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Understanding Genetic Disease Resistance

Understanding Genetic Disease Resistance
Recovery from disease depends on an effective and protective immune response. Identifying genes that control immunity may help identify pigs with faster or better responses to disease challenges

Traditional genetic approaches have been used effectively in the swine industry to enhance feed efficiency, carcass leanness and reproductive traits. In the last decade, swine genome research has expanded from identifying genes associated with production and reproduction traits to swine health, well-being and disease-resistance traits (illustrated above).

Recovery from disease depends on an effective and protective immune response. Identifying genes that control immunity may help identify pigs with faster or better responses to disease challenges.

Geneticists began addressing genetic disease resistance by assessing whether immune traits were highly heritable, then they mapped the genes involved — referred to as the quantitative trait loci (QTL). These studies confirmed that the proportion of several blood immune cell subsets and serum antibody levels were heritable.

More recent efforts have affirmed that QTL for specific anti-disease antibody levels could be identified and mapped, along with the location of immune proteins that are associated with anti-disease responses (e.g., cytokines, such as anti-viral interferons). Other research determined that the percentage of certain blood cell subsets were correlated with improved growth rates during the entire productive life of the pig.

Overall, this data indicated the potential for selecting pigs for health traits as well as performance traits. To perform these analyses, geneticists have their tools, parentage for inheritance determination, and molecular genomic markers, such as single nucleotide polymorphisms or SNPs, which can be measured using pig SNP chips. With these tools, mapping can proceed from the chromosome to the gene or locus to the exact DNA sequence.

Canadian researchers focused on selecting pigs for high or low immune responses. They concluded that pigs could be bred for higher immune responses. However, their data showed that higher may not be better, because high-immune-response pigs also had higher arthritis symptoms associated with Mycoplasma hyorhinis infection. In the end, there was no indication of a consistent, line-related health

Disease Resistance Defined

Research on genetic resistance to infectious pathogens of pigs aims to identify the genetic differences, termed alleles, that are associated with resistance/susceptibility. Unfortunately, there are few infectious pathogens for which complete disease resistance has been found.

True resistance for certain pig bacterial (e.g., E. coli) infections is due to the genetic allele for not encoding the intestinal receptor. No receptor means no binding upon infection and no bacterial diarrhea.

Scientists typically look for pigs that clear pathogens quickly with limited clinical signs and growth delays. Pigs may appear to be resistant to viral infection, but some may replicate the virus, show no clinical symptoms, and be tolerant to viral pathology. Such pigs would be sources of viral shedding.

Age, previous infection and vaccination status, nutritional plane, stress, reproductive status, and external environment can alter the interactions between the pig host and pathogens.

Breeding for disease resistance is a balancing act. In the past, breeders may have inadvertently created selection indices for improved growth that included undesirable effects, such as viral susceptibility.

In the future, genomics will hold the promise of providing powerful DNA markers for the desired production traits, as well as selection for markers aimed at minimizing disease effects.

New Genomic Tools

Modern genomic tools have radically improved the potential for determining which genes may be involved in controlling immune responses and, thus, disease responses. SNP chips with each pig DNA sample can test for genetic variation (alleles) at each of 60,000 SNPs (Figure 1).

Similarly, next-generation sequencing analyses (NGSA) technologies have transformed how analyses of pig post-infection or vaccination responses and gene expression studies can be performed. Scientists have advanced from testing expression of single “candidate genes,” to analyzing more than 20,000 genes simultaneously using tools called microarrays, to performing in-depth NGSA, or RNA sequencing, with millions of sequences analyzed.

Finally, the full swine genome sequence ( was completed in December 2010. With NGSA technology, it is now possible to fully sequence the genome of selected pigs, although this would be quite expensive and data-intensive.

Genome-Wide Studies

With these new tools for pig mapping and expression analyses, researchers can now determine which genes or genomic regions may correlate with improved pig health, vaccine responses, or resistance to specific infectious agents. Genetic studies will now use genome-wide association studies (GWAS) to map disease resistance genes. These results, in turn, will enable breeders to use this information for genome-wide selection studies.
Results from genomic studies should provide researchers with new tools, such as sets of DNA markers, molecular pathways, and blood protein biomarkers. These will help producers and breeders to identify pigs that exhibit better disease resistance against specific pathogens, resulting in healthier pigs and fewer therapeutic treatments.

