The efficacy of aerosol treatment with non-ionic surfactant vesicles containing amphotericin B in rodent models of leishmaniasis and pulmonary aspergillosis infection.

Amphotericin B (AMB) is used to treat both fungal and leishmanial infections, which are of major significance to human health. Clinical use of free AMB is limited by its nephrotoxicity, whereas liposomal AMB is costly and requires parenteral administration, thus development of novel formulations with enhanced efficacy, minimal toxicity and that can be applied via non-invasive routes is required. In this study we analysed the potential of non-ionic surfactant vesicles (NIV) given by nebulisation to deliver AMB to the lungs, liver and skin. Treatment with AMB-NIV resulted in significantly higher drug levels in the lungs and skin (p<0.05) compared to similar treatment with AMB solution but significantly lower plasma levels (p<0.05). Treatment with AMB-NIV resulted in a significant reduction in fungal lung burdens in a rat model of invasive pulmonary aspergillosis (p<0.05) compared to treatment with the carrier alone. Treatment with AMB-NIV but not AMB solution significantly suppressed Leishmania donovani liver parasite burdens (p<0.05) but could not inhibit the growth of cutaneous Leishmania major lesions. The results of this study indicate that aerosolised NIV enhanced pulmonary and hepatic delivery whilst minimising systemic exposure and toxicity.

Virulence spectra of typed strains of Campylobacter jejuni from different sources: a blinded in vivo study.

Campylobacter jejuni is a major cause of human diarrhoeal disease, but specific virulence mechanisms have not been well defined. The aims of the present blinded study were to measure and compare the in vivo properties of 40 serotyped, biotyped and genotyped C. jejuni isolates from different sources and genetic makeup. An 11-day-old chick embryo lethality assay, which measured embryo deaths and total viable bacteria over 72 h following inoculation of bacteria into the chorioallantoic membrane, revealed a spectrum of activity within the C. jejuni strains. Human and chicken isolates showed similar high virulence values for embryo deaths while the virulence of the bovine isolates was less pronounced. A one-way ANOVA comparison between the capacity of the strains to kill the chick embryos after 24 h with cytotoxicity towards cultured CaCo-2 cells was significant (P=0.025). After inoculation with a Campylobacter strain, mouse ligated ileal loops were examined histologically and revealed degrees of villous atrophy, abnormal mucosa, dilation of the lumen, congestion and blood in lumen, depending on the isolate examined. A 'total pathology score', derived for each C. jejuni strain after grading the pathology features for degree of severity, showed no apparent relationship with the source of isolation. Some relationship was found between amplified fragment length polymorphism groups and total ileal loop pathology scores, and a one-way ANOVA comparison of the mouse pathology scores against total chick embryo deaths after 72 h was significant (P=0.049).

Mixed herbs drugs: inhibitory effect on growth of the endogenous mycoflora and aflatoxin production.

Twenty commercial mixed herbal drugs were examined for mycological profile. Aspergillus species were the predominant fungi found in the drugs. Other fungi harboured in the drugs with less frequency were Paecilomyces species, Eurotium species, Monascus species, Acremonium species, Penicillium species, Cladosporium species, Scopulariopsis species, Phialophora species and Fonseceae species. Fungal count was between 1.0 log(10) CFU and 2.4 log(10) CFU per gram of sample. When the drugs were incubated in 85% humidity at 25 degrees C, fungal colonies grew on only two of the drugs. The mixed herbal drugs were extracted with water and the extracts were used to grow Aspergillus parasiticus. All extracts reduced aflatoxin B(1) and aflatoxin G(1) production by 62-97%. All but two of the extracts reduced aflatoxin B(2) and aflatoxin G(2) production by 39-95%. It can be concluded that the commercial powdered mixed herbal drugs contained low number of endogenous fungi, and these drugs are inhibitory to the growth of its endogenous fungi and aflatoxins production by aflatoxigenic fungi.

Comparison of virulence-associated in vitro properties of typed strains of Campylobacter jejuni from different sources.

