Zoonotic Bacterial Pathogens Research Articles

Human and bovine adenoviruses for the detection of source-specific fecal pollution in coastal waters in Australia

In this study, the host-specificity and -sensitivity of human- and bovine-specific adenoviruses (HS-AVs and BS-AVs) were evaluated by testing wastewater/fecal samples from various animal species in Southeast, Queensland, Australia. The overall specificity and sensitivity of the HS-AVs marker were 1.0 and 0.78, respectively. These figures for the BS-AVs were 1.0 and 0.73, respectively. Twenty environmental water samples were collected during wet conditions and 20 samples were colleted during dry conditions from the Maroochy Coastal River and tested for the presence of fecal indicator bacteria (FIB), host-specific viral markers, zoonotic bacterial and protozoan pathogens using PCR/qPCR. The concentrations of FIB in water samples collected after wet conditions were generally higher compared to dry conditions. HS-AVs was detected in 20% water samples collected during wet conditions and whereas BS-AVs was detected in both wet (i.e., 10%) and dry (i.e., 10%) conditions. Both Campylobacter jejuni mapA and Salmonella invA genes detected in 10% samples collected during dry conditions. The concentrations of Salmonella invA ranged between 3.5 × 10 2 and 4.3 × 10 2 genomic copies per 500 ml of water Giardia lamblia β-giardin gene was detected only in one sample (5%) collected during the dry conditions. Weak or significant correlations were observed between FIB with viral markers and zoonotic pathogens. However, during dry conditions, no significant correlations were observed between FIB concentrations with viral markers and zoonotic pathogens. The prevalence of HS-AVs in samples collected from the study river suggests that the quality of water is affected by human fecal pollution and as well as bovine fecal pollution. The results suggest that HS-AVs and BS-AVs detection using PCR could be a useful tool for the identification of human sourced fecal pollution in coastal waters.

Yersinia pseudotuberculosisandY. enterocoliticaInfections, FoodNet, 1996–2007

<i>Yersinia pseudotuberculosis</i>and<i>Y. enterocolitica</i>Infections, FoodNet, 1996–2007

Vaccination with attenuated Salmonella enterica Dublin expressing E coli O157:H7 outer membrane protein Intimin induces transient reduction of fecal shedding of E coli O157:H7 in cattle

BackgroundEscherichia coli serogroup O157:H7 has emerged as an important zoonotic bacterial pathogen, causing a range of symptoms from self-limiting bloody diarrhea to severe hemorrhagic colitis and hemolytic-uremic syndrome in humans. Beef and dairy cattle are considered the most important animal reservoirs for this pathogen. One of the important virulence characteristics of E. coli O157:H7 is the eaeA gene encoding the 97 kDa surface protein intimin. Intimin is required for attachment and effacement during the interaction of enterohemorrhagic E. coli with human and bovine neonatal enterocytes. The present study was undertaken to test the hypothesis that an adaptive mucosal immune response directed against intimin will reduce or prevent enteric colonization and fecal shedding of E. coli O157:H7 in cattle.ResultsCattle were orally inoculated with either milk (control), milk with live attenuated Salmonella enterica serovar Dublin (vector), or milk with live attenuated recombinant S. Dublin expressing intimin (vaccinated) on days 0, 14 and 28. On day 98, all calves were challenged orally with E. coli O157:H7 to evaluate whether vaccination with the recombinant S. Dublin expressing intimin would reduce the level of E. coli O157:H7 fecal shedding.During the first 28 days, vaccinated calves shed both the vector strain and the intimin-expressing S. Dublin strain at a similar level. The vector strain was shed for a significantly longer period as compared to the level of recombinant vaccine strain. Calves that received the intimin-expressed vaccine ceased shedding S. Dublin from day 28 to day 63. All calves were challenged with E. coli O157:H7 on day 98 to determine the effect on fecal shedding of E. coli O157:H7. The amount of E. coli O157:H7 in feces was measured for 30 days post-challenge. We observed a transient clearance of E. coli O157:H7 from the feces in the vaccinated calves. The magnitude of fecal E. coli O157:H7 shedding did not correlate with the presence of intimin-specific fecal IgA.ConclusionOral vaccination with live attenuated recombinant S. Dublin expressing intimin reduced enteric colonization and fecal shedding of E. coli O157:H7. However, the transient clearance of E. coli O157:H7 was not associated with an enhanced IgA-mediated mucosal immune response.

