International Journal of Aquaculture, 2017, Vol.7, No. 3, 15
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22
20
rRNA genes of the isolates was amplified using the 27F:5‟AGAGTTTGATCMTGGCTCAG3‟and1492R:5‟
TACGGYTACCTTGTTACGACTT 3‟ primers on an ABI 9700 Applied Biosystems thermal cycler at a f
inal volume of 50 microliters for 35 cycles. The PCR mix included: the X2 Dream taq Master mix sup
plied by Inqaba, South Africa (taq polymerase, DNTPs, MgCl), the primers at a concentration of 0.4 M
and the extracted DNA as template. The PCR conditions were as follows: Initial denaturation of DNA te
mplate strands at 95ºC for 5 minutes; subsequent denaturation at 95ºC for 30 seconds; annealing of prim
ers at 52ºC for 30 seconds; elongation at 72ºC for 30 seconds for 35 cycles and final elongation at 72º
C for 5 minutes. The product was resolved on a 1% Agarose gel at 120V for 15 minutes and visualize
d on a UV Trans illuminator. The amplified 16S products were sequenced on a 3 500 genetic analyser
using the Bigdye-Termination technique by Inqaba South Africa. The sequences were edited using the bi
oinformatics algorithm Bioedit. Similar sequences were downloaded from the National Biotechnology Info
rmation Center (NCBI) data base using Blast-N, and these sequences were aligned using ClustalX. Neigh
bor-Joining mechanism was used to infer the history of evolution in MEGA 6.0 and Jukes-Cantor metho
d was used to compute the distances in evolution.
3.3 Pathogenicity tests of
Pseudomonas
aeruginosa
and
Vibrio mimicus
to shrimp culture
A stock concentration of
Pseudomonas
aeruginosa
suspension grown on cetrimide agar for 24 hours and that of
Vibrio mimicus
suspension grown on TCBS agar for 24 hours were prepared by diluting each bacterial suspension
and adjusting to McFarland No.1 standard turbidity (equivalent to 3 x 10
8
cfu/ml). Ten-fold serial dilutions with
normal saline were carried out to obtain 3x10
7
, 3x10
6
, 3x10
5
, 3x10
4
and 3x10
3
cfu/ml. Different tanks of 4 L
capacity, each containing 2 L habitat water buffered using sterile bicarbonate powder and constantly aerated were
set up. Fifty (50) healthy shrimp were stocked into each of the tanks. Then 0.1 ml of each suspension was
introduced into each tank. Shrimp grown in habitat water alone served as control. The experiment was carried out
in triplicate. The shrimp mortalities, if any, were monitored and recorded daily for 4 days. The mortality rate of
the animal was calculated for each treatment. Probit Analysis Package (SPSS version 20) was used to determine
LC
50
of
Vibrio mimicus
.
3.4 Histopathological examination of the hepatopancreas of experimental shrimp
The five-step procedure (fixation,
dehydration and clearing, embedding, sectioning, and staining and mounting)
for histological study of the test shrimp after pathogenicity test was carried out according to the method described
by Wiss et al. (2010). The sections were viewed and the micrographs of the tissues of the test animal were
compared with the control.
3.5 Probiotic application and infection of shrimp culture by immersion method
Three experimental groups were set up using 4 L tanks each containing 2 L of habitat water. The tanks were
buffered using sterile bicarbonate powder and constantly aerated. Fifty (50) healthy shrimp were stocked into each
of the tanks. To shrimp culture in treatment group 1, 0.1 ml 3x10
5
cfu/ml fresh culture of
Pseudomonas
aeruginosa
was added and challenged with 0.1 ml 3x10
5
cfu/ml fresh cultures of pathogenic
Vibrio mimicus
on
the third day. There was no addition of bacteria in treatment group 2 shrimp culture (control). Treatment group 3
was infected with 0.1 ml 3x10
5
cfu/ml fresh culture of
Vibrio mimicus
. Mortalities were recorded daily for 12 days.
The experiment was conducted in three replicates. Water quality parameters (pH, temperature, ammonia, nitrite,
nitrate, salinity and dissolved oxygen) were monitored during the culture period.
3.6 Determination of water quality
Water quality parameters such as pH, temperature, dissolved oxygen (DO) and salinity were measured daily,
while the concentrations of nitrate, nitrite and ammonia were determined at two days interval. Water samples were
collected in plastic bottles and examined according to the methods described in APHA (2005).
3.7 Statistical analysis
One way ANOVA was used to analyze the differences in shrimp mortality rates and Duncan’s multiple range test
was used to test significant differences among means (p ˂ 0.05). Two Sample t-test was used to determine
