《渔业科学进展》虚拟专辑——对虾疾病：虾肝肠胞虫(EHP)
虾肝肠胞虫(Enterocytozoon hepatopenaei, EHP)是一种严格细胞内寄生的微孢子虫，成熟的孢子呈椭圆形，大小为(1.4±0.3)×(0.8±0.1) μm，后端有1个空泡，胞内含1个细胞核，5~6圈极丝，1个与极丝相连的锚定盘和1层电子致密的孢子壁。EHP于2009年首次从斑节对虾(Penaeus monodon)中发现并命名，感染EHP的对虾往往正常进食，但生长缓慢甚至停滞，直接影响到养殖产量，给养殖户带来严重的经济损失，如何预防和治疗EHP已经成为对虾养殖产业急需解决的科学问题。
编辑部整理了近年来发表在本刊上的8篇关于虾肝肠胞虫研究论文组成本期虚拟专辑，欢迎阅读、引用。
关键词：暂无
1 Validation of a highly sensitive kit for the rapid detection of Enterocytozoon hepatopenaei in field
2022, 43(4):218-225.
DOI: 10.19663/j.issn2095-9869.20210422001
Abstract:
Enterocytozoon hepatopenaei (EHP) has caused serious losses in shrimp aquaculture in China in recent years. For cultured shrimp, disease prevention cannot be carried out by vaccination because of the lack of specific immune mechanisms. The most effective measure to prevent disease outbreaks is to conduct on-site rapid pathogen detection as far as possible for relatives or seedlings or to carry out on-site detection early in the onset of shrimp, timely detection, and identification of pathogens. There is, therefore, an urgent demand for rapid detection techniques and kits for controlling and preventing EHP. A novel, highly sensitive kit, developed in our laboratory, can achieve rapid detection of EHP in the field by optimizing the three steps of tissue nucleic acid extraction, nucleic acid amplification, and nucleic acid detection, and provide a practical solution for early rapid screening and detection of shrimp EHP disease. To validate the newly developed highly sensitive kit for rapid EHP detection in the field, a systematic evaluation of the six performance parameters of the kit was carried out in this study. The analytical specificity results showed that the kit did not cross-react with the DNA/RNA extracted from healthy shrimp or shrimp infected with five other pathogens, including WSSV, CMNV, SHIV, VpAHPND, and IHHNV. The analytical sensitivity analysis showed that the detection limit was 101 copies/μL with shrimp DNA preparation. The diagnostic sensitivity and diagnostic specificity were determined to be 91.7% and 99.2%, respectively, in tests of 298 clinical samples, in comparison with the TaqMan qPCR protocol of EHP. The repeatability was 100% for strongly positive and negative samples and 95.8% for weakly positive samples. The period of validity of the kit was tested and found to be over 7 months at –20℃ storage and over 12 months at –40℃. The study results demonstrated that the kit is a simple, sensitive, specific, and accurate tool for the rapid detection of EHP in practical applications.
Enterocytozoon hepatopenaei (EHP) has caused serious losses in shrimp aquaculture in China in recent years. For cultured shrimp, disease prevention cannot be carried out by vaccination because of the lack of specific immune mechanisms. The most effective measure to prevent disease outbreaks is to conduct on-site rapid pathogen detection as far as possible for relatives or seedlings or to carry out on-site detection early in the onset of shrimp, timely detection, and identification of pathogens. There is, therefore, an urgent demand for rapid detection techniques and kits for controlling and preventing EHP. A novel, highly sensitive kit, developed in our laboratory, can achieve rapid detection of EHP in the field by optimizing the three steps of tissue nucleic acid extraction, nucleic acid amplification, and nucleic acid detection, and provide a practical solution for early rapid screening and detection of shrimp EHP disease. To validate the newly developed highly sensitive kit for rapid EHP detection in the field, a systematic evaluation of the six performance parameters of the kit was carried out in this study. The analytical specificity results showed that the kit did not cross-react with the DNA/RNA extracted from healthy shrimp or shrimp infected with five other pathogens, including WSSV, CMNV, SHIV, VpAHPND, and IHHNV. The analytical sensitivity analysis showed that the detection limit was 101 copies/μL with shrimp DNA preparation. The diagnostic sensitivity and diagnostic specificity were determined to be 91.7% and 99.2%, respectively, in tests of 298 clinical samples, in comparison with the TaqMan qPCR protocol of EHP. The repeatability was 100% for strongly positive and negative samples and 95.8% for weakly positive samples. The period of validity of the kit was tested and found to be over 7 months at –20℃ storage and over 12 months at –40℃. The study results demonstrated that the kit is a simple, sensitive, specific, and accurate tool for the rapid detection of EHP in practical applications.
