Rapid detection of porcine sapelovirus by reverse transcription recombinase polymerase amplification assay.

BACKGROUND: Porcine Sapelovirus (PSV) infection has been confirmed in pigs worldwide, mostly asymptomatic, but in some cases, it can lead to significant issues in the gastrointestinal, respiratory, neurological, or reproductive systems. PSV is considered an emerging pathogen of porcine species. Recombinase polymerase amplification (RPA) is a simple and fast isothermal technique that uses three enzymes for amplification without the use of any sophisticated equipment. METHODS AND RESULTS: The reverse transcription recombinase polymerase amplification (RT-RPA) assay was developed and optimized for field based detection of PSV. The assay was developed by targeting 5´UTR region of PSV genome and optimized for reaction time, temperature, primer and MgOAc concentration. The analytical sensitivity and specificity of assay was determined. The assay was evaluated on 85 porcine faecal samples collected from field. In addition to conventional format, this assay was also optimized for visual dye-based detection format and lateral flow strips based detection (in combination with probe). The developed assay works at constant temperature of 35 °C for 20 min with forward primer concentration 20pm, reverse primer concentration 10pm and MgOAc concentration of 14mM. This assay is highly sensitive and detects up to 28 copies of viral nucleic acid both in the conventional as well as in fluorescent dye based detection format. Using the newly developed assay 21 samples out of 85 samples were found positive, showing positivity rate of 24.7%. The positivity rate of RT-RPA assay corroborated with the gold standard RT-PCR test. CONCLUSIONS: This study presented the development of an RT-RPA isothermal assay for rapid and accurate detection of PSV. The assay is highly sensitive, specific, works at a low and constant temperature, does not require any high-end instrument and can be a potential diagnostics tool for pen-side testing of PSV in the field conditions. The newly developed RT-RPA assay could successfully detect PSV circulating in swine population of Haryana, India. This is a first report of this kind from the region.

Identification and assessment of stress and associated stressors among veterinary students in India using a cross-sectional questionnaire survey.

Background: Veterinary education, is a rigorous professional training program, which exposes students to significant academic and non-academic pressures. The identification of stressors and stress levels among veterinary students mighty help the designing and implementation of coping strategies to protect the students' mental health. Methods: A 44-item based cross-sectional questionnaire survey was prepared and disseminated among veterinary students in India to identify the stressors responsible, measure the amount of stress, and relate stress to characteristics like gender, degree year, and family income. A total of n = 611 veterinary students across 14 states including 27 colleges/universities participated in the study. The collected data was evaluated for sampling adequacy, construct validity, and reliability using a set of statistical tests. Results: The analysis revealed high sampling adequacy with a KMO value of 0.957 and a highly significant anti-image correlation (p < 0.001). The principal component analysis generated six factors or subscales which effectively explained 51.98% of the variance in the data, depicting high construct validity. The Cronbach's alpha value of 0.957 revealed high internal consistency for the questionnaire. Analysis revealed more than 94% of pupils under stress, with levels ranging from moderate to severe. Academic-related stressor (95.58%) was the leading cause of overall stress in the present study followed by inter- and intrapersonal and career related-stressors (93.12%) and exams and evaluation-related stressor (90.99%). In comparison to male students, female students reported significantly higher levels of overall stress, academic stress, and intrapersonal and interpersonal stress (p < 0.001) using Chi-square. The students from lower-income families experienced significantly higher overall stress as well as stress due to family responsibilities (p < 0.001). The first-year undergraduate students reported significantly higher (p < 0.001) stress due to family responsibilities-related stressors whereas second-year students due to social activities-related stressors. The hierarchal regression model predicted that gender, family income, academic-related stressors, inter- and intrapersonal and career-related stressors, and social activities-related stressors can be employed to evaluate overall stress among students, as they ensured the maximum variance in the data (p < 0.001). Conclusions: To the best of our knowledge, this is the first Indian study to identify stressors, quantify associated stress and predict major attributes to be targeted in future studies for veterinary students.