Evaluation of an indirect hemagglutination inhibition technique for identifying antibody in animals experimentally and naturally exposed to fungi

Rabbits were exposed to aerosols containing spores of Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, or of a Penicillium sp. Sera from these rabbits were tested by indirect hemagglutination (IHA) and by IHA inhibition. The serologic reactions with the rabbit sera were compared to reactions with sera from cattle naturally exposed to airborne microorganisms. By three months of age, most cattle had positive IHA reactions to A. fumigatus and Penicillium antigens. The IHA inhibition tests indicated that antibody production in 12 of the 20 cattle probably resulted from exposure to A. flavus. One calf reacted as if sensitized by A. niger. Two were totally nonreactive. Five of the cattle had reactions that were not identifiable relative to the reactions in rabbits.Purchased by U.S. Department of Agriculture for Official Use.     

Production of gliotoxin during the pathogenic state in turkey poults byAspergillus fumigatus Fresenius

Turkey poults were given either of two different dosages of two different gliotoxin-producing strains ofAspergillus fumigatus. Infected lung tissue was examined postmortem for the presence of gliotoxin. Gliotoxin was found in lung tissue of ten poults infected with one strain and in seven of ten poults infected with the other strain. Concentrations of gliotoxin in the tissue exceeded 6 ppm in some of the infected tissues. The concentration of gliotoxin found in infected tissue did not appear to be correlated with the dosage of organism given. Considering the pathologic changes observed in turkey poults with aspergillosis and the production of gliotoxin during the pathogenic state in turkey poults, gliotoxin is considered likely to be involved in avian aspergillosis. Disclaimer: Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.     

Influence of formic acid on fungal flora of barley and on aflatoxin production inAspergillus flavus link

In recent yearsAspergillus flavus and aflatoxin production have been noted on several occasions in grain preserved with formic acid. Samples of mouldy barley treated with formic acid and stored in an open bin were investigated for the presence of fungi. In the lower part of the bin there was a clear dominance ofFusarium sporotrichioides, and deoxynivalenol and neosolaniol were detected.A. flavus andA. fumigatus were also present.Paecilomyces variotii occurred, almost as a pure culture, in the upper part of the bin, but no patulin was found. Cultivation of four fungal isolates from these genera on laboratory substrates containing formic acid showedP. variotii to be the most tolerant to formic acid, withstanding 150 mM, but still without patulin production.F. sporotrichioides andA. fumigatus tolerated only 6 mM formic acid. The growth ofA. flavus was reduced and atypical at 60 mM formic acid. Pretreatment ofA. flavus spores with formic acid increased aflatoxin production about 800 times.     

Some extracellular enzymes associated with two tomato fruit spoilage molds

The production of amylolytic, cellulolytic and pectinolytic enzymes by Aspergillus flavus and A. fumigatus was investigated. The two fungi were cultured on wheat offal and liquid crystalline carboxymethylcellulose media. A. flavus produced amylases on basal and starch containing media while A. fumigatus could only produce amylases on starch medium. The cellulolytic activities of filtrates from culture or infected fruits showed that A. flavus produced lesser quantities of cellulolytic enzymes than A. fumigatus. At 25 °C and at a pH range of 6–8, A. flavus best produces amylases and cellulases, while A. fumigatus showed highest activities of the two enzymes at 35–40 °C and at pH 7.0. Two pectinolytic enzymes — polymethylgalacturonase and pectinmethyltrans-eliminase — were identified in vivo with the two molds. An endopolygalacturonase in addition to these two pectinolytic enzymes was well associated with A. fumigatus.     

Fungi that cause mouldiness of processed sheet rubber in Western Nigeria

The fung isolated from mouldy processed sheet rubber in Western Nigeria wereAspergillus fumigatus Fres,Aspergillus flavus Link andAspergillus aculeatus Iisuka. When inoculated on sterilized, uninfected sheet rubber, bothA. fumigatus andA. flavus produced symptoms which were similar to those originally observed on mouldy processed sheet from the rubber estate.

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Zusammenfassung Die folgenden Pilze sind vom verschimmelten, bearbeiteten Rubber isoliert worden:Aspergillus fumigatus Fres,A. flavus Link andA. aculeatus Iisuka. WennA. fumigatus undA. flavus an sterilisiertem, nicht infiziertem Rubber verimpft worden sind, haben sie identische Läsionen am sterilen Rubber verursacht.

