Haemolytica Serotype A1 Research Articles

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.

Stimulating immunoglobulin response by intramuscular delivery of exopolysaccharides-adjuvanted mannheimiosis vaccine in goats.

Pneumonic mannheimiosis (PM) is a common respiratory bacterial disease among small ruminants. Despite numerous management methods, vaccination remains a suitable strategy to combat or reduce PM in goats and sheep. Thus, a study was conducted in Malaysia to evaluate the immunogenicity of exopolysaccharide-adjuvanted Mannheimia haemolytica A2 vaccine (EPS-MHA2) under laboratory and field conditions for its potential use as an efficient vaccine against PM. This study induced immunoglobulin (Ig) responses following intramuscular (IM) delivery of the EPS-MHA2 vaccine on 12 goats for about 7 months. Goats were divided into three groups, with three goats per group, and they were vaccinated intramuscularly as follows: Group 1 was vaccinated with an adjuvanted vaccine prepared from formalin-killed M. haemolytica serotypes A2 and EPS excipient; Group 2 was vaccinated with formalin-killed M. haemolytica seed only, whereas Group 3 was injected with phosphate-buffered saline (PBS) as the negative control. Measures of specific immunity included serum IgM, IgG, and IgA as well as bronchoalveolar lavage fluid secretory IgA and the size and number of the bronchus-associated lymphoid tissue (BALT). From the 1st day of vaccination, Groups 1 and 2 showed a significant (p < 0.05) increase in serum IgM, IgG, and IgA levels. However, the antibodies started to decline 5-week post-vaccination, indicating that the booster dose was necessary. On the second exposure to the same vaccine (booster), the level of antibodies showed a significant increase (p < 0.05), particularly IgG. All groups were challenged intratracheally by virulent MHA2 2 weeks after the decline of second antibodies on the administration of booster. All goats were euthanatized and necropsied 4-week post-challenge. The number and size of the BALT in Group 1 goats significantly increased compared with those in Group 2 and the unvaccinated control. Bacteriological parameters were evaluated, in which MHA2 was reisolated successfully from lung samples in Group 3. The IgA level produced by the group vaccinated with EPS-MHA2 was significantly (p < 0.001) higher than that the MHA2 vaccine and PBS groups. All data obtained were analyzed statistically using a one-way analysis of variance. The results indicate that IM injection of EPS-MHA2 vaccine significantly enhanced the immune response against MHA2. Therefore, the addition of EPS to MHA2 (EPS-MHA2 vaccine) can effectively protect goats from lethal mannheimiosis infection. Factors such as the ideal concentration of EPS should be further studied to verify its application potential as a vaccine adjuvant, and the extraction of EPS from different microalgae species should be further investigated. This study showed a novel and exciting set of data and a vaccination system, in which the suppressive effects of mannheimiosis may be further investigated.

Distinct Mannheimia haemolytica serotypes isolated from fatal infections in veal calves and dairy cows

Fatal Mannheimia haemolytica (M. haemolytica) infections in cattle, which emerged in the Netherlands between 2004 and 2018, showed two distinct disease presentations: acute fibrinous polyserositis (FPS) in veal calves, and acute fibrinous pleuro-pneumonia (FPP) in adult dairy cattle. To determine whether these presentations were caused by different M. haemolytica genotypes, whole genome sequencing was performed on 96 isolates cultured after necropsy from inflamed sites of veal calves that died of M. haemolytica-associated FPS (n = 49) or with FPP lesions (n = 2), and from dairy cows that died of M. haemolytica-associated FPP (n = 45). Among the 96 M. haemolytica isolates, 93 were shown to belong to either of two large clusters, with 48/51 calf isolates belonging to one, and 43/45 cow isolates and two calf isolates from cases of FPP to the other. All M. haemolytica isolates from veal calves with FPS were of serotype A2, whereas the isolates from dairy cows and two calves with FPP were predominantly of serotypes A1 and A6. Most serotype A2 isolates from veal calves with FPS (95.6 %) contained multiple antibiotic resistance genes (ARGs) against three to five antimicrobial classes (phenicols, sulphonamides, tetracyclines, aminoglycosides or beta-lactams). In contrast, these ARGs were only present in 10.8 % of M. haemolytica A1 and A6 isolates from pneumonic adult cattle and absent in isolates from the two calves with FPP. These two disease presentations appear to be caused by genetically distinct strains with different antimicrobial resistance gene patterns. While M. haemolytica serotype A2 is generally considered to be a commensal microorganism of cattle, it was clearly associated with fatal FPS in veal calves in the Netherlands.

