Diseased Shrimp Research Articles

Isolation of a pathogenic strain of Vibrio alginolyticus from necrotic larvae of Macrobrachium rosenbergii (de Man)

Giant freshwater prawn, Macrobrachium rosenbergii (de Man), is an important commercial species with considerable export value, ideal for cultivation under low saline conditions and in freshwater zones (Kurup 1994). However, despite more than a decade of research on its larval production systems, vibriosis still hampers seed production resulting in high mortality rates. Among the different species of vibrios, Vibrio alginolyticus has been isolated frequently from diseased shrimp as the aetiological agent of vibriosis and has been described as a principal pathogen of both penaeids and nonpenaeids (Lightner 1988; Baticados, Cruz-Lacierda, de la Cruz, Duremdez-Fernandez, Gacutan, LavillaPitogo & Lio-Po 1990; Mohney, Lightner & Bell 1994; Lee, Yu, Chen, Yang & Liu 1996). Vibrio fluvialis, V. alginolyticus, V. cholerae non-O1 (Fujioka & Greco 1984), Aeromonas liquifaciens and V. anguillarum (Colorni 1985) have been isolated from the larvae of M. rosenbergii. A profound relationship between the abundance of members of the family Vibrionaceae and larval mortality (Singh 1990) and the predominance of Vibrio in eggs, larvae and post-larvae of M. rosenbergii (Hameed, Rahaman, Alagan & Yoganandhan 2003) was reported. The present paper reports the isolation, characterization, pathogenicity and antibiotic sensitivity of V. alginolyticus associated with M. rosenbergii larvae during an occurrence of severe mass mortality at the ninth larval stage. Moribund larvae (about 500) were collected from M/s Rosen Fisheries (Trichur, Kerala). The larvae were reared in 15& sea water, fed on freshly hatched Artemia nauplii until stage 6, and supplemented with egg custard subsequently. Water quality parameters monitored were pH (7.5–8.0), temperature (25–28 C), total ammonia (<0.1 ppm) and nitrite (<1.0 ppm). Partial water exchange (30%) was provided daily. The diseased samples were obtained from a tank (5-tonne capacity with 500 000 mysis as the initial stocking density) where the majority of the larvae at stage 9 had displayed anorexia, inactivity, poor growth, morbidity and necrotic appendages. Collection of moribund larvae and the rearing water was made in sterile polypropylene bottles (autoclaved at 121 C for 15 min) with sterile sea water (15&) as the transport medium. The samples were transported in an ice-chest at 4 C and analysed in the laboratory within 2 h of collection. Larvae (n 1⁄4 30) were washed in a 20-mL volume of sterile (autoclaved at 121 C for 15 min) sea water (15&) three times for a duration of 1 min each, and macerated to a fine paste in a sterile glass Journal of Fish Diseases 2006, 29, 187–191

Molecular epidemiology of Vibrio nigripulchritudo, a pathogen of cultured penaeid shrimp ( Litopenaeus stylirostris) in New Caledonia

A collection of 57 isolates of Vibrio nigripulchritudo from either diseased or healthy shrimp and from shrimp farms environment was studied in order to gain a better understanding of the epidemiology of this pathogen, notably isolated from two distinct shrimp disease complexes. Molecular typing using two different techniques, arbitrarily primed PCR (AP-PCR) and multi-locus sequence typing (MLST), studied together with experimental pathology data allowed a relevant epidemiological insight into this possibly emerging pathogen. Additionally, results obtained with the two molecular typing techniques were congruent and allowed discriminating the strains associated with the “Summer Syndrome” from strains isolated from other contexts, especially the other shrimp vibriosis “Syndrome 93”. These results highlight that the “Summer Syndrome” is most probably caused by an emergent clonal pathogen that therefore deserves surveillance and that AP-PCR can satisfactorily be used for that purpose.

