Aquaculture Vol. 164 (1-4) pp. 201-220
Shrimp diseases and current diagnostic methods

^a D.V. Lightner
^a R.M. Redman
^a , Department of Veterinary Science, University of Arizona, , Tucson, AZ 85721, USA
Abstract:  In less than 30 yr, the penaeid shrimp culture industries of the world developed from their experimental beginnings into major industries providing hundreds of thousands of jobs, billions of U.S. dollars in revenue, and augmentation of the world's food supply with a high value crop. Concomitant with the growth of the shrimp culture industry has been the recognition of the ever increasing importance of disease, especially those caused by infectious agents. Major epizootics have plagued the world's shrimp culture industries. The most important diseases of cultured penaeid shrimp have had viral or bacterial etiologies, but a few important diseases have fungal and protozoan agents as their cause. Diagnostic methods for these pathogens include the traditional methods of morphological pathology (direct light microscopy, histopathology, electron microscopy), enhancement and bioassay methods, traditional microbiology, and the application of serological methods. While tissue culture is considered to be a standard tool in medical and veterinary diagnostic labs, it has never been developed as a useable, routine diagnostic tool for shrimp pathogens. The need for rapid, sensitive diagnostic methods led to the application of modern biotechnology to penaeid shrimp disease. The industry now has modern diagnostic genomic probes with nonradioactive labels for viral pathogens like IHHNV, HPV, TSV, WSSV, MBV, and BP. Additional genomic probes for viruses, for bacterial pathogens like NHP and certain Vibrio spp., and Microsporidia have also been developed. Highly sensitive detection methods for some pathogens that employ DNA amplification methods based on the polymerase chain reaction (PCR) now exist, and more PCR methods are being developed for additional agents. These advanced molecular methods promise to provide badly needed diagnostic and research tools to an industry reeling from catastrophic epizootics and which must become poised to go on with the next phase of its development as an industry that must be better able to understand and manage disease. © 1998 Elsevier Science B.V.

Aquaculture Vol. 164 (1-4) pp. 243-251
Detection of white-spot syndrome in cultured penaeid shrimp in Asia: Microscopic observation and polymerase chain reaction

^a J. Kasornchandra
^a S. Boonyaratpalin
^b T. Itami
^a , National Institute of Coastal Aquaculture, Koaseng, , Songkhla 90000, Thailand
^b , National Fisheries University, 2-7-1 Nagata-Honmachi, , Shimonoseki, 759-65, Japan
Abstract:    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. © 1998 Elsevier Science B.V. All rights reserved.

Aquaculture Vol. 164 (1-4) pp. 233-242
Detection of white spot syndrome associated baculovirus in experimentally infected wild shrimp, crab and lobsters by in situ hybridization

^a Poh-Shing Chang
^a Hsiao-Chao Chen
^a Yu-Chi Wang
^a , Department of Aquaculture, National Kaohsiung Institute of Marine Technology, , Kaohsiung, Taiwan
Abstract:    The techniques of detecting white spot syndrome associated baculovirus (WSBV) by in situ hybridization are already well established. A DNA probe specific to PmNOB III, an isolate of WSBV from Penaeus monodon, was labeled with digoxigenin and used to detect WSBV DNA in experimentally infected wild shrimps, crabs and lobsters that are native to Taiwan. WSBV could be detected in tissue sections of all examined specimens by in situ hybridization. In marine shrimps, Trachypenaeus curvirostris, Metapenaeus ensis and Exopalaemon orientalis, WSBV DNA positive cells were observed in the gills, stomach, cuticular epidermis, antennal gland, hepatopancreas, compound eye, muscle, heart and reproductive tissues. WSBV DNA was observed only in the gills, cuticular epidermis and hepatopancreas in the freshwater shrimps Macrobrachium sp. and Procambarus clarkii. In crabs Portunus sanguinolentus and Charybdis granulata, WSBV DNA could be detected in the gills, stomach, hepatopancreas, muscle and reproductive tissues. In lobsters Panulirus versicolor and Panulirus penicillatus, the positive cells were observed in the gills, stomach, cuticular epidermis and hepatopancreas. © 1998 Elsevier Science B.V. All rights reserved.

