Effects in air-exposed corn silage of medium chain fatty acids on select spoilage microbes, zoonotic pathogens, and in vitro rumen fermentation.

Medium chain fatty acid (MCFA) treatment (0.75% C6, hexanoic; C8, octanoic; C10, decanoic; or equal proportion mixtures of C6:C8:C10:C12 or C8:C10/g; C12 = dodecanoic acid) of aerobically-exposed corn silage on spoilage and pathogenic microbes and rumen fermentation were evaluated in vitro. After 24 h aerobic incubation (37 °C), microbial enumeration revealed 3 log10 colony-forming units (CFU)/g fewer (P = 0.03) wild-type yeast and molds in C8:C10-treated silage than controls. Compared with controls, wild-type enterococci decreased (P < 0.01) in all treatments except the C6:C8:C10:C12 mixture; lactic acid bacteria were decreased (P < 0.01) in all treatments except C6 and the C6:C8:C10:C12 mixture. Total aerobes and inoculated Staphylococcus aureus or Listeria monocytogenes were unaffected by treatment (P > 0.05). Anaerobic incubation (24 h at 39 °C) of ruminal fluid (10 mL) with 0.02 g overnight air-exposed MCFA-treated corn silage revealed higher hydrogen accumulations (P = 0.03) with the C8:C10 mixture than controls. Methane, acetate, propionate, butyrate, or estimates of fermented hexose were unaffected. Acetate:propionate ratios were higher (P < 0.01) and fermentation efficiencies were marginally lower (P < 0.01) with C8- or C8:C10-treated silage than controls. Further research is warranted to optimize treatments to target unwanted microbes without adversely affecting beneficial microbes.

High-Resolution Genomic Comparisons within Salmonella enterica Serotypes Derived from Beef Feedlot Cattle: Parsing the Roles of Cattle Source, Pen, Animal, Sample Type, and Production Period.

Salmonella enterica is a major foodborne pathogen, and contaminated beef products have been identified as one of the primary sources of Salmonella-related outbreaks. Pathogenicity and antibiotic resistance of Salmonella are highly serotype and subpopulation specific, which makes it essential to understand high-resolution Salmonella population dynamics in cattle. Time of year, source of cattle, pen, and sample type (i.e., feces, hide, or lymph nodes) have previously been identified as important factors influencing the serotype distribution of Salmonella (e.g., Anatum, Lubbock, Cerro, Montevideo, Kentucky, Newport, and Norwich) that were isolated from a longitudinal sampling design in a research feedlot. In this study, we performed high-resolution genomic comparisons of Salmonella isolates within each serotype using both single-nucleotide polymorphism-based maximum-likelihood phylogeny and hierarchical clustering of core-genome multilocus sequence typing. The importance of the aforementioned features in clonal Salmonella expansion was further explored using a supervised machine learning algorithm. In addition, we identified and compared the resistance genes, plasmids, and pathogenicity island profiles of the isolates within each subpopulation. Our findings indicate that clonal expansion of Salmonella strains in cattle was mainly influenced by the randomization of block and pen, as well as the origin/source of the cattle, i.e., regardless of sampling time and sample type (i.e., feces, lymph node, or hide). Further research is needed concerning the role of the feedlot pen environment prior to cattle placement to better understand carryover contributions of existing strains of Salmonella and their bacteriophages. IMPORTANCESalmonella serotypes isolated from outbreaks in humans can also be found in beef cattle and feedlots. Virulence factors and antibiotic resistance are among the primary defense mechanisms of Salmonella, and are often associated with clonal expansion. This makes understanding the subpopulation dynamics of Salmonella in cattle critical for effective mitigation. There remains a gap in the literature concerning subpopulation dynamics within Salmonella serotypes in feedlot cattle from the beginning of feeding up until slaughter. Here, we explore Salmonella population dynamics within each serotype using core-genome phylogeny and hierarchical classifications. We used machine learning to quantitatively parse the relative importance of both hierarchical and longitudinal clustering among cattle host samples. Our results reveal that Salmonella populations in cattle are highly clonal over a 6-month study period and that clonal dissemination of Salmonella in cattle is mainly influenced spatially by experimental block and pen, as well by the geographical origin of the cattle.

Astragallus mollissimus plant extract: a strategy to reduce ruminal methanogenesis.

