Screening and quantification of antibiotic residues in broiler chicken meat and milk in the central region of Algeria. / Recherche et quantification des résidus d'antibiotiques dans le muscle du poulet de chair et dans le lait dans la région centre d'Algérie.

Antibiotics used in animals may be found in food of animal origin and pose a risk to human health. The aim of this study was to screen for antibiotic residues in broiler chickens and milk. Two hundred and twenty-one samples were collected (71 samples of chicken breast meat, 117 samples of raw cow's milk and 33 samples of raw goat's milk). The chicken meat samples underwent a microbiological analysis, followed by a physical/chemical analysis using high-performance liquid chromatography (HPLC). The milk samples were screened using a commercial microbiological test, followed by a further test for residues of beta-lactams and tetracyclines. For chicken meat, 32.39% of the samples were positive, with 56.52% of these samples containing aminoglycosides, 52.17% containing sulphonamides, 30.43% containing beta-lactams and/or tetracyclines and 21.73% containing macrolides. The concentrations of amoxicillin, penicillin G, erythromycin and sulfisoxazole exceeded the maximum residue limits laid down in European regulations in 28.57%, 85.71%, 80% and 91.66% of samples, respectively. The results of the test on milk showed that 12.6% of samples were contaminated by inhibiting substances. Betalactams and tetracyclines were present in 26.32% and 15.79% of the samples analysed, respectively. These results indicate that the contamination of chicken meat and milk is due to non-compliance with administration procedures and inappropiate use of antibiotics.

Les antibiotiques utilisés chez les animaux sont susceptibles de se retrouver dans les denrées alimentaires provenant de ces derniers et constituent un risque pour la santé humaine. Cette étude a pour but la recherche des résidus d'antibiotiques dans le muscle du poulet de chair et le lait. Deux cent vingt et un échantillons ont été collectés (71 échantillons de muscle du bréchet, 117 échantillons de lait cru de vache et 33 échantillons de lait cru de chèvre). Les muscles de poulet ont été analysés par la méthode microbiologique suivie d'une quantification par la méthode physico-chimique de chromatographie en phase liquide à haute performance (HPLC). Les échantillons de lait ont subi un criblage au moyen d'un test microbiologique commercial, suivi d'un autre test pour la recherche des résidus de ß-lactamines et de tétracyclines. Pour le poulet de chair, les résultats ont montré que 32,39% des échantillons sont positifs, dont 56,52% contenant des aminosides, 52,17% des sulfamides, 30,43% des ß-lactamines et/ou des tétracyclines et 21,73% des macrolides. Les concentrations en amoxicilline, en pénicilline G, en érythromycine et en sulfisoxazole sont supérieures aux limites maximales de résidus test fixées par la réglementation européenne dans respectivement 28,57%, 85,71%, 80% et 91,66% des échantillons. Pour le lait, les résultats du test ont montré que 12,67% d'échantillons sont contaminés par des substances inhibitrices. Les ß-lactamines et les tétracyclines sont présentes dans respectivement 26,32% et 15,79% des échantillons analysés. Ces résultats montrent que la contamination de la viande de poulet et du lait est une conséquence du non-respect des modalités d'utilisation des antibiotiques et du mauvais usage de ces derniers.

