Isolation,characterization, and in vitro differentiation of ovine amniotic stem cells

Challenge tests with Artemia four different development stages (nauplii, metanauplii, pseudoadults and adults) to white spot syndrome virus was carried out by immersion challenge and virus-phytoplankton adhesion route in order to asses the possibility of Artemia acting as a vector of WSSV to penaeid shrimp Litopenaeus vannamei postlarvae. The WSSV succeeded in infecting four stages Artemia, and nested-PCR detection for WSSV revealed positive results to virus-phytoplankton adhesion route. No mass mortalities were observed in penaeid shrimp postlarvae fed with WSSV-positive Artemia which exposed to WSSV by virus-phytoplankton adhesion route, whereas WSSV DNA detected in penaeid shrimp postlarvae by nested-PCR. By contrary, no WSSV-positive was detected in any animal fed with WSSV-negative Artemia. These results indicated that Artemia could serve as a vector in WSSV transmission.     

Unique Physicochemical Properties of the Occluded Penaeid Shrimp Baculoviruses and Their Use in Diagnosis of Infections

Abstract

Occlusion bodies in wet mounts of phloxine-stained tissue squashes and paraffin sections of penaeid shrimp infected with Baculovirus penaei and Penaeus monodon-type baculovirus fluoresced when viewed with an epifluorescent microscope. Inclusions of hepatopancreatic parvo-like virus, infectious hypodermal and hematopoietic necrosis virus, and Autographa californica nuclear polyhedrosis virus did not fluoresce. The use of phloxine stain (alone or as a component of an eosin preparation) and fluorescent microscopy may enhance detection of lowgrade infections by these disease-causing agents.     

Evaluation of Difloxacin for Shrimp Aquaculture: In Vitro Minimum Inhibitory Concentrations,Medicated Feed Palatability,and Toxicity to the Shrimp Penaeus vannamei

Abstract

Standard in vitro minimum inhibitory concentrations (MICs) were determined for difloxacin and compared with the MICs of several other antimicrobials, against a standardized battery of 13 gram-negative bacterial isolates associated with shrimp disease. The palatability and safety (toxicity) of difloxacin to the shrimp Penaeus vannamei were also evaluated during 15 d of medicated feeding at 1× (100 mg/kg of feed), 2×, and 4× treatment levels to give doses of approximately 5, 10, and 20 mg difloxacin/kg body weight. A significant reduction (P ≤ 0.05) in difloxacin-medicated feed palatability was noted in the 2× and 4× trials. However, differences were still acceptable, because more than 80% of the feeds were consumed in both treatments relative to the control diet. Shrimp mortality rates increased with difloxacin level from 7% for the control treatment to 20% for the 4× treatment. Differences in percent survival were not significant (P > 0.05) by the Williams test; however, analysis of mean survival time indicated that difloxacin significantly (P ≤ 0.05) reduced survival time at the highest dose. Signs of animal stress, characterized by extreme lethargy, were noted in the 4× treatment. An actual therapeutic dose for difloxacin in penaeid shrimp is unknown; however, the 1× treatment (100-mg/kg) was acceptable with respect to both palatability and toxicity, whereas 400 mg/kg of feed or more may be unpalatable and toxic to shrimp.     

Communications: Comparison of Traditional and Molecular Detection Methods for Baculovirus penaei Infections in Larval Penaeus vannamei

