Source: LOUISIANA STATE UNIVERSITY submitted to
NATIONAL ANIMAL GENOME RESEARCH PROGRAM
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
0198550
Grant No.
(N/A)
Project No.
LAB93658
Proposal No.
(N/A)
Multistate No.
NRSP-8
Program Code
(N/A)
Project Start Date
Oct 1, 2003
Project End Date
Sep 30, 2008
Grant Year
(N/A)
Project Director
Miller, J. E.
Recipient Organization
LOUISIANA STATE UNIVERSITY
202 HIMES HALL
BATON ROUGE,LA 70803-0100
Performing Department
VETERINARY SCIENCE
Non Technical Summary
Gastrointestinal nematode parasitism in small ruminants can cause production losses and even death. The purpose of this study is to find genetic markers that will aid in identification and selection of animals resistant to gastrointestinal nematode parasitism.
Animal Health Component
100%
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
3043610108050%
3113610109050%
Knowledge Area
311 - Animal Diseases; 304 - Animal Genome;

Subject Of Investigation
3610 - Sheep, live animal;

Field Of Science
1080 - Genetics; 1090 - Immunology;
Goals / Objectives
1. Enhance and integrate genetic and physical maps of agriculturally important animals for cross species comparisons and sequence annotation. 2. Facilitate integration of genomic, transcriptional, proteomic and metabolomic approaches toward better understanding of biological mechanisms underlying economically important traits.
Project Methods
Continue to screen the ovine genome to identify markers that are associated with resistance to gastrointestinal nematode infection in sheep. Fifty additional markers will be used to cover regions that were not covered previously. The 2 regions of the genome associated with resistance that were identified previously will be further refined in an attempt to map the gene(s) involved.

Progress 10/01/03 to 09/30/08

Outputs
OUTPUTS: The ovine genome was screened with microsatellite, single nucleotide polymorphic (SNP) and radiation hybrid (RH) markers for association with resistance to Haemonchus contortus (Hc) infection in sheep. The established F2 families consisted of grandparent Suffolk (S, susceptible) and Gulf Coast Native (N, resistant), F1 parent and F2 lambs. 184 new lambs were added for a total of 399 on the project. During each year, lambs grazed until weaned and again for six weeks during the summer. Infection level was evaluated after both grazing periods by fecal egg count (FEC) and blood PCV. There continued to be a clear segregation of FEC/PCV for all 5 years. The selective genotyping strategy included markers screened across all F2 lambs and their parents (done at Utah State University). Within-family linkage analysis (done at Utah State University) suggested preliminary QTLs for parasite resistance on ovine chromosomes 1, 3, 6, 9 and 19. Interval mapping showed a highly significant potential QTL region on chromosome 19 at marker OARAE119 (FEC) and CSSM06/OARAE119 (PCV). A high-resolution RH map of OAR19 showed two linkage groups that aligned with the genetic linkage map. These experiments allowed resolution of the parasite resistance QTL region to a 3.5 Mb interval around position 22.17 Mb on OAR19. PARTICIPANTS: James E. Miller (Co-PI), Louisiana State University; Noelle E. Cockett (Co-PI), Utah State University. TARGET AUDIENCES: Small ruminant researchers and producers; veterinary parasitologists. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Two experiments were conducted to evaluate host response to Hc infection. 1) Artificial infection. N and S lambs were cleared of infection and re-infected artificially with Hc infective larvae. Animals were necropsied on Day 14 (early infection) and Day 21 (late infection). Fecal and blood samples were collected on Days 0, 2, 7, 14 and 21. At necropsy, nematodes were recovered and abomasal tissue samples were collected. PCV remained higher in N lambs. FEC in late infection N lambs was significantly lower at Day 21. At both Day 14 and Day 21, abomasal mucosal eosinophil and mast cell numbers were significantly greater (p<0.05) in N. 2) Natural infection. N and S lambs were cleared of infection and allowed to graze. Fecal and blood samples were collected on Days 0, 2, 7, 14, 21, 28, 35 and 42. mRNA was extracted from PBMCs; cDNA was made; and IFN-gamma, IL-4 and IL-10 were quantified. At necropsy, nematodes were recovered; and abomasal tissue samples were collected. FEC and PCV were significantly lower and higher in N, respectively. Hc was the predominant nematode, and infection was significantly lower in N. Peripheral blood eosinophil levels and abomasal mucosal eosinophil, mast cell and globule leukocyte levels were significantly higher in N. There was no difference between breeds for IFN-gamma, and levels decreased at the same time IL-10 levels increased. IL-10 was significantly lower in N which coincided with a significantly higher level of IL-4. IL-10 down regulates some Th2 response, and this could have resulted in the depressed IL-4 activity and reduced mucosal response (eosinophils, mast cells and globule leukocytes) in S. There was no significant difference between breeds for serum IgM, IgG1 and IgG2. S serum IgE levels remained unchanged, and N levels were consistently and significantly higher which correlated with the spike in IL-4 expression. Phenotyping of F2 lambs resulted in a relatively large range of infection, based on FEC and PCV, which indicates that nematode resistance segregates nicely. Potential candidate regions on a number of chromosomes have been associated with resistance, which could lead to identification of genetic markers for use in marker-assisted selection programs. Results indicate that there is a more enhanced immune response in Gulf Coast Native than Suffolk lambs and that response may account for their relative resistance to infection with H. contortus.

