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Analysis of the genomic regions associated to response to coccidiosis caused by Eimeria maxima in broiler chickens. 36. Conference of the International Society for Animal Genetics (ISAG)

Coccidiosis is a parasitic disease caused by species from Eimeria genus, widespread in poultry farming, leading to important losses by its direct impact on production and the associated control measures. The integrative management against this disease should lead to include new parameters of animal’s response to coccidiosis in selection schemes. Our goal was to study the variability of broilers’ response to coccidiosis caused by Eimeria maxima, to identify associated genomic regions and to understand underlying biological mechanisms. We performed an experimental infection with 2 024 animals and measured phenotypes of animals’ response (body weight, temperature, lesions and many blood parameters). Animals have been genotyped using a 580K SNP panel, and the genome-wide association study has revealed genomic regions highly significantly associated to body weight gain, plasma colour and plasma β2-globuline concentration. The analysis of biological functions of genes has shown the major role played by biological pathways of metabolism and some innate immune parameters. The analysis of interactions has shown very significant networks centred on gut repair or cardiovascular functions. The genetic markers identified during this study can be used in selection programmes.; La coccidiose est une parasitose intestinale causée par des espèces du genre Eimeria, très répandue dans les élevages avicoles à l’origine de pertes importantes par son impact direct sur la production et les mesures de lutte associées. La gestion intégrée de cette pathologie devrait conduire à inclure dans les programmes de sélection des critères relatifs à la réponse des animaux à la coccidiose. Notre objectif a donc été d’étudier la variabilité de la réponse de poulets de chair à Eimeria maxima et d’identifier les régions génomiques associées, puis de comprendre les mécanismes biologiques sous-jacents. Pour cela, nous avons réalisé une infection expérimentale sur 2 024 animaux et mesuré des phénotypes (poids, température, lésions et de nombreux paramètres sanguins) de la réponse des animaux. Afin de comprendre les bases génétiques de cette réponse, ces animaux ont été génotypés à l’aide d’un panel de 580K marqueurs SNP. L’étude d’association génomique a permis d’identifier des régions du génome très significativement associées avec le gain de poids, la coloration plasmatique ou le taux plasmatique de β2-globuline. Une analyse des fonctions biologiques des gènes a révélé la grande importance des voies biologiques de métabolisme énergétique et de certains paramètres de réponse innée. Une analyse des interactions a identifié des réseaux très significatifs centrés sur des fonctions de réparation intestinale ou cardiovasculaires.Les marqueurs génétiques identifiés lors de cette étude peuvent être utilisés dans des programmes de sélection.

Serum IL-10, a novel diagnostic biomarker of resistance in the domestic chicken to coccidiosis. 14. Avian Immunology Research Group (AIRG) Meeting 2016

Genome-wide association study of the [i]Eimeria maxima[/i] response in Cobb500 broilers. 67. Annual Meeting of the European Association for Animal Production (EAAP)

The objective of this thesis was to improve the understanding of molecular mechanisms controlling the response to two major pathogens, Eimeria maxima (coccidiosis) and infectious bronchitis virus (IBV), in broiler and layer chickens, respectively. Breeding for the improved response to the two pathogens is a possible complementary approach for their control in poultry production. A key step is to understand the molecular mechanisms controlling the host’s response to a disease. For E. maxima, a large-scale challenge study on Cobb500 broilers and a follow-up genome-wide association study (GWAS) were performed, which aimed at evaluating the effects of the challenge on the measured parameters, then identifying genomic regions and biological pathways explaining variation of these parameters. Several candidate genes were identified after the performed GWAS. Moreover, biological pathways involved in tissue repair, general robustness and early immune response seem to play an important role during the initial stage of the infection. For IBV, an experimental infection was performed on two chicken lines which were divergently selected for low and high levels of serum mannose-binding lectin (MBL), respectively. MBL plays a key role in innate immunity which is crucial for improving disease resistance. We observed large differences in the immune parameters and the spleen transcriptome profiles for uninfected and IBV infected birds between the two MBL lines. The large differences in expression of genes involved in the adaptive cellular immune response, therefore the ability to respond to the infection, explain probably the contrasted viral loads observed between lines. These findings provide additional knowledge for both potential improvements of animal robustness to IBV infection as well as vaccine outcome.

