Neutralizing antibody response after immunization with a COVID-19 bivalent vaccine: Insights to the future.

The raising of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants led to the use of COVID-19 bivalent vaccines, which include antigens of the wild-type (WT) virus, and of the Omicron strain. In this study, we aimed to evaluate the impact of bivalent vaccination on the neutralizing antibody (NAb) response. We enrolled 93 volunteers who had received three or four doses of monovalent vaccines based on the original virus (n = 61), or a booster shot with the bivalent vaccine (n = 32). Serum samples collected from volunteers were subjected to neutralization assays using the WT SARS-CoV-2, and Omicron subvariants. In addition, immunoinformatics to quantify and localize highly conserved NAb epitopes were performed. As main result, we observed that the neutralization titers of samples from individuals vaccinated with the bivalent vaccine were higher for the original virus, in comparison to their capacity of neutralizing the Omicron variant and its subvariants. NAb that recognize epitopes mostly conserved in the WT SARS-CoV-2 were boosted, while those that recognize epitopes mostly present in the Omicron variant, and subvariants were primed. These results indicate that formulation of future vaccines shall consider to target present viruses, and not viruses that no longer circulate.

Immune Responses to Influenza D Virus in Calves Previously Infected with Bovine Viral Diarrhea Virus 2.

Bovine viral diarrhea virus (BVDV) induces immunosuppression and thymus depletion in calves. This study explores the impact of prior BVDV-2 exposure on the subsequent immune response to influenza D virus (IDV). Twenty 3-week-old calves were divided into four groups. Calves in G1 and G3 were mock-treated on day 0, while calves in G2 and G4 received BVDV. Calves in G1 (mock) and G2 (BVDV) were necropsied on day 13 post-infection. IDV was inoculated on day 21 in G3 calves (mock + IDV) and G4 (BVDV + IDV) and necropsy was conducted on day 42. Pre-exposed BVDV calves exhibited prolonged and increased IDV shedding in nasal secretions. An approximate 50% reduction in the thymus was observed in acutely infected BVDV calves (G2) compared to controls (G1). On day 42, thymus depletion was observed in two calves in G4, while three had normal weight. BVDV-2-exposed calves had impaired CD8 T cell proliferation after IDV recall stimulation, and the α/ß T cell impairment was particularly evident in those with persistent thymic atrophy. Conversely, no difference in antibody levels against IDV was noted. BVDV-induced thymus depletion varied from transient to persistent. Persistent thymus atrophy was correlated with weaker T cell proliferation, suggesting correlation between persistent thymus atrophy and impaired T cell immune response to subsequent infections.

Immunogenicity of an inactivated vaccine for intravaginal application against bovine alphaherpesvirus type 5 (BoHV-5).

The present study was carried out to evaluate the intravaginal vaccine potential against bovine alphaherpesvirus type 5 (BoHV-5). Sixty three cows were divided into seven groups (n: 9) and inoculated intravaginally (VA) or intramuscularly (IM) with inactivated BoHV-5, associated with the recombinant B subunit of the heat-labile enterotoxin of E. coli (rLTB), 2-hydroxyethylcellulose (Drug Delivery System A - DDS-A) or Poloxamer 407 (Drug Delivery System B - DDS-B) as follows: G1 (DDS-A + BoHV-5 + rLTB), G2 (DDS-A + BoHV-5), G3 (DDS-B + BoHV-5 + rLTB), G4 (DDS-B + BoHV-5), G5 (BoHV-5 + rLTB), G6 (Negative control) e G7 (Positive control). The local and systemic humoral responses were measured by indirect ELISA (IgA and IgG) and serum neutralization tests, and the cellular response was measured by a quantitative direct ELISA (IL-2 and IFN-Gamma). The results showed the group inoculated by the IM route, G5, demonstrated the highest levels of IgG in the vaginal mucosa among the experimental groups (p < 0.05). In the groups tested with polymers (G1 and G3) in the vaginal mucosa, even higher levels of IgG were seen in comparison to the positive control (G7; p < 0.01). Higher levels of IgA were also noted in relation to the other groups (p < 0.05) on days 30, 60 and 90 post-inoculations. The groups G1 and G3 also provided higher titers of neutralizing antibodies (Log2) in relation to other treatments (p < 0.01) 90 days after inoculation. In the nasal mucosa, there was an increase in the levels of IgA and IgG with the use of vaccines from groups G1 and G3, in relation to the positive control, G7 (p < 0.05) at 60 and 90 days after the first inoculation. Moreover, neutralizing antibodies titers were detected at 60 and 90 days by serum neutralization. The inclusion of the evaluated polymers resulted in a superior response (p < 0.05) of immunoglobulins and IL-2 and IFN-Gamma in relation to the treatment using only rLTB (G5). This data demonstrates the capabilities of a vaccine with an intravaginal application in cattle to stimulate a local and systemic immune response.

