Effects of lactobacillus plantarum, pediocococcus pentosaceus, and lactobacillus fermentum supplemented diet on survival rate, resistance to vibrio spp. and to acute hepatopancreatic necrosis disease (ahpnd) in white leg shrimp (litopenaeus vannamei).

AGU International Journal of Sciences – 2019, Vol 7 (3), 47 – 55<br /> <br /> <br /> <br /> <br /> EFFECTS OF LACTOBACILLUS PLANTARUM, PEDIOCOCOCCUS PENTOSACEUS, AND<br /> LACTOBACILLUS FERMENTUM SUPPLEMENTED DIET ON SURVIVAL RATE,<br /> RESISTANCE TO VIBRIO SPP. AND TO ACUTE HEPATOPANCREATIC NECROSIS<br /> DISEASE (AHPND) IN WHITE LEG SHRIMP (Litopenaeus vannamei).<br /> <br /> Nguyen Thi Truc Linh1, Nguyen Thi Hong Nhi2, Duong Hoang Oanh2, Dang Thi Hoang Oanh3,<br /> Truong Quoc Phu3<br /> 1<br /> Tra Vinh University<br /> 2<br /> Master. Tra Vinh University<br /> 3<br /> Can Tho University<br /> <br /> Information: ABSTRACT<br /> Received: 04/06/2019<br /> The experiment was conducted to determine the effects of L. plantarum, P.<br /> Accepted: 11/07/2019<br /> pentosaceus, and L. fermentum supplemented diet on survival, resistance to<br /> Published: 11/2019<br /> AHPND in white leg shrimp. The experimental results showed that the density<br /> Keywords: of Vibrio spp. in the gut lumen of shrimp was significant decreased during the<br /> AHPND, Litopenaeus experimental period in the treatments of which the lactic acid bacteria (LAB)<br /> vannamei, white leg shrimp, used as supplemented diet and without challenged with Vibrio<br /> Vibrio spp, Vibrio parahaemolyticus. The survival rate of shrimp was very high (88,9-92,23%) in<br /> parahaemolyticus.<br /> these treatments. In addition, hepatopancreas tissues of shrimp did not have the<br /> typical symptoms of AHPND. In the treatments that the shrimps were challenged<br /> to V. parahaemolyticus, the survival rate of the shrimps was significant<br /> increased to (79,8%) and (75,8%) when L. plantarum, and P. pentosaceus,<br /> respectively, were supplemented into the diet of the shrimps. The density of<br /> Vibrio spp. in the gut lumen of the shrimps was also significant decreased during<br /> the experimental period. The infection rate of AHPND was highest in positive<br /> control treatment (77,8%) and lowest in L. plantarum supplemented diet<br /> treatment (11%). In conclusion, the survival, resistance to Vibrio spp. and to<br /> AHPND on white leg shrimp were improved with L. plantarum and P.<br /> pentosaceus supplemented diet.<br /> <br /> <br /> 1. INTRODUCTION to prevent Viriosis by applied chemicals,<br /> antibiotics, biological control agents. However,<br /> Vibrio are described as the common pathogens<br /> the long term application of chemicals and<br /> of cultured shrimp on the world (Lightner, 1996;<br /> antibiotics in aquaculture can cause the presence<br /> Flegel, 2012), in which V. parahaemolyticus is<br /> of resistant bacteria and accumulation of the<br /> known as the bacterium caused the tremendous<br /> residual of chemicals, antibiotics in the final<br /> losses of 1 billion USD per year in the shrimp<br /> aquaculture products. The application of<br /> farming in brackish water (Zorriehzahra, 2015).<br /> chemicals and antibiotics to kill bacteria,<br /> Several solutions have been recently developed<br /> therefore, should be minimised in aquaculture.<br /> <br /> <br /> <br /> 47<br /> AGU International Journal of Sciences – 2019, Vol 7 (3), 47 – 55<br /> <br /> The LAB have been widely applied as probiotics was then treated by chlorine 30 mg/L with strong<br /> to stimulate digestive activities and to prevent aeration in 24h. Chlorine residual was<br /> bacterial diseases in human and animals. LAB neutralized by Na2S2O3. Freshwater was added<br /> have been recently added into aquafeed as into the seawater to reduce the salinity to 20‰.