Isolation, Characterization and Genetic Diversity of Aspergillus flavus in Animal Feed

Sixty representative pelleted animal and poultry feed samples were collected from Assiut governorate farms for aflatoxins (B1, B2, G1, and G2) analysis by thin layer chromatography (TLC) and ultra-performance liquid chromatography (UPLC) in the EGAC-accredited central lab. of the Faculty of Veterinary Medicine, Assiut University, as well as for their aflatoxigenic fungi prevalence assessment. TLC results showed that 35% of the analyzed feed samples were positive for AFs. UPLC results showed that means ± SE of AFB1, AFB2, AFG1, AFG2 and total AFs were 23.36±13.12, 2.88±1.07, 1.33±0.64, 0.52±0.25 and 28.08±13.98 μg kg-1 in animal feed and 34.88±25.18, 2.55±0.78, 1.80±1.51, 1.30±0.52 and 40.83±25.59 μg kg-1 in poultry feed, respectively. The prevalence of aflatoxins was 70% and 100% in animal feed and poultry feed samples, respectively. Aspergillus flavus isolates from the analyzed feed samples showed different degrees of aflatoxigenic ability on coconut agar medium, and the poultry feed samples were more contaminated with AFs and aflatoxigenic fungi than animal feed samples. Key words: Aflatoxins; animal feed; poultry feed; TLC; UPLC

Preliminary study for evaluation of some fungicides against Aspergillus flavus isolated from historical illuminated paper manuscript dated back to the Mamluk period

Mycotoxin production and predictive modelling kinetics on the growth of Aspergillus flavus and Aspergillus parasiticus isolates in whole black peppercorns (Piper nigrum L)

One of the most promising biologically based nanomanufacturing processes is the production of selenium nanoparticles (SeNPs) by fungi. The use of these biosynthesized nanoparticles in agricultural practices has emerged as a new approach for controlling pathogen growth and mycotoxin production. In the present study, different chemical and physical parameters were investigated for the growth of Fusarium oxysporum (CCASU-2023-F9) to increase selenite reduction and obtain the highest yield of selenium nanoparticles (SeNPs). Fusarium oxysporum (CCASU-2023-F9) exhibited tolerance to up to 1 mM sodium selenite (Na2SeO3), accompanied by red coloration of the medium, which suggested the reduction of selenite and the formation of selenium nanoparticles (SeNPs). Reduced selenite was quantified using inductively coupled plasma‒mass spectrometry (ICP-MS), and the results revealed that Fusarium oxysporum (CCASU-2023-F9) is able to transform 45.5% and 50.9% of selenite into elemental selenium by using fructose and urea as the best carbon and nitrogen sources, respectively. An incubation temperature of 30°C was the best physical condition at which 67.4% of the selenite was transformed into elemental selenium. The results also indicated that pH 7 was the optimum pH, as it displayed 27.2% selenite reduction with a net dry weight of 6.8mg/mL. Increasing the concentration of sulfate resulted in a significant increase in selenite reduction, as it reached a maximum value of 75.3% at 0.15% g/ml sulfate. The maximum reduction in sodium selenite content was 85.2% at a C/N ratio of 2:1. The biosynthesized SeNPs exhibited antifungal activity against several fungi, such as Aspergillus flavus, Aspergillus niger, and Fusarium oxysporum, that were isolated from animal and poultry feed. Elevated SeNP concentrations (10500 ppm) significantly inhibited fungal growth. SeNPs at a concentration of 5000 ppm inhibited aflatoxin production (B1, B2, G1, and G2) by A. flavus, in addition to inhibiting mycotoxin production (T2 toxin, fumonisin B1, zearaleone, fusarin C, and moniliformin) by F. oxysporum. In conclusion, the results revealed favorable nutritional conditions for the maximum production of SeNPs by Fusarium oxysporum (CCASU-2023-F9) and indicated the marked inhibitory effect of SeNPs on mycotoxins that contaminate animal feed, causing serious consequences for animal health, and that lead to improving the quality of commercially produced animal feed. The obtained results can serve as a basis for commercial applicability.

