《渔业科学进展》虚拟专辑——对虾疾病：副溶血弧菌(Vibrio parahaemolyticus)和对虾急性肝胰腺坏死病(AHPND)
副溶血弧菌(Vibrio parahaemolyticus)是一种革兰氏阴性嗜盐菌，广泛存在于养殖水域、底泥和沉积物中，附着于海洋生物的体表进行生长繁殖，为重要的病原性弧菌，其引发的水产动物疾病不仅会对水产养殖业造成重大的经济损失，还对人体健康构成潜在威胁。目前，副溶血弧菌已成为世界上多个沿海国家和地区急性胃肠炎的重要病原菌之一，严重者可引起伤口感染和败血症等。
对虾急性肝胰腺坏死病(AHPND)是由致AHPND副溶血弧菌(VpAHPND)携带的pVA1-like质粒所表达的PirAVp和PirBVp毒力蛋白对对虾肝胰腺的急性毒性所致。
编辑部整理了近年来发表在本刊上的10篇关于副溶血弧菌和对虾急性肝胰腺坏死病的研究论文，组成本期虚拟专辑，欢迎阅读、引用。
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2020, 41(6):174-180.
DOI: 10.19663/j.issn2095-9869.20190820003
Abstract:
To understand the drug resistance and virulence genes of Vibrio parahaemolyticus in shrimp culture ponds, we collected and isolated V. parahaemolyticus from shrimp culture ponds in four different areas of Shandong Province in 2018. The Kirby-Bauer disk method was used to detect the resistance of V. parahaemolyticus to 12 antibiotics, and PCR was used to detect the heat-resistant direct hemolysin gene (tdh) and heat-resistant-related hemolysin gene (trh). A total of 50 V. parahaemolyticus strains were isolated from the shrimp culture ponds. Drug susceptibility test showed that the resistance of V. parahaemolyticus to gentamicin, neomycin sulfate, and ampicillin were high at 98%, 90%, and 86%, respectively. The susceptibility rates to florfenicol, chloramphenicol, and ceftazidime were higher, and the resistance rates were 10%, 10%, and 20%, respectively. Overall, 88% of the strains showed multidrug resistance. The virulence gene test showed that all the strains lacked the tdh gene and 4% of the strains were trh-positive. This study suggests that the antibiotic resistance of V. parahaemolyticus in a shrimp aquaculture environment is concerning. Etiological surveillance of V. parahaemolyticus should be strengthened, and antibiotics should be reasonably used in the aquaculture process to realize the healthy development of the aquaculture industry.
To understand the drug resistance and virulence genes of Vibrio parahaemolyticus in shrimp culture ponds, we collected and isolated V. parahaemolyticus from shrimp culture ponds in four different areas of Shandong Province in 2018. The Kirby-Bauer disk method was used to detect the resistance of V. parahaemolyticus to 12 antibiotics, and PCR was used to detect the heat-resistant direct hemolysin gene (tdh) and heat-resistant-related hemolysin gene (trh). A total of 50 V. parahaemolyticus strains were isolated from the shrimp culture ponds. Drug susceptibility test showed that the resistance of V. parahaemolyticus to gentamicin, neomycin sulfate, and ampicillin were high at 98%, 90%, and 86%, respectively. The susceptibility rates to florfenicol, chloramphenicol, and ceftazidime were higher, and the resistance rates were 10%, 10%, and 20%, respectively. Overall, 88% of the strains showed multidrug resistance. The virulence gene test showed that all the strains lacked the tdh gene and 4% of the strains were trh-positive. This study suggests that the antibiotic resistance of V. parahaemolyticus in a shrimp aquaculture environment is concerning. Etiological surveillance of V. parahaemolyticus should be strengthened, and antibiotics should be reasonably used in the aquaculture process to realize the healthy development of the aquaculture industry.
2020, 41(3):133-141.
