[Antimicrobial in vitro effects of eight essential oils on Pasteurella multocida and Mannheimia haemolytica from nasopharyngeal swab samples of fattening calves]. / Antimikrobielle in-vitro-Wirkung acht ätherischer Öle auf Pasteurella multocida und Mannheimia haemolytica aus Nasenrachentupferproben von Mastkälbern.

INTRODUCTION: Essential oils are secondary metabolites of aromatic plants and are used in phytotherapy to treat various diseases. In the present study, eight selected essential oils - ajwain oil (Trachyspermum ammi L.), fennel oil (Foeniculum vulgare Mill. subsp. vulgare var. vulgare), thyme oil chemotype (ct.) thymol (Thymus vulgaris L.), tea tree oil (Melaleuca alternifolia Cheel.), oregano oil (Origanum vulgare L.), mountain savory oil (Satureja montana L.), lemongrass oil (Cymbopogon citratus (DC.) Stapf.) and eucalyptus oil (Eucalyptus globulus Labill.) -were examined for their antibacterial effect against Pasteurella (P.) multocida and Mannheimia (M.) haemolytica isolates from deep nasopharyngeal swab samples of fattening calves using agar diffusion and microdilution. All eight essential oils were effective against the tested isolates. Lemongrass oil proved to be the most potent of all eight essential oils, while fennel oil was only weakly effective. Different antimicrobial effects were observed between the two research methods. The effectiveness of ajwain, thyme, oregano and mountain savory oils was comparable in agar diffusion. However, this could not be reproduced using the microdilution method. P. multocida was found to be more sensitive to all essential oils tested than M. haemolytica. This study shows that the tested essential oils have antimicrobial in-vitro effects on P. multocida and M. haemolytica isolates and that the examination method is associated with the test result.

INTRODUCTION: Les huiles essentielles sont des métabolites secondaires de plantes aromatiques et sont utilisées en phytothérapie pour le traitement de différentes maladies. Dans la présente étude, huit huiles essentielles sélectionnées ­ huile d'ajowan (Trachyspermum ammi L.), huile de fenouil (Foeniculum vulgare Mill. subsp. vulgare var. vulgare), huile de thym chémotype (ct.) thymol (Thymus vulgaris L.), huile d'arbre à thé (Melaleuca alternifolia Cheel.), huile d'origan (Origanum vulgare L.), huile de sarriette de montagne (Satureja montana L. ), huile de citronnelle (Cymbopogon citratus (DC.) Stapf.) et huile d'eucalyptus (Eucalyptus globulus Labill.) ­ ont été étudiées par diffusion sur gélose et microdilution pour leur effet antibactérien sur des isolats de Pasteurella (P.) multocida et de Mannheimia (M.) haemolytica provenant d'échantillons d'écouvillons nasaux profonds de veaux d'engraissement. Les huit huiles essentielles se sont révélées efficaces sur les isolats testés. L'huile de citronnelle s'est avérée être la plus puissante des huit huiles essentielles, tandis que l'huile de fenouil n'était que faiblement efficace. Des effets différents ont été observés entre les deux méthodes de recherche utilisées. Par exemple, l'efficacité des huiles d'ajowan, de thym, d'origan et de sarriette de montagne était comparable dans la diffusion sur gélose. Cependant, cela n>a pas pu être reproduit avec la méthode de microdilution. P. multocida s'est révélée plus sensible que M. haemolytica à toutes les huiles essentielles testées. Cette étude montre premièrement que les huiles essentielles testées ont une efficacité antimicrobienne in vitro sur des isolats cliniques de P. multocida et de M. haemolytica. Deuxièmement, elle montre que la méthode d'examen est associée au résultat du test.

Antibacterial Activity and Cytotoxicity of the Novel Bacteriocin Pkmh.

