Robin C. Anderson

Incidence and ecology of Campylobacter jejuni and coli in animals

Since its initial emergence in the 1970s, Campylobacter has become one of the most common causative agents of bacterial foodborne illness. Campylobacter species readily colonize the gastrointestinal tracts of domestic, feral and wild animals and while they rarely cause clinical disease in food animals, they can produce severe acute gastroenteritis in humans. Prevalence of Campylobacter in food animals can exceed 80% thus challenging processors to employ post-harvest pathogen reduction strategies. Reduction of pathogens before arrival to the abattoir is also of interest because the implementation of pre-harvest interventions may compliment existing post-harvest control techniques to further diminish possible retail sources of infection. Such multiple hurdle approaches that simultaneously utilize pre- and post-harvest control techniques are expected to be the most effective approach for decreasing human illness associated with foodborne pathogens.

Bacteriophage isolated from feedlot cattle can reduce Escherichia coli O157:H7 populations in ruminant gastrointestinal tracts.

Escherichia coli O157:H7 can live undetected in the gut of food animals and be spread to humans directly and indirectly. Bacteriophages are viruses that prey on bacteria, offering a natural, nonantibiotic method to reduce pathogens from the food supply. Here we show that a cocktail of phages isolated from commercial cattle feces reduced E. coli O157:H7 populations in the gut of experimentally inoculated sheep. A cocktail of phages was used in order to prevent the development of resistance to the phages. In our first in vivo study we found that our cocktail of phages reduced E. coli O157:H7 populations in the feces of sheep (p < 0.05) by 24 hours after phage treatment. Upon necropsy, populations of inoculated E. coli O157:H7 were reduced by phage treatment in both the cecum (p < 0.05) and rectum (p < 0.1). In our second in vivo study, several ratios of phage plaque-forming units (PFU) to E. coli O157:H7 colony-forming units (CFU) were used (0:1, 1:1, 10:1, and 100:1 PFU/CFU) to determine the most efficacious phage dose. A 1:1 ratio of phage to bacteria was found to be more effective (p < 0.05) than either of the higher ratios used (10:1 or 100:1). Ruminal levels of E. coli O157:H7 were not significantly reduced (p > 0.10) in any of the studies due to relatively low inoculated E. coli O157:H7 ruminal populations. Our results demonstrate that phage can be used as a preharvest intervention as part of an integrated pathogen reduction scheme.

Bactericidal effect of sodium chlorate on Escherichia coli O157:H7 and Salmonella Typhimurium DT104 in rumen contents in vitro.

Escherichia coli O157:H7 and Salmonella Typhimurium DT104 are important foodborne pathogens affecting the beef and dairy industries and strategies are sought to rid these organisms from cattle at slaughter. Both pathogens possess respiratory nitrate reductase that also reduces chlorate to the lethal chlorite ion. Because most anaerobes lack respiratory nitrate reductase, we hypothesized that chlorate may selectively kill E. coli O157:H7 and Salmonella Typhimurium DT104 but not potentially beneficial anaerobes. In support of this hypothesis, we found that concentrations of E. coli O157:H7 and Salmonella Typhimurium DT104 were reduced from approximately 1,000,000 colony forming units (CFU) to below our level of detection (< or = 10 CFU) following in vitro incubation (24 h) in buffered ruminal contents (pH 6.8) containing 5 mM added chlorate. In contrast, chlorate had little effect on the most probable number (mean +/- SD) of total culturable anaerobes (ranging from 9.9 +/- 0.72 to 10.7 +/- 0.01 log10 cells/ml). Thus, chlorate was bactericidal to E. coli O157:H7 and Salmonella Typhimurium DT104 but not to potentially beneficial bacteria. The bactericidal effect of chlorate was concentration dependent (less at 1.25 mM) and markedly affected by pH (more bactericidal at pH 6.8 than pH 5.6).

