RNA-sequencing analysis of shell gland shows differences in gene expression profile at two time-points of eggshell formation in laying chickens.

BACKGROUND: Eggshell formation takes place in the shell gland of the oviduct of laying hens. The eggshell is rich in calcium and various glycoproteins synthesised in the shell gland. Although studies have identified genes involved in eggshell formation, little is known about the regulation of genes in the shell gland particularly in a temporal manner. The current study investigated the global gene expression profile of the shell gland of laying hens at different time-points of eggshell formation using RNA-Sequencing (RNA-Seq) analysis. RESULTS: Gene expression profiles of the shell gland tissue at 5 and 15 h time-points were clearly distinct from each other. Out of the 14,334 genes assessed for differential expression in the shell gland tissue, 278 genes were significantly down-regulated (log2 fold change > 1.5; FDR < 0.05) and 413 genes were significantly up-regulated at 15 h relative to the 5 h time-point of eggshell formation. The down-regulated genes annotated to Gene Ontology (GO) terms showed anion transport, synaptic vesicle localisation, organic anion transport, secretion and signal release as the five most enriched terms. The up-regulated gene annotation showed regulation of phospholipase activities, alanine transport, transmembrane receptor protein tyrosine kinase signalling pathway, regulation of blood vessels diameter and 3, 5-cyclic nucleotide phosphodiesterase activity as the five most enriched GO terms. The putative functions of genes identified ranged from calcium binding to receptor activity. Validation of RNA-Seq results through qPCR showed a positive correlation. CONCLUSIONS: The down-regulated genes at 15 h relative to the 5 h time-point were most likely involved in the transport of molecules and synthesis activities, initiating the formation of the eggshell. The up-regulated genes were most likely involved in calcium transportation, as well as synthesis and secretory activities of ions and molecules, reflecting the peak stage of eggshell formation. The findings in the current study improve our understanding of eggshell formation at the molecular level and provide a foundation for further studies of mRNA and possibly microRNA regulation involved in eggshell formation in the shell gland of laying hens.

Infectious bronchitis virus and brown shell colour: Australian strains of infectious bronchitis virus affect brown eggshell colour in commercial laying hens differently.

The aim of the current study was to assess any effect of wild and vaccine Australian infectious bronchitis virus (IBV) strains on shell colour in brown-shelled eggs. In Experiment 1, eggs were collected from day 1 to day 13 post-inoculation (p.i.) from unvaccinated laying hens challenged with IBV wild strains T and N1/88 and from a negative control group of hens. In Experiment 2, eggs were collected from 2 to 22 days p.i. from unvaccinated and vaccinated laying hens challenged with either a wild or a vaccine strain of IBV. In Experiment 1, there was a significant effect (P < 0.05) of day p.i. and of viral strain on shell reflectivity, L* and protoporphyrin IX (PP IX) in eggshells, with and without cuticle. The mean PP IX/g of shell with and without cuticle was significantly higher on day 1 p.i. compared to day 7, after which PP IX increased with day p.i. In Experiment 2, shell reflectivity and L* increased and PP IX decreased with increased day p.i. until day 12. Shell reflectivity and L* decreased slightly after day 12 and increased again towards day 22. Shell reflectivity, L* and PP IX were not significantly different for eggshells from unvaccinated and vaccinated laying hens in the intact eggshell, but were significantly different in shells from which cuticle had been removed. In conclusion, the IBV strains reduced the intensity of brown shell colour to different extents with a lower amount of PP IX in eggshells.

Joint population pharmacokinetic/pharmacodynamic model for the heart rate effects at rest and at the end of exercise for cilobradine.

PURPOSE: To develop a semi-mechanistic population pharmacokinetic/pharmacodynamic (PKPD) model for the selective bradycardic agent cilobradine describing simultaneously the heart rate (HR) measured at rest and just after the end of exercise sharing the same set of PKPD parameters. METHODS: Healthy subjects received cilobradine orally once daily over 2 weeks at 0.25-5 mg doses or placebo. Plasma drug concentrations and HR were measured at rest and following 3 min of exercise over the entire study period. PK and HR data were analyzed using the population approach with NONMEM VII. RESULTS: Plasma disposition of cilobradine was described with a three compartment model. Cilobradine showed dose proportional and time independent pharmacokinetics. HR response was drug concentration dependent and appeared with a significant delay with respect to PK profiles, a phenomenon modeled using two transit compartments. Perturbation in HR at rest as a consequence of exercise was described assuming that physiological processes controlling cardiac frequency were constantly increased over the period of exercise only. CONCLUSIONS: The selected model provides a useful modeling tool for cases where the PD response measured is the result of a temporal experimental induced perturbation.