PRRS Virus Challenges
Based on these advances, geneticists wanted to apply these genetic tools to identify disease-resistant pigs for the most economically important disease worldwide — porcine reproductive and respiratory syndrome (PRRS). The PRRS virus is a stealthy, infectious agent that can efficiently enter any stage of a production unit.

As illustrated in Figure 2 on page 34, PRRS virus possesses a complex pattern of movement through a production system, including efficient horizontal (pig-to-pig) and vertical (dam-to-fetus) transmission, by intranasal, intramuscular, oral or vaginal routes.

During early infection stages, the virus subverts the innate response. Immunologists and geneticists expect they will be able to use genomic technologies to identify QTL and alleles for selecting pigs with the best anti-PRRS virus immune responses.

Herd immunity against PRRS, conferred by vaccination or recovery following infection with field virus, is tenuous because continuous virus mutation and evolution of several viral genes may allow the virus to escape host immunity and persist in a population. Within infected herds, there may be pigs with subclinical or persistent infection (viral carriers). This can result in perpetual circulation of virus within infected herds.

The problems posed by PRRS virus challenge traditional approaches to disease control and virus elimination. The PRRS Host Genomic Consortium was formed to search for pigs which are genetically resistant to PRRS virus infection.

When addressing infectious disease problems such as PRRS, the plan of attack is often to go after the so-called low-hanging fruit by supporting low-cost research that can deliver solutions to producers relatively quickly.

Investigations of immunity during PRRS virus infection have produced a wealth of knowledge describing the elements of protective immunity, serum-neutralizing antibodies and anti-viral interferon responses, and prevention of virus escape mechanisms and viral persistence. However, investigations so far have not yielded significant improvements over current vaccines, which were first introduced in the 1990s. Immunopharmaceuticals, which may enhance the immune response, also have yet to show widespread commercial use.

Evidence for Genetic Resistance

Studies in the late ’90s at Iowa State University (ISU) first showed that breeds did differ in their resistance to PRRS. This has been affirmed for both type I (European) and type II (North American) PRRS. Data indicated that pigs selected for improved growth were more susceptible to PRRS than those selected for improved reproductive traits, which were more resistant.

Selection for certain immune traits, such as activity of macrophages (the cell targets for PRRS virus infection), did not improve resistance. Sows with higher numbers of anti-viral, interferon-producing cells appeared more resistant in most herds. Studies showed that, contrary to predictions, severity of PRRS clinical signs is not a good indicator of genetic susceptibility.

By 2005, veterinarians, producers and scientists agreed that much of the lower hanging fruit of PRRS solutions had been picked. New solutions to nagging problems would require long-term, riskier, and more expensive efforts requiring a greater level of commitment from stakeholders.

PRRS Host Genetics Consortium

With the support of the National Pork Board and the USDA PRRS Coordinated Agricultural Project (CAP), Lunney and Rowland led the formation in 2007 of the PRRS Host Genetics Consortium (PHGC), a national effort to identify genes linked to PRRS disease resistance, susceptibility and related growth effects.

The fundamental plans for the consortium were formulated with input from PRRS researchers, pig genome and genetic researchers, National Pork Board, veterinarians, producers and commercial companies representing breeders, animal health, nutrition and diagnostics.

In four years, the PHGC has exceeded many of the early milestones and expectations, and has laid the groundwork for breeding programs that can reduce the impact of PRRS and other infectious diseases.

The PHGC represents a first-of-its-kind approach for doing “big science.” The basic tenet of the consortium philosophy is that data is developed as a team effort; members receive a benefit after making a contribution. Within the consortium, the scientific benchwork is performed at a variety of research institutions, including Kansas State University (KSU), Iowa State University (ISU), Purdue University, Michigan State University, Washington State University and USDA’s Agricultural Research Service at Beltsville, MD (BARC).

The hub in the PHGC wheel of research activity is the secure relational database developed and maintained at ISU. All data derived from consortium participants is deposited in the database so that all qualified investigators can access raw data and basic information related to the project.

The consortium is a vehicle for breeding and animal health companies to make donations of pigs and supplies as a funding support. The reward for their contribution is access to the larger PHGC resource database.

One of the daunting obstacles in supporting long-term research efforts, like the PHGC, was finding the necessary resources, including funding. Pork Checkoff provided the initial funding, which supported the infection of pigs at KSU and the development of a relational database at ISU. The U.S. national genome research program, (NRSP-8), supported a major portion of the SNP genotyping and provided backup resources for the PHGC database.

