Cloning and Expression of PirA gene of Vibrio parahaemolyticus Strain K5 Causing Acute Hepatopancreatic Necrosis Disease in Whiteleg Shrimp in E. coli Host Cell

Background: Acute hepatopancreatic necrosis disease (AHPND), is a bacterial disease of whiteleg shrimp, which has a high mortality rate (100%) and incurs economic losses. Our objective was to identify the genes which lead to cell and organ damage and investigate bioproducts to prevent and treat. Methods: Litopenaeus vannamei shrimp in Thua Thien Hue province, Vietnam were collected from an infected pond and analysed at the Institute of Biotechnology, Hue University. The PirA gene of Vibrio parahaemolyticus strain K5 was isolated and analyzed for nucleotide sequence and paired with the expression vector pQE30. The expression vector was transformed into E. coli strain M15, the PirA recombinant protein was expressed in the form of 6xHis-PirA fusion protein of about 15 kDa. PirA recombinant protein was purified and determined the PirAvp binding ratio, cloning and sequencing of PirA gene from Vibrio parahaemolyticus strain K5 causing AHPND by PCR method with specific primers and molecular weights of PirAvp and the PirAvp complex. Results: PirA gene from Vibrio parahaemolyticus strain K5 was cloned into pGEM-T easy vector (Promega, USA) and screened E. coli TOP10 colonies containing pGEM T easy/PirA recombinant plasmid on LB agar/ampicillin/IPTG/X-Gal medium. PCR showing a band of about 347 bp, matching the size of PirA gene and two nucleotide sequences (BamHI and HindIII). The results showed that PirA gene has a length of 336 bp and similar to PirA gene on GenBank (Code: KU556825.1). The results of protein extracted from E. coli M15 recombinant cells and 6xHis-PirA target protein was collected in elution fractions from EF2 to EF6, showed that the concentration of 6xHis-PirA protein and EF3 elution fraction collected a highest protein concentration (1,586.54 µg/ml). Conclusions: The purified PirA recombinant protein will provide materials for development research to create biological products to prevent and treat AHPND.

A Review of the Functional Annotations of Important Genes in the AHPND-Causing pVA1 Plasmid

Acute hepatopancreatic necrosis disease (AHPND) is a lethal shrimp disease. The pathogenic agent of this disease is a special Vibrio parahaemolyticus strain that contains a pVA1 plasmid. The protein products of two toxin genes in pVA1, pirAvp and pirBvp, targeted the shrimp’s hepatopancreatic cells and were identified as the major virulence factors. However, in addition to pirAvp and pirBvp, pVA1 also contains about ~90 other open-reading frames (ORFs), which may encode functional proteins. NCBI BLASTp annotations of the functional roles of 40 pVA1 genes reveal transposases, conjugation factors, and antirestriction proteins that are involved in horizontal gene transfer, plasmid transmission, and maintenance, as well as components of type II and III secretion systems that may facilitate the toxic effects of pVA1-containing Vibrio spp. There is also evidence of a post-segregational killing (PSK) system that would ensure that only pVA1 plasmid-containing bacteria could survive after segregation. Here, in this review, we assess the functional importance of these pVA1 genes and consider those which might be worthy of further study.

Selection of Lactic Acid Bacteria (LAB) Antagonizing Vibrio parahaemolyticus: The Pathogen of Acute Hepatopancreatic Necrosis Disease (AHPND) in Whiteleg Shrimp (Penaeus vannamei)

Acute hepatopancreatic necrosis disease (AHPND) has recently emerged as a serious disease of cultured shrimp. A total of 19 lactic acid bacteria (LAB) strains isolated from shrimp samples were characterized based on morphological characteristics, biochemical tests, sequencing analysis, and their ability to antagonize Vibrio parahaemolyticus, which causes AHPND in whiteleg shrimp. Results from the agar well diffusion method indicated that 3 out of 19 isolated LAB strains showed the highest antagonizing ability against AHPND V. parahaemolyticus strain with an inhibition zone diameter ranging from 18 to 20 mm. Experiments where shrimps were given feed supplemented with these LAB strains and challenged with AHPND strain showed high survival rates (approximately 80.0%), which were not significantly different as compared to those recorded in the negative control treatment (86.6%), but significantly different to those recorded in the positive control treatment (40.6%) after 16 days of the experiment. However, the histological images of shrimp hepatopancreas indicated that the infection rate significantly reduced from 60.0% to 11.1% in shrimps fed with LAB-supplemented feeds and challenged with AHPND V. parahaemolyticus strain as compared to those in the positive control treatment. A polymerase chain reaction (PCR) and 16S rRNA gene sequencing confirmed the identification of LAB strain. These results can be applied in further experiments to investigate the ability of L. plantarum in preventing AHPND in intensively cultured whiteleg shrimp.

