Dynamics of Humoral Immunity to Myxoma and Rabbit Hemorrhagic Disease Viruses in Wild European Rabbits Assessed by Longitudinal Semiquantitative Serology.

Myxoma virus (MYXV) and rabbit hemorrhagic disease virus (RHDV) are important drivers of the population decline of the European rabbit, an endangered keystone species. Both viruses elicit strong immune responses, but the long-term dynamics of humoral immunity are imperfectly known. This study aimed to assess the determinants of the long-term dynamics of antibodies to each virus based on a longitudinal capture-mark-recapture of wild European rabbits and semiquantitative serological data of MYXV and RHDV GI.2-specific IgG. The study included 611 indirect enzyme-linked immunosorbent assay (iELISA) normalized absorbance ratios for each MYXV and RHDV GI.2 from 505 rabbits from 2018 to 2022. Normalized absorbance ratios were analyzed using log-linear mixed models, showing a significant positive relationship with the time since the first capture of individual rabbits, with monthly increases of 4.1% for antibodies against MYXV and 2.0% against RHDV GI.2. Individual serological histories showed fluctuations over time, suggesting that reinfections boosted the immune response and likely resulted in lifelong immunity. Normalized absorbance ratios significantly increased with the seroprevalence in the population, probably because of recent outbreaks, and with body weight, highlighting the role of MYXV and RHDV GI.2 in determining survival to adulthood. Juvenile rabbits seropositive for both viruses were found, and the dynamics of RHDV GI.2 normalized absorbance ratios suggest the presence of maternal immunity up to 2 months of age. Semiquantitative longitudinal serological data provide epidemiological information, otherwise lost when considering only qualitative data, and support a lifelong acquired humoral immunity to RHDV GI.2 and MYXV upon natural infection. IMPORTANCE This study addresses the long-term dynamics of humoral immunity to two major viral pathogens of the European rabbit, an endangered keystone species of major ecological relevance. Such studies are particularly challenging in free-ranging species, and a combination of longitudinal capture-mark-recapture and semiquantitative serology was used to address this question. Over 600 normalized absorbance ratios of iELISA, obtained from 505 individual rabbits in 7 populations over 5 years, were analyzed using linear mixed models. The results support a lifelong acquired humoral immunity to myxoma virus and rabbit hemorrhagic disease virus upon natural infection and suggest the presence of maternal immunity to the latter in wild juvenile rabbits. These results contribute to understanding the epidemiology of two viral diseases threatening this keystone species and assist in developing conservation programs.

Multi-event capture-recapture models estimate the diagnostic performance of serological tests for myxoma and rabbit haemorrhagic disease viruses in the absence of reference samples.

Estimation of the diagnostic performance of serological tests often relies on another test assumed as a reference or on samples of known infection status, yet both are seldom available for emerging pathogens in wildlife. Longitudinal disease serological data can be analysed through multi-event capture-mark-recapture (MECMR) models accounting for the uncertainty in state assignment, allowing us to estimate epidemiological parameters such as incidence and mortality. We hypothesized that by estimating the uncertainty in state assignment, MECMR models estimate the diagnostic performance of serological tests for rabbit haemorrhagic disease virus (RHDV) and myxoma virus (MYXV). We evaluated this hypothesis on longitudinal serological data of three tests of RHDV and one test of MYXV in two populations of the European rabbit (Oryctolagus cuniculus algirus). First, we selected the optimal cut-off threshold for each test using finite mixture models, a reference method not relying on reference tests or samples. Second, we used MECMR models to compare the diagnostic sensitivity (Se) and specificity (Sp) of the three tests for RHDV. Third, we compared the estimates of diagnostic performance by MECMR and finite mixture models across a range of cut-off values. The MECMR models showed that the RHDV test employing GI.2 antigens (Se: 100%) outperformed two tests employing GI.1 antigens (Se: 21.7% ± 8.6% and 8.7% ± 5.9%). At their selected cut-offs (2.0 for RHDV GI.2 and 2.4 for MYXV), the estimates of Se and Sp were concordant between the MECMR and finite mixture models. Over the duration of the study (May 2018 to September 2020), the monthly survival of European rabbits seropositive for MYXV was significantly higher than that of seronegative rabbits (82.7% ± 4.9% versus 61.5% ± 12.7%) at the non-fenced site. We conclude that MECMR models can reliably estimate the diagnostic performance of serological tests for RHDV and MYXV in European rabbits. This conclusion could extend to other diagnostic tests and host-pathogen systems. Longitudinal disease surveillance data analysed through MECMR models allow the validation of diagnostic tests for emerging pathogens in novel host species while simultaneously estimating epidemiological parameters.