DOI: 10.19663/j.issn2095-9869.20190317001
Abstract:
In this culture experiments of Litopenaeus vannamei, we explore the effect of compound probiotics on the ability of L. vannamei to resist Vibrio parahaemolyticus infection at different experimental stages. One strain of Bacillus licheniformis (BL-9), one B. subtilis (BS-12), and one Pseudoalteromonas flavipulchra (CDM8) were selected to make up a compound of probiotics at the same concentration (107 CFU/ml). The effect of compound probiotics on the V. parahaemolyticus infection of L. vannamei was observed by adding the compound probiotics to the culture water of the L. vannamei. The experiment took place over 30 d, including a temporary period (7 d), a probiotics immersion period (15 d), and a V. parahaemolyticus infection period (10 d). The experiment results showed that the addition of compound probiotics in the culture water could significantly increase the total number of bacteria culture in the water and the intestinal tract of L. vannamei (P<0.05). The cumulative survival rate of shrimp in the experimental group was (73.33±6.83)% at the end of the infection periodwhich was significantly higher than that of the positive control group (25.33±15.43)%. The disease-resistant-related gene: heat shock protein 70 (Hsp70), β-1,3-glucan binding protein-lipoprotein (βGBP-HDL), lipopolysaccharide-β-1, 3-glucan binding protein(LGBP), crustin, and immune related enzymes catalase (CAT), was up-regulated by different degrees in the probiotics immersion phase. And all the genes up-regulated extensively during the V. parahaemolyticus infection stage, but expressed at different levels. The results suggested that the compound probiotics with BL-9, BS-12, and CDM8 in water could improve the ability of shrimp to resist V. parahaemolyticus infection. The increase of disease resistance of L. vannamei may be related to the colonization of probiotics in the intestinal tract, and the expression level of disease resistance related genes and catalase activity in L. vannamei. It is hoped that the results of this study can provide a reference for the application of compound probiotics in the cultivation of L. vannamei.
In this culture experiments of Litopenaeus vannamei, we explore the effect of compound probiotics on the ability of L. vannamei to resist Vibrio parahaemolyticus infection at different experimental stages. One strain of Bacillus licheniformis (BL-9), one B. subtilis (BS-12), and one Pseudoalteromonas flavipulchra (CDM8) were selected to make up a compound of probiotics at the same concentration (107 CFU/ml). The effect of compound probiotics on the V. parahaemolyticus infection of L. vannamei was observed by adding the compound probiotics to the culture water of the L. vannamei. The experiment took place over 30 d, including a temporary period (7 d), a probiotics immersion period (15 d), and a V. parahaemolyticus infection period (10 d). The experiment results showed that the addition of compound probiotics in the culture water could significantly increase the total number of bacteria culture in the water and the intestinal tract of L. vannamei (P<0.05). The cumulative survival rate of shrimp in the experimental group was (73.33±6.83)% at the end of the infection periodwhich was significantly higher than that of the positive control group (25.33±15.43)%. The disease-resistant-related gene: heat shock protein 70 (Hsp70), β-1,3-glucan binding protein-lipoprotein (βGBP-HDL), lipopolysaccharide-β-1, 3-glucan binding protein(LGBP), crustin, and immune related enzymes catalase (CAT), was up-regulated by different degrees in the probiotics immersion phase. And all the genes up-regulated extensively during the V. parahaemolyticus infection stage, but expressed at different levels. The results suggested that the compound probiotics with BL-9, BS-12, and CDM8 in water could improve the ability of shrimp to resist V. parahaemolyticus infection. The increase of disease resistance of L. vannamei may be related to the colonization of probiotics in the intestinal tract, and the expression level of disease resistance related genes and catalase activity in L. vannamei. It is hoped that the results of this study can provide a reference for the application of compound probiotics in the cultivation of L. vannamei.
2020, 41(2):150-158.
DOI: 10.19663/j.issn2095-9869.20190215001
Abstract:
The identification of reference genes is critical for the establishment of sensitive and reproducible qRT-PCR-based assays. The current study was designed to explore the effects on acute hepatopancreatic necrosis disease (AHPND)-causing Vibrio parahaemolyticus strains (VPAHPND) on the growth of shrimp in the presence of different concentrations of Sanguisorba officinalis L. alcoholic extracts and to select the optimal reference genes suitable for the evaluation of the inhibitory effect of S. officinalis L. on VPAHPND. The expression of six common candidate reference genes (rec A, pvs A, pvu A, gapdh, 16S rRNA, and rpo S) of VPAHPND under stress induced by S. officinalis L. alcoholic extracts were detected by qRT-PCR. Data analysis was conducted using the GeNorm, Norm Finder, Best Keeper, Delta CT, and Ref Finder software packages. The results showed that S. officinalis L. alcoholic extracts had a strong inhibitory effect on V. parahaemolyticus. The amplicons of these six genes had good specificity under the stress induced by different concentrations of S. officinalis L. extract. The lowest variation in Ct value was found for 16S rRNA (CV=3.88), and the highest variation occurred in pvs A (CV=12.53). The stability of the six reference genes judged by the five methods was as follows: The stability order results were: rpo S = 16S rRNA > gapdh > rec A > pvu A > pvs A from GeNorm; gapdh > rpo S > pvu A > 16S rRNA> rec A > pvs A from Norm Finder; 16S rRNA > rpo S > gapdh > rec A > pvu A > pvs A from Best Keeper; and gapdh > rpo S > pvu A > 16S rRNA > rec A > pvs A from Delta Ct. The comprehensive ranking result from by Ref Finder was rpo S > gapdh > 16S rRNA > pvu A >pvs A > rec A. After consideration of the pairwise variations, it is recommended to use both rpo S and gapdh as reference genes in these conditions. It was also revealed that the stability of reference genes differed between different strains and under different experimental conditions. With the improved experimental accuracy requirements, screening and verification of the appropriate reference gene has become an essential part of the experimental methodology. The results provide a foundation to support the study of the inhibitory mechanism of S. officinalis L. on VPAHPND through the perspective of gene expression. It is of great significance for the establishment of AHPND-prevention technology using S. officinalis L. as the core drug.