Campylobacter jejuni is a major cause of human diarrhoeal disease, but specific virulence mechanisms have not been well defined. This blinded study was undertaken with 40 C. jejuni isolates from different sources to determine their haemolytic, cytotoxic and adhesion and invasion activities towards mammalian cells. The results were correlated with source of isolation and genetic makeup by amplified fragment length polymorphism (AFLP) typing. The isolates had variable degrees of haemolytic activity against rabbit erythrocytes and cytotoxicity towards CaCo-2, HeLa and Vero cells. The data indicated that the haemolytic and cytotoxic activities were due to separate factors. A range of cytotoxicity was exhibited, whereby some strains had no activity against the target cells and others had activity against all three cell lines. Certain strains had activity against CaCo-2 cells but little or no activity against the other cells, while others exhibited the opposite phenotype. The data suggested that the cytotoxicity assay with the different cell lines may have detected more than one cytotoxin. A wide variation between isolates was observed for both adherence and invasion with all three cell lines, yet, overall, the strains showed a significantly greater invasion capacity for CaCo-2. There was no clear relationship between source of isolation or disease manifestation and possession of statistically significantly higher levels of particular virulence-associated factors although, in some cases, a correlation between cytotoxicity and cell invasion was evident. Five AFLP clusters, each representing two to eleven isolates with similar profiles, were observed at the 90 % similarity level. Some AFLP groups contained isolates with a common serotype, but each group had C. jejuni isolates from more than one source with the exception of group IV, which contained only human isolates. Isolates with high cytotoxic activity against CaCo-2 cells were confined to groups I, III and IV and a group of unrelated strains (U). Group II isolates had uniformly low cytotoxicity. Isolates in groups I, V and U were more invasive for CaCo-2 cells than isolates in groups II, III and IV. The strain differences in cytotoxicity or invasion did not correlate with source of isolation.

Comparison of in vitro cytotoxicity of Fusarium mycotoxins,deoxynivalenol, T-2 toxin and zearalenone on selected human epithelial cell lines.

Three human epithelial cell lines (CaCo-2, HEp-2 and HeLa) implicated as potential targets for three Fusarium toxins were tested for the extent of survival on exposure to increasing toxin concentration and incubation periods. Cytotoxicity assay using 3(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) was carried out with deoxynivalenol (DON), T-2 toxins and zearalenone (ZON) on CaCo-2, HEp-2 and HeLa cell lines. Of the three cell lines used, HeLa was the most sensitive, eliciting cell death after 2 days exposure at 100 ng ml(-1)with T-2 toxin. HeLa was the only cell line to exhibit cytotoxicity towards ZON showing cell death at 1000 ng ml(-1)after 2 days which increased to 4 days, showing substantial cell death at 200 ng ml(-1). HEp-2 was sensitive to DON showing cell death after 2 days (100 ng ml(-1)) with complete cell death occurring at 200 ng ml(-1) after 4 days of exposure. Substantial cytoxicity of T-2 towards HEp-2 occurred after 2 days at 1000 ng ml(-1) and complete cell death occurred with 100 ng ml(-1) at day 4. The CaCo-2 cell line was generally resistant to the mycotoxins tested between 100 and 1000 ng ml(-1). This study shows that cytotoxicity of Fusarium toxins to epithelium cell lines is concentration- and time- dependant and results from ZON-HeLa interaction indicate possible cell type-mycotoxin specificity.

Occurrence of aflatoxin M1 in randomly selected North African milk and cheese samples.

