Pasteurella multocida is a pathogen that causes bovine respiratory disease, and the development of an effective vaccine is important for improving animal health. Live-attenuated vaccines induce a long-lasting immune response with minimal side effects. The objective of this study was to evaluate potential live vaccine candidates from three P. multocida mutants produced by separately disrupting the genes of filamentous hemagglutinin 2 (fhaB2), hydrogenase-1 operon (hyaE), and n-acylneuraminate-9-phosphatase (nanP) of a serogroup 3 strain (P1062, WT) by clinical testing and transcriptome analysis. Challenge with WT and the three mutants conferred protection against P. multocida, with less lung lesions (4.7-6.2%) compared to 22.4% in the sham group. Transcriptome analysis identified 807 differentially expressed protein-coding transcripts (DETs) in the blood and 6473 DETs in the liver compared to the sham, WT, and each of the mutants. In total, 15 and 64 differentially expressed microRNAs (DEmiRNAs) and 12 and 74 differentially expressed long non-coding RNAs (DElncRNAs) were identified in blood and liver, respectively. The DEmiRNAs were not significantly associated with the DETs within each comparison. DElncRNAs were associated with 12 and 170 DETs in blood and liver respectively. The greatest number of unique DETs were found between hyaE and sham groups in the liver, which agreed with the low colonization rate in the nares and palatine tonsils. For the DETs between sham and WT the under-enriched gene ontology terms in blood were all included in the liver for the DETs identified by WT vs. sham, nanP vs. sham, and hyaE vs. sham, and were related to the signaling pathway, stimulus, and sensory perceptions in biological processes with the molecular function of olfactory receptor activity. The number of identified DETs, decreased percentage of lung lesions, and colonization rates indicate that fhaB2 could be a promising vaccine candidate.

The aim of this study was to assess the role of leukotoxin in Mannheimia haemolytica on colonization dynamics in the nasopharynx and bacterial shedding in calves concurrently infected with bovine herpesvirus 1 (BHV-1). Eight calves were assigned to two treatment groups of four and housed in isolation rooms. After acclimation, BHV-1 was administered intranasally to both groups. On the same day, calves in one group received feed containing leukotoxin (LktA) deleted mutants of the M. haemolytica serotype (ST1) and ST6 strains. Five days post-BHV-1 exposure, this group was re-exposed to LktA mutants intranasally. On day six, both groups were intranasally inoculated with wild-type ST1 and ST6 parent strains, and the animals were euthanized on day 10. Results indicate that both ST1 and ST6 can establish high-level colonization in the nasopharynx of BHV-1 infected calves. LktA mutants exhibited approximately 10-fold lower nasopharyngeal colonization compared to their wild-type counterparts. The higher shedding levels observed in wild-type strains compared to LktA mutants suggested a clear advantage in spreading the infection to other susceptible animals. These findings provide valuable insight into the nature of M. haemolytica infection in bovine hosts. They also indicate that leukotoxin expression may contribute to the competitive fitness M. haemolytica strains, potentially influencing their adaptation within the host.

Introduction:Mycoplasma bovis causes chronic respiratory disease with high mortality rates in American bison (Bison bison). A recent study showed that a subunit vaccine containing M. bovis elongation factor thermal unstable (EFTu) and heat shock protein 70 (Hsp70) antigens induced immunity and enhanced protection in bison, resulting in reduced lung lesions and bacterial loads following experimental M. bovis challenge. This study aimed to characterize the transcriptional responses underlying this protection in vaccinated (n = 5) compared to unvaccinated control (n = 4) bison following M. bovis infection. Methods:Two doses of vaccines were administered on day 0 and at 21 days post-vaccination (DPV), followed by intranasal inoculation with bovine herpesvirus-1 (BHV-1) at 36 DPV and M. bovis at 40 DPV. RNA sequencing was performed on liver, palatine tonsil (PT), retropharyngeal lymph node (RPLN), tracheobronchial lymph node (TBLN), spleen, and whole blood samples. Blood was collected at 1st vaccination (Day 0), 2nd vaccination (21 days post-vaccination), BHV-1 inoculation (36 DPV), M. bovis inoculation (40 DPV), and 1 week post M. bovis inoculation (47 DPV). Results and discussion:The greatest number of differentially expressed transcripts (DETs) (≤0.05 FDR) were found in blood at 36 DPV (123 total DETs) and in spleen (57 DETs). At 36 DPV, vaccinated animals showed upregulation of transcripts involved in in cell adhesion, T-helper cell (Th1/Th2/Th17) differentiation, and antigen processing and presentation. This signifies a robust response to the 2nd vaccine dose, which caused increased expression of CD3E, CD4, and CD8B correlating to increased T cell proliferation. Notably, transcription factors TBX21 and GATA3 were upregulated in vaccinated animals. Spleen-specific regulation included transcripts involved in innate immune response, such as LGALS3 and GBP-1. These findings highlight the robust immune response induced by the vaccine, particularly through T-cell mediated responses, demonstrating its potential to enhance protective immunity against M. bovis in bison.

