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.

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.

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.

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.

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.

Effect of on-arrival bovine respiratory disease vaccination on ultrasound-confirmed pneumonia and production parameters in male dairy calves: A randomized clinical trial.

The high degree of commingling and accumulation of stressors during and after transport makes prevention of bovine respiratory disease (BRD) extremely challenging in the veal and dairy beef industry. Upon arrival, vaccination for agents involved in BRD is practically most achievable, but its efficacy under such conditions in dairy veal calves is unknown. Given the high prevalence of subclinical pneumonia in these settings, the primary objective of the present study was to determine the effect of 2 vaccination protocols administered upon arrival against bovine respiratory syncytial virus (BRSV), bovine parainfluenza type 3 virus (BPI-3), and Mannheimia haemolytica on clinical BRD and lung ultrasonographic findings in dairy veal calves. In addition, the effects of vaccination on average daily live weight gain and cold carcass weight were determined. In this randomized clinical trial, 443 male dairy calves were assigned to one of 3 groups: a negative, placebo-controlled group (n = 151), a vaccination group with 2 subcutaneous injections 4 wk apart with an inactivated vaccine containing BRSV, BPI-3, and M. haemolytica (parenteral [PE] group; n = 149) and a second vaccination group receiving an intranasal live-attenuated vaccine containing BRSV and BPI-3 and 2 subcutaneous vaccinations with the same inactivated vaccine as the PE vaccination group (intranasal-parenteral [IN-PE] group; n = 143). Clinical scoring and quick thoracic ultrasonography (qTUS) were performed on all calves on arrival (wk 0), at the peak of respiratory disease (outbreak; wk 1), at the end of the first antimicrobial group treatment (wk 3), and at a long-term evaluation point (wk 10). Culture and nanopore sequencing on nonendoscopic bronchoalveolar lavage (nBAL) samples were used to identify pathogens involved in the outbreak. Upon arrival, 15.1% of the calves had lung consolidation ≥1cm and incidence quickly rose to 42.8% during the outbreak. In both the PE and IN-PE group, the odds of pneumonia in wk 10 were reduced by 62% (odds ratio [OR] = 0.38; 95% confidence interval [CI] = 0.23-0.64) and 41% (OR = 0.59; 95% CI = 0.37-0.96), respectively. Short-term cure rate (50.3%), as determined immediately after the first group antimicrobial treatment, was not influenced by vaccination. In contrast, long-term cure rate, determined at wk 10, was affected by vaccination with higher cure in the PE group compared with the control group (69.4% vs. 51.2%; OR = 2.2; 95% CI = 1.1-5.0). Average daily gain in the first 10 wk of production was not affected by vaccination. Vaccination resulted in an increase in cold carcass weight of 3.5 and 4.3 kg in the PE (95% CI = -0.9-7.9) and IN-PE group (95% CI = -0.17-8.7), respectively. In conclusion, under the conditions of the present study, vaccination upon arrival resulted in a reduced prevalence of pneumonia at wk 10 of production, likely caused both by an improved cure rate of secondary infections and a reduced incidence of new cases between outbreak and long-term evaluation. The present protocol, using qTUS for pneumonia detection and nBAL diagnostics for pathogen identification adds a new dimension to randomized clinical trials on respiratory disease in calves.

Pro-inflammatory cytokines and reproductive hormone responses in bucks post-challenge with Mannheimia haemolytica A2 and its outer membrane protein.