Antibacterial Activity of Extracts from Dried and Fresh Herbal Plant (Phyllanthus amarus) Against Pathogens Causing Acute Hepatopancreatic Necrosis Disease (Ahpnd) in White Leg Shrimp (Litopenaeus vannamei) at Thua Thien Hue Province, Vietnam

The study aimed to determine extract yield (%), antibacterial activity, and minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of plant extracts from dried and fresh herbal plants (Phyllanthus amarus) against Vibrio parahaemolyticus strain causing acute hepatopancreatic necrosis disease (AHPND) in white leg shrimp (L. vannamei). The result showed that the extract yields of dry and fresh herbs reached 11.50% and 2.75%, respectively and the antibacterial activity of the two extracts both are good at concentrations from 250 to 1,000 mg/mL at the same bacterial density of 106 CFU/mL. Specifically, the diameter of the inhibition zone at 250; 500; 750 and 1,000 mg/mL concentration of dried herbal extracts reached 16.75±0.96; 18.50±1.29; 20.75±0.96 and 21.25±0.50 mm, while that of fresh herbal extracts reached 14.50±1.29; 16.25±0.50; 16.75±0.50 and 17.00±0.00 mm, respectively, with a statistically significant difference p<0.05. The result also showed that MIC values of dried and fresh extracts were defined at 125 mg/mL and 250 mg/mL, respectively and that MBC values of the extracts were 500 and 1,000 mg/mL respectively. The GC-MS analysis revealed that there were 19 natural compounds in the dried extract, in which Ethyl Linoleolate (C20H36O2) compound occupied the highest ratio (22.43%), while 2.3-Dihydro-3.5-dihydroxy-6-methy-4H-pyran-4-one (C6H8O4) was the lowest (0.24%).

Draft Genome Sequence of Vibrio parahaemolyticus Strain PH1339, Which Causes Acute Hepatopancreatic Necrosis Disease in Shrimp in the Philippines

We report the first draft genome sequence of an acute hepatopancreatic necrosis disease (AHPND)-causing Vibrio parahaemolyticus strain isolated from a Penaeus vannamei sample from the Philippines. The strain carries the genes encoding the Pir-like toxin pair PirAvp and PirBvp.

Molecular Identification of Cellulolytic Bacteria From Mangrove Sediment at Tin Minning Region In West Bangka

Cellulose as an abundant source of glucose in Indonesia requires acceleration of decomposition utilizing cellulolytic bacteria. Cellulolytic bacteria can be obtained from the isolation of mangrove organic matter, such as sediments. Muntok Sub-district is one of the regions with the most tin mining in West Bangka Regency also has mangroves in the coastal area. Exploration of cellulolytic bacteria in mangroves with different environmental characteristics encourages researchers to find new bacterial strains that produce cellulase enzymes with new properties. Thirteen isolates were successfully isolated from three locations. Tembelok mangrove sediments produced Seven bacterial isolates, Peltim Mangrove samples produced three isolates and from Sukal Mangrove three isolates were obtained. Seven isolates showed clear zones in the Lugol test and three isolates including were gram-positive bacteria. Molecular test with 16S rRNA analysis showed TBL1 isolate has 85% similar identity of Vibrio parahaemolyticus strain HY3 and TBL2 isolate has 98% similar identity of Bacillus amyloliquefaciens strain HS8. Bacillus amyloliquefaciens potential to further study as cellulose degrading bacteria for feed ingredients.

Draft Genome Sequence of a Vibrio parahaemolyticus Strain, KS17.S5-1, with Multiple Antibiotic Resistance Genes, Which Causes Acute Hepatopancreatic Necrosis Disease in Penaeus monodon in the West Coast of Peninsular Malaysia

We report the first draft genome sequence of a Vibrio parahaemolyticus strain (Vp AHPND), which causes acute hepatopancreatic necrosis disease (AHPND) in Penaeus monodon. The strain has a pVA1-like plasmid carrying pirAvp and pirBvp genes.