2022, 43(3):75-83.
DOI: 10.19663/j.issn2095-9869.20210302001
Abstract:
Enterocytozoon hepatopenaei (EHP) is a highly infectious intracellular parasite that primarily parasitizes the hepatopancreas, intestine, and muscle of shrimp. It can reproduce by consuming ATP from the host cells, resulting in growth retardation or even growth cessation of the host and increasing individual differences within a population. In recent years, we discovered EHP infection in the breeding process of Exopalaemon carinicauda culture, which has caused losses to the E. carinicauda culture industry. Intestinal microorganisms, which play a very important role in the growth and development of shrimp, can regulate nutritional metabolism, resist pathogen infection, and also have an important impact on the host immune function. Therefore, it is helpful to clarify the pathogenesis of EHP by exploring the differences and functions of the intestinal microflora between healthy and diseased shrimp. To screen potential probiotics for inhibiting or slowing down the spread of EHP, this study analyzed the intestinal microflora structure of shrimp based on 16s rRNA gene sequencing, and further explored the effect of EHP infection on the intestinal microflora of E. carinicauda. The results showed that the intestinal microflora of infected shrimp was significantly different from that of healthy individuals, and the structural diversity of the intestinal microflora was significantly lower than that of the healthy shrimp. Proteobacteria, including Desulfovibrionaceae, Vibrionaceae, unidentified Cyanobacteria, Mycoplasmataceae, and unidentified Alphaproteobacteria were the dominant bacteria in diseased shrimp, whereas Firmicutes including Lactobacillaceae, Bifidobacteria, Bacillaceae, and Chitinophagaceae were dominant in healthy shrimp. Infection with EHP significantly increased the potential pathogenic bacteria level in the intestines of the infected shrimp (P<0.05), and increased their susceptibility to other diseases. In addition, through the Tax4Fun function prediction, we found that the primary function of the intestinal microflora in infected shrimp was metabolism to resist EHP infection, whereas the intestinal microflora of healthy shrimp was primarily involved in individual growth and environmental information processing to ensure growth and survival.
Enterocytozoon hepatopenaei (EHP) is a highly infectious intracellular parasite that primarily parasitizes the hepatopancreas, intestine, and muscle of shrimp. It can reproduce by consuming ATP from the host cells, resulting in growth retardation or even growth cessation of the host and increasing individual differences within a population. In recent years, we discovered EHP infection in the breeding process of Exopalaemon carinicauda culture, which has caused losses to the E. carinicauda culture industry. Intestinal microorganisms, which play a very important role in the growth and development of shrimp, can regulate nutritional metabolism, resist pathogen infection, and also have an important impact on the host immune function. Therefore, it is helpful to clarify the pathogenesis of EHP by exploring the differences and functions of the intestinal microflora between healthy and diseased shrimp. To screen potential probiotics for inhibiting or slowing down the spread of EHP, this study analyzed the intestinal microflora structure of shrimp based on 16s rRNA gene sequencing, and further explored the effect of EHP infection on the intestinal microflora of E. carinicauda. The results showed that the intestinal microflora of infected shrimp was significantly different from that of healthy individuals, and the structural diversity of the intestinal microflora was significantly lower than that of the healthy shrimp. Proteobacteria, including Desulfovibrionaceae, Vibrionaceae, unidentified Cyanobacteria, Mycoplasmataceae, and unidentified Alphaproteobacteria were the dominant bacteria in diseased shrimp, whereas Firmicutes including Lactobacillaceae, Bifidobacteria, Bacillaceae, and Chitinophagaceae were dominant in healthy shrimp. Infection with EHP significantly increased the potential pathogenic bacteria level in the intestines of the infected shrimp (P<0.05), and increased their susceptibility to other diseases. In addition, through the Tax4Fun function prediction, we found that the primary function of the intestinal microflora in infected shrimp was metabolism to resist EHP infection, whereas the intestinal microflora of healthy shrimp was primarily involved in individual growth and environmental information processing to ensure growth and survival.
