Efficacy of chestnut and quebracho wood extracts to control Salmonella in poultry.

AIMS: The study was aimed to evaluate the antibacterial activity and efficacy of chestnut and quebracho wood extracts against Salmonella by in vitro assays and in vivo trials. METHODS AND RESULTS: The extracts showed inhibitory activity against Salmonella determined by the minimum inhibitory concentration method as well as on the adhesion and invasion of S. Gallinarum (SG) and S. Enteritidis (SE) in Caco-2 cells. Also, transmission electron microscopy revealed that extract-treated Salmonella showed disruption of cell walls and membranes, damage of the cytoplasm and tannin-protein aggregations. In addition, efficacy of the extracts to control SG and SE was evaluated in experimental infection trials in laying hens and broilers respectively. SE excretion was significantly reduced on days 5 (P < 0·01) and 12 (P < 0·025) only in the quebracho group. In the fowl typhoid infection model, hens that received the chestnut extract showed a significantly reduced mortality (P < 0·05). CONCLUSIONS: Our results evidence that these alternative natural products may be a useful tool to control Salmonella in poultry. SIGNIFICANCE AND IMPACT OF THE STUDY: Salmonella is a zoonotic pathogen usually associated with poultry production. This study provides information about the mechanism of antibacterial effects of chestnut and quebracho wood extracts to control Salmonella in poultry.

Use of Plant Extracts as an Effective Manner to Control Clostridium perfringens Induced Necrotic Enteritis in Poultry.

Necrotic enteritis (NE) is an important concern in poultry industry since it causes economic losses, increased mortality, reduction of bird welfare, and contamination of chicken products for human consumption. For decades, the use of in-feed antimicrobial growth promoters (AGPs) has been the main strategy to control intestinal pathogens including Clostridium perfringens (CP), the causative agent of NE. However, the use of AGPs in animal diet has been linked to the emergence and transmission of antimicrobial resistance through food-borne microorganisms, which has led to the ban of AGPs in many countries. This scenario has challenged the poultry industry to search for safer alternative products in order to prevent NE. In this context, the utilization of natural plant extracts with antimicrobial properties appears as a promising and feasible tool to control NE in chicken. In this paper, we review the scientific studies analyzing the potential of plant extracts as alternative feed additives to reduce NE in poultry, with focus on two types of plant products that arise as promising candidates: tannins and essential oils. Some of these products showed antimicrobial activity against CP and coccidia in vitro and in vivo and are able to increase productive performance, emulating the bioactive properties of AGPs.

Hydrolyzable and condensed tannins resistance in Clostridium perfringens.

Tannins added in the diet are being used to improve nutrition and health in farm animals as an alternative to antibiotic growth promoters and to control enteric clostridial diseases. However, the capacity of Clostridium perfringens to develop resistance under the selective pressure of tannins is unknown. The purpose of this study was to determine if C. perfringens possess the ability to develop resistance against tannins in comparison with antimicrobial agents. Susceptibility for 7 AGPs (antimicrobial growth promoters), 9 therapeutic antimicrobials and 2 tannin based extracts was determined for 30 C. perfringens strains isolated from poultry and cattle. Two susceptible strains were selected and cultured in presence of sub-inhibitory concentrations of tannins and AGPs for resistant sub-populations selection. Tannin resistance of C. perfringens isolates from both animal species revealed no statistically significant differences in MICs (minimum inhibitory concentration). Poultry isolates showed higher MICs to several AGPs compared with cattle isolates. All isolates were susceptible to the therapeutic antimicrobials tested, but avian isolates showed a significantly lower susceptibility to these antimicrobials which was highly correlated with an increased resistance to bacitracin and others AGPs. In-vitro selection of resistant clones suggests that C. perfringens was unable to develop resistance against tannins at least compared to AGPs like bacitracin and avilamycin. Avian origin strains, which were previously exposed to antibiotics showed higher resistance, compared to cattle origin strains. These results suggest that the evolution of resistance against tannins in C. perfringens would be more difficult and slower than to the determined AGPs.