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 2mL of PBS pH 7.0, group 2 received 2mL of 1.2 × 109CFU/mL M. haemolytica A2 intranasally, and group 3 received 2mL of LPS extracted from 1.2 × 109CFU/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.

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

BackgroundMannheimia 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. MethodologyTwelve 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. ResultsCompared 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-1β 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. ConclusionThe 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.

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-1β, 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 2ml of sterile phosphate-buffered saline (PBS) and served as a negative control group. Group 2 was challenged intranasally with 2ml of bacterial inoculum containing 109 colony-forming units (CFU)/ml of M. haemolytica serotype A2, while Group 3 was challenged intravenously with 2ml of LPS endotoxin extracted from 109CFU/ml of M. haemolytica serotype A2. Following that, blood samples were collected serially at pre-determined intervals for serological analyses. All does were euthanised 60days 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-1β, 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.

Attempts to Stimulate Immunology Responses by Intramuscular and Intraperitoneal Delivery of EPS-Adjuvanted Mannheimiosis Vaccine

This study was conducted on 48 male Sprague Dawley rats to determine the immunological responses of experimental adjuvanted vaccines of Mannheimia haemolytica A2 (M. haemolytica A2) injection and to observe their protection level upon live M. haemolytica A2 challenge. Forty-eight clinically healthy Sprague Dawley rats were divided into four groups; Groups 1 and 2 were vaccinated intramuscularly and intraperitoneally, respectively, with an Exo-Polysaccharide (EPS)-adjuvanted vaccine prepared from formalin-killed M. haemolytica serotypes A2, Group 3 with formalin killed M. haemolytica seed. At the same time, Group 4 received intraperitoneally Phosphate Buffer Saline (PBS) as a control. After the first vaccination dose, Groups 1,2, and 3 showed a gradual increase in IgM, IgG, and IgA levels significantly (p<0.05). However, their level started to decline six weeks post-vaccination, indicating that the booster dose was needed. Upon the delivery of the second booster dose, antibodies titer showed a persistent increase significantly (p<0.05), especially the serum IgG level. All groups were challenged with live virulent Mannheimia haemolytica A2 after the level of antibodies declined twice after the second booster was delivered. No rat deaths were found in the Combined EPS - M. haemolytica adjuvant vaccine after 14 days following the challenge (0%). In unvaccinated rats, higher mortality and morbidity were noted (100%) and less was reported in rats receiving M. haemolytica A2 Vaccine seed (8%). Histologically at two weeks, post-challenge revealed negligible lung lesions in groups 1 and 2 and mild lesions in group 3. Lung lesions were recorded in all unvaccinated control rats. Furthermore, M. haemolytica A2 re-isolated successfully from lung samples in groups 3 and 4. In conclusion, Rats receiving adjuvant-M. haemolytica vaccine confers reasonably high preventive efficacy compared to M. haemolytica A2 by itself. Further studies should be conducted on measuring the antibody titer in different vivo, such as goats or sheep.

Response of Reproductive Hormones in Vaccinated and Non-Vaccinated Pneumonic Female Goats Via Experimental Infection of Mannheimia haemolytica Serotype A2 Under Rainy and Hot Seasons

Response of Reproductive Hormones in Vaccinated and Non-Vaccinated Pneumonic Female Goats Via Experimental Infection of Mannheimia haemolytica Serotype A2 Under Rainy and Hot Seasons

Bovine apo-lactoferrin affects the secretion of proteases in Mannheimia haemolytica A2.

