Experimental Chlorate Research Articles

Evaluation of an Experimental Chlorate Product as a Preslaughter Feed Supplement to Reduce Salmonella in Meat-Producing Birds

A study was conducted to evaluate the effect of experimental chlorate product (ECP) feed supplementation on Salmonella Typhimurium (ST) in the crop and ceca of market-age broilers. In trial 1, 160 market-age broilers were randomly assigned to 8 treatment groups and replicated twice, with 20 broilers per pen for 1 wk. Trial 2 used the same design, but used 80 market-age broilers with 10 broilers per pen. Treatments were as follows: 1) control feed + double-distilled drinking water (dd H2 O); 2) control + 18.5% experimental zeolite carrier with dd H2 O; 3 to 7) control feed supplemented with 0.5, 1.0, 5.0, 10.0, or 18.5% of a feed grade ECP + dd H2 O; 8) control feed + 1× ECP (0.16% w/v; containing 15 mM chlorate ion equivalent) added to dd H2 O. Seven-week-old broilers were provided experimental treatments for 7 d, killed, and then ceca and crops were removed and evaluated for ST. Broilers fed 5 to 18.5% ECP or water ECP had a significantly lower (P < 0.05) incidence of ST in the crop (36 to 38% and 14%, respectively) when compared with the control (60%). Broilers fed 10% ECP or water ECP had significantly lower ST crop concentrations (1.03 log10 and 0.38 log10 ST/g, respectively) when compared with broilers fed a control diet (1.54 log10 ST/g). Crop and ceca ST incidence (32 to 48%) and concentration (1.00 to 1.82 log10 ST/g) were significantly lower in broilers fed 5 to 18.5% ECP as compared with the control (78%; 2.84 log10 ST/g). Broilers fed 5% or greater ECP had significantly higher water consumption (380 to 580 mL water/d) and litter moisture (31 to 56%) when compared with the control (370 mL water/d; 23% moisture). Only broilers fed 18.5% ECP had significantly lower 7-wk BW (2.77 kg of BW) when compared with the controls (3.09 kg of BW). Average daily gains were significantly depressed in broilers fed 10 or 18.5% ECP compared with the controls. These results indicate broilers supplemented with feed ≤ 5% ECP or water ECP 7 d before slaughter reduced ST without affecting growth parameters.

Effects of Nitrate or Nitro Supplementation, with or without Added Chlorate, on Salmonella enterica Serovar Typhimurium and Escherichia coli in Swine Feces

The effects of coincubating the active agent of an experimental chlorate product with nitrate or select nitro compounds, possible inducers and competing substrates for the targeted respiratory nitrate reductase, on concentrations of experimentally inoculated Salmonella enterica serovar Typhimurium and indigenous Escherichia coli were determined. Studies were completed in swine fecal suspensions as a prelude to the administration of these inhibitors to pigs. Results confirmed the bactericidal effect of chlorate (5 to 10 mM) against these fecal enterobacteria, reducing (P < 0.05) concentrations by >2 log CFU ml−1after 3 to 6 h of incubation. An effect (P < 0.05) of pH was observed, with considerable regrowth of Salmonella and E. coli occurring after 24 h of incubation in suspensions buffered to pH 7.1 but not in suspensions buffered to pH 6.5 or 5.6. A 24-h coincubation of fecal suspensions with 5 to 10mM chlorate and as little as 2.5mM nitrate or 10 to 20mM 2-nitro-1-propanol, 2-nitroethanol, and, sometimes, nitroethane decreased (P < 0.05) Salmonella but not necessarily E. coli concentrations. 2-Nitro-1-propanol and 2-nitroethanol exhibited inhibitory activity against Salmonella and E. coli by an undetermined mechanism, even in the absence of added chlorate.