Homogeneous heating of a sample space by a modified heating assembly in a belt-type high-pressure apparatus.

To create homogeneous heating in the sample space in a belt-type high-pressure apparatus, modified heating assemblies under pressure of 2.5 GPa and temperature up to 1700 °C were examined. Counterbores (with several diameters) were made at both ends of a cylindrical graphite heater to suppress the temperature gradient along the cylindrical axis of the heater. Temperature distributions within the heaters were measured by thermocouples and geothermometers. Both sets of measurements revealed that the temperature distribution in the sample space (6.9 mm outside diameter/12 mm length) was homogenized (i.e., variation of less than 10 °C under heating at 1700 °C) by optimizing the heater shape.

In vivo time-resolved DOT measurement and image reconstruction of human forearm under exercises.

In vivo measurement of time-resolved diffuse optical tomography (TR-DOT) were performed for human forearms under the exercises. The DOT images of oxygenation state were reconstructed, and the activities of the inner muscles were assessed.

Sudden death syndrome in adult cows associated with Clostridium perfringens type E.

Clostridium perfringens type E is considered a rare toxinotype and an infrequent cause of enterotoxemia of lambs, calves, and rabbits. Until now, only cases of young animal of C. perfringens type E bovine enterotoxemia, characterized by hemorrhagic enteritis and sudden death, have been reported. The present report details the genotypic characterization of C. perfringens type E isolates obtained from intestinal samples of adult cattle during an outbreak of enterotoxemia in Argentina. The sequences of several housekeeping genes of these isolates were analyzed and compared with those obtained from calves in North America showing a clonal unique lineage.

Detection of Shiga toxin-producing Escherichia coli by sandwich enzyme-linked immunosorbent assay using chicken egg yolk IgY antibodies.

Enterohemorrhagic Escherichia coli (EHEC), a subset of Shiga toxin producing E. coli (STEC) is associated with a spectrum of diseases that includes diarrhea, hemorrhagic colitis and a life-threatening hemolytic-uremic syndrome (HUS). Regardless of serotype, Shiga toxins (Stx1 and/or Stx2) are uniformly expressed by all EHEC, and so exploitable targets for laboratory diagnosis of these pathogens. In this study, a sandwich ELISA for determination of Shiga toxin (Stx) was developed using anti-Stx2B subunit antibodies and its performance was compared with that of the Vero cell assay and a commercial immunoassay kit. Chicken IgY was used as capture antibody and a HRP-conjugated rabbit IgG as the detection antibody. The anti-Stx2B IgY was harvested from eggs laid by hens immunized with a recombinant protein fragment. Several parameters were tested in order to optimize the sandwich ELISA assay, including concentration of antibodies, type and concentration of blocking agent, and incubation temperatures. Supernatants from 42 STEC strains of different serotypes and stx variants, including stx(2EDL933), stx(2vha), stx(2vhb), stx(2g), stx(1EDL933), and stx(1d) were tested. All Stx variants were detected by the sandwich ELISA, with a detection limit of 115 ng/ml Stx2. Twenty three strains negative for stx genes, including different bacteria species, showed no activity in Vero cell assay and produced negative results in ELISA, except for two strains. Our results show that anti-Stx2B IgY sandwich ELISA could be used in routine diagnosis as a rapid, specific and economic method for detection of Shiga toxin-producing E. coli.

Antihypertensive effect of a fixed-dose combination of losartan/hydrochlorothiazide in patients with uncontrolled hypertension: a multicenter study.

