Role of Genetic Variation SNPs in Host Resistance (Swine) to S. Suis

Role of genetic variation SNPs in host resistance (Swine) to S. suis

TABLE OF CONTENTS

LITERATURE REVIEW

General introduction – infectious disease in swine

Genetic resistance

Symptoms and background

Epidemiology

Capsule

Transmission between pigs and survival

Virulence Factors and Pathogenesis

Biosecurity

Direct Contact Transmission

Airborne Transmission

Transmission by birds, rodents, and other animals

Transmission by trucks, boots, and equipment

Transmission by people

Monitoring

Antibiotics

Genetic resistance in pigs

General mechanisms of genetic resistance

(Swine) Host genes involved in resistance to bacterial pathogens

Genetic resistance conditions – Porcine brucellosis; success in controlling for genetic resistance

Mechanisms of genetic resistance

(Swine) Host genes involved in resistance to bacterial pathogens

THESIS PROPOSAL

Objectives and hypotheses

Experimental approach

Limitations

REFERENCES

TABLES AND FIGURES

Infectious Disease in Swine

Pork is the most widely eaten meat in the world accounting for more than a third of global meat consumption at ~36% (Nations, 2018). As such, swine production is an important factor contributing to food security and the entire agricultural economy. With a rapidly growing global population, the demand for pork production has to increased correspondingly. Therefore, farms have proportionately intensified farming protocols by increasing the density of animals in production to meet demands; some farms contain hundreds or even thousands of pigs (Martinez, Rhodes, & Upper, 2002). Hence this increased stress to pigs raises the potential for proliferation of pathogens in the farm (Martinez et al., 2002). Some notorious swine pathogens include but are not limited to: Salmonella spp.  Escherichia coli, influenza, Pasteurella multocida, andporcine reproductive and respiratory virus (PRRS), and foot-and-mouth disease (FMD). These infectious diseases present an immense economic burden that could result from the following: mortality, production downtime, increased veterinary costs, costs form biosecurity programs, disruption of trade, and limitations imposed by food safety (Dehove, Commault, Petitclerc, Teissier, & Macé, 2012). For example, PRRS in the United States accounted for a total of US $560.32 million in losses during 2005; US $66.75 million in breeding herds and US$93.57 million in growing-pig populations (link 1). In 1997, Taiwan suffered a major FMD outbreak that caused a ban on exports of pork to Japan resulting in an estimated total economic loss of US $1.6 billion (P. C. Yang, Chu, Chung, & Sung, 1999).

Some of these infectious diseases pose not only a threat to swine but also humans. are These zoonotic pathogens can be transferred to consumers across the world. A number of the infamous zoonotic swine diseases include but are not limited to the following: Yersinia enterocolitica, Staphylococcus aureus, Salmonella spp., Campylobacter spp., and Streptococcus suis. Salmonella spp. in particular, Salmonella spp. cause ~1.3 billion cases of nontyphoidal salmonellosis worldwide (Thapaliya et al., 2015). The resulting economic losses was estimated to be US $0.5 billion to 2.3 billion (Thapaliya et al., 2015). Finish this paragraph. Wrap it up and talk about s. suis

Genetic resistance – What about mental health impacts – this is a new area of research has well.

Classical research on genetics has had a major impact on the swine industry improving factors that are not limited to: breeding, feed efficiency, carcass leanness, and reproductive traits over the past century. And more recently, swine genomics have been applied to improve swine health, general well-being, and host disease-resistant traits. Host disease-resistance is particularly important because there are many pigs of the same species that could all be susceptible to the same disease challenges. Thus, the innate ability to endure environmental conditions or diseases that could be either lethal or non-lethal are especially important when raising a herd. With all this considered, there is a branch of disease resistance research, host-genetic resistance, in which its goal is to identify disease-resistant traits that could affect host -tolerance of disease challenges.

