Comparative study of antibacterial activity and stability of D-enantiomeric and L-enantiomeric bovine NK-lysin peptide NK2A.

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.

Bactericidal activity and recovery effect of hydroxyl radicals generated by ultraviolet irradiation and silver ion application on an infected titanium surface.

This study investigated the bactericidal effect, the underlying mechanisms of treatment, and recovery of biocompatibility of the infected titanium surface using a combination treatment of silver ion application and ultraviolet-A (UV-A) light irradiation. Streptococcus mutans and Aggregatibacter actinomycetemcomitans were used in suspension and as a biofilm on a titanium surface to test for the bactericidal effect. The bactericidal effect of the combination treatment was significantly higher than that of silver ion application or UV-A light irradiation alone. The bactericidal effect of the combination treatment was attributable to hydroxyl radicals, which generated from the bacterial cell wall and whose yield increased with the silver concentration. To assess the biocompatibility, proliferation and calcification of MC3T3E1 cells were evaluated on the treated titanium surface. The treated titanium screws were implanted into rat tibias and the removal torques were measured 28 days post-surgery. The titanium surface that underwent the combination treatment exhibited recovery of biocompatibility by allowing cellular proliferation or calcification at levels observed in the non-infected titanium surfaces. The removal torque 28 days after surgery was also comparable to the control values. This approach is a novel treatment option for peri-implantitis.

Characterization of emergent Avibacterium paragallinarum strains and the protection conferred by infectious coryza vaccines against them in China.

Infectious coryza (IC), an acute respiratory disease of chickens, is caused by Avibacterium paragallinarum. Here, the current epidemiological status of IC was investigated in China over 5 yr (2013 to 2018). A total of 28 Av. paragallinarum field isolates were identified by PCR tests and by sequence analysis of the hemagglutinin gene. The pathogenicities of 4 field isolates, the efficacy of 2 commercial inactivated oil-emulsion IC vaccines and vaccines containing different Av. paragallinarum isolates were also evaluated. The PCRs revealed a high rate (51.5%) of sample positivity for Av. paragallinarum during 2013 to 2018. Phylogenetic analysis showed that most field strains fell into the same cluster and had a farther genetic relationship with the early isolates from China. Pathogenicity testing revealed that the Chinese Av. paragallinarum isolates were able to induce the typical clinical signs of IC; hence, they were clearly pathogenic to chickens. Vaccine efficacy tests revealed that the 2 commercial inactivated oil-emulsion IC vaccines we tested had low protection rates against 2 selected Av. paragallinarum isolates after a single immunization, whereas the inactivated vaccine containing the Av. paragallinarum BJ26 isolate generated a relatively high protection rate against the field isolates compared with other three tested vaccines. The results indicate that IC is currently prevalent in China, and that commercial vaccines have not counteracted its presence in this country.

Designing a polytopic complex vaccine candidate against Gallibacterium anatis: an In-silico study.

The haemolytic biovar of Gallibacterium anatis (G. anatis) is responsible for urogenital, gastrointestinal, and respiratory diseases in chickens. There are numerous reports on the resistance of G. anatis to antibiotics and recurrence of the disease, which raise concerns about antimicrobial treatment efficiency. Vaccination has been considered as the most feasible procedure of prevention in high risk farms. Subunit vaccines containing immunogenic components can have practical protective value in preventive measures regarding the infection. The present study aimed to introduce a polytopic vaccine candidate based on epitope detection. All registered sequences of four immunogenic proteins, includig Flfa, GTxA, Gab_1309, and Gab_2348 were retrieved and directed for variational analysis. A vaccine isolate was selected for each protein and tested for B-cell epitope mapping using different tools. Furthermore, consensus selected immunogenic regions with special patterns fused together by flexible linkers were integrated into two constructs and checked for the best status of proteasomal cleavage sites, as well as hydropathy plot. Moreover, back translations, along with codon optimization were performed, and then some tags were added to the constructs. The selected consensus B-cell immunogenic epitopes were for 12656: AA114-181, 7990: AA114-181, Avicor: AA42-77, 134-197, and IPDH: 61-155 for Flfa protein, AA185-235, AA372-457, and AA807-941 for GtxA-N, AA260-305, AA340-400, and AA110-146 for Gab-1309, and AA125-AA175 for Gab-2348. Two suitable patterns of attachment were selected from the different fusion patterns of epitopes in B-cell polytopic vaccinal constructs. Finally, the examination of these constructs showed their effect and efficacy for immune system stimulation. Based on bioinformatics results, these immunogens could be utilized as potential candidates to develop polytopic protective vaccines and design diagnostic kits.

