Yersinia enterocolitica of porcine origin: carriage of virulence genes and genotypic diversity.

Yersinia enterocolitica is an important foodborne pathogen, and pigs are recognized as a major reservoir and potential source of pathogenic strains to humans. A total of 172 Y. enterocolitica recovered from conventional and antimicrobial-free pig production systems from different geographic regions (North Carolina, Ohio, Michigan, Wisconsin, and Iowa) were investigated to determine their pathogenic significance to humans. Phenotypic and genotypic diversity of the isolates was assessed using antibiogram, serogrouping, and amplified fragment length polymorphism (AFLP). Carriage of chromosomal and plasmid-borne virulence genes were investigated using polymerase chain reaction. A total of 12 antimicrobial resistance patterns were identified. More than two-thirds (67.4%) of Y. enterocolitica were pan-susceptible, and 27.9% were resistant against ß-lactams. The most predominant serogroup was O:3 (43%), followed by O:5 (25.6%) and O:9 (4.1%). Twenty-two of 172 (12.8%) isolates were found to carry Yersinia adhesion A (yadA), a virulence gene encoded on the Yersinia virulence plasmid. Sixty-nine (40.1%) isolates were found to carry ail gene. The ystA and ystB genes were detected in 77% and 26.2% of the strains, respectively. AFLP genotyping of isolates showed wide genotypic diversity and were grouped into nine clades with an overall genotypic similarity of 66.8-99.3%. AFLP analysis revealed that isolates from the same production system showed clonal relatedness, while more than one genotype of Y. enterocolitica circulates within a farm.

Prevalence and antimicrobial resistance profile of Campylobacter spp. isolated from conventional and antimicrobial-free swine production systems from different U.S. regions.

We conducted a study to compare the prevalence and antimicrobial resistance profile of Campylobacter isolated from 34 farm-slaughter pair cohorts of pigs raised in conventional and antimicrobial-free (ABF) production systems. Isolates originated from four different states of two geographic regions (region 1--Ohio and Michigan; region 2--Wisconsin and Iowa). A total of 838 fecal and 1173 carcass samples were examined. Campylobacter isolates were speciated using multiplex polymerase chain reaction targeting ceuE and hipO genes. The minimum inhibitory concentration was determined using agar dilution to a panel of six antimicrobials: chloramphenicol, erythromycin, gentamicin, ciprofloxacin, nalidixic acid, and tetracycline. Campylobacter spp. was isolated from 472 of 838 pigs (56.3%). Campylobacter prevalence did not vary significantly based on production system (conventional [58.9%] and ABF [53.7%], odds ratio [OR] 1.4, 95% confidence interval [CI] 0.8-2.6, p = 0.24) or geographic region (region 1 [54.1%] and region 2 [58.2%], OR 1.02, 95% CI 0.6-1.9, p = 0.92). At slaughter plant, Campylobacter prevalence varied based on processing stages (19.4% at pre-evisceration, 25.3% at postevisceration, and 3.2% at postchill). Resistance was common to tetracycline (64.5%), erythromycin (47.9%), and nalidixic acid (23.5%). Campylobacter isolates from conventional production systems were more likely to be erythromycin resistant than from ABF (OR 3.2, 95% CI 1.4-7.2, p = 0.01). The proportion of ciprofloxacin-resistant Campylobacter coli isolates were 3.7% and 1.2% from ABF and conventional production systems, respectively. Thirty-seven out of 1257 C. coli (2.9%) were resistant to both erythromycin and ciprofloxacin, drugs of choice for treatment of invasive human campylobacteriosis. The finding of ciprofloxacin resistance, particularly from ABF herds, has significant implications on the potential role of risk factors other than mere antimicrobial use for production purposes.

Seroprevalence of Trichinella, Toxoplasma, and Salmonella in antimicrobial-free and conventional swine production systems.

