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JDS Communications May 2023Appropriate sample collection, storage conditions, and time for transport to the laboratory are important for an accurate diagnostic result. We evaluated the effects of...
Appropriate sample collection, storage conditions, and time for transport to the laboratory are important for an accurate diagnostic result. We evaluated the effects of transport storage medium type, time of storage, and storage temperatures on (MH) and (PM) recovery using an in vitro model simulation. A quantitative culture method, using colony-forming units per milliliter, was used to recover MH or PM by an in vitro model with cotton swabs. Three independent trials were conducted, in which cotton swabs were inoculated with MH or PM and placed in either (1) a sterile 15-mL polypropylene tube without transport medium (dry), (2) Amies culture medium with charcoal (ACM), or (3) Cary-Blair transport agar (CBA). Swabs were evaluated for recovery of MH or PM when stored at 3 temperatures (4°C, 23°C, or 36°C) and after storage for 8 h, 24 h, or 48 h. From all study group combinations, a total of 162 individual independent swabs were evaluated. The nonparametric Dunn all-pairs approach was used to compare the proportion of culturable bacteria, between the various storage media, temperature, and time point combinations. The proportion of MH in samples stored at 4°C was significantly higher for ACM and CBA than dry storage at 24 and 48 h. The MH samples stored at 36°C had a significantly higher proportion for ACM and CBA than dry storage at 24 h. The proportion of PM in samples stored at 4°C was significantly lower for ACM compared with dry at 8 h but significantly higher at 48 h. The PM samples stored at 23°C in ACM had a significantly higher proportion than dry samples at 24 h, and, at 48 h, ACM and CBA had a significantly higher proportion than the dry group. All swabs stored at 36°C for 48 h had a proportion close to zero, indicating decreasing diagnostic efficacy. These results support the use of transport media such as ACM and CBA for increasing the detection of PM and MH from samples, especially when samples are exposed to high temperatures. The combination of longer periods from collection of samples to diagnostic evaluation (>24 h) and higher storage temperatures (>23°C) were shown to significantly impair diagnostic accuracy.
PubMed: 37360122
DOI: 10.3168/jdsc.2022-0329 -
Veterinary Microbiology Jan 2024Mannheimia haemolytica is known as one of the major bacterial contributors to Bovine Respiratory Disease (BRD) syndrome. This study sought to establish a novel...
Mannheimia haemolytica is known as one of the major bacterial contributors to Bovine Respiratory Disease (BRD) syndrome. This study sought to establish a novel species-specific PCR to aid in identification of this key pathogen. As well, an existing multiplex PCR was used to determine the prevalence of serovars 1, 2 or 6 in Australia. Most of the 65 studied isolates originated from cattle with a total of 11 isolates from small ruminants. All problematic field isolates in the identification or serotyping PCRs were subjected to whole genome sequencing and bioinformatic analysis. The field isolates were also subjected to rep-PCR fingerprinting. A total of 59 out of the 65 tested isolates were conformed as M. haemolytica by the new species-specific PCR which is based on the rpoB gene. The confirmed M. haemolytica field isolates were assigned to serovars 1 (24 isolates), 2 (seven isolates) and 6 (26 isolates) while two of the isolates were negative in the serotyping PCR. The two non-typeable isolates were assigned to serovar 7 and 14 following whole genome sequencing and bioinformatic analysis. The rep-PCR typing resulted in five major clusters with serovars 1 and 6 often within the same cluster. The M. haemolytica-specific PCR developed in this work was species specific and should be a valuable support for frontline diagnostic laboratories. The serotyping results support the relative importance of serovars 1 and 6 in bovine respiratory disease.
Topics: Cattle; Animals; Mannheimia haemolytica; Bacteria; Serotyping; Cattle Diseases; Ruminants; Multiplex Polymerase Chain Reaction; Respiratory Tract Diseases
PubMed: 38086163
DOI: 10.1016/j.vetmic.2023.109930 -
Biomaterials Apr 2022Emerging antimicrobial resistance in infections asks for novel intervention strategies. Galacto-oligosaccharides (GOS) might be attractive alternatives to antibiotics...
