'Bright-red' syndrome in Pacific white shrimp Litopenaeus vannamei is caused by Vibrio harveyi

Since July 2005, recurrent outbreaks of vibriosis have occurred in shrimp farms in northwestern Mexico. Moribund Litopenaeus vannamei associated with mass mortalities were lethargic and displayed red discoloration spots on their abdomen, and hence were called 'bright-reds' by farmers. Shrimp submitted for diagnosis were examined using wet tissue mounts, bacteriological assays and their respective minimum inhibitory concentration (MIC), and histology. A dominant yellow bacterial colony was isolated in thiosulphate citrate bile salts-sucrose (TCBS) agar and identified by molecular methods as Vibrio harveyi strain CAIM 1792. Pathogenicity of the V. harveyi strain was demonstrated in L. vannamei. The lowest MIC against Vibrio isolates from bright-red shrimp was obtained with enrofloxacine (3.01, SD = 5.96 pg ml(-1)). Histology detected severe necrosis in lymphoid organ tubules, muscle fibers, and connective tissue, as well as melanization and hemocytic nodules associate with microcolonies of Gram-negative bacilli. Bacteria from severely affected shrimp were dispersed from the haemocoel to other tissues causing a systemic vibriosis. The data indicate that V. harveyi strain CAIM 1792 is the cause of bright-red syndrome (BRS) and represents a threat to the Mexican shrimp farming industry.     

The immune response of white shrimp Litopenaeus vannamei following Vibrio alginolyticus injection

White shrimp Litopenaeus vannamei injected with saline, and injected with tryptic soy broth (TSB)-grown Vibrio alginolyticus at 1.0 x 10(5) and 1.8 x 10(5) colony-forming units (cfu) shrimp(-1) were examined for hyaline cell (HC) counts, granular cell (GC) counts, total haemocyte counts (THCs), phenoloxidase (PO) activity, respiratory burst (RB) and superoxide dismutase (SOD) activity after 1-168 h. Shrimp that received no injection served as the control. The shrimps which received V. alginolyticus at both doses showed significant decreases in these parameters after 6-96 h. The values for HC and SOD activity decreased earlier and then RB. The time to cause maximum depletion of haemocytes (haemocytopenia), PO activity, RB, and SOD activity were 12, 72, 48, and 24 h post-injection, respectively. The HC, GC, and RB returned to the original values earlier at 72 h, followed by SOD activity at 96 h, and then PO activity at 168 h post-infection. It was concluded that an injection of V. alginolyticus rapidly reduced the shrimp's immunity by decreasing HC, GC, SOD activity, RB, and PO activity within 3-24 h, followed by a slow recovery during 72-168 h post-injection. Furthermore, white shrimp L. vannamei which received V. alginolyticus showed a 6-9 h later response in PO activity, and a 72-96 h later recovery of PO activity, compared to the responses in RB and SOD activity indicating their roles in shrimp defence and immunity.     

Virulence of Vibrio harveyi responsible for the "Bright-red" Syndrome in the Pacific white shrimp Litopenaeus vannamei

Vibrio harveyi (Vh) CAIM 1792 strain was isolated from Litopenaeus vannamei affected with "Bright-red" Syndrome (BRS). The strain grew in 1-10% NaCl, at 15-35°C and was resistant to ampicillin (10 μg), carbenicillin (100 μg) and oxytetracycline (30 μg). The lowest MIC was for enrofloxacine (0.5 μgml(-1)). The in vivo and in vitro toxicity of bacterial cells and the extracellular products (ECPs) of Vh CAIM 1792 grown at 1.0%, 2.0% and 4.0% NaCl were evaluated. Adherence ability, enzymatic activities and siderophore production of bacterial cell was tested. The ECPs exhibited several enzymatic activities, such as gelatinase, amylase, lipase, phospholipase and caseinase. These ECPs displayed a strong cytotoxic effect on HELA cell line at 6 and 24 h. Challenges using 10(3) CFU g(-1) caused opacity at the site of injection and over 80% shrimp mortality before 24 h p.i. (post-injection). Mortality caused by the ECPs was higher than mortalities with bacteria, especially in the first hours p.i. Bacteria were re-isolated from hemolymph samples of moribund shrimp and identified as Vh CAIM 1792 by rep-PCR. Histological analysis of shrimp L. vannamei injected with Vh CAIM 1792 revealed generalized necrosis involving skeletal muscle (MU) at the injection site, the lymphoid organ (LO), heart and connective tissues. Melanization within the MU at the site of injection was also observed as well as hemocytic nodules within the hearth and MU at 168 h p.i. LO was the target organ of BRS. Necrosis of the MU at the injection site was the main difference in comparison to other shrimp vibriosis.     