Bacteriocins are a class of proteins produced by bacteria that are toxic to other bacteria. These bacteriocins play a role in bacterial competition by helping to inhibit potential competitors. In this study, we isolated and purified a novel bacteriocin Pkmh, different from the previously reported bacteriocin PA166, from Pseudomonas sp. strain 166 by ammonium sulfate precipitation, dialysis membrane method, ion exchange chromatography, and gel filtration chromatography. SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) revealed that the molecular weight of Pkmh is approximately 35 kDa. Pkmh exhibited potent antimicrobial activity against bovine Mannheimia haemolytica (M. haemolytica) with low cytotoxicity, and lower hemolytic activity was observed. In addition, Pkmh retained antimicrobial activity at different pH ranges (2-10) and temperature conditions (40, 60, 80, 100 °C). Our analysis of its antimicrobial mechanism showed that Pkmh acts on bacterial cell membranes, increasing their permeability and leading to cell membrane rupture and death. In conclusion, Pkmh exhibited low hemolytic activity, low toxicity, and potent antibacterial effects, suggesting its potential as a promising candidate for clinical therapeutic drugs.

Effectiveness of an experimental subunit ovine Mannheimia haemolytica respiratory vaccine in reducing pneumonia in lambs.

Background: Pneumonia is the largest cause of mortality in Canadian lambs. Currently there are no licensed ovine vaccines in Canada to reduce economic losses from this production-limiting disease. Objective animals and procedure: The effectiveness of an experimental subunit Mannheimia haemolytica leukotoxin A (LtxA) and transferrin binding protein B (TbpB) vaccine was evaluated in lambs for reduction of clinical disease in an experimental challenge study and in a controlled randomized field trial in a large commercial sheep operation. Results: Following an experimental challenge of parainfluenza 3 virus and M. haemolytica, the subunit vaccine induced significantly higher LtxA and TbpB antibody titers at 48 d post-challenge compared to the adjuvant and Ovipast Plus bacterin (Merck Animal Health), but there were no significant differences in clinical signs or mortality among vaccine groups. Following vaccination of commercial ewes and their lambs at weaning, the only significant difference in health, growth, and carcass traits between vaccinates and non-vaccinates was a slightly higher pneumonia treatment rate in vaccinated preweaned lambs (25.7%) compared to unvaccinated preweaned lambs (23.4%) (P = 0.04). Conclusion and clinical relevance: Although vaccination with the experimental subunit M. haemolytica vaccine induced high LtxA and TbpB antibodies, it did not reduce clinical disease in lambs following an experimental challenge study or in a controlled randomized field trial in a commercial sheep operation. Further research is required to identify additional protective antigens for a safe and effective ovine respiratory vaccine to reduce pneumonia losses in commercial sheep flocks.

Efficacité d'un vaccin respiratoire sous-unitaire expérimental de Mannheimia haemolytica ovin à réduire la pneumonie chez les agneaux. Contexte: La pneumonie est la principale cause de mortalité chez les agneaux canadiens. Présentement, il n'y a aucun vaccin ovin homologué au Canada pour réduire les pertes économiques associées à cette pathologie limitant la production. Objectif animaux et procédure: L'efficacité d'un vaccin sous-unitaire expérimental à base de la leucotoxine A (LtxA) et de la protéine B liant la transferrine (TbpB) de Mannheimia haemolytica a été évalué chez des agneaux pour la réduction de la maladie clinique lors d'une infection expérimentale et lors d'un essai de champs randomisé et contrôlé dans un grand élevage commercial de moutons. Résultats: À la suite d'une infection expérimentale avec le virus parainfluenza 3 et M. haemolytica, le vaccin sous-unitaire a induit des titres d'anticorps significativement plus élevés contre LtxA et TbpB à 48 j post-infection comparativement à l'adjuvant et à la bactérine Ovipast Plus (Merck Santé Animale), mais il n'y avait aucune différence significative dans les signes cliniques ou la mortalité parmi les groupes vaccinés. À la suite de la vaccination de brebis commerciales et de leurs agneaux au moment du sevrage, la seule différence significative dans la santé, la croissance et les caractéristiques des carcasses entre les animaux vaccinés et non-vaccinés était un taux légèrement plus élevé de traitement de la pneumonie chez les agneaux vaccinés pré-sevrage (25,7 %) comparativement aux agneaux non-vaccinés au présevrage (23,4 %) (P = 0,04). Conclusion et pertinence clinique: Bien que la vaccination avec le vaccin sous-unitaire expérimental M. haemolytica ait induit des taux d'anticorps élevés contre LtxA et TbpB, il n'a pas réduit la maladie clinique chez les agneaux à la suite d'une infection expérimentale ou lors d'un essai clinique randomisé contrôlé dans un élevage ovin commercial. Des recherches supplémentaires sont requises pour identifier des antigènes protecteurs additionnels pour un vaccin respiratoire ovin efficace pour réduire les pertes associées à la pneumonie dans les troupeaux ovins commerciaux.(Traduit par Dr Serge Messier).

