Advances in molecular and proteomic technologies and methods have enabled new diagnostic tools for bovine respiratory pathogens that are high-throughput, rapid, and extremely sensitive. Classically, diagnostic testing for these pathogens required culture-based approaches that required days to weeks and highly trained technical staff to conduct. However, new advances such as multiplex hydrolysis probe-based real-time PCR technology have enabled enhanced and rapid detection of bovine respiratory disease (BRD) pathogens in a variety of clinical specimens. These tools provide many advantages and have shown superiority over culture for co-infections/co-detections where multiple pathogens are present. Additionally, the integration of matrix-assisted laser desorption ionization time of flight mass spectrometry (MS) into veterinary diagnostic labs has revolutionized the ability to rapidly identify bacterial pathogens associated with BRD. Recent applications of this technology include the ability to type these opportunistic pathogens to the sub-species level (specifically Mannheimia haemolytica) using MS-based biomarkers, to allow for the identification of bacterial genotypes associated with BRD versus genotypes that are more likely to be commensal in nature.

Bovine respiratory disease (BRD) is the most common cause of morbidity and mortality in North American beef cattle. In recent years, isolation of strains of Mannheimia haemolytica that are resistant to multiple different classes of antimicrobials has become commonplace. New research would suggest that the routine use of antimicrobials by some cattle operations might be driving emerging resistance patterns, with the majority of the spread observed due to propagation of strains of M. haemolytica that have acquired integrative conjugative elements. To date, there is little information evaluating the impact of antimicrobial resistance on clinical outcome in cattle with BRD.

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

The objective of this paper was to perform a critical review of the literature as it pertains to the current status of antimicrobial resistance in pathogens associated with bovine respiratory disease (BRD) in beef cattle and to provide a concise yet informative narrative on the most relevant publications available. As such, the scientific literature contained in PubMed, AGRICOLA, and CAB were searched in February of 2014 for articles related to susceptibility testing of Mannheimia haemolytica, Pasteurella multocida, and Histophilus somni from cases of BRD. Titles and abstracts were read and 105 articles that were relevant to the subject of BRD antibiotic resistance were attained for further review. After the application of exclusion criterion (publications must have originated from North America, be in English, adhere to standards set forth by the Clinical and Laboratory Standards Institute, and be concerning antimicrobial resistance in BRD in beef cattle), 16 articles remained and are the focus of this publication. Due to the disparate data from the few studies that investigate susceptibility testing of BRD pathogens, a quantitative assessment or meta-analysis was not performed on the studies presented in this review. However, considering diagnostic lab data, there appears to be a clear trend of a decrease in susceptibility of the three major BRD pathogens to the antimicrobials used commonly for treatment and control of BRD. Studies performing sensitivity testing on healthy cattle report much lower resistance, but it remains unclear if this is because of a true lack of resistance mechanisms, or if the isolates do contain quiescent genes for resistance that are only phenotypically expressed following the administration of an antimicrobial for either treatment or control of BRD. Future research to address this question of genotype and phenotypic expression before and after antimicrobial administration will further advance our knowledge in this area.

Bovine respiratory disease complex (BRDC) is a major animal health and economic issue that affects cattle industries worldwide. Within the USA, the beef cattle industry loses up to an estimated 1 billion dollars a year due to BRDC. There are many contributors to BRDC, including environmental stressors and viral and/or bacterial infections. One species of bacteria in particular, Mannheimia haemolytica, is recognized as the major cause of severe fibrinonecrotic pneumonia in cattle. M. haemolytica is an opportunistic pathogen that normally populates the upper respiratory tract of cattle, and invades the lower respiratory tract in stressed and/or virally infected cattle by mechanisms that are not completely understood. However, not all M. haemolytica appear to be equally pathogenic to cattle. Thus, a test could be developed to distinguish M. haemolytica genetic subtypes by their propensity to cause respiratory disease, allowing isolation and/or treatment of cattle harboring strains with an increased propensity to cause disease. To that end, the genomes of over 300 M. haemolytica strains are being sequenced.

