| dc.description | INTRODUCTION
Low genetic potential and high prevalence of diseases
are among of the major factors limiting productivity of the
local chickens in the tropics (Yongolo, 1996; Alexander,
2001; Otim, 2005). Among many diseases of poultry
endemic in Tanzania and other developing countries,
Newcastle disease (ND) has been reported to be the
most important (Rahman et al., 2002; Illango et al., 2005;
Otim, 2005). When Newcastle disease strikes, 60 to
100% of chickens in a household can be lost (Yongolo,
1996; Alexander, 2001; Acamovic et al., 2005). Although
the strategy of mass vaccination has largely been an
*Corresponding author. E-mail: kifaro@suanet.ac.tz
effective control of the Newcastle disease, however,
combining vaccination programs and development of
genetically resistant stocks will further maximize
protection of the chickens from the disease. Improving
disease resistance in poultry by direct selection or by
selection for immune response may hardly be feasible
due to quantitative nature of these traits, their low to
moderate heritability, and the difficulties associated with
obtaining reliable measurements (Yonash et al., 2000). In
this situation, marker assisted selection (MAS) is expec ted to be a more effective breeding approach. Genes
located within the Major Histocompatibility Complex
(MHC) B region on chicken Micro-chromosome 16 has
been demonstrated by many workers to be associated
with immune response and disease resistance as well as
productivity (Heller et al., 1991; Dunnington et al., 1992;
Caron et al., 1997; Liu et al., 2002; Juul-Madsen et al.,
2002; Parmentier et al., 2004; Taylor, 2004; Joiner et al.,
2005; Boonyanuwat et al., 2006; Fulton et al., 2006).
Results from these studies further showed Restriction
Fragment Length Polymorphism (RFLP), and micro satellites linked to this region (LEI0258 in chickens) to be
promising DNA markers in characterizing MHC B genes.
Identifying marker alleles (bands) associated with supe riority is important before MAS can be used. Using MHC RFLP, results from some of MHC studies revealed that
marker alleles associated with superiority vary depending
on population under consideration i.e. background
genome (Dunnington et al., 1992). Therefore there is a
need for identifying bands associated with superiority in a
population of interest before MAS can be used to improve
immunocompetence of that population.
Past research on local chicken MHC in Tanzania
(Lawrence, 1998) and other countries such as Bolivia and
India (Baelmans et al., 2005) has only tested the pre sence of different MHC haplotypes using specific
alloantisera. Similar work has been done on Brazilian
local chickens using LEI0258 microsatellite (Lima-Rosa
et al., 2005). However, no study has been carried out to
identify MHC alleles/haplotypes responsible for either
high or low immune responses (a pre-requisite for MAS)
in the local chicken populations of Tanzania. Therefore
this study was carried out to identify LEI0258 micro satellite alleles associated with antibody response
against NDV vaccine and body weight in two Tanzania
chicken ecotypes viz. Kuchi and Tanzania Medium
(Medium). Previous studies (Lawrence, 1998; Msoffe et
al., 2001) have shown these ecotypes to be relatively
superior to other Tanzania chicken ecotypes in terms of
body weight and egg production.
MATERIALS AND METHODS
Study site and experimental materials
This study was carried out at Sokoine University of Agriculture
Poultry Research Unit, Morogoro, Tanzania. The place is located at
an altitude of about 525 m above sea level. The relative humidity at
the location is about 81%, while the monthly mean and maximum
temperatures are 18.7 and 30.1
oC, respectively. The area has a
mean annual rainfall of 846 mm. Experimental chicks were derived
from two parent stocks, one representing Kuchi ecotype obtained
from drier parts of north west Tanzania, and another representing
Tanzania Medium (Medium) ecotype obtained from central part of
the country. A total of 85 and 88 chicks for Kuchi and Medium
ecotypes, respectively randomly sampled from five batches were
involved in this study.
Management of experimental birds
Birds were fed a starter ration (20% CP and 2800 Kcal ME/kg) from
day old to 8
th week of age, growers ration (16% CP and 2750 Kcal
ME/kg) from 9
th
to 16
th week of age, and layers ration (17% CP and
2700 Kcal ME/kg) from 17
th week of the age to the rest of the
period. Water was supplied on ad libitum basis. Furthermore, birds
Lwelamira et al. 715
were vaccinated against Gumboro disease when they were 10 to
14 days of age, and also due to the experimental set-up they were
first vaccinated against Newcastle disease when they were 4 weeks
of age, and vaccinations were repeated 3 weeks post vaccination,
and later on after every 3 months.
