African Swine Fever Virus (ASFV) is a high-consequence transboundary animal pathogen that often causes hemorrhagic disease in swine with a case fatality rate close to 100%. Lack of treatment or vaccine for the disease makes it imperative that safe and efficacious vaccines are developed to safeguard the swine industry. In this study, we evaluated the immunogenicity of seven adenovirus-vectored novel ASFV antigens, namely A151R, B119L, B602L, EP402RΔPRR, B438L, K205R and A104R. Immunization of commercial swine with a cocktail of the recombinant adenoviruses formulated in adjuvant primed strong ASFV antigen-specific IgG responses that underwent rapid recall upon boost. Notably, most vaccinees mounted robust IgG responses against all the antigens in the cocktail. Most importantly and relevant to vaccine development, the induced antibodies recognized viral proteins from Georgia 2007/1 ASFV-infected cells by IFA and by western blot analysis. The recombinant adenovirus cocktail also induced ASFV-specific IFN-γ-secreting cells that were recalled upon boosting. Evaluation of local and systemic effects of the recombinant adenovirus cocktail post-priming and post-boosting in the immunized animals showed that the immunogen was well tolerated and no serious negative effects were observed. Taken together, these outcomes showed that the adenovirus-vectored novel ASFV antigen cocktail was capable of safely inducing strong antibody and IFN-γ+ cell responses in commercial swine. The data will be used for selection of antigens for inclusion in a multi-antigen prototype vaccine to be evaluated for protective efficacy.

(1) Background: Hepatitis E virus (HEV) is a causative agent of acute viral hepatitis, predominantly transmitted by the fecal–oral route. In developed countries, HEV is considered to be an emerging pathogen since the number of autochthonous cases is rising. Hepatitis E is a viral disease with a proven zoonotic potential for some of its genotypes. The main viral reservoirs are domestic pigs and wild boar. Consumption of undercooked meat, as well as occupational exposure, are key factors for the spread of HEV. In order to evaluate the risks of future viral evolution, a detailed examination of the ecology and distribution of the virus is needed. The aim of the present study is to investigate the prevalence of anti-HEV IgG Ab in domestic pigs and wild boar in Bulgaria; (2) Methods: In this study, during the period of three years between 2017 and 2019, 433 serum samples from 19 different pig farms and 1 slaughterhouse were collected and analyzed. In addition, 32 samples from wild boar were also collected and analyzed during the 2018–2019 hunting season. All samples were analyzed by commercial indirect ELISA; (3) Results: Overall, HEV seroprevalence was 60% (95% CI 42.7–77.1) in domestic pigs and 12.5% (4/32) in wild boar. The observed seroprevalence of the slaughter-aged pigs was 73.65% (95% Cl 58.7–87.3). Prevalence in domestic pigs was significantly higher in the samples collected during 2019 (98% (95% Cl 96.1–99.9)) compared to those collected during 2017 (45.33% (95% CI 2.7–87.3)) and 2018 (38.46% (95% CI 29.1–49.7.); (4) Conclusions: Our findings suggest that domesticated pigs and wild boar might be the reason for the increased HEV transmission across Bulgaria. The genotypic characterization of HEV found in pigs, wild boar and humans will give a more accurate view of the zoonotic transmission of this virus.

BackgroundHepatitis E virus (HEV) causes epidemics in developing countries and is primarily transmitted through the fecal-oral route. There have been recent reports on the zoonotic spread of the virus, and several animal species, primarily pigs, have been recognized as reservoirs of HEV. Because of its possible spread, there is an urgent need of a method for the cost-effective production of HEV proteins that can be used as diagnostic antigens for the serological detection of anti-HEV antibodies.MethodsThe HEV open reading frame (ORF)2 protein was purified from plant tissue by using immobilized metal-anion chromatography (IMAC). The recombinant protein was used to develop an in-house ELISA for testing anti-HEV antibodies in both human and swine sera. Thirty-six serum samples collected from patients with serologically proven HEV infection with commercial kits were tested for anti-HEV IgG antibodies by using the plant-expressed protein. Forty-five serum samples collected from apparently healthy pigs in Bulgarian farms were also tested.ResultsWe confirmed the transient expression and purification of a truncated version of the HEV genotype 3 capsid protein in Nicotiana benthamiana and its usefulness as a diagnostic antigen. ELISA showed the presence of anti-HEV IgG antibodies in 29 of the 36 human samples. The in-house ELISA showed anti-HEV IgG antibodies in 34 of the 45 pigs.ConclusionsWe describe a method for the production of HEV ORF2 protein in N. benthamiana and the usefulness of this protein for the serological detection of anti-HEV antibodies in both humans and swine.

