Time course of necrotizing hepatopancreatitis (NHP) in experimentally infected Litopenaeus vannamei and quantification of NHP-bacterium using real-time PCR

Necrotizing hepatopancreatitis (NHP), a severe bacterial disease affecting penaeid shrimp aquaculture, is caused by a gram-negative, pleomorphic, intracellular alpha-proteobacterium referred to as the NHP-bacterium (NHPB). The time course of NHP was investigated in experimentally infected juveniles of Kona stock Litopenaeus vannamei. Susceptible animals were individually isolated in 41 of aerated artificial seawater at salinity 30 +/- 1 ppt and maintained in a water bath at 30 +/- 1 degree C for 60 d. A total of 120 individuals were exposed per os to a 0.05 g piece of NHPB-infected hepatopancreas and 100 controls were exposed to uninfected tissue. At intervals of 3, 6, 9, 16, 23, 30, 37, 44, and 53 d post-exposure, 6 shrimp exposed to NHPB-infected tissue and 4 controls were randomly removed from the experiment; hepatopancreas samples were processed for histological and molecular analysis, and feces were processed for molecular diagnosis of NHPB infection. NHPB was first detected in the hepatopancreas through histology at 6 d post-exposure. All control shrimp were diagnosed as NHPB negative. NHPB infections classified as stage I (scattering of hepatopancreatic tubules with adjacent epithelial cells containing NHPB) and stage II (numerous infected tubules with occasional hemocyte infiltration) were observed from 6 to 37 d post-exposure. All animals that experienced NHPB-induced mortality from 16 to 51 d post-exposure were at stage III (numerous necrotic tubules, dense hemocyte infiltration, and presence of granulomas). NHPB is capable of infecting all hepatopancreatic cell types including embryonic, resorptive, fibrillar and blister-like cells. The percent of hepatopancreatic tubules containing NHPB in epithelial cells increased over time, representing bacteria multiplication and spread. Real-time PCR allowed for quantification of NHPB in hepatopancreas and feces. Over the course of infection, NHPB was present at 10(3) to 10(7) copies mg(-1) of hepatopancreas and 10(1) to 10(5) copies mg(-1) of feces. Lethal infections contained 10(6) to 10(7) copies mg(-1) of hepatopancreas and 10(3) to 10(6) copies mg(-1) of feces.     

Effect of salinity on transmission of necrotizing hepatopancreatitis bacterium (NHPB) to Kona stock Litopenaeus vannamei

Elevated salinity and temperature have been observed prior to devastating necrotizing hepatopancreatitis (NHP) outbreaks in several geographically isolated shrimp ponds. These observations have led to the hypothesis that the NHP-bacterium (NHPB) is hindered by reduced salinity, even though the mechanism is not understood. The objective of this research was to examine the effect of salinity on transmission of NHPB. The transmission rate of NHPB was estimated through laboratory experiments whereby individuals of Kona stock Litopenaeus vannamei were orally exposed to a dead NHPB-infected shrimp. For each replicate, 12 susceptible shrimp were placed with a dead NHPB-infected shrimp in a 1 m2 bottom area cylindrical tank maintained at 30 degrees C for a period of 24 h. Four salinities of 10, 20, 30, and 40 per thousand were replicated 2 times in 2 trials, giving a total of 192 shrimp exposed per os to infective material. In each trial, a negative control group was included at each salinity, giving a total of 96 shrimp exposed orally to uninfected material. After the 24 h exposure period, susceptible shrimp were individually isolated at the same physical conditions for up to 60 d to determine NHPB transmission. The NHPB was transmissible regardless of salinity: nearly a quarter of susceptible shrimp exposed to NHPB at the lowest (10 per thousand) and highest (40 per thousand) salinity examined acquired NHPB. Transmission rates were highest at the intermediate salinities of 20 and 30 per thousand, suggesting that those salinities are optimal for NHPB transmission. The observed association between high salinity and NHP outbreak in a shrimp pond is not explained by these results because reduced transmission occurred at very low and very high salinities.     