Innate immunity of the white shrimp Litopenaeus vannamei weakened by the combination of a Vibrio alginolyticus injection and low-salinity stress

White shrimp Litopenaeus vannamei were reared at a salinity of 35‰ without a Vibrio alginolyticus injection (unchallenged group), and other shrimp were reared at 35‰, injected with tryptic-soy broth (TSB)-grown V. alginolyticus at 1.8 × 10⁵ colony-forming units (cfu) shrimp^?1 (challenged group), and then examined for the hyaline cell (HC) count, granular cell (GC, including semi-granular cell) count, total haemocyte count (THC), phenoloxidase (PO) activity, respiratory burst (RB) and superoxide dismutase (SOD) activity after transfer to 35‰ (control), 25‰, 20‰, and 15‰ for 1, 6, 12, 24, 72, and 120 h. Results indicated that the haemocyte count, PO activity, RB, and SOD activity of unchallenged shrimp and challenged shrimp that were transferred to low-salinity levels all began to significantly decrease at 6, 6, 6, and 1 h, respectively, and reached the lowest levels at 12 h. HC, GC, the THC, PO activity, RB, and SOD activity of unchallenged shrimp that were transferred to 15‰ decreased by 53%, 41%, 49%, 68%, 39%, and 62%, whereas those parameters of challenged shrimp that were transferred to 15‰ decreased by 79%, 78%, 79%, 82%, 54%, and 72%, respectively after 12 h compared to control shrimp. These immune parameters began to recover after 24–72 h for both unchallenged shrimp and challenged shrimp. We concluded that the innate immunity was weakened in white shrimp L. vannamei that received combined stresses of a V. alginolyticus injection, and low-salinity transfer. It was also concluded that shrimp with respectively 21%, 18%, 46%, and 28% lower THC, PO activity, RB, and SOD activity of the original values would be killed due to decreases in their immunity, and resistance to V. alginolyticus infection. Shrimp farming should be maintained at a constant high salinity level to prevent exacerbated decreases in innate immune parameters of shrimp when infected by a pathogen coupled with low-salinity stress leading to mortality.     

凡纳滨对虾繁殖中不同亲本对子代遗传贡献率的差异

利用5个含有稀有等位基因的高度多态性微卫星位点比较了凡纳滨对虾繁殖中不同亲本对子代遗传贡献率的差异。通过稀有等位基因的5个微卫星位点能够对亲代和子代的谱系进行明确的鉴别。10个亲代个体中有8个个体对子代群体的基因库有贡献,不同个体之间的贡献率存在差别,最高为54.28%,最低为8.57%。在亲代和子代群体遗传结构的分析中,子代等位基因的数目与亲代相比降低了11.11%。子代的平均期望杂合度(He)、平均观测杂合度(Ho)和平均多态性信息含量(PIC)等指标均低于亲代。实验结果表明:亲本对子代基因库的贡献率的差异也是造成子代群体遗传变异水平降低的原因之一;微卫星标记可作为一种有效的工具用于对虾系谱的确认、人工繁育群体遗传多样性水平的监测等方面     

Gene expression of apoptosis-related genes,stress protein and antioxidant enzymes in hemocytes of white shrimp Litopenaeus vannamei under nitrite stress

Apoptotic cell ratio and mRNA expression of caspase-3, cathepsin B (CTSB), heat shock protein 70 (HSP70), manganese superoxide dismutase (MnSOD), catalase (CAT), glutathione peroxidase (GPx) and thioredoxin (TRx) in hemocytes of white shrimp Litopenaeus vannamei exposed to nitrite-N (20 mg/L) was investigated at different stress time (0, 4, 8, 12, 24, 48 and 72 h). The apoptotic cell ratio and mRNA expression level of CTSB were significantly increased in shrimp exposed to nitrite-N for 48 and 72 h. Caspase-3 mRNA expression level significantly increased by 766.50% and 1811.16% for 24 and 48 h exposure, respectively. HSP70 expression level significantly increased at 8 and 72 h exposure. MnSOD mRNA expression in hemocytes up-regulated at 8 and 48 h, while CAT mRNA expression level increased at 24 and 48 h. GPx expression showed a trend that increased first and then decreased. Significant increases of GPx expression were observed at 8 and 12 h exposure. Expression level of TRx reached its highest level after 48 h exposure. These results suggest that nitrite exposure induces expression of apoptosis-related genes in hemocytes, and subsequently caused hemocyte apoptosis. Meanwhile, expression levels of HSP70 and antioxidant enzymes up-regulated to protect the hemocyte against nitrite stress.     