2020, 41(6):165-173.
DOI: 10.19663/j.issn2095-9869.20190810002
Abstract:
Secreted proteins are synthesized in cells and secreted to function outside the cell. They play an important role in the manipulation of host cells and the virulence of many eukaryotic protozoan parasites. Enterocytozoon hepatopenaei (EHP) is an obligate intracellular parasite that can infect a variety of economically valuable shrimp species, and is one of the most serious diseases affecting global shrimp production. Here, we predict the secreted proteins of the EHP genome with the EuSecPred2.0 pipeline and analyzed the length of these secreted proteins, the length of the signal peptide, and the amino acid distribution at the cleavage site, and annotated the function of secreted proteins. The results show that the length of the secreted proteins ranged from 30 to 400 amino acid residues, the signal peptides covered approximately 9~32 amino acids, and the cleavage sites of the signal peptides were mainly composed of hydrophobic amino acids. Motif analysis revealed a NV[VT][IK]CA[ED][SA] motif in signal peptides. Functional annotation of proteins revealed that a variety of key proteins are involved in adhesion and infection of microsporidia, regulation of cell cycle, and immune response. The results shed light on the infection mechanism of EHP and provide a theoretical basis for further defining the pathogenic factors of EHP.
Secreted proteins are synthesized in cells and secreted to function outside the cell. They play an important role in the manipulation of host cells and the virulence of many eukaryotic protozoan parasites. Enterocytozoon hepatopenaei (EHP) is an obligate intracellular parasite that can infect a variety of economically valuable shrimp species, and is one of the most serious diseases affecting global shrimp production. Here, we predict the secreted proteins of the EHP genome with the EuSecPred2.0 pipeline and analyzed the length of these secreted proteins, the length of the signal peptide, and the amino acid distribution at the cleavage site, and annotated the function of secreted proteins. The results show that the length of the secreted proteins ranged from 30 to 400 amino acid residues, the signal peptides covered approximately 9~32 amino acids, and the cleavage sites of the signal peptides were mainly composed of hydrophobic amino acids. Motif analysis revealed a NV[VT][IK]CA[ED][SA] motif in signal peptides. Functional annotation of proteins revealed that a variety of key proteins are involved in adhesion and infection of microsporidia, regulation of cell cycle, and immune response. The results shed light on the infection mechanism of EHP and provide a theoretical basis for further defining the pathogenic factors of EHP.
SONG Zenglei
,
DONG Xuan
,
ZHAO Ruoheng
,
WANG Xiuhua
,
WU Heying
,
YU Danghui
,
XIE Guosi
,
HUANG Jie
2019, 40(3):122-132.
DOI: 10.19663/j.issn2095-9869.20180421002
Abstract:
Two populations of Litopenaeus vannamei from Haiyang and Weifang in Shandong Province were sampled. TaqMan qPCR was used to measure Enterocytozoon hepatopenaei (EHP) in the shrimp hepatopancreas one by one, and then the extracted DNA samples were pooled by 5-pool (5∶1), 25-pool (25∶1), 50-pool (50∶1), 100-pool (100∶1), and 150-pool (150∶1) to test the EHP in the pooled samples. The amplification used a special threshold cycle value (Ct) of samples lower/higher than an assumed critical cycle value (Ca), differentiated as an analyzed positive/negative ratio in the single sample test and pooled sample test. The relationship between different pooling modes and the analyzed positive rate, diagnostic sensitivity, diagnostic specificity, and quantitative accuracy were compared. The results showed that the detection for high pooling rate samples could be reduced in very low analytic sensitivity. When the positive rate of individuals in the pooled samples was above 30%, the detection results of the high pooling rate were consistent with that of the single sample detection. If the positive rate of individuals with heavy infections in the pooled sample were not less than 6.7%, satisfying results could be obtained in the sample with a pooling rate below 50∶1; while the positive rate of individuals with light infections in the pooled sample were not less than 16%, the satisfying result could be obtained in the sample with a pooling rate below 25∶1. The pooled samples with a positive rate below 1.3% of individuals with heavy infection, or a positive rate below 8% of individuals with light infection, might lead to false negative results in all pooling rates. All of the pooling modes have good diagnostic specificity. The sample with a 50∶1 pooling rate had a similar diagnostic sensitivity to the sample with a 5∶1 pooling rate, which is the highest pooling rate recommended by the OIE standard. It had a very significant correlation between the mean of the detected EHP load of pooled sample, and the mean of the known EHP load calculated according to the single sample, with a ratio of 0.27~2.83. The quantitative test results of the pooled samples could roughly reflect the average EHP load of the single sample at the order of magnitude. This study provides a reference for sample detection in aquatic animal disease diagnosis, and epidemiological investigations.