Ruminal methanogenesis is considered an inefficient process as it can result in the loss of 4 to 12% of the total energy consumed by the ruminant. Recent studies have shown that compounds such as nitroethane, 2-nitroethanol, 2-nitro-1-propanol, and 3-nitro-1-propionic acid are capable of inhibiting methane production during in vitro studies. However, all of these nitrocompounds came from a synthetic origin, which could limit their use. In contrast, some plants of the Astragallus genus produce a natural nitrocompound, although its anti-methanogenic effect has not been evaluated. To determine the anti-methanogenic effect, in vitro cultures of freshly collected mixed populations of ruminal microbes were supplemented with A. mollissimus extracts (MISER). Cultures supplemented with 2-nitroethanol, ethyl 2-nitroacetate, or nitroethane were used as positive controls whereas distilled water was added to the untreated control tubes. After a 24 h incubation period, the methane production was reduced by more than 98% for the samples treated with A. mollissimus extract (P < 0.05) compared to the untreated controls (10.2 ± 0.1 mmol mL-1 incubated liquid). Cultures supplemented with MISER produced a greater (P < 0.05) amount of total VFA, compared to the rest of treated and untreated cultures. Considering that there are significant differences between MISER treatment, positive controls and untreated cultures (P < 0.05) regarding the amounts of total gas, gas composition (CH4 and H2), and the amount of VFA produced, it is concluded that Astragallus mollissimus poses an alternative strategy to reduce ruminal methanogenesis. To further explore such alternative, it is necessary to determine if the metabolization byproducts are safe and/or useful for the animal.

Population Dynamics of Salmonella enterica within Beef Cattle Cohorts Followed from Single-Dose Metaphylactic Antibiotic Treatment until Slaughter.

Antibiotic use in cattle can select for multidrug-resistant Salmonella enterica, which is considered a serious threat by the U.S. Centers for Disease Control and Prevention. A randomized controlled longitudinal field trial was designed to determine the long-term effects of a single dose of ceftiofur or tulathromycin on Salmonella population characteristics in cattle feces and peripheral lymph nodes and on hides. A total of 134 beef cattle from two sources were divided among 12 pens, with cattle in each of the 3-pen blocks receiving a single dose of either ceftiofur or tulathromycin or neither (control) on day 0. Fecal samples were collected before treatment (day 0) and repeatedly following treatment until slaughter (day 99+). Hide and lymph node samples were collected at slaughter age. Salmonella prevalence, phenotypic antimicrobial resistance, serotype, and phylogenetic relationships were examined. Multilevel mixed logistic regression models indicated no significant effects (P ≥ 0.218) of metaphylactic antibiotics on the prevalence of Salmonella across sample types. However, there was a significant time effect observed, with prevalence increasing from spring through the midsummer months (P < 0.0001) in feces. The majority of Salmonella isolates were pansusceptible to a panel of 14 antibiotics both before and after treatment. Highly prevalent Salmonella serotypes were Salmonella enterica serovar Montevideo, Salmonella enterica serovar Anatum, Salmonella enterica serovar Cerro, and Salmonella enterica serovar Lubbock across all sample types. Strong pen and cattle source serotype clustering effects were observed among Salmonella isolates originating from fecal, lymph node, and hide samples; however, the potential role of Salmonella isolates from the pen environment prior to animal placement was not assessed in this study.IMPORTANCESalmonella is a leading bacterial foodborne pathogen, causing a significant number of human infections and deaths every year in the United States. Macrolides and 3rd-generation cephalosporins play critical roles in the treatment of human salmonellosis. Use of these antibiotics in beef cattle can select for resistant bacteria that may enter the food chain or spread from the farm via manure. There is a lack of longitudinal research concerning the long-term effects of metaphylactic antibiotic administration. Here, we assessed Salmonella population dynamics during the feeding period until slaughter following single-dose antibiotic treatment. We found no long-term effects of antibiotic use early in the cattle-feeding period on Salmonella prevalence and antimicrobial resistance at slaughter. We identified the pens in which cattle were housed as the factor that contributed most to Salmonella serotypes being shared; importantly, the dominant strain in each pen changed repeatedly over the entire feeding period.

Effects of thymol and carvacrol, alone or in combination, on fermentation and microbial diversity during in vitro culture of bovine rumen microbes.

Two essential oils (EO), thymol and carvacrol, were used in six ratio (100:00, 80:20, 60:40, 40:60, 20:80 and 00:100) combinations of both EO and in a dose of 0.2 g L-1 in bovine ruminal culture medium, 24-h cultures, to evaluate effects on total gas production (TGP), methane production, in vitro dry matter digestibility (IVDMD) and in vitro culture population dynamics of methanogenic and total bacteria. Total DNA extracted from ruminal microorganisms was subjected to denaturing gradient gel electrophoresis (DGGE)-polymerase chain reaction (PCR) to examine effects on bacterial populations. The effect of EO on TGP and IVDMD were assessed by comparison to untreated control cultures. In general, methane production by the microbial populations appeared to be higher with treatments containing the highest concentration of thymol than with treatments containing more carvacrol resulting in a tendency for greater methane-inhibiting activity achieved as the thymol concentration in the thymol:carvacrol mixtures decreased linearly. The population of total bacteria with a 74.5% Dice similarity coefficient for comparison of DGGE band patterns indicating shifts in bacterial constituents as EO ratios changed. No effects on TGP, IVDMD while only slight shifts in the methanogenic populations were seen with an overall 91.5% Dice similarity coefficient.