Effects of inflammatory stimuli on the development of Mycoplasma bovis pneumonia in experimentally challenged calves.

Many cattle infected with Mycoplasma bovis remain healthy while others develop severe chronic respiratory disease. We hypothesized that inflammatory stimuli such as co-pathogens worsen disease outcomes in M. bovis-infected calves. Calves (n=24) were intrabronchially inoculated with M. bovis and either killed bacterial lysate, transient M. haemolytica infection, or saline. Caseonecrotic lesions developed in 7/7 animals given M. haemolytica and M. bovis compared to 2/8 given M. bovis with no inflammatory stimulus, and 6/9 animals given bacterial lysate and M. bovis (P=0.01). Animals receiving M. haemolytica and M. bovis had more caseonecrotic foci in lungs than those receiving M. bovis with no inflammatory stimulus (median = 21 vs 0; P = 0.01), with an intermediate response (median = 5) in animals given bacterial lysate. In addition to caseonecrotic foci, infected animals developed neutrophilic bronchiolitis that appeared to develop into caseonecrotic foci, peribronchiolar lymphocytic cuffs that were not associated with the other lesions, and 4 animals with bronchiolitis obliterans. The data showed that transient lung inflammation at the time of M. bovis infection provoked the development of caseonecrotic bronchopneumonia, and the severity of inflammation influenced the number of caseonecrotic foci that developed. In contrast, caseonecrotic lesions were few or absent in M. bovis-infected calves without a concurrent inflammatory stimulus. These studies provide insight into how caseonecrotic lesions develop within the lung of M. bovis-infected calves. This and other studies suggest that controlling co-pathogens and harmful inflammatory responses in animals infected with M. bovis could potentially minimize development of M. bovis caseonecrotic bronchopneumonia.

Apo-Lactoferrin Inhibits the Proteolytic Activity of the 110 kDa Zn Metalloprotease Produced by Mannheimia haemolytica A2.

Mannheimia haemolytica is the main etiological bacterial agent in ruminant respiratory disease. M. haemolytica secretes leukotoxin, lipopolysaccharides, and proteases, which may be targeted to treat infections. We recently reported the purification and in vivo detection of a 110 kDa Zn metalloprotease with collagenase activity (110-Mh metalloprotease) in a sheep with mannheimiosis, and this protease may be an important virulence factor. Due to the increase in the number of multidrug-resistant strains of M. haemolytica, new alternatives to antibiotics are being explored; one option is lactoferrin (Lf), which is a multifunctional iron-binding glycoprotein from the innate immune system of mammals. Bovine apo-lactoferrin (apo-bLf) possesses many properties, and its bactericidal and bacteriostatic effects have been highlighted. The present study was conducted to investigate whether apo-bLf inhibits the secretion and proteolytic activity of the 110-Mh metalloprotease. This enzyme was purified and sublethal doses of apo-bLf were added to cultures of M. haemolytica or co-incubated with the 110-Mh metalloprotease. The collagenase activity was evaluated using zymography and azocoll assays. Our results showed that apo-bLf inhibited the secretion and activity of the 110-Mh metalloprotease. Molecular docking and overlay assays showed that apo-bLf bound near the active site of the 110-Mh metalloprotease, which affected its enzymatic activity.

Lactoferrin Affects the Viability of Bacteria in a Biofilm and the Formation of a New Biofilm Cycle of Mannheimia haemolytica A2.