Vibrio alginolyticus infection in the white shrimp Litopenaeus vannamei confirmed by polymerase chain reaction and 16S rDNA sequencing

A gram-negative, rod-shaped bacterium identified as Vibrio alginolyticus was isolated from diseased Litopenaeus vannamei (also called Penaeus vannamei) in Taiwanese culture ponds. The diseased shrimp displayed poor growth, anorexia, inactivity, reddish pleural borders of antennae, uropods and telson, opaque and whitish musculature, and mortality. In histological preparations, melanized hemocytic granulomas were observed in the connective tissue around hemal sinuses together with hemocytic aggregation in necrotic musculature. Six isolates of Vibrio were collected from diseased shrimp at 3 farms, and these were evaluated for characteristics including morphology, physiology, biochemistry and sensitivity to antibiotics. The results indicated that the isolates belonged to a single species that grew in 1 to 8% NaCl, at 10 to 40 degrees C and on TCBS (thiosulfatecitrate-bile sucrose) agar, and that gave positive catalase, O/F (Oxidation/Fermentation), lysine decarboxylase, gelatinase and cytochrome-oxidase tests. Identification of CH003 (1 of 6 isolates) was confirmed by PCR assay for V. alginolyticus (expected amplicon 1486 bp). The 16S rDNA sequence (GenBank accession number AY373027) gave 99.9% sequence identity to V. alginolyticus (GenBank accession number X74690). The calculated 96 h LD50 dose of the isolated strain was 3.0 x 10(5) colony forming units (CFU) shrimp(-1) (6.6 x 10(4) CFU g(-1)).

THE EVIDENCE OF BACILLIFORM VIRUS A CAUSATIVE AGENT OF WHITE SPOT SYNDROME OF WHITE SHRIMP Penaeus merguiensis

The rod-shape virus particles were found in the hyperthrophied nucleus and cytoplasm of diseased white shrimp Penaeus merguiensis naturally infected by White spot syndrome (WSBV). In natural infection cumulative mortality of shrimp were very high, 95 percent of population were dead in 3-7 days, and 5 percent of shrimp population survived. The disease was outbreak in intensive culture farms used the closed circulation sea water system. Others crustacean such as crabs and bentatos were not infected by WSBV at time of sampling. The virus particle were rod-shape ranging from 78 10 nm in diameter and 280 10 nm in length. The pathogenic bacteria mainly dominated by genus Vibrio sp were isolated from shrimp.

Preliminary Study on Genetic Distance of Vibrio parahaemolyticus Isolates from Diseased Fish and Shrimp Brackishwater Ponds by Random Amplified Polymorphic DNA (RAPD) in Malaysia

This study was conducted to determine the genetic distance of V. parahaemolyticus isolates from diseased fish (clinical) and shrimp brackishwater ponds (environmental) in Malaysia;four clinical isolates (numbered F1 to F4) and six environmental isolates (W1 to W6) from shrimp brackishwater ponds. The isolates were identified using conventional biochemical tests in combination with BBL Crystal Kit TM. Two published 10-mer arbitrary primers (Gen1-50-01 and Gen1-50-02) were used for detecting DNA polymorphisms. RAPDistance program (version 1.04) was used to analyze the RAPD patterns. Dendrogram was then constructed from the combined results of two primers using Neighbour-Joining Tree (NJTREE) algorithm. The primers detected DNA polymorphisms in all isolates except W1 and W2, which were DNA monomorphic. Each primer generated fingerprints of up to 20 bands, ranging from 100 to 6000 bp. Dendrogram elucidated a tree of two main clusters (A and B) where a close genetic distance between the clinical isolates (F2, F3 and F4) except isolate F1 was observed to be closer to environmental isolates W3 and W5. Isolates W1, W2 and W4 were found to be genetically far from any clinical isolates.