Detection of pathogenic Streptococcus suis bacteria using magnetic glycoparticles

Detection of the zoonotic bacterial pathogen Streptococcus suis was achieved using magnetic glycoparticles. The bacteria contain an adhesion protein for the carbohydrate sequence Galalpha1,4Gal. After incubation with various amounts of the pathogen, magnetic concentration and ATP detection, bacterial levels down to 10(5) cfu could be detected. Submicrometer particles were needed, since with the larger microparticles the method did not succeed.

The occurrence and sources of Campylobacter spp., Salmonellaenterica and Escherichia coli O157:H7 in the Salmon River, British Columbia, Canada

In this study, we wished to assess the prevalence and determine the sources of three zoonotic bacterial pathogens (Salmonella, Campylobacter, and Escherichia coli O157:H7) in the Salmon River watershed in southwestern British Columbia. Surface water, sewage, and animal faecal samples were collected from the watershed. Selective bacterial culture and PCR techniques were used to isolate these three pathogens and indicator bacteria from these samples and characterize them. Campylobacter was the most prevalent pathogen in all samples, followed by Salmonella, and E. coli O157:H7. E. coli O157:H7 and Salmonella isolation rates from water, as well as faecal coliform densities correlated positively with precipitation, while Campylobacter isolation rates correlated negatively with precipitation. Analysis of DNA extracted from water samples for the presence of Bacteroides host-species markers, and comparisons of C. jejuni flaA-RFLP types and Salmonella serovars from faecal and water samples provided evidence that human sewage and specific domestic and wild animal species were sources of these pathogens; however, in most cases the source could not be determined or more than one source was possible. The frequent isolation of these zoonotic pathogens in the Salmon River highlights the risks to human health associated with intentional and unintentional consumption of untreated surface waters.

Evaluation of live-attenuated Salmonella vaccines expressing Campylobacter antigens for control of C. jejuni in poultry

Campylobacter jejuni is a zoonotic bacterial pathogen of worldwide importance. It is estimated that 460,000 human infections occur in the United Kingdom per annum and these involve acute enteritis and may be complicated by severe systemic sequelae. Such infections are frequently associated with the consumption of contaminated poultry meat and strategies to control C. jejuni in poultry are expected to limit pathogen entry into the food chain and the incidence of human disease. Toward this aim, a total of 840 Light Sussex chickens were used to evaluate a Salmonella enterica serovar Typhimurium ΔaroA vaccine expressing the C. jejuni amino acid binding protein CjaA as a plasmid-borne fusion to the C-terminus of fragment C of tetanus toxin. Chickens were given the vaccine at 1-day-old and two weeks later by oral gavage, then challenged after a further two weeks with C. jejuni. Across six biological replicates, statistically significant reductions in caecal C. jejuni of c. 1.4log10 colony-forming units/g were observed at three and four weeks post-challenge relative to age-matched unvaccinated birds. Protection was associated with the induction of CjaA-specific serum IgY and biliary IgA. Protection was not observed using a vaccine strain containing the empty plasmid. Vaccination with recombinant CjaA subcutaneously at the same intervals significantly reduced the caecal load of C. jejuni at three and four weeks post-challenge. Taken together these data imply that responses directed against CjaA, rather than competitive or cross-protective effects mediated by the carrier, confer protection. The impact of varying parameters on the efficacy of the S. Typhimurium ΔaroA vaccine expressing TetC-CjaA was also tested. Delaying the age at primary vaccination had little impact on protection or humoral responses to CjaA. The use of the parent strain as carrier or changing the attenuating mutation of the carrier to ΔspaS or ΔssaU enhanced the protective effect, consistent with increased invasion and persistence of the vaccine strains relative to the ΔaroA mutant. Expression in the ΔaroA strain of a TetC fusion to Peb1A, but not TetC fusions to GlnH or ChuA, elicited protection against intestinal colonisation by C. jejuni that was comparable to that observed with the TetC-CjaA fusion. Our data are rendered highly relevant by use of the target host in large numbers and support the potential of CjaA- and Peb1A-based vaccines for control of C. jejuni in poultry.