DNA isolation and MHC haplotyping
For each chicken, DNA was isolated from 200 l packed cells from
EDTA-stabilized blood using a salt protocol, as described by Juul Madsen et al. (1993). The MHC haplotypes were determined by
PCR-based genotyping of the LEI0258 microsatellite locus
(Dalgaard et al., 2005; Lima-Rosa et al., 2005; Fulton et al., 2006;
Schou et al., 2006). The PCR amplification from genomic DNA was
carried out in 25 l reaction volumes using standard buffer (Amer sham) containing 0.05 M of each primer (the forward primer
having been labelled with fluorescein), 0.4 mM of each dNTP, 1.5
mM MgCl2 and 1unit of Taq DNA polymerase (Amersham). After an
initial 5 min of denaturation at 94
oC, the amplification went through
25 cycles of denaturation at 94
oC for 1 min, annealing at 56
oC for 1
min and extension at 72
oC for 2 min. The amplification was
completed with a final extension for 10 min at 72
oC. The amplicons
were determined by electrophoresis on a denaturing polyacrylamide
gel in an ALF DNA sequencer (Amersham) to detect allelic poly morphism. A mixture of 10 fluorescein-labelled fragments of 50-500
bp (Amersham) was used as a size marker. Furthermore, DNA
samples from three well-characterised lines of White Leghorn
homozygous for the MHC haplotypes B13, B19 and B21 (Miller et
al., 2004) were included on a gel as control samples. The different
MHC haplotypes were finally classified according to the repeat motif
of the LEI0258 microsatellite (Fulton et al., 2006).
Traits studied
The traits considered were primary antibody response against NDV
vaccination, and body weight at 16 weeks of age. Egg production
and related traits were not included in the association analysis as
there were very few observations per allele for statistical analyses
in nearly all the alleles considered.
Assessment of antibody response against NDV vaccine
The chicks were vaccinated with Newcastle disease virus vaccine
(La Sota) according to manufacturer’s instructions at the age of 4
weeks and antibody levels were assessed just prior and 2 weeks
post vaccination. Blood from each chick was collected from wing
vein using syringes. Samples were titrated for Newcastle disease
virus (NDV) specific antibodies by the microtitre method of the
haemagglutination inhibition (HI) test (Allan and Gough, 1974)
using NDV antigen. Four haemagglutination (HA) units were used
and twofold serial dilutions of sera added with a starting dilution of
1:2. The titres were expressed in log210 form of the highest dilution
causing HI. Since antibody titre prior to vaccination were almost
zero in nearly all chicks, then only antibody titre 2 weeks post
vaccination (primary antibody response) was considered in
subsequent analyses.
Statistical analyses
Since the distribution of different alleles were similar in the two
ecotypes, the data for the two ecotypes were pooled together, and
six most frequent alleles in both ecotype (i.e. 205, 215, 234, 307,
321, and 345 bp size alleles) were chosen for the association study.
Choosing the most frequent alleles was based on the concept that
716 Afr. J. Biotechnol.
0,00
5,00
10,00
15,00
20,00
25,00
191 193 203 205 215 234 241 249 261 273 285 295 307 308 321 333 345 357 381 405 420 443 472 487
Allele
Percent Figure 1. Allele frequencies in Kuchi ecotype.
for a random mating population, at a particular locus, frequency of a
certain allele in a population is expected to be increased by natural
selection if it plays a significant role in the survival of the individuals
in the environment (Jeffery and Bangham, 2000; Sabeti et al., 2002;
Saunders et al., 2002; Verrelli et al., 2002). For each band (allele),
all individuals were scored as a carrier (1) or non carrier (0) of the
allele. Then single band analysis was carried out to determine the
association of each band with the traits considered by regression
analysis using REG procedures of SAS (2000). In this analysis it
was assumed that the mode of gene action for these alleles is
complete dominance (Schou et al., 2006). Furthermore, before
analysis data were adjusted for the fixed effects of ecotype, sex and
hatch using GLM procedures of SAS (2000).