Mycoplasma bovis (M. bovis) is an emerging pathogen in American bison (Bison bison) responsible for high mortality epizootics of severe pneumonia and systemic disease. Though M. bovis poses a significant threat to bison conservation and ranching, there are no commercial vaccines licensed for use in this species. To this end, novel modified-live Mannheimia haemolytica (M. haemolytica) vaccine strains, serotypes 1 and 6, secreting inactive leukotoxin fused to truncated M. bovis Elongation Factor Thermal unstable (EFTu) and Heat shock protein (Hsp) 70 (EFTu-Hsp70-ΔlktCAV4) were evaluated for efficacy in bison. Modified-live M. haemolytica were administered intranasally and both serotypes were sporadically recovered from bison nasal swabs. Bison inoculated with the two M. haemolytica EFTu-Hsp70- ΔlktCAV4 strains produced systemic and mucosal antibodies to the M. bovis antigens and M. haemolytica surface antigens prior to intranasal challenge with M. bovis. Following intranasal M. bovis challenge no differences were observed in lung bacterial counts nor in lung lesion formation between bison that received the M. haemolytica EFTu-Hsp70-ΔlktCAV4 strains or the ΔlktCAV4, lacking the M. bovis antigens. Despite this, fewer M. bovis positive tissue swabs were collected from animals administered the M. haemolytica EFTu-Hsp70-ΔlktCAV4. These results demonstrate that modified-live M. haemolytica vaccine strains expressing M. bovis antigens failed to provide full protection against M. bovis infection in bison.

Mannheimia haemolytica (Mh) is a normal inhabitant of the upper respiratory tract of ruminants and is associated with bovine respiratory disease. Polysaccharide capsule and surface adhesins are suggested to function in adherence and colonization of M. haemolytica to the mucosa of the upper respiratory tract. M. haemolytica serotype 1 mutant strains containing deletions of either the capsule biosynthetic gene cluster (∆cap) or putative adhesin genes (∆adh123) were created using a temperature-sensitive plasmid and tested for colonization in a calf challenge model. Two treatment groups were used in the study: Sham-Mh-BHV-1 (SMB; intranasal administration of uninfected cell culture lysate/supernatant [sham; S] 4 days before intranasal M. haemolytica inoculation, and intranasal inoculation of bovine-herpesvirus-1 [BHV-1] 20 days post-Mh) and BHV-1-Mh-Sham (BMS; intranasal inoculation of BHV-1 4 days before intranasal Mh inoculation and intranasal sham administration 20 days post-Mh). A mixture of wild-type M. haemolytica parent strain, ∆cap, and ∆adh123 mutants was included in the Mh inoculum. Animals were observed for clinical signs and nasal colonization for approximately 7 weeks. The ∆adh123 mutant and parent strain colonized the nasopharynx, whereas the ∆cap mutant was not detected after 1 day post-inoculation. The ∆adh123 mutant colonized the nasopharynx at significantly higher levels (P < 0.0001) compared to wild type. Higher colonization of ∆adh123 was also found in palatine tonsils. These findings suggest a requirement of capsule in long-term colonization and an advantage for ∆adh123 in colonization over the parent strain.IMPORTANCEUnderstanding the colonization dynamics of Mannheimia haemolytica is crucial for developing effective prevention and treatment strategies for bovine respiratory disease (BRD), a significant cause of economic loss in the cattle industry. This study highlights the role of capsular polysaccharide and surface adhesins in nasopharyngeal colonization. These findings demonstrate that the deletion of putative surface adhesins leads to enhanced colonization compared to the wild-type strain, while mutants containing a deletion of the capsule biosynthetic gene cluster failed to establish long-term colonization. These results suggest that targeting bacterial adhesion mechanisms could influence bacterial persistence and immune response, offering potential avenues for controlling BRD.

