<i>In vitro</i> model to assess the adsorption of oral veterinary drugs to mycotoxin binders in a feed- and aflatoxin B1-containing buffered matrix

<p>Mycotoxin binders are feed additives which are mixed in the feed to adsorb mycotoxins and thereby reducing their toxic effects on animals. Interactions with orally administered veterinary medicinal products, such as antimicrobials or coccidiostats, have been reported previously. This paper describes an <i>in vitro</i> model to screen the interaction between mycotoxin binders and veterinary drugs with respect to the non-specific binding of drugs. It is designed as a static setup using a single concentration of drug and binder in a feed-containing or a feed-plus-mycotoxin-containing matrix, buffered at different pH values. The model was applied to two frequently used antimicrobials in veterinary medicine, doxycycline (DOX) and tylosin (TYL), one major mycotoxin, aflatoxin B1 (AFB1), and four mycotoxin binders. Proportions of feed, DOX or TYL, AFB1, and binder are equivalent to the <i>in vivo</i> situation for broiler chickens, while pH and volume of the buffer are representative of the gastrointestinal tract of chickens. A substantial binding of DOX (~ 88%) and TYL (~ 66%) to the feed-matrix was observed. For the mycotoxin binders, similar results were obtained for DOX and TYL; more specifically up to an inclusion rate of 20 g binder/kg feed, no significant binding was demonstrated, determined as the free concentration of DOX and TYL. A single exception was noticed for TYL and one specific bentonite-based mycotoxin binder, for which no significant interaction could be demonstrated up to 10 g binder/kg but there was an effect at 20 g/kg. In all cases, there was no competition between the tested drugs DOX or TYL and the mycotoxin AFB1 for binding to the bentonite-based mycotoxin binder.</p

Comparative Oral Bioavailability, Toxicokinetics, and Biotransformation of Enniatin B1 and Enniatin B in Broiler Chickens

A toxicokinetic study of the Fusarium mycotoxins enniatin B1 (ENN B1) and enniatin B (ENN B) was performed in broiler chickens. Each animal received ENN B1 or B orally via an intracrop bolus and intravenously at a dose of 0.2 mg/kg body weight. Both enniatins were poorly absorbed after oral administration, with absolute oral bioavailabilities of 0.05 and 0.11 for ENNs B1 and B, respectively. Both enniatins were readily distributed to the tissues, with mean volumes of distribution of 25.09 and 33.91 L/kg for ENNs B1 and B, respectively. The mean total body clearance was rather high, namely, 6.63 and 7.10 L/h/kg for ENNs B1 and B, respectively. Finally, an UHPLC-HRMS targeted approach was used to investigate the phase I and II biotransformations of both mycotoxins. Oxygenation was the major phase I biotransformation pathway for both ENNs B1 and B. Neither glucuronide nor sulfate phase II metabolites were detected

Effects of DON and an adsorbent on oxidative stress in the liver of broiler chickens.

<p>Results are presented as mean (± SEM) mRNA expression. Fold change in gene expression levels of the chicken liver relative to control group, which is considered 1. * Indicates significant differences between treated and control animals.</p

Length of villi (µm) and crypt depth (µm) in duodenum, jejunum and ileum after 3 weeks feeding a control diet or feed contaminated with DON, either or not supplemented with an adsorbing agent. Results are presented as mean values and standard deviations of fifteen villi or crypts measured from 8 chickens per treatment group.

<p>a,b mean values within a row with unlike superscript letters are significantly different (p≤0.05).</p

Effects of DON and an adsorbent on intestinal barrier in broiler chickens.

<p>Results are presented as mean (± SEM) mRNA expression. Fold change in gene expression levels of the chicken intestines relative to control group, which is considered 1. ^a–c Different lower-case letters indicate significant differences between groups.</p

TEM overview of the development of <i>Bd</i> in skin explants of <i>Xenopus laevis</i>.

<p>(<b>A</b>) adhesion of an encysted zoospore (ZS) to the superficial mucus layer (M) on top of the stratum corneum (SC); at the site where adhesion occurs the cell wall of the encysted zoospore is remarkably thickened (arrow); scale bar = 500 nm; (<b>B</b>) initiation of germ tube development (arrow); note the polarisation of the cell cytoplasm (*); scale bar = 2 µm; (<b>C</b>) germ tube (GT) elongating upon the epidermis of <i>X. laevis</i>, with the presence of numerous lipid globules (LG) in the germ tube; scale bar = 1 µm; (<b>D</b>) a growing germ tube protruding the stratum corneum; scale bar = 2 µm; (<b>E</b>) invasion of a host cell resulting in the loss of cell cytoplasm; remnants of the host cell cytoplasm (arrow) are seen at the tip of a protruded germ tube; note the presence of a collapsed sporangium (ZS) due to cell polarisation (*); (SS): stratum spinosum; scale bar = 2 µm; (<b>F</b>) infected epidermal cell with digested cell content (*) alternated by an uninfected normal epidermal cell; note the presence of lipid globules in the infected host cell; scale bar = 1 µm.</p

