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.

Identification of serotypes of Mannheimia haemolytica and Pasteurella multocida from pneumonic cases of sheep and goats and their antimicrobial sensitivity profiles in Borana and Arsi zones, Ethiopia.

Respiratory diseases caused by Mannheimia haemolytica (M. haemolytica) and Pasteurella multocida (P. multocida) have been known to result in a considerable loss due to mortality and reduced production. This study aimed at isolation and identification of M. haemolytica and P. multocida associated with pneumonic pasteurellosis in sheep and goats using bacteriological and molecular techniques. Identification of serotypes of M. haemolytica and P. multocida was done using indirect haemagglutination test. The in vitro antimicrobial sensitivity profiles of the M. haemolytica were tested using standard disk diffusion method. A total of 52 and 78 nasal swabs were collected from pneumonic cases for bacterial isolation and identification in Borana and Arsi zone, respectively. Four hundred sera samples were collected for identification of serotypes. The results showed that 17 of 52 (32.69%; 95% CI 20.33, 47.11) nasal swabs collected from pneumonic animals in Borana yielded positive results for Pasteurella/Mannheimia species, 13 (25.00%; 95% CI 14.03, 38.95) of which were M. haemolytica. None of the samples yielded P. multocida. Twenty-three of 78 (29.49%; 95% CI 19.69, 40.89) nasal swabs collected at Arsi from pneumonic animals yielded positive results for M. haemolytica (17) and P. multocida (6). Secondary biochemical characterization revealed that 14 of the 17 isolates conform to M. haemolytica whereas none of the 6 isolates suspected to be P. mutocida were confirmed. Eleven (84.62%) isolates from Borana and 4 (28.57%) from Arsi were confirmed to be M. haemolytica using PCR targeting the Rpt2 genes. Assay for M. haemolytica serotype A1 revealed all belong to A1. None of the isolates with cultural and morphological features of P. multocida gave positive results by molecular assay. Serological assay identified three serotypes of M. haemolytica namely A1, A2 and A7 almost in all of the samples whereas P. multocida serotype A was detected in 78.75% of the samples. The M. haemolytica isolates tested for susceptibility to antibiotics showed resistance against Bacitracin (83.33%) and Penicillin (50.00%) while they were found susceptible to Gentamycin (100%), Chloramphenicol (100%) and Sulfamethoxazole (100%) and Tetracycline (83.33%). In conclusion, the results of the present study revealed the association of M. haemolytica with pneumonic pasteurellosis in sheep and goats and can be of use in vaccine development in Ethiopia. Nevertheless, further investigations and continuous monitoring of antimicrobial resistance and appropriate selection and prudent use of antimicrobials in livestock sector are required.

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.

Toxigenic and non-toxigenic Pasteurella multocida genotypes, based on capsular, LPS, and virulence profile typing, associated with pneumonic pasteurellosis in Iran.

Pasteurella multocida is an important cause of pneumonic pasteurellosis in small ruminants. Its prevalence was investigated in 349 pneumonic lungs from sheep (n = 197) and goats (n = 152), and genotypes of isolates were determined by capsular and lipopolysaccharide (LPS) typing as well as by virulotyping based on the detection of 12 virulence-associated genes. P. multocida was isolated from 29.4 % of sheep lungs and 13.8 % of goat lungs. A (78.5 %) and D (21.5 %) capsular types, as well as L3 (41.8 %) and L6 (57.0 %) LPS genotypes, were detected, with the A:L6 genotype being the most prevalent in both sheep (59.6 %) and goat (52.4 %) isolates. A total of 19 virulence profiles (VP) were detected, seven non-toxigenic and 12 toxigenic, which correlated with the capsular-LPS genotype. All isolates of each VP belonged to the same LPS and capsular genotype, except for one isolate of VP1. The diversity in VP was higher among toxigenic (0.29) than non-toxigenic (0.18) isolates. Moreover, the toxigenic VPs showed more diversity in their capsular-LPS genotypes, with the two main toxigenic VPs belonging to genotypes D:L3 (VP2) and A:L3 (VP3). Therefore, the abundance of toxigenic isolates among sheep and goat isolates does not seem to correspond to the expansion of a more virulent lineage associated with pneumonic pasteurellosis in small ruminants. The most prevalent genotypes among sheep isolates were the non-toxigenic VP1:A:L6 (41.4 %) and the toxigenic VP3:A:L3 (17.2 %) genotypes, whereas the most prevalent among goat isolates were the toxigenic VP2:D:L3 (33.3 %) and the non-toxigenic VP1:A:L6 (14.3 %) and VP4:A:L6 (14.3 %) genotypes. These prevalent toxigenic and non-toxigenic genotypes seem to be epidemiologically relevant in pneumonic pasteurellosis of small ruminants.