Two hundreds and fifty samples of feeds (125 of each of ingredients of plant origin yellow corn, white corn, soya bean, soya bean meal, wheat and beans and compound manufactured feed and animal protein concentrates (meat-bone meal, fish meal, poultry offal and mixed feed). Samples were collected from various poultry farms at Giza and Cairo Governorates. Fungal isolation from ingredients of plant origin revealed lower rate of mould contamination in comparison to compound manufactured feed and animal protein concentrates. For instance, Aspergillus species was isolated from 48-68% and 80-100% of feed ingredients and animal concentrates respectively. This findings was correlated with higher levels of mycotoxins in manufactured feed and animal protein concentrates than feed ingredients of plant origin. For aflatoxins and ochratoxin A the mean levels in feed ingredients were ranged from (18-31 ppb and 15-28 ppb, respectively), while in manufactured feed and animal protein concentrates they ranged from (23-42 ppb and 32-47 ppb) respectively.The isolated Aspergillus flavus and A. ochraceus produced significant levels of respective mycotoxins. The induction of aflatoxicosis and ochratoxicosis in quails with particular reference to their partial elimination by garlic and molasses were carried out. The changes in biochemical parameters (AST, ALT, ALP, LDH, total lipid, triglyceride, cholesterol, LDL, HDL and VLDL), also in serum total protein and protein electrophoresis due to aflatoxicosis and ochratoxicosis were significantly improved under supplementation of garlic extract and molasses in diet.

An experiment with a full factorial design was used to study the effects of and interactions among temperature, water activity (a(infw)), incubation period, and substrate on coproduction of aflatoxins (AF) and cyclopiazonic acid (CPA) by an isolate of Aspergillus flavus. Analysis of variance showed that there was a complex interaction among all of these factors and that this influenced the relative concentrations of the mycotoxins produced. The optimum temperatures for the production of AF and CPA were 30(deg)C and 25(deg)C, respectively. Both mycotoxins were maximally produced (0.306 to 0.330 (mu)g of AF(middot)ml of medium(sup-1), 4.040 to 6.256 (mu)g of CPA(middot)ml of medium(sup-1)) at an a(infw) of 0.996 and after 15 days of incubation. No AF were produced in either yeast extract agar or Czapek yeast autolysate agar medium at an a(infw) of 0.90 at 20 or 37(deg)C after 15 days (minimum conditions), while 0.077 to 0.439 (mu)g of CPA(middot)ml of medium(sup-1) was produced under the same conditions. Yeast extract agar favored maximum AF production, and Czapek yeast autolysate agar favored maximum CPA production.

Aflatoxins are toxic carcinogenic secondary metabolite produced by Aspergillus flavus and are responsible for contamination in animal feed. The aim of the study was to determine the prevalence of aflatoxin contamination in animal feed in Karnataka state, India. The screening was performed by desiccated coconut agar and quantification of aflatoxin by liquid ammonia vapor test, TLC and ELISA. A total of 29 samples received from different places of Karnataka were analysed for aflatoxin B1. Out of 29 animal feed sample aflatoxin B1 detected in 12 samples representing 41.38% at average concentration of 288.50 μg/kg. Out of 42 isolates screened in animal feed, Aspergillus flavus was found to be in 86.2% and Aspergillus niger was 24.1%. It was observed that out of 42 isolates analyzed from animal feed, aflatoxin B1 was detected in 12 samples. Aflatoxin B1 is the most common contaminant and the method is more sensitive in screening and detection of aflatoxin B1 in the animal feed.

Aflatoxin contamination is a major problem in maize, groundnut, chillies, cotton and tree nuts. These aflatoxins are low molecular weight toxic and carcinogenic secondary metabolites produced by Aspergillus flavus, A. parasiticus and A. nomius. In the present study, a total of 11 isolates of A. flavus isolated from groundnut, maize and chilli collected from different locations of Tamil Nadu, India were tested for their ability to produce aflatoxin B1 (AFB1) in vitro by indirect competitive enzyme-linked immunosorbent assay. The results show that the isolates vary in their level of toxin production. The amount of AFB1 produced by the toxigenic isolates of A. flavus ranged from 6.6 to 108.1 ng ml−1. Among the various isolates of A. flavus, the isolate VKR produced the highest amount (108.1 ng ml−1) of AFB1. The isolates viz. CBE1, CBE2, BSR1, BSR3 and BSR4 were found to be non-toxigenic. The genetic variability among these isolates was assessed by Random amplified polymorphic DNA (RAPD) analysis. DNA fragments of between 0.15 and 3.0 kb were obtained using 13 random primers, and each isolate differed in the size and number of PCR products indicating considerable polymorphism. Cluster analysis using Unweighted Pair Group Method with Arithmetic Mean clearly separated the isolates into four main clusters confirming the genetic diversity among the isolates of A. flavus. Both toxigenic and non-toxigenic isolates were intermingled in these four groups, indicating that no relationship exists between RAPD profile and the production of aflatoxin by A. flavus.