Prolonged Viability of Senecavirus A in Exposed House Flies (Musca domestica).

House flies (Musca domestica) are often present in swine farms worldwide. These flies utilize animal secretions and waste as a food source. House flies may harbor and transport microbes and pathogens acting as mechanical vectors for diseases. Senecavirus A (SVA) infection in pigs occurs via oronasal route, and animals shed high virus titers to the environment. Additionally, SVA possesses increased environmental resistance. Due to these reasons, we investigated the tenacity of SVA in house flies. Five groups of flies, each composed of ten females and ten males, were exposed to SVA, titer of 109.3 tissue culture infectious dose (TCID50/mL). Groups of male and female flies were collected at 0, 6, 12, 24, and 48 h post-exposure. For comparison purposes, groups of flies were exposed to Swinepox virus (SwPV). Infectious SVA was identified in all tested groups. Successful isolation of SVA demonstrated the titers varied between 106.8 and 102.8 TCID50/mL in female groups and varied from 105.85 to 103.8 TCID50/mL in male groups. In contrast, infectious SwPV was only detected in the female group at 6 h. The significant SVA infectious titer for prolonged periods of time, up to 48 h, indicates a potential role of flies in SVA transmission.

Jabuticaba [Plinia peruviana (Poir.) Govaerts]: a Brazilian fruit with a promising application against itraconazole-susceptible and -resistant Sporothrix brasiliensis.

Hydroalcoholic extract from Jabuticaba peels was evaluated for the chemical profile, antioxidant potential, cytotoxicity, and anti-Sporothrix brasiliensis activities against both dimorphic phases. Higher phenolic content (14.91 ± 0.97) compared to flavonoid (2.05 ± 1.00) associated with the ellagic acid (1.93 ± 0.03; LC-MS), and a good scavenging ability against ABST and DPPH radicals was noted. On MDBK cells, elevated cell viability (>90%) was demonstrated between 0.39 and 0.097 mg/ml (MTT assay). Mycelial (CLSI M38-A2) and yeast (CLSI M27-A3) phases of 18 isolates of Sporothrix brasiliensis from cats (n = 8), dogs (n = 8) and humans (n = 2) were used. They were identified itraconazole-susceptible and itraconazole-resistant isolates in both phases, which were all inhibited (MIC of ≤1.56-6.25 mg/ml for both phases) and killed (MFC of ≤1.56-12.5 mg/ml for mycelial; ≤1.56-50 mg/ml for yeast) by Jabuticaba. For the first time, these findings highlighted the potential usefulness of hydroalcoholic extract from Jabuticaba peel as a promising antifungal against sporotrichosis.

Alternatives for obtaining a continuous cell line from Apis mellifera / Alternativas para a obtenção de uma linhagem celular contínua de abelhas Apis mellifera

ABSTRACT: In worldwide there are reports of a significant decrease in colonies of the species Apis mellifera, caused by several factors, including viral infections. In order to study and diagnose illnesses caused by viruses, in vitro cell culture is used as a valuable tool. Yet, there are still no immortalized cell lines of honey bee Apis mellifera. Primary cell cultures are promising for this purpose and can supply the lack of continuous strains, but their establishment is difficult and laborious, which often makes them unfeasible for many research centers. Through the use of cell immortalization techniques, it is possible to develop continuous cell lines and thus benefit, in different ways, research related to different species of bees. The choice of technique is challenging, since in addition to the ability to remain viable for countless passages, cells must keep the genotype and phenotype similar or identical to the original tissue. This review intends to present methodologies that can be used to immortalize Apis mellifera cells, aiming to establish a cell line. The genotypic and phenotypic implications of each technique are evaluated, and the purpose of the cell line to be developed.