Emergence of fatal Mannheimia haemolytica infections in cattle in the Netherlands.

In the Dutch national surveillance system, outbreaks of fatal infections by Mannheimia haemolytica (M. haemolytica) in dairy cows and veal calves have become apparent in recent years. These observations prompted an in-depth analysis of available pathology data over the period 2004-2018 to investigate changes in the occurrence and/or expression of M. haemolytica-associated cattle disease. With multilevel logistic regression models, time trends were identified and corrected for farm, season, pathologist and region. Deaths associated with M. haemolytica infection increased over time with dairy cows and veal calves diagnosed with fatal M. haemolytica infections 1.5 and 1.4 times more frequently every following 3-year period between 2004 and 2018, respectively. M. haemolytica-associated disease showed two distinct disease presentations: acute pleuropneumonia in dairy cows and polyserositis in veal calves. The prevalence of both disease presentations with M. haemolytica confirmed increased in each 3-year time period between 2004 and 2018, with an odds ratio (OR) of 1.5 for acute pleuropneumonia in dairy cows and an OR of 1.7 for polyserositis in veal calves. No change was found for M. haemolytica-associated disease in dairy calves. Although M. haemolytica is considered an opportunist bovine pathogen, and the presence of primary pathogens such as BHV-1, BVDV and Mycoplasma species was not completely ruled out in our study, substantial evidence is provided to indicate infections with M. haemolytica were the most likely cause of death. M. haemolytica-associated diseases occurred more often in October-June than July-September, and were detected more often in necropsied animals from the North, South and East Netherlands than the West Netherlands.

Strength of association between isolation of Pasteurella multocida and consolidation lesions in ovine pneumonic pasteurellosis.

This study investigated the association of Pasteurella multocida isolation and the molecular characteristics of the isolates with the presence of pneumonic lesions in lambs at slaughter to assess its importance as a causative agent of pneumonic pasteurellosis compared with Mannheimia haemolytica. P. multocida was isolated from the 13.9% and 2.7%, and M. haemolytica from the 36.4% and 26.8%, of lungs with and without lesions, respectively (P < 0.05). Both microorganisms were frequently coisolated (23.2% and 12.5% from lungs with and without lesions, respectively). Isolation of P. multocida alone exhibited greater strength of association with pneumonic lesions (OR 11.4; 95% CI 3.2-40.6) than that exhibited by M. haemolytica alone (OR 3.0; 95% CI 1.6-5.4). Cluster analysis grouped the lungs into four clusters characterized by the isolation of M. haemolytica or P. multocida alone (clusters 1 and 4), coisolation of both microorganisms (cluster 3), and isolation of neither (cluster 2). Cluster 4 lungs exhibited higher frequencies of pneumonic lesions (87.5%) and severe (20.8%) and moderate (25.0%) lesions. Lungs coinfected with both pathogens (cluster 3) did not exhibit a higher frequency of severe and moderate consolidation lesions (6.1% and 14.3%, respectively), suggesting that P. multocida and M. haemolytica do not act synergically to cause more severe pneumonic infections. The greater strength of association of P. multocida isolation with pneumonic lesions together with the higher severity of the lesions caused could indicate a greater role played by this pathogen in the aetiopathogenesis of pneumonic pasteurellosis in sheep than is commonly assumed.

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.

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.

The effects of Mannheimia haemolytica and albendazole on marbofloxacin pharmacokinetics in lambs.