One of the greatest challenges to overcome in the swine industry is the PRRS virus because it occurs in all age groups world-wide. There is no single strategy that has yielded complete removal of PRRS. Should PRRS be tested positive in a herd however, a program is usually adopted to either eliminate or regulate PRRS to decrease disease. In addition to management strategies in these programs, a vaccine is normally administered as both live and killed variants, but its use has been inconsistent with very limited success. As such, host genetic resistance has been widely considered as an alternative long-term solution to not only PRRS but for other diseases as well. With the recent decrease in cost and increase in efficiency, new tools such as next generation sequencing and SNP chips have become feasible and even essential tools in genomics research. Although it is unlikely that a diseased-free pig will be bred, it is likely that genetic resistance conferred towards PRRS could also overlap with other viruses. However, the overlap between viral resistance and resistance towards pathogens have yet to be determined.

Symptoms and background

Streptococcus suis is an encapsulated, facultatively anaerobic, gram-positive, opportunistic pathogen that can be found in the tonsils, intestines, and genital tracts of clinically healthy pigs. In countries with a highly developed porcine industry, certain serotypes of S. suis have been known to be lethal that resulted in pyrexia and sudden death in pigs (Wertheim, Nghia, Taylor, & Schultsz, 2009). Currently, S. suis is present worldwide and has a range of 35 serotypes, in which its virulence towards pigs and humans remain poorly understood (Brousseau et al., 2001). Most studies have been performed on serotype 2 and despite extensive research, there exist nonvirulent and virulent strains. Furthermore,S. suis is a zoonotic disease that is transmittable from pigs to humans through infected carcasses and contaminated meat; a common route of human infection is small skin abrasions but are not the only way for bacterial entry (Fittipaldi, Segura, Grenier, & Gottschalk, 2012). To reduce infection, previous literature has suggested that hand-washing with liquid soap inactivated S. suis serotype 2 in less than one minute; this proposes that soap and water would be sufficient in removing skin contamination (F. A. Clifton-Hadley & Enright, 1984). In humans, meningitis is the most common disease from S. suis infection, but all infections result in disease. Other diseases that stem from S. suis infection the following: endocarditis, cellulitis, peritonitis, rhabdomyolysis, pneumonia, and septicemia (Gottschalk, Segura, & Xu, 2007; Lun, Wang, Chen, Li, & Zhu, 2007). 

Epidemiology

Cases of S. suis infections in humans spiked dramatically in the last two decades, with two epidemics recorded in China in 1998 and 2005. Previous reports indicated that approximately 14 in 25 individuals and 32 in 215 individuals infected with S. suis died during the 1998 and 2005 epidemic, respectively (Yu et al., 2006). Despite the prevalence of S. suis in Western and Asian countries, the number of diseased cases that resulted from human infections differed greatly but the primary cause for disease remains poorly understood. For example, S. suis is the third most common cause of bacterial meningitis in Hong Kong and the top cause in Vietnam (Huong et al., 2014; Ip et al., 2007). Comparatively, in Western countries such as the United States and Canada, S. suis serotype 2infection is considered a rare event with only four human cases reported in North America—two cases in Canada (one endocarditis, one meningitis) and two cases of meningitis in the United States (Lee et al., 2008).

Similarly, in pigs, S. suis is widespread in areas that have developed swine industries with serotypes 1–9 representing over 70% of S. suis isolates extracted from diseased pigs. Within the 1-9 serotype subset, serotype 2 remains the most common in worldwide while serotype 9 is the most common in Spain, Germany, and Netherlands.

Capsule

The bacterial capsule represents a large portion of the surface on many bacteria and it is composed of many polysaccharides. The capsule is widely considered a virulence factor because its ability to cause disease by adhering to cell surfaces and to prevent engulfment. To date, there 35 serotypes have been identified based on the capsular polysaccharides (CPS) of S. suis (Brousseau et al., 2001). Some serotypes are considered more dominant and virulent in certain countries than in others. For instance, serotypes 1, 9, and 14 are more prominent in some European countries while serotype 2 is considered the most dominant and virulent in Asian countries (Goyette-Desjardins, Auger, Xu, Segura, & Gottschalk, 2014; Wertheim et al., 2017). Because of the dominance and relatively recent outbreak of the S. suis serotype 2 strain, nearly all studies have used this strain as the model to understand virulence factors and pathogenesis. Interestingly, the serotype 2 strain does not exert the same level of dominance in North American countries, which could suggest a different set of virulence factors and a potential for disease in Asian strains (Messier, Lacouture, & Gottschalk, 2008). Additionally, variation within serotypes based on genetics, which could make subsets of one serotype virulent or avirulent. For example, there exists virulent and avirulent serotype 2 S. suis strains.

