Examination of the horizontal gene transfer dynamics of an integrative and conjugative element encoding multidrug resistance in Histophilus somni.

Integrative and conjugative elements (ICEs) are self-transferable mobile genetic elements that play a significant role in disseminating antimicrobial resistance between bacteria via horizontal gene transfer. A recently identified ICE in a clinical isolate of Histophilus somni (ICEHs02) is 72 914 base pairs in length and harbours seven predicted antimicrobial resistance genes conferring resistance to tetracycline (tetR-tet(H)), florfenicol (floR), sulfonamide (Sul2), aminoglycosides (APH(3″)-Ib, APH(6)-Id, APH(3')-Ia), and copper (mco). This study investigated ICEHs02 host range, assessed effects of antimicrobial stressors on transfer frequency, and examined effects of ICEHs02 acquisition on hosts. Conjugation assays examined transfer frequency of ICEHs02 to H. somni and Pasteurella multocida strains. Polymerase chain reaction assays confirmed the presence of a circular intermediate, ICE-associated core genes, and cargo genes in recipient strains. Susceptibility testing examined ICEHs02-associated resistance phenotypes in recipient strains. Tetracycline and ciprofloxacin induction significantly increased the transfer rates of ICEHs02 in vitro. The copy numbers of the circular intermediate of ICEHs02 per chromosome exhibited significant increases of ∼37-fold after tetracycline exposure and ∼4-fold after ciprofloxacin treatment. The acquisition of ICEHs02 reduced the relative fitness of H. somni transconjugants (TG) by 28% (w = 0.72 ± 0.04) and the relative fitness of P. multocida TG was decreased by 15% (w = 0.85 ± 0.01).

Biofilm formation of Salmonella enterica serovar Enteritidis cocultured with Acanthamoeba castellanii responds to nutrient availability / La formación de biopelículas de Salmonella enterica serovar Enteritidis cocultivada con Acanthamoeba castellanii responde a la disponibilidad de nutrientes

Acanthamoeba spp. and Salmonella share common habitats, and their interaction may influence the biofilm-forming ability of Salmonella. In this study, biofilm formation of Salmonella enterica serovar Enteritidis cocultured with Acanthamoeba castellanii was examined in nutrient-rich and nutrient-deficient media. Furthermore, transcript copy number of biofilm-related genes in the biofilm cells of S. Enteritidis in monoculture was compared to those in coculture with A. castellanii. Results demonstrated that the presence of A. castellanii in the culture media activates the genes involved in the biofilm formation of S. Enteritidis, regardless of the nutrient availability. However, biofilm formation of S. Enteritidis cocultured with A. castellanii was not consistent with the transcript copy number results. In nutrient-rich medium, the number of Salmonella biofilm cells and the contents of the three main components of the biofilms including eDNA, protein, and carbohydrates were higher in the presence of A. castellanii compared to monocultures. However, in nutrient-deficient medium, the number of biofilm cells, and the amount of biofilm components in coculture conditions were less than the monocultures. These results indicate that despite activation of relevant genes in both nutrient-rich and nutrient-deficient media, biofilm formation of S. Enteritidis cocultured with A. castellanii responds to nutrient availability.(AU)

Biofilm formation of Salmonella enterica serovar Enteritidis cocultured with Acanthamoeba castellanii responds to nutrient availability.

Acanthamoeba spp. and Salmonella share common habitats, and their interaction may influence the biofilm-forming ability of Salmonella. In this study, biofilm formation of Salmonella enterica serovar Enteritidis cocultured with Acanthamoeba castellanii was examined in nutrient-rich and nutrient-deficient media. Furthermore, transcript copy number of biofilm-related genes in the biofilm cells of S. Enteritidis in monoculture was compared to those in coculture with A. castellanii. Results demonstrated that the presence of A. castellanii in the culture media activates the genes involved in the biofilm formation of S. Enteritidis, regardless of the nutrient availability. However, biofilm formation of S. Enteritidis cocultured with A. castellanii was not consistent with the transcript copy number results. In nutrient-rich medium, the number of Salmonella biofilm cells and the contents of the three main components of the biofilms including eDNA, protein, and carbohydrates were higher in the presence of A. castellanii compared to monocultures. However, in nutrient-deficient medium, the number of biofilm cells, and the amount of biofilm components in coculture conditions were less than the monocultures. These results indicate that despite activation of relevant genes in both nutrient-rich and nutrient-deficient media, biofilm formation of S. Enteritidis cocultured with A. castellanii responds to nutrient availability.

