Salmonella Prevalence and Antimicrobial Susceptibility Among Dairy Farm Environmental Samples Collected in Texas

Abstract

Dairy cattle are a reservoir of several Salmonella serovars that are leading causes of human salmonellosis. The objectives of this study were to estimate the environmental prevalence of Salmonella on dairy farms in Texas and to characterize the antimicrobial susceptibility of the isolates. Eleven dairy farms throughout Texas were sampled from August through October 2013, using a cross-sectional approach. Samples were collected from four locations within each farm (hospital pen, maternity pen, cow housing area, and calf housing area), and feces were collected from cull cows as available. Environmental and fecal samples were processed for Salmonella, and isolates were tested for susceptibility to 15 antimicrobial agents. Serovar characterization was performed on a subset of these isolates. Salmonella was isolated from 67.0% (236/352) of the environmental samples and 64.2% (43/67) of the cull cow fecal samples. Environmental samples from the maternity pen were significantly more likely to be Salmonella positive than samples from the cow and calf housing areas. Multidrug resistance was evident in 11.9% (27/226) of environmental isolates and 19.5% (8/41) of fecal isolates. Salmonella isolates from the calf housing area and maternity pen were significantly more likely to be multidrug resistant (MDR) than isolates from the cow housing area. The most common serovars found among the MDR isolates were Newport, Muenchen, and Typhimurium. These results help provide a focus for efforts to mitigate the burden of antimicrobial-resistant Salmonella at the preharvest level.

Introduction

S almonella enterica poses an important threat to public health, causing an estimated 1.2 million illnesses, 20,000 hospitalizations, and 400 deaths annually in the United States (Scallan et al., 2011). Efforts to reduce the incidence of human salmonellosis have been minimally successful over the last 15 years (CDC, 2013; Crim et al., 2014). Although primarily a cause of self-limiting acute enteritis, Salmonella infections can be invasive and potentially fatal. Children under the age of 5 years, elderly adults, and immunocompromised persons are at increased risk for invasive disease (Sirinavin et al., 1988; Chiu et al., 1999; Arshad et al., 2008; Jones et al., 2008). Antimicrobial therapy is required in these cases, with drugs of choice being ciprofloxacin in adults and ceftriaxone in children (Guerrant et al., 2001; Hohmann, 2001). However, antimicrobial resistance among Salmonella strains has been associated with adverse patient outcomes, including higher frequency of treatment failures, increased risk and duration of hospitalization, and higher mortality (Helms et al., 2002; Mølbak, 2005; Varma et al., 2005).

Dairy cattle are a reservoir of Salmonella serovars that are leading causes of human salmonellosis, including multidrug-resistant (MDR) S. enterica serovars Newport and Typhimurium (Gupta et al., 2003; Dechet et al., 2006; Varma et al., 2006; Karon et al., 2007). Foodborne transmission is the most common route (Scallan et al., 2011) and can occur through vehicles such as undercooked ground beef and unpasteurized dairy products. Salmonella can also be transmitted by direct contact with feces from infected dairy cattle (Cummings et al., 2012), underscoring the importance of occupational and environmental exposure. According to the U.S. Department of Agriculture (USDA) National Animal Health Monitoring System (NAHMS) Dairy 2007 study, 13.7% of cows representing 121 herds in 17 major dairy states were Salmonella positive based on fecal culture results from a single sampling visit (USDA, 2011). Dairy cattle can shed Salmonella for extended durations following clinical disease (Cummings et al., 2009), resulting in environmental contamination and increased risk of within-herd transmission. Salmonella has been shown to survive for prolonged periods in suitable conditions outside the host (You et al., 2006; Toth et al., 2011).