Respiratory diseases in ruminants are responsible for enormous economic losses for the dairy and meat industry. The main causative bacterial agent of pneumonia in ovine is Mannheimia haemolytica A2. Due to the impact of this disease, the effect of the antimicrobial protein, bovine lactoferrin (bLf), against virulence factors of this bacterium has been studied. However, its effect on biofilm formation has not been reported. In this work, we evaluated the effect on different stages of the biofilm. Our results reveal a decrease in biofilm formation when bacteria were pre-incubated with bLf. However, when bLf was added at the start of biofilm formation and on mature biofilm, an increase was observed, which was visualized by greater bacterial aggregation and secretion of biofilm matrix components. Additionally, through SDS-PAGE, a remarkable band of ~80 kDa was observed when bLf was added to biofilms. Therefore, the presence of bLf on the biofilm was determined through the Western blot and Microscopy techniques. Finally, by using Live/Dead staining, we observed that most of the bacteria in a biofilm with bLf were not viable. In addition, bLf affects the formation of a new biofilm cycle. In conclusion, bLf binds to the biofilm of M. haemolytica A2 and affects the viability of bacteria and the formation a new biofilm cycle.

Mapping Heterogeneous Population Structure of Mannheimia haemolytica Associated with Pneumonic Infection of Sheep in Southern State Karnataka, India.

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.

Identification of genetic markers of resistance to macrolide class antibiotics in Mannheimia haemolytica isolates from a Saskatchewan feedlot.

Mannheimia haemolytica 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 M. haemolytica 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 M. haemolytica to macrolide class antibiotics commonly used for control of BRD. Genome sequences were determined for 99 M. haemolytica isolates with six different susceptibility phenotypes collected over 2 years from a feedlot in Saskatchewan, Canada. Known macrolide resistance genes estT, msr(E), and mph(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 Mannheimia haemolytica, 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.

Improving the detection of integrative conjugative elements in bovine nasopharyngeal swabs using multiplex recombinase polymerase amplification.

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.

Isolation and characterization of a Mannheimiahaemolytica secreted serine protease that degrades sheep and bovine fibrinogen.

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.

Antimicrobial Activities of α-Helix and ß-Sheet Peptides against the Major Bovine Respiratory Disease Agent, Mannheimia haemolytica.

Bovine respiratory disease (BRD) is the leading cause of morbidity and mortality in cattle raised in North America. At the feedlot, cattle are subject to metaphylactic treatment with macrolides to prevent BRD, a practice that may promote antimicrobial resistance and has resulted in an urgent need for novel strategies. Mannheimia haemolytica is one of the major bacterial agents of BRD. The inhibitory effects of two amphipathic, α-helical (PRW4, WRL3) and one ß-sheet (WK2) antimicrobial peptides were evaluated against multidrug-resistant (MDR) M. haemolytica isolated from Alberta feedlots. WK2 was not cytotoxic against bovine turbinate (BT) cells by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. All three peptides inhibited M. haemolytica, with WK2 being the most efficacious against multiple isolates. At 8-16 µg/mL, WK2 was bactericidal against Mh 330 in broth, and at 32 µg/mL in the presence of BT cells, it reduced the population by 3 logs CFU/mL without causing cytotoxic effects. The membrane integrity of Mh 330 was examined using NPN (1-N-phenylnaphthylamine) and ONPG (o-Nitrophenyl ß-D-galactopyranoside), with both the inner and outer membranes being compromised. Thus, WK2 may be a viable alternative to the use of macrolides as part of BRD prevention and treatment strategies.

Isolation and Identification of Pasteurella multocida and Mannheimia haemolytica from Pneumonic Small Ruminants and Their Antibiotic Susceptibility in Haramaya District, Eastern Ethiopia.

