Immunome differences between porcine ileal and jejunal Peyer's patches revealed by global transcriptome sequencing of gut-associated lymphoid tissues.

The epithelium of the intestinal mucosa and the gut-associated lymphoid tissues (GALT) constitute an essential physical and immunological barrier against pathogens. In order to study the specificities of the GALT transcriptome in pigs, we compared the transcriptome profiles of jejunal and ileal Peyer's patches (PPs), mesenteric lymph nodes (MLNs) and peripheral blood (PB) of four male piglets by RNA-Seq. We identified 1,103 differentially expressed (DE) genes between ileal PPs (IPPs) and jejunal PPs (JPPs), and six times more DE genes between PPs and MLNs. The master regulator genes FOXP3, GATA3, STAT4, TBX21 and RORC were less expressed in IPPs compared to JPPs, whereas the transcription factor BCL6 was found more expressed in IPPs. In comparison between IPPs and JPPs, our analyses revealed predominant differential expression related to the differentiation of T cells into Th1, Th2, Th17 and iTreg in JPPs. Our results were consistent with previous reports regarding a higher T/B cells ratio in JPPs compared to IPPs. We found antisense transcription for respectively 24%, 22% and 14% of the transcripts detected in MLNs, PPs and PB, and significant positive correlations between PB and GALT transcriptomes. Allele-specific expression analyses revealed both shared and tissue-specific cis-genetic control of gene expression.

Rapid Discovery of De Novo Deleterious Mutations in Cattle Enhances the Value of Livestock as Model Species.

In humans, the clinical and molecular characterization of sporadic syndromes is often hindered by the small number of patients and the difficulty in developing animal models for severe dominant conditions. Here we show that the availability of large data sets of whole-genome sequences, high-density SNP chip genotypes and extensive recording of phenotype offers an unprecedented opportunity to quickly dissect the genetic architecture of severe dominant conditions in livestock. We report on the identification of seven dominant de novo mutations in CHD7, COL1A1, COL2A1, COPA, and MITF and exploit the structure of cattle populations to describe their clinical consequences and map modifier loci. Moreover, we demonstrate that the emergence of recessive genetic defects can be monitored by detecting de novo deleterious mutations in the genome of bulls used for artificial insemination. These results demonstrate the attractiveness of cattle as a model species in the post genomic era, particularly to confirm the genetic aetiology of isolated clinical case reports in humans.

Preimplantation genetic diagnosis in Welsh pony embryos after biopsy and cryopreservation.

Preimplantation genetic diagnosis and embryo cryopreservation are important tools to improve genetic management in equine species with marked consequences on the economic value, health, biodiversity, and preservation of the animals. This study aimed to develop a biopsy method at the blastocyst stage that provides viable genotyped cryopreserved Welsh pony embryos. Embryos were collected at d 6.75 to 7 after ovulation. Biopsies were performed with either a microblade or a micropipette. After biopsy, embryos were cryopreserved. The survival rate of biopsied embryos was evaluated on fresh and cryopreserved embryos either 24 h after in vitro culture or after transfer to recipients. Fresh and nonbiopsied embryos were used as controls. Sex, coat color genes, myotony (neuromuscular disorder) diagnosis, and markers of parentage were investigated using PCR on biopsied cells after whole-genome amplification and on remaining embryos. The embryo survival rate after transfer was not affected by the micropipette biopsy (50%, = 8; 43%, = 7; and 50%, = 12, at d 30 for fresh biopsied embryos, vitrified biopsied embryos, and control embryos, respectively) but was significantly reduced by the use of microblade biopsy: 9 ( = 11) vs. 67% ( = 12) for control embryos. Successful sex determination was achieved for 82% ( = 28) of the micropipette biopsies and 100% ( = 50) of the microblade biopsies. Sex determined on biopsied cells was found to correspond completely (100%) with that determined on the remaining embryo ( = 37). More than 90% of the parentage checking markers, coat color, and myotony diagnosis were successfully determined on biopsies obtained with either a micropipette or a microblade. Mendelian incompatibility (7.5 and 5.5%) and embryo genotyping errors (6.6 and 8.6%) were low and not significantly different between the 2 methods. In conclusion, for the first time, pregnancy at Day 30 was obtained after transfer of Welsh pony biopsied and vitrified embryos >300 µm in diameter to recipient pony mares. The biopsied cells collected enabled multigenetic embryo diagnoses to be performed to a high degree of accuracy. The micropipette biopsy is the better method to apply on Welsh pony embryos.

