Location:2009 Annual Report
1a. Objectives (from AD-416)
1. Characterize the interaction of virus replication and macrophage responses. 2. Identify natural genetic variation associated with disease susceptibility.
1b. Approach (from AD-416)
Identification of specific pathways that associate with variation in porcine reproductive and respiratory syndrome virus (PRRSV) replication and macrophage function leading to novel gene targets for the control of PRRSV infection. Alveolar macrophages will be obtained from diverse populations of swine and evaluated for their ability to support replication of PRRS viruses. Replication parameters will be estimated and macrophages that support either high or low levels of virus replication will be selected for studies of gene expression. Identifying PRRSV genotypes that confer fitness in macrophages, and host genes that respond to PRRSV fitness, to provide novel targets for intervention and control of PRRSV infections. These studies will use adapted isolates to identify viral genotypes that correlate with fitness of PRRSV in porcine alveolar macrophages and corresponding changes in macrophage transcriptional profiles. Genetic variation in specific ovine genes influences predisposition to ovine lentivirus (OLV) and the associated disease, ovine progressive pneumonia (OPP). We will thoroughly evaluate the most obvious candidate genomic regions for effects on lentiviral disease, like that containing CCR5. Our aim is to evaluate important regions of the genome for allelic association with the OLV disease susceptibility and progression phenotypes. Selection of regions will be based on a variety of scientific observations including, but not limited to, comparative mammalian biology. A selected set of 90 single nucleotide polymorphism (SNP) markers will be identified that are highly-informative in beef and dairy cattle. The development of this marker set represents non-hypothesis-driven research. The markers and genotyping assays for the markers will be readily available for any traceback needs. The same markers are also ideal for animal identification (i.e., sample matching) and routine parentage analysis. After ear tags and other physical identification devices have been removed, an animal’s DNA remains as a stable, accessible, integral, and identifiable component of its products and, thus, provides a gold standard for auditing the fidelity of physical labels and associated records.
3. Progress Report
Bovine Respiratory Disease Complex (BRDC) accounts for 75% of morbidity and 50% of mortality in feedlots. Its cost is cumulative and includes: treatment, lost production, as well as death. These losses make BRDC one of the most costly diseases affecting cattle. BRDC is composed of an assortment of bacterial and viral pathogens. One of these bacteria, Mannheimia haemolytica (formerly Pasteurella haemolytica), causes pneumonia in ruminants and produces a leukotoxin that is cytotoxic only for ruminant leukocytes (white blood cells). When viral and bacterial agents break down the antimicrobial barrier in the lungs, M. haemolytica--which is usually a commensal organism--can become pathogenic. In collaboration with scientists in the Department of Veterinary Biosciences at the University of Nebraska – Lincoln, we have initiated in vitro studies using bovine macrophages infected with M. haemolytica to determine if we can alter the levels of leukotoxin produced under different cell-culture conditions. We have determined that succinate is an important metabolite in the growth of M. haemolytica that can be used in place of supplemental serum in macrophage/bacterial cultures. We plan to exploit this finding in future metabolic studies of this important bovine pathogen. In collaboration with scientists in the Department of Homeland Security (DHS), U. S. Meat Animal Research Center (USMARC) scientists have been testing bovine, ovine, and porcine cell lines developed at USMARC for their suitability to support protein expression of a Foot and Mouth Disease (FMD) molecular vaccine. Four cell lines (MARC-BGCF2, MARC-BOE, Cdelta 2+, and Cdelta 2-) were screened by electron microscopy (EM) for their ability to support FMD protein expression; unfortunately, none of the cell lines supported detectable protein expression. These same four cell lines were screened using EM and virus isolation for their ability to support FMD virus (FMDV) expression. Cells were infected with two different FMDV strains. One of the cell lines, MARC-BOE, supported excellent replication of one of the FMDV isolates. Virus titers were similar to that achieved in the gold-standard cell line currently used for FMDV propagation. Development of informatics for studying the transcriptome of swine infected by porcine reproductive and respiratory syndrome virus (PRRSV). Collaborated with scientists at the National Animal Disease Center (NADC) to investigate the genes involved in the progression of PRSSV in infected swine. Scientists at NADC and USMARC were awarded a National Pork Board grant (effective 11/01/2008 to 11/01/2010) entitled “Gene expression in lymph nodes of PRRSV-infected pigs,” CRIS Project: 3625-32000-088-28R. Over the past year, NADC and USMARC scientists have collaborated to coordinate the collection of data generated from this grant at NADC with its analysis at USMARC. NADC and USMARC scientists have adapted pre-existing data processing pipelines to analyze transcriptomic data from this project to gain new insights into the molecular mechanisms involved in PRRSV infection.
1. Development of a new typing system for Escherichia coli O157:H7. E. coli O157:H7 bacteria are carried by cattle and can cause serious disease in humans. For reasons that are not well understood, some E. coli O157:H7 strains are more virulent to humans than others. To determine the extent of genetic variation within the serotype and to develop a new method for identifying genetically diverse strains, the genomes of 193 E. coli O157:H7 strains were sequenced. Sequence analyses identified key nucleotide polymorphisms that effectively classify strains of cattle or human origin. Assays that detect the polymorphisms were developed as part of a new typing system for E. coli O157:H7. This system is expected to become the gold standard for characterizing E. coli O157:H7 genetic diversity, as it can be used to distinguish strains involved in epidemiological investigations and to map E. coli O157:H7 genetic determinants that are responsible for increased human virulence.
2. Ovine reference materials and assays for scrapie genotyping. Genetic predisposition to scrapie in sheep is associated with variation in the peptide sequence of the ovine prion protein encoded by Prnp. Codon variants implicated in scrapie susceptibility or disease progression include those at amino acid positions 112, 136, 141, 154, and 171. A DNA sequencing strategy was developed to unambiguously determine the full length Prnp coding sequence for any sheep and, thereby, produce a consensus sequence for any population. The strategy was applied to more than 900 sheep DNAs, including those from two sets of reference sheep (one set for standardizing Prnp genotyping and another set for discovering SNPs) estimating allele frequencies, and analyzing inheritance patterns. DNA sequence analysis revealed four previously unreported SNPs, including a rare L237P codon variant on the 141F haplotype. Mass spectrometry assays were developed to simultaneously score codons 112, 136, 141, 154, and 171. The ability to identify Prnp polymorphisms in any sheep provides critical information for designing efficient population-based scrapie genotyping systems. Combined with reference DNA panels, these resources facilitate training, certification, and the development of new tests and knowledge that may expedite the eradication of sheep scrapie.
Kijas, J.W., Townley, D., Dalrymple, B.P., Heaton, M.P., Maddox, J.F., McGrath, A., Wilson, P., Ingersoll, R.G., McCulloch, R., McWilliam, S., Tang, D., McEwan, J., Cockett, N., Oddy, V.H., Nicholas, F.W., Raadsma, H. 2009. A Genome Wide Survey of SNP Variation Reveals the Genetic Structure of Sheep Breeds. PLoS One [serial online]. 4(3):e4668. Available: http://www.plosone.org.