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

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

Monitoring of bacterial agents isolated from small cattle on the territory of certain regions of the Russian Federation

The paper presents the results of bacterial screening of goat and sheep breeding enterprises in certain regions of Russia (Tver, Moscow, Smolensk regions, as well as the Republic of Mari-El and Tatarstan), conducted in the period from 2018 to 2021. In the course of this work, 556 samples of sectional material (heart, lungs, gastrointestinal tract, spleen, kidneys, liver, lymph nodes, breast, flushes from the genitourinary system, as well as exudate from purulent lesions) were subjected to a comprehensive bacteriological study. As a result of the conducted studies, 1223 isolates belonging to 25 families (111 bacterial species) were isolated and identified (by the method of time-of-flight mass spectrometry MALDI-ToF). According to the data obtained, the incidence of Escherichia coli isolation was 10.95%, Trueperella pyogenes – 5.47%, Staphylococcus aureus – 5.31%, Proteus mirabilis – 4.08%, Mannheimia haemolytica – 4.00%, Enterococcus faecalis – 3.76%, Enterobacter cloacea and Staphylococcus haemolyticus – 3.59%, Streptococcus dysgalactiae – 3.51%, Pasteurella multocida – 3.27%, Acinetobacter lwoffii – 2.78%, Staphylococcus cohnii – 2.61%, Bibersteinia trehalosi – 2.29%, Pseudomonas aeruginosa – 2.12%, Bacillus cereus and Micrococcus luteus – 1.96%, Corynebacterium pseudotuberculosis and Staphylococcus equorum – 1.88%, Aerococcus viridans – 1.80%, Corynebacterium xerosis – 1.72%, Clostridium perfringens, Streptococcus mitis and Streptococcus pyogenes – 1.39%, Staphylococcus chromogenes and Streptococcus entericus – 1.14%, respectively. The incidence of isolation of other types of microorganisms was below 1%. The data obtained indicate the circulation of a wide range of bacteria in goat and sheep breeding enterprises of the Russian Federation, some of which should be positioned as pathogenic flora (for example, Pasteurella multocida, Listeria monocytogenes, Salmonella enteritidis, Salmonella typhimurium, Clostridium perfringens, etc.), some as conditionally pathogenic (Trueperella pyogenes, Staphylococcus aureus, Bibersteinia trehalosi, Mannheimia haemolytica, Pseudomonas aeruginosa, Moraxella bovis, Moraxella bovoculi, etc.), as well as the normal flora of the animal body. It is worth focusing on these data when conducting a survey of livestock enterprises in order to establish an objective epizootic situation, including taking into account the possibility of circulating pathogens of factor diseases.

The Effects of Ursolic Acid Treatment on Immunopathogenesis Following Mannheimia haemolytica Infections

Bovine respiratory disease complex (BRDC) is a costly economic and health burden for the dairy and feedlot cattle industries. BRDC is a multifactorial disease, often involving viral and bacterial pathogens, which makes it difficult to effectively treat or vaccinate against. Mannheimia haemolytica (MH) are common commensal bacteria found in the nasopharynx of healthy cattle; however, following environmental and immunological stressors, these bacteria can rapidly proliferate and spread to the lower respiratory tract, giving rise to pneumonic disease. Severe MH infections are often characterized by leukocyte infiltration and dysregulated inflammatory responses in the lungs. IL-17A is thought to play a key role in this inflammatory response by inducing neutrophilia, activating innate and adaptive immune cells, and further exacerbating lung congestion. Herein, we used a small molecule inhibitor, ursolic acid (UA), to suppress IL-17A production and to determine the downstream impact on the immune response and disease severity following MH infection in calves. We hypothesized that altering IL-17A signaling during MH infections may have therapeutic effects by reducing immune-mediated lung inflammation and improving disease outcome. Two independent studies were performed (Study 1 = 32 animals and Study 2 = 16 animals) using 4-week-old male Holstein calves, which were divided into 4 treatment group including: (1) non-treated and non-challenged, (2) non-treated and MH-challenged, (3) UA-treated and non-challenged, and (4) UA-treated and MH-challenged. Based on the combined studies, we observed a tendency (p = 0.0605) toward reduced bacterial burdens in the lungs of UA-treated animals, but did not note a significant difference in gross (p = 0.3343) or microscopic (p = 0.1917) pathology scores in the lungs. UA treatment altered the inflammatory environment in the lung tissues following MH infection, reducing the expression of IL-17A (p = 0.0870), inflammatory IL-6 (p = 0.0209), and STAT3 (p = 0.0205) compared to controls. This reduction in IL-17A signaling also appeared to alter the downstream expression of genes associated with innate defenses (BAC5, DEFB1, and MUC5AC) and lung remodeling (MMP9 and TIMP-1). Taken together, these results support our hypothesis that IL-17A signaling may contribute to lung immunopathology following MH infections, and further understanding of this inflammatory pathway could expand therapeutic intervention strategies for managing BRDC.