Molecular identification of pathogenic and nonpathogenic strains of Vibrio harveyi using PCR and RAPD

Fifteen environmental samples of Vibrio spp. isolated from healthy and diseased shrimps were tested for pathogenicity to juvenile shrimps. Two isolates, strains Z2 and Z3, were observed to be pathogenic, causing 100% mortality of the target host compared to the control strain Vibrio harveyi ATCC 14126. Environmental and type strains were subjected to molecular characterization by restriction fragment length polymorphism (RFLP) and PCR using primers targeted to different virulence, transcriptional regulator, or quorum sensing genes from V. harveyi. Primers designed for luxN were specific and identified all the environmental strains as V. harveyi. The random amplified polymorphic DNA (RAPD) method was used to differentiate between pathogenic and nonpathogenic strains of V. harveyi. These methodologies allowed us to detect and distinguish strains virulent and avirulent to juvenile shrimp.

Virulence of luminous vibrios to Artemia franciscana nauplii.

From healthy and diseased penaeid shrimp from Asia and the Americas, 25 luminous and 2 non-luminous bacterial strains were isolated, and 14 were phenotypically identified as Vibrio harveyi; 9 isolates produced significant mortalities (45 to 80%) in Artemia franciscana nauplii at inoculation densities of 10(5) to 10(6) CFU ml(-1) compared to the controls (unchallenged nauplii). The maximum number of bacteria ingested (bioencapsulated) by the Artemia nauplii varied from less than 10 to 10(3) CFU nauplius(-1) and no significant relationship was observed between the density of bacteria inoculated, the amount of bacteria ingested, and naupliar mortality. Significant correlations were obtained between naupliar mortality and production of proteases, phospholipases or siderophores, but not between mortality and lipase production, gelatinase production, hydrophobicity or hemolytic activity. The results suggest that virulence of the strains tested was more related to the production of particular exoenzymes than to the measured colonization factors.

Characteristics, Pathogenicity and Antibiotic Sensitivity of Bacterial Isolates from White Spot Diseased Shrimp

A bacteriological study was undertaken on white spot diseased shrimp collected from extensive, modified-extensive and semi-intensive ponds located at Visakhapatnam and East Godavari districts of North Coastal Andhra Pradesh. The diseased shrimp exhibited white spots and red discoloration of the body. Four species of bacteria, V. alginolyticus, V. parahaemolyticus, V. anguillarum and Pseudomonas aeruginosa were isolated from the hemolymph of the diseased shrimp. V. alginolyticus was found to be the most dominant and virulent species. All the bacterial isolates showed sensitivity towards oxytetracycline which is a commonly used antibiotic in culture ponds.

Detection of white spot syndrome virus (WSSV) ofPenaeus chinensis by in situ hybridization

White Spot Syndrome Virus (WSSV) was purified from hemolymph of infected shrimp. After nucleic acid extraction from the purified virus particles, EcoR I-digested fragments of the WSSV genome were cloned; three of these fragments were used as non-radioactive probes labeled with DIG-11-dUTP. The probes hybridized in situ, with sections located in the nuclei of all WSSV-infected tissues. The virus was detected in the gill, stomach, epidermis, and connective tissue and so on, but not detected in healthy shrimp tissues and epithelial cells of hepatopancreatic tubules of diseased shrimp.

White spot baculovirus syndrome in the Indian shrimp Penaeus monodon and P. indicus

Sporadic occurrences of white spot baculovirus (WSBV) infections have been reported in shrimp farms throughout the maritime states of India. WSBV presents as a reddish discolouration with white spots on the exoskeleton and epidermis with muscle opacity. Onset of the disease is extremely rapid with mass mortalities. Infected juveniles and sub-adults of Penaeus indicus and P. monodon become lethargic surface frequently, exhibit loss of balance, with reduced feeding and preening activities. The nuclei of WSBV-infected epithelial (hypodermal), septal and secretory cells of the gill filaments exhibit basophilic hypertrophied nuclei with a reduced cytoplasmic volume. Massive tissue disintegration occurred in the ectodermal and mesodermal tissues. The electron-dense nucleoplasm of the gill epithelial cells is mostly replaced with virions. Electron microscopic examination revealed the presence of double-enveloped, non-occluded, rod-shaped virions with a tube-like or branched extension and empty capsids. The numbers of mitochondria, endoplasmic reticulum (ER) and Golgi were also reduced, as were the numbers of secretory or storage vesicles. WSBV is considered to be the main causative agent responsible for mass mortalities of juveniles and sub-adults in the cultured Indian penaeid shrimp, P. monodon and P. indicus. WSBV is highly pathogenic and readily transmitted from diseased shrimp to healthy susceptible shrimp via, contaminated water, faeces and by scavenging on dead infected shrimp. It may affect all stages of shrimp. The spread of the disease from cultured to natural systems and vice versa cannot be dismissed.

