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Pathogens (Basel, Switzerland) Jun 2024This study aimed to enhance our understanding of the agreement between two sampling methods for the detection of bovine respiratory disease (BRD) pathogens in calves...
Comparing Occurrence of Bovine Respiratory Pathogens Detected by High-Throughput Real-Time PCR in Nasal Swabs and Non-Endoscopic Bronchoalveolar Lavage Samples from Dairy and Veal Calves.
This study aimed to enhance our understanding of the agreement between two sampling methods for the detection of bovine respiratory disease (BRD) pathogens in calves using high-throughput real-time qPCR (ht-RT-qPCR). In total, 233 paired nasal swab (NS) and non-endoscopic bronchoalveolar lavage (nBAL) samples were collected from 152 calves from 12 Danish cattle herds. In 202 of the observations, the calves were examined using a standardized clinical protocol. Samples were tested for three viruses (bovine respiratory syncytial virus, bovine corona virus, and influenza D virus) and six bacteria (, , , Mycoplasma species, , and ). The results showed age-related differences in disease and pathogen occurrence, with the highest detection rates in calves aged 35 days or older. Poor to moderate agreement was found between the NS and nBAL results. The presence of in both NS and nBAL in younger calves and in nBAL in older calves was associated with clinical BRD. There was a potential link between BRD and influenza D virus in older calves, although it was only found in one herd in a small sample size. Overall, NS was a relatively poor predictor of pathogens in the lower respiratory tract. The present study confirms the complexity of pathogen detection in BRD, with marked influences of age and the sampling method on pathogen detection and disease associations.
PubMed: 38921777
DOI: 10.3390/pathogens13060479 -
Applied and Environmental Microbiology Jun 2024is a major contributor to bovine respiratory disease (BRD), which causes substantial economic losses to the beef industry, and there is an urgent need for rapid and...
is a major contributor to bovine respiratory disease (BRD), which causes substantial economic losses to the beef industry, and there is an urgent need for rapid and accurate diagnostic tests to provide evidence for treatment decisions and support antimicrobial stewardship. Diagnostic sequencing can provide information about antimicrobial resistance genes in more rapidly than conventional diagnostics. Realizing the full potential of diagnostic sequencing requires a comprehensive understanding of the genetic markers of antimicrobial resistance. We identified genetic markers of resistance in to macrolide class antibiotics commonly used for control of BRD. Genome sequences were determined for 99 isolates with six different susceptibility phenotypes collected over 2 years from a feedlot in Saskatchewan, Canada. Known macrolide resistance genes , (E), and (E) were identified in most resistant isolates within predicted integrative and conjugative elements (ICEs). ICE sequences lacking antibiotic resistance genes were detected in 10 of 47 susceptible isolates. No resistance-associated polymorphisms were detected in ribosomal RNA genes, although previously unreported mutations in the L22 and L23 ribosomal proteins were identified in 12 and 27 resistant isolates, respectively. Pangenome analysis led to the identification of 79 genes associated with resistance to gamithromycin, of which 95% (75 of 79) had no functional annotation. Most of the observed phenotypic resistance was explained by previously identified antibiotic resistance genes, although resistance to the macrolides gamithromycin and tulathromycin was not explained in 39 of 47 isolates, demonstrating the need for continued surveillance for novel determinants of macrolide resistance.IMPORTANCEBovine respiratory disease is the costliest disease of beef cattle in North America and the most common reason for injectable antibiotic use in beef cattle. Metagenomic sequencing offers the potential to make economically significant reductions in turnaround time for diagnostic information for evidence-based selection of antibiotics for use in the feedlot. The success of diagnostic sequencing depends on a comprehensive catalog of antimicrobial resistance genes and other genome features associated with reduced susceptibility. We analyzed the genome sequences of isolates of , a major bovine respiratory disease pathogen, and identified both previously known and novel genes associated with reduced susceptibility to macrolide class antimicrobials. These findings reinforce the need for ongoing surveillance for markers of antimicrobial resistance to support improved diagnostics and antimicrobial stewardship.