Assessment of bovine respiratory disease progression in calves challenged with bovine herpesvirus 1 and Mannheimia haemolytica using point-of-care and laboratory-based blood leukocyte differential assays

Blood leukocyte differentials can be useful for understanding changes associated with bovine respiratory disease (BRD) progression. By improving turnaround time, point-of-care leukocyte differential assays (PCLD) may provide logistical advantages to laboratory-based assays. Our objective was to assess BRD progression in steers challenged with bovine herpesvirus 1 and Mannheimia haemolytica using point-of-care and laboratory-based blood leukocyte differentials. Thirty Holstein steers (average body weight of 211 kg + 2.4 kg) were inoculated intranasally on day 0 with bovine herpesvirus 1 and intrabronchially on day 6 with Mannheimia haemolytica. Blood leukocytes differentials were measured using both assays from study day 0 to 13. Linear mixed models were fitted to evaluate the associations between: 1) the type of assay (laboratory-based or PCLD) with respect to leukocyte, lymphocyte, and neutrophil concentrations, 2) study day with cell concentrations, and 3) cell concentrations with lung consolidation measured at necropsy. Point-of-care leukocyte, lymphocyte, and neutrophil concentrations were significantly associated (P < 0.05) with the respective cell concentrations obtained from the laboratory-based leukocyte differential. Cell concentrations reported by both assays differed significantly (P < 0.05) over time, indicating shifts from healthy to viral and bacterial disease states. Lymphocyte concentrations, lymphocyte / neutrophil ratios obtained from both assays, and band neutrophil concentrations from the laboratory-based assay were significantly associated (P < 0.05) with lung consolidation, enhancing assessments of disease severity. The PCLD may be a useful alternative to assess BRD progression when laboratory-based leukocyte differentials are impractical.

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

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.

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-1β 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.

On-farm colorimetric detection of Pasteurella multocida, Mannheimia haemolytica, and Histophilus somni in crude bovine nasal samples

This work modifies a loop-mediated isothermal amplification (LAMP) assay to detect the bovine respiratory disease (BRD) bacterial pathogens Pasteurella multocida, Mannheimia haemolytica, and Histophilus somni in a colorimetric format on a farm. BRD causes a significant health and economic burden worldwide that partially stems from the challenges involved in determining the pathogens causing the disease. Methods such as polymerase chain reaction (PCR) have the potential to identify the causative pathogens but require lab equipment and extensive sample processing making the process lengthy and expensive. To combat this limitation, LAMP allows accurate pathogen detection in unprocessed samples by the naked eye allowing for potentially faster and more precise diagnostics on the farm. The assay developed here offers 66.7–100% analytical sensitivity, and 100% analytical specificity (using contrived samples) while providing 60–100% concordance with PCR results when tested on five steers in a feedlot. The use of a consumer-grade water bath enabled on-farm execution by collecting a nasal swab from cattle and provided a colorimetric result within 60 min. Such an assay holds the potential to provide rapid pen-side diagnostics to cattle producers and veterinarians.

Biological and molecular characterization of a sheep pathogen isolate of Mannheimia haemolytica and leukotoxin production kinetics

Background and Aim: Mannheimia haemolytica (Mha) is a common agent of pneumonia in ruminants globally, causing economic losses by morbidity, mortality, and treatment costs. Infection by Mha is often associated with or promoted by respiratory viral pathogens and environmental conditions. Infections due to Mha have rarely been described in small ruminants. This study reports the biological and molecular characteristics of a new Moroccan Mha isolate from small ruminants presenting typical respiratory symptoms. We also studied the cultural parameters, growth kinetics, and Lkt excretion of the isolate and its pathogenicity on laboratory animals and small ruminants. Materials and Methods: Suspected pasteurellosis cases in sheep and goat flocks in Morocco were investigated. A local strain of Mha was isolated and identified using biochemical and molecular methods. Polymerase chain reaction-targeting specific genes were used for serotyping and phylogenetic analyses; further, leukotoxin production, cytotoxicity, and pathogenicity of the isolate in mice, goats, and sheep were investigated. Results: Phylogeny analysis revealed 98.76% sequence identity with the USA isolate of 2013; the strain growth with a cycle of 9-10 h with leukotoxin secretion was detected by NETosis and quantified by cytotoxicity and mortality of mice. Goat and sheep infections cause hyperthermia, with characteristic postmortem lesions in the trachea and lung. Conclusion: A local isolate of Mha from sheep that died of pneumonia was characterized for the 1st time in North Africa using biological and molecular methods. Although growth on appropriate culture media is accompanied by intense leukotoxin secretion, experimental infections of sheep and goats cause hyperthermia and typical lesions of pneumonia.

Differences between Predicted Outer Membrane Proteins of Pasteurella multocida, Histophilus somni, and Genotype 1 and 2 Mannheimia haemolytica Strains Isolated from Cattle

Common bacterial causes of bovine respiratory disease (BRD) include Histophilus somni, Mannheimia haemolytica, and Pasteurella multocida. Within M. haemolytica, two major genotypes are commonly found in cattle (1 and 2), however, genotype 2 strains are isolated from diseased lungs much more frequently than genotype 1 strains. Outer membrane proteins (OMPs) of H. somni, P. multocida, and genotype 2 M. haemolytica may be important factors for acquired host immunity. Predicted OMP differences between genotype 1 and 2 M. haemolytica have been previously identified. In this study, we expanded that focus to include bovine-isolated strain genomes representing all three species and the two M. haemolytica genotypes. Reported here are the core genomes unique to each of them, core genomes shared between some or all combinations of the three species and two M. haemolytica genotypes, and predicted OMPs within these core genomes. The OMPs identified in this study are potential candidates for further study and the development of interventions against BRD.