Transmission between pigs and survival

S. suis transmissions usually occur in asymptomatic healthy pigs that carry a virulent strain into non-infected herds. However, it is possible for sows to transmit S. suis to their piglets during parturition and nursing as very few piglets infected with a virulent strain survive to infect a herd. As previous reports have shown, pigs that carry the serotype 2 variant asymptomatically can act as a reservoir for multiple unknown S. suis serotypes (Monter Flores et al., 1993). For nursery pigs, this poses as a window of vulnerability because asymptomatic nursey pigs are mixed or comingled with other piglets in the nursey, when maternal immunity is no longer present. Infections of S. suis are far more common in nursery pigs (2-5 weeks after weaning) and grower pigs. Contrastingly, fewer infections occur in suckling pigs and finisher pigs. As a result, infection and disease will first be exhibited in nursery or grower pigs.

Many researchers have studied the resilience of S. suis serotype 2 because of sudden disease outbreaks that occurred in healthy herds.  It has been reported that S. suis serotype 2 is viable in 4 ^ºC in water for 1-2 weeks, in experimentally inoculated feces at 0 ^ºC, 9 ^ºC, and 22–25 ^ºC for 104, 10, and 8 days respectively (F. Clifton-Hadley & Alexander, 1988). In dusty conditions, S. suis serotype 2 bacteria were exposed to the same temperatures as feces. These were viable for 54, 25, and 0 days respectively (F. Clifton-Hadley & Alexander, 1988). In pig carcasses, S. suis serotype 2 survived for 6 weeks at 4 ^ºC and 12 days at 22–25 ^ºC, which allowed for the continued spread of the bacteria (F. Clifton-Hadley & Alexander, 1988). Although resilience in harsh conditions allows the bacteria to thrive and divide, previous research has been shown that tolerance in harsh conditions can potentially serve as an underlying virulence factor. For example another gram-positive bacterial species, Streptococcus pneumoniae, desiccation tolerance has been demonstrated to be an essential virulence factor that is independent of the major cause of virulence, the polysaccharide capsule (Walsh & Camilli, 2011).

Virulence Factors and Pathogenesis

Most studies on virulence factors and pathogenesis have used the serotype 2 variant as a model for S. suis infection. It should be done with caution however, to generalize virulence and avirulence findings of serotype 2 on other serotypes. This is because some virulence factors described in recent literature have been found in both virulent and avirulent variants. As such, there lies a great deal of discrepancy regarding the pathogenesis of S. suis infections and disease. For serotype 2, a CPS locus responsible for the synthesis of CPS has been identified; the inactivation of cps2E and cps2G genes results in impaired capsule production (Zhang et al., 2016).

Biosecurity

Is this specific for S. suis or all infectious diseases?  You just jumped from virulence factors to biosecurity – need to work on the flow/organization as well.

Biosecurity programs usually includes two stages: external biosecurity and internal biosecurity. External biosecurity focuses on limiting pathogen entry into the farm while internal biosecurity concentrates on preventing the spread of pathogens within the farm. Biosecurity in Canada is far more manageable compared to other countries because of a number of fundamental characteristics. Hot and dry summers in Canada usually eliminated most viruses and bacteria while cold winters forced farmers to practice high density confinement animal farming, which reduced parasitism. In Canada, farms tend to be far apart, which reduces the possibility for the spread of disease. Moreover, Canada is also free of major swine pathogens including: cholera, African swine fever, foot and mouth disease (FMD), swine vesicular disease, and pseudorabies. Furthermore, the US acts as a buffer zone to limit the spread of disease from Mexico and the Caribbean by preventing diseases from moving farther north. Most finisher pigs from Canada move south to abattoirs in the US.

