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