There has been a growing niche-market, outdoor, antimicrobial-free (ABF) swine production system in the last few years prompted by consumers' demand for a more "natural" pork product. The impact of such production systems on reemergence of current and historically significant swine-associated pathogens has not been determined. The objectives of the current study were to determine and compare Salmonella, Toxoplasma, and Trichinella seropositivity in two swine production systems: outdoor ABF and intensive indoor production systems. These three foodborne pathogens represent those with the highest importance for pork consumption. A total of 675 serum samples from three participating states, Wisconsin, North Carolina, and Ohio, were investigated. We found significantly higher seroprevalence of Salmonella and Toxoplasma from ABF herds (54% and 7%, respectively) than conventional (39% and 1%, respectively) (p = 0.001). Two pigs, both from ABF herds, were found to be seropositive for Trichinella. The results from this preliminary study suggest that all three pathogens were more commonly present in pigs that were reared in an ABF, outdoor, niche-market type of environment than the conventional, indoor-reared herds though there were some geographical variation in Salmonella. This warrants a robust epidemiologic study to determine the role of various risk factors in the two production systems that may lead to persistence of bacterial (Salmonella) pathogens and reemergence of parasites (such as Trichinella) of historical significance.

Oral sodium chlorate, topical disinfection, and younger weaning age reduce Salmonella enterica shedding in pigs.

Salmonella enterica subsp. enterica can cause swine illness or human foodborne disease. Although nontoxic to mammalian cells, chlorate can be converted to cytotoxic chlorite by salmonellae. To test whether chlorate is effective at reducing Salmonella shedding in weaned pigs exposed to shedding dams, a chlorate-nitrate-lactate (chlorate) oral dose was administered daily for 5 days following weaning, and this treatment was evaluated in combination with two weaning ages and a topical disinfectant. A total of 80 pigs were weaned at 10 or 21 days of age. Half within each age group were topically disinfected at weaning. Piglets were selected from dams for which Salmonella was detected in feces shortly after giving birth. Chlorate treatment reduced Salmonella prevalence and estimated Salmonella concentration in feces, cecal contents, and ileocolic lymph nodes. Younger weaning age (10 days of age) was associated with reduced shedding (lower concentration and prevalence) in samples collected at 10 days postweaning (DPW) and later. Chlorate treatment reduced the concentration of Salmonella in fecal samples at 5 DPW and in cecal samples at 14 DPW. The protective effects persisted through the end of the study at 14 DPW, 9 days after the final administration of chlorate. Disinfectant treatment reduced shedding in fecal samples at 14 DPW. Interactions were detected between the effects of chlorate and disinfection and between chlorate and weaning age. Chlorate treatment, topical disinfection, and younger weaning age may be useful tools for reducing Salmonella shedding on farms that practice segregated weaning and where sow-to-piglet transfer of Salmonella is an important source of infection in nursery pigs.

Testing an ecological model for transmission of Salmonella enterica in swine production ecosystems using genotyping data.

An ecological model for transmission of Salmonella enterica in swine production ecosystems was developed, identifying host species, environmental reservoirs, and temporal, spatial, and functional (i.e., stage of production) dimensions. It was hypothesized that transmission was most likely within spatial and functional compartments, between hosts of the same species and abiotic compartments of the same type. Eighteen swine production systems in Illinois, USA, were sampled in four collection cycles (1998, 1999, 2000, 2003). There were 11,873 samples collected, including feces from swine and other mammals and birds, and samples from insects, pen floors, boots, feed, and water. The 460 Salmonella isolates obtained were genotyped using repetitive sequence PCR with three primers-REP, BOX, and ERIC. All isolates from 2000 and 2003 were serotyped, as well as a subsample from 1998 and 1998. Genetic relatedness was estimated from the similarity of fragmentation patterns after gel electrophoresis of PCR products. Cluster analysis identified genetically related isolates. Linking of isolates in tight clusters (similarity >or=85%) was viewed as evidence for transmission. Five farms had a sufficient number of tight clusters for hypothesis testing. The factors most differentiating isolates genetically were farm of origin and time of sampling. Isolates were also differentiated genetically by site, building, room, and pen. There was no consistent association of genotype with stage of production or host/environment reservoir. Serotype analysis confirmed that Salmonella lineages were differentiated by visit and site. Thus, Salmonella transmission was primarily over short distances, i.e., within the same pen or room, with some transmission between rooms and buildings on the same site, but with limited transmission between sites. Transmission was observed across a variety of ecological niches represented by different host species and environmental reservoirs. Genetic differences over time reflected multiple introductions into the ecosystem of different Salmonella genotypes, as well as evolutionary changes within lineages. Intervention strategies to reduce Salmonella prevalence within swine production ecosystems would be best targeted at maintaining spatial barriers to transmission, whereas intervention targeted at specific biological hosts or environmental reservoirs is less likely to be effective.