Administration of antimicrobial agents to dairy cattle and other food animals is considered to be a driving factor for antimicrobial resistance among Salmonella and other enteric pathogens (Holmberg et al., 1984; Cohen and Tauxe, 1986; Angulo et al., 2000; Threlfall et al., 2000; White et al., 2001). Antimicrobial agents that are currently licensed for use in dairy cattle in the United States include enrofloxacin, florfenicol, and various penicillins, cephalosporins, macrolides, sulfonamides, and tetracyclines (enrofloxacin and florfenicol are only approved for use in dairy cattle less than 20 months of age). Common uses for antimicrobial agents on dairy farms include feeding of medicated milk replacer to preweaned heifers, treatment of respiratory and gastrointestinal diseases in preweaned heifers, treatment of respiratory disease in weaned heifers, prevention and treatment of mastitis in cows, and treatment of respiratory disease, reproductive disorders, and lameness in cows (USDA, 2008). However, a number of studies have failed to find conclusive evidence that the use of antimicrobial agents in animal production systems leads to a sustained increase in antimicrobial resistance among pathogens within the gastrointestinal tract (Ray et al., 2006; Singer et al., 2008; Daniels et al., 2009; Heider et al., 2009; Mann et al., 2011; Morley et al., 2011). In fact, recent experimental work suggests that the environment might play a central role in antimicrobial selection pressure. Excreted ceftiofur metabolites were found to exert selection pressure that promoted resistance in the environment, and cattle subsequently became infected with resistant bacteria through environmental exposure (Subbiah et al., 2012; Call et al., 2013). This suggests that there is likely to be a discrepancy among environmental reservoirs in their ability to promote the emergence and persistence of antimicrobial resistance on dairy farms.

The objectives of this study were to estimate the environmental prevalence of Salmonella on dairy farms in Texas and to determine the antimicrobial susceptibility of the isolates. Identification of high-risk environmental reservoirs within dairies would provide an opportunity to mitigate the burden of antimicrobial resistance at the preharvest level.

Materials and Methods

Study design

Data for this study were collected prospectively from a convenience sample of dairy farms throughout Texas; these farms were located in the two major dairy areas of the state, in northwest and central Texas. Dairy farms were sampled from August through October 2013, using a cross-sectional approach. To estimate the environmental prevalence of Salmonella within ±5% with 95% confidence (anticipating a prevalence of 40%), the target was ∼370 environmental samples. Eight samples were collected from each of four locations per farm for Salmonella culture: hospital pen, maternity pen, cow housing area, and calf housing area. Fecal samples were also collected from cows that were due to be culled from each of the dairies at the time of environmental sampling.

Sample collection

Environmental samples were collected using sterile 4 × 4-inch gauze pads saturated in double-strength skim milk (Becton Dickinson and Company, Franklin Lakes, NJ), which had been placed into a sterile flip-top container (Capitol Vials, Auburn, AL). Eight individual drag-swab samples were collected from each of the four locations per farm, with the gauze pad moved across the surface in a single sweeping arc. A new glove was used to collect each sample, and the gauze was placed back into its respective flip-top container. Hospital and maternity pen samples consisted of either eight swabs of the floor in group pens or eight swabs of the bedding in individual pens. In the case of group pens, samples were collected with the goal of attaining broad representative coverage. Locations sampled in each cow housing area included eight sites on the floor within high-traffic sections of the barn. Calf housing samples consisted of either eight swabs of the floor in group housing areas or eight swabs of the bedding in individual hutches or pens. All environmental samples were maintained at ∼4°C until processing.

Fecal samples from cull cows were collected through free catch or rectal retrieval, with a new glove being used to collect each sample. Approximately 10 g of feces was placed into a Para-Pak C & S bottle (Meridian Bioscience, Inc., Cincinnati, OH) and sealed. All samples were either immediately transported or shipped overnight to the research laboratory for bacteriologic culture.

Microbiologic procedure for Salmonella detection

Standard bacteriologic culture methods were used to isolate Salmonella (Andrews and Hammack, 2007). Individual gauze pads and fecal samples were placed into Whirl-Pak bags (Nasco, Fort Atkinson, WI) for enrichment in 90 mL of tetrathionate broth (Difco, Detroit, MI) containing 1.8 mL of iodine solution; the mixture was incubated at 37°C for 18–24 h. Twenty microliters was then transferred into 5 mL of Rappaport-Vassiliadis (RV) broth (Difco) for incubation at 42°C for 18–24 h. After incubation, the RV broth was subcultured to Brilliant Green agar (Oxoid, Cambridge, UK) with novobiocin (Sigma-Aldrich, Saint Louis, MO) and incubated at 37°C for 18–24 h. Presumptive Salmonella colonies were inoculated into triple sugar iron slants and lysine iron agar slants (Difco) and incubated at 37°C for 18–24 h. Colonies that exhibited the biochemical properties of Salmonella were frozen at −80°C in 15% glycerol for subsequent characterization.

