David J. Nisbet

Evaluation of bacterial diversity in the rumen and feces of cattle fed different levels of dried distillers grains plus solubles using bacterial tag-encoded FLX amplicon pyrosequencing.

Dietary components and changes cause shifts in the gastrointestinal microbial ecology that can play a role in animal health and productivity. However, most information about the microbial populations in the gut of livestock species has not been quantitative. In the present study, we utilized a new molecular method, bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP) that can perform diversity analyses of gastrointestinal bacterial populations. In the present study, cattle (n = 6) were fed a basal feedlot diet and were subsequently randomly assigned to 1 of 3 diets (n = 2 cows per diet). In each diet, 0, 25, or 50% of the concentrate portion of the ration was replaced with dried distillers grain (DDGS). Ruminal and fecal bacterial populations were different when animals were fed DDGS compared with controls; ruminal and fecal Firmicute:Bacteroidetes ratios were smaller (P = 0.07) in the 25 and 50% DDG diets compared with controls. Ruminal pH was decreased (P < 0.05) in ruminal fluid from cattle fed diets containing 50% compared with 0% DDGS. Using bTEFAP, the normal microbiota of cattle were examined using modern molecular methods to understand how diets affect gastrointestinal ecology and the gastrointestinal contribution of the microbiome to animal health and production.

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.

Probiotics, prebiotics and competitive exclusion for prophylaxis against bacterial disease

Abstract The microbial population of the intestinal tract is a complex natural resource that can be utilized in an effort to reduce the impact of pathogenic bacteria that affect animal production and efficiency, as well as the safety of food products. Strategies have been devised to reduce the populations of food-borne pathogenic bacteria in animals at the on-farm stage. Many of these techniques rely on harnessing the natural competitive nature of bacteria to eliminate pathogens that negatively impact animal production or food safety. Thus feed products that are classified as probiotics, prebiotics and competitive exclusion cultures have been utilized as pathogen reduction strategies in food animals with varying degrees of success. The efficacy of these products is often due to specific microbial ecological factors that alter the competitive pressures experienced by the microbial population of the gut. A few products have been shown to be effective under field conditions and many have shown indications of effectiveness under experimental conditions and as a result probiotic products are widely used in all animal species and nearly all production systems. This review explores the ecology behind the efficacy of these products against pathogens found in food animals, including those that enter the food chain and impact human consumers.

Effect of supplementation of prebiotic mannan-oligosaccharides and probiotic mixture on growth performance of broilers subjected to chronic heat stress

The present study was aimed at elucidating the effects of supplementing mannan-oligosaccharides (MOS) and probiotic mixture (PM) on growth performance, intestinal histology, and corticosterone concentrations in broilers kept under chronic heat stress (HS). Four hundred fifty 1-d-old chicks were divided into 5 treatment groups and fed a corn-soybean diet ad-libitum. The temperature control (CONT) group was held at the normal ambient temperature. Heat stress broilers were held at 35 ± 2°C from d 1 until the termination of the study at d 42. Heat stress groups consisted of HS-CONT fed the basal diet; HS-MOS fed the basal diet containing 0.5% MOS; HS-PM fed the basal diet containing 0.1% PM; and HS-SYN (synbiotic) fed 0.5% MOS and 0.1% PM in the basal diet. Broilers were examined at d 21 and 42 for BW gain, feed consumption, feed conversion ratio (FCR), serum corticosterone concentrations, and ileal microarchitecture. The results revealed that the CONT group had higher (P < 0.01) feed consumption, BW gain, and lower FCR on d 21 and 42, compared with the HS-CONT group. Among supplemented groups, the HS-MOS had higher (P < 0.05) BW gain and lower FCR compared with the HS-CONT group. On d 21 and 42, the HS-CONT group had higher (P < 0.05) serum corticosterone concentrations compared with the CONT and supplemented groups. The CONT group had higher (P < 0.05) villus height, width, surface area, and crypt depth compared with the HS-CONT group. On d 21, the HS-PM had higher (P < 0.05) villus width and surface area compared with HS-CONT group. On d 42, the HS-SYN had higher (P < 0.05) villus width and crypt depth compared with the HS-CONT group. These results showed that chronic HS reduces broiler production performance, intestinal microarchitecture, and increases adrenal hormone concentrations. Also, supplementation of the MOS prebiotic and the PM can partially lessen these changes.

