Transcriptomic profiles of Mannheimia haemolytica planktonic and biofilm associated cells.

Mannheimia haemolytica is the principal agent contributing to bovine respiratory disease and can form biofilms with increased resistance to antibiotic treatment and host immune defenses. To investigate the molecular mechanisms underlying M. haemolytica biofilm formation, transcriptomic analyses were performed with mRNAs sequenced from planktonic and biofilm cultures of pathogenic serotypes 1 (St 1; strain D153) and St 6 (strain D174), and St 2 (strain D35). The three M. haemolytica serotypes were cultured in two different media, Roswell Park Memorial Institute (RPMI) 1640 and brain heart infusion (BHI) to form the biofilms. Transcriptomic analyses revealed that the functions of the differentially expressed genes (DEGs) in biofilm associated cells were not significantly affected by the two media. A total of 476 to 662 DEGs were identified between biofilm associated cells and planktonic cells cultured under BHI medium. Functional analysis of the DEGs indicated that those genes were significantly enriched in translation and many biosynthetic processes. There were 234 DEGs identified in St 1 and 6, but not in St 2. The functions of the DEGs included structural constituents of ribosomes, transmembrane proton transportation, proton channels, and proton-transporting ATP synthase. Potentially, some of the DEGs identified in this study provide insight into the design of new M. haemolytica vaccine candidates.

Enhanced phagocytosis and complement-mediated killing of Mannheimia haemolytica serotype 1 following in-frame CMP-sialic acid synthetase (neuA) gene deletion.

IMPORTANCE: The Gram-negative coccobacillus Mannheimia haemolytica is a natural inhabitant of the upper respiratory tract in ruminants and the most common bacterial agent involved in bovine respiratory disease complex development. Key virulence factors harbored by M. haemolytica are leukotoxin, lipopolysaccharide, capsule, adhesins, and neuraminidase which are involved in evading innate and adaptive immune responses. In this study, we have shown that CMP-sialic acid synthetase (neuA) is necessary for the incorporation of sialic acid onto the membrane, and inactivation of neuA results in increased phagocytosis and complement-mediated killing of M. haemolytica, thus demonstrating that sialylation contributes to the virulence of M. haemolytica.

Assessing shared respiratory pathogens between domestic (Ovis aries) and bighorn (Ovis canadensis) sheep; methods for multiplex PCR, amplicon sequencing, and bioinformatics to characterize respiratory flora.

Respiratory disease is responsible for dramatic population declines in bighorn sheep (Ovis canadensis), and respiratory pathogen diagnostics contribute to the management of bighorn populations. To create a comprehensive and consistent approach to bighorn sheep respiratory diagnostics, we created a culture-independent assay to detect and strain type Mannheimia haemolytica, Bibersteinia trehalosi, Pasteurella multocida, and Mycoplasma ovipneumoniae. The assay also detects and characterizes the Pasteurellaceae leukotoxin A gene, and broadly assesses the bacterial composition of each sample based on 16S rRNA sequences. The assay is based on a three-step approach: 1) Multiplex PCR to amplify targets including eight loci for each bacterial species, the Pasteurellaceae lktA gene, and the 16S rRNA gene 2) Library preparation, barcoding, and short-read Illumina sequencing to determine the genetic sequences of each target, and 3) Bioinformatics in the form of automated software to analyze genetic sequences. The assay was designed to assess shared pathogens between domestic and bighorn sheep, but could be useful for many applications in bighorn sheep respiratory disease research and management.

Differential identification of Mannheimia haemolytica genotypes 1 and 2 using colorimetric loop-mediated isothermal amplification.

OBJECTIVE: Mannheimia haemolytica is the primary bacterial pathogen associated with bovine respiratory disease complex (BRDC). While M. haemolytica has been subdivided into 12 capsular serotypes (ST), ST1, ST2 and ST6 are commonly isolated from cattle. More recently, M. haemolytica strains isolated from North American cattle have been classified into genotypes 1 (ST2) and 2 (ST1 and ST6). Of the two genotypes, genotype 1 strains are frequently isolated from healthy animals whereas, genotype 2 strains are predominantly isolated from BRDC animals. However, isolation of both genotypes from pneumonic lung samples can complicate diagnosis. Therefore, the aim of this study was to develop a colorimetric loop-mediated isothermal amplification (LAMP) assay to differentiate M. haemolytica genotypes. RESULTS: The genotype specificity of the LAMP was tested using purified genomic DNA from 22 M. haemolytica strains (10 genotype 1, 12 genotype 2) and strains from four related Pasteurellaceae species; Bibersteinia trehalosi, Mannheimia glucosida, Pasteurella multocida, and Histophilus somni. Genotype 1 (adhesin pseudogene B1) specific-LAMP reactions amplified DNA only from genotype 1 strains while genotype 2 (adhesin G) reactions amplified DNA only from genotype 2 strains. The overall detection sensitivity and specificity of the newly developed colorimetric LAMP assay for each genotype were 100%. The limits of detection of two LAMP assays were 1-100 target gene copies per reaction. LAMP primers designed in this study may help the differential identification of M. haemolytica genotypes 1 and 2.