Chlorine-stressed Salmonella cells are significantly more engulfed by Acanthamoeba trophozoites and have a longer intracystic survival than the non-stressed cells.

In the present study, the effect of sublethal chlorine-induced oxidative stress on the subsequent interaction of Salmonella enterica serovars Enteritidis and Typhimurium with Acanthamoeba castellanii and A. polyphaga was evaluated. Sublethal chlorine concentration was determined using the lag phase extension information and used to prepare chlorine-stressed Salmonella cells. Coculture experiments of Acanthamoeba and Salmonella cells were performed in Page's amoeba saline (PAS) at 25 °C for 2 h. The results showed that the chlorine-stressed Salmonella cells were significantly more engulfed by A. castellanii and A. polyphaga trophozoites than the non-stressed cells. The uptake rates of the chlorine-stressed and non-stressed Salmonella cells were in the range of 14.17-27.34 and 6.51-11.52% for A. castellanii, and in the range of 8.32-17.76 and 2.28-6.12% for A. polyphaga trophozoites, respectively. Moreover, intracystic survival time of chlorine-stressed cells of S. Enteritidis and S. Typhimurium was significantly longer than that of non-stressed cells. While, non-stressed Salmonella cells survived within A. castellanii and A. polyphaga cysts for 13-20 and 8-15 days, chlorine-stressed cells were recovered from A. castellanii and A. polyphaga cysts after 22-32 and 15-24 days, respectively. These results underscore the importance of bacterial exposure to sublethal chlorine concentrations in their interaction with free-living amoebae, and may lead to a better understanding of the parameters affecting the persistence of Salmonella enterica serovars in food-related environments.

The Role of Whole Genome Sequencing in the Surveillance of Antimicrobial Resistant Enterococcus spp.: A Scoping Review.

Enterococcus spp. have arisen as important nosocomial pathogens and are ubiquitous in the gastrointestinal tracts of animals and the environment. They carry many intrinsic and acquired antimicrobial resistance genes. Because of this, surveillance of Enterococcus spp. has become important with whole genome sequencing emerging as the preferred method for the characterization of enterococci. A scoping review was designed to determine how the use of whole genome sequencing in the surveillance of Enterococcus spp. adds to our knowledge of antimicrobial resistance in Enterococcus spp. Scoping review design was guided by the PRISMA extension and checklist and JBI Reviewer's Guide for scoping reviews. A total of 72 articles were included in the review. Of the 72 articles included, 48.6% did not state an association with a surveillance program and 87.5% of articles identified Enterococcus faecium. The majority of articles included isolates from human clinical or screening samples. Significant findings from the articles included novel sequence types, the increasing prevalence of vancomycin-resistant enterococci in hospitals, and the importance of surveillance or screening for enterococci. The ability of enterococci to adapt and persist within a wide range of environments was also a key finding. These studies emphasize the importance of ongoing surveillance of enterococci from a One Health perspective. More studies are needed to compare the whole genome sequences of human enterococcal isolates to those from food animals, food products, the environment, and companion animals.

Genetic Characterization of AmpC and Extended-Spectrum Beta-Lactamase Phenotypes in Escherichia coli and Salmonella From Alberta Broiler Chickens.