Abstract

Penaeid shrimp Penaeus vannamei were experimentally infected at a mysis II stage with the penaeid shrimp virus Baculovirus penaei (BP). The larvae were sampled at 0, 8, 12, 18, 24, 48, and 72 h postinfection and were examined for evidence of infection by three different methods: wet mounts, histology, and nucleic acid probes. Wet-mount squashes of the hepatopancreas were prepared and examined for BP tetrahedral occlusion bodies (TOBs) or larvae were fixed in Davidson's fixative and processed for routine histology, then either stained with hematoxylin and eosin (H&E) or reacted with nonradioactive BP gene probes by in situ hybridization. Sections stained with H&E or reacted with labeled gene probes were examined for signs of BP infection, such as cell cytopathology, the presence of TOBs, or the presence of a purple precipitate (which indicates BP-specific nucleic acid in the tissue). Occlusion bodies were initially observed in the wet-mount squash of the hepatopancreas of one shrimp at 18 h postinfection, and advanced infections were commonly seen at 48 h postinfection. The first definitive positive by H&E histology was detected at 24 h postinfection, whereas the probes detected infections at 12 h postinfection. The results indicate that the BP gene probes have the capability of detecting a BP infection before TOBs are readily observable in wet-mount squashes of the hepatopancreas and before the formation of TOBs in infected tissue.     

二温式PCR检测对虾白斑综合征病毒

本研究设计了一对能扩增大小为306bp对虾白斑综合征病毒(WSSV)某段基因的特异性引物，优化建立了能快速检测WSSV的二温式PCR，在对包括105份临床样品、10份SPF南美白对虾组织样品和其他对虾病害病原在内的样品检测结果中，有65份临床样品呈现WSSV阳性，而10份SPF南美白对虾组织样品和其他对虾病害病原的PCR结果为阴性。该二温式PCR最低能检测到1pg的WSSV感染对虾组织样品总DNA。这些结果表明，该PCR具有高度的特异性和敏染性。     

Shrimp Antimicrobial Testing. II. Toxicity Testing and Safety Determination for Twelve Antimicrobials with Penaeid Shrimp Larvae

Abstract

The need for federally approved chemotherapeutants for use in domestic shrimp culture in the USA is acute. A summary of toxicity testing of 12 antimicrobials against penaeid shrimp larvae is presented. In addition, the toxicity data are examined in concert with previously reported data on minimum inhibitory concentrations to establish a therapeutic index (a measure of margin of safety) for each compound tested. The 11 prospective antimicrobials were compared with the reference compound, chloramphenicol. The antimicrobials tested were enrofloxacin, erythromycin, florfenicol, oxytetracycline, paromomycin, four experimental fluoroquinolines (PD124816, PD127391, PD131628, PD132133), Romet-30®, and Sarafin®. The top six compounds (in their relative order of safety, from the safest to the least safe) were PD127391, oxytetracycline, chloramphenicol, enrofloxacin, florfenicol, and Romet-30.     

White Spot Syndrome Virus Infection of Cultured Shrimp in China

Abstract

In 1993 and 1994, an epizootic of disease (white spot) occurred among cultured shrimp in China, resulting in mass mortality. During the periods of outbreak epizootiological surveys were undertaken. Based on these surveys it is known that the disease occurred among populations of cultured Penaeus chinensis, P. japonicus, and P. monodon, infecting shrimp from 2.4 cm to adult. The disease presumably spread among farms as a result of transport of contaminated shrimp seedlings and seawater. Water temperatures exceeding 25°C caused the disease to spread more rapidly. Challenge experiments showed the causative agent was highly virulent. Both naturally and experimentally infected shrimp exhibited white spots on the carapace. Moribund shrimp contained turbid hemolymph, hypertrophied lymphoid organ, and a necrotic hepatopancreas. By electron microscopy, viral particles were observed in gills, stomach, lymphoid organ, and hypodermal tissue of infected shrimp. The virions were slightly ovoid with an envelope and averaged 350 × 150 nm and nucleocapsids measured 325 × 120 nm in size. By use of negative stains, complete virions were 375 × 157 nm and unenveloped nucleocapsids averaged 395 × 83 nm.     