Antimicrobial susceptibility testing

Antimicrobial susceptibility of Salmonella isolates was determined using a broth microdilution method. Minimal inhibitory concentrations (MICs) were determined using the National Antimicrobial Resistance Monitoring System (NARMS) Gram-negative panel of 15 antimicrobial agents (Sensititre plate code CMV2AGNF; TREK Diagnostic Systems, Cleveland, OH): amoxicillin/clavulanic acid, ampicillin, azithromycin, cefoxitin, ceftiofur, ceftriaxone, chloramphenicol, ciprofloxacin, gentamicin, kanamycin, nalidixic acid, streptomycin, sulfisoxazole, tetracycline, and trimethoprim/sulfamethoxazole. Clinical and Laboratory Standards Institute (CLSI) guidelines were used to interpret MIC values when available (CLSI, 2008, 2012). Otherwise, MIC values were interpreted using NARMS breakpoints (FDA, 2013). Isolates were classified as resistant or susceptible to each agent. Quality control was performed using Escherichia coli ATCC 25922 as recommended by the CLSI.

Serovar determination

Serovar was determined for Salmonella isolates demonstrating multidrug resistance, defined here as in vitro resistance to two or more antimicrobial classes. The xMAP^® Salmonella Serotyping Assay (Luminex, Austin, TX) was used to identify Salmonella isolates. The assay simultaneously determines O and H antigen genes and also identifies serovar-specific markers in the AT (additional targets) test. Salmonella template DNA was extracted using the InstaGene Matrix as described by the manufacturer (Bio-Rad, Hercules, CA). Multiplex PCR was performed, and the amplicons were hybridized with the oligonucleotide probe-coupled bead mixture and then labeled with streptavidin-R-phycoerythrin reporter. The assay plate was analyzed on a Luminex^® LX200™ platform, and the data were exported to Excel (Microsoft, Redmond, WA) for analysis.

Statistical analyses

Data were imported into a commercial statistical software program (SAS, version 9.4; SAS Institute, Inc., Cary, NC) for variable coding and analysis. Descriptive analysis was performed on all variables, and 95% confidence intervals (CIs) for Salmonella prevalence were calculated using variance estimation that accounted for clustered data. Chi-squared testing was used to characterize the distribution of Salmonella by farm and by environmental location. Among samples, a multivariable logistic regression model was used to determine if environmental location was associated with positive Salmonella status. Among Salmonella isolates, a multivariable logistic regression model was used to determine if environmental location was associated with MDR status. The generalized estimating equations method was used to account for the clustering of samples by farm. For all analyses, p < 0.05 was considered significant.

Results

Eleven dairy farms participated in this study; seven were located in northwest Texas and four in central Texas. The median herd size was ∼3000 lactating cows (range: 400–6300). These herds were all conventionally managed and they represented a range of housing types, including drylot facilities (7 farms), free-stall barns (3 farms), and Saudi barns (1 farm). Salmonella was isolated from 67.0% (236/352; 95% CI, 53.4–80.6%) of environmental samples and 64.2% (43/67; 95% CI, 52.9–75.5%) of fecal samples (Table 1). All farms yielded positive samples. Environmental prevalence of Salmonella varied significantly by farm (p < 0.0001), with proportions of positive samples ranging from 31.3% to 96.9%. Salmonella prevalence also varied significantly by environmental location (p = 0.001), with higher proportions of positive samples from the maternity (83.0%, 73/88) and hospital pens (68.2%, 60/88) and lower proportions from the calf housing (58.0%, 51/88) and cow housing areas (59.1%, 52/88). Samples from the maternity pen were significantly more likely to be Salmonella positive than samples from the cow housing area (odds ratio [OR], 3.4; 95% CI, 1.7–6.5; p = 0.0003), although the OR overestimates the risk ratio in light of the high prevalence of positive samples. The odds of being Salmonella positive did not differ significantly among samples from the hospital pen, calf housing area, and cow housing area. Maternity pen samples were significantly more likely to be positive compared with calf housing samples (p = 0.04) but not hospital pen samples (p = 0.2).