Horizontal gene transfer is an important mechanism which facilitates bacterial populations in overcoming antimicrobial treatment. In this study, a total of 120 Escherichia coli and 62 Salmonella enterica subsp. enterica isolates were isolated from broiler chicken farms in Alberta. Fourteen serovars were identified among Salmonella isolates. Thirty one percent of E. coli isolates (37/120) were multiclass drug resistant (resistant to ≥ 3 drug classes), while only about 16% of Salmonella isolates (10/62) were multiclass drug resistant. Among those, eight E. coli isolates had an AmpC-type phenotype, and one Salmonella isolate had an extended-spectrum beta-lactamase (ESBL)-type beta-lactamase phenotype. We identified both AmpC-type (blaCMY-2) and ESBL-type (blaTEM) genes in both E. coli and Salmonella isolates. Plasmids from eight of nine E. coli and Salmonella isolates were transferred to recipient strain E. coli J53 through conjugation. Transferable plasmids in the eight E. coli and Salmonella isolates were also transferred into a lab-made sodium azide-resistant Salmonella recipient through conjugation. The class 1 integrase gene, int1, was detected on plasmids from two E. coli isolates. Further investigation of class 1 integron cassette regions revealed the presence of an aadA gene encoding streptomycin 3''-adenylyltransferase, an aadA1a/aadA2 gene encoding aminoglycoside 3''-O-adenyltransferase, and a putative adenylyltransferase gene. This study provides some insight into potential horizontal gene transfer events of antimicrobial resistance genes between E. coli and Salmonella in broiler chicken production.

Salmonella spp. prevalence and antimicrobial resistance in broiler chicken and turkey flocks in Canada from 2013 to 2018.

Salmonella infections are a major human health concern. In the elderly and immunocompromised, infections can be life-threatening and may require antibiotic therapy. Where antibiotic therapy is required, antimicrobials of choice include fluoroquinolones and extended-spectrum cephalosporins (ESC). The aim of this study is to utilize data from the Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS) to compare the prevalence of Salmonella serovars between broiler chicken and turkey flocks across Canada, and to gain an understanding of the prevalence of resistance to antimicrobials categorized as important to human health. There were 1,596 Salmonella isolates obtained from 514 broiler chicken flocks, and 659 Salmonella isolates obtained from 217 turkey flocks (2013-2018). All isolates were obtained from pooled faecal samples. Among broiler chicken flocks, the top three serovars were Kentucky (n = 573, 36%), Enteritidis (n = 314, 20%) and Heidelberg (n = 127, 8%). Resistance to ceftriaxone among Salmonella ser. Kentucky decreased from 27% in 2013 to 22% in 2018. There was no resistance among Salmonella ser. Enteritidis reported until 2018 when one isolate from British Columbia was resistant to ampicillin, streptomycin, sulphisoxazole and tetracycline. Salmonella ser. Heidelberg resistance to ceftriaxone decreased from 19% in 2013 to 14% in 2018. Among turkey flocks the top three serovars were Uganda (n = 109, 16.5%), Hadar (n = 85, 12%) and Muenchen (n = 66, 10%). No isolates of Salmonella ser. Uganda or Salmonella ser. Muenchen were resistant to any ß-lactams. Salmonella ser. Hadar (34/81, 42%) exhibited resistance to ampicillin. There was no resistance to quinolones among turkey isolates. Emerging resistance among Salmonella ser. Enteritidis, and resistance to ß-lactams and fluoroquinolones among Salmonella ser. Kentucky from broilers are cause for concern as these classes of antimicrobials are important for treatment of salmonellosis.

A cross-sectional study of the prevalence factors associated with fluoroquinolone resistant Campylobacter jejuni in broiler flocks in Canada.