An animal health diagnostic company donated 10,000 polymerase chain tests and five different breeding companies donated high-health pigs. The core Pork Board funding was matched by support from the USDA-funded PRRS CAP, USDA ARS, and USDA AFRI functional genomics grant funding. The most recent infusion of funding will come from Canada’s Genome Alberta. In total, the PHGC has accumulated an operational budget in the millions of dollars.

Infection Model for PRRS

From the start, it was clear in the PHGC project that any study of the genetic components of host defenses during PRRS infection would require hundreds, and potentially thousands, of PRRS-infected pigs. For this, numerous groups have explored field data. However, such data is complicated by numerous factors, including unknown timing of original infection, effects of underlying infections on PRRS severity, plus age, environmental and nutritional issues.

The PHGC wanted to develop an infection model that would reproduce relevant aspects of PRRS disease, at a reasonable cost, and provide sufficiently large numbers of samples that could then be used for genetic analysis of disease-related parameters, the phenotypic disease traits.

One important impact of PRRS virus infection is on the sow farm, where it can cause abortions, stillbirths and weak-born pigs. These disease traits are highly relevant and can be accurately measured. There are several well-characterized sow infection models; however, attaining sufficient numbers of pigs would take a long time, and it was clear that a sow model of PRRS would be too costly.

Another model of PRRS infection incorporates the calculation of lung lesion scores in infected pigs. This model is designed to assess the impact of acute respiratory disease, a major disease sign associated with PRRS in nursery pigs. For this model, young pigs are infected and lungs removed for evaluation of pathology at 7-14 days after infection. However, determining lung scores is subjective. Such a model, combined with quantitation of serum PRRS virus levels, enabled University of Nebraska researchers to show improved genetic control of PRRS in pig lines selected for reproductive traits.

The model selected for the PHGC is a nursery pig model. Pigs are infected at 3-4 weeks of age with a well-characterized PRRS virus isolate, and followed for 42 days, covering both the acute and persistent stages of PRRS infection. Any virus present after 42 days is the result of “persistence.” Blood and weight data are collected regularly and tonsils collected at the end of the study. The phenotypic disease traits include virus load in serum, immune-associated cytokines in serum, weight, mortality and persistence (amount of virus in tonsil).

Research Spinoffs

Loss of performance as a result of dead and light pigs covers a significant portion of the estimated $600 million loss annually from PRRS. One spinoff from the PHGC is the collection of oral fluid samples, which are being used by PRRS scientists to develop and validate new diagnostic tests. Samples collected from PHGC are catalogued, distributed to appropriate testing labs, or stored in two locations (KSU and BARC) for future research.

Control of PRRS virus infection is a dynamic process. Thus, as part of the PHGC sampling, blood samples were collected in Tempus tubes for later use in analysis of anti-viral RNA gene expression. This RNA data will enable PHGC researchers to probe differences in early (viral replication, innate immune) as well as later (viral persistence, acquired immune) PRRS virus responses in resistant vs. susceptible pigs. The work may reveal unique PRRS clearance mechanisms used by resistant pigs or missing responses in susceptible pigs.

Once identified, such novel anti-PRRS mechanisms may provide the basis for animal health companies to develop new vaccines or biotherapeutics. Geneticists will use statistical analyses to probe RNA gene expression for critical regulatory control points, genes and pathways involved in anti-PRRS responses. Results should reveal expression QTL (eQTL) for which genes can be identified and mapped. Overall, these mapped eQTLs should reinforce SNP mapping data.

What We’ve Learned

Measurements of weight and virus loads in PHGC trials have already revealed some dramatic effects. The distribution of weights for 600 PRRS-infected pigs from trials 1-3 is shown in Figure 3. Ideally, the weight distribution of finishing pigs should be relatively homogeneous. In the PRRS-infected population, the weight of pigs varies widely, with most of the infected pigs lagging behind.

Virus load shows the early appearance of PRRS virus in the blood, peaking at 7-10 days, followed by its disappearance from the blood in most pigs at 21-28 days after infection. Some pigs do not clear their original virus even by 42 days after infection.

Approximately 20% of pigs actually showed a reactivation of PRRS virus infection. This effect of PRRS virus was first visualized in the PHGC study and explains why PRRS virus can remain in a herd for an extended period of time. The effect of PRRS virus on other disease traits, such as immune antibody and anti-viral cytokine responses, are being determined.

