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

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

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

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

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.

Histophilus somni disease conditions with simultaneous infections by ovine gammaherpesvirus 2 in cattle herds from Southern Brazil.

This report investigated the cause of cattle mortality in two farms in Southern Brazil. The tissues of one animal from each farm (animals #1 and #2) respectively were used in pathological and molecular investigations to determine the possible cause of death. The principal pathological findings observed in animal #1 were pulmonary, myocardial, and encephalitic hemorrhages with vasculitis, and lymphoplasmacytic interstitial pneumonia with proliferative vascular lesions (PVL). The main pathological findings observed in animal #2 were purulent bronchopneumonia, hemorrhagic myocarditis, and lymphoplasmacytic interstitial pneumonia with PVL. An immunohistochemical assay detected intralesional antigens of a malignant catarrhal fever virus (MCFV) from multiple tissues of animal #2 while PCR confirmed that the MCFV amplified was ovine gammaherpesvirus 2 (OvGHV2), genus Macavirus, subfamily Gammaherpesvirinae; OvGHV2 was also amplified from multiple tissues of animal #1. Furthermore, PCR assays amplified Histophilus somni DNA from multiple fragments of both animals. However, the nucleic acids of Mannheimia haemolytica, Pasteurella multocida, Mycoplasma bovis, bovine respiratory syncytial virus, bovine alphaherpesvirus virus 1 and 5, bovine coronavirus, and bovine parainfluenza virus 3 were not amplified from any of the tissues analyzed, suggesting that these pathogens did not participate in the development of the lesions herein described. These findings demonstrated that both animals were concomitantly infected by H. somni and OvGHV2 and developed the septicemic and encephalitic manifestations of H. somni. Furthermore, the interstitial pneumonia observed in cow #2 was more likely associated with infection by OvGHV2.

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.

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.

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.

Anti-Inflammatory Properties of Fructo-Oligosaccharides in a Calf Lung Infection Model and in Mannheimia haemolytica-Infected Airway Epithelial Cells.

Emerging antimicrobial-resistant pathogens highlight the importance of developing novel interventions. Here, we investigated the anti-inflammatory properties of Fructo-oligosaccharides (FOS) in calf lung infections and in airway epithelial cells stimulated with pathogens, and/or bacterial components. During a natural exposure, 100 male calves were fed milk replacer with or without FOS for 8 weeks. Then, immune parameters and cytokine/chemokine levels in the bronchoalveolar lavage fluid (BALF) and blood were measured, and clinical scores were investigated. Calf primary bronchial epithelial cells (PBECs) and human airway epithelial cells (A549) were treated with Mannheimia haemolytica, lipopolysaccharides (LPS), and/or flagellin, with or without FOS pretreatment. Thereafter, the cytokine/chemokine levels and epithelial barrier function were examined. Relative to the control (naturally occurring lung infections), FOS-fed calves had greater macrophage numbers in BALF and lower interleukin (IL)-8, IL-6, and IL-1ß concentrations in the BALF and blood. However, FOS did not affect the clinical scores. At slaughter, FOS-fed calves had a lower severity of lung lesions compared to the control. Ex vivo, FOS prevented M. haemolytica-induced epithelial barrier dysfunction. Moreover, FOS reduced M. haemolytica- and flagellin-induced (but not LPS-induced) IL-8, TNF-α, and IL-6 release in PBECs and A549 cells. Overall, FOS had anti-inflammatory properties during the natural incidence of lung infections but had no effects on clinical symptoms.

Zn-Enhanced Asp-Rich Antimicrobial Peptides: N-Terminal Coordination by Zn(II) and Cu(II), Which Distinguishes Cu(II) Binding to Different Peptides.