The lipopolysaccharide (LPS) endotoxin and outer membrane protein (OMP) are among the virulence factors of Gram-negative bacteria responsible for inducing pathogenicity in the infected host. OMP and LPS occur on the outer membrane of M. haemolytica A2, the primary aetiological agent of pneumonic mannheimiosis in small ruminants. While the LPS is known to mediate Gram-negative bacterial infection by activating downstream inflammatory pathways, the potential role of OMP during inflammatory responses remained unclear. Hence, this study determined the effect of the OMP of M. haemolytica A2 on the serum concentration of pro-inflammatory cytokines and the male reproductive hormones (testosterone and Luteinizing Hormone). We randomly assigned twelve bucks to three groups (n = 4 bucks each): Group 1 was challenged with 2 mL PBS buffer (pH 7.0) intranasally; Group 2 received 2 mL of 1.2 X 109 CFU/mL whole M. haemolytica A2 intranasally; and Group 3 received 2 mL of OMP extract obtained from 1.2 X 109 CFU/mL M. haemolytica A2 intramuscularly. Serum samples collected at pre-determined intervals were used for the quantitative determination of the pro-inflammatory cytokines (IL-1ß, IL-6, and TNFα) and reproductive hormones (testosterone and LH) using commercial sandwich enzyme-linked immunosorbent assay (ELISA). The serum concentration of IL1ß was initially increased within the first-hour post-challenge in Groups 2 and 3, followed by a significant decrease in concentration at 21d and 35d (p < 0.05) in Group 3. Only mild fluctuations in IL-6 occurred in group 2, as opposed to the 1.7-fold rapid increase in TNFα within 2 h post-challenge before decreasing at 6 h. An increase in pro-inflammatory cytokines was accompanied by an acute febrile response of 39.5 ± 0.38 °C (p < 0.05) at 2 h and 40.1 ± 0.29 °C (p < 0.05) at 4 h in Group 2 and Group 3, respectively. Serum testosterone decreased significantly (p < 0.05) in both treatment groups but remained significantly (p > 0.05) lower than in Group 1 throughout the study. There was a moderate negative association between testosterone and IL1ß (r = -0.473; p > 0.05) or TNFα (r = -0.527; p < 0.05) in Group 2. Serum LH also showed moderate negative associations with TNFα in Group 2 (r = -0.63; p < 0.05) and Group 3 (r = -0.54; p > 0.05). The results of this study demonstrated that M. haemolytica A2 and its OMP produced marked alterations in serum levels of pro-inflammatory cytokines and male reproductive hormones. The negative correlations between serum testosterone and inflammatory cytokines would suggest the potential role of OMP in causing male infertility by mediating innate inflammatory responses to suppress testosterone production in bucks.

Mannheimia haemolytica OmpH binds fibrinogen and fibronectin and participates in biofilm formation.

Mannheimia haemolytica is the causal agent of the shipping fever in bovines and produces high economic losses worldwide. This bacterium possesses different virulence attributes to achieve a successful infection. One of the main virulence factors expressed by a pathogen is through adhesion molecules; however, the components participating in this process are not totally known. The present work identified a M. haemolytica 41 kDa outer membrane protein (Omp) that participates in bacterial adhesion. This protein showed 100% identity with the OmpH from M. haemolytica as determined by mass spectrometry and it interacts with sheep fibrinogen. The 41 kDa M. haemolytica OmpH interacts with bovine monocytes; a previous incubation of M. haemolytica with a rabbit hyperimmune serum against this Omp diminished 45% cell adhesion. The OmpH was recognized by serum from bovines affected by acute or chronic pneumonia, indicating its in vivo expression; moreover, it showed immune cross-reaction with the serum of rabbit infected with Pasteurella multocida. The OmpH is present in biofilms and previous incubation of M. haemolytca with rabbit serum against this protein diminished biofilm, indicating this protein's participation in biofilm formation. M. haemolytica OmpH is proposed as a relevant immunogen in bovine pneumonia protection.

Responses of selected biomarkers, female reproductive hormones and tissue changes in non-pregnant does challenged with Mannheimia haemolytica serotype A2 and its outer membrane protein (OMP) immunogen.

