Raising pigs that are healthy and disease free is a goal of every pork producer. Biosecure facilities help meet this goal, yet enteric and respiratory diseases persist.
Porcine reproductive and respiratory syndrome (PRRS), the associated porcine respiratory disease complex (PRDC), and enteric diseases continue to cause major economic losses worldwide. Can the genetic components of immunity and disease resistance help pork producers prevent these diseases and cut their losses?
Mapping Pig Genes
Pig genetic maps have been developed internationally (see www.genome.iastate.edu/maps/pigbase.html). The major mapping goal over the last decade has been to identify pigs with genetic alleles (alternative forms of a gene) that result in better pork meat quality, growth and improved reproductive traits.
Molecular genetic tests have been developed, and are now used routinely, for selecting pigs with improved traits. The estrogen receptor (ESR) alleles and the porcine stress syndrome (RYR1) are examples. Yet, for disease work, there has been limited progress.
Mapping Bacterial Diarrhea Resistance
Historical precedence says identification of disease-resistant pigs should be possible. Pigs that are fully resistant to bacteria-induced diarrhea (Escherichia coli) were identified as early as 1978; their resistance was due to a total lack of expression of the intestinal E. coli K88 receptor. Pigs without this receptor do not bind the bacteria as it passes through their intestine; thus, they are completely resistant to E. coli K88 infection.
However, the genetic test for identifying these resistant pigs was very difficult. They could be identified either by their ability to live through an infectious episode or by a bacterial binding test with pieces of their intestines after death. Lack of adherence to intestinal tissues resulted in the identification of the K88 adhesin or the genetic locus, K88.
Despite mapping studies and use of modern genomic tools, scientists have only localized this K88 gene to a general area on swine chromosome 13 (SSC13). To date, there is no publicly available, quick molecular test for blood cell DNA to identify K88 resistant pigs.
Resistance to another bacterial infection, also associated with presence or absence of an intestinal receptor, was the E. coli F18 receptor. Molecular research showed that this receptor was associated with alleles of the FUT1 gene on chromosome 6 (SSC6).
In 1999, Polish researchers developed a molecular test for FUT1 alleles and demonstrated that allele inheritance was associated with resistance to postweaning diarrhea due to E. coli F18 infections. Because there is a molecular test, breeding companies do offer E. coli F18-resistant breeding stock.
Genetic Mapping of Disease Immunity
Direct mapping approaches have been used to define genetic markers associated with immunity and disease resistance. Iowa State University researchers proved that different vaccine responses could be attributed to pig genetics.
Work at our Immunology and Disease Resistance Lab showed preliminary data for the genetic basis of resistance to foodborne parasite infections, Trichinella spiralis and Toxoplasma gondii.
German researchers are attempting to map genes associated with pseudorabies virus resistance. They have also targeted differences in susceptibility/resistance against the parasite, Sarcocystis miescheriana, in the European Pietrain and the Chinese Meishan pig breeds.
Case Study: Salmonella Resistance
Recent studies in England, on mapping resistance and susceptibility genes for salmonellosis, illustrate the difficulty of most infectious disease mapping studies. The team began by identifying salmonella-resistant and salmonella-susceptible breeding stock, then bred them to develop a reference family. All progeny were challenged with a defined dose of salmonella bacteria.
A large amount of data (phenotypes) on pig immunity to infection, including serum and blood cell activity and tissue bacterial burden, was collected. Additionally, genomic mapping information (genotypes) on each pig still has to be generated.
These studies suggested a role for several phenotypic markers in resistance to salmonellosis, including blood neutrophil function and cell proliferation. Much more data is needed to identify the exact genetic alleles encoding resistance.
Indeed, when some pigs bred to be resistant turned out to be relatively susceptible, the potential complexity of genetic inheritance of resistance was revealed. Salmonellosis, like most infectious diseases, will likely require favorable alleles at several genes to generate substantial disease resistance.
Key Questions Remain
Still very much in its early stages, research to date has raised serious questions that must be answered as targets for genetic resistance to infectious disease are established. For example:
Should research focus on a single or multiple disease agents?
Is there data indicating that disease resistance is heritable?
What disease responses (phenotypes) need to be targeted for best resistance — mortality? morbidity? carrier status? shedding capacity? sow transmission? boar transmission?
How much genotypic data is needed?
Would identifying highly susceptible animals be useful for targeted drug or vaccination treatments?
Would selection for faster recovery from disease be an advantage?
How will production traits be affected by selection for disease resistance?
Can pigs with improved immunity be identified? The above questions outline several issues that need to be addressed for genetic studies of infectious disease to advance.