PubMed: 38864630
DOI: 10.1128/aem.00502-24 -
Current Microbiology Jun 2024Mannheimia haemolytica is recognized as principal pathogen associated with pneumonic pasteurellosis leading to huge economic losses to small ruminant farmers. Even...
Mannheimia haemolytica is recognized as principal pathogen associated with pneumonic pasteurellosis leading to huge economic losses to small ruminant farmers. Even though the disease causes huge economic losses, epidemiology of M. haemolytica is less studied, hindering the formulation of effective control strategies. Current study aimed to highlight molecular characterisation of M. haemolytica strains isolated from ovine pneumonic infection. M. haemolytica 27 isolates with two reference strains were characterised using capsular and virulence gene typing, multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) methods. M. haemolytica serotype A2 recognized as predominant serotype (74%) followed by A6 (11%) and A1 (5%) serotypes. Virulence gene profiling by PCRs showed dominance of all five virulent genes [such as adh and gcp (100% each)] followed by gs60 (88.8%), lktC (85.2%), tbpB (51.9%) and least nmaA gene (14.8%). MLST profiling delineated M. haemolytic isolates into 11 sequence types (STs) with most prevalent being ST37 (27.9%) and ST16 (23%) and nine new STs (ST37, 38, 39, 40, 41, 42, 47, 48, and 49). These new STs did not belong to any of the three clonal complexes (CC4, CC8 and CC28). ST16 was exclusively noted in A1 and A6 serotypes. Amongst 25 isolates, 22 pulsotypes (GD 0.88) recorded indicated variability of the M. haemolytica isolates in PFGE analysis. In conclusion, the study suggested dominance of M. haemolytica serotype A2 harbouring different virulent genes, diverse STs and pulsotypes responsible for pneumonic pasteurellosis frequently encountered in sheep.
Topics: Animals; Mannheimia haemolytica; Sheep; Sheep Diseases; India; Multilocus Sequence Typing; Pasteurellosis, Pneumonic; Serogroup; Electrophoresis, Gel, Pulsed-Field; Virulence Factors; Virulence; Phylogeny
PubMed: 38862704
DOI: 10.1007/s00284-024-03740-7 -
Biological Trace Element Research Jun 2024The study aimed to assess the impact of injectable trace mineral ("ITM"; Multimin90; Fort Collins, CO) supplementation on bacterial infection in cattle. Angus-crossbred...
The study aimed to assess the impact of injectable trace mineral ("ITM"; Multimin90; Fort Collins, CO) supplementation on bacterial infection in cattle. Angus-crossbred steers (n = 32) were organized into two blocks by initial body weight. Steers were maintained on a ryelage and dry-rolled corn-based growing diet without supplementation of Zn, Cu, Mn, and Se for the duration of the study. The steers were transported 6 h, then randomized into three treatment groups: control received sterile saline ("CON"), ITM administered 1 day after transport (6 days before infection, "ITMPRE"), and ITM administered 2 days post infection (dpi) concurrent with antibiotic treatment ("ITMPOST"). Steers were infected with Mannheimia haemolytica on day 0, and all were treated with tulathromycin at 2 dpi. Plasma levels of Zn, Cu, and Se did not differ among treatments (P ≥ 0.74). Liver Se was higher in ITMPRE at 2 dpi (P < 0.05), and both ITM groups had higher liver Se at 5 dpi (P < 0.05) compared to CON. A time × treatment interaction was detected for liver Cu (P = 0.02). Clinical scores were lower (P < 0.05) in ITMPRE on 1 and 8 dpi and ITMPOST on 8 dpi compared to CON. Thoracic ultrasonography scores were lower in ITMPRE at 2 dpi compared to CON (P < 0.05) and ITMPOST (P < 0.1). No treatment effects (P > 0.10) were observed for bacterial detection from bronchoalveolar lavage (BAL) or nasopharyngeal swabs. At 5 dpi, both ITMPRE and ITMPOST showed higher frequencies of γδ T cells and NK cells in BAL compared to CON (P < 0.05). Before infection, leukocytes from ITMPRE steers produced more IL-6 (P < 0.01) in response to stimulation with the TLR agonist, Pam3CSK4. Use of ITM may be an effective strategy for improving disease resistance in feedlot cattle facing health challenges.
