Detection of Brucella suis,Campylobacter jejuni,and Escherichia coli Strains in Feral Pig ( Sus scrofa ) Communities of Georgia

Abstract

Feral pigs (Sus scrofa) are an environmentally destructive invasive species that act as a reservoir for zoonotic pathogens. The aim of this study was to determine the presence of Brucella suis, Campylobacter jejuni, and of Escherichia coli in feces of feral pigs from Georgia. Fecal samples were collected from 87 feral pigs from forested and agricultural regions of Georgia. DNA was extracted from the fecal samples and quantitative PCR (qPCR) was used to screen for each of the four pathogens. The qPCR assays indicated that B. suis and eaeA-containing strains of E. coli was present in about 22% and 28% of the samples, respectively. C. jejuni was undetected in any of the feral pig fecal samples. The incidence of B. suis was higher in the pigs from forested region, whereas E. coli strains possessing eaeA gene incidence was higher in the pigs from agricultural regions. In Georgia, feral pigs harbor infectious agents and are a growing threat to the transmission of pathogens to native wildlife, humans, and food crops.

Introduction

Feral pigs (Sus scrofa) were introduced in the United States in the 1500s and their range has been expanding ever since. Today, their population is estimated to be over 5 million and is considered as one of the most widespread and destructive invasive mammals in the country (Pimental 2007). The annual damage caused by feral pigs is estimated to be billions of dollars, including $800 million damage to agricultural crops (Ferretti et al. 2015, Schlichting et al. 2015). These destructive animals harbor a wide range of zoonotic pathogens, feast on field crops (e.g., corn, soy beans, rice, watermelon, peanuts, and wheat), and have a tendency to prey upon young livestock and other small animals (USDA APHIS 2005).

Studies have shown that feral pigs can carry over 65 disease-causing agents that affect livestock and humans (USDA APHIS 2005, Meng et al. 2009). Their activities of wallowing and defecating can contaminate soil and water resources with pathogens, including bacteria, viruses, parasites, etc. and spread diseases to other wild animals, domestic swine, and humans (Meng et al. 2009). Feral pigs are reported to live in all of Georgia's 159 counties. It is likely that the number of wild pigs in Georgia at this time exceeds 200,000 and is increasing at an alarming rate (USDA APHIS 2005). Therefore, these free-living swine and their ability to harbor infectious agents that are transmissible to humans and animal population is becoming a growing public health concern (Meng et al. 2009, Paredes et al. 2017).

The prevalence of zoonotic pathogens carried by feral pigs and their potential to transmit pathogens to humans and domesticated animals in Georgia is not well studied. Some of the most economically important and widespread bacterial pathogens carried by feral pigs in the United States include Brucella suis, Campylobacter jejuni, and virulent strains of Escherichia coli (e.g., E. coli O157:H7), which can be transmitted by direct or indirect contact with feral pigs to farm animals and humans (USDA APHIS 2005, Meng et al. 2009). In recent years, PCR technology has allowed researchers to detect the presence of B. suis, C. jejuni, and virulent strains of E. coli in fecal samples (Chaban et al. 2009, Winchell et al. 2010, Bachoon et al. 2012).

B. suis is a Gram-negative coccobacillus that is related with most cases of swine brucellosis, a zoonotic disease that can induce failure of reproductive organs and consequently sterility in animals (Winchell et al. 2010, Ridoutt et al. 2014). Brucellosis has been eliminated from domestic swine herds in the United States (USDA APHIS 2005, Meng et al. 2009). The main reservoir for B. suis is feral pigs and this agent can be easily transmitted to domestic pigs or humans from direct contact with infected animals or inhalation of aerosol droplets (Pappas et al. 2005). Campylobacter is a helical-shaped, nonspore-forming, Gram-negative, microaerophilic bacteria found in animal feces and is one of the most common causal pathogens of gastrointestinal disorders (Keramas et al. 2004). Pigs seem to be a natural reservoir of Campylobacter spp. with prevalence between 50% and 100% (Jensen et al. 2006). C. jejuni is the dominant cause of human cases of campylobacteriosis and may constitute a majority (up to 87%) of the Campylobacter spp. detected on hog farms (Nielsen et al. 1996, Harvey et al. 1999). Feral pigs, cattle, and other ruminants are natural reservoirs of virulent strains of E. coli, including E. coli O157:H7 that can introduce pathogenic E. coli into the environment through fecal shedding (Booher et al. 2002, Jay et al. 2007). For example, in 2006, a large outbreak of E. coli O157:H7 in California was attributed to contamination of spinach with cattle manure along with feces of feral pigs (Jay et al. 2007). The aim of this study was to determine the tendency of fecal samples of feral pigs in Georgia to contain B. suis, C. jejuni, and potentially virulent strains of E. coli.