Direct Contact Transmission

Diseases are usually transmitted by direct contact between an infected or shedding pig and a susceptible pig; this usually occurs when introducing new breeding stock into a herd. As such, there are two major steps in minimizing or preventing the spread of disease. First, proper records regarding the health and the biosecurity of the breeding must be known. Second, in the case of infection an appropriate quarantine procedure must be in place. Typically, the health of the breeding stock is assessed by veterinarians and is given a score based on factors including but not limited to: location away from farms, locked doors, presence of live animals, wash and disinfection protocols. In addition to this assessment, serology and nasal swabs are performed to determine antibody titers and the presence of pathogens. Producers are also not allowed to use drugs or vaccines while testing that could potentially cover the symptoms of disease. Although these tests allow producers to select breeding stock that best suits their herd, this does not completely eliminate the risk of disease that could derive from a boar or gilt. Breeding stock is not usually transferred immediately to the producer after testing and as a result, lab results may be inaccurate because disease can be acquired during this transitional period. Some pigs can produce false negative results, be asymptomatic to disease, or even carriers or disease. Transporting pigs remains a very sensitive period for susceptible pigs because of the stressed induced by loading and mixing, which could increase the chance of disease transmission. When receiving breeding stock, quarantining and acclimating pigs to the new herd are two essential aspects when reducing the spread of disease. Usually, incoming pigs are isolated or quarantined for a 60-day period; this period is used to detect and control for any new problems associated with the new herd and it allows for the new herd to be acclimatized with the conditions of the existing herd. An alternative approach is to mix the new herd with healthy pigs to observe any signs of disease; this approach limits the interaction between susceptible individuals of the herd with those of the new herd. A closed-herd approach involves the use of artificial insemination, embryo transfer, or Caesarian sections; the benefit of these methods is that it reduces the risk of introducing disease, but diseases and pathogens can still be transmitted. Off-site early weaning is another approach that is normally adopted by smaller systems because it is usually effective at reducing specific diseases. Weaning piglets from the sow at a young age depends on the stability of the herd. If the sow population have high antibody titers to a certain pathogen but do not yield any clinical signs of disease, then it is highly likely that the piglets would be passively protected for about 3 weeks after birth. As a result, weaning at less than 2 weeks of age after birth has been widely successful in preventing diseases from being transferred from the sow to piglets.

Airborne Transmission

Some pathogens travel short distances (< 5 m) via droplets in the air to cause respiratory bacterial infections. Some examples include pleuropneumonia, streptococcal meningitis, atrophic rhinitis, and Glässer’s disease. Other respiratory pathogens can travel far distances ( > 1 km) if blown through the wind. Some examples include footh and mouth disease, pseudorabies, PRRS virus, and influenza virus.

Transmission by birds, rodents, and other animals

It is highly discouraged to introduce animals other than swine to farms because birds, rodents, house pets, and flies are all known to transfer pathogens that affect swine. A pest control program should be in place to frequently monitor for the presence of these animals.

Transmission by trucks, boots, and equipment

Many pathogens can persist for long periods of time in manure and urine and because of that, trucking live animals is one of the most critical points in external biosecurity. Similar to animals, barn supplies and equipment can act as fomites to transfer pathogens from one site to another. As such, it is extremely important to have a protocol in place to effectively clean and quarantine equipment to prevent the spread of disease.

Transmission by people

There have been records detailing the transfer of foot and mouth disease (FMD) and transmissible gastroenteritis (TGEV) from veterinarians and farm workers to other facilities. To reduce the risk for the spread of pathogens, farms should adopt a Danish entry because it acts as barrier between the facility and outside. The Danish entrance require hand-washing, a clean change of clothes and footwear. The effectiveness of showering before entering a swine facility remains uncertain. Usually, veterinarians take 24 hours before entering another swine facility to reduce the risk for the spread of disease.

Monitoring

Once a biosecurity program is in within a herd, the next step is to implement routine monitoring to determine the efficacy of the program. Routine monitoring will help in early diagnosis of disease. The steps for disease monitoring include: visual assessment of clinical disease, assessment of disease using production data, serological monitoring, and feedback from slaughter houses.

