In shrimp, Acute Hepatopancreatic Necrosis Disease (AHPND) has caused remarkable mortality (up to 100%) in populations of shrimp cultured in South East Asian and Latin American countries. This disease is referred to as early mortality syndrome (EMS). AHPND is caused by several
The Tam Giang lagoon in Thua Thien Hue province of Vietnam is the biggest lagoon in the Southeast Asia. Aquaculture has developed in the Tam Giang Lagoon since the late 1970s, and has become the most important livelihood activity since the early 1990s. However, aquaculture production decreased continuously since 2009 until now. The main reason is because of the disease outbreak, natural disasters, climate change and the environment issues, especially water pollution has serious impacts on the health of the aquatic animals. In addition, the increased intensity of the flood seriously affects aquaculture production through changing of water quality and salinity. Many farmers lost their production due to serious Vibrio bacterial and viral shrimp diseases related to increasing water temperature
Randomly amplified polymorphic DNA (RAPD) is a PCR-based genotyping technique, using random primers to detect changes in the DNA sequence. According to Behura et al (2015), RAPD was a reliable and fast technique for discriminating between the species of Vibrio, therefore, this is a powerful tool for these prawn pathogens study
In Thua Thien Hue, to our knowledge there is limited published data available on the genetic diversity and toxic genes analysis of
Seventy samples of shrimp (body weight of 0,64 g ± 0,22 and length of 4,37 cm ±0.29 Hepatopancreatic Necrosis Disease syndromes were collected from white-leg shrimp (
Thirty-two samples of fishes including seabass (
After 24 hours, the development of colonies was checked. The types of colonies on plates were distinguished based on color, shape and size of colonies. The dominant colonies were transferred to TCBS medium to select pure colonies for further studies.
A total of 120 Vibrio spp. colonies were isolated on TBSC medium from shrimps (54 colonies) and fishes (66 colonies) in Thua Thien Hue province, Vietnam for toxic genes determination.
The presence of toxin genes in
PCR procedure: 50 ng of total DNA, 10 pmol of each primer, 6 µL 2× Go Taq® Green Master Mix (M7502, Promega, USA), and sterile distilled water (total volume of 12 µL). PCR amplification was performed in MJ Mini ™ Thermal Cycler (Bio-Rad, USA) as follow: 95°C for 10 minutes; followed by 30 cycles at 95°C for 30 seconds, 53°C for 30 seconds, and 72°C for 1 minute; last cycle was 72°C for 10 minutes. PCR products were used for electrophoresis on 0.8% agarose gel, stained by SafeView™ Classic Nucleic Acid Stain (Applied Biological Materials Inc., Canada) and determined by Ultra Slim LED Illuminator system.
Genes | Primer names | Nucleotide sequences | Size (bp) | References |
---|---|---|---|---|
pirAvp | VpPirA-284 | 5’-TGACTATTCTCACGATTGGACTG-3’ 5’-CACGACTAGCGCCATTGTTA-3’ | 284 | 1 |
pirBvp | VpPirB-392 | 5’-TGATGAAGTGATGGGTGCTC-3’ 5’-TGTAAGCGCCGTTTAACTCA-3’ | 392 | 1 |