Materials and Methods

Sample collection

Fecal samples from corral-trapped feral pigs were provided by the United States Department of Agriculture–Animal and Plant Health Inspection Service (USDA-APHIS), Athens, GA, and by JAGER PRO™, LLC, Hog Control Systems (commercial hog hunting company, Fortson, GA). The fecal samples were collected from March 2015 to August 2016 in 13 Georgia counties (Table 2) as part of ongoing feral pig eradication operations. Eighty-seven fecal samples from 29 males, 23 females, and 35 unknowns of weight ranging from 30 to 180 lbs. were collected. The collected samples were stored at −20°C and transported on ice to the laboratory for analysis. In addition, fecal samples were collected from dairy cattle from Putnam County, organic farm-raised pigs (Berkshire, Mulefoot, and Ossabaw) in Newton County, and human (sewage) from Baldwin County.

DNA extraction and quantitative PCR analysis

DNA was extracted from about 0.25 g of fecal samples using the MO BIO PowerSoil DNA Isolation Kit (Carlsbad, CA) following the manufacturer's protocol. Extracted DNA was then quantified using a NanoDrop, ND-1000 Spectrophotometer (Wilmington, DE) and stored at −20°C until further analysis. Quantitative PCR (qPCR) assays were conducted in a Bio-Rad Thermal Cycler (CFX96, Hercules, CA) for the detection of transposase, cpn60, and eaeA genes from B. suis, C. jejuni, and virulent E. coli strains, respectively. Positive controls used for PCR assays were B. suis 1330 bv1 (CDC, Atlanta, GA), C. jejuni (MK7 ATCC^® 43432D-5™), and E. coli O157:H7 (genomic DNA from the European Commission Institute for Reference Materials and Measurements IRMM-449). The PCR assay for each gene was optimized by running an annealing temperature gradient for each primer pair with alternating positive (target bacteria), negative (remaining three groups), and no-template controls. The PCR assay for each sample was performed in duplicates, a total volume of the PCR mixtures was 25 μL, which contained 2× SsoFast™ EvaGreen^® Supermix (Bio-Rad, Hercules, CA), 25 mM of magnesium chloride, 200 nM of primer (Table 1), and 1 μL of template DNA. In the analysis of Yersinia enterocolitica, TaqMan assay was used, which comprised a total volume of 20 μL PCR mixture: 2 × TaqMan Probe PCR master mix (Qiagen, Valencia, CA), 500 μM primers, and 100 μM probe. The PCR primer set for each target bacteria and their associated annealing temperatures are listed in Table 1. B. suis transposase gene consisted qPCR conditions of 1 cycle of 50°C for 2 min and 1 cycle of 95°C for 10 min, followed by 40 cycles of 95°C for 5 s and annealing temperature for 30 s (Winchell et al. 2010). For the C. jejuni cpn60 chaperonin gene, the qPCR conditions were initial denaturation at 95°C for 3 min followed by 40 cycles of 95°C for 15 s and annealing temperature for 15 s and at 72°C for 15 s (Chaban et al. 2009). For E. coli O157:H7, eaeA gene, PCR conditions were initial denaturation at 98°C for 50 s followed by 25 cycles of 95°C for 15 s and annealing temperature for 20 s followed by 72°C for 30 s and 75°C for 1 s (Guion et al. 2008). Comparisons of melt curve peaks and Cq cycles to the positive control for each bacterium were used to indicate the presence of a specific pathogen in EvaGreen-based and probe-based assays, respectively. The lower limit of each assay was determined using serial dilutions of positive control DNA and was between 10 and 100 fg (Winchell et al. 2010). Fecal DNA extracts were evaluated for the presence of PCR inhibitors by evaluating shifts in Ct-values between a sample and its 10-fold diluent (Dick et al. 2010).

