RESUMO
Acute hepatopancreatic necrosis disease (AHPND) is an emerging shrimp (Penaeus monodon) disease caused by Vibrio parahaemolyticus (VP) since 2013 in Bangladesh. The aim of this work was to evaluate a PCR and RT-PCR techniques as rapid methods for detecting V. parahaemolyticus AHPND-positive P. monodon using genetic markers. Healthy and diseased shrimp (P. monodon) samples were collected from three monitoring stations. The samples were enriched in TCBS plates and DNA extraction from the cultured bacteria. DNA quantifications, PCR amplification, RT-PCR, and gene sequencing were done for the detection of V. parahaemolyticus AHPND-positive P. monodon. The sequence of PCR amplicons showed 100% identity and significant alignment with V. parahaemolyticus. The primers used provided high specificity for V. parahaemolyticus in PCR detection compared with another Vibrio species. In the PCR, amplification resulted positive amplicons, whereas, non-AHPND isolates showed no amplicons. Neighbor-joining methods indicated that all genes evolved from a common ancestor and clades have different traits with very low genetic distance and low variability. The pairwise alignment scores of atpA, tox, blaCARB, 16S rRNA, and pirA genes were 100.0, 98.90, 98.89, 95.53, and 41.42, respectively. The RT-qPCR exposed variable expression levels for all genes in the AHPND-positive strain. Homology analysis and distance matrix exhibited all genes to have the lowest similarity and most divergence, offering the highest specificity. In this study, the expression and variability of target genes confirmed the presence of V. parahaemolyticus in all sampling sites. The results suggested that PCR amplification, RT-qPCR, and gene sequencing can be used for the rapid detection of V. parahaemolyticus in AHPND-positive P. monodon that may lead to subsequent prevention and treatment research in the future for managing this disease.
RESUMO
BACKGROUND: Using formative research to guide the planning and implementation of home fortification programs is critical if they are to achieve the desired level of acceptance and coverage by the target beneficiaries. OBJECTIVE: To explore contextual factors that could influence acceptance, delivery, and use of micronutrient powders (MNP) in Aileu District, Timor-Leste. METHODS: Two focus group discussions were conducted with mothers of children 6 to 23 months of age, and 56 in-depth interviews were conducted with mothers (n = 18), fathers (n = 14), grandmothers (n = 14), health workers (n = 8), and Catholic catechists (n = 2). These were followed by a 14-day usability trial during which 45 mothers fed their children MNP daily and were interviewed about their experience. Participants were selected from three villages. RESULTS: The findings revealed limited exclusive breastfeeding and early introduction of complementary foods due to traditional beliefs and poor knowledge. MNP was generally liked by the respondents. Thirty of the 45 children in the trial consumed all of the 14 MNP sachets provided to them. The majority of mothers (n > or = 30) split and fed the daily dose of MNP at different times of the day. They gave several reasons for this practice, including changes in the color of food when a whole sachet of MNP was added. Only six mothers shared MNP-fortified food among siblings. The participants suggested contextual attributes that could influence their adoption of MNP including preferred name, packaging design, and delivery channel. They preferred orange-colored sachets with a picture of a "healthy" Timorese baby, the logo of the Ministry of Health, and instructions on how to use the product. CONCLUSIONS: The findings offer context-specific knowledge that could guide the success of the MNP program in this district and similar settings.