Prevalence and occurrence of zoonotic bacterial pathogens in surface waters determined by quantitative PCR

The prevalence and concentrations of Campylobacter jejuni, Salmonella spp. and enterohaemorrhagic Escherichia coli (EHEC) were investigated in surface waters in Brisbane, Australia using quantitative PCR (qPCR) based methodologies. Water samples were collected from Brisbane City Botanic Gardens (CBG) Pond, and two urban tidal creeks (i.e., Oxley Creek and Blunder Creek). Of the 32 water samples collected, 8 (25%), 1 (3%), 9 (28%), 14 (44%), and 15 (47%) were positive for C. jejuni mapA, Salmonella invA, EHEC O157 LPS, EHEC VT1, and EHEC VT2 genes, respectively. The presence/absence of the potential pathogens did not correlate with either E. coli or enterococci concentrations as determined by binary logistic regression. In conclusion, the high prevalence, and concentrations of potential zoonotic pathogens along with the concentrations of one or more fecal indicators in surface water samples indicate a poor level of microbial quality of surface water, and could represent a significant health risk to users. The results from the current study would provide valuable information to the water quality managers in terms of minimizing the risk from pathogens in surface waters.

Francisella-Like Endosymbiont DNA andFrancisella tularensisVirulence-Related Genes in Brazilian Ticks (Acari: Ixodidae)

Ticks are vectors of a variety of pathogens, including Francisella tularensis. Bacteria in the genus Francisella have been identified mostly in the Northern Hemisphere and include tick endosymbionts. Francisella has never been described in Brazil, where Amblyomma spp. ticks are known as the vector of many bacterial zoonotic pathogens. In the present work, we have identified bacterial DNA sequences with identity to Francisella genes in Amblyomma dubitatum Neumann Dermacentor nitens (Neumann), and Rhipicephalus microplus (Canestrini) in Brazil. DNA fragments with homology to Francisella spp. 16S rDNA and the tul4 gene were polymerase chain reaction amplified from tick DNA samples collected in Minas Gerais and Mato Grosso states. These sequences were 96-99% identical to the reported sequences for Francisella-like tick endosymbionts (FLEs). Sequences similar to the tularemia agent F. tularensis pathogenicity island gene iglC and its regulatory gene mglA also were identified in FLEs.

Quinolone resistance in the food chain

Antimicrobials are used in pet animals and in animal husbandry for prophylactic and therapeutic reasons and also as growth promoters, causing selective pressure on bacteria of animal origin. The impact of quinolones or quinolone-resistant bacteria on the management of human infections may be associated with three different scenarios. (i) Quinolone-resistant zoonotic bacterial pathogens are selected and food is contaminated during slaughter and/or preparation. (ii) Quinolone-resistant bacteria non-pathogenic to humans are selected in the animal. When the contaminated food is ingested, the bacteria may transfer resistance determinants to other bacteria in the human gut (commensal and potential pathogens). And (iii) quinolones remain in residues of food products, which may allow the selection of antibiotic-resistant bacteria after the food is consumed. In this review, we analyse the abovementioned aspects, emphasising the molecular basis of quinolone resistance in Escherichia coli, Salmonella spp. and Campylobacter spp.