Background: Pasteurella species are frequently encountered as serious diseases in small ruminants. It is the main cause of respiratory pasteurellosis in sheep and goats of all age groups. Methods: The cross-sectional study was conducted from December 2022 to April 2023 in Haramaya district, eastern Ethiopia, to isolate and identify Pasteurella multocida and Mannheimia haemolytica and estimate their prevalence, associated risk factors, and antimicrobial sensitivity of isolates in small ruminants using a purposive sampling method. A total of 384 samples (156 nasal swabs from clinic cases and 228 lung swabs from abattoir cases) were collected. STATA 14 software was used to analyze the data. In addition, multivariable logistic regression analysis was performed to assess an association of risk factors. Results: Out of the 384 samples examined, 164 were positive for pasteurellosis, resulting in a 42.70% prevalence. Similarly, 63 (38.4%) of the 164 positive results were from nasal swabs, while 101 (61.6%) came from lung samples. M. haemolytica accounted for 126 (76.82%) of the isolates, while P. multocida accounted for 38 (23.17%). Of the 63 nasal swab isolates, 33 (37%) were from goats and 30 (42.8%) were from sheep. And 17 (10.89%) and 46 (29.58%), respectively, were P. multocida and M. haemolytica. Of the 46 (40%) of the 101 (44.3%) isolates of the pneumonic lung, samples were from goats, while 55 (48.47%) were from sheep. In this study, the risk factors (species, age, and body condition score) were found to be significant (p < 0.05). Pasteurella isolates evaluated for antibiotic susceptibility were highly resistant to oxacillin (90.90%), followed by gentamycin (72.72%), and penicillin (63.63%). However, the isolates were highly sensitive to chloramphenicol (90.90%), followed by tetracycline (63.63%), and ampicillin (54.54%). Conclusion: This study showed that M. haemolytica and P. multocida are the common causes of mannheimiosis and pasteurellosis in small ruminants, respectively, and isolates were resistant to commonly used antibiotics in the study area. Thus, an integrated vaccination strategy, antimicrobial resistance monitoring, and avoidance of stress-inducing factors are recommended.

Mannheimia haemolytica-associated fibrinonecrotizing abomasitis in lambs.

Mannheimia haemolytica-associated abomasitis has been clinically described as a cause of sudden death in lambs, but it is poorly characterized. We describe the pathological features of a severe fibrinonecrotizing abomasitis in 3 lambs that died suddenly. All 3 abomasums had a thickened submucosa due to edema and necrotic areas delimited by bands of degenerate neutrophils with slender nuclei (oat cells) and angiocentric distributions. The overlying mucosa was congested. Myriads of gram-negative coccobacilli were observed within the oat cell bands. M. haemolytica was isolated from the abomasum in all 3 animals and was serotyped as A2 in one of them. Pericarditis and pleuritis were observed in 2 of the lambs. Clostridium spp. were isolated in 1 lamb and detected by immunohistochemistry in the 3 animals, suggesting clostridial co-infection. M. haemolytica should be considered among the differential diagnoses of necrotizing abomasitis in lambs.

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.

Identification, Biochemical Characterization, and In Vivo Detection of a Zn-Metalloprotease with Collagenase Activity from Mannheimia haemolytica A2.

Respiratory diseases in ruminants are a main cause of economic losses to farmers worldwide. Approximately 25% of ruminants experience at least one episode of respiratory disease during the first year of life. Mannheimia haemolytica is the main etiological bacterial agent in the ruminant respiratory disease complex. M. haemolytica can secrete several virulence factors, such as leukotoxin, lipopolysaccharide, and proteases, that can be targeted to treat infections. At present, little information has been reported on the secretion of M. haemolytica A2 proteases and their host protein targets. Here, we obtained evidence that M. haemolytica A2 proteases promote the degradation of hemoglobin, holo-lactoferrin, albumin, and fibrinogen. Additionally, we performed biochemical characterization for a specific 110 kDa Zn-dependent metalloprotease (110-Mh metalloprotease). This metalloprotease was purified through ion exchange chromatography and characterized using denaturing and chaotropic agents and through zymography assays. Furthermore, mass spectrometry identification and 3D modeling were performed. Then, antibodies against the 110 kDa-Mh metalloprotease were produced, which achieved great inhibition of proteolytic activity. Finally, the antibodies were used to perform immunohistochemical tests on postmortem lung samples from sheep with suggestive histology data of pneumonic mannheimiosis. Taken together, our results strongly suggest that the 110-Mh metalloprotease participates as a virulence mechanism that promotes damage to host tissues.

Detection of immune effects of the Mannheimia haemolytica gamma irradiated vaccine in sheep.

