| dc.description.abstract | Unequivocal identification of fresh meat and heat-treated
meat is of special concern to food analysts and game
conservation authorities. The food analyst has to provide
proof of fraudulent substitution of more expensive meats with
cheaper ones. Such adulteration represents unfair trading
practices at either international or national level and is
objectionable for reasons of culture, health or religion.
The game conservation authorities need to differentiate meat
of protected species from that of unprotected and domestic
animals in their endeavour to curb poaching and protect
endangered wildlife species.
It is imperative, therefore, that reliable analytical
methods be developed to detect and quantify the adulteration
in order to implement legislative measures necessary to
achieve these objectives.
In this study, species identification of fresh meat and
heat-treated meat was investigated using antisera to
thermostable muscle antigens (TMA) obtained from the meat of
13 wild and 7 domestic animal species.
Antisera to TMA were raised in rabbits and goats.
Unabsorbed rabbit antisera showed a wider range of
cross-reactions than goat antisera. In immunodiffusion
tests, rabbit and goat antisera showed two precipitin lines
with homologous TMA and reacted also with serum, giving a
reaction of identity with one of the two precipitin lines
given by TMA of the same species. Thermostable muscle
antigens of other species reacted with unabsorbed antisera to
TMA, giving a reaction of identity with the precipitin line
given by serum. This shows that serum contains a component
of TMA which is identical or similar to one present in T~~ of
several species.
Absorption of rabbit antisera to TMA with copolymerized
serum proteins rendered the rabbit antisera to TMA of oryx
and camel species-specific, while the other antisera produced
in rabbits retained cross-reactivities with TMA of several
species. It was concluded that extracts of TMA contain
related or similar antigenic components common to several
animal species, especially within the bovidae, as shown by
the wide range of reactivities of antisera produced in
rabbits. It was virtually impossible to render these antisera
species-specific by absorptions.
Although rabbit antisera to TMA lacked sufficient
specificity to differentiate TMA of closely related species,
the antisera could be used to speciate TMA from distantly
related species. In addition, antisera raised in rabbits to
TMA of closely related species can be used to screen for the
presence or absence of meat from bovidae and non-bovidae
species. Rabbit antisera to TMA of topi, warthog and Grant's
gazelle were proved to be useful in this regard.
The same absorption procedure rendered antisera produced
in goats specific for all species, except the antisera to TMA
of kongoni, topi, Thomson's gazelle and Grant's gazelle. Goat
antiserum to TMA of kongoni reacted with the lMA of topi and
wildebeest, while goat antiserum to TMA of topi reacted only
with T~~ of kongoni. Goat antisera to T~~ of Thomson's
gazelle and Grant's gazelle reacted with the TMA of both
species. These results demonstrate the importance of
selecting the animal species in which the antiserum is to be
raised, in order to reduce the cross-reactions with antigens
from closely related species.
Goat antisera also identified the species of origin of
fresh meat using fresh meat antigens (FMA), and cooked meat
using cooked meat antigens (CMA) in immunodiffusion tests.
However, goat antiserum to TMA of cattle reacted with CMA and
FMA of buffalo, thus failing to differentiate them from those
of cattle. Cattle CMA and FMA could be differentiated from
those of buffalo by using goat antiserum to TMA of buffalo.
The CMA and fllA of Thomson's gazelle and Grant's gazelle, -
topi and kongoni, - pig and bushpig, - were not easily
distinguishable using goat antisera to T~~ of these species
in immunodiffusion tests. While antisera to TMA produced in
rabbits possessed limited capability to distinguish between
species, goat antisera to TMA were capable either singly or
in combination to identify all the F~~, CMA and TMA from the
twenty animal species investigated in this study.
The results of immunodiffusion tests using TMA, CMA and
FMA of a single species and goat antiserum to TMA, suggest
that TMA are naturally occuring antigens of muscle tissue and
are not novel antigens arising from heat treatment.