Aquaculture Vol. 164 (1-4) pp. 253-262
Detection of white spot baculovirus (WSBV) in giant freshwater prawn, Macrobrachium rosenbergii, using polymerase chain reaction

^a S.E. Peng
^a C.F. Lo
^a C.H. Ho
^b C.F. Chang
^a G.H. Kou
^a , Department of Zoology, National Taiwan University, , Taipei, Taiwan
^b , Tung Kang Marine Laboratory, Taiwan Fisheries Research Institute, Tung Kang, , Ping Tung, Taiwan
Abstract:    White spot baculovirus (WSBV) is the causative agent of a disease which decimated some cultured penaeid shrimp populations and inflicted severe economic damage in Taiwan. Until very recently, the giant freshwater prawn Macrobrachium rosenbergii was thought to be unaffected by this virus, but now signs closely resembling white spot syndrome (WSS) have been observed on its exoskeleton. In this paper, WSBV was established as the causative agent by using the diagnostic polymerase chain reaction (PCR) with WSBV-specific primers. WSBV was found in M. rosenbergii larvae, postlarvae, juveniles, and adults. The amplified product from the DNA of the naturally-infected WSS M. rosenbergii was similar to that of WSBV-infected Penaeus monodon. Furthermore, comparison of the restriction profiles of these two PCR products by HaeIII, HpaII, RsaI, and Sau3AI revealed no differences, suggesting that WSBVs from the infected P. monodon and M. rosenbergii are closely related, if not identical. A homogenate positive in one-step WSBV diagnostic PCR was prepared from frozen P. monodon for the challenge experiment. Dilutions were added to tanks of healthy M. rosenbergii larvae and postlarvae. After 2 days, some of the dead specimens were positive for WSBV by diagnostic PCR. © 1998 Elsevier Science B.V.

DAO 43:175-181 (2000) Abstract 
Identification of genomic variations among geographic isolates of white spot syndrome virus using restriction analysis and Southern blot hybridization 
Qiong Wang1,*, Linda M. Nunan**, Donald V. Lightner 
Department of Veterinary Science and Microbiology, University of Arizona, Tucson, Arizona 85721, USA 
*Present address: Max Planck Institute for Biology, Corrensstrasse 38, 72076 Tübingen, Germany **Corresponding author. E-mail: lmn@u.arizona.edu 

ABSTRACT: White spot syndrome virus (WSSV) is widely distributed in most of the Asian countries where penaeid shrimp are cultured, as well as in some regions of the USA. Six geographic isolates of WSSV--1 each from penaeid shrimp from China, India, Thailand, and the US states of Texas and South Carolina, and 1 isolated from crayfish at the National Zoological Park in Washington, DC--were compared by combining the methods of restriction analysis and Southern blot hybridization. DNA was extracted from purified viruses and then digested with selected endonucleases: AccI, BglII, ClaI, BamHI, EcoRI, HindII, HaeI, SacI and XhoI. The blots were detected with digoxigenin-11-dUTP-labeled WSSV genomic probes: LN4, C42 and A6. No distinctive differences among the 5 WSSV isolates from penaeid shrimp were detected; however, differences in the WSSV isolate from crayfish were observed. A 2.8 kb DNA fragment originating from the crayfish isolate and encompassing the LN4 region was subcloned into pBluescript and sequenced for comparison with the LN4 fragment from the Thailand WSSV isolate. The results indicate that some genomic components of WSSV from different geographic regions share a high degree of homology. This method can be used to distinguish between the WSSV isolate from crayfish and the WSSV isolates from penaeid shrimp.