BACKGROUND: Achieving adequate blood pressure (BP) control often requires more than one antihypertensive agent. The purpose of this study was to determine whether a fixed-dose formulation of losartan (LOS) plus hydrochlorothiazide (HCTZ) (LOS/HCTZ) is effective in achieving a greater BP lowering in patients with uncontrolled hypertension. METHODS: The study was a prospective, multicenter, observational trial exploring the antihypertensive effect of a single tablet of LOS 50 mg/HCTZ 12.5 mg. A total of 228 patients whose BP had previously been treated with more than one antihypertensive agents without having achieved BP goal below 130/80 mmHg enrolled in the study. RESULTS: A significant decrease in systolic and diastolic BP was observed in both clinic and home measurement after switching from the previous treatment to LOS/HCTZ. There was a significant decrease in both B-type natriuretic peptide (BNP) and urinary albumin creatinine (Cr) excretion ratio (ACR), especially in patients with elevated values. In contrast, there was a significant increase in serum Cr concentration in conjunction with a decrease in estimated glomerular filtration rate (eGFR). Overall serum uric acid (UA) concentration increased, whereas in patients with hyperuricemia there was a significant reduction in this value. CONCLUSION: Switching to LOS/HCTZ provides a greater reduction in clinic and home BP in patients with uncontrolled hypertension. This combination therapy may lead to cardio-, reno protection and improve UA metabolism.

Antibodies anti-Shiga toxin 2 B subunit from chicken egg yolk: isolation, purification and neutralization efficacy.

Shiga toxins (Stx1 and Stx2) are the main virulence factors of enterohemorrhagic Escherichia coli (EHEC), a foodborne pathogen associated with diarrhea, hemorrhagic colitis and hemolytic uremic syndrome. The aim of this study was to evaluate the antibodies against Stx2 obtained from egg yolks of laying hens immunized with a recombinant Stx2B subunit. A high specific response in serum was observed 25 days after the first immunization and IgY antibodies were extracted from day 47th and purified from egg yolk. A concentration of 0.84 mg of total IgY/ml of egg yolk was obtained, of which 8% were antigen specific. The ability of anti-Stx2B IgY to recognize Stx2B and Stx2 either in solid-phase or in solution were evaluated and compared with anti-Stx2B rabbit antibodies by Western blotting and ELISA. The protective efficacy of IgY against Stx2 was determined by in vitro and in vivo experiments. The results show that IgY was able to recognize Stx2B and Stx2 in denatured conditions, attached to a solid-phase and free in solution. The anti-Stx2B IgY could effectively block the biological activity of Stx2 on Vero cells and protect mice from Stx2 challenge. The data suggest that immunization of hens with Stx2B could be a strategy to obtain at low cost a relatively high concentration of anti-Stx2 egg yolk IgY, able to neutralize Stx2 lethal activity. IgY technology could be an useful tool for research, diagnosis and therapy of EHEC infection.

Clostridium perfringens epsilon toxin inhibits the gastrointestinal transit in mice.

Epsilon toxin produced by Clostridium perfringens type B and D is a potent toxin that is responsible for a highly fatal enterotoxemia in sheep and goats. In vitro, epsilon toxin produces contraction of the rat ileum as the result of an indirect action, presumably mediated through the autonomic nervous system. To examine the impact of epsilon toxin in the intestinal transit, gastric emptying (GE) and gastrointestinal transit (GIT) were evaluated after intravenous and oral administration of epsilon toxin in mice. Orally administered epsilon toxin produced a delay on the GIT. Inhibition of the small intestinal transit was observed as early as 1 h after the toxin was administered orally but the effects were not observed after 1 week. Epsilon toxin also produced an inhibition in GE and a delay on the GIT when relatively high toxin concentrations were given intravenously. These results indicate that epsilon toxin administered orally or intravenously to mice transitorily inhibits the GIT. The delay in the GIT induced by epsilon toxin could be relevant in the pathogenesis of C. perfringens type B and D enterotoxemia.

Toxinas de Clostridium perfringens / Toxins of Clostridium perfringens

Clostridium perfringens es un bacilo grampositivo anaerobio con capacidad de formar esporas. Es uno de los patógenos bacterianos con mayor distribución en el medio ambiente, ya que puede ser aislado de muestras de suelo y de agua y además forma parte de la microbiota intestinal de animales y humanos. Sin embargo, en ciertas ocasiones puede actuar como patógeno oportunista y causar enfermedades como la gangrena gaseosa, la enterotoxemia del ovino y del caprino y la disentería del cordero, entre otras. En humanos, está asociado a enfermedades como la intoxicación por alimentos, la enterocolitis necrotizante en niños y la enteritis necrótica o pigbel de las tribus de Papúa-Nueva Guinea. El renovado interés que existe actualmente en el estudio de C. perfringens como patógeno veterinario y humano, junto con el avance de la biología molecular, han hecho posible que la ciencia tenga hoy un conocimiento más profundo sobre la biología y la patogenia de esta bacteria. En esta revisión bibliográfica se discuten y actualizan los principales aspectos de la patogenia intestinal de C. perfringens teniendo en cuenta las toxinas con mayor importancia médica descritas hasta el presente.