Forty-nine samples of raw cow's milk and 20 samples of fresh white soft cheese were collected directly from 20 local dairy factories in the north-west of Libya and analysed for the presence of aflatoxin M1 (AFM1). The samples were analysed using a high-performance liquid chromatography technique for toxin detection and quantification. Thirty-five of the 49 milk samples (71.4%) showed AFM1 levels between 0.03 and 3.13 ng ml(-1) milk. Multiple analyses of five milk samples free of AFM1 artificially contaminated with concentrations of AFM1 at 0.01, 0.05, 0.1, 1.0 and 3.0 ng ml(-1) showed average recoveries of 66.85, 72.41, 83.29, 97.94 and 98.25%, with coefficients of variations of 3.77, 4.11, 1.57, 1.29 and 0.54%, respectively. Fifteen of 20 white soft cheese samples (75.0%) showed the presence of AFM1 in concentrations between 0. 11 and 0.52 ng g(-1) of cheese. Multiple assays of five cheese samples free of AFM1 spiked with different concentration of AFM1 (0.1, 0.5, 1.0 and 3.0 ng g(-1)) showed average recoveries of 63.23, 78.14,83.29 and 88.68%, with coefficients of variation of 1.53, 9.90, 4.87 and 3.79%, respectively. The concentrations of AFM1 were lower in the cheese products than in the raw milk samples.

Microflora of date fruits and production of aflatoxins at various stages of maturation.

Twenty-five varieties of dates (Phoenix dactylifera) were examined at different maturation stages for total microbial counts, aflatoxins and aflatoxigenic Aspergillus sp. and lactic acid bacteria. The samples were examined as fresh and under simulated storage condition of high humidity. Microbial counts were high at the first stage of maturation (Kimri) and increased sharply at the second stage (Rutab), then decrease significantly at the final dried stage of maturation (Tamr). Aflatoxins were detected in 12% of the samples although aflatoxigenic Aspergillus were detected in 40% of the varieties examined, all at Kimri stage only. Lactic acid bacteria were present only at the Rutab stage in some varieties including all varieties in which aflatoxins or aflatoxigenic Aspergillus were detected. No aflatoxins or aflatoxigenic Aspergillus were detected at the final edible stage of maturation.

Cellular morphology of rough forms of Listeria monocytogenes isolated from clinical and food samples.

Transmission electron microscopy (TEM) studies revealed that rough cell-forms of L. monocytogenes (designated FR variants), isolated from clinical and food samples (and under conditions of sublethal heat stress), consist of either single or paired long-filaments. These FR variants markedly contrast in cell morphology from other previously described avirulent rough-mutants of L. monocytogenes that form long chains consisting of multiple cells of similar size (designated MCR variants). The identity of these Listeria isolates was determined using a commercially available, anti-Listeria polyclonal KPL antibody and by the API Listeria biochemical gallery. This study shows that filamentous rough-forms of L. monocytogenes may occur in clinical and food samples that are of undetermined pathogenicity.

Virulent rough filaments of Listeria monocytogenes from clinical and food samples secreting wild-type levels of cell-free p60 protein.

Atypical rough cell filaments of Listeria monocytogenes (designated FR variants), isolated from clinical and food samples, form long filaments up to 96 microm in length and demonstrated wild-type levels of adherence, invasion, and cytotoxicity to human epithelial HEp-2, Caco-2, and HeLa cells. Unlike previously described avirulent rough mutants of L. monocytogenes that secrete diminished levels of the major extracellular protein p60 and that form long chains that consist of multiple cells of similar size (designated MCR variants), FR variants secreted wild-type or greater levels of p60. This study shows that virulent filamentous forms of L. monocytogenes occur in clinical and food environments and have atypical morphological characteristics compared to those of the wild-type form.

A limited survey of aflatoxins and fumonisins in retail maizebased products in the UK using immunoassay detection.

A total of 27 maize-based products destined for human consumption were collected from retail outlets within the city of Glasgow in the UK and were analysed for the presence of aflatoxins using immunoaftinity column chromatography with fluorescence detection and for fumonisins by competitive ELISA. Aflatoxins were detected at a trace level below 4 in eight (30%) of the 27 samples tested, no sample contained aflatoxins at a high level although one sample of sweetcorn did contain aflatoxins at a level of 5-10 Fumonisins were detected in eight (30%) of the samples at levels from 1 to 8mgkg(-1) and a further eight samples contained fumonisin at a level below 1 mgkg(-1) but above the detectable level. The highest concentration of fumonisins was found in a sample of fine corn meal at 8-12mgkg(-1).

