A review on aflatoxin contamination and its implications in the developing world: a sub-Saharan African perspective.

Mycotoxins contamination in some agricultural food commodities seriously impact human and animal health and reduce the commercial value of crops. Mycotoxins are toxic secondary metabolites produced by fungi that contaminate agricultural commodities pre- or postharvest. Africa is one of the continents where environmental, agricultural and storage conditions of food commodities are conducive of Aspergillus fungi infection and aflatoxin biosynthesis. This paper reviews the commodity-wise aetiology and contamination process of aflatoxins and evaluates the potential risk of exposure from common African foods. Possible ways of reducing risk for fungal infection and aflatoxin development that are relevant to the African context. The presented database would be useful as benchmark information for development and prioritization of future research. There is need for more investigations on food quality and safety by making available advanced advanced equipments and analytical methods as well as surveillance and awareness creation in the region.

Selected pharmacokinetic parameters for Cefovecin in hens and green iguanas.

The third generation cephalosporin cefovecin has been shown to have an exceptionally long elimination half-life in dogs and cats, making it suitable for antibacterial treatment with a 14-day dosing interval in these species. Pharmacokinetic parameters for cefovecin were investigated in juvenile hens and green iguanas, following subcutaneous injections with 10 mg cefovecin/kg bodyweight. Preliminary studies in eight additional species of birds and reptiles were performed and results were compared with the parameters found in hens and green iguanas. The kinetics were characterized by rapid absorption with peak plasma concentration of 6 +/- 2 microg/mL in hens and 35 +/- 12 microg/mL in green iguanas. The mean plasma half-life for cefovecin was 0.9 +/- 0.3 h for hens and 3.9 h in green iguanas. Volume of distribution was 1.6 +/- 0.5 L/kg for hens and 0.3 L/kg for green iguanas and clearance was 1252 +/- 185 mL.h/kg for hens and 53 mL.h/kg for green iguanas. Results from preliminary studies did not differ notably from those seen in hens and green iguanas. Cefovecin is not suitable for the treatment of bacterial infections with a 14-day dosing interval in hens or green iguanas and seems not to be in a number of other bird and retile species either.

Influence of the calcium content of the diet offered to leopard tortoises (Geochelone pardalis).

Twenty-four juvenile leopard tortoises were divided into four groups of six; one group was fed a basic low-calcium feed for six months, and the other three groups were fed the same basic diet supplemented with one, three and nine times the amount of calcium recommended as a supplement to the diet of reptiles. The animals' bone mineral content and bone mineral density were estimated by dual energy x-ray absorptiometry, and blood samples were taken at the start and at the conclusion of the study. One tortoise from each group was examined postmortem. There was a clear depletion of calcium in the body of the tortoises receiving no calcium supplement, and the shell of the tortoises receiving the recommended calcium supplement did not calcify to the extent expected. The tortoises that received three times the recommended calcium supplementation had the highest growth rate and were thriving. However, metastatic calcifications were observed postmortem in the two groups that were given the highest doses of calcium.

Factors that influence weight loss in the puerperium.

A study group of 795 women was followed with frequent weight measurements and questionnaires about their activities for 6 months postpartum. The mean (+/- SD) net weight gain from the first prenatal visit to 6 months postpartum was 1.4 +/- 4.8 kg. Weight gain during prenatal care was the variable most highly correlated to weight loss. Return to work outside the home, parity, and smoking also correlated significantly to weight loss. Breast-feeding, exercise, season of the year, age, and marital status were not correlated. Route of delivery was related to weight loss at 2 and 6 weeks, but not at 6 months. Counseling women about weight gain during pregnancy and weight loss requires an understanding of these variables with a long-term perspective of at least 6 months.

Microbial degradation of amygdalin of bitter apricot seeds (Prunus armeniaca).