Hepatitis E is an emerging global disease, mainly transmitted via the fecal–oral route in developing countries, and in a zoonotic manner in the developed world. Pigs and wild boar constitute the primary Hepatitis E virus (HEV) zoonotic reservoir. Consumption of undercooked animal meat or direct contact with infected animals is the most common source of HEV infection in European countries. The purpose of this study is to develop an enzyme immunoassay (EIA) for the detection of anti-hepatitis E virus IgG in pig serum, using plant-produced recombinant HEV-3 ORF2 as an antigenic coating protein, and also to evaluate the sensitivity and specificity of this assay. A recombinant HEV-3 ORF2 110-610_6his capsid protein, transiently expressed by pEff vector in Nicotiana benthamiana plants was used to develop an in-house HEV EIA. The plant-derived HEV-3 ORF2 110-610_6his protein proved to be antigenically similar to the HEV ORF2 capsid protein and it can self-assemble into heterogeneous particulate structures. The optimal conditions for the in-house EIA (iEIA) were determined as follows: HEV-3 ORF2 110-610_6his antigen concentration (4 µg/mL), serum dilution (1:50), 3% BSA as a blocking agent, and secondary antibody dilution (1:20 000). The iEIA developed for this study showed a sensitivity of 97.1% (95% Cl: 89.9–99.65) and a specificity of 98.6% (95% Cl: 92.5–99.96) with a Youden index of 0.9571. A comparison between our iEIA and a commercial assay (PrioCHECK™ Porcine HEV Ab ELISA Kit, ThermoFisher Scientific, MA, USA) showed 97.8% agreement with a kappa index of 0.9399. The plant-based HEV-3 ORF2 iEIA assay was able to detect anti-HEV IgG in pig serum with a very good agreement compared to the commercially available kit.

Honey bees (Apis mellifera L.) are crucial pollinators for many crops and natural ecosystems. However, honey bee colonies have been experiencing heavy overwinter mortality in almost all parts of the world. In the present study we have investigatеd, for the first time, the effects from the application of the herbal supplements NOZEMAT HERB® (NH) and NOZEMAT HERB PLUS® (NHP) on overwintering honey bee colony survival and on total protein and lysozyme content. To achieve this, in early autumn 2019, 45 colonies were selected and treated with these herbal supplements. The total protein and lysozyme content were evaluated after administration of NH and NHP twice the following year (June and September 2020). The obtained results have shown that both supplements have a positive effect on overwintering colony survival. Considerable enhancement in longevity of “winter bees” has been observed after the application of NHP, possibly due to the increased functionality of the immune system and antioxidant detoxification capacity. Although the mechanisms of action of NH and NHP are yet to be completely elucidated, our results suggest a new holistic approach on overwintering honey bee colony survival and welfare.

The aim of this work was to investigate the effect of Ochratoxin A (OTA) and Silymarin on serum lysozyme concentrations, complement and betalysin activity in broiler chickens. In this experiment 144 one-day-old Ross 308 male broiler chicks were used. All chicks were divided in four groups of 36 birds each: Group 1: Basal diet (BD) with no supplementation of Ochratoxin A (OTA) and Silymarin; Group 2: BD with 1.0% Silymarin; Group 3: BD with 3.0 mg/kg OTA; Group 4: BD with 3.0 mg/kg OTA plus 1.0% Silymarin. It was found that lysozyme concentration in the 2nd group there is a significant difference to groups 3 and 4. The immunosuppressive effect of Ochratoxin A is underlined but no protective effect of Silymarin in the group 4 was found. The alternative pathway of complement activation (APCA) is affected in the group 4. Betalysine there is a significant decreasing in the group 3 but slightly increasing of Betalysine in 4th group. Based on these results it can be concluded that OTA there is an immunosuppressive effect on the studied traits and there is a positive effect of Silymarin only on serum betalysine.

Solidified floating organic drop microextraction (SFODME) in combination with gas chromatography with an electron capture detector was used for separation/enrichment and determination of 2,5-dichloronitrobenzene, 2,3-dichloro-nitrobenzene, and 2,6-dichloro-4-nitroaniline in water samples. The main parameters affecting the performance of SFOME, such as solvent volume, extraction time, stirring rate, extraction temperature, sample volume, nature of the solvent and ionic strength were optimized. Under the optimum experimental conditions, good relative standard deviations of 7.5%, 6.9%, and 9.3% for 7 extractions and determination of 50 ng L −1 of 2,5-dichloro-nitrobenzene, 2,3-dichloro-nitrobenzene and 2,6-dichloro-4-nitroaniline were obtained, respectively. Enrichment factors of 1390, 1398, and 1362 and detection limits of 1.4, 1.2, and 10 ng L −1 for 2,5-dichloro-nitrobenzene, 2,3-dichloro-nitrobenzene, and 2,6-dichloro-4-nitroaniline were found, respectively.

SummaryThe experiments for evaluation of the effect of Immunobeta were performed with turkeys and organically reared layer hens. Turkeys received Immunobeta at a dose of 4 g/kg feed during the beginning of lay (cold period), thermoneutral period and hot summer period. In hens, two dietary levels of Immunobeta were tested: 2 g/kg and 4 g/kg. Blood serum and egg lysozyme levels, alternative pathway of complement activation (APCA) and beta-lysins concentrations were determined. It was found out that Immunobeta had a beneficial effect on serum lysozyme, APCA activity and beta-lysins in turkeys and hens. The lysozyme concentration in egg white was statistically significantly higher in eggs produced by birds treated with both doses of the immunomodulator.