Genes expression in Penaeus monodon of Bangladesh; challenged with AHPND-causing Vibrio parahaemolyticus

Vibrio parahaemolyticus, the causative agent of Acute hepatopancreatic necrosis disease (AHPND), was discovered in 2013 as a unique isolate that produces toxins and kills penaeid shrimps in devasting nature in Bangladesh and causes severe economic losses. This research aimed to understand the expressions of immune genes in different stages of the host species, Penaeus monodon, against virulence and toxin genes upon being challenged with V. parahaemolyticus. Healthy post-larvae (PL) samples were collected from southwestern of Bangladesh from July 2021 to August 2022. The tryptic soy agar with 1.5% sodium chloride (NaCl) was used to inoculate the cells of V. parahaemolyticus, and the tryptic soy broth (TSB) with 1.5% NaCl was used to transfer the colonies. The spectrophotometry measured bacteria density. PCR, qPCR, SDS-PAGE, and Western blot measured gene expression and survivability after the immersion challenge. The 1 × 105CFU/mL of V. parahaemolyticus was used for 144 h.p.i (hours post-infection) challenge to six stages of post-larvae (PL) of P. monodon (PL20, PL25, PL30, PL35, PL40, and PL45), PL30 and PL35 showed 100% mortality by day 72 (h.p.i.) after exposure that indicated most vulnerable to V. parahaemolyticus. The expression of immune and toxic genes was confirmed by qPCR. The immune genes toll-like receptors (TLR), prophenoloxidase (ProPO), lysozyme (lyso), and penaeidin (PEN) of PL20 and PL25 of P. monodon were expressed robustly up-trends. PL30 and PL35 showed the lowest gene expression at the end of 72 (h.p.i.). At the end of the 144 (h.p.i.) exposure, the immune genes TLR, ProPO, lyso, and PEN expressed highest in PL45 than other post-larvae stages of P. monodon. The toxic genes (pirA, ToxR, ToxA, ToxB, tlh, tdh, and trh) in PL30 and PL35 of P. monodon after exposure of V. parahaemolyticus were expressed highest at the end of the 72 (h.p.i.). The lowest toxic genes expressions were revealed in PL20 and PL45 at the end of the 144 (h.p.i.). The SDS-PAGE analysis of proteins from the bacterium revealed identical protein profiles with toxic genes, and those toxins were further confirmed by Western blot. The 20 kDa, 78 kDa (ToxR), 20 kDa, 25 kDa (ToxA), 25 kDa (ToxB), 20 kDa, 27 kDa, 75 kDa (tdh), and 20 kDa, 27 kDa, 75 kDa, and 78 kDa (trh) proteins were strong responses in Western blot, indicating the crucial involvement of these immune-related genes in the defense and recovery of the first-line defense mechanisms during V. parahaemolyticus infection to shrimp. The all-toxic genes showed a unique homology and those derived from the common ancestor compared with V. parahaemolyticus (NCBI accession no. AP014859.1). All clades were derived with different traits with very low genetic distance, where the overall mean distance was 3.18 and showed a very uniform and homogenous pattern among the lineages. The V. parahaemolyticus infection process in different PL stages in P. monodon revealed novel insights into the immune responses. The responses may lead to the subsequent production of a DNA vaccine, enhancing shrimp health management to minimize the economic losses due to AHPND experiencing an outbreak of early mortality syndrome (EMS) toward sustainable production P. monodon (shrimp)

Diversity of bacterial communities in the Sundarbans mangrove, Bangladesh, with special focus on pathogens affecting aquatic organisms