Emerging antimicrobial resistance in infections asks for novel intervention strategies. Galacto-oligosaccharides (GOS) might be attractive alternatives to antibiotics due to their anti-inflammatory and anti-adhesive properties. Mannheimia haemolytica is one of the major Pasteurellaceae associated with bovine lung infections. Using M. haemolytica, we demonstrated that GOS have the capacity to reduce bacterial viability and can be used as adjuvant to improve antibiotic efficacy. Using M. haemolytica-treated primary bronchial epithelial cells (PBECs) of calves, we identified the anti-adhesive and anti-invasive activities of GOS. The observed inhibition of cytokine/chemokine release and the prevention of airway epithelial barrier dysfunction in M. haemolytica-treated PBECs by GOS might be related to the downregulation of "toll-like receptor 4/nuclear factor-κB" pathway and the anti-invasive and anti-adhesive properties of GOS. Particularly, GOS lowered lipopolysaccharides- but not flagellin-induced cytokine/chemokine release in calf and human airway epithelial cells. Finally, we performed in vivo experiments in calves and demonstrated for the first time that intranasal application of GOS can relieve lung infections/inflammation and lower M. haemolytica positivity in the lungs without affecting clinical performance. These findings not only shed light on the anti-inflammatory mechanisms of GOS during lung infections, but GOS might also be a promising anti-bacterial agent for preventing (lung) infections.
Topics: Animals; Anti-Bacterial Agents; Bacteria; Cattle; Humans; Lung; Mannheimia haemolytica; Oligosaccharides; Pneumonia
PubMed: 35286857
DOI: 10.1016/j.biomaterials.2022.121461 -
Antibiotics (Basel, Switzerland) Apr 2022Numerous antimicrobial resistance (AMR) surveillance studies have been conducted in North American feedlot cattle to investigate the major bacterial pathogens of the... (Review)
Review
Numerous antimicrobial resistance (AMR) surveillance studies have been conducted in North American feedlot cattle to investigate the major bacterial pathogens of the bovine respiratory disease (BRD) complex, specifically: , , , and . While most bacterial isolates recovered from healthy cattle are susceptible to a repertoire of antimicrobials, multidrug resistance is common in isolates recovered from cattle suffering from BRD. Integrative and conjugative elements (ICE) have gained increasing notoriety in BRD- as they appear to play a key role in the concentration and dissemination of antimicrobial resistant genes. Likewise, low macrolide susceptibility has been described in feedlot isolates of . Horizontal gene transfer has also been implicated in the spread of AMR within mycoplasmas, and experiments have shown that exposure to antimicrobials can generate high levels of resistance in mycoplasmas via a single conjugative event. Consequently, antimicrobial use (AMU) could be accelerating AMR horizontal transfer within all members of the bacterial BRD complex. While metagenomics has been applied to the study of AMR in the microbiota of the respiratory tract, the potential role of the respiratory tract microbiome as an AMR reservoir remains uncertain. Current and prospective molecular tools to survey and characterize AMR need to be adapted as point-of-care technologies to enhance prudent AMU in the beef industry.
PubMed: 35453238
DOI: 10.3390/antibiotics11040487 -
Scientific Reports Oct 2015The bovine nasopharyngeal tract plays an important role in animal health and welfare by acting as a site for the carriage of pathogens causing bovine respiratory...
The bovine nasopharyngeal tract plays an important role in animal health and welfare by acting as a site for the carriage of pathogens causing bovine respiratory disease, a condition which results in significant morbidity and mortality in feedlot cattle. We characterized the bacterial nasopharyngeal microbiota in cattle at feedlot entry (day 0) and day 60 using 454 pyrosequencing. We also identified the most frequently isolated aerobic bacteria from nasopharyngeal swabs after plating onto three types of media. The cattle nasopharyngeal microbiota was composed primarily of Proteobacteria (68.9%) and Firmicutes (19.2%). At the genus-level, there was more inter-individual variability and a total of 55 genera were identified. The genera Pseudomonas (23.7%), Shewanella (23.5%), Acinetobacter (17.5%), and Carnobacterium (12.2%) were most prevalent at entry, while after 60 days in the feedlot, Staphylococcus (20.8%), Mycoplasma (14.9%), Mannheimia (10.4%), and Moraxella (9.4%) were dominant. The nasopharyngeal microbiota also became more homogenous after 60 days in the feedlot and differed in structure at day 0 and 60. Using culture-based methods, the most frequently isolated bacteria from nasopharyngeal swabs were Bacillus, Staphylococcus, Moraxella, Pasteurella, and Mannheimia. These results provide insight into the nasopharyngeal microbiota of cattle and demonstrate that specific changes take place during feedlot production.
