Transcriptome analysis of the uterus of hens laying eggs differing in cuticle deposition.

BACKGROUND: Avian eggs have a proteinaceous cuticle. The quantity of cuticle varies and the deposition of a good cuticle in the uterus (Shell-gland) prevents transmission of bacteria to the egg contents. RESULTS: To understand cuticle deposition, uterus transcriptomes were compared between hens with i) naturally good and poor cuticle and, ii) where manipulation of the hypothalamo-pituitary-gonadal-oviduct axis produced eggs with or without cuticle. The highest expressed genes encoded eggshell matrix and cuticle proteins, e.g. MEPE (OC-116), BPIFB3 (OVX-36), RARRES1 (OVX-32), WAP (OVX-25), and genes for mitochondrial oxidative phosphorylation, active transport and energy metabolism. Expression of a number of these genes differed between hens laying eggs with or without cuticle. There was also a high expression of clock genes. PER2, CRY2, CRY1, CLOCK and BMAL1 were differentially expressed when cuticle deposition was prevented, and they also changed throughout the egg formation cycle. This suggests an endogenous clock in the uterus may be a component of cuticle deposition control. Cuticle proteins are glycosylated and glycosaminoglycan binding genes had a lower expression when cuticle proteins were deposited on the egg. The immediate early genes, JUN and FOS, were expressed less when the cuticle had not been deposited and changed over the egg formation cycle, suggesting they are important in oviposition and cuticle deposition. The uterus transcriptome of hens with good and poor cuticle deposition did not differ. CONCLUSIONS: We have gained insights into the factors that can affect the production of the cuticle especially clock genes and immediate early genes. We have demonstrated that these genes change their expression over the period of eggshell formation supporting their importance. The lack of differences in expression between the uterus of hens laying eggs with the best and worse cuticle suggest the genetic basis of the trait may lie outside the oviduct.

Genotype-associated differences in bursal recovery after infectious bursal disease virus (IBDV) inoculation.

T-cell immune responses were shown to play an important role in the regulation of infectious bursal disease virus (IBDV) replication and development of lesions in the bursa of Fabricius (BF) (bursal lesions) but also in the recovery from the infection. Studies suggested that the host-genotype influences T-cell responses during the acute phase of infection. Genotype-related differences in the recovery phase were not investigated so far. The present study used commercial broiler- (BT), layer- (LT), dual-purpose type (DT) chicken lines as well as a specific pathogen free (SPF) LT chicken as a reference for comparison of T-cell related differences in IBDV-immunopathogenesis not only in the early phase post inoculation (pi) but also in the recovery phase. The Deventer formula was used to determine the optimal time point of inoculation with an intermediate plus IBDV strain when maternally derived antibody (MDA) titers were below the calculated breakthrough level of the virus for all genotypes. Differences in the bursal lesion development, intrabursal CD4+ and CD8+ T-cell accumulation and numbers of IBDV-positive cells were determined. In addition, anti-IBDV antibody development and the relative amount of anti-inflammatory cytokine mRNA were recorded until 28 days post IBDV inoculation. Differences between the genotypes were observed in the duration and magnitude of bursal lesions, CD4+ and CD8+ T-cell infiltration as well as the presence of anti-inflammatory Interleukin (IL)-10 and Transforming growth factor (TGF) ß4 cytokine mRNA (P < 0.05). While the investigated immune parameters were comparable between the genotypes at seven days pi, during 14, 21 and 28 days pi a delayed recovery process in LT and DT chickens compared to BT chickens was observed (P < 0.05). Furthermore, the age and residual MDA levels had a genotype-dependent influence on the onset of the anti-IBDV specific humoral and T-cell mediated immune responses. This study suggests, that the impact of T-cell immunity on the recovery process after IBDV infection may need to be considered further for the development of new breeding programs for disease resistant chicken lines.

Genetic variation and potential for genetic improvement of cuticle deposition on chicken eggs.