Los antibióticos utilizados en los animales pueden acabar encontrándose en los productos alimentarios obtenidos a partir de ellos, razón por la cual suponen un riesgo para la salud humana. Los autores describen un estudio encaminado a detectar residuos de antibióticos en la carne de pollos asaderos y en la leche. Para empezar se obtuvieron 221 muestras (71 muestras del músculo de la quilla, 117 muestras de leche cruda de vaca y 33 muestras de leche cruda de cabra). Los músculos de pollo fueron analizados primero por métodos microbiológicos, seguidos de una cuantificación por la técnica físico-química de cromatografía líquida de alto rendimiento (HPLC). Las muestras de leche fueron sometidas a cribado por una prueba microbiológica comercial, seguida de otra prueba para la detección de residuos de ß-lactaminas y tetraciclinas. En el caso de los pollos, los resultados arrojaron un 32,39% de muestras positivas, de las que un 56,52% contenía aminósidos, un 52,17% sulfamidas, un 30,43% ß-lactaminas y/o tetraciclinas y un 21,73% macrólidos. Las concentraciones de amoxicilina, penicilina G, eritromicina y sulfisoxazol superaban los límites máximos de residuos (LMR) fijados en la normativa europea en un 28,57%, un 85,71%, un 80% y un 91,66% de las muestras respectivamente. En el caso de la leche, la prueba Delvotest® SP puso de manifiesto que un 12,67% de las muestras presentaba contaminación por sustancias inhibidoras. Las ß-lactaminas y las tetraciclinas estaban presentes, respectivamente, en un 26,32% y un 15,79% de las muestras analizadas. Tales resultados demuestran que la contaminación de la carne de pollo y la leche es consecuencia del incumplimiento de los procedimientos de administración de antibióticos y del uso inadecuado de estos fármacos.

Quantification of red blood cell fragmentation: usefulness of heating cells and of automatic counting devices.

Spontaneous red blood cell (RBC) fragmentation occurs in some membrane erythropathies like hereditary elliptocytosis (HE); this phenomenon is produced in normal RBC by heating at 49 degrees C, but not at temperatures below this limit; fragmentation is usually quantified by counting the number of fragments/1000 RBC under light microscopic examination. The present work demonstrates: i) that enumeration of fragments is performed more precisely with an automatic blood cells counter on the 'platelet' channel; and ii) that heating at 48 degrees C enhances the fragmentation of RBC when they have a severe disruption of skeletal lattice, like in HE.

Occurrence and characteristics of hereditary spherocytosis in Algeria.

In a survey of more than 12,000 persons referred to a hematological outpatient clinic in Algiers, we estimated that the incidence of hereditary spherocytosis (HS) is 1/1000. Another 9 cases were found in nine of the corresponding families. Anemia was present in a total of 44 subjects (81%). The transmission was dominant in five of eight informative families (63%). No firm conclusion could be reached concerning the amount of spectrin and ankyrin in nine families; however two-dimensional peptide maps ruled out any alphaII domain abnormality in these families. We estimate that HS has roughly the same incidence and features among Algerians as in Europeans or people of European descent.

Sp alpha V/41: a common spectrin polymorphism at the alpha IV-alpha V domain junction. Relevance to the expression level of hereditary elliptocytosis due to alpha-spectrin variants located in trans.

Spectrin alpha-chain mutants associated with hereditary elliptocytosis are highly variable in their level of expression. It has been assumed that the degree of elliptocytosis can be increased when the spectrin alpha chain, encoded by the alpha gene in trans to the variant, is expressed at a low level. We now provide strong evidence for the existence of low-level expression of spectrin alpha chains. This condition is referred to as the alpha V/41 polymorphism. It has been observed in 15 different families or individuals of French, North African, and African ancestry in which seven distinct elliptocytogenic alpha-spectrin variants were co-inherited. Whenever the alpha V/41 polymorphism was present, the severity of the biochemical, morphological, and, sometimes, the clinical phenotype of elliptocytosis was increased. The alpha V/41 polymorphism was also frequently encountered among 36 unrelated control subjects in the heterozygous or homozygous states, and was entirely asymptomatic in both cases. The main biochemical feature was an increased susceptibility to proteolysis of the alpha IV-alpha V domain junction. Alteration of the facing beta IV domain of spectrin was demonstrated by in vitro spectrin dimer reconstitution experiments. It appears that the alpha V/41 polymorphism is often required for alpha-spectrin elliptocytogenic variants to become manifest in the heterozygous state. Thus, alpha-spectrin-related elliptocytosis may be viewed as a bifactorial condition.

Evidence that expression of Sp alpha I/65 hereditary elliptocytosis is compounded by a genetic factor that is linked to the homologous alpha-spectrin allele.