PubMed: 38853197
DOI: 10.1007/s12011-024-04251-z -
Microorganisms May 2024Pradofloxacin is the newest of the veterinary fluoroquinolones to be approved for use in animals-initially companion animals and most recently food animals. It has a...
Comparative In Vitro Killing by Pradofloxacin in Comparison to Ceftiofur, Enrofloxacin, Florfenicol, Marbofloxacin, Tildipirosin, Tilmicosin and Tulathromycin against Bovine Respiratory Bacterial Pathogens.
Pradofloxacin is the newest of the veterinary fluoroquinolones to be approved for use in animals-initially companion animals and most recently food animals. It has a broad spectrum of in vitro activity, working actively against Gram-positive/negative, atypical and some anaerobic microorganisms. It simultaneously targets DNA gyrase (topoisomerase type II) and topoisomerase type IV, suggesting a lower propensity to select for antimicrobial resistance. The purpose of this study was to determine the rate and extent of bacterial killing by pradofloxacin against bovine strains of and , in comparison with several other agents (ceftiofur, enrofloxacin, florfenicol, marbofloxacin, tildipirosin, tilmicosin and tulathromycin) using four clinically relevant drug concentrations: minimum inhibitory and mutant prevention drug concentration, maximum serum and maximum tissue drug concentrations. At the maximum serum and tissue drug concentrations, pradofloxacin killed 99.99% of cells following 5 min of drug exposure (versus growth to 76% kill rate for the other agents) and 94.1-98.6% of following 60-120 min of drug exposure (versus growth to 98.6% kill rate for the other agents). Statistically significant differences in kill rates were seen between the various drugs tested depending on drug concentration and time of sampling after drug exposure.
PubMed: 38792823
DOI: 10.3390/microorganisms12050996 -
Pathogens (Basel, Switzerland) May 2024Pradofloxacin-a dual-targeting fluoroquinolone-is the most recent approved for use in food animals. Minimum inhibitory and mutant prevention concentration values were...
Pradofloxacin-a dual-targeting fluoroquinolone-is the most recent approved for use in food animals. Minimum inhibitory and mutant prevention concentration values were determined for pradofloxacin, ceftiofur, enrofloxacin, florfenicol, marbofloxacin, tildipirosin, tilmicosin, and tulathromycin. For strains, MIC values were ≤0.016/≤0.016/≤0.016 and MPC values were 0.031/0.063/0.063; for strains, the MIC values ≤0.016/≤0.016/0.031 and MPC ≤ 0.016/0.031/0.063 for pradofloxacin. The pradofloxacin C/MIC and C/MPC values for and strains, respectively, were 212.5 and 53.9 and 212.5 and 109.7. Similarly, AUC/MIC and AUC/MPC for were 825 and 209.5, and for they were 825 and 425.8. Pradofloxacin would exceed the mutant selection window for >12-16 h. Pradofloxacin appears to have a low likelihood for resistance selection against key bovine respiratory disease bacterial pathogens based on low MIC and MPC values.
PubMed: 38787251
DOI: 10.3390/pathogens13050399 -
Frontiers in Microbiology 2024Bovine respiratory disease (BRD) is one of the most important animal health problems in the beef industry. While bacterial culture and antimicrobial susceptibility...
Bacterial enrichment prior to third-generation metagenomic sequencing improves detection of BRD pathogens and genetic determinants of antimicrobial resistance in feedlot cattle.
