-
PloS One 2017Respiratory disease has been a persistent problem for the recovery of bighorn sheep (Ovis canadensis), but has uncertain etiology. The disease has been attributed to...
Respiratory disease has been a persistent problem for the recovery of bighorn sheep (Ovis canadensis), but has uncertain etiology. The disease has been attributed to several bacterial pathogens including Mycoplasma ovipneumoniae and Pasteurellaceae pathogens belonging to the Mannheimia, Bibersteinia, and Pasteurella genera. We estimated detection probability for these pathogens using protocols with diagnostic tests offered by a fee-for-service laboratory and not offered by a fee-for-service laboratory. We conducted 2861 diagnostic tests on swab samples collected from 476 bighorn sheep captured across Montana and Wyoming to gain inferences regarding detection probability, pathogen prevalence, and the power of different sampling methodologies to detect pathogens in bighorn sheep populations. Estimated detection probability using fee-for-service protocols was less than 0.50 for all Pasteurellaceae and 0.73 for Mycoplasma ovipneumoniae. Non-fee-for-service Pasteurellaceae protocols had higher detection probabilities, but no single protocol increased detection probability of all Pasteurellaceae pathogens to greater than 0.50. At least one protocol resulted in an estimated detection probability of 0.80 for each pathogen except Mannheimia haemolytica, for which the highest detection probability was 0.45. In general, the power to detect Pasteurellaceae pathogens at low prevalence in populations was low unless many animals were sampled or replicate samples were collected per animal. Imperfect detection also resulted in low precision when estimating prevalence for any pathogen. Low and variable detection probabilities for respiratory pathogens using live-sampling protocols may lead to inaccurate conclusions regarding pathogen community dynamics and causes of bighorn sheep respiratory disease epizootics. We recommend that agencies collect multiples samples per animal for Pasteurellaceae detection, and one sample for Mycoplasma ovipneumoniae detection from at least 30 individuals to reliably detect both Pasteurellaceae and Mycoplasma ovipneumoniae at the population-level. Availability of PCR diagnostic tests to wildlife management agencies would improve the ability to reliably detect Pasteurellaceae in bighorn sheep populations.
Topics: Animals; DNA, Bacterial; Mycoplasma ovipneumoniae; Pasteurellaceae; Population Density; Prevalence; Real-Time Polymerase Chain Reaction; Respiratory Tract Infections; Sheep; Sheep Diseases; Sheep, Bighorn; Specimen Handling
PubMed: 28708832
DOI: 10.1371/journal.pone.0180689 -
Virulence 2018Bacterial lipooligosaccharide (LOS) is an important virulence-associated factor, and its sialylation largely confers its ability to mediate cell adhesion, invasion,...
Bacterial lipooligosaccharide (LOS) is an important virulence-associated factor, and its sialylation largely confers its ability to mediate cell adhesion, invasion, inflammation, and immune evasion. Here, we investigated the function of the Haemophilus parasuis α-2,3-sialyltransferase gene, lsgB, which determines the terminal sialylation of LOS, by generating a lsgB deletion mutant as well as a complementation strain. Our data indicate a direct effect of lsgB on LOS sialylation and reveal important roles of lsgB in promoting the pathogenicity of H. parasuis, including adhesion to and invasion of porcine cells in vitro, bacterial load and survival in vivo, as well as a contribution to serum resistance. These observations highlight the function of lsgB in mediating LOS sialylation and more importantly its role in H. parasuis infection. These findings provide a more profound understanding of the pathogenic mechanism of this disease-causing bacterium.
Topics: Amino Acid Sequence; Animals; Cells, Cultured; Gene Deletion; Genetic Complementation Test; Haemophilus parasuis; Lipopolysaccharides; Mutation; Sialyltransferases; Swine; Virulence; Virulence Factors
PubMed: 30036124
DOI: 10.1080/21505594.2018.1502606 -
Scientific Reports Aug 2019We investigated three bovine respiratory pathobionts in healthy cattle using qPCR optimised and validated to quantify Histophilus somni, Mannheimia haemolytica and...