Clostridium perfringens is an anaerobic gram-positive spore-forming bacillus. It is one of the pathogens with larger distribution in the environment; it can be isolated from soil and water samples, which also belongs to the intestinal flora of animals and humans. However, on some occasions it can act as an opportunistic pathogen, causing diseases such as gas gangrene, enterotoxemia in sheep and goats and lamb dysentery, among others. In human beings, it is associated to diseases such as food poisoning, necrotic enterocolitis of the infant and necrotic enteritis or pigbel in Papua-New Guinea tribes. The renewed interest existing nowadays in the study of C. perfringens as a veterinarian and human pathogen, together with the advance of molecular biology, had enabled science to have deeper knowledge of the biology and pathology of these bacteria. In this review, we discuss and update the principal aspects of C. perfringens intestinal pathology, in terms of the toxins with major medical relevance at present.

Intracellular uptake of a new boronated porphyrin EC032.

We measured the toxicity and intracellular uptake of a newly developed boronated porphyrin EC032, and verified the fluorescence-based boron concentration measuring methods. Toxicity study showed that concentration required to produce a 50% reduction in viability (IC(50)) of EC032 was more than 0.25 mM. Fluorescence study showed the intracellular uptake of EC032 increased up until 24 h after its exposure to C6, 9L, U87, and U251 cells. There was also a linear correlation between ICP-AES and fluorescence intensity as an arbitrary unit about measurement of boron concentration. Fluorescence-based boron concentration measuring methods are very simple and useful methods, especially for screening of slight test dose of porphyrin compounds.

[Toxins of Clostridium perfringens]. / Toxinas de clostridium perfringens.

Clostridium perfringens is an anaerobic gram-positive spore-forming bacillus. It is one of the pathogens with larger distribution in the environment; it can be isolated from soil and water samples, which also belongs to the intestinal flora of animals and humans. However, on some occasions it can act as an opportunistic pathogen, causing diseases such as gas gangrene, enterotoxemia in sheep and goats and lamb dysentery, among others. In human beings, it is associated to diseases such as food poisoning, necrotic enterocolitis of the infant and necrotic enteritis or pigbel in Papua-New Guinea tribes. The renewed interest existing nowadays in the study of C. perfringens as a veterinarian and human pathogen, together with the advance of molecular biology, had enabled science to have deeper knowledge of the biology and pathology of these bacteria. In this review, we discuss and update the principal aspects of C. perfringens intestinal pathology, in terms of the toxins with major medical relevance at present.

Clostridium perfringens epsilon toxin is absorbed from different intestinal segments of mice.

Clostridium perfringens epsilon toxin is a potent toxin responsible for a rapidly fatal enterotoxaemia in several animal species. The pathogenesis of epsilon toxin includes toxicity to endothelial cells and neurons. Although epsilon toxin is absorbed from the gastrointestinal tract, the intestinal regions where the toxin is absorbed and the conditions favoring epsilon toxin absorption are unknown. The aim of this paper was to determine the toxicity of epsilon toxin absorbed from different gastrointestinal segments of mice and to evaluate the influence of the intestinal environment in the absorption of this toxin. Epsilon toxin diluted in one of several different saline solutions was surgically introduced into ligated stomach or intestinal segments of mice. Comparison of the toxicity of epsilon toxin injected in different sections of the gastrointestinal tract showed that this toxin can be absorbed from the small and the large intestine but not from the stomach of mice. The lethality of epsilon toxin was higher when this toxin was injected in the colon than in the small intestine. Low pH, and Na(+) and glucose added to the saline solution increased the toxicity of epsilon toxin injected into the small intestine. This study shows that absorption of epsilon toxin can occur in any intestinal segment of mice and that the physicochemical characteristics of the intestinal content can affect the absorption of this toxin.

