Influenza A virus (IAV) in swine is a significant economic concern, and there is a critical need to improve vaccine efficacy. Commercial and experimental vaccine platforms are effective against homologous infection but may not reliably provide protection against drifted or heterologous viruses. Live attenuated influenza A virus (LAIV) vaccines induce mucosal antibody and localized cellular immune responses that may provide partial protection from drifted IAV. However, limited data exist on the induction of mucosal antibody and cellular immune responses and heterologous protection induced by RNA-based vaccines in swine. In this work, experimental, non-adjuvanted hemagglutinin-based replicon particle (RP-HA), and live attenuated influenza A virus (LAIV) vaccines were assessed for induction of mucosal antibody, cellular immune responses, and heterologous protection. LAIV reduced viral shedding and viral lung load while RP-HA limited macroscopic lung lesions. Both vaccines induced similar homologous systemic antibody and mucosal IgG, while only LAIV induced high levels of mucosal IgA. Both vaccines stimulated ex vivo virus-specific T cell proinflammatory cytokine production and proliferation. LAIV induced greater CD8+ T cell responses in the blood and the lungs, and CD4+ T cells in the blood, though RP-HA induced higher lung CD4+ T cell cytokine responses. Together, these results demonstrate that LAIV and RP-HA IAV vaccines induce differential antibody and T cell responses that are likely impacted by vaccine platform and route of exposure. A better understanding of correlates of protection, such as cellular immunity and mucosal antibody induction, will aid in the development of improved swine IAV vaccination strategies.

BackgroundMycoplasmopsis (Mycoplasma) bovis is a significant pathogen in North American bison (Bison bison), causing severe disease with high morbidity and mortality. Effective serological diagnostics are essential for the surveillance and management of disease, yet commercially available ELISAs for M. bovis have only been developed for use in cattle and have not been validated in bison. In this study we evaluated a P48-based indirect ELISA for M. bovis detection and compared its diagnostic performance to a commercially available ELISA developed for use in cattle. Serum samples from bison were tested using both assays, and diagnostic metrics were assessed in comparison to infection status as confirmed by PCR and/or culture.ResultsThe commercial ELISA demonstrated superior sensitivity (77.8%) and specificity (98.0%) compared to the P48 ELISA (63.9% sensitivity, 94.9% specificity). Combining both ELISAs slightly improved sensitivity (83.3%) but did not significantly enhance overall diagnostic performance. Both the P48 and commercial ELISAs also exhibited cross-reactivity with other Mycoplasma species.ConclusionsWe identify significant challenges in serological diagnostics of M. bovis in bison with implications for interpretation of previous serological studies. Future research should focus on multiple immunogenic targets to enhance sensitivity and specificity for M. bovis serological surveillance.

Mycoplasma bovis is a growing threat to American bison (Bison bison) health and restoration efforts, causing significant mortality and disease in affected bison herds. Despite this, little is known about the epidemiology or clinical course of M. bovis infection in bison. In this study, we present continued observations from a cohort of naturally infected American bison, in which maintenance of subclinical M. bovis infections was previously reported. Most (8/11) surviving previously infected animals mounted a detectable immunoglobulin G (IgG) response that waned within 6-24 mo. Two bison mounted and maintained robust IgG antibody responses throughout the study period; one of these also remained quantitative PCR and culture positive throughout the study. One animal failed to mount a detectable IgG response despite becoming infected with M. bovis during the study. Also, naïve animals (n=4) were added to the environment where positive animals were previously kept, shared a water tank with known positive animals, and were finally added to the cohort and sampled at 3-mo intervals for a 2-yr follow-up period. The four naïve animals, and a calf born to one of them, remained M. bovis negative despite commingling with known positive animals in the cohort. We discuss limitations of current antemortem test approaches and the need for more accurate testing to support healthy bison restoration and management.

Bovine respiratory syncytial virus (BRSV) is a major viral pathogen frequently associated with bovine respiratory disease complex. Recent studies identified P2Y6, a purinergic receptor to be involved in the recruitment of leukocytes as part of the host response to viral infections. P2Y6 is a G-protein-coupled purinergic receptor expressed by leukocytes and epithelial cells that recognizes uridine diphosphate, a danger-associated molecular pattern. P2Y6 signaling upregulates CCL-2, CXCL8, CXCL9, and CXCL10 expression. CXC chemokines were previously shown to be upregulated during BRSV infection. Adenosine receptors, G-protein-coupled purinergic receptors expressed on immune cell subsets, have immunoregulatory functions. To examine the expression of purinergic receptors and chemokines during BRSV infection, challenged Holstein calves were euthanized on 7 and 14 days post-infection (DPI) at peak and convalescing stages of infection, respectively. Real-time PCR and RNA in-situ hybridization were utilized to evaluate the expression of purinergic receptors and chemokines in lung samples. On 7 DPI, P2Y6, CXCL9, and CXCL10 were significantly upregulated. In contrast, adenosine A3 receptor gene expression was lower than controls. On 14 DPI, P2Y6 expression trended higher compared to controls, while chemokine expression was decreased. Future studies are needed to examine the potential role of P2Y6 in regulating chemokine induction during BRSV infection.