Vaccination with outer membrane vesicles and the fimbrial protein FlfA offers improved protection against lesions following challenge with Gallibacterium anatis.

Gallibacterium anatis is an opportunistic poultry pathogen belonging to the Pasteurellaceae family. It has been shown to cause oophoritis, salpingitis and peritonitis in hens, as well as being associated with reduced semen quality in cockerels. Widespread multidrug resistance and substantial antigenic variation among strains of Gallibacterium anatis is a major constraint to treatment with antimicrobials and prevention of infection by vaccination. Novel vaccine strategies targeting G. anatis are therefore necessary. Outer membrane vesicles (OMVs) are nanosized vesicles formed from the outer membrane of Gram-negative bacteria. These vesicles have shown promising potential as both adjuvants and as vaccine candidates against numerous bacterial species. A high vesiculating mutant of G. anatis (G. anatis ΔtolR) has previously been made, enabling production of OMVs in large scale. In this study, we elucidated the potential of G. anatis ΔtolR OMVs as adjuvant for the conserved antigens GtxA-N (the N-terminal part of the RTX like toxin Gallibacterium toxin A) and FlfA (F17-like fimbria), as well as evaluated if combinations of OMVs together with antigens could facilitate cross-protective immunity against three different strains of G. anatis. We showed that ΔtolR OMVs function as an adjuvant for GtxA-N by inducing antigen specific antibody production. However, OMVs in combination with GtxA-N failed to induce protection against lesions after challenge infection. In contrast, vaccination with OMVs in combination with FlfA protected against lesions, especially in the salpinx, caused by two diverse strains of G. anatis, thereby indicating a cross-protective potential. No protection against the third G. anatis strain 7990 could be obtained in any of the experimental settings. In conclusion, ΔtolR OMVs and FlfA could serve as potential future vaccine components againt G. anatis.

In silico identification and high throughput screening of antigenic proteins as candidates for a Mannheimia haemolytica vaccine.

This study examined the use of comparative genomic analysis for vaccine design against Mannheimia haemolytica, a respiratory pathogen of ruminants. A total of 2,341genes were identified in at least half of the 23 genomes. Of these, a total of 240 were identified to code for N-terminal signal peptides with diverse sub-cellular localizations (78 periplasmic, 52 outer membrane, 15 extracellular, 13 cytoplasmic membrane and 82 unknown) and were examined in an ELISA assay using a coupled-cell free transcription/translation system for protein expressionwith antisera from cattle challenged with serovars 1, 2 or 6 of M. haemolytica. In total, 186 proteins were immunoreactive to at least one sera type and of these, 105 were immunoreactive to all sera screened. The top ten antigens based on immunoreactivity were serine protease Ssa-1 (AC570_10970), an ABC dipeptid transporter substrate-binding protein (AC570_04010), a ribonucleotide reductase (AC570_10780), competence protein ComE (AC570_11510), a filamentous hemagglutinin (AC570_01600), a molybdenum ABC transporter solute-binding protein (AC570_10275), a conserved hypothetical protein (AC570_07570), a porin protein (AC569_05045), an outer membrane assembly protein YeaT (AC570_03060), and an ABC transporter maltose binding protein MalE (AC570_00140). The framework generated from this research can be further applied towards rapid vaccine design against other pathogens involved in complex respiratory infections in cattle.