对虾病毒病研究进展

对虾病毒病造成全世界对虾养殖业的巨大经济损失,因此已成为国内外学者研究的热点之一。本文就近年对虾病毒病的研究动态进行了概述,并对对虾病毒种类及性质,传播途径、诊断技术和防治措施进行了介绍。     

Communications: Bacteria in the Hemolymph of the Freshwater Prawn Macrobrachium rosenbergii

Abstract

Bacteria from the hemolymph of the freshwater prawn Macrobrachium rosenbergii were identified and quantified. Total bacterial counts ranged from 0.0 to 4.6 × 10⁵ cells/1.0 mL of hemolymph. Predominant bacteria isolated included Aeromonas spp., Bacillus sp., and Pseudomonas spp. No bacteria were found in the hemolymph of prawns without lesions. The predominant species of bacteria isolated from water samples of prawn culture ponds was a chitinoclastic Bacillus sp.     

A Biopsy Procedure for the Nondestructive Determination of Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV) Infection in Penaeus vannamei

Abstract

The shrimp Penaeus vannamei is frequently an asymptomatic carrier of infectious hypodermal and hematopoietic necrosis virus (IHHNV). In addition, there is increasing evidence that P. vannamei may also suffer severe disease from infection by IHHNV under some culture conditions. Hence, a nondestructive technique has been developed that has the potential to replace the old method of destructive subsampling, at least with this penaeid species. The new procedure is simpler, faster, less labor intensive, and equally precise when compared with the old method. The procedure involves the excision of the first pereiopod, followed by standard histological examination of the appendage nerve cord for the presence of pathognomonic Cowdry type A intranuclear inclusions. The nondestructive nature of this new technique may be its most important asset; animals tested for IHHNV via this method need not be sacrificed and are thus still available for use as brood stock.     

Isolation and Identification of Mycobacterium peregrinum from the Pacific White Shrimp Penaeus vannamei

Abstract

Mycobacterium peregrinum was isolated from multifocal, melanized nodular lesions in the carapace of the cultured marine shrimp Penaeus vannamei. This is the first identification of this species of Mycobacterium in shrimp. The lesions appeared to be the result of opportunistic infections in otherwise healthy animals. The presence of M. peregrinum in cultured shrimp has a direct negative impact on the marketability of shrimp because it causes obvious black lesions. The potential for accidental infections of shrimp farm or packing plant workers from handling infected shrimp is of concern since nodular skin lesions induced by the bacterium are difficult to treat.     

Evidence of existence of infectious hypodermal and hematopoietic necrosis virus in penaeid shrimp cultured in China

Infectious hypodermal and hematopoietic necrosis virus is the causative agent of a shrimp disease which causes economic losses on a global scale. A pair of primers, I2814F/I3516R, was designed from the IHHNV genomic sequence (GenBank) that encodes for structural protein corresponding to nucleotides 2814-3516, which amplifies a 703 base pair (bp) region from the virus genome. PCR amplification with the primers generated a product of the expected size from the purified IHHNV DNA of Litopenaeus vannamei and IHHNV-infected penaeid populations but not from the IHHNV-free shrimp, white spot syndrome virus (WSSV) and hepatopancreatic parvovirus (HPV). The PCR amplicon described above was labeled with digoxigenin (DIG)-11-dUTP as a probe used for dot blot hybridization and in situ hybridization test. Under the optimized PCR conditions, the primers were detected by as little as 20 fg of purified IHHNV DNA, which contained only 8.83 x 10(3) copies of IHHNV, a 1000-fold greater than using dot blot hybridization. Sections of histopathology showed eosinophilic intranuclear inclusions (Cowdry type A inclusions or CAIs) in infected tissues while in situ hybridization, cells displayed an intense reaction with the DIG-labeled probe. PCR assay was developed to detect IHHNV in penaeid shrimp and other crustaceans from the rearing ponds of China (March 2001-June 2004). The positive rate was 51.5% (154 out of 299) and 8.3% (2 out of 24) for penaeid shrimp and crab samples, respectively. The survey demonstrated the presence of IHHNV in China.     