Effect of waste milk pasteurization on fecal shedding of Salmonella in preweaned calves.

The objective of the current research was to determine if pasteurization of nonsaleable waste milk influences fecal Salmonella concentrations and prevalence, or antimicrobial susceptibility and serotype of the cultured isolates. Holstein dairy calves (n = 211) were housed on a single commercial dairy in the southwestern United States and randomly allotted to be fed either pasteurized (PWM; n = 128 calves) or nonpasteurized waste milk (NPWM; n = 83 calves). Fecal samples were collected via rectal palpation or from freshly voided, undisturbed fecal pats, weekly during the first 4 wk of the animal's life and then again at weaning. Eight total collections were made and 1,117 fecal samples cultured for Salmonella. One isolate from each culture-positive fecal sample was preserved for antimicrobial susceptibility screening and serotyping. Sixty-nine percent of the fecal samples were culture positive for Salmonella with no difference due to treatment (67.7 and 69% Salmonella positive for PWM and NPWM treatments, respectively). Few fecal samples (178/1,117; 15.9%) contained Salmonella concentrations above the limit of detection (∼1 cfu/g of feces) with concentrations ranging from 1.0 to 6.46 cfu (log10)/g of feces. Concentration was not affected by treatment. Seventeen different serotypes were identified, the majority of which were Montevideo and Anatum. A greater percentage of Typhimurium (87 vs. 13%), Muenchen (88 vs. 12%), and Derby (91 vs. 9%) were recovered from calves fed PWM compared with NPWM-fed calves. Conversely, Newport (12.5 vs. 86%), Bredeney (22.2 vs. 77.8%), and Muenster (12.5 vs. 87.5%) were lower in PWM compared with NPWM treatments. The majority (66.7%) of isolates were susceptible to all of the antibiotics examined. Results from this one commercial dairy suggest that milkborne Salmonella is not an important vector of transmission in dairy neonates, nor does pasteurization of waste milk influence fecal shedding of this pathogen. Caution should be used, however, when extrapolating results to other farms as Salmonella contamination of milk on farm is well documented. The potential benefits of pasteurization in disease prevention outweigh the potential risks of feeding a nonpasteurized product and warrants incorporation into any calf-rearing program using nonsaleable waste milk for feeding young dairy neonates.

Bacterial communities related to 3-nitro-1-propionic acid degradation in the rumen of grazing ruminants in the Qinghai-Tibetan Plateau.

The objectives of this current study were to characterize the overall rumen bacterial community in grazing yak and two sheep species (Tibetan and Small Tail Han sheep) reared in the unique environmental conditions of the Qinghai-Tibetan Plateau, as well as the bacterial community associated with the detoxification of a phytotoxin, 3-nitro-1-propionic acid (NPA), during in vitro culture with 4.2 mM NPA. Using 16S rRNA gene high-throughput sequencing, it was found that the yak rumen harbored populations showing a higher bacterial diversity compared to Tibetan sheep. The rumen bacterial community in the three ruminant species differed from each other. PICRUSt analysis identified that the pathway involved in nitrogen metabolism was enriched in Tibetan sheep while that related to fatty acid biosynthesis was over-represented in the yak. The methane metabolism pathway was dominant in bacterial populations from the Small Tail Han sheep. Comparisons between freshly collected rumen fluid and populations subjected to consecutive 72 h batch cultures revealed substantial decreases in alpha diversity in populations cultured with NPA. Moreover, the relative abundances of some bacterial taxa changed significantly, with increased abundance of Proteobacteria and Actinobacteria. In addition, the overall community structure of the bacterial population in the freshly collected ruminal fluid was clearly different than that within populations observed in the 72 h batch cultures likely due to the impact of NPA treatments and the more restrictive growth conditions of the culture medium. In regard to PICRUSt analysis, the methane metabolism pathway became scarce in Tibetan and Small Tail Han sheep, whereas the energy and carbohydrate metabolic pathways such as nitrogen metabolism, ABC transporters and glycolysis/gluconeogenesis were found to be maintained across all populations. Results from the present study provide new information on the bacterial and functional composition within ruminal populations adapted to three economically important grazing ruminant species prominent on the Qinghai-Tibetan Plateau. The results further reveal that effects of NPA treatment on community structure can have an impact not only the metabolism of NPA but on other digestive functions as well.

