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Genetic resistance to infectious diseases in sheep and goats

IWMGQSG Toulouse-2003. Genetic resistance to infectious diseases in sheep and goats. Santé Animale. F Lantier 1 , C Moreno 2 , I Lantier 1 , P Berthon 1 , P Sarradin 1 , D Marc 1 , O Andréoletti 3 , F Schelcher 3 , E Cribiu 4 and JM Elsen 2.

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Genetic resistance to infectious diseases in sheep and goats

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  1. IWMGQSG Toulouse-2003 Genetic resistance to infectious diseases in sheep and goats Santé Animale F Lantier1, C Moreno2, I Lantier1, P Berthon1, P Sarradin1, D Marc1, O Andréoletti3, F Schelcher3, E Cribiu4 and JM Elsen2 1 INRA, Laboratoire de Pathologie infectieuse et immunologie, Nouzilly 2 INRA, Station d’Amélioration Génétique des Animaux, Castanet-Tolosan 3 INRA-ENVT, Physiopathologie infectieuse et parasitaire des ruminants, Toulouse 4 INRA, Laboratoire de Génétique Biochimique et Cytogénétique, Jouy-en-Josas

  2. General Objectives • To study the host resistance mechanisms through the identification of implicated genes as a mean to increase our knowledge of: • resistance/susceptibility pathways • inductors and effectors of the immune response • regulation of defence mechanisms (vs. immuno-pathology) • To develop tools allowing genetic selection for animal resistance to one or several diseases of economic or/and public health importance 2

  3. In farm animals ... • Infectious diseases are of major economic importance, inducing direct and indirect losses • A number of them are a challenge for human health • In a number of cases we have no means or imperfect ones to control these diseases: • therapeutics are too expensive and may have detrimental effects on the environment • we have few efficient vaccines • hygiene measures are often difficult to apply • Breeding for genetic resistance should be one more mean to reduce their incidence 3

  4. The idea is that an increase of (genetic) resistance to invasion, dissemination, and/or multiplication of the pathogen into the host should reduce both: • the level of contamination of the host tissues • the risk of chronic infection (carrier status) • the excretion of the pathogen, and consequently its horizontal and vertical transmission (reservoir) 4

  5. Infectious diseases in sheep and goat • Mastitis (Staphylococus, Streptococcus) • Neonatal diarrhea (Coccidia, E. Coli) • Infectious abortion (Brucella, Chlamydia, Coxiella, Salmonella, List, Toxo) • Neurological diseases (Listeria, Rabies, Spongiform encephalopathies) • Respiratory diseases (Pasteurella, Mycoplasma, ...) • Maedi-Visna et CAEV (lentivirus) • Jaagsiekte (adenomatosis retrovirus) • Tuberculosis and Paratuberculosis (Mycobacterium …) • Caseous lymphadenitis (Corynebacterium pseudotuberculosiss) • Echtyma, Footrot, Fleecerot …. 5

  6. Although molecular tools have progressed, few genetic controls of infectious disease resistance/susceptibility have been investigated or even suggested in sheep and goat (Lantier, Vu Tien Khang, 1988) • Maedi visna (Berritua et al 2003) • CAEV (Dolf et al, 1994) • Trypanosoma (Whitelaw et al, 1982, Faye et al, 2002) • Mastitis (Barillet et al, 2001) • Flystrike and Foot rot (Raadsma et al, 1998, Escayg et al, 1997) • Cowdriosis (Matheron et al, 1987, Camus et al, 1995) • Corynebacterium pseudotuberculosis (Pepin et al, 1988) • Intra-cellular bacteria (Brucella, Mycobaterium, Salmonella) • Scrapie One of the main difficulty resides in the definition of traits that have to be investigated and the complexity of the host-pathogen interaction 6