The objective of this thesis was to improve the understanding of molecular mechanisms controlling the response to two major pathogens, Eimeria maxima (coccidiosis) and infectious bronchitis virus (IBV), in broiler and layer chickens, respectively. Breeding for the improved response to the two pathogens is a possible complementary approach for their control in poultry production. A key step is to understand the molecular mechanisms controlling the host’s response to a disease. For E. maxima, a large-scale challenge study on Cobb500 broilers and a follow-up genome-wide association study (GWAS) were performed, which aimed at evaluating the effects of the challenge on the measured parameters, then identifying genomic regions and biological pathways explaining variation of these parameters. Several candidate genes were identified after the performed GWAS. Moreover, biological pathways involved in tissue repair, general robustness and early immune response seem to play an important role during the initial stage of the infection. For IBV, an experimental infection was performed on two chicken lines which were divergently selected for low and high levels of serum mannose-binding lectin (MBL), respectively. MBL plays a key role in innate immunity which is crucial for improving disease resistance. We observed large differences in the immune parameters and the spleen transcriptome profiles for uninfected and IBV infected birds between the two MBL lines. The large differences in expression of genes involved in the adaptive cellular immune response, therefore the ability to respond to the infection, explain probably the contrasted viral loads observed between lines. These findings provide additional knowledge for both potential improvements of animal robustness to IBV infection as well as vaccine outcome., L’objectif de cette thèse était de mieux comprendre les mécanismes moléculaires contrôlant la réponse à deux pathogènes majeurs, Eimeria maxima (cocciodiose) et le virus IBV (bronchite infectieuse), chez le poulet de chair et la poule pondeuse. La sélection d’animaux avec une meilleure réponse à ces deux pathogènes est une voie complémentaire de leur contrôle en élevage. Une étape clé est la compréhension des mécanismes moléculaires contrôlant la réponse de l’hôte à une pathologie. Pour E. maxima, une infection expérimentale à grande échelle sur des poulets Cobb500 et une étude d’association génomique (GWAS) ont été conduites, afin d’évaluer les effets de l’infection sur les paramètres mesurés, puis d’identifier les régions génomiques et les voies biologiques expliquant la variation de ces paramètres. Plusieurs gènes candidats ont été identifiés avec le GWAS et des voies biologiques impliquées dans la réparation tissulaire, la robustesse générale et la réponse immune précoce semblent jouer un rôle important durant la phase initiale de l’infection. Pour IBV, une infection expérimentale a été réalisée sur deux lignées divergentes de poules sélectionnées pour un taux sérique haut ou bas de mannose-binding lectin (MBL). MBL a un rôle clé dans l’immunité innée, ce qui est essentiel pour l’amélioration de la résistance aux pathogènes. De fortes différences dans les paramètres immunitaires et les transcriptomes de rate ont été observés entre les deux lignées, pour les animaux infectés par IBV ou non. Les grandes différences dans l’expression de gènes impliqués dans la réponse cellulaire, donc la capacité de répondre à l’infection, expliquent probablement les différences de charges virales observées entre lignées. Ces résultats apportent de nouvelles connaissances qui pourraient permettre d’améliorer à la fois la robustesse des animaux à l’IBV et la stratégie vaccinale.