tlh | tl | 5’-AAAGCGGATTATGCAGAAGCACTG-3’ 5’-GCTACTTTCTAGCATTTTCTCTGC-3’ | 450 | 7 |
trh | L-trh R-trh | 5’-TTGGCTTCGATATTTTCAGTATCT-3’ 5’-CATAACAAACATATGCCCATTTCCG-3’ | 410 | 5 |
tdh | L-tdh R-tdh | 5’-GTAAAGGTCTCTGACTTTTGGAC-3’ 5’-TGGAATAGAACCTTCATCTTCACC-3’ | 245 | 5 |
Genomic DNA were used as template for RAPD analysis, including 14 strains with
PCR-RAPD amplicons were scored as presence (1) or absence (0) of a band in each sample. The sizes of the RAPD markers were estimated by using the DNA size standard (GeneRuler 1kb DNA Ladder, Thermo Scientific). The genetic identity and genetic distance between populations were expressed using Nei’s (1972) genetic distance
From 120
In shrimp, the pVPA3-1 plasmid causes AHPND was identified in
Sample codes | Strain | Colonies morphology | Location | Genes | ||
---|---|---|---|---|---|---|
pirA | pirB | |||||
1 | VT19 | V. parahaemolyticus TX07-3/3 | Green, small center, serrated edges | Phu Thuan | + | + |
2 | VT23 | V. shilonii TX05-3/3 | Green, big center, non-round edges | Phu Thuan | + | + |
3 | VT26 | V. shilonii TX03-3/3 | Green, non-center, round edges | Phu Thuan | + | + |
4 | VT27 | V. shilonii TX02-3/3 | Green, big center, round, medium from green to yellow | Phu Thuan | + | + |
5 | VT29 | V. shilonii TX01-3/3 | Green, dark center, round | Phu Thuan | + | + |
6 | VT33 | V. shilonii TV02-3/3 | Yellow, big center, round, medium from green to yellow | Phu Thuan | + | + |
7 | VT34 | V. parahaemolyticus 29X2 - 13/5 | Green, round, smooth, 2 mm diameter | Dien Huong | - | - |
8 | VT41 | V. parahaemolyticus K31-X-13/6/2019 | Green to yellow, round, smooth, 2 mm diameter, medium from green to yellow | Dien Huong | + | + |
9 | VT44 | V. parahaemolyticus K39-X-13/6 | Green to yellow, round, smooth, 2 mm diameter | Dien Huong | + | + |
10 | VT47 | V. communis R-PD-K4-V-13/6 | Yellow, round, smooth, 2 mm diameter, medium from green to yellow | Dien Mon | + | + |
11 | VT56 | V. parahaemolyticus PD-K1-10/6 | Green, round, smooth, 2 mm diameter | Dien Mon | - | - |
12 | VT59 | V. parahaemolyticus R-PD-K3-10/6 | Yellow, round,1.5 mm diameter, medium from green to yellow | Dien Mon | + | + |
13 | VT62 | V. parahaemolyticus R-PD-K3-10/6 | Green, round, smooth, 2 mm diameter | Dien Mon | + | + |
14 | VT65 | V. furnissii R-K39-13/6 | Yellow, round, 2 mm diameter | Dien Huong | + | + |
15 | VX01 | V. parahaemolyticus VX01 | Green, viscous, round, smooth, 2 mm diameter | Dien Huong | + | + |
16 | K5 | V. parahaemolyticus K5 | Green, round, convex | Dien Huong | + | + |
In fish, the majority of
The previous studies in Malaysia showed that the most virulent of the non-cholera vibrios (
Sample codes | Strains | Colonies morphology | Location | Organs | Host | Genes | |||
---|---|---|---|---|---|---|---|---|---|
tlh | tdh | trh | |||||||
1 | VC15 | V. vulnificus HM-TA-D2-L2-V2 | Yellow, medium from green to yellow | Thuan An | Skin | Sciaenops ocellatus | + | - | - |
2 | VC17 | V. vulnificus HM-TA-G2-V1-Đ2 | Yellow, medium from green to yellow | Thuan An | Liver | Sciaenops ocellatus | + | - | - |