Periodic occurrence of epithelial viral necrosis outbreaks in Penaeus vannamei in Ecuador.

Epizootics of an infectious cuticular epithelial necrosis virus (ICENV) occurred in cultured Penaeus vannamei in Ecuadorian shrimp farms from 1994 to 1996. There were few reports of outbreaks during 1997, but in the second half of 1998 epizootics were again reported. Histopathological examination revealed extensive tissue changes and necrosis as described for infections by what others have called Taura syndrome virus (TSV). Infiltration of haemocytes in the cuticular epithelium was also one of the characteristics in the subacute and acute forms of this disease. Electron microscopy of affected tissues demonstrated the presence of a single type of virus particle in the cytoplasm of diseased shrimps from these outbreaks and it corresponded to the published descriptions for TSV. The epizootics of ICENV were periodic in occurrence, and data indicated that they might be related to the oceanographic and climatic variations reported in the eastern Pacific from 1994 to 1998. Using 3 mo rolling averages, a statistically significant negative correlation was found between prevalence of ICENV and temperature but not temperature change. By contrast, there was a statistically significant positive correlation between prevalence of ICENV and salinity change but not salinity value. Although these data do not establish causal relationships, they suggest that laboratory tests should be conducted to determine whether low temperature and upward changes in salinity can increase shrimp susceptibility to infection and mortality by ICENV.

Rapid and sensitive PCR detection of Vibrio penaeicida, the putative etiological agent of syndrome 93 in New Caledonia.

Experimental infections of Penaeus (Litopenaeus) stylirostris were performed with a Vibrio penaeicida strain (AM101) isolated in New Caledonia from Syndrome 93 diseased shrimp. Cumulative mortalities resulting from intramuscular injection or immersion of shrimp in bacterial suspensions demonstrated high virulence for this bacterial strain and suggested that V. penaeicida could be the etiological agent of Syndrome 93. The median lethal dose (LD50) for AM101 was 1.3 x 10(4) CFU (colony forming units) ml-1 by immersion and less than 5 CFU shrimp-1 by intramuscular challenge, with mortality outbreaks at 48 and 22 h after challenge, respectively. A polymerase chain reaction (PCR) detection assay using a primer set designed from the 16S ribosomal RNA gene of V. penaeicida was developed. It gave an expected amplicon of approximately 310 bp in ethidium bromide-stained agarose gels. The specificity of these primers was assessed with different Vibrio species. Furthermore, DNA extracted by the Chelex method could be used to detect fewer than 20 cultured Vibrio cells in sea-water or shrimp hemolymph by this assay. It appears to be a reliable screening method for detecting V. penaeicida in shrimp and from the aquatic environment.

Diagnosis of Penaeus monodon-type baculovirus by PCR and by ELISA of occlusion bodies.