The objective of this thesis was to improve the understanding of molecular mechanisms controlling the response to two major pathogens, Eimeria maxima (coccidiosis) and infectious bronchitis virus (IBV), in broiler and layer chickens, respectively. Breeding for the improved response to the two pathogens is a possible complementary approach for their control in poultry production. A key step is to understand the molecular mechanisms controlling the host’s response to a disease. For E. maxima, a large-scale challenge study on Cobb500 broilers and a follow-up genome-wide association study (GWAS) were performed, which aimed at evaluating the effects of the challenge on the measured parameters, then identifying genomic regions and biological pathways explaining variation of these parameters. Several candidate genes were identified after the performed GWAS. Moreover, biological pathways involved in tissue repair, general robustness and early immune response seem to play an important role during the initial stage of the infection. For IBV, an experimental infection was performed on two chicken lines which were divergently selected for low and high levels of serum mannose-binding lectin (MBL), respectively. MBL plays a key role in innate immunity which is crucial for improving disease resistance. We observed large differences in the immune parameters and the spleen transcriptome profiles for uninfected and IBV infected birds between the two MBL lines. The large differences in expression of genes involved in the adaptive cellular immune response, therefore the ability to respond to the infection, explain probably the contrasted viral loads observed between lines. These findings provide additional knowledge for both potential improvements of animal robustness to IBV infection as well as vaccine outcome.; L’objectif de cette thèse était de mieux comprendre les mécanismes moléculaires contrôlant la réponse à deux pathogènes majeurs, Eimeria maxima (cocciodiose) et le virus IBV (bronchite infectieuse), chez le poulet de chair et la poule pondeuse. La sélection d’animaux avec une meilleure réponse à ces deux pathogènes est une voie complémentaire de leur contrôle en élevage. Une étape clé est la compréhension des mécanismes moléculaires contrôlant la réponse de l’hôte à une pathologie. Pour E. maxima, une infection expérimentale à grande échelle sur des poulets Cobb500 et une étude d’association génomique (GWAS) ont été conduites, afin d’évaluer les effets de l’infection sur les paramètres mesurés, puis d’identifier les régions génomiques et les voies biologiques expliquant la variation de ces paramètres. Plusieurs gènes candidats ont été identifiés avec le GWAS et des voies biologiques impliquées dans la réparation tissulaire, la robustesse générale et la réponse immune précoce semblent jouer un rôle important durant la phase initiale de l’infection. Pour IBV, une infection expérimentale a été réalisée sur deux lignées divergentes de poules sélectionnées pour un taux sérique haut ou bas de mannose-binding lectin (MBL). MBL a un rôle clé dans l’immunité innée, ce qui est essentiel pour l’amélioration de la résistance aux pathogènes. De fortes différences dans les paramètres immunitaires et les transcriptomes de rate ont été observés entre les deux lignées, pour les animaux infectés par IBV ou non. Les grandes différences dans l’expression de gènes impliqués dans la réponse cellulaire, donc la capacité de répondre à l’infection, expliquent probablement les différences de charges virales observées entre lignées. Ces résultats apportent de nouvelles connaissances qui pourraient permettre d’améliorer à la fois la robustesse des animaux à l’IBV et la stratégie vaccinale.

Background Coccidiosis is the most common and costly disease in the poultry industry and which caused by protozoans from the genus of Eimeria. The current control of coccidiosis, based on the use of anticoccidial drugs and vaccination, faces serious obstacles such as drug resistance and the high costs for development of efficient vaccines, respectively. Therefore, the present control programs must be expanded with complementary approaches such as the use of genetics for improvement of the host’s response to Eimeria infections. Recently, we have performed a large-scale challenge study on Cobb500 broilers using E. maxima where we investigated variability among animals in response to the challenge. Taking advantage of size and structure of the large-scale challenge study, we performed a genome-wide association study (GWAS) with the aim to identify genomic regions underlying variability of the measured traits in the response to Eimeria maxima in broilers. Furthermore, we conducted a post-GWAS functional analysis with the aim of gaining a better biological understanding of the underlying response to Eimeria maxima challenge in broilers. Results In total, we identified 22 single nucleotide polymorphisms (SNP) with q-value

absent

Use of genetic tools for improvement of host’s response is considered as a promising complementary approach for coccidiosis control. Therefore, we performed genome wide association study (GWAS) for response to Eimeria maxima challenge in broilers. The challenge was done on 2024 Cobb500 broilers. We phenotyped animals on two levels: global phenotyping performed on all animals and deep phenotyping on a subset of 184 extreme animals. The global phenotyping included: body weight gain, hematocrit, plasma coloration and rectal temperature and the deep phenotyping included: blood cell count, plasma proteins profiles, oocyst count and lesion score. GWAS was conducted on 1975 animals which were genotyped using 580K Affymetrix® Axiom® HD genotyping array. Quality control procedures involved the following criteria: SNP call rate (1%). GWAS was carried out in GEMMA software using standard univariate linear mixed model. The false-discovery rates (FDR) were calculated using the R package q-value and the cut-off of the significant association was set at q-value ≤ 0.05. We identified several genomic regions associated with measured parameters. Among the most promising results, robust associations have been identified between albumin levels and one region on GGA2, alpha 1 globulin and one region on GGA1, alpha 2 globulin and two regions on GGA1 and GGA6 and plasma coloration and one region on GGA6. To our knowledge this is the largest association study that has been conducted in a commercial broiler product to ascertain the genetic components driving the response of chickens to coccidiosis.