Publications

  • Pena, M.T., Miller, J.E., Horohov, D.W., 2004. Effect of dexamethasone treatment on the immune response of Gulf Coast Native lambs to Haemonchus contortus infection. Vet Parasitol 119: 223-235.
  • Burke, J.M., Miller, J.E., 2004. Resistance to gastrointestinal parasites in Dorper, Katahdin, and St. Croix lambs in the southeastern United States. Small Rum Res 54, 43-51.
  • Miller, J.E., Waller, P.J., 2004. Novel approaches to control of parasites - a workshop. Vet. Parasitol. 125, 59-68.
  • Miller, J.E., Horohov, D.W., 2006. Immunological aspects of nematode parasite control. J Anim Sci 84 Suppl E, 124-32.
  • Pena, M.T., Miller, J.E., Horohov, D.W., 2006. Effect of CD4+ T lymphocytes depletion on resistance of Gulf Coast Native lambs to Haemonchus contortus infection. Vet Parasitol 138, 240-246.
  • Jackson, F., Miller, J.E., 2006. Alternative approaches to control - Quo vadis. Vet Parasitol 139, 371-384.
  • Miller, J.E., Bishop, S.C., Cockett, N.E., McGraw, R.A., 2006. Segregation of natural and experimental gastrointestinal nematode infection in F2 progeny from susceptible Suffolk and resistant Gulf Coast Native sheep and its usefulness in assessing genetic variation. Vet Parasitol 140, 83-89.
  • Hunt, P.W., McEwan, J.C., Miller, J.E., 2008. Future perspectives for the implementation of genetic markers for parasite resistance in sheep. Trop Biomed 25, 18-33.
  • Miller, J.E., Fernandez, J.M., 2005. Relative susceptibility of Suffolk, Gulf Coat Native, Katahdin and St. Croix lambs to naturally acquired gastrointestinal nematode infection. Proc Hair Sheep Workshop, 24.
  • Miller, J.E., Horohov, D.W., 2005. Immunological aspects of nematode parasite control. J Anim Sci 83 (Suppl. 1), 128.
  • Cockett, N., Bishop, S., Davies, G., Hadfield, T., Eng, S., Miller, J., 2005. Use of QTL to determine parasite resistance in sheep. J Anim Sci 83 (Suppl. 1), 128.
  • Shakya, K.P., Miller, J.E., Horohov, D.W., 2007. Evaluation of selected immune responses to Haemonchus contortus infection in Gulf Coast Native compared with Suffolk lambs. Proc 52ndAnn Meet Amer Assoc Vet Parasitol: 54.
  • Shakya, K.P., Miller, J.E., Horohov, D.W., 2008. Comparative evaluation of immune responses to Haemonchus contortus infections in relatively resistant Gulf Coast Native and susceptible Suffolk lambs. Proc 53rdAnn Meet Amer Assoc Vet Parasitol: 40.