Nigella sativa L. as an alternative antibiotic feed supplement and effect on growth performance in weanling pigs.

BACKGROUND: Nigella sativa L. (NS) is a plant containing bioactive constituents such as thymoquinone. Extracts of NS improve performance and reduce enteropathogen colonization in poultry and small ruminants, but studies with swine are lacking. In two different studies oral administration of NS extracts at doses equivalent to 0, 1.5 and 4.5 g kg-1 diet was assessed on piglet performance and intestinal carriage of wildtype Escherichia coli and Campylobacter, and Salmonella Typhimurium. RESULTS: Wildtype E. coli populations in the jejunal and rectal content collected 9 days after treatment began were decreased (P ≤ 0.05). Populations recovered from pigs treated with extract at 1.5 and 4.5 g kg-1 diet were 0.72-1.31 log10 units lower than the controls (ranging from 6.05 to 6.61 log10 CFU g-1 ). Wildtype Campylobacter and Salmonella Typhimurium were unaffected by NS treatment. Feed efficiency over the 9 days improved linearly (P < 0.05) from 3.88 with 0 NS-treated pigs to 1.47 and 1.41 with pigs treated with NS at 1.5 and 4.5 g kg-1 diet, respectively, possibly due to high glutamine/glutamic acid content of the NS extract. CONCLUSION: NS supplementation of weanling pigs improved feed efficiency and helped control intestinal E. coli during this vulnerable production phase. © 2017 Society of Chemical Industry.

Pseudomonas sp. BUP6, a novel isolate from Malabari goat produces an efficient rhamnolipid type biosurfactant.

This study describes the characteristics of a biosurfactant produced by Pseudomonas sp. BUP6, a rumen bacterium, and optimization of parameters required for its production. Initial screening of five parameters (pH, temperature, agitation, incubation, and substrate concentration) was carried out employing Plackett-Burman design, which reduced the number of parameters to 3 (pH, temperature, and incubation) according to their significance on the yield of biosurfactant. A suitable statistical model for the production of biosurfactant by Pseudomonas sp. BUP6 was established according to Box-Behnken design, which resulted in 11% increase (at pH 7, 35 °C, incubation 75 h) in the yield (2070 mg L-1 ) of biosurfactant. The biosurfactant was found stable at a wide range of pH (3-9) with 48 mg L-1 critical micelle concentration; and maintained over 90% of its emulsification ability even after boiling and in presence of sodium chloride (0.5%). The highest cell hydrophobicity (37%) and emulsification (69%) indices were determined with groundnut oil and kerosene, respectively. The biosurfactant was found to inhibit the growth and adhesion of E. coli and S. aureus significantly. From the phytotoxicity studies, the biosurfactant did not show any adverse effect on the germinating seeds of rice and green gram. The structural characterization of biosurfactant employing orcinol method, thin layer chromatography and FT-IR indicated that it is a rhamnolipid (glycolipid). Thus, Pseudomonas sp. BUP6, a novel isolate from Malabari goat is demonstrated as a producer of an efficient rhamnolipid type biosurfactant suitable for application in various industries.

Short chain nitrocompounds as a treatment of layer hen manure and litter; effects on in vitro survivability of Salmonella, generic E. coli and nitrogen metabolism.

The current study was conducted to assess the bactericidal effectiveness of several nitrocompounds against pathogens in layer hen manure and litter. Evidence from an initial study indicated that treatment of layer hen manure with 12 mM nitroethane decreased populations of generic E. coli and total coliforms by 0.7 and 2.2 log10 colony forming units (CFU) g-1, respectively, after 24 h aerobic incubation at ambient temperature when compared to untreated populations. Salmonella concentrations were unaffected by nitroethane in this study. In a follow-up experiment, treatment of 6-month-old layer hen litter (mixed with 0.4 mL water g-1) with 44 mM 2-nitroethanol, 2-nitropropanol or ethyl nitroacetate decreased an inoculated Salmonella typhimurium strain from its initial concentration (3 log10 CFU g-1) by 0.7 to 1.7 log10 CFU g-1 after 6 h incubation at 37°C in covered containers. After 24 h incubation, populations of the inoculated S. Typhmiurium in litter treated with 44 mM 2-nitroethanol, 2-nitropropanol, ethyl nitroacetate or nitroethane were decreased more than 3.2 log10 CFU g-1 compared to populations in untreated control litter. Treatment of litter with 44 mM 2-nitroethanol, 2-nitropropanol, ethyl nitroacetate decreased rates of ammonia accumulation more than 70% compared to untreated controls (0.167 µmol mL-1 h-1) and loses of uric acid (< 1 µmol mL-1) were observed only in litter treated with 44 mM 2-nitropropanol, indicating that some of these nitrocompounds may help prevent loss of nitrogen in treated litter. Results warrant further research to determine if these nitrocompounds can be developed into an environmentally sustainable and safe strategy to eliminate pathogens from poultry litter, while preserving its nitrogen content as a nutritionally valuable crude protein source for ruminants.

