Screening and selection of essential oils for an intranasal spray against bovine respiratory pathogens based on antimicrobial, antiviral, immunomodulatory, and antibiofilm activities.

Introduction: The rise in antibiotic resistant pathogens associated with bovine respiratory disease (BRD) poses a serious challenge, particularly to the beef feedlot industry, as they currently depend on antibiotics to prevent BRD to mitigate the financial burden (approx. $1 billion annual loss) inflicted by BRD-associated high mortality and morbidity in feedlot cattle. Thus, there is an impetus need for the development of antimicrobial alternative strategies against BRD. This study aimed to screen and select candidate essential oils (EOs) for the development of an intranasal EO spray that can inhibit BRD pathogens and promote microbiota-mediated respiratory health. Methods: The effects of selected EOs (ajowan, cinnamon leaf, citronella, grapefruit, fennel, and thyme) on a bovine nasopharyngeal microbiota culture were evaluated using 16S rRNA gene sequencing. The microbiota culture was enriched by incubating nasopharyngeal swabs obtained from finishing beef heifers in brain heart infusion broth with and without EOs (0.025%, v/v). These EOs were then also evaluated for their immunomodulatory effects on bovine turbinate (BT) cells by analyzing the concentrations of 15 cytokines and chemokines in cell culture after 24 h incubation. The crystal violet assay was done to assess the antibiofilm activity of EOs against Escherichia coli UMN026 strain. Finally, 15 EOs were screened for their antiviral activity against the bovine viral diarrhea virus 1 (BVDV-1) using BT cells and a fluorescence-based method. Results: Ajowan, fennel, and thyme resulted in a moderate reduction of overall nasopharyngeal microbiota growth with significant alterations of both alpha and beta diversity, and the relative abundance of predominant bacterial families (e.g., increasing Enterobacteriaceae and decreasing Moraxellaceae) compared to the control (p < 0.05). Co-incubation of BT cells with selected EOs resulted in minimal alterations in cytokine and chemokine levels (p > 0.05). Ajowan, thyme, fennel, and cinnamon leaf exhibited antibiofilm activity at concentrations of 0.025 and 0.05%. Reduction of BVDV-1 replication in BT cells was observed with thyme (strong), and ajowan and citronella (moderate) at 0.0125% concentration. Discussion: Accordingly, ajowan, thyme, fennel, cinnamon leaf, and citronella EOs were selected for further development as an intranasal EO spray to prevent and control of BRD pathogens in feedlot cattle.

A single intranasal dose of essential oil spray confers modulation of the nasopharyngeal microbiota and short-term inhibition of Mannheimia in feedlot cattle: a pilot study.

Five essential oils (EOs) were previously characterized in vitro and identified as candidate EOs for the development of an intranasal EO spray to mitigate bovine respiratory disease (BRD) pathogens. In the present study, these EOs were evaluated for their potential to (i) reduce BRD pathogens, (ii) modulate nasopharyngeal microbiota, and (iii) influence animal performance, feeding behavior and immune response when a single dose administered intranasally to feedlot cattle. Forty beef steer calves (7-8 months old, Initial body weight = 284 ± 5 kg [SE]) received either an intranasal EO spray (ajowan, thyme, fennel, cinnamon leaf, and citronella) or PBS (Control; n = 20/group) on day 0. Deep nasopharyngeal swabs were collected on days (d) -1, 1, 2, 7, 14, 28, and 42 and processed for 16S rRNA gene sequencing, qPCR, and culturing. Significant effects of EO on community structure (d1), microbial richness and diversity, relative abundance of some dominant phyla (d1, d2, and d14), and the overall interaction network structure of the nasopharyngeal microbiota were detected. The relative abundance of Mannheimia was lower in the EO calves (4.34%) than in Control calves (10.4%) on d2, and M. haemolytica prevalence on d7 as compared to control calves. Feed intake, average daily gain, feeding behavior, and blood cell counts were not affected by EO treatment. Overall, a single intranasal dose of EO spray resulted in moderate modulation of nasopharyngeal microbiota and short-term inhibition of Mannheimia while not influencing animal performance, feeding behavior or immune response. Our study, for the first time, shows the potential use of intranasal EO to mitigate BRD in feedlot cattle.

Genome sequences of 11 Alkalihalobacillus clausii, Bacillus safensis, and Escherichia coli bacteriophages isolated from bovine rumen and vagina.

Here, we present the coding-complete genomes of 11 lytic bacteriophages isolated from bovine ruminal fluid and vaginal swabs that can infect the bacterial hosts Alkalihalobacillus clausii, Bacillus safensis, and Escherichia coli.

Genome Sequences of Alkalihalobacillus clausii, Bacillus safensis, Escherichia coli, and Pasteurella multocida Isolates from the Rumen, Nasopharynx, Vagina, and Uterus of Healthy Beef Cattle.

