Evaluation of the endotoxin binding efficiency of clay minerals using the Limulus Amebocyte lysate test: an in vitro study

Endotoxins are part of the cell wall of Gram-negative bacteria. They are potent immune stimulators and can lead to death if present in high concentrations. Feed additives, which bind endotoxins in the gastrointestinal tract of animals, could help to prevent their negative impact. The objective of our study was to determine the potential of a bentonite (Bentonite 1),a sodium bentonite (Bentonite 2),a chemically treated smectite (Organoclay 1) and a modified attapulgite (Organoclay 2) to bind endotoxins in vitro. Polymyxin B served as positive control. The kinetic chromogenic Limulus Amebocyte lysate test was adapted to measure endotoxin activity. Firstly, a single sorption experiment (10 endotoxin units/mL (EU/mL)) was performed. Polymyxin B and organoclays showed 100% binding efficiency. Secondly, the adsorption efficiency of sorbents in aqueous solution with increasing endotoxin concentrations (2, 450 - 51, 700 EU/mL) was investigated. Organoclay 1 (0.1%) showed a good binding efficiency in aqueous solution (average 81%),whereas Bentonite 1 (0.1%) obtained a lower binding efficiency (21-54%). The following absorbent capacities were calculated in highest endotoxin concentration: 5.59 mg/g (Organoclay 1) > 3.97 mg/g (Polymyxin B) > 2.58mg/g (Organoclay 2) > 1.55 mg/g (Bentonite 1) > 1.23 mg/g (Bentonite 2). Thirdly, a sorption experiment in artificial intestinal fluid was conducted. Especially for organoclays, which are known to be unspecific adsorbents, the endotoxin binding capacity was significantly reduced. In contrast, Bentonite 1 showed comparable results in artificial intestinal fluid and aqueous solution. Based on the results of this in vitro study, the effect of promising clay minerals will be investigated in in vivo trials

Milk Thistle Extract and Silymarin Inhibit Lipopolysaccharide Induced Lamellar Separation of Hoof Explants in Vitro

The pathogenesis of laminitis is not completely identified and the role of endotoxins (lipopolysaccharides, LPS) in this process remains unclear. Phytogenic substances, like milk thistle (MT) and silymarin, are known for their anti-inflammatory and antioxidant properties and might therefore have the potential to counteract endotoxin induced effects on the hoof lamellar tissue. The aim of our study was to investigate the influence of endotoxins on lamellar tissue integrity and to test if MT and silymarin are capable of inhibiting LPS-induced effects in an in vitro/ex vivo model. In preliminary tests, LPS neutralization efficiency of these phytogenics was determined in an in vitro neutralization assay. Furthermore, tissue explants gained from hooves of slaughter horses were tested for lamellar separation after incubation with different concentrations of LPS. By combined incubation of explants with LPS and either Polymyxin B (PMB; positive control), MT or silymarin, the influence of these substances on LPS-induced effects was assessed. In the in vitro neutralization assay, MT and silymarin reduced LPS concentrations by 64% and 75%, respectively, in comparison PMB reduced 98% of the LPS concentration. In hoof explants, LPS led to a concentration dependent separation. Accordantly, separation force was significantly decreased by 10 µg/mL LPS. PMB, MT and silymarin could significantly improve tissue integrity of explants incubated with 10 µg/mL LPS. This study showed that LPS had a negative influence on the structure of hoof explants in vitro. MT and silymarin reduced endotoxin activity and inhibited LPS-induced effects on the lamellar tissue. Hence, MT and silymarin might be used to support the prevention of laminitis and should be further evaluated for this application

Concentration Dependent Influence of Lipopolysaccharides on Separation of Hoof Explants and Supernatant Lactic Acid Concentration in an Ex Vivo/In Vitro Laminitis Model.

Laminitis is one of the most common diseases in horses. It is not only painful for the animal, but also has a significant financial impact on the equine industry. This multifactorial disease affects the connective tissue of the hoof. However, the pathogenesis of laminitis is still not fully understood. Endotoxins, also known as lipopolysaccharides (LPS), and bacterial exotoxins seem to play an important role during the development of laminitis. The aim of our study was to investigate the effect of increasing LPS concentrations (0, 2.5, 5, 10, and 100 μg/mL) on cell viability of isolated epidermal and dermal hoof cells as well as on the tissue integrity of hoof explants. Furthermore, glucose, acetic acid, lactic acid, and propionic acid concentrations in explant supernatants were measured to evaluate the energy metabolism in the hoof tissue. LPS did not exhibit cytotoxic effects on epidermal or dermal cells. Force required to separate LPS treated hoof explants decreased in a concentration dependent manner. Specifically, explants incubated with 10 and 100 μg/mL needed significantly less force to separate compared to control explants. Lactic acid concentrations were significantly decreased in explants incubated with 5, 10, or 100 μg/mL LPS, while glucose, acetic acid and propionic acid concentrations were unaffected by LPS treatment. Our study indicates that LPS has no cytotoxic effect on epidermal and dermal cells isolated from hoof tissue, but impairs integrity of hoof explants. In addition, LPS led to an alteration of the lactic acid production in the lamellar tissue. Since our data highlight that LPS can affect the integrity of the equine hoof tissue in vitro, endotoxins should be further explored for their contribution to facilitate the development of laminitis