Achromobacter denitrificans strain SP1 efficiently remediates di(2-ethylhexyl)phthalate.

This study describes how Achromobacter denitrificans strain SP1, a novel isolate from heavily plastics-contaminated sewage sludge efficiently consumed the hazardous plasticizer, di(2-ethylhexyl)phthalate (DEHP) as carbon source supplemented in a simple basal salt medium (BSM). Response surface methodology was employed for the statistical optimization of the process parameters such as temperature (32°C), agitation (200 rpm), DEHP concentration (10 mM), time (72 h) and pH (8.0). At these optimized conditions, experimentally observed DEHP degradation was 63%, while the predicted value was 59.2%; and the correlation coefficient between them was 0.998, i.e., highly significant and fit to the predicted model. Employing GC-MS analysis, the degradation pathway was partially deduced with intermediates such as mono(2-ethylhexyl)phthalate and 2-ethyl hexanol. Briefly, this first report describes A. denitrificans strain SP1 as a highly efficient bacterium for completely remediating the hazardous DEHP (10 mM) in 96 h in BSM (50% consumed in 60 h), which offers great potentials for efficiently cleaning the DEHP-contaminated environments such as soil, sediments and water upon its deployment.

Comparative effect of thymol or its glucose conjugate, thymol-ß-D-glucopyranoside, on Campylobacter in avian gut contents.

Campylobacter jejuni is an important human food-borne pathogen that can contaminate meat and poultry during processing. Consequently, strategies are sought to reduce the carriage of C. jejuni in food animals before they arrive at the abattoir. Thymol is a natural product that reduces survivability of Campylobacter in vitro, but its rapid absorption from the proximal alimentary tract limits its bactericidal efficacy in vivo. Thymol-ß-D-glucopyranoside is more resistant to absorption than free thymol, but its administration to chickens has not been reported. In the present studies, 1 mM thymol-ß-D-glucopyranoside was shown to exhibit near equal anti-Campylobacter activity as 1 mM thymol when incubated anaerobically in avian crop or cecal contents in vitro, resulting in reductions of 1.10-2.32 log10 colony forming units mL(-1) in C. jejuni concentrations after 24 h incubation. In a follow-up live animal study, oral administration of thymol-ß-D-glucopyranoside, but not free thymol, significantly lowered (>10-fold) recovery of Campylobacter from the crop of market-aged broilers when compared to placebo-treated controls (n = 6 broilers/treatment). Neither thymol-ß-D-glucopyranoside nor thymol affected recovery of Campylobacter from cecal contents of the treated broilers. These results indicate that rapid absorption or passage of free thymol from the crop precluded its anti-Campylobacter activity at this site and throughout the entire gastrointestinal tract. Conversely, lower recovery of Campylobacter from the crop of birds treated with thymol-ß-D-glucopyranoside indicates this conjugate was retained and able to be hydrolyzed to biologically active free thymol at this site as intended, yet was not sufficiently protected to allow passage of efficacious amounts of the intact glycoside to the lower gut. Nevertheless, these results warrant further research to see if higher doses or encapsulation of thymol-ß-D-glucopyranoside or similar glycosides may yield an efficacious additive to reduce carriage of Campylobacter as well as other pathogens throughout the avian gut.

Characterization of bovine ruminal and equine cecal microbial populations enriched for enhanced nitro-toxin metabolizing activity.

