Paula J. Fedorka-Cray

Antimicrobial Use and Resistance in Animals

Food animals in the United States are often exposed to antimicrobials to treat and prevent infectious disease or to promote growth. Many of these antimicrobials are identical to or closely resemble drugs used in humans. Precise figures for the quantity of antimicrobials used in animals are not publicly available in the United States, and estimates vary widely. Antimicrobial resistance has emerged in zoonotic enteropathogens (e.g., Salmonella spp., Campylobacter spp.), commensal bacteria (e.g., Escherichia coli, enterococci), and bacterial pathogens of animals (e.g., Pasteurella, Actinobacillus spp.), but the prevalence of resistance varies. Antimicrobial resistance emerges from the use of antimicrobials in animals and the subsequent transfer of resistance genes and bacteria among animals and animal products and the environment. To slow the development of resistance, some countries have restricted antimicrobial use in feed, and some groups advocate similar measures in the United States. Alternatives to growth-promoting and prophylactic uses of antimicrobials in agriculture include improved management practices, wider use of vaccines, and introduction of probiotics. Monitoring programs, prudent use guidelines, and educational campaigns provide approaches to minimize the further development of antimicrobial resistance.

Use of a Genus- and Species-Specific Multiplex PCR for Identification of Enterococci

ABSTRACT The 16S rRNA gene has previously been used to develop genus-specific PCR primers for identification of enterococci. In addition, the superoxide dismutase gene (sodA) has been identified as a potential target for species differentiation of enterococci. In this study, Enterococcus genus-specific primers developed by Deasy et al. (E1/E2) were incorporated with species-specific primers based upon the superoxide dismutase (sodA) gene for development of a multiplex PCR. This assay provides simultaneous genus and species identification of 23 species of enterococci using seven different reaction mixtures. Accuracy of identification of the multiplex PCR was determined by comparisons to standard biochemical testing, the BBL Crystal kit, VITEK, and API Rapid ID 32 Strep. Isolates from swine feces, poultry carcasses, environmental sources, and retail food were evaluated and, overall, results for 90% of the isolates tested by PCR agreed with results obtained using standard biochemical testing and VITEK. Eighty-five percent and 82% of PCR results agreed with results from the API Rapid ID 32 Strep and BBL Crystal tests, respectively. With the exception of concurrence between identification using standard biochemical testing and VITEK (85%) and between BBL Crystal and VITEK (83%), the percent agreement for PCR was higher than or equal to any other pairwise comparison. Multiplex PCR for genus and species determination of enterococci provides an improved, rapid method for identification of this group of bacteria.

Fecal Shedding of Salmonella spp. by Dairy Cows on Farm and at Cull Cow Markets

As part of a national study of the U.S. dairy cow population, fecal samples were collected from representative cows on 91 dairies and 97 cull dairy cow markets in 19 states. Salmonella spp. were recovered from 5.4% of milk cows, 18.1% of milk cows expected to be culled within 7 days, and 14.9% of culled dairy cows at markets. On a premise basis, Salmonella shedding in milk cows was detected on 21.1% of dairies and 66% of cull dairy cow markets. The percentage of herds with at least one cow with detectable Salmonella fecal shedding was higher during the sampling period from May through July, in herds with at least 100 milk cows, and in herds in the South region. The most common Salmonella serogroups isolated were E (30.8% of isolates) and C1 (28.6%); the most common serotypes isolated were Salmonella Montevideo (21.5% of isolates), Salmonella Cerro (13.3%), and Salmonella Kentucky (8.5%). Fecal shedding of Salmonella Typhimurium or Salmonella Typhimurium var. copenhagen was infrequent (2.8% of isolates). Most isolates (88.9%) were susceptible to all 17 antimicrobials evaluated; multiple resistance was an infrequent occurrence. This study provides information describing the distribution of Salmonella fecal shedding from dairy cows on farm and at markets and will serve as a baseline for future studies.

Distribution of Campylobacter spp. in selected U.S. poultry production and processing operations.

A study was conducted of 32 broiler flocks on eight different farms, belonging to four major U.S. producers. The farms were studied over I complete calendar year. Overall, 28 (87.5%) of the flocks became Campylobacter positive, and only four (12.5%) remained negative throughout the 6- to 8-week rearing period. In the majority of flocks, sampled every 2 weeks throughout production, Campylobacter-positive fecal and cecal samples were not detected until 4 to 8 weeks of age. In only six of the flocks were environmental samples found to be positive before shedding of Campylobacter was detected in the birds. Even in some of the Campylobacter-negative flocks, contamination of the rearing environment was positive for Campylobacter but did not result in the birds subsequently excreting the organism. These findings are discussed in relation to U.S. husbandry practices and present uncertainty about sources of Campylobacter infection for poultry flocks. Birds were often transported to the processing plant in coops that were already contaminated with Campylobacter, and the organisms were sometimes found in samples of scald water and chill water. After chilling, the proportions of Campylobacter-positive carcasses from different producers ranged from 21.0 to 40.9%, which is lower than in other studies, and possible reasons are considered.