Zoonotic bacterial populations, gut fermentation characteristics and methane production in feedlot steers during oral nitroethane treatment and after the feeding of an experimental chlorate product

Nitroethane inhibits the growth of certain zoonotic pathogens such as Campylobacter and Salmonella spp., foodborne pathogens estimated to cause millions of human infections each year, and enhances the Salmonella- and Escherichia coli-killing effect of an experimental chlorate product being developed as a feed additive to kill these bacteria immediately pre-harvest. Limited studies have shown that nitroethane inhibits ruminal methane production, which represents a loss of 2–12% of the host's gross energy intake and contributes to global warming and destruction of the ozone layer. The present study was conducted to assess the effects of 14-day oral nitroethane administration, 0 (0X), 80 (1X) or 160 (2X) mg nitroethane/kg body weight per day on ruminal and fecal E. coli and Campylobacter, ruminal and fecal methane-producing and nitroethane-reducing activity, whole animal methane emissions, and ruminal and fecal fermentation balance in Holstein steers ( n = 6 per treatment) averaging 403±26 (SD) kg BW. An experimental chlorate product was fed the day following the last nitroethane administration to determine effects on E. coli and Campylobacter. The experimental chlorate product decreased ( P < 0.001 ) fecal, but not ruminal ( P > 0.05 ) E. coli concentrations by 1000- and 10-fold by 24 and 48 h, respectively, after chlorate feeding when compared to pre-treatment concentrations (>5.7 log 10 colony forming units/g). No effects ( P > 0.05 ) of nitroethane or the experimental chlorate product were observed on fecal Campylobacter concentrations; Campylobacter were not recovered from ruminal contents. Nitroethane treatment decreased ( P < 0.01 ) ruminal (8.46, 7.91 and 4.74±0.78 μmol/g/h) and fecal (3.90, 1.36 and 1.38±0.50 μmol/g/h) methane-producing activity for treatments 0X, 1X and 2X, respectively. Administration of nitroethane increased ( P < 0.001 ) nitroethane-reducing activity in ruminal, but not fecal samples. Day of study affected ruminal ( P < 0.0001 ) but not fecal ( P > 0.05 ) methane-producing and nitroethane-reducing activities ( P < 0.01 ); treatment by day interactions were not observed ( P > 0.05 ). Ruminal accumulations of acetate decreased ( P < 0.05 ) in 2X-treated steers when compared with 0X- and 1X-treated steers, but no effect ( P > 0.05 ) of nitroethane was observed on propionate, butyrate or the acetate to propionate ratio. Whole animal methane emissions, expressed as L/day or as a proportion of gross energy intake (%GEI), were unaffected by nitroethane treatment ( P > 0.05 ), and were not correlated ( P > 0.05 ) with ruminal methane-producing activity. These results demonstrate that oral nitroethane administration reduces ruminal methane-producing activity but suggest that a microbial adaptation, likely due to an in situ enrichment of ruminal nitroethane-reducing bacteria, may cause depletion of nitroethane, at least at the 1X administration dose, to concentrations too low to be effective. Further research is warranted to determine if the optimization of dosage of nitroethane or related nitrocompouds can maintain the enteropathogen control and anti-methanogen effect in fed steers.

The Effect of an Experimental Chlorate Product on Salmonella Recovery of Turkeys when Administered Prior to Feed and Water Withdrawal

Previously, an experimental chlorate product (ECP) has been observed to reduce Escherichia coli and Salmonella infections in swine, cattle, and broilers. The following studies were performed to investigate the effects of different concentrations and durations of administering ECP on crop and ceca Salmonella typhimurium (ST) colonization of turkeys. In 2 separate trials, each conducted with 2 replicates, 15-wk-old turkey toms were challenged with 107 to 109 cfu of ST. In Experiment 1, toms were administered 0, 0.5, 1.0, 2.0, or 4.0× of ECP (a 1.0× concentration is equivalent to a 15 mM chlorate ion concentration) in the drinking water for 38 h. In Experiment 2, toms were administered a 2× concentration of ECP in the drinking water for 0, 14, 26, or 38 h prior to water withdrawal. All treatments were followed by a 10-h water withdrawal and an 8-h feed withdrawal prior to organ sampling. In Experiment 1, turkeys provided ECP had significantly (P < 0.05) lower populations and incidences of crop (>1.4 log reduction) and ceca (>0.6 log reduction) ST as compared with control birds (2.1 and 0.94 log ST average for all trials, respectively), with little or no additional benefit from administration of higher ECP concentrations. In Experiment 2, toms provided ECP had lower populations of crop (>2.2 log reduction) and ceca (>1.5 log reduction) ST when compared with controls (3.1 and 1.8 log ST, respectively). Again, there appeared to be little benefit in longer administration intervals on quantitative reduction of ST. These experiments suggest that the ECP significantly reduces Salmonella colonization in commercial turkeys when administered prior to feed and water withdrawal.

