Identification of abundant and informative microsatellites from shrimp (Penaeus monodon) genome

Microsatellites were isolated from P. monodon genomic libraries by direct sequencing of recombinant clones without probe screening. Forty-nine out of 83 clones sequenced contained 99 microsatellite arrays of three or more repeats. When five or more and ten or more repeats were considered, 28 and 14 microsatellites were detected, respectively. The 99 microsatellites were classified as perfect (75%), imperfect (6%), compound perfect (3%) and compound imperfect (16%). The abundance of di-, tri-, tetra- and hexanucleotide repeats were 67%, 20%, 9% and 3%, respectively. The dinucleotide repeats included 36 (CT)n, 31 (GT)n, 17(AT)n and 3 (CG)n. One octanucleotide repeat (ATTTATTC)5 was found within a large repeat sequence. Optimal annealing temperatures were determined for PCR using 11 primer sets encompassing 15 microsatellites. Ten primer sets provided successful amplifications with allele sizes generally ranging from 139 to 410 bp. All these primers amplified polymorphic loci with PIC values ranging from 0.63 to 0.96. Two primer sets amplified additional bands which can easily be distinguished from the bands of the main locus. Three out of 10 P. monodon microsatellites also amplified alleles in P. vannamei. The abundance and informative nature of P. monodon microsatellites and their potential for cross-species amplification make them useful for genetic studies.     

Distribution of Virulence-Associated Genes and Genetic Relationships in Non-O1/O139 Vibrio cholerae Aquatic Isolates from China

Non-O1/O139 Vibrio cholerae is naturally present in aquatic ecosystems and has been linked with cholera-like diarrhea and local outbreaks. The distribution of virulence-associated genes and genetic relationships among aquatic isolates from China are largely unknown. In this study, 295 aquatic isolates of V. cholerae non-O1/O139 serogroups from different regions in China were investigated. Only one isolate was positive for ctxB and harbored a rare genotype; 10 (3.4%) isolates carried several types of rstR sequences, eight of which carried rare types of toxin-coregulated pili (tcpA). Furthermore, 16 (5.4%) isolates carried incomplete (with partial open reading frames [ORFs]) vibrio seventh pandemic island I (VSP-I) or VSP-II clusters, which were further classified as 11 novel types. PCR-based analyses revealed remarkable variations in the distribution of putative virulence genes, including mshA (95.6%), hlyA (95.3%), rtxC (89.8%), rtxA (82.7%), IS1004 (52.9%), chxA (30.2%), SXT (15.3%), type III secretion system (18.0%), and NAG-ST (3.7%) genes. There was no correlation between the prevalence of putative virulence genes and that of CTX prophage or TCP genes, whereas there were correlations among the putative virulence genes. Further multilocus sequence typing (MLST) placed selected isolates (n = 70) into 69 unique sequence types (STs), which were different from those of the toxigenic O1 and O139 counterparts, and each isolate occupied a different position in the MLST tree. The V. cholerae non-O1/O139 aquatic isolates predominant in China have high genotypic diversity; these strains constitute a reservoir of potential virulence genes, which may contribute to evolution of pathogenic isolates.     

Prevalence of Cholera Toxin Genes (ctxA and zot) among Non-O1/O139 Vibrio cholerae Strains from Newport Bay, California

The examination of 137 non-O1/O139 Vibrio cholerae isolates from Newport Bay, California, indicated the presence of diverse genotypes and a temporal succession. Unexpectedly, the cholera toxin gene (ctxA) was found in 17% of the strains, of which one-third were also positive for the zot gene. This suggests that ctxA is prevalent in the region of nonepidemicity and is likely to have an environmental origin.     