  7. Pathogenesis of Salmonella typhimurium oral infection (JC Sirard, 2003) • Resistance to the pH barrier of stomach, biliary salts and gut enzymes Stress response • Adherence to mucus and epithelial cells Various adhesive systems (fim, lpf, pef, agf) + SPI-1 invasins + ... • Resistance to antimicrobial mechanisms Modification of parietal structures (pmr) • Invasion of epithelial intestinal cells and/or CD18+ cells SPI-1 invasion machinery • Diversion of epithelial signalling (pro-inflammatory cytokines)  diarrhea LPS-independent. SPI-1 + SPI-5 effectors + … • Resistance to killing by phagocytic cells SPI-2 + SPI-3 + SPI-4 machinery + … • Intracellular replication in phagocytes and spreading  sepsis 7

  8. ANTIGEN CAPTURE ANTIGEN PRESENTATION Induction of immune response: trafficking and maturation of dendritic cells Banchereau et al. Ann. Rev. Immunol. (2000) 8

  9. This complexity could be approached by measuring relative gene expression by using DNA microarrays. • Capillary printing is used to array DNA fragments • RNA is prepared from the 2 samples to be compared, • labeled cDNA is prepared by RT, incorporating either Cy3 (green) or Cy5 (red). • The two labeled cDNA mixtures are mixed and hybridized to the microarray • In the pseudocolor image resulting from scanning, the green Cy3 and red Cy5 signals are overlaid--yellow spots indicate equal intensity. • With the use of image analysis software, signal intensities are determined for each dye Craig A. Cummings* and David A. Relman*† Stanford University, Stanford, California, USA; VA Palo Alto Health Care System, Palo Alto, California, USA 9 (Illustration by J. Boldrick, Stanford University).

  10. Selection criteria • the response to natural or artificial infection • (e.g. in case of strongilid infestation ) • predictors of the intensity of the immune response • (e.g. antibody response to antigen, cytokine level) • genetic markers associated to several genes (or QTL) • ( e.g. in case of Salmonellosis: candidate genes and QTL) • “causal” mutation(s) in a major gene • (e.g. in case of Scrapie: the PRNP gene) 10

  11. Part 1: Comparative mapping to look for candidate genes and QTLs: lessons from salmonellosis 11

  12. Lessons from salmonellosis: candidate genes and QTLs • In mice, several genes controlling innate resistance (polygenic control) to salmonellosis have been cloned (Ity/Nramp1/Slc11a1, Lps/TolR4, Xid/Btk, Nu/Foxn1 ) or localised (H-2, several QTLs) • The mouse Nramp1 gene has been identified using a positional cloning strategy (Vidal et al, 1993) • Gene inactivation have demonstrated that it controls the early multiplication of Salmonella, Mycobacterium, Leishmania and other intracellular pathogens in mouse macrophages (Vidal et al, 1995, 1996) 12

  13. NRAMP1 (Slc11a1 ) is a membrane bivalent cation transporter Predicted membrane topology of Nramp1: the number, position and polarity of the 12 predicted trans-membrane domains is shown with respect to the cytoplasmic face and the luminal face of the lysosome (inset) and the phagosome. Lam-Yuk-Tseung, Steven & Gros, Philippe ; Genetic control of susceptibility to bacterial infections in mouse models. Cellular Microbiology5 (5), 299-313. 13

  14. From mice to sheep …comparative genetics - VIL1, FN1, TNP1, CHNRG as a conserved synteny group (sheep/Hamster hybrids) (Tabet-Aoul et al, 1992; Pitel et al, 1994) - PCR amplification of a fragment of the sheep NRAMP1 gene using the mouse sequence (Pitel et al, 1995) • In situ hybridization (Pitel et al, 1995 b) • Sequence of the complete ovine gene from a BAC clone (Bussmann et al, 1998) • two associated microsatellites OAR2 14

  15. From mice to sheep ... comparative pathology • Salmonella abortusovis • is a sheep natural pathogen (abortion, mortality of young) • is not transmissible to other species • can be controlled by a live vaccine (strain Rv6, Pardon et al, 1990), that we used as a model of Salmonella infection in field conditions • has been studied in mice where its multiplication is controlled by the host resistance gene Nramp1 15