Preservation of necrotizing hepatopancreatitis bacterium (NHPB) by freezing tissue collected from experimentally infected Litopenaeus vannamei

Necrotizing hepatopancreatitis (NHP), a severe disease of penaeid shrimp, is caused by bacteria (NHPB) that have previously been demonstrated to reside in tubular epithelial hepatopancreatic (HP) cells of infected shrimp. There has yet to be a successful in vitro culture method to grow the intracellular organism; therefore, it must be propagated in vivo via transmission from NHPB-infected shrimp to healthy individuals. In our studies, NHPB propagation tanks containing infected shrimp were used to maintain a constant supply of organisms for experiments. In order to develop a method for storing infectious NHPB material for future challenge studies, we collected HP tissue containing NHPB by flash freezing whole, fresh HPs at -80 degrees C for up to 80 d and used it to successfully infect specific pathogen-free Litopenaeus vannamei per os in controlled experiments. HP tissue samples were collected from dead shrimp, and PCR was performed to confirm the presence of NHPB. Our results demonstrate that the infectivity of NHPB in tissue is not altered after being frozen at -80 degrees C when compared to NHPB in fresh tissue. Thus, the continual propagation of NHPB in vivo is not required to assure a source of the infectious agent.     

Epidemiological parameters of White Spot Syndrome Virus infections in Litopenaeus vannamei and L. setiferus

An experimental protocol based on a mathematical epidemiology model was developed to study the transmission, virulence, and recovery rates of White Spot Syndrome Virus (WSSV). Two modes of transmission were compared for WSSV in Litopenaeus vannamei. We compared transmission by ingestion of infected cadavers to transmission by cohabitation with infected animals. In addition, we compared the ingestion transmission of WSSV in L. vannamei and in L. setiferus. Finally, we compared the virulence and recovery rates of WSSV in L. vannamei and L. setiferus. The transmission rate of WSSV to L. vannamei by cohabitation was 0.01. The transmission rate by ingestion of infected cadavers was over an order of magnitude larger at 0.46, suggesting that cohabitation is a much less important mode of transmission for WSSV. A statistically significant difference was detected between the estimates of ingestion transmission of L. vannamei (0.46) and those of L. setiferus (0.84), yet no differences in the virulence or recovery rates were detected between hosts. The overall estimated virulence rate was 0.34, and the overall estimated recovery rate from a WSSV infection was 0.007 for both species. According to epidemiological theory the threshold density of hosts necessary for an epidemic to occur is directly related to the virulence and recovery rates and inversely related to the transmission rate. Therefore, the epidemic threshold density may be lower for ingestion transmission than cohabitation transmission and lower for L. setiferus than for L. vannamei.     

In situ hybridization demonstrates that Litopenaeus vannamei, L. stylirostris and Penaeus monodon are susceptible to experimental infection with infectious myonecrosis virus (IMNV)

Infectious myonecrosis virus (IMNV) was recently found to be the cause of necrosis in the skeletal muscle of farm-reared Litopenaeus vannamei from northeastern Brazil. Nucleic acid extracted from semi-purified IMN virions showed that this virus contains a 7.5 kb RNA genome. A cDNA library was constructed, and a clone, designated as IMNV-317, was labeled with digoxigenin-11-dUTP and used as a gene probe for in situ hybridization (ISH). This probe specifically detected IMNV in infected tissues. To determine the susceptibility of 3 species of penaeid shrimp (L. vannamei, L. stylirostris, Penaeus monodon) to IMNV infection, juveniles were injected with purified virions and observed for clinical signs of infection and mortality over a 4 wk period. All L. vannamei exhibited typical lesions after 6 d, and lesions were visible in all L. stylirostris by Day 13. The clinical signs of opaque muscle were not seen in P. monodon, due to their highly pigmented exoskeleton precluding visual detection of lesions. Moderate mortality (20%) occurred in infected L. vannamei. No mortalities were observed in either L. stylirostris or P. monodon. Histological examination and ISH indicated that all 3 species are susceptible to IMNV infection. Using ISH, IMNV was detected in tissues including the skeletal muscle, lymphoid organ, hindgut, and phagocytic cells within the hepatopancreas and heart. In all 3 species, skeletal muscle cells produced the strongest ISH reactions. Based on the onset of clinical signs of infection and mortality, L. vannamei appears to be the most susceptible of these 3 species to IMNV infection.     

