Virus infection in cultured abalone, Haliotis diversicolor reeve in Guangdong Province, China.

ABSTRACT A serious disease occurred in many Holiotis diversicolor Reeve farms during 1999 and 2002. The symptoms of the infected abalone were as follows: the pleopod became stiff, its surface became black, and the mantle shrank. Ali sizes of abalone became infected and mortality was high. A spherical virus was detected in the tissues of the digestive gland, mantle, gill, and intestine of infected animals. It was observed in the cytoplasm and had a diameter about ~100-130 nm. Electron microscope examination showed that organelles of infected cells, such as the endoplasmic reticulum; expanded, mitochondria dilated, nuclear membranes dissolved or disappeared, and the edges of the nucleolus swelled. The pathogenicity of the virus was confirmed by experimental infection.

KEY WORDS: virus infection, pathogen, Haliotis diversicolor Reeve

INTRODUCTION

Numerous pathogens that cause serious abalone diseases have been reported from different abalone species around the world. Virus-like particles have been observed in the cytoplasm of cells near the nerve trunk of diseased Haliotis discus discus, examined with an electron microscope (Otsu & Sasaki 1997). Virus-like particles were also isolated from diseased H. discus discus in primary cultures of abalone hemocytes (Nakatsugawa et al. 1999). A spherical virus was detected in "crack shell disease" of Haliotis discus hannai (Wang & Li 1997, Li et al. 1998). A similar disease and virus in Haliotis diversicolor Reeve were also studied (Wang et al. 2000). The pathogen of an epidemic disease that resulted in mass mortality of Haliotis diversicolor aquatilis was studied. Artificially infected abalone revealed that the virus was the lethal pathogen (Song et al. 2000). Three forms of spherical viruses were observed when an epidemic disease of cultured Haliotis diversicolor supertexm occurred in Dongshan, Fujian province in 1999 (Zhang et al. 2001).

A bacterial infection was reported in juveniles of red abalone Haliotis rafescens (Elston & Lockwood 1983). Mass mortalities of 4-day-old red abalone juveniles were caused by Vibrio alginolyticu (Anguiano-Beltran et al. 1998). A disease of young Haliotis diversicolor surpertexta was also caused by Vibrio spp. The abalone were more easily infected when the temperature was higher (Lee et al. 2001). Other bacteria that have been isolated from Haliotis discus hannai are Vibro fluvialis-II (Liu et al. 1995, Nie et al. 1995, Li et al. 1996), V. campbellii (Ma et al. 1996), and Psuedomonas fluoresces (Ye et al. 1997). Parasites that infect different species of abalone include ciliates in H. midae and H. spadicea (Botes et al. 1998), kidney Coccidia in Haliotis rufescens and Haliotis midae (Mouton 2000, Sales & Britz 2000), Perkinsus spp. in Haliotis rubra, Haliotis laevigata (Goggin & Lester 1995), sabellid polychaetes in Haliotis midae (Cook 1998, Ruck & Cook 1998, Sales & Britz 2000), and trematode metacercariae in Haliotis ruber (Bores et al. 1999).

Haliotis divetMcolor Reeve has been increasingly cultured along the southern coastal area of China and has become one of the most exploited and valuable mariculture species. Mass mortalities have been observed in many commercial farms since a serious disease occurred. All sizes of abalone could be infected, and the mortality may reach 100% in a few days. The culture tanks were filled with foam. The pleopod of the diseased abalone became stiff with a black surface and the mantle and foot shrank. The dead abalone attached to the bottom of the culture cage. Here we study the pathogen, the influence of temperature on its infectivity, and the ultrastructure pathology of the infected abalone.

MATERIALS AND METHODS

Specimens

Diseased abalone, of shell length 4-6 cm, were collected from several commercial farms located in the eastern area of Guangdong Province during 2000-2002.

Healthy abalone, of shell length 4-6 cm, were collected from a farm located in an isolated bay that had never experienced the same disease.

Preparation of Solution for Injection

Ten grams of tissue of diseased abalone was cut into small pieces and homogenized, using a mortar, in the presence of liquid nitrogen. The homogenate was suspended with phosphate-buffered saline (PBS) (Ph = 7.6) (W: W = 1:10) and the suspension was centrifuged at x2500g for 30 min at 4[degrees]C. The supernatant fluid was centrifuged at x6000g for 30 min at 4[degrees]C, and the supernatant fluid was collected and filtered through a 0.22-[micro]m filter membrane.

The suspension from the healthy abalone was prepared using the same procedure.

Challenge Test

Four groups were used for challenge test 1, and were treated as indicated in Table 1. The animals in group1 were injected with 0.05 mL virus suspension each; group 2 were injected with 0.1 mL virus suspension each; the animals in control group l were injected with 0.1 mL suspension of healthy abalone each; the animals in control group 2 were injected with 0.1 mL PBS each. All animals were maintained in the aquaria with recirculating water system at 17[degrees]C. The salinity of the water was 30 ppt. The mortality and clinical signs were observed and recorded every day.