RESUMO: Ao redor do mundo há relatos da diminuição significativa de colônias da espécie Apis mellifera, causada por diversos fatores, incluindo infecções virais. Para estudo e diagnóstico de enfermidades causadas por vírus utiliza-se, como uma ferramenta valiosa, o cultivo celular in vitro. Contudo, ainda não existem linhagens celulares imortalizadas de abelhas Apis mellifera. Os cultivos celulares primários são promissores para este fim e podem suprir a falta de linhagens contínuas, porém seu estabelecimento é difícil e laborioso o que, muitas vezes, os torna inviáveis para muitos centros de pesquisa. Através do uso de técnicas de imortalização celular é possível desenvolver linhagens contínuas de células e assim beneficiar, de diversas formas, as pesquisas relacionadas às diferentes espécies de abelhas. A escolha da técnica é desafiadora, visto que, além da capacidade de permanecer viável por inúmeras passagens, as células devem manter o genótipo e fenótipo semelhante ou idêntico ao tecido original. O objetivo deste trabalho é apresentar metodologias que podem ser utilizadas para imortalização de células de Apis mellifera, visando o estabelecimento de uma linhagem celular. São avaliadas as implicações genotípicas e fenotípicas de cada técnica, e a finalidade da linhagem celular a ser desenvolvida.

Isolation of Salmonella Typhimurium, Listeria monocytogenes and coagulase-positive Staphylococcus from salami sold at street fairs in Porto Alegre, Brazil / Isolamento de Salmonella Typhimurium, Listeria monocytogenes e Staphylococcus coagulase positiva de salames comercializados em feiras livres de Porto Alegre, Brasil

Salami is a ready-to-eat (RTE) product frequently purchased at street fairs in Porto Alegre. Salmonella enterica, Listeria monocytogenes, and coagulase-positive Staphylococcus (CPS) are important causes of foodborne disease and can be transmitted through the consumption of RTE foods. The aim of this study was to evaluate the presence of these pathogens in salami sold at street fairs. Ninety salami samples from three commercial brands available at street fairs were analyzed by routine bacteriological methods for Salmonella spp. and Listeria spp., as well as enumeration of CPS. In addition, two samples from each commercial brand were analyzed for water activity (aw). Samples of brand A showed aw values (0.938 and 0.942) above those set by the legislation, while brand B (0.849 and 0.860) and brand C (0.826 and 0.854) were compliant. Microbiological analyses showed that 67.7% were negative to all investigated bacteria. Salmonella Typhimurium was isolated from 4.4% (4/90) of salami samples, all from commercial brand A. ­Listeria monocytogenes was detected in 3.3% (3/90) of samples, from commercial brands B and C. Moreover, 7.7% (7/90) of samples contained CPS populations non-compliant with legislation. Although the great majority of salami sold at street fairs of Porto Alegre was compliant with standards, S. enterica, L. monocytogenes, and CPS ≥ 5 × 103 cfu.g-1 could be found in this RTE product. Therefore, control measures in the processing industry and consumer's education about foodborne illness prevention should be maintained.(AU)