Table 1.

Salmonella Prevalence Among Dairy Farm Environmental Samples and Cull Cow Fecal Samples in Texas

Dairy farm	Environmental sample-level Salmonella prevalence	Cow-level Salmonella prevalence
A	59.4% (19/32)	0% (0/2)
B	31.3% (10/32)	66.7% (10/15)
C	37.5% (12/32)	40.0% (2/5)
D	56.3% (18/32)	57.1% (4/7)
E	50.0% (16/32)	70.0% (7/10)
F	68.8% (22/32)	33.3% (2/6)
G	96.9% (31/32)	N/A
H	65.6% (21/32)	N/A
I	84.4% (27/32)	71.4% (5/7)
J	90.6% (29/32)	87.5% (7/8)
K	96.9% (31/32)	85.7% (6/7)

N/A denotes farms where no cull cow samples were collected.

Antimicrobial susceptibility testing was performed on 95.7% (267/279) of the Salmonella isolates from environmental and fecal samples. Resistance to individual antimicrobial agents ranged from 0% (azithromycin, ciprofloxacin, gentamicin, and nalidixic acid) to 11.6% (tetracycline) of all isolates tested. Resistance among isolates ranged from zero to 11 drugs, and nine phenotypes were observed (Table 2). Multidrug resistance was evident in 11.9% (27/226) of environmental isolates and 19.5% (8/41) of fecal isolates. Resistance to three or more antimicrobial classes was evident in 5.8% (13/226) of environmental isolates and 9.8% (4/41) of fecal isolates. Five (45.5%) farms yielded MDR Salmonella isolates from the environment, cull cattle, or both (Table 3). Isolates from the calf housing area (OR, 3.4; 95% CI, 1.3–8.9; p = 0.01) and maternity pen (OR, 2.9; 95% CI, 1.1–8.1; p = 0.04) were significantly more likely to be MDR than isolates from the cow housing area. The odds of being MDR did not differ significantly between isolates from the hospital pen and isolates from the cow housing area. Calf housing isolates (p = 0.1) and maternity pen isolates (p = 0.1) were not significantly more likely to be MDR compared with hospital pen isolates.

Table 2.

Multidrug-Resistant Isolates by Serovar and Resistance Phenotype

Serovar	No. of isolates ^a	Resistance phenotype	Farms on which the isolates were recovered
Newport	12	AUG, AMP, FOX, XNL, AXO, CHL, STR, FIS, TET	C, E, I
Muenchen	11	FIS, TET	D
Typhimurium	6	AUG, AMP, FOX^b, XNL, AXO	C
Heidelberg	3	AUG, AMP, FOX, XNL, AXO, CHL, KAN, STR, FIS, TET, SXT	D
Muenster	2	CHL, CIP^b, STR, FIS, TET, SXT	C
No ID^c	1	FIS, TET, SXT	G

All the isolates within a serovar represented the same resistance phenotype.

These isolates showed intermediate susceptibility to the antimicrobial agent.

Serogroup C1, antigenic formula: 6,7:z4:-.

AMP, ampicillin; AUG, amoxicillin/clavulanic acid; AXO, ceftriaxone; CHL, chloramphenicol; CIP, ciprofloxacin; FIS, Sulfisoxazole; FOX, cefoxitin; KAN, kanamycin; STR, streptomycin; SXT, trimethoprim/sulfamethoxazole; TET, tetracycline; XNL, ceftiofur.

Table 3.