ABSTRACT Bovine respiratory disease complex (BRDC) is a multifactorial syndrome that involves complex interactions between environment, bacterial/viral pathogens, and the host. Mannheimia haemolytica is the most significant bacterial pathogen associated with BRDC. This study investigated the virulence of a M. haemolytica serotype 1 capsular-deficient (Δcap) mutant and a M. haemolytica lipopolysaccharide (LPS)-sialylation-deficient (ΔneuA, cytidine monophosphate-sialic acid synthetase) mutant in a calf lung challenge model. Twelve colostrum-deprived calves were divided into three groups (four calves per group) and intratracheally administered inoculum of M. haemolytica wild-type (WT), M. haemolytica Δcap, or M. haemolytica ΔneuA strains (~5 × 108 CFU per animal). Animals were observed for signs of pneumonia and were humanely euthanized 2 to 3 days post-bacterial challenge. Lungs were examined for gross pulmonary lesions, histopathological changes, and bacterial culture. Calves administered WT M. haemolytica exhibited severe lung lesions characterized by extensive consolidation and hemorrhage. In contrast, calves administered M. haemolytica Δcap or M. haemolytica ΔneuA mutants displayed significantly reduced lung lesions (P < 0.05). The most severely affected lung lobes were the right cranial and right middle lobes, with ~50% consolidation. The WT group exhibited significantly higher lung tissue bacterial loads than either of the groups receiving the mutant strains (P < 0.05). The reduced clinical signs, pneumonic lung lesions, and bacterial recovery in the lungs of the calves challenged with either the Δcap or the ΔneuA M. haemolytica mutant strains indicated that these mutations were significantly less virulent than the parent strain.IMPORTANCEWhile Mannheimia haemolytica leukotoxin is well recognized as a major virulence factor, the roles of other possible virulence factors, such as capsule and sialic acid (sialylation of LPS) in M. haemolytica, have not been investigated in animal models. This study revealed that the abolishment of capsule (Δcap) or LPS sialylation (ΔneuA) significantly reduced the virulence of each mutant in calf lung challenges. These results demonstrate that both M. haemolytica capsule and sialylated LPS are important virulence factors that play a key role in the evasion of host defenses.

Pasteurella multocida is a Gram-negative coccobacillus and is the causative agent of fowl cholera in avian species. P. multocida expresses two large filamentous hemagglutinin (FhaB) proteins encoded by fhaB1 and fhaB2 genes. Previously, it was demonstrated that P. multocida FhaB2 is an important virulence factor in the development of fowl cholera disease. In the current study, we examined the potential role of FhaB1 in fowl cholera disease development. An fhaB1 deletion mutant, devoid of foreign DNA, was constructed using a temperature sensitive plasmid in a well-characterized P. multocida avian strain P-1059 (A:3). Real-time PCR assay confirmed the expression of full-length fhaB1 mRNA in the wild-type parent strain and truncated fhaB1 mRNA in the ΔfhaB1 mutant strain. Both parent and the mutant strain produced biofilm; however, the ΔfhaB1 mutant produced significantly lower amounts of biofilm. Turkey poults were challenged intranasally and intramuscularly to assess the virulence of the fhaB1 mutant and the wild-type parent strains. Contrary to our expectation, inactivation of fhaB1 did not reduce virulence by either challenge route. These findings indicate that this large and highly conserved FhaB1 protein is not necessary for the development of acute fowl cholera disease in turkeys.