Putative Virulence Traits and Pathogenicity of Vibrio cholerae Non-O1, Non-O139 Isolates from Surface Waters in Kolkata, India

Vibrio cholerae non-O1, non-O139 was isolated from natural surface waters from different sites sampled in diarrhea endemic zones in Kolkata, India. Twenty-one of these isolates were randomly selected and included in the characterization. The multiserogroup isolates were compared by their virulence traits with a group of clinical non-O1, non-O139 isolates from the same geographic area. Of the 21 environmental isolates, 6 and 14 strains belonged to Heiberg groups I and II, respectively. Three of the environmental isolates showed resistance to 2,2-diamine-6,7-diisopropylpteridine phosphate. All of the non-O1, non-O139 strains were positive for toxR, and except for one environmental isolate, none of them were positive for tcpA in the PCR assay. None of the isolates were positive for genes encoding cholera toxin (ctxA), heat-stable toxin (est), heat-labile toxin (elt), and Shiga toxin variants (stx) of Escherichia coli. Additionally, except for one environmental isolate (PC32), all were positive for the gene encoding El Tor hemolysin (hly). The culture supernatants of 86% (18 of 21) of the environmental isolates showed a distinct cytotoxic effect on HeLa cells, and some of these strains also produced cell-rounding factor. The lipase, protease, and cell-associated hemagglutination activities and serum resistance properties of the environmental and clinical isolates did not differ much. However, seven environmental isolates exhibited very high hemolytic activities (80 to 100%), while none of the clinical strains belonged to this group. The environmental isolates manifested three adherence patterns, namely, carpet-like, diffuse, and aggregative adherence, and the clinical isolates showed diffuse adherence on HeLa cells. Of the 11 environmental isolates tested for enteropathogenic potential, 8 (73%) induced positive fluid accumulation (≥100) in a mouse model, and the reactivities of these isolates were comparable to those of clinical strains of non-O1, non-O139 and toxigenic O139 V. cholerae. Comparison of the counts of the colonized environmental and clinical strains in the mouse intestine showed that the organisms of both groups had similar colonizing efficiencies. These findings indicate the presence of potentially pathogenic V. cholerae non-O1, non-O139 strains in surface waters of the studied sites in Kolkata.     

Haliphthoros milfordensis isolated from black tiger prawn larvae (Penaeus monodon) in Vietnam

A marine fungus was isolated from the black tiger prawn Penaeus monodon at Nha Trang, Vietnam, on March 20, 2001 and named isolate NJM 0131. The fungus was identified as Haliphthoros milfordensis from the characteristics of asexual reproduction, and its physiological characteristics were investigated. Although the optimum temperature for growth of the isolate was 25°–30°C, the fungus grew at a wide range of temperatures (15°–40°C). H. milfordensis grew well in 50%–100% seawater, but poorly in PYG agar containing 1.0%–5.0% NaCl and KCl. The fungus grew at a wide range of pH (4.0–11.0) with the optimum pH value of 7.0–9.0. The isolate also showed pathogenicity to swimming crab larvae (Portunus trituberculatus) by artificial infection, but mortality was not high. This is the first report of disease in the black tiger prawn P. monodon in Vietnam caused by H. milfordensis.     

Cloning and molecular characterization of heat shock cognate 70 from tiger shrimp (Penaeus monodon)

We cloned the complementary deoxyribonucleic acid (cDNA) of the heat shock cognate 70 (hsc70) gene of tiger shrimp (Penaeus monodon). It was 2207 bp long and included a 1959-bp coding region, a 40-bp flanking region at the 5' end, and a 208-bp flanking region at the 3' end. The deduced, 652-amino acid sequence had a molecular mass of 71 481 Da and an estimated isoelectric point (pI) of 5.2. Based on phylogenetic analysis, the gene is clustered with the hsc70 proteins of invertebrates and vertebrates. In native gel electrophoresis, recombinant P. monodon hsc70 expressed in an Escherichia coli system is tightly associated with carboxymethylated alpha-lactalbumin (CMLA), which indicates that hsc70 probably functions as a chaperone. In an in vitro adenosine triphosphatase assay, recombinant hsc70 hydrolyzed adenosine triphosphate to adenosine-5'-diphosphate and increased hydrolysis activity by binding to unfolded peptide, CMLA. In situ hybridization using an antisense riboprobe revealed that the hsc70 gene was active in most tissues of unstressed shrimp. The expression of hsc70 messenger ribonucleic acid (mRNA) in hemocytes increased 2- to 3-fold at the first hour after shrimp experienced heat shock and 0.5-hour recovery. Hsc70 mRNA decreased gradually to the background level. Cloning and characterizing the P. monodon hsc70 gene is the first, crucial step in studying the relationship of heat shock proteins with the stress or immune responses of shrimp.     