The development of Weinstein's noise sensitivity scale.

Many studies have shown the significant correlation between noise annoyance and noise sensitivity identified by Weinstein's noise sensitivity scale (WNS). However, the validity of the scale has not been sufficiently assessed. This study was designed to investigate the validity of each question in WNS and to develop a more valid noise sensitivity measurement scale. A questionnaire study was conducted in a residential area along trunk roads in Kusatsu, Japan, and 301 responses were collected. In this paper, noise sensitivity was defined as the factor that induced individual variability in reactions caused by noise exposure and that is not affected by the noise exposure. The relationship between noise exposure and answers to each question in WNS was investigated by multiple logistic regression analysis, and the influence of response bias on the score of WNS was examined. The results showed that WNS contained some questions that were inappropriately related to noise exposure level and that the score was affected by response bias. The reported correlation between annoyance and the score of WNS could be confounded by noise exposure and response bias. A noise sensitivity measurement scale named WNS-6B was newly developed, excluding the biased questions from the original WNS and applying binary coding to six-response options in order to reduce the response bias. WNS-6B seemed to be more appropriate to assess noise sensitivity than the original scale.

Clostridium perfringens enterotoxin damages the human intestine in vitro.

In vitro, Clostridium perfringens enterotoxin (CPE) binds to human ileal epithelium and induces morphological damage concurrently with reduced short-circuit current, transepithelial resistance, and net water absorption. CPE also binds to the human colon in vitro but causes only slight morphological and transport changes that are not statistically significant.

Epsilon-toxin is required for most Clostridium perfringens type D vegetative culture supernatants to cause lethality in the mouse intravenous injection model.

Clostridium perfringens type D enterotoxemias have significant economic impact by causing rapid death of several domestic animal species. Consequently, domestic animals are commonly vaccinated, at varying efficacy, with inactivated type D vegetative supernatants. Improved type D vaccines might become possible if the lethal toxins produced by type D isolates were characterized and the contributions of those toxins to supernatant-induced lethality were established. Therefore, the current study evaluated the presence of lethal toxins in supernatants prepared from late-log-phase vegetative cultures of a large collection of genotype D isolates. Under this growth condition, most genotype D isolates produced variable levels of at least three different lethal toxins, including epsilon-toxin (ETX). To model the rapid lethality of type D enterotoxemias, studies were conducted involving intravenous (i.v.) injection of genotype D vegetative supernatants into mice, which were then observed for neurotoxic distress. Those experiments demonstrated a correlation between ETX (but not alpha-toxin or perfringolysin O) levels in late-log-phase genotype D supernatants and lethality. Consistent with the known proteolytic activation requirement for ETX toxicity, trypsin pretreatment was required for, or substantially increased, the lethality of nearly all of the tested genotype D vegetative supernatants. Finally, the lethality of these trypsin-pretreated genotype D supernatants could be completely neutralized by an ETX-specific monoclonal antibody but not by an alpha-toxin-specific monoclonal antibody. Collectively, these results indicate that, under the experimental conditions used in the present study, ETX is necessary for the lethal properties of most genotype D vegetative supernatants in the mouse i.v. injection model.

Clostridium perfringens epsilon-toxin increases permeability of single perfused microvessels of rat mesentery.