Hepatopancreatic alterations in Litopenaeus vannamei (Boone, 1939) (Crustacea: Decapoda: Penaeidae) experimentally infected with a Vibrio alginolyticus strain

As in other countries, the presence of disease has affected and limited the development of shrimp culture in Venezuela. Vibriosis is one of the most prevalent, causing high mortality not only in larval cultures but also in shrimp production. The present work shows the histological changes in hepatopancreas of Litopenaeus vannamei experimentally induced by a strain of Vibrio alginolyticus. The shrimps were infected by the immersion technique, being exposed to a concentration of 5.2 x 10(7) bacteria/ml. As soon as their behavior indicated a moribund condition, the animals were sacrificed, and their hepatopancreata were fixed and processed for histological observation. Light microscopical observations revealed loss of the acinar structure of the digestive gland and sloughing off of cellular lining because of cytolisis. The acinar cellular content was dispersed within the lumina and among the acini. Clusters of hemocytes, in the majority loaded with bacteria, were observed under the connective tissue capsule surrounding the hepatopancreatic lobes and in the hemal spaces of the connective tissue sheath surrounding the tubules.     

Factors affecting abundance of Triaenophorus infection in Cyclops strenuus, and parasite-induced changes in host fitness

Factors affecting the abundance of Triaenophorus crassus and Triaenophorus nodulosus procercoids in their copepod first intermediate host, Cyclops strenuus, and effects of infection on feeding behaviour, reproduction and survival of the host were studied experimentally. When exposed to the same number of coracidia, copepods harboured considerably less procercoids in the trials where ciliates or Artemia salina nauplii were given as alternative food items. The prevalence of infection was higher in adult copepods as compared with copepodite stage IV and stage V, and higher in stage V than in stage IV. The prevalences in adult females and males did not differ significantly from each other. The frequency of females carrying egg sacs was lower among infected than among exposed uninfected and unexposed copepods. The rate of feeding on Artemia nauplii remained at the same level in uninfected copepods, but decreased strongly in infected copepods during 7 days p.i. The survival of unexposed, exposed uninfected and infected copepods did not differ significantly from each other for the first 11 days post-exposure, but the mortality of infected copepods increased significantly after 3 weeks post-exposure. However, the rate of development and mortality of copepods might have been affected by the apparently arrested development of stage IV copepodites found in the experiment. Some of the contradictions between these results and earlier observations are suggested to be caused by the differences in the duration of exposure, intensity of infection and duration of observation post-exposure in the present study as compared with other experiments.     

Association of bovine embryos produced by in vitro fertilization with a noncytopathic strain of bovine viral diarrhea virus type II

In the first experiment, heifers were infected experimentally with bovine viral diarrhea virus type II (BVDV-type II, strain CD87; characterized by high morbidity and mortality). Subsequently, in vitro fertilized embryos were produced from oocytes collected on Day 4, 8, and 16 post infection. In a total of 29 heifers, the infectious virus was detected in 55% of the samples of the follicular fluid, in 10% of the oviductal cells, in 10% of the uterine flushes and in 41% of the in vitro fertilized embryos. The highest number of embryos associated with the virus was detected in the group of animals slaughtered on Day 8 post infection (58%). The amount of the virus (10(1.5-2.0) TCID50/mL) associated with the washed single embryos generated from oocytes of heifers 8 and 16 d post infection was sufficient for disease transmission by intravenous inoculation to the seronegative recipients (6/15). In the second experiment, uninfected oocytes were exposed in vitro to BVDV (10(5) TCID50/mL) in the maturation medium and then fertilized and cultured prior to viral assay. Virus was detected in 4 of 7 samples containing embryos but not in samples of embryos produced from the control group of uninfected oocytes. The presence of BVDV in the IVF system did not affect embryonic development in vitro. In conclusion, it appears that BVDV-type II has the ability to be transferred with oocytes through the IVF system, resulting in infectious embryos with normal morphological appearance which may have a potential for disease transmission.     

