Differential regulation of porcine beta-defensins 1 and 2 upon Salmonella infection in the intestinal epithelial cell line IPI-2I

Intestinal epithelial cells represent the first line of defence against pathogenic bacteria in the lumen of the gut. Besides acting as a physical barrier, epithelial cells orchestrate the immune response through the production of several innate immune mediator molecules including beta-defensins. Here, we establish the porcine intestinal cell line IPI-2I as a new model system to test the regulation of porcine beta-defensins 1 and 2. Gene expression of both defensins was highly upregulated by foetal calf serum components in normal growth medium. In serum-free medium, baseline expression remained low, but pBD-2 gene expression was increased 10-fold upon infection with Salmonella Typhimurium. Arcobacter cryaerophilus and Salmonella Enteritidis, pathogenic bacteria with comparable adhesion and invasion characteristics, failed to increase pBD-2 mRNA levels. Heat killed or colistin-treated Salmonella Typhimurium had no effect, showing that the upregulation of pBD-2 was dependent on the viability of the Salmonella Typhimurium. Gene expression of pBD-1 was regulated differently since an increase in pBD-1 mRNA was observed by Salmonella Enteritidis infection. We conclude that the IPI-2I cells can serve as a new model to study porcine beta-defensin regulation and that pBD-1 and pBD-2 are differentially regulated in this cell line.     

The isoflavone content of commercially-available feline diets in New Zealand

AIM: To identify and quantify concentrations of the isoflavones genistein, daidzein, biochanin A and formononetin in commercially- prepared feline diets sold in New Zealand. METHODS: Feline diets (n=138) were collected from supermarkets, pet stores and veterinary clinics in New Zealand. Diets were classified into five categories based on the following criteria: the presence/absence of soy, the presence/absence of non-soy plant material, and dry matter (DM) content. A high performance liquid chromatography (HPLC)-based assay was developed and validated to identify and quantify concentrations of the isoflavones genistein, daidzein, biochanin A and formononetin. RESULTS: Isoflavones were detected in all categories of diet, and at quantifiable concentrations in 104/138 (75%) of the diets tested. More dry diets (127/138; 92%) contained isoflavones at quantifiable concentrations than moist diets (83/138; 60%, p<0.001). Of the isoflavone-positive diets, moist diets that contained soy had the highest median isoflavone content (71.1 mg/kg DM and 0.018 mg/kcal metabolisable energy; ME) whilst moist meat-only diets had the lowest (3.24 mg/kg DM and 0.0004 mg/kcal ME). Isoflavone contents varied similarly between categories of diet whether evaluated on a DM or ME basis. High isoflavone contents were associated with diets containing soy and those of low cost. Biochanin A and formononetin were found at concentrations above the detection limit of the assay in 25/138 (18%) and 7/138 (5%) of the diets analysed, respectively. The estimated median daily isoflavone exposure for cats consuming feline diets sold in New Zealand ranged from 0.03 mg/kg bodyweight (BW) for moist meat-only diets to 1.47 mg/kg BW for moist diets containing soy, whilst the diet with the highest isoflavone content would provide daily exposure of 8.13 mg/kg BW. CONCLUSIONS: Isoflavones are present in commercially-prepared feline diets sold in New Zealand at concentrations that have elicited physiological responses in the reproductive, endocrine, and immune systems of other mammalian species. Since isoflavones appear to be a common constituent of feline diets, further investigation of the biological activities of these dietary compounds in cats is warranted.     

The influence of diets supplemented with conjugated linoleic acid, selenium, and vitamin E, with or without animal protein, on the composition of pork from female pigs

The objective of this study was to evaluate the effect of dietary manipulations on the fatty acid composition, Se content, and vitamin E content of pork. Sixty Duroc-cross gilts were randomly allocated at weaning to 1 of 4 dietary treatment groups (n = 15 per group). The 4 experimental diets were based on animal plus plant components or plant components only, with or without the inclusion of a dietary supplement (0.614%) containing CLA, Se, and vitamin E. The growth performance to approximately 100 kg of BW was similar with diets containing animal plus plant components or only plant components. Growth was also similar when either of these diets included the supplement. Inclusion of the supplement led to expected increases in Se and vitamin E contents (P < 0.001) of the LM. The differences found in the fatty acid profile of the lipid in LM, loin subcutaneous fat, and the belly cut (pork belly) between the groups with and without animal components in their diets largely reflected differences in the diet composition. Inclusion of the supplement led to greater CLA contents in all 3 tissues (P < 0.001), and also to lower contents of oleic acid (P < 0.001) and greater contents of stearic acid (P < 0.05), possibly due to an inhibition of stearoyl-CoA desaturase enzyme. The supplement also led to an increase in LM intramuscular fat (P < 0.05), but did not affect P2 fat depths (65 mm lateral to the midline of the spine at the last rib; mean depth of 11.8 mm). It is concluded that changing from a part animal component diet to an all plant diet will not change the growth performance of pigs but changes in the fatty acid profile of pork are likely to occur. It is further concluded that the nutritional value of pork may be successfully enhanced by simultaneously supplementing the diet with CLA, selenium, and vitamin E.     