Aspergillus flavus is most often associated with acute or chronic aflatoxicosis due to its ability to produce aflatoxin. These recent years, many studies have been conducted to identify biopesticides able to mitigate the aflatoxinogenic strains in soils and crops. Then, essential oils naturally contain various bioactive molecules whose antifungal properties offer promising prospects for mitigating the dangers posed by aflatoxins. This study aims to determine the chemical composition of Lippia alba essential oil and to assess its effectiveness on 2 strains of A. flavus isolated from peanut seeds in Senegal. Chemical analysis by GC-MS of the essential oil (EO) of Lippia alba revealed more oxygenated monoterpenes (84.3%) than hydrocarbons (8.4%) and oxygenated (2.4%) sesquiterpenes. Neral (34.6%) and geranial (46.6%) were also identified as major and distinctive biochemical components present in the essential oil extracted from Lippia alba plants in Senegal. Furthermore, the biological activity of this EO, at three different doses (100, 500, and 1000 ppm), was more effective in inhibiting the mycelial growth of the two isolates of Aspergillus flavus (TN and V), compared to Azoxystrobin (Positive control). The inhibition rates recorded with the highest dose (1000 ppm) reached 91.4% on the TN isolate (Peanut Basin isolate) and 84.6% on the V isolate (Casamance isolate). In contrast, after 11 days of incubation, Azoxystrobin at 1000 ppm produced 34.3% and 66.9% inhibition rates, respectively, on the TN and V isolates. Due to their chemical composition, essential oils are positioned as an alternative to synthetic pesticides and in the fight against crop pests and mycotoxins. However, for the practical use of this EO as a fungicide, future research would need to include searching for a suitable carrier appropriate inert material, allowing the development of a biopesticide formula directly applicable to peanut seeds without any negative impact on their physico-chemical, functional, and organoleptic properties.

Studies on the microflora in the air of a seed germination laboratory indicated that the highest numbers of airborne spores as measured with a Burkard spore trap occurred during working hours. When measured on a monthly basis, spore counts were greatest in winter months, when approximately 90% of all corn and soybean samples were tested. Qualitative measurements on the Andersen air sampler showed that Aspergillus flavus was the most prevalent fungus found, with A. niger and Penicillium sp. occurring in trace amounts. A significant correlation was obtained between spore counts on the day the tests were prepared and the degree of fungal growth on germination tests (r = 0.68). Germination values were negatively correlated with the amount of fungal growth (r = -0.83). Air in the laboratory and seed were identified as significant sources of these fungi, but not water and/or substrates. The quality of the seed per se might also affect contamination;Isolates of Aspergillus flavus, A. niger, Penicillium sp. and Rhizopus sp. when applied to germination tests had no effect on corn, whether it was treated with Captan or untreated, regardless of substrate. Penicillium oxalicum, A. flavus, A. niger and Rhizopus sp. caused significant reduction of soybean germinations in Kimpak and paper towels, but not on sand. Aspergillus flavus and A. niger were associated with increased cotyledonary decay, A. niger with glassy (translucent) hypocotyls and Penicillium sp. with stubby roots. According to the Rules of the Association of Official Seed Analysts, these malformations could be interpreted as abnormals in soybean germination tests. The described effects were more pronounced in poorer quality seeds and were reduced by Captan seed treatment. When inoculated onto seeds before aging, all fungi reduced germination by at least 50% after an accelerated aging period of 72 hours at 41C and 100% relative humidity. Captan and Nystatin, however, reduced fungal contaminants and increased germination.

Different species of Aspergillus growth on food and feed and by producing of secondary metabolites including aflatoxin, cause a lot of damage and reduce the value of their food. In this study the effect of different concentrations of citric acid, propionic acid and aloe vera extract on colony growth of the Aspergillus flavus and Aspergillus parasiticus were evaluated, for this purpose, the desired concentration of the compounds prepared and was added to to Czapek media. Then this environment was inoculated by five-millimeter discs containing fresh culture Aspergillus parasiticus, and the results showed that propionic acid at concentrations of 0.5, 0.8, 1 and 1.5 % completely prevented the growth of the colonies. Citric acid at concentrations of 0.5, 0.8, and 1 % has moderate anti-fungal properties, and the concentration of 1.5 % had no effect on reducing the diameter of the colony. Aloe vera plant extract with a concentration of 2, 4 and 6 % showed a deterrent effect. The relationship between aflatoxin production and sclerotia formation, isolates of Aspergillus flavus and Aspergillus parasiticus were cultured in medium Chapk, And high-pressure liquid chromatography was used to measure aflatoxin level. Compared with the total amount of aflatoxin produced by the isolates, a small sclerotium group produced more aflatoxin than the group without sclerotium and large sclerotium.

Objective: To determine the antimycotic activity of the four medicinal plant extracts, kalachuchi bark (Plumeria acuminata Ait.), atsuete bark (Bixa orellana Linn.), akapulko leaves (Cassia alata Linn.), and neem leaves (Azadirachta indica Adr. Juss), when compared to the standard clotrimazole in the treatment of otomycosis.
 