Host age and pathogen exposure level as factors in the susceptibility of the house fly,Musca domestica (Diptera: Muscidae) to Beauveria bassiana

Adult house flies, Musca domestica L., of four ages, <1, 3, 7, and 14 day post-eclosion, were exposed to three strains of Beauveria bassiana (P89, L90 and 447). Flies were exposed to moistened filter paper treated with either a low (1.57×10⁴ conidia/cm²) or high (1.57×10⁵ conidia/cm²) concentration of each fungal strain for 6 h. Strain 447 was superior to the two house fly-derived B. bassiana strains in inducing host infection and mortality. Significant spikes in infection and mortality occurred as early as 5 days post-exposure with higher concentration exposures acting more quickly. Few differences were observed in either infection or mortality among the four fly age classes. On Day 10 post-exposure, 77% of the high-concentration, 447-exposed flies were infected, compared with only 24% of the flies from the P89 low-concentration exposure. Potential applications of these results in integrated house fly management programs are discussed.     

Experimental infections reveal that common Thai crustaceans are potential carriers for spread of exotic Taura syndrome virus

Taura syndrome virus (TSV) was first reported as a serious cause of shrimp mortality limited to reared Penaeus (Litopenaeus) vannamei in the Americas, where it spread principally through regional and international transfer of live post larvae (PL) and broodstock. Subsequently, through importation of infected broodstock, TSV outbreaks spread to Asia, first to Taiwan and China and then to Thailand, Indonesia and Korea. Since its introduction to Thailand, outbreaks have occasionally been reported from rearing ponds stocked with batches of specific pathogen free (SPF) P. vannamei PL that tested negative for TSV by nested RT-PCR assay. Since it was possible that the outbreaks may have occurred via horizontal transfer of TSV from wild carrier species, we tested 5 common native crustaceans that live in and around shrimp ponds (2 palaemonid shrimp species, Palaemon styliferus and Macrobrachium lanchesteri, and 3 species of crabs, Sesarma mederi, Scylla serrata and Uca vocans) for susceptibility to TSV in experimental challenges. We found that U. vocans, S. serrata and S. mederi did not die but, respectively, gave strong RT-PCR reactions indicating heavy viral load at 5, 10 and 15 d post-injection of TSV and 10, 15 and up to 50 d after feeding with TSV-infected P. vannamei carcasses. Also after feeding, P. styliferus did not die, but a high proportion gave strong RT-PCR reactions at 5 d post-challenge and no reactions at 15 d. Similarly after feeding, M. lanchesteri showed no mortality and gave only light RT-PCR reactions at 2 d, moderate reactions at 5 d and no reaction at 15 d. By contrast, transmission experiments from the TSV-infected crabs and palaemonid shrimp via water or feeding resulted in death of all the exposed P. vannamei from 8 to 12 d post-challenge and all were positive for heavy viral load by RT-PCR assay. Despite the results of these laboratory challenge tests, natural TSV infections were not detected by nested RT-PCR in samples of these species taken from the wild. These results indicated that transmission of TSV from infected crabs and palaemonid shrimp via water or feeding might pose a potential risk to shrimp aquaculture.     

Chronic and persistent viral hemorrhagic septicemia virus infections in Pacific herring

Chronic viral hemorrhagic septicemia virus (VHSV) infections were established in a laboratory stock of Pacific herring Clupea pallasii held in a large-volume tank supplied with pathogen-free seawater at temperatures ranging from 6.8 to 11.6 degrees C. The infections were characterized by viral persistence for extended periods and near-background levels of host mortality. Infectious virus was recovered from mortalities occurring up to 167 d post-exposure and was detected in normal-appearing herring for as long as 224 d following initial challenge. Geometric mean viral titers were generally as high as or higher in brain tissues than in pools of kidney and spleen tissues, with overall prevalence of infection being higher in the brain. Upon re-exposure to VHSV in a standard laboratory challenge, negligible mortality occurred among groups of herring that were either chronically infected or fully recovered, indicating that survival from chronic manifestations conferred protection against future disease. However, some survivors of chronic VHS infections were capable of replicating virus upon re-exposure. Demonstration of a chronic manifestation of VHSV infection among Pacific herring maintained at ambient seawater temperatures provides insights into the mechanisms by which the virus is maintained among populations of endemic hosts.     

