2016-08-15T11:39:21+05:302016-08-15T11:39:16+05:302016-08-15T11:39:21+05:30Acrobat PDFMaker 11 for Worduuid:aaea3d8a-fa1e-429d-80af-4a5aeb6a44f3uuid:e332b921-bf99-45c4-83af-a5932baa3b169xmlThe Identification of Vibrio SppWin 7Adobe PDF Library 11.0D:20160808110047
International Journal of ChemTech Research CODEN (USA): IJCRGG, ISSN: 0974-4290, ISSN(Online):2455-9555 Vol.9, No.07 pp 448-452, 2016
The identification of Vibrio Spp. Bacteria from Litopenaeus Vannamei infected by White Feces Syndrome Maskar Jayadi*, Arief Prajitno, Maftuch Faculty of Fisheries and Marine Sciences, Universitas Brawijaya, Jl. Veteran, Malang 65145, Jawa Timur Province, Indonesia Abstract : The vannamei shrimp ponds in Indonesia have infected by White Feces Syndrome (WFS) intensively in Sumatra, Java, Sulawesi, Bali, Lombok and Sumbawa. This research is aimed to know the density and type of Vibrio spp. from hemolymph, hepatopancreas and intestines of vannamei shrimp which is infected by WFS. The bacteria identification test was performed in the Microbiology Laboratory of Balai Besar Perikanan Budidaya Air Payau Jepara (a brackish water aquaculture office in Jepara, Central Java). The research method in this study is an explorative descriptive research by using a simple random sampling. Conventional method is used for the identification of the bacteria. The indication of the shrimp which is infected by WFS is already exposed to the Vibrio spp. genus bacteria, it consists of V. Parahaemolyticus; V. Vulnificus; V. chollera and V. anguillarum. The total average of Vibrio spp. in hemolymph is 2,9 x 10while in hepatopancreas is 4,4x10and in intestines 3,9x10. 4 CFU/ml, 7 CFU/g 7 CFU/gKeywords: WFS, Vibrio, Vannamei Shrimp. Introduction The case of White Feces Syndrome (WFS) on the vannamei shrimp cultivation is characterized by a decreased appetite levels, abnormal growth, and white feces2
. The symptoms of WFS occurred after the cultivation above 50 days since the first day of stocking. 1. The level of vannamei shrimp life which is severely infected by WFS is 20-30%. The daily weight gain (ADG) of the infected vannamei shrimp is ≤ 0.1 g while the ADG of normal vannamei shrimp is 0.2 g. The value of the feed conversion ratio (FCR) on the infected vannamei shrimp is 1.7-2.5 while on the normal vannamei shrimp is 1,5
The vannamei shrimp which is infected by WFS is due to Vibrio spp. type V. vulnificus, V. fluvialis, V. parahaemolyticus, V. alginolyticus, V. mimicus, V. chollerae and V. Damselae. WFS is caused by the V. Chollerae bacteria, it is based on the comparison between the genetic and phenotype characteristics of the V. Chollerae bacteria which is isolated from the infected vannamei shrimp and the other isolated V. chollerae bacteria3. This research is aimed to find the density and type of the Vibrio bacteria from hemolymph, hepatopancreas and intestines of the infected vannamei shrimp. The research is located in the Microbiology Laboratory of Balai Besar Perikanan Budidaya Air Payau Jepara (a brackish water aquaculture office in Jepara Regency, Jawa Tengah Province, Indonesia). Methods The research method in this study is an explorative descriptive research, and the sampling was done by using a simple random sampling. Conventional method is used for the identification of the bacteria The main parameter observed in this study is the density and type of the Vibrio bacteria in hemolymph, hepatopancreas and intestines. 4.Sample The vannamei shrimp samples are obtained from the WFS investigation results at the vannamei shrimp cultivation area in Kragan, Rembang Regency, Jawa Tengah Province. Vannamei shrimp cultivation technologies are developed at the location of the semi-intensive and intensive sampling. The average cultivation area is 2000 up to 2500 m2. The stocking density is between 60-100 tail/m2 and the feed protein is about 36%. The water quality at the moment of sampling is 29.8oC up to 31.8oC and it has a dissolved oxygen of 6.2-7 mg/L; water pH of 7.5-8.4; and 23-30 ppt for the salinity. Vibrio spp. Total Identification There are 30 samples of vannamei shrimp. The hemolymph of the shrimp is taken by using syrige, and then disseminated on TCBS to be incubated at 32. oC for 24 hours. The surgery of the vannamei shrimp is performed aseptically in order to take the hepatopancreas and intestines. Each hepatopancreas and intestines is weighed as much as 1 gram, then it mashed with sterile mortar and added with 9 ml of tri-salt solution. Next, 1 ml samples are taken by using a 1 ml micropipette and poured on the reaction tubes. The samples in reaction tubes are noted for 10-1, they are homogenized with the help of vortex. This step is done until the 6th reaction tubes and recorded as 10-6 . The samples of 10-4, 10-5 and 10-6 are taken by using 0.1 ml micropipette and poured on the petri dish that already contains TCBS, then it gets flattened by using a spatula. The petri dish which has the bacteria within is coated with plastic wrap and stored in the incubator for 24 hours, so that the bacteria can live and grow maximallyResult and Discussion Based on the observation results of the infected ponds, white and long feces which has similar appearance with threads is floating on the surface of the water (Figure 1A) and the water color of the ponds is thick-green (Figure 1B). The results also showed about the clinical symptoms of the infected vannamei shrimp such as decreased appetite, white hepatopancreas (Figure 1C), and the average cultivation time (age) which is >50-75 days. The white feces which floats on the surface of the water is the result of the shrimp hepatopancreas peeling which is issued by the shrimp through the intestines in the form of Aggregated Transformed Microvilli (ATM). 2The intestines of the shrimp looks empty and white due to the lack of food (Figure 1D). The peeling of the hepatopancreas makes the intestines full, it caused the appetite of the shrimp decreased and the process of nutrients absorption interrupted5
.