Campylobacter infections in humans are usually self-limiting; however, antibiotic intervention may be necessary in the case of severe infection. Fluoroquinolones are often the drug of choice for treatment of campylobacteriosis; however, resistance to these drugs can develop rapidly, complicating treatment protocols. Increasing resistance to fluoroquinolones in human infections has coincided with approval of use of fluoroquinolones in animals, therefore, isolation of fluoroquinolone resistant (FQr) Campylobacter in broiler flocks is concerning. This cross-sectional study utilized data collected from 2013-2018 by the Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS) on-farm surveillance program to investigate prevalence factors associated with the isolation of FQr C. jejuni from broiler faecal samples. Mixed effects logistic regression models accounting for clustering of flocks within hatcheries, with and without a fixed effect for the presence of flock level tetracycline resistance were used to assess prevalence factors among 536 C. jejuni isolates from 158 flocks. Both models indicated that the type of bird used (Ross versus Cobb or mixed), the use of virginiamycin as a feed additive, the use of traps to control rodent populations in the barn, and the total number of birds in the barn were significant prevalence factors for increased FQr C. jejuni in a flock. In the model where flock level tetracycline resistance was included as a fixed effect, the odds of FQr C. jejuni increased by 16 (95% CI: 3.74, 68), and the magnitude of the effect of each of the identified prevalence factors was larger. Both models indicated that methods of disinfection of water lines between production cycles is important, with the use of chlorine being protective in the model where tetracycline resistance was included as a fixed effect, and the use of hydrogen peroxide being a risk factor in the model where tetracycline resistance was not included as a fixed effect. The use of hot water to wash the barn between production cycles was also a significant protective factor in the model where tetracycline resistance was not included as a fixed effect. These results indicate that biosecurity and sanitation procedures play a role in the dissemination of FQr C. jejuni in broiler flocks. Future analysis should seek to understand the effect of different disinfectant products on the isolation of FQr C. jejuni. Gaining a better understanding of the management of these critical practices may allow for the reduction of this enteric pathogen in broiler flocks in Canada.

Draft Genome Sequences of 12 Histophilus somni Strains Isolated from Feedlot Cattle in Alberta, Canada.

Histophilus somni is a Gram-negative opportunistic pathogen associated with respiratory disease in cattle. Here, we report the draft genome sequences of 12 Histophilus somni strains isolated from feedlot cattle in Alberta, Canada, which were diagnosed with respiratory disease.

Integrative Conjugative Element ICEHs1 Encodes for Antimicrobial Resistance and Metal Tolerance in Histophilus somni.

The objectives of this study were to determine antimicrobial resistance and metal tolerance, and identify associated genes and mobile genetic elements in clinical strains of Histophilus somni isolated from feedlot cattle in Alberta during years 2012-2016 (contemporary isolates, n = 63) and years 1980-1990 (historical isolates, n = 31). Comparison of antimicrobial resistance (AMR) showed a significant increase in resistance among contemporary isolates compared to historical isolates (P < 0.001). Tolerance to copper (Cu) and zinc (Zn) concentrations above 1 mM was observed in 68 and 52% of the contemporary isolates, respectively. The tet(H) gene associated with oxytetracycline resistance and multicopper oxidase (mco) and cation efflux (czcD) genes associated with Cu and Zn tolerance were identified. An integrative conjugative element; ICEHs1, was identified in whole genome sequences of strains resistant to oxytetracycline, which had Cu and Zn minimum inhibitory concentrations (MIC) >1 mM. The length of ICEHs1 was 64,932 bp and it contained 83 genes, including tetracycline resistance gene tetH, a multidrug efflux pump gene ebrB, and metal tolerance genes mco, czcD, and acr3. Comparative genomics of ICEs revealed that ICEHs1 shares high homology with previously described ICEs of Histophilus somni, Pasteurella multocida, and Mannheimia haemolytica. The ICEHs1 is an active element capable of intra- and inter-genus transfer as demonstrated by successful transfer to H. somni and P. multocida recipients. All isolates carrying ICEHs1 were resistant to tetracycline, a commonly used antibiotic in feedlots, and had Cu and Zn MIC higher than 1 mM. Since Cu and Zn are routinely used in feedlots, there is the possibility of co-selection of AMR in H. somni due to selection pressure created by Cu and Zn. Based on results of in-vitro conjugation experiments, ICEHs1 mediated transmission of antimicrobial and metal resistance genes is possible between BRD pathogens in the respiratory tract, potentially undermining treatment options available for histophilosis and BRD.

Diversity of Antimicrobial Resistance Phenotypes in Salmonella Isolated from Commercial Poultry Farms.