<br /> supplemented diets to (1) eliminate bacteria Culture tanks: Glass tanks 30L containing 20L<br /> causing diseases in aquatic animals (Garriques & the seawater 20‰ were used to grow the shrimp<br /> Arevalo, 1995; Moriarty, 1997; Gomez-Gil et during our experiment. The empty glass tanks<br /> al., 2000; Balca´-zar et al., 2004; Vine et al., were chlorinated at 30mg/L and exposed to<br /> 2004), (2) provide digestic enzymes and nutrient sunlight 5 hours before using in the experiment.<br /> agents (Sakata, 1990; Garriques & Arevalo,<br /> 1995) and (3) other benefits such as improving Shrimp seeds: White leg shrimp seeds post<br /> immune systems in aquatic animals, that can larvae 10 (PL10) were reared in recirculation<br /> help to minimise risks causing by harmful aquaculture systems (RAS) at Department of<br /> bacteria (Andlid et al., 1995; Rengpipat et al., Pathology, Aquaculture Faculty, CanTho<br /> 2000; Gullian & Rodríguez, 2002; Irianto & University. The shrimp seeds approximatelly<br /> Austin, 2002; Balcázar, 2003; Balcázar et al., 1g/species tested for WSSV (OIE, 2006) and V.<br /> 2004) and virus (Kamei et al., 1988; Girones et parahaemolyticus (Sirikharin et al., 2014) by<br /> al., 1989; Direkbusarakom et al., 1998). PCR with AP3 primer. The WSSV- and V.<br /> parahaemolyticus negative shrimp seeds were<br /> In vitro trials showed that the bacteria strains L. used as the experimental animals. The shrimp<br /> plantarum, P. pentosaceus, and L. fermentum seeds were gradually acclimated to the culture<br /> can inhibit the growth of the bacterium V. conditions in 3 days before using as the<br /> parahaemolyticus causing AHPND (Nguyen et experimental animals.<br /> al., 2018). Recent studies also focused on the<br /> application of benefit bacteria with inhibitory V. parahaemolyticus: The bacterial strain V.<br /> activity against Vibrio spp. in shrimp farming, parahaemolyticus causing AHPND was stored<br /> especially the benefit bacteria can be mixed at -80˚C in laboratory of aquaculture faculty,<br /> directly into shrimp feed (Soccol et al., 2010). CanTho university (Nguyen et al., 2015). The<br /> Therefore, we propose the hypothesis that the strain was activated in nutrient broth (NB,<br /> bacterial strains L. plantarum, P. pentosaceus, Merck) adding 1,5% NaCl (NB+) and incubated<br /> and L. fermentum can inhibit the growth of at 28˚C in 18h. Colour and shape of coliform was<br /> Vibrio spp. and the cultured shrimps fed checked and gram staining was also applied to<br /> suplemented diets with L. plantarum, P. test for the pure line strain. The bacterial strain<br /> pentosaceus, and L. fermentum can be resistant was grown in NB+ medium at 28˚C in 18 - 24h<br /> to AHPND, which can result in high survival rate and the density of the bacterial strain was<br /> of the shrimps against V. parahaemolyticus. determined by spectrophotometer at 610 nm.<br /> <br /> 2. MATERIALS AND METHODS Feed preparation: The bacterial strains L.<br /> plantarum (LAB1), P. pentosaceus (LAB2) and<br /> 2.1 Materials L. fermentum (LAB3) were isolated in digestive<br /> Water supply: Sea water 72 - 85‰ transferred systems of the shrimps and fishes collected from<br /> from Vinh Chau district, Soc Trang province, semi-intensive ponds in TraVinh and BenTre<br /> Viet Nam was filtered via filter bag. The water Provinces. The bacterial strains were then grown<br /> <br /> <br /> <br /> 48<br /> AGU International Journal of Sciences – 2019, Vol 7 (3), 47 – 55<br /> <br /> in nutrient broth de Man Rogosa Sharpe (MRS, 4) Treatment LAB3: The shrimp seeds were fed<br /> Merck) agar adding 1,5% NaCl in 48h. The diets supplemented with LAB3 (L. fermentum)<br /> biomass of the bacterial strains were collected by and were not challenged with V.<br /> centrifugation of 700 rpm in 5 minutes. Gram parahaemolyticus (non-infection).