Low Level Nitrate or Nitroethane Preconditioning Enhances the Bactericidal Effect of Suboptimal Experimental Chlorate Treatment AgainstEscherichia coliandSalmonellaTyphimurium but NotCampylobacterin Swine

An experimental chlorate product that targets the respiratory nitrate reductase enzyme of bacteria such as Salmonella and Escherichia coli has shown promising results in reducing concentrations of these bacteria in the gut of food animals. Because expression of the target enzyme is induced by nitrate, we administered short-duration, low level nitrate or nitroethane preconditioning treatments to finishing swine to see if these would enhance the ability of an experimental chlorate product to kill these bacteria. Results from these studies showed that preconditioning the gut microflora of swine with low levels of nitrate or nitrocompounds enhanced (more than tenfold) the ability of the chlorate product to kill Salmonella and E. coli, but not Campylobacter. Further studies are needed before these compounds can be fed as feed additives to animals, although it is likely that nitrate preconditioning may be more near to market than the nitrocompounds, which may require more comprehensive review by regulatory authorities.

Evaluation of Salmonella enteritidis in molting hens after administration of an experimental chlorate product (for nine days) in the drinking water and feeding an alfalfa molt diet

The method most commonly used to induce molting and stimulate multiple egg-laying cycles in laying hens for commercial egg production is to fast the hens. Unfortunately, increased risk of Salmonella enteritidis (SE) infection may result from the use of this method. Methods to stimulate multiple egg-laying cycles without increasing the risk of SE infection are needed. Hens over 50 wk of age were divided into 12 groups of 11 hens each and placed in individual laying cages. One week prior to dietary changes, hens were placed on an 8-h light and 16-h dark photoperiod that continued for the 9-d molt. All hens were challenged orally with 106 cfu of SE on the fourth day of the molt. Treatments were nonfed hens with distilled water (NFD), nonfed hens with the experimental chlorate product (ECP, which provided 15 mM chlorate ion) water (NFECP), alfalfa diets with distilled water (ALD), and alfalfa diets with ECP water (ALECP). In the NFD hens, 67% (log10 2.74) of the crops and 94% (log10 5.62) of the ceca were colonized, whereas for the NFECP hens significant reductions to 22% (log10 1.05) of the crops and 61% (log10 2.44) of the ceca were observed. In the ALD hens, 61% (log10 2.52) of the crops and 94% (log10 4.06) of the ceca were colonized. In the ALECP hens, highly significant reductions to 11% (log10 1.26) of the crops and 39% (log10 1.12) of the ceca were observed. When compared with the NFD hens, significant reductions in SE invasion of the ovary, liver, and spleen occurred in all other treatments, except the ovary in the ALD hens. The low alfalfa intake is probably a factor in our lowered protection against SE when compared with previous results. For several parameters, these results suggest that ECP or the combination of ECP and alfalfa may be a useful tool to reduce the risk of SE during an induced molt.

Novel preharvest strategies involving the use of experimental chlorate preparations and nitro-based compounds to prevent colonization of food-producing animals by foodborne pathogens

Foodborne diseases caused by enterohemorrhagic Escherichia coli, Salmonella, and Campylobacter species are of public health and economic significance. Shedding of these pathogens during production and slaughter are risks for contamination of products for human consumption. Consequently, strategies are sought to prevent or reduce the carriage of these pathogens in food animals before slaughter. Experimental products containing chlorate salts have been proven efficacious in reducing concentrations of E. coli and Salmonella Typhimurium in the gut of cattle, sheep, swine, and poultry when administered as feed or water additives. Mechanistically, chlorate selectively targets bacteria expressing respiratory nitrate reductase activity, such as most members of the family Enterobacteriaceae, as this enzyme catalyzes the reduction of chlorate to lethal chlorite. Most beneficial gut bacteria lack respiratory nitrate reductase activity, and thus the technology appears compatible with many bacteria exhibiting competitive exclusion capabilities. More recently, select nitrocompounds have been investigated as potential feed additives, and although these nitrocompounds significantly reduce pathogens on their own, evidence indicates that they may most effectively be used to complement the bactericidal activity of chlorate. A particularly attractive aspect of the nitrocompound technology is that, as potent inhibitors of ruminal methanogenesis, they may allow producers the opportunity to recoup costs associated with their use. At present, neither chlorate nor the nitrocompounds have been approved as feed additives by the US Food and Drug Administration, and consequently they are not yet available for commercial use.