Salmonella remains the leading cause of foodborne illness in the United States, and the dissemination of drug-resistant Salmonellae through the food chain has important implications for treatment failure of salmonellosis. We investigated the ecology of Salmonella in integrated broiler production in order to understand the flow of antibiotic susceptible and resistant strains within this system. Data were analyzed from a retrospective study focused on antimicrobial resistant Salmonella recovered from commercial broiler chicken farms conducted during the initial years of the US FDA's foray into retail meat surveillance by the National Antimicrobial Resistance Monitoring System (NARMS). Sixty-three percentage of Salmonella were pan-susceptible to a panel of 19 antimicrobials used by the NARMS program. Twenty-five antimicrobial resistance phenotypes were observed in Salmonella isolated from two broiler chicken farms. However, Salmonella displaying resistance to streptomycin, alone, and in combination with other antibiotics was the most prevalent (36.3%) antimicrobial resistance phenotype observed. Resistance to streptomycin and sulfadimethoxine appeared to be linked to the transposon, Tn21. Combinations of resistance against streptomycin, gentamicin, sulfadimethoxine, trimethoprim, and tetracycline were observed for a variety of Salmonella enterica serovars and genetic types as defined by pulsed-field gel electrophoresis. There were within and between farm differences in the antibiotic susceptibilities of Salmonella and some of these differences were linked to specific serovars. However, farm differences were not linked to antibiotic usage. Analysis of the temporal and spatial distribution of the endemic Salmonella serovars on these farms suggests that preventing vertical transmission of antibiotic-resistant Salmonella would reduce carcass contamination with antibiotic-resistant Salmonella and subsequently human risk exposure.

Genome Sequences of Two Salmonella enterica Serovar Kentucky Isolates Recovered from Poultry Carcasses in the United States.

We report here the draft genome sequences of two Salmonella enterica serovar Kentucky eBurstGroup 15 isolates collected from poultry carcasses in Georgia (USA).

Evaluation of two quantitative PCR assays using Bacteroidales and mitochondrial DNA markers for tracking dog fecal contamination in waterbodies.

This study describes a comparative performance evaluation of two qPCR assays targeting a dog-associated Bacteroidales 16S rRNA genetic marker (CanBac-UCD) and a dog mitochondrial DNA (mtDNA) marker. The same fecal and environmental samples were assayed for the two markers thereby allowing direct comparison. A wide range of non-target species including, human, pig, horse, deer, mountain goat, bison, caribou, and moose were tested. Marker persistence was also monitored in freshwater microcosms. Both markers were prevalent in the canine samples collected in Regina, Saskatchewan and Calgary, Alberta, Canada (91% and 98% sensitivity, respectively). The mtDNA marker was detected exclusively in the target species while the CanBac-UCD marker was detected in all the non-target species (31% specificity). The CanBac-UCD marker exhibited faster decay in freshwater microcosms. The markers were rarely detected in the water samples collected from dog parks in Calgary and in Regina as well as from waterbodies and sewage influents in Saskatchewan, indicating possibly low to negligible levels of dog fecal contamination in the sampling areas. Altogether, the results of this study support the utility of the dog mtDNA assay in detecting dog fecal contamination in waterbodies.

Comparison of dkgB-linked intergenic sequence ribotyping to DNA microarray hybridization for assigning serotype to Salmonella enterica.

Two DNA-based methods were compared for the ability to assign serotype to 139 isolates of Salmonella enterica ssp. I. Intergenic sequence ribotyping (ISR) evaluated single nucleotide polymorphisms occurring in a 5S ribosomal gene region and flanking sequences bordering the gene dkgB. A DNA microarray hybridization method that assessed the presence and the absence of sets of genes was the second method. Serotype was assigned for 128 (92.1%) of submissions by the two DNA methods. ISR detected mixtures of serotypes within single colonies and it cost substantially less than Kauffmann-White serotyping and DNA microarray hybridization. Decreasing the cost of serotyping S. enterica while maintaining reliability may encourage routine testing and research.

Cross reactive cellular immune responses in chickens previously exposed to low pathogenic avian influenza.