Large-scale investigation of the parameters in response to Eimeria maxima challenge in broilers.

Coccidiosis, a parasitic disease of the intestinal tract caused by members of the genera Eimeria and Isospora, is one of the most common and costly diseases in chicken. The aims of this study were to assess the effect of the challenge and level of variability of measured parameters in chickens during the challenge with Eimeria maxima. Furthermore, this study aimed to investigate which parameters are the most relevant indicators of the health status. Finally, the study also aimed to estimate accuracy of prediction for traits that cannot be measured on large scale (such as intestinal lesion score and fecal oocyst count) using parameters that can easily be measured on all animals. The study was performed in 2 parts: a pilot challenge on 240 animals followed by a large-scale challenge on 2,024 animals. In both experiments, animals were challenged with 50,000 Eimeria maxima oocysts at 16 d of age. In the pilot challenge, all animals were measured for BW gain, plasma coloration, hematocrit, and rectal temperature and, in addition, a subset of 48 animals was measured for oocyst count and the intestinal lesion score. All animals from the second challenge were measured for BW gain, plasma coloration, and hematocrit whereas a subset of 184 animals was measured for intestinal lesion score, fecal oocyst count, blood parameters, and plasma protein content and composition. Most of the parameters measured were significantly affected by the challenge. Lesion scores for duodenum and jejunum (P < 0.001), oocyst count (P < 0.05), plasma coloration for the optical density values between 450 and 490 nm (P < 0.001), albumin (P < 0.001), α1-globulin (P < 0.01), α2-globulin (P < 0.001), α3-globulin (P < 0.01), and ß2-globulin (P < 0.001) were the most strongly affected parameters and expressed the greatest levels of variation. Plasma protein profiles proved to be a new, reliable parameter for measuring response to Eimeria maxima. Prediction of intestinal lesion score and fecal oocyst count using the other parameters measured was not very precise (R2 < 0.7). The study was successfully performed in real raising conditions on a large scale. Finally, we observed a high variability in response to the challenge, suggesting that broilers' response to Eimeria maxima has a strong genetic determinism, which may be improved by genetic selection.

Comparison of SNPs and microsatellites for assessing the genetic structure of chicken populations.

Many studies in human genetics compare informativeness of single-nucleotide polymorphisms (SNPs) and microsatellites (single sequence repeats; SSR) in genome scans, but it is difficult to transfer the results directly to livestock because of different population structures. The aim of this study was to determine the number of SNPs needed to obtain the same differentiation power as with a given standard set of microsatellites. Eight chicken breeds were genotyped for 29 SSRs and 9216 SNPs. After filtering, only 2931 SNPs remained. The differentiation power was evaluated using two methods: partitioning of the Euclidean distance matrix based on a principal component analysis (PCA) and a Bayesian model-based clustering approach. Generally, with PCA-based partitioning, 70 SNPs provide a comparable resolution to 29 SSRs. In model-based clustering, the similarity coefficient showed significantly higher values between repeated runs for SNPs compared to SSRs. For the membership coefficients, reflecting the proportion to which a fraction segment of the genome belongs to the ith cluster, the highest values were obtained for 29 SSRs and 100 SNPs respectively. With a low number of loci (29 SSRs or ≤100 SNPs), neither marker types could detect the admixture in the Gödöllö Nhx population. Using more than 250 SNPs allowed a more detailed insight into the genetic architecture. Thus, the admixed population could be detected. It is concluded that breed differentiation studies will substantially gain power even with moderate numbers of SNPs.

A global analysis of molecular markers and phenotypic traits in local chicken breeds in Taiwan.