  16. 8 6 6 4 4 3 2 2 1 0 Level of splenic infection with S. abortusovis in two congenic mouse strains Virulent strain 15/5 Vaccinal strain Rv6 IV BALB/c - S C.CB - R SC/P Log 10 Salmonella per spleen 0 2 6 10 15 25 0 1 3 6 10 days after inoculation 16

  17. Measure of the NRAMP1effect in sheep and search for other QTLs Sheep population • belonging to the INRA 401 synthetic line of sheep which is a mixture of Berrichon du Cher (meat breed, salmonella “resistant”) and Romanov (prolific breed, salmonella “susceptible”) • large enough for the detection of a QTL of 1 s.d. effect close to a marker (64 microsatellites) • 30 sire families /40 progenies in 3 batches (1200 lambs) • allowing search for QTL controlling other traits 17

  18. Experimental protocol • Bacteria : Vaccinal Salmonella abortusovis strain Rv6 • Mouse susceptibility criterions • Bacterial colonisation of spleen/antibody synthesis • Sheep susceptibility criterions: • Lymph-nodes colonisation (LogBgLN, LogBLN) • Spleen colonisation (LogBS, LogBgS) • Antibody synthesis (IgM0, var IgM, IgG10, var IgG1) • Body and organ weights (WtLN, WtS, varWt) 18

  19. Log - Log - Var Log - Log - Log - Log - Log - Log - Log - IgM 0 IgG1 VarIgG1 IgM BgLN BgRN BgS WtLN WtRN WtS VarWt 0 0.14 Log - IgG1 - 0.53 0.27 0 0.30 Log - VarIgG 1 0.55 0.27 - 0.24 - 0.60 0.64 IgM 0.25 0.38 0 0.13 0.37 VarIgM 0.33 0.29 0.25 0.31 - 0.45 0.22 L og - BgLN 0.18 0.93 0.25 - 0.41 0.27 Log - BgRN 0.19 0.45 0.37 - 0.21 0.06 Log - BgS 0.19 - 0.58 0.34 Log - WtLN 0.89 0.26 Log - WtRN 0.52 0.25 - 0.13 0.56 Log - WtS 0.13 0.10 - 0.21 - 0.25 0.10 Log - Var - Wt - 0.40 Heritability of the sheep response to Salmonella abortus ovis Rv6 vaccine strain(Moreno et al, 2002) 19

  20. CMH NRAMP1 QTLs Detected using an interval mapping method ?? 20

  21. Main results • A sire effect has been observed on all measured variables • Significant heritabilities of these variables have been detected • A low effect of NRAMP1 markers, as in chicken and human • QTLs with a significant effect (p<0.01) on salmonella resistance criteria have been detected • A large part of the observed genetic variance has still to be explained • QTLs for growth, body conformation and wool traits 21

  22. Further prospects • Complete genotyping (64 markers presently) • Other candidate genes (TolR4, cytokines, mouse/chicken QTLs) • Effects detected with the live vaccine are tested with a virulent strain of S.abortusovis • Carrier state of resistant animals ? 22

  23. Part 2Lessons from Transmissible Spongiform Encephalopathies (TSE): the major effect of the PRNP gene on sheep susceptibility to scrapie 23

  24. Transmissible Spongiform encephalopathies (TSEs) are • a group of fatal neurodegenerative diseases • affecting human • sporadic CJD (known since 1920, 85% of case) • familial CJD, FFI, GSS (PRNP mutations, 13%) • Iatrogenic CJD (surgery, grafts, growth hormone, 2%) • variant CJD (148 cases in GB, 6 in France, …) • and animals • Scrapie in sheep (1732) • Mink encephalopathie (1947) • CWD in deer and elk (1967) • BSE in cattle (1986) • FSE in cat and other Felidae 24

  25. TSEs are characterized : • by a long incubation period (from months in PrP transgenic mice to years in animals and human) • the accumulation in lymphoid and nervous tissues of an abnormal protease resistant form (PrPsc = PrPres) of a normal cellular protein (PrPc) of the host • PrPc function is unknown (oxydative stress, signaling, …) • PrPsc is enriched in beta sheet, aggregates and partly purified as Scrapie associated fibrils (SAF) alpha-helix: 43% Beta-sheet: 3% alpha-helix: 30% Beta-sheet: 43% PrPc PrPsc 25 Alper et al., 1967; Griffith et al., 1967; Prusiner, 1982