The study aimed to define the effects of M. haemolytica and a single oral dose of albendazole on the single-dose pharmacokinetics of marbofloxacin in lambs. The pharmacokinetic-pharmacodynamic integration of marbofloxacin was applied to describe a 3 mg/kg intramuscular dose in lambs. The 6 healthy and 12 naturally infected with M. haemolytica lambs (Akkaraman, males weighing 10-15 kg and aged 2-3 months) were used in this study. In the marbofloxacin group, 6 healthy lambs received marbofloxacin. In the albendazole group after 2 weeks washout period, the same animals received marbofloxacin on 1 h after albendazole. In the diseased marbofloxacin group, 6 lambs naturally infected with M. haemolytica received marbofloxacin. In the diseased albendazole group, 6 lambs naturally infected with M. haemolytica received marbofloxacin on 1 h after albendazole. The marbofloxacin and albendazole were administered each as a single dose of 3 mg/kg intramuscular and 7.5 mg/kg oral, respectively, in the respective groups. Plasma concentration of marbofloxacin was measured with HPLC-UV and pharmacokinetic parameters were analyzed by non-compartmental model. Albendazole did not change the pharmacokinetic profiles of marbofloxacin in healthy and diseased lambs. However, M. haemolytica affected the pharmacokinetics of marbofloxacin in diseased lambs, AUC0-24/MIC90 ratio was not found to be higher than 125, but Cmax/MIC90 ratios was found to be higher than 10 for an MIC value of 0.25 µg/mL in all groups. The marbofloxacin dose described in this study may not be effective for the treatment of infections due to M. haemolytica in lambs, with MIC ≤ 0.25 µg/mL.

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.

Essential oils inhibit the bovine respiratory pathogens Mannheimia haemolytica, Pasteurella multocida and Histophilus somni and have limited effects on commensal bacteria and turbinate cells in vitro.

AIMS: The objective of this study was to determine antimicrobial activities of essential oils (EOs) against bovine respiratory disease (BRD) pathogens and nasopharyngeal commensal bacteria, as well as cytotoxicity in bovine turbinate (BT) cells in vitro. METHODS AND RESULTS: The chemical composition of 16 EOs was determined using gas chromatography-mass spectrometry. All EOs were first evaluated for growth inhibition of a single BRD pathogen Mannheimia haemolytica serotype 1 strain (L024A). The most inhibitory EOs (n = 6) were then tested for antimicrobial activity against multidrug-resistant strains of M. haemolytica (serotypes 1, 2 and 6); the BRD pathogens Pasteurella multocida and Histophilus somni, as well as commensal bacteria that were isolated from the nasopharynx of feedlot cattle. The cytotoxicity of 10 EOs was also evaluated using a BT cell line. The EOs ajowan, thyme and fennel most effectively inhibited all BRD pathogens tested including multidrug-resistant strains with minimum inhibitory concentrations (MIC) of ≤0·025% (volume/volume, v/v). For these EOs, the MIC was 2-32 fold greater against commensal bacteria, compared to BRD-associated pathogens. No cytotoxic effects of EOs against BT cells were observed within the tested range of concentrations (0·0125-0·4%, v/v). CONCLUSIONS: The EOs ajowan, thyme and fennel inhibited M. haemolytica, P. multocida and H. somni at a concentration of 0·025% and had minimal antimicrobial activity against nasopharyngeal commensal bacteria and cytotoxicity against BT cells. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated that EOs may have potential for intra-nasal administration to mitigate bovine respiratory pathogens in feedlot cattle.

Molecular characterization of Mannheimia haemolytica isolates associated with pneumonic cases of sheep in selected areas of Central Ethiopia.