BACKGROUND: Avian influenza (AI) infection in poultry can result in high morbidity and mortality, and negatively affect international trade. Because most AI vaccines used for poultry are inactivated, our knowledge of immunity against AI is based largely on humoral immune responses. In fact, little is known about cellular immunity following a primary AI infection in poultry, especially regarding cytotoxic T lymphocytes (CTL's). METHODS: In these studies, major histocompatibility complex (MHC)-defined (B2/B2) chickens were infected with low pathogenic AI (LPAI) H9N2 and clinical signs of disease were monitored over a two weeks period. Splenic lymphocytes from infected and naïve birds were examined for cross reactivity against homologous and heterologous (H7N2) LPAI by ex vivo stimulation. Cellular immunity was determined by cytotoxic lysis of B2/B2 infected lung target cells and proliferation of T cells following exposure to LPAI. RESULTS: Infection with H9N2 resulted in statistically significant weight loss compared to sham-infected birds. Splenic lymphocytes derived from H9N2-infected birds displayed lysis of both homologous (H9N2) and heterologous (H7N2) infected target cells, whereas lymphocytes obtained from sham-infected birds did not. T cell proliferation was determined to be highest when exposed to the homologous virus. CONCLUSIONS: Taken together these data extend the findings that cellular immunity, including CTL's, is cross reactive against heterologous isolates of AI and contribute to protection following infection.

Mucosal vaccination with a codon-optimized hemagglutinin gene expressed by attenuated Salmonella elicits a protective immune response in chickens against highly pathogenic avian influenza.

The purpose of this study was to evaluate clinical protection from challenge conferred by two attenuated Salmonella enteria serovar typhimurium vaccine strains expressing the hemagglutinin (HA1) gene from a highly pathogenic avian influenza (HPAI) H5N1 (A/whooper swan/Mongolia/3/2005), under control of the anaerobically inducible nir15 promoter. Two-week-old White Leghorn chickens were immunized by oral gavage with one milliliter doses of >109 Salmonella colony-forming units once weekly for 4 weeks prior to challenge. Expression of recombinant protein was confirmed via Western blot. Serum and mucosal gavage samples were collected prior to, and following immunization and antibodies against avian influenza HA were confirmed by Western blot and hemagglutination-inhibition (HI) assay. Chickens were challenged with homologous (A/whooper swan/Mongolia/3/2005), or heterologous (A/Chicken/Queretaro/14588-19/95) HPAI virus strains. Chickens immunized with attenuated Salmonella strains containing plasmid expression vector (pTETnir15HA) demonstrated a statistically significant increase in survival compared to control groups. Results provide evidence of effectiveness of attenuated Salmonella strains for delivery of recombinant avian influenza HA antigens and induction of mucosal and systemic immune responses protective against lethal challenge with HPAI.

Vaccine-induced protection from egg production losses in commercial turkey breeder hens following experimental challenge with a triple-reassortant H3N2 avian influenza virus.

Infections of avian influenza virus (AIV) in turkey breeder hens can cause a decrease in both egg production and quality, resulting in significant production losses. In North Carolina in 2003, a triple-reassortant H3N2 AIV containing human, swine, and avian gene segments was isolated from turkey breeder hens (A/turkey/NC/16108/03). This viral subtype was subsequently isolated from both turkeys and swine in Ohio in 2004, and in Minnesota in 2005, and was responsible for significant losses in turkey production. The objective of this study was to determine if currently available commercial, inactivated avian influenza H3 subtype oil-emulsion vaccines would protect laying turkey hens from egg production losses following challenge with the 2003 H3N2 field virus isolate from North Carolina. Laying turkey hens were vaccinated in the field with two injections of either a commercial monovalent (A/duck/Minnesota/79/79 [H3N4]) or autogenous bivalent (A/turkey/North Carolina/05 (H3N2)-A/turkey/North Carolina/88 [H1N1]) vaccine, at 26 and 30 wk of age, and subsequently challenged under BSL 3-Ag conditions at 32 wk of age. Vaccine-induced efficacy was determined as protection from a 50% decrease in egg production and from a decrease in egg quality within 21 days postchallenge. Results indicate that, following a natural route of challenge (eye drop and intranasal), birds vaccinated with the 2005 North Carolina H3N2 subtype were significantly protected from the drop in egg production observed in both the H3N4 vaccinated and sham-vaccinated hens. The results demonstrate that groups receiving vaccines containing either H3 subtype had a decreased number of unsettable eggs, increased hemagglutination inhibition titers following challenge, and decreased virus isolations from cloacal swabs as compared to the sham-vaccinated group. Phylogenetic analysis of the nucleotide sequence of the HA1 gene segment from the three H3 viruses used in these studies indicated that the two North Carolina turkey isolates had 90.4% similarity in HA1 nucleotide sequence, but had only 77.4% and 76.1% sequence similarity to the HA1 of the H3N4 duck isolate. This study provides the first detailed description of the clinical protection afforded to laying turkey hens by vaccination against challenge with a circulating field isolate of a H3N2 triple-reassortant AIV.