3 | VC21 | V. vulnificus CTX1-3/3 | Green, non-center, round | Hai Duong | Kidney | Lates calcarifer | + | - | - |
4 | VC24 | V. harveyi CDV1-3/3 | Yellow, non-center, serrated edges, medium from green to yellow | Hai Duong | Skin | Lates calcarifer | + | - | - |
5 | VC25 | V. harveyi CTV1-3/3 | Yellow | Hai Duong | Kidney | Lates calcarifer | + | - | - |
6 | VC28 | V. vulnificus CTX3-3/3 | Green, small center, non-round edges | Hai Duong | Kidney | Lates calcarifer | + | - | - |
7 | VC35 | V. vulnificus LTA.CC.X.01-24/3 | Green, non-center, non-round edges, smooth | Thuan An | Kidney | Lates calcarifer | - | - | - |
8 | VC37 | V. vulnificus LTA.CC.X.02 | Green, non-center, non-round edges, smooth, medium from green to yellow | Thuan An | Kidney | Lates calcarifer | + | - | - |
9 | VC39 | V. parahaemolyticus LTA.CC.X.03 | Green, big-center, round, smooth, medium from green to yellow | Thuan An | Kidney | Lates calcarifer | + | - | - |
10 | VC41 | V. parahaemolyticus LTA.CC.X.04 | Green, big-center, non-round edges, smooth | Thuan An | Kidney | Lates calcarifer | + | - | - |
11 | VC43 | V. cholerae LTA.CC.V.01 | Yellow, small-center, round, smooth, medium from green to yellow | Thuan An | Kidney | Lates calcarifer | + | - | - |
12 | VC45 | V. harveyi LTA.CC.V.02 | Yellow, dark center, non-round edges, rough, medium from green to yellow | Thuan An | Kidney | Lates calcarifer | + | - | - |
13 | VC52 | V. brasiliensis HM-X-13/6 | Green, round, 2 mm | Hai Duong | Kidney | Sciaenops ocellatus | + | - | - |
14 | VC53 | V. cholerae V-13/6 | Yellow, round, smooth, 3 mm diameter | Hai Duong | Spleen | Sciaenops ocellatus | + | - | - |
15 | VC59 | V. parahaemolyticus HM-17/6 | Green, round, 2 mm diameter | Hai Duong | Gut | Sciaenops ocellatus | + | - | - |
16 | VC61 | V. cholerae HM-V-13/6 | Green, round, 1 mm diameter, medium from green to yellow | Hai Duong | Skin | Sciaenops ocellatus | + | - | - |
17 | VC67 | V. vulnificus HM-X-13/6 | Green, round, 4 mm diameter | Hai Duong | Skin | Sciaenops ocellatus | + | - | - |
18 | VC72 | V. fluvialis ML2-13/8 | Yellow, non-center, 4 mm diameter, medium from green to yellow | Thuan An | Spleen | Epinephelus fuscoguttatus | + | - | - |
19 | VC81 | V. natriegens MT6-13/8 | Yellow, non-round edges, 4 mm diameter, medium from green to yellow | Thuan An | Kidney | Epinephelus fuscoguttatus | + | - | - |
20 | VC85 | V. parahaemolyticus MD8-13/8 | Yellow, round, 2 mm diameter, medium from green to yellow | Thuan An | Skin | Epinephelus fuscoguttatus | - | - | - |
Vibrio spp. that caused disease in shrimp and marine fish are widely distributed in brackish and saline waters worldwide. They exist in the aquatic environment and can be harmful to shrimp and fish in favorable conditions.
In shrimp, the presence of
Although bacteria can be infected in different parts, they often attack the heart and muscles of the fish. Because they have a strong ability to attack the muscular system, hence it is very serious and difficult to treat, bacteria can cause chronic diseases in adults. If the disease occurs acute, the consequences of damage are very heavy.