BACKGROUND: Mannheimia haemolytica has been recognized as the principal cause of pneumonic pasteurellosis in sheep and goats. It is one of the important diseases of small ruminants in Ethiopia. While annual vaccination using a monovalent vaccine (inactivated Pasteurella multocida biotype A) is common, respiratory diseases are still reported in various parts of Ethiopia. This suggests the need for further investigation into the species and strains responsible for the disease, which is vital information for development of a multivalent vaccine. The objective of the current study was to isolate M. heamolytica associated with pneumonic cases of sheep in selected areas of Central Ethiopia, determine its role and the strains/genotypes of the bacterium circulating in the study area. RESULTS: Bacteriological analysis of nasal swab samples collected from a total of 76 pneumonic cases of sheep showed that M. haemolytica was isolated from 26 of them while B.trehalosi from two cases. Further molecular analyses of the isolates using M. haemolytica species-specific and M.haemolytica serotype-1 antigen specific PCR assays revealed, 26 of the isolates were identified as M. haemolytica of which 21 of them were M. haemolytica serotype-1. Both M. haemolytica and B.trehalosi isolates were not detected in a PCR assay targeting capsular biosynthesis gene (capA) of P.multocida despite the non-specific products observed in M. haemolytica isolates. Phylogenetic analysis of M. haemolytica isolates included in this study in comparison with the reference strains with respect to PHSSA and Rpt2 genes revealed that the Ethiopian M. haemolytica isolates constituted three distinct genotypes consistent with site of origin. CONCLUSION: The study indicated that M.haemolytica is commonly associated with cases of pneumonia in sheep in the study areas of central Ethiopia although the remaining other pathogens responsible for majority of the cases are yet to be determined. Molecular characterization revealed the existence of three genotypes of M. haemolytica circulating in the study areas consistent to the site of isolation. The findings suggest further extensive work to determine all pathogens associated with sheep pneumonia and the strain distribution of M. heamolytica to understand its molecular epidemiology at national level and design cost effective prevention and control methods.

Evolution of the nasopharyngeal bacterial microbiota of beef calves from spring processing to 40 days after feedlot arrival.

The composition of the nasopharyngeal bacterial microbiota has been shown to play a role in cattle respiratory health. However, previous studies are narrow in scope regarding longitudinal observations, limiting our understanding of how respiratory bacteria evolve over time. The objective was therefore to characterize this microbiota and its evolution over time in beef calves. A total of 120 crossbred beef-breed steer calves were enrolled in a study in southern Alberta at the time of first vaccination (spring processing), comprising three groups (40 calves/group) that originated from different ranches and were placed in different feedlots. Deep nasopharyngeal swab samples were collected from the calves at the time of spring processing, arrival at the feedlot, and a targeted 40 days after feedlot arrival. The swabs were processed for DNA extraction and the V4 region of the 16S rRNA gene was sequenced to evaluate the microbiota. The composition of the microbiota differed among groups of calves, with each group showing different relative abundances of 963 observed sequence variants. Mycoplasma was the most abundant genus and M. dispar the most abundant species across all groups. There was a distinct shift in the composition of the microbiota over time for all calf groups; however, changes in sequence variants differed by group. Variations in both microbiota composition and temporal changes of sequence variants according to calf group indicates that the respiratory microbiota of beef cattle may lack a common pattern of evolution from ranch to feedlot, and that future studies should account for potential group effects.

Ovine Mannheimia haemolytica isolates from lungs with and without pneumonic lesions belong to similar genotypes.

This study investigated the genetic characteristics of 121 ovine Mannheimia haemolytica isolates from lungs with (n = 75) and without pneumonic lesions (n = 46) using multilocus sequence typing (MLST), virulence-associated gene typing and pulsed-field gel electrophoresis (PFGE). Twelve STs were identified with most isolates (81%) belonged to ST16, ST28 and ST8. Analysis of the M. haemolytica MLST Database indicate a wide distribution of these genotypes in small ruminants, never reported in bovine isolates. This could suggest the adaptation of certain genetic lineages of M. haemolytica to small ruminants. e-BURST analysis grouped most STs into three clonal complexes (CC2, CC8 and CC28), consistent with a clonal population structure of M. haemolytica. Virulence-associated gene typing identified five virulence profiles in 64% and 65.1% of the M. haemolytica isolates from lungs with and without pneumonic lesions, respectively. These data suggest that M. haemolytica isolates from the lungs with and without pneumonic lesions are genetically homogeneous. By PGFE analysis a high level of genetic diversity was observed not only within isolates from lungs without pneumonic lesions but also among isolates from pneumonic lesions (GD 0.69 and GD 0.66, respectively; P > 0.05). These results indicate that multiple strains of M. haemolytica may be associated with individual cases of pneumonia in sheep.

Mannheimia haemolytica in bovine respiratory disease: immunogens, potential immunogens, and vaccines.