In vitro gas and methane production in rumen fluid from dairy cows fed grass silages differing in plant maturity,compared to in vivo data

The relationship between in vitro rumen CH₄ production of grass silages, using the gas production technique, and in vivo data obtained with the same cows and rations in respiration chambers was investigated. Silages were made from grass harvested in 2013 on May 6th, May 25th, July 1st and July 8th. The grass silages were used to formulate four different rations which were fed to 24 cows in early and late lactation, resulting in a slightly different dry matter intake (DMI; 16.5 kg/day vs. 15.4 kg/day). The experimental rations consisted of 70% grass silage, 10% maize silage, and 20% concentrates on a dry matter basis. Cows were adapted to the rations for 17 days before rumen fluid was collected via oesophageal tubing, and in vitro gas and CH₄ production were analysed. In vitro total gas and CH₄ production of the (ensiled) grass expressed as ml/g OM decreased with advancing maturity of the grass. The in vitro CH₄ production after 48 hr of incubation expressed in ml/g OM did not correlate with the in vivo CH₄ production expressed in g/kg organic matter intake or g/kg DMI (R² = .00–.18, p ≥ .287). The differences in CH₄ emission per unit of intake observed in vivo were rather small between the different rations, which also contributed to the observed poor relationship. Utilizing stepwise multiple regression improved the correlation only slightly. In vitro gas and CH₄ production varied based on whether donor cows were previously adapted to the respective ration or not, suggesting that careful adaption to the experimental diet should be envisaged in in vitro gas and CH₄ production experiments.     

Stability of creatine monohydrate and guanidinoacetic acid during manufacture (retorting and extrusion) and storage of dog foods

The stability of creatine monohydrate (CrMH), crystallised guanidinoacetic acid (GAA‐C) and granulated GAA (GAA‐G) in a moist retorted and a dry extruded dog food formulation during production and storage was investigated. Commercial food mixtures were supplemented with CrMH, GAA‐C or GAA‐G. Uniformity after mixing and retorting or extrusion was determined based on replicate samples (moist n = 8, dry n = 10). Storage stability was evaluated at 25°C/60% relative humidity for 15 months and 40°C/75% for 6 months. Foods with CrMH were analysed for creatine (Cr) and creatinine (Crn), whereas GAA‐C and GAA‐G foods were analysed for GAA concentrations. Coefficients of variation (CV) for uniformity of the additives after mixing of moist and dry pet food formulations were below 15%, and the CV was lower in processed mixtures. Recoveries after retorting and extrusion were higher for GAA‐G (79 and 99%) and GAA‐C (89 and 86%) compared to CrMH (36 and 85%) foods. In moist CrMH food, Cr concentrations re‐increased by 54% whilst Crn concentrations decreased by 39% after storage at 25°C for 15 months. With total molar Cr + Crn remaining stable throughout storage, Crn and Cr appeared to effectively interconvert. Storage of the extruded CrMH food at 25°C for 15 months resulted in a 63% decrease in Cr and a 39% increase in Crn concentration. The decrease in Cr concentration was larger at 6 months storage at 40°C compared to 15 months storage at 25°C. Both GAA‐C and GAA‐G moist and dry foods were stable during storage (<10% decrease). This study showed that GAA is highly stable during production and storage of moist and dry canine foods whilst CrMH is relatively unstable, particularly during storage. The latter makes it difficult to establish a guaranteed Cr content in finished moist retorted and dry extruded foods with CrMH.     

The prevalence of lead shot ingestion in wild mallards (Anas platyrhynchos) in the Netherlands

Summary

During the hunting season 1986–1987, 2859 gizzards from mallards shot in the Netherlands were collected. Gizzards were selected radiographically and examined visually for ingested lead shot. The 95% confidence interval of the prevalence of lead shot ingestion was calculated to be 1.7 to 2.9%. In some gizzards a large number of lead shot (12–16) were seen. This is an indication for locally heavily contaminated areas.