Table 1.

Primers Used for Polymerase Chain Reaction Detection of Brucella suis, Campylobacter jejuni, and Escherichia coli

Target organism	Target gene	Annealing temp. (°C)	Amplicon size (bp)	Primer sequence	Reference
Brucella suis	transposase	60.8	163	F-TGCGCTATGATCTGGTTAGTT	Winchell et al. (2010)
Brucella suis	transposase	60.8	163	R-AGCGCGGTTTTCTGAAGGT	Winchell et al. (2010)
Campylobacter jejuni	cpn60	59	174	F-GAGCTTTCAAGCCCTTATATCC	Chaban et al. (2009)
Campylobacter jejuni	cpn60	59	174	R-AAGAACACCGCGAAGTTTATTT	Chaban et al. (2009)
Escherichia coli	eaeA	59	248	F-ATGCTTAGTGCTGGTTTAGG	Guion et al. (2008)
Escherichia coli	eaeA	59	248	R-GCCTTCATCATTTCGCTTTC	Guion et al. (2008)

Results

Sample collection

Feral pigs are the most abundant free-roaming and destructive ungulates in the state of Georgia. In recent years, federal (USDA-APHIS), state (Georgia EPD) agencies, and contracted hunters (e.g., Jager Pro LLC) have been trying to eradicate or manage the feral pig population in Georgia. These agencies often use baited corral traps in their efforts to capture and remove animals from an area. We were fortunate that these groups were able to supply us with fecal samples of trapped pigs from 13 counties (Fig. 1). However, it was sometimes difficult to collect more than one or two fecal samples from some counties. The majority of the fecal samples came from southwestern counties: Calhoun (15), Dooly (18), Sumter (8), Terrell (8), and Morgan (19) county in north Georgia (Fig. 1). The southwestern counties were dominated by row crop farmlands (cotton, corn, peanuts, etc.) and the samples from Morgan County were from a forested area (mixed hardwood/pine forest). For comparisons, fecal samples from human (sewage samples), free-range farm pigs in Newton County, and dairy cattle in Putnam County were analyzed.

FIG. 1.

Georgia counties from which fecal samples of feral pigs were collected, number of samples.

DNA extraction and qPCR

Fecal DNA extracts from 5 to 15 ng/μL were recovered from 0.25 g of samples using the MO BIO PowerSoil DNA Isolation Kit (Carlsbad, CA). To establish the optimal conditions for each qPCR assay, a series of reactions were performed with each primer set using the assay's corresponding positive-control bacterial DNA (5 ng) and a panel of DNA from the other three pathogenic bacterial groups as negative controls. For comparison, fecal DNA were collected from human (sewage samples), free-range farm pigs in Newton County, and dairy cattle in Putnam County. Although all primers and qPCR assays listed in Table 1 were previously optimized to detect their target bacteria, we revalidated each qPCR assay for their specificity on our thermocycler and reaction mixtures. The optimal annealing temperatures for each assay was determined by running a gradient PCR and selecting the highest temperature that generated significant specific product, as determined from their respective melt curve (Table 1). In general, any sample with qPCR Ct-value greater than 37 cycles were considered to be negative.