The antimicrobial activity of surfactant-associated anionic peptides (SAAPs), which are isolated from the ovine pulmonary surfactant and are selective against the ovine pathogen Mannheimia haemolytica, is strongly enhanced in the presence of Zn(II) ions. Both calorimetry and ITC measurements show that the unique Asp-only peptide SAAP3 (DDDDDDD) and its analogs SAAP2 (GDDDDDD) and SAAP6 (GADDDDD) have a similar micromolar affinity for Zn(II), which binds to the N-terminal amine and Asp carboxylates in a net entropically-driven process. All three peptides also bind Cu(II) with a net entropically-driven process but with higher affinity than they bind Zn(II) and coordination that involves the N-terminal amine and deprotonated amides as the pH increases. The parent SAAP3 binds Cu(II) with the highest affinity; however, as shown with potentiometry and absorption, CD and EPR spectroscopy, Asp residues in the first and/or second positions distinguish Cu(II) binding to SAAP3 and SAAP2 from their binding to SAAP6, decreasing the Cu(II) Lewis acidity and suppressing its square planar amide coordination by two pH units. We also show that these metal ions do not stabilize a membrane disrupting ability nor do they induce the antimicrobial activity of these peptides against a panel of human pathogens.

Engineering and characterization of human ß-defensin-3 and its analogues and microcin J25 peptides against Mannheimia haemolytica and bovine neutrophils.

Mannheimia haemolytica-induced bovine respiratory disease causes loss of millions of dollars to Canadian cattle industry. Current antimicrobials are proving to be ineffective and leave residues in meat. Antimicrobial peptides (AMPs) may be effective against M. haemolytica while minimizing the risk of drug residues. Cationic AMPs can kill bacteria through interactions with the anionic bacterial membrane. Human ß-Defensin 3 (HBD3) and microcin J25 (MccJ25) are AMPs with potent activity against many Gram-negative bacteria. We tested the microbicidal activity of wild-type HBD3, three HBD3 peptide analogues (28 amino acid, 20AA, and 10AA) derived from the sequence of natural HBD3, and MccJ25 in vitro against M. haemolytica. Three C-terminal analogues of HBD3 with all cysteines replaced with valines were manually synthesized using solid phase peptide synthesis. Since AMPs can act as chemoattractant we tested the chemotactic effect of HBD3, 28AA, 20AA, and 10AA peptides on bovine neutrophils in Boyden chamber. Minimum bactericidal concentration (MBC) assay showed that M. haemolytica was intermediately sensitive to HBD3, 28AA and 20AA analogues with an MBC of 50 µg/mL. The 10AA analogue had MBC 6.3 µg/mL which is likely a result of lower final inoculum size. MccJ25 didn't have significant bactericidal effect below an MBC < 100 µg/mL. Bovine neutrophils showed chemotaxis towards HBD3 and 20AA peptides (P < 0.05) but not towards 28AA analogue. Co-incubation of neutrophils with any of the peptides did not affect their chemotaxis towards N-formyl-L-methionyl-L-leucyl-phenylalanine (fMLP). The data show that these peptides are effective against M. haemolytica and are chemotactic for neutrophils in vitro.

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

BACKGROUND: We 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. RESULTS: Regardless 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-1ß, 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)-1ß, 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. CONCLUSION: Host responses to pneumonic mannheimiosis based on increased serum levels of biomarkers (cortisol, HSP70, IL-1ß 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)-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.

Detection of Mycoplasma capricolum subsp. capripneumoniae and Mannheimia haemolytica as causative agents of pleuropneumonia in goats / Detección de Mycoplasma capricolum subsp. capripneumoniae y Mannheimia haemolytica como agentes causales de pleuro-neumonia en cabras

Abstract Objective: The aim of the study was detection of two major causative agents of pleuropneumonia, Mycoplasma capricolum subsp. capripneumoniae (Mccp) and Mannheimia haemolytica, in goats. To the best of our knowledge, this study is the first investigation of Mccp in Iran. Methods: 50 grossly suspected lungs to pleuropneumonia and 10 healthy samples were collected from Shiraz abattoir. Results: Histopathological evaluation of tissue samples showed various diagnosed pneumonias including 40% bronchointerstitial pneumonia (20 samples), 34% interstitial pneumonia (17 samples), 10% fibrinopurulent bronchopneumonia (5 samples), 12% purulent bronchopneumonia (6 samples) and 4% chronic pneumonia (2 samples). In molecular study, all 50 suspected samples and 10 healthy ones by PCR showed no Mccp positive sample, but the detection rate of M. haemolytica in suspected samples was 14% and in healthy lungs was zero. Conclusions: It may be concluded that goats referred to Shiraz abattoir is free of Mccp. Further sampling and molecular testing at the level of suspected herds to CCPP can be useful.