Salame é um alimento pronto para o consumo frequentemente adquirido pela população em feiras livres de Porto Alegre. Salmonella enterica, Listeria monocytogenes e Staphylococcus coagulase positiva são importantes causas de doenças transmitidas por alimentos e podem ser veiculadas por alimentos prontos para o consumo. O objetivo desse estudo foi avaliar a presença desses patógenos em salames vendidos em feiras livres. Noventa amostras de salame pertencentes a três marcas comerciais foram analisados por métodos bacteriológicos de rotina quanto à presença de Salmonella spp. e Listeria spp., bem como enumeração de Staphylococcus coagulase positiva (SCP). Além disso, foi determinada a Atividade de Água (aw) de duas amostras de cada marca comercial. Amostras da marca A apresentaram valores de aw (0,938 e 0,942) acima do permitido na legislação, enquanto as amostras da marca B (0,849 e 0,860) e C (0,826 e 0,854) não violaram esse parâmetro. A análise microbiológica demonstrou que 67,7% das amostras foram negativas para todas as bactérias investigadas. Salmonella Typhimurium foi isolada de 4,4% (4/90) das amostras de salame, todas da marca comercial A. Listeria monocytogenes foi detectada em 3,3% (3/90) das amostras das marcas B e C. Além disso, 7,7% (7/90) das amostras apresentaram SCP acima do número permitido pela legislação. Apesar da grande maioria dos salames comercializados em feiras livres estarem de acordo com a legislação, S. enterica, L. monocytogenes e SCP ≥ 5 × 103 cfu.g-1 podem estar presentes nesse alimento pronto para o consumo. Dessa forma, o controle nas indústrias e a educação dos consumidores sobre a prevenção de doenças transmitidas por alimentos devem ser mantidos.(AU)

Melittin and its potential in the destruction and inhibition of the biofilm formation by Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa isolated from bovine milk.

Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa stand out in veterinary and human medicine for their role in opportunistic infections and their pathogenic mechanisms, including the biofilms formation. It was investigated the antibacterial activity of melittin and antibiofilm of such bacteria. Twelve strains of these microorganisms isolated from bovine milk were used, as well as the strains S. aureus ATCC 12600, E. coli ATCC 8739 and Pseudomonas aeruginosa ATCC 15442. The minimum inhibitory concentrations (MIC) and minimum bactericidal concentration (MBC) were determined by broth microdilution technique. The biofilms were formed in 96-well plates and melittin on these colonies was added at different concentrations and times. Bacteria previously exposed to melittin were evaluated for inhibition of biofilm production. The MIC and MBC were respectively in µg/mL: S. aureus (6-7 and 32-64), E. coli (40-42.5 and 64-128) and P. aeruginosa (65-70 and 64-128). S. aureus biofilms were more sensitive to the action of melittin, since upon exposure to a concentration 10 times lower than the MIC for 4 h, was completely destroyed. In Gram negative bacteria, the pre-formed biofilm was destroyed only when exposed for 4 h under the MIC. With respect to inhibition of biofilm production, S. aureus was the most sensitive again because produced only 37.2% of the biofilm formed by the control (without previous exposure to melittin), when exposed to the MIC, and at a concentration hundred times smaller than MIC, this microorganism produced 75.2% of the biofilm. E. coli was the most resistant bacteria and produced 56.3% of the biofilm, even if previously exposed to melittin MIC. Melittin presents desirable effects in combating microorganisms studied both at your disposal, biofilm destruction and inhibition of the formation, and maybe used in future studies of new strategies to combat infections caused by these pathogens.

Atividade antiviral e virucida de extratos hidroalcoólicos de própolis marrom, verde e de abelhas Jataí (Tetragonisca angustula) frente ao herpersvírus bovino tipo 1 (BoHV-1) e ao vírus da diarreia viral bovina (BVDV) / Antiviral and virucidal activity of hydroalcoholic extracts of propolis brown, green and jataí bees (Tetragonisca angustula) against Bovine Herpesvirus Type-1 (BoHV-1) and Bovine Viral Diarrhea Virus (BVDV)