Molecular and phenotypic data have been combined to characterize the genetic diversity of six local chicken breeds maintained with a long-term conservation programme. Hua-Tung, Hsin-Yi, Ju-Chi and Quemoy originated from Taiwan, Shek-Ki is from South China, and Nagoya is from Japan. Molecular tools included 24 microsatellite markers, melanocortin 1 receptor (alpha melanocyte stimulating hormone receptor) (MC1R), the LEI0258 marker located within the major histocompatibility complex (MHC), and mitochondrial DNA. Performance was recorded on the same individuals for body weight, panting rate in summer and antibody response (antigens: Newcastle disease virus and sheep red blood cells). A multivariate method previously proposed for taxonomy was used to combine the different data sets. Melanocortin 1 receptor (alpha melanocyte stimulating hormone receptor) and the MCW330 marker contributed the most to the first axis of the multiple coinertia analysis of molecular markers. Melanocortin 1 receptor (alpha melanocyte stimulating hormone receptor) showed evidence of selection, probably related to its effect on feather colour. The MHC exhibited a large diversity, with 16 alleles of the LEI0258 marker. Immune response traits contributed the most to the principal component analysis of phenotypic data. Eight mitochondrial DNA haplotypes related to clades A, B, C and E were distributed across breeds and revealed an important contribution of Indian and European breeds to Ju-Chi, Quemoy and Hsin-Yi. Phenotypic data contributed less than molecular data to the combined analysis, and two markers, LEI0258 and LEI0228, contributed the most. The combined analysis could clearly discriminate all breeds, except Ju-Chi, which was similar to Quemoy for many criteria, except immune response.

Sex and PRNP genotype determination in preimplantation caprine embryos.

The objective of this study was to test the accuracy of genotype diagnosis after whole amplification of DNA extracted from biopsies obtained by trimming goat embryos and to evaluate the viability of biopsied embryos after vitrification/warming and transfer. Whole genome amplification (WGA) was performed using Multiple Displacement Amplification (MDA). Sex and prion protein (PRNP) genotypes were determined. Sex diagnosis was carried out by PCR amplification of ZFX/ZFY and Y chromosome-specific sequences. Prion protein genotype determination was performed on codons 142, 154, 211, 222 and 240. Embryos were collected at day 7 after oestrus and biopsied either immediately after collection (blastocysts and expanded blastocysts) or after 24 h of in vitro culture (compacted morulae). Biopsied embryos were frozen by vitrification. Vitrified whole embryos were kept as control. DNA of biopsies was extracted and amplified using MDA. Sex diagnosis was efficient for 97.4% of biopsies and PRNP genotyping was determined in 78.7% of biopsies. After embryo transfer, no significant difference was observed in kidding rate between biopsied and vitrified control embryos, whereas embryo survival rate was different between biopsied and whole vitrified embryos (p = 0.032). At birth, 100% of diagnosed sex and 98.2% of predetermined codons were correct. Offspring PRNP profiles were in agreement with parental genotype. Whole genome amplification with MDA kit coupled with sex diagnosis and PRNP genotype predetermination are very accurate techniques to genotype goat embryos before transfer. These novel results allow us to plan selection of scrapie-resistant genotypes and kid sex before transfer of cryopreserved embryo.

Vietnamese chickens: a gate towards Asian genetic diversity.

BACKGROUND: Chickens represent an important animal genetic resource and the conservation of local breeds is an issue for the preservation of this resource. The genetic diversity of a breed is mainly evaluated through its nuclear diversity. However, nuclear genetic diversity does not provide the same information as mitochondrial genetic diversity. For the species Gallus gallus, at least 8 maternal lineages have been identified. While breeds distributed westward from the Indian subcontinent usually share haplotypes from 1 to 2 haplogroups, Southeast Asian breeds exhibit all the haplogroups. The Vietnamese Ha Giang (HG) chicken has been shown to exhibit a very high nuclear diversity but also important rates of admixture with wild relatives. Its geographical position, within one of the chicken domestication centres ranging from Thailand to the Chinese Yunnan province, increases the probability of observing a very high genetic diversity for maternal lineages, and in a way, improving our understanding of the chicken domestication process. RESULTS: A total of 106 sequences from Vietnamese HG chickens were first compared to the sequences of published Chinese breeds. The 25 haplotypes observed in the Vietnamese HG population belonged to six previously published haplogroups which are: A, B, C, D, F and G. On average, breeds from the Chinese Yunnan province carried haplotypes from 4.3 haplogroups. For the HG population, haplogroup diversity is found at both the province and the village level (0.69).The AMOVA results show that genetic diversity occurred within the breeds rather than between breeds or provinces. Regarding the global structure of the mtDNA diversity per population, a characteristic of the HG population was the occurrence of similar pattern distribution as compared to G. gallus spadiceus. However, there was no geographical evidence of gene flow between wild and domestic populations as observed when microsatellites were used. CONCLUSIONS: In contrast to other chicken populations, the HG chicken population showed very high genetic diversity at both the nuclear and mitochondrial levels. Due to its past and recent history, this population accumulates a specific and rich gene pool highlighting its interest and the need for conservation.