The Sundarbans, located in Bangladesh, is the world's largest mangrove forest with a rich and diverse microbial community. The main objective of this study was to use 16S rRNA sequencing to identify the bacterial community in the Sundarban mangrove forest, particularly pathogenic bacteria to aquatic organisms. This study conducted in Bangladesh collected 225 samples of soil, water, fish, and shellfish from five locations with a typical mangrove environment. All samples underwent genomic DNA extraction, quantification, gel documentation, and subsequent sequencing. Sanger sequencing was then carried out, followed by sequencing 11 concentrated samples using the universal bacterial primer 16S rRNA. The study found 872 bacterial species, 94 genera, 38 families, and 6 phyla. Pseudomonades or proteobacteria accounted for 92% of the phylum composition, with firmicutes, bactericides, cyanobacteria, Verrucomicrobia, and actinobacteria also present at smaller percentages. Chromatiaceae had the highest species count (20.3%), followed by Ectothiorhodospiraceae (15.5%), Methylococcaceae (12.3%), Moraxellaceae (10.7%), Thiotrichaceae (8.7%), Idiomarinaceae (5.4%), and smaller groups. All the species that were identified were gram-negative bacteria and were found in different environments, mainly halophilic ones. Based on 16 s rRNA sequencing, additional experimental and clinical data, as well as NCBI (BLAST), approximately 49.3% of the 872 bacterial species are typically harmless and not classified as pathogenic. The pathogenic characteristics of 24.9% of the species are still unidentified. 13.4% showed either pathogenic or non-pathogenic characteristics (a bacterium as having the potential to exhibit pathogenic or non-pathogenic characteristics in certain circumstances), while 8.4% were exclusively pathogenic, potentially capable of causing disease. Also, the bacterial species are categorized as 2.6% non-highly pathogenic (a bacterium can still have a significant impact on health, especially in weakened immune systems or other underlying health conditions), 1.3% sometimes pathogenic and 0.1% potentially pathogenic (a bacterium don't always cause disease). Sequence analysis, combination of phylogenetic, biochemical characterization (data were not shown), and other experimental validation conclude a genetic baseline of bacterial community, this research may serve as a valuable reference for future ecological and taxonomical studies as well as addressing further research scope on bacterial diversity found in the Sundarbans mangrove of Bangladesh

Bacteriophage and non-pathogenic Vibrio to control diseases in shrimp aquaculture

The study aimed to address the recurring outbreaks of microbial diseases in shrimp aquaculture in Bangladesh the study focused on the utilization of bacteriophages and non-pathogenic Vibrio. The bacteriophages were isolated from sewage water sample collected from shrimp farm, hatchery, and the JUST campus. The bacteriophages were tested for their ability to infect different Vibrio strains in order to assess their bacteriolytic activity. Non-pathogenic Vibrio strains were obtained from suspected diseased isolates collected from the south-western region of Bangladesh through PCR amplification. In laboratory tests, the bacteriophages successfully infected 91 % of the tested Vibrio strains (19 out of 21 strains). In the experimental unit, shrimp treated with phage prophylaxis and phage treatment demonstrated notable protection against AHPND and was able to survive a deadly bacterial challenge. A total of 35 suspected diseased isolates were tested, and PCR amplification revealed 6 non-pathogenic Vibrio strains. In field trials, cultured bacteriophages were applied at a concentration of 1.5×106 PFU/ml, while non-pathogenic Vibrio was applied at 5×105 CFU/ml. The trials showed increased protection against infections and no severe deaths during the adaptive research phase. The cultured shrimp were analyzed morphologically and showed a muscle gut ratio greater than 4:1. No abnormal deformities were observed in their appendages or overall body, suggesting their overall health and well-being. The bacteriological tests conducted on the shrimp samples (application of bacteriophages and non-pathogenic Vibrio) revealed that 18 % of them were infected with bacteria, primarily Vibrio cholerae, Vibrio parahaemolyticus, and other bacterial species. Despite this, the infections did not lead to a disease outbreak; PCR amplification showed negative results for AHPND, White Spot Syndrome Virus (WSSV), and Enterocytozoon hepatopenaei (EHP). The results highlight the potential of using bacteriophages and non-pathogenic Vibrio as a sustainable solution for preventing and controlling microbial diseases in shrimp aquaculture. Furthermore, this study will contribute valuable insights into the development of alternative strategies to combat antibiotic resistance and promote the growth of the shrimp industry in Bangladesh