New biomarkers for increased intestinal permeability induced by dextran sodium sulphate and fasting in chickens

Increased intestinal permeability (IP) can lead to compromised health in chickens. As there is limited literature on in vivo biomarkers to assess increased IP in chickens, the objective of this study was to identify a reliable biomarker of IP using DSS ingestion and fasting models. Male Ross chickens (n = 48) were reared until day 14 on the floor pen in an animal care facility, randomized into the following groups: control, DSS and fasting (each with n = 16), and then placed in metabolism cages. DSS was administered in drinking water at 0.75% from days 16 to 21, while controls and fasted groups received water. All birds had free access to feed and water except the birds in the fasting group that were denied feed for 19.5 h on day 20. On day 21, all chickens were given two separate oral gavages comprising fluorescein isothiocyanate dextran (FITC‐d, 2.2 mg in 1 ml/bird) at time zero and lactulose, mannitol and rhamnose (LMR) sugars (0.25 g L, 0.05 g M and 0.05 g R in 2 ml/bird) at 60 min. Whole blood was collected from the brachial vein in a syringe 90 min post‐LMR sugar gavage. Serum FITC‐d and plasma LMR sugar concentrations were measured by spectrophotometry and high‐performance ion chromatography respectively. Plasma concentrations of intestinal fatty acid binding protein, diamine oxidase, tight junction protein (TJP), d ‐lactate and faecal α‐antitrypsin inhibitor concentration were also analysed by ELISA. FITC‐d increased significantly (p < 0.05) after fasting compared with control. L/M and L/R ratios for fasting and L/M ratio for DSS increased compared with control chickens (p < 0.05). TJP in plasma was significantly increased due to fasting but not DSS treatment, compared with controls. Other tests did not indicate changes in IP (p > 0.05). We concluded that FITC‐d and LMR sugar tests can be used in chickens to assess changes in IP.     

Antimicrobial Susceptibility of 41 Burkholderia mallei Isolates From Spontaneous Outbreaks of Equine Glanders in Punjab,Pakistan

Minimum inhibitory concentrations (MICs) of 20 antimicrobial agents for 41 isolates of Burkholderia mallei from natural outbreaks of equine glanders were determined by agar dilution. All isolates were intrinsically resistant to ampicillin (MIC₉₀ ≥128). Resistance to other antimicrobials was as follows: 95.1% to amoxicillin and cephradine, 85.4% to cefuroxime and norfloxacin, 68.3% to ceftizoxime and ceftriaxone, 61.1% to ceftiofur, 58.5% to oxytetracycline, 41.5% to ciprofloxacin, 58.5% to roxithromycin, 17.1% cefotaxime, and 12.2% clarithromycin. Overall resistance patterns revealed that 17% of isolates were simultaneously resistant to amoxicillin, cephradine, cefuroxime, ceftizoxime, ceftriaxone and norfloxacin. None of the isolates were resistant to amoxicillin-clavulanic acid, doxycycline, chloramphenicol, gentamicin or trimethoprim-sulphadiazine. Mode MICs for these antimicrobials were 2, 1, 8, 4 and 1 μg/ml, respectively. A majority of the isolates (∼ 94%) were susceptible to both enrofloxacin and ofloxacin. These data provide an updated perspective on susceptibility profiles of current strains of B. mallei in an endemic area.     

In Vitro Chemopreventive Potential of Phlorotannins-Rich Extract from Brown Algae by Inhibition of Benzo[a]pyrene-Induced P2X7 Activation and Toxic Effects

Phlorotannins are polyphenols occurring exclusively in some species of brown algae, known for numerous biological activities, e.g., antioxidant, antiproliferative, antidiabetic, and antiallergic properties. Their effects on the response of human lung cells to benzo[a]pyrene (B[a]P) has not been characterized. Our objective was to in vitro evaluate the effects of a phlorotannin-rich extract obtained from the brown algae Ascophyllum nodosum and Fucus vesiculosus on B[a]P cytotoxic effects. The A549 cell line was incubated with B[a]P for 48 and 72 h in the presence or absence of the brown algae extract. Cytochrome P450 activity, activation of P2X7 receptor, F-actin disorganization, and loss of E-cadherin expression were assessed using microplate cytometry and fluorescence microscopy. Relative to control, incubation with the brown algae extract was associated with lower B[a]P-induced CYP1 activity, lower P2X7 receptor activation, and lower reactive oxygen species production. The brown algae extract inhibited the alterations of F-actin arrangement and the downregulation of E-cadherin expression. We identified a phlorotannins-rich extract that could be deeper investigated as a cancer chemopreventive agent to block B[a]P-mediated carcinogenesis.     