Haemolymph parameters of Pacific white shrimp (Litopenaeus vannamei) infected with Taura syndrome virus

Pacific white shrimp (Litopenaeus vannamei) were injected with Taura syndrome virus (TSV) to assess shrimp immune responses and survival. TSV-infected shrimp suffered high mortality, but mock-infected and untreated shrimp experienced no mortality. Moribund shrimp were a pale, reddish colour and were lethargic and soft-shelled. Their haemolymph was clear red and coagulated poorly. In TSV-infected shrimp, the total haemocyte count (THC), hyalinocyte and granulocyte counts, and total plasma protein decreased significantly to 21%, 24%, 17% and 56% of untreated control values, respectively. Haemocyanin decreased to 67%, and clottable proteins to 80% of control values (P< 0.01). Copper and calcium ions, haemocytic transglutaminase (TGase) activity and plasma growth inhibitory activity against Vibrio harveyi also decreased significantly. Generation of intrahaemocytic superoxide anion, O(-2), in TSV-infected shrimp was significantly greater (P< 0.05) than in both control groups, no matter whether glucan stimulated or unstimulated. But the relative increase of intrahaemocytic O(-2) generation in TSV-infected shrimp response to glucan stimulation was lower in both controls. Plasma phenoloxidase (PO) activity increased significantly in TSV-infected shrimp. The plasma bacterial agglutinin titre against E. coli and V. harveyi, growth inhibition of E. coli and the concentration of magnesium ions in TSV-infected shrimp did not change significantly.In conclusion, ten of thirteen haemolymph parameters changed significantly during the host-TSV interaction. These parameters might be valuable references of shrimp health status.     

Isolation and characterization of microsatellites in Pacific white shrimp Penaeus (Litopenaeus) vannamei

Five polymorphic microsatellite loci were characterized for Penaeus (Litopenaeus) vannamei. Loci were isolated using a partial Sau3A1 genomic library by the sequencing of randomly selected clones and by a biotinylated (CT)₁₀ and (GT)₁₀ probes screening procedure. The last strategy resulted in the most useful data. About 40% of the clones showed a previously reported satellite/microsatellite (PVS1), reducing the chance of finding new microsatellite regions. Whereas two of the microsatellite loci with more than 10 alleles will be useful for mating analysis in a breeding program, the others might prove useful for population genetic studies.     

Isolation and characterization of microsatellite markers from Pacific white shrimp (Litopenaeus vannamei)

We report the development of 11 polymorphic microsatellite loci in pacific white shrimp (Litopenaeus vannamei) using an unenriched genomic library. The number of the alleles ranged from two to 18 and observed hererozygosity ranged from 0.0286 to 0.9429, indicating that these markers will be useful for population studies and mapping in pacific white shrimp. Seven loci were detected deviated from Hardy–Weinberg, caused by deficiency of heterozygote, suggesting population genetic structure across the sampled population. No evidence for linkage disequilibrium was found.     

Effects of hypoxia and hypercapnic hypoxia on the localization and the elimination of Vibrio campbellii in Litopenaeus vannamei, the Pacific white shrimp

Low oxygen (hypoxia) and elevated CO2 (hypercapnia, are characteristic of estuarine environments. Although hypoxia and hypercapnic hypoxia decrease the resistance of shrimp to bacterial pathogens, their direct effects on the immune system are unknown. Here we present evidence demonstrating in the penaeid shrimp Litopenaeus vannamei that both hypoxia and hypercapnic hypoxia affect the localization of bacteria, their conversion from culturable to non-culturable status (bacteriostasis), and their elimination from hemolymph and selected tissues. Shrimp were injected with a sublethal dose of a pathogenic strain of Vibrio campbellii expressing green fluorescent protein and resistance to kanamycin. Real-time polymerase chain reaction was used to determine the number of intact V. campbellii in hemolymph, gills, hepatopancreas, heart, and lymphoid organ. Selective plating was used to quantify the injected bacteria that remained culturable. We found that both hypercapnic hypoxia and hypoxia increased the percentage of culturable bacteria recovered from the hemolymph and tissues, suggesting an overall decrease in bacteriostatic activity. Hypoxia and hypercapnic hypoxia generally increased the distribution of intact V. campbellii to the hepatopancreas and the gills, which are major targets for the pathogenic effects of Vibrio spp., without affecting the number of intact bacteria in the lymphoid organ, a main site of bacterial accumulation and bacteriostatic activity.     