Expression of Histophilus somni IbpA DR2 protective antigen in the diatom Thalassiosira pseudonana.

Increasing demand for the low-cost production of valuable proteins has stimulated development of novel expression systems. Many challenges faced by existing technology may be overcome by using unicellular microalgae as an expression platform due to their ability to be cultivated rapidly, inexpensively, and in large scale. Diatoms are a particularly productive type of unicellular algae showing promise as production organisms. Here, we report the development of an expression system in the diatom Thalassiosira pseudonana by expressing the protective IbpA DR2 antigen from Histophilus somni for the production of a vaccine against bovine respiratory disease. The utilization of diatoms with their typically silicified cell walls permitted development of silicon-responsive transcription elements to induce protein expression. Specifically, we demonstrate that transcription elements from the silicon transporter gene SIT1 are sufficient to drive high levels of IbpA DR2 expression during silicon limitation and growth arrest. These culture conditions eliminate the flux of cellular resources into cell division processes, yet do not limit protein expression. In addition to improving protein expression levels by molecular manipulations, yield was dramatically increased through cultivation enhancement including elevated light and CO2 supplementation. We substantially increased recombinant protein production over starting levels to 1.2% of the total sodium dodecyl sulfate-extractable protein in T. pseudonana, which was sufficient to conduct preliminary immunization trials in mice. Mice exposed to 5 µg of diatom-expressed DR2 in whole or sonicated cells (without protein purification) exhibited a modest immune response without the addition of adjuvant.

A recombinant subunit vaccine for bovine RSV and Histophilus somni protects calves against dual pathogen challenge.

Bovine respiratory syncytial virus (BRSV) and Histophilus somni synergize to cause respiratory disease in cattle. These pathogens cause enhanced disease during dual-infection and an IgE response to antigens of H. somni in dual-infected but not singly infected calves. Vaccines containing whole inactivated BRSV or H. somni have been associated with IgE responses A vaccine strategy that avoids stimulation of IgE antibodies would provide superior protection from dual infection. We hypothesized that a subunit vaccine consisting of the nucleoprotein (NP) from BRSV and the recombinant antigen IbpA DR2 (a surface antigen of H. somni with two toxic fic motifs) in Quil A adjuvant would elicit protection without disease enhancement. Three groups of calves were vaccinated twice with either: Formalin inactivated BRSV (FI) plus Somnivac®, NP & IbpA DR2 plus Quil A or Quil A alone, followed by BRSV and H. somni challenge. Clinical scores and antibody levels (to whole pathogens and to the subunits) were evaluated. Lungs were examined at necropsy on day 23 after infection. Clinical scores were significantly greatest for the FI & Somnivac® group and both clinical scores and lung pathology were lowest for the subunit group. All calves shed BRSV in nasal secretions. FI & Somnivac® induced IgE antibodies to H. somni and BRSV, but not to NP or DR2. The subunit vaccine did not induce an IgE antibody response to IbpA DR2 antigen and induced little IgE to H. somni. It did not induce an IgG antibody response to BRSV and H. somni, but stimulated production of IgG antibodies against the subunits. In summary, the subunit vaccine, consisting of the BRSV NP and H. somni IbpA DR2 in Quil A, protected against severe clinical signs and decreased lung pathology but did not prevent viral shedding. Importantly it prevented synergistic disease expression in response to dual infection.

Coinfection of Avibacterium paragallinarum and Gallibacterium anatis in Specific-Pathogen-Free Chickens Complicates Clinical Signs of Infectious Coryza, Which Can Be Prevented by Vaccination.