Changes in the phenotypic susceptibility of Mannheimia haemolytica isolates to macrolide antimicrobials during the early feeding period following metaphylactic tulathromycin use in western Canadian feedlot calves.

Cattle at high-risk for bovine respiratory disease on entry to western Canadian feedlots are often treated metaphylactically with antimicrobials from the macrolide class. High levels of resistance to macrolides have been reported in Mannheimia haemolytica isolates from clinical samples, but it is less clear whether this trend extends to the broader feedlot population. The objective was to describe near-term [< 40 days on feed (DOF)] changes in the recovery and susceptibility of M. haemolytica isolates from healthy feedlot calves after metaphylactic exposure to tulathromycin. Eight cohorts of 100 calves (n = 800) were sampled via deep nasopharyngeal swab at entry processing (i.e., before metaphylaxis, at 1 DOF) and again at 13 DOF. Ten calves from each cohort (n = 80) were randomly sampled a third time at 36 DOF. Recovery of M. haemolytica isolates across all cohorts increased over the study period, from 33% (95% CI: 26.5 to 40.2%) at 1 DOF to 75% (95% CI: 71.4 to 78.3%) at 36 DOF. A significant shift in the minimum inhibitory concentration (MIC) distribution of tulathromycin from 1 DOF (MIC90 ≤ 8 µg/mL) to 13 DOF (MIC90 > 64 µg/mL) was observed. A subset of 36 isolates from 13 DOF screened for macrolide resistance genes via multiplex polymerase chain reaction all harbored the msrE and mphE genes. Recovery of M. haemolytica at 13 and 36 DOF did not decline in response to metaphylactic use of tulathromycin; conversely, we inferred the potential for rapid inter-pen spread of a macrolide-resistant clone by 13 DOF in 6 of 8 pens under selective pressure from antimicrobial use.

Changements dans la sensibilité phénotypique des isolats de Mannheimia haemolytica aux a ntibiotiques de la classe des macrolides au début de la période d'alimentation après l'utilisation m étaphylactique de tulathromycine chez les veaux des parcs d'engraissement de l'Ouest canadien. Les bovins à risque élevé de maladies respiratoires bovines à leur entrée dans les parcs d'engraissement de l'Ouest canadien sont souvent traités métaphylactiquement avec des antimicrobiens de la classe des macrolides. Des taux élevés de résistance aux macrolides ont été signalés chez les isolats de Mannheimia haemolytica provenant d'échantillons cliniques, mais il est moins clair si cette tendance s'étend à la population plus large des parcs d'engraissement. L'objectif était de décrire les changements à court terme [< 40 jours d'alimentation (DOF)] dans la récupération et la sensibilité des isolats de M. haemolytica provenant de veaux sains en parc d'engraissement après une exposition métaphylactique à la tulathromycine. Huit cohortes de 100 veaux (n = 800) ont été échantillonnées via un prélèvement nasopharyngé profond lors du traitement d'entrée (i.e., avant la métaphylaxie, à 1 DOF) et à nouveau à 13 DOF. Dix veaux de chaque cohorte (n = 80) ont été échantillonnés au hasard une troisième fois à 36 DOF. La récupération des isolats de M. haemolytica dans toutes les cohortes a augmenté au cours de la période d'étude, passant de 33 % (IC 95 % : 26,5 à 40,2 %) à 1 DOF à 75 % (IC 95 % : 71,4 à 78,3 %) à 36 DOF. Un changement significatif dans la distribution de la concentration minimale inhibitrice (MIC) de la tulathromycine de 1 DOF (MIC90 ≤ 8 µg/mL) à 13 DOF (MIC90 > 64 µg/mL) a été observé. Un sous-ensemble de 36 isolats de 13 DOF criblés pour les gènes de résistance aux macrolides via une réaction d'amplification en chaîne par polymérase multiplex hébergeaient tous les gènes msrE et mphE. L'isolement de M. haemolytica à 13 et 36 DOF n'a pas diminué en réponse à l'utilisation métaphylactique de la tulathromycine; à l'inverse, nous avons suggéré le potentiel de propagation rapide entre les enclos d'un clone résistant aux macrolides par 13 DOF dans 6 des 8 enclos sous la pression sélective de l'utilisation d'antimicrobiens.(Traduit par Dr Serge Messier).

