Luminescent Vibrio harveyi is a natural microflora of marine and coastal water bodies and is associated with mortality of larval shrimp in penaeid shrimp hatcheries. It is also known that the bacteriophages occur virtually in all places where their hosts exist. In this study, distribution of luminescent V. harveyi and the bacteriophages affecting these hosts was examined in a commercial Penaeus monodon hatchery during three shrimp larval production cycles, including a cycle affected by luminescent bacterial (LB) disease outbreak.Out of a total of 1195 samples drawn from seawater source, sand-filtered water, nauplius, zoea, mysis and post larval rearing tanks, maturation and spawning tanks, Artemia hatching tank and algal culture tanks processed using conventional microbiological techniques, 21.4% of the samples harboured luminescent bacteria. During the larval production cycle affected by LB disease (LBD), luminescent V. harveyi could be recovered from 52% of the hatchery samples, whereas during luminescent bacterial disease-free larval production cycle (LBDF), these bacteria could be recovered from only about 9% of the samples. The predominant source of luminescent bacteria was the brood shrimp and their rearing tanks in maturation and spawning facilities. 73% of the maturation and 80% of the spawning tank water samples harbored LB during LBD, whereas, only 20% and 32% of the maturation and spawning tanks respectively harbored LB during LBDF. LB could be isolated from 17% of the water samples in tanks from nauplius stage onwards with increasing counts that subsequently lead to LB disease.Bacteriophages affecting the luminescent V. harveyi could be isolated from as many as 36% (21% and 43% of the samples analysed during LBDF and LBD respectively) of a total of 181 water samples drawn from various sources in the hatchery, using 27 luminescent V. harveyi hosts by agar overlay technique. The maturation tank water samples were found to be the predominant source of bacteriophages, followed by spawning tank water samples as observed with the LB. Sixty five bacteriophages, 13 during LBDF and 52 during LBD were isolated, which were grouped in to seven types based on their plaque morphology.The study has indicated that the brooders, maturation and spawning facilities in the shrimp hatchery are the main source of luminescent V. harveyi and their bacteriophages and that occurrence of LB even in low counts during early larval stages can possibly lead to development of LB disease despite presence of bacteriophages in the larval rearing tanks.
Four lytic bacteriophages designated as φVh1, φVh2, φVh3, and φVh4 were isolated from commercial shrimp hatcheries, possessing broad spectrum of infectivity against luminescent Vibrio harveyi isolates, considering their potential as biocontrol agent of luminescent bacterial disease in shrimp hatcheries, and were characterized by electron microscopy, genomic analysis, restriction enzyme analysis (REA), and pulsed-field gel electrophoresis (PFGE). Three phages φVh1, φVh2, and φVh4 had an icosahedral head of 60-115 nm size with a long, noncontractile tail of 130-329 × 1-17 nm, belonged to the family Siphoviridae. φVh3 had an icosahedral head (72 ± 5 nm) with a short tail (27 × 12 nm) and belonged to Podoviridae. REA with DraI and PFGE of genomic DNA digested with ScaI and XbaI and cluster analysis of their banding patterns indicated that φVh3 was distinct from the other three siphophages. PFGE-based genome mean size of the four bacteriophages φVh1, φVh2, φVh3, and φVh4 was estimated to be about 85, 58, 64, and 107 kb, respectively. These phages had the property of generalized transduction as demonstrated by transduction with plasmid pHSG 396 with frequencies ranging from 4.1 × 10(-7) to 2 × 10(-9) per plaque-forming unit, suggesting a potential ecological role in gene transfer among aquatic vibrios.
The current study was conducted for 75 days to evaluate the impact of Potassium diformate (KDF), Sodium formate (SF) and organic acid blend (KDF + SF) on the growth, immunity and disease resistance in Penaeus vannamei against Vibrio harveyi. Pacific white shrimps (2.11 ± 0.01g) were fed with organic acids and organic acid blend in two different inclusion levels, such as 1 & 2%. After 60 days of feeding trial, the higher body weight gain and survival rate was observed in shrimps fed KDF 2% diet compared to the control groups. Challenge study conducted for 15 days with V. harveyi (108) has shown significantly lower cumulative mortality rate in shrimps fed KDF 2%, KDF 1% and SF 2% treatment groups compared to the control groups. At the end of challenge study, total cultivable bacteria and presumptive vibrio spp. count in shrimp fed KDF2 and all treatments were significantly lower than control diet. The histopathology results were observed with less hepatopancreatic damage in shrimps fed with KDF at 2% level compared to the control. The immune parameters, Total haemocyte count (THC), Prophenoloxidase activity (proPO), Respiratory burst activity (RBA), Superoxide dismutase (SOD), and Catalase activity (CAT) were found to be significantly higher than the control.
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