North American bison (Bison bison) are keystone herbivores that shaped the ecology and evolution of North American prairies and peoples alike. Bison populations were pushed to near-extinction at the turn of the 20th century. Today, bison remain highly susceptible to newly introduced pathogens to which they have not evolved immunity, and Mycoplasma bovis is a significant threat to bison health. Although M. bovis is frequently associated with multifactorial bovine respiratory disease complex in its reservoir host, domestic cattle, M. bovis is a devastating primary pathogen in bison. As a fastidious, insidious, and rapidly mutating organism that lacks a cell wall, M. bovis is difficult to diagnose in an infected animal, and the lack of bison-specific knowledge and diagnostic tools further limits options for herd managers. Here we present a review of the current state of the field of M. bovis in bison, identify gaps in our understanding of bison physiology and M. bovis ecology, and we highlight the unique evolutionary differences of bison from domestic livestock. Dedicated bison research is urgently needed to improve prevention, surveillance, response, and management of M. bovis in this iconic North American wildlife species.

The bacterial pathogen Mycoplasma bovis (M. bovis) emerged as the etiologic agent of mastitis in cattle during the early 1960’s. Concurrent with the emergence of M. bovis, Holsteins in the US were undergoing intensive genetic selection for increased milk production. Comparison studies with unselected Holsteins have found an association with selection and increased susceptibility to disease. More recently, M. bovis was identified as the cause of epizootics of severe, chronic respiratory disease in American bison (Bison bison). In cattle, M. bovis causes chronic respiratory disease and is associated with the bovine respiratory disease complex while in bison, M. bovis is considered a primary pathogen. The objective of this study was to characterize and compare the transcriptomic response of peripheral blood mononuclear cells (PBMCs) from contemporary Holsteins (CH), unselected Holsteins (UH), and bison at 24 hr following in vitro M. bovis infection. The greatest and fewest number of differentially expressed genes (DEG) were observed in UH and bison, respectively. Ingenuity pathway analysis of DEG identified the genotype-specific expression of pathways involved in cytokine and chemokine response, neutrophil degranulation, and antigen processing and presentation. This study is the first to characterize how PBMC transcriptomic responses to in vitro M. bovis infection differ by genotype (CH vs. UH) and species (Holstein vs. bison). These findings provide insight into observed differences in clinical disease between bison and cattle and highlight the effects of primary selection for milk production on Holstein responses to M. bovis.

Bovine viral diarrhea virus (BVDV) is a pathogen of economic concern for the cattle industry due to reproductive losses, persistently infected animals, and for contributing to the bovine respiratory disease complex. Bovine interferon lambda 3 (IFN-λ3) is a type III interferon and a glycosylated cytokine with potent antiviral activity. The recombinant glycosylated bovine IFN-λ3 (rbIFN-λ3) has antiviral activity against BVDV in Madin-Darby bovine kidney (MDBK) cells. Bacterial expression systems provide a higher-yield and more cost-effective alternative to eukaryotic expression systems. The antiviral properties of bacterially expressed non-glycosylated rbIFN-λ3 against BVDV in cell culture was determined in this study. The coding sequence for the mature bovine IFN-λ3 was cloned into a bacterial expression vector and non-glycosylated rbIFN-λ3 was expressed and purified. Cells were pre-treated with serial dilutions of rbIFN-λ3 one day prior to infection with BVDV. The rbIFN-λ3 treatment was repeated daily, and viral infection status was assessed three days post-infection using immunohistochemistry with a monoclonal antibody specific to the BVDV E2 glycoprotein. A single band corresponding to non-glycosylated rbIFN-λ3 with the expected molecular mass was observed on SDS-PAGE, and the identity of rbIFN-λ3 was confirmed via western blotting. Our results indicated that bacterially expressed rbIFN-λ3 without glycosylation demonstrated concentration-dependent antiviral activity, effectively reducing BVDV replication. These results indicate that glycosylation is not required for the antiviral function of bovine IFN-λ3, and supports the potential of using bacterially expressed, non-glycosylated bovine IFN-λ3 in antiviral therapies against BVDV infections.