Identification of the species of origin of internal organs
(liver, kidney and heart) was easily achieved using
thermostable organ antigens (TOA) and goat antisera to TMA in
immunodiffusion tests. The reactions of TI1A, TOA with goat
antisera to TMA, revealed that there exist (i) a
species-specific thermostable antigen common to muscles and
organs, (ii) a species and striated muscle-specific antigen,
found in skeletal and cardiac muscles and (iii) a species and
tissue-specific antigen restricted to skeletal muscles.
Hydroxyproline was demonstrated in the TMA extracts,
indicating the presence of gelatin in these preparations.
Other protein contaminants were also shown to be present in
the TMA extracts. These contaminants inhibited the adsorption
of the TMA to micro titre plates. The inhibitory effect of
gelatin was demonstrated using commercial gelatin. Five
milligrams of gelatin per millilitre resulted in complete
inhibition of the adsorption of partially purified TMA (PTMA).
Gelatin and the other contaminants are, therefore, undesirable
in the enzyme immunoassay (EIA). A modification of the
extraction procedure incorporating a partial purification
step was adopted and yielded PTMA suitable for use in EIA.
The molecular weight of the PTMA, as determined using
buffalo PTMA"in gel filtration experiments, was calculated to
be 210 kd.
Inspite of the apparent high specificity of goat antisera
in immunodiffusion tests, several antisera showed
cross-reactions with PTMA of several species in quantitative
EIA. However, goat antisera to TMA of buffalo, topi, pig,
camel and horse were shown to be specific for these species.
Nine other antisera showed various degrees of cross-reactions
with 2 to 10 heterologous PTMA. Some of these reactions were
nearly equal to those given by the homologous sytems, thus
failing to differentiate homologous from heterologous PTMA.
Goat antisera to TMA of cattle and eland cross-reacted
with 13 and 15 heterologous PTMA respectively, giving
reactions which did not allow unequivocal differentiation of
the homologous from the heterologous PTMA. In addition, the
goat antiserum to TMA of cattle gave only weak reactions with
PTMA of topi, pig, horse and Thomson's gazelle, which enabled
differentiation between the homologous and these heterologous
PTMA at P< 0.05. Similarly, goat antiserum to TMA of eland
gave weak reactions with PTMA of horse and cattle which
allowed differentiation between the PTMA of eland and those
of cattle and horse at P~0.05.
In instances where a single antiserum failed to distinguish
between homologous and heterologous PTMA, species
identification could be achieved in enzyme immunoassay by
employing a number of goat antisera to TMA of several
species. Goat antisera to TMA provided reagents that were
capable of correctly identifying the PTMA of eighteen species
examined.
Because of their exquisite specificity, monoclonal
antibodies to TMA would be more suitable for species
identification than the polyclonal antisera used in this
study. However, it is conceivable that immunization of
additional animals of different species, especially within
the bovidae, would yield polyclonal antisera with sufficient
specifity for use in enzyme immunoassays.
Indirect EIA was also used to detect the presence of beef
in laboratory prepared beef/pork sausages. The test detected
beef at 5% level in pork sausages.
Milk powder commonly used as a binder in meat products did
not give false positive reactions for beef in EIA, using goat
antisera to TMA. Antisera to TMA can therefore be used to
detect adulterants in fresh and heat-treated meat products
containing milk proteins.
Detection of beef and pork in 90 commercially prepared beef
and pork products from two factories were analysed using EIA.
Fifty per cent of the pork products from both factories were
found to contain beef, while 78.6% of the beef products from
factory A and 6.3% from factory B, were found to contain pork.
This may not be regarded as adulteration since specifications
by the Kenya Bureau of Standards for fresh and heat-treated
beef and pork products allow inclusion of flesh from other
species, so long as the stipulated content of either beef or
pork is maintained in the products. The specifications of
the Kenya Bureau of Standards also state that the actual
ingredients used in the manufacture of any food product must
be shown on the attached label.
This study has shown that antisera to TMA produced in
selected animal species possessed sufficient specificity to
allow species identification of meat which haa been subjected
to heat-treatment which destroys the more abundant and easily ,
identifiable heat-labile species-specific antigens. The same
antisera also made possible the determination of species of
origin of fresh meat as well as of internal organs such as
kidney, liver and heart. | en |