     

Aspergillus fumigatus Densities in Relation to Forest Succession and Edge Effects: Implications for Wildlife Health in Modified Environments

The hihi (or stitchbird, Notiomystis cincta) is a New Zealand endemic nectivorous forest bird now restricted to one pristine island. Relocation to establish viable hihi populations on other islands has been the main conservation action since the early 1980s. To date, hihi reintroductions to young growth islands have had poor success despite the absence of mammalian predators. It was thought that past failures were due to food limitation, but research suggests that food limitation alone cannot account for their poor survivorship. Post-mortems of dead hihi has shown that aspergillosis caused by Aspergillus fumigatus is a major mortality factor and there is current concern regarding their susceptibility to this fungal disease. In this paper we develop and assess the hypothesis that A. fumigatus limits hihi population viability on modified islands, and suggest that A. fumigatus is a potential indicator species for habitat disturbance. We report that the prevalence of A. fumigatus spores in the soil is much higher in young growth forests and forest edge habitats. Results suggest that hihi mortality rates between islands are potentially due to differential exposure to A. fumigatus spores. We assess relationships between habitat disturbance, A. fumigatus contamination and hihi mortality rates by testing the following predictions: (1) that densities of A. fumigatus spores will be higher on modified islands, (2) that densities of A. fumigatus spores on islands will be correlated with hihi mortality rates and (3) that densities of A. fumigatus spores will be higher at the forest edge than in the interior. We test each of these predictions using soil samples, air samples and samples of nectar from plant species fed on by hihi.     

A Murine Inhalation Model to Characterize Pulmonary Exposure to Dry Aspergillus fumigatus Conidia

Most murine models of fungal exposure are based on the delivery of uncharacterized extracts or liquid conidia suspensions using aspiration or intranasal approaches. Studies that model exposure to dry fungal aerosols using whole body inhalation have only recently been described. In this study, we aimed to characterize pulmonary immune responses following repeated inhalation of conidia utilizing an acoustical generator to deliver dry fungal aerosols to mice housed in a nose only exposure chamber. Immunocompetent female BALB/cJ mice were exposed to conidia derived from Aspergillus fumigatus wild-type (WT) or a melanin-deficient (Δalb1) strain. Conidia were aerosolized and delivered to mice at an estimated deposition dose of 1×10⁵ twice a week for 4 weeks (8 total). Histopathological and immunological endpoints were assessed 4, 24, 48, and 72 hours after the final exposure. Histopathological analysis showed that conidia derived from both strains induced lung inflammation, especially at 24 and 48 hour time points. Immunological endpoints evaluated in bronchoalveolar lavage fluid (BALF) and the mediastinal lymph nodes showed that exposure to WT conidia led to elevated numbers of macrophages, granulocytes, and lymphocytes. Importantly, CD8⁺ IL17⁺ (Tc17) cells were significantly higher in BALF and positively correlated with germination of A. fumigatus WT spores. Germination was associated with specific IgG to intracellular proteins while Δalb1 spores elicited antibodies to cell wall hydrophobin. These data suggest that inhalation exposures may provide a more representative analysis of immune responses following exposures to environmentally and occupationally prevalent fungal contaminants.     

FT-IR Spectroscopy for Rapid Differentiation of Aspergillus flavus, Aspergillus fumigatus, Aspergillus parasiticus and Characterization of Aflatoxigenic Isolates Collected from Agricultural Environments

In agricultural areas, Aspergillus flavus, Aspergillus fumigatus and Aspergillus parasiticus are commonly identified in various feedstuffs and bioaerosols originated from feed handling. Some isolates belonging to these fungal species could produce mycotoxins and constitute a risk factor for human and animal health. In this study, Fourier-transform infrared spectroscopy was used for a rapid detection and characterization of 99 isolates collected from agricultural areas. The results showed a first cluster corresponding to strains previously attributed to the A. fumigatus group according to current taxonomic concepts, and a second cluster divided in 2 groups around reference strains of A. flavus and A. parasiticus species. The toxigenic capacity of isolates was evaluated by high performance liquid chromatography coupled to mass spectrometry. In the A. flavus group, only 6 strains of A. parasiticus and 4 strains of A. flavus were able to produce aflatoxins on culture media. FT-IR spectroscopy, respectively, allowed the differentiation of non-toxigenic and toxigenic A. flavus and A. parasiticus isolates at 75 and 100%. Discrimination between toxigenic and non-toxigenic A. fumigatus was not possible because all of the isolates produced at least one mycotoxin.     