Exposure to gamma rays from cobalt 60 (Co60) can induce a complete inactivation of Mannheimia haemolytica. The inactivated bacterial pathogen is a potential vaccine candidate for immunization of ruminants such as sheep. The subcutaneous administration of irradiated vaccine in a two-dose regimen (4.0 × 109 colony forming unit (CFU) per dose) results in no mortality in any of the vaccinated sheep during immunization and after subsequent challenge of the live bacteria of the same strain of M. haemolytica. A significant rise in serum IgG titer, detected through ELISA, is observed after the passage of two weeks from the inoculation of the first dose whereas, the peak of the mean serum antibody titer occurred after two weeks of booster dose. The vaccination does not bring significant change to the IFN-γ levels in serum. The bacterial challenge of the vaccinated sheep does not induce a further seroconversion relative to serum antibody titer. In conclusion, the vaccinated sheep are protected by the elevated IgG titer and increased levels of IL-4 (Th-2 response) compared to the non-vaccinated sheep. Radiation technology can provide the opportunity for mass production of immunologically safe vaccines against animal and zoonotic diseases. Ethics Approval by the National Research Center Ethics Committee (Trial Registration Number (TRN) no 13,602,023, 13/5/2023) was obtained.

Molecular identification and characterisation of Mannheimia haemolytica.

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.

Antimicrobial Metaphylaxis and its Impact on Health, Performance, Antimicrobial Resistance, and Contextual Antimicrobial Use in High-Risk Beef Stocker Calves.

The objective of this blinded, cluster-randomized, complete block trial was to evaluate the impact of metaphylaxis on health, performance, antimicrobial resistance, and contextual antimicrobial use (AMU) in high-risk beef stocker calves. Calves (n = 155) were randomly assigned to receive either saline or tulathromycin at the time of arrival processing. Deep nasopharyngeal swabs were collected from each calf at arrival and 14 d later. Calves were monitored for bovine respiratory disease (BRD) for 42 d. Body weights were obtained at arrival, days 14, 28, and 42. Contextual antimicrobial use (AMU) was calculated using dose and mass-based metrics. Calves given tulathromycin had a greater average daily gain (0.96 ±â€…0.07 kg vs. 0.82 ±â€…0.07 kg; P = 0.034) and lower prevalence of BRD than controls (17% vs. 40%; P = 0.008). Proportions of calves with BRD pathogens identified at arrival were similar between treatment groups [17%; P = 0.94]. Proportions of calves with BRD pathogens identified at day 14 were lower for calves receiving tulathromycin compared to controls (15% vs. 60%, P < 0.001). Overall, 81% of Pastuerella multocida isolates and 47% of Mannheimia haemolytica isolates were pansusceptible. When measured as regimens per head in, AMU in calves receiving tulathromycin was higher than calves receiving saline (P = 0.01). Under the conditions of this study, metaphylaxis had positive impacts on the health and performance of high-risk beef stocker calves, did not contribute to the selection of resistant bacterial isolates in the nasopharynx of treated cattle, and increased AMU.

In this study, we investigated the impact of metaphyactic antimicrobial administration on health, performance, and antimicrobial use in high-risk beef stocker calves. Our findings demonstrated that metaphylaxis improves performance and has positive effects on animal health and well-being but increases total antimicrobial use. Additionally, our study revealed that metaphylaxis alone does not contribute to the selection of antimicrobial-resistant pathogens in the upper airway of treated cattle.

Probing enzymatic properties of N-glycosyltransferase isoforms from Mannheimia haemolytica.

N-Glycosyltransferase (NGT) is an inverting glycosyltransferase for an unusual pathway of N-linked protein glycosylation and glycosylates polypeptides in the consensus sequon (N-(X≠P)-T/S) with hexose monosaccharides. Here, we expressed and characterized a novel N-glycosyltransferase from Mannheimia haemolytica (named MhNGT). RP-HPLC and Mass Spectrometry were used to assay and quantify glycopeptide formation by MhNGT and determine its substrate specificities. MhNGT could utilize a variety of nucleotide-activated sugar donors, including UDP-Glc, UDP-Gal and UDP-Xyl, to glycosylate the tested peptides DANYTK, GGNWTT and PAVGNCSSALR with higher efficiency than ApNGT which was comprehensive studied. The optimum temperature of MhNGT was about 30 °C and the optimum pH was 7.5-8.0 in PBS-NaOH buffer. MhNGT exhibited a different position-specific residue preference of substrate peptides from the NGT of Actinobacillus pleuropneumoniae (ApNGT). The effective glycosylation of common short peptides by MhNGT demonstrated the enzyme's potential to alter therapeutically significant mammalian N-glycoproteins.

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.