Development and validation of an insulated isothermal PCR assay for the rapid detection of Mannheimia haemolytica.

We developed a rapid insulated isothermal PCR (iiPCR) assay for on-site detection of Mannheimia haemolytica using a primer and probe set targeting the superoxide dismutase (sodA) gene. Our iiPCR assay detected M. haemolytica clinical isolates successfully and produced negative results on other bovine or ovine respiratory pathogens, including Histophilus somni, Bibersteinia trehalosi, Trueperella pyogenes, Streptococcus suis, and Mycoplasma spp., indicating that the PCR reactions were specific. Additionally, our iiPCR assay detected as few as 21 copies of genomic DNA and 17.2 cfu/mL of bacterial culture, which was 10 and 100 times more sensitive than conventional PCR, respectively. Our iiPCR assay can be performed on a portable device in a total of 58 min and may be a useful tool for the detection of M. haemolytica in bovine and ovine respiratory disease in the field.

Differences between predicted outer membrane proteins of Pasteurella multocida, Histophilus somni, and genotype 1 and 2 Mannheimia haemolytica strains isolated from cattle.

Common bacterial causes of bovine respiratory disease (BRD) include Histophilus somni, Mannheimia haemolytica, and Pasteurella multocida. Within M. haemolytica, 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 H. somni, P. multocida, and genotype 2 M. haemolytica may be important factors for acquired host immunity. The predicted OMP differences between genotypes 1 and 2 M. haemolytica have been previously identified. In this study, we expanded the focus to include bovine-isolated strain genomes representing all three species and the two M. haemolytica 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 M. haemolytica 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.

Prediction of Mannheimia haemolytica serotypes based on whole genomic sequences.

The aim of the investigation was to predict the serotypes of M. haemolytica based on whole genomic sequences with the capsular gene region as target. A total of 22 strains selected to have been serotyped and to represent all serotypes were investigated by whole genomic sequencing. The BIGSdb (Bacterial Isolate Genome Sequence Database) was downloaded and installed on a Linux server. Here the sequence database was setup with unique loci at serotype level. The server allows serotypes of M. haemolytica to be predicted from whole genomic sequences and the service is available to the public for free from https://ivsmlst.sund.root.ku.dk.

Occurrence of major and minor pathogens in calves diagnosed with bovine respiratory disease.

Bovine respiratory disease (BRD) is caused by a mixture of viruses and opportunistic bacteria belonging to Pasteurellaceae and Mycoplasma bovis. However, these organisms are also commonly isolated from healthy calves. This study aimed to determine whether the organisms are present in higher numbers in calves sick with acute BRD than in clinically healthy calves, and further to genetically characterize bacteria of the family Pasteurellaceae to understand whether particular types are associated with disease. Forty-six clinically healthy and 46 calves with BRD were sampled by broncheoalveolar lavage (BAL) method in 11 herds geographically spread over Denmark to determine presence and quantity of microorganisms by culture and quantitative real time qPCR. Isolates of Pasteurellaceae were tested for antibiotic resistance and were whole genome sequenced to determine genotypes. Histophilus somni was in particular positively associated with BRD, suggesting particular importance of this organism as likely aetiology of BRD. In addition, quantification of bacteria revealed that higher counts of H. somni as well as of M. haemolytica was also a good indicator of the disease. Pasteurellaceae isolates were susceptible to the commonly used antibiotics in treatment of BRD, and genotypes were shared between isolates from clinically healthy and sick calves.

Molecular characterization of Mannheimia haemolytica associated with ovine and caprine pneumonic lung lesions.