BACKGROUND: The cuticle is an invisible glycosylated protein layer that covers the outside of the eggshell and forms a barrier to the transmission of microorganisms. Cuticle-specific staining and in situ absorbance measurements have been used to quantify cuticle deposition in several pure breeds of chicken. For brown eggs, a pre-stain and a post-stain absorbance measurement is required to correct for intrinsic absorption by the natural pigment. For white eggs, a post-stain absorbance measurement alone is sufficient to estimate cuticle deposition. The objective of the research was to estimate genetic parameters and provide data to promote adoption of the technique to increase cuticle deposition and reduce vertical transmission of microorganisms. RESULTS: For all pure breeds examined here, i.e. Rhode Island Red, two White Leghorns, White Rock and a broiler breed, the estimate of heritability for cuticle deposition from a meta-analysis was moderately high (0.38 ± 0.04). In the Rhode Island Red breed, the estimate of the genetic correlation between measurements recorded at early and late times during the egg-laying period was ~ 1. There was no negative genetic correlation between cuticle deposition and production traits. Estimates of the genetic correlation of cuticle deposition with shell color ranged from negative values or 0 in brown-egg layers to positive values in white- or tinted-egg layers. Using the intrinsic fluorescence of tryptophan in the cuticle proteins to quantify the amount of cuticle deposition failed because of complex quenching processes. Tryptophan fluorescence intensity at 330 nm was moderately heritable, but there was no evidence of a non-zero genetic correlation with cuticle deposition. This was complicated furthermore by a negative genetic correlation of fluorescence with color in brown eggs, due to the quenching of tryptophan fluorescence by energy transfer to protoporphyrin pigment. We also confirmed that removal of the cuticle increased reflection of ultraviolet wavelengths from the egg. CONCLUSIONS: These results provide additional evidence for the need to incorporate cuticle deposition into breeding programs of egg- and meat-type birds in order to reduce vertical and horizontal transmission of potentially pathogenic organisms and to help improve biosecurity in poultry.

Immune responses upon in ovo HVT-IBD vaccination vary between different chicken lines.

The genotype of chickens is assumed to be associated with variable immune responses. In this study a modern, moderate performing dual-purpose chicken line (DT) was compared with a high-performing layer-type (LT) as well as a broiler-type (BT) chicken line. One group of each genotype was vaccinated in ovo with a recombinant herpesvirus of turkeys expressing the virus protein VP2 of the infectious bursal disease virus (HVT-IBD) while one group of each genotype was left HVT-IBD unvaccinated (control group). Genotype associated differences in innate and adapted immune responses between the groups were determined over five weeks post hatch. HVT-IBD vaccination significantly enhanced humoral immune responses against subsequently applied live vaccines compared to non-HVT-IBD vaccinated groups at some of the investigated time points (P < 0.05). In addition HVT-IBD vaccination had depending on the genotype a significant impact on splenic macrophage as well as bursal CD4+ T-cell numbers (P < 0.05). On the other hand, the detectable genotype influence on Interferon (IFN) γ and nitric oxide (NO) release of ex vivo stimulated spleen cells was independent of HVT-IBD vaccination. The results of our study suggest considering a genotype specific vaccination regime in the field.

Cuticle deposition improves the biosecurity of eggs through the laying cycle and can be measured on hatching eggs without compromising embryonic development.

The cuticle is part of the egg's natural defense and it can be improved by genetic selection. Prior to adoption of this measurement in breeding programs, questions that need to be addressed are whether improved cuticle deposition will result in a reduced risk of eggs becoming contaminated and whether selection for this trait will have any unintended consequences on the incubation process. Bacterial penetration experiments were carried out using eggs from a pedigree line of broiler breeders (BB) and Rhode Island Red (RIR) layers. Within the natural variation in cuticle deposition in each line, a good cuticle was shown to reduce an egg's susceptibility to penetration by Escherichia coli (BB, P = 0.023) and Salmonella typhimurium (RIR, P < 0.001). Deglycosylation of cuticle proteins had little effect on their antimicrobial activity. The effect of bird age on cuticle deposition was also examined. Shell color decreased with age as anticipated; however, we found no evidence that cuticle deposition decreases with age, at least up to 50 wk. A thicker cuticle could affect the water vapor conductance (WPC) of hatching eggs. The WPC of eggs was, therefore, measured on eggs selected from the top and tail of the cuticle distribution, this time in a Lohmann Selected Leghorn (LSL) pedigree line. Broiler breeder eggs were also tested. No evidence of a relationship between cuticle deposition and WPC was found for LSL or BB eggs. Cuticle deposition measurements require eggs to be stained. Here, we show that this has no adverse effect on embryo development at d 12 of incubation. Thus, we conclude that cuticle deposition is important in preventing bacterial penetration of eggs in genetically divergent breeds of chicken and that the measurement can be practically incorporated into breeding programs. This will contribute to improving the biosecurity of eggs by reducing vertical and horizontal transmission of potentially zoonotic and pathogenic organisms from parent to offspring.

