Table 1. Time of appearance of clinical signs and mortality in 3 species of penaeid shrimp after IMNV injection, and detection of IMNV infection by histology and ISH. Species Day of appearance Day of initial No. dead shrimp IMNV infection b of clinical signs a death L. vannamei 6 1 3 2/6 Positive L. stylirostris 13 no death 0/6 Positive P monodon no signs c no death 0/6 Positive a Days after injection when all the shrimp showed clinic signs 6 Determined by both H&E and in situ hybridization c Lesions obscured by highly pigmented exoskeleton of P ,nonodon use of ISH, infection of other tissues could also be detected. With the probe, positive signals, as expected, were most intense in the skeletal muscle. The signals were also localized, suggesting the virus did not readily spread to neighboring muscle bundles. However, other tissues were also found to be infected. In L. vannamei and L. stylirostris, the reacting tissues were skeletal muscle, lymphoid organ, normal tubules and spheroids, hindgut and the phagocytic cells within the hepatopancreas and heart. In P monodon, the probe reacted to those tissues and also to gill tissue. The tissues that were ISH negative for IMNV in all three species included the stomach, ventral nerve cord, ganglia, and hematopoietic organ. Conclusion We developed an ISH method for the definitive diagnosis of IMNV infection in histological sections. The ISH diagnosis of IMNV infection corresponded with that of histology, which was based on the presence of lesions. However, ISH diagnosis has a greater sensitivity, and tissues without visible signs of infection by histological examination were found to be infected when tested with the probe. Also, the probe provides the specificity needed for reliable diagnosis. For example, samples of L. vannamei (from Belize) and P monodon (from 20 MARCH 2007 Australia) exhibiting muscle necrosis suspected to be caused by IMNV infection were found to be ISH negative. Thus, without the availability of the ISH test, diagnosis based on gross signs and/or histology alone can be erroneous. All three major species of farmed penaeid shrimp (L. vannamei, L. stylirostris and P monodon) tested in the bioassay were susceptible to IMNV infection. L. vannamei, the natural host of IMNV, appears to be most susceptible based on the onset of clinical signs and degree of mortality. However, IMNV is not particularly virulent compared to other shrimp viruses such as TSV, YHV, and WSSV. In the bioassay described here, no mortalities occurred until 13 days after exposure to IMNV. In similar bioassays with TSV, YHV, or WSSV, significant mortality usually occurs within 1-3 days. The relatively long delay in mortalities from IMNV infections is possibly because skeletal muscle, the primary target tissue of IMNV infection, can withstand more damage than the other tissues before survival is threatened. However, losses from IMNV infection can be significant; over 40 percent mortality often occurs late in the production cycle. In addition, reduction in market value of the survivors, which have necrotic tail muscles, adds to the economic loss. Note 1Department of Veterinary Science and Microbiology, University of Arizona, Tucson, Arizona 85721 USA. Fax: + 1 -520-621 -4899, E-mail: fengjyu@ u.arizona.edu Acknowledgments This work was supported by Gulf Coast Research Laboratory Consortium Marine Shrimp Farming Program, CSRS, USDA, and a special grant from the National Fishery Institute. We thank T. P. Andrade of the Brazilian Shrimp Farms Association (ABCC) for providing the IMNV-infected shrimp samples, and Stephen Nelson for assistance in editing this manuscript. References Lightner D. V., C. R. Pantoja, B. T. Poulos, K. F. J. Tang, R. M. Redman, T. Pasos-de-Andrade and J. R. Bonami. 2004. Infectious myonecrosis: New disease in Pacific white shrimp. Global Aquaculture Advocate 7: 85. Nunes, A. J. P., P. C. C. Martins and T. C. V. Gesteira. 2004. Produtores sofrem com as mortalidades decorrentes do virus da mionecrose infeccisa (IMNV). Panorama da AQUICULTURA, Maio/Junho.
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