Use of a Recombinant Vaccinia Virus Expressing Interferon Gamma for Post-Exposure Protection against Vaccinia and Ectromelia Viruses

Post-exposure vaccination with vaccinia virus (VACV) has been suggested to be effective in minimizing death if administered within four days of smallpox exposure. While there is anecdotal evidence for efficacy of post-exposure vaccination this has not been definitively studied in humans. In this study, we analyzed post-exposure prophylaxis using several attenuated recombinant VACV in a mouse model. A recombinant VACV expressing murine interferon gamma (IFN-γ) was most effective for post-exposure protection of mice infected with VACV and ectromelia virus (ECTV). Untreated animals infected with VACV exhibited severe weight loss and morbidity leading to 100% mortality by 8 to 10 days post-infection. Animals treated one day post-infection had milder symptoms, decreased weight loss and morbidity, and 100% survival. Treatment on days 2 or 3 post-infection resulted in 40% and 20% survival, respectively. Similar results were seen in ECTV-infected mice. Despite the differences in survival rates in the VACV model, the viral load was similar in both treated and untreated mice while treated mice displayed a high level of IFN-γ in the serum. These results suggest that protection provided by IFN-γ expressed by VACV may be mediated by its immunoregulatory activities rather than its antiviral effects. These results highlight the importance of IFN-γ as a modulator of the immune response for post-exposure prophylaxis and could be used potentially as another post-exposure prophylaxis tool to prevent morbidity following infection with smallpox and other orthopoxviruses.     

Quantity and infectivity of embryo-associated bovine viral diarrhea virus and antiviral influence of a blastocyst impede in vitro infection of uterine tubal cells

In previous studies, bovine viral diarrhea virus (BVDV) remained associated with IVF embryos after viral exposure and washing. However, uterine tubal cells (UTC) were not infected when exposed embryos were washed and individually co-cultured with them. The objective of this study was to evaluate quantity and infectivity of embryo-associated virus and antiviral influence of a blastocyst as possible explanations for failure to infect the UTC in vitro. Morulae and blastocysts were produced in vitro and washed. A portion of the embryos were incubated for 2 h in medium containing 10(6) to 10(8) cell culture infective doses (50%, CCID50) of a genotype I, noncytopathic BVDV per milliliter and then washed again. Virus isolation was attempted on sonicated negative (virus unexposed) and positive (virus exposed) control embryo groups after washing. The influence of quantity and infectivity of embryo-associated virus was evaluated by transferring exposed, washed embryo groups (2, 5, and 10 embryos/group) or sonicate fluid of exposed, washed, sonicated embryo groups (2, 5, and 10 embryos/group) to cultures containing bovine UTC in IVC medium that was free of BVDV neutralizing activity. The antiviral influence of an embryo was evaluated by adding 1 to 10(5) CCID50 of BVDV to UTC in the presence or absence of a single unexposed blastocyst in IVC medium. After 2 d in co-culture, the UTC, IVC medium and washed embryos (when present) were tested separately for the presence of BVDV using virus isolation. Virus was isolated from sonicate fluids of all positive but no negative controls. Virus was not isolated from any UTC following 2 d of culture with virally exposed groups of intact embryos. However, virus was isolated from UTC cultured with sonicate fluids from some groups of 5 (60%) and 10 (40%) embryos. Infective virus also remained associated with some groups of 2 (20%), 5 (40%) and 10 (60%) intact embryos after 48 h of post-exposure culture. Finally, primary cultures of UTC were more susceptible to infection with BVDV in the absence of a blastocyst (P = 0.01). Results indicate that insufficient quantity and reduced infectivity of embryo-associated virus as well as an antiviral influence of intact IVF blastocysts may all contribute to failure of embryo-associated virus to infect UTC in vitro.     