The identification of reference genes is critical for the establishment of sensitive and reproducible qRT-PCR-based assays. The current study was designed to explore the effects on acute hepatopancreatic necrosis disease (AHPND)-causing Vibrio parahaemolyticus strains (VPAHPND) on the growth of shrimp in the presence of different concentrations of Sanguisorba officinalis L. alcoholic extracts and to select the optimal reference genes suitable for the evaluation of the inhibitory effect of S. officinalis L. on VPAHPND. The expression of six common candidate reference genes (rec A, pvs A, pvu A, gapdh, 16S rRNA, and rpo S) of VPAHPND under stress induced by S. officinalis L. alcoholic extracts were detected by qRT-PCR. Data analysis was conducted using the GeNorm, Norm Finder, Best Keeper, Delta CT, and Ref Finder software packages. The results showed that S. officinalis L. alcoholic extracts had a strong inhibitory effect on V. parahaemolyticus. The amplicons of these six genes had good specificity under the stress induced by different concentrations of S. officinalis L. extract. The lowest variation in Ct value was found for 16S rRNA (CV=3.88), and the highest variation occurred in pvs A (CV=12.53). The stability of the six reference genes judged by the five methods was as follows: The stability order results were: rpo S = 16S rRNA > gapdh > rec A > pvu A > pvs A from GeNorm; gapdh > rpo S > pvu A > 16S rRNA> rec A > pvs A from Norm Finder; 16S rRNA > rpo S > gapdh > rec A > pvu A > pvs A from Best Keeper; and gapdh > rpo S > pvu A > 16S rRNA > rec A > pvs A from Delta Ct. The comprehensive ranking result from by Ref Finder was rpo S > gapdh > 16S rRNA > pvu A >pvs A > rec A. After consideration of the pairwise variations, it is recommended to use both rpo S and gapdh as reference genes in these conditions. It was also revealed that the stability of reference genes differed between different strains and under different experimental conditions. With the improved experimental accuracy requirements, screening and verification of the appropriate reference gene has become an essential part of the experimental methodology. The results provide a foundation to support the study of the inhibitory mechanism of S. officinalis L. on VPAHPND through the perspective of gene expression. It is of great significance for the establishment of AHPND-prevention technology using S. officinalis L. as the core drug.
HUANG Mengshi
,
YANG Qianqian
,
ZHANG Yan
,
JIANG Xiaoyu
,
ZHAO Jun
,
ZHANG Xuzhi
,
DING Dongsheng
,
QU Keming
2019, 40(2):132-140.
DOI: 10.19663/j.issn2095-9869.20180305002
Abstract:
Vibrio parahaemolyticus (VP), which is ubiquitous in the marine environment, is one of the key foodborne pathogens affecting humans. It is thus of great significance for both scientific research and public safety that we understand how and what environmental factors affect its growth. To determine the functional relationship between a combination of environmental factors and VP growth, in this study the effects of temperature, salinity, pH, phosphate, nitrate, and ammonium on the growth of this kind of bacteria were investigated using response surface methodology (RSM). First, a Box-Behnken design was used to optimize the three critical internal factors of temperature, salinity and pH for this species, using optical density (OD600 nm) as a marker for the growth of VP. It was found that for these factors, the optimum parameters were 34.5℃, 3%, and 8.0, respectively. Based on these results, the combined effects of phosphate, nitrate, and ammonium on the growth of VP were then further studied with the same method to develop a response model. The coefficient of determination (R2) of the model equation was 0.9217, while the correction coefficient (R2) was 0.8211. The regression model had an F-value of 9.16, and was thus statistically significant (P=0.004). These results indicated that the model proposed herein had satisfactory accuracy. Results obtained from verified testing were also in good agreement with those obtained with the model. Clearly, phosphate, nitrate, and ammonium all exhibited negative effects on the growth of VP. These outcomes are beneficial to the culture of VP, and even to the establishment of early warning systems for marine environments and seafood safety.