Mannheimia haemolytica serotype A2 is the main bacterial causative agent of ovine mannheimiosis, a disease that leads to substantial economic losses for livestock farmers. Several virulence factors allow M. haemolytica to colonize the lungs and establish infection. Virulence factors can be directly secreted into the environment by bacteria but are also released through outer membrane vesicles (OMVs). In addition, due to the abuse of antibiotics in the treatment of this disease, multidrug-resistant bacterial strains of M. haemolytica have emerged. One therapeutic alternative to antibiotics or an adjuvant to be used in combination with antibiotics could be lactoferrin (Lf), a multifunctional cationic glycoprotein of the mammalian innate immune system to which no bacterial resistance has been reported. The aim of this work was to determine the effect of bovine iron-free Lf (apo-BLf) on the production and secretion of proteases into culture supernatant (CS) and on their release in OMVs. Zymography assays showed that addition of sub-MIC concentrations of apo-BLf to M. haemolytica cultures inhibited protease secretion without affecting culture growth. Biochemical characterization revealed that these proteases were mainly cysteine- and metalloproteases. The secretion of a 100 kDa metalloprotease was inhibited by sub-MIC concentrations of apo-BLf since this protease was present in the cytoplasm and OMVs but not in CS proteins, as corroborated by Western blotting. On the other hand, proteases produced by M. haemolytica caused cleavage of apo-BLf. However, when Lf is cleaved, peptides known as lactoferricins, which are more bactericidal than natural Lf, can be produced. M. haemolytica A2 protease-mediated degradation of host tissue proteins could be an important virulence factor during the infectious process of pneumonia in ovines. The mechanism of M. haemolytica protease secretion could be inhibited by treatment with apo-BLf in animals.

PSXVI-28 Late-Breaking: Effects of Preconditioning (Value Added Programs) on the Health, Performance, Mannheimia haemolytica, and Pasteurella multocida in Cattle Received on Winter Wheat Pasture

Abstract Bovine respiratory disease (BRD) is a persistent health problem impacting the beef industry. Research shows improved health and performance in preconditioned (PRECON) calves compared with nonpreconditioned (NONPRE) or commingled (COMM) calves received in the feedlot but little research has been focused on calves received on winter wheat pasture prior to feedlot entry. Our objective for this presentation is to investigate the effects of preconditioning on the health and performance of newly received beef calves on winter wheat pasture. Mixed breed steers (n = 145) were purchased from an auction barn in Dalhart, Texas, as PRECON (n = 70) or NONPRE (n = 75) and were transported to the Clayton Livestock Research Center in Clayton, New Mexico, for this 112-d study trial. Three treatments were used in this completely randomized design: PRECON (n = 50), NONPRE (n = 50) and COMM (n = 45). Upon arrival, steers were offloaded into separate pens. On d 0, steers were processed using a standard health protocol along with collection of nasopharyngeal (NP) swabs, randomly allocated to treatment, and released onto a 120-acre winter wheat pasture split into three paddocks with a common water source; weights were collected again on d 2, 90, and 112. There were no statistical differences in morbidity and mortality rates between treatments. Weight gain was analyzed using PROC GLM of SAS from d 0 to d 90. COMM steers had greater weight gains than PRECON (P = 0.04) and NONPRE (P = 0.02) steers. NP swabs were used to show the distribution of Mannheimia haemolytica (MH) serotype A1, A2, and A6 and Pasteurella multocida (PM) by day and by treatment. No statistical differences were observed in serotype distribution of MH A1, A2, or A6 or in PM. PRECON steers displayed no health or performance advantage over NONPRE or COMM steers.

Biomarkers, immune responses and cellular changes in vaccinated and non-vaccinated goats during experimental infection of M. haemolytica A2 under tropical conditions