We investigated three bovine respiratory pathobionts in healthy cattle using qPCR optimised and validated to quantify Histophilus somni, Mannheimia haemolytica and Pasteurella multocida over a wide dynamic range. A longitudinal study was conducted to investigate the carriage and density of these bacteria in the nasal passages of healthy beef calves (N = 60) housed over winter in an experimental farm setting. The three pathobiont species exhibited remarkably different carriage rates and density profiles. At housing, high carriage rates were observed for P. multocida (95%), and H. somni (75%), while fewer calves were positive for M. haemolytica (13%). Carriage rates for all three bacterial species declined over the 75-day study, but not all individuals became colonised despite sharing of environment and airspace. Colonisation patterns ranged from continuous to intermittent and were different among pathobiont species. Interval-censored exponential survival models estimated the median duration of H. somni and P. multocida carriage at 14.8 (CI: 10.6-20.9) and 55.5 (CI: 43.3-71.3) days respectively, and found higher density P. multocida carriage was associated with slower clearance (p = 0.036). This work offers insights into the dynamics of pathobiont carriage and provides a potential platform for further data collection and modelling studies.
Topics: Animals; Bacterial Load; Carrier State; Cattle; Cattle Diseases; DNA, Bacterial; Longitudinal Studies; Male; Mannheimia haemolytica; Nasal Cavity; Pasteurella multocida; Pasteurellaceae; Pasteurellaceae Infections; Polymerase Chain Reaction
PubMed: 31420565
DOI: 10.1038/s41598-019-48007-5 -
International Journal of Molecular... Jul 2021(1) Background: Short-read sequencing allows for the rapid and accurate analysis of the whole bacterial genome but does not usually enable complete genome assembly.... (Comparative Study)
Comparative Study
(1) Background: Short-read sequencing allows for the rapid and accurate analysis of the whole bacterial genome but does not usually enable complete genome assembly. Long-read sequencing greatly assists with the resolution of complex bacterial genomes, particularly when combined with short-read Illumina data. However, it is not clear how different assembly strategies affect genomic accuracy, completeness, and protein prediction. (2) Methods: we compare different assembly strategies for , which causes Glässer's disease, characterized by fibrinous polyserositis and arthritis, in swine by using Illumina sequencing and long reads from the sequencing platforms of either Oxford Nanopore Technologies (ONT) or SMRT Pacific Biosciences (PacBio). (3) Results: Assembly with either PacBio or ONT reads, followed by polishing with Illumina reads, facilitated high-quality genome reconstruction and was superior to the long-read-only assembly and hybrid-assembly strategies when evaluated in terms of accuracy and completeness. An equally excellent method was correction with Homopolish after the ONT-only assembly, which had the advantage of avoiding hybrid sequencing with Illumina. Furthermore, by aligning transcripts to assembled genomes and their predicted CDSs, the sequencing errors of the ONT assembly were mainly indels that were generated when homopolymer regions were sequenced, thus critically affecting protein prediction. Polishing can fill indels and correct mistakes. (4) Conclusions: The assembly of bacterial genomes can be directly achieved by using long-read sequencing techniques. To maximize assembly accuracy, it is essential to polish the assembly with homologous sequences of related genomes or sequencing data from short-read technology.
Topics: Animals; Genome, Bacterial; Haemophilus parasuis; High-Throughput Nucleotide Sequencing; Nanopore Sequencing; Phylogeny; Sequence Alignment; Sequence Analysis, DNA; Swine
PubMed: 34299288
DOI: 10.3390/ijms22147668 -
Clinical Microbiology and Infection :... Jul 2020To compare the determinants of trimethoprim-sulfamethoxazole resistance with established susceptibility values for fastidious Haemophilus spp., to provide... (Comparative Study)
Comparative Study
OBJECTIVES
To compare the determinants of trimethoprim-sulfamethoxazole resistance with established susceptibility values for fastidious Haemophilus spp., to provide recommendations for optimal trimethoprim-sulfamethoxazole measurement.