Revealing fine scale subpopulation structure in the Vietnamese H'Mong cattle breed for conservation purposes.

BACKGROUND: During the last decades, there has been an acceleration of the loss of domestic animal biodiversity. For conservation purposes, the genetic diversity of the H'Mong cattle, an indigenous local breed was studied. Single-nucleotide polymorphisms (SNP) of the SRY gene and mtDNA D-Loop sequence were analysed to clarify the origin of the breed. The genetic diversity was assessed through genetic data with twenty-five FAO microsatellites, and morphometric data with five body measurements from 408 animals sampled from eight districts of the Ha Giang province. RESULTS: The SRY genes were all of the zebu type. Among the 27 mtDNA haplotypes, 12 haplotypes were of the taurine type and the remaining 15 of the zebu type. This indicates female taurine introgression in the zebu H'Mong. The observed and expected heterozygosity ranged from 0.616 to 0.673 and from 0.681 to 0.729 respectively according to district, with low genetic differentiation (F ST = 0.0076). Multivariate analysis on morphometric and genetic data shows a separation of districts into two groups following a south-west/north-east cline and admixture analysis confirmed the two clusters, but no differentiation of taurine introgression between clusters was observed. A possible admixture with the Yellow cattle breed from a neighbouring province was suggested through genetic data and householder interviews. CONCLUSIONS: In this study we demonstrate the interest of fine-scale sampling for the study of genetic structure of local breeds. Such a study allows avoiding erroneous conservation policies and on the contrary, proposes measures for conserving and limiting crossbreeding between the H'Mong and the Yellow cattle breeds.

Genetic and morphometric characterization of a local Vietnamese swamp buffalo population.

The water buffalo plays a key role in the socio-economy of South-East Asia as it is the main draught power for paddy rice cultivation. While in the Indian subcontinent the water buffalo is the riverine type, in South-East Asia the majority of buffaloes are of the swamp type. In the poor remote northern province of Ha Giang in Vietnam, improvement of the swamp buffalo breed may be one of the best ways to increase sustainability of farming systems. Therefore, analysis of the genetic structure of the province buffalo population is a prerequisite to any conservation or improvement project. A total of 1122 animals were described for 11 body and horn measurements for morphometric characterization. From this sample set, 744 animals were genotyped for 17 microsatellite markers. Also 17 animals from southern provinces of Vietnam were genotyped as a comparative sample. The results showed that genetic diversity as well as inbreeding value in the Ha Giang was high. The F(ST) values within the province and across Vietnam were low indicating that most of the population variation is explained by individual variability. Bayesian clustering analysis did not highlight the presence of subdivided populations. These results are useful for the implementation of a conservation and improvement strategy of the swamp buffalo in order to guarantee the householders' needs for sustainability of the farming system in the Ha Giang province.

Genetic analysis of local Vietnamese chickens provides evidence of gene flow from wild to domestic populations.