Use of immunoaffinity chromatography as a purification step for the determination of aflatoxin M1 in cheeses.

A method using immunoaffinity as a purification step for the determination of aflatoxin M1 in cheese is described. A simple solvent extraction with dichloromethane followed by a washing step with N-hexane gives a prepurified extract. A comparison between two ways of aflatoxin M1 purification, by solid-phase extraction clean-up and by immunoaffinity, was carried out. The use of immunoaffinity columns containing monoclonal antibodies against aflatoxin M1 gives the best result, i.e. a very clean preparation containing purified aflatoxin M1. The quantification of aflatoxin M1 is then performed by high performance liquid chromatography using fluorometric detection. This method was successfully carried out on naturally-contaminated and spiked cheeses. Recoveries are about 75%. The limit of quantification is 0.020 microgram of aflatoxin M1 per kg of cheese. This method seems suitable for use in monitoring programmes for aflatoxin M1 contamination in dairy products such as cheese.

Determination of ochratoxin A by immunoaffinity column clean-up and HPLC In wheat and pig liver.

Determination of ochratoxin A (OTA) by immunoaffinity column clean-up and HPLC detection was performed on wheat and pig liver. Several extraction protocols involving methanol and ethyl acetate were investigated. The optimum experimental conditions for analysis of OTA in artificially contaminated wheat (87.4% recovery) using immunoaffinity column clean-up was found to be the methanol: PBS (1∶1 v/v) protocol. These conditions, however, gave low recoveries for pig liver (40.4%).

Immunoassay of ochratoxin and other mycotoxins from a single extract of cereal grains utilizing monoclonal antibodies.

Immunoassays provide rapid, specific, sensitive and inexpensive methods for analysing mycotoxins but have generally been tested individually. Mycotoxigenic fungi rarely occur in pure culture in nature, and different mycotoxins may occur together. It would therefore be advantageous if several immunoassays for different mycotoxins could utilize a single extract. Monoclonal antibodies specific for ochratoxin A, aflatoxin B1 and T-2 toxin, raised at the University of Strathclyde, allow detection limits of 1 ng/ml ochratoxin A, 0.1 ng/ml aflatoxin B1 and 10 ng/ml T-2 toxin, when used in competitive enzyme-linked immunosorbent assays. These antibodies have been used to assay ochratoxin A. aflatoxin B1 and T-2 toxin in a single acetonitrile: 0.5% KCl:6% H2SO4 (89:10:1) extract of cereal grain. Extracts were either diluted 1:10 for direct assay or subjected, before assay, to a simple liquid-liquid clean-up procedure, which removed interfering substances and resulted in a 5:1 concentration. Recoveries from barley to which mycotoxins had been added averaged 95.8% for ochratoxin A, 93.8% for aflatoxin B1 and 80.6% for T-2 toxin, and the detection limits were 5 ng/g for ochratoxin A, 4 ng/g for aflatoxin B1 and 50 ng/g for T-2 toxin. Mean coefficients of variation within and between assays and between subsamples were less than 12% for ochratoxin A and aflatoxin B1 but up to 17% for T-2 toxin in assays of barley inoculated with toxigenic fungi.(ABSTRACT TRUNCATED AT 250 WORDS)

Aflatoxin monoclonals: academic development to commercial production.

A monoclonal antibody (mAb) has been produced to aflatoxin B1 (AF B1) after successful immunization of mice and fusion of sensitized spleen cells with myeloma cancer cells. The mice were immunized with AF B1-oxime-protein conjugate. Positive mAbs were screened using an indirect ELISA specific for AF B1. The selected mAb was then developed in direct competitive ELISA and immunoaffinity column chromatography methods for aflatoxin detection in foods and feeds. Both assays are rapid, sensitive, specific and require only the minimum of sample preparation. Both immunological assays have now been commercialized and are produced in convenient ready-made kit formats.

Monoclonal antibody-based enzyme linked immunosorbent assay of aflatoxin B1, T-2 toxin, and ochratoxin A in barley.