Here, we present the first draft genome sequences of 10 bacterial strains that were isolated from the rumen, nasopharynx, vagina, or uterus of healthy beef cattle. These genomes are from one Alkalihalobacillus clausii isolate, three Bacillus safensis isolates, five Escherichia coli isolates, and one Pasteurella multocida isolate.

Characterization of Salmonella enterica Isolates from Selected U.S. Swine Feed Mills by Whole-Genome Sequencing.

Every year salmonellosis is responsible for $2.3 billion in costs to the U.S. food industry, with nearly 6% of the reported cases associated with pork and/or pork products. Several studies have demonstrated the role of pigs as Salmonella reservoirs. Furthermore, this pathogen has been identified as a potential biological hazard in many livestock feeds. The overall objective of this research was to characterize Salmonella enterica isolates in selected U.S. swine feed mills by whole-genome sequencing (WGS) and evaluate isolates in association with the season and feed production stages. Salmonella isolates were collected from 11 facilities during a previous study. Samples were analyzed for Salmonella prevalence following the U.S. Department of Agriculture guidelines and confirmed by PCR. WGS was carried out on either the MiSeq or NextSeq sequencer. De novo genome assemblies were obtained with the Shovill pipeline, version 0.9. ResFinder and SPIFinder were used to identify antibiotic resistance genes and pathogenicity islands. Finally, their phylogenetic relationship and diversity were determined by core genome multilocus sequence typing. Overall, our analysis showed the presence of S. enterica in the feed mill environment. Isolates belonged to 16 different serotypes. Salmonella Agona, Salmonella Mbandaka, Salmonella Senfenberg, and Salmonella Scharzengrund were the most frequently found, and 18 single-nucleotide polymorphism clusters were identified. In silico analysis showed that 40% of the strains carried at least one antimicrobial resistance gene. All isolates in this study could be considered of public health concern and pathogenic potential. Our findings underscore the potential role of the feed mill environment as the pathogen entry route into the human food value chain.

Seasonal Presence of Salmonella spp., Salmonella Typhimurium and Its Monophasic Variant Serotype I 4,[5],12:i:-, in Selected United States Swine Feed Mills.

This study evaluated the seasonal prevalence and distribution of Salmonella spp., Salmonella enterica serovar Typhimurium (ST) and its monophasic variant 4,[5],12:i:- (STM), in selected swine feed mills across the United States. Eleven facilities were selected for this study and 12 sites were sampled within each mill during fall 2016, early spring 2017, and summer 2017. Samples were evaluated following the USDA-FSIS guidelines for Salmonella isolation and culture positive samples were analyzed by polymerase chain reaction (PCR). A multiplex real-time PCR was used to differentiate ST and STM from other serotypes. Associations between season, mill, and sample site with Salmonella presence were investigated using generalized linear mixed models. Both season (p < 0.007) and mill (p < 0.005) were significantly associated with Salmonella spp. presence. Fall months were associated with a higher Salmonella prevalence (13.2%) compared with early spring and summer. A total of five isolates, among the 383 samples were serotyped as ST and STM. These two serotypes showed a similar seasonal presence throughout the study, being found during fall and summer seasons. These findings demonstrated the seasonal presence of Salmonella spp. in feed mills and the role of these environments as potential pathogen entry route into the human food chain.

Effects of different moisture and temperature levels on Salmonella survival in poultry fat.

Fat products have been historically thought to have too low water activity to harbor pathogens. However, it has been recently reported that high moisture levels in fats may lead to Salmonella presence and growth. Limited research on strategies to eliminate pathogens in these environments is available, and the mechanisms contributing to microbial presence and growth are not yet well understood. The purpose of this research was to evaluate the effects of moisture levels and storage temperatures on the growth and survival of Salmonella in poultry fat. Samples were stored for 7 d at 48°C or 76°C and remaining Salmonella was evaluated. When poultry fat was challenged with a wet high inoculum, more than a 4 log CFU/mL difference in Salmonella population was observed with 1% and 3% moisture levels at 48°C after 5 d (P < 0.05). No differences between moisture levels (P > 0.05) were observed when samples were tested with a wet low inoculum. Counts below detectable limits were observed after 24 h in samples challenged at 76°C, regardless of inoculum level. When poultry fat was stored at 48°C and inoculated with low levels of Salmonella spp., bacterial growth was influenced only by time (P < 0.05) and not affected (P > 0.05) by moisture level. However, when poultry fat was stored at 48°C and inoculated with high levels of Salmonella spp., bacterial decrease was easier (P < 0.05) in samples containing greater moisture. This research suggests that residual moisture in containers during transportation of poultry fat largely does not affect Salmonella spp. growth.

Evaluation of Salmonella presence in selected United States feed mills.