 

Vaccination

When deciding whether to vaccinate a herd, there are several factors to consider that could potentially interfere with the effectiveness of the vaccine. One of the most common reasons for vaccine failure comes from the specificity of the vaccine that is being used; the correct bacterial or viral strain must be used for maximum success. Vaccines typically provide little overlap between bacterial or viral strains thus other strains can still result in disease. Another reason for vaccine failure is the timing of vaccine administration. The immunity resulting from vaccines are temporary and thus it is especially important to administer vaccines during periods of greatest risk of infection or susceptibility. For example, colibacillosis is administered before a sow farrows to increase antibody titres in the sow’s colostrum; this provides passive immunity onto suckling pigs. Immunity achieved by vaccines can be optimized with multiple treatments as well allowing at least one to two weeks for the body to develop antibody titres.

With all else considered, there are also financial risks that could deter one from implementing certain vaccines in a vaccination program. For example, the cost of the vaccine, the plausibility of getting the disease, the labour required to administer vaccines, economic loss resulting from disease, and the stress that it could have on the pigs.

Antibiotics You go almost 100 lines without a reference.  The lit review has to be referenced fully.  While there may be some things that are “common knowledge” that don’t need referencing, certainly 3 pages of biocontainment and disease transmission wouldn’t be.

Genetic resistance in pigs

Despite solid breeding practices, genetic screening, and responsible antibiotic use, several pathogens are able to persist and cause disease. Disease usually occurs in low proportions relative to the herd population, but sporadic outbreaks occur that pique concern. In recent years, host genetic resistance has been prompted the topic of research to iron out these outbreaks by selectively breeding individuals that have innate resilience towards the pathogen.

In previous studies, host genetic resistance has been successful in ruling out Salmonella pullorum in poultry and Brucella suis in swine (H. E. H. & G. P. W. Cameron H.S., 1942; L.E., 1926). Studies on host genetics usually involves identification and characterization of candidate genes, microsatellite markers, single-nucleotide polymorphisms (SNPs), and comparative gene mapping. As such, identifying these genes that are essential and impactful for combatting bacterial diseases will allow for practical solutions.

An animal genome is vast and complex, which involves usually involves multiple pathways and mechanisms when in response to a pathogen. And it all begins at the genetic level. As a result, multiple genes, loci, and alleles are usually involved that would account for these significant changes in disease resistance.

In cases where bacterial infections are low relative to the population of the herd, it is important to understand the co-evolutionary relationship between the pathogen and its host. More specifically, each the host and the pathogen evolved from competition into a stalemate, which allowed for the coexistence between the pathogen and the hosts’ immune system (5). This process known as known as natural disease resistance took thousands of years for the domestication of livestock to influence the host-pathogen relationship. Natural disease resistance is the inherent level of resistance towards a disease without prior prompt with a vaccine or exposure to the pathogen (Clyde, 1958). However, the process in which an animal is resistant towards a disease can be related to its immunity and barriers. There have been several studies that suggested natural resistance is related to environmental factors, but a significant portion of it is attributed to genetics and eventually, heritability. A previous study on mycobacteria characterized two steps in forming an immunological response to a disease (Mackaness, 1962, 1964). The first step is the host exposure to the disease followed by an innate and adaptive immune response; this process results in completely ridding the disease. Secondly, these immune responses are meticulously coordinated by the genetics of the host where each gene is responsible at different stages.

General mechanisms of genetic resistance

There are three major stages in host resistance against pathogens. The first is the epithelial barrier that prevents a pathogen from penetration. This phase is critical because some pathogens can adhere to the cell-surface receptors on the barrier, which usually marks the beginning of bacterial infection. The second stage is innate immunity and it is a non-specific resistant mechanism against pathogens. Some general mechanisms include: generating impenetrable host barriers, inability for the pathogen to multiply, lack of receptors for pathogen binding, failure for pathogen to survive in the host, and elimination by host phagocytes. Despite the term non-specific, this resistance mechanism is heavily dependent upon the genetic makeup of the host. The third and last stage is adaptive or acquired immunity that delivers a specific response that involves cytokines, natural killer (NK) cells, macrophages, T cells, and B cells (Wakelin D. & Blackwell J.M., 1988)In both innate and adaptive immunity, there is a specific category of genes that govern the functionality of innate and adaptive immunity; non-major histocompatibility complex (MHC) genes for innate and MHC genes for adaptive immunity. Although there are clear mechanistic differences between both immune responses, many common genes are expressed in aggregation to protect the host from bacterial infection (Buschman E., Schurr E., Gros P, 1990).