Temperature Influence on Disease Outbreak

The abalone were divided into 5 groups with 10 individuals each. They were treated as indicated in Table 2. The animals in group 3, group 4, group 5, and group 6 were injected with 0.1 mL virus suspension respectively; the animals in control group3 were injected with 0.1 mL PBS. All animals were maintained in aquaria with recirculating water, the salinity of the water was 30 ppt. Mortality and clinical signs were observed and recorded every day.

Transmission Electron Microscopy 

The tissues of digestive gland, mantle, gill, and intestine of both naturally and experimentally infected abalone were cut into pieces about 1 [mm.sup.3] in size, and fixed in 2.5% glutaraldehyde in 0.1 PBS buffer at 7.4. After being washed in PBS buffer for 1 h at 4[degrees]C, samples was postfixed in 1% osmium tetroxide in the same buffer at 4[degrees]C. The samples were dehydrated in ascending concentrations of ethanol (50% to 100%), and then embedded in Epon812 resin. Ultra-thin sections were cut and stained with uranyl acetate and lead citrate, and examined in a PhilipsCM10 transmission electron microscope.

Negative Stain

Twenty grams of tissue of diseased abalone was cut into small pieces and homogenized using a mortar, in the presence of liquid nitrogen. The homogenate was suspended in phosphate-buffered saline (PBS) (W: W = 1:10) and the suspension was centrifuged at x6000g for 30 min at 4[degrees]C. The supernatant fluid was centrifuged at x200000g for 60 min at 4[degrees]C, and the deposit was resuspended with PBS and stained with 2.5% sodium phosphotungustate (pH 7.0), and then examined in a Philips CM10 transmission electron microscope.

RESULTS

Challenge Test

Several dead abalone were observed in group 1 and group 2 that were injected with virus suspension within 48 h and all died within 96 h (Table 3). The aquarium was filled with foam, which also occurred when the infection occurred naturally (Fig. 1a). The clinical symptoms of infected abalone were also similar to naturally infected abalone (Fig. 1b). No mortality was recorded in the control groups.

[FIGURE 1 OMITTED]

Temperature Influence on Disease Outbreak

The disease outbreak was associated with low temperature (Table 4). All the abalone died within 96 h when the temperature of the water was at 17[degrees]C and 20[degrees]C. No abalone died when the temperature of water was 23[degrees]C or 26[degrees]C. All abalone in the control group survived.

Electron Microscopy

Infected cells in the connective tissue showed hypertrophied or irregular shaped nuclei and marginated chromatin (Fig. 1c, d), which indicated the condensation of chromatin and DNA. Some nuclei were highly electron dense. Chromatin was densely packed around the nuclear envelope, and the membrane of the nucleus disintegrated (Fig. 1c). The nucleus collapsed in seriously affected cells. The nuclear membrane totally dissolved, and some highly electron dense material could be seen in the collapsed nucleus (Fig. 1e).

Pathologic changes were observed in the organelles of the infected cells. The endoplasmic reticulum expanded, mitochondria dilated and became irregularly shaped, and mitochondrial cristae shrank or disappeared (Fig. 1f). In the cytoplasm, irregularly shaped spore-like bodies were observed. The bodies appeared to be mineral concretions, composed of numerous alternating electron dense and electron lucent layers (Fig. 2a).

[FIGURE 2 OMITTED]

Virus particles were mainly observed in connective tissue cells and occasionally in hemocytes. The particles were circular in shape, 100-130 nm in diameter (Fig. 2b), contained an electrondense nucleocapsid, and the outer layer was the thick envelope. Between the envelope and nucleocapsid, there was an electron lucent layer about 10-15 nm in thickness. The nucleocapsid was electron-dense and 80-100 nm in diameter. In some cells, numerous virus particles aggregated together and were surrounded by a double-layer membrane (Fig. 2c, d). The virus particles scattered in the cytoplasm in some infected cells (Fig. 2e, f). Figure 3a shows a magnification of the virus particles. Among the aggregating viruses, there were empty and deformed capsid-like structures (Fig. 2e). These may be abnormally assembled viral proteins. TEM examination revealed that virus particles existed in challenged individuals, but not in nonchallenged ones.

[FIGURE 3 OMITTED]

Negative stain showed the virus particle was about 100-130 nm in diameter (Fig. 3b), similar to the size of virns particle observed in ultra-thin section (Fig. 3a).

DISCUSSION

Virus are some of the most important agents that cause mass mortalities of animals. Viruses reported in marine shellfish include Herperviridae, Iridoviridae, Papovaridae, Togaviridae, Retroviridae, Reviridae, and Paramyxoviridae.