Comparative investigation of prevalence and antimicrobial resistance of Salmonella between pasture and conventionally reared poultry.

The objective of this study was to compare Salmonella prevalence and antimicrobial resistance between pasture and conventional poultry farms. We collected the first Salmonella prevalence and antimicrobial resistance data on pasture poultry farms. Fecal droppings were collected from 31 farms from Wisconsin (nine farms from each production type) and the Southeast (North Carolina, Virginia, and South Carolina; five conventional and 10 pasture poultry farms) in a 1-yr period. The specimens were cultured for Salmonella and tested for resistance to 12 antimicrobials. A univariate analysis was conducted to determine the significant differences in prevalence and resistance. At the farm level, no significant difference in Salmonella prevalence was found on 33% pasture and 47% conventional poultry farms (P= 0.4928). On an individual specimen level, flocks reared conventionally had higher prevalence than in pasture (P < 0.0001). Of all the isolates found to have resistance, 80% were from the Southeast. Of all the pasture isolates tested, 5% (8 of 162) were resistant to ceftriaxone, which is higher than previously reported from any production setting. None of the isolates from conventional flocks showed resistance to ceftriaxone. Multidrug resistance (resistance to three or more classes of antimicrobials) was found in 69% of the isolates from conventional farms and 11% on pasture farms in the Southeast (P < 0.0001), with the predominant resistance type of AmCSSuTeAx (ampicillin, chloramphenicol, streptomycin, sulfasoxazole, tetracycline, amoxicillin/clavulanic acid; 62%). About 5% of the pasture isolates from the Southeast showed the AmAxCFCRO (ampicillin, amoxicillin/clavulanic acid, cephalothin, ceftriaxone) multidrug resistance pattern. None of the isolates from Wisconsin were found to be multidrug resistant.

Molecular epidemiologic investigation of Campylobacter coli in swine production systems, using multilocus sequence typing.

Multilocus sequence typing of 151 Campylobacter coli isolates from swine reared in conventional (n = 74) and antimicrobial-free (n = 77) production systems revealed high genotypic diversity. Sequence type (ST) 1413 was predominant and observed among ciprofloxacin-resistant strains. We identified a C. coli ST 828 clonal complex consisting of isolates from both production systems.

Salmonella enterica in superficial cervical (prescapular) and ileocecal lymph nodes of slaughtered pigs.

Because certain lymph nodes may be incorporated in food products, the presence of Salmonella enterica in these tissues could pose a food safety risk. We designed this two-part study to assess the prevalence of Salmonella in prescapular lymph nodes from normal slaughtered swine. Prescapular lymph nodes were collected from 300 systematically selected pigs in study 1 and, in study 2, from 75 pigs distributed among 10 herds. For study 2, pooled bacterial cultures were also completed on ileocecal lymph nodes, combining tissue from five pigs per pool (n = 60 pools). No Salmonella was detected in study 1 among prescapular lymph nodes (95% confidence interval, 0.0 to 1.16%). Salmonella was not detected in 75 prescapular lymph nodes from study 2, although Salmonella was detected in 5 of 10 herds in ileocecal lymph nodes. We conclude that prescapular lymph nodes posed a limited food safety risk in this population of pigs.