 (Swine) Host genes involved in resistance to bacterial pathogens

When considering host resistance against pathogens, many of the pathways that result in a response is controlled by a series of proteins. These structures and functions of these proteins are predetermined by the transcription of messenger RNAs (mRNA) and its corresponding gene. Even at the gene level, there exists an added level of variation with different alleles at each site. As such, changes at any level of gene expression would ultimately change the hosts’ resistance against bacterial infections. Selective pressures have maintained these genes across multiple generations. Given the ease and efficiency in growing mice, many genetic resistance related genes have been identified to be essential against bacterial pathogens. Many of these genes observed in mice are likely to be present in other animals as well. Notably, the microbicidal natural resistance-associated macrophage protein 1 (NRAMP1) gene discovered in mice involved in the resistance against the pathogens: Salmonella typhimurium, and Mycobacterium paratuberculosis has also been mapped in the pig genome. However, more research is needed to determine its functional role (Sun, Wang, Rothschild, & Tuggle, 1998).

There is far more out there on genetic resistance work in swine.  You need to hit the literature hard and review all the current stuff. I would think there would be some references on this in the last 20 years.  Need to be as current as possible.

Genetic resistance conditions – Porcine brucellosis; success in controlling for genetic resistance

When conducting studies regarding genetic resistance, it is important to identify whether a single gene or multiple genes are associated with combatting the disease. In most cases, multiple genes are generally associated with genetic resistance and it is one of many considerations that need to be defined in the study. Another factor to consider is pathogen exposure to the herd. A classic example is Porcine brucellosis in which breeding for genetic resistance resulted in a major increase in herd resistance. In a previous study by Cameron et al. (G. P. W. & H. E. H. Cameron H.S., 1940b, 1940a; H. E. H. & G. P. W. Cameron H.S., 1942), Brucellosis suis genetic resistance was selected for by breeding two sows and a boar; antibodies for B. suis were not previously developed. The resulted offspring were compared against a control group that were andomly selected. When both parties were exposed to B. suis, in a second encounter, 24 of 33 piglets were more resistant to B. suis from the resistant group compared to the more susceptible 3 of 24 piglets from the control group. These results sparked interest in exploring whether other pathogens could be selected against based on resistant groups. Clearly the results showed that by breeding parties resilient to the pathogen, the next generation of offspring developed far more resistance than random breeding.

Mechanisms of genetic resistance

There are three major stages in host resistance against pathogens. The first is the epithelial barrier that prevents a pathogen from penetration. This phase is critical because some pathogens can adhere to the cell-surface receptors on the barrier, which usually marks the beginning of bacterial infection. The second stage is innate immunity and it is a non-specific resistant mechanism against pathogens. Some general mechanisms include: generating impenetrable host barriers, inability for the pathogen to multiply, lack of receptors for pathogen binding, failure for pathogen to survive in the host, and elimination by host phagocytes. Despite the term non-specific, this resistance mechanism is heavily dependent upon the genetic makeup of the host. The third and last stage is adaptive or acquired immunity that delivers a specific response that involves cytokines, natural killer (NK) cells, macrophages, T cells, and B cells (Wakelin D. & Blackwell J.M., 1988)In both innate and adaptive immunity, there is a specific category of genes that govern the functionality of innate and adaptive immunity; non-major histocompatibility complex (MHC) genes for innate and MHC genes for adaptive immunity. Although there are clear mechanistic differences between both immune responses, many common genes are expressed in aggregation to protect the host from bacterial infection (Buschman E., Schurr E., Gros P, 1990).

(Swine) Host genes involved in resistance to bacterial pathogens

When considering host resistance against pathogens, many of the pathways that result in a response is controlled by a series of proteins. These structures and functions of these proteins are predetermined by the transcription of messenger RNAs (mRNA) and its corresponding gene. Even at the gene level, there exists an added level of variation with different alleles at each site. As such, changes at any level of gene expression would ultimately change the hosts’ resistance against bacterial infections. Selective pressures have maintained these genes across multiple generations. Given the ease and efficiency in growing mice, many genetic resistance related genes have been identified to be essential against bacterial pathogens. Many of these genes observed in mice are likely to be present in other animals as well. Notably, the microbicidal natural resistance-associated macrophage protein 1 (NRAMP1) gene discovered in mice involved in the resistance against the pathogens: Salmonella typhimurium, and Mycobacterium paratuberculosis has also been mapped in the pig genome. However, more research is needed to determine its functional role (Sun et al., 1998).

