R. S. E. BASHIR ABASS ET AL
OPEN ACCESS JGIS
3
(Ministry of Animal Resources, River Nile State).
2.2. Samples Collection
Fifty three blood samples were collected randomly from
cattle in River Nile state, on October 2012, locations
identified using GPS (GARMIN-GPSMAP 60CSx).The
blood samples were collected in plain vacutainers, cen-
trifuged to separate the serum and stored at –20˚C till
used. Collected sera were tested for the presence of lep-
tospira antibodies using indirect Leptospira hardijo
ELLISA kit (Cat. No.79038923, prioCHECK L. hardijo
AB, Germany).
2.3. Detection of Leptospira Antibodies
Briefly; the test uses 96-well microtitration plates sensi-
tized by purified antigen Leptospira hardjo. The plate’s
odd columns (1, 3, 5, 7, 9, and 11) contain the LPS,
whereas the even columns (2, 4, 6, 8, 10, and 12) contain
a control antigen.
The sera were diluted in the dilution buffer. The plate
was incubated and the LPS washed, then the conjugate,
protein G peroxidase-labelled, is added to the wells. The
plate was incubated a second time at 21˚C +/− 3˚C. After
the second incubation, the plate was washed again and
the chromogen (tetramethylbenzidine) is added. This
chromogen has the advantages of being more sensitive
than the other peroxidase chromogens and not being car-
cinogenic. If specific Leptospira hardjo immunoglobu-
lins are present in the test sera, the conjugate remains
bound to the microwell that contains the bacterial antigen
and the enzyme catalyses the transformation of the
colorless chromogen into a pigmented compound. The
intensity of the resulting blue color is proportionate to the
titer of the specific antibody in the sample.
All the reagents were brought to 21˚C +/− 3˚C before
the use. For sera, one milliliter aliquots were placed of
the dilution buffer, prepared as instructed in the “Com-
position of the Kit” section, in 5 or 10 ml hemolysis
tubes. 10 ml of the serum samples was added to each of
these tubes (dilution 1/100) and were shacked briefly on
a mechanical agitator.
Positive and negative sera were diluted 1/100 in dilu-
tion buffer. Sera samples were distributed and the posi-
tive and negative sera (100 ml/well) as follows: positive
serum in wells A1 and A2, negative serum in wells B1
and B2, sample 1 in wells C1 and C2, sample 2 in wells
D1 and D2 etc. The plate was incubated at 21˚C +/− 3˚C
for one hour. The plate was rinsed with the washing
solution, was prepared as instructed in the “Composition
of the Kit” section, as follows: the microplate was emp-
tied of its contents by flipping it over sharply above a
sink. Tap the microplate upside down against a piece of
clean absorbent paper to remove all the liquid. The used
wells were filled with the washing solution using a
squeeze bottle or by plunging the plate in a vessel of the
right dimensions, then empty the wells once more by
turning the plate over above a sink. Repeat the entire
operation two more times, taking care to avoid the for-
mation of bubbles in the microwells. After the plate has
been washed three times the conjugate was diluted 1:50
in the dilution buffer. 100 ml of the conjugate solution
was added to each well. Incubated for 1 hour at 21˚C
+/−3˚C, the plate was washed as described in step 6
above.
100 ml of the chromogen solution was added to each
well on the plate and incubated for 10 minutes at 21˚C
+/− 3˚C protected from the light and uncovered. 50 ml of
stop solution was added to each microwell.
The optical densities in the microwells were read using
a plate reader at 450 nm. Results were read fairly soon
after the stopping solution has been added since the
chromogen may cristallize in the wells with strong sig-
nals and thereby distort the data.
The test can be validated only if the positive serum
yields a difference in optical density at 10 minutes that is
greater than 0.8 and the negative serum yields a differ-
ence in optical density that is lower than 0.3.
Divide the signal read for each sample well by the
corresponding positive control serum signal and multiply
this result by 100 to express it as a percentage.
ValDelta OD Sample100Delta OD positive
Table 1 can be used to determine the serum’s degree
of positivity.
2.4. GIS Software
An Arc View 9.3 software system was used to map sam-
ples locations. The data used were: animals population
2011(Ministry of Animal Resources-River Nile state),
human population 2008 (Sudan Central Bureau of Statis-
tics), Samples location by GPS (River Nile State), hafairs
locations where animals contacts, Animals routs. The
buffering method was used to determine the risk area.
3. Results
This study demonstrated the use of GIS in the study of
Table 1. The serum’s degree of positivity.
0 + ++ +++ ++++ +++++
Val <= 20% < Val <= 40% < Val <= 60% < Val <= 80% < Val <= 100% < Val