Amygdalin is a cyanogenic glycoside occurring among others in almonds and bitter apricot seeds with interesting levels of dietary protein. Utilization of seeds for human or animal nutrition requires adequate detoxification. In the present paper, selected filamentous fungi (Mucor circinelloides, Penicillium nalgiovense) and yeasts (Hanseniaspora valbyensis, Endomyces fibuliger) were tested for their in-situ ability to decompose amygdalin. The latter (Endomyces fibuliger) was best able to grow on autoclaved bitter apricot seeds and detoxify them from 30 microMol CN/g dry matter to less than 1 microMol CN/g dry matter after 48 h of incubation at 27 degrees C.

Degradation of cyanogenic glycosides by Lactobacillus plantarum strains from spontaneous cassava fermentation and other microorganisms.

Strains of Lactobacillus plantarum, Leuconostoc mesenteroides, Candida tropicalis and Penicillium sclerotiorum were screened for 19 enzymatic activities using the commercial kit API zym (Bio Mérieux). This activity was compared to the ability of degrading the toxic cyanogenic glycosides amygdalin, linamarin, and linseed cyanogens (a mixture of linustatin and neolinustatin). Good correlation between the beta-glucosidase activity found in the API zym screening and the ability to degrade the cyanogenic glycosides was found for the first three species mentioned. P. sclerotiorum strains exhibited very high activity in the API zym test (substrate: 6-Br-2-naphthyl-beta-D-glucopyranoside), but proved unable to degrade any of the cyanogenic substrates. Among the seven strains of L. plantarum tested, a great variation was seen in the beta-glucosidase activity as well as in the ability to degrade the cyanogens. This was also the case for the strains of C. tropicalis. However, all the glucosidase positive strains of these species were also able to degrade all of the cyanogens tested and at approximately the same rate. A co-culture of the most active strain of L. plantarum and C. tropicalis seemed to degrade linamarin faster than the mono cultures. L. plantarum LPI (originally isolated from fermented cassava) was investigated in further detail. The hydrolytic activity of this strain was intracellular or cell bound, and beta-bis-glycosides such as amygdalin were hydrolysed by a two-stage sequential mechanism as follows: (1) amygdalin to prunasin and (2) prunasin to cyanohydrin. Finally, inoculation of extracted linseed meal (containing linustatin and neolinustatin) with L. plantarum LPI resulted in hydrolysis of the glycosides.

Toxicological, nutritional and microbiological evaluation of tempe fermentation with Rhizopus oligosporus of bitter and sweet apricot seeds.

Bitter and sweet apricot seeds are by-products of the apricot processing industry. Bitter seeds, in particular, contain toxic levels of the cyanogenic substance amygdalin. Tempe was made from both kinds of seeds. The bitter seeds contain antimicrobial substances which must be removed by leaching and boiling prior to tempe fermentation. Apricot seed tempe had an agreeable taste. It contained approx. 21% (w/w) crude protein, 52% (w/w) crude fat, 1.5% (w/w) crude fibre and 25.5% (w/w) carbohydrates based on dry matter. The extent of biological acidification during soaking prior to fungal inoculation was inadequate to prevent growth of Bacillus cereus, and requires further optimisation. Bitter seeds were detoxified by the tempe process (approx. 70% of total cyanide was removed). However, additional improvement of the detoxification process is required to obtain a completely safe product.

Microdiffusion method with solid state detection of cyanogenic glycosides from cassava in human urine.

A method for quantitative determination of cyanogenic glycosides in human urine is described. It is based on enzymatic cleavage of the glycosides, microdiffusion of the hydrogen cyanide formed, solid state detection by colour formation on a picrate-impregnated sheet, and subsequent rating of the coloured spot by the absorption of transmitted light at 540 nm with a thin-layer (TLC) densitometer. The method has been tested using normal as well as pathological urines containing glucose, protein, leucocytes, blood and bacteria. The method allows quantification of urinary linamarin above 70 mumol/litre, in 40 microliters urine. In Mozambican subjects consuming insufficiently processed cassava the mean urinary linamarin levels were 211 mumol/litre, indicating for the first time that substantial amounts of the main cyanogenic glycoside in cassava may be absorbed from the human gut and excreted intact in the urine.