Aflatoxin B1 (B1), T-2 toxin (T2), and ochratoxin A (OA) were assayed in a single extract from barley grain by using competitive enzyme linked immunosorbent assays (ELISAs) with monoclonal antibodies. B1 and T2 monoclonal antibodies were conjugated to horseradish peroxidase for direct competitive ELISA while an indirect competitive ELISA was used for OA determination. The competitive ELISA detected 0.1 ng/mL of B1, 10 ng/mL of T2, or 1 ng/mL of OA. Acetonitrile-0.5% KCl-6% H2SO4 (89 + 10 + 1) extracts of barley grain either were diluted 1:10 for direct assay or were subjected to a simple liquid-liquid cleanup procedure to concentrate the extract 10:1 before assay. For cleanup, water was added to the acetonitrile extract to partition water-soluble interfering substances, and then the mycotoxins were re-extracted with chloroform. The chloroform extract was evaporated to dryness and redissolved in Tris HCl buffer for ELISA. The mean recoveries from barley spiked with 4-60 ng/g of B1, 50-5000 ng/g of T2, and 5-500 ng/g of OA were, respectively, 93.8, 80.6, and 95.8%. The mean within-assay, inter-assay, and subsample coefficients of variation by ELISA of barley grain colonized with toxigenic fungi were less than 12% for B1 and OA but as high as 17% for T2.

Monoclonal antibody technology for mycotoxins.

Specific monoclonal antibodies (MABs) against aflatoxins, ochratoxin A, zearalenone, diacetoxyscirpenol and T-2 toxin have been prepared in various laboratories by the application of hybridoma technology to mycotoxins. These antibodies can be selected for sensitivity, reduced cross-reactivity, reliability and ease of production. When a suitable antibody is chosen it can then be used in a rapid immunological method such as an enzyme-linked or radio-immunoassay or immunoaffinity chromatography system. These assays have a lower limit of mycotoxin detection in the ng/ml range and have been applied to the determination of mycotoxins in samples such as maize, peanuts, peanut butter, milk and porcine kidneys. Using these immunoassay techniques, sample preparation has generally been simplified to a matter of solvent extraction of mycotoxins from the sample followed by dilution; under these conditions, levels of 1-5ug of mycotoxins/kg of sample can be found. The application and advantages of MABs to mycotoxins and the use of these antibodies in various assay techniques is discussed.

Determination of ochratoxin A by monoclonal antibody-based enzyme immunoassay.

A simple and rapid indirect enzyme-linked immunosorbent assay was developed for the quantitative determination of ochratoxin A in barley after the successful production of a high affinity, specific monoclonal antibody. A rapid sample cleanup was achieved by extracting ochratoxin A from barley with chloroform and partitioning the toxin into bicarbonate buffer; the buffer solution was then added directly to the assay plate and ochratoxin A content was assessed. Recoveries were greater than 85% and detection limits were 5 micrograms ochratoxin A/kg barley.

Detection of ochratoxin A in porcine kidneys by a monoclonal antibody-based radioimmunoassay.

By using an indirect enzyme-linked immunosorbent assay, eight monoclonal antibodies (MAbs) were selected. Mice were immunized with ochratoxin A that was conjugated to bovine serum albumin. The hybridoma cell line designated 10G2 was grown in tissue culture and as an ascites tumor. The MAb was characterized to be specific to ochratoxin A and of the immunoglobulin G (IgG) class. Subsequently, the ascites fluid of this hybridoma was used in a competitive solid-phase IgG radioimmunoassay on protein A-Sepharose CL-4B, with [14C]ochratoxin A as tracer. Porcine kidneys were extracted with 0.5% phosphoric acid in chloroform. A two-step cleanup was achieved on a Sep-Pak C18 cartridge and a Sep-Pak silica cartridge. Radioimmunoassay with MAbs coupled to protein A-Sepharose CL-4B allowed the detection of ochratoxin A in porcine kidneys at a concentration as low as 0.2 ng/g.