In fish larvae, fry or fingerlings, when seriously infected they can be killed up to 50%. In adult fish, the rate of damage is lower but the fish will develop anorexia or stop eating or ceases in growth and at harvest it is possible to observe necrotic wounds on the skin and muscles of the fish. When the fish were infected with Vibrio, fish eats less or stops eating, they swim on the surface layer and around the cage edge. On the fish body, often appear various small reddish ulcers with the skin bulging surrounding it and a lot of viscous. The internal organs, liver, kidney, spleen with hemorrhage would have occurred and empty gastrointestinal tract
Restrepo et al. (2018) reported that the gene transfer capacity of
A total of 20 RAPD primers were assayed for their specificity in detecting
Primer code | Nucleotide sequence (5’-3’) | Number of amplification genotypes | Size range of amplified bands (bp) | Number of amplified bands |
---|---|---|---|---|
OPA-03 | AGTCAGCCAC | 32 | 250-3500 | 15 |
OPA-11 | CAATCGCCGT | 35 | 350-3100 | 16 |
OPB-01 | GTTTCGCTCC | 33 | 500-4000 | 16 |
OPB-18 | CCACAGCAGT | 34 | 200-2500 | 15 |
OPC-13 | AAGCCTCGTC | 31 | 320-3500 | 13 |
OPD-02 | GGACCCAACC | 34 | 300-3300 | 17 |
OPD-07 | TTGGCACGGG | 35 | 320-2700 | 11 |
OPG-17 | ACGACCGACA | 36 | 320-3000 | 15 |
OPN-03 | GGTACTCCCC | 30 | 350-4000 | 13 |
OPN-06 | GAGACGCACA | 35 | 250-3000 | 17 |
Overall | 200-4000 | 148 | ||
Mean | 14.80 |
In this study, the percentage of polymorphic bands (PPB) of each population was ranged from 25.80% (
Species | Number of PCR amplicons | Number of polymorphic loci | Percentage of polymorphic bands | Number of specific loci |
---|---|---|---|---|
V. parahaemolyticus | 105 | 105 | 100 | 0 |
V. vulnificus | 61 | 61 | 100 | 0 |
V. shilonii | 31 | 8 | 25.80 | 1 |
V. harveyi | 55 | 42 | 76.36 | 3 |
V. cholerae | 45 | 33 | 73.33 | 0 |
Others* | 97 | 96 | 98.97 | 0 |
Mean | 65.67 | 57.5 | 79.08 | 0.67 |
Overall | 4 |
*Others: samples including
Data for observed number of alleles (na), effective number of alleles (ne), Nei’s (1973) genetic diversity (h), Shannon’s information index (I), for all the
Populations | na | ne | h | I |
---|---|---|---|---|
V. parahaemolyticus | 1.7095 | 1.2440 | 0.1645 | 0.2695 |
V. vulnificus | 1.4122 | 1.2047 | 0.1247 | 0.1925 |
V. shilonii | 1.0405 | 1.0221 | 0.0136 | 0.0207 |
V. harveyi | 1.2838 | 1.1782 | 0.1054 | 0.1574 |
V. cholerae | 1.2230 | 1.1558 | 0.0885 | 0.1298 |
Others | 1.6419 | 1.2560 | 0.1719 | 0.2768 |
Overall | 2.0000 | 1.2885 | 0.2046 | 0.3435 |
Standard deviation (SD) | 0.0000 | 0.2139 | 0.1195 | 0.1612 |
The average diversity within populations (Hs) was 0.1114, which accounted for 51.72% of the total diversity found in the populations (Ht = 0.2154). The mean coefficient of gene differentiation (Gst) value of 0.4827 indicated a very high degree of genetic differentiation among species. The gene flow (Nm) was 0.5359, which indicated that it was small among the species (
Parameters | Ht | Hs | Gst | Nm |
---|---|---|---|---|
Across all populations | 0.2154 | 0.1114 | 0.4827 | 0.5359 |
SD | 0.0176 | 0.0048 |
The values of genetic identity and genetic distance between
Populations | V. parahaemolyticus | V. vulnificus | V. shilonii | V. harveyi | V. cholerae | Others |
---|---|---|---|---|---|---|
V. parahaemolyticus | **** | 0.9545 | 0.8196 | 0.8840 | 0.9024 | 0.9582 |
V. vulnificus | 0.0465 | **** | 0.8375 | 0.8838 | 0.8791 | 0.9474 |
V. shilonii | 0.1989 | 0.1774 | **** | 0.7416 | 0.7579 | 0.8381 |
V. harveyi | 0.1233 | 0.1235 | 0.2989 | **** | 0.7897 | 0.8670 |
V. cholerae | 0.1027 | 0.1289 | 0.2772 | 0.2361 | **** | 0.8794 |
Others | 0.0427 | 0.0541 | 0.1766 | 0.1427 | 0.1285 | **** |
In the genetic similarity dendrogram constructed on the basis of comparative analysis of the total loci obtained with the ten RAPD primers, the
In our study, 120
This study was financial supported by the grant from Vietnam Ministry of Education and Training (Project No. CT-2018-DHH-01).