First Characterization and Emergence of SHV-60 in Raw Milk of a Healthy Cow in Japan

During monitoring of raw milk samples from healthy cows for the presence of antibiotic resistant bacteria, one isolate of Klebsiella pneumoniae strain HUF-100 was found to be resistant to oxyimino-cephalosporins and aztreonam. It was found to carry a chromosomally-encoded extended-spectrum beta-lactamase that has not been described previously, namely SHV-60. Thus, it must be expected that this strain will spread further among food-producing animals and thereby constitute a reservoir of this resistant strain and resistance gene that can transfer to and cause treatment problems for humans. The present study confirms the hypothesis that some of novel multiple antibiotic resistant zoonotic bacterial pathogens may initially emerge from food animals and reports, for the first time, this type of emergence in Japan.

Incidence and Persistence of Zoonotic Bacterial and Protozoan Pathogens in a Beef Cattle Feedlot Runoff Control–Vegetative Treatment System

Determining the survival of zoonotic pathogens in livestock manure and runoff is critical for understanding the environmental and public health risks associated with these wastes. The occurrence and persistence of the bacterial pathogens Escherichia coli O157:H7 and Campylobacter spp. in a passive beef cattle feedlot runoff control-vegetative treatment system were examined over a 26-mo period. Incidence of the protozoans Cryptosporidium spp. and Giardia spp. was also assessed. The control system utilizes a shallow basin to collect liquid runoff and accumulate eroded solids from the pen surfaces; when an adequate liquid volume is attained, the liquid is discharged from the basin onto a 4.5-ha vegetative treatment area (VTA) of bromegrass which is harvested as hay. Basin discharge transported E. coli O157, Campylobacter spp., and generic E. coli into the VTA soil, but without additional discharge from the basin, the pathogen prevalences decreased over time. Similarly, the VTA soil concentrations of generic E. coli initially decreased rapidly, but lower residual populations persisted. Isolation of Cryptosporidium oocysts and Giardia cysts from VTA samples was infrequent, indicating differences in sedimentation and/or transport in comparison to bacteria. Isolation of generic E. coli from freshly cut hay from VTA regions that received basin discharge (12 of 30 vs. 1 of 30 control samples) provided evidence for the risk of contamination; however, neither E. coli O157 or Campylobacter spp. were recovered from the hay following baling. This work indicates that the runoff control system is effective for reducing environmental risk by containing and removing pathogens from feedlot runoff.

Multiple Antimicrobial Resistance in Plague: An Emerging Public Health Risk

Antimicrobial resistance in Yersinia pestis is rare, yet constitutes a significant international public health and biodefense threat. In 1995, the first multidrug resistant (MDR) isolate of Y. pestis (strain IP275) was identified, and was shown to contain a self-transmissible plasmid (pIP1202) that conferred resistance to many of the antimicrobials recommended for plague treatment and prophylaxis. Comparative analysis of the DNA sequence of Y. pestis plasmid pIP1202 revealed a near identical IncA/C plasmid backbone that is shared by MDR plasmids isolated from Salmonella enterica serotype Newport SL254 and the fish pathogen Yersinia ruckeri YR71. The high degree of sequence identity and gene synteny between the plasmid backbones suggests recent acquisition of these plasmids from a common ancestor. In addition, the Y. pestis pIP1202-like plasmid backbone was detected in numerous MDR enterobacterial pathogens isolated from retail meat samples collected between 2002 and 2005 in the United States. Plasmid-positive strains were isolated from beef, chicken, turkey and pork, and were found in samples from the following states: California, Colorado, Connecticut, Georgia, Maryland, Minnesota, New Mexico, New York and Oregon. Our studies reveal that this common plasmid backbone is broadly disseminated among MDR zoonotic pathogens associated with agriculture. This reservoir of mobile resistance determinants has the potential to disseminate to Y. pestis and other human and zoonotic bacterial pathogens and therefore represents a significant public health concern.