Characterization of Enterococcus faecalis isolates by chicken embryo lethality assay and ERIC-PCR.

Enterococcus faecalis is the major causative agent of amyloid arthropathy in chickens. Given the difficulty of estimating the risk from field strains, the embryo lethality assay (ELA) is proposed in this study as a model to predict the virulence of 68 avian E. faecalis strains. Additionally, Enterobacterial Repetitive Intergenic Consensus Polymerase Chain Reaction (ERIC-PCR) was used to characterize the genetic diversity of the E. faecalis strains. The ELA was performed 10 times with subsets of 7-8 E. faecalis strains each on a sample of 9987 eggs, including control groups. An estimated 3-24 colony-forming units were inoculated into the allantoic cavity of 10-day-old embryos. The embryonic mortality rate (EMR) was determined by means of candling the eggs over a period of seven days. The ELA was able to distinguish the virulence of the E. faecalis strains. Twenty-six strains were considered as avirulent strains with an EMR of below 40%. Five strains were highly virulent with an EMR above 80%. The remaining 37 strains were classified as strains of moderate virulence, causing an EMR between 40% and 80%. The highest EMR occurred three and four days post-inoculation (p.i.). From the fourth day p.i., almost no embryonic mortality was observed. Therefore, the ELA could be optimized by reducing experiment duration to four days p.i. ERIC-PCR did not cluster the strains according to its virulence, although ERIC banding patterns revealed a considerable genetic diversity. In conclusion, the ELA can be considered a reliable and useful tool to predict the virulence of avian E. faecalis strains.

Understanding avian egg cuticle formation in the oviduct: a study of its origin and deposition.

The cuticle is a unique invisible oviduct secretion that protects avian eggs from bacterial penetration through gas exchange pores. Despite its importance, experimental evidence is lacking for where, when, and what is responsible for its deposition. By using knowledge about the ovulatory cycle and oviposition, we have manipulated cuticle deposition to obtain evidence on these key points. Cuticle deposition was measured using staining and spectrophotometry. Experimental evidence supports the location of cuticle deposition to be the shell gland pouch (uterus), not the vagina, and the time of deposition to be within the final hour before oviposition. Oviposition induced by arginine vasotocin or prostaglandin, the penultimate and ultimate factors for the induction of oviposition, produces an egg with no cuticle; therefore, these factors are not responsible for cuticle secretion. Conversely, oviposition induced by GNRH, which mimics the normal events of ovulation and oviposition, results in a normal cuticle. There is no evidence that cuticle deposition differs at the end of a clutch and, therefore, there is no evidence that the ovulatory surge of progesterone affects cuticle deposition. Overall, the results demonstrate that the cuticle is a specific secretion and is not merely an extension of the organic matrix of the shell. Cuticle deposition was found to be reduced by an environmental stressor, and there is no codependence of the deposition of pigment and cuticle. Defining the basic facts surrounding cuticle deposition will help reduce contamination of hen's eggs and increase understanding of the strategies birds use to protect their eggs.

Chicken embryo lethality assay for determining the lethal dose and virulence of Enterococcus faecalis.

Enterococcus faecalis is the major pathogen found in field cases of amyloid arthropathy in chickens. Given the need for a better understanding of the virulence mechanisms of the causative strains, the embryo lethality assay (ELA) is proposed in the present study as a model to evaluate the virulence of E. faecalis strains, specifically the pathogenic avian strain K923/96, which was previously related with amyloid arthropathy. Hence, 0.2 ml of five doses of the cited strain (from 2.5 to 2500 colony-forming units (CFU) per ml) were inoculated into the allantoic cavity of 10-day-old embryos. The embryo mortality rate (EMR) was determined by daily candling of the eggs over a period of seven days and based on this information the median lethal dose (LD50) was calculated. The ELA was repeated four times on a sample of 3443 eggs. The infectious dose showed a significant effect on the EMR. The EMR with the doses of 2.5, 5, 25, 250 and 2500 CFU/ml was 43%, 45%, 63%, 90% and 93%, respectively. The estimated dose at LD50 was 6.6 CFU/ml. As expected, the higher the infectious dose, the greater the EMR and the lower the embryo survival time. The highest EMR was recorded after three and four days post-inoculation in all doses. In conclusion, these results can be used as a basis for further researches on the E. faecalis virulence. In order to corroborate its model capacity to predict the virulence of this bacterium, more ELAs with different E. faecalis strains are required.