Bovine respiratory disease (BRD) involves complex interactions amongst viral and bacterial pathogens that can lead to intense pulmonary inflammation (fibrinous pleuropneumonia). Viral infection greatly increases the susceptibility of cattle to secondary infection of the lung with bacterial pathogens like Mannheimia haemolytica and Histophilus somni. The underlying reason for this viral/bacterial synergism, and the manner in which cattle respond to the virulence strategies of the bacterial pathogens, is incompletely understood. Bovine herpesvirus type 1 (BHV-1) infection of bronchial epithelial cells in vitro enhances the binding of M. haemolytica and triggers release of inflammatory mediators that attract and enhance binding of neutrophils. An exotoxin (leukotoxin) released from M. haemolytica further stimulates release of inflammatory mediators and causes leukocyte death. Cattle infected with H. somni frequently display vasculitis. Exposure of bovine endothelial cells to H. somnii or its lipooligosaccharide (LOS) increases endothelium permeability, and makes the surface of the endothelial cells pro-coagulant. These processes are amplified in the presence of platelets. The above findings demonstrate that bovine respiratory pathogens (BHV-1, M. haemolytica and H. somni) interact with leukocytes and other cells (epithelial and endothelial cells) leading to the inflammation that characterizes BRD.

Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, Mycoplasma bovis and Arcanobacterium pyogenes are all frequently implicated in bovine respiratory disease (BRD). M. haemolytica is considered the most important of the group. These bacteria are commensals in the nasopharynx and establish infection in the lungs of cattle that are subjected to a variety of stresses. Factors that permit adherence to and proliferation in the lungs and factors that cause tissue destruction and inflammation have been identified as having major roles in pathogenesis. These virulence factors include protein adhesins, capsular polysaccharide, outer membrane proteins, iron-binding proteins, lipopolysacharide or lipooligosaccharide, enzymes and toxins. These bacterial products function to evade the immune system, damage the immune system and induce a severe inflammatory response.

The objective was to describe the physicochemical changes during the early phase of subclinical mastitis and to associate them with pathological findings. A Mannheimia haemolytica strain was deposited into one teat duct of 25 ewes and the clinical, bacteriological, cytological, physicochemical (pH, milk composition), gross-pathological and histological findings were subsequently recorded. The organism was consistently isolated from samples of teat duct material (140/150) but not from mammary secretion (50/150). California Mastitis Test (CMT) scores increased (>1) and remained high (143/150 samples) after challenge; polymorphonuclear neutrophils (PMN) predominated in milk films, but the proportion of lymphocytes and macrophages progressively increased. Increased pH values (>7·0) were recorded in the mammary secretion from the challenged side. Furthermore, content of fat, total proteins and lactose therein decreased markedly. Histological changes (leucocytic infiltration, destruction of epithelial cells) were observed in the mammary parenchyma of the ewes. The present results confirm that the reduction of milk constituents is the effect of cellular damage and can occur soon after infection.

Mannheimia haemolytica is the principal bacterium isolated from respiratory disease in feedlot cattle and is a significant component of enzootic pneumonia in all neonatal calves. A commensal of the nasopharynx, M. haemolytica is an opportunist, gaining access to the lungs when host defenses are compromised by stress or infection with respiratory viruses or mycoplasma. Although several serotypes act as commensals, A1 and A6 are the most frequent isolates from pneumonic lungs. Potential virulence factors include adhesin, capsular polysaccharide, fimbriae, iron-regulated outer membrane proteins, leukotoxin (Lkt), lipopolysaccharide (LPS), lipoproteins, neuraminidase, sialoglycoprotease and transferrin-binding proteins. Of these, Lkt is pivotal in induction of pneumonia. Lkt-mediated infiltration and destruction of neutrophils and other leukocytes impairs bacterial clearance and contributes to development of fibrinous pneumonia. LPS may act synergistically with Lkt, enhancing its effects and contributing endotoxic activity. Antibiotics are employed extensively in the feedlot industry, both prophylactically and therapeutically, but their efficacy varies because of inconsistencies in diagnosis and treatment regimes and development of antibiotic resistance. Vaccines have been used for many decades, even though traditional bacterins failed to demonstrate protection and their use often enhanced disease in vaccinated animals. Modern vaccines use culture supernatants containing Lkt and other soluble antigens, or bacterial extracts, alone or combined with bacterins. These vaccines have 50–70% efficacy in prevention of M. haemolytica pneumonia. Effective control of M. haemolytica pneumonia is likely to require a combination of more definitive diagnosis, efficacious vaccines, therapeutic intervention and improved management practices.