Avibacterium paragallinarum and Gallibacterium anatis are recognized bacterial pathogens both infecting the respiratory tract of chickens. The present study investigated outcomes of their coinfection by elucidating clinical signs, pathologic lesions, and bacteriologic findings. Additionally, the efficacy of a commercially available vaccine to prevent diseases caused by A. paragallinarum and G. anatis was evaluated. Birds inoculated with G. anatis alone did not present any clinical signs and gross pathologic lesions in the respiratory tract. However, clinical signs of infectious coryza were reproduced in nonvaccinated birds that were challenged with A. paragallinarum alone or together with G. anatis . Such clinical signs were more severe in the coinfected group, including the death of four birds. Some of the birds that were vaccinated and challenged showed mild clinical signs at 7 days postinfection (dpi). Inflammation of sinus infraorbitalis was the most prominent gross pathologic lesion found in the respiratory tract of nonvaccinated birds inoculated either with A. paragallinarum and G. anatis or A. paragallinarum alone. In the reproductive tract, hemorrhagic follicles were observed in nonvaccinated birds that were infected either with G. anatis alone or together with A. paragallinarum . In vaccinated birds, no gross pathologic lesions were found except in one bird that was coinfected with both the pathogens characterized by mucoid tracheitis. Bacteriologic investigations revealed that multiplication of G. anatis at 7 dpi was supported by the coinfection with A. paragallinarum . Altogether, it can be concluded that simultaneous infection of A. paragallinarum and G. anatis can increase the severities of disease conditions in chickens. In such a scenario, vaccination appears to be an effective tool for prevention of the disease, as protection was conferred based on clinical, pathologic, bacteriologic, and serologic data.

Melanocortin agonism as a viable strategy to control alveolar bone loss induced by oral infection.

Alveolar bone loss is a result of an aggressive form of periodontal disease (PD) associated with Aggregatibacter actinomycetemcomitans (Aa) infection. PD is often observed with other systemic inflammatory conditions, including arthritis. Melanocortin peptides activate specific receptors to exert antiarthritic properties, avoiding excessing inflammation and modulating macrophage function. Recent work has indicated that melanocortin can control osteoclast development and function, but whether such protection takes place in infection-induced alveolar bone loss has not been investigated. The purpose of this study was to evaluate the role of melanocortin in Aa-induced PD. Mice were orally infected with Aa and treated with the melanocortin analog DTrp8-γMSH or vehicle daily for 30 d. Then, periodontal tissue was collected and analyzed. Aa-infected mice treated with DTrp8-γMSH presented decreased alveolar bone loss and a lower degree of neutrophil infiltration in the periodontium than vehicle-treated animals; these actions were associated with reduced periodontal levels of TNF-α, IFN-γ, and IL-17A. In vitro experiments with cells differentiated into osteoclasts showed that osteoclast formation and resorptive activity were attenuated after treatment with DTrp8-γMSH. Thus, melanocortin agonism could represent an innovative way to tame overexuberant inflammation and, at the same time, preserve bone physiology, as seen after Aa infection.-Madeira, M. F. M., Queiroz-Junior, C. M., Montero-Melendez, T., Werneck, S. M. C., Corrêa, J. D., Soriani, F. M., Garlet, G. P., Souza, D. G., Teixeira, M. M., Silva, T. A., Perretti, M. Melanocortin agonism as a viable strategy to control alveolar bone loss induced by oral infection.

Recombinant proteins from Gallibacterium anatis induces partial protection against heterologous challenge in egg-laying hens.

Gallibacterium anatis is a Gram-negative bacterium and major cause of salpingitis and peritonitis in egg-laying hens, thereby contributing to decreased egg production and increased mortality among the hens. Due to widespread drug resistance and antigenic diversity, novel prophylactic measures are urgently required. The aim of the present study was to evaluate the cross-protective capacity of three recombinant proteins recently identified as potential vaccine candidates; GtxA-N, GtxA-C, and FlfA, in an in vivo challenge model. Nine groups of birds were immunized twice with each protein, respectively, with 14 days separation. Additionally, three groups served as non-immunized controls. After 3 weeks, the birds were challenged with either of three G. anatis strains: 12656-12, 7990 or IPDH 697-78, respectively. Blood samples were taken at three different time points prior to challenge, as well as 48 h after challenge. All birds were euthanized and subjected to a post mortem procedure including scoring of lesions and sampling for bacterial growth. Moreover, ELISA assays were used to quantify antigen-specific IgG titers in serum. The results showed that all three proteins induced protection against the homologous strain 12656-12. No protein induced complete protection against strain 7990, although FlfA reduced the bacterial re-isolation rate. Moreover, immunization with GtxA-N and FlfA induced protection, while GtxA-C reduced the bacterial re-isolation, against strain IPDH 697-78. Thus although complete cross-protection against all three strains was not achieved, the results hold great promise for a new generation of immunogens in the search for novel prophylactic measures against G. anatis.