Abundance and Survival Rates of the Hawai’i Island Associated Spinner Dolphin (Stenella longirostris) Stock

Reliable population estimates are critical to implement effective management strategies. The Hawai’i Island spinner dolphin (Stenella longirostris) is a genetically distinct stock that displays a rigid daily behavioural pattern, foraging offshore at night and resting in sheltered bays during the day. Consequently, they are exposed to frequent human interactions and disturbance. We estimated population parameters of this spinner dolphin stock using a systematic sampling design and capture–recapture models. From September 2010 to August 2011, boat-based photo-identification surveys were undertaken monthly over 132 days (>1,150 hours of effort; >100,000 dorsal fin images) in the four main resting bays along the Kona Coast, Hawai’i Island. All images were graded according to photographic quality and distinctiveness. Over 32,000 images were included in the analyses, from which 607 distinctive individuals were catalogued and 214 were highly distinctive. Two independent estimates of the proportion of highly distinctive individuals in the population were not significantly different (p = 0.68). Individual heterogeneity and time variation in capture probabilities were strongly indicated for these data; therefore capture–recapture models allowing for these variations were used. The estimated annual apparent survival rate (product of true survival and permanent emigration) was 0.97 SE±0.05. Open and closed capture–recapture models for the highly distinctive individuals photographed at least once each month produced similar abundance estimates. An estimate of 221±4.3 SE highly distinctive spinner dolphins, resulted in a total abundance of 631±60.1 SE, (95% CI 524–761) spinner dolphins in the Hawai’i Island stock, which is lower than previous estimates. When this abundance estimate is considered alongside the rigid daily behavioural pattern, genetic distinctiveness, and the ease of human access to spinner dolphins in their preferred resting habitats, this Hawai’i Island stock is likely more vulnerable to negative impacts from human disturbance than previously believed.     

Differences in susceptibility of anadromous and resident stocks of Arctic charr to infections of Gyrodactylus salaris, under experimental conditions

The ability of Gyrodactylus salaris , an important pathogen of the Atlantic salmon Salmo salar , in Norway, to infect anadromous and resident stocks of the Arctic charr, Salvelinus alpinus , has been examined in the laboratory. Resident charr (Korssjoen stock) exposed to heavily infected salmon, were considered innately resistant as they lost their infections within 21 days when individually isolated. Isolated anadromous harr (Hammerfest stock) remained infected for up to 150 days, although most infections disappeared within 30–50 days. In many cases the parasite population grew initially, but growth was limited after 20–30 days and infections subsequently disappeared. At the same time, shoals of 50 anadromous charr, swimming in the tanks containing the individually isolated fish in floating cages, remained infected for up to 280 days. Charr isolated from these shoals after 115 days and subsequently monitored individually lost their infections within 30 days, although the parasite persisted within the shoals for a further 75–135 days. This suggests that G. salaris , persisted on shoaling charr despite an immune response which led to the elimination of parasites from isolated hosts. The Hammerfest stock of anadromous charr supports G. salaris , in the laboratory, and the extended period of survival on this host suggests that charr may be important in the epidemiology of G. salaris , in northern Norway.     

Embryotoxic effects adjacent and opposite to the early regressing bovine corpus luteum