Evaluation of an in vitro method for acaricidal effect. Activity of parathion, phosmet and phoxim against Sarcoptes scabiei.

An in vitro test to determine the acaricidal effect of organophosphorous insecticides (OP) is described. The effect of parathion, phoxim and phosmet against the pig mange mite Sarcoptes scabiei var. suis was evaluated. The test is based on the migration ability of mites on the surface of agar gels containing the acaricide. The mite activity is expressed as a migration index (MI) and compared with the OP concentration in the agar. Good dose-response data were obtained for all three OPs tested, although the instability of phosmet required special precautions concerning the analysis of the agar. The test was found to be accurate, sensitive, easy to carry out and applicable for routine determinations. However, the test requires that the actual concentrations of the OPs in the gel batches are determined. For the three OPs used analytical methods were developed. While the lower threshold for acaricidal effect in vitro was approximately 1-2 micrograms g-1 for all three OPs tested, a significant difference in the higher concentration range was seen between the dose-response curve for parathion and the curves for phoxim and phosmet. While the latter curves decreased only slightly at concentrations above 3-6 micrograms g-1 (corresponding to MI values around 5-10), the curve for parathion was linear down to an MI value of 1, corresponding to a parathion concentration of approximately 30 micrograms g-1. This discrepancy was ascribed to different rates of uptake through the cuticula due to differences in the lipophilicity of the OPs.

A method for in vitro determination of the acaricidal effect of ivermectin using Sarcoptes scabiei var. suis as test organism.

An in vitro assay for the acaricidal effect of ivermectin is described. The test is based on the migration ability and survival of Sarcoptes scabiei var. suis mites on the surface of agar gels containing the acaricide. The effect of a given concentration of ivermectin is expressed as an activity index (AI) for the mites, calculated from observed migration and mite survival after 24 h of incubation. In order to allow a concentration-effect curve to be based on true values of the acaricide, a method for the determination of ivermectin in agar gels was developed. Employing this method, ivermectin was demonstrated to be stable in the gels at room temperature for a month, thus allowing storage of gels for later use. The test was found to be accurate, sensitive, easy to carry out and applicable for routine determinations. The lower threshold for acaricidal effect was found to be 50-100 ng ml-1 with the AI showing a reverse linear dependence on the log concentrations above this value.

Two-step hydrolysis of amygdalin in molds.

Mucor circinelloides LU M40 and Penicillium aurantiogriseum P 35, characterized by extracellular beta-glucosidase activity on cyanogenic glycosides, hydrolyse amygdalin by a two-step reaction mechanism being the first step of hydrolysis, from amygdalin to prunasin, very rapid (15 min) and the second one, from prunasin to mandelonitrile, much slower (120 min).

Chemical safety of cassava products in regions adopting cassava production and processing--experience from Southern Africa.

The cassava belt area in Southern Africa is experiencing an unforeseen surge in cassava production, processing and consumption. Little documentation exists on the effects of this surge on processing procedures, the prevailing levels of cyanogenic glucosides of products consumed and the levels of products commercially available on the market. Risk assessments disclose that effects harmful to the developing central nervous system (CNS) may be observed at a lower exposure than previously anticipated. We interviewed farmers in Zambia and Malawi about their cultivars, processing procedures and perceptions concerning cassava and chemical food safety. Chips, mixed biscuits and flour, procured from households and markets in three regions of Zambia (Luapula-North, Western and Southern) as well as products from the Northern, Central and Southern regions of Malawi, were analyzed for total cyanogenic potential (CNp). Processed products from Luapula showed a low CNp, <10 mg HCN equiv./kg air dried weight, while samples from Mongu, Western Province, exhibited high levels of CNp, varying from 50 to 290 mg HCN equiv./kg. Even the lowest level is five times higher than the recommended safety level of 10mg/kg decided on for cassava flour. Our results call for concerted efforts in promoting gender oriented processing technologies.