Expression Analysis of RNA-Binding Motif Gene on Y Chromosome (RBMY) Protein Isoforms in Testis Tissue and a Testicular Germ Cell Cancer-Derived Cell Line (NT2)

Background: RNA-binding motif gene on Y chromosome (RBMY), a germ cell-specific nuclear protein, is known as a key factor in spermatogenesis and disorders associated with this protein have been recognized to be related to male infertility. Although it was suggested that this protein could have different functions during germ cell development, no studies have been conducted to uncover the mechanism of this potential function yet. Here, we analyzed the expression pattern of RBMY protein isoforms in testis compared to NT2, a testicular germ cell cancer-derived cell line, to test probability of differential expression of RBMY protein isoforms at different spermatogenesis stages. Methods: Full length and a segment of RBMY gene were cloned and expressed in E. coli. Anti-human RBMY antibody was produced in rabbit using the recombinant proteins as antigen. Western-blot and immunofluorescence were conducted for detection and comparison of RBMY protein isoforms. Results: Selected segment of RBMY protein resulted in producing a mono-specific antibody. As results shows, only the longest isoform of RBMY was expressed at protein level in NT2 cell line, while three isoforms of this protein were detected in the whole testis lysate. Conclusion: The results imply that different alternative splicing may happen in testis cells and probably difference of RBMY function during spermatogenesis is due to the differential expression of RBMY protein isoforms. These results and further experiments on RBMY isoforms can help to obtain a better understanding of the function of this protein, which may increase our knowledge about spermatogenesis and causes of male infertility. Key Words: Protein isoforms, Spermatogenesis, Male infertility     

Effect of different levels of rice bran processed by various techniques on performance of broiler chicks

1. Rice bran processed by extrusion cooking, roasting or pelleting and treated with antioxidant was used in broiler starter and finisher diets at various concentrations up to 500 g/kg. 2. Extrusion cooking gave the best performance, followed by roasting, while non-significant differences were observed between raw and pelleted bran. 3. Increasing the amount of rice bran in broiler diets resulted in significantly negative effects on growth performance. Non-significant differences were observed in mortality and dressing percentage due to different processes and concentrations of rice bran in the diet. Organ weights were significantly higher on raw and pelleted rice bran as compared to extruded and roasted bran. Liver and heart weights significantly increased with increasing contents of rice bran above 200g/kg, while pancreas weight increased with an addition of rice bran. 4. Treating the rice bran with antioxidant up to 250ppm had non-significant effects on broiler performance.     

Gene expression and morphological changes in the intestinal mucosa associated with increased permeability induced by short‐term fasting in chickens

Short‐term fasting for 4.5 and 9 hr has been demonstrated to increase intestinal permeability (IP) in chickens. This study aimed to investigate the effects of 0, 4.5, 9 and 19.5 hr fasting on intestinal gene expression and villus‐crypt architecture of enterocytes in jejunal and ileal samples. On day 38, Ross‐308 male birds were fasted according to their group and then euthanised. Two separate intestinal sections (each 2 cm long, jejunum and ileum) were collected. One section was utilised for villus height and crypt depth measurements. The second section was snap‐frozen in liquid nitrogen for quantitative polymerase chain reaction (qPCR) analysis of tight junction proteins (TJP) including claudin‐1, claudin‐3, occludin, zonula occludens (ZO‐1, ZO‐2), junctional adhesion molecules (JAM) and E‐cadherin. Additionally genes involved in enterocyte protection including glucagon‐like peptide (GLP‐2), heat‐shock protein (HSP‐70), intestinal alkaline phosphatase (IAP), mammalian target of rapamycin (mTOR), toll‐like receptors (TLR‐4), mucin (MUC‐2), cluster differentiation (CD‐36) and fatty acid‐binding protein (FABP‐6) were also analysed. Normally distributed data were analysed using one‐way analysis of variance ANOVA. Other data were analysed by non‐parametric one‐way ANOVA. Villus height and crypt depth were increased (p < .05) only in the ileum after fasting for 4.5 and 9 hr compared with non‐fasting group. mRNA expression of claudin‐3 was significantly reduced in the ileum of birds fasted for 9 and 19.5 hr, suggesting a role in IP modulation. However, all other TJP genes examined were not statistically different from control. Nevertheless, ileal FABP‐6 of all fasted groups was significantly reduced, which could possibly be due to reduced bile acid production during fasting.     