Prevalence of Multidrug Resistance Among Salmonella Isolates, Stratified by Farm and Environmental Location

Farm ID	Hospital pen	Maternity pen	Cow housing	Calf housing
A	0% (0/1)	0% (0/6)	0% (0/4)	0% (0/5)
B	0% (0/5)	0% (0/3)	0% (0/1)	0% (0/0)
C	50.0% (2/4)	57.1% (4/7)	0% (0/0)	100% (1/1)
D	50.0% (1/2)	100% (5/5)	33.3% (1/3)	71.4% (5/7)
E	33.3% (1/3)	40.0% (2/5)	100% (1/1)	100% (2/2)
F	0% (0/8)	0% (0/5)	0% (0/3)	0% (0/6)
G	0% (0/8)	0% (0/8)	12.5% (1/8)	0% (0/7)
H	0% (0/4)	0% (0/8)	0% (0/7)	0% (0/2)
I	0% (0/8)	0% (0/7)	0% (0/7)	20.0% (1/5)
J	0% (0/7)	0% (0/7)	0% (0/8)	0% (0/7)
K	0% (0/8)	0% (0/8)	0% (0/7)	0% (0/8)
All farms	6.9% (4/58)	15.9% (11/69)	6.1% (3/49)	18.0% (9/50)

The 35 MDR isolates belonged to five serovars: Newport (n = 12), Muenchen (n = 11), Typhimurium (n = 6), Heidelberg (n = 3), Muenster (n = 2), in addition to one isolate classified within serogroup C₁ with the antigenic formula 6,7:z4:– (Table 2).

Discussion

Salmonella was ubiquitous on Texas dairy farms. The prevalence results can be partially explained by the sample collection period (i.e., August through October) as previous studies have found prevalence of fecal Salmonella shedding among dairy cattle to peak during summer and autumn (Fossler et al., 2005; Pangloli et al., 2008; Cummings et al., 2009). Furthermore, observations from previous studies suggest that there is regional prevalence variation, in which the Salmonella burden increases across a southerly gradient (Edrington et al., 2004b; Kunze et al., 2008; Gragg et al., 2013). The Salmonella prevalence among dairy farm environmental samples in our study was greater (67.0%; 236/352) than that in a study (28.3%; 402/1420) conducted in the northeastern United States (Rodriguez-Rivera et al., 2014), a discrepancy likely influenced by geographic location. Salmonella shedding by cull cows was higher (64.2%) than in cull dairy cows on other Texas dairy farms (32.6%) (Loneragan et al., 2012). Differences in Salmonella prevalence among farms in the same geographic region might be due to farm management practices and herd characteristics (Ruzante et al., 2010). Additionally, Loneragan et al. (2012) reported that three of the farms in their study implemented whole-herd vaccination with a commercially available vaccine to protect the cattle against Salmonella Newport. The Salmonella prevalence was lower (8.0%) than in herds that were not vaccinated (36.8%). Future research with more dairy farms would be necessary to adequately evaluate the effects of herd-level factors such as farm management practices, biosecurity protocols, and vaccination programs. This information would help determine factors associated with Salmonella shedding and thus enable development of improved preharvest control strategies in this region of the country.

Samples from the maternity pen were significantly more likely to be Salmonella positive than samples from the cow and calf housing areas, implying either a higher probability of Salmonella occurrence or a higher concentration of Salmonella organisms in maternity pens. Evidence suggests that periparturient cows are the adult cattle group that is most susceptible to Salmonella infection (Fossler et al., 2005; Cobbold et al., 2006) based on immunosuppressive mechanisms that prevail during late gestation, parturition, and early lactation (Madsen et al., 2002; Mehrzad et al., 2002; Lippolis et al., 2006). These factors could also make recrudescence of Salmonella shedding more likely among periparturient cows that are already infected. It is important to note that none of the dairies used a joint hospital-maternity pen, a management strategy that has been associated with a higher prevalence of Salmonella contamination relative to other dairy farm environments (Cobbold et al., 2006).