Determination of cyanide and cyanogenic compounds in biological systems.

A survey of methods for the qualitative and quantitative determination of cyanide and cyanogenic compounds is presented. Particular attention is paid to determination in complex matrices. Chromatographic methods able to separate mixtures of closely related structures, such as glycosides of enantiomeric hydroxynitriles or lipids with slightly differing fatty acid spectra, are included, as are highly selective methods of detection, such as enzymic post-column cleavage combined with electrochemical detection as used in high-performance liquid chromatography. Details of thin-layer chromatography, including methods for detection, are given for both straight-phase and reverse-phase systems. The survey includes simple field methods as well as automated laboratory methods for the determination of 'free', 'bound' and 'total' cyanide, for example in processed food products from Manihot esculenta Cranz. Sources of enzymes are listed and attention is given to problems of sample storage and preparation. References are given to review articles which include data from methods such as ultraviolet, infrared and proton and carbon-13 nuclear magnetic resonance spectroscopies.

Electrochemical detection of cyanogenic glycosides after enzymatic post-column cleavage.

Crude and partly purified extracts from Helix pomatia and linamarase from cassava were immobilized on columns packed with porous glass or silica and used as post-column reactors in the high-performance liquid chromatography of cyanogenic glycosides. Sodium hydroxide (2 M) was added to the flowstream after the enzyme-reactor resulting in the formation of cyanide, which was then detected at a silver electrode by an amperometric measurement at 0 V with reference to a silver-silver chloride electrode. The selective detection of cyanide allows measurements in a complex matrix. The response is linear and the detection limit is in the low picomole range.

Cyanogenic glycosides and cyanohydrins in plant tissues. Qualitative and quantitative determination by enzymatic post-column cleavage and electrochemical detection, after separation by high-performance liquid chromatography.

A rapid, simple and reproducible high-performance liquid chromatography procedure is described for the qualitative and quantitative analysis of mixtures of cyanogenic glycosides. The separation is achieved by means of a reversed-phase (C8) column eluted with a phosphate buffer, pH 5.0, containing either 15 or 7.5% (v/v) methanol, 7.5% being necessary for resolution of epimeric pairs of the more hydrophilic glycosides. When this separation is combined with enzymatic post-column cleavage and electrochemical detection of the cyanide formed, a highly specific and very sensitive system is obtained. The method was applied to cyanogenic glycosides in crude plant tissue extracts, and compared with both a thin-layer chromatographic method and to a traditional determination of total cyanide released after hydrolysis. Sensitivity, selectivity and accuracy were found sufficient to enable its routine use for analysis of food and fodder samples, for example. Cyanohydrins could be detected qualitatively.

Beta-glycosidase (amygdalase and linamarase) from Endomyces fibuliger (LU677): formation and crude enzyme properties.