Many cases of hereditary elliptocytosis (HE) result from mutated spectrin alpha-chains. It has repeatedly been observed that the amount of a mutant alpha-chain is different in various affected individuals, resulting in clinical pictures of variable severity. The different levels are thought to result from different percentages of the alpha-spectrin allele in trans. Such percentages, in turn, could be under genetic control. We tested this hypothesis in a large Algerian family with Sp alpha I/65 HE. In an informative sibship, we found three persons with a distinctly high level of expression of the Sp alpha I/65 variant, suggesting the existence, in trans, of a low percentage alpha-allele. The alpha-spectrin gene haplotype associated with the latter was constantly - + -, based on the XbaI, PvuII, and MspI polymorphic sites. In contrast, a basal level of expression of the Sp alpha I/65 variant in the same sibship indicated, in trans, the existence of a normal percentage alpha-allele. The haplotype corresponding to this other alpha-allele was + - +. Study of another generation of the family showed, however, that the - + - haplotype could also be linked to a normal percentage alpha-allele. These results are consistent with the view that the expression level of alpha I/65 spectrin (and of other types of alpha-variants) is compounded by a genetic factor that is linked to the normal alpha-allele in trans. The low percentage allele itself remains silent in the simple heterozygous state.

A study of membrane protein defects and alpha hemoglobin chains of red blood cells in human beta thalassemia.

The soluble pool of alpha hemoglobin chains present in blood or bone marrow cells was measured with a new affinity method using a specific probe, beta A hemoglobin chain labeled with [3H]N-ethylmaleimide. This pool of soluble alpha chains was 0.067 +/- 0.017% of hemoglobin in blood of normal adult, 0.11 +/- 0.03% in heterozygous beta thalassemia and ranged from 0.26 to 1.30% in homozygous beta thalassemia intermedia. This elevated pool of soluble alpha chains observed in human beta thalassemia intermedia decreased 33-fold from a value of 10% of total hemoglobin in bone marrow cells to 0.3% in the most dense red blood cells. The amount of insoluble alpha chains was measured by using the polyacrylamide gel electrophoresis in urea and Triton X-100. In beta thalassemia intermedia the amount of insoluble alpha chains was correlated with the decreased spectrin content of red cell membrane and was associated with a decrease in ankyrin and with other abnormalities of the electrophoretic pattern of membrane proteins. The loss and topology of the reactive thiol groups of membrane proteins was determined by using [3H]N-ethylmaleimide added to membrane ghosts prior to urea and Triton X-100 electrophoresis. Spectrin and ankyrin were the major proteins with the most important decrease of thiol groups.

Molecular basis of Sp alpha I/65 hereditary elliptocytosis in North Africa: insertion of a TTG triplet between codons 147 and 149 in the alpha-spectrin gene from five unrelated families.

Hereditary elliptocytosis in North Africa is frequently associated with the alpha I/65 spectrin variant, characterized by an abnormal alpha I 65-kD instead of the normal alpha I 80-kD peptide following limited trypsin digestion of whole spectrin. A similar variant (although it yielded a 68-kD fragment) has been shown recently, in two black patients, to result from the insertion of a leucyl residue at position 148 (Marchesi et al: J Clin Invest 80:191, 1987). In order to determine if the underlying molecular defect was the same in North Africans and blacks (who originate from both sides of the Sahara Desert), we performed analysis directly at the DNA level. Starting from the DNA of an Algerian alpha I/65 heterozygote in whom the mutation was associated with identifiable RFLPs, we cloned and sequenced the alpha-spectrin gene region, which includes the mutation. We thus identified an extra leucine codon (TTG) between codons 147 and 149, the coding sequence becoming CAG TTG TTG CTG instead of CAG TTG CTG. We then used the polymerase chain reaction (PCR) method and dot-blot hybridization of the amplified DNA with mutant and normal allele-specific oligonucleotides to screen the DNA from four other unrelated North African subjects with Sp alpha I/65 hereditary elliptocytosis. In all families we studied, these subjects were heterozygous for the TTG insertion. These results demonstrate that Sp alpha I/65 hereditary elliptocytosis has the same molecular basis in North Africans and blacks.

Sp alpha I/65 hereditary elliptocytosis in North Africa.