Comparative study of Aspergillus mycotoxin production on enriched media and construction material

Isolates of Aspergillus flavus and Aspergillus fumigatus from indoor air were compared with a known mycotoxin producer for their capacity to produce mycotoxins on a variety of enrichment media and with growth on indoor substrates such as ceiling tile and wall board. In enrichment media, four of seven isolates of A. flavus produced at least one aflatoxin and both isolates of A. fumigatus produced mycotoxins. The spectrum of mycotoxins and their concentrations varied with the strain and medium. When the mycotoxin-positive strains were grown to a dense concentration on indoor construction and finishing materials such as ceiling tile and wall boards, mycotoxins were not detected in extracts of the materials. Colonization of indoor surfaces by mycotoxin-producing strains of A. flavus and A. fumigatus may not necessarily expose inhabitants to mycotoxins or result in production of mycotoxins. Received 09 February 1999/ Accepted in revised form 28 June 1999     

Effects of Conidia of Various Aspergillus Species on Apoptosis of Human Pneumocytes and Bronchial Epithelial Cells

Aspergillus species can cause mycoses in human and animals. Previously, we demonstrated that A. fumigatus conidia from a human isolate inhibited apoptosis in human pneumocytes and bronchial epithelial cells. In the current study, we studied the effects of A. fumigatus conidia non-human origin and A. flavus, A. nidulans, A. niger and A. oryzae conidia on human cells apoptosis. Human pneumocytes or bronchial epithelial cells were simultaneously exposed to apoptotic inductors and aspergilli conidia. The cell cultures were analyzed by flow cytometry, immunoblotting, and examination of nuclear morphology. Similar to A. fumigatus conidia, A. flavus conidia inhibited cellular apoptosis while A. nidulans, A. niger and A. oryzae conidia did not affect apoptosis. We further studied the species specificity of conidia: there were no differences in the inhibition of apoptosis by A. fumigatus conidia from either human or bird isolates. In order to determine whether the inhibition of apoptosis by conidia is limited to certain strains, the effect on human cell apoptosis of different A. fumigatus human clinical isolates and A. fumigatus of environmental origin was evaluated. All A. fumigatus isolates inhibited apoptosis; an anti-apoptotic factor was released by conidia. For TNF-induced apoptosis, the anti-apoptotic effect of conidia of all isolates was found to be associated with a reduction of caspase-3 in human cells. The results suggest that suppression of apoptosis may play a role in reducing the efficacy of host defense mechanisms during infection with Aspergillus species. F. Féménia and D. Huet made an equal contribution to this work.     

Prevalence of parasitism by Amblyomma americanum on wild turkey poults as influenced by prescribed burning.

Captive-raised eastern wild turkey (Meleagris gallopavo silvestris) poults were allowed to forage on either recently burned plots or on plots that had not been burned during the previous 3 years. Following a two hour exposure on the study plots, external parasites were recovered from all poults. The louse (Menacanthus stramineus) and the lone star tick (Amblyomma americanum) were the only parasites recovered. Fifty-nine poults exposed to the burned plots had a significantly (P less than 0.001) lower prevalence of A. americanum infestation than did 57 poults exposed to the unbured plots. Thirty-five percent of the poults exposed to the unburned plots were infested by A. americanum. A single A. americanum was recovered from one poult exposed to burned plots.     

Prevalence of airborne Aspergillus flavus in Khartoum (Sudan) Airspora with reference to dusty weather and inoculum survival in simulated summer conditions

Khartoum air was scanned for airborne Aspergillus flavus for 12 months using the horizontal gravitational settling method. Frequency of occurrence was related to total fungal catch and dusty weather. The Aspergilli were prevalent (68% of total isolated/plate/month) and A. flavus constituted 31% of the total Aspergilli. In June (hot, dry & dusty) Aspergilli constituted 79% of the total isolates, whilst A. flavus represented 30% from amongst the other Aspergilli. A. flavus, A. niger, A. nidulans (conidial & ascosporic states), A. terreus, Eurotium amstelodami and A. fumigatus, in descending order of prevalence were isolated in June. Other pathogenic or potentially pathogenic forms, isolated, were Cladosporium, Curvularia and Penicillium. Amongst winter isolations A. flavus was sporadic to absent in occurrence. A. flavus spore inocula that underwent hourly intermitted exposure to 45 °C, showed a decrease in spore germinability as well as reduced germ length.     