Dentre as propriedades biológicas da própolis, a atividade antimicrobiana tem merecido destacada atenção. No presente trabalho, descreve-se a ação antiviral e virucida de três extratos hidroalcoólicos de própolis (marrom, verde e de abelhas jataí (Tetragonisca angustula), frente ao Herpesvírus Bovino tipo (BoHV-1) e ao Vírus da Diarreia Viral Bovina (BVDV). Os três extratos hidroalcoólicos foram obtidos de extração etanólica e são oriundos do sul do Brasil. A composição química dos extratos de própolis foi determinada pela cromatografia líquida de alta eficiência acoplada a espectrômetro de massas (UFLC-PDA-ESI-TOF/MS) que identificou e quantificou compostos como: ácido cafeico e ácido p-cumárico, ácido clorogênico, ácido ferúlico, além de flavonoides como a rutina. A toxicidade celular bem como a atividade antiviral dos extratos de própolis em monocamadas de células MDBK (Madin-Darby Bovine Kidney) foi avaliada através de observação microscópica e quantificada pelo teste de MTT (3-(4,5 dimetiltiazol-2yl)-2-5-difenil-2H tetrazolato de bromo). O extrato de própolis de abelhas jataí demonstrou ser menos citotóxico (1,57µg/mL), quando comparado aos extratos verde (0,78µg/mL) e marrom (0,39µg/mL). Quanto a atividade antiviral, a própolis verde demostrou maior eficácia em ambos os tratamentos celulares (pós e pré-exposição) frente ao BoHV-1 em relação aos outros extratos, ou seja, houve maior viabilidade celular quando comparada aos controles de células e vírus. Já a de jataí apresentou atividade frente aos dois vírus (BoHV-1 e BVDV) no método pré-infecção, enquanto a própolis marrom demonstrou ação apenas frente ao BoHV-1 também no método pré-infecção. Para determinação da atividade virucida foram utilizadas diferentes diluições dos vírus, bem como temperaturas e tempos distintos de incubação. A própolis verde a 37°C propiciou a maior redução no título viral (4,33log) em relação a marrom (log = 3,5log) e de jataí (log = 3,24log). No entanto, frente ao BVDV a própolis jataí apresentou os melhores resultados em ambas as temperaturas (22oC e 37oC). Portanto, os extratos avaliados apresentaram atividade antiviral e virucida frente ao BoHV-1 e BVDV, o que os torna alvo para o desenvolvimento de novos biofármacos como alternativa ao uso de antivirais comerciais em Medicina Veterinária.(AU)

Among the biological properties of propolis, the antimicrobial activity has received prominent attention. In this paper, we describe the antiviral and virucidal effect of three hydroalcoholic extracts of propolis (brown, green and jataí bees (Tetragonisca angustula), against bovine herpesvirus type-1 (BoHV-1) and bovine viral diarrhea Virus (BVDV). All hydroalcoholic extracts were obtained from ethanol extraction. The chemical composition of propolis extracts was determined by high-performance liquid chromatography coupled to mass spectrometer (UFLC-PDA-ESI-TOF/MS) to identify and quantify compounds such as caffeic acid and p-coumaric acid, chlorogenic acid, ferulic, and flavonoids such as rutin. Cell toxicity and antiviral activity of propolis extracts in monolayers of MDBK cells (Madin-Darby Bovine Kidney) were assessed by microscopic observation and quantified by the MTT assay (3- (4.5 dimethylthiazol-2yl) -2- 5-diphenyl-2H-tetrazolato bromine). Propolis extract from Jataí bees proved to be less cytotoxic (1.57mg / ml) when compared to green extracts (0.78mg / ml) and brown (0.39mg/mL). Regarding antiviral activity, propolis has shown greater efficacy in both cellular treatments (post and pre-exposure) against BoHV-1 when compared to other extracts, ie, there was increased cell viability compared to cell and virus controls. Extracts from Jataí showed activity against both viruses (BoHV-1 and BVDV) infection in the pre-test, whereas brown propolis demonstrated action only against the BoHV-1 in the pre-infection method. To determine the virucidal activity, it were used different dilutions of virus, as well as different temperatures and incubation times. The green propolis at 37°C led to a greater reduction in viral titer (4.33log) compared to brown (3.5log) and jataí (3.24log). Jataí propolis showed the best results in both temperatures (22oC and 37oC) when tested against BVDV. In summary, the evaluated extracts showed antiviral and virucidal activity against BoHV-1 and BVDV, and may be important targets for the development of new compounds as an alternative to commercial antivirals.(AU)