonstrate the need for vaccine producers to use new robust methodologies in the context of their routine vaccine quality controls prior to market release.