METHODS
We collected 50 strains each of Haemophilus influenzae and Haemophilus parainfluenzae at Bellvitge University Hospital. Trimethoprim-sulfamethoxazole susceptibility was tested by microdilution, E-test and disc diffusion using both Mueller-Hinton fastidious (MH-F) medium and Haemophilus test medium (HTM) following EUCAST and CLSI criteria, respectively. Mutations in folA, folP and additional determinants of resistance were identified in whole-genome-sequenced isolates.
RESULTS
Strains presented generally higher rates of trimethoprim-sulfamethoxazole resistance when grown on HTM than on MH-F, independent of the methodology used (average MIC 2.6-fold higher in H. influenzae and 1.2-fold higher in H. parainfluenzae). The main resistance-related determinants were as follows: I95L and F154S/V in folA; 3- and 15-bp insertions and substitutions in folP; acquisition of sul genes; and FolA overproduction potentially linked to mutations in -35 and -10 promoter motifs. Of note, 2 of 19 H. influenzae strains (10.5%) and 9 of 33 H. parainfluenzae strains (27.3%) with mutations and assigned as resistant by microdilution were inaccurately considered susceptible by disc diffusion. This misinterpretation was resolved by raising the clinical resistance breakpoint of the EUCAST guidelines to ≤30 mm.
CONCLUSIONS
Given the routine use of disc diffusion, a significant number of strains could potentially be miscategorized as susceptible to trimethoprim-sulfamethoxazole despite having resistance-related mutations. A simple modification to the current clinical resistance breakpoint given by the EUCAST guideline for MH-F ensures correct interpretation and correlation with the reference standard method of microdilution.
Topics: Anti-Bacterial Agents; Bacterial Proteins; Culture Media; Drug Resistance, Multiple, Bacterial; Haemophilus Infections; Haemophilus influenzae; Haemophilus parainfluenzae; High-Throughput Nucleotide Sequencing; Humans; Microbial Sensitivity Tests; Mutation; Promoter Regions, Genetic; Trimethoprim, Sulfamethoxazole Drug Combination; Whole Genome Sequencing
PubMed: 31811916
DOI: 10.1016/j.cmi.2019.11.022 -
PloS One 2023Small RNAs (sRNA), in association with the global chaperone regulator Hfq, positively or negatively regulate gene expression in bacteria. For this study, Histophilus...
Small RNAs (sRNA), in association with the global chaperone regulator Hfq, positively or negatively regulate gene expression in bacteria. For this study, Histophilus somni sRNAs that bind to Hfq were identified and then partially characterized. The Hfq-associated sRNAs in H. somni were isolated and identified by co-immunoprecipitation using anti-Hfq antibody, followed by sRNA sequencing. Sequence analysis of the sRNA samples identified 100 putative sRNAs, out of which 16 were present in pathogenic strain 2336, but not in non-pathogenic strain 129Pt. Bioinformatic analyses suggested that the sRNAs HS9, HS79, and HS97 could bind to many genes putatively involved in virulence/biofilm formation. Furthermore, multi-sequence alignment of the sRNA regions in the genome revealed that HS9 and HS97 could interact with sigma 54, which is a transcription factor linked to important bacterial traits, including motility, virulence, and biofilm formation. Northern blotting was used to determine the approximate size, abundance and any processing events attributed to the sRNAs. Selected sRNA candidates were confirmed to bind Hfq, as determined by electrophoretic mobility shift assays using sRNAs synthesized by in vitro transcription and recombinant Hfq. The exact transcriptional start site of the sRNA candidates was determined by RNA ligase-mediated rapid amplification of cDNA ends, followed by cloning and sequencing. This is the first investigation of H. somni sRNAs that show they may have important regulatory roles in virulence and biofilm formation.