BACKGROUND: Mannheimia haemolytica causative agent of pneumonic mannheimiosis, a common respiratory disease of goat and sheep, which cause huge economic losses to farmers worldwide. Pneumonic mannheimiosis caused by M. haemolytica serotype A2 has been reported among small ruminants in Malaysia. The lipopolysaccharide (LPS) and outer membrane protein (OMP) are major virulence determinants for M. haemolytica serotype A2. Although pneumonic mannheimiosis is known to cause poor reproductive performance in small ruminants under field conditions, there is a dearth of published information on the specific effects of M. haemolytica serotype A2 infection on the female reproductive physiology. In this experiment, we explored the impact of M. haemolytica serotype A2 and its OMP immunogen on selected pro-inflammatory cytokines, acute phase proteins, female reproductive hormones, and cellular changes in visceral and female reproductive organs of non-pregnant does. METHODOLOGY: Twelve healthy, non-pregnant, Boer crossbreds does were divided equally into three groups (n = 4); Group 1 served as the negative control and was challenged with 2 ml of sterile PBS intranasally. Group 2 served as the positive control and was challenged with 2 ml of 109 colonies forming unit (CFU) of M. haemolytica serotype A2 suspension intranasally. Group 3 was challenged with 2 ml of OMP extracted from 109 CFU of M. haemolytica A2 intramuscularly. The experimental does were monitored for clinical signs and responses periodically. Blood samples were collected at 0, 1, 2, 4, 6, 12 and 24 h and 3, 7, 21, 35 and 56 days post treatment for serological analyses. All does were euthanised using the halal slaughter method on day 60 post challenge/treatment. Tissues from the uterus, liver, lung and associated bronchial lymph nodes were collected and fixed in 10% formalin for 14 days for histopathological study. RESULTS: Compared to the control group, the challenged/treated groups showed significant (p < 0.05) increase in the rectal temperature, respiratory rate, heart rate, and rumen motility. Serum analyses revealed that the concentrations of progesterone and estrogen hormones were significantly (p < 0.05) decreased in groups 2 & 3. In contrast, the concentrations of pro-inflammatory cytokines (IL-1ß and IL-6) and acute phase proteins (Hp and SAA) were significantly increased (p < 0.05) in the challenged/treated groups compared to the control group. Histopathological lesion scoring revealed mild to moderate cellular changes characterised by congestion, haemorrhage, degeneration, leucocytic cellular infiltration, and cellular necrosis in the tissues of does from the OMP treatment and bacterial challenge groups compared to the control group. CONCLUSION: The findings from this study suggests that M. haemolytica serotype A2 and its OMP immunogen induced mild to moderate inflammatory and degenerative changes which may potentially interfere with fertilization through hormonal imbalances and cause temporary loss of fertility in infected does.

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.

Isolation of the various serotypes of Mannheimia haemolytica and preparation of the first vaccine candidate in Iran.

BACKGROUND: Mannheimia haemolytica is one of the main agents of domestic pneumonic mannheimiosis, but a proper vaccine has not been explored in IRAN. METHODS AND RESULTS: 362 lung and nasal samples from sick domestic animal were detected by culture and PCR methods. Totally, 71 M. haemolytica isolates were identified in three main serotypes (A1, A2, and A6). Serotypes A2 (38/71; 54%) and A1 (25/71; 39%) were the most frequently detected, whereas the A6 serotype was detected with a frequency of less than 1% (1/71; 1%) and 7 isolates remained unknown (7/71; 10%). Subsequently, M. haemolytica vaccinal strain was developed and then formalin-killed vaccine was prepared. It provided the best protection against mannheimiosis in sheep which was proved by indirect ELISA. CONCLUSIONS: Our results suggest that the efficacy and safety of vaccine strain are remarkable and may serve as a new therapeutic target in mannheimiosis.

Mannheimia haemolytica serovars associated with respiratory disease in cattle in Great Britain.