Weight gain and virus load are being used by the PHGC as examples of important traits to monitor following PRRS virus infection. To identify markers on the pig genome linked to low weight gain and high virus loads is a complex process. It starts with the phenotypic data collected at KSU, followed by genomic DNA preparation at BARC; DNA is then transferred to a commercial source for SNP chip analyses to generate the SNP genotypic data. The labeled DNA was placed on the chip and unique genomic markers for each pig are identified.

Results are stored in the PHGC database at ISU. Once this data was available, ISU scientists began their genome-wide association studies (GWAS) using sophisticated models and deep computer power. Alleles at each of the more than 60,000 SNP markers are interrogated for correlation with each phenotypic trait. Within the region surrounding each SNP marker, genes of interest would be found and termed “candidate genes.” Based on the phenotypic and genotypic data from the first three trials of 600 experimentally infected pigs, the ISU researchers have identified several chromosomal regions that control weight gain and virus load. Figure 4 on page 42 shows the results for SNP marker location on swine chromosome 4.

One concern was the possibility that hundreds of markers might be associated with each disease trait implicating the participation of multiple genes, perhaps randomly scattered across the whole genome. This would make the implementation of breeding programs as the means to increase disease resistance an impossible task.

Results from the ISU lab provided the first glimpse into the number of markers associated with virus load and weight gain. It turns out that a small number of SNP markers are associated with each trait; in fact, both traits map to an overlapping location in the same region in swine Chromosome 4. This confirms that some genes influencing weight gain may be the same genes that affect lower PRRS virus load. These early results suggest that breeding programs to increase PRRS resistance are a real possibility.

One benefit to the identification of genetic markers that confer disease susceptibility is the ability to avoid some of the unintended consequences of breeding.

For example, genes involved in improving feed efficiency and growth may be linked to nearby genes that increase disease susceptibility; the PHGC results suggest this problem may be prevented.

Predicting the Outcome

There are two genomes that participate in the outcome of infection: the host (pig) and the pathogen (virus) genomes. The interaction between pathogen and host is akin to a “death dance.” Both partners twirl wildly across the room, but only one survives. Identifying genes on the host genome that confer resistance can help improve the survival of the host. But understanding both genomes will allow producers to apply diagnostic tests that will predict the outcome even before infection.

Two new areas of focus within the PHGC are addressing this challenge. The first characterizes the PRRS virus genes that contribute to disease severity. PRRS virus genetic markers can identify strains of virus which are highly virulent. The second area is referred to as “ultra-deep” phenotyping. The PRRS-related phenotypic disease traits measured so far include weight gain and virus load, with limited data on antibodies and cytokines.

In reality, there likely exist numerous relevant disease traits that can be accurately measured and analyzed for their prediction of PRRS disease responses. The PHGC provides the benefit of stored samples that can be tested for new traits to add information on the best anti-PRRS phenotypes.

Just like the identification of genes in the pig genome that influence disease outcomes, these PRRS studies are long-term, risky endeavors. But PHGC researchers are determined to pursue efforts to improve the phenotypic and genotypic markers used to evaluate PRRS resistance and susceptibility.

In the future, good genetics combined with good vaccines, diagnostic strategies and biosecurity will form an integrated and comprehensive strategy for disease control and elimination.

Frequently Asked Questions about Swine Genetics
Are we on the cusp of developing genetic lines that will usher in a new era of disease-free swine herds?

It is unlikely that we will have completely disease-free pigs. It is possible that many of the genetic traits that confer improved resistance to PRRS will likely confer resistance to other viruses.

Would genetic selection for resistance to viral infections improve resistance to bacterial infections?

This would be likely, especially if those traits are involved in innate immunity, that is, the induction of early factors that block infection. However, the amount of gene overlap for resistance to different pathogens remains to be determined.

How can swine genome mapping (marker-assisted selection) help enhance future disease prevention efforts?

Breeding companies will use genetic information on PRRS resistance to inform their marker-assisted selection efforts. If effective, this approach will help to decrease the viral pathogen burden for the herd. This combined with other biosecurity and vaccination strategies should enable producers to prevent, or at least control, new infectious disease problems.

How can gene markers help in identifying swine diseases to which vaccines may provide a viable health strategy?

Understanding mechanisms for disease control enables animal health researchers and vaccine companies to design more effective vaccines. One idea related to the genetics of disease control is the so-called “vaccine-ready pig.” Breeding to select pigs with enhanced responses to vaccination would help producers in their disease control efforts.

TAGS: Reproduction