Genetic resistance conditions – Porcine brucellosis; success in controlling for genetic resistance

When conducting studies regarding genetic resistance, it is important to identify whether a single gene or multiple genes are associated with combatting the disease. In most cases, multiple genes are generally associated with genetic resistance and it is one of many considerations that need to be defined in the study. Another factor to consider is pathogen exposure to the herd. A classic example is Porcine brucellosis in which breeding for genetic resistance resulted in a major increase in herd resistance. In a previous study by Cameron et al. (G. P. W. & H. E. H. Cameron H.S., 1940b, 1940a; H. E. H. & G. P. W. Cameron H.S., 1942), Brucellosis suis genetic resistance was selected for by breeding two sows and a boar; antibodies for B. suis were not previously developed. The resulted offspring were compared against a control group that were randomly selected. When both parties were exposed to B. suis, in a second encounter, 24 of 33 piglets were more resistant to B. suis from the resistant group compared to the more susceptible 3 of 24 piglets from the control group. These results sparked interest in exploring whether other pathogens could be selected against based on resistant groups. Clearly the results showed that by breeding parties resilient to the pathogen, the next generation of offspring developed far more resistance than random breeding.

Single Nucleotide Polymorphisms (SNPs) and Disease Resistance

Single nucleotide polymorphisms (SNPs) are the most common type of genetic variation in individuals. Each SNP represents a variation in one of the four nucleotides: Adenine, Cytosine, Thymine, or Guanine, which occurs roughly once in every 300 nucleotides. SNPs usually occur between genes, which could act as biological markers but if a SNP were to occur within a gene, it could alter its function. Since SNPs are located between genes, most SNPs tend to not have any effect. However, some SNPs have been determined to have phenotypic effects. In regard to agriculture and livestock, SNPs are investigated with the goal of discovering SNPs that can influence highly desired production traits such as litter size, growth, disease resistance, carcass weight, leanness, temperament, and health. Previously, animal breeding programs selected for individuals based on breeding values derived from intricate linear mixed models (Russo et al., 2008). There have been a number of studies involving SNPs in swine. For example, SNPs in porcine a protease gene, cathepsin, was associated with economically favourable traits such as growth, carcass, and production traits in Italian Large White pigs (Russo et al., 2008). Another study found SNPs that were critical in influencing litter size in Chinese Erhualian pigs (He et al., 2017). In a study that investigated PRRS challenge in fetal pigs, found 21 candidate genomic regions that were associated with viral load in thymus (VLT), viral load in endometrium (VLE), fetal death (FD), and fetal viability (FV) (T. Yang et al., 2016).

GWAs studies

Genome-wide association studies (GWAs) have recently become the standard to discover new genes related to disease. The fundamental approach with GWAs is to investigate SNPs in individuals that occur with a frequency greater than 1% are responsible for complex genetic diseases or traits such as diseases and disorders or genetic resistance (Cantor, Lange, & Sinsheimer, 2010).

THESIS PROPOSAL

Streptococcus suis is an important pathogen in pigs that is endemic in nearly all countries with a developed pig industry.  It usually causes septicemia, meningitis, endocarditis, arthritis, and other infections (Gottschalk et al., 2007).  S. suis is also zoonotic through cuts and abrasions when handling with infected pig or pork-derived products (Goyette-Desjardins et al., 2014). There have been reports of fatal cases in humans and has had resistance towards penicillin (Palmieri, Varaldo, & Facinelli, 2011). A number of studies have investigated the pathogen virulence factors, regulatory genes, surface-associated or secreted proteins, or metabolic pathways but its virulence remains a highly debated topic (Segura, Fittipaldi, Calzas, & Gottschalk, 2017). Conversely, little is known about host genetic resistance against S. suis in Ontari. There have been however, studies on swine host resistance against E. coli and PRRS (Fu et al., 2012; Lunney & Chen, 2010).