Carriage of four bacterial pathogens by beef cattle in Northern Ireland at time of slaughter

To determine the prevalence of four bacterial zoonotic pathogens in beef cattle at time of slaughter in Northern Ireland (NI), in order to assess their potential for reducing beef safety. Faeces were collected postmortem from beef cattle (n =220) at seven EU registered abattoirs. Standard enrichment culturing methods were employed, plus immunomagnetic enrichment in the case of Escherichia coli O157:H7. Campylobacter spp. were found in 52 samples (24.8%), Listeria monocytogenes in 10 (4.8%), E. coli O157:H7 in 2 (0.9%) whilst Salmonella spp. were isolated from six out of 200 samples (3.0%). Five salmonellas were Salmonella Chandans and one was Salmonella Liverpool. Campylobacter spp. were the most frequently isolated pathogen, despite being relatively rare in beef. Genotyping showed the campylobacters to be very diverse, indicating cattle encounter campylobacters from many sources. The remaining three pathogens, which are associated with meats, occurred at relatively low frequencies, especially E. coli O157:H7. The Salmonella serovars found rarely infect humans. The low prevalence of E. coli O157:H7 in NI beef cattle was confirmed and the reasons for this merit further study. The four pathogens should have little impact on beef quality.

Influence of genotype and diet on steer performance, manure odor, and carriage of pathogenic and other fecal bacteria. II. Pathogenic and other fecal bacteria1,2

This study assessed the influence of cattle genotype and diet on the carriage and shedding of zoonotic bacterial pathogens and levels of generic Escherichia coli in feces and ruminal contents of beef cattle during the growing and finishing periods. Fifty-one steers of varying proportions of Brahman and MARC III [0 (15), 1/4 (20), 1/2 (7), and 3/4 Brahman (9)] genotypes were divided among 8 pens, such that each breed type was represented in each pen. Four pens each were assigned to 1 of 2 diets [100% chopped bromegrass hay or a diet composed primarily of corn silage (87%)] that were individually fed for a 119-d growing period, at which time the steers were switched to the same high-concentrate, corn-based finishing diet and fed to a target weight of 560 kg. Feces or ruminal fluid were collected and analyzed at alternating intervals of 14 d or less. Generic E. coli concentrations in feces or ruminal fluid did not differ (P > 0.10) by genotype or by growing diet in the growing or finishing periods. However, the concentrations in both feces and ruminal fluid increased in all cattle when switched to the same high-corn diet in the finishing period. There was no effect (P > 0.25) of diet or genotype during either period on E. coli O157 shedding in feces. Forty-one percent of the steers were positive for Campylobacter spp. at least once during the study, and repeated isolations of Campylobacter spp. from the same steer were common. These repeated isolations from the same animals may be responsible for the apparent diet (P = 0.05) and genotype effects (P = 0.02) on Campylobacter in feces in the finishing period. Cells bearing stx genes were detected frequently in both feces (22.5%) and ruminal fluid (19.6%). The number of stx-positive fecal samples was greater (P < 0.05) for 1/2 Brahman steers (42.9%) than for 1/4 Brahman (25.0%) or 3/4 Brahman steers (22.2%), but were not different compared with MARC III steers (38.3%). The greater feed consumption of 1/2 Brahman and MARC III steers may have resulted in greater starch passage into the colon, accompanied by an increase in fecal bacterial populations, which may have further improved the ability to detect stx genes in these cattle. There was no correlation between either ADG or daily DMI and the number of positive samples of E. coli O157, Campylobacter spp., or stx genes, which agrees with our current understanding that these microorganisms occur commonly in, and with no measurable detriment to, healthy cattle.