Facilitadores para a prática de atividade física no lazer em adolescentes / Facilitators for physical activity at leisure in adolescents

O objetivo do estudo foi identificar os facilitadores para a prática de atividade física no lazer em adolescentes do ensino médio de São José dos Pinhais (PR). Foi realizada a técnica de grupos focais, da pesquisa qualitativa, seguindo um roteiro semiestruturado, com adolescentes de 14 a 17 anos de idade (50% do sexo feminino, média de idade de 15,5 ±1,5) de escolas públicas e privadas. Ao total foram realizados 12 grupos focais, sendo metade para cada sexo e tendo adolescentes ativos e inativos fisicamente. Para a análise dos dados foi utilizado a análise de conteúdo e a estatística descritiva para contabilizar os relatos. Os resultados foram analisados separadamente por tipo de escola e sexo. Os facilitadores mais relatados foram: apoio social dos amigos (12,2%), apoio social dos pais (11,7%), ter habilidades (11,1%), educação física trabalhar as habilidades motoras e físicas (10,3%) e praticar atividade física no contraturno das aulas (7,6%). Para maximizar os níveis de atividade física no lazer em adolescentes, deve-se levar em consideração os facilitadores encontrados nesse estudo, que dizem respeito ao apoio social e a prática efetivamente, seja ela para o aprendizado de habilidades motoras e físicas, ou para o aprimoramento, tanto nas aulas de educação física escolar quanto nas atividades físicas no contraturno.

The objective of the study was to identify the facilitators for the practice of leisure physical activity in high school adolescents from São José dos Pinhais (PR). A focus group technique was used for qualitative research, followed by a semi-structured script, with adolescents between 14 and 17 years of age (50% females, mean age 15.5 ± 1.5) from public and private schools. Twelve focus groups were performed, half for each sex and adolescents with different levels of physical activity. For the analysis of the data, we used the content analysis and the descriptive statistics to account for the reports. The results were stratified by type of school, gender and general, with the most reported being: social support from friends (12.2%), parental social support (11.7%), having skills (11.1%), education physical and motor skills (10.3%) and practice physical activity in the back of class (7.6%). In order to maximize physical activity levels in adolescent leisure, the facilitators found in this study, which concern social support and practice, should be taken into account. Whether it is for the learning of physical and motor skills or for improvement, both in physical education classes and in physical activities in the back of class.

Flutriafol and pyraclostrobin residues in Brazilian green coffees.

The aim of this work was to monitor flutriafol and pyraclostrobin residues in Brazilian green coffees. More than 10,000 samples were analyzed. The pesticides were extracted using the QuEChERS method and analyzed by LC-MS/MS. The validated method is fast, with 5 min runs, and efficient, as precision and accuracy showed RSD no greater than 5% and recoveries within the 88-119% range. LOQ for flutriafol and pyraclostrobin were 0.005 mg/kg. The results of the analyzed samples showed that the percentage of nonconformities regarding flutriafol increased throughout the years, with over 1200 samples (11.8%). On the other hand, just 15 samples (0.15%) presented residues above 10 µg/kg for pyraclostrobin. Considering that flutriafol is a toxic and carcinogenic pesticide, as well as the increase in the number of irregularities throughout the years, it becomes important to implement public actions to assure consumer safety.

Evaluation of storage and evaporation in the removal efficiency of D-norgestrel and progesterone in human urine.

Pharmaceuticals are emerging contaminants and it must be noted that approximately 70 % of them are excreted via urine. Therefore, urine usage implies the risk of transfer of pharmaceutical residues to agricultural fields and environment contamination. Thus, this study aimed on the development and validation of a LC-MS/MS method for D-norgestrel (D-NOR) and progesterone (PRO) determination in human urine, as well as the evaluation of the removal efficiency of two methods (storage and evaporation), and the effects of acidification with sulfuric acid. The storage process was evaluated for 6 weeks, while the evaporation was assessed at three different temperatures (50, 75, and 100 °C). All experiments were done with normal urine (pH = 6.0) and acidified urine (pH = 2.0, with sulfuric acid). The results of validation showed good method efficiency. In the second week of storage, higher hormone degradation was observed. In the evaporation method, both D-NOR and PRO were almost completely degraded when the volume was reduced to the lowermost level. Results also indicate that acidification did not affect degradation. Overall, the results showed that combination of two methods can be employed for more efficient hormone removal in urine.