Lysine acetylation contributes to development,aflatoxin biosynthesis and pathogenicity in Aspergillus flavus

Aspergillus flavus is a pathogenic fungus that produces carcinogenic aflatoxins, posing a great threat to crops, animals and humans. Lysine acetylation is one of the most important reversible post-translational modifications and plays a vital regulatory role in various cellular processes. However, current information on the extent and function of lysine acetylation and aflatoxin biosynthesis in A. flavus is limited. Here, a global acetylome analysis of A. flavus was performed by peptide pre-fractionation, pan-acetylation antibody enrichment and liquid chromatography–mass spectrometry. A total of 1313 high-confidence acetylation sites in 727 acetylated proteins were identified in A. flavus. These acetylation proteins are widely involved in glycolysis/gluconeogenesis, pentose phosphate pathway, citric acid cycle and aflatoxin biosynthesis. AflO (O-methyltransferase), a key enzyme in aflatoxin biosynthesis, was found to be acetylated at K241 and K384. Deletion of aflO not only impaired conidial and sclerotial developments, but also dramatically suppressed aflatoxin production and pathogenicity of A. flavus. Further site-specific mutations showed that lysine acetylation of AflO could also result in defects in development, aflatoxin production and pathogenicity, suggesting that acetylation plays a vital role in the regulation of the enzymatic activity of AflO in A. flavus. Our findings provide evidence for the involvement of lysine acetylation in various biological processes in A. flavus and facilitating in the elucidation of metabolic networks.     

Can it be all more simple? Manufacturing aflatoxin biocontrol products using dry spores of atoxigenic isolates of Aspergillus flavus as active ingredients

Aflatoxin contamination of staple crops, commonly occurring in warm areas, negatively impacts human and animal health, and hampers trade and economic development. The fungus Aspergillus flavus is the major aflatoxin producer. However, not all A. flavus genotypes produce aflatoxins. Effective aflatoxin control is achieved using biocontrol products containing spores of atoxigenic A. flavus. In Africa, various biocontrol products under the tradename Aflasafe are available. Private and public sector licensees manufacture Aflasafe using spores freshly produced in laboratories adjacent to their factories. BAMTAARE, the licensee in Senegal, had difficulties to obtain laboratory equipment during its first year of production. To overcome this, a process was developed in Ibadan, Nigeria, for producing high-quality dry spores. Viability and stability of the dry spores were tested and conformed to set standards. In 2019, BAMTAARE manufactured Aflasafe SN01 using dry spores produced in Ibadan and sent via courier and 19 000 ha of groundnut and maize in Senegal and The Gambia were treated. Biocontrol manufactured with dry spores was as effective as biocontrol manufactured with freshly produced spores. Treated crops contained safe and significantly (P < 0.05) less aflatoxin than untreated crops. The dry spore innovation will make biocontrol manufacturing cost-efficient in several African countries.     

Gliotoxin inhibits transformation and its cytotoxic to turkey peripheral blood lymphocytes

Gliotoxin, an epipolythiodioxopiperizine mycotoxin, has been shown to be produced by, among other fungi,Aspergillus fumigatus Fresenius. This organism is the major causative agent of the respiratory disease aspergillosis in avian species, especially turkeys. Because gliotoxin has been shown to be immunosuppressive and has the potential for being involved in the pathogenesis of aspergillosis, the in vitro activity of this compound with avian lymphocytes was investigated. Immunosuppression was investigated using peripheral blood lymphocytes from turkeys in a lymphoblastogenesis assay and a cytotoxicity assay using conversion of the tetrazolium salt MTT to MTT formazan by the mitochondrial succinate dehydrogenase enzyme elaborated only by living cells. Gliotoxin appeared to have a threshold level in both tests because little or no response or stimulation was evident when cells were exposed to concentrations of the toxin below 100 ng/ml, but at 100 ng/ml, all cells appeared to be dead. Using T-2 mycotoxin as a known cytotoxic agent, the response in the MTT bioassay using turkey peripheral lymphocytes was linear with increasing concentrations of toxin. Gliotoxin may potentially cause immunosuppression in turkey poults through action on the lymphocytes or if this toxin were present in low concentrations stimulation could possibly occur.     