RESULTS AND DISCUSSION
LEI0258 microsatellite allele frequencies in the two
Tanzania chicken ecotypes
Results from the current study revealed that 22 and 23
alleles of LEI0258 were identified in Kuchi and Medium
ecotypes, respectively (Figures 1 and 2). In a study by
Schou et al. (2006) in local chickens of Vietnam and
Lima-Rosa et al. (2005) in local chickens of Brazil, a total
of 19 and 15 alleles were identified in their populations,
respectively, using the same microsatellite, which are
somewhat lower than the number obtained in the current
study. However, the number of alleles in these studies
including the current work are much higher than those
reported for commercial breeds such as Lohman Silver
Line (3 alleles) (Fink et al., 2005), and in Lohaman brown
line (5 alleles) (Schou et al., 2006). In most cases local
chicken are kept under free range conditions in which a
variety of diseases are prevalent compared to intensive
management in which commercial chickens are kept
(Pinard-van der Laan, 2002). The observed high number
of alleles in free range local chickens in the present study
and the study by Lima-Rosa et al. (2005) and Schou et
al. (2006) are therefore not surprising. Increased
polymorphism at MHC increases their ability to respond
to various disease antigens and hence high chance of
surviving in their environments. Furthermore, apart from
rearing environment, reduced polymorphism at MHC in
commercial chickens is also likely being contributed by
selection for productivity, as opposed to the out-bred
populations.
Results from current study also revealed high level of
heterozygosity in the studied populations, in which the
proportion of heterozygous individuals in Kuchi and
Medium ecotypes were 88.2 and 86.4%, respectively,
and the difference between the two ecotypes was not
significant (P>0.05) (Table 1). These values are in close
agreement with that of 91% reported by Schou et al.
(2006) in one population of Vietinamese local chickens,
but higher than those of 50% and 75% reported by Lima Rosa et al. (2005) in two populations of Brazilian local
chickens, typed using the same microsatellite. As review ed by Wegner et al. (2004), MHC heterozygosity seems
to be advantageous in MHC mediated disease resistance
due to increased diversity of antigens capable of being
presented to T cells. Therefore the frequency of heterozy gosity at the MHC is expected to be higher in out-bred
populations exposed to all kinds of infectious agents as
Lwelamira et al. 717
Table 1. Frequency of homozygous and heterozygous individuals summarized by ecotype.
Kuchi (n = 85) Medium (n = 88)
Status
Frequency % Frequency %
Homozygous 10 11.8 12 13.6
Heterozygous 75 88.2 76 86.4
2
(Chi-square) value = 0.136, P > 0.05.
0,00
5,00
10,00
15,00
20,00
25,00
191 193 203 205 215 234 241 249 261 273 285 295 307 308 321 333 345 357 381 405 420 443 472 487
Allele
Percent
Figure 2. Allele frequencies in Medium ecotype.
observed in the current study. The low degree of
heterozygosity reported for Brazilian local chickens by
Lima-Rosa et al. (2005) compared to the results of the
current study could probably be attributed to relatively low
antigenic diversity prevailing in the environments in which
their chickens have evolved compared to those of
populations in the current study (Kuchi and Medium).
This is also reflected in the degree of polymorphism
(number of alleles), in which lower number of alleles (15)
were reported in these Brazilian chickens compared to 22
and 23 alleles found in the present study for Kuchi and
Medium ecotypes, respectively.
Association of LEI0258 microsatellite alleles with
primary antibody response and body weight
Association between LEI0258 and the performance was
also evaluated in the current study. As started earlier,
since distribution of the different alleles among the two
studied ecotypes were very similar (Figures 1 and 2), the
data for the two ecotypes were pooled together and six
most frequent alleles in both ecotypes (205, 215, 234,
307, 321, and 345 bp size alleles) were chosen for the
association analysis.
Results from Table 2 indicate that alleles 205 bp and
307 bp were of special interest. The allele 205 bp was
significantly (P<0.001) positively associated with the
elevated primary antibody responses against NDV
vaccine, while the allele 307 bp was significantly (P<0.05)
negatively associated with this trait. Significant influence
of LEI0258 microsatellite alleles on fitness parameters in
chickens were also demonstrated in a study by Schou et
al. (2006) in which allele 276 bp which is not found in the
populations under current study was found to be
associated to resistance to some species of worms in
Vietnamese local chickens. Results from Table 2 further
show that body weight was only influenced by allele 307
bp in which its presence was associated with increased
body weight at 16 weeks of age. Association of some
alleles of microsatellites located within MHC region with
performance were also reported in other livestock
species such as sheep (Bot et al., 2004).