Vibrio parahaemolyticus (VP), which is ubiquitous in the marine environment, is one of the key foodborne pathogens affecting humans. It is thus of great significance for both scientific research and public safety that we understand how and what environmental factors affect its growth. To determine the functional relationship between a combination of environmental factors and VP growth, in this study the effects of temperature, salinity, pH, phosphate, nitrate, and ammonium on the growth of this kind of bacteria were investigated using response surface methodology (RSM). First, a Box-Behnken design was used to optimize the three critical internal factors of temperature, salinity and pH for this species, using optical density (OD600 nm) as a marker for the growth of VP. It was found that for these factors, the optimum parameters were 34.5℃, 3%, and 8.0, respectively. Based on these results, the combined effects of phosphate, nitrate, and ammonium on the growth of VP were then further studied with the same method to develop a response model. The coefficient of determination (R2) of the model equation was 0.9217, while the correction coefficient (R2) was 0.8211. The regression model had an F-value of 9.16, and was thus statistically significant (P=0.004). These results indicated that the model proposed herein had satisfactory accuracy. Results obtained from verified testing were also in good agreement with those obtained with the model. Clearly, phosphate, nitrate, and ammonium all exhibited negative effects on the growth of VP. These outcomes are beneficial to the culture of VP, and even to the establishment of early warning systems for marine environments and seafood safety.
2018, 39(5):106-113.
Abstract:
In this study, the effect of beta-glucan on survival rates and the mRNA expression of Lectin and TLR2 in both uninfected and infected Manila clams Ruditapes philippinarum were studied. The results showed that beta-glucan could effectively improve the survival rate of Manila clams infected by Vibrio parahaemolyticus. The survival rate was highest at a beta-glucan concentration of 1000 mg/L. In the gill tissues of the uninfected group (A1 and A2), the TLR2 peaked at 6 h, which was significantly higher than that of other times (P<0.05). In the infected group, TLR2 increased to a peak at 1.5 h and then decreased. Lectin expression in both the infected and the uninfected group increased first and then decreased. The relative expression of Lectin in the A1 group was significantly higher than that of the B1 group at 3 h (P<0.05). In the mantle, the expression of TLR2 in both infected and uninfected groups decreased gradually between 3~12 h. At 24 h, the expression of group A2 was highest. However, in the infected group, the expression of Lectin in 1000 mg/L beta-glucan was higher than at 100 mg/L in the mantle, but there was only a significant difference at 0 h and 24 h (P<0.05). The expression patterns of Lectin in the gill and in the mantle were different, but the feeding of beta-glucan promoted the expression of Lectin during the early stages of infection. From these results, beta-glucan soaking can increase the relative expression of the two genes, and TLR2 and Lectin are expressed more quickly after infection by V. parahaemolyticus when soaked with beta-glucan. The aim of this study is to understand the dose dependent effect conferred by beta-glucan on the immune system and the survival rate of Manila clams, which might provide some theoretical basis for the stock culture, seed breeding and disease control in pond culture of Manila clams.
In this study, the effect of beta-glucan on survival rates and the mRNA expression of Lectin and TLR2 in both uninfected and infected Manila clams Ruditapes philippinarum were studied. The results showed that beta-glucan could effectively improve the survival rate of Manila clams infected by Vibrio parahaemolyticus. The survival rate was highest at a beta-glucan concentration of 1000 mg/L. In the gill tissues of the uninfected group (A1 and A2), the TLR2 peaked at 6 h, which was significantly higher than that of other times (P<0.05). In the infected group, TLR2 increased to a peak at 1.5 h and then decreased. Lectin expression in both the infected and the uninfected group increased first and then decreased. The relative expression of Lectin in the A1 group was significantly higher than that of the B1 group at 3 h (P<0.05). In the mantle, the expression of TLR2 in both infected and uninfected groups decreased gradually between 3~12 h. At 24 h, the expression of group A2 was highest. However, in the infected group, the expression of Lectin in 1000 mg/L beta-glucan was higher than at 100 mg/L in the mantle, but there was only a significant difference at 0 h and 24 h (P<0.05). The expression patterns of Lectin in the gill and in the mantle were different, but the feeding of beta-glucan promoted the expression of Lectin during the early stages of infection. From these results, beta-glucan soaking can increase the relative expression of the two genes, and TLR2 and Lectin are expressed more quickly after infection by V. parahaemolyticus when soaked with beta-glucan. The aim of this study is to understand the dose dependent effect conferred by beta-glucan on the immune system and the survival rate of Manila clams, which might provide some theoretical basis for the stock culture, seed breeding and disease control in pond culture of Manila clams.