 Study Design: Experimental Study
 
 Methods: Taxonomically identified plants, kalachuchi, atsuete, akapulko, and neem tree were collected and deposited in an herbarium. Extracts of these plants and the standard clotrimazole were tested against isolates of Aspergillus flavus, Aspergillus niger, and Candida albicans taken from patients with otomycosis. Three trials were made for each extract using different solvents and results subjected to statistical analysis.
 
 Result: Of the four medicinal plant extracts studied, only kalachuchi bark extract exhibited antifungal activity against Aspergillus flavus and Aspergillus niger using methylethylketone as solvent when compared to the standard clotrimazole. It was equally effective in inhibiting the growth of A. flavus and A. niger. However, all plant extracts using all types of solvents were equally ineffective in inhibiting the growth of Candida albicans.
 
 Conclusion: This in vitro study suggested that kalachuchi (Plumeria acuminata Linn.) bark extract inhibits the growth of Aspergillus species and was comparable to the standard clotrimazole. Following appropriate further studies and clinical trials, it may be a potential alternative treatment option for otomycosis caused by Aspergillus species.
 
 
 Key words: otomycosis; kalachuchi (Plumeria acuminata Linn.) bark; antimycotic; Aspergillus flavus; Aspergillus niger

Aflatoxin B(1) (0.05 muM per larvae) was detected in silkworm larvae artificially attacked by an Aspergillus flavus isolate from a sericultural farm in Japan.

The article presents the data on the quantitative and qualitative composition of micromycetes of wheat grain grown in the Polissya region. During the research period, an average of 2.88-104 ± 3.62-103 colony forming units (CFU) per 1 g of grain was found in wheat grain samples collected in the Polissya region. Twenty species of microscopic fungi belonging to 9 genera were isolated from the wheat grain of the Polissya zone. Among them were the genera Alternaria (92.5 %), Mucor (92.5 %), Aspergillus (83.0 %), Penicillium (47.2 %), Fusarium (60.4 %), Phoma (15,.1 %), Mycelia (15.1 %), Trichotecium (1.9 %) and Monascus (1.9 %) of the samples. Aspergillus niger (17.0 %), Aspergillus candidus (9.4 %), and Aspergillus terreus (1.9 %) were detected less frequently among Aspergillus. Fusarium spp. (17.0 %), Fusarium oxysporum, Fusarium moniliforme, Fusarium semitectum in (5.7 %), and Fusarium culmorum (3.8 %) of the samples were identified among Fusarium. A smaller number of microscopic fungi species represents the endophytic mycobiota of wheat grain. Among the isolates of micromycetes isolated from the Polissya zone, pure cultures were obtained from F. sporotrichiella var. tricinctum isolate 1218/4, and F. sporotrichiella isolate 1218/5. These isolates were atoxic against the test culture Candida pseudotropicalis strain 44 PC, but F. sporotrichiella var. tricinctum 1218/4 produced a growth retardation zone with Rf 0.05 and produced an unidentified trichothecene mycotoxin (TTMT). Among the isolated fungi, Aspergillus flavus isolate 1219/3 and Aspergillus flavus isolate 1221/1 were the first to produce kojic and aspergillic acids and the second to synthesize penicillic and aspergillic acids. To reduce the negative effect of the mycotoxin deoxynivalenol on the body of chickens of the meat and egg breed Adler Silver, the birds were fed the sorbent “Mikosorb”. It was found that when added to the diet, the sorbent “Mikosorb” in the amount of 2.0 % of the total feed weight reduces the negative effect of deoxynivalenol on the body of chickens of the experimental group. This was confirmed by a 12.0 % reduction in bird mortality. Feeding “Microsorb” in the amount of 2.0 % by weight of complete feed contributed to an increase in the average daily weight gain of poultry during the experiment by 5.43 % compared to the experimental group that consumed feed with the toxin deoxynivalenol (DON). During the experiment, the birds of the experimental group No. 2 consumed 28.91 kg of feed more than group No. 2. Feeding the sorbent “Mikosorb” in the amount of 2.0 % of the feed weight to chickens of the meat and egg breed Adler silver contributes to an increase in gross weight gain by 9.69 kg during the experiment. The use of “Mikosorb” in the technology of feeding chickens of experimental group No. 2 contributed to an increase in the profitability of poultry production by up to 12.0 % compared to poultry that consumed feed affected by DON toxin. After analyzing the scientific results from the literature and our research results, we concluded that the abovementioned studies on wheat grain should be carried out throughout Ukraine during harvesting and storage in warehouses or storages.

In Vitro Susceptibility of Filamentous Fungal Isolates From a Corneal Ulcer Clinical Trial