Several viruses associated with the mass mortalities of the host species have been reported. Irido-like virus attacked the velar epithelium of Crassostrea gigas and caused severe hatchery losses (nearly 100%), which usually appeared from March to May, but also occurred throughout the summer (Elston, 1979). Icosahedral DNA virus caused the Portuguese oyster Crassostrea angulata Lamarck velar virus disease and hemocyte infection virus disease (HIV), extensive gill erosion corresponding with high mortalities. Gill disease was regarded as one factor in the elimination of the Portuguese oyster from important culture areas on the Atlantic coast of France (Comps 1988). In Tiostrea chilensis larvae and spat, the interstitial cells, mantle, and digestive tract of epithelial cells were infected with herpes-like viruses and infection seemed to be associated with about 95% mortality, over 3 to 4 days, among experimentally exposed veligers (Hine et al. 1998). Herpes-like viruses were the main agents that led to high mortalities of the Pacific oyster Crassostrea gigas larvae (Renault et al. 2001). The herpes-like viral infection in larval Ruditapes philippinarum was associated with sporadic high mortalities in a commercial hatchery (Renault et al. 2001). Electron microscopic examination revealed the presence of spherical virus-like particles in the digestive gland, kidney, and intestine of moribund scallop Chlamys farreri in 2000. The virus was approximately 130-170 nm in diameter and always observed in the cytoplasmic vesicles of infected cells. It was the main cause of the mass mortality of scallops, from July to August since 1997, in the northern area of China (Liu 2002).

With the development of abalone culture industries, the impact of diseases on abalone have become more and more serious. We described, for the first time, the virus infection in abalone Haliotis diversicolor Reeve associated with the mass mortalities in Guangdong Province. With respect to the size, morphologic features, and syndrome of the infected abalone, it was similar to the 100 nm diameter spherical virus that was tbund in the cytoplasm of liver cells of diseased Haliotis diversicolor Aqualitis from Fujian Province (Song et al. 2000). In our study, the virus particles were mainly observed in the cytoplasm of the infected cells, but never in the nucleus.

Pathologic changes in infected cells included expanded endoplasmic reticulum and dilated or dissolved mitochondria. In the cytoplasm, the endoplasmic reticulum is associated with the Golgi body and mitochondria and functions in synthesis, transportation, and secretion (Wu Zh 1990). The impact of viruses on cells included the destrnction of membrane structures of organelles such as endoplasmic reticulum and mitochondria. The viruses were enveloped by the membrane systems of these organelles (Fig. 2d).

The disease infected all sizes of abalone when the temperature was <20[degrees]C. It seemed to break out more easily when the temperature became lower. When the water temperature was at [greater than or equal to]23[degrees]C, mortality did not occur. It is suspected that the viruses propagate only in low temperatures. The optimal temperature for Haliotis diversicolor Reeve to grow and develop is about 25[degrees]C, and it may be that physiologic changes that occur in the species at lower water temperatures may make the animals more susceptible to infection.

TABLE 1.

Preparation of challenge test in haliotis diversicolor Reeve.

                  Number of                              Temperature
                   Healthy    Dose of                        for
Group             Abalones    Injection                  Maintaining

Group 1              10       0.05 ml virus suspension   17[degrees]C
Group 2              10       0.1 ml virus suspension    17[degrees]C
Control Group 1      10       0.1 ml suspension of       17[degrees]C
                                healthy abalone
Control Group 2      10       0.1 ml PBS                 17[degrees]C

TABLE 2.

Temperature influence on disease outbreak.

                  Number of                             Temperature
                   Healthy            Dose of               for
Group             Abalones           Injection          Maintaining

Group 3              10       0.1 ml virus suspension   17[degrees]C
Group 4              10       0.1 ml virus suspension   20[degrees]C
Group 5              10       0.1 ml virus suspension   23[degrees]C
Group 6              10       0.1 ml virus suspension   26[degrees]C
Control Group 3      10       0.1 ml PBS                17[degrees]C

TABLE 3.

The cumulative mortality of challenge test.

                        Time  (h)          Mortality

Group              24    48    72    96      Rate

Group 1             0     2     7    10      100%
Group 2             0     3     9    10      100%
Control Group 1     0     0     0     0        0
Control Group 2     0     0     0     0        0

TABLE 4.

The cumulative mortality of infected abalones at
different temperature

                       Time (h)
                                         Mortality
Group            24    48    72    96      Rate

Group 3           0     2     7    10      100%
Group 4           0     4     8    10      100%
Group s           0     0           0        0
Group 6           0     0           0        0
Control Group    30     0           0        0

ACKNOWLEDGMENTS

This research was supported by the Department of Science and Technology of Guangdong Province. The project number is 2KB05301N.

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JIANGYONG WANG, (1,2) ZHIXUN GUO, (1) JUAN FENG, (1) GUANGFENG LIU, (1) LIWEN XU, (1) BISHENG CHEN, (1) * AND JINPEI PAN (2)

(1) South China Sea Fisheries Research Institute, Guangzhou 510300, People's Republic of China; (2) South China Sea Institute of Oceanology, The Chinese Academy of Sciences, Guangzhou 510301, People's Republic of China

* Corresponding author. E-mail: [e-mail].