Objectives and Hypotheses

Objective 1: Using a genome-wide approach, determine if genetic variants associated with increased risk of infectious disease in pigs. I hypothesize that there will be genetic variants that occurs more frequently in pigs with infectious disease.

Objective 2: Identify SNPs associated with a broad range of diseases and/or pathogens. I hypothesize that there will be at least one genetic variant common across diseased pigs

Objective 3: Identify genetic variants associated with S. suis. I expect that at least one genetic variant that was common in a broad range of diseases will be present in pigs infected with S. suis.

Experimental Approach

Study design and sample collection – Farms that raised weaning pigs were asked to report symptoms of meningitis. When a report was filed, farms were asked to quarantine piglets. A total of ten different farms reported meningitis across southern Ontario totaling ___ cases. Visits occurred within 24 hours accompanied by a veterinarian to clinically diagnose each weaning pig. It was preferred to draw blood samples via the jugular vein but if it was unsuccessful, retroorbital bleeding was used instead. Healthy control piglets also were selected from the same room as the sick piglets and its blood was drawn using the same method as the sick piglets. All blood was collected using an anticoagulant tube containing EDTA and stored at 4ºC for a maximum of 24 hours.

White blood cell isolation and DNA extraction – Whole blood samples were collected from each weaner pig. The whole blood was centrifuged for ~2000 X g for 10 min at room temperature and the white blood cells (WBC) were recovered. The WBC was then centrifuged again at 2000 X g for 10 min and the plasma was removed. Remaining red blood cells (RBC) were lysed by vortex and then centrifuged for 3000 X g for 10 mins. The WBC sample was then washed using PBS and stored at -20ºC until DNA is ready to be extracted. DNA was extracted using the DNA kit: OMEGA E.Z.NA. Poly-Gel DNA Extraction kit according to the manufacturer’s blood and body fluids protocol.

SNP Genotyping – The DNA was sent to Eurofins Genomics for genotyping ~54,000 variants via a custom SNP array.

Data quality control and data analysis – The resulted CEL files from the custom SNP array were pruned for quality reads by Axiom™ Analysis Suite 2.0 using Best Practices Workflow with threshold settings suggested from the manufacturer. The SNP list was exported to PLINK format. PLINK was used from Anderson et. al. that further pruned SNPs that had an excessive missing data rate and different genotype call rates between cases and controls (data quality control in genetic case-control). The resulted BED will be analyzed using GEMMA (Genome-wide mixed-model association) (Hao, Ye, & Zhao, 2013; Zhou, 2018; Zhou, Carbonetto, & Stephens, 2013; Zhou & Stephens, 2013)

 

Limitations

A major limitation is the sample size as it limits the power to detect real associations in this GWAs study (Ioannidis, 2005). This study was also a case-control association test, which makes it susceptible to Type 1 errors due to population stratification. Thus, it is critical to set an appropriate p-value threshold to minimize Type 1 error and also maximize the power when there are true associations.

GWAs studies are often compared with fishing expeditions and by its very nature have some fundamental limitations. For instance, potential genomic regions, genes, or pathways that have been identified by GWAs need to be further validated with other methods (MacPherson, Otto, & Nuismer, 2018). Candidate genes that have been identified by GWAs studies may heavily depend on the sampled environment, which introduces bias. (Thomas, 2010). The inherent nature of pathogen evolution or host-pathogen coevolution could render the host resistance genes irrelevant or changed over time (Lambrechts, 2010). Because resistance genes are in response to infectious disease, it is likely that identified genes heavily depend on the genetic architecture of the pathogen (Newport & Finan, 2011). Furthermore, certain genetic combinations between the host and pathogen could yield resistance or susceptibility (G x G interactions) (Newport & Finan, 2011).

Future Studies

Most researching findings are false but there are ways to increase the probability that a researching finding is true (Ioannidis, 2005). By replicating this study in different populations, it increases the likelihood that the research findings are true (Moonesinghe, Khoury, & Janssens, 2007). Better causal inferences can be drawn by comparing the most compelling SNPs in this current GWAs study with another population (i.e. S. suis post-mortem or other disease conditions). Consequently, the most significant SNPs can be analyzed further for the biology of these variants and its potential relationship with favourable production traits.