Pharmacokinetics and tissue distribution of enrofloxacin after single intramuscular injection in Pacific white shrimp

The pharmacokinetic properties and tissue distribution of enrofloxacin (EF) were investigated after single intramuscular (i.m.) dose of 10 mg/kg body weight (b.w.) in Pacific white shrimp at 22 to 25°C. EF and its metabolite ciprofloxacin (CF) were determined by high‐performance liquid chromatography. After i.m. administration, EF was absorbed quickly, and the peak of EF concentration (C_max) reached at first time point in hemolymph. The volume of distribution V_d(area) of EF was 3.84 L/kg, indicating that the distribution of EF was good. The area under the concentration–time curve (AUC) of EF was 90.1 and 274.2 μg hr/ml in muscle and hepatopancreas, respectively, which was higher than 75.8 μg hr/ml in hemolymph. The EF elimination was slow in muscle and hepatopancreas with the half‐life (T_1/2β) of 52.3 and 75.8 hr, respectively. CF, the mainly metabolite of EF, was detected in hemolymph, muscle and hepatopancreas. The C_max was 0.030, 0.013 and 0.218 μg/ml, respectively. Based on a minimum inhibitory concentration (MIC) of 0.006–0.032 μg/ml for susceptible strains, EF i.m. injected at a dose 10 mg/kg could be efficacious against common pathogenic bacteria of Pacific white shrimp.     

Demonstration of Infectious Taura Syndrome Virus in the Feces of Seagulls Collected during an Epizootic in Texas

Abstract

Laughing gulls Larus atricilla were observed feeding on diseased shrimp Penaeus vannamei during an epizootic of the Taura syndrome virus (TSV) at a south Texas farm in May 1995. Fresh fecal samples were collected from a gull roosting site on a pond levee where gulls that fed on diseased shrimp had roosted overnight. The feces samples were tested for the presence of TSV by injection bioassay. A cell-free clarified homogenate of the fecal material was injected into indicator P. vannamei, and the shrimp were observed for 9 d. Cumulative mortalities of the two replicate groups of indicator shrimp were 45% and 70%. Moribund indicator shrimp collected and preserved for histological examination exhibited lesions pathognomonic for acutephase infections by TSV. The presence of TSV in the induced lesions was confirmed with in situ hybridization with cDNA probes specific for a portion of the TSV genome. The presence of infectious TSV in the gulls' feces supports the hypothesis that these birds are a probable transport vector of the virus within and among nearby shrimp farms.     

Farm Level and Geographic Predictors of Antibiotic Use in Sri Lankan Shrimp Farms

Abstract

Black tiger shrimp Penaeus monodon farming is important for Sri Lanka's rural development plans. Consumer confidence is critical for the development and maintenance of export and domestic shrimp markets. Public concern about the use of antimicrobial drugs and chemicals on shrimp farms, however, could threaten market access. We sought to identify high-risk areas and farm-level risk factors for antimicrobial use to inform the core messages and strategic placement of extension programs to help farmers develop best management practices for antimicrobial use. We undertook a survey of 603 operating farms within the Puttalam district over 42 weeks. Lower stocking density and early harvest were associated with a lower risk of antimicrobial use, whereas standard management practices, including water treatment, feed supplements, probiotic use, pond fertilizing, disinfectant use, and pesticide use, were associated with increased risk. Spatial cluster detection found three significant clusters of antimicrobial-using farms. Antimicrobials were more likely to be used in areas with lower farm density. Some of our counterintuitive findings are discussed from a socioecological perspective. A comprehensive understanding of why antimicrobials are used on shrimp farms requires an evaluation of the physical, epidemiological, and socioeconomic factors.

Received June 10, 2011; accepted December 6, 2011     

Osmotic Responses of Red Swamp Crawfish to Trapping,Sorting, and Storage in a Cooler

Abstract

Hemolymph osmotic pressures and chloride concentrations were measured in red swamp crawfish Procambarus clarkii at harvest and after storage in a cooler in March and May. Male and female crayfish had similar hemolymph concentrations regardless of treatment, and osmotic pressure and chloride levels were generally stable for a storage period of up to 5 d after harvest. These data suggest that trapping or storage in a cooler causes no dramatic physiological change in red swamp crawfish.     