Receptor for Activated C Kinase-1 protein from Penaeus monodon (Pm-RACK1) participates in the shrimp antioxidant response

Cellular oxidative stress responses are caused in many ways, but especially by disease and environmental stress. After the initial burst of reactive oxygen species (ROS), the effective elimination of ROS is crucial for the survival of organisms and is mediated by antioxidant defense mechanisms. In this paper, we investigate the possible antioxidant function of Penaeus monodon Receptor for Activated C Kinase-1 (Pm-RACK1). When Pm-RACK1 was over-expressed in Escherichia coli cells or Spodoptera frugiperda (Sf9) insect cells exposed to H₂O₂, it significantly protected the cells from oxidative damage induced by H₂O₂. When recombinant Pm-RACK1 protein was expressed as a histidine fusion protein in E. coli and purified with a Ni²⁺-column it possessed antioxidant functions that protected DNA from metal-catalyzed oxidation. Shrimp (Penaeus vannamei) held at an alkaline pH had a much higher hepatopancreatic expression of Pm-RACK1 than in those held at pH 7.4. The exposure of shrimp to alkaline pH is also known to increase ROS production. These results provide strong evidence that Pm-RACK1 can participate in the shrimp antioxidant response induced by the formation of ROS.     

Isolation and characterization of cDNA encoding Argonaute, a component of RNA silencing in shrimp (Penaeus monodon)

We have identified a cDNA clone that encodes a protein with high sequence homology to Argonaute proteins of mammals and Drosophila melanogaster. The cDNA of Penaeus monodon (Pm Ago) consisted of 3178 nucleotides encoding 939-amino acid residues with a calculated molecular weight of 104 kDa. The primary structure of Pm Ago showed the presence of two signature domains, PAZ and PIWI domains that exhibit highest homology to their counterparts in D. melanogaster. The inferred protein sequence of Pm Ago was 80.8% identical with D. melanogaster and 82.1% identical with Anopheles gambiae Ago proteins. Phylogenetic analysis of Pm Ago with other invertebrate and vertebrate Argonaute proteins suggested that Pm Ago belongs to the Ago1 subfamily that plays crucial roles in stem cell differentiation or RNA interference (RNAi). Semi-quantitative RT-PCR analysis showed that the gene is highly expressed in the lymphoid organ and moderately expressed in intestine, muscle, pleopods and hemocytes. The expression of Pm Ago1 mRNA was 2-3-fold increased during the early period of viral infection but declined rapidly at 30 hour post infection. By contrast, infection of shrimp by a bacterial pathogen, Vibrio harveyi did not induce a reduction of Pm Ago1 mRNA suggesting that its expression is associated with virus infection.     

Isolation and characterization of 23 polymorphic microsatellite markers for diversity and stock analysis in tiger shrimp (Penaeus monodon)

The tiger shrimp (Penaeus monodon) is an important marine crustacean in terms of biological diversity and aquaculture resource. The shrimp is widespread across the Indo‐Pacific region and shows apparent genetic differentiation among geographical populations. It is common practice to transport female brooders between different countries to seed the shrimp farms, posing potential problems of unwanted population admixture. We developed 23 polymorphic microsatellites for P. monodon (average H_E = 0.936) and these microsatellites were applicable for studying population differentiation, identifying valid stocks and tagging nonindigenous farmed shrimps.     

Antimicrobial effect of Moringa oleifera seed powder against Vibrio cholerae isolated from the rearing water of shrimp (Penaeus vannamei) postlarvae