BackgroundWe investigated the biomarkers, immune responses and cellular changes in vaccinated and non-vaccinated goats experimentally challenged with M. haemolytica serotype A2 under rainy and hot tropical conditions. A total of twenty-four clinically healthy, non-pregnant, female goats randomly allocated to 2 groups of 12 goats each were used for the study. The 12 goats in each season were subdivided into three groups (n = 4), which served as the control (G-NEG), non-vaccinated (G-POS), and vaccinated (G-VACC). In week-1, the G-VACC received 2 mL of alum-precipitated pasteurellosis vaccine while G-POS and G-NEG received 2 ml of sterile PBS. In week 2, the G-POS and G-VACC received 1 mL intranasal spray containing 105 CFU of M. haemolytica serotype A2. Inoculation was followed by daily monitoring and weekly bleeding for eight weeks to collect data and serum for biomarkers and immune responses using commercial ELISA test kits. The goats were humanely euthanised at the end of the experiments to collect lungs and the submandibular lymph nodes tissue samples for gross and histopathological examinations. ResultsRegardless of the season, we have observed a significant (p < 0.05) increase in serum concentrations of acute-phase proteins (haptoglobin, serum amyloid A), proinflammatory cytokines (interleukine-1β, interleukin-6), antibodies (immunoglobulin M, immunoglobulin G), and stress markers (cortisol and heat shock protein 70) in the G-POS goats compared to G-VACC and G-NEG. With regards to seasons, there was a significantly (p < 0.05) higher serum concentration with 1.5, 2 and 1-folds increase in the serum interleukin (IL)-1β, cortisol, and heat shock protein (HSP)-70 in the G-POS during rainy compared to the hot season. Histopathology of the lungs in G-POS goats revealed inflammatory cell infiltration, degeneration, haemorrhage/congestion, and pulmonary oedema in the alveoli spaces; thickening of the interstitium, and desquamation of bronchiolar epithelium. Cellular changes in the lymph node were characterized by a marked hypercellularity in G-POS goats. ConclusionHost responses to pneumonic mannheimiosis based on increased serum levels of biomarkers (cortisol, HSP70, IL-1β and IL-6) and severe cellular changes seen in the lungs and lymph nodes of G-POS goats compared to vaccinated goats and control group are influenced by the high environmental humidity recorded in the rainy season. Increased relative humidity in the rainy season is a significant stress factor for the higher susceptibility and severity of pneumonic mannheimiosis of goats in the tropics. Vaccination of goats using the alum precipitated Pasteurella multocida vaccine before the onset of the rainy season is recommended to minimise mortality due to potential outbreaks of pneumonia during the rainy season.

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)-1β, 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.

Effect of apo-lactoferrin on leukotoxin and outer membrane vesicles of Mannheimia haemolytica A2

Mannheimia haemolytica serotype A2 is the principal cause of pneumonic mannheimiosis in ovine and caprine livestock; this disease is a consequence of immune suppression caused by stress and associated viruses and is responsible for significant economic losses in farm production worldwide. Gram-negative bacteria such as M. haemolytica produce outer membrane (OM)-derived spherical structures named outer membrane vesicles (OMVs) that contain leukotoxin and other biologically active virulence factors. In the present study, the relationship between M. haemolytica A2 and bovine lactoferrin (BLf) was studied. BLf is an 80 kDa glycoprotein that possesses bacteriostatic and bactericidal properties and is part of the mammalian innate immune system. Apo-BLf (iron-free) showed a bactericidal effect against M. haemolytica A2, with an observed minimal inhibitory concentration (MIC) of 16 µM. Sublethal doses (2–8 µM) of apo-BLf increased the release of OMVs, which were quantified by flow cytometry. Apo-BLf modified the normal structure of the OM and OMVs, as observed through transmission electron microscopy. Apo-BLf also induced lipopolysaccharide (LPS) release from bacteria, disrupting OM permeability and functionality, as measured by silver staining and SDS and polymyxin B cell permeability assays. Western blot results showed that apo-BLf increased the secretion of leukotoxin in M. haemolytica A2 culture supernatants, possibly through its iron-chelating activity. In contrast, holo-BLf (with iron) did not have this effect, possibly due to differences in the tertiary structure between these proteins. In summary, apo-BLf affected the levels of several M. haemolytica virulence factors and could be evaluated for use in animals as an adjuvant in the treatment of ovine mannheimiosis.

Association between Mannheimia haemolytica infection with reproductive physiology and performance in small ruminants: A review.