A widely used transformed bovine peritoneal macrophage cell line (BoMac) is known to be contaminated with bovine viral diarrhea virus of the 2a sub-genotype (BVDV2a). Studies have asserted that the culture and serial passage of BoMac cells in the presence of the antiviral compound 2-(2-benzimidazolyl)-5-[4-(2-imidazolino)phenyl]furan dihydrochloride (DB772) is sufficient to clean the cell line of the persistent contaminating infection. Here, we report that a mutation of P262A/H in the NS5B/RNA-dependent RNA polymerase region of the BVDV2a viral genome, that confers resistance against DB772 in vivo, also occurs when persistently infected BoMac cells were treated with DB772 in vitro and escapes the generation of a BVDV-free clean cell culture stock. 3D-biomolecular complex prediction of wildtype and mutant NS5B in complex with DB772 suggests that the P262A and P262H mutations generate structural changes that interfere with DB772 from accessing its binding site. These mutations lead to increased BVDV proliferation when DB722 treatment is stopped.

The American bison (Bison bison) is an ecologically, economically, and culturally significant species that is exceptionally vulnerable to disease caused by Mycoplasma bovis. In contrast to livestock in which M. bovis is one of many infectious agents comprising the bovine respiratory disease complex, infection in bison is characterized by severe pneumonia and potential for systemic disease in the absence of coinfecting pathogens. In bison, morbidity and mortality are highest in adult cows, whereas calves and yearlings infrequently present with clinical disease. The infection dynamics of M. bovis in young bison exposed during an outbreak have not been fully characterized. Herein, we describe a severe outbreak of M. bovis in a closed, extensively managed herd from which we established a cohort of young bison for longitudinal observation, sampling, and testing. Our findings indicate that M. bovis can colonize the nasopharynx of calves and yearlings during an outbreak, often without causing apparent clinical signs. Although some animals cleared the infection during a 12-mo follow-up study, others remained PCR and culture positive, highlighting the potential for asymptomatic carriage in bison calves as a source of subsequent outbreaks. Using a paired swabbing approach, we show that sampling the superficial nasal cavity is adequate for detection of M. bovis during an outbreak. Over time, however, deep sampling of the nasopharynx is necessary to maximize detection of subclinical infections. Uncertainty in detection using PCR on nasal swab samples can complicate herd assessments and limit the ability to fully assess risk. This study emphasizes the difficulty of identifying chronic carriers following an outbreak and underscores the need for further research to inform M. bovis management and minimize risk in the sensitive and iconic American bison.

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.

Highly pathogenic avian influenza (HPAI) H5N1 virus vaccines typically yield lower neutralizing antibody titers in animals than influenza A virus (IAV) vaccines derived from other viral subtypes. To understand these differences, we compared the cellular immune responses in the draining lymph nodes (dLNs) of mice vaccinated with an inactivated whole H5N1 vaccine to those in mice vaccinated with seasonal H1N1pdm09, H7N9, or H9N2 IAV vaccines. H5N1-vaccinated mice exhibited reduced serum neutralizing antibody titers, despite the hemagglutinin-binding immunoglobulin production being similar to that with other IAV vaccines. Although bulk RNA sequencing showed no differences in B-cell populations after H5N1 and H1N1pdm09 vaccination, H5N1 vaccination resulted in fewer, but larger, dLN germinal centers and significantly more extrafollicular B cells, which are known to produce lower neutralizing antibody titers. Furthermore, H5N1-vaccinated mice had significantly more follicular helper and regulatory T cells. Therefore, differences in neutralizing antibody production in mice after IAV vaccination correlate with subtype-dependent germinal center reactions in the dLNs.

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.

Cattle macrophages possess an enhanced ability to phagocyte some type of bacteria compared to American bison (Bison bison) macrophages. However, it remains unclear whether observed phagocytic differences between the two related ruminant species are restricted to E. coli. Although not frequently reported, bison are susceptible to pneumonia caused by Mannheimia haemolytica. Previously, we showed that M. haemolytica LPS sialylation-deficient mutant (ΔneuA) was more sensitive to phagocytic (neutrophils and monocytes) and complement-mediated killing than were the wildtype parent strain. Therefore, the goal of this study was to compare the phagocytic- and complement-mediated killing of M. haemolytica wildtype and ΔneuA strains between bison and cattle. Relatively higher percentages of reactive oxygen species positive neutrophils and monocytes were found in cattle compared to bison. Significant uptake of M. haemolytica wildtype and ΔneuA mutant strains by both monocytes and neutrophils in cattle were observed, compared to bison as assessed by flow cytometry (p < 0.05). However, both species showed similar phagocytic- and complement-mediated killing in which ΔneuA mutant was more sensitive to killing than the wildtype strain (p < 0.0001). Complement-mediated ΔneuA mutant killing disappeared in both species when the serum was heat inactivated. Taken together, these findings suggested that despite differences in phagocytosis efficiencies and uptake, there is no significant difference between phagocytic- and complement-mediated killing of M. haemolytica ΔneuA mutant between two related ruminant species.

