Prevalence of STEC in domestic pigs reared outdoors on diversified small-scale farms from Chapter 1 was lower than this current study, but had a similar sample size . Samples were collected in 2018 for Chapter 3 and 2015-16 for Chapter 1. Differences in STEC prevalence between 2015-2016 and 2018 may be due to different laboratory processing methods or environmental factors. Both study periods were drought years in California; however, 2017 was a very wet year, which may have affected 2018. Three farms participated in both studies and all three farms saw increases in STEC prevalence between 2015-16 and 2018: Farm 1 had a 5.13% STEC prevalence in 2015-16 compared to 20.00% in 2018, Farm 2: 0% STEC prevalence increased to 83.33% and Farm 3: 11.11% to 66.67% . However, a smaller number of samples and animals for Farm 2 and 3 accounts for some of this seemingly large increase between studies. A 2018 study conducted in Georgia reported 62.5% STEC in organic “free ranging” domestic swine, but reported a small sample size of eight. Differences between STEC prevalence may be due to different study designs, laboratory tests, environmental factors or farm management practices, such as the density of pigs raised in each paddock. The scarcity of data regarding STEC in swine raised outdoors indicates a need for future studies. Studies measuring the prevalence of STEC in feral pig populations in the US are infrequent, unlike European studies. A 2006 US study sampled swine necropsy and fecal samples and reported 0 – 23.4% prevalence E. coli O157:H7 in feral pigs. A 2018 study conducted in Georgia detected an overall STEC prevalence of 19.5% in feral swine and they identified a higher prevalence of STEC in feral pigs sampled in agricultural counties.

Feral pigs are attracted to agricultural areas because of resource availability , grow raspberries in a pot and their direct or indirect contact with livestock may create a risk of food borne pathogen transmission. The risk of pathogen sharing between feral pigs and domestic swine has been studied, but only a small subset of these studies investigated the risks to outdoor based pigs, even though there have been multiple cases of feral pigs transmitting pathogens, such as Brucella suis, to domestic swine raised outdoors. Wyckoff et al concluded that increasing populations of feral swine are a risk for the reintroduction of eradicated diseases as well as emerging TBD, especially for backyard operations that allow domestic swine outdoor access, because male feral pigs are attracted to female pens. In a Corsica study that focused on traditional pig farms that raise their animals outdoors, the authors determined that a significant risk factor for the spread of diseases between wild boars and domestic swine was interactions between these two swine groups. Our study results indicated that 45.45% of farm participants had seen evidence of feral pig presence on their farms. Schembri et al conducted a questionnaire of backyard and small-scale swine producers in Australia and found that a third of producers, both indoor and outdoor, had seen feral pigs on their farms. Understanding the prevalence of STEC in feral pigs, combined with the aforementioned study results indicating that these animals reside near resource-rich farms, highlights the need for further studies to address the risk of disease transmission associated with feral pig presence near operations that raise swine outdoors. Serotypes identified in this study that can cause severe human illness included E. coli O157:H7 , O26:H11 and O103:H11 . The serogroups O26:H11 and O103:H11 contained only the stx1 gene, not stx2.

The only O103:H11 serotype contained both eae and ehxA and all the O26:H11 isolates contained the eae gene, with five O26:H11serotypes also containing the ehxA gene. A study by Cha et al also found O26 with stx1 and eae in commercial swine raised indoors in Ohio, US. A study conducted in finishing swine, measured 6.9% of positive samples were O26 and 2.4% contained O103. In 2017, the US Food Safety and Inspection Service conducted a Raw Pork Baseline Study to determine the prevalence of STEC in various types of pork products at slaughterhouses and processing facilities and measured a prevalence of 0.2% STEC, mostly in comminuted pork products. However, this study only looked for the top seven STEC serogroups, even though 309 other samples were positive for key virulence factors like stx and eae genes. Additionally, on-farm or slaughterhouse swine samples may reflect different prevalence ranges than meat products. Considering most studies identified E. coli O157:H7 and non-O157 STEC serotypes that cause human illness in swine samples, pigs should be considered an important reservoir of STEC, and mitigation strategies established to prevent the spread of food borne pathogens from farm to consumer. Significant risk factors associated with the presence of STEC in fecal samples collected during this study included distance from the nearest surface water and whether domestic swine had access to wild areas, such as forest or wetlands. These variables were measured as a proxy for suitable feral pig habitat that borders farms. Feral pigs are reservoirs of STEC, and surface water and/or wild areas provide habitat for these animals to exist near OPO. For instance, a study by Rutten et al predicted suitable habitat for wild boar in Belgium and identified forest , as a significant predictor. Additionally, Wu et al reported distance from a forest to be a significant risk factor for contact with wild boars in Switzerland, especially those domestic pigs less than 500 meters from a forest. A 2017 study reported that distance to water affects feral pigmovement mostly in states where water is scarce versus states where water is more prevalent .