Two populations of Litopenaeus vannamei from Haiyang and Weifang in Shandong Province were sampled. TaqMan qPCR was used to measure Enterocytozoon hepatopenaei (EHP) in the shrimp hepatopancreas one by one, and then the extracted DNA samples were pooled by 5-pool (5∶1), 25-pool (25∶1), 50-pool (50∶1), 100-pool (100∶1), and 150-pool (150∶1) to test the EHP in the pooled samples. The amplification used a special threshold cycle value (Ct) of samples lower/higher than an assumed critical cycle value (Ca), differentiated as an analyzed positive/negative ratio in the single sample test and pooled sample test. The relationship between different pooling modes and the analyzed positive rate, diagnostic sensitivity, diagnostic specificity, and quantitative accuracy were compared. The results showed that the detection for high pooling rate samples could be reduced in very low analytic sensitivity. When the positive rate of individuals in the pooled samples was above 30%, the detection results of the high pooling rate were consistent with that of the single sample detection. If the positive rate of individuals with heavy infections in the pooled sample were not less than 6.7%, satisfying results could be obtained in the sample with a pooling rate below 50∶1; while the positive rate of individuals with light infections in the pooled sample were not less than 16%, the satisfying result could be obtained in the sample with a pooling rate below 25∶1. The pooled samples with a positive rate below 1.3% of individuals with heavy infection, or a positive rate below 8% of individuals with light infection, might lead to false negative results in all pooling rates. All of the pooling modes have good diagnostic specificity. The sample with a 50∶1 pooling rate had a similar diagnostic sensitivity to the sample with a 5∶1 pooling rate, which is the highest pooling rate recommended by the OIE standard. It had a very significant correlation between the mean of the detected EHP load of pooled sample, and the mean of the known EHP load calculated according to the single sample, with a ratio of 0.27~2.83. The quantitative test results of the pooled samples could roughly reflect the average EHP load of the single sample at the order of magnitude. This study provides a reference for sample detection in aquatic animal disease diagnosis, and epidemiological investigations.
2018, 39(4):83-92.
Abstract:
Growth-related parameters of individuals from four populations of Litopenaeus vannamei from Huanghua of Heibei Province (HH), Pingdu of Shandong Province (PD), Wujiang of Jiangsu Province (WJ), and Rizhao of Shandong Province (RZ) were assessed. Amount of Enterocytozoon hepatopenaei (EHP) in the hepatopancreas of individuals from all populations and EHP in multiple tissues of shrimp from the RZ population were assessed with TaqMan-based quantitative PCR. The results showed that the RZ population possessed optimal growth-related parameters and the lowest EHP among the four populations. The histograms of case-logarithmic EHPs of the four populations presented different modes. The case distribution of logarithmic EHPs from the HH and PD populations showed double peaks, while those of the WJ and RZ populations showed a single peak. The different distribution modes may indicate a different EHP spread in the four populations. The population with a single peak mode or the higher logarithmic EHP subpopulation isolated from the population with a multiple peak mode showed a significant negative correlation with shrimp body length or body weight to logarithmic EHP. The EHP detected in different tissues of the RZ population followed the order (from highest to lowest), EHP in hepatopancreas > EHP in midgut > EHP in hemolymph > EHP in gills > EHP in muscle. The logarithmic EHP in the hepatopancreas–midgut, hepatopancreas–gills, and midgut–gills had a significant correlation level above 99.9% (P<0.001), while the logarithmic EHP of the other two tissues had a significant correlation level above 99.0% (P<0.01) or above 99.5% (P<0.05), except for midgut–hemolymph and hepatopancreas–hemolymph. In-situ hybridization of a DIG-labeled EHP probe in the hepatopancreas, muscle, gills, and midgut showed that the hepatopancreas is the major target tissue of EHP infection in shrimp. Minor and weak hybridization signals were also observed in other tissues, which indicated that a few cells in those tissues were also susceptible to EHP infection in L. vannamei.
