Bacterial load and 16s RRNA sequence based identification of Vibrio spp. in fish and shrimp hatcheries of Bangladesh

Abstract

The presence of Vibrio spp., one of the deadliest fish and shrimp pathogen in aquaculture facilities worldwide for which hatchery owners often suffer hectic economic losses, were observed in this study with species level identification in three shrimp and fish hatcheries of Cox’s Bazar, Mymensingh and Bogra, Bangladesh. Bacterial enumeration was done in nutrient agar (NA), marine agar (MA) and thiosulphate citrate bile salt sucrose agar (TCBS) plate to understand the microbial load in the corresponding shrimp, tilapia, shing, magur and pangas fry rearing environment which will provide an insight into the environmental management implications and need for further initiatives. Artemia hatching tank of Zomzom hatchery, Cox’s Bazar had similar total bacterial build up (2.59 ± 0.10×107 cfu/g) in the water sampled and in the shrimp post larvae (PL) sampled at stage 10 and 12 (2.37 ± 0.11×107 cfu/g and 2.42 ± 0.10×107 cfu/g respectively). However, bacterial load determined from the samples of water corresponding to the stages of PL were similar but different from the samples of Artemia tank and PL stages of 10 and 12. In MA plate, no significant differences were observed in the bacterial count of these samples. Similar result was observed for the total presumptive vibrio count in TCBS plates which ranged from 3.8 ± 0.60×103 cfu/g to 1.62 ± 0.50×103 cfu/g. Total bacterial load (7.5 ± 0.11×107) measured in the water sampled from 25 day old fry rearing pond of tilapia, from Reliance Tilapia Hatchery, Mymensingh, was similar to that of 33 day old fry (8.6 ± .66×107). The bacterial density found in the 25 (1.6 ± 0.50×107), 28 (3.12 ± 0.14×107) and 40 day old fry (6.46 ± 1.52×106) samples were similar but significantly different from the sample of 33 day old fry and the water sample of the pond of 25 day old fry. In TCBS plate, bacterial abundance detected in the samples across all four age groups were similar (25 day old fry: 4.21 ± 3.79×103; 28 day old fry: 4.90 ± 3.50×103; 33 day old fry: 1.08 ± 0.12×103; 40 day old fry: 7.04 ± 2.08×103). No bacterial count was found in the water sampled from 25 day old fry rearing pond. In GM Aquaculture Ltd, Bogra, the overall bacterial build up (2.03 ± 0.31×108) found in the samples of zeol fish fry in NA plate was significantly higher than that of thecorresponding rearing pond water (2.11 ± 0.459×107) and the water of the live food rearing tank ( 8.43 ± 0.57×106). Similar to that, TCBS plates had 2.3-, and 5.09-folds higher bacterial load (1.08± 0.25×103) in the samples of fish fry than in the samples of the corresponding water samples and water samples of the live food rearing tank, respectively (4.70 ± 1.67×102 and 2.12 ± 0.28×102). 37 Vibrio colonies, selected based on their morphological dissimilarities in TCBS plate, were subjected to amplified 16S ribosomal DNA restriction analysis (ARDRA) using AluI restriction enzymefollowing their DNA extraction and amplification of 16S rRNA (1450 bp). From this analysis, ultimately 8 groups (representative isolates), named as ARH 1, ARH 2, ARH 3, ARH 4, ARH 5, ARH 6, ARH 7 and ARH 8, of different band pattern were sequenced and identified as Vibrio alginolyticus, Aeromonas veronii, Aeromonas hydrophila, Vibrio vulnificus, Vibrio cholera, Edwardsiella hoshinae, Bacillus methylotrophicus and Aeromonas veronii,respectively. Polymorphic sites among the sequenced strains were studied by multiple sequence alignment considering 1320 bp nucleotides where 12.9% dissimilarities were observed among the identified Aeromonas and Vibrio species which is 5.7% among 3 Vibrio species. Phylogenetic analysis also confirmed the taxonomic relation among the identified species. Vibrio species identified in this study, are pathogenic for human and aquatic organisms, and were found only in shrimp hatchery with the dominance of V. alginolyticus. Findings of this study indicate the poor quality of water treatment and management of the hatchery. It is also observed that all these 3 Vibrio species were present in the Artemia rearing tank which also indicates the possible source of pathogens.