Effect of chitosan on Salmonella Typhimurium in broiler chickens.

Public concern with the incidence of antibiotic-resistant bacteria, particularly among foodborne pathogens such as Salmonella, has been challenging the poultry industry to find alternative means of control. The purposes of the present study were to evaluate in vitro and in vivo effects of chitosan on Salmonella enterica serovar Typhimurium (ST) infection in broiler chicks. For in vitro crop assay experiments, tubes containing feed, water, and ST were treated with either saline as a control or 0.2% chitosan. The entire assay was repeated in three trials. In two independent in vivo trials, 40 broiler chicks were assigned to an untreated control diet or dietary treatment with 0.2% chitosan for 7 days (20 broiler chicks/treatment). At day 4, chicks were challenged with 2×105 colony-forming units (CFU) ST/bird. In a third in vivo trial, 100 broiler chicks were assigned to untreated control diet or dietary treatment with 0.2% chitosan for 10 days (50 broiler chicks/treatment) to evaluate ST horizontal transmission. At day 3, 10 birds were challenged with 105 CFU ST/bird, and the remaining nonchallenged birds (n=40) were kept in the same floor pen. In all three in vitro trials, 0.2% chitosan significantly reduced total CFU of ST at 0.5 and 6 h postinoculation compared with control (p<0.05). In two in vivo trials, at 7 days, dietary 0.2% chitosan significantly reduced total CFU of recovered ST in the ceca in both experiments. Dietary 0.2% chitosan significantly reduced total ST CFU recovered in the ceca of horizontally challenged birds in the third in vivo trial. Chitosan at 0.2% significantly reduced the CFU of recovered ST in vitro and in vivo, proving to be an alternative tool to reduce crop, ceca, and consequently carcass ST contamination as well as decreasing the amount of ST shed to the environment.

Delineating liver events in trichloroethylene-induced autoimmune hepatitis.

Exposure to the environmental pollutant trichloroethylene (TCE) has been linked to autoimmune disease development in humans. Chronic (32-week) low-level exposure to TCE has been shown to promote autoimmune hepatitis in association with CD4(+) T cell activation in autoimmune-prone MRL+/+ mice. MRL+/+ mice are usually thought of as a model of systemic lupus rather than an organ-specific disease such as autoimmune hepatitis. Consequently, the present study examined gene expression and metabolites to delineate the liver events that skewed the autoimmune response toward that organ in TCE-treated mice. Female MRL+/+ mice were treated with 0.5 mg/mL TCE in their drinking water. The results showed that TCE-induced autoimmune hepatitis could be detected in as little as 26 weeks. TCE exposure also generated a time-dependent increase in the number of antibodies specific for liver proteins. The gene expression correlated with the metabolite analysis to show that TCE upregulated the methionine/homocysteine pathway in the liver after 26 weeks of exposure. The results also showed that TCE exposure altered the expression of selective hepatic genes associated with immunity and inflammation. On the basis of these results, future mechanistic studies will focus on how alterations in genes associated with immunity and inflammation, in conjunction with protein alterations in the liver, promote liver immunogenicity in TCE-treated MRL+/+ mice.

Evidence for Clostridium septicum as a primary cause of cellulitis in commercial turkeys.

Etiology and methods of immunoprophylaxis against common field cellulitis in commercial turkeys were evaluated. It was determined that intravenous administration ( approximately 10(8) cells/ml) of 1 of 4 isolates of Clostridium septicum from cellulitis lesions rapidly caused the classic lesions of cellulitis followed by death within 36 hr at high doses. When the supernatant alone was injected into turkey poults, signs of depression and ataxia were temporarily observed for up to 20 hr after injection, but no cellulitis lesions were detected. An enzyme-linked immunosorbent assay was developed to measure antibody levels against the isolated C. septicum. This assay was used to predict susceptibility to infection. An experimental formalin-killed bacterin/toxoid was produced from the challenge strain of C. septicum with inactivation timed to allow toxin accumulation and approximately 10(8) cells/ml. This bacterin/toxoid given at day of hatch generated a rapid and persistent antibody response against the homologous C. septicum in the vaccinated birds at 9 weeks (P < 0.001). The ability of this experimental vaccine to protect birds in the field as well as its ability to evaluate unvaccinated flocks to establish the time of seroconversion and the relationship to clinical disease are currently under evaluation.

Environmental contaminant and disinfection by-product trichloroacetaldehyde stimulates T cells in vitro.