The qPCR assay for B. suis (10) indicated that the sewage, cattle, and domesticated pigs were free of B. suis, but 52% of the feral pig sample from Morgan County had detectable levels of B. suis (Table 2). Overall, B. suis was detected in 21.84% ± 1.3% of the total feral pigs studied (Table 2). qPCR of the eaeA gene indicated that one of the five cattle fecal samples and many of the domesticated pigs (5 of 8) were harboring virulent E. col strains (Table 2). The majority of the feral pig samples that carried E. coli containing the eaeA gene came from Morgan, Dooly, and Sumter Counties. Overall, there was a higher prevalence of virulent E. coli in feral pigs from the southeastern counties of Georgia (Fig. 1). C. jejuni marker genes were not detected in the feral pigs' fecal samples through qPCR analysis (Table 2).

Table 2.

Quantitative Polymerase Chain Reaction Detection and Percentage of Brucella suis and Escherichia coli in Fecal Samples of Domestic Pigs, Dairy Cattle, Human, and Feral Pigs in Georgia

Organism	County (sample no.)	Brucella suis, n (%) ^a	Escherichia coli, n (%) ^a
Domestic pigs	Newton (8)	0	5 (7.45)
Dairy cattle	Putnam (5)	0	1 (1.1)
Human	Baldwin (10)	1 (1.1)	1 (1.1)
Feral pigs	Bleckley (1)	0	0
Feral pigs	Burke (2)	2 (2.29)	0
Feral pigs	Calhoun (15)	4 (4.59)	3 (4.47)
Feral pigs	Chatham (4)	0	0
Feral pigs	Dooly (18)	0	3 (4.47)
Feral pigs	Elbert (5)	0	0
Feral pigs	Morgan (19)	10 (14.90)	6 (8.94)
Feral pigs	Oglethorpe (1)	0	0
Feral pigs	Randolf (3)	0	1 (1.1)
Feral pigs	Sumter (8)	1 (1.1)	3 (4.47)
Feral pigs	Terrell (8)	0	1 (1.1)
Feral pigs	Washington (2)	1 (1.1)	0
Feral pigs	Wilkinson (1)	0	0
Total feral pigs (% ± SD)	87	19 (21.84 ± 1.3%)^b	24 (27.58 ± 2.9%)^b

Values within column represent number of positive samples (% of total pig samples).

Values at the end of each column represents total number of positive samples (% positive ± standard deviation).

Discussion

Previous studies relied on using blood or tissue samples of animals for the isolation of B. suis, C. jejuni, and virulent E. coli followed by time-consuming biochemical, antigenic, and metabolic profiling for identification of these pathogens (Chaban et al. 2009, Winchell et al. 2010). The groups involved in the trapping of the feral pigs were able to provide us with fecal samples, but we were not able to collect blood or tissue samples from the pigs. However, recent molecular approaches, such as PCR, have greatly enhanced the ability to rapidly detect many zoonotic pathogens carried by feral pigs from fecal shedding (Winchell et al. 2010, Leiser et al. 2013). Therefore, the fecal DNA samples collected in this study were used for detection of B. suis, C. jejuni, and E. coli strains.

The cattle and swine commercial production in the United States is currently free of brucellosis (The Center for Food Security & Public Health [CFPH], 2007). The primary route of transmission of B. suis is venereal, but it can be transmitted through infected milk or exposure to infected tissue (CFPH 2007). The qPCR assay for B. suis indicated that the sewage, cattle, and domesticated pigs were free of B. suis. However, feral pigs in forested and agricultural areas from five counties harbored B. suis, which was 21.84% of the total feral pigs studied (Table 2). The findings agree with Pedersen et al. (2012) who reported that up to 14% of feral swine in the United States have been exposed to brucellosis, depending on the state. In contrast, serological testing indicated that there was a 1.7% prevalence of swine brucellosis detected in feral pigs from Glynn, Chatham, and Oglethorpe counties (Pedersen et al. 2012). The increased amount of B. suis detected in our study was attributed to small sample size, the detection methods, geographical differences in the study sites, and the fact that incidence brucellosis often occurs in clusters of feral pig populations (Pedersen et al. 2012). Therefore, it is not irrational to find some regions of Georgia with higher incidence of B. suis. For example, 52% of the feral pig samples from forested regions of Morgan County had detectable levels of B. suis (Table 2). Consequently, high prevalence of B. suis in some counties of Georgia represents a growing threat to humans (e.g., hunters), domesticated cattle, and pigs (Gaskamp et al. 2016).