BACKGROUND: Mannheimia haemolytica is commonly associated with respiratory disease in cattle worldwide as a cause of fibrinous pneumonia, bronchopneumonia and pleuritis. M. haemolytica is further subdivided into 12 serovars, however not all are considered to be pathogenic in cattle. The study aim was to determine the most common serovars of M. haemolytica associated with respiratory disease in cattle in Great Britain, which is currently unknown and could be useful information for clinicians when considering preventative strategies. RESULTS: One hundred four M. haemolytica isolates isolated from bovine clinical pathology and post-mortem samples from pneumonia cases between 2016 and 2018 were tested using a multiplex PCR assay to identify M. haemolytica serovars A1, A2 and A6. 46 isolates (44.2%) typed as M. haemolytica serovar A1, 31 (29.8%) as M. haemolytica serovar A2 and 18 isolates (17.3%) as M. haemolytica serovar A6. Nine isolates (8.7%) were not A1, A2 or A6 so were considered to belong to other serovars or were not typable. CONCLUSION: This study highlights the importance of M. haemolytica serovars other than A1 which may be responsible for respiratory disease in cattle and could help guide the veterinarian when making choices on preventative vaccination programmes.

Seminal and histopathological alterations in bucks challenged with Mannheimia haemolytica serotype a2 and its LPS endotoxin.

Pneumonic mannheimiosis is a widespread respiratory bacterial disease of small ruminants caused by Mannheimia haemolytica serotype A2. The disease is known to affect the respiratory organs of infected animals, but its effect on other vital and reproductive organs has not been fully explored. Previous studies have demonstrated increased serum pro-inflammatory cytokine concentration post-challenge with M. haemolytica A2 and its LPS, indicating systemic inflammation in the host. This study determined the potential tissue changes and alterations of sperm parameters due to infection of M. haemolytica A2 and its LPS endotoxin. In this study, twelve experimental bucks were randomly assigned to three groups of four bucks each: group 1 (control group) were intranasally inoculated with 2 mL of PBS pH 7.0, group 2 received 2 mL of 1.2 × 109 CFU/mL M. haemolytica A2 intranasally, and group 3 received 2 mL of LPS extracted from 1.2 × 109 CFU/mL of M. haemolytica A2 intravenously. Semen samples were collected at pre-determined intervals using an electro-ejaculator and analysed immediately after collection. All experimental bucks were slaughtered via exsanguination on day 60 to collect their vital and reproductive organs at necropsy, and the samples were processed and analysed for histopathological changes. The current study has revealed that bucks challenged with M. haemolytica A2 and its LPS exhibited alterations in semen parameters such as motility, wave pattern, viability, and morphological abnormalities. Mild to moderate histopathological changes of the lung, liver, testis, epididymis, vas deferens, prostate, and lymph nodes were also observed in both challenged groups. Therefore, this study revealed the potential harmful effects of respiratory mannheimiosis on the reproductive organs of the infected bucks and sheds light on the expanse of systemic effects of this disease.

Changes in selected cytokines, acute-phase proteins, gonadal hormones and reproductive organs of non-pregnant does challenged with Mannheimia haemolytica serotype A2 and its LPS endotoxin.