It had been shown previously that MRL+/+ mice exposed to occupationally relevant doses of the environmental contaminant trichloroethylene in their drinking water developed lupus-like symptoms and autoimmune hepatitis in association with activation of Interferon-gamma (IFN-gamma)-producing CD4+ T cells. Since trichloroethylene must be metabolized in order to promote the T-cell activation associated with autoimmunity, the present study was initiated to determine whether the immunoregulatory effects of trichloroethylene could be mimicked by one of its major metabolites, trichloroacetaldehyde (TCAA). At concentrations ranging from 0.04 to 1 mM TCAA co-stimulated proliferation of murine T-helper type 1 (Th1) cells treated with anti-CD3 antibody or antigen in vitro. TCAA at similar concentrations also induced phenotypic alterations commensurate with activation (upregulation of CD28 and downregulation of CD62L) in both cloned memory Th1 cells, as well as naïve CD4+ T cells from MRL+/+ mice. TCAA-induced Th1 cell activation was accompanied by phoshorylation of activating transcription factor 2 (ATF-2) and c-Jun, two components of the activator protein-1 (AP-1) transcription factor. TCAA at higher concentrations was also shown to form a Schiff base on T cells, and inhibition of Schiff base formation suppressed the ability of TCAA to phosphorylate ATF-2. Taken together, these results suggest that TCAA promotes T-cell activation via stimulation of the mitogen-activated protein (MAP) kinase pathway in association with Schiff base formation on T-cell surface proteins. By demonstrating that TCAA can stimulate T-cell function directly, these results may explain how the environmental toxicant trichloroethylene promotes T-cell activation and related autoimmunity in vivo.

Evaluation of respiratory route as a viable portal of entry for Salmonella in poultry.

With increasing reports of Salmonella infection, we are forced to question whether the fecal-oral route is the major route of infection and consider the possibility that airborne Salmonella infections might have a major unappreciated role. Today's large-scale poultry production, with densely stocked and enclosed production buildings, is often accompanied by very high concentrations of airborne microorganisms. Considering that the upper and lower respiratory lymphoid tissue requires up to 6 weeks to be fully developed, these immune structures seem to have a very minor role in preventing pathogen infection. In addition, the avian respiratory system in commercial poultry has anatomic and physiologic properties that present no challenge to the highly adapted Salmonella. The present review evaluates the hypothesis that transmission by the fecal-respiratory route may theoretically be a viable portal of entry for Salmonella in poultry. First, we update the current knowledge on generation of Salmonella bioaerosols, and the transport and fate of Salmonella at various stages of commercial poultry production. Further, emphasis is placed on survivability of Salmonella in these bioaerosols, as a means to assess the transport and subsequent risk of exposure and infection of poultry. Additionally, the main anatomic structures, physiologic functions, and immunologic defense in the avian respiratory system are discussed to understand the potential entry points inherent in each component that could potentially lead to infection and subsequent systemic infection of poultry by Salmonella. In this context, we also evaluate the role of the mucosal immune system as essentially one large interconnected network that shares information distally, since understanding of this sort of communication between mucosal sites is fundamental to establish the next phase of disease characterization, and perhaps immunization and vaccine development. Further characterization of the respiratory tract with regard to transmission of Salmonella under field conditions may be of critical importance in developing interventional strategies to reduce transmission of this important zoonotic pathogen in poultry.

Identification and characterization of lactic Acid bacteria in a commercial probiotic culture.

The aim of the present study was to describe the identification and characterization (physiological properties) of two strains of lactic acid bacteria (LAB 18 and 48) present in a commercial probiotic culture, FloraMax(®)-B11. Isolates were characterized morphologically, and identified biochemically. In addition, the MIDI System ID, the Biolog ID System, and 16S rRNA sequence analyses for identification of LAB 18 and LAB 48 strains were used to compare the identification results. Tolerance and resistance to acidic pH, high osmotic concentration of NaCl, and bile salts were tested in broth medium. In vitro assessment of antimicrobial activity against enteropathogenic bacteria and susceptibility to antibiotics were also tested. The results obtained in this study showed tolerance of LAB 18 and LAB 48 to pH 3.0, 6.5% NaCl and a high bile salt concentration (0.6%). Both strains evaluated showed in vitro antibacterial activity against Salmonella enterica serovar Enteritidis, Escherichia coli (O157:H7), and Campylobacter jejuni. These are important characteristics of lactic acid bacteria that should be evaluated when selecting strains to be used as probiotics. Antimicrobial activity of these effective isolates may contribute to efficacy, possibly by direct antimicrobial activity in vivo.