Noradrenaline modulates the immunity of white shrimp Litopenaeus vannamei

The total haemocyte count (THC), phenoloxidase activity, respiratory burst, superoxide dismutase (SOD) activity, phagocytic activity and clearance efficiency in response to pathogen Vibrio alginolyticus were measured when the white shrimp Litopenaeus vannamei (18.4 +/- 1.2 g) were injected individually with noradrenaline at 10(-8), 10(-7) and 10(-6) mol shrimp(-1). For the shrimp that received noradrenaline at 10(-8), 10(-7) and 10(-6) mol shrimp(-1), the THC decreased by 15%, 21% and 32%, phenoloxidase activity decreased by 15%, 31% and 31%, respiratory burst decreased by 13%, 21% and 32%, and SOD activity decreased by 46%, 56% and 55%, respectively, after 2 h. The phagocytic activity and clearance efficiency of shrimp that received noradrenaline at either dose decreased significantly after 2 h. The THC, phenoloxidase activity, respiratory burst, SOD activity, phagocytic activity and clearance efficiency returned to normal values after 4, 4, 8, 24, 16 and 8 h, respectively, in the shrimp that received noradrenaline at either dose. In another experiment, L. vannamei which had received noradrenaline at 10(-8), 10(-7) and 10(-6) mol shrimp(-1) were challenged after 1h by injection with V. alginolyticus at 1.0 x 10(5) colony-forming units (cfu)shrimp(-1) and then placed in seawater of 20 per thousand. The cumulative mortality of shrimp that received noradrenaline at either dose was significantly higher than that of shrimp that received saline after 4 h, and at the termination of the experiment (48 h after the challenge). It is therefore concluded that noradrenaline administration at 10(-6) mol shrimp(-1) or less causes immune modulation of L. vannamei.     

The protective effect of chitin and chitosan against Vibrio alginolyticus in white shrimp Litopenaeus vannamei

White shrimp, Litopenaeus vannamei, which had been injected with chitin at 4, 6 and 8 microg g(-1) or chitosan at 2, 4 and 6 microg g(-1), were challenged with pathogen Vibrio alginolyticus at 2 x 10(6) colony-forming units (cfu) shrimp(-1) and then placed in seawater of 34 per thousand. The survival of shrimp that received chitin or chitosan at either dose was significantly higher than that of control shrimp after 1 day, and at the termination of the experiment (6 days after the challenge). In another experiment, the total haemocyte count (THC), phenoloxidase activity, respiratory burst, superoxide dismutase (SOD) activity, and phagocytic activity to V. alginolyticus were measured when L. vannamei (10.4 +/- 0.7 g) were injected individually with chitin at 4 and 6 microg g(-1) or chitosan at 2 and 4 microg g(-1). L. vannamei received chitin at 6 microg g(-1) or chitosan at 2 and 4 microg g(-1) increased significantly its THC and respiratory burst after 2 days. L. vannamei received chitin at 6 microg g(-1) or chitosan at 2 and 4 microg g(-1) still maintained significantly higher phenoloxidase activity after 6 days. L. vannamei received chitin at 4 and 6 microg g(-1) or chitosan at 2 and 4 microg g(-1) increased its phagocytic activity against V. alginolyticus after 1 day, respectively. It is therefore concluded that L. vannamei that received chitin at 6 microg g(-1) or chitosan at 4 microg g(-1) or less increased its immune ability and resistance to V. alginolyticus infection.     

Experimental infection of Pacific white shrimp Litopenaeus vannamei with Necrotizing Heptopancreatitis (NHP) bacterium by per os exposure

Necrotizing Hepatopancreatitis Bacterium (NHPB), which causes Necrotizing Hepatopancreatitis, was successfully transmitted in individually isolated Kona stock Litopenaeus vannamei through per os exposure. Animals (140) were individually exposed orally to a 0.05 g piece of an NHPB-infected hepatopancreas and 120 control animals were each exposed to a 0.05 g piece of NHPB-negative hepatopancreas. Shrimp were maintained in Sterilite containers with approximately 41 of artificial seawater at 30 per thousand salinity and 30 degrees C for 60 d. Mortality of infected shrimp was observed from Day 16 to Day 51 post-exposure. Infected animals sustained reduced feeding activity and displayed empty guts. Some infected animals developed a pale hepatopancreas noticeable through the carapace. Survival probabilities fit a Weibull distribution and parametric survival analysis revealed lowered survival due to NHPB infection. Median survival time of NHPB-infected animals was 34.5 d. After correcting for background daily mortality in the controls, mean acute daily mortality of NHPB was estimated at 0.09, a value much lower than that estimated for other diseases in Kona stock L. vannamei such as White Spot Syndrome Virus (0.40) and Taura Syndrome Virus (0.30). A chronic, or carrier, state was not demonstrated in NHPB epizootics because all NHPB-positive animals experienced mortality and no animals surviving to 60 d post-exposure were diagnosed NHPB-positive through PCR or histology.