Mannheimia haemolytica is the major cause of severe pneumonia in bovine respiratory disease (BRD). Early M. haemolytica bacterins were either ineffective or even enhanced disease in vaccinated cattle, which led to studies of the bacterium's virulence factors and potential immunogens to determine ways to improve vaccines. Studies have focused on the capsule, lipopolysaccharide, various adhesins, extracellular enzymes, outer membrane proteins, and leukotoxin (LKT) resulting in a strong database for understanding immune responses to the bacterium and production of more efficacious vaccines. The importance of immunity to LKT and to surface antigens in stimulating immunity led to studies of individual native or recombinant antigens, bacterial extracts, live-attenuated or mutant organisms, culture supernatants, combined bacterin-toxoids, outer membrane vesicles, and bacterial ghosts. Efficacy of several of these potential vaccines can be shown following experimental M. haemolytica challenge; however, efficacy in field trials is harder to determine due to the complexity of factors and etiologic agents involved in naturally occurring BRD. Studies of potential vaccines have led current commercial vaccines, which are composed primarily of culture supernatant, bacterin-toxoid, or live mutant bacteria. Several of those can be augmented experimentally by addition of recombinant LKT or outer membrane proteins.

Mannheimia haemolytica A2 secretes different proteases into the culture medium and in outer membrane vesicles.

Respiratory diseases in ruminants have a significantly negative impact on the worldwide economy. The bacterium Mannheimia haemolytica is involved in pneumonic infections in bovine and ovine. In gram-negative bacteria, six secretion systems related to the colonization process and host tissue damage have been reported. In addition, in the last two decades, the production of outer membrane vesicles has been studied as a different bacterial strategy to release virulence factors, such as exotoxins, lipopolysaccharides, and proteases. However, in M. haemolytica serotype A2, protease secretion and release in vesicles have not been reported as virulence mechanisms. The aim of this work was to identify proteases released into the culture supernatant and in vesicles of M. haemolytica A2. Our results showed evident differences in the molecular mass and activity of proteases present in culture supernatants and outer membrane vesicles based on zymography assays. The biochemical characterization of M. haemolytica proteases revealed that the main types were cysteine and metalloproteases. A specific metalloprotease of 100 kDa was active in the culture supernatants, but it was not active and was found in low quantities in vesicles. Proteases could be an important virulence factor during the infectious pneumonic process led by M. haemolytica.

Bacterial, PCR and clinico-pathological diagnosis of naturally occurring pneumonic pasturellosis (mannheimiosis) during subtropical climate in sheep.

Mannheimia haemolytica is causative agent of pneumonic pasteurellosis (mannheimiosis) that causes huge economic losses to livestock farmers. We investigated the microbial and clinico-pathological patterns associated with ovine pneumonic pasturellosis during an outbreak. Prior to death, infected sheep revealed clinical signs including dyspnoea, salivation, pyrexia and mucopurulent nasal discharge. Mortality was significantly (p < 0.05) high in young sheep as compared to adults. Necropsy findings revealed presence of froth in trachea, congestion and consolidation of lungs, pulmonary edema, severe pleural adhesions, pericarditis, hemorrhages on mucosa of jejunum and kidneys. Histopathological examination revealed circumscribed and centrally calcified necrotic areas punctuated with chronic inflammatory cells and interstitial pneumonia. Moreover, bronchial epithelial hyperplasia, edema, congestion, mononuclear cell infiltration, thick interlobular septae and peri-vascular cuffing were the striking changes in lungs. Furthermore, lungs showed severe fibrin depositions along with abundant amount of fibrin meshwork on pleura infiltrated with chronic inflammatory cells. Histologically, liver, kidneys and lymph nodes showed degenerative changes. Mannheimia haemolytica and Pasteurella multocida were differentially identified on the basis of culture characteristics and biochemical tests. M. haemolytica was further confirmed by using polymerase chain reaction. From the findings of current study, it is concluded that M. haemolytica is a major respiratory threat in small ruminants that causes severe pneumonic changes in infected animals.

OmpA protein sequence-based typing and virulence-associated gene profiles of Pasteurella multocida isolates associated with bovine haemorrhagic septicaemia and porcine pneumonic pasteurellosis in Thailand.