Previous investigations have revealed that lipopolysaccharide (LPS) endotoxin from certain Gram-negative bacteria could adversely affect the reproductive system of female animals. However, it is unknown whether LPS endotoxin of Mannheimia haemolytica serotype A2, the principal causative bacteria that cause pneumonic mannheimiosis in small ruminants, may also induce similar insidious effects. Therefore, this study aimed to investigate the effects of M. haemolytica serotype A2 and its LPS endotoxin on the responses of female gonadal hormones (progesterone and oestrogen), pro-inflammatory cytokines (interleukin-1ß, interleukin-6), acute-phase proteins (haptoglobin and serum amyloid A) and cellular changes via histopathology study of female reproductive organs of the treatment does. Twelve clinically healthy, non-pregnant, crossbred does were randomly allocated into three equal groups. Group 1 was administered intranasally with 2 ml of sterile phosphate-buffered saline (PBS) and served as a negative control group. Group 2 was challenged intranasally with 2 ml of bacterial inoculum containing 109 colony-forming units (CFU)/ml of M. haemolytica serotype A2, while Group 3 was challenged intravenously with 2 ml of LPS endotoxin extracted from 109 CFU/ml of M. haemolytica serotype A2. Following that, blood samples were collected serially at pre-determined intervals for serological analyses. All does were euthanised 60 days post-challenges, and tissue samples from the ovaries, oviducts, uterine horns, uterine body, cervix and vagina were collected for histopathological study. The serological result revealed a significant increase (p < 0.05) in the mean concentrations of progesterone, oestrogen, interleukin-1ß, interleukin-6, haptoglobin and serum amyloid A for both challenged groups. Histopathologically, all reproductive organs (except the cervix and vagina) from both challenged groups displayed significant cellular alterations (p < 0.05) characterised by haemorrhage and congestion, necrosis and degeneration, inflammatory cell infiltration and oedema. This study provides new information that elucidates the potential role of pneumonic mannheimiosis in the pathogenesis of female infertility amongst small ruminants.

Protection against Mycoplasma bovis infection in calves following intranasal vaccination with modified-live Mannheimia haemolytica expressing Mycoplasma antigens.

Novel live vaccine strains of Mannheimia haemolytica serotypes (St)1 and St6, expressing and secreting inactive yet immunogenic leukotoxin (leukotoxoid) fused to antigenic domains of Mycoplasma bovis Elongation Factor Tu (EFTu) and Heat shock protein (Hsp) 70 were constructed and tested for efficacy in cattle. Control calves were administered an intranasal mixture of M. haemolytica St1 and St6 mutants (ΔlktCAV4) expressing and secreting leukotoxoid while vaccinated calves were administered an intranasal mixture of like M. haemolytica St1 and St6 leukotoxoid mutants coupled to M. bovis antigens (EFTu-Hsp70-ΔlktCAV4). Both M. haemolytica strains were recovered from palatine tonsils up to 34 days post intranasal exposure. On day 35 all calves were exposed to bovine herpes virus-1, four days later lung challenged with virulent M. bovis, then euthanized up to 20 days post-challenge. Results showed all cattle produced systemic antibody responses against M. haemolytica. The vaccinates also produced systemic antibody responses to M. bovis antigen, and concurrent reductions in temperatures, middle ear infections, joint infection and lung lesions versus the control group. Notably, dramatically decreased lung loads of M. bovis were detected in the vaccinated cattle. These observations indicate that the attenuated M. haemolytica vaccine strains expressing Mycoplasma antigens can control M. bovis infection and disease symptoms in a controlled setting.

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.

Responses of testosterone hormone and important inflammatory cytokines in bucks after challenge with Mannheimia haemolytica A2 and its LPS endotoxin.

Previous studies have shown that Mannheimia haemolytica A2 is the principal microorganism causing pneumonic mannheimiosis, a major bacterial respiratory disease among sheep and goats. The effect of this bacteria on the respiratory system is well-established. However, its effect on the reproductive physiology remains unclear. Therefore, this study aimed to determine the alterations in the level of pro-inflammatory cytokines and testosterone hormone post-inoculation with M. haemolytica serotype A2 and its lipopolysaccharide (LPS) endotoxin which were hypothesized to affect the reproductive functions of bucks. Twelve clinically healthy adult male goats were divided equally into three groups. Goats in group 1 were treated with 2 ml of sterile phosphate-buffered saline (PBS) pH 7.0 intranasally (negative control), group 2 with 2 ml of 109 colony-forming unit (CFU) of M. haemolytica serotype A2 intranasally (positive control), and group 3 were treated with 2 ml of lipopolysaccharide extracted from 109 CFU of M. haemolytica serotype A2 intravenously. Following inoculation, blood samples were collected via jugular venipuncture into plain tubes at pre-determined intervals for serum collection to determine the concentration of interleukin (IL)-1ß, IL6, tumor necrosis factor (TNF)-α, and testosterone hormone by using commercial ELISA test kits. Results from this study demonstrated that the inoculation of M. haemolytica A2 and its LPS increases the concentration of pro-inflammatory cytokines but decreases the concentration of testosterone hormone in challenged animals at most time points throughout the 56 days experimental period (p < 0.05). This study suggests that the M. haemolytica A2 and its LPS could alter the concentration of pro-inflammatory cytokines and testosterone hormone, which in turn, may negatively affect the reproductive functions of bucks.