Survey of Salmonella serotypes in feedlot cattle.

A national study of health and management of cattle in feedlots was conducted. Within this study, the prevalence of Salmonella spp. in fecal samples was determined. Fifty fecal samples were collected from each of 100 feedlots. Within each feedlot, 25 fresh fecal samples were collected from the floor of the pens of cattle which had been on feed the shortest and 25 from those on feed the longest periods of time. The total number of samples collected was 4,977; 2,484 and 2,495 from pens of cattle on feed the shortest and longest times, respectively. Salmonella spp. were recovered from 38% (38 of 100) of the feedlots. Salmonella spp. were recovered from 5.5% (273 of 4,977) of all samples and from 3.5% (88 of 2,484) and 7.4% (185 of 2,495) of samples from pens of cattle shortest and longest on feed, respectively. The most common serotype recovered was S. anatum (27.9%), followed by S. montevideo (12.9%), S. muenster (11.8%), S. kentucky (8.2%), and S. newington (4.3%). The most common serogroups identified were E1 (39.6%), C1 (20.7%), and B (10.4%). Shedding of the serotypes most commonly associated with human illness occurred infrequently (13 of 273: 4.8%). This study provides information on the status of Salmonella spp. from cattle in feedlots and may serve as baseline information for future studies.

Determination of the Incidence of Salmonella spp., Campylobacter jejuni, and Clostridium perfringens in Wild Birds near Broiler Chicken Houses by Sampling Intestinal Droppings

Several methods were evaluated for collecting fecal and intestinal samples from wild birds found near broiler chicken houses. A few intestinal samples and cloacal swabs were obtained from European starlings and house sparrows. Most of the samples collected consisted of wild bird droppings found on or near the houses. Samples were collected from each of four farms of a broiler integrator during a grow-out cycle: a cycle in the summer for farm A, fall for farm B, and spring, summer, fall, and winter for farms C and D. Of the 25 wild bird intestinal and fecal samples collected from a broiler house on farm A during a grow-out cycle in July-August 1997, 24% were positive for Salmonella spp., 4% for Campylobacter jejuni, and 28% for Clostridium perfringens. Of the nine fecal samples collected from broiler house B in a grow-out cycle in September-November 1997, 33% were positive for Salmonella spp., 11% for C. jejuni, and 22% for C. perfringens. For farms C and D, of the 23 samples collected in March-April 1998, 0 were positive for Salmonella spp., 11% for C. jejuni, and 52% for C. perfringens; of 27 samples collected in June-July 1998, 4% were positive for Salmonella spp., 0 for C. jejuni, and 13% for C. perfringens; of 24 samples collected in August-October 1998, 14% were positive for Salmonella spp., 5% for C. jejuni, and 4% for C. perfringens; of 14 samples collected December 1998-January 1999, 0 were positive for Salmonella, 50% for C. jejuni, and 14% for C. perfringens. The incidence of these bacterial enteropathogens in wild birds near the broiler chicken houses suggests that wild birds that gain entry to poultry grow-out houses have the potential to transmit these pathogens to poultry.

Multiplex PCR-Based Method for Identification of Common Clinical Serotypes of Salmonella enterica subsp. enterica

ABSTRACT A multiplex PCR method has been developed to differentiate between the most common clinical serotypes of Salmonella enterica subsp. enterica encountered in Washington State and the United States in general. Six genetic loci from S. enterica serovar Typhimurium and four from S. enterica serovar Typhi were used to create an assay consisting of two five-plex PCRs. The assays gave reproducible results with 30 different serotypes that represent the most common clinical isolates of S. enterica subsp. enterica. Of these, 22 serotypes gave unique amplification patterns compared with each other and the other 8 serotypes were grouped into four pairs. These were further resolved by two additional PCRs. We compared the data from PCR serotyping with conventional serotyping and found that PCR serotyping was nearly as discriminatory as conventional serotyping was. The results from a blind test screening 111 clinical isolates revealed that 97% were correctly identified using the multiplex PCR assay. The assay can be easily performed on multiple samples with final results in less than 5 h and, in conjunction with pulsed-field gel electrophoresis, forms a very robust test method for the molecular subtyping of Salmonella enterica subsp. enterica.

Sources and movement of Salmonella through integrated poultry operations: a multistate epidemiological investigation.