We used a Mannheimia haemolytica isolate to study differences in susceptibility to experimental mastitis between two breeds of dairy sheep. The isolate was deposited into the teat duct of Karagouniko (K, n=8) or Frisarta (F, n=8) ewes. The animals were monitored by means of clinical, bacteriological, cytological and pathological methods. K ewes did not develop any systemic or mammary clinical signs, whilst F ewes became ill and developed acute clinical mastitis 12 h later (P<0·001). Bacteria were isolated from 34/48 samples from K ewes and from 46/46 samples from F ewes. Positive California mastitis test (CMT) results were 17/24 samples from K ewes and 23/23 samples from F ewes; leucocytes were seen in Giemsa-stained films. Total pathology score summed over all group K ewes was 41 (maximum possible: 128); Man. haemolytica was isolated from 12/24 tissue samples. Total pathology score summed over all group F ewes was 93; Man. haemolytica was isolated from 24/24 tissue samples. Hyperplastic lymphoid nodules consisting of lymphocytes and plasma cells with germinal activity were characteristically present at the border between teat duct–teat cistern of group K ewes; no such structures were observed in teats of group F ewes. The results identified differences in susceptibility/resistance to a mastitis pathogen among animals of the two breeds. Defence mechanisms of the teat appeared to be inadequate against the invading organisms; as lymphoid nodules have been considered important defensive mechanisms of the ovine teat, their observed lack in Frisarta ewes might have predisposed them to development of mastitis.

Progress in producing improved vaccines against bacterial diseases of cattle is limited by an incomplete understanding of the pathogenesis of these agents. Our group has been involved in investigations of two members of the family Pasteurellaceae, Mannheimia haemolytica and Haemophilus somnus, which illustrate some of the complexities that must be confronted. Susceptibility to M. haemolytica is greatly increased during active viral respiratory infection, resulting in rapid onset of a severe and even lethal pleuropneumonia. Despite years of investigation, understanding of the mechanisms underlying this viral–bacterial synergism is incomplete. We have investigated the hypothesis that active viral infection increases the susceptibility of bovine leukocytes to the M. haemolytica leukotoxin by increasing the expression of or activating the β2 integrin CD11a/CD18 (LFA-1) on the leukocyte surface. In vitro exposure to proinflammatory cytokines (i.e. interleukin-1β, tumor necrosis factor-α and interferon-γ) increases LFA-1 expression on bovine leukocytes, which in turn correlates with increased binding and responsiveness to the leukotoxin. Alveolar macrophages and peripheral blood leukocytes from cattle with active bovine herpesvirus-1 (BVH-1) infection are more susceptible to the lethal effects of the leukotoxin ex vivo than leukocytes from uninfected cattle. Likewise, in vitro incubation of bovine leukocytes with bovine herpesvirus 1 (BHV-1) potentiates LFA-1 expression and makes the cells more responsive to leukotoxin. A striking characteristic of H. somnus infection is its propensity to cause vasculitis. We have shown that H. somnus and its lipo-oligosaccharide (LOS) trigger caspase activation and apoptosis in bovine endothelial cells in vitro. This effect is associated with the production of reactive oxygen and nitrogen intermediates, and is amplified in the presence of platelets. The adverse effects of H. somnus LOS are mediated in part by activation of endothelial cell purinergic receptors such as P2X7. Further dissection of the pathways that lead to endothelial cell damage in response to H. somnus might help in the development of new preventive or therapeutic regimens. A more thorough understanding of M. haemolytica and H. somnus virulence factors and their interactions with the host might identify new targets for prevention of bovine respiratory disease.