from those, we selected one clone, which contained a 1.2 kb viral insert, for use in probe development. The sequence of this insert is located within the capsid protein-coding region of the viral genome. We selected one pair of primers, within this insert, to produce a labeled probe. We used PCR (polymerase chain reaction) to amplify the cDNA in the presence of digoxigenin-labeled dUTP to prepare a 1.0 kb probe. When used during in situ hybridization, the probe should anneal to the corresponding viral sequence present in infected tissue sections. The resulting probe was used to detect IMNV in histological sections of tissue from infected L. vannamei. In the stained section, the clumps of viral particles are revealed as basophilic inclusion bodies in the cytoplasm (Figure 2A). In histological analysis, the presence of these inclusion bodies is indicative of a viral infection. When exposed to the newly developed probe through in situ hybridization, the inclusion bodies reacted intensely (Figure 2B). This shows that the probe can be used to detect the IMNV genome. The in situ hybridization procedure with the new probe was optimized through testing on denaturation of viral genome. To detect IMNV through in situ hybridization, the double-stranded RNA viral genome must first be denatured to separate the strands and allow them to hybridize to the probe. Thus, IMNV in situ hybridization needs to include a denaturation step prior to hybridization. We found a strong signal (dark blue precipitate) in tissue sections that were denatured at 84°C for 10 min, while the signal was either absent or faint when this step was omitted. Fig. 2. (A) H&E histology, arrows indicate inclusions; (8) in situ hybridization detection of IMNV in infected Litopenaeus vannamei. To be useful, the probe must also be specific; that is, it should not react to either the shrimp genome or to other viruses. We tested the specificity of the optimized procedure with other shrimp tissue samples. The probe was shown to be highly specific; it did not react to tissues of un-infected shrimp or those of shrimp known to be infected with white spot syndrome virus (WSSV), infectious hypodermal and hematopoietic necrosis virus (IHHNV), Taura syndrome virus (TSV) or yellow head virus (YHV) . Fig. 3. In situ hybridization of skeletal muscle section of (A) Litopenaeus vannamei, (8) L. stylirostris and (C) Penaeus monodon infected with INMV in a laboratory Susceptibility of Penaeid Shrimp to IMNV We used the new probe to test the susceptibility of three species of penaeid shrimp to IMNV in a laboratory bioassay. In this experiment, seven individuals (mean weight 3 g) of each species (L. vannamei, L. stylirostris, and P monodon) were injected with purified IMNV virions prepared from infected shrimp. Each shrimp was injected with 0.1 mL of inoculum. Shrimp were then maintained in three aquaria on a pelleted ration for 28 days. All of the species proved to be susceptible to the virus. The major clinical sign of infection, the appearance of whitish lesions in tail muscle, was seen first in the L. vannamei group (Table 1), in which all of the injected shrimp had obvious lesions after 6 days. In the L. stylirostris group, the lesions, developed slowly, with only three showing signs of infection after seven days; all of the shrimp had lesions after 13 days. No clinical signs of infection were observed in any of the individuals of the Penaeus monodon group. However, lesions could be obscured because of the highly pigmented exoskeleton of this species. On day 14 post-injection, one individual from each species was killed for ISH; and they all tested positive for IMNV (Figure 3). Although in the group of L. vannamei there were two mortalities (days 13 and 21), no mortalities occurred in either the L. stylirostris or P monodon groups during the bioassay period. Using the probe, we were also able to gain more detailed information on the type of tissues that were infected. With histological analysis, we found that for infected shrimp of each species the viral infection was evident only in the skeletal muscle and lymphoid organ. Through the WORLD AQUACULTURE 19
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