2018, 39(4):93-100.
Abstract:
Acute hepatopancreatic necrosis disease (AHPND) results from acute toxicity in the hepatopancreas of infected shrimp caused by the toxic proteins PirAVp and PirBVp, which are expressed by the pVA1 plasmid carried by AHPND-causing Vibrio parahaemolyticus (VpAHPND). In this study, Litopenaeus vannamei were exposed to 2.19×105 CFU/ml VpAHPND strain 20130629002S01 by immersion to explore the dynamic changes of VpAHPND in the tissues of shrimp. The hepatopancreas, gills, midgut, and muscle of infected shrimp were collected 2~9 days after immersion infection, and the quantity of pirAVP was measured by qPCR. The results showed that VpAHPND could be detected in all sampled tissues of infected shrimp. The amount of VpAHPND in the hepatopancreas reached a peak on day 4 post-infection at 8.71×104 copies/mg, while the gills, muscle, and midgut reached peaks on day 3, 4, and 5 post-infection at 9.08×103、2.59×104、5.76×104 copies/mg, respectively. The highest amount of VpAHPND was detected in the gills during the early stage of infection, followed by the hepatopancreas and midgut in sequence during heavy disease, with frequent deaths. Subsequently, the amount of VpAHPND declined rapidly in all tissues, with similar levels in the midgut, hepatopancreas, and muscle. Histopathology revealed that AHPND lesions were denser in hepatopancreas samples from moribund shrimp compared with those from morbid shrimp, when taken at the same time from infection. Furthermore, the histopathologic symptoms of both became more severe along the infection process, but with decreasing levels of VpAHPND. The results showed that the copy number of pirAVp in tissues of VpAHPND-infected shrimp does not represent the real-time condition of diseased shrimp and the quantity of VpAHPND may not be high in a severe AHPND sample.
Acute hepatopancreatic necrosis disease (AHPND) results from acute toxicity in the hepatopancreas of infected shrimp caused by the toxic proteins PirAVp and PirBVp, which are expressed by the pVA1 plasmid carried by AHPND-causing Vibrio parahaemolyticus (VpAHPND). In this study, Litopenaeus vannamei were exposed to 2.19×105 CFU/ml VpAHPND strain 20130629002S01 by immersion to explore the dynamic changes of VpAHPND in the tissues of shrimp. The hepatopancreas, gills, midgut, and muscle of infected shrimp were collected 2~9 days after immersion infection, and the quantity of pirAVP was measured by qPCR. The results showed that VpAHPND could be detected in all sampled tissues of infected shrimp. The amount of VpAHPND in the hepatopancreas reached a peak on day 4 post-infection at 8.71×104 copies/mg, while the gills, muscle, and midgut reached peaks on day 3, 4, and 5 post-infection at 9.08×103、2.59×104、5.76×104 copies/mg, respectively. The highest amount of VpAHPND was detected in the gills during the early stage of infection, followed by the hepatopancreas and midgut in sequence during heavy disease, with frequent deaths. Subsequently, the amount of VpAHPND declined rapidly in all tissues, with similar levels in the midgut, hepatopancreas, and muscle. Histopathology revealed that AHPND lesions were denser in hepatopancreas samples from moribund shrimp compared with those from morbid shrimp, when taken at the same time from infection. Furthermore, the histopathologic symptoms of both became more severe along the infection process, but with decreasing levels of VpAHPND. The results showed that the copy number of pirAVp in tissues of VpAHPND-infected shrimp does not represent the real-time condition of diseased shrimp and the quantity of VpAHPND may not be high in a severe AHPND sample.
JIA Dan
,
HI Chengyin
,
HUANG Jie
,
ZHANG Qingli
,
WAN Xiaoyuan
,
XU Hua
,
LIU Ranyang
,
WANG Haibo
,
GUO Chengcheng
,
XIE Guosi
2018, 39(3):103-111.