Symposium: Antibiotics in Animal Feeds: Are There Viable Alternatives?

Antibiotic resistance in microorganisms has become a significant problem worldwide. Although use of antimicrobial growth promoters (AGP) remains controversial, the data for implicating a direct link to human health hazards from feeding AGP remain uncertain. Nonetheless, the public sector is building pressure to outlaw the practice. Thus, it seems inevitable that the use of AGP will decline in the future. In view of this situation, this symposium was organized to briefly review the history of the issue, assess the current status, and examine possible AGP alternatives.Dibner and Richards provide readers with a review of AGP use in the commercial animal industry over the years as an overview of the history of legislation in the United States and Europe. In addition, they examine the biological basis for antibiotic effects on animal growth efficiency. Finally, they discuss possible replacement compounds for AGP and conclude that it is “… unlikely that a single replacement will be found that will prove to be economically viable.”By water reviews evidence that antimicrobial use in animal production has resulted in harm to human health and concludes that such evidence remains elusive. The author suggests that well-designed surveillance programs are essential in assessing the qualitative and quantitative risks associated with individual AGP agents.Anderson et al. consider the use of experimental chlorate preparations as a preharvest intervention technology to prevent the colonization of food producing animals with pathogens. Huff and co-authors examine the bacteriophage administration for controlling foodborne pathogens and Escherichia coli infections. Berghman et al. discuss...

Effects of experimental chlorate preparations as feed and water supplements on Escherichia coli colonization and contamination of beef cattle and carcasses

The effects of feed or water administration of experimental chlorate preparations on recovery of generic Escherichia coli and E. coli O157:H7 from the gut, hide and carcasses of feedlot cattle were tested. Sixty-four naturally colonized cattle were randomly assigned to one of four feed treatments with or without a 12 h chlorate-containing water treatment. An analysis of variance revealed a main effect of feed treatment ( P = 0.002 ) on generic E. coli concentrations in feces collected before shipment to slaughter. Main effect means were 5.57, 4.75, 5.00 and 4.08 log 10 cfu/g for animals fed an experimental chlorate product at 0, 0.01% in last meal, 0.01% for last 5 d and 0.05% of body weight in last meal, respectively. A main effect of feed treatment was observed ( P = 0.041 ) on generic E. coli concentrations in feces collected at slaughter (means for the respective treatments were 4.92, 3.57, 3.98 and 3.20 log 10 cfu/g) as well as on numbers of generic E. coli recovered ( P = 0.034 ) from hide swabs collected at the rump (means for the respective treatments were 4.86, 3.92, 3.87 and 4.06 log 10 cfu/swab). A main effect of water treatment was observed ( P < 0.016 ) on generic E. coli concentrations in rumen contents (3.44 vs. 2.72 cfu log 10 cfu/g for animals administered 0 or 2500 ppm active chlorate ion, respectively). Logistic regression analysis revealed a main effect of feed treatment ( P < 0.001 ) on the incidence of E. coli O157:H7 recovered from feces collected at slaughter (75%, 33%, 20% and 25% for animals fed an experimental chlorate product at 0%, 0.01% in last meal, 0.01% for last 5 d and 0.05% of body weight in last meal, respectively). Animals exhibited no symptoms of chlorate toxicity and negative effects on feed or water intake or animal performance were not observed.

Utilization of the nitrate reductase enzymatic pathway to reduce enteric pathogens in chickens