Resumen Objetivo: El objetivo del estudio fue la detección de dos agentes causantes principales de pleuroneumonía, Mycoplasma capricolum subsp. Capripneumoniae (Mccp) y Mannheimia haemolytica, en cabras. Hasta donde sabemos, este estudio es la primera investigación de Mccp en Irán. Métodos: 50 pulmones muy sospechosos de pleuroneumonía y 10 muestras sanas se obtuvieron del matadero de Shiraz. Resultados: La evaluación histopatológica de muestras de tejido mostró varias neumonías diagnosticadas, incluyendo 40% de neumonía broncointersticial (20 muestras), 34% de neumonía intersticial (17 muestras), 10% de bronconeumonía fibrinopurulenta (5 muestras), 12% de bronconeumonía purulenta (6 muestras) y 4% neumonía crónica (2 muestras). En un estudio molecular, las 50 muestras sospechosas y 10 sanas por PCR no mostraron una muestra positiva de Mccp, pero la tasa de detección de M. haemolytica en muestras sospechosas fue del 14% y en pulmones sanos fue cero. Conclusiones: se puede concluir que las cabras referidas al matadero Shiraz están libres de Mccp. La realización de muestreo adicional y pruebas moleculares a nivel de rebaños sospechosos para CCPP puede ser útil.

Differentiated ovine tracheal epithelial cells support the colonisation of pathogenic and non-pathogenic strains of Mannheimia haemolytica.

Mannheimia haemolytica is the primary bacterial species associated with respiratory disease of ruminants. A lack of cost-effective, reproducible models for the study of M. haemolytica pathogenesis has hampered efforts to better understand the molecular interactions governing disease progression. We employed a highly optimised ovine tracheal epithelial cell model to assess the colonisation of various pathogenic and non-pathogenic M. haemolytica isolates of bovine and ovine origin. Comparison of single representative pathogenic and non-pathogenic ovine isolates over ten time-points by enumeration of tissue-associated bacteria, histology, immunofluorescence microscopy and scanning electron microscopy revealed temporal differences in adhesion, proliferation, bacterial cell physiology and host cell responses. Comparison of eight isolates of bovine and ovine origin at three key time-points (2 h, 48 h and 72 h), revealed that colonisation was not strictly pathogen or serotype specific, with isolates of serotype A1, A2, A6 and A12 being capable of colonising the cell layer regardless of host species or disease status of the host. A trend towards increased proliferative capacity by pathogenic ovine isolates was observed. These results indicate that the host-specific nature of M. haemolytica infection may result at least partially from the colonisation-related processes of adhesion, invasion and proliferation at the epithelial interface.

Mannheimia haemolytica and lipopolysaccharide induce airway epithelial inflammatory responses in an extensively developed ex vivo calf model.

Pulmonary infection is associated with inflammation and damage to the bronchial epithelium characterized by an increase in the release of inflammatory factors and a decrease in airway barrier function. Our objective is to optimize a method for the isolation and culture of primary bronchial epithelial cells (PBECs) and to provide an ex vivo model to study mechanisms of epithelial airway inflammation. PBECs were isolated and cultured from the airways of calves in a submerged cell culture and liquid-liquid interface system. A higher yield and cell viability were obtained after stripping the epithelium from the bronchial section compared to cutting the bronchial section in smaller pieces prior to digestion. Mannheimia haemolytica and lipopolysaccharide (LPS) as stimulants increased inflammatory responses (IL-8, IL-6 and TNF-α release), possibly, by the activation of "TLR-mediated MAPKs and NF-κB" signaling. Furthermore, M. haemolytica and LPS disrupted the bronchial epithelial layer as observed by a decreased transepithelial electrical resistance and zonula occludens-1 and E-cadherin expression. An optimized isolation and culture method for calf PBECs was developed, which cooperated with animal use Replacement, Reduction and Refinement (3R's) principle, and can also contribute to the increased knowledge and development of effective therapies for other animal and humans (childhood) respiratory diseases.