Despite the presence of significant association between
718 Afr. J. Biotechnol.
Table 2. Association between LEI0258 microsatellite alleles and body weight at 16 weeks of age and primary
antibody response against NDV vaccine.
Trait Allele (bp) S.E Sig. R
2
205 1.34 0.26 0.000
215 0.03 0.27 0.900
234 -0.10 0.29 0.730
0.082
307 -0.92 0.29 0.001
321 0.08 0.18 0.652
Ab
345 -0.16 0.28 0.557
0.028
205 21.53 46.19 0.641
215 -65.32 46.94 0.165
234 -18.80 50.07 0.708
307 135.33 49.51 0.007
321 -12.40 31.17 0.691
Bwt16
345 -7.41 48.01 0.877
0.002
some of the alleles and the performance in the present
study, results for R
2
in Table 2 indicate that the proportion
of total phenotypic variance explained by these alleles is
too low (less than 0.10). Using RFLP, studies by Yonash
et al. (1999, 2000) have also reported low proportion of
total phenotypic variation that is explained by single MHC
allele/band with regard to primary antibody response to
Escherichia Coli, Sheep Red Blood Cells (SRBC), and
NDV vaccination in broilers. Low R
2
could be due to the
fact that antibody response (humoral immune response)
and body weight are controlled by many loci, and some of
these loci map outside the MHC region (Yonash et al.,
2000, 2001; Zhou and Lamont, 2003).
Due to low total phenotypic variation explained by
significant alleles, incorporating these markers in breed ing programs would result into marginal additional
response. The method used for microsatellite typing in
the current study (automated microsatellite typing) involv ed use of expensive equipments (ALF DNA Sequencer
®
,
Amersham), which might be difficult to obtain in develop ing countries like Tanzania. Hence, additional response
by use of these markers and costs involved in typing
could sometimes not be justified, unless cheap methods
of MHC typing which does not require expensive equip ments (serological method and manual microsatellite
typing) are used. The problem of serological method for
MHC typing in local chickens is that currently available
alloantisera have been derived from inbred lines of
commercial chickens, that is, White Leghorn (Baelmans
et al., 2005; Fulton et al., 2006). Inbred lines contain a
limited combination of BG, BF and BL genes. In contrast,
in out-bred populations (local chickens), novel alleles and
combinations of alleles are likely to exist. Therefore,
available alloantisera might not be able to identify all the
existing haplotypes in local chickens (some chickens may
not show reaction to any of the available haplotype
specific antisera) as it has been demonstrated in studies
by Lawrence (1998) and Baelmans et al. (2005). There fore this method requires development of alloantisera
which can adequately type our populations of local
chickens. To start with, haplotypes/groups obtained from
LEI0258 microsatellite typing can be used in initial
development of serological reagents using procedures
described by Juul-Madsen et al. (2006). However, since
novel alleles and combination of alleles are likely to exist
in these out-bred populations (local chickens), this
process may result into large panel of alloantisera which
could be difficult to manage. The situation can further be
complicated by the problem cross-reactivity (Lawrence,
1998; Fulton et al., 2006). In this regard, therefore,
manual microsatellite typing currently remains as the
method of choice (Msoffe et al., 2005).
Conclusion
Significant associations existed between some LEI0258
microsatellite alleles and antibody response against NDV
vaccine and body weight, hence prospects for using MAS
in improving these traits in breeding programs. However,
considering high costs of automated typing, and marginal
additional response (based on R
2
) expected from these
alleles when incorporated into breeding programs, their
use in developing countries could sometimes not be
justified, and hence use of cheaper methods for MHC
typing is required. Finally, it can be recommended that
further studies on association between MHC haplotypes
and phenotype should be carried out to explore other
components of immune system, which are also involved
in immune response to Newcastle disease. Furthermore,
other diseases which are also a problem to local chickens
should be considered. These studies should go further by
challenging prospective MHC haplotypes/groups with the
diseases to further investigate the nature of resistance.
ACKNOWLEDGEMENT
The authors are very grateful for the financial support
from Production and Health of Smallholder Livestock
(PHSL) project funded by DANIDA which sponsored the
senior author in his Ph.D. programme.
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