<br /> staining was applied to test for pure line 5) Positive control: the shrimp seeds were fed<br /> selection. The sterilized saline solution was used diets without LAB and were challenged with V.<br /> to clean and to dilute the biomass the bacterial parahaemolyticus (infection).<br /> strains to 1010 CFU/mL. An aliquot of 10mL<br /> solution of each bacterial strain was added into 6) Treatment VP+LAB1: the shrimp seeds were<br /> 100mg shrimp feed (109CFU/g) covered by fed diets supplemented with LAB1 (L.<br /> squid oil. The shrimp feed was packed and plantarum) and were challenged with V.<br /> labelled and stored at 4˚C before feeding. In the parahaemolyticus (infection).<br /> treatment added LAB and challenged AHPND 7) Treatment VP+LAB2: the shrimp seeds were<br /> V. parahaemolyticus, shrimps were fed with fed diets supplemented with LAB2<br /> Lactobacillus plantarum (VPL1), Lactobacillus (Pediocococcus pentosaceus) and were<br /> fermentum (VPL2), and Pediococcus challenged with V. parahaemolyticus<br /> pentosaceus (VPL3), respectively (infection).<br /> supplementation for 7 days before experiment. 8) Treatment LAB3: the shrimp seeds were fed<br /> 2.2 Methods diets supplemented with LAB3 (L. fermentum)<br /> The 30 shrimp seeds were grown in each glass and were challenged with V. parahaemolyticus<br /> tanks 30L containing 20L as mentioned in 2.1. (infection).<br /> The CP 40% protein was used as the shrimp feed The shrimp was fed 3 times per day at 7, 13 and<br /> in this experiment. The shrimp feed was fed with 17 h and amount of feed was depended on<br /> or without LAB as described in the following demand of the shrimps. In treatment 6, 7 and 8,<br /> treatments and controls. There are 6 treatments, shrimps were continued to feed with<br /> 1 negative and 1 positive controls in this Lactobacillus plantarum, Lactobacillus<br /> experiment and each treatment, each control has fermentum, and Pediococcus pentosaceus,<br /> three replicates, including: respectively, and challenged with AHPND V.<br /> 1) Negative control: the shrimp seeds were fed parahaemolyticus. Challenge method was<br /> diets without LAB and were not challenged with following guidelines described by Tran et al.<br /> V. parahaemolyticus (non-infection). (2013). Shrimps were applied AHPND V.<br /> parahaemolyticus at 106 CFU/mL. Water was<br /> 2) Treatment LAB1: the shrimp seeds were fed exchanged at 30% after 3 days of challenge.<br /> diets supplemented with LAB1 (L. plantarum) After that, water was exchanged 30% every day.<br /> and were not challenged with V. The experiment was lasted in 14 days.<br /> parahaemolyticus (non-infection).<br /> Sampling<br /> 3) Treatment LAB2: The shrimp seeds were fed<br /> diets supplemented with LAB2 (Pediocococcus The three shrimps were randomly collected from<br /> pentosaceus) and were not challenged with V. all culture tanks as the samples to determine<br /> parahaemolyticus (non-infection). Vibrio density and histopathological analysis.<br /> The samples were collected first time before the<br /> <br /> <br /> <br /> 49<br /> AGU International Journal of Sciences – 2019, Vol 7 (3), 47 – 55<br /> <br /> infection and each 3 days after infection during were put in 45 - 50˚C water before sticking on<br /> the experimental period. There were 5 samplings glass slides to make microscopic samples. The<br /> in total. slides were stained with Haematoxylin và Eosin<br /> Vibrio spp. density in the shrimp gut lumen was (H&E) before observing under a microscope.<br /> determined by counting the number of colony 2.3 Data analysis<br /> grown on agar plate (Hadi et al., 2009). The One-way ANOVA was applied to test the data of<br /> shrimp guts were collected, weighed and grinded the current experiment, in which Duncan’s Test<br /> in the saline water to make solution in a and Tukey Test were used to determine<br /> microbiological safety cabinet to avoid significant difference (p