The color of the water which is used for the cultivation is thick-green due to the high organic material which comes from the unconsumed feed. Organic materials such as detritus and mineral can be used as a food source to heterotrophic organisms like plankton to stimulate the autotrophic production6. There are only about 22% nitrogen of the feed which are used to form the body of the shrimp and the rest are disposed into the environment in the form of organic nitrogen and ammonium (NH4 +). Those materials are good for the growth of plankton7. Figure 1. Clinical signs of WFS infection. (A) Long-white feces which looks like threads floating on the surface of the water, (B) the water color which is thick-green, (C) pale-white hepatopancreas, (D) empty intestines. Table 1. Vibrio spp total on the hemolymph, hepatopancreas and intestines of the infected vannamei shrimp.
Replication
Fish Pond
Vibrio spp. total
Hemolymph (CFU/ml)
Hepatopancreas ( CFU/g)
Intestines (CFU/g)
1
2,5 x 103
8,3 x 107
3,3 x 105
I
2
1,3 x 103
4,24 x 107
3,36 x 107
3
8,0 x 104
5,8 x 107
9,2 x 107
1
1,5 x 103
3,3 x 107
5,1 x 107
II
2
1,1 x 103
2,5 x 107
2,3 x 107
3
8,59 x 104
2,3 x 107
4,0 x 107
Average Value
2,9 x 104
4,4 x 107
3,9 x 107
The average total of the Vibrio spp. in hemolymph is 2.9 x 104 CFU/ml, whereas in hepatopancreas is 4, 4x107 CFU/ml and intestines is 3, 9x107 (Table 1). The Vibrio spp. total on the infected vannamei shrimp on average is 8.505 x 104 CFU/g for hemolymph and 3.508 x 107 CFU/g for intestines. It is possible that the number and types of the Vibrio spp. bacteria can be found in Litopenaeus vannamei shrimp which is normal and healthy8. Various population of Vibrio spp are found in the intestines, stomach and hepatopancreas, ranging from 2x102– 3x103 CFU/ml. 2x106 CFU/g8. It is also reported that the high amount of bacteria in hemolymph shows a common septicemia which is found on a sick animal9. Table 2. Bio chemical test on hemolymph isolates, intestines and hepatopancreas of the infected vannamei shrimp.
Biochemical Test
HM.1
HM.2
SS.1
SS.2
HP.1
HP.2
TCBS
+
+
+
+
+
+
Shape
btg
btg
btg
btg
btg
btg
Cat Gram
─
─
─
─
─
─
Swaming
─
─
─
─
─
─
Growth with 0% NaCl
─
─
─
─
+
─
Arginine decarboxilase
─
+
─
─
─
─
Lysine decarboxilase
+
─
+
+
+
+
Ornithine decarboxilase
+
─
─
+
+
+
Nitrat reduced
+
+
+
+
+
+
Oxidase
+
+
+
+
+
+
Gas from Glucose
─
─
─
─
─
─
Indol
+
+
+
+
+
+
ONPG
+
+
+
─
+
─
MR
+
+
+
+
+
+
VP
─
─
─
─
─
─
Simmon citrat
+
+
+
─
+
─
Gelatinase
+
+
+
+
+
+
Resisten to :
0/129 10 µg
+
+
+
+
+
+
0/129 150 µg
─
─
─
─
─
─
ampicillin 10 µg
─
─
─
─
─
─
Starch Hydrolysis
─
+
─
+
+
+
Urea Hydrolysis
─
─
─
─
+
─
Acid from :
L-arabinose
─
─
─
+
─
─
Arbutin
─
─
─
─
+
─
Salicin
+
─
+
─
+
+
Sucrose
+
+
+
─
+
─
Xylose
─
─
+
─
─
─
Growth on :
Ethanol
─
─
─
─
─
─
Propanol
─
─
─
─
─
─
The identification result of the Vibrio spp. bacteria from 6 isolates (Table 2) is HM1 and HM2 isolates, it is close with the strains of V. vulnificus and V. anguillarum, the isolates from SS1 and SS2 is identified as V. vulnificus, V. parahaemolyticus, while Hp.1 and Hp.2. has the similarities with V. parahaemolyticus and V. chollera. It is reported that V. parahaemolyticus once attacked the vannamei shrimp in China and Thailand10
. Vibrio parahaemolyticus is a species of shrimp pathogenic bacteria which is injected with v. parahaemolyticus with the concentration of 104 CFU/ml to kill half of the shrimp population in 7 days11
. It is also reported that V. parahaemolyticus has infected the vannamei shrimp with the clinical symptoms such as black stripes on the side of the chephalothorax12