Is there an alternate approach? For PRDC, several infectious agents contribute to this disease complex — PRRS virus and viral and/or bacterial infections. With so many infectious organisms there must be numerous receptors and genes that would encode resistance. Thus, different approaches are required. The genetic questions then become:
Can pigs that resist respiratory infections be identified?
Could pigs with improved immune responses to infections be identified and shown to be genetically more resistant to respiratory infections?
- Is it possible that pigs that make higher levels of the anti-viral cytokine, interferon-gamma (IFNg), would be genetically more resistant to PRDC?
Canadian researchers have developed lines of pigs selected for high/low levels of a suite of immune traits: antibody production and cell-mediated immune responses. Their high-responder pigs did develop higher vaccine responses, grew faster, and had lower disease scores following infection with mycoplasma. The exception, however, was their arthritis scores were greater in the high line and likely due to differences in cytokine production between the selected lines. These pigs have not been directly tested for resistance to a wide range of diseases; this is important information to know.
Innate vs. Specific Immunity
The mammalian immune system is complex, as has been noted in the other articles in this issue. It is important to understand the difference between immediate, innate immune response and the slower, cell-mediated, specific immune response when a disease challenge occurs.
The innate immune system acts immediately in response to infectious insults (see Figure 1). It is dependent on immune factors, termed cytokines, and immune cells, including macrophages, neutrophils and natural killer (NK) cells. While innate immunity may be quick, it is not specific and may not be potent enough to control most infectious organisms.
To effectively battle viral and bacterial infections, animals require cell-mediated immune responses. Animals need “B” cells to make antibodies to bind up viruses or bacteria. “T” cells are needed to kill infected cells or to produce the immune cytokines to activate and enhance immune responses. “T” cells are also essential for long-term memory, such as the memory response to infection after vaccination.
Molecular Gene Expression Data
How can an animal's immune system be quantitated? Like your personal physician, scientists can now test swine blood cell numbers and subsets. As was found for salmonella resistance, some immune cell activities can be associated with resistance.
Indeed, molecular tests can be used to compare how active immune cells are. Molecular “gene expression patterns” can be generated and quantities of different immune factors compared for pigs at different ages or times after infections.
The Immunology and Disease Resistance Lab has worked with researchers at the Beltsville Human Nutrition Center to develop molecular gene expression patterns to help identify genes controlling numerous immune, disease and vaccine responses. In the future, even broader gene expression systems, referred to as microarrays, will enable scientists to test thousands of genes for simultaneous analyses.
Finding the Pigs
We must ask ourselves — can pigs with enhanced innate immunity be identified? Will they be healthier?
Previously, our lab and others had shown that newborn pigs have poorly developed immune systems. It takes several months for pigs to have an adult level of immune cytokines and cells. Using molecular assays, it is now possible to compare pigs at the same age and potentially identify those with higher levels of innate immune factors.
We might find pigs whose genetically determined level of innate immune response helps them to jump-start their infectious disease responses and/or quickly shift their responses to anti-viral, cell-mediated immune responses.
However, immunity needs to be controlled so that it does not overreact and cause pathology to take over. The “best” pigs, therefore, might be those that respond quickly to an infectious agent, prevent it from replicating, and then stop their immune reaction.
Alternatives to Antibiotics
Continued consumer demands for decreased antibiotic usage in food animals increases the need for alternative genetic disease resistance approaches to be developed. Identifying pigs with desirable immune responses should enable producers to decrease dependence on drugs.
A major issue for scientists now is to identify exactly which genetic alleles result in “better immunity.” As new, serious disease organisms are identified, aggressive efforts to identify pig genes that control resistance, and stimulate protective immunity will be required.
Alternately, genomic research efforts can be targeted at the infectious organisms (microbes). Thus, microbial genomic efforts should lead to disease-specific diagnostics, and high throughput screening assays for cost-effective, targeted drug testing.
Vaccinations help pigs respond more quickly to known infectious agents, thus preventing disease-related losses. Although it may be impossible to select pigs with resistance to a wide range of diseases, it may be possible to identify pigs that have better innate immune system or better anti-viral cell-mediated immune response levels. In other words, we may find pigs that are genetically more responsive to anti-viral vaccines — that is, “vaccine ready.”
Will we select/produce disease-resistant stock for specific tasks? The high expense of disease resistance studies means that producers and regulators have to determine which are the highest priority diseases to address with their limited resources.
Should we select for respiratory disease resistance, or is food safety, and salmonella resistance, more important?