UNLABELLED: Previous studies suggested that multiple domestication events in South and South-East Asia (Yunnan and surrounding areas) and India have led to the genesis of modern domestic chickens. Ha Giang province is a northern Vietnamese region, where local chickens, such as the H'mong breed, and wild junglefowl coexist. The assumption was made that hybridisation between wild junglefowl and Ha Giang chickens may have occurred and led to the high genetic diversity previously observed. The objectives of this study were i) to clarify the genetic structure of the chicken population within the Ha Giang province and ii) to give evidence of admixture with G. gallus. A large survey of the molecular polymorphism for 18 microsatellite markers was conducted on 1082 chickens from 30 communes of the Ha Giang province (HG chickens). This dataset was combined with a previous dataset of Asian breeds, commercial lines and samples of Red junglefowl from Thailand and Vietnam (Ha Noï). Measurements of genetic diversity were estimated both within-population and between populations, and a step-by-step Bayesian approach was performed on the global data set. RESULTS: The highest value for expected heterozygosity (> 0.60) was found in HG chickens and in the wild junglefowl populations from Thailand. HG chickens exhibited the highest allelic richness (mean A = 2.9). No significant genetic subdivisions of the chicken population within the Ha Giang province were found. As compared to other breeds, HG chickens clustered with wild populations. Furthermore, the neighbornet tree and the Bayesian clustering analysis showed that chickens from 4 communes were closely related to the wild ones and showed an admixture pattern. CONCLUSION: In the absence of any population structuring within the province, the H'mong chicken, identified from its black phenotype, shared a common gene pool with other chickens from the Ha Giang population. The large number of alleles shared exclusively between Ha Giang chickens and junglefowl, as well as the results of a Bayesian clustering analysis, suggest that gene flow has been taking place from junglefowl to Ha Giang chickens.

Delayed-type hypersensitivity response to KLH in F2 and backcrosses of two immune selected chicken lines: effect of immunisation and selection.

Cell-mediated immune response to keyhole limpet hemocyanin (KLH) was studied in 13 different progeny groups belonging to a second generation cross between two lines of White Leghorn previously selected for 10 generations for high antibody response to Newcastle disease vaccine (ND3) and high cell-mediated response to phytohemagglutinin (PHA) measured as wing web swelling. The cutaneous hypersensitivity response to KLH was assessed in KLH immunized and non-immunized birds. Wing thickness was measured at three time points: before the injection and 4 and 24 hours after the injection. The effect of previous immunization and genetic background was assessed. While no differences were present between progeny types or to a randomly bred control line at any of the three time points, significant differences were present between immunized and not immunized birds 24 hours after challenge. Results demonstrate that KLH immunized birds perform better and that the previous selection and crossbreeding scheme has not influenced the intensity of the skin swelling response to KLH. Apparently, neither selection for antibody responsiveness to ND3 nor selection for enhanced responsiveness to a T cell mitogen affected delayed-type hypersensitivity (DTH) to the Th 2 antigen KLH. These results may serve to facilitate further selection based on independent immune parameters.

Molecular genotype investigation of the Gallus gallus major histocompatibility complex.

The association between genetic markers and serological major histocompatibility complex (MHC) definition was studied in experimental White Leghorn lines selected for different immune traits. Different markers, three variable number tandem repeats (VNTRs) and 10 single nucleotide polymorphisms (SNPs), distributed along the MHC sequence were genotyped. Correlation between VNTR marker alleles and serology was studied in more than 800 animals and correspondence between SNP haplotypes and allele size was defined. A complete table of correspondence between VNTR allele sizes, SNP genotypes and serology was then drawn. This research shows that integrating new molecular tools can definitely help in MHC haplotype characterization and that, since the chicken MHC locus has been associated with disease resistance, these tools are becoming more and more needed for disease resistance and immunogenomics studies.

Using molecular markers and multivariate methods to study the genetic diversity of local European and Asian chicken breeds.

French and Asian subsets of chicken breeds were first analysed using 22 microsatellites and then compared to the AVIANDIV European set using 14 loci. Positive correlations were observed between F(IT) or F(ST) and typological values or variance of markers using the multivariate analysis mcoa. The first axis of the multivariate representation separated Asian from European breeds, revealing breeds with Asian ancestor. Using all or 14 loci, correct assignation rate was always higher than 93%. The Weitzman index and the aggregate diversity D were calculated using 22 loci within French and Asian breeds. The French breed Coucou de Rennes and the Hua-Tung breed seemed to contribute the most to the global diversity of each subset. This approach on French-only breeds and then on French with AVIANDIV domestic breeds (14 loci) showed that the Marans breed contributed the most. The AVIANDIV framework could be useful to evaluate the genetic diversity of local breeds and to help in connecting national and regional conservation policies.