Dabigatran vs. placebo in patients with acute coronary syndromes on dual antiplatelet therapy: a randomized, double-blind, phase II trial.

AIM: After an acute coronary syndrome, patients remain at risk of recurrent ischaemic events, despite contemporary treatment, including aspirin and clopidogrel. We evaluated the safety and indicators of efficacy of the novel oral direct thrombin inhibitor dabigatran. METHODS AND RESULTS: In this double-blind, placebo-controlled, dose-escalation trial, 1861 patients (99.2% on dual antiplatelet treatment) in 161 centres were enrolled at mean 7.5 days (SD 3.8) after an ST-elevation (60%) or non-ST-elevation (40%) myocardial infarction and randomized to twice daily treatment with dabigatran 50 mg (n = 369), 75 mg (n = 368), 110 mg (n = 406), 150 mg (n = 347), or placebo (n = 371). Primary outcome was the composite of major or clinically relevant minor bleeding during the 6-month treatment period. There were 96 primary outcome events and, compared with placebo, a dose-dependent increase with dabigatran, hazard ratio (HR) 1.77 (95% confidence intervals 0.70, 4.50) for 50 mg; HR 2.17 (0.88, 5.31) for 75 mg; HR 3.92 (1.72, 8.95) for 110 mg; and HR 4.27 (1.86, 9.81) for 150 mg. Compared with placebo, D-dimer concentrations were reduced in all dabigatran dose groups by an average of 37 and 45% at weeks 1 and 4, respectively (P< 0.001). Fourteen (3.8%) patients died, had a myocardial infarction or stroke in the placebo group compared with 17 (4.6%) in 50 mg, 18 (4.9%) in 75 mg, 12 (3.0%) in 110 mg, and 12 (3.5%) in the 150 mg dabigatran groups. CONCLUSIONS: Dabigatran, in addition to dual antiplatelet therapy, was associated with a dose-dependent increase in bleeding events and significantly reduced coagulation activity in patients with a recent myocardial infarction.

Regulation of Immunity-Related Genes by Infectious Bronchitis Virus Challenge in Spleen of Laying Chickens.

Understanding of host pathogen interactions is important in planning strategies for effective control of the pathogen. The present study investigated the regulation of genes involved in the activation of splenic immune system in mature laying chickens challenged with T strain of infectious bronchitis virus (IBV). Among all the genes studied, the relative expression levels of Fas cell surface death receptor (FAS), interleukin 7 (IL7), IL18, proteasome subunit alpha 3 (PSMA3), major histocompatibility complex, class II (MHCII), interferon alpha (IFNα), immunoglobulin A (IgA), and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) were significantly (p < 0.05) upregulated, while Toll-like receptor 7 (TLR7) and TLR5 were significantly downregulated in the challenge compared with the control group. Genes such as vascular cell adhesion molecule 1 (VCAM1), FK506-binding protein 1B (FKBP1B), transforming growth factor-beta 3 (TGFB3), NLR family pyrin domain containing 3 (NLRP3), TYRO3 protein tyrosine kinase (TYRO3), TNF receptor-associated factor 3 (TRAF3), C-X-C motif chemokine receptor 4 (CXCR4), macrophage inflammatory protein-3 (MIP3A), TLR2-1, TLR3, and TLR21 were not altered in mRNA expression levels between the challenge and control groups. In conclusion, the splenic immune response to IBV infection involved the regulation of cytokines, TLRs and NF-κB.

Genes involved in mitochondrial biogenesis and function may not show synchronised responses to mitochondria in shell gland of laying chickens under infectious bronchitis virus challenge.

BACKGROUND: Egg formation takes place in the oviduct of laying hens over a 24 h period. Infectious bronchitis virus (IBV) causes pathological lesions in the chicken oviduct. In the current study, mitochondrial counts were determined in three different segments of the oviduct during egg formation in laying chickens challenged with IBV T strain. Nuclear DNA encoded genes that are involved in mitochondrial biogenesis, fission and function were studied in the shell gland of the oviduct undergoing virus multiplication. RESULTS: In the shell gland, the mitochondrial count was significantly lower (P < 0.05) in the challenged group, compared with the control group. However, it did not vary in response to IBV challenge in the isthmus and magnum regions of the oviduct. The gene succinate dehydrogenase complex, subunit A, flavoprotein variant (SDHA) was down-regulated in the shell gland by IBV challenge (P < 0.05), while other genes being studied did not show responses to the challenge (P > 0.05). Differential expression of the genes was observed at different time-points of egg-shell formation. The expression levels of citrate synthase (CS), cytochrome C, somatic (CYC, S) and sodium-potassium adenosine triphosphatase (Na+-K+ATPase) genes were significantly higher, while those of SDHA and dynamin related protein 1 (Drp1) genes were significantly lower, at 15 h compared with 5 h following oviposition of the previous egg. The expression level of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) did not show significant change at different time-points. CONCLUSIONS: It was concluded that IBV T strain infection in laying hens reduced mitochondrial counts only in the shell gland region of the oviduct. The genes involved in mitochondrial biogenesis or function may not show synchronised responses to that of mitochondria in the shell gland of chickens under T strain of IBV challenge.