Especially places where mallards are attracted for hunting purposes, by daily feeding of corn, predispose these birds to lead poisoning. A ban with regard to these hunting practices will probably drastically reduce the prevalence of lead shot ingestion. A change from lead shot to steel shot will solve the problem completely.     

Fibrin–alginate hydrogel supports steroidogenesis,in vitro maturation of oocytes and parthenotes production from caprine preantral follicles cultured in group

This study aimed to establish a culture system that improves the in vitro development of caprine preantral follicles. In a first experiment, follicles were encapsulated as a single unit per bead and cultured singly or in groups or with five follicles in the same alginate (ALG) bead for 18 days. In a subsequent experiment, the “five follicles per bead” design was chosen to culture in ALG, fibrin–alginate (FA) or hyaluronate (HA) for 18 days. In a third experiment, we chose the five follicles per bead in FA to culture for 30 days. The culture set‐up of five follicles per ALG bead increased antrum formation and follicle diameter compared to the other culture designs (p < .05). Moreover, under this condition, 44.44% of the oocytes from in vitro cultured preantral follicles reached meiotic resumption. A significant increase of follicle diameter occurred in attachment system and FA (p < .05), but the ALG condition reached the highest among all groups on day 18 (p < .05). Follicles encapsulated in matrix produced more estradiol and progesterone than attachment system (p < .05). The expression of MMP‐9 mRNA was higher in FA than in other groups (p < .05) and similar to antral follicles from in vivo control (p > .05). Only FA group resulted in oocytes matured. After 30 days, oocytes from preantral follicles in vitro grown in FA developed to eight‐cell parthenotes. In conclusion, a culture system using FA supported the development of caprine preantral follicles cultured in group and included in the same bead of hydrogel, improving the oocyte maturation and producing parthenotes.     

Comparison of the in vitro pathogenicity of two Salmonella Typhimurium phage types

The in vitro pathogenicity of Salmonella enterica serovar Typhimurium phage type (pt) 90 and pt 506 (also known as DT 104) isolates from human and porcine origin was studied in adhesion and invasion assays to the human cell line Caco-2 and the porcine cell line IPI-2. Interleukin-8 (IL-8) production by these two cell lines in response to stimulation by the two Salmonella phage types was also measured. Generally, Salmonella Typhimurium pt 506 and pt 90 adhered to and invaded Caco-2 cells and IPI-2 cells equally well. The release of IL-8 by Caco-2 cells or by IPI-2 cells was similar, independent of the Salmonella phage type used for stimulation of the cells. These data suggest that Salmonella Typhimurium pt 90 has a similar ability to cause Salmonella infections as Salmonella Typhimurium DT 104.     

Cloning, sequencing and expression of white rhinoceros (Ceratotherium simum) interferon-gamma (IFN-gamma) and the production of rhinoceros IFN-gamma specific antibodies

Bovine tuberculosis (BTB) is endemic in African buffalo (Syncerus caffer) in the Kruger National Park (KNP). In addition to buffalo, Mycobacterium bovis has been found in at least 14 other mammalian species in South Africa, including kudu (Tragelaphus strepsiceros), Chacma baboon (Papio ursinus) and lion (Panthera leo). This has raised concern about the spillover into other potentially susceptible species like rhinoceros, thus jeopardising breeding and relocation projects aiming at the conservation of biodiversity. Hence, procedures to screen for and diagnose BTB in black rhinoceros (Diceros bicornis) and white rhinoceros (Ceratotherium simum) need to be in place. The Interferon-gamma (IFN-gamma) assay is used as a routine diagnostic tool to determine infection of cattle and recently African buffalo, with M. bovis and other mycobacteria. The aim of the present work was to develop reagents to set up a rhinoceros IFN-gamma (RhIFN-gamma) assay. The white rhinoceros IFN-gamma gene was cloned, sequenced and expressed as a mature protein. Amino acid (aa) sequence analysis revealed that RhIFN-gamma shares a homology of 90% with equine IFN-gamma. Monoclonal antibodies, as well as polyclonal chicken antibodies (Yolk Immunoglobulin-IgY) with specificity for recombinant RhIFN-gamma were produced. Using the monoclonals as capture antibodies and the polyclonal IgY for detection, it was shown that recombinant as well as native white rhinoceros IFN-gamma was recognised. This preliminary IFN-gamma enzyme-linked immunosorbent assay (ELISA), has the potential to be developed into a diagnostic assay for M. bovis infection in rhinoceros.