BACKGROUND: Pasteurella multocida is a Gram-negative bacterium that causes economically significant infections of a broad range of animal species. Pneumonic and septicaemic pasteurellosis caused by this bacterium remain important problems in pigs, cattle, and water buffaloes in Thailand. The aim of this study was to characterise the virulence-associated gene profiles and to develop an OmpA molecular typing scheme for classifying 191 bovine and porcine isolates of P. multocida collected between 1989 and 2012 in Thailand using polymerase chain reactions (PCRs), nucleotide sequencing, and sequence and structural bioinformatics analyses. RESULTS: PCR screening successfully characterised the profiles of 25 virulence-associated genes in all isolates. The gene profiles separated these isolates into bovine and porcine clusters based on eight genes (hgbB, hsf1, tadD, nanH, pfhA, plpE, pmHAS, and tbpA). Phylogenetic analyses of the nucleotide and protein sequences corresponding to the ompA gene, which encodes a major outer membrane surface protein, showed two major bovine and porcine clusters. Structural prediction and analysis of the dN/dS ratio revealed four hypervariable extracellular loops of the OmpA transmembrane domains. These four loops were used to develop an OmpA typing scheme. This scheme classified 186 isolates into five major loop sequence types (LST8, LST12, LST15, LST18, and LST19), consistent with the phylogenetic results. The loop regions of the bovine isolates were predicted to be more antigenic than those of the porcine isolates. Thus, molecular evolution of the OmpA proteins could be used to classify P. multocida isolates into different capsular types, host types, and, possibly, pathogenicity levels. CONCLUSIONS: Together with the virulence-associated gene profiles, the typing reported in this work provides a better understanding of P. multocida virulence. Effective monitoring and potential strain-specific subunit vaccines could be developed based on these loop oligopeptides.

Prevalence of different OmpH-types among Pasteurella multocida isolated from lungs of calves with respiratory problems.

Pasteurella multocida is among the most important respiratory pathogens of cattle. Outer-membrane protein (OmpH) constitutes an essential bacterial antigen and is well studied in avian bacterial strains. Studies on isolates from cattle with signs of respiratory disease caused by Pasteurella multocida serotypes A and D have not yet been covered in the literature. The objective of this study was a comparative analysis of the ompH gene sequences from 83 isolates and four Russian reference strains of P. multocida to assign them to the allelic variants of the gene (OmpH-types). In addition, the above P. multocida strains have been characterized on the basis of capsular serotypes and virulence-associated genes. The isolates were classified into the OmpH -types based on allele specific PCR and gene fragment sequencing. The isolates of capsular serotype A have been subdivided into 6 OmpH -types, of which the most common types identified were A1 and A2. All capsular serotype D isolates belong to the same OmpH-type (D1). On 16 of a total of 23 farms all isolates belong to only one OmpH-type, on 4 farms - to 2, and on 3 farms - to three OmpH-types. The tbpA and pfhA genes were found more often in the isolates of capsular group А as compared to capsular group D (p ≤ 0.05). OmpH-types of serogroup А differ significantly (p ≤ 0.05) among themselves by the prevalence of the pfhA and hgbB genes.

Seroprevalence of respiratory viral pathogens of indigenous calves in Western Kenya.

Most studies of infectious diseases in East African cattle have concentrated on gastro-intestinal parasites and vector-borne diseases. As a result, relatively little is known about viral diseases, except for those that are clinically symptomatic or which affect international trade such as foot and mouth disease, bluetongue and epizootic haemorrhagic disease. Here, we investigate the seroprevalence, distribution and relationship between the viruses involved in respiratory disease, infectious bovine rhinotracheitis virus (IBR), bovine parainfluenza virus Type 3 (PIV3) and bovine viral diarrhoea virus (BVDV) in East African Shorthorn Zebu calves. These viruses contribute to the bovine respiratory disease complex (BRD) which is responsible for major economic losses in cattle from intensive farming systems as a result of pneumonia. We found that calves experience similar risks of infection for IBR, PIV3, and BVDV with a seroprevalence of 20.9%, 20.1% and 19.8% respectively. We confirm that positive associations exist between IBR, PIV3 and BVDV; being seropositive for any one of these three viruses means that an individual is more likely to be seropositive for the other two viruses than expected by chance.