Non-specific protection from respiratory tract infections in cattle generated by intranasal administration of an innate immune stimulant.

Alternatives to antibiotics for prevention of respiratory tract infections in cattle are urgently needed given the increasing public and regulatory pressure to reduce overall antibiotic usage. Activation of local innate immune defenses in the upper respiratory tract is one strategy to induce non-specific protection against infection with the diverse array of viral and bacterial pathogens associated with bovine respiratory disease complex (BRDC), while avoiding the use of antibiotics. Our prior studies in rodent models demonstrated that intranasal administration of liposome-TLR complexes (LTC) as a non-specific immune stimulant generated high levels of protection against lethal bacterial and viral pathogens. Therefore, we conducted studies to assess LTC induction of local immune responses and protective immunity to BRDC in cattle. In vitro, LTC were shown to activate peripheral blood mononuclear cells in cattle, which was associated with secretion of INFγ and IL-6. Macrophage activation with LTC triggered intracellular killing of Mannheimia hemolytica and several other bacterial pathogens. In studies in cattle, intranasal administration of LTC demonstrated dose-dependent activation of local innate immune responses in the nasopharynx, including recruitment of monocytes and prolonged upregulation (at least 2 weeks) of innate immune cytokine gene expression by nasopharyngeal mucosal cells. In a BRDC challenge study, intranasal administration of LTC prior to pathogen exposure resulted in significant reduction in both clinical signs of infection and disease-associated euthanasia rates. These findings indicate that intranasal administration of a non-specific innate immune stimulant can be an effective method of rapidly generating generalized protection from mixed viral and bacterial respiratory tract infections in cattle.

Pre-weaning plane of nutrition and Mannheimia haemolytica dose influence inflammatory responses to a bovine herpesvirus-1 and Mannheimia haemolytica challenge in post-weaning Holstein calves.

The objectives of this study were to determine whether plane of nutrition (PON) of milk replacer previously provided to calves, and dosage level of Mannheimia haemolytica (MH), influenced inflammatory responses to a combined viral-bacterial respiratory challenge. Holstein calves (1 d of age; n = 30) were assigned to treatments in a 2 × 3 factorial with pre-weaning PON and MH dose as main effects (n = 5 per treatment). Calves were fed either a low (LPN; n = 15) or a high PON (HPN; n = 15) from birth through weaning. Calves fed LPN were fed 436 g of dry matter (DM) per day of milk replacer until weaning, and HPN calves were fed 797 g of DM per day of milk replacer from d 1 to 10 and 1,080 g of DM per day from d 11 until weaning. Calf starter and water were offered ad libitum. Calves were step-down weaned beginning at d 54 and moved into an enclosed barn at d 70. Indwelling rectal temperature (RT) recording devices and jugular catheters were inserted at d 80. Calves were challenged with 1.5 × 108 plaque-forming units (pfu) per mL of bovine herpesvirus-1 (BHV-1) in each nostril at d 81 and with either 106, 107, or 108 cfu of MH at d 84. Blood samples were collected at varying intervals relative to BHV-1 and MH challenges. Four LPN calves either died or were euthanized soon after the 144-h observation period, whereas all HPN calves survived the entire observation period. As dosage of MH administered increased, acute and systemic inflammatory responses increased. Higher doses of MH resulted in increased leukocyte, neutrophil, and haptoglobin concentrations in infected calves. Data from the current study suggest that the highest dose, 108 cfu, triggered weaned calves' acute disease response, whereas the lower doses, 106 and 107 cfu, caused more moderate inflammation and disease. The effects of PON on inflammation responses to the disease challenge indicated that calves previously fed the LPN diet had more severe pathophysiological responses. Calves fed LPN showed higher peripheral neutrophil and leukocyte counts and serum haptoglobin concentrations following the BHV-1 challenge. Additionally, following the MH challenge, LPN calves had higher peripheral neutrophil counts, neutrophil-to-lymphocyte ratios, and serum tumor necrosis factor-α concentrations. These data demonstrate that higher doses of MH increase the acute inflammatory response and prolong inflammation, and that calves previously fed LPN responded more severely to the combined viral-bacterial respiratory challenge.

