Species identification of fresh meat, heat-treated meat and internal organs using antisera to thermostable antigens
Kang'ethe, Erastus Kiambi
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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.