  26. PrPSCaggregates are associated to lesions (vacuoles) 26

  27. The unconventional agent of TSE(s) or Prion • Co-purify with SAF (PrPsc) • Is highly resistant to classical disinfectants (formol, ethanol…), RNAse, DNAse, ionising radiations • Does not induce disease in Prnp KO mice. • Mouse susceptibility is restored and even increased (shorter incubation period) in transgenic mice (over-) expressing PrPc SAF: Scrapie associated fibrils • PrPsc or PrPres is a proteinaceous infectious organism • = prion hypothesis, Prusiner 1982 • or is associated to the agent (virinohypothesis, virus receptor) • is presently the only specific signature of the infection 27

  28. The PRNP gene which encodes the PrP protein • has been cloned through classical techniques (cDNA bank) in mice (Carlson et al, 1986), then in many species • is composed of 2-3 exons and large introns • is highly conserved in mammalian species (human, cattle, sheep, goat, elk, cat, mink, etc …) and has been found in fish • has been duplicated as PRND (doppel) and PRNT (testis) • contains polymorphisms associated to TSE susceptibility in: • Human (predisposition to sCJD, vCJD, GSS, FFI …) • Mouse (duration of incubation period) • Sheep (predisposition to Scrapie and experimental BSE) 28

  29. Ovine susceptibility to scrapie is controlled by the PRNP gene A high correlation with Scrapie susceptibility PrP polymorphism 112 136 141 154 171 Codons Met Ala Leu Arg Gln Wild allele Thr Ala Leu Arg Gln MetVal LeuArg Gln Mutations Met Ala Phe Arg Gln MetAlaLeuHis Gln Met Ala LeuArg Arg VRQ VRQ ARQ ARR AHQ ARR AHQ AHQ AHQ ARR VRQ ARQ ARQ VRQ VRQ ARQ ARQ AHQ ARR ARR 74 272 184 78 54 96 91 11 37 47 Elsen et al, 1999. Arch Virol, 144: 431-445. 29

  30. Experimental protocol: kinetics of natural contamination by the scrapie agent • Investigated by serial autopsies: • groups of 4 sheep of each PRNP-genotype • raised in a contaminated environment (Langlade flock) - from birth to appearance of clinical symptoms - more than 50 organs sampled per animal (lymphoid and nervous) • PrPsc detection by IHC (formalin fixed tissues, parafin embeded sections are PK treated and autoclaved) using anti-PrP MAb • Double-immunolabelling protocol for phenotyping PrPsc-accumulating cells using Mab selected for recognition of Ag in parafin sections. (Andréoletti et al, 2000. J Gen Virol, 81: 3115-3126) 30

  31. PrPsc within GALT of VRQ/VRQ sheep(Andréoletti et al, 2000) 31

  32. Number of sheep with at least one PrPsc-positive sampled organ Age (month) PrP-genotype 2 3 4 5 6 9 18* ARR/ARR 0/4 -- 0/4 -- 0/4 0/4 0/4 VRQ/ARR 0/4 -- 0/4 -- 0/4 0/4 1/4 VRQ/VRQ 1/4 3/4 4/4 4/4 4/4 4/4 4/4 * appearance of clinical symptoms in VRQ/VRQ sheep 32

  33. PrPsc-positive organs in VRQ/VRQ sheep Age (month) Organs 2 3 4 5 6 9 Ileal Peyer’s patch ++ +++ +++ +++ +++ +++ Ileal MLN + +++ +++ +++ +++ +++ GALT - +++ +++ +++ +++ +++ Spleen and LN - + +++ +++ +++ +++ ENS- - - - -+++ Obex (DMNV)- - - - -+ 33

  34. Pathogenesis variations according to the PRNP genotype : no PrPsc in ARR/ARR sheep Brain Nervous route Tonsils Spinal cord Lymph nodes + Nerves Nervous plexus Intestine Lymphoid tissues Peyer’s patches Spleen In VRQ/ARR heterozygous sheep, as in bovine and human sporadic CJD, only nervous tissues are involved 34