The Sp alpha I/65 variant of the spectrin has been recently described in black people with hereditary elliptocytosis (HE). The present study reports on a similar Sp alpha I/65 variant in nine North African persons belonging to four unrelated families. The abnormality was associated with a variable degree of elliptocytosis. In one case, red cell morphology was normal. In the nine carriers of the biochemical abnormality, the spectrin dimer self-association was defective. The association constant was reduced: 0.65 to 1.7 X 10(5) M-1 (controls: 4.6 +/- 0.5 X 10(5) mM-1 (n = 21)); in six cases, there was a higher level of spectrin dimer in the low ionic strength extract at 4 degrees C: 13.0 to 19.7% (controls: 6.4 +/- 2.1% (n = 7)). Limited tryptic digests of spectrin from the nine persons revealed a decrease of the 80,000-dalton alpha-1 domain, and the concomitant appearance of a peptide with a molecular weight of 65,000 daltons and an isoelectric point ranging from 5.0 to 5.1. There was a correlation between the proportion of the 65,000-dalton fragments, the defect of spectrin self-association, and the extent of morphological alteration. This is the first large series concerning a spectrin abnormality in non-black persons. In North Africa, cases of HE that are not due to a protein 4.1 defect have turned out so far to be associated with the Sp alpha I/65 variant.

Red cell membrane sialoglycoprotein beta in homozygous and heterozygous 4.1(-) hereditary elliptocytosis.

Sialoglycoprotein beta, a minor sialoglycoprotein of the red cell membrane, was studied in homozygous and heterozygous 4.1(-) hereditary elliptocytosis, a variety of hereditary elliptocytosis characterized by total or partial absence of protein 4.1. Erythrocytes were treated with the periodic acid-NaB3H4 procedure. Following polyacrylamide gel electrophoresis in the presence of SDS, labelled sialoglycoproteins were revealed by fluorography. (i) In the ghosts from the 4.1(-) homozygote, sialoglycoprotein beta was sharply decreased. It is not sure whether the residual material is sialoglycoprotein beta itself, or a distinct sialoglycoprotein migrating in the same place. In long exposure fluorograms, sialoglycoprotein gamma (a sialoglycoprotein related to sialoglycoprotein beta) also turned out to be reduced. In the homozygote's Triton-shells, sialoglycoprotein beta and gamma appeared completely absent. (ii) In the 4.1(-) heterozygote, sialoglycoprotein beta appeared slightly reduced, whereas sialoglycoprotein gamma appeared normal. Both of these proteins were extracted in seemingly normal amounts in the Triton-shells. These observations bring further support to the view that there is an interaction between skeletal membrane protein 4.1 and sialoglycoprotein beta, that is additional to other interactions between the former protein and the lipid bilayer and/or other transmembrane proteins.

The heterozygous form of 4.1(-) hereditary elliptocytosis [the 4.1(-) trait].

Using clinical, morphological, genetic, and biochemical criteria, we studied ten white and North African families with hereditary elliptocytosis (HE). In four families, elliptocytic individuals displayed a highly significant reduction of band 4.1, which was recorded using two electrophoretic procedures. The 4.1a/4.1b ratio was also significantly reduced, as is usually observed in suspensions enriched in young red cells. This form of HE was invariably associated with the following characteristics: absence of clinical signs; numerous, smooth and well-elongated elliptocytes; dominant transmission; and, when investigated, normal osmotic fragility. Its frequency, among all forms of HE, is about one third as a first estimate, at least in whites and North Africans. In the other six families studied, elliptocytic subjects presented normal 4.1 bands. Again, the 4.1a/4.1b ratio was decreased, reflecting the red cell age-dependent changes in these two components. In three of these families, elliptocytosis was accompanied by clinical signs of variable intensity, and the mode of inheritance could not be unequivocally determined. Therefore, HE with a partially reduced band 4.1 defines a homogeneous variety of HE that can be isolated from other forms of HE. We suggest that it be termed the 4.1 (-) trait, so as to correspond with a previously proposed terminology.