Foodborne Campylobacter: Infections, Metabolism, Pathogenesis and Reservoirs

Campylobacter species are a leading cause of bacterial-derived foodborne illnesses worldwide. The emergence of this bacterial group as a significant causative agent of human disease and their propensity to carry antibiotic resistance elements that allows them to resist antibacterial therapy make them a serious public health threat. Campylobacter jejuni and Campylobacter coli are considered to be the most important enteropathogens of this genus and their ability to colonize and survive in a wide variety of animal species and habitats make them extremely difficult to control. This article reviews the historical and emerging importance of this bacterial group and addresses aspects of the human infections they cause, their metabolism and pathogenesis, and their natural reservoirs in order to address the need for appropriate food safety regulations and interventions.

Effect of sodium chlorate on Salmonella Typhimurium concentrations in the weaned pig gut

Salmonella cause economic losses to the swine industry due to disease and compromised food safety. Since the gut is a major reservoir for Salmonella, strategies are sought to reduce their concentration in pigs immediately before processing. Respiratory nitrate reductase activity possessed by Salmonella also catalyzes the intracellular reduction of chlorate (an analog of nitrate) to chlorite, which is lethal to the microbe. Since most gastrointestinal anaerobes lack respiratory nitrate reductase, we conducted a study to determine if chlorate may selectively kill Salmonella within the pig gut. Weaned pigs orally infected with 8 x 10(7) CFU of a novobiocin- and nalidixic acid-resistant strain of Salmonella Typhimurium were treated 8 and 16 h later via oral gavage (10 ml) with 0 or 100 mM sodium chlorate. Pigs were euthanized at 8-h intervals after receiving the last treatment. Samples collected by necropsy were cultured qualitatively and quantitatively for Salmonella and for most probable numbers of total culturable anaerobes. A significant (P < 0.05) chlorate treatment effect was observed on cecal concentrations of Salmonella, with the largest reductions occurring 16 h after receiving the last chlorate treatment. An observed treatment by time after treatment interaction suggests the chlorate effect was concentration dependent. Chlorate treatment may provide a means to reduce foodborne pathogens immediately before harvest.

Identification and Quantification of Methanogenic Archaea in Adult Chicken Ceca

ABSTRACT By using molecular methods for the identification and quantification of methanogenic archaea in adult chicken ceca, 16S rRNA genes of 11 different phylotypes, 10 of which were 99% similar to Methanobrevibacter woesei, were found. Methanogen populations, as assessed by cultivation, and the 16S rRNA copy number were between 6.38 and 8.23 cells/g (wet weight) and 5.50 and 7.19 log10/g (wet weight), respectively.

Escherichia coli O157:H7 populations in sheep can be reduced by chlorate supplementation

Ruminant animals are a natural reservoir of the foodborne pathogen Escherichia coli O157:H7. Some foodborne pathogens (e.g., E. coli) are equipped with a nitrate reductase that cometabolically reduces chlorate. The intracellular reduction of chlorate to chlorite kills nitrate reductase-positive bacteria; however, species that do not reduce nitrate are not affected by chlorate. Therefore, it has been suggested that ruminants be supplemented with chlorate prior to shipment for slaughter in order to reduce foodborne illnesses in human consumers. Sheep (n = 14) were fed a high-grain ration and were experimentally infected with E. coli O157:H7. These sheep were given an experimental product (XCP) containing the equivalent of either 2.5 mM NaNO3 and 100 mM NaCl (control sheep; n = 7) or 2.5 mM NaNO3 and 100 mM NaClO3 (chlorate [XCP]-treated sheep; n = 7). Control and XCP-treated sheep were treated for 24 h; XCP treatment reduced the population of inoculated E. coli O157:H7 (P < 0.05) from 10(2), 10(5), and 10(5) CFU/g in the rumen, cecum, and rectum, respectively, to < 10(1) CFU/g in all three sections of the gastrointestinal tract. The number of sheep testing positive for E. coli O157:H7 was significantly reduced by XCP treatment. In a similar fashion, total E. coli and coliforms were also reduced (P < 0.05) in all three compartments of the intestinal tract. Intestinal pH, total volatile fatty acid production, and the acetate/propionate ratio were unaffected by XCP treatment. On the basis of these results, it appears that chlorate treatment can be an effective method for the reduction of E. coli O157:H7 populations in ruminant animals immediately prior to slaughter.

