Calcium Montmorillonite-Based Dietary Supplement Attenuates Necrotic Enteritis Induced by Eimeria maxima and Clostridium perfringens in Broilers.

Necrotic enteritis (NE) is a poultry disease caused by Clostridium perfringens and characterized by severe intestinal necrosis. The incidence of avian NE has been progressively increasing following the removal of antibiotics from poultry feed. We evaluated the effect of diets supplemented with the thermally-processed clays, calcium montmorillonite (CaMM) on clinical signs, immunopathology, and cytokine responses in broiler chickens using an experimental model of NE consisting of co-infection with Eimeria maxima and C. perfringens. In Trial 1, Ross/Ross chickens were fed from hatch with a normal basal diet or a CaMM-supplemented diet with or without a fermentable fiber, an organic acid, and/or a plant extract, and co-infected with E. maxima and C. perfringens under conditions simulating clinical infection in the field. Chickens fed a diet supplemented with CaMM plus a fermentable fiber and an organic acid had increased body weight gain, reduced gut lesions, and increased serum antibody levels to C. perfringens α-toxin and NetB toxin compared with chickens fed the basal diet alone. Levels of transcripts for interleukin-1ß (IL-1ß), IL-6, inducible nitric oxide synthase, and tumor necrosis factor-α superfamily-15 were significantly altered in the intestine and spleen of CaMM-supplemented chickens compared with unsupplemented controls (p<0.05). In Trial 2, Cobb/Cobb chickens were fed an unsupplemented diet or a diet supplemented with CaMM or Varium®, each with a fermentable fiber and an organic acid, and co-infected with E. maxima and C. perfringens under subclinical infection conditions. Compared with unsupplemented controls, broilers fed with CaMM plus a fermentable fiber and an organic acid had increased body weight gain, and reduced feed conversion ratio, mortality, and intestinal lesions, compared with chickens fed an unsupplemented diet (p<0.05). Dietary supplementation of broiler chickens with CaMM plus a fermentable fiber and an organic acid might be useful to control avian NE in the field.

Immunopathology and cytokine responses in broiler chickens coinfected with Eimeria maxima and Clostridium perfringens with the use of an animal model of necrotic enteritis.

The incidence of necrotic enteritis (NE) due to Clostridium perfringens (CP) infection in commercial poultry has been increasing at an alarming rate. Although pre-exposure of chickens to coccidia infections is believed to be one of the major risk factors leading to NE, the underlying mechanisms of CP virulence remain undefined. The objectives of this study were to utilize an experimental model of NE produced by Eimeria maxima (EM) and CP coinfection to investigate the pathologic and immunologic parameters of the disease. Broilers coinfected with EM plus CP exhibited more severe gut pathology compared with animals given EM or CP alone. Additionally, EM/CP coinfection increased the numbers of intestinal CP bacteria compared with chickens exposed to an identical challenge of CP alone. Coinfection with EM and CP repressed nitric oxide synthase gene expression that was induced by EM alone, leading to lower plasma NO levels. Intestinal expression of a panel of cytokine and chemokine genes following EM/CP coinfection showed a mixed response depending on the transcript analyzed and the time following infection. In general, IFN-alpha, IFN-gamma, IL-1beta, IL-2, IL-12, IL-13, IL-17, and TGF-beta4 were repressed, whereas IL-8, IL-10, IL-15, and LITAF were increased during coinfection compared with challenge by EM or CP alone. These results are discussed in the context of EM and CP to act synergistically to create a more severe disease phenotype leading to an altered cytokine/chemokine response than that produced by infection with the individual pathogens.

Disruption of dopamine homeostasis underlies selective neurodegeneration mediated by alpha-synuclein.