The nitrotoxins 3-nitro-1-propionic acid (NPA) and 3-nitro-1-propanol (NPOH) are produced by a wide variety of leguminous plants, including over 150 different species and varieties of Astragalus potentially grazed by livestock. These toxins are known to be detoxified by at least one ruminal bacterium but detoxification by bacteria from other gut habitats is not known. In the present study, mixed populations of bovine ruminal and equine cecal microbes were enriched for NPA-metabolizing bacteria via consecutive 24-72 h culture in a basal minimal rumen fluid-based medium supplemented with 4.2 mM NPA and H2 as the energy source. Rates of NPA metabolism by the respective populations increased from 58.4 ± 4.8 and 8.6 ± 11.6 nmol NPA/mL per h during initial culture to 88.9 ± 30.6 and 50.2 ± 30.9 nmol NPA/mL per h following enrichment. Results from 3-tube most probable number tests indicated that numbers of NPA-degrading microbes increased 2.1 and 1.8 log10 units during enrichment from numbers measured pre-enrichment (3.9 × 10³ and 4.3 × 10¹ cells/mL for ruminal and equine cecal populations, respectively). Hydrogen, formate, and to a lesser extent, DL-lactic acid, served as electron donors to the enriched populations and CO2 or formate were needed to maintain high rates of NPA-metabolism. The NPA-enriched populations were able to metabolize nitrate which, being a preferred electron acceptor, was antagonistic to NPA metabolism. Supplemental NPA was inhibitory to methanogenesis. Fermentation balance estimates indicated that only 47.6% of carbon available in potential substrates was recovered in headspace CO2, volatile fatty acids or unmetabolized NPA after 72 h incubation of NPA-enriched populations that had metabolized 98% of 8.4 mM added NPA. Overall, these results reveal low level carriage of NPA-metabolizing, CO2 or formate-requiring bacterial populations in the equine cecum yet support the concept that Denitrobacterium detoxificans-like organisms may well be the functional agents of NPA and NPOH detoxification in the populations studied here.

Evaluation of linalool, a natural antimicrobial and insecticidal essential oil from basil: effects on poultry.

Linalool is a natural plant-product used in perfumes, cosmetics, and flavoring agents. Linalool has proven antimicrobial and insect-repellent properties, which indicate it might be useful for control of enteropathogens or insect pests in poultry production. However, there are no published reports that linalool may be safely administered to or tolerated by chickens. Linalool was added to the diets of day-of-hatch chicks, and they were fed linalool-supplemented diets for 3 wk. We studied the effects of linalool on serum chemistry, gross pathology, feed conversion, and relative liver weights. Linalool had a dramatic negative dose-dependent effect on feed conversion at concentrations in the feed exceeding 2% linalool, but not on gross pathology. Liver weights were significantly increased in the 5% linalool-treated birds. There was a statistical effect on blood glucose, but this parameter remained below the cut-offs for elevated serum glucose, and the result is likely of no biological significance. Linalool caused serum aspartate aminotransferase (AST) levels to increase, but it did not increase serum gamma-glutamyl transferase levels. The linalool effect on AST was dose-dependent, but in linalool doses between 0.1 and 2% of the feed, AST was not elevated beyond normal parameters. Linalool at 2% or less may be safely added to chicken feed. We suggest future studies to evaluate the addition of linalool to the litter, where it may be used as an antimicrobial or an insect repellent or to produce a calming effect.

Bacterial proximity effects on the transfer of antibiotic resistance genes within the alimentary tract of yellow mealworm larvae.

The arthropod intestinal tract and other anatomical parts naturally carry microorganisms. Some of which are pathogens, secrete toxins, or carry transferable antibiotic-resistance genes. The risks associated with the production and consumption of edible arthropods are dependent on indigenous microbes, as well as microbes introduced during the processes of rearing. This mass arthropod production puts individual arthropods in close proximity, which increases the possibility of their exposure to antibiotic-resistant bacteria carried by bacteria from fellow insects, industry workers, or rearing hardware and substrates. The purpose of this study was to determine if the alimentary tract of the yellow mealworm provided an environment permitting horizontal gene transfer between bacteria. The effect of the concentration of bacterial exposure was also assessed. Antibiotic resistance gene transfer between marker Salmonella Lignières (Enterobacterales: Enterobacteriaceae) and Escherichia coli (Migula) (Enterobacterales: Enterobacteriaceae) introduced into the larval gut demonstrated that the nutrient-rich environment of the yellow mealworm gut provided favorable conditions for the transfer of antibiotic resistance genes. Conjugation frequencies were similar across inoculum concentrations; however, transconjugant production correlated positively to increased exposure concentration. The lowest concentration of bacterial exposure required enrichment to detect and thus may have been approaching a threshold level for the 2 bacteria to colocate within the expanse of the larval gut. While many factors can affect this transfer, the simple factor of the proximity of donor and recipient bacteria, as defined by the concentration of bacteria within the volume of the insect gut, likely primarily contributed to the efficiency of antibiotic gene transfer.

Selection for high and low antibody responses to sheep red blood cells influences cytokine and chemokine expression in chicken peripheral blood leukocytes and splenic tissue.