Salmonella is a pathogen of public health concern. Each year, Salmonella infections cost to the food industry approximately $2.3 billion and 33% of the reported cases are associated with beef, poultry, or pork. Pathogen presence in feed mills can represent one of the many potential routes for entry and transmission into the food production chain. Nevertheless, little is known about Salmonella incidence and association with these types of environments. The objective of this study was to investigate Salmonella presence in different feed mills across the United States. Eleven facilities were selected in eight states and 12 sites were sampled within each feed mill. Samples were analyzed following the FSIS guidelines for isolation and identification of Salmonella. Positive isolates were further investigated by a PCR analysis targeting the invA gene to differentiate for Salmonella enterica. The total number of environmental samples collected was 237: 66% resulted culture positive and 13.1% were PCR positive. All sampled feed mills had at least one culture positive site and following production flow the number of positive samples decreased from ingredient receiving to final product. These preliminary results demonstrate the presence of Salmonella in selected United States feed mills and suggest their potential role as vehicle for pathogen transmission and spread into the food production chain.

Draft Genome Sequences of 57 Salmonella enterica Strains from Selected U.S. Swine Feed Mills.

The number of Salmonella infection cases linked to pork products has increased. Pathogen presence in the feed mill environment is one of the many potential transmission routes into the food production chain. Here, we describe the draft genome sequences of 57 Salmonella enterica isolates from selected U.S. swine feed mills.

Comparison data of a two-target real-time PCR assay with and without an internal control in detecting Salmonella enterica from cattle lymph nodes.

A real-time PCR (qPCR) assay targeting on invA and pagC genes was developed and validated for the detection and quantification of Salmonella enterica strains (Bai et al., 2018) [1]. A host gene, normally an endogenous housekeeping gene (Beer-Davidson et al., 2018; Poon et al., 2004) [2,3], or an irrelevant exogenous gene (Cheng et al., 2015; Sedlak et al., 2014) [4,5] has been widely used as an internal control to monitor nucleic acid extraction efficiencies and potential PCR inhibitions in PCR-based detection assays. An endogenous internal control designed based on the 18S rRNA gene was used in the above-mentioned qPCR assay. This 18S rRNA internal control amplifies the target gene in multiple species including bovine, swine, ovine, caprine and cervine. Data was generated by the duplex qPCR assay on 138 enriched cattle lymph node samples without the internal control, and compared with data on the same samples tested by the triplex qPCR assay that has the 18S rRNA gene as internal control. Threshold cycle (Ct) data for the duplex and the triplex qPCR on the 138 samples were similar, and are presented in this brief report.

A multiplex real-time PCR assay, based on invA and pagC genes, for the detection and quantification of Salmonella enterica from cattle lymph nodes.

Cattle lymph nodes can harbor Salmonella and potentially contaminate beef products. We have developed and validated a new real-time PCR (qPCR) assay for the detection and quantification of Salmonella enterica in cattle lymph nodes. The assay targets both the invA and pagC genes, the most conserved molecular targets in Salmonella enterica. An 18S rRNA gene assay that amplifies from cattle and other animal species was also included as an internal control. Available DNA sequences for invA, pagC and 18S rRNA genes were used for primer and probe selections. Three Salmonella serotypes, S. Typhimurium, S. Anatum, and S. Montevideo, were used to assess the assay's analytical sensitivity. Correlation coefficients of standard curves generated for each target and for all three serotypes were >99% and qPCR amplification efficiencies were between 93% and 110%. Assay sensitivity was also determined using standard curve data generated from Salmonella-negative cattle lymph nodes spiked with 10-fold dilutions of the three Salmonella serotypes. Assay specificity was determined using Salmonella culture method, and qPCR testing on 36 Salmonella strains representing 33 serotypes, 38 Salmonella strains of unknown serotypes, 252 E. coli strains representing 40 serogroups, and 31 other bacterial strains representing 18 different species. A collection of 647 cattle lymph node samples from steers procured from the Midwest region of the US were tested by the qPCR, and compared to culture-method of detection. Salmonella prevalence by qPCR for pre-enriched and enriched lymph nodes was 19.8% (128/647) and 94.9% (614/647), respectively. A majority of qPCR positive pre-enriched samples (105/128) were at concentrations between 104 and 105 CFU/mL. Culture method detected Salmonella in 7.7% (50/647) and 80.7% (522/647) of pre- and post-enriched samples, respectively; 96.0% (48/50) of pre-enriched and 99.4% (519/522) of post-enriched culture-positive samples were also positive by qPCR. More samples tested positive by qPCR than by culture method, indicating that the real-time PCR assay was more sensitive. Our data indicate that this triplex qPCR can be used to accurately detect and quantify Salmonella enterica strains from cattle lymph node samples. The assay may serve as a useful tool to monitor the prevalence of Salmonella in beef production systems.