Cystic hydatidosis in slaughtered goats from various municipal abattoirs in Oman

A passive surveillance study was conducted from April 2012 to April 2013 to determine the incidence, the affect of age, sex and cyst location, fertility, and viability on the occurrence of cystic hydatidosis in slaughtered goats from six municipal abattoirs around Oman. Morphologic characterization of suspected visceral organs has shown that Echinococcos granulosus (E. granulosus) metacestodes were present in 9.2 % of the total samples (682). Incidence (per 10,000) varied from 1.4 to 1.5. There was a significant difference between abattoirs, with Salalah having the highest infection (63.2 %), followed by Nizwa (27.2 %), Al Buraimi (10.8 %), Bousher (7.2 %), and the lowest in Sohar (1.6 %). Infection was significantly higher in indigenous goats (22.1 %) compared with imported breeds (2.5 %) from Somalia. The visceral organs were found harboring one or more hydatid cysts. Greatest cyst infection was recorded in lungs (53.4 %), followed by heart (50 %), liver (6.2 %), and multiple organs (14 %). A significant difference was observed between females (25 %) and male goats (6 %). Furthermore, the fertility of the hydatid cysts was found higher in heart and lungs (100 %, 51.6 %) and the viability rate of protoscolices was high (64 %). The findings of this study demonstrated that cystic hydatidosis is common and widely distributed in local goats, and they might play an important role in the life cycle and transmission of this zoonosis in Oman.     

Expression of selected genes encoding mechanistic pathways,nutrient and amino acid transporters in jejunum and ileum of broiler chickens fed a reduced protein diet supplemented with arginine,glutamine and glycine under stress stimulated by dexamethasone

Reducing crude protein and supplementation with synthetic amino acids in poultry nutrition is a recent trend to avoid wastage of protein and ammonia in production systems. Stress has been shown to impair intestinal barrier and increase inflammatory response. This study was performed on intestinal tissues of broiler chickens to understand the mechanism of stress induced by a synthetic glucocorticoid, dexamethasone (DEX) and the effect of supplementation of arginine, glutamine and glycine in reduced protein diets. Intestinal tissue samples from a previous study were utilized. Male Ross 308 chickens received a basal diet for the first seven days and then fed with crude protein that was reduced to 194 g/kg in grower experimental diets supplemented with glutamine, glycine and additional arginine at 10, 10 and 5 g/kg respectively. Half of the 96 individual birds were injected with DEX (0.5 mg/kg body weight) or saline on days 14, 16, 18 and 20 of age. mRNA expression for jejunum and ileum for amino acid transporters (y+LAT-1, B^o,+AT, EAAT-3 and CAT-1), mechanistic genes (SGLT-1, mTOR, IAP and FABP-2) and pro-inflammatory genes (MUC-2, NF-κB, iNOS, IL-8 and IL-1β) were analysed using real-time PCR. The results showed that DEX decreased y+ LAT1 in jejunum, B^o,+AT and EAAT-3 in ileum. Arginine increased CAT-1 in the jejunum and ileum under DEX treatment. Through an interaction, DEX reduced IAP in jejunum of glycine and arginine supplemented group and reduced mTOR in jejunum independently. DEX reduced MUC-2 and iNOS in jejunum and increased iNOS and IL8 in the ileum. Amino acid supplementation did not appear to ameliorate these effects; however, there were some positive effects of glycine on NF-κB and arginine through increased CAT-1. Mechanistic understanding of amino acid supplementation in broiler diets warrants further research particularly when dietary protein is reduced below the level tested in the present study.     

In vivo anthelmintic activity of Butea monosperma against Trichostrongylid nematodes in sheep

Seeds of Butea monosperma administered as crude powder (CP) at doses of 1, 2 and 3 g/kg to sheep naturally infected with mixed species of gastrointestinal nematodes exhibited a dose and a time-dependent anthelmintic effect. The maximum reduction of 78.4% in eggs per gram of feces (EPG) was recorded on day 10 after treatment with 3 g/kg. Levamisole (7.5 mg/kg), a standard anthelmintic agent, exhibited 99.1% reduction in EPG.