Progress 01/01/07 to 12/31/07

Outputs
OUTPUTS: 1. Station report at annual meeting of USDA NRSP-8, January 14, 2007, San Diego, CA.; Poster presentation at Plant and Animal Genome XV Conference, January 13-17, 2007, San Diego, CA.; Poster presentation at 21st International Conference World Association Advancement Veterinary Parasitology, August 20-23, 2007, Gent, Belgium.; Poster presentation at Phi Zeta Research Emphasis Symposium, September 26, 2007. Baton Rouge, LA. 2. Oral presentation at the annual meeting of the American Association of Veterianry Parasitologists, July 16, 2007, Washington DC.; Poster presentation at Phi Zeta Research Emphasis Symposium, September 26, 2007. Baton Rouge, LA. PARTICIPANTS: Miller, J (PI); NE Cockett TARGET AUDIENCES: Small ruminant researchers

Impacts
1. An additional 39 F2 lambs from 20 F1 (Suffolk x Gulf Coast Native) ewes that were bred to the fifth F1 sire on the project were added to the project. That makes a total of 66 lambs from that sire. Blood and DNA from all new F2 lambs and their parents is stored ( 20'C) for future work. Genotyping and QTL analysis are being done at Utah State University. As with previous lambs, resistance to nematode infection segregated based on fecal egg count (FEC). After a summer pasture challenge, mean FEC was 6298 eggs per gram (EPG) and ranged from 1433 to 30917 EPG. Genetic markers associated with parasite resistance have the potential to be applied across breeds/flocks to enhance resistance and/or eliminate susceptible animals. 2. The serum immunoglobulin isotyping that was pending for the previous report has been completed, except for IgA (problem with reagents).There was no difference between breeds for serum IgM, IgG1 and IgG2 and levels were not affected by infection. Serum IgE level of Suffolk lambs remained unchanged during the study and GCN levels increased substantially from 7 to 14 days of infection and were consistently and significantly (p<0.05) higher than Suffolk lambs. This increase correlated nicely with the spike in IL-4 mRNA expression on day 10 of infection. IL-5 mRNA expression is pending. Elucidation of the specific immune components involved in resistance of GCN lambs to infection may be useful in the search for QTL.

Publications

  • Shakya, K.P., Miller, J.E., Horohov, D.W., 2007. Evaluation of selected immune responses to Haemonchus contortus infection in Gulf Coast Native compared to Suffolk lambs. Proc 52nd Ann Meeting Amer Assoc Vet Parasitol: 54.
  • Shakya, K.P., 2007. Evaluation of selected immune response to Haemonchus contortus in Gulf Coast Native compared to Suffolk lambs. PhD dissertation, Louisiana State University, Baton Rouge, LA, 135 pp.
  • Hadfield, T.S, Miller, J.E., Wu, C., Bishop, S., Davies, G., Cockett, N.E, 2007. Identification of putative QTL for parasite resistance in sheep. Ann Conf Plant and Animal Genome (XV): 239.
  • Hadfield, T., Miller, J., Wu, C., Bishop, S., Davies, G., Cockett, N., 2007.Identification of putative QTL for nematode parasite resistance in sheep. Proc. 21st Inter Conf World Assoc Adv Vet Parasitol, 397.


