A study that screened livestock at the California state fair in 2005, which usually hosts livestock raised on small farms or in backyards, observed a 3% prevalence of E. coli O157:H7 in pigs, but did not find O157:H7 in any other livestock samples including dairy cows, whereas our study identified O157:H7 in cattle but not pigs. A 2002 study that also collected fecal samples at fairs in three states, identified an E. coli O157:H7 prevalence of 11.4% in cattle, 1.2% in swine and 3.6% in sheep and goats, whereas we measured a 5.31% E. coli O157:H7 in all cattle . Differing STEC prevalence in these aforementioned studies may reflect different management practices on farms or other climate or animal-level factors. Additionally, since ruminants are the main reservoirs for STEC, our results indicating that STEC prevalence in swine is lower comparatively than the other sampled species is in agreement with previous research, however, pigs are still a livestock species of public health concern, as they harbor E. coli O157:H7 as indicated by many studies. Our model results also indicated that cattle and sheep are a significant factor in STEC presence on farms, as compared to goats and pigs. However, differences in location, laboratory methods and sampling methods make comparison between studies challenging. More than half of the identified O-serogroups in this study are on CDC’s list of the top 7 STEC of concern for public health, including six O157:H7, twenty-two O26, nine O103 and one O111. Stx2, blueberry production which is the more virulent form of the Shiga toxin gene that has been implicated in severe human disease, was identified in 16.46% of O-serogroups; 13.92% contained both stx1 and stx2.
The eaeA gene, which allows STEC bacteria to attach to human host cells, was detected in 55.69% of positive STEC samples, contrary to a study conducted by Dewbury et al, which rarely discovered eaeA in their non-O157 isolates from cattle fecal samples. The ehxA gene, which is reported in severe human cases of STEC, was detected in 88.61% of the positive isolates . Compared to a study conducted by Djordjevic et al in adult sheep and lambs, they detected stx1, stx2 and ehxA in 78.2% of their positive serogroups, versus our study which only identified those three genes in 1.27% of positive serogroups. However, they reported 0.8% of their serogroups had just stx2 and ehxA genes, whereas in this current study, 11.39% of the positive isolates contained these two virulence genes. The pathogenic STEC O-serogroups, genes and virulence factors identified in this study highlight the need for continued studies on DSSF, as well as outreach to stakeholders regarding pre-harvest food safety risks and development of onfarm mitigation strategies. Significant risk factors identified by the final mixed effect model included daily maximum temperature °C. The data in our study ranged from 11.7°C – 39.80°C. An experiment that measured the decline of E. coli O157:H7 in inoculated manure at four temperatures, 7°C, 16°C, 23°C and 33°C, reported that E. coli O157:H7 declined significantly faster in manure at 23°C and 33°C, than at 7°C and 16°C, for both oscillating and constant temperatures. This study confirms our model result, which suggested that as the daily maximum temperature increased, the odds of finding STEC in a fecal sample was less likely. A study by Franklin et al also identified daily maximum temperature as a significant risk factor, when conducting a study of STEC in wild ungulates in Colorado.
They detected a positive association between temperature and STEC presence in fecal samples, whereas our model identified a negative association with the daily maximum temperature. However, the range of daily maximum temperatures displayed in their analysis were narrower than our recorded daily maximum temperatures, which may account for this difference. Although many studies indicate that STEC sheds more in summer months, California microclimates differ from each other and from the majority of seasons in other states. California valleys and foothills experience low humidity and temperatures above 37.78°C in the summer and autumn, which may affect STEC shedding from livestock raised on California farms located in different microclimates. For instance, to compensate for the numerous microclimates in California in our study on Campylobacter spp., which included the same farms included in this current STEC study, we divided the California summer season into Coastal and Inland and season was a significant risk factor in that final multilevel logistic regression model. Interestingly, our Campylobacter study also found a significant association between presence of Campylobacter spp. and a farm owning swine, with 13.76% prevalence of Campylobacter spp. measured in pigs raised outdoors. Difference in climate conditions between states in the US reveal a need to report the full range of temperatures and other environmental factors measured for studies estimating the effect of weather on food borne pathogen shedding in livestock. For instance, a study that collected samples from conventional dairy and beef cattle in Michigan revealed that high average temperatures measured one to five days before sampling had a 2.5 times greater odds of STEC than lower temperatures, which differs from our study results that suggested that STEC survival is less likely at higher maximum temperatures.