Growth-related parameters of individuals from four populations of Litopenaeus vannamei from Huanghua of Heibei Province (HH), Pingdu of Shandong Province (PD), Wujiang of Jiangsu Province (WJ), and Rizhao of Shandong Province (RZ) were assessed. Amount of Enterocytozoon hepatopenaei (EHP) in the hepatopancreas of individuals from all populations and EHP in multiple tissues of shrimp from the RZ population were assessed with TaqMan-based quantitative PCR. The results showed that the RZ population possessed optimal growth-related parameters and the lowest EHP among the four populations. The histograms of case-logarithmic EHPs of the four populations presented different modes. The case distribution of logarithmic EHPs from the HH and PD populations showed double peaks, while those of the WJ and RZ populations showed a single peak. The different distribution modes may indicate a different EHP spread in the four populations. The population with a single peak mode or the higher logarithmic EHP subpopulation isolated from the population with a multiple peak mode showed a significant negative correlation with shrimp body length or body weight to logarithmic EHP. The EHP detected in different tissues of the RZ population followed the order (from highest to lowest), EHP in hepatopancreas > EHP in midgut > EHP in hemolymph > EHP in gills > EHP in muscle. The logarithmic EHP in the hepatopancreas–midgut, hepatopancreas–gills, and midgut–gills had a significant correlation level above 99.9% (P<0.001), while the logarithmic EHP of the other two tissues had a significant correlation level above 99.0% (P<0.01) or above 99.5% (P<0.05), except for midgut–hemolymph and hepatopancreas–hemolymph. In-situ hybridization of a DIG-labeled EHP probe in the hepatopancreas, muscle, gills, and midgut showed that the hepatopancreas is the major target tissue of EHP infection in shrimp. Minor and weak hybridization signals were also observed in other tissues, which indicated that a few cells in those tissues were also susceptible to EHP infection in L. vannamei.
2017, 38(4):96-103.
DOI: 10.11758/yykxjz.20160418002
Abstract:
Microsporidia Enterocytozoon hepatopenaei (EHP) was detected using probe based real-time PCR from 442 shrimp coming from 5 farmed Litopenaeus vannamei populations in Tianjin, Zhejiang, and Shandong Province. The biological length and body weight of shrimp were measured individually. The Rohrer’s ponderal index (PI, W/L3) originally used in medical science was introduced in this study to establish the fuction of body weight (W) and biological length (L) of shrimp. The results showed that the PI of 4 EHP positive populations with an average biological length at (5.37±1.19) cm was calculated as (5.19±0.26)× 10–3 g/cm3, while that of the EHP negative population with an average biological length was (7.96±0.51)×10–3 g/cm3. After adjusting of the difference of PI caused by biological length according to the function PI=a×L(b–3), the PI of EHP positive populations was about (70.5±8.7)% of that of the EHP negative populations at the same biological length. It indicated that the average body weight of shrimp in an EHP positive population was about 30% smaller than that of shrimp with same size in biological length in an EHP negative population. The coefficient of variation (CV) of biological length and body weight in EHP positive populations was (2.39±0.93) times and (2.05±0.86) times of those in the EHP population, respectively, suggesting significant size variation in the EHP positive populations. The deviation ratio of actual body weight to body weight estimated on biological length in the EHP positive populations was 2.34 to 3.45 times of that in the EHP negative population, indicating the shrimp with same size in the EHP positive populations had greater fluctuation in body weight.