Shrimp farming has experienced rising costs as a result of disease outbreaks associated with Vibrio spp. Suitable strategies for disease prevention and control are therefore urgently needed. This study aimed to evaluate the antimicrobial effect of Moringa oleifera seed powder against Vibrio cholerae in the rearing water of Pacific white shrimp (Penaeus vannamei) postlarvae. In vitro assays included the determination of minimum inhibitory concentration (MIC) of M. oleifera seed powder against V. cholerae, whereas in vivo assays included the effect of M. oleifera seed powder on bacterial load and water quality parameters in the rearing tanks, as well as its effect on shrimp postlarvae survival. M. oleifera seed powder inhibited the growth of V. cholerae with MIC values of 62·5 µg ml⁻¹. Moreover, seawater pH of treated tanks (8·66) was significantly lower (P < 0·01) than pH of the control tanks (9·02), whereas the visibility of treated tanks (37·08 cm) was significantly higher (P < 0·01) as compared to control tanks (35·37 cm). Likewise, V. cholerae load was significantly reduced (P < 0·01) from 4·7 × 10⁴ to 3·1 × 10³ CFU per ml in tanks treated with M. oleifera seed powder. Altogether, this study demonstrates the antimicrobial activity of M. oleifera against V. cholerae in shrimp culture.     

Characterization of four lytic transducing bacteriophages of luminescent Vibrio harveyi isolated from shrimp (Penaeus monodon) hatcheries

Four lytic bacteriophages designated as φVh1, φVh2, φVh3, and φVh4 were isolated from commercial shrimp hatcheries, possessing broad spectrum of infectivity against luminescent Vibrio harveyi isolates, considering their potential as biocontrol agent of luminescent bacterial disease in shrimp hatcheries, and were characterized by electron microscopy, genomic analysis, restriction enzyme analysis (REA), and pulsed-field gel electrophoresis (PFGE). Three phages φVh1, φVh2, and φVh4 had an icosahedral head of 60-115 nm size with a long, noncontractile tail of 130-329 × 1-17 nm, belonged to the family Siphoviridae. φVh3 had an icosahedral head (72 ± 5 nm) with a short tail (27 × 12 nm) and belonged to Podoviridae. REA with DraI and PFGE of genomic DNA digested with ScaI and XbaI and cluster analysis of their banding patterns indicated that φVh3 was distinct from the other three siphophages. PFGE-based genome mean size of the four bacteriophages φVh1, φVh2, φVh3, and φVh4 was estimated to be about 85, 58, 64, and 107 kb, respectively. These phages had the property of generalized transduction as demonstrated by transduction with plasmid pHSG 396 with frequencies ranging from 4.1 × 10(-7) to 2 × 10(-9) per plaque-forming unit, suggesting a potential ecological role in gene transfer among aquatic vibrios.     

Tissue-specific expression and regulation of the haemolymph clottable protein of tiger shrimp (Penaeus monodon)

The clottable protein (CP) involved in Penaeus monodon haemolymph coagulation has previously been characterized and cloned. Polyclonal antibodies against purified CP were also prepared from rabbit serum. By Western blot analyses, we showed occurrence of CP in the shrimp central nervous system, gill, and lymphoid organ. Results of RT-PCR further indicated that the central nervous system, gill, and lymphoid organ transcribed more CP, heart and hepatopancreas transcribed less, while the haemocytes and the muscle did not. We further analyzed the CP distribution within shrimp lymphoid organ by immunohistochemical method, CP was found to localise in stromal cells of lymphoid organ rather than in the developing haemocytes. In addition, concentrations and regulation of the plasma CP under normal and artificially traumatic conditions were studied with rocket immunoelectrophoresis. The average plasma CP concentration in normal intermolt shrimps was elevated from 3 mg ml(-1) to above 12 mg ml(-1) after successive blood-withdrawing for a week. The production and secretion of CP apparently were increased more than 4 folds to compensate its loss. Our result also suggested that the shrimp sinus gland endocrine system is not directly required for the expression and up-regulation of CP.     

