Multiplex real-time PCR SYBR Green for detection and typing of group III Clostridium botulinum

Clostridium botulinum type C and type D belonging to the group III organisms, are mainly responsible for animal botulism outbreaks. Clinical signs alone are often insufficient to make a diagnosis of botulism and a laboratory confirmation is required. Laboratory confirmation can be performed by demonstrating the presence of botulinum neurotoxins in serum, gastrointestinal contents, liver, wound of sick or dead animals, or by demonstrating the presence of C. botulinum in gastrointestinal contents, liver, and wound. Demonstration of spores in gastrointestinal contents or tissue of animals with clinical signs indicative of botulism reinforces the clinical diagnosis. With the aim of detecting and typing C. botulinum group III organisms, a multiplex real-time PCR SYBR Green was developed and in-house validated. Selectivity, limit of detection, relative accuracy, relative specificity, relative sensitivity, and repeatability of the method were investigated. The multiplex real-time PCR SYBR green used showed a 100% selectivity, 100% relative accuracy, 100% relative specificity, 100% relative sensitivity and a limit of detection of 277 and 580 DNA copies for C. botulinum type C and C. botulinum type D, respectively. The method reported here represents a suitable tool for laboratory diagnosis of type C and D botulism and for testing a large number of samples collected during the animal botulism surveillance and prevention activities.     

Escherichia coli lipopolysaccharides and Staphylococcus aureus enterotoxin B differentially modulate inflammatory microRNAs in bovine monocytes

MicroRNAs (miRNAs) are a family of regulatory molecules involved in many physiological processes, including activation of cells of the immune system. This study investigated the effect of Escherichia coli lipopolysaccharide (LPS) and Staphylococcus aureus enterotoxin B (SEB) on the expression of five miRNAs involved in the inflammatory response, including miR-9, miR-125 b, miR-155, miR-146 a and miR-223, in bovine CD14(+) cells (monocytes). Incubation of monocytes with SEB induced down-regulation of miR-155, miR-223 and miR-125 b, but not the anti-inflammatory miRNA miR-146 a. Conversely, incubation with LPS upregulated both miR-155 and miR-146 a. In vitro incubation of isolated CD14(+) bovine monocytes with LPS and SEB elicited different and opposite expression of miRNAs reportedly involved in inflammatory reactions.     

Grafts of porcine small intestinal submucosa seeded with cultured homologous smooth muscle cells for bladder repair in dogs

Background

Due to numerous complications associated to gastrointestinal augmented cystoplasty, this study aimed to analyze the anatomic repair of the bladder of 10 female dogs using grafts of porcine small intestinal submucosa (SIS) seeded with cultured homologous smooth muscle cells, and compare them with the acellular SIS grafts.

Results

We assessed the possible side effects and complications of each type of graft by clinical examination, abdominal ultrasound and laboratory findings. Anatomic repair of neoformed bladder was assessed by histological staining for H/E and Masson''s Trichrome, analyzed with a Nikon Photomicroscope connected to the system of image analysis Image J.

Conclusions

We propose that SIS associated to homologous smooth cells can improve the quality of tissue repair, and consequently decrease the potential complications inherent to acellular SIS.     

Municipal Waste and Poultry Manure Compost Affect Biomass Production,Nitrate Reductase Activity and Heavy Metals in Tomato Plants

Organic wastes can be usefully recovered to produce organic amendments, for example, compost, to be used for crop production, thus reducing impacts through efficient waste management. The aim of this work was to study the effects of compost obtained from municipal waste in combination with poultry manure on plant growth, nitrate reductase (NR) activity and absorption and distribution of heavy metals (HM) in plant tissues of tomatoes, grown in pots in greenhouses. Two compost types obtained from municipal waste mixed with poultry manure (C1?=?3:2 and C2?=?2:3) were used at two different ages (105 d and 173 d) and at two mix rates with soil (32.5?g pot^?1 and 65?g pot^?1); soil with no compost amendment was used as control. The experiment was conducted using tomato plants in pots and plant growth and nutrient plant uptake was determined after 65?days from plant transplanting. Results obtained indicated that compost type and compost rate affected biomass production. However, compost age did not influence the development of plants. Nutrient status of tomato plants was also investigated with reference to the N cycle. Nitrite accumulation in the leaves increased with the increase in compost doses. The accumulation of NO₂^? was associated with a significant increase in NR activity. HM content in leaves decreased with compost use. HM accumulated preferentially in roots and leaves and the soil to root metals transfer was in this order: Fe (1.08–2.14)> Co (0.53–4.10)>Cu (0.28–2.28) >Mn (0.3–1.34) >Zn (0.87–1.21)>Cr (0.12–1.64). The highest and lowest dynamic bioaccumulation factors (BAF_dyn) were observed in roots and stems, respectively. The root system acted as a barrier for Cd and Pb. It was concluded that compost use is beneficial for tomato plants, with particular reference to the compost obtained by using a higher amount of poultry manure (C2) in the mix.     

Stomatocytosis of Standard Schnauzers is not associated with stomatin deficiency

Stomatocytosis resembles human overhydrated hereditary stomatocytosis (OHSt), a disease characterised by a reduced or absent stomatin expression. The objective of this report was to investigate the expression level of stomatin in erythrocytes from Standard Schnauzers with stomatocytosis. Routine haematology, intraerythrocytic Na(+)/K(+) concentration and stomatin expression were evaluated in blood from twelve Standard Schnauzers and from three controls. SDS-PAGE and Western blotting on isolated integral membrane proteins were used to investigate stomatin expression. Circulating stomatocytes, macrocytosis, anisocytosis, increased erythrocyte fragility and high intracellular sodium and potassium concentrations were found in 10/12 dogs from the same breeding line although stomatin levels were similar to those of controls. In spite of the clinico-pathological similarities between human and canine stomatocytosis, erythrocytes from affected dogs do not lack stomatin and the expression level of this protein cannot therefore be used to diagnose hereditary stomatocytosis in Standard Schnauzers.