Aerobiological,biochemical and immunological studies on some of the dominant <Emphasis Type="Italic">Aspergillus</Emphasis> species of South Assam (India)

Two years atmospheric survey of air-borne Aspergillus was carried out in the environmental conditions of South Assam. The survey revealed a total of 16 different species of Aspergillus with marked seasonal and annual variations. Aspergillus fumigatus was found to be the dominant atmospheric fungal species followed by Aspergillus flavus, Aspergillus niger, etc. Among the sample extracts tested, highest quantity of soluble protein was recorded in Aspergillus fumigatus (95.0 mg/g) whereas highest quantity of soluble carbohydrate (40.8 mg/g) and free amino acid (135.0 mg/g) was recorded in the sample extract of Aspergillus niger per gram of dry weight, respectively. The highest numbers of protein polypeptide bands were detected in the sample extract of Aspergillus fumigatus followed by Aspergillus flavus and lowest in Aspergillus niger. The maximum numbers of immunoglobulin E binding protein fractions were found in Aspergillus fumigatus, followed by Aspergillus flavus, Aspergillus clavatus, etc.     

Melanin targets LC3-associated phagocytosis (LAP): A novel pathogenetic mechanism in fungal disease

Intracellular swelling of conidia of the major human airborne fungal pathogen Aspergillus fumigatus results in surface exposure of immunostimulatory pathogen-associated molecular patterns (PAMPs) and triggers activation of a specialized autophagy pathway called LC3-associated phagocytosis (LAP) to promote fungal killing. We have recently discovered that, apart from PAMPs exposure, cell wall melanin removal during germination of A. fumigatus is a prerequisite for activation of LAP. Importantly, melanin promotes fungal pathogenicity via targeting LAP, as a melanin-deficient A. fumigatus mutant restores its virulence upon conditional inactivation of Atg5 in hematopoietic cells of mice. Mechanistically, fungal cell wall melanin selectively excludes the CYBA/p22phox subunit of NADPH oxidase from the phagosome to inhibit LAP, without interfering with signaling regulating cytokine responses. Notably, inhibition of LAP is a general property of melanin pigments, a finding with broad physiological implications.     

Effect of Bile on Infectivity of Histomonas meleagridis of Liver Origin

SYNOPSIS After 30 min exposure to fresh turkey bile at 99 F, 100,000 Histomonas meleagridis of turkey liver origin failed to infect any of 20 poults inoculated rectally. Only one of 20 poults similarly inoculated with histomonads exposed to fresh turkey bile for only 5 min at 99 F became infected. Fifteen of 20 poults became infected following rectal inoculation with 100,000 H. meleagridis of liver origin held in physiologic saline for 30 min at 99 F. All infected birds developed liver lesions and died. Altho H. meleagridis was very abundant in the livers of all potential donor poults and all infected recipient poults, histomonads were never found in the gall bladders of any of these birds. In some such birds, a few apparently degenerating histomonads were detected in the duodenum near the entrance of the bile ducts. Histomonads of liver origin are regarded as of little or no consequence in the transmission of this protozoon.     

Rye Affects Bacterial Translocation,Intestinal Viscosity,Microbiota Composition and Bone Mineralization in Turkey Poults

Previously, we have reported that rye significantly increased both viscosity and Clostridium perfringens proliferation when compared with corn in an in vitro digestive model. Two independent trials were conducted to evaluate the effect of rye as a source of energy on bacterial translocation, intestinal viscosity, gut microbiota composition, and bone mineralization, when compared with corn in turkey poults. In each experiment, day-of-hatch, turkey poults were randomly assigned to either a corn or a rye diet (n = 0 /group). At 10 d of age, in both experiments, 12 birds/group were given an oral gavage dose of fluorescein isothiocyanate dextran (FITC-d). After 2.5 h of oral gavage, blood and liver samples were collected to evaluate the passage of FITC-d and bacterial translocation (BT) respectively. Duodenum, ileum and cecum gut sections were collected to evaluate intestinal viscosity and to enumerate gut microbiota. Tibias were collected for observation of bone parameters. Broilers fed with a rye diet showed increased (p<0.05) intestinal viscosity, BT, and serum FITC-d. Bacterial enumeration revealed that turkey poults fed with rye had increased the number of total lactic acid bacteria (LAB) in all three sections of the gastrointestinal tract evaluated when compared to turkey poults fed with corn. Turkey poults fed with rye also had significantly higher coliforms in duodenum and ileum but not in the ceca, whereas the total number of anaerobes increased only in duodenum. A significant reduction in bone strength and bone mineralization was observed in turkey poults fed with rye when compared with corn fed turkey poults. In conclusion, rye evoked mucosal damage in turkey poults that increased intestinal viscosity, increased leakage through the intestinal tract, and altered the microbiota composition and bone mineralization. Studies to evaluate dietary inclusion of selected Direct-Fed Microbial (DFM) candidates that produce exogenous enzymes in rye fed turkey poults are currently being evaluated.