Mycoplasma bovis (M. bovis) is the etiologic agent of high mortality epizootics of chronic respiratory disease in American bison (Bison bison). Despite the severity of the disease, no efficacious commercial vaccines have been licensed for the prevention of M. bovis infection in bison. Elongation factor thermal unstable (EFTu) and Heat Shock Protein 70 (Hsp70, DnaK) are highly conserved, constitutively expressed proteins that have previously been shown to provide protection against M. bovis infection in cattle. To assess the suitability of EFTu and Hsp70 as vaccine antigens in bison, the immune response to and protection conferred by an injectable, adjuvanted subunit vaccine comprised of recombinantly expressed EFTu and Hsp70 was evaluated. Vaccinates developed robust antibody and cellular immune responses against both EFTu and Hsp70 antigens. To assess vaccine efficacy, unvaccinated control and vaccinated bison were experimentally challenged with bovine herpes virus-1 (BHV-1) 4 days prior to intranasal infection with M. bovis. Vaccinated bison displayed reductions in joint infection, lung bacterial loads, and lung lesions compared to unvaccinated controls. Together, these results showed that this subunit vaccine reduced clinical disease and bacterial dissemination from the lungs in M. bovis challenged bison and support the further development of protein subunit vaccines against M. bovis for use in bison.

Histophilus somni is an important causative agent of bovine respiratory disease complex. Here, we report the complete genome sequence of a Histophilus somni strain 91, which was isolated from a pneumonic lung tissue sample collected from a beef calf.

Histophilus somni is an important pathogen of the bovine respiratory disease complex, yet the mechanisms underlying its virulence remain poorly understood. It is known that H. somni can incorporate sialic acid into lipooligosaccharide (LOS), and sialylated H. somni is more resistant to phagocytosis and complement-mediated killing by serum compared to non-sialylated bacteria in vitro. However, the virulence of non-sialylated H. somni has not been evaluated in vivo using an animal model. In this study, we investigated the contribution of sialic acid to virulence by constructing an H. somni sialic acid uptake mutant (ΔnanP-ΔnanU) and comparing the parent and mutant strains in a mouse septicemia and mortality model. Intraperitoneal challenge of mice with wildtype H. somni (1 × 108 colony forming units/mouse, CFU) was lethal to all animals. Mice challenged with three different doses (1, 2, or 5 × 108 CFU/mouse) of an H. somni ΔnanP-ΔnanU sialic acid uptake mutant exhibited survival rates of 90 %, 60 %, and 0 % respectively. High-performance anion exchange chromatography analyses revealed that LOS prepared from both parent and the ΔnanP-ΔnanU mutant strains of H. somni were sialylated. These findings suggest the presence of de novo sialic acid synthesis pathway, although the genes associated with de novo sialic acid synthesis (neuB and neuC) were not identified by genomic analysis. The lower attenuation in mice is most likely attributed to the sialylated LOS of H. somni nanPU mutant.

Mannheimia haemolytica is the principal agent contributing to bovine respiratory disease and can form biofilms with increased resistance to antibiotic treatment and host immune defenses. To investigate the molecular mechanisms underlying M. haemolytica biofilm formation, transcriptomic analyses were performed with mRNAs sequenced from planktonic and biofilm cultures of pathogenic serotypes 1 (St 1; strain D153) and St 6 (strain D174), and St 2 (strain D35). The three M. haemolytica serotypes were cultured in two different media, Roswell Park Memorial Institute (RPMI) 1640 and brain heart infusion (BHI) to form the biofilms. Transcriptomic analyses revealed that the functions of the differentially expressed genes (DEGs) in biofilm associated cells were not significantly affected by the two media. A total of 476 to 662 DEGs were identified between biofilm associated cells and planktonic cells cultured under BHI medium. Functional analysis of the DEGs indicated that those genes were significantly enriched in translation and many biosynthetic processes. There were 234 DEGs identified in St 1 and 6, but not in St 2. The functions of the DEGs included structural constituents of ribosomes, transmembrane proton transportation, proton channels, and proton-transporting ATP synthase. Potentially, some of the DEGs identified in this study provide insight into the design of new M. haemolytica vaccine candidates.

Mannheimia haemolytica is a major bacterial pathogen associated with broncho- and fibrinous pneumonia in ruminants. Here, we report the complete genome sequence of an isolate of serotype A2 M. haemolytica (D95) recovered from a pneumonic ovine lung. The D95 genome has a size of 2.7 Mb and contains 2,720 genes.