Effect of select nitrocompounds on ruminal fermentation; an initial look at their potential to reduce economic and environmental costs associated with ruminal methanogenesis

Methane production by ruminal microbes during the digestion of feedstuffs is an inefficient process resulting in losses of 2-12% of the gross energy consumed by ruminants. Presently, we report the effect of three inhibitors on ruminal methane production in vitro. Mixed populations of ruminal microbes collected from cannulated cows maintained on an alfalfa hay:corn diet (50:50) were incubated at 39 degrees C for 24 h under a 100% carbon dioxide gas phase in closed tubes with 72 mM added sodium formate. Cultures were supplemented with 12 mM 2-nitropropanol, nitroethane or nitroethanol (experiment 1) or with 2, 12 or 24 mM nitroethane or a combination of 12 mM nitroethane and 4 mM nitroethanol (experiment 2). Control cultures containing no added nitrocompound were incubated simultaneously with treated incubations. Methane concentrations were reduced (P<0.05) from those measured in control incubations (27.6 +/- 2.1 and 17.7 +/- 0.8 micromol/ml; mean +/- SD for experiments 1 and 2, respectively) by at least 57% and as much as 94% in the nitrocompound supplemented incubations. By comparison, the widely fed methane inhibitor, monensin, typically reduces ruminal methane production by about 33%. Concentrations of volatile fatty acids and ammonia that accumulated in the nitrocompound supplemented incubations were not markedly affected compared to those produced by control cultures despite the reductions in methane produced. Hydrogen accumulated only slightly in cultures supplemented with the nitrocompounds. These results demonstrate that 2-nitropropanol, nitroethane and nitroethanol inhibit ruminal methane production. Further research is warranted to determine the mechanisms responsible for this inhibition and to see if these inhibitors can be used in practical application to reduce economic and environmental costs associated with ruminal methanogenesis.

Evaluation of feeding glycerol on free-fatty acid production and fermentation kinetics of mixed ruminal microbes in vitro

Ruminant-derived foods contain high proportions of saturated fats as a result of ruminal biohydrogenation that rapidly saturates and thus limits the availability of free unsaturated fatty acids for assimilation. The objective of this study was to evaluate the effects of glycerol on ruminal free-fatty acid (FFA) production rates and in vitro fermentation kinetics of alfalfa hay. In vitro incubations demonstrated 48% and 77% reductions in rates of FFA accumulation in incubations supplemented with 2% and 20% glycerol as compared to controls. In vitro incubations with alfalfa hay demonstrated that increasing levels of glycerol did not affect NDF digestibility of the hay. Additionally, increasing amounts of glycerol decreased the acetate to propionate ratio in the rumen. These results suggest that inhibiting bacterial fat degradation may promote ruminal passage of total lipid, thereby providing greater proportions of beneficial unsaturated fat for incorporation into beef products.

Denitrobacterium detoxificans gen. nov., sp. nov., a ruminal bacterium that respires on nitrocompounds

A new group of anaerobic, Gram-positive, high G + C (56-60 mol%) bacteria was isolated from the bovine rumen. Of four strains characterized, all were non-motile and none produced spores. The isolates did not produce indole or H2S and did not hydrolyse gelatin. Cells of each strain exhibited similar rod-shaped morphology (0.5-1.0 x 1.0-1.5 microns) although bulbous ends were sometimes present. None of the four strains were able to grow via oxidation of a variety of potentially fermentable substrates but rather obtained energy for growth via anaerobic respiration processes, oxidizing hydrogen, formate or lactate for reduction of various oxidized nitrogen compounds. Trimethylamine oxide and DMSO were also used as electron acceptor. All four strains shared greater than 99% 16S rRNA gene sequence identity. The closest match found between the 16S rRNA gene sequence of all four strains, NPOH1T, NPOH2, NPOH3 and MAJ1, to sequences available in GenBank was that of Coriobacterium glomerans (86% sequence similarity), a phenotypically dissimilar anaerobe within the class Actinobacteria. To accommodate these bacteria the creation of a new genus and species, Denitrobacterium detoxificans, for placement within the family Coriobacteriaceae is proposed. The type strain, NPOH1T (ATCC 700546T), grew equally well over a narrow range of incubation temperatures tested (32-39 degrees C).