Topics: Blotting, Northern; Cell Aggregation; Computational Biology; Pasteurellaceae; RNA, Small Untranslated
PubMed: 37220152
DOI: 10.1371/journal.pone.0286158 -
Microbiology Spectrum Dec 2021Histophilus somni is a Gram-negative bacterial organism that acts as an opportunistic pathogen and is a fastidious member of the family associated with diseases of...
Histophilus somni is a Gram-negative bacterial organism that acts as an opportunistic pathogen and is a fastidious member of the family associated with diseases of respiratory, reproductive, cardiac, and other tissues of ruminants. We identified an intervening sequence (IVS) embedded in all five copies of the 23S rRNA gene in the closed genome sequence of the H. somni isolate USDA-ARS-USMARC-63250 that may play an important role in affecting the biology of the organism. Sequencing the RNA from this isolate shows that most of the IVS is cleaved from the transcript, resulting in independent fragments of this structural rRNA that remain functional within the bacterial ribosome. The IVS lies between positions 1170 and 1278 bp of the 3,017-bp gene and exhibits self-complementarity between its 5' and 3' ends that predicts a stem-loop structure interrupting helix-45 in the transcribed 23S rRNA. Excision removes a 94-nucleotide (nt) stem-loop structure that displays an unusual 1-nt 3' end overhang instead of the more typical 2-nt overhang commonly observed at the ends of other excised IVS stem-loops. A comparison with genomes of other H. somni isolates indicates that this IVS is highly conserved, with 31 of 32 complete genomes having similar interruptions of canonical 23S rRNA genes. The potential biological effects of either the released IVS or the fragmentation of the functional 23S rRNA are unknown, but fragmentation may enhance rRNA degradation in ways that contribute to the regulation of gene expression. The genome biology underlying H. somni virulence, pathogenicity, environmental adaptability, and broad tissue tropism is understood poorly. We identified a novel H. somni 109-nt IVS stem-loop structure, of which the central portion is excised from the 23S rRNA transcript, resulting in the fragmentation of this rRNA in the H. somni isolate USDA-ARS-USMARC-63250 and the release of a 94-nt structured RNA of unknown function. We determined that this peculiar rRNA biology is widespread among sequenced H. somni isolates, suggesting it has importance to organism biology. The fragmented 23S rRNA molecules remain functional in the ribosome, given that the isolate grows in culture. The structured excised portion of the IVS, presumably due to the action of the endoribonuclease III, has an unusual 1-nt 3' end overhang. This newly discovered H. somni 23S rRNA fragmentation may enhance rRNA degradation providing a previously unrecognized avenue for regulating H. somni biological processes.
Topics: Animals; Base Sequence; Cattle; Cattle Diseases; Introns; Inverted Repeat Sequences; Nucleic Acid Conformation; Pasteurellaceae; Pasteurellaceae Infections; RNA, Bacterial; RNA, Ribosomal, 23S; Respiratory Tract Infections; Ribosomes; Sequence Analysis, RNA
PubMed: 34851158
DOI: 10.1128/Spectrum.01431-21 -
Veterinary Medicine and Science Mar 2022We report Avibacterium paragallinarum and Klebsiella pneumoniae coinfection in a grey crowned crane (Balearica regulorum). The crane was recovered from illegal captivity...