Antibody responses to keyhole limpet hemocyanin, lipopolysaccharide, and Newcastle Disease virus vaccine in F2 and backcrosses of white Leghorn lines selected for two different immune response traits.

Planned crosses were designed to produce an F(2) and 2 backcross populations from 2 lines of White Leghorn chickens previously selected over 10 generations for 2 different in vivo immune responses. The selection criteria applied on the 2 grandparental lines were as follows: high antibody response to Newcastle disease virus vaccine 3 wk after vaccination (ND3) and high cell-mediated immune response [response to phytohemagglutinin]. Furthermore a control line was kept by random breeding. The objective of the study was to estimate if the 2 selection criteria applied on the pure lines had changed the level of and type of immune (humoral) response to a new antigen, keyhole limpet hemocyanin (KLH), in the various second-generation progeny groups. In addition, correlations between parameters of acquired and innate immunity were tested. Primary total (IgT) and isotype-specific (IgG and IgM) antibody response to KLH 1 wk after immunization and levels of natural antibodies (NAB) binding to Salmonella enteriditis-derived lipopolysaccharide (LPS) were measured. Although no differences were present between IgM and IgG antibodies to KLH and the phytohemagglutinin skin-swelling response, significant differences were present between all the progeny groups for IgT to KLH and ND3 and NAB binding to LPS. The mean values for IgT to ND3 and KLH were significantly different between the crosses using the selected lines compared with the control line, indicating a contribution of the previous selection. In addition, a sex effect was found for IgM to KLH and NAB to LPS, for which females had a higher response than males in both cases. No interaction between progeny type and sex was found. Furthermore, significant positive correlations were found between NAB to LPS and specific antibody titers to KLH. Finally, the results of the present study demonstrated an interaction between innate and acquired immunity under this strategy of selection and crossbreeding and confirmed the effect of selection on general immune response to a new antigen in second-generation crosses.

Cytogenetic repartition of chicken CR1 sequences evidenced by PRINS in Galliformes and some other birds.

Chicken repeat 1 (CR1) belongs to the non-long repeat class of retrotransposons. Nearly 100000 repeats interspersed in the chicken genome are subdivided into at least six distinct subfamilies, each 300 bp long and all sharing substantial sequence similarity. CR1-like elements were found in genomes from invertebrates to mammals, suggesting their importance for genome structure and/or function. Moreover, numerous data support the hypothesis of their implication in regulation of gene expression. So, the chromosomal distribution of these CR1 sequences in vertebrates is of great interest to improve our knowledge about the genome structure, function and evolution. A comparison of the cytogenetic distribution of CR1 sequences was performed by PRINS using consensus chicken primers on the chromosomes of chicken and species of several bird orders: Galliformes, Anseriformes, Passeriformes and Falconiformes. The study revealed that CR1 repeats are spread over nearly all chicken chromosomes with a higher density on the macrochromosomes and in particular with hot spots on subtelomeric regions of chromosome 1, 2, 3q, 4q, 5q. Their distribution on the macrochromosomes forms a kind of banding pattern, which was not systematically matched with R- or G-banding. This banding pattern appears to be conserved on the chromosomes of the Galliformes studied, irrespective of their karyotypes, rearranged or not. CR1 primers also show similar signals on the chromosomes of birds phylogenetically more distant (Anseriformes, Passeriformes and Falconiformes). This fact confirms the importance of these sequences at the large scale of bird evolution and in the chromosomal structure. The location of CR1 sequences, and in particular of the hot spots, mainly within the richest CG areas are in conformity with the data on an epigenetic role of these highly conserved sequences.

Chromosome banding studies in the Bateleur (Terathopius ecaudatus, Aves, Accipitridae).

The karyotype of the Bateleur (Terathopius ecaudatus) was studied with conventional and Ag-NOR staining, and using GTG and CBG banding. The karyotype organization is typically accipitrid, with satellites and few microchromosomes, close to the karyotypes of true vultures.