Characterization of Argonaute2 gene from black tiger shrimp (Penaeus monodon) and its responses to immune challenges

Argonaute2 binds to a short guide RNA (microRNA or short interfering RNA) and guides RNAs direct RISC to complementary mRNAs that are targets for RISC-mediated gene silencing. Here we identified and characterized Argonaute2 from black tiger shrimp Penaeus monodon (designated as PmAgo2). The full-length cDNA of PmAgo2 contained a 5′ untranslated region (UTR) of 106 bp, an open reading frame (ORF) of 2616 bp and a 3′ UTR of 123 bp. The predicted PmAgo2 protein is 99.4 KDa with the theoretical isoelectric point of 9.54. PmAgo2 shared the highest similarity of amino acid with Marsupenaeus japonicus Argonaute2 and Litopenaeus vannamei Argonaute2, at 69.0% and 68.5%, respectively. Phylogenic analysis showed PmAgo2 clustered with shrimp Argonaute2, and closed to the group of insects. Real-time quantitative PCR showed that PmAgo2 was widely expressed in almost all examined tissues except eyestalk, with high expression in lymph and haemocyte. mRNA expression also revealed that PmAgo2 was significantly up-regulated by Staphylococcus aureus and White Spot Syndrome Virus (WSSV) in hepatopancreas. Furthermore, our study also confirmed that dsRNA and ssRNA homologous poly (I:C) and R848 activated the expression of PmAgo2. The result indicated that PmAgo2 responded to both bacterial infection and viral infection, especially, it may induce an ssRNA-mediated RNAi with other core members of siRNA pathway in black tiger shrimp.     

Immunological responses of Penaeus monodon to DNA vaccine and its efficacy to protect shrimp against white spot syndrome virus (WSSV)

White spot disease is an important viral disease caused by white spot syndrome virus (WSSV) and is responsible for huge economic losses in the shrimp culture industry worldwide. The VP28 gene encoding the most dominant envelope protein of WSSV was used to construct a DNA vaccine. The VP28 gene was cloned in the eukaryotic expression vector pcDNA3.1 and the construct was named as pVP28. The protective efficiency of pVP28 against WSSV was evaluated in Penaeus monodon by intramuscular challenge. In vitro expression of VP28 gene was confirmed in sea bass kidney cell line (SISK) by fluorescence microscopy before administering to shrimp. The distribution of injected pVP28 in different tissues of shrimp was studied and the results revealed the presence of pVP28 in gill, head soft tissue, abdominal muscle, hemolymph, pleopods, hepatopancreas and gut. RT-PCR and fluorescence microscopy analyses showed the expression of pVP28 in all these tissues examined. The results of vaccination trials showed a significantly higher survival rate in shrimp vaccinated with pVP28 (56.6-90%) when compared to control groups (100% mortality). The immunological parameters analyzed in the vaccinated and control groups revealed that the vaccinated shrimp showed significantly high level of prophenoloxidase and superoxide dismutase (SOD) when compared to the control groups. The high levels of prophenoloxidase and superoxide dismutase (SOD) might be responsible for developing resistance against WSSV in DNA vaccinated shrimp.     

O1或O139霍乱疫苗对异群霍乱缺乏交叉保护

O1和O139霍乱弧菌是引起急性腹泻的病原微生物,用这两群菌的灭活全菌体与重组霍乱毒素B亚单位构建的亚单位／菌体型疫苗免疫队CA小鼠,对本群菌的攻击可提供良好免疫保护,而对异群霍乱菌则缺乏交叉保护作用。     

Detection of cholera (ctx) and zonula occludens (zot) toxin genes in Vibrio cholerae O1, O139 and non-O1 strains

Vibrio cholerae O1 and V. cholerae non-O1 strains isolated from environmental samples collected in São Paulo, Brazil, during cholera epidemics and pre-epidemic periods were examined for the presence of toxin genes. V. cholerae O1 strains isolated from clinical samples in Peru and Mexico, and V. cholerae O139 strains from India were also examined for the presence of ctx (cholera toxin gene) and zot (zonula occludens toxin gene) by polymerase chain reaction (PCR). A modified DNA-extraction method applied in this study yielded satisfactory recovery of genomic DNA from vibrios. Results showed that strains of V. cholerae O1 isolated during the preepidemic period were ctx ^-/zot ^- whereas strains isolated during the epidemic were ctx ⁺/zot ⁺. All V. cholerae non-O1 strains tested in the study were ctx ^-/zot ^-, whereas all V. cholerae O139 strains were ctx ⁺/zot ⁺. Rapid detection of the virulence genes (ctx and zot) can be achieved by PCR and this can serve as an important tool in the epidemiology and surveillance of V. cholerae.