Rotaviruses in oysters by cell culture techniques . . .
cubated at 37 ꢁC for 60 min. After this adsorption period, the in- Table 1—Semi-quantitative evaluation of the non-inoculated
oyster extract toxicity towards MA104 cells and cytopathic
effects of rotavirus isolated from oysters extracts using MA
oculum was removed by suction and 0.2 ml maintenance medi-
um (complete MEM without FCS) supplemented with 10 mg/ml
trypsin added to the infected cells. Chamber slides were then in-
1
04 cells incubated for 48 h.
Oysters
Oyster extract
(non-inoculated)
toxicity
cubated for 24 h at 37 ꢁC in a 5% CO atmosphere. At the end of
2
Extract
Extract
A
Extract
B
the infection period, the medium was decanted and monolayer
washed twice with PBS. Cells were then fixed twice in chilled
methanol for 5 min, and air-dried. Fixed slides were rehydrated
for 5 min in PBS, and incubated for 15 min with 0.3 ml (each
chamber) PBS containing 1% BSA (w/v), and 0.05% (v/v) Tween-
Dilutions
1
1
1
1
:32
:64
:128
:256
2.75 ꢅ 0.43
1.25 ꢅ 0.43
1 ꢅ 0
2.5 ꢅ 0.57
2.0 ꢅ 0.81
2.25 ꢅ 0.5
1.75 ꢅ 0.5
2.0 ꢅ 0.0
2.25 ꢅ 0.5
1.75 ꢅ 0.5
1.75 ꢅ 0.5
1.25 ꢅ 0.5
1.0 ꢅ 0.0
1.0 ꢅ 0.0
1 ꢅ 0
1 ꢅ 0
1 ꢅ 0
2
0 to block non-specific reactions. Then, each chamber was 1:500
1
:1000
1.0 ꢅ 0.0
treated with 100 ml of tissue culture supernatant containing Mab
M60 (specific for the rotavirus outer capsid protein, VP7), gener- *Values refer to means and standard deviations (n = 4).
ously donated by Dr. H. Greenberg from Stanford University (1:1
dilution) at 37 ꢁC for 60 min (Barardi and others 1998). The slides
were washed and stained with goat anti-mouse IgG conjugated
Table 2—Variance analysis (ANOVA) of the cytopathic effects
on MA104 cells after 48 h of incubation with experimentally
infected oyster extracts (A and B).
to FITC (fluorescein isothiocyanate) for 15 min. FITC-stained
cells were then washed, and slides mounted properly for immer-
sion (applying 5 ml of a mounting medium composed by 40%
PBS, 50% glycerol, 5% formalin, 5% NaCl, and 2.5% DABCO) and
observed under epifluorescence microscopy (Olympus America
BX40, Inc., Melville, N.Y., U.S.A.) using a filter and beam splitter
combination for blue-green excitation using 400ꢀ magnifica-
tion. The number of fluorescent cells were counted in triplicates
in each chamber.
Variation Fonts
SQ
GL
QM
F-test
Among groups (e)
In the groups (d)
Total
12.42
7.5
19.92
11
36
47
1.13
0.21
5.42*
SQ = squares sum; GL = freedom grades; QM = Qui-squares, F-tests = QMe/QMd;
significant difference at level of 5% probability.
Results and Discussion
YTOTOXICITY EVALUATION OF UNSEEDED OYSTER EXTRACTS IN
Table 3—Numbers of infected cells (ffu) per well and virus
recovery by IFA using oyster extracts experimentally inocu-
C
MA104 cells has been performed according to the method
described here. In this assay, semi-quantitative observation of lated with simian rotavirus SA11 (“A” and “B” extracts)
the morphological alteration of tissue culture cell monolayer was
Nr of
Nr of
performed for each extract dilution after different periods of in- Dilution
cubation (24, 48, and 72 h). From the 1:64 dilution, the negative of oyster
infected
cells/well
(ffu/50ml
infected
cells/well
extract or
Virus
(ffu/50ml
inoculum) % virus
Extract B recovery
extract did not produce any discernable toxicity against cells
virus
within 24 h (not shown) and low toxicity after 48 h incubation
control
concentration inoculum)
(ffu/)
Extract A
(
Table 1). The period of 72 h of incubation showed high cytotox-
1
1
1
:50
:100
:200
2.0 ꢀ 105
166 ꢅ 67.80* 101 ꢅ 1.52*
966 ꢅ 58.07* 833 ꢅ 1.52*
126.6 ꢅ 73.3* 106.3 ꢅ 8.14*
60.84
86.2
83.9
icity even in high dilutions of oyster extracts. So this period of in-
cubation was not considered for further CPE analysis (not
shown). Hence a 1:32 dilution was considered the limit between
the non-cytotoxic and toxic dilution of oyster extracts and was
used as the first dilution for CPE assay.
5
1.0 ꢀ 10
5.0 ꢀ 104
*
= Values represent means and standard deviations (n = 3)
Oyster extracts “A” and “B” were serially diluted and inoculat-
ed on to MA104 cells and incubated for 24 and 48 h. From the
semi-quantitative values (Table 1) an ANOVA single-factor analy-
sis was performed for the incubation period of 48 h. This showed
statistically significant differences (P < 0.05) between the 2 peri-
ods of time (indicated by F-test) (Table 2). Serial dilution of non-
cytotoxic oysters extracts “A” and “B” were also performed for IFA
assay, with the goal of evaluating virus viability using another
protocol involving cell culture. Table 3 shows the results obtained
using this assay expressed as the number of infected and fluores-
cent cells for oyster extract “B”. Table 3 also shows the percent vi-
rus recovery defining the recovery with extract “A” (seeded with
virus at the end of the process) as 100%.
were about 100% of the initial seeding dose, indicating little loss
of virus infectivity during extract preparation. Our data strength-
en the hypothesis that the laboratory methods used to isolate en-
teric viruses from oysters tissues do not substantially interfere
with virus infectivity, and can be used for detection of loads with
enteric viruses in oysters for monitoring purposes. Screening
oysters for virus contamination should help protect those con-
suming raw oysters. We conclude that the method for oyster ex-
tract preparation described here does not interfere with the via-
bility of virus particles as shown by extracts “A” and “B” in CPE
and IFA assays.
Conclusion
References
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implications and depuration. J. Food Protect 60(6):677-681.
T
not inactivated in oysters, remaining viable even after prep-
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Med Virol 13:377-383.
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Atmar RL, Metcalf TG, Neill FH, Estes MK. 1993. Detection of enteric viruses in
study using cytopathic effects observations (CPE) and immunof-
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extract preparation is very efficient. Recoveries of infective virus
oysters by using the polymerase chain reaction. Appl. Environ. Microbiol.
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870 JOURNAL OF FOOD SCIENCE—Vol. 67, Nr. 5, 2002