ABSTRACT Mannheimia haemolytica is the most significant bacterial pathogen associated with the bovine respiratory disease complex. Although sialic acid is a known virulence factor in other members of Pasteurellaceae, such as Histophilus somni and Pasteurella multocida, the significance of sialic acid to the virulence of M. haemolytica is currently unknown. Therefore, the role of sialic acid as a virulence determinant of M. haemolytica was investigated by constructing an in-frame neuA [CMP-N-acetylneuraminic acid (Neu5Ac/sialic) synthetase] mutant, which was shown by high-performance anion exchange chromatographic analysis (HPAEC) to be devoid of sialic acid on the lipopolysaccharide (LPS). Both the neuA mutant and wild-type parent strains exhibited similar growth rates in the growth curve assay. Real-time qPCR and ELISA evaluation showed no differences in proinflammatory cytokine expressions (IL-1β, IL-6, and IL-8) between the neuA mutant and parent strain when peripheral blood mononuclear cells were incubated with LPS. Interestingly, the neuA mutant was three to four logs more sensitive to a whole-blood bacterial killing assay than the parent strain. Similar results were also observed in plasma and serum bacterial killing assays. Flow cytometry analyses showed higher uptake of neuA mutant by phagocytes, compared to the parent strain, in the whole-blood phagocytosis assay; however, no difference in reactive oxygen species production in neutrophils or monocytes was detected for either strain. Taken together, these results indicate that sialylation of M. haemolytica LPS plays a vital role in reducing complement-mediated and phagocytic killing. IMPORTANCE The Gram-negative coccobacillus Mannheimia haemolytica is a natural inhabitant of the upper respiratory tract in ruminants and the most common bacterial agent involved in bovine respiratory disease complex development. Key virulence factors harbored by M. haemolytica are leukotoxin, lipopolysaccharide, capsule, adhesins, and neuraminidase which are involved in evading innate and adaptive immune responses. In this study, we have shown that CMP-sialic acid synthetase (neuA) is necessary for the incorporation of sialic acid onto the membrane, and inactivation of neuA results in increased phagocytosis and complement-mediated killing of M. haemolytica, thus demonstrating that sialylation contributes to the virulence of M. haemolytica.

Abstract Objective Mannheimia haemolytica is the primary bacterial pathogen associated with bovine respiratory disease complex (BRDC). While M. haemolytica has been subdivided into 12 capsular serotypes (ST), ST1, ST2 and ST6 are commonly isolated from cattle. More recently, M. haemolytica strains isolated from North American cattle have been classified into genotypes 1 (ST2) and 2 (ST1 and ST6). Of the two genotypes, genotype 1 strains are frequently isolated from healthy animals whereas, genotype 2 strains are predominantly isolated from BRDC animals. However, isolation of both genotypes from pneumonic lung samples can complicate diagnosis. Therefore, the aim of this study was to develop a colorimetric loop-mediated isothermal amplification (LAMP) assay to differentiate M. haemolytica genotypes. Results The genotype specificity of the LAMP was tested using purified genomic DNA from 22 M. haemolytica strains (10 genotype 1, 12 genotype 2) and strains from four related Pasteurellaceae species; Bibersteinia trehalosi, Mannheimia glucosida, Pasteurella multocida, and Histophilus somni. Genotype 1 (adhesin pseudogene B1) specific-LAMP reactions amplified DNA only from genotype 1 strains while genotype 2 (adhesin G) reactions amplified DNA only from genotype 2 strains. The overall detection sensitivity and specificity of the newly developed colorimetric LAMP assay for each genotype were 100%. The limits of detection of two LAMP assays were 1–100 target gene copies per reaction. LAMP primers designed in this study may help the differential identification of M. haemolytica genotypes 1 and 2.

L-enantiomers of antimicrobial peptides (AMPs) are sensitive to proteolytic degradation; however, D-enantiomers of AMPs are expected to provide improved proteolytic resistance. The present study aimed to comparatively investigate the in vitro antibacterial activity, trypsin and serum stability, toxicity, and in vivo antibacterial activity of L-enantiomeric bovine NK2A (L-NK2A) and its D-enantiomeric NK2A (D-NK2A). Circular dichroism spectroscopy of D-NK2A and L-NK2A in anionic liposomes showed α-helical structures and the α-helical conformation of D-NK2A was a mirror image of L-NK2A. Both D-NK2A and L-NK2A displayed minimal in vitro and in vivo toxicities. RP-HPLC and mass spectrometry analyses revealed that D-NK2A, but not L-NK2A, was resistant to trypsin digestion. D-NK2A and L-NK2A showed similar in vitro bacterial killing activities against Histophilus somni. Slightly reduced antibacterial activity was observed when D-NK2A and L-NK2A were pre-incubated with serum. Confocal and transmission electron microscopic findings confirmed that both peptides induced disruption of bacterial inner- and outer-membranes. Improved survivals with D-NK2A treatment were observed when compared to L-NK2A in a murine model of acute H. somni septicemia. We conclude that antibacterial activity and mode of action of NK2A are not chiral specific. With further optimization, D-NK2A may be a viable AMP candidate to combat bacterial 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.