This study investigated via polymerase chain reaction (PCR) three main serotypes (A1, A2, and A6) and nine virulence-associated genes in 71 ovine and caprine Mannheimia haemolytica isolates obtained from lungs (n = 349) with pneumonic lesions from a slaughterhouse in Iran. The lung specimens were collected from sheep (n = 197) and goats (n = 152) between December 2018 and January 2020. A total of 71 M. haemolytica isolates were identified in sheep (37/197; 18.8%) and goat (34/152; 22.4%) pneumonic lungs. Serotypes A2 (30/71; 42.3%) and A6 (29/71; 40.9%) were the most frequently detected, whereas the A1 serotype was detected with a frequency of less than 10% (7/71; 9.9%) and five isolates remained unknown. The virulence genes lkt, pomA, and nanH were present in all the isolates. The detection rates for the remaining virulence-associated genes were: gcp (95.8%), lpsA (93%), fhaC (90%), irp (70.4%), hf (57.7%), and sodC (21%). The sodC gene was exclusively detected among A2 isolates (50%), while the irp gene was more prevalent among A2 isolates and the hf gene among A1 and A6 isolates. These data may be useful for the typing of isolates in epidemiological studies. This study provides information about the main serotypes and the prevalence of virulence-associated genes among M. haemolytica ovine and caprine isolates in Iran.

Differences between predicted outer membrane proteins of genotype 1 and 2 Mannheimia haemolytica.

BACKGROUND: Mannheimia haemolytica strains isolated from North American cattle have been classified into two genotypes (1 and 2). Although members of both genotypes have been isolated from the upper and lower respiratory tracts of cattle with or without bovine respiratory disease (BRD), genotype 2 strains are much more frequently isolated from diseased lungs than genotype 1 strains. The mechanisms behind the increased association of genotype 2 M. haemolytica with BRD are not fully understood. To address that, and to search for interventions against genotype 2 M. haemolytica, complete, closed chromosome assemblies for 35 genotype 1 and 34 genotype 2 strains were generated and compared. Searches were conducted for the pan genome, core genes shared between the genotypes, and for genes specific to either genotype. Additionally, genes encoding outer membrane proteins (OMPs) specific to genotype 2 M. haemolytica were identified, and the diversity of their protein isoforms was characterized with predominantly unassembled, short-read genomic sequences for up to 1075 additional strains. RESULTS: The pan genome of the 69 sequenced M. haemolytica strains consisted of 3111 genes, of which 1880 comprised a shared core between the genotypes. A core of 112 and 179 genes or gene variants were specific to genotype 1 and 2, respectively. Seven genes encoding predicted OMPs; a peptidase S6, a ligand-gated channel, an autotransporter outer membrane beta-barrel domain-containing protein (AOMB-BD-CP), a porin, and three different trimeric autotransporter adhesins were specific to genotype 2 as their genotype 1 homologs were either pseudogenes, or not detected. The AOMB-BD-CP gene, however, appeared to be truncated across all examined genotype 2 strains and to likely encode dysfunctional protein. Homologous gene sequences from additional M. haemolytica strains confirmed the specificity of the remaining six genotype 2 OMP genes and revealed they encoded low isoform diversity at the population level. CONCLUSION: Genotype 2 M. haemolytica possess genes encoding conserved OMPs not found intact in more commensally prone genotype 1 strains. Some of the genotype 2 specific genes identified in this study are likely to have important biological roles in the pathogenicity of genotype 2 M. haemolytica, which is the primary bacterial cause of BRD.

Mannheimia haemolytica and Pasteurella multocida in Bovine Respiratory Disease: How Are They Changing in Response to Efforts to Control Them?

The bacteria Mannheimia haemolytica and Pasteurella multocida contribute to bovine respiratory disease (BRD), which is often managed with antimicrobials. Antimicrobial resistance in these bacteria has been rare, but extensively drug-resistant strains have recently become common. Routine antimicrobial use may be driving this resistance. Resistance spread is caused in part by propagation of strains harboring integrative conjugative elements. The impact of antimicrobial resistance on treatment outcomes is not clear, but clinical observations suggest that response to first treatment has decreased over time, possibly because of resistance. Clinicians should consider antimicrobial resistance when designing BRD treatment and control programs.

Differentiation of Mannheimia haemolytica genotype 1 and 2 strains by visible phenotypic characteristics on solid media.

Genotype 2 Mannheimia haemolytica associate with the lungs of cattle with bovine respiratory disease more frequently than genotype 1 strains. Different colony colors and morphologies were identified between genotype 1 and 2 solid media cultures. Genotype of strains, and frequency differences between them in mixed cultures are discernable by visual inspection.

Mannheimia haemolytica IgA-specific proteases.