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.

Influenza A viruses (IAV) of subtypes H1N1, H1N2, and H3N2 are endemic in US domestic swine populations and contribute to significant economic losses annually and pose a persistent pandemic threat. Adjuvanted, wholeinactivated virus (WIV) vaccines are the primary countermeasure to control IAV in swine. The compositions of these vaccines are matched for hemagglutinin (HA) strain and content, often ignoring the other IAV glycoprotein, the neuraminidase (NA). The IAV NA is immunogenic and antibodies targeting epitopes adjacent to the active site have been shown to inhibit the sialidase activity of NA thereby reducing virus replication and shedding. To assess the ability of neuraminidase inhibiting (NAI) antibodies induced from WIV administration to protect swine from challenge with IAV containing homologous and heterologous NA, we produced WIV composed of viruses with an irrelevant mismatched H9 HA but expressing NA proteins from two predominant clades (N2-2002A.2 and N2-2002B.2) currently circulating in US domestic swine populations. Pigs that received two doses of H9N2 WIV developed vaccine-specific neuraminidase inhibition antibodies and when challenged with a wild-type H3N2 virus containing homologous NA, displayed reduced virus shedding in the upper respiratory tract and decreased virus titers in the lung compared to unvaccinated controls. Pigs challenged with H3N2 containing a heterologous NA also had reduced virus titers in the nasal swab and BALF samples. Together these results show that NAI antibodies cross-protected across phylogenetic clades and reduced virus replication and shedding in swine.

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.

Mycoplasma bovis (M. bovis) is an important pathogen of American bison (Bison bison), associated with high morbidity and mortality epizootics of respiratory and reproductive disease. Despite the significant negative impact on bison health, little is known about the kinetics of disease and the host immune response to infection. To address these questions, a cohort of bison calves was created and serially sampled 5 times, once every 2-3 mo, over a 12-mo period. At each sampling period nasal swab samples were collected and tested by PCR for the presence of M. bovis. Serum samples were also collected and assessed for M. bovis-specific antibodies using both a commercial and an in-house ELISA. Overall, 19/41 bison (46.3%) had positive PCR tests, and 31/41 (75.6%) were seropositive. Over the course of the study, the frequency of PCR-positive nasal swabs and the ELISA scores decreased, although serum samples remained positive for at least 6 mo following the final positive PCR test. Bison were grouped according to results from the in-house ELISA into high-responder (n=7), low-responder (n=5), and seronegative (n=7) groups. M. bovis-specific IgG antibody levels were significantly elevated in the high-responder group compared to the low-responder and seronegative groups. The differences were statistically significant for 3/5 sampling periods. A trend toward increased IgG2 levels was observed in the high-responder group. High total IgG responses correlated with a decline in positive PCR tests from nasal swabs. These data provide evidence that a strong humoral response is beneficial and is probably involved in the clearance of M. bovis from bison.

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.

Although Human Respiratory Syncytial Virus (HRSV) is a significant cause of severe respiratory disease with high morbidity and mortality in pediatric and elderly populations worldwide there is no licensed vaccine. Bovine Respiratory Syncytial Virus (BRSV) is a closely related orthopneumovirus with similar genome structure and high homology between structural and nonstructural proteins. Like HRSV in children, BRSV is highly prevalent in dairy and beef calves and known to be involved in the etiology of bovine respiratory disease, in addition to being considered an excellent model for HRSV. Commercial vaccines are currently available for BRSV, though improvements in efficacy are needed. The aims of this study were to identify CD4 + T cell epitopes present in the fusion glycoprotein of BRSV, an immunogenic surface glycoprotein that mediates membrane fusion and a major target of neutralizing antibodies. Overlapping peptides representing three regions of the BRSV F protein were used to stimulate autologous CD4 + T cells in ELISpot assays. T cell activation was observed only in cells from cattle with the DRB3*011:01 allele by peptides from AA249-296 of the BRSV F protein. Antigen presentation studies with C-terminal truncated peptides further defined the minimum peptide recognized by the DRB3*011:01 allele. Computationally predicted peptides presented by artificial antigen presenting cells further confirmed the amino acid sequence of a DRB3*011:01 restricted class II epitope on the BRSV F protein. These studies are the first to identify the minimum peptide length of a BoLA-DRB3 class II-restricted epitope in BRSV F protein.