. Vibrio parahaemolyticus is the cause of the intestinal epithelium damage of the penaeidae shrimp (Martin et al., 2004). Cao et. al,. (2015concluded that the White Feces Syndrome is caused by the v. chollerae bacteria, it is based on the comparison between the genetic and phenotype characteristics of the v. chollerae bacteria which is isolated from the infected vannamei shrimp and the other isolated v. chollerae bacteria. There are three (3) types of Vibrio spp which are found from the bacteriological analysis on the hemolymph of the tiger prawn which is infected by white gut disease, such as V. harveyi, V. alginolyticus, and V. Anguillarum. The average total density of the Vibrio is 0,2 x 105 CFU/ml (Jayasree et al., 2006).
To conclude, these studies provide such indication that the infected vannamei shrimp is due to the Vibrio spp. bacteria which consists of V. parahaemolyticus, V. vulnificus, V.chollerae and V. anguillarum. The average total of Vibrio spp. on hemolymph is 2.9 x 104 CFU/ml, whereas in hepatopancreas is 4,4x107 CFU/ g and in intestines is 3,9x107 CFU/g. References
1. Limsuwan, C. White Faeces Disease in Thailand. 2010.
www.nicovita.com
. Accessed on April 23, 2015.
2. Sriurairatana, S., V. Boonyawiwat, W. Gangnonngiw, C. Laosutthipong. J. Hiranchan, T. Flegel. White feces syndrome of shrimp arises from transformation, sloughing and aggregation of hepatopancreatic microvilli into vermiform bodies superficially resembling Gregarines. PLOS ONE\www.plosone.org, 2014, Vol. 9 issue 6.
3. Cao, H., L. Wen., S. He., L. Lu., X. Yang., B. Chen. Vibrio chollerae: a causal agent for the white feces syndrome in freshwater cultured whiteleg shrimp (Penaeus vannamei). The Israeli Journal of Aquaculture – Bamidgeh, 2015, p.1-11.
4. Cowan, S.T. Manual for the identification of medical bacteria, 2nd edition. Cambridge. University Press. London, 1974, p.238.
5. Mastan, S. A. Incidences of White Feces Syndrome (WFS) in Farm-Reared Shrimp, Litopenaeusvannamei, Andhra Pradesh. Indo American Journal of Pharmaceutical Research, 2015, Vol 5(9), p.3044-3047.
6. Boyd C.E., C.S Tucker. Pond Aquaculture Water Quality Management. Kluwer Academic Publishers. Masachusetts, USA, 1998.
7. Burford, M. A., K.C. Williams. The fate of nitrogenous waste from shrimp feeding. Aquaculture, 2001, 198, 79-93.
8. Gomes-gill, B.,Tron-maye’n, A.Roque, J.F Turnbull, V. Inglis, A.L. Guerra-Flores. Species of vibrio isolated from hepatopancreas, haemolymph and digestive tract of population of heathy juvenile penaeus vannamei. Aquaculture, 1998, 163:1-9
9. Lightner, D.V. and R.M. Redman. Shrimp disease and current diagnostic methods. Aquaculture, 1998, 164,201-220
10. Sudheesh P.S., K. Jie, and H.S. Xu. Random Amplified Polymorphic DNA-PCR Typing of Vibrio parahaemolyticus and V. alginolyticus Isolated from Cultured Shrimps. Aquaculture, 2002, 207:11–17.
11. Martin G.G., Rubin, N., Swanson, E. Vibrio parahaemolyticus and V. harveyi cause detachment of the epithelium from the midgut trunk of the penaeid shrimp Sicyonia ingentis. Department of Biology, Occidental College, Los Angeles, California 90041, USA. Diseases of Aquatic Organisms Dis Aquat Org, 2004, Vol. 60: 21–29, 2004
12. Longyant, S., S. Rukpratanporn, P. Chaivisuthangkura, P. Suksawad, C. Srisuk, W. Sithigorngul, S. Piyatiratitivorakul, and P. Sithigorngul. Identification of Vibrio spp. in Vibriosis Penaeus vannamei Using Developed Monoclonal Antibodies. Journal of Invertebrate Pathology, 2008, 98: 63–68.