Microsporidia Enterocytozoon hepatopenaei (EHP) was detected using probe based real-time PCR from 442 shrimp coming from 5 farmed Litopenaeus vannamei populations in Tianjin, Zhejiang, and Shandong Province. The biological length and body weight of shrimp were measured individually. The Rohrer’s ponderal index (PI, W/L3) originally used in medical science was introduced in this study to establish the fuction of body weight (W) and biological length (L) of shrimp. The results showed that the PI of 4 EHP positive populations with an average biological length at (5.37±1.19) cm was calculated as (5.19±0.26)× 10–3 g/cm3, while that of the EHP negative population with an average biological length was (7.96±0.51)×10–3 g/cm3. After adjusting of the difference of PI caused by biological length according to the function PI=a×L(b–3), the PI of EHP positive populations was about (70.5±8.7)% of that of the EHP negative populations at the same biological length. It indicated that the average body weight of shrimp in an EHP positive population was about 30% smaller than that of shrimp with same size in biological length in an EHP negative population. The coefficient of variation (CV) of biological length and body weight in EHP positive populations was (2.39±0.93) times and (2.05±0.86) times of those in the EHP population, respectively, suggesting significant size variation in the EHP positive populations. The deviation ratio of actual body weight to body weight estimated on biological length in the EHP positive populations was 2.34 to 3.45 times of that in the EHP negative population, indicating the shrimp with same size in the EHP positive populations had greater fluctuation in body weight.
2017, 38(2):158-166.
DOI: 10.11758/yykxjz.20160123001
Abstract:
Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is one of the pathogens causing the significant diseases of crustaceans listed by the OIE (Office internaptional despizooties) Manual of Diagnostic Tests for Aquatic Animals. Infection with IHHNV does not produce any pathognomonic gross clinical signs, although runt-deformity syndrome (RDS) has been noted in infected juvenile Litopenaeus vannamei and Penaeus monodon. Enterocytozoon hepatopenaei (EHP) was first found and isolated in Thailand in 2009, which caused slow growth of P. monodon. IHHNV and EHP were found in the samples of L. vannamei postlarvae which were collected from hatchery farms in Tianjin and Hebei Province in 2013. The diseased shrimps showed some clinical signs with high mortality, slow growth and difference of individual growth rate. The IHHNV and EHP individual detection was conducted on these samples using the real-time PCR technique. In total, 108 out of 108 (100%) L. vannamei postlarvae samples were IHHNV positive, and 53 out of 108 (49.1%) L. vannamei postlarvae samples were EHP positive. Positive samples contained approximately 103–107 copies/(μg DNA) of IHHNV and big size of individual shrimp carried higher number of virus copies. Positive samples contained approximately 103–105 copies/(μg DNA) of EHP and mainly the small sized individual shrimp. The analysis showed that the IHHNV loads were positively correlated with the shrimp growth rate. IHHNV loads above 8.51×104 copies/μg DNA represented a high risk level. EHP loads were negatively correlated with the shrimp growth rate. EHP loads above 2.19×104 copies/μg DNA represented a high risk level. Our study showed that the diseased L. vannamei postlarvae samples occurred in the hatchery farms of Tianjin and Hebai Province infected with shrimp pathogens of IHHNV and EHP. Data provides scientific basis on the effects of pathogens infection on the growth of cultured penaeid shrimp.