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 two 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 days 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 were measured for intestinal lesion scores, fecal oocyst count, blood parameters, plasma protein content and composition. Most of the parameters measured were significantly affected by the challenge. Lesion scores for duodenum and jejunum (P

For successful genetic dissection of disease resistance it is of great importance to accurately identify the respective phenotypes. In case of coccidiosis some of the conventional phenotypes don’t fully reflect the animal health status. The objective of this study was large-scale evaluation of plasma components as potential parameters for describing health status of broilers challenged with Eimeria maxima. The challenge was performed on 2024 Cobb500 broilers. Plasma coloration was measured on all the animals while measurement of plasma protein content and analysis of plasma protein composition were performed on subset of 184 extreme animals selected based on body weight gain. All fractions of plasma proteins associated with acute phase proteins, except β1-globulin, have been significantly elevated. We observed that the best estimation of plasma coloration variation under a coccidiosis challenge can be obtained by measuring optical density between 450 and 490 nm

Coccidia are mainly intestinal parasites affecting animals and leading to substantialeconomic losses in poultry production. A genetic variability for resistance to coccidiosis in chicken hasbeen evidenced in different studies and suggests that applying a selection for this natural resistancecould limit the effects of the disease. In this study we focused on the impact of Eimeria maxima ondifferent traits (e.g., weight gain, body temperature, blood composition, intestinal lesions scores,oocysts count) in two challenge experiments on commercial broilers (pilot then larger experiment with200 and 2000 animals challenged, respectively). We demonstrated that it was possible to replicate thechallenge effects observed across the two experiments. As some traits are difficult to measure on largescale, we tried to predict those from traits easier to phenotype using partial least squares models, withmoderate success. Finally, we demonstrated that the challenged population presents a geneticvariability for resistance to Eimeria maxima. The next step will be to identify genomic regionsinvolved in the resistance determinism, for further selection use.

Conventionally, levels of inbreeding for livestock animals are estimated analyzing pedigree data. The recent availability of high density SNP arrays for the bovine genome has provided the opportunity for investigation of levels of autozygosity based on runs of homozygosity (ROH). A run of homozygosity is a continuous segment of DNA sequence without heterozygosity in the diploid state. Levels of homozygosity derived from ROH are recognized as potential inbreeding measure in livestock animals. Here we compare levels of homozygosity derived from proportion of ROH in genome (FROH) and pedigree inbreeding estimates (Fped). We analyzed genotype and pedigree data from 1687 animals from four cattle breeds (Brown Swiss, Norwegian Red, Tyrol Grey and Fleckvieh – dual purpose Simmental). Distribution of ROH was obtained by PLINK software, while proportions of genome in ROH were calculated for five cut-off lengths (1Mb, 2Mb, 4Mb, 8Mb, 16Mb). Pedigree data was analyzed by PEDIG software and inbreeding coefficients for complete pedigree (average complete generation equivalents ranging from 6.46 to 9.02 for the four breeds) and over 5 generations were calculated. Levels of autozygosity from pedigree information were similar to those derived from ROH >8Mb. The strongest correlations with pedigree inbreeding coefficients were obtained between levels of homozygosity derived from ROH for cut-off length of 4Mb (FROH4 ) and inbreeding coefficients for complete pedigrees (FpedT) varying from 0.619 for Norwegian Red up to 0.705 for Tyrol Grey. We conclude that proportion of the genome arranged in long homozygous segments provides a good indication of inbreeding levels and that the choice of cut-off lengths of ROH allows determining autozygosity derived from recent or remote ancestors.