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.

Pathology and immunohistochemical evaluation of lungs of cattle slaughtered at metropolitan abattoirs in Nigeria and Ghana.

There was a dearth of information on pathology and causal agents of bovine pneumonia in West Africa. This cross-sectional study conducted at four major metropolitan abattoirs in Nigeria and Ghana was to evaluate the pathology and to immunohistochemically demonstrates viral and bacterial pathogens of bovine pneumonia in West Africa. Out of the 20,605 cattle lungs examined at post-mortem using standard inspection procedures, 136 samples grossly showed pneumonic lesions and 99 randomly selected lung samples were fixed in 10% neutral buffered formalin for histopathological and immunohistochemical examination. The overall prevalence of pneumonia was 0.66%, with 0.72% prevalence in Ibadan, Nigeria and 9.68% prevalence in Ghana. Age and breed were observed to be among the predisposing factors to pneumonia in cattle. Histologically, bronchopneumonia (0.65%), broncho-interstitial pneumonia (0.13%), and interstitial pneumonia (0.08%) were the prominent type of pneumonias observed. Immunohistochemically, 0.8% was positive for bovine PI-3, 0.9% for bovine RSV, 1.0% for Mannheimia haemolytica (MH), and 0.6% for Pasteurella multocida (PM). There were a few interactions of pathogens: PI3 and MH (0.01%), RSV and MH (0.01%), PM and MH (0.02%). This was the first study that immunohistochemically demonstrated bacterial and viral antigens in naturally occurring pneumonia in cattle in Nigeria and Ghana.

Pathogenic Mannheimia haemolytica Invades Differentiated Bovine Airway Epithelial Cells.

The Gram-negative bacterium Mannheimia haemolytica is the primary bacterial species associated with bovine respiratory disease (BRD) and is responsible for significant economic losses to livestock industries worldwide. Healthy cattle are frequently colonized by commensal serotype A2 strains, but disease is usually caused by pathogenic strains of serotype A1. For reasons that are poorly understood, a transition occurs within the respiratory tract and a sudden explosive proliferation of serotype A1 bacteria leads to the onset of pneumonic disease. Very little is known about the interactions of M. haemolytica with airway epithelial cells of the respiratory mucosa which might explain the different abilities of serotype A1 and A2 strains to cause disease. In the present study, host-pathogen interactions in the bovine respiratory tract were mimicked using a novel differentiated bovine bronchial epithelial cell (BBEC) infection model. In this model, differentiated BBECs were inoculated with serotype A1 or A2 strains of M. haemolytica and the course of infection followed over a 5-day period by microscopic assessment and measurement of key proinflammatory mediators. We have demonstrated that serotype A1, but not A2, M. haemolytica invades differentiated BBECs by transcytosis and subsequently undergoes rapid intracellular replication before spreading to adjacent cells and causing extensive cellular damage. Our findings suggest that the explosive proliferation of serotype A1 M. haemolytica that occurs within the bovine respiratory tract prior to the onset of pneumonic disease is potentially due to bacterial invasion of, and rapid proliferation within, the mucosal epithelium. The discovery of this previously unrecognized mechanism of pathogenesis is important because it will allow the serotype A1-specific virulence determinants responsible for invasion to be identified and thereby provide opportunities for the development of new strategies for combatting BRD aimed at preventing early colonization and infection of the bovine respiratory tract.