Topics: Animals; Bacteria; Cattle; DNA Barcoding, Taxonomic; Microbiota; Nasopharynx
PubMed: 26497574
DOI: 10.1038/srep15557 -
Frontiers in Cellular and Infection... 2023Bovine respiratory disease (BRD) is the most devastating disease affecting beef and dairy cattle producers in North America. An emerging area of interest is the... (Meta-Analysis)
Meta-Analysis
BACKGROUND
Bovine respiratory disease (BRD) is the most devastating disease affecting beef and dairy cattle producers in North America. An emerging area of interest is the respiratory microbiome's relationship with BRD. However, results regarding the effect of BRD on respiratory microbiome diversity are conflicting.
RESULTS
To examine the effect of BRD on the alpha diversity of the respiratory microbiome, a meta-analysis analyzing the relationship between the standardized mean difference (SMD) of three alpha diversity metrics (Shannon's Diversity Index (Shannon), Chao1, and Observed features (OTUs, ASVs, species, and reads) and BRD was conducted. Our multi-level model found no difference in Chao1 and Observed features SMDs between calves with BRD and controls. The Shannon SMD was significantly greater in controls compared to that in calves with BRD. Furthermore, we re-analyzed 16S amplicon sequencing data from four previously published datasets to investigate BRD's effect on individual taxa abundances. Additionally, based on Bray Curtis and Jaccard distances, health status, sampling location, and dataset were all significant sources of variation. Using a consensus approach based on RandomForest, DESeq2, and ANCOM-BC2, we identified three differentially abundant amplicon sequence variants (ASVs) within the nasal cavity, ASV5_, ASV19_, and ASV37_ However, no ASVs were differentially abundant in the other sampling locations. Moreover, based on SECOM analysis, ASV37_ had a negative relationship with ASV1_, ASV5_, and ASV4_. ASV19_ had negative relationships with ASV1_, ASV4_, ASV54_, ASV7_, and ASV8_.
CONCLUSIONS
Our results confirm a relationship between bovine respiratory disease and respiratory microbiome diversity and composition, which provide additional insight into microbial community dynamics during BRD development. Furthermore, as sampling location and sample processing (dataset) can also affect results, consideration should be taken when comparing results across studies.
Topics: Cattle; Animals; Respiratory Tract Diseases; Cattle Diseases; Clostridiales; Microbiota; Mycoplasma hyorhinis
PubMed: 37743862
DOI: 10.3389/fcimb.2023.1223090 -
BMC Veterinary Research Aug 2017Outbreaks of a Haemorrhagic Septicaemia (HS) like disease causing large mortalities in camels (Camelus dromedarius) in Asia and in Africa have been reported since 1890....
BACKGROUND
Outbreaks of a Haemorrhagic Septicaemia (HS) like disease causing large mortalities in camels (Camelus dromedarius) in Asia and in Africa have been reported since 1890. Yet the aetiology of this condition remains elusive. This study is the first to apply state of the art molecular methods to shed light on the nasopharyngeal carrier state of Pasteurellaceae in camels. The study focused on HS causing Pasteurella multocida capsular types B and E. Other Pasteurellaceae, implicated in common respiratory infections of animals, were also investigated.
METHODS
In 2007 and 2008, 388 nasopharyngeal swabs were collected at 12 locations in North Kenya from 246 clinically healthy camels in 81 herds that had been affected by HS-like disease. Swabs were used to cultivate bacteria on blood agar and to extract DNA for subsequent PCR analysis targeting P. multocida and Mannheimia-specific gene sequences.
RESULTS
Forty-five samples were positive for P. multocida genes kmt and psl and for the P. multocida Haemorrhagic Septicaemia (HS) specific sequences KTSP61/KTT72 but lacked HS-associated capsular type B and E genes capB and capE. This indicates circulation of HS strains in camels that lack established capsular types. Sequence analysis of the partial 16S rRNA gene identified 17 nasal swab isolates as 99% identical with Mannheimia granulomatis, demonstrating a hitherto unrecognised active carrier state for M. granulomatis or a closely related Mannheimia sp. in camels.
CONCLUSIONS
The findings of this study provide evidence for the presence of acapsular P. multocida or of hitherto unknown capsular types of P. multocida in camels, closely related to P. multocida strains causing HS in bovines. Further isolations and molecular studies of camelid P. multocida from healthy carriers and from HS-like disease in camels are necessary to provide conclusive answers. This paper is the first report on the isolation of M. granulomatis or a closely related new Mannheimia species from camelids.
Topics: Animals; Camelus; Carrier State; DNA, Bacterial; Nasopharynx; Pasteurella Infections; Pasteurella multocida; Pasteurellaceae; Pasteurellaceae Infections; Pilot Projects; Polymerase Chain Reaction
PubMed: 28830429
DOI: 10.1186/s12917-017-1189-y -
Genome Feb 2022Common bacterial causes of bovine respiratory disease (BRD) include , , and . Within , two major genotypes are commonly found in cattle (1 and 2); however, genotype 2...