Reference gene selection for the shell gland of laying hens in response to time-points of eggshell formation and nicarbazin.

Ten reference genes were investigated for normalization of gene expression data in the shell gland of laying hens. Analyses performed with geNorm revealed that hypoxanthine phosphoribosyltransferase 1 (HPRT1) and hydroxymethylbilane synthase (HMBS) were the two most stable reference genes in response to post-oviposition time alone (POT) or with nicarbazin treatment (POT+N) of laying hens. NormFinder analyses showed that the two most stable reference genes in response to POT and POT+N were 18S ribosomal RNA (18S rRNA), ribosomal protein L4 (RPL4) and HMBS, RPL4, respectively. BestKeeper analyses showed that 18S rRNA, RPL4 and HPRT1, HMBS were the two most stable reference genes for POT, and POT+N, respectively. Of the ten reference genes, all except B2M showed geNorm M <0.5, suggesting that they were stably expressed in the shell gland tissue. Consensus from these three programs suggested HPRT1 and HMBS could be used as the two most stable reference genes in the present study. Expression analyses of four candidate target genes with the two most and the two least stable genes showed that a combination of stable reference genes leads to more discriminable quantification of expression levels of target genes, while the least stable genes failed to do so. Therefore, HMBS and HPRT1 are recommended as the two most stable reference genes for the normalization of gene expression data at different stages of eggshell formation in brown-egg laying hens. Available statistical programs for reference gene ranking should include more robust analysis capability to analyse the gene expression data generated from factorial design experiments.

Downregulation of ALAS1 by nicarbazin treatment underlies the reduced synthesis of protoporphyrin IX in shell gland of laying hens.

Shell colour is an important trait for eggs and an understanding of pigment deposition will assist potential management of egg shell colour loss. We demonstrated that nicarbazin feeding down-regulated ALAS1 and reduced protoporphyrin IX (PP IX) in both shell gland and eggshell, indicating the role of nicarbazin in inhibiting the synthesis of PP IX. Additionally, the expression levels of the genes did not show sequential upregulation in the same order of diurnal time-points (TP) during egg formation. The gene SLC25A38, responsible for transporting glycine from cytoplasm to mitochondria, and the gene ALAS1, encoding rate-limiting enzyme (delta-aminolevulinic acid synthase 1), had higher expression at 15 hr, as compared with 2, 5 and 23.5 hrs postoviposition. Interestingly, ABCB6, a gene encoding an enzyme responsible for transporting coproporphyrinogen III, showed higher expression level at 2 and 5 hrs. However, the expression of CPOX that converts coproporphyrinogen III to protoporphyrinogen III, and ABCG2 that transports PP IX out from mitochondria did not alter. Nevertheless, mitochondrial count per cell did not show consistent change in response to time-points postoviposition and nicarbazin feeding. The information obtained in the study sheds light on how nicarbazin disrupts the synthesis of PP IX.

Reference gene selection for gene expression study in shell gland and spleen of laying hens challenged with infectious bronchitis virus.

Ten reference genes were investigated for normalisation of candidate target gene expression data in the shell gland and spleen of laying hens challenged with two strains of infectious bronchitis virus (IBV). Data were analysed with geNorm, NormFinder and BestKeeper, and a comprehensive ranking (geomean) was calculated. In the combined data set of IBV challenged shell gland samples, the comprehensive ranking showed TATA-box binding protein (TBP) and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ) as the two most stable, and succinate dehydrogenase complex flavoprotein subunit A (SDHA) and albumin (ALB) as the two least stable reference genes. In the spleen, and in the combined data set of the shell gland and spleen, the two most stable and the two least stable reference genes were TBP and YWHAZ, and ribosomal protein L4 (RPL4) and ALB, respectively. Different ranking has been due to different algorithms. Validation studies showed that the use of the two most stable reference genes produced accurate and more robust gene expression data. The two most and least stable reference genes obtained in the study, were further used for candidate target gene expression data normalisation of the shell gland and spleen under an IBV infection model.

Effect of production system and flock age on eggshell and egg internal quality measurements.