Fumonisin B1 (FB1) Induces Lamellar Separation and Alters Sphingolipid Metabolism of In Vitro Cultured Hoof Explants

One of the most important hoof diseases is laminitis. Yet, the pathology of laminitis is not fully understood. Different bacterial toxins, e.g. endotoxins or exotoxins, seem to play an important role. Additionally, ingestion of mycotoxins, toxic secondary metabolites of fungi, might contribute to the onset of laminitis. In this respect, fumonsins are of special interest since horses are regarded as species most susceptible to this group of mycotoxins. The aim of our study was to investigate the influence of fumonisin B1 (FB1) on primary isolated epidermal and dermal hoof cells, as well as on the lamellar tissue integrity and sphingolipid metabolism of hoof explants in vitro. There was no effect of FB1 at any concentration on dermal or epidermal cells. However, FB1 significantly reduced the separation force of explants after 24 h of incubation. The Sa/So ratio was significantly increased in supernatants of explants incubated with FB1 (2.5–10 µg/mL) after 24 h. Observed effects on Sa/So ratio were linked to significantly increased sphinganine concentrations. Our study showed that FB1 impairs the sphingolipid metabolism of explants and reduces lamellar integrity at non-cytotoxic concentrations. FB1 might, therefore, affect hoof health. Further in vitro and in vivo studies are necessary to elucidate the effects of FB1 on the equine hoof in more detail

Morphology of primary isolated hoof cells.

<p>Epidermal (A) and dermal (B) cells stained with FITC conjugated wheat germ agglutinin.</p

Correlation of lactic acid concentration in supernatants and separation force of explants.

<p>Correlation of lactic acid concentration in supernatants and separation force of explants incubated with medium or LPS [0–100 μg/mL] for 24 hours (n = 3 hooves).</p

Current challenges in the diagnosis of zearalenone toxicosis as illustrated by a field case of hyperestrogenism in suckling piglets

Abstract Background The mycotoxin zearalenone (ZEN) causes functional and morphological alterations in reproductive organs of pigs. In the field, diagnosis of ZEN-induced disorders is often challenging, as relevant feed lots are no longer available, or feed analysis results are not conclusive. Here, we report a field case of hyperestrogenism in newborn piglets. Surprisingly, more than 50 fungal metabolites were detected in hay pellets fed to gestating sows, including ZEN and its modified form zearalenone-14-sulfate (ZEN-14-S). Despite the broad contamination range in this unconventional feed component, a definite diagnosis of mycotoxicosis could not be achieved. In this context, current limitations regarding the confirmation of suspected cases of ZEN-induced disorders are discussed, covering both feed analysis and the biomarker approach. Case presentation A piglet producer with 200 sows experienced a sudden increase in suckling piglet losses up to 30% by lower vitality and crushing. Predominant clinical signs were splay legs and signs of hyperestrogenism such as swollen and reddened vulvae in newborn piglets. The first differential diagnosis was ZEN mycotoxicosis although feed batches had not been changed for months with the exception of ground hay pellets, which had been included in the diet five months before. Analysis of hay pellets resulted in a sum value of ZEN and its modified forms of more than 1000 μg/kg, with ZEN-14-S alone accounting for 530 μg/kg. Considering the inclusion rate of 7% in the diet for gestating sows, the severe impact of the additional ZEN load due to the contaminated hay pellets seemed unrealistic but could not be completely excluded either. One month after hay pellets had been removed from the diet no further clinical signs were observed. Conclusions Enrichment materials and other fibre sources can contain significant amounts of mycotoxins and should be therefore included in feed analysis. Adequate methods for broad spectrum mycotoxin determination, including modified mycotoxins, are important. As highlighted by this field case, there is a need to establish reliable biomarkers for ZEN exposure in pigs. Currently, available biomarkers do not allow a solid prediction of the ZEN intake of pigs under field conditions, which limits their application to experimental studies

Glucose, acetic acid, lactic acid, and propionic acid concentration of supernatants of hoof explants incubated withLPS.

<p>Glucose, acetic acid, lactic acid, and propionic acid concentration measured of culture medium and supernatant of explants (n = 12 explants) incubated with medium or LPS [0–100 μg/mL] for 24 hours.</p><p>^ab Superscripts indicate significant difference p < 0.05.</p><p>Glucose, acetic acid, lactic acid, and propionic acid concentration of supernatants of hoof explants incubated withLPS.</p

Viablity of primary isolated hoof cells incubated with LPS.

<p>Absorbance values (450 nm) of epidermal (A, blue) and dermal (B, green) cells incubated with medium or LPS (2.5–100 μg/mL) for 24 hours measured with the WST-1 assay (n = 4). Error bars display standard deviation.</p

Viability of hoof explants incubated with LPS.

<p>Absorbance values (450 nm) of supernatants of explants incubated with medium or LPS (2.5–100 μg/mL) for 24 hours measured with the WST-1 assay (n = 3). Error bars display standard deviation.</p