The prevalence of Salmonella from numerous sources in 32 integrated broiler operations of high- and low-performing broiler houses was characterized from four states across four seasons. Previous studies of Salmonella in broilers have been limited in scope, offering only a snapshot of pathogen prevalence as seen on a small number of individual farms. Twenty-six different sample types were collected from the hatchery to the end of processing, and Salmonella was found in all sample types. A total of 10,740 samples were analyzed for Salmonella, and 973 (9.1%) of these samples, including 49 of 798 (6.1%) carcass rinse samples, were Salmonella positive. Hatchery transport pads (389 of 765, 50.8%), flies (28 of 150, 18.7%), drag swabs (57 of 402, 14.2%), and boot swabs (20 of 167, 12%) were samples from which Salmonella was most frequently isolated. Thirty-six different serotypes were identified, and the most frequently encountered serotypes were Salmonella Senftenberg, Salmonella Thompson, and Salmonella Montevideo. Determining critical contaminating sources and following the movement of Salmonella through integrated poultry operations will help researchers and the industry develop practical intervention strategies.

Antimicrobial Resistance-Conferring Plasmids with Similarity to Virulence Plasmids from Avian Pathogenic Escherichia coli Strains in Salmonella enterica Serovar Kentucky Isolates from Poultry

ABSTRACT Salmonella enterica, a leading cause of food-borne gastroenteritis worldwide, may be found in any raw food of animal, vegetable, or fruit origin. Salmonella serovars differ in distribution, virulence, and host specificity. Salmonella enterica serovar Kentucky, though often found in the food supply, is less commonly isolated from ill humans. The multidrug-resistant isolate S. Kentucky CVM29188, isolated from a chicken breast sample in 2003, contains three plasmids (146,811 bp, 101,461 bp, and 46,121 bp), two of which carry resistance determinants (pCVM29188_146 [strAB and tetRA] and pCVM29188_101 [blaCMY-2 and sugE]). Both resistance plasmids were transferable by conjugation, alone or in combination, to S. Kentucky, Salmonella enterica serovar Newport, and Escherichia coli recipients. pCVM29188_146 shares a highly conserved plasmid backbone of 106 kb (>90% nucleotide identity) with two virulence plasmids from avian pathogenic Escherichia coli strains (pAPEC-O1-ColBM and pAPEC-O2-ColV). Shared avian pathogenic E. coli (APEC) virulence factors include iutA iucABCD, sitABCD, etsABC, iss, and iroBCDEN. PCR analyses of recent (1997 to 2005) S. Kentucky isolates from food animal, retail meat, and human sources revealed that 172 (60%) contained similar APEC-like plasmid backbones. Notably, though rare in human- and cattle-derived isolates, this plasmid backbone was found at a high frequency (50 to 100%) among S. Kentucky isolates from chickens within the same time span. Ninety-four percent of the APEC-positive isolates showed resistance to tetracycline and streptomycin. Together, our findings of a resistance-conferring APEC virulence plasmid in a poultry-derived S. Kentucky isolate and of similar resistance/virulence plasmids in most recent S. Kentucky isolates from chickens and, to lesser degree, from humans and cattle highlight the need for additional research in order to examine the prevalence and spread of combined virulence and resistance plasmids in bacteria in agricultural, environmental, and clinical settings.

Comparison of Subtyping Methods for Differentiating Salmonella enterica Serovar Typhimurium Isolates Obtained from Food Animal Sources

ABSTRACT Molecular characterization (e.g., DNA-based typing methods) of Salmonella isolates is frequently employed to compare and distinguish clinical isolates recovered from animals and from patients with food-borne disease and nosocomial infections. In this study, we compared the abilities of different phenotyping and genotyping methods to distinguish isolates of Salmonella enterica serovar Typhimurium from different food animal sources. One hundred twenty-eight S. enterica serovar Typhimurium strains isolated from cattle, pigs, chickens, and turkeys or derived food products were characterized using pulsed-field gel electrophoresis (PFGE), repetitive element PCR (Rep-PCR), multilocus sequence typing (MLST), plasmid profiling, and antimicrobial susceptibility testing. Among the 128 Salmonella isolates tested, we observed 84 Rep-PCR profiles, 86 PFGE patterns, 89 MLST patterns, 36 plasmid profiles, and 38 susceptibility profiles. The molecular typing methods, i.e., PFGE, MLST, and Rep-PCR, demonstrated the best discriminatory power among Salmonella isolates. However, no apparent correlation was evident between the results of one molecular typing method and those of the others, suggesting that a combination of multiple methods is needed to differentiate S. enterica serovar Typhimurium isolates that genetically cluster according to one particular typing method.