The black tiger prawn Penaeus monodon is a valuable aquaculture product in Taiwan. Two specific diagnostic methods were established for P. monodon-type baculovirus, one using polymerase chain reaction (PCR) technology and the other enzyme-linked immunosorbent assay (ELISA) technology. Monodon-type baculovirus (MBV) was purified by sucrose gradient centrifugation from occlusion bodies of MBV-infected postlarvae of P. monodon. MBV DNA was subsequently purified from the occlusion bodies and its presence was confirmed by PCR using primers of the polyhedrin gene. Based on conserved sequences of the DNA polymerase genes of Autographa californica nuclear polyhedrosis virus (AcMNPV) and Lymantria dispar nuclear polyhedrosis virus (LdMNPV), primers were designed and synthesized to yield a 714 bp PCR fragment from MBV. However, the sequence of this fragment revealed low homology with that of LdMNPV and AcMNPV. From the DNA sequence of this fragment, a second set of primers was designed, and using these primers, a 511 bp DNA fragment was amplified only when MBV DNA was the template. DNA templates from AcMNPV, white spot syndrome diseased shrimp, or PMO cells (a cell line derived from the Oka organ of Penaeus monodon) did not give any amplified DNA fragment. Therefore, this primer pair was specific for the diagnosis of MBV. By using intraspleenic immunization of rabbits with purified MBV occlusion bodies, a polyclonal rabbit antiserum against MBV was obtained. This antiserum could detect nanogram levels of MBV, but did not cross react with white spot syndrome virus (WSSV), homogenates of PMO cells, postlarvae, hepatopancreatic tissue or intestinal tissue of black tiger prawns by competitive ELISA. This sensitive method could detect MBV even in tissue homogenates.

Molecular characterization of the glycoproteins from two warm water rhabdoviruses: snakehead rhabdovirus (SHRV) and rhabdovirus of penaeid shrimp (RPS)/spring viremia of carp virus (SVCV)

We have determined the complete coding sequences for the glycoprotein (G) genes from two rhabdoviruses that infect warm water aquatic animals, the snakehead rhabdovirus (SHRV) and rhabdovirus of penaeid shrimp (RPS). Surprisingly, the G nucleotide sequence from RPS, a virus which has been isolated from diseased shrimp in Hawaii on numerous occasions, was over 99% identical to the G nucleotide sequence from spring viremia of carp virus (SVCV), a fish virus from Europe and Asia. This is the first report of SVCV isolation outside of Europe and Asia, and it is also the first report of SVCV infecting a non-vertebrate species. The G gene from SHRV was most closely related to the G genes from the three Novirhabdoviruses, viral hemorrhagic septicemia virus (VHSV), infectious hematopoietic necrosis virus (IHNV), and hirame rhabdovirus (HIRRV), with 47, 37, and 36% amino acid identity, respectively. In addition, a phylogenetic analysis using the amino acid sequence from rhabdovirus G genes indicated that SHRV should be classified within the Novirhabdovirus genus. Finally, the SHRV-G gene was successfully expressed in mammalian cells under the control of the cytomegalovirus (CMV) promoter, establishing that it can potentially be used in the production of pseudotyped retroviruses designed to infect fish.

Infection of IHHN virus in two species of cultured penaeoid shrimp Litopenaeus vannamei (Boone) and Litopenaeus stylirostris (Stimpson) in Ecuador during El Niño 1997-98

Infectious hypodermal and haematopoietic necrosis virus (IHHNV) was first noted in blue shrimp Litopenaeus stylirostris (Stimpson) in mid-1981. Since that time, at least 12 species of penaeoid shrimp have been reported to be infected with IHHNV. Pacific white shrimp Litopenaeus vannamei (Boone) represents a shrimp species highly refractory to the disease, whereas L. stylirostris was highly susceptible to the disease. Since the beginning of the shrimp farming industry in Ecuador, viral diseases have been observed in L. vannamei and L. stylirostris. Of these, L. vannamei represents ≈ 80% of cultured shrimp. Histopathology, ultrastructure and in situ DNA hybridization confirm the presence and assess the prevalence of IHHNV in pond-reared shrimp, and especially in abnormally small animals of both species. Although IHHNV may be considered enzootic in cultured L. vannamei in Ecuador, we did not find high prevalence (Cowdry A bodies) in specimens of diseased pond shrimp before 1996. From that time to 1998, a higher prevalence of IHHNV has been observed in both species. The epizootic of the IHHNV disease has been related to the oceanographic and climatological conditions caused by El Niño. In addition, it has been suggested that large quantities of wild shrimp post-larvae of both species that were stocked in shrimp farms, infected as latent carriers in 1997, from which the virus could spread to a larger population of these shrimp in 1998.