Foodborne Salmonella ecology in the avian gastrointestinal tract

Foodborne Salmonella continues to be a major cause of salmonellosis with Salmonella Enteritidis and S. Typhimurium considered to be responsible for most of the infections. Investigation of outbreaks and sporadic cases has indicated that food vehicles such as poultry and poultry by-products including raw and uncooked eggs are among the most common sources of Salmonella infections. The dissemination and infection of the avian intestinal tract remain somewhat unclear. In vitro incubation of Salmonella with mammalian tissue culture cells has shown that invasion into epithelial cells is complex and involves several genetic loci and host factors. Several genes are required for the intestinal phase of Salmonella invasion and are located on Salmonella pathogenicity island 1 (SPI 1). Salmonella pathogenesis in the gastrointestinal (GI) tract and the effects of environmental stimuli on gene expression influence bacterial colonization and invasion. Furthermore, significant parameters of Salmonella including growth physiology, nutrient availability, pH, and energy status are considered contributing factors in the GI tract ecology. Approaches for limiting Salmonella colonization have been primarily based on the microbial ecology of the intestinal tract. In vitro studies have shown that the toxic effects of short chain fatty acids (SCFA) to some Enterobacteriaceae, including Salmonella, have resulted in a reduction in population. In addition, it has been established that native intestinal microorganisms such as Lactobacilli provide protective mechanisms against Salmonella in the ceca. A clear understanding of the key factors involved in Salmonella colonization in the avian GI tract has the potential to lead to better approach for more effective control of this foodborne pathogen.

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).

Defined competitive exclusion cultures in the prevention of enteropathogen colonisation in poultry and swine.

A competitive exclusion culture (CE) containing a mixture of 29 different bacterial isolates obtained from the cecae of broiler chickens was developed utilizing continuous-flow culture techniques. This culture (CF3) has been efficacious in controlling gut colonization by enteropathogens in both experimentally infected broilers and under commercial field conditions. In day-old broiler chicks provided CF3, and challenged with 10,000 CFU Salmonella typhimurium greater than a 99% reduction in Salmonella cecal colonization levels was observed compared to control chicks. Similarly, CF3 has also been shown to protect experimentally infected broiler chicks from cecal colonization by S. enteritidis (Phage types 4 and 13), S. gallinarum, Listeria Monocytogenes, and Escherichia coli O157:H7. A commercial product was developed from CF3 and is sold under the tradename PREEMPT™. In a Food and Drug Administration approved, double blinded, pivotal field trial, chicks treated with PREEMPTT™ had significantly fewer salmonellae than untreated chicks at end-of-growout. This product is the first of its kind available to the U.S. poultry industry. Using similar technology a product has also been developed that decreases shedding of salmonellae in neonate and weaned pigs, and also has been shown to reduce mortality associated with enteropathogens in young pigs both in the laboratory and in a commercial swine herd.

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.

Characterization of Chloramphenicol Resistance in Beta-Hemolytic Escherichia coli Associated with Diarrhea in Neonatal Swine

ABSTRACT Ninety beta-hemolytic Escherichia coli isolates associated with diarrhea in neonatal pigs from multiple farms in Oklahoma were investigated for known associated disease serotypes, virulence factors, ribotypes, and antimicrobial susceptibility phenotypes. Fifteen different serotypes were observed, with 58% of isolates belonging to groups that produce one of three major enterotoxins: O149, O147, and O139. Thirty percent of the swine E. coli isolates possessed a combination of F4 fimbriae and the heat-labile toxin and heat-stable toxin B enterotoxins. Seventy-three percent of the E. coli isolates were resistant to five or more antibiotics. Interestingly, 53% of swine E. coli isolates exhibited resistance to chloramphenicol (CHL), an antibiotic whose use in food animals has been prohibited in the United States since the mid-1980s. The cmlA gene, which encodes a putative CHL efflux pump, was detected by PCR in 47 of the 48 CHL-resistant isolates, and 4 of these also possessed the cat2 gene, which encodes a chloramphenicol acetyltransferase. The one CHL-resistant isolate that did not contain either cmlA or cat-2 possessed the flo gene, which confers resistance to both florfenicol and CHL. To determine whether CHL-resistant swine E. coli isolates represented dissemination of a clonal strain, all 90 isolates were analyzed by ribotyping. Seventeen distinct E. coli ribogroups were identified, with CHL resistance observed among the isolates in all except one of the major ribogroups. The identification of the cmlA gene among diverse hemolytic enterotoxigenic E. coli strains demonstrates its broad dissemination in the swine production environment and its persistence even in the absence of CHL selection pressure.