INTRODUCTION
Bovine respiratory disease (BRD) is one of the most important animal health problems in the beef industry. While bacterial culture and antimicrobial susceptibility testing have been used for diagnostic testing, the common practice of examining one isolate per species does not fully reflect the bacterial population in the sample. In contrast, a recent study with metagenomic sequencing of nasal swabs from feedlot cattle is promising in terms of bacterial pathogen identification and detection of antimicrobial resistance genes (ARGs). However, the sensitivity of metagenomic sequencing was impeded by the high proportion of host biomass in the nasal swab samples.
METHODS
This pilot study employed a non-selective bacterial enrichment step before nucleic acid extraction to increase the relative proportion of bacterial DNA for sequencing.
RESULTS
Non-selective bacterial enrichment increased the proportion of bacteria relative to host sequence data, allowing increased detection of BRD pathogens compared with unenriched samples. This process also allowed for enhanced detection of ARGs with species-level resolution, including detection of ARGs for bacterial species of interest that were not targeted for culture and susceptibility testing. The long-read sequencing approach enabled ARG detection on individual bacterial reads without the need for assembly. Metagenomics following non-selective bacterial enrichment resulted in substantial agreement for four of six comparisons with culture for respiratory bacteria and substantial or better correlation with qPCR. Comparison between isolate susceptibility results and detection of ARGs was best for macrolide ARGs in reads but was also substantial for sulfonamide ARGs within and reads and tetracycline ARGs in reads.
DISCUSSION
By increasing the proportion of bacterial DNA relative to host DNA through non-selective enrichment, we demonstrated a corresponding increase in the proportion of sequencing data identifying BRD-associated pathogens and ARGs in deep nasopharyngeal swabs from feedlot cattle using long-read metagenomic sequencing. This method shows promise as a detection strategy for BRD pathogens and ARGs and strikes a balance between processing time, input costs, and generation of on-target data. This approach could serve as a valuable tool to inform antimicrobial management for BRD and support antimicrobial stewardship.
PubMed: 38779502
DOI: 10.3389/fmicb.2024.1386319 -
Microbial Pathogenesis Jul 2024Mannheimiahaemolytica is an opportunistic agent of the respiratory tract of bovines, a member of the Pasteurellaceae family, and the causal agent of fibrinous...
Mannheimiahaemolytica is an opportunistic agent of the respiratory tract of bovines, a member of the Pasteurellaceae family, and the causal agent of fibrinous pleuropneumonia. This bacterium possesses different virulence factors, allowing it to colonize and infect its host. The present work describes the isolation and characterization of a serine protease secreted by M. haemolytica serotype 1. This protease was isolated from M. haemolytica cultured media by precipitation with 50 % methanol and ion exchange chromatography on DEAE-cellulose. It is a 70-kDa protease able to degrade sheep and bovine fibrinogen or porcine gelatin but not bovine IgG, hemoglobin, or casein. Mass spectrometric analysis indicates its identity with protease IV of M. haemolytica. The proteolytic activity was active between pH 5 and 9, with an optimal pH of 8. It was stable at 50 °C for 10 min but inactivated at 60 °C. The sera of bovines with chronic or acute pneumonia recognized this protease. Still, it showed no cross-reactivity with rabbit hyperimmune serum against the secreted metalloprotease from Actinobacilluspleuropneumoniae, another member of the Pasteurellaceae family. M. haemolytica secreted proteases could contribute to the pathogenesis of this bacterium through fibrinogen degradation, a characteristic of this fibrinous pleuropneumonia.
Topics: Animals; Mannheimia haemolytica; Sheep; Cattle; Fibrinogen; Hydrogen-Ion Concentration; Serine Proteases; Temperature; Proteolysis; Molecular Weight; Gelatin; Enzyme Stability; Bacterial Proteins; Mass Spectrometry; Chromatography, Ion Exchange; Swine; Virulence Factors
PubMed: 38763316
DOI: 10.1016/j.micpath.2024.106706 -
Journal of Microbiological Methods Jun 2024Bovine respiratory disease (BRD) is an important health and economic burden to the cattle industry worldwide. Three bacterial pathogens frequently associated with BRD...