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.

Novel live vaccine strains of Mannheimia haemolytica serotypes (St)1 and St6, expressing and secreting inactive yet immunogenic leukotoxin (leukotoxoid) fused to antigenic domains of Mycoplasma bovis Elongation Factor Tu (EFTu) and Heat shock protein (Hsp) 70 were constructed and tested for efficacy in cattle. Control calves were administered an intranasal mixture of M. haemolytica St1 and St6 mutants (ΔlktCAV4) expressing and secreting leukotoxoid while vaccinated calves were administered an intranasal mixture of like M. haemolytica St1 and St6 leukotoxoid mutants coupled to M. bovis antigens (EFTu-Hsp70-ΔlktCAV4). Both M. haemolytica strains were recovered from palatine tonsils up to 34 days post intranasal exposure. On day 35 all calves were exposed to bovine herpes virus-1, four days later lung challenged with virulent M. bovis, then euthanized up to 20 days post-challenge. Results showed all cattle produced systemic antibody responses against M. haemolytica. The vaccinates also produced systemic antibody responses to M. bovis antigen, and concurrent reductions in temperatures, middle ear infections, joint infection and lung lesions versus the control group. Notably, dramatically decreased lung loads of M. bovis were detected in the vaccinated cattle. These observations indicate that the attenuated M. haemolytica vaccine strains expressing Mycoplasma antigens can control M. bovis infection and disease symptoms in a controlled setting.

Mannheimia haemolytica (MH) is a common commensal bacteria found in the nasopharynx of healthy cattle, and its colonization is generally well controlled by innate and adaptive immune defenses. Following immunological stressors, MH can migrate into the lungs and develop into lower respiratory tract infections or pneumonia. These infections are often exacerbated by infiltrating cytotoxic leukocytes and excessive inflammation. IL-17A is thought to play an integral part in MH pathogenesis by driving inflammatory pathways, invoking neutrophilia, and modulating innate immune defenses. Therefore, to better understand the immune-induced tissue damage that follows MH infection, this study employed the use of an IL-17A inhibitor, ursolic acid (UA), and determined its impact on immune responses and disease severity. Two independent experiments were performed using 4 week old Holstein calves; each of these studies challenged a non-treated group (UA−/MH+; n = 8) and a prophylactic UA treated group (UA+/MH+; n=8). Serum samples, whole blood, and nasal swabs were collected throughout the course of both studies, with bronchoalveolar lavage fluid and lung tissue sections collected at necropsy. UA treated calves had reduced lung pathology and bacterial loads in the lungs compared to control calves. Subsequent analysis of lung tissue confirmed UA treatment modulated host immune responses, including decreased expression of the inflammatory mediators IL-6, IL-17, and STAT3; furthermore, expression of innate defense molecules, such as antimicrobial peptides, mucins, and metalloproteases, was altered. Taken together, these results outline a mechanistic role for IL-17A-mediated immunity to contribute to the pathogenesis of MH infection.

Septicemic pasteurellosis is an acute and fatal bacterial disease of cattle and wild ungulates caused by certain serotypes of Pasteurella multocida. Here we report a single case of septicemic pasteurellosis in a 6-month-old, Red Angus heifer from a cow-calf operation in Alberta, Canada. Postmortem examination revealed necrotizing and hemorrhagic myositis, fibrinous pericarditis and multisystemic bacterial emboli. Pasteurella multocida was isolated from muscle in pure culture, and the capsular antigen group was identified as serogroup B using polymerase chain reaction. To the best of our knowledge, this is the first reported case of septicemic pasteurellosis in beef cattle in Canada. Key clinical message: Veterinary practitioners and diagnosticians should include septicemic pasteurellosis on their list of differential diagnoses when they encounter similar presentations of peracute death and severe necrotizing myositis in cattle in Canada.