Common bacterial causes of bovine respiratory disease (BRD) include , , and . Within , two major genotypes are commonly found in cattle (1 and 2); however, genotype 2 strains are isolated from diseased lungs much more frequently than genotype 1 strains. Outer membrane proteins (OMPs) of , , and genotype 2 may be important factors for acquired host immunity. The predicted OMP differences between genotypes 1 and 2 have been previously identified. In this study, we expanded the focus to include bovine-isolated strain genomes representing all three species and the two genotypes. Reported here are the core genomes unique to each of them, core genomes shared between some or all combinations of the three species and two genotypes, and predicted OMPs within these core genomes. The OMPs identified in this study are potential candidates for further studies and the development of interventions against BRD.
Topics: Animals; Bacterial Outer Membrane Proteins; Cattle; Genotype; Mannheimia haemolytica; Pasteurella multocida
PubMed: 34348051
DOI: 10.1139/gen-2021-0038 -
Archives of Razi Institute Jun 2019Mannheimia haemolytica is responsible for considerable economic losses to cattle, sheep, and goat industries in many parts of the world. This bacterium isone of the...
Mannheimia haemolytica is responsible for considerable economic losses to cattle, sheep, and goat industries in many parts of the world. This bacterium isone of the causative agents of shipping fever in cattle. Current vaccines against M. haemolytica are moderately efficacious since they do not provide complete protection against the disease. Production of an economic vaccine for protecting farm animals against M. haemolytica has attracted the attention of many scientists. The outer membrane proteins (OMPs) play a major role in the pathogenesis and immunogenicity of M. haemolytica. Research on M. haemolytica OMPs has shown that antibodies to a particular OMP may be important in immune protection. In the current study, the gene for M. haemolytica OMP PlpE was cloned into the expression vector pET26-b, and then expressed in Escherichia coli BL21. The expression of the protein was carried out by the induction of cultured Escherichiacoli Bl21 cells with 1mM isopropyl-β-D-thiogalactopyranoside. The recombinant PlpE was purified using Ni-NTA agarose resin, and then subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. The identity of the expressed protein was analyzed by western blotting. It was revealed that rPlpE was expressed and produced properly. To assess the immunogenicity of the recombinant protein, the purified rPlpE was used as an antigen for antibody production in goats. The observations suggested that the produced recombinant protein can be used as a antigen for developing diagnostic tests and or as a vaccine candidate.
Topics: Antibody Formation; Bacterial Outer Membrane Proteins; Blotting, Western; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Gene Expression; Mannheimia haemolytica; Microorganisms, Genetically-Modified; Recombinant Proteins
PubMed: 31232560
DOI: 10.22092/ari.2018.116479.1169 -
Veterinary World May 2018is a Gram-negative, non-motile, and non-spore-forming rod-shaped coccobacillus bacterium. On blood agar plate, it shows complete hemolysis. This bacterium constitutes a...
BACKGROUND AND AIM
is a Gram-negative, non-motile, and non-spore-forming rod-shaped coccobacillus bacterium. On blood agar plate, it shows complete hemolysis. This bacterium constitutes a part of normal flora of the upper respiratory system of ruminants. It is considered as the opportunistic pathogen and the main factor of pneumonic pasteurellosis, which has caused a severe economic loss in sheep and cattle industries. Considering the prevalence of the disease in sheep and goat population in the dry and hot regions of the country in general and in Fars province in particular in the form of pneumonia, the purpose of this study was to isolate and identify the bacterium from the lung tissues of sheep slaughtered in Shiraz abattoir through culturing and polymerase chain reaction (PCR) methods.
MATERIALS AND METHODS
In this study, a total of 2500 sheep's lungs were evaluated for finding pneumonic effects. Then, 161 infected pneumonic samples of lung tissues were investigated by culture and PCR methods.
RESULTS
After cultivation, purification, and DNA extraction, 38 samples were found positive for by cultivation and then all the 38 isolates were confirmed by PCR and multiplex PCR (mPCR). Results of this study indicated that culture and PCR are both practical in identification and isolation of this bacterium though culture is more time-consuming. The utilized mPCR has been more successful in the identification of the bacteria since it requires less time and cost.
CONCLUSION
In this study, PCR as a superior method among other methods of bacteriology for fast examination of infectious diseases and mPCR, which is a valuable tool for identification of . in clinical samples of animals, was used.
PubMed: 29915502
DOI: 10.14202/vetworld.2018.636-641