A key challenge in Parkinson's disease research is to understand mechanisms underlying selective degeneration of dopaminergic neurons mediated by genetic factors such as alpha-synuclein (alpha-Syn). The present study examined whether dopamine (DA)-dependent oxidative stress underlies alpha-Syn-mediated neurodegeneration using Drosophila primary neuronal cultures. Green fluorescent protein (GFP) was used to identify live dopaminergic neurons in primary cultures prepared on a marked photoetched coverslip, which allowed us to repeatedly access preidentified dopaminergic neurons at different time points in a non-invasive manner. This live tracking of GFP-marked dopaminergic neurons revealed age-dependent neurodegeneration mediated by a mutant human alpha-Syn (A30P). Degeneration was rescued when alpha-Syn neuronal cultures were incubated with 1 mm glutathione from Day 3 after culturing. Furthermore, depletion of cytoplasmic DA by 100 microm alpha-methyl-p-tyrosine completely rescued the early stage of alpha-Syn-mediated dopaminergic cell loss, demonstrating that DA plays a major role in oxidative stress-dependent neurodegeneration mediated by alpha-Syn. In contrast, overexpression of a Drosophila tyrosine hydroxylase gene (dTH1) alone caused DA neurodegeneration by enhanced DA synthesis in the cytoplasm. Age-dependent dopaminergic cell loss was comparable in alpha-Syn vs dTH1-overexpressed neuronal cultures, indicating that increased DA levels in the cytoplasm is a critical change downstream of mutant alpha-Syn function. Finally, overexpression of a Drosophila vesicular monoamine transporter rescued alpha-Syn-mediated neurodegeneration through enhanced sequestration of cytoplasmic DA into synaptic vesicles, further indicating that a main cause of selective neurodegeneration is alpha-Syn-induced disruption of DA homeostasis. All of these results demonstrate that elevated cytoplasmic DA is a main factor underlying the early stage of alpha-Syn-mediated neurodegeneration.

Functional characterization of tumor necrosis factor superfamily 15 (TNFSF15) induced by lipopolysaccharides and Eimeria infection.

A full-length cDNA encoding chicken tumor necrosis factor superfamily 15 (TNFSF15) was isolated and its functional role was investigated. TNFSF15 transcripts were primarily expressed in spleen, liver, intestinal intraepithelial lymphocytes (IEL), peripheral blood lymphocytes and bursa. In vitro infection of HTC macrophages with three species of Eimeria sporozoites induced TNFSF15 gene expression. In vivo experiments revealed that TNFSF15 gene was highly increased following primary infections with Eimeria acervulina or Eimeria maxima. In contrast, no consistent changes in transcript levels were seen following primary infection with Eimeria tenella, or following secondary infection with any of the three Eimeria species. Following infection with E. acervulina and E. maxima, TNFSF15 transcripts were primarily expressed in intestinal CD4(+) and TCR2(+) IEL, respectively. A dose-dependent cytotoxic effect of recombinant TNFSF15 protein was observed on HTC and LSCC-RP9 tumor cells. These results indicate that TNFSF15 plays an important role in local inflammatory response to Eimeria.

Selective loss of dopaminergic neurons and formation of Lewy body-like aggregations in alpha-synuclein transgenic fly neuronal cultures.

A key pathological feature of Parkinson's disease (PD) is the selective loss of dopaminergic neurons accompanied by the formation of Lewy bodies (LB). Given the complex nature of the disease, it is imperative to develop a model system suitable for molecular and cellular manipulation in order to study the mechanisms underlying the pathogenesis of PD. Here, we report that a new in vitro model of PD has been developed by using Drosophila melanogaster primary neuronal cultures expressing a human mutant alpha-synuclein (alpha-Syn; A30P). The selective loss of dopaminergic (DA) neurons was observed when alpha-Syn was pan-neuronally expressed while non-dopaminergic neurons (e.g. GABAergic) were not influenced. This degeneration was also observed even when alpha-Syn was specifically expressed in DA neurons, demonstrating alpha-Syn toxicity is DA cell-autonomous. In all experiments, cultures 5 days or older showed clear degeneration of DA neurons whereas this degeneration was not significant in 3-day-old cultures. In addition, there were intracellular aggregations in 5-day or older alpha-Syn neurons that were recognized by anti-alpha-Syn or ubiquitin antibodies, demonstrating the formation of LB-like inclusions. By contrast, no such aggregations were found in 3-day-old neurons. The results demonstrate that mutated human alpha-Syn expressed in Drosophila primary neuronal cultures causes the selective loss of DA neurons and the formation of cellular aggregations. Therefore, this is one of the first in vitro models recapitulating two important cellular features of PD and will be useful in examining mechanisms underlying selective neurodegeneration mediated by alpha-Syn.