Abstract:
Acute hepatopancreatic necrosis disease (AHPND) is an emerging shrimp disease causing great losses for the shrimp culture industry worldwide since 2010. In the present study, a bacterial strain 20160303005-1 was isolated from the hepatopancreas tissue of Litopenaeus vannamei with early mortality syndrome (EMS), and was identified as Vibrio parahaemolyticus based on its physiological and biochemical characteristics; and the analysis of both 16S rRNA and groEL gene sequence. The serotype of the bacterium is O1:KUT (K untypeable). It revealed positive amplification of the genes pirAVP and pirBVP which is related to cause AHPND in a virulence plasmid harboring this strain. However, the isolate examined showed negative amplification results for the virulent clinical V. parahaemolyticus strain markers—thermostable direct hemolysin gene tdh and TDH-related hemolysin gene trh. The immersion challenge test with L. vannamei was also employed to study pathogenicity and histopathology. The results showed that the isolate was highly virulent, with a median lethal dose (LD50) value of 7.96×103 CFU/ml. The empty gut in shrimp was observed at 6 h post-challenged. The hepatopancreas appeared pale and atrophic at 9 h. More than half of the shrimps died at 12 h, and up to 100% died at 24 h. Subsequent histological analyses showed that the hepatopancreas tubules collapsed with massive sloughing of hepatopancreas epithelial cells, which was the typical pathological characteristics of AHPND. Among 21 antibiotics tested, the isolate was resistant to amoxicillin, cefalotin, ticarcillin, cefuroxime, and cotrimoxazol; however, it was sensitive to gentamicin, ciprofloxacin, and other 14 antibiotics tested. These results provide basic data for epidemiology and drug control research on V. parahaemolyticus in aquaculture.
Acute hepatopancreatic necrosis disease (AHPND) is an emerging shrimp disease causing great losses for the shrimp culture industry worldwide since 2010. In the present study, a bacterial strain 20160303005-1 was isolated from the hepatopancreas tissue of Litopenaeus vannamei with early mortality syndrome (EMS), and was identified as Vibrio parahaemolyticus based on its physiological and biochemical characteristics; and the analysis of both 16S rRNA and groEL gene sequence. The serotype of the bacterium is O1:KUT (K untypeable). It revealed positive amplification of the genes pirAVP and pirBVP which is related to cause AHPND in a virulence plasmid harboring this strain. However, the isolate examined showed negative amplification results for the virulent clinical V. parahaemolyticus strain markers—thermostable direct hemolysin gene tdh and TDH-related hemolysin gene trh. The immersion challenge test with L. vannamei was also employed to study pathogenicity and histopathology. The results showed that the isolate was highly virulent, with a median lethal dose (LD50) value of 7.96×103 CFU/ml. The empty gut in shrimp was observed at 6 h post-challenged. The hepatopancreas appeared pale and atrophic at 9 h. More than half of the shrimps died at 12 h, and up to 100% died at 24 h. Subsequent histological analyses showed that the hepatopancreas tubules collapsed with massive sloughing of hepatopancreas epithelial cells, which was the typical pathological characteristics of AHPND. Among 21 antibiotics tested, the isolate was resistant to amoxicillin, cefalotin, ticarcillin, cefuroxime, and cotrimoxazol; however, it was sensitive to gentamicin, ciprofloxacin, and other 14 antibiotics tested. These results provide basic data for epidemiology and drug control research on V. parahaemolyticus in aquaculture.
2016, 37(3):78-84.
DOI: 10.11758/yykxjz.20150519001
Abstract:
Three bacterial strains, 20131023A00, 20131023A01, and 20131023A05, were isolated from shrimp guts and the farming ponds using inorganic selecting medium. When cultured in the liquid selecting medium (pH=7.2) containing 0.12% ammonia nitrogen at 28℃ for 24 h, the ammonia conversion rates of the three strains were (38.9±0.1)%, (43.1±0.4)%, and (49.9±0.5)% respectively. Using the paper disk method, we identified that 20131023A05 was the only strain that antagonized Vibrio parahaemolyticus. We measured the diameters of inhibition zones produced by Strain 20131023A05 on the plates coated with V. parahaemolyticus at the densities of 1.57×105 CFU/cm2, 1.57×104 CFU/cm2, and 1.57×103 CFU/cm2, and the diameters were (9.14±0.05) mm, (11.57±0.03) mm, and (13.59±0.02) mm respectively. It was identified that the strain had the closest genetic relationship with Pseudoalteromonas piscicida according to 16S rDNA sequencing. Marsupenaeus japonicus immersed with Strain 20131023A05 at 2.5×105 CFU/ml showed resistance to intramuscular-injected V. parahaemolyticus and the relative survival rate (RPS) was 35%. We applied strain 20131023A05 at 3.13×104 CFU/ml once every three days as well as brown sugar (70% of diet) every day in the culture water of Litopenaeus vannamei for 60 days. Subsequently the concentration of ammonia nitrogen in the water became significantly lower than the control and other groups. Our results suggested that Strain 20131023A05 could function in both ammonia removal and V. parahaemolyticus inhibition, therefore may have great potentials in shrimp farming industry.