Mannheimia haemolytica colonizes the nasopharynx of cattle and can cause severe fibrinous pleuropneumonia. IgA proteases are metalloendopeptidases released by bacteria that cleave IgA, enhancing colonization of mucosa. The objectives of these studies were to characterize M. haemolytica IgA1 and IgA2 proteases in vitro and in silico, to clone and sequence the genes for these proteases, and to demonstrate immunogenicity of components of the entire IgA protease molecule. Both IgA protease genes were cloned, expressed, and sequenced. Sequences were compared to other published sequences. Components were used to immunize mice to determine immunogenicity. Sera from healthy cattle and cattle that recovered from respiratory disease were examined for antibodies to IgA proteases. In order to assay the cleavage of bovine IgA with IgA1 protease, M. haemolytica culture supernatant was incubated with bovine IgA. Culture supernatant cleaved purified bovine IgA in the presence of ZnCl2. Both IgA proteases contain three domains, 1) IgA peptidase, 2) PL1_Passenger_AT and 3) autotransporter. IgA1 and IgA2 peptidases have molecular weights of 96.5 and 87 kDa, respectively. Convalescent bovine sera with naturally high anti-M. haemolytica antibody titers had high antibodies against all IgA1 & IgA2 protease components. Mouse immunizations indicated high antibodies to the IgA peptidases and autotransporters but not to PL1_Passenger_AT. These data indicate that M. haemolytica produces two IgA proteases that are immunogenic, can cleave bovine IgA, and are produced in vivo, as evidenced by antibodies in convalescent bovine sera. Further studies could focus on IgA protease importance in pathogenesis and immunity.

Genetic characterization of susceptible and multi-drug resistant Mannheimia haemolytica isolated from high-risk stocker calves prior to and after antimicrobial metaphylaxis.

Bovine Respiratory Disease (BRD) is a major threat to animal health and welfare in the cattle industry. Strains of Mannheimia haemolytica (Mh) that are resistant to multiple classes of antimicrobials are becoming a major concern in the beef industry, as the frequency of isolation of these strains has been increasing. Mobile genetic elements, such as integrative conjugative elements (ICE), are frequently implicated in this rapid increase in multi-drug resistance. The objectives of the current study were to determine the genetic relationship between the isolates collected at arrival before metaphylaxis and at revaccination after metaphylaxis, to identify which resistance genes might be present in these isolates, and to determine if they were carried on an ICE. Twenty calves culture positive for Mh at arrival and revaccination were identified, and a total of 48 isolates with unique susceptibility profiles (26 from arrival, and 22 from revaccination) were submitted for whole-genome sequencing (WGS). A phylogenetic tree was constructed, showing the arrival isolates falling into four clades, and all revaccination isolates within one clade. All revaccination isolates, and one arrival isolate, were positive for the presence of an ICE. Three different ICEs with resistance gene modules were identified. The resistance genes aphA1, strA, strB, sul2, floR, erm42, tetH/R, aadB, aadA25, blaOXA-2, msrE, mphE were all located within an ICE. The gene bla-ROB1 was also present in the isolates, but was not located within an ICE.

Cloning and Expression of Mannheimia haemolytica PlpE Gene in Escherichia coli and its Immunogenicity Assessment.

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-&beta;-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.

Nanopore ultra-long read sequencing technology for antimicrobial resistance detection in Mannheimia haemolytica.

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.

Plasmid-located extended-spectrum ß-lactamase gene blaROB-2 in Mannheimia haemolytica.

OBJECTIVES: To identify and analyse the first ESBL gene from Mannheimia haemolytica. METHODS: Susceptibility testing was performed according to CLSI. Plasmids were extracted via alkaline lysis and transferred by electrotransformation. The sequence was determined by WGS and confirmed by Sanger sequencing. RESULTS: The M. haemolytica strain 48 showed high cephalosporin MICs. A single plasmid, designated pKKM48, with a size of 4323 bp, was isolated. Plasmid pKKM48 harboured a novel blaROB gene, tentatively designated blaROB-2, and was transferred to Pasteurella multocida B130 and to Escherichia coli JM107. PCR assays and susceptibility testing confirmed the presence and activity of the blaROB-2 gene in the P. multocida and in the E. coli recipient carrying plasmid pKKM48. The transformants had high MICs of all ß-lactam antibiotics. An ESBL phenotype was seen in the E. coli transformant when applying the CLSI double-disc confirmatory test for E. coli. The blaROB-2 gene from plasmid pKKM48 differed in three positions from blaROB-1, resulting in two amino acid exchanges and one additional amino acid in the deduced ß-lactamase protein. In addition to blaROB-2, pKKM48 harboured mob genes and showed high similarity to other plasmids from Pasteurellaceae. CONCLUSIONS: This study described the first ESBL gene in Pasteurellaceae, which may limit the therapeutic options for veterinarians. The transferability to Enterobacteriaceae with the functional activity of the gene in the new host underlines the possibility of the spread of this gene across species or genus boundaries.