Published in DAO Vol. 39, No. 1 (1999) on December 22

Results from black tiger shrimp Penaeus monodon culture ponds stocked with postlarvae PCR-positive or -negative for white-spot syndrome virus (WSSV)

Boonsirm Withyachumnarnkul*

Department of Anatomy, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand

*E-mail: pimfern@asianet.co.th

ABSTRACT: Commercial, intensive, earthen shrimp ponds (188) in southern Thailand were stocked with postlarvae (PL) of Penaeus monodon that had tested positive or negative for white-spot syndrome virus (WSSV) infection by polymerase chain reaction (PCR) assay. All the PL were grossly healthy. At 2 wk intervals after stocking, shrimp from each pond were examined for gross WSSV lesions and tested for WSSV by PCR. Shrimp from all the ponds stocked with WSSV-PCR-positive PL (Group 0, n = 43) eventually showed gross signs of white-spot disease (WSD) at an average of 40 d after stocking. Of the remaining ponds stocked with WSSV-PCR-negative PL (n = 145), some remained WSSV-PCR-negative throughout the study (Group 5, n = 52), while others (93) became WSSV-PCR-positive after stocking, during the first month (Group 1, n = 23), second month (Group 2, n = 40), third month (Group 3, n = 24), or fourth month (Group 4, n = 6). Crop failure was defined as a pond drain or forced harvest before 14 wk or 98 d of cultivation. For Group 0 the proportion of ponds failing was 0.953, while it was only 0.019 for Group 5. Thus, the relative risk of failure for Group 0 was approximately 50 times that of Group 5. The relative risk of failure for Group 0 was also 3 times that for ponds stocked with WSSV-PCR-negative PL. Obviously, not all WSSV outbreaks resulted in crop failure. Of the 93 ponds stocked with PCR-negative PL that later yielded WSSV-PCR-positive shrimp, 53% reached successful harvest. The study showed that PCR screening of PL and rejection of WSSV-positive batches before stocking could greatly improve the chances of a successful harvest.

Studies on effective PCR screening strategies for white spot syndrome virus (WSSV) detection in Penaeus monodon brooders

Hui-Chen Hsu¹, Chu-Fang Lo¹, Shan-Ching Lin¹, Kuan-Fu Liu², Shao-En Peng¹, Yun-Shiang Chang¹, Li-Li Chen¹, Wang-Jing Liu¹, Guang-Hsiung Kou^1,*

¹Department of Zoology, National Taiwan University, Taipei, Taiwan, ROC
²Tung-Kang Marine Laboratory, Taiwan Fisheries Research Institute, Tung-Kang, Ping-Tung, Taiwan, ROC ghkou@ccms.ntu.edu.tw

ABSTRACT: We re-tested stored (frozen) DNA samples in 5 independent polymerase chain reaction (PCR) replicates and confirmed that equivocal test results from a previous study on white spot syndrome virus (WSSV) in brooders and their offspring arose because amounts of WSSV DNA in the test samples were near the sensitivity limits of the detection method. Since spawning stress may trigger WSSV replication, we also captured a fresh batch of 45 brooders for WSSV PCR testing before and after spawning. Replicates of their spawned egg batches were also WSSV PCR tested. For these 45 brooders, WSSV prevalence before spawning was 67% (15/45 1-step PCR positive, 15/45 2-step PCR positive and 15/45 2-step PCR negative). Only 27 (60%) spawned successfully. Of the successful spawners, 56% were WSSV PCR positive before spawning and 74% after. Brooders (15) that were heavily infected (i.e. 1-step PCR positive) when captured mostly died within 1 to 4 d, but 3 (20%) did manage to spawn. All their egg batch sub-samples were 1-step PCR positive and many failed to hatch. The remaining 30 shrimp were divided into a lightly infected group (21) and a 2-step PCR negative group (9) based on replicate PCR tests. The spawning rates for these 2 groups were high (81 and 78%, respectively). None of the negative spawners (7) became WSSV positive after spawning and none gave egg samples positive for WSSV. In the lightly infected group (21), 6 brooders were 2-step WSSV PCR negative and 15 were 2-step WSSV PCR positive upon capture. However, all of them were WSSV PCR positive in replicate tests and after spawning or death. Four died without spawning. The remaining 17 spawned but only 2 gave egg samples PCR negative for WSSV. The other 15 gave PCR positive egg samples, but they could be divided into 2 spawner groups: those (7) that became heavily infected (i.e. 1-step PCR positive) after spawning and those (8) that remained lightly infected (i.e. became or remained 2-step PCR positive only). Of the brooders that became heavily infected after spawning, almost all egg sample replicates (91%) tested 2-step PCR positive. One brooder even gave heavily infected (i.e. 1-step PCR positive) egg samples. For the brooders that remained lightly infected after spawning, only 27% of the egg sample replicates were 2-step PCR positive. Based on these results, we recommend that to avoid false negatives in WSSV PCR brooder tests screening tests should be delayed until after spawning. We also recommend, with our PCR detection system, discarding all egg batches from brooders that are 1-step PCR positive after spawning. On the other hand, it may be possible with appropriate monitoring to use eggs from 2-step PCR positive brooders for production of WSSV-free or lightly infected postlarvae. These may be used to stock shrimp ponds under low-stress rearing conditions.