Changes in the composition of Vibrio communities in pond water during tiger shrimp ( Penaeus monodon) cultivation and in the hepatopancreas of healthy and diseased shrimp

Two batches of shrimp were cultivated in a total of 4 culture ponds from March 28 to June 13, and July 17 to September 11, 1997. Vibrio spp. were isolated from the pond water and the hepatopancreas of healthy and diseased shrimp using thiosulfate–citrate–bile salt–sucrose agar plate, and then identified using fatty acid methyl ester (FAME) analysis. In each culture pond, decreases in the diversity of the Vibrio community were observed prior to outbreaks of vibriosis. FAME analysis suggested that in diseased pond water (i.e., water from a pond in which there had been an outbreak of disease) and the hepatopancreas of diseased shrimp, V. furnissii was the major component of the Vibrio community. However, since identification confidence levels were too low (≤0.500), the identity of 48 randomly selected putative V. furnissii was further checked using the Biolog system. Of these, 27 were identified as V. harveyi/V. carchariae, six as V. parahaemolyticus, five as V. anguillarum, four as V. alginolyticus, four as V. vulnificus, and two as V. damsela, the majority of which species are known to be associated with diseased shrimp. Similar results were also found for Vibrio communities in the hepatopancreas of diseased shrimps, with 68.2% of the isolated vibrios eventually identified as V. harveyi/ V. carchariae.

The geographic distribution of Taura Syndrome Virus (TSV) in the Americas: determination by histopathology and in situ hybridization using TSV-specific cDNA probes

Representative archived Litopenaeus vannamei samples (117 total), originating from 13 different countries and collected between 1992 to 1996, were analyzed by in situ hybridization to verify the presence of Taura Syndrome Virus (TSV) within pathodiagnostic acute phase TS histological lesions. The in situ assay results showed that TSV was present in one or more representative samples analyzed from each country (76 of 117 samples or 65%), thus, confirming the original histological diagnosis of TSV infection. The false negative in situ hybridization results, obtained for 35% of the samples assayed (41 in total), were attributed to over-fixation with Davidson's AFA (acetic acid, formaldehyde, alcohol) solution and consequent acid hydrolysis of TSV genomic RNA within pathodiagnostic TSV lesions. The collective findings of this disease survey assisted in documenting the spread and current distribution of TSV over a 5-year period and definitively established the presence of TSV within TS diseased shrimp originating from Ecuador when and where the disease was first recognized in 1992. These findings further strengthen the existing evidence that TS has a viral, not a toxic, etiology and indicate that either a single TSV strain, or very similar strains of the same virus, are responsible for the TSV panzootic that has been expanding in the Americas since 1992.

Investigations of Penaeus stylirostris disease (Syndrome 93) in New Caledonia, exploring a viral hypothesis

Semi-intensive farming of Penaeus stylirostris (Mexican strain) is a developing industry in New Caledonia. Since 1993 crops have experienced mortality episodes (named `Syndrome 93') which are strongly affecting the industry. A search for bacteria in the hemolymph of moribund shrimps revealed bacterial septicemia. The Vibrio spp. initially isolated were variable and usually weakly pathogenic. Therefore, another research was investigated. Histological sections of moribund shrimps revealed not only classical vibriosis lesions, but also numerous pycnotic cells and intracytoplasmic basophilic bodies, which resemble those described in the case of `yellow head disease' and `Taura syndrome'. These observations suggested a possible participation of viral agents in `Syndrome 93'. To explore this viral hypothesis, five cephalothorax from moribund shrimps caught in 1994 were ground, centrifuged and filtered on a 0.22 μm membrane. Filtered aliquots were kept at −76°C, then injected into groups of presumably healthy P. stylirostris (1 g), from New Caledonia and specific pathogen-free P. stylirostris and P. vannamei from Tucson, AZ. While 100% mortality was observed in groups injected with the filtrate 6 h to 5 days after injection, no significant mortality occurred in the control groups. Basophilic bodies similar to those described above were observed in experimentally infected moribund shrimps. This demonstrates the existence of ultrafiltrable entities in diseased shrimps which can experimentally reproduce the lesions, symptoms and mortalities characteristic of `Symdrome 93'. Specific gene probe were used (dot blot) to detect Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV) and showed that 94 out of 95 moribund shrimps were positive but the detected levels were very low. Purification trials using centrifugation on sucrose and cesium chloride gradients failed to detect IHHNV which is probably present in small quantities. However, some fractions revealed an abundance of calibrated particles of about 100 mm of variable shape following examination of negative staining under transmission electron microscopy. Nucleic acid extraction trials and electrophoresis of these fractions on agarose gel revealed small quantities of a monocatenary DNA molecule of about 10 Kb, probably of viral origin. Results suggest the presence of the IHHNV, and probably of a second virus in shrimp crops in New Caledonia. The etiological role of each of those entities in `Syndrome 93' is currently being studied.