Epsilon-toxin, the primary virulence factor of Clostridium perfringens type D, causes mortality in livestock, particularly sheep and goats, in which it induces an often-fatal enterotoxemia. It is believed to compromise the intestinal barrier and then enter the gut vasculature, from which it is carried systemically, causing widespread vascular endothelial damage and edema. Here we used single perfused venular microvessels in rat mesentery, which enabled direct observation of permeability properties of the in situ vascular wall during exposure to toxin. We determined the hydraulic conductivity (L(p)) of microvessels as a measure of the response to epsilon-toxin. We found that microvessels were highly sensitive to toxin. At 10 microg ml(-1) the L(p) increased irreversibly to more than 15 times the control value by 10 min. At 0.3 microg ml(-1) no increase in L(p) was observed for up to 90 min. The toxin-induced increase in L(p) was consistent with changes in ultrastructure of microvessels exposed to the toxin. Those microvessels exhibited gaps either between or through endothelial cells where perfusate had direct access to the basement membrane. Many endothelial cells appeared necrotic, highly attenuated, and with dense cytoplasm. We showed that epsilon-toxin, in a time- and dose-dependent manner, rapidly and irreversibly compromised the barrier function of venular microvessel endothelium. The results conformed to the hypothesis that epsilon-toxin interacts with vascular endothelial cells and increases the vessel wall permeability by direct damage of the endothelium.

The Shiga toxin 2 B subunit inhibits net fluid absorption in human colon and elicits fluid accumulation in rat colon loops.

Shiga toxin (Stx)-producing Escherichia coli (STEC) colonizes the large intestine causing a spectrum of disorders, including watery diarrhea, bloody diarrhea (hemorrhagic colitis), and hemolytic-uremic syndrome. It is estimated that hemolytic-uremic syndrome is the most common cause of acute renal failure in infants in Argentina. Stx is a multimeric toxin composed of one A subunit and five B subunits. In this study we demonstrate that the Stx2 B subunit inhibits the water absorption (Jw) across the human and rat colonic mucosa without altering the electrical parameters measured as transepithelial potential difference and short circuit current. The time-course Jw inhibition by 400 ng/ml purified Stx2 B subunit was similar to that obtained using 12 ng/ml Stx2 holotoxin suggesting that both, A and B subunits of Stx2 contributed to inhibit the Jw. Moreover, non-hemorrhagic fluid accumulation was observed in rat colon loops after 16 h of treatment with 3 and 30 ng/ml Stx2 B subunit. These changes indicate that Stx2 B subunit induces fluid accumulation independently of A subunit activity by altering the usual balance of intestinal absorption and secretion toward net secretion. In conclusion, our results suggest that the Stx2 B subunit, which is non-toxic for Vero cells, may contribute to the watery diarrhea observed in STEC infection. Further studies will be necessary to determine whether the toxicity of Stx2 B subunit may have pathogenic consequences when it is used as a component in an acellular STEC vaccine or as a vector in cancer vaccines.

The Shiga toxin 2 B subunit inhibits net fluid absorption in human colon and elicits fluid accumulation in rat colon loops

Shiga toxin (Stx)-producing Escherichia coli (STEC) colonizes the large intestine causing a spectrum of disorders, including watery diarrhea, bloody diarrhea (hemorrhagic colitis), and hemolytic-uremic syndrome. It is estimated that hemolytic-uremic syndrome is the most common cause of acute renal failure in infants in Argentina. Stx is a multimeric toxin composed of one A subunit and five B subunits. In this study we demonstrate that the Stx2 B subunit inhibits the water absorption (Jw) across the human and rat colonic mucosa without altering the electrical parameters measured as transepithelial potential difference and short circuit current. The time-course Jw inhibition by 400 ng/ml purified Stx2 B subunit was similar to that obtained using 12 ng/ml Stx2 holotoxin suggesting that both, A and B subunits of Stx2 contributed to inhibit the Jw. Moreover, non-hemorrhagic fluid accumulation was observed in rat colon loops after 16 h of treatment with 3 and 30 ng/ml Stx2 B subunit. These changes indicate that Stx2 B subunit induces fluid accumulation independently of A subunit activity by altering the usual balance of intestinal absorption and secretion toward net secretion. In conclusion, our results suggest that the Stx2 B subunit, which is non-toxic for Vero cells, may contribute to the watery diarrhea observed in STEC infection. Further studies will be necessary to determine whether the toxicity of Stx2 B subunit may have pathogenic consequences when it is used as a component in an acellular STEC vaccine or as a vector in cancer vaccines.