In vivo hypotensive effect and in vitro inhibitory activity of some Cyperaceae species

In 1820, French naturalist August Saint Hillaire, during a visit in Espírito Santo (ES), a state in southeastern Brazil, reported a popular use of Cyperaceae species as antidote to snake bites. The plant may even have a hypotensive effect, though it was never properly researched. The in vitro inhibitory of the angiotensin converting enzyme (ACE) activity of eigth ethanolic extracts of Cyperaceae was evaluated by colorimetric assay. Total phenolic and flavonoids were determined using colorimetric assay. The hypotensive effect of the active specie (Rhychonospora exaltata, ERE) and the in vivo ACE assay was measured in vivo using male Wistar Kyoto (ERE, 0.01-100mg/kg), with acetylcholine (ACh) as positive control (5 µg/kg, i.v.). The evaluation of ACE in vivo inhibitory effect was performed comparing the mean arterial pressure before and after ERE (10 mg/kg) in animals which received injection of angiotensin I (ANG I; 0,03, 03 and 300 µg/kg, i.v.). Captopril (30 mg/kg) was used as positive control. Bulbostylis capillaris (86.89 ± 15.20%) and ERE (74.89 ± 11.95%, ERE) were considered active in the in vitro ACE inhibition assay, at 100 µg/mL concentration. ACh lead to a hypotensive effect before and after ERE's curve (-40±5% and -41±3%). ERE showed a dose-dependent hypotensive effect and a in vivo ACE inhibitory effect. Cyperaceae species showed an inhibitory activity of ACE, in vitro, as well as high content of total phenolic and flavonoids. ERE exhibited an inhibitory effect on both in vitro and in vivo ACE. The selection of species used in popular medicine as antidotes, along with the in vitro assay of ACE inhibition, might be a biomonitoring method for the screening of new medicinal plants with hypotensive properties.

Em 1820, o naturalista francês August Saint Hillaire, durante uma visita ao Espírito Santo (ES), Estado do sudeste do Brasil, relatou o uso popular de espécies de Cyperaceae como antídoto para picadas de cobra. As espécies podem possuir efeito hipotensor, embora nunca tenham sido devidamente pesquisadas. A inibição in vitro da atividade da enzima conversora da angiotensina (ECA) de oito extratos etanólicos de Cyperaceae foi avaliada por ensaio colorimétrico. Totais de fenólicos e flavonóides foram determinados utilizando ensaio colorimétrico. O efeito hipotensor da espécie ativa (Rhychonospora exaltata, ERE) no ensaio de ECA in vitro foi avaliada in vivo utilizando-se machos Wistar Kyoto (ERE, 0.01-100 mg/kg), com a acetilcolina (ACh), como controle positivo (5 µg/kg, iv). A avaliação do efeito inibidor da ECA in vivo foi realizado comparando-se a pressão arterial média, antes e após ERE (10 mg/kg) nos animais que receberam injeção de angiotensina I (Ang I, 0,03, 03 e 300 µg/kg, iv). Captopril (30 mg/kg) foi utilizado como controle positivo. Bulbostylis capillaris (86,89±15,20%) e ERE (74,89±11,95%, ERE) foram considerados ativas no ensaio de inibição da ECA in vitro, na concentração de 100 µg/mL. A ACh gerou um efeito hipotensor, antes e depois da curva de ERE (-40 ± 5% -41 ± 3%). ERE apresentou efeito hipotensivo dependente da dose e um efeito inibidor da ECA in vivo. As espécies de Cyperaceae mostraram uma atividade inibidora de ACE in vitro, bem como elevado conteúdo de substâncias fenólicas e flavonóides. ERE exibiu um efeito inibidor da ECA tanto in vitro como in vivo. A seleção das espécies utilizadas na medicina popular como antiofídicos, juntamente com o ensaio in vitro de inibição da ECA, pode ser um método de biomonitoramento para a seleção de novas plantas medicinais com propriedades hipotensores.