Feral pigs and other ruminants that are harboring E. coli can shed this bacterium into fields and surface water for several months (Booher et al. 2002). Although cattle are the major reservoir of virulent strains of E. coli (e.g., E. coli O157:H7) in the United States, in some countries such as Chile the prevalence of E. coli O157:H7 in pigs (10.8%) was greater than that reported from cattle (2.9%) (Cornick and Helgerson 2004). Previous studies have reported the presence of E. coli O157:H7 in water samples from the cattle farming regions of middle Georgia (Bachoon et al. 2012). qPCR of the eaeA gene that is often associated with virulent strains of this bacterium indicated that one of the five cattle fecal samples and many of the domesticated pigs (five of eight) were harboring E. coli strains (Table 2). The high incidence of eaeA E. coli strains in farm-raised pigs was attributed to their management practices; raised in a small fenced area, sharing the same food and water, and the high density of animals in each enclosure. In contrast, ∼28% of the feral pigs' fecal samples had detectable levels of E. coli containing the eaeA gene (Table 2). The majority of the feral pig samples that carried eaeA gene came from Morgan, Dooly, and Sumter Counties. Overall, there was a higher prevalence of virulent E. coli strains in feral pigs from the agricultural southwestern counties of Georgia (Fig. 1 and Table 2). This is alarming because food crop (corn, soy, tomatoes, etc.) is a major part of the economy in Morgan, Dooly, and Sumter counties and feral pigs can contaminate these crops with potentially virulent E. coli strains. Most cases of E. coli O157:H7 illness in humans in the United States occurs from ingestion of contaminated food or water (Cornick and Helgerson 2004, Jay et al. 2007).

C. jejuni was not detected in any of the feral pigs' fecal samples. Studies found that C. jejuni may constitute a majority (up to 87%) of the Campylobacter spp. detected on hog farms (Harvey et al. 1999). However, Jensen et al. (2006) reported that there was only a 10% incidence of C. jejuni in free-range pigs. Furthermore, it was observed that the prevalence of C. jejuni was only 0.8% (2/243) of the feral pigs sampled in the United States (Research, Education, and Economics Information System [REEIS], 2014). Therefore, our failure to detect C. jejuni in this study was attributed to the sample size (87 pigs) and the low prevalence of C. jejuni harbored by feral pigs in the United States. It has been suggested that the lower animal density probably reduces the infection pressure and roughage stimulates the intestinal flora, which is likely to reduce the susceptibility of pigs to C. jejuni infections (Mikkelsen et al. 2004).

Conclusion

The rapidly growing feral pig populations in the State of Georgia and throughout the United States can increase the dissemination of zoonotic pathogens to humans and farm animals. Feral pigs in Georgia can act as reservoirs of B. suis and E. coli and can potentially increase the spread of disease through fecal contamination of food crops, water, and transmission of disease to livestock. As the feral pig population continues to grow in the United States, more studies are required to understand the risk of pathogen transmission from feral pigs to humans and animals.

Footnotes

Acknowledgments

This work was supported by the USDA National Institute of Food and Agriculture (NIFA), Agriculture and Food Research Initiative Competitive [grant no. 2015-680007-23137]. The authors appreciate Matthew Ondovchik (USDA-APHIS, Athens, GA) and Rod Pinkston (JAGER PRO™, LLC, Fortson, GA) for help with the sample collection. They also thank the laboratory of Dr. Alex Hoffmaster (CDC) and Rebekah Tiller (CDC) for providing positive DNA control of Brucella suis as well as Marirosa Molina (USEPA, Athens, GA) for Campylobacter jejuni control contribution.

Author Disclosure Statement

No conflicting financial interests exist.

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