As national bio-safety and bio-defense issues are defined, our priorities may be substantially altered. Limited research funds and facilities necessitate that we choose our priorities wisely. Tough questions remain.
If foot and mouth disease- (FMD) resistant pigs were available, would we use them to stock areas close to active infection or to restock previously infected farms after depopulation and decontamination?
The Potential is Great
Studies targeting the genetics of disease resistance are essential for improved pig health and pork quality. As scientists identify breeding stock that is healthier, by virtue of its innate disease-resistant properties, they will also help decrease our dependence on antibiotics.
Studies which incorporate specific production parameters and nutritional concerns in their design will help producers select the breeding stock which possesses the necessary resistance characteristics: higher respiratory disease resistance for confined pigs, higher parasite resistance for pasture pigs. There is an enormous benefit that will come from such studies.
There is great potential for genetic studies of disease resistance to reveal novel host effector mechanisms that will lead to new biotherapeutics and cost-effective approaches for disease control and growth optimization.
Figure 1. Cells Regulating Immune Responses
All immune cells in the blood, the hematopoietic cells, are derived from bone marrow stem cells. These hematopoietic stem cells give rise to two main lineages: one for lymphoid cells (lymphoid progenitor) and one for myeloid cells (myeloid progenitor). The common lymphoid progenitor will differentiate into either T cells or B cells depending on the tissue to which it travels (homes). In mammals, T cells develop in the thymus while B cells develop in the fetal liver and bone marrow. Pigs use special areas of their intestines, termed the Peyer's patches, for B cell maturation. B cells produce the antibodies so crucial to immune and vaccine responses. To produce antibodies, B cells must become antibody-forming cells (AFC), or plasma cells. Innate immune responses are carried out by natural killer (NK) cells that also derive from the common lymphoid progenitor cell. The myeloid cells differentiate into the committed cells on the left. The platelets help blood to clot and thus heal injured tissue. Three other myeloid-derived cell types, the monocyte, macrophage and dendritic cells are critical in helping the immune system recognize what is foreign, and thus stimulating specific immune system responses. Finally, the “granulocytes,” a term used for eosinophils, neutrophils and basophils, have specialized functions, e.g., neutrophils will use antibodies to trap and kill invading bacteria.
Adapted from http://www.ed.sc.edu:85/book/immunol-sta.htm. Courtesy of Department of Pathology & Microbiology, University of South Carolina School of Medicine, Columbia, SC
Fig 2. Making Road Maps of Genes
Scientists worldwide are learning more about the genetic makeup of pigs. The eventual payoff could mean less expensive meat products, lower levels of fat, higher milk production and improved health and resistance to disease. Genome mapping could take the guesswork out of breeding the most productive animals.
The genome maps, comprised of “genetic markers,” are 95% covered for the pig. These markers, equivalent to route markers on a road map, give scientists an idea of where they are in the pig genome and what they are near when attempting to find genes that are linked to important traits.
The goal is to eventually enable scientists to pinpoint the exact location of every gene in an animal. The genome maps will someday allow the livestock industry to produce animals that are genetically resistant to certain diseases or parasites.
Within each cell nucleus are chromosomes grouped in pairs (one inherited from the father, one from the mother). Pigs have 19 pairs of chromosomes.
Chromosomes consist of tightly coiled strands of DNA.
Within the DNA are nucleotide bases commonly abbreviated by the letters A, T, C and G, which stand for adenine, thymine, cystosine, and guanine. Adenine always pairs with thymine, while cystosine always attaches to guanine. There are approximately 3 billion base pairs of nucleotines in a single cell of a pig.
The small section of the chromosome in the box shown here, for example, could contribute some information toward determining an animal's blood groups or interferon genes. It is this unique sequence of nucleotide bases and their lengths that spell out genetic instructions.
For more complex traits, such as milk production, disease resistance, antibody production, or reproductive capacity, the information would be contained in several genes on many different chromosomes.
Scientists use different methods to break up the long strands of DNA and the nucleotide sequences on them. They then designate “markers” or reference points located on or near genes.
This is the map of swine chromosome 6 from the USDA funded swine genome mapping site: http//www.genome.iastate.edu/pig.html. The RYR1 gene locus on chromosome 6 in the pig, for example, encodes the “stress” gene syndrome. The RYR1 position is also known as the Ryanodine receptor, the calcium release channel, or the Halothane (HAL) locus. Next is TGFB1 (transforming growth factor beta 1), which is an immune system regulatory factor. Further down chromosome 6 is the EAH (erythrocyte antigen H) gene for the blood group H antigen.
Adapted from U.S. Department of Agriculture Agricultural Research Service display.