  35. In VRQ/VRQ homozygous sheep, as in human vCJD (BSE strain), lymphoid tissues allow amplification of the agent Brain Nervous route Tonsils Spinal cord + Nerves Nervous plexus Lymph nodes Intestine Lymphoid tissues MLN Peyer’s patches Spleen 35

  36. PrPsc in placenta is controlled by the fœtal genotype (Andreoletti et al, 2002) PrPsc in Placenta Ram genotype Ewe genotype Infect Status Lamb genotype + - + - - - VRQ/VRQ VRQ/VRQ ARR/ARR ARR/ARR VRQ/VRQ ARR/ARR ARQ/VRQ (n=7), VRQ/VRQ (n=6) ARQ/VRQ (n=1), VRQ/VRQ (n=4) ARR/ARQ (n=3), ARR/VRQ (n=5) ARR/ARQ (n=4), ARR/VRQ (n=4) ARR/VRQ (n=5) ARR/ARR (n=9) ARQ/VRQ (n=9) ARQ/VRQ (n=3) ARQ/VRQ (n=5) ARQ/VRQ (n=3) ARR/ARR (n=5) ARR/ARR (n=6) + - _ - - - 36

  37. Further prospects • Challenge of prion free sheep with BSE using the IC or oral route (in progress) • Selection for sheep carrying the resistant ARR allele (see Palhiere et al) • Research for new QTLs in mice and sheep (see Moreno et al) • PRNP polymorphism(s) linked to scrapie susceptibility in goat? (see Martin-Burriel et al) 37

  38. There is still need for research on TSEs ! • Today : progress in diagnosis and in genetic resistance of the host • Elimination of infected animals and selection (sheep only) • Research topics : • Genetic /pathogenesis : caprine, bovine • Other susceptibility genes • Improve diagnostic methods: an earlier and a better detection of PrPsc in living animals (other approaches : incubation phase markers, gene expression profiles …). • Excretion (milk, urine, feces ?) • Reservoirs? • Infected cells : how restrict the dissemination of the agent ? • Mechanism of neuronal death : therapeutics ? • Nature of the agent and genetic support of strains … 38

  39. Co-evolution of Host and Pathogens • A general miss-understanding of the host-pathogen relationship is that the parasite has to kill the host. • Although it has to by-pass the host defence mechanisms and has developed a number strategies for this purpose, one can imagine that the pathogen population interest is that their favourite host survives as long as possible. • This might help the immunologist to think that selection for efficient immune mechanisms simply consist to accelerate natural selection for the optimal equilibrium. • (see Zaharic et al, 2002 ; NRAMP1 vs SPI2) 39

  40. Pathologie Infectieuse et Immunologie, INRA, centre de Tours, 37380 Nouzilly Ph BERNARDET, V. BUSSMANN, P. BERTHON, R. BOIVIN, A. GAUTIER, F. LANTIER, I. LANTIER, P. LECHOPIER, P SARRADIN, K. TABET-AOUL La Sapinière, INRA, domaine expérimental de Bourges, 18390 Osmoy J-C. BRUNEL, J-L. WEISBECKER Génétique cellulaire, INRA, centre de Toulouse, 31326 Castanet-Tolosan F. PITEL, J. GELIN Génétique biochimique et cytogénétique, INRA, centre de Jouy-en-Josas E.P. CRIBIU, L SCHIBLER, A OUSTRY-VAIMAN, D. VAIMAN ENVT- INRA, Ecole Nationale Vétérinaire de Toulouse O. ANDREOLETTI, F. SCHELCHER Domaine expérimental de Langlade, INRA, Centre de Toulouse F EYCHENNE SAGA, INRA, centre de Toulouse, 31326 Castanet-Tolosan C. MORENO, D. FRANCOIS, J. BOUIX, J. VU TIEN KHANG and J-M. ELSEN 40

  41. Thank you for Your attention ... 41

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