Papel de las actividades superóxido dismutasa y catalasa en la ...
Papel de las actividades superóxido dismutasa y catalasa en la ...
Papel de las actividades superóxido dismutasa y catalasa en la ...
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2.5. Chall<strong>en</strong>ge with Photobacterium damse<strong>la</strong>e subsp. piscicida<br />
Groups of 10 fish (15-30 g weight) were chall<strong>en</strong>ged at the <strong>en</strong>d of the feeding trial,<br />
after 60 days of beginning. For the chall<strong>en</strong>ge, three groups of fish were assayed, fed<br />
with diets A, B and C.<br />
Soles were intraperitoneally injected with a dose of P. damse<strong>la</strong>e subsp. piscicida<br />
(Lg h41/01 ) of 5 x 10 4 cfu g -1 from a bacterial susp<strong>en</strong>sion in PBS (phosphate buffer saline)<br />
adjusted to 5 x 10 8 cfu ml -1 . As control, the same number of fish was inocu<strong>la</strong>ted with 0.1<br />
ml PBS. Inocu<strong>la</strong>ted fish were followed daily for 10 days, and all mortalities were<br />
recor<strong>de</strong>d, consi<strong>de</strong>ring only those that could be linked to the bacterial chall<strong>en</strong>ge by reiso<strong>la</strong>tion<br />
in pure culture from internal organs of <strong>de</strong>ad fish.<br />
2.6. Analysis of the sole intestinal microbiota<br />
2.6.1. Sample collection<br />
Three groups of fish were analyzed for intestinal microbiota composition, i.e. fish<br />
fed with diets A, B and C.<br />
Per treatm<strong>en</strong>t group, three specim<strong>en</strong>s of S<strong>en</strong>egalese sole were sacrificed at the <strong>en</strong>d<br />
of the feeding trial (60 days), and whole intestines were collected and stored at -80 ºC<br />
until further analysis.<br />
2.6.2. DNA iso<strong>la</strong>tion from intestinal cont<strong>en</strong>t and probiotic cultures<br />
The gut cont<strong>en</strong>ts were homog<strong>en</strong>ized in 4 ml PBS (phosphate buffer saline) pH<br />
7.2, and a 1 ml aliquot was c<strong>en</strong>trifuged at 1000 × g for 5 min. The supernatant was pretreated<br />
by <strong>en</strong>zymatic digestion with 40 μl proteinase K (20 mg/ml) and 50 μl do<strong>de</strong>cyl<br />
sulphate (SDS) 10% and incubated at 65 ºC for 30 min. Subsequ<strong>en</strong>tly, DNA extraction<br />
from the susp<strong>en</strong>sion was performed with the Fast DNA Spin kit for soil (Qbiog<strong>en</strong>e, Inc.,<br />
Carlsbad, CA) according to manufacturer’s instructions. Agarose gel (1.5% [wt/vol])<br />
electrophoresis in the pres<strong>en</strong>ce of ethidium bromi<strong>de</strong> was used to check visually for<br />
DNA quality and yield.<br />
Pure cultures of probiotic strains were grown until expon<strong>en</strong>tial phase in TSBs,<br />
and th<strong>en</strong> c<strong>en</strong>trifuged at 2500 × g for 15 min. Pellets were washed with PBS and<br />
c<strong>en</strong>trifuged again. DNA was extracted from the resulting pellet resusp<strong>en</strong><strong>de</strong>d in 500 μl<br />
PBS with the Fast DNA Spin kit (Qbiog<strong>en</strong>e, Inc., Carlsbad, CA).<br />
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