Communications: Clearance of Amyloodinium ocellatum Dinospores by Artemia salina

Abstract

The potential for using nauplii of brine shrimp Artemia salina to remove the dinoflagellate Amyloodinium ocellatum from aquaculture systems for red drum Sciaenops ocellatus was investigated. Dinospores of A. ocellatum were dispensed in cell culture plates at a concentration of approximately 10,000/mL and were exposed to brine shrimp nauplii at concentrations of l, 2, 4, or 8 nauplii/mL over a 24-h period. 1n the presence of 8 nauplii/mL, dinospores were eliminated in 8 h. In another study, 300,000 dinospores were placed in 3 L of saltwater in two tanks, and l,000 nauplii were added to one tank. After 12 h, three 202-g red drums were placed in both tanks for an additional 12-h-exposure period, after which the fish were euthanized and the trophont load on gill filaments was assessed. Fish from the tank with brine shrimp nauplii had 65% fewer trophonts on their gills than those from the nontreated tank (10.75 versus 3.75 trophonts/filament). These data and the observation that dinospore remains could be observed in naupliar fecal casts suggest the potential value of brine shrimp as a bioremediation measure for this serious gill parasite.     

Surveillance of Francisella tularensis in surface water of Kurdistan province,west of Iran

BackgroundThe etiologic agent of tularemia, Francisella tularensis, is transmitted to humans via ingestion of contaminated water or food, arthropods bite, respiratory aerosols, or direct contact with infected animals body fluids or tissues. In the current study, due to the importance of water in transmitting the disease and the report of the disease in different regions of Iran, surface water of Kurdistan province were evaluated for the presence of F.tularensis.Materials and MethodsSampling was carried out in five-counties of Kurdistan province. Sixty-six specimens of surface water were collected. The detection was carried out by targeting ISFtu2 and fopA genes using TaqMan real-time PCR. Moreover, the samples were both cultured and inoculated into NMRI inbreed mice. Spleens of inoculated mice and bacterial isolates were tested by TaqMan real-time PCR.ResultsDespite the lack of isolation of F. tularensis, the results of the molecular testing indicate the presence of bacteria in surface water. Molecular positivity of one sample (1.51%) was confirmed using a real-time PCR for both ISFtu2 and fopA genes. Moreover, 4.54% of the samples were positive for ISFtu2.ConclusionSince the in vitro isolation of bacteria from environmental samples is associated with a very low success rate and depends on various environmental parameters, the use of molecular techniques for monitoring of the bacteria in the contaminated areas is fully recommended.     

A review of viral diseases of cultured shrimp

The diagnosis, prevention and control of viral infections of penaeid shrimp are discussed in the light of a review of the basic pathologic knowledge available on this important resource.     

Identification of Genes Involved in Taura Syndrome Virus Resistance in Litopenaeus Vannamei

Abstract

The goal of the present research was to identify the genes that are differentially expressed between two lineages of Pacific white shrimp Litopenaeus vannamei displaying different susceptibilities to Taura syndrome virus (TSV) and to understand the molecular pathways involved in resistance to the disease. An oligonucleotide microarray was constructed and used to identify several genes that were differentially expressed in the two L. vannamei lineages following infection with TSV. Individual L. vannamei from either resistant or susceptible lineages were exposed via injection to TSV. Individuals were removed at 6 and 24 h postinfection, and gene expression was assessed with the in-house microarray. The microarray data resulted in the selection of a set of 397 genes that were altered by TSV exposure between the different lineages. Significantly differentially expressed genes were subjected to hierarchical clustering and revealed a lineage-dependent clustering at 24 h postinoculation, but not at 6 h postinoculation. Discriminant analysis resulted in the identification of a set of 11 genes that were able to correctly classify Pacific white shrimp as resistant or susceptible based on gene expression data.

Received June 21, 2013; accepted October 24, 2013