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.

Histophilus somni-associated syndromes in sheep from Southern Brazil

Abstract Histophilus somni is a Gram-negative bacterium that is associated with a disease complex (termed histophilosis) that can produce several clinical syndromes predominantly in cattle, but also in sheep. Histophilosis is well described in North America, Canada, and in some European countries. In Brazil, histophilosis has been described in cattle with respiratory, reproductive, and systemic disease, with only one case described in sheep. This report describes the occurrence of Histophilus somni-associated disease in sheep from Southern Brazil. Eight sheep with different clinical manifestations from five farms were investigated by a combination of pathological and molecular diagnostic methods to identify additional cases of histophilosis in sheep from Brazil. The principal pathological lesions were thrombotic meningoencephalitis, fibrinous bronchopneumonia, pulmonary abscesses, and necrotizing myocarditis. The main clinical syndromes associated with H. somni were thrombotic meningoencephalitis (n = 4), septicemia (n = 4), bronchopneumonia (n = 4), and myocarditis (n = 3). H. somni DNA was amplified from multiple tissues of all sheep with clinical syndromes of histophilosis; sequencing confirmed the PCR results. Further, PCR assays to detect Pasteurella multocida and Mannheimia haemolytica were negative. These findings confirmed the participation of H. somni in the clinical syndromes investigated during this study, and adds to the previous report of histophilosis in sheep from Brazil.

Leukotoxin of Bibersteinia trehalosi Contains a Unique Neutralizing Epitope, and a Non-Neutralizing Epitope Shared with Mannheimia haemolytica Leukotoxin.

Bibersteinia trehalosi and Mannheimia haemolytica, originally classified as Pasteurella haemolytica biotype T and biotype A, respectively, under Genus Pasteurella has now been placed under two different Genera, Bibersteinia and Mannheimia, based on DNA-DNA hybridization and 16S RNA studies. While M. haemolytica has been the predominant pathogen of pneumonia in ruminants, B. trehalosi is emerging as an important pathogen of ruminant pneumonia. Leukotoxin is the critical virulence factor of these two pathogens. While the leukotoxin of M. haemolytica has been well studied, the characterization of B. trehalosi leukotoxin has lagged behind. As the first step towards addressing this problem, we developed monoclonal antibodies (mAbs) against B. trehalosi leukotoxin and used them to characterize the leukotoxin epitopes. Two mAbs that recognized sequential epitopes on the leukotoxin were developed. One of them, AM113, neutralized B. trehalosi leukotoxin while the other, AM321, did not. The mAb AM113 revealed the existence of a neutralizing epitope on B. trehalosi leukotoxin that is not present on M. haemolytica leukotoxin. A previously developed mAb, MM601, revealed the presence of a neutralizing epitope on M. haemolytica leukotoxin that is not present on B. trehalosi leukotoxin. The mAb AM321 recognized a non-neutralizing epitope shared by the leukotoxins of B. trehalosi and M. haemolytica. The mAb AM113 should pave the way for mapping the leukotoxin-neutralizing epitope on B. trehalosi leukotoxin and the development of subunit vaccines and/or virus-vectored vaccines against this economically important respiratory pathogen of ruminants.