Genomics and Applied Biology 2016, Vol.7, No.4, 1-8
3
incubator. At hatching, each chick was wing tagged with an identification number. Brooding was from hatching to
6 weeks. Brooding of chicks from each ecotype was separated in deep litter brooders using infra-red electric bulbs.
The population density was 12birds/m
2
. At the beginning of the 7th week, chicks were transferred to randomly
selected deep litter rearing pens within the same house. Sex was determined by phenotypic appearance.
2.2 Health and disease management
The chicken received routine vaccinations against Marek’s disease (d 1), New Castle disease (NCD; wk 2, 6, 12,
15), infectious bronchitis (d1, wk 2, 10, 12, 15), infectious bursal disease (wk 3, 15) and fowl pox (wk 15).
2.3 Overt natural antibodies isotype assays
Blood samples (2 ml in EDTA) from 24 IC for variation and repeatability studies was drawn from the wing vein
of each bird and serum separated by centrifugation at 2000rpm for 10 minutes. These birds were all female, same
age, genetic background and under same management. Isotype specific IgA, IgM and IgG antibody titres to
keyhole limpet hemocyanin (KLH) in serum from the IC were determined by indirect enzyme-linked
immunosorbent assay (ELISA) as described by (Khobondo et al., 2015c). Briefly, 96 well plates were coated
with 2μg/ml KLH (MP Biomedicals Inc., Aurora, OH) and incubated overnight at 4°C. The plates were washed
using a washing/dilution buffer (phosphate buffered saline (PBS) containing 0.05% Tween) and incubated for
1.5 hours at 25°C with IC serum diluted 1:10 in dilution fluid. The plates were washed using washing buffer to
remove unbound serum. To detect IgA, IgM and IgG antibodies binding to KLH, a secondary 1:20,000 diluted
affinity purified goat anti-chicken IgM (Fc specific), conjugated with horseradish peroxidase (GACh/IgM (Fc)
/PO) antibody, or 1:20,000 diluted whole rabbit anti-chicken IgG (heavy and light chains) conjugated with
horseradish peroxidase (RACh/IgG(H+L)/PO) antibody or 1:20,000 diluted affinity purified goat anti chicken IgA
(Fc specific) conjugated with horseradish peroxidase (GACh/IgA (Fc) /PO) (Nordic Immunological Laboratories,
Eindhoven, The Netherlands ) was added and incubated for 1.5 hours at 25°C. The plates were washed again
and 100 μL substrate-buffer (containing aqua dest, 10% tetramethylbenzidine-buffer and 1.33%
tetramethylbenzidine) was added in each well and incubated for 10 minutes at room temperature. The reaction was
stopped with 1.25 M H
2
SO
4
. Absorbance levels were measured with a spectrophotometer (mrc Scientific
Instrument-UT- 6100, Israel) at 450 nm.
2.4 Statistical analysis
Descriptive statistics were used to explore the data and linear model in SAS (SAS Institute Inc., Cary;Version 9.1)
used for initial analysis. Because Isotype of antibodies (IgA, IgG, IgM) were treated as different parameters,
further analysis were performed using separate isotypes. Variation was estimated using the following mixed
model;
Y
ij
= µ+ time
i
+ IC
j
+e
ij
Where Y
ij
is the Nab titre (IgM or IgG or IgA) of IC
j
at time
i
, µis the common mean. Time is the fixed effect of
time of measurement i (i = 1, 2, 3, 4). IC is the random effect of the IC
j
(j = 1...24; normal, independent and
identically distributed (0 = σ
2
IC) and e
ij
is the random residual (normal, independent and identically distributed).
Repeated measures analyses was performed using PROC MIXED of SAS (SAS Institute Inc., Cary;Version 9.1).
Covariance structure used was compound symmetry (αI= δ
2
|i=j). Model assumptions regarding normality were
evaluated by examining whether skewedness and kurtosis were close to 0 and whether a probability plot did not
show deviation from a straight line.
Repeatability (r) along time and within IC was calculated as:
σ
2
IC
e
IC
I
r
2
2
2