Michigan results contradict ours, however the highest maximum daily temperature measured in our study is not a temperature normally observed in many areas of the US. The range of daily maximum temperatures for the Michigan study was 22.78 – 32.2°C, with one 36.11°C outlier. Additionally, our study included winter temperatures, while their study was only conducted in summer . Extreme temperature, heat index or humidity values observed in different parts of the world may affect conclusions and interpretations of results, especially between studies. Stanford et al reported the effects of severe weather events on STEC shedding in Canadian cattle. Although they also observed that STEC prevalence increased when ambient temperatures were higher than 28.9°C, a separate finding indicated that the O-serogroup O26 had a significant reduction in prevalence during extreme heat in July and August. Almost 28% of the O-serogroups in our study were O26, and the final model results may have been influenced by this strain. The ways that different non-O157 STEC strains react to varying environmental conditions, such as temperature or humidity, may account for variations in results between studies. Moreover, changes in the host species during various temperature fluctuations or extreme weather events should also be studied. For instance, Dawson et al measured behavioral changes in cattle during increased temperatures, as a possible driver of changes in STEC prevalence, such as increased water consumption or change in grazing habits. Their simulation results indicated that higher summer temperatures may encourage more resting by cattle in crowded areas, such as under shade trees, which can lead to direct transmission of STEC. Since the aforementioned studies differ in conclusions regarding the direction of environmental effects on STEC shedding in livestock, this risk factor needs further investigation, as perhaps there are underlying mechanisms accounting for the difference between results, including microclimates or animal level factors. Our multi-variable model also indicated that livestock sharing a barn with other animals resulted in 3.5 greater odds of a positive STEC sample. Multiple livestock housed in a barn could share pathogens by cross-contamination of food or water troughs or persistence of STEC in a barn environment that may not be subjected to regular cleaning. Other studies have indicated that STEC persists for long periods of time in barns or on surfaces within the farm environment. For instance, blueberry in container one study swabbed multiple barn surfaces at a dairy ranch and measured 14.9% – 19.1% STEC in samples from cattle or calf feeders, and 11.3% – 18% on other surfaces. Another study implicated water troughs as harboring E. coli O157:H7, and inferred that shared water troughs play a key role in the persistence and maintenance of continued E. coli O157:H7 infections in cattle. A British study reported that housed beef cattle shed more STEC than unhoused and suggested that this may be due to shared water sources or feeding bins and an accumulation of pathogens in a shared environment. Finally, the last significant risk factor from the multi-variable final model indicated that livestock in contact with wild areas, such as forests or wetlands, have a higher likelihood of STEC presence in their feces. Wildlife, including feral pigs, deer, rodents and birds are known reservoirs of STEC.
A study conducted in California identified a low prevalence of E. coli O157:H7 in rodents , however, they did not test for non-O157 STEC in samples, which may have a higher prevalence in rodents. A 2016 published study discovered the stx2 gene in over twenty percent of Canada geese fecal samples and seven percent of nearby water samples from Lake Eric bordering Ohio, USA. A case-control study conducted after 15 human cases of E. coli O157:H7, identified the source of STEC as those who ate fresh strawberries contaminated by deer feces. Livestock that graze in wild areas may be exposed to indirect sources of STEC, for instance through environmental contamination of soil or water, or because wildlife that live in these bordering wild areas enter agricultural areas and contaminate the pastures grazed by farm animals. Limitations of this study include the small sample size of farms that were convenience sampled, so the model results are not generalizable to other regions and farms. Moreover, because we collected the freshest fecal samples available and did not randomize sample collection, we may have added bias to the study results. Unmeasured variables that should be included in future studies include the age of the animal and whether livestock have direct or indirect contact with neighboring livestock. Although a majority of commercial swine production in the United States occurs indoors with high levels of biosecurity, the US is currently experiencing a return to raising domestic pigs outdoors. Before the 1950s, most swine operations in the US were small scale family farms and either a hybrid of indoor/outdoor or solely outdoor-based. Beginning in the 1960s, commercial swine production began transitioning to indoor systems, based on goals to increase efficiency and reduce swine disease transmission as well as a public health mandate to decrease human trichinosis cases.However, consumer demand for sustainable or pasture-raised animal products within the past few decades has revived traditional methods of raising swine outdoors or on pasture . While primarily considered a niche production method in the US, outdoor-raised pig operations are broadly distributed throughout California. A challenge in raising pigs outdoors is the possibility of these animals interacting with wildlife disease reservoirs, such as feral pigs, and the associated risk of zoonotic and/or swine pathogen transmission Both domestic and feral pigs share the same genus and species and can be reservoirs for zoonotic pathogens ,Also, swine diseases eradicated in conventional indoor-raised herds have been documented in feral swine in California and contact between feral pigs and outdoor-raised swine herds is a risk factor for the reintroduction of these diseases to domestic herds in the US. For example, a 2016 human case of brucellosis in New York state was traced to a feral pig intrusion event on a pasture-raised pig farm. Brucella suis was then transmitted to domestic pigs raised outdoors in 13 other states through animal sales. Feral pigs could also play a significant role in the transmission and maintenance of transboundary animal diseases introduced to North America., For instance, African Swine Fever is actively spreading in eastern Europe, with wild boars transmitting this devastating disease between and within countries. Similarly, wild boars abet the transmission of ASF in South Korea, spreading the virus to outdoor-raised swine. And most recently, ASF was identified in domestic swine in the Dominican Republic, which is the closest to the US that ASF as spread in this century. During the past few decades, feral pig populations have greatly increased in the US from 17 to 41 states. California has one of the largest and widest geographic distributions of feral pigs and this invasive species has the broadest habitat range of any large mammal except humans, which is in part due to their ability to adapt to a diverse range of ecological habitats and their opportunistic omnivore diet. Feral pig population distribution and abundance is dynamic yet has not been documented at fine spatial units. Previous presence maps reported feral pigs for an entire county, even if there had only been a single occurrence recorded countywide.