Streptobacillus moniliformis: a mouse trying to become a rat

Streptobacillus moniliformis is a rare but emerging zoonotic bacterial pathogen. It causes febrile illness associated with a rash, arthritis, and carditis, typically following rat bites or exposure to food or water contaminated with rat feces. In some cases, a clear exposure history cannot be confirmed. We report a case of sepsis caused by S. moniliformis in a pet owner. Specialized laboratory technique is necessary for the isolation and recognition of S. moniliformis. Rapid diagnostic methods using PCR allow earlier diagnosis and tailored antibiotic therapy for S. moniliformis infections.

Identification of transposon insertion mutants of Francisella tularensis tularensis strain Schu S4 deficient in intracellular replication in the hepatic cell line HepG2

BackgroundFrancisella tularensis is a zoonotic intracellular bacterial pathogen that causes tularemia. The subspecies tularensis is highly virulent and is classified as a category A agent of biological warfare because of its low infectious dose by an aerosol route, and its ability to cause severe disease. In macrophages F. tularensis exhibits a rather novel intracellular lifestyle; after invasion it remains in a phagosome for three to six hours before escaping to, and replicating in the cytoplasm. The molecular mechanisms that allow F. tularensis to invade and replicate within a host cell have not been well defined.MethodsWe constructed a stable transposon mutagenesis library of virulent strain Schu S4 using a derivative of the EZ::TN transposon system®. Approximately 2000 mutants were screened for the inability to invade, and replicate in the hepatic carcinoma cell line HepG2. These mutants were also tested for replication within the J774.1 macrophage-like cell line.ResultsEighteen mutants defective in intracellular replication in HepG2 cells were identified. Eight of these mutants were auxotrophs; seven had mutations in nucleotide biosynthesis pathways. The remaining mutants had insertions in genes that were predicted to encode putative transporters, enzymes involved in protein modification and turnover, and hypothetical proteins. A time course of the intracellular growth of a pyrB mutant revealed that this mutant was only able to grow at low levels within HepG2 cells but grew like wild-type bacteria in J774.1 cells. This pyrB mutant was also attenuated in mice.ConclusionThis is the first reported large-scale mutagenesis of a type A strain of F. tularensis and the first identification of mutants specifically defective in intracellular growth in a hepatic cell line. We have identified several genes and pathways that are key for the survival and growth of F. tularensis in a hepatic cell line, and a number of novel intracellular growth-defective mutants that have not been previously characterized in other pathogens. Further characterization of these mutants will help provide a better understanding of the pathogenicity of F. tularensis, and may have practical applications as targets for drugs or attenuated vaccines.

Molecular mechanisms of resistance in multidrug-resistant serovars of Salmonella enterica isolated from foods in Germany

The objectives of this study were to determine antimicrobial susceptibility and to characterize the molecular mechanisms of multidrug resistance among German food-borne Salmonella isolates of different serovars. A total of 319 epidemiologically independent multidrug-resistant isolates from German foodstuffs comprising 25 different serovars were tested for their antimicrobial susceptibility by broth microdilution. The presence of antimicrobial resistance genes, integrons of classes 1 and 2 and their integrated resistance gene cassettes as well as the Salmonella genomic island 1 (SGI1) was investigated by PCR and DNA sequencing. Localization of integrons and relevant resistance genes was done by Southern hybridization. Sequence analysis revealed mutations in the quinolone resistance-determining region of the gyrA gene. The most prevalent resistances found in the multidrug-resistant serovars of Salmonella enterica from foods were to streptomycin (94%), sulfamethoxazole (92%), tetracycline (81%), ampicillin (73%), spectinomycin (72%), chloramphenicol (48%) and trimethoprim (27%). Twenty-four resistance genes covering six antimicrobial families (beta-lactams, aminoglycosides, phenicols, sulphonamides, tetracycline, and trimethoprim) were identified in the food isolates, many of them integrated as gene cassettes in class 1 and class 2 integrons. Class 1 integrons were detected in 65% of the multidrug-resistant Salmonella isolates comprising 16 different serovars, while class 2 integrons were found in 10% of the isolates belonging to two serovars only. The results demonstrate a clear predominance of both SGI1-borne resistance genes and class 1 integrons in Salmonella serovar Typhimurium DT104 and of class 2 integrons in Salmonella serovar Paratyphi B (d-tartrate positive). Nalidixic acid resistance found in 15% of the isolates was associated with single mutations in the gyrA gene. This study confirms the role of foods of animal and other origin as a reservoir of multidrug-resistant Salmonella and underlines the need for continuing surveillance of food-borne zoonotic bacterial pathogens along the food chain.