We report Avibacterium paragallinarum and Klebsiella pneumoniae coinfection in a grey crowned crane (Balearica regulorum). The crane was recovered from illegal captivity and released at a grey crowned crane (GCC) rehabilitation facility located at Akagera National Park in Rwanda. One year after being transferred, the bird presented with clinical signs suggesting a respiratory disease. Those signs included severe dyspnoea with mouth breathing, sneezing and nasal discharge. The crane was put on a 3-day treatment with antibiotics (ceftiofur 200 mg/ml at 50 mg/kg intramuscularly) and anti-inflammatory drug (meloxicam, intramuscular injection at a dose of 2 mg/kg), after which the crane seemed to have recovered. A month later, the same crane presented similar clinical signs and was treated with enrofloxacin at 10 mg/kg intramuscularly. Despite the treatment, the crane died 19 h later. At necropsy, adhesive air sacculitis and hydroperitoneum were observed, and a reddish fluid in air sacs and in the abdominal cavity was found. Also, a marked hepatomegaly and splenomegaly were observed. Samples were collected for laboratory examination. Molecular tests done on the tracheal and cloacal swabs revealed A. paragallinarum and K. pneumoniae, respectively. This is the first case of A. paragallinarum and K. pneumoniae coinfection reported in a grey crowned crane. Our study contributes to knowledge on the ecological distribution of both these pathogens in wild birds. It provides an opportunity to investigate further the clinical significance of infectious coryza in Rwanda's wild and domestic birds.
Topics: Animals; Birds; Coinfection; Haemophilus paragallinarum
PubMed: 35143715
DOI: 10.1002/vms3.766 -
Frontiers in Cellular and Infection... 2022In the management of otitis media (OM), identification of causative bacterial pathogens and knowledge of their biofilm formation can provide more targeted treatment...
In the management of otitis media (OM), identification of causative bacterial pathogens and knowledge of their biofilm formation can provide more targeted treatment approaches. Current clinical diagnostic methods rely on the visualization of the tympanic membrane and lack real-time assessment of the causative pathogen(s) and the nature of any biofilm that may reside behind the membrane and within the middle ear cavity. In recent years, optical coherence tomography (OCT) has been demonstrated as an improved diagnostic tool for visualization and morphological characterization of OM biofilms and middle ear effusions; but lacks specificity about the causative bacterial species. This study proposes the combination of OCT and Raman spectroscopy (RS) to examine differences in the refractive index, optical attenuation, and biochemical composition of , , , and ; four of the leading otopathogens in OM. This combination provides a dual optical approach for identifying and differentiating OM-causing bacterial species under three different growth environments (i.e., agar-grown colonies, planktonic cells from liquid cultures, and biofilms). This study showed that RS was able to identify key biochemical variations to differentiate all four OM-causing bacteria. Additionally, biochemical spectral changes (RS) and differences in the mean attenuation coefficient (OCT) were able to distinguish the growth environment for each bacterial species.
Topics: Bacteria; Biofilms; Haemophilus influenzae; Humans; Otitis Media; Spectrum Analysis, Raman; Tomography, Optical Coherence
PubMed: 36034696
DOI: 10.3389/fcimb.2022.869761 -
The Canadian Veterinary Journal = La... Jan 2022This study compared changes in prevalence and antimicrobial susceptibility of , and in feedlot calves derived from the auction market (AUCT; = 299) and from a...
This study compared changes in prevalence and antimicrobial susceptibility of , and in feedlot calves derived from the auction market (AUCT; = 299) and from a single-ranch source (RANCH; = 300). In the AUCT calves, the prevalence of decreased, whereas increased over the feeding period. The AUCT calves showed an increase in isolates not susceptible to tulathromycin for all bovine respiratory disease (BRD) pathogens, an increase in and isolates not susceptible to oxytetracycline, and an increase in isolates not susceptible to florfenicol. In the RANCH calves, the prevalence of all 3 BRD pathogens was high at feedlot entry and decreased significantly during the study period. In RANCH calves, there was a significant increase in isolates not susceptible to oxytetracycline, tulathromycin, and florfenicol. Surprisingly, there was a significant decrease in isolates that were not susceptible to oxytetracycline, tilmicosin, and tulathromycin.
Topics: Animals; Anti-Bacterial Agents; Bovine Respiratory Disease Complex; Cattle; Cattle Diseases; Drug Resistance, Bacterial; Mannheimia haemolytica; Microbial Sensitivity Tests; Pasteurella multocida
PubMed: 34975167
DOI: No ID Found