White Leghorn chickens from a common founder population have been divergently selected for high (HAS) or low (LAS) antibody responses to sheep red blood cells (SRBC) for 49 generations resulting in 2 diverse lines for this trait. Much has been studied in these two lines; however, the impact of these selection pressures on cytokine and chemokine expression is not fully understood. The purpose of this study is to determine if selection for antibody response to SRBC impacts cytokine and chemokine expression in peripheral blood leukocytes (PBL) and spleen from HAS and LAS chickens. Total RNA was isolated from PBL and spleen after which mRNA expression of cytokines (IL4, IL6, IL10, TGF-ß4) and chemokines (CXCL8, CCL4) were determined by quantitative real-time RT-PCR (qRT-PCR). The data were analyzed using Student's t test comparing HAS and LAS (P < 0.05) and are reported as corrected 40-CT. PBL and spleen samples were analyzed separately. With respect to PBL, expression of IL6 was higher (P < 0.05) in PBL isolated from LAS chickens compared to those from the HAS line whereas there were no differences (P > 0.05) in IL4, IL10, CXCL8, CCL4, or TGF-ß4. The cytokine and chemokine mRNA expression profiles were different in the spleen between the two lines. IL4 and CXCL8 expression were higher (P < 0.05) in spleen samples from HAS chickens than LAS. The expression of IL6, IL10, CCL4, or TGF-ß4 in the spleens did not differ (P > 0.05) between the lines. The data indicate that selection for specific antibody responses to SRBC impacts the cytokine and chemokine expression profile in PBL and spleens but in different ways in HAS and LAS. These studies provide insight into the influence that selection pressures for antibody responses have on different immune response components, specifically cytokines and chemokines typically involved in the innate response.

Chlorophyllin Supplementation of Medicated or Unmedicated Swine Diets Impact on Fecal Escherichia coli and Enterococci.

Considering that certain catabolic products of anaerobic chlorophyll degradation inhibit efflux pump activity, this study was conducted to test if feeding pigs a water-soluble chlorophyllin product could affect the antibiotic resistance profiles of select wild-type populations of fecal bacteria. Trial 1 evaluated the effects of chlorophyllin supplementation (300 mg/meal) on fecal E. coli and enterococcal populations in pigs fed twice daily diets supplemented without or with ASP 250 (containing chlortetracycline, sulfamethazine and penicillin at 100, 100 and 50 g/ton, respectively). Trial 2, conducted similarly, evaluated chlorophyllin supplementation in pigs fed diets supplemented with or without 100 g tylosin/ton. Each trial lasted 12 days, and fecal samples were collected and selectively cultured at 4-day intervals to enumerate the total numbers of E. coli and enterococci as well as populations of these bacteria phenotypically capable of growing in the presence of the fed antibiotics. Performance results from both studies revealed no adverse effect (p > 0.05) of chlorophyllin, antibiotic or their combined supplementation on average daily feed intake or average daily gain, although the daily fed intake tended to be lower (p = 0.053) for pigs fed diets supplemented with tylosin than those fed diets without tylosin. The results from trial 1 showed that the ASP 250-medicated diets, whether without or with chlorophyllin supplementation, supported higher (p < 0.05) fecal E. coli populations than the non-medicated diets. Enterococcal populations, however, were lower, albeit marginally and not necessarily significantly, in feces from pigs fed the ASP 250-medicated diet than those fed the non-medicated diet. Results from trial 2 likewise revealed an increase (p < 0.05) in E. coli and, to a lesser extent, enterococcal populations in feces collected from pigs fed the tylosin-medicated diet compared with those fed the non-medicated diet. Evidence indicated that the E. coli and enterococcal populations in trial 1 were generally insensitive to penicillin or chlortetracycline, as there were no differences between populations recovered without or with antibiotic selection. Conversely, a treatment x day of treatment interaction observed in trial 2 (p < 0.05) provided evidence, albeit slight, of an enrichment of tylosin-insensitive enterococci in feces from the pigs fed the tylosin-medicated but not the non-medicated diet. Under the conditions of the present study, it is unlikely that chlorophyllin-derived efflux pump inhibitors potentially present in the chlorophyllin-fed pigs were able to enhance the efficacy of the available antibiotics. However, further research specifically designed to optimize chlorophyll administration could potentially lead to practical applications for the swine industry.

The bacterial and archaeal communities of flies, manure, lagoons, and troughs at a working dairy.