Previous reports have shown that some bacteria, including Salmonella, use a dissimilatory nitrate reductase enzyme pathway (NREP) in anaerobic environments. This enzyme reduces nitrate to nitrite and has been shown to cometabolize chlorate to cytotoxic chlorite. The present investigations were performed to evaluate the susceptibility of a competitive exclusion culture (CE) to the experimental chlorate product (ECP). A commercially available CE product was evaluated for its nitrate reductase activity and therefore its chlorate sensitivity. Individual isolates (in triplicate) were cultured in 10 mL of Viande Levure broth containing 5 mM sodium nitrate or 10 mM sodium chlorate. Bacterial growth (optical density at 625 nm) was measured and 1-mL aliquots were removed concurrently for colorimetric determination of nitrate content at 0, 3, 6, and 24 h. Of the 15 different facultative strains, 11 had slight NREP utilization, 3 had moderate NREP utilization, and the remainder were NREP negative (with slight and moderate NREP utilization: >0.1 to <1.0 mM and >1.0 mM nitrate used within 6 h, respectively). Of the obligate anaerobes evaluated, 3 had slight NREP utilization and the remainder were NREP negative. In vivo studies utilizing both products (CE and ECP) in a horizontal transmission challenge model (seeders + contacts) showed significant reductions in Salmonella from 5.37 to 1.76 log10 cfu/g and 3.94 to 0.07 log10 cfu/g, respectively. The combined effect of the CE culture and an ECP are effective in killing these food-borne pathogens.

Effect of drinking-water administration of experimental chlorate ion preparations on Salmonella enterica serovar Typhimurium colonization in weaned and finished pigs.

Foodborne disease caused by Salmonella is of public health and economic significance. In order to assess the practical effectiveness of a new intervention strategy, experimental chlorate preparations (ECP) were administered via the drinking water to weaned and finished pigs that had been orally challenged the previous day with 10(9)-10(10) colony-forming units of Salmonella serovar Typhimurium. After 24 or 36 h ad libitum access to 0X, 1X or 2X ECP treatment (where X is the concentration estimated to deliver a minimal daily effective dose), the pigs were euthanized and gut contents and lymph tissue collected at necropsy were cultured for the challenge Salmonella. Drinking water administration of ECP effectively reduced (p < 0.05) caecal Salmonella concentrations and, with the weaned pigs, tended (p < or = 0.10) to reduce rectal Salmonella concentrations. No negative effects of ECP treatment on water intake and animal wellbeing were observed and only marginal effects on gut fermentation characteristics occurred. The bactericidal effect of administering ECP in drinking water was relatively rapid, with reductions in caecal Salmonella concentrations occurring within 24 h. These results suggest that ECP administered to pigs just days before slaughter may reduce gut concentrations of Salmonella; however, the impacts of such reductions on slaughter hygiene have yet to be determined.

Effect of experimental chlorate product administration in the drinking water on Salmonella typhimurium contamination of broilers

The crop is a known source of Salmonella and Campylobacter contamination. Previously, we evaluated lactic acid in the drinking water during a simulated pretransport feed withdrawal (FW) and reported 0.44% lactic acid significantly (P < 0.05) reduced the number of Salmonella recovered in market-age broiler crops. However, total consumption of the organic acid-treated drinking water was reduced. Presently, we evaluated the effect of experimental chlorate product (ECP; 1x ECP is equivalent to a 15 mM chlorate ion concentration) during a 10-h pretransport FW. Market-age broilers were obtained from a commercial processing plant and randomly assigned to ECP-treated or control (nontreated) groups. Broilers were challenged by crop gavage with 10(8) Salmonella Typhimurium (ST) immediately upon arrival and 1 d prior to termination of the experiment. One day later, broilers were killed for ST enumeration (cfu) in the crop and ceca. Broilers provided ECP 24 h prior to slaughter consumed slightly more ECP water than broilers provided distilled water. Treatment with ECP caused a significant decrease (P < 0.05) in the incidence of ST in crop contents (2%) as compared to the controls (36.7%). Similarly, ECP treatment caused a significant decrease (P < 0.05) in number of ST (0.96 log10 ST/g cecal content) detected in the ceca when compared to controls (2.52 log10 ST). This study suggested that incorporation of ECP in the drinking water 24 to 48 h prior to slaughter could reduce Salmonella contamination in broilers.