Germ Tube Mediated Invasion of <em>Batrachochytrium dendrobatidis</em> in Amphibian Skin Is Host Dependent

<div><p><em>Batrachochytrium dendrobatidis</em> (<em>Bd</em>) is the causative agent of chytridiomycosis, a fungal skin disease in amphibians and driver of worldwide amphibian declines.</p> <p>We focussed on the early stages of infection by <em>Bd</em> in 3 amphibian species with a differential susceptibility to chytridiomycosis. Skin explants of <em>Alytes muletensis</em>, <em>Litoria caerulea</em> and <em>Xenopus leavis</em> were exposed to <em>Bd</em> in an Ussing chamber for 3 to 5 days. Early interactions of <em>Bd</em> with amphibian skin were observed using light microscopy and transmission electron microscopy. To validate the observations <em>in vitro</em>, comparison was made with skin from experimentally infected frogs. Additional <em>in vitro</em> experiments were performed to elucidate the process of intracellular colonization in <em>L. caerulea</em>.</p> <p>Early interactions of <em>Bd</em> with amphibian skin are: attachment of zoospores to host skin, zoospore germination, germ tube development, penetration into skin cells, invasive growth in the host skin, resulting in the loss of host cell cytoplasm. Inoculation of <em>A. muletensis</em> and <em>L. caerulea</em> skin was followed within 24 h by endobiotic development, with sporangia located intracellularly in the skin. Evidence is provided of how intracellular colonization is established and how colonization by <em>Bd</em> proceeds to deeper skin layers. Older thalli develop rhizoid-like structures that spread to deeper skin layers, form a swelling inside the host cell to finally give rise to a new thallus.</p> <p>In <em>X. laevis</em>, interaction of <em>Bd</em> with skin was limited to an epibiotic state, with sporangia developing upon the skin. Only the superficial epidermis was affected. Epidermal cells seemed to be used as a nutrient source without development of intracellular thalli. The <em>in vitro</em> data agreed with the results obtained after experimental infection of the studied frog species. These data suggest that the colonization strategy of <em>B. dendrobatidis</em> is host dependent, with the extent of colonization most likely determined by inherent characteristics of the host epidermis.</p> </div

TEM overview of the development of <i>Bd</i> in skin explants of <i>Alytes muletesis and Litoria caerulea</i>.

<p>(<b>A</b>) infected epidermis of <i>A. mulentensis</i> at 1 dpi, with loss of the host cell cytoplasma and the presence of germ tube fragments inside the infected cell in cross and longitudinal section (arrow); scale bar = 2 µm; (<b>B</b>) infected epidermis of <i>L. caerulea</i> at 2 dpi showing colonization of the stratum corneum, loss of the host cell cytoplasm and the presence of germ tube fragments (arrow); intracellular chytrid sporangia are observed in the stratum spinosum; scale bar = 2 µm; GT; germ tube, SC: stratum corneum, SP: sporangium, SS: stratum corneum, ZS: encysted zoospore.</p

Light microscopical overview of the development of <i>Bd</i> in skin explants of <i>Alytes muletensis</i> and <i>Litoria caerulea</i>.

<p>(<b>A</b>) at 1 day post infection (dpi) germlings have developed germ tubes (arrow) that invade the epidermis of <i>A. muletensis</i>; Gomori methenamine silver (GMS) stain; scale bar = 10 µm; (<b>B</b>) at 1 dpi both <i>Bd</i> germlings (black arrow) attached upon the epidermal surface as intracellular chytrid thalli (white arrow) in the stratum corneum of <i>L. caerulea</i> are observed; haematoxylin and eosin stain; scale bar = 10 µm.</p

Light microscopical overview of the development of <i>Bd</i> in skin explants of <i>Xenopus laevis</i>.

<p>(<b>A</b>) adhesion of encysted zoospores (arrow) to the host epidermis at 1 dpi; (1) stratum corneum, (2) stratum spinosum; haematoxylin and eosin (HE) stain; scale bar = 20 µm; (<b>B</b>) at 1 dpi <i>Bd</i> germlings have developed germ tubes, that penetrate the stratum corneum and develop into a branched mesh work of rhizoids (arrow) in heavily infected epidermis; Gomori methenamine silver stain; scale bar = 10 µm; (<b>C</b>) at 2 dpi the infected host cells have lost their cytoplasm (arrow) subsequent to invasion by <i>Bd</i>, only the cell membrane remains; HE stain; scale bar = 20 µm; (<b>D</b>) at 4 dpi germlings have developed into mature zoosporangia (arrow), the upper layer of the stratum corneum is shed; HE stain; scale bar = 20 µm.</p