Although the Salmonella prevalence was high relative to other regions of the country, antimicrobial resistance among environmental and fecal isolates was uncommon. Only 13.1% (35/267) of the isolates tested were resistant to two or more antimicrobial classes. Similarly, 11% of Salmonella isolates from cows culled from other dairy farms in Texas were resistant to two or more antimicrobial agents. In contrast, the NAHMS Dairy 2007 study reported that only 2.7% of 556 Salmonella isolates from fecal samples from healthy cattle were resistant to two or more antimicrobial agents. Resistant organisms can be shed directly from the gastrointestinal tract into the environment. Alternatively, the dairy farm environment might play a key role in antimicrobial selection pressure (Subbiah et al., 2012; Call et al., 2013; Andersson and Hughes, 2014). Despite the low prevalence of antimicrobial resistance, a number of MDR phenotypes were observed (Table 2). Farm C yielded the most MDR phenotypes (n = 3; Table 2). One MDR phenotype was present on more than one farm, and all the isolates belonged to serovar Newport (n = 12; Table 2). Several factors have been investigated for association with antimicrobial-resistant Salmonella on dairy farms, including manure management practices (Habing et al., 2012). The use of composted manure for bedding was significantly associated with isolation of resistant Salmonella from dairy farms (Habing et al., 2012). Two antimicrobial agents of particular concern are ciprofloxacin and ceftriaxone because of their importance in treating invasive salmonellosis among adults and children, respectively (Guerrant et al., 2001; Hohmann, 2001). Resistance to quinolones was not detected in this study, although two isolates showed decreased susceptibility to ciprofloxacin (breakpoint = 0.5 μg/mL). However, 21 isolates resistant to ceftriaxone were obtained from four of the 11 farms.

Calf housing areas and maternity pens were more likely to yield MDR isolates, relative to cow housing areas. Salmonella strains isolated from calf housing areas and maternity pens could be subjected to greater selection pressure because antimicrobial agents are used in those locations for various reasons, such as medicated milk replacer for preweaned heifers (USDA, 2008) and treatment of periparturient adult cattle (Cobbold et al., 2006). It is important to define which dairy farm locations are more likely to contain MDR strains because of the implications for animal management. For example, it has been observed that calves are more likely to die from salmonellosis than adult cows (Cummings et al., 2009), and appropriate measures must be taken to minimize morbidity and mortality within dairy herds. Furthermore, certain dairy farm environments might pose an increased risk to public health, such as direct contact transmission of resistant strains to farm personnel. Infection of adult cattle with MDR Salmonella through environmental exposure could also facilitate downstream foodborne transmission of resistant strains. Calves are further removed from the food production chain than adult cattle but could serve as a reservoir for persistent MDR phenotypes on dairy operations. Previous research conducted on dairies in the southwestern United States suggested that calves and cattle in sick–fresh pens were the main groups of concern regarding MDR Salmonella (Edrington et al., 2008).

The most common MDR serovars were Newport, Muenchen, and Typhimurium (Table 2). Similarly, the NAHMS Dairy 2007 study found the most common MDR serovars among healthy dairy cattle to be Newport, Montevideo, and Typhimurium (USDA, 2011). Salmonella Typhimurium and Salmonella Newport are the second and third most frequently isolated serovars from humans with laboratory-confirmed salmonellosis in the United States, respectively (CDC, 2014). Other serovars that are frequently identified among Salmonella-positive dairy cattle in the southwestern United States, regardless of susceptibility status, include Montevideo, Mbandaka, Senftenberg, Anatum, and Give (Edrington et al., 2004a).

Limitations of this study include a relatively small sample of enrolled dairy farms. In addition, the results are likely to be representative of dairy herds in Texas or the southern United States only. Nevertheless, these data indicate that dairy farm environments in northwest and central Texas have a high burden of Salmonella contamination relative to other areas of the country. Of all sampled environments, the maternity pen yielded the highest prevalence of Salmonella isolates and MDR strains, highlighting the potential role of this environmental niche in promoting the emergence and persistence of MDR Salmonella within dairy farms. Further research is needed to determine if hygiene and other management practices focused on maternity pens are associated with decreased prevalence and diversity of antimicrobial resistance within dairy farms and along the food production chain. Additional study of appropriate antimicrobial use practices on dairy farms is also necessary to reduce the occurrence of drug-resistant Salmonella.

Footnotes

Acknowledgments

The authors thank the dairy farm owners who participated in this study as well as Kate Andrews for her excellent technical assistance. This research was funded by The Beef Checkoff and the U.S. Department of Agriculture National Institute of Food and Agriculture's National Integrated Food Safety Initiative award no. 2011-51110-31081.

Disclosure Statement

In terms of products mentioned in this article, G.H.L. has provided scientific advice to Zoetis and Epitopix, LLC, and has on occasion billed for this service. G.H.L. has also received honoraria for service on advisory boards and presentations for these companies. No other financial conflicts of interests are reported.

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