Mannheimiosis or pneumonic pasteurellosis commonly occurs in small ruminants. Mannheimiosis is caused by Mannheimia haemolytica (M. haemolytica) a Gram-negative coccobacillus producing acute febrile and infectious condition resulting in death of animal if not diagnosed and treated promptly. M. haemolytica serotype A2 is a commensal of the nasopharynx, gaining access to the lungs when host defenses are compromised by stress or infection in small ruminants. Till date, there is a vast literature and research that has been conducted on the pathogenesis of M. haemolytica invariably on respiratory system and its related immune system and mechanisms. From the clinical point of view, infection or diseases involving vital organs will systemically affect the production and performance of the infected animal. Therefore, there is a huge gap of knowledge and research to answer the question whether there is any association between M. haemolytica infection with reproductive physiology and performance in small ruminants and how it affects the productivity level. This review will explore the possibilities of involvement and new potential research to be carried out to determine the involvement of male and female reproductive system with M. haemolytica infection among small ruminants.

Effect of suckler cow vaccination against glycoprotein E (gE)-negative bovine herpesvirus type 1 (BoHV-1) on passive immunity and physiological response to subsequent bovine respiratory disease vaccination of their progeny

The study objectives were: 1) to characterise the development of immunocompetence in beef suckler calves from birth to three months of age, and 2) to trace glycoprotein E (gE)-negative bovine herpesvirus type 1 (BoHV-1) antibodies from dam to calf and subsequent vaccination against pneumonia. Thirty multiparous beef suckler, spring-calving cows, consisting of two genotypes were involved; Limousin×Friesian (LF) and Charolais×Limousin (CL). Cows were immunised against the inactivated antigen strain of BoHV-1 (gE- (IBR marker vaccine) at day −84 and received a booster at day −56 relative to the expected calving date (d 0). Calves were immunised at 14 and 42days of age against PI-3 virus, BRSV and Mannheimia (Pasteurella) haemolytica serotype A1 using a commercial vaccine administered subcutaneously. Additionally, calves were immunised against BoHV-1 at 42days of age, using 1 dose of a live commercial vaccine administered intranasally. Blood samples were collected from all calves (n=30) via jugular venipuncture at birth, prior to colostrum feeding (0h), at 12h (h), 24h, 72h and 168h after the initial feeding of colostrum, and at d 7, 14, 28, 42, 56 and 84 post birth. The mean ratio of gE negative antibodies circulating in the blood of LF and CL dams pre-partum scored negative to gE ab (S/N≥0.70). Antibody levels of BoHV-1 (wild type (wt)) peaked at 12h post-birth in calves and declined thereafter, as the maternal antibodies decayed. There was no difference in BoHV-1 and BRSV antibody levels in calves post vaccination.

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.

X-ray crystallographic structure of a bacterial polysialyltransferase provides insight into the biosynthesis of capsular polysialic acid

Polysialic acid (polySia) is a homopolymeric saccharide that is associated with some neuroinvasive pathogens and is found on selective cell types in their eukaryotic host. The presence of a polySia capsule on these bacterial pathogens helps with resistance to phagocytosis, cationic microbial peptides and bactericidal antibody production. The biosynthesis of bacterial polySia is catalysed by a single polysialyltransferase (PST) transferring sialic acid from a nucleotide-activated donor to a lipid-linked acceptor oligosaccharide. Here we present the X-ray structure of the bacterial PST from Mannheimia haemolytica serotype A2, thereby defining the architecture of this class of enzymes representing the GT38 family. The structure reveals a prominent electropositive groove between the two Rossmann-like domains forming the GT-B fold that is suitable for binding of polySia chain products. Complex structures of PST with a sugar donor analogue and an acceptor mimetic combined with kinetic studies of PST active site mutants provide insight into the principles of substrate binding and catalysis. Our results are the basis for a molecular understanding of polySia biosynthesis in bacteria and might assist the production of polysialylated therapeutic reagents and the development of novel antibiotics.

Comparative analysis of multiple inducible phages from Mannheimia haemolytica.