Mechanisms of quinolone resistance in Escherichia coli and Salmonella: Recent developments

Fluoroquinolones are broad-spectrum antimicrobials highly effective for treatment of a variety of clinical and veterinary infections. Their antibacterial activity is due to inhibition of DNA replication. Usually resistance arises spontaneously due to point mutations that result in amino acid substitutions within the topoisomerase subunits GyrA, GyrB, ParC or ParE, decreased expression of outer membrane porins, or overexpression of multidrug efflux pumps. In addition, the recent discovery of plasmid-mediated quinolone resistance could result in horizontal transfer of fluoroquinolone resistance between strains. Acquisition of high-level resistance appears to be a multifactorial process. Care needs to taken to avoid overuse of this important class of antimicrobial in both human and veterinary medicine to prevent an increase in the occurrence of resistant zoonotic and non-zoonotic bacterial pathogens that could subsequently cause human or animal infections.

Use of Transposon-Transposase Complexes To Create Stable Insertion Mutant Strains of Francisella tularensis LVS

Francisella tularensis is a highly virulent zoonotic bacterial pathogen capable of infecting numerous different mammalian species, including humans. Elucidation of the pathogenic mechanisms of F. tularensis has been hampered by a lack of tools to genetically manipulate this organism. Herein we describe the use of transposome complexes to create insertion mutations in the chromosome of the F. tularensis live vaccine strain (LVS). A Tn5-derived transposon encoding kanamycin resistance and lacking a transposase gene was complexed with transposase enzyme and transformed directly into F. tularensis LVS by electroporation. An insertion frequency of 2.6 x 10(-8) +/- 0.87 x 10(-8) per cell was consistently achieved using this method. There are 178 described Tn5 consensus target sites distributed throughout the F. tularensis genome. Twenty-two of 26 transposon insertions analyzed were within known or predicted open reading frames, but none of these insertions was associated with the Tn5 target site. Analysis of the insertions of sequentially passed strains indicated that the transposons were maintained stably at the initial insertion site after more than 270 generations. Therefore, transformation by electroporation of Tn5-based transposon-transposase complexes provided an efficient mechanism for generating random, stable chromosomal insertion mutations in F. tularensis.

Recent pre-harvest supplementation strategies to reduce carriage and shedding of zoonotic enteric bacterial pathogens in food animals.

Food-borne bacterial illnesses strike more than 76 million North Americans each year. Many of these illnesses are caused by animal-derived foodstuffs. Slaughter and processing plants do an outstanding job in reducing bacterial contamination after slaughter and during further processing, yet food-borne illnesses still occur at an unacceptable frequency. Thus, it is imperative to widen the window of action against pathogenic bacteria. Attacking pathogens on the farm or in the feedlot will improve food safety all the way to the consumer's fork. Because of the potential improvement in overall food safety that pre-harvest intervention strategies can provide, a broad range of preslaughter intervention strategies are currently under investigation. Potential interventions include direct anti-pathogen strategies, competitive enhancement strategies and animal management strategies. Included in these strategies are competitive exclusion, probiotics, prebiotics, antibiotics, antibacterial proteins, vaccination, bacteriophage, diet, and water trough interventions. The parallel and simultaneous application of one or more preslaughter strategies has the potential to synergistically reduce the incidence of human food-borne illnesses by erecting multiple hurdles, thus preventing entry of pathogens into the food chain. This review emphasizes work with Escherichia coli O157:H7 to illustrate the various strategies.