ABSTRACT
Disruptive innovations in long-range, cost-effective direct template nucleic acid sequencing are transforming clinical and diagnostic medicine. A multidrug resistant strain and a pan-susceptible strain of Mannheimia haemolytica, isolated from pneumonic bovine lung samples, were sequenced at 146× and 111× coverage, respectively with Oxford Nanopore Technologies MinION. De novo assembly produced a complete genome for the non-resistant strain and a nearly complete assembly for the drug resistant strain. Functional annotation using RAST (Rapid Annotations using Subsystems Technology), CARD (Comprehensive Antibiotic Resistance Database) and ResFinder databases identified genes conferring resistance to different classes of antibiotics including ß-lactams, tetracyclines, lincosamides, phenicols, aminoglycosides, sulfonamides and macrolides. Resistance phenotypes of the M. haemolytica strains were determined by minimum inhibitory concentration (MIC) of the antibiotics. Sequencing with a highly portable MinION device corresponded to MIC assays with most of the antimicrobial resistant determinants being identified with as few as 5437 reads, except for the genes responsible for resistance to Fluoroquinolones. The resulting quality assemblies and AMR gene annotation highlight the efficiency of ultra-long read, whole-genome sequencing (WGS) as a valuable tool in diagnostic veterinary medicine.
Subject(s)
Drug Resistance, Bacterial , Mannheimia haemolytica/drug effects , Mannheimia haemolytica/genetics , Nanopore Sequencing/methods , Pneumonia of Calves, Enzootic/microbiology , Animals , Anti-Bacterial Agents/pharmacology , Cattle , Genome, Bacterial , Mannheimia haemolytica/isolation & purification , Microbial Sensitivity Tests , Pneumonia of Calves, Enzootic/diagnosis , Sequence Analysis, DNA , Whole Genome SequencingABSTRACT
Mannheimia haemolytica is the most important bacterial pathogen isolated from cases of Bovine Respiratory Disease (BRD). Routine identification of these bacteria is usually performed using phenotypic methods. Our study showed that MALDI-TOF MS is a reliable alternative to these methods. All of the strains analyzed were identified as M. haemolytica. The identification results were compared to those obtained using conventional methods commonly used in microbiological diagnostics, based on detection and analysis of biochemical properties of microorganisms. The degree of agreement between the two methods for identifying M. haemolytica was 100%.
Subject(s)
Mannheimia haemolytica , Pneumonia of Calves, Enzootic/diagnosis , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/veterinary , Animals , Cattle , Pneumonia of Calves, Enzootic/microbiology , Reproducibility of Results , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methodsABSTRACT
BACKGROUND: Serum haptoglobin (Hp) and haptoglobin matrix metalloproteinase 9 complexes (Hp-MMP 9) have been identified as biomarkers with diagnostic potential in cattle with conditions resulting in an acute inflammatory response. The purpose of this study was to evaluate potential diagnostic applications of serum Hp and Hp-MMP 9 concentrations in calves with BRD and establish a timeline for their detection in calves experimentally challenged with Bibersteinia trehalosi and Mannheimia haemolytica. Thirty-five cross bred dairy calves were inoculated via tracheal catheterization with either a PCR confirmed leukotoxin negative B. trehalosi isolate, a PCR confirmed leukotoxin positive B. trehalosi isolate, a Mannheimia haemolytica isolate, a combination of leukotoxin negative B. trehalosi and M. haemolytica, or a negative control. Serum samples were collected throughout the study. Calves were euthanized and necropsy performed on day 10 post inoculation. RESULTS: M. haemolytica inoculated calves had increased lung involvement. Serum Hp and Hp- MMP 9 concentrations were elevated compared to the other treatment groups. Increases in serum Hp and Hp-MMP 9 concentrations for the M. haemolytica group were significantly different from other study groups on day 7 of the study. B. trehalosi inoculated calves did not have increased lung involvement compared to control calves, but the leukotoxin positive B. trehalosi group demonstrated increased serum Hp-MMP 9 concentrations from day 3 to the end of the study compared to the pre-inoculation concentrations. CONCLUSION: Serum Hp-MMP 9 concentration is a useful diagnostic tool for detecting early pulmonary inflammation in calves challenged with B. trehalosi and M. haemolytica. Serum Hp-MMP 9 may also be a useful tool in detecting subclinical pulmonary inflammation in challenged calves.