Early luteal regression in cattle has an embryotoxic effect that is not overcome by replacement with progesterone, but is prevented by removal of the regressing CL. Two experiments were designed to test the null hypothesis that the luteal component of the embryotoxic effect is delivered by a systemic pathway. Beef heifers and cows (n = 39) received two good quality embryos, one placed into each uterine horn on Day 6 or 7 of the estrous cycle. Treated animals (n = 20) received 15 mg of PGF2alpha three times per day from Day 7 (n = 11; Experiment 1) or 5 (n = 9; Experiment 2) through 8; controls (n = 19) received saline. Progestogen replacement therapy (12 mg flurogestone acetate daily, s.c.) was provided from Day 6 (Experiment 1) or 4 (Experiment 2) until ultrasonographic diagnosis of embryo survival on Day 35 after estrus. The effects of treatment, location of the embryo and location by treatment interaction on embryo survival were tested by Chi square. In Experiment 1, there was no significant difference in embryo survival rate between PGF2alpha-treated and control recipients. In Experiment 2, only 6 of 18 embryos survived to Day 35 when transferred to animals treated with PGF2alpha compared to 12 of 18 in control animals (P< 0.05). The survival of embryos did not differ with location (adjacent or opposite to the regressing CL) or location by treatment interaction. Thus no evidence was obtained to support a local effect of the regressing CL. The embryo mortality associated with luteolytic doses of PGF2alpha in cows receiving replacement therapy with progestogen probably involves compounds that either act systemically or are transported via the uterine lumen to the uterine horn contralateral to the regressing CL.     

凡纳滨对虾DNaseⅠ基因的克隆及原核表达

利用RT-PCR和RACE技术,从凡纳滨对虾(Litopenaeus vannamei)肝胰腺中克隆了DNaseⅠ基因的全长cDNA序列。该序列全长1614bp,包含1209bp的开放阅读框,编码一个含403个氨基酸的蛋白;5′非翻译区为116bp,3′非翻译区为289bp。实时定量PCR分析结果表明,DNaseⅠ基因在肝胰腺的表达量是其他器官表达量的16～162倍,表明凡纳滨对虾DNaseⅠ基因属于胰腺型表达。本研究还利用酶切重组构建原核表达载体,并在大肠杆菌(Escherichia coli)中成功表达出了有活性的重组DNaseⅠ蛋白。     

Short Communication

Anaerobic metabolism and oxygen carrying-capacity of white shrimp ( Penaeus vannamei ) exposed to short term (three days) and long term (two weeks) moderate hypoxia (2-2.6 mg/L) was investigated. Glucose and lactate levels in hemolymph increased under both hypoxic conditions, indicating an activation of anaerobic pathways during the two-weeks exposure period. In muscle, no differences of glucose and lactate levels were observed between the control group and the exposed groups. In animals exposed to hypoxia for two weeks, hemocyanin and copper in hemolymph were higher than in animals under normoxic conditions or exposed for three days. These results indicate that an increase in oxygen carrying-capacity in shrimp is evident only after a sustained condition of hypoxia. Copper levels in the hepatopancreas decreased in both hypoxic conditions, suggesting a mobilization of copper stores for hemocyanin synthesis. These results indicate that penaeid shrimp can tolerate moderate hypoxic conditions by physiological adaptations, such as anaerobic metabolism and increased oxygen carrying-capacity. These adaptations require an adequate dietary supply of proteins and copper for hemocyanin synthesis and of carbohydrates for anaerobic metabolism.     

Viral interference between infectious hypodermal and hematopoietic necrosis virus and white spot syndrome virus in Litopenaeus vannamei

White spot syndrome virus (WSSV) is highly virulent and has caused significant production losses to the shrimp culture industry over the last decade. Infectious hypodermal and hematopoietic necrosis virus (IHHNV) also infects penaeid shrimp and, while being less important than WSSV, remains a major cause of significant production losses in Litopenaeus vannamei (also called Penaeus vannamei) and L. stylirostris (also called Penaeus stylirostris). These 2 viruses and their interactions were previously investigated in L. stylirostris. We report here laboratory challenge studies carried out to determine if viral interference between IHHNV and WSSV also occurs in L. vannamei, and it was found that experimental infection with IHHNV induced a significant delay in mortality following WSSV challenge. L. vannamei infected per os with IHHNV were challenged with WSSV at 0, 10, 20, 30, 40 and 50 d post-infection. Groups of na?ve shrimp infected with WSSV alone died in 3 d whereas shrimp pre-infected with IHHNV for 30, 40 or 50 d died in 5 d. Real-time PCR analysis showed that the delay correlated to the IHHNV load and that WSSV challenge induced a decrease in IHHNV load, indicating some form of competition between the 2 viruses.