Progress 01/01/06 to 12/31/06

Outputs
An additional 27 F2 lambs from 20 F1 (Suffolk x Gulf Coast Native, GCN) ewes that were bred to a new F1 ram were added to the project. Resistance to nematode infection continued to segregate based on fecal egg count (FEC). After a summer pasture challenge, mean FEC was 6906 eggs per gram (EPG) and ranged from 50-29100 EPG. Blood and DNA from all new F2 lambs and their parents is stored (-20'C) for future work. Genotyping and QTL analysis are being done at Utah State University. Ten GCN and 10 Suffolk lambs were dewormed and grazed infective pasture. Animals were necropsied on Days 35 and 42. Fecal and blood samples were collected on Days 0, 2, 7, 14, 21, 28, 35 and 42. Blood was used to determine PCV and eosinophil levels and mRNA was extracted from PBMCs, cDNA was made and IFN-gamma, IL-4 and IL-10 was quantified. Serum was stored for immunoglobulin isotyping (IgG1. IgG2, IgM, IgA and IgE). Fecal samples were used to determine FEC. At necropsy, nematodes were recovered and abomasal tissue samples were collected for histopathology. Results indicated that FEC increased in both breeds with significantly (p<0.05) lower levels attained for GCN lambs compared to Suffolk lambs. PCV decreased in both breeds and the decrease was significantly (p<0.05) less in GCN compared to Suffolk lambs. H. contortus was the predominant nematode recovered at necropsy and infection was significantly (p<0.05) lower in GCN lambs. Peripheral blood eosinophil levels and abomasal mucosal eosinophil, mast cell and globule leukocyte levels were significantly (p<0.05) higher in GCN lambs. There was no difference between breeds for IFN-gamma, and levels decreased at the same time IL-10 levels increased which would be expected as Il-10 down regulates Th1 responses. IL-10 was significantly (p<0.05) lower in GCN lambs on days 7, 10 and 14 which coincided with a significantly (p<0.05) higher level of IL-4 on day 10. IL-10 also down regulates some Th2 response and since Suffolk lambs had consistently higher IL-10 levels, this could have resulted in the depressed IL-4 activity and reduced mucosal response in Suffolk lambs. Immunoglobulin isotyping is pending. Results indicate that there is a more enhanced immune response in GCN than Suffolk lambs and that response may account for their relative resistance to infection with H. contortus. There are 2 microsatellite makers (IFN-gamma and CSRD2138) reported in the literature as being associated with resistance to nematode infection. DNA was extracted from blood taken at weaning and these 2 markers were genotyped across 156 weaned lambs that were exposed to a summer pasture challenge infection at 4 locations (LA, GA, VA, AR). All animals were dewormed to remove existing infections before grazing. Infection level was monitored by fecal egg count. Grazing at each location was for the same 5-6 week period. FEC at all locations ranged from low to high as would be expected. The CSRD2138 marker did not work well and there was no definitive correlation of IFN-gamma with fecal egg count.

Impacts
Genetic markers associated with parasite resistance has the potential to be applied across breeds/flocks to enhance resistance and/or eliminate susceptible animals. Elucidation of the specific immune components involved in resistance of GCN lambs to infection may be useful in the search for QTL. Neither IFN-gamma nor CSRD2138 appear to be very useful for marker assisted selection under the conditions of this work.

Publications

  • Miller, J.E., Horohov, D.W., 2006. Immunological aspects of nematode parasite control. J Anim Sci 84 Suppl E124-32.
  • Pena, M.T., Miller, J.E., Horohov, D.W., 2006. Effect of CD4' T lymphocytes depletion on resistance of Gulf Coast Native lambs to Haemonchus contortus infection. Vet Parasitol 138, 240-246.
  • Miller, J.E., Bishop, S.C., Cockett, N.E., McGraw, R.A., 2006. Segregation of natural and experimental gastrointestinal nematode infection in F2 Progeny from susceptible Suffolk and resistant Gulf Coast Native sheep and its usefulness in assessing genetic variation. Vet Parasitol 140, 83-89.