Three bacterial strains, 20131023A00, 20131023A01, and 20131023A05, were isolated from shrimp guts and the farming ponds using inorganic selecting medium. When cultured in the liquid selecting medium (pH=7.2) containing 0.12% ammonia nitrogen at 28℃ for 24 h, the ammonia conversion rates of the three strains were (38.9±0.1)%, (43.1±0.4)%, and (49.9±0.5)% respectively. Using the paper disk method, we identified that 20131023A05 was the only strain that antagonized Vibrio parahaemolyticus. We measured the diameters of inhibition zones produced by Strain 20131023A05 on the plates coated with V. parahaemolyticus at the densities of 1.57×105 CFU/cm2, 1.57×104 CFU/cm2, and 1.57×103 CFU/cm2, and the diameters were (9.14±0.05) mm, (11.57±0.03) mm, and (13.59±0.02) mm respectively. It was identified that the strain had the closest genetic relationship with Pseudoalteromonas piscicida according to 16S rDNA sequencing. Marsupenaeus japonicus immersed with Strain 20131023A05 at 2.5×105 CFU/ml showed resistance to intramuscular-injected V. parahaemolyticus and the relative survival rate (RPS) was 35%. We applied strain 20131023A05 at 3.13×104 CFU/ml once every three days as well as brown sugar (70% of diet) every day in the culture water of Litopenaeus vannamei for 60 days. Subsequently the concentration of ammonia nitrogen in the water became significantly lower than the control and other groups. Our results suggested that Strain 20131023A05 could function in both ammonia removal and V. parahaemolyticus inhibition, therefore may have great potentials in shrimp farming industry.
2016, 37(2):105-110.
DOI: 10.11758/yykxjz.20150417002
Abstract:
Acute hepatopancreatic necrosis disease (AHPND) is caused by infection with Vibrio parahaemolyticus. In this study, five strains of V. parahaemolyticus were isolated from Litopenaeus vannamei with AHPND. The primer set AP2 targeting the plasmid harbored in AHPND-causing V. parahaemolyticus (VPAHPND) was used in the PCR amplification, and it was found that all five isolates carried the AHPND related plasmid. Mitomycin C was used to induce and screen the lysogenic phages in VPAHPND isolates. The growth curves showed that two isolates of V. parahaemolyticus, 20130629002S01 and 20130726001S01, contained lysogenic phages. Phage 1 and Phage 2 were released from 20130629002S01 and 20130726001S01 respectively after the induction with 0.5 μg/ml mitomycin C. Transmission electron microscopy revealed that Phage 1 had a tailed shape and Phage 2 had a spherical shape. The challenge test on Artimia nauplii suggested that the virulence was significantly different between the 5 strains of V. parahaemolyticus. The pathogenicity of two lysogenic phages-containing strains, 20130629002S01 and 20130726001S01, was significantly lower than that of phage-free 20130721001S02. Our study suggested that the presence of lysogenic phage might not necessarily correlate with the pathogenicity of the host bacterium V. parahaemolyticus.
Acute hepatopancreatic necrosis disease (AHPND) is caused by infection with Vibrio parahaemolyticus. In this study, five strains of V. parahaemolyticus were isolated from Litopenaeus vannamei with AHPND. The primer set AP2 targeting the plasmid harbored in AHPND-causing V. parahaemolyticus (VPAHPND) was used in the PCR amplification, and it was found that all five isolates carried the AHPND related plasmid. Mitomycin C was used to induce and screen the lysogenic phages in VPAHPND isolates. The growth curves showed that two isolates of V. parahaemolyticus, 20130629002S01 and 20130726001S01, contained lysogenic phages. Phage 1 and Phage 2 were released from 20130629002S01 and 20130726001S01 respectively after the induction with 0.5 μg/ml mitomycin C. Transmission electron microscopy revealed that Phage 1 had a tailed shape and Phage 2 had a spherical shape. The challenge test on Artimia nauplii suggested that the virulence was significantly different between the 5 strains of V. parahaemolyticus. The pathogenicity of two lysogenic phages-containing strains, 20130629002S01 and 20130726001S01, was significantly lower than that of phage-free 20130721001S02. Our study suggested that the presence of lysogenic phage might not necessarily correlate with the pathogenicity of the host bacterium V. parahaemolyticus.