Associations between on-farm and slaughter plant detection of Salmonella in market-weight pigs.

Thirty swine production units in the midwestern United States were studied to assess the relationship of herd-level prevalence of Salmonella on the farm prior to slaughter versus at slaughter. Fecal samples were collected from 30 pigs on each farm within 48 h of slaughter, and 30 ileocecal lymph node samples were collected in the same shipment cohort at slaughter. Samples were cultured by conventional methods, and Salmonella identity was confirmed by serotyping. Overall, 11.7% (n = 105) of the fecal samples and 14.9% (n = 133) of the ileocecal lymph node samples were positive for Salmonella. Seventeen of the farms (56.7%) had one or more positive fecal samples, and 24 (80.0%) had one or more positive ileocecal lymph node samples. Twenty-four recognized serotypes and three additional distinct antigenic types were identified. Among all isolates, 56.5% had serotypes that were duplicated both on the farm and at slaughter for a particular cohort, whereas the remaining samples lacked a duplicate serotype in the other sample type. There was a positive correlation in the prevalence of Salmonella between fecal samples and ileocecal lymph node samples (Spearman's p = 0.75; 95% confidence interval [CI], 0.62 to 0.89). Linear regression analysis was used to identify two farms that biased the regression estimates. Excluding these farms, 62% of the variance in farm slaughter Salmonella prevalence was accounted for by on-farm prevalence. The analyses suggest that the prevalence of Salmonella spp. at slaughter can be predicted from preslaughter on-farm sampling and vice versa.

Effects of Mycoplasma hyopneumoniae vaccine on pigs naturally infected with M. hyopneumoniae and porcine reproductive and respiratory syndrome virus.

The effects of a single-dose Mycoplasma hyopneumoniae vaccine was studied in growing pigs. Each of 24 vaccinated cohorts of approximately 1200 pigs reared in separate barns was matched by time, farm site, and sex with unvaccinated cohorts. Pigs were naturally exposed to M. hyopneumoniae and porcine reproductive respiratory syndrome virus (PRRSv). Daily weight gain was 42 g per pig per day higher and mortality rate was 15.2/1000 pigs lower for vaccinated cohorts. Age at PRRSv onset was associated with mortality rate, but did not modify vaccine effects. M. hyopneumoniae vaccination was effective in promoting growth in spite of concurrent PRRSv infection.

Distribution of Salmonella in swine production ecosystems.

The objective of this 2-year field survey was to sample multiple ecological compartments within swine production systems to identify potential sources of Salmonella infection for swine. Twelve single-site production systems within Illinois were identified by slaughter sampling to have detectable Salmonella in swine and therefore selected for study. There were four visits to each farm during a 5-month period. Fecal samples were obtained from swine and other wild and domestic mammals. Arthropods and environmental samples of feed, water, pen floors, boots, and bird feces were also collected. All 8,066 samples obtained were cultured to detect Salmonella. Salmonella was detected on 11 of the 12 farms. There were 206 positive cultures, including samples from swine (83), pen floors (54), boots (32), flies (16), mice (9), cats (3), and birds (3). Swine shedding Salmonella in feces were detected on 9 of the 12 farms. The more Salmonella-abundant ecological compartments were cats (12% of samples positive), boots (11%), bird feces (8%), flies (6%), and mice (5%); 2.1% of 4,024 swine samples were positive. All 221 feed samples were negative for Salmonella. There was a correlation between a farm having a high prevalence of shedding Salmonella in pigs and a high abundance on pen floors, flies, and boots. The most common serotypes detected were Derby, Agona, Worthington, and Uganda, which were distributed throughout the ecosystem, suggesting widespread transmission across ecological compartments. The ubiquitous distribution of Salmonella suggests that an effective control strategy must target multiple compartments of the swine production ecosystem.