Progress 01/01/05 to 12/31/05

Outputs
Analysis of the QTL search to date has identified QTL on chromosomes 1, 6, 9 and 19 with about 28% genome coverage (858 cM). Subsequent to this analysis, 15 new markers have been added. An additional 35 F2 lambs from 30 F 1 (Suffolk x Gulf Coast Native) ewes and the same F 1 ram used in 2004 were added to the project. As with previous lambs, resistance to nematode infection segregated based on fecal egg count (FEC). After a summer pasture challenge, mean FEC was 1457 eggs per gram (EPG) and ranged from 383-3600 EPG. Blood and DNA from all new F2 lambs and their parents is stored (-20'C) for future work. Host response to Haemonchus contortus infection in Gulf Coast Native lambs was compared to Suffolk lambs. 30 Gulf Coast Native and 30 Suffolk weaned lambs were cleared of existing infection by deworming. Subsequently, 10 lambs of each breed were inoculated with a single dose of 18,000 H. contortus infective larvae, 10 of each breed with trickle inoculations of 500 infective larvae 3 times a week and 10 of each breed remained free of infection (control). Half of the animals from each group were necropsied on Day 14 (early infection) and the other half on Day 21 (late infection). The original protocol was to carry the late infections out to Day 35, but life threatening infections in Suffolk lambs resulted in early termination. Fecal and blood samples were collected on Days 0, 2, 7, 14 and 21. Blood was used to determine PCV and samples are frozen for cytokine (IFN-gamma, EL-4, EL-5, IIL-13) quantification (pending). Fecal samples were used to determine egg count. At necropsy, nematodes were recovered for enumeration and identification (pending), and abomasal tissue samples were collected to determine eosinophil and mast cell levels in response to infection. Results indicated that PCV remained consistent for all control animals and decreased for all infected animals, more so for Suffolk lambs. Fecal egg counts for control and early infection animals remained essentially zero, which was expected in that egg counts do not become evident until 3 weeks post infection. Egg counts in late infection animals were substantially greater in Suffolk lambs by Day 21 than in Gulf Coast Native lambs. At both Day 14 and Day 21, abomasal mucosal eosinophil and mast cell numbers were 2-3 times greater (p<0.05) in Gulf Coast Native lambs than Suffolk lambs. There are 2 microsatellite markers (IFN-gamma and CSRD2138) reported in the literature as being associated with resistance to nematode infection. DNA was extracted from blood taken at weaning and these 2 markers were genotyped across 156 weaned lambs that were exposed to a summer pasture challenge infection at 4 locations (LA, GA, VA, AR). All animals were dewormed to remove existing infections before grazing. Infection level was monitored by fecal egg count. Grazing at each location was for the same 5-6 week period. Fecal egg counts at all locations ranged from low to high as would be expected. The CSRD213 8 marker did not work well so only IFN-gamma will be initially used.

Impacts
Genetic markers associated with parasite resistance has the potential to be applied across breeds/flocks to enhance resistance and/or eliminate susceptible animals. Elucidation of the mechanism(s) of resistance to infection in Gulf Coast Native lambs may be useful in the search for QTL. IFN-gamma and/or CSRD2138 may be useful for marker assisted selection.

Publications

  • No publications reported this period


Progress 01/01/04 to 12/31/04

Outputs
An additional 36 F2 lambs from 30 F1 (Suffolk x Gulf Coast Native) ewes and a new F1 ram were added to the project. As with previous lambs, resistance to nematode infection segregated based on fecal egg count (FEC). After a summer pasture challenge, mean FEC was 8200 eggs per gram (EPG) and ranged from 417-21,667 EPG. The selective genotyping strategy used in this project has been expanded to cover an additional 33 microsatellite markers. The genome scan is being continued and resolution of the two previously identified QTLs (chromosomes 1 and 3) is being made within a 5 cM region. The most significant results were detected on chromosome 1, with a putative QTL localized on the central region of this chromosome. Linkage analysis is being updated (Roslin Institute) with the addition of the new markers. Blood and DNA from all new F2 lambs and their parents is stored (-20 degrees C) for future work.

Impacts
Genetic markers associated with parasite resistance has the potential to be applied across breeds/flocks to enhance resistance and/or eliminate susceptible animals.

Publications

  • No publications reported this period