Background: Fundamental investigations into the location, load, and persistence of microbes, whether beneficial or detrimental, are scarce. Many questions about the retention and survival of microbes on various surfaces, as well as the load necessary for spread, exist. To answer these questions, we must know more about where to find various microbes and in what concentrations, the composition of the microbial communities, and the extent of dissemination between various elements. This study investigated the diversity, composition, and relative abundance of the communities associated with manure, lagoons, troughs, house flies, and stable flies present at a dairy, implementing two different free-stall management systems: flow-through and cross-vent. Shotgun metagenomics at the community level was used to compare the microbiomes within the dairy, allowing confident interpretation at the species level. Results: The results showed that there were significant difference in microbial composition between not only each of the dairy elements but also management styles. The primary exceptions were the microbiomes of the house fly and the stable fly. Their compositions heavily overlapped with one another, but interestingly, not with the other components sampled. Additionally, both species of flies carried more pathogens than the other elements of the dairy, indicating that they may not share these organisms with the other components, or that the environments offered by the other components are unsatisfactory for the survival of some pathogens.. Conclusion: The lack of overlapping pathogen profiles suggests a lack of transfer from flies to other dairy elements. Dairy health data, showing a low incidence of disease, suggests minimal sharing of bacteria by the flies at a level required for infection, given the health program of this dairy. While flies did carry a multitude of pathogenic bacteria, the mere presence of the bacteria associated with the flies did not necessarily translate into high risk leading to morbidity and mortality at this dairy. Thus, using flies as the sole sentinel of dairy health may not be appropriate for all bacterial pathogens or dairies.

Campylobacter jejuni Response When Inoculated in Bovine In Vitro Fecal Microbial Consortia Incubations in the Presence of Metabolic Inhibitors.

Infection with the foodborne pathogen Campylobacter is the leading bacterial cause of human foodborne illness in the United States. The objectives of this experiment were to test the hypothesis that mixed microbial populations from the bovine rumen may be better at excluding Campylobacter than populations from freshly voided feces and to explore potential reasons as to why the rumen may be a less favorable environment for Campylobacter than feces. In an initial experiment, C. jejuni cultures inoculated without or with freshly collected bovine rumen fluid, bovine feces or their combination were cultured micro-aerobically for 48 h. Results revealed that C. jejuni grew at similar growth rates during the first 6 h of incubation regardless of whether inoculated with the rumen or fecal contents, with rates ranging from 0.178 to 0.222 h-1. However, C. jejuni counts (log10 colony-forming units/mL) at the end of the 48 h incubation were lowest in cultures inoculated with rumen fluid (5.73 log10 CFUs/mL), intermediate in cultures inoculated with feces or both feces and rumen fluid (7.16 and 6.36 log10 CFUs/mL) and highest in pure culture controls that had not been inoculated with the rumen or fecal contents (8.32 log10 CFUs/mL). In follow-up experiments intended to examine the potential effects of hydrogen and hydrogen-consuming methanogens on C. jejuni, freshly collected bovine feces, suspended in anaerobic buffer, were incubated anaerobically under either a 100% carbon dioxide or 50:50 carbon dioxide/hydrogen gas mix. While C. jejuni viability decreased <1 log10 CFUs/mL during incubation of the fecal suspensions, this did not differ whether under low or high hydrogen accumulations or whether the suspensions were treated without or with the mechanistically distinct methanogen inhibitors, 5 mM nitrate, 0.05 mM 2-bromosulfonate or 0.001 mM monensin. These results suggest that little if any competition between C. jejuni and hydrogen-consuming methanogens exists in the bovine intestine based on fecal incubations.

Chlortetracycline Concentration Impact on Salmonella Typhimurium Sustainability in the Presence of Porcine Gastrointestinal Tract Bacteria Maintained in Continuous Culture.

Concern exists that the continued use of antibiotics in animal feeds may lead to an increased prevalence of resistant bacteria within the host animal's gastrointestinal tract. To evaluate the effect of chlortetracycline on the persistence of Salmonella enterica serotype Typhimurium within a diverse population of porcine cecal bacteria, we cultured a mixed population of cecal bacteria without or with added chlortetracycline. When grown at a 24 h vessel turnover rate, chlortetracycline-susceptible S. Typhimurium exhibited more than 2.5 times faster (p < 0.05) disappearance rates than theoretically expected (0.301 log10 colony-forming unit/mL per day) but did not differ whether treated or not with 55 mg of chlortetracycline/L. Chlortetracycline-resistant S. Typhimurium was not recovered from any of these cultures. When the mixed cultures were inoculated with a chlortetracycline-resistant S. Typhimurium, rates of disappearance were nearly two times slower (p < 0.05) than those observed earlier with chlortetracycline-susceptible S. Typhimurium, and cultures persisted at >2 log10 colony-forming units/mL for up to 14 days of treatment with 110 mg of chlortetracycline/L. Under the conditions of this study, chlortetracycline-resistant S. Typhimurium was competitively enabled to persist longer within the mixed populations of porcine gut bacteria than chlortetracycline-susceptible S. Typhimurium, regardless of the presence or absence of added chlortetracycline.