Perkinsus marinus is a highly contagious pathogen of the eastern oyster Crassostrea virginica. Until recently, transmission studies have employed wild-type parasites isolated directly from infected oysters. Newly developed methods to propagate P. marinus in vitro have led to using cultured parasites for infection studies, but results suggest that cultured parasites are less virulent than wild-type parasites. In this paper, we report results of experiments designed to quantify differences between wild-type and cultured P. marinus virulence and to test the following hypotheses: (1) in vitro-cultured parasites are less virulent than wild-type parasites; (2) virulence decreases gradually during in vitro culture; (3) virulence of in vitro cultures can be restored by in vivo passage; (4) virulence changes with culture phase. Our results demonstrate that parasites freshly isolated from infected hosts are much more virulent than those propagated in culture, indicating a potential deficiency in the culture medium used. Virulence was lost immediately in culture and, for that reason, the practice of repassing cultured cells through the host to restore virulence does not work for P. marinus. Virulence was also associated with culture phase: log-phase parasites were significantly more virulent than those obtained from lag-or stationary-phase cultures.KEY WORDS: Disease · Parasite · Oyster · Crassostrea virginica · Loss of virulence · Culture phase Resale or republication not permitted without written consent of the publisherDis Aquat Org 51: [187][188][189][190][191][192][193][194][195][196][197][198][199][200][201] 2002 at different temperatures (Dungan & Hamilton 1995, Gauthier & Vasta 1995; however, the same does not appear true for virulence, the degree to which the pathogen causes disease. Studies using P. marinus isolated from infected oysters have shown that lethal infections can be generated with as few as 50 to 100 parasites injected into the shell cavity (Mackin & Sparks 1962, Valiulis & Haskin 1972, Chu & Volety 1997. Further, in repeated experiments, Ray (1954) obtained deaths in about 30 d when oysters were fed tissue minces of highly infected oysters. In contrast, Bushek et al. (1997) were unable to establish infections in oysters by feeding them up to 10 7 cultured parasites. When the same concentration was injected into the shell cavity, infections developed, but no oysters died during a 2 mo experiment. Similarly, La Peyre et al. (1993) found only light infections after 8 wk when 10 6 cultured parasites were injected into the shell cavity. Gauthier & Vasta (1993), on the other hand, reported heavy infections in 4 to 5 wk, followed by mortality, after 2 biweekly shell-cavity injections of 2 × 10 5 P. marinus. Loss of virulence in cultured pathogens is well known. One possible contributing factor is the culture phase. Morphologically different forms of Perkinsus marinus occur in oysters (Mackin & Boswell 1955) and most of these have been observed in vitro (La Peyre et al. 1993) where their relat...

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Perkinsus marinus, a pathogen of the eastern oyster Crassostrea virginica, is transmitted directly among oysters. Previous studies found viable P. marinus parasites in the feces and pseudofeces of oysters within hours of injection with parasites, suggesting that the parasite may be voided from live oysters and subsequently dispersed in the water column. The experiments described here were designed to quantify P. marinus shed in the feces and pseudofeces of experimentally infected oysters. The results indicated that parasites were shed in 2 phases. A 'decreasing' phase occurred within 2 wk of challenge and before net parasite proliferation began in the host. An 'increasing' phase occurred after P. marinus had begun replicating. The quantity of P. marinus recovered in the feces and pseudofeces of exposed oysters was only about 5% of the dose administered. In vitro-cultured P. marinus were eliminated at a greater rate than wild-type P. marinus and the fraction discharged was not associated with culture phase. Oysters that were continuously dosed with P. marinus in their food gradually lost the ability to discard the parasite in pseudofeces. The quantity of P. marinus shed in feces of infected oysters was correlated with both the P. marinus body burden and subsequent survival time, suggesting that noninvasive fecal counts could predict infection intensity and survival. The results indicate that in an epizootic, shedding of P. marinus via feces is relatively small compared to the potential number released by cadavers of heavily infected oysters, but that fecal discharge may be important in transmission before infections become lethal. KEY WORDS: Disease · Parasite · Oyster · Crassostrea virginica · Infection · Modes of transmission Resale or republication not permitted without written consent of the publisherDis Aquat Org 51: [217][218][219][220][221][222][223][224][225] 2002 oysters frequently coincides with P. marinus-caused deaths of native oysters. In addition, experiments in which positions of infected and uninfected oysters were fixed showed that mortalities due to P. marinus were accelerated in individuals near dead and dying infected oysters (Ray 1954, Andrews 1988. Decay of infected tissue, with consequent release of parasites into the water column, has thus been considered the main source of infective cells for P. marinus transmission (Mackin 1962, Andrews 1988.Oyster deaths caused by Perkinsus marinus are seasonal, and typically highest in the late summer and fall; however, oysters can become infected earlier in the summer (Andrews 1988, Burreson & Ragone Calvo 1996. Observations of an initial infection increase in June may be attributable to the proliferation of parasites that survived in vivo overwinter (Ragone Calvo & Burreson 1994, Burreson & Ragone Calvo 1996, but it is possible that some parasites are able to survive overwinter outside the host and can infect oysters as soon as the oysters begin to filter in the spring (Mackin 1962). Because oysters are metabolically active and parasites...

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41Land-based management has reduced nutrient discharges, however, many coastal waterbodies waterbodies without circulation models. The value of removed nitrogen was estimated using