碩士 / 國立臺灣大學 / 動物學系 / 85 / White spot syndrome virus (WSSV) is the causative agent of a
disease which has been causing mass mortalities of cultured
shrimps. The principle clinical sign of this disease is the
presence of white spots on the exoskeleton, especially on the
carapace of moribund shrimp. A Two-step WSSV diagnostic
polymerase chain reaction (PCR), based upon the specific primer
sets, was routinely used to detect WSSV in shrimps. Recently,
white spots had also been observed on the 4th pleopod
(ambulatory leg) of cultured crabs (Scylla serrata) and the
carapace of wild-caught portunid crabs (Charybdis feriatus).
When DNA was extracted from crabs collected from three sources:
cultured crabs, wild-caught crabs and a non-cultured pest crab
(Helice tridens), the PCR products showed the expected mobility,
that is, they were coincident with the products amplified from
the DNA prepared from WSSV-infected Penaeus monodon. The
restriction profiles of these PCR products cleaved with HaeIII,
HpaII, RsaI and Sau3AI were also the same. This is already
strong evidence for the presence of WSSV in these various
population. WSSV diagnostic PCR was also used to investigate
the infection rate and the infected tissues of WSSV in wild-
caught crabs. Almost all the tissues or organs of the seriously
infected crabs showed WSSV positive after only one-step of
amplification while tissues or organs of the lightly infected
crabs were only positive in two-step WSSV PCR. The infection
rate was particularly high in the gill, ambulatory leg and
hemolymph, and only slightly less high in the stomach, eyestalk
and maxilliped. The histopathological changes in the seriously
infected tissues or organs were characterized by degenerated
cells with hypertrophied nuclei. These nuclei were stained
homogeneously by H & E. WSSV was confirmed as the causative
agent by using in situ hybridization with a WSSV-specific probe.
Various tissues from the mesoderm and ectoderm, such as
connective tissue, epithelium, nervous tissue and muscle, could
be infected by WSSV. Based on the number of the positive
signals, the grades of infection could be distinguished. The
gill, stomach and heart were the most seriously infected organs
in crabs. Again, as with wild-caught shrimps which sometimes
developed a patent infection after capture, the stress inherent
in the cultured environment probably contributed to the wild-
caught crabs﹐which were originally only lightly infected
becoming seriously infected. In transmission electron
micrographs of ultra-thin sections of infected tissues taken
from infected wild-caught crabs (Portunus sanguinolentus),
enveloped viral particles were readily observed in the nucleus
of the infected cells. The viral agent was purified by CsCl
gradient centrifugation. Negatively stained preparations showed
the virus to be rod-shaped. The nucleocapsid measured 60-70 nm
at its broadest point and was 325-350 nm long. The capsid was
apparently composed of rings of subunits in a stacked series.
The rings were aligned perpendicular to the longitudinal axis of
the capsid. The thickness of the rings was very constant,
usually being 20 nm. The morphological characteristics of this
virus were thus very the similar to the viral agent purified
from WSSV infected Penaeus monodon. Finally, healthy
juvenile cultured crab, Scylla serrata, were exposed by
immersion to epidermal filtrate from diseased P. monodon.
Cumulative mortalities reached 40% within 2 weeks and WSSV was
detected to be present in those experimentally infected crabs as
early as 2 days post-infection. In situ hybridization with a
WSSV-specific probe gave positive signals at 48 hr post-
infection in the stomach, gill, cuticular epidermis and
hepatopancreas. By 6 days post infection, almost all organs were
heavily infected with WSSV. All of these results indicated
that the causative agents of white spot syndrome of shrimps and
crabs were in fact the same or closely related.
| Identifer | oai:union.ndltd.org:TW/085NTU00312004 |
| Date | January 1997 |
| Creators | Chiu, Ya-Lin, 邱雅琳 |
| Contributors | Guang-Hsiung Kou, Chu-Fang Lo, 郭光雄, 羅竹芳 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 95 |
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