In our previous studies, the yeast Endomyces fibuliger LU677 was found to degrade amygdalin in bitter apricot seeds. The present investigation shows that E. fibuliger LU677 produces extracellular beta-glycosidase activity when grown in malt extract broth (MEB). Growth was very good at 25 degrees C and 30 degrees C and slightly less at 35 degrees C. When grown in MEB of pH 5 and pH 6 with addition of 0, 10 or 100 ppm amygdalin, E. fibuliger produced only slightly more biomass at pH 5, and was only slightly inhibited in the presence of amygdalin. Approximately, 60% of the added amygdalin was degraded (fastest at 35 degrees C) during an incubation period of 5 days. Supernatants of cultures grown at 25 degrees C and pH 6 for 5 days were tested for the effects of pH and temperature on activity (using amygdalin, linamarin and prunasin as substrates). Prunase activity had two pH optima (pH 4 and pH 6), amygdalase and linamarase only one each at pH 6 and pH 4-5 respectively. The linamarase activity evolved earlier than amygdalase (2 days and 4 days respectively). The data thus indicate the presence of at least two different glycosidases having different pH optima and kinetics of excretion. In the presence of amygdalin, lower glycosidase activities were generally produced. However, the amygdalin was degraded from the start of the growth, strongly indicating an uptake of amygdalin by the cells. The temperature optimum for all activities was at 40 degrees C. Activities of amygdalase (assayed at pH 4) and linamarase (at pH 6) evolving during the growth of E. fibuliger were generally higher in cultures grown at 25 degrees C and 30 degrees C. TLC analysis of amygdalin degradation products show a two-stage sequential mechanism as follows: (1) amygdalin to prunasin and (2) prunasin to cyanohydrin.

Production of ß-glycosidases (linamarase and amygdalase) and pectolytic enzymes by Penicillium spp.

Fifty-eight strains, representing 31 species of Penicillium, were screened for extracellular ß-glycosidase (amygdalase/linamarase) and pectolytic (polygalacturonase, pectin lyase) enzymes. One strain each of P. turbatum, P. piceum and P. paxilli showed very high ß-glycosidase activity and slightly lower activities were found in P. crustosum, P. expansum, P. oxalicum and P. aurantiogriseum. Generally, maximum ß-glycosidase activity showed reached during the stationary phase of growth. The seven species with highest ß-glycosidase activity showed different patterns of pectolytic activities, indicating that different species or combinations of species could be selected for different potential applications.

Disposition of parathion after dermal application in pigs.

Pharmacokinetic parameters of parathion were studied in pigs after intravenous (i.v.) and dermal administration of unlabelled and 14C-parathion. Plasma concentration-time data were subjected to non-compartmental analysis. Intravenous injection studies showed a mean residence time (MRT) of 2.15 h, a body clearance (ClB) of 4.4 l/kg/h and a volume of distribution (Vss) of 9.8 l/kg. Dermal application led to a mean absorption time (MAT) of 78 h, and a bioavailability of 9.9%. Plasma levels of 14C-parathion (parathion + metabolites) were much higher and more persistent than those of parathion itself. After i.v. administration, recovery of 14C-parathion from urine plus faeces was almost 100% within 3 d, while it was less than 20% after dermal application. Ten days after dermal application high 14C concentrations remained in the back skin, i.e. the application area. In skin samples from areas where contamination from the application area could not have occurred, the 14C-parathion concentration was as low as 2 micrograms/g. It is concluded that in view of the low dermal bioavailability for organophosphorus insecticides it is unlikely that pour-on preparations containing these insecticides reach the ectoparasites through absorption and systemic distribution, but rather that this happens after spreading on the surface of the skin.

Rapid quantitative assay for acaricidal effects on Sarcoptes scabiei var. suis and Otodectes cynotis.

Brimer et al. (Vet. Parasitol. 51: 123-135, 1993 and 59: 249-255, 1995) developed a migration assay for acaricidal effect of acetylcholinesterase inhibitors and macrocyclic lactones utilising Sarcoptes scabiei var. suis mites. In contrast to many others, this assay is fully quantitative but quite time-consuming. The aim of the present investigation was to modify this assay to become faster and simpler. As a result accurate determinations can now be obtained within 6h, as opposed to 24h. Furthermore it was demonstrated that also Otodectes cynotis mites can be used with only minor modifications of the procedures. The cholinesterase inhibitor diazinon and the formamide amitraz were used as acaricides. Thus, the mite migration assay now has been proven useful for acaricidal compounds belonging to three chemical groups with different modes of action, namely organophosphorous cholinesterase inhibitors, macrocyclic lactones acting on the glutamanergic/GABAegic motoneurons, and formamide inhibitors of the octopamine systems of arthropods.