DAO 41:9-18 (2000)

A new bacterial white spot syndrome (BWSS) in cultured tiger shrimp Penaeus monodon and its comparison with white spot syndrome (WSS) caused by virus

Y. G. Wang*, K. L. Lee, M. Najiah, M. Shariff*, M. D. Hassan

Aquatic Animal Health Unit, Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia

E-mail: wangyingeng@hotmail.com or shariff@vet.upm.edu.my
ABSTRACT: This paper describes a new bacterial white spot syndrome (BWSS) in cultured tiger shrimp Penaeus monodon. The affected shrimp showed white spots similar to those caused by white spot syndrome virus (WSSV), but the shrimp remained active and grew normally without significant mortalities. The study revealed no evidence of WSSV infection using electron microscopy, histopathology and nested polymerase chain reaction. Electron microscopy indicated bacteria associated with white spot formation, and with degeneration and discoloration of the cuticle as a result of erosion of the epicuticle and underlying cuticular layers. Grossly the white spots in BWSS and WSS look similar but showed different profiles under wet mount microscopy. The bacterial white spots were lichen-like, having perforated centers unlike the melanized dots in WSSV-induced white spots. Bacteriological examination showed that the dominant isolate in the lesions was Bacillus subtilis. The occurrence of BWSS may be associated with the regular use of probiotics containing B. subtilis in shrimp ponds. The externally induced white spot lesions were localized at the integumental tissues, i.e., cuticle and epidermis, and connective tissues. Damage to the deeper tissues was limited. The BWS lesions are non-fatal in the absence of other complications and are usually shed through molting

Published in DAO Vol. 40, No. 2 (2000)

Natural and experimental infection of white spot syndrome virus (WSSV) in benthic larvae of mud crab Scylla serrata

Li-Li Chen¹, Chu-Fang Lo¹, Ya-Lin Chiu¹, Chen-Fang Chang², Guang-Hsiung Kou^1,*

¹Department of Zoology, National Taiwan University, Taipei, Taiwan, ROC
²Tung Kang Marine Laboratory, Taiwan Fisheries Research Institute, Tung Kang, Ping Tung, Taiwan, ROC

E-mail: ghkou@ccms.ntu.edu.tw
ABSTRACT: White spot syndrome virus (WSSV), the causative agent of white spot syndrome in shrimp, has a wide host range which extends to crabs, copepods and other arthropods. In this study, benthic larvae of the mud crab Scylla serrata were captured from Taiwan's coastal waters and screened for the presence of WSSV by polymerase chain reaction (PCR) and in situ hybridization. WSSV was detected in around 60% of the larvae, and this prevalence rate remained fairly constant when the captured larvae were subsequently maintained in an aerated system in the laboratory. WSSV-free larvae obtained from a hatchery were challenged by immersion in a WSSV inoculum. Fifteen days after challenge, cumulative mortality in the experimental group reached 43% compared to 20% in the control group. PCR detection of WSSV in both moribund and surviving specimens clearly implicated the virus as the cause of death in most cases. Histological and in situ hybridization data confirmed that WSSV tissue tropism in Scylla serrata crab larvae is similar to that found in shrimp.