Infectious hypodermal and hematopoietic necrosis virus (IHHNV) is one of the pathogens causing the significant diseases of crustaceans listed by the OIE (Office internaptional despizooties) Manual of Diagnostic Tests for Aquatic Animals. Infection with IHHNV does not produce any pathognomonic gross clinical signs, although runt-deformity syndrome (RDS) has been noted in infected juvenile Litopenaeus vannamei and Penaeus monodon. Enterocytozoon hepatopenaei (EHP) was first found and isolated in Thailand in 2009, which caused slow growth of P. monodon. IHHNV and EHP were found in the samples of L. vannamei postlarvae which were collected from hatchery farms in Tianjin and Hebei Province in 2013. The diseased shrimps showed some clinical signs with high mortality, slow growth and difference of individual growth rate. The IHHNV and EHP individual detection was conducted on these samples using the real-time PCR technique. In total, 108 out of 108 (100%) L. vannamei postlarvae samples were IHHNV positive, and 53 out of 108 (49.1%) L. vannamei postlarvae samples were EHP positive. Positive samples contained approximately 103–107 copies/(μg DNA) of IHHNV and big size of individual shrimp carried higher number of virus copies. Positive samples contained approximately 103–105 copies/(μg DNA) of EHP and mainly the small sized individual shrimp. The analysis showed that the IHHNV loads were positively correlated with the shrimp growth rate. IHHNV loads above 8.51×104 copies/μg DNA represented a high risk level. EHP loads were negatively correlated with the shrimp growth rate. EHP loads above 2.19×104 copies/μg DNA represented a high risk level. Our study showed that the diseased L. vannamei postlarvae samples occurred in the hatchery farms of Tianjin and Hebai Province infected with shrimp pathogens of IHHNV and EHP. Data provides scientific basis on the effects of pathogens infection on the growth of cultured penaeid shrimp.
2016, 37(2):119-126.
DOI: 10.11758/yykxjz.20150512003
Abstract:
In this study we developed fluorescence SYBR Green I real-time quantitative PCR (qPCR) to detect shrimp microsporidian Enterocytozoon hepatopenaei (EHP). A pair of specific primers was designed according to the SSU rDNA sequences of shrimp EHP published in GenBank and the optimized annealing temperature was determined to be 60℃. The melting curve of amplified products exhibited only one specific peak. Between 8.3×101–8.3×108 copies/µl the test results were linearly correlated with the titers of EHP. The cycles of amplification threshold (Ct) and the logarithmic of the initial template quantity [log(Sq)] conformed to Ct = –3.369 log(Sq) + 39.364, of which the coefficient of association R2 was 0.992 and the amplification efficiency was 98.1%. This method had high sensitivity (8.3×101 copies/µl), and generated duplicable results both within a group and between different groups. The test of 31 samples of farmed L. vannamei suggested that the qPCR method was 4 times more sensitive than the previously reported nested PCR method. To further verify this method, we tested hepatopancreatic DNA (HpDNA) samples extracted from 94 samples of L. vannamei that were collected from farms in Jiangsu, Hainan, and Shandong provinces. The results showed that the EHP loads in the hepatopancreas were negatively correlated with the shrimp growth rates. EHP load above 103 copies/(ng HpDNA) indicated high risk. In conclusion, we developed a highly specific, sensitive, rapid and quantitative method, which could be a useful tool in the prevention and control of shrimp microsporidian E. hepatopenaei.
In this study we developed fluorescence SYBR Green I real-time quantitative PCR (qPCR) to detect shrimp microsporidian Enterocytozoon hepatopenaei (EHP). A pair of specific primers was designed according to the SSU rDNA sequences of shrimp EHP published in GenBank and the optimized annealing temperature was determined to be 60℃. The melting curve of amplified products exhibited only one specific peak. Between 8.3×101–8.3×108 copies/µl the test results were linearly correlated with the titers of EHP. The cycles of amplification threshold (Ct) and the logarithmic of the initial template quantity [log(Sq)] conformed to Ct = –3.369 log(Sq) + 39.364, of which the coefficient of association R2 was 0.992 and the amplification efficiency was 98.1%. This method had high sensitivity (8.3×101 copies/µl), and generated duplicable results both within a group and between different groups. The test of 31 samples of farmed L. vannamei suggested that the qPCR method was 4 times more sensitive than the previously reported nested PCR method. To further verify this method, we tested hepatopancreatic DNA (HpDNA) samples extracted from 94 samples of L. vannamei that were collected from farms in Jiangsu, Hainan, and Shandong provinces. The results showed that the EHP loads in the hepatopancreas were negatively correlated with the shrimp growth rates. EHP load above 103 copies/(ng HpDNA) indicated high risk. In conclusion, we developed a highly specific, sensitive, rapid and quantitative method, which could be a useful tool in the prevention and control of shrimp microsporidian E. hepatopenaei.