2014, 35(6):76-82.
DOI: 10.11758/yykxjz.20140611
Abstract:
Acute Hepatopancreatic Necrosis Syndrome (AHPNS), also know as Early Mortality Syndrome (EMS), was officially reported in China in 2010 and has caused large-scale die-offs of cultivated shrimp in several Asian countries. A research team of the University of Arizona has identified that the pathogen is Vibrio parahaemolyticus which is a strain of a bacterium commonly found in brackish coastal waters around the globe. Because V. parahaemolyticus is a conditioned pathogen, it is very important to study the effects of ammonia nitrogen on the toxicity of this pathogen to Litopenaeus vannamei. To evaluate the effects of total ammonia nitrogen during the outbreak of AHPNS in L. vannamei, we examined the susceptibility to the pathogen, the non-specific immunity and the expression of LvLT mRNA in the hepatopancreas of the shrimp. Shrimp underwent the ammonia stress for 20 days with different ammonia nitrogen concentrations: 2.5 mg/L, 5.0 mg/L, 7.5 mg/L, 10.0 mg/L and the control concentration. After the exposure to V. parahaemolyticus, the death rate of shrimp perked in 6–24 h. The accumulative mortality rates of the treated groups were 0, 8%, 12%, 20% and 36% respectively at 120 h. The activities of phenoloxidase (PO) reached the lowest at 12 h in the control, 2.5 and 5.0 mg/L groups, and at 24 h in the 7.5 and 10.0 mg/L groups. The activity of superoxide dismutase (SOD) increased at first and then gradually declined, and lysozyme (LSZ) exhibited the same trend. The expression of LvLT mRNA in the hepatopancreas increased at 6 h in all groups and was significantly higher in the control, 2.5, 5.0 and 7.5 mg/L group than that in the 10.0 mg/L group. The LvLT mRNA expression reached the maximum at 12 h and then declined gradually at 24 h. The results indicated that high ammonia nitrogen could cause depression in the immunity of L. vannamei, and subsequently increase their susceptibility to V. parahaemolyticu and the resultant mortality rate. Therefore it is crucial to regulate the concentration of total ammonia nitrogen in aquatic environment to prevent the breakout of Acute Hepatopancreas Necrosis Syndrome.
Acute Hepatopancreatic Necrosis Syndrome (AHPNS), also know as Early Mortality Syndrome (EMS), was officially reported in China in 2010 and has caused large-scale die-offs of cultivated shrimp in several Asian countries. A research team of the University of Arizona has identified that the pathogen is Vibrio parahaemolyticus which is a strain of a bacterium commonly found in brackish coastal waters around the globe. Because V. parahaemolyticus is a conditioned pathogen, it is very important to study the effects of ammonia nitrogen on the toxicity of this pathogen to Litopenaeus vannamei. To evaluate the effects of total ammonia nitrogen during the outbreak of AHPNS in L. vannamei, we examined the susceptibility to the pathogen, the non-specific immunity and the expression of LvLT mRNA in the hepatopancreas of the shrimp. Shrimp underwent the ammonia stress for 20 days with different ammonia nitrogen concentrations: 2.5 mg/L, 5.0 mg/L, 7.5 mg/L, 10.0 mg/L and the control concentration. After the exposure to V. parahaemolyticus, the death rate of shrimp perked in 6–24 h. The accumulative mortality rates of the treated groups were 0, 8%, 12%, 20% and 36% respectively at 120 h. The activities of phenoloxidase (PO) reached the lowest at 12 h in the control, 2.5 and 5.0 mg/L groups, and at 24 h in the 7.5 and 10.0 mg/L groups. The activity of superoxide dismutase (SOD) increased at first and then gradually declined, and lysozyme (LSZ) exhibited the same trend. The expression of LvLT mRNA in the hepatopancreas increased at 6 h in all groups and was significantly higher in the control, 2.5, 5.0 and 7.5 mg/L group than that in the 10.0 mg/L group. The LvLT mRNA expression reached the maximum at 12 h and then declined gradually at 24 h. The results indicated that high ammonia nitrogen could cause depression in the immunity of L. vannamei, and subsequently increase their susceptibility to V. parahaemolyticu and the resultant mortality rate. Therefore it is crucial to regulate the concentration of total ammonia nitrogen in aquatic environment to prevent the breakout of Acute Hepatopancreas Necrosis Syndrome.