Egg quality was measured in eggs from different flocks that were reared together and then allocated to different production systems. Eggs were processed for measurements of eggshell and egg internal quality variables, scoring of ultrastructural mammillary layer features, completeness of cuticle cover, and protoporphyrin IX (PP IX) quantification. There was a significant main effect (P < 0.05) of production system on shell reflectivity, egg weight, and egg internal quality and significant effects of flock age on most measurements. The mammillary layer ultrastructural variables showed no clear relationship with production system and flock age. However, there was a significant interaction between production system and flock age for mammillary cap, early and late fusions. Cuticle cover ([Formula: see text]), was significantly higher in barn eggs (19.20), followed by free range (17.57), and cage eggs (15.99). Completeness of cuticle cover was significantly higher in eggs from the 44 week old flock than for 64 week and 73 week old flocks. For eggshells with cuticle intact, there was a significant main effect of both production system and flock age, and significant interaction between the two, for shell reflectivity, L*a*b* values and amount of PP IX. For PP IX, when this difference was calculated for the cuticle alone, there were no statistically significant differences. In 1 g of shell with and without cuticle, there was more PP IX in cage eggs (9.49 × 10-8, 7.90 × 10-8 mM) followed by free range (8.24 × 10-8, 6.90 × 10-8 mM), and barn eggs (8.64 × 10-8, 7.28 × 10-8 mM). Similar trends were recorded for the amount of PP IX in 1 g of cuticle, but the difference was not statistically significant. The amount of PP IX decreased significantly with increasing flock age. Comparing the cage and barn production systems at 68 week of flock age, there was no difference for the amount of PP IX in shell with or without cuticle, or in the cuticle alone. Eggs from the cage production system were darker in color and contained more PP IX mainly within the calcareous part of the shell. For the barn production system, the completeness of cuticle cover was higher and egg weight generally lower.

Effect of egg washing and correlation between cuticle and egg penetration by various Salmonella strains.

In Australia, Europe and the United States, eggs and egg products are frequently associated with Salmonella food poisoning outbreaks. Many of the egg-associated Salmonella outbreaks have been due to the products such as mayonnaise, ice-cream and cold desserts which are eaten without cooking following the addition of raw egg. The ability of four Salmonella isolates (one each of S. Singapore, S. Adelaide, S. Worthington and S. Livingstone) to penetrate washed and unwashed eggs using whole egg and agar egg penetration methods was investigated in the current study. The results of the agar penetration experiment indicated that all the isolates used in the present study have the capacity to penetrate the eggshell. Eggshell penetration by the S. Worthington isolate was higher but not significant (p=0.06) in washed eggs compared to unwashed eggs. However, for all other isolates (S. Singapore, S. Adelaide and S. Livingstone), there was no significant difference in penetration of washed and unwashed eggs. Statistical analysis indicated that cuticle score was a significant linear predictor of Salmonella eggshell penetration. Whole egg penetration results showed that all of the Salmonella isolates used in the present study were capable of surviving on the eggshell surface after 21days of incubation (at 20°C) following a high dose of inoculation (10(5)CFU/mL). The combined data of all isolates demonstrated that, the survival rate of Salmonella on eggshells (inoculated with 10(5)CFU/mL) was significantly higher (p=0.002) at 20°C as compared to 37°C. S. Singapore, S. Worthington, and S. Livingstone were not detected in egg internal contents whereas S. Adelaide was detected in one egg's internal contents.

Effect of egg washing and correlation between eggshell characteristics and egg penetration by various Salmonella Typhimurium strains.

Salmonella is an important foodborne pathogen, causing an estimated 11,992 cases of infection in Australia per year. Egg or egg product related salmonellosis is a major concern for the egg industry. Worldwide, S. Typhimurium is one of the most common serovars identified in Salmonella food poisoning cases. The current study investigated the ability of five S. Typhimurium strains to penetrate washed and unwashed eggs using whole egg and agar egg penetration methods. All S. Typhimurium strains were able to penetrate eggshells and survive in egg albumen (at 20°C) according to whole egg penetration results. Polymerase Chain Reaction results demonstrated that S. Typhimurium strain 2 (10(3) and 10(5) CFU/mL), and strain 5 (10(3) and 10(5) CFU/mL) egg penetration was significantly higher (p<0.05) in washed eggs when compared to unwashed eggs. Statistical analysis of the agar penetration experiment indicated that S. Typhimurium was able to penetrate washed eggs at a significantly higher rate when compared to unwashed eggs (p<0.05). When compared to unwashed eggs, washed eggs also had significantly damaged cuticles. Statistical analysis also indicated that eggshell penetration by S. Typhimurium was related to various eggshell ultrastructural features such as cap quality, alignment, erosion, confluence, Type B bodies and cuticle cover.