Reduction of E. coli O157:H7 populations in sheep by supplementation of an experimental sodium chlorate product

Ruminant animals are naturally infected with the pathogen Escherichia coli O157:H7, annually responsible for numerous meat recalls, foodborne illnesses and deaths. E. coli are equipped with the enzyme nitrate reductase, which not only enables this bacteria to respire anaerobically, but also converts chlorate to the toxic metabolite chlorite. This enzyme system is particular to only a few intestinal bacteria, therefore the vast majority are not affected by chlorate. Sodium chlorate has been shown to effectively decrease foodborne pathogens in several livestock species, including ruminants. However, because infection and proliferation of E. coli occurs primarily in the lower intestine, there is interest in “by-passing” the rumen, thereby, delivering chlorate directly to the largest population of pathogens. The objective of the current study was to evaluate the ability of an experimental sodium chlorate product (ECP II), designed to by-pass the rumen, in reducing fecal shedding and gut concentrations of E. coli O157:H7. Twenty crossbred mature ewes were adapted to a high grain ration and experimentally inoculated with E. coli O157:H7. Thirty-six hours following inoculation, sheep received in their feed one of the following ECP treatments: (1) control (CON), no chlorate; (2) 1 X (LOW); (3) 2 X (MED); and (4) 4 X (HIGH) where X=1.1 g chlorate ion equivalents/kg BW (five sheep per treatment). Fecal samples were collected every 12 h following inoculation and 24 h following the feeding of chlorate, all animals were euthanized and tissue samples and their respective contents collected from the rumen, cecum and rectum. The MED and HIGH chlorate treatments significantly reduced fecal shedding of E. coli O157:H7 compared to the CON treatment [1.53, 1.11, and 3.89 CFU/g feces (log 10), respectively]. Ruminal contents were similar among treatments, while chlorate tended to decrease ( P=0.08) and reduced ( P<0.05) E. coli O157:H7 populations in the cecum and rectum, respectively. Populations of generic E. coli in the cecal contents were numerically lower ( P=0.11) in the LOW treatment and tended to decrease ( P=0.06) in the MED and HIGH chlorate treatments, respectively. Fermentation profiles through the gastrointestinal tract were unaffected as indicated by slight, but not significant, changes in volatile fatty acids (VFA) profiles in sheep fed chlorate. Results from this study indicate that this experimental chlorate product, administered in the feed, was effective in reducing E. coli O157:H7 from the lower gut of sheep as evidenced by the lower cecal and rectal but not ruminal concentrations. Feeding chlorate may be an effective method to decrease E. coli O157:H7 populations in ruminant animals prior to slaughter.

Reduction of Salmonella Typhimurium in Experimentally Challenged Broilers by Nitrate Adaptation and Chlorate Supplementation in Drinking Water

The effects of two feed supplements on Salmonella Typhimurium in the ceca of market-age broilers were determined. Broilers orally challenged 6 days before slaughter with a novobiocin- and nalidixic acid–resistant strain of Salmonella Typhimurium were divided into one of four groups (20 birds each). The first group (the control group) received no treatment, the second group received sodium nitrate (SN) treatment (574 mg of NaNO3 per kg of feed), the third group received experimental chlorate product (ECP) treatment (15mM NaClO3 equivalents), and the fourth group received ECP treatment in combination with SN treatment. The SN treatment was administered via feed for 5 days immediately before slaughter, and ECP was provided via ad libitum access to drinking water for the last 2 days before slaughter. Cecal contents were subjected to bacterial analysis. Significant (P < 0.05) Salmonella Typhimurium reductions (ca. 2 log units) relative to levels for untreated control broilers were observed for broilers receiving ECP in combination with SN. The ECP-only treatment resulted in significant (P < 0.05) reductions (ca. 0.8 log) of Salmonella Typhimurium in trial 2. We hypothesize that increasing Salmonella Typhimurium nitrate reductase activity resulted in increased enzymatic reduction of chlorate to chlorite, with a concomitant decrease in cecal Salmonella Typhimurium levels. On the basis of these results, preadaptation with SN followed by ECP supplementation immediately preharvest could be a potential strategy for the reduction of Salmonella Typhimurium in broilers.

Individual ionic activities and mass transfer in anodic chlorate formation

The individual activity of hypochlorite ion in concentrated neutral salt (NaCl-NaClO3) solutions has been estimated kinetically by measurement of the Faradaic efficiency in a chlorate producing system comprised of a cell and external reactor, with provision for brine re-circulation. By use of this activity in the diffusion relationships developed by Ibl and Landolt [1–3], reasonable agreement has been achieved between the experimental chlorate yield and the yield expected based on the combined hydrolytic and diffusional flux of active chlorine. In this way it has been shown as expected from basic theory that the gradient of chemical potential rather than the concentration difference represents the driving force of diffusion. A formal activity coefficient for active chlorine species is suggested as being a useful term when considering the chlorate cell system.