Vertical and horizontal transmission of salmonella within integrated broiler production system.

Salmonella remains one of the leading causes of food-borne illness in the United States, and many key questions regarding the introduction and persistence in animal production systems still remain. In order to understand the ecology of Salmonella within an integrated commercial broiler production system, 289 Salmonella enterica were recovered from two integrated poultry farms during the production and processing of seven consecutive flocks. The variety and prevalence of Salmonella serotypes differed between farms. Overall, 15 serotypes were identified, with the most common being Typhimurium (55%), Montevideo (7.9%), Kentucky (9%), and Enteritidis (9.7%). Salmonella Typhimurium and Enteritidis isolates recovered from processed carcasses from Farm One were further characterized using pulsed-field gel electrophoresis (PFGE), and were shown to be indistinguishable from isolates recovered from the poultry house environment and mice trapped on this farm. Additionally, the same broiler S. Typhimurium and S. Enteritidis strains, identified by PFGE, were also isolated from samples taken at a company breeder farm, suggesting vertical transmission of these Salmonella serotypes in this poultry production system. Results indicate that management practices at the breeder level may have a profound effect on the transmission and persistence of salmonellae within an integrated production system, as well as on the potential contamination of poultry-derived products.

Investigation and control of an outbreak of salmonellosis caused by multidrug-resistant Salmonella typhimurium in a population of hospitalized horses.

An outbreak of salmonellosis in a population of hospitalized horses resulted in the closure of a teaching hospital for a period of 10 weeks. Fecal samples were collected from suspected cases and cultured for Salmonella. Salmonella isolates were characterized using antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE) and phage typing. Thirty-three cases of infection by a multidrug-resistant strain of S. typhimurium were detected. The index case was admitted on 26 August 1999. Fifteen (45%) cases occurred between April and June 2000. PFGE results suggested that this strain of S. typhimurium might have been introduced into the hospital environment by a foal presenting with diarrhea. The hospital was closed on June 13, and intensive environmental cleaning and disinfection were completed. Enforcement of infectious disease control protocols in hospitals and environmental and patient surveillance is needed to prevent outbreaks of salmonellosis.

Application of nested polymerase chain reaction to detection of Salmonella in poultry environment.

Isolation of Salmonella from environmental and processing-plant poultry samples requires the sampling of large numbers of areas within the poultry house or plant. Subsequently, the required number of samples necessitates a large volume of work for a microbiology laboratory, especially when the protocol requires the inclusion of a delayed secondary enrichment for the isolation of Salmonella. This study examined the use of the polymerase chain reaction (PCR) to identify those secondary enrichments containing Salmonella. The unique Salmonella virulence gene invA was chosen as the target for the development of a nested PCR because of its uniform distribution among Salmonella serotypes. The use of nested PCR primers increased the sensitivity of detection 100-fold, resulting in the detection of as few as four cells. There was a strong, statistically significant positive correlation between PCR and culture results as determined by chi-square (P < 0.001) and kappa (kappa = 0.915; excellent agreement) tests. Using PCR to screen primary enrichments for presumptive Salmonella contamination, we improved our efficiency at isolating Salmonella upon secondary enrichment by 20%, and no false negatives were observed. This method will not only validate the use of secondary enrichment procedures but also reduce costs and manpower required for the surveillance of Salmonella.