BackgroundMannheimia haemolytica is a commensal bacterium that resides in the upper respiratory tract of cattle that can play a role in bovine respiratory disease. Prophages are common in the M. haemolytica genome and contribute significantly to host diversity. The objective of this research was to undertake comparative genomic analysis of phages induced from strains of M. haemolytica serotype A1 (535A and 2256A), A2 (587A and 1127A) and A6 (1152A and 3927A).ResultsOverall, four P2-like (535AP1, 587AP1, 1127AP1 and 2256AP1; genomes: 34.9–35.7 kb; G+C content: 41.5–42.1 %; genes: 51–53 coding sequences, CDSs), four λ-like (535AP2, 587AP2, 1152AP2 and 3927AP1; genomes: 48.6–52.1 kb; 41.1–41.4 % mol G+C; genes: 77–83 CDSs and 2 tRNAs) and one Mu-like (3927AP2; genome: 33.8 kb; 43.1 % mol G+C; encoding 50 CDSs) phages were identified. All P2-like phages are collinear with the temperate phage φMhaA1-PHL101 with 535AP1, 2256AP1 and 1152AP1 being most closely related, followed by 587AP1 and 1127AP1. Lambdoid phages are not collinear with any other known λ-type phages, with 587AP2 being distinct from 535AP2, 3927AP1 and 1152AP2. All λ-like phages contain genes encoding a toxin-antitoxin (TA) system and cell-associated haemolysin XhlA. The Mu-like phage induced from 3927A is closely related to the phage remnant φMhaMu2 from M. haemolytica PHL21, with similar Mu-like phages existing in the genomes of M. haemolytica 535A and 587A.ConclusionsThis is among the first reports of both λ- and Mu-type phages being induced from M. haemolytica. Compared to phages induced from commensal strains of M. haemolytica serotype A2, those induced from the more virulent A1 and A6 serotypes are more closely related. Moreover, when P2-, λ- and Mu-like phages co-existed in the M. haemolytica genome, only P2- and λ-like phages were detected upon induction, suggesting that Mu-type phages may be more resistant to induction. Toxin-antitoxin gene cassettes in λ-like phages may contribute to their genomic persistence or the establishment of persister subpopulations of M. haemolytica. Further work is required to determine if the cell-associated haemolysin XhlA encoded by λ-like phages contributes to the pathogenicity and ecological fitness of M. haemolytica.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0494-5) contains supplementary material, which is available to authorized users.

Complete Closed Genome Sequences of a Mannheimia haemolytica Serotype A1 Leukotoxin Deletion Mutant and Its Wild-Type Parent Strain.

Mannheimia haemolytica is a bacterial pathogen that secretes leukotoxin (LktA) which binds to leukocyte membranes via CD18, causing bacterial pneumonia in ruminants. We report the complete closed genome sequences of a leukotoxin mutant and its parent strain that are frequently used in respiratory disease studies.

English

This experimental study was done on a total of 40 male lambs with the objectives of developing experimental vaccines from Mannheimia haemolytica serotypes A2 and A7 that express iron regulated outer membrane protein and in vivo evaluation of their efficacy. Lambs were categorized in to four experimental groups and vaccinated with 1 ml of vaccine containing 5 × 108 CFU/ml. Group 1 was vaccinated with M. haemolytica A2, group 2 with A7, group 3 with serotype A2 and A7 combination, and group 4 received saline as control. They were challenged intratracheally by the respective homologous serotype after 35 days of vaccination. Post challenge clinical investigation showed that significant higher rate of morbidity was seen in control group which was demonstrated by raised rectal temperature (by 0.5-1°C) and respiratory signs. From the total of 26 lambs challenged with live M. haemolytica A2 and A7, 6 (23.1%) and 4 (15.3%) lambs were found dead and sick, respectively. Higher mortality and morbidity were observed in unvaccinated control group; however, lesser was recorded in combined vaccinated group. Lung lesions of variable severity were observed in 13 (50.0%) lambs following challenge. From vaccinated groups, 5 (27.8%) lambs were found to have a +1 lung lesion score. All of the lambs in unvaccinated control group had scores between +2 and +3. There was a statistically significant difference (p 0.05) was seen among vaccinated groups concerning lung lesion scores. Furthermore, the respective serotypes of M. haemolytica were successfully re-isolated from pneumonic lungs at a mean titre range of 102.2 – 108.1 CFU/g. In conclusion, lambs which received combined vaccine confer relatively good protective efficacy than M. haemolytica A2 or A7 vaccinated groups. Therefore, further study should be done on evaluation of antibody titer at different time points. Key words: Challenge, efficacy, lambs, lung lesion score, Mannheimia heamolytica, morbidity, mortality.