Additionally, feral pigs may contaminate these habitat areas, which may lead to indirect STEC transmission to swine raised outdoors, as studies have shown that STEC can be transmitted through contaminated surface water sources and the environment. A 2014 study conducted in the Central Coast of California detected E. coli O157:H7 and non-O157 in many water sources. These results indicate a need to separate domestic swine raised outdoors from wild areas to avoid direct or indirect transmission of pathogens from feral pigs. In this current study, only the juvenile age group, which included weaners, finishing and market swine , was significant when compared to adults. Many US and international studies have tested similar-aged pigs at slaughterhouses and reported a wide range of STEC prevalence. A study by Tseng et al sampled finishing pigs , which are included in our juvenile category, and determined that the highest prevalence amongst three cohorts occurred between 14-18 weeks of age. At 24 weeks, STEC prevalence in all cohorts had dropped and ranged from 0 – 6.7% in the three groups. This same study mentions that the finishing age group are most susceptible to STEC oedema, which is caused by E. coli strains carrying the stx2 gene and may be associated with detecting STEC in this juvenile age category. A longitudinal study conducted by Cha et al in commercial indoor domestic swine found that 68.3% of finishing pigs shed STEC at least once during the study period, which showcases the intermittent nature of STEC shedding in swine. The high prevalence identified in this study might be due to repeated sampling over a longer period of time than conducted in our study. Additionally, our study sampled all ages of swine only once, best grow pots which might indicate an under reporting of STEC in our results. The effect of age on STEC shedding is more frequently reported in cattle versus swine. For instance, a Raies et al study sampled beef cattle and reported that STEC prevalence was highest during the first six months of life and then decreased toward adulthood. Another study by Cho et al also detected that calves over one month old were two times more likely to shed STEC than those younger than one month, except for pre-weaned calves. If age is a risk factor for STEC shedding in swine, then targeting key age groups for STEC mitigation strategies to reduce the overall bacterial load in slaughtered swine may reduce the risk of these pathogens in the food supply. Limitations of this study included a small sample size for the total number of farm participants as well as the final number of feral pig samples collected, as we could only gather feral pig feces in three of the six targeted counties. The post-hoc power calculation results were 0.12 for feral pigs and 0.69 for OPO, which indicated that the prevalence estimates are inexact. Moreover, many of the significant variables in the final logistic regression model had wide confidence intervals, which indicates less precise estimates.

Since this was a cross-sectional study conducted only during two seasons and only one season per farm, we may have missed STEC positive farms due to seasonality of shedding or other factors that affect STEC detection in feces, including the intermittent nature of shedding in pigs. Our study participants volunteered and therefore we could not conduct random sampling; our study results contain selection bias and are not generalizable to other OPO in California or the US. Strengths of our study included measuring STEC in both feral pig and outdoor reared pigs in California. This study is an innovative approach toward evaluating areas of contact between feral and domestic pigs reared outdoors, by targeting STEC surveillance based on a risk map built in Chapter 2. Moreover, assessing STEC prevalence in feral pigs near OPO serves as a proxy for the risk of exposure and transmission of other zoonotic pathogens to domestic pigs reared outdoors. Future research studies could enhance our current study results by comparing STEC strains between the two swine groups using WGS bio-informatic analyses. Similarity of STEC isolates can be used as a biological indicator to track possible transmission of diseases between feral and outdoor-raised swine, as noted in a few recent studies.The three scientific research projects in this dissertation added important epidemiological information to the body of knowledge regarding STEC detected on DSSF in California and the risk of potential disease transmission from suitable feral pig habitat located near domestic pigs raised outdoors. Although consumers perceive small-scale farms or outdoor-raised meat as safer and more natural, these studies together demonstrate that even livestock raised outdoors on small-scale farms are reservoirs for STEC, including serogroups that cause severe illness in humans, including O157:H7, O26, O103 and O111. Interestingly, Chapter 1 and 3 models indicated that livestock raised outdoors that have access to wild areas, such as wetlands or forest, was a key risk factor for the presence of STEC. Chapter 2 results revealed that nearly 50% of domestic pigs raised outdoors are located near suitable feral pig habitat, and this overlap of feral and domestic swine could be a risk factor for potential emerging or reemerging disease transmission. Also, STEC was detected in domestic swine in both Chapter 1 and 3, even though pigs are currently considered a low risk key species for STEC outbreaks by the US FSIS. These study results indicate the need for further studies on DSSF to ascertain risk factors for food borne pathogens. The objective of Chapter 1 entailed conducting an overall assessment of prevalence and risk factors of STEC on diversified small-scale farms in California, while also describing the unique characteristics of DSSF. Temperature was a key risk factor identified in the final multilevel logistic regression model. Many food borne pathogen studies indicate season as a risk factor for STEC, however, seasons vary across the US. For instance, California summers are characterized by dry heat whereas summers in most states are humid and hot. Measuring and monitoring temperature during field sampling may be a more precise indicator of risk than season and allow for more accurate comparisons between studies. Additionally, as weather patterns shift due to climate change, assessing environmental factors, , as a risk factor for the presence of STEC on farms will be useful for stakeholders, to understand how weather affects the presence of food borne pathogens in livestock raised on DSSF. Studies elucidating whether ambient temperature affects survival of STEC in a farm environment or whether temperature affects the host animal harboring STEC will be useful, especially as extreme climate events become more common.