Bovine respiratory disease (BRD) is an important health and economic burden to the cattle industry worldwide. Three bacterial pathogens frequently associated with BRD (Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni) can possess integrative and conjugative elements (ICEs), a diverse group of mobile genetic elements that acquire antimicrobial resistance (AMR) genes (ARGs) and decrease the therapeutic efficacy of antimicrobial drugs. We developed a duplex recombinase polymerase amplification (RPA) assay to detect up to two variants of ICEs in these Pasteurellaceae. Whole genome sequence analysis of M. haemolytica, P. multocida, and H. somni isolates harbouring ICEs revealed the presence of tnpA or ebrB next to tet(H), a conserved ARG that is frequently detected in ICEs within BRD-associated bacteria. This real-time multiplex RPA assay targeted both ICE variants simultaneously, denoted as tetH_tnpA and tetH_ebrB, with a limit of detection (LOD) of 29 (95% CI [23, 46]) and 38 genome copies (95% CI [30, 59]), respectively. DNA was extracted from 100 deep nasopharyngeal swabs collected from feedlot cattle on arrival. Samples were tested for ICEs using a real-time multiplex RPA assay, and for M. haemolytica, P. multocida, H. somni, and Mycoplasma bovis using both culture methods and RPA. The assay provided sensitive and accurate identification of ICEs in extracted DNA, providing a useful molecular tool for timely detection of potential risk factors associated with the development of antimicrobial-resistant BRD in feedlot cattle.
Topics: Animals; Cattle; Nasopharynx; Recombinases; Multiplex Polymerase Chain Reaction; Interspersed Repetitive Sequences; Cattle Diseases; DNA, Bacterial; Drug Resistance, Bacterial; Bovine Respiratory Disease Complex; Conjugation, Genetic; Sensitivity and Specificity; Mannheimia haemolytica; Pasteurellaceae
PubMed: 38705209
DOI: 10.1016/j.mimet.2024.106943 -
Vaccines Apr 2024is a bovine respiratory pathogen commonly associated with bacterial bronchopneumonia. Current vaccine strategies have shown variable efficacy in feedlot cattle, and...
BACKGROUND
is a bovine respiratory pathogen commonly associated with bacterial bronchopneumonia. Current vaccine strategies have shown variable efficacy in feedlot cattle, and therefore novel vaccines are needed. spores have been investigated as a mucosal vaccine platform, due to their ability to bind and present antigens to the mucosa and act as an adjuvant. The aim of this study was to develop two spore-based mucosal vaccines targeting and evaluate their immunogenicity in mice.
METHODS
Two antigen constructs composed of cholera toxin B subunit, leukotoxin, and either the outer membrane protein PlpE (MhCP1) or GS60 (MhCP2) were synthesized, purified and then bound to spores as vaccines. In two separate mice trials, the spore-bound vaccines (Spore-MhCP1 and Spore-MhCP2) were administered to mice through intranasal and intragastric routes, while free antigens were administered intranasally and intramuscularly. Unbound spores were also evaluated intranasally. Antigen-specific serum IgG and mucosal IgA from bronchoalveolar lavage, feces, and saliva were measured after vaccination. Mice sera from all treatment groups were assessed for their bactericidal activity against .
RESULTS
In both mice experiments, intramuscular immunization induced the strongest serum IgG antibody response. However, the intranasal administration of Spore-MhCP1 and Spore-MhCP2 elicited the greatest secretory IgA-specific response against leukotoxin, PlpE, and GS60 in bronchoalveolar lavage, saliva, and feces ( < 0.05). Compared to the intranasal administration of free antigen, spore-bound antigen groups showed greater bactericidal activity against ( < 0.05).
CONCLUSIONS
Since intranasally delivered Spore-MhCP1 and Spore-MhCP2 elicited both systemic and mucosal immune responses in mice, these vaccines may have potential to mitigate lung infection in cattle by restricting colonization and proliferation in the respiratory tract. The efficacy of these mucosal spore-based vaccines merits further assessment against in cattle.
PubMed: 38675757
DOI: 10.3390/vaccines12040375