`Syndrome 93' in New Caledonian outdoor rearing ponds of Penaeus stylirostris: history and description of three major outbreaks

Farming of Penaeus stylirostris (Mexican strain) in New Caledonia is a developing industry conducted in flow-through semi-intensive system, at 20 to 40 animals/m 2 with a theoretical extrapolated yield of 4.2 t/ha/yr. In 1995, approximately 878 t of shrimps were produced from 352 ha of ponds. From the 1980s to 1992, production increased progressively (538 t in 1990, 648 t in 1991 and 734 t in 1992). The increase was the result of improved techniques and greater production surface area. In 1993, production decreased (621 t in 1993), then increased again in 1994 and 1995 due to increased grow out surface only. Low yields and survival rates in 1993, 1994 and 1995 were associated with abnormal mortalities. This pathology, given the name `Syndrome 93', appeared during a period of drought and was more pronounced in winter (mid-May to mid-August). The mortalities were chronic or acute. High pond biomass and sudden water temperature drop seemed to have an impact on the development of the disease. Moribund shrimps were weak, sensitive to stress and swam near the water surface. Shrimps were soft with dark and dull exoskeleton and whitish muscles. Their hemolymph clotted slowly. The hemolymph of diseased shrimps was generally infected with one predominant strain of bacteria, Vibrio sp. The hemolymph of presumed healthy shrimp was sometimes sterile or contained variable but generally limited numbers of different bacteria of the genera Vibrio, Pseudomonas, Flavobacterium and Cytophaga. Moribund shrimps exhibited classical anatomopathologic septicemic vibriosis lesions. However, numerous pycnotic hemocytes and atypical basophilic granulations were also observed in most tissues, especially the lymphoid organ, heart, connective tissue of the stomach and cuticular epithelium. These basophilic granulations seemed to correspond partly to nuclear debris and partly to intracytoplasmic inclusions which are similar to those described in penaeid viral infections (`yellow head disease', `Taura syndrome') indicating that pathogens other than Vibrio sp. could be involved.

Detection of white-spot syndrome in cultured penaeid shrimp in Asia: Microscopic observation and polymerase chain reaction

Serious disease outbreak caused by a new virus has been occurring among cultured penaeid shrimps in Asian countries since 1993. Typical signs include white spots or patches on the inner surface of the shell and carapace and/or reddish coloration of the body. Histopathological changes observed among diseased shrimps collected from various countries exhibited widespread cellular degeneration and severe nuclear hypertrophy in cells of most tissues derived from ectodermal and mesodermal origin. Similar rod-shaped to elliptical virus particles of various sizes of 70–120 nm×240–340 nm surrounded by typical trilaminar envelope were found in the hypertrophied nuclei of affected cells. This virus was tentatively classified as a member of genus non-occluded baculovirus (NOB) of the subfamily Nudibaculovirinae of Baculovirus. A portion of the sequences of specific DNA fragment of an isolate from Thailand was used as a primer and compared to the other isolates collected from various countries. Results indicate that closely related strains of putative baculovirus were the causative agent of the white-spot syndrome of cultured penaeid shrimps occurring in six Asian countries.