Physiological Properties and Salmonella Growth Inhibition of Probiotic Bacillus Strains Isolated from Environmental and Poultry Sources.

The objective of the present study was to describe the physiological properties of seven potential probiotic strains of Bacillus spp. Isolates were characterized morphologically, biochemically, and by 16S rRNA sequence analyses for identification. Tolerance to acidic pH, high osmotic concentrations of NaCl, and bile salts were tested. Isolates were also evaluated for their ability to metabolize different carbohydrates sources. The antimicrobial sensitivity profiles were determined. Inhibition of gastrointestinal Salmonella colonization in an avian model was also evaluated. Five strains of Bacillus were tolerant to acidic conditions (pH 2.0) and all strains were tolerant to a high osmotic pressure (NaCl at 6.5%). Moreover, all strains were able to tolerate concentration of 0.037% bile salts after 24 h of incubation. Three strains were able to significantly reduce Salmonella Typhimurium levels in the crop and in the ceca of broiler-type chickens. Among the 12 antibiotics tested for antibiotic resistance, all strains were resistant to bacitracin and susceptible to gentamycin, neomycin, ormethoprim, triple sulfa, and spectinomycin. Bacterial spore formers have been shown to prevent gastrointestinal diseases in animals and humans. The results obtained in this study show important characteristics to be evaluated when selecting Bacillus spp. candidates to be used as probiotics.

Environmental contaminant trichloroethylene promotes autoimmune disease and inhibits T-cell apoptosis in MRL(+/+) mice.

The ability of environmental contaminant trichloroethylene to alter immune function and promote autoimmunity was tested in female MRL(+/+) mice. MRL(+/+) mice exposed to occupationally relevant doses of trichloroethylene in their drinking water for 32 weeks developed autoantibodies and pathological evidence of autoimmune hepatitis. The ability of trichloroethylene (TCE) to promote autoimmunity was associated with the expansion of activated (CD44(hi) CD62L(lo)) CD4(+) T-lymphocytes that produced increased levels of the pro-inflammatory cytokine interferon (IFN)-gamma. Activated T-lymphocytes can accumulate if activation-induced apoptosis is suppressed. Consequently, the effect of TCE on apoptosis in CD4(+) T-lymphocytes was investigated. These experiments were conducted with TCE and one of the major oxidative metabolites of trichloroethylene, namely trichloroacetaldehyde hydrate (TCAH). CD4(+) T-lymphocytes isolated from MRL(+/+) mice exposed to TCE or TCAH in their drinking water for 4 weeks were resistant to activation-induced apoptosis in vitro. The TCE-or TCAH-induced decrease in activation-induced apoptosis was associated with decreased expression of FasL, one of the cell surface molecules that mediate apoptosis. These results suggest that exposure to the common water contaminant TCE or its metabolite TCAH inhibits activation-induced apoptosis in CD4+ T-lymphocytes, thereby promoting autoimmune disease by suppressing the process that would otherwise delete activated self-reactive T-lymphocytes.

Activation and attenuation of apoptosis of CD4+ T cells following in vivo exposure to two common environmental toxicants, trichloroacetaldehyde hydrate and trichloroacetic acid.

Exposure to occupationally relevant concentrations of the environmental pollutant, trichloroethylene (TCE), in the drinking water of autoimmune-prone MRL+/+ mice has been shown to promote the generation of lupus and autoimmune hepatitis in association with the activation of Interferon-gamma (IFN-gamma)-producing CD4+ T cells. Since blocking TCE metabolism suppressed the TCE-induced alteration in immune function, the present study was initiated to determine whether the major metabolites of TCE, trichloroacetaldehyde hydrate (TCAH) and trichloroacetic acid (TCA) could also mediate these immunoregulatory affects in vivo. TCAH and TCA were administered to the drinking water of MRL+/+ mice for 4 weeks. CD4+ T cells from TCAH and TCA-treated MRL+/+ mice, unlike CD4+ T cells from control mice, demonstrated functional and phenotypic signs of activation, as evidenced by increased IFN-gamma production in association with the increased percentage of CD62L(lo) CD4+ T cells. Interestingly, it was also found that the CD4+ T cells from the TCAH and TCA-treated mice showed a decreased susceptibility to the activation-induced cell death (AICD) form of apoptosis following re-stimulation in vitro. By demonstrating that TCAH and TCA can activate CD4+ T cells and inhibit their apoptosis following in vivo exposure represents a mechanism by which environmental toxicants may induce or accelerate the development of autoimmune disease.