Immunogenic and protective efficacy of recombinant protein GtxA-N against Gallibacterium anatis challenge in chickens.

Gallibacterium anatis is a major cause of reproductive tract infections in chickens. Here, we aimed to evaluate the efficacy of the recombinant protein GtxA-N at protecting hens, by addressing three objectives; (i) evaluating the antibody response following immunization (ii) scoring and comparing lesions, following challenge with G. anatis, in immunized and non-immunized hens and (iii) investigating if the anti-GtxA-N antibody titre in individual hens correlated with the observed lesions. Two consecutive experiments were performed in hens. In the first experiment hens were immunized with GtxA-N on day 0 and day 14, infected with G. anatis on day 28 and euthanized on day 56. The GtxA-N antibody response was assessed in pooled serum samples throughout the experiment, using an indirect enzyme-linked immunosorbent assay (ELISA). In the second experiment the GtxA-N antibody titres were assessed in individual hens before and after immunization. Subsequently, the hens were inoculated with G. anatis and finally all hens where euthanized and submitted for post mortem examination 48 h after inoculation. Immunization elicited strong antibody responses that lasted at least 8 weeks (P < .0001). The individual antibody titres observed in response to immunization varied considerably among hens (range: 174,100-281,500). Lesion scores following G. anatis infection were significantly lower in immunized hens compared to non-immunized hens (P = .004). Within the immunized group, no correlation was found between the individual antibody titres and the lesion scores. This study clearly demonstrated GtxA-N as a vaccine antigen able of inducing protective immunity against G. anatis.

Mastitis in sheep--The last 10 years and the future of research.

Bacterial mastitis is a significant welfare and financial problem in sheep flocks. This paper reviews the recently published literature, including publications that highlight the significance and virulence factors of the causal agents, especially Staphylococcus aureus and Mannheimia haemolytica, the primary causes of the disease. Research has also contributed to the understanding of risk factors, including genetic susceptibility of animals to infections, supporting future strategies for sustainable disease control. Pathogenetic mechanisms, including the role of the local defenses in the teat, have also been described and can assist formulation of strategies that induce local immune responses in the teat of ewes. Further to well-established diagnostic techniques, i.e., bacteriological tests and somatic cell counting, advanced methodologies, e.g., proteomics technologies, will likely contribute to more rapid and accurate diagnostics, in turn enhancing mastitis control efforts.

Antagonistic effect of peptidoglycan of Streptococcus sanguinis on lipopolysaccharide of major periodontal pathogens.

Streptococcus sanguinis is often found in subgingival biofilm including periodontopathogens, and is correlated with a delay in colonization by periodontopathogens. However, the effect of S. sanguinis on inflammation induced by periodontopathogens is poorly understood. Thus, this study investigated the effect of S. sanguinis peptidoglycan (PGN) on induction of TNF-α, IL-6, and IL-8 expression by lipopolysaccharide (LPS) of periodontal pathogens. LPS was extracted from Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Tannerella forsythia, and PGN was isolated from S. sanguinis. THP-1 cells, a monocytic cell-line, were cotreated with LPS of the periodontal pathogens and S. sanguinis PGN, and then the expression of inflammatory cytokines was analyzed by real-time RT-PCR. To analyze the underlying mechanism, the binding assay of the LPS to CD14 or LPS-binding protein (LBP) was performed in the presence or absence of the PGN after coating recombinant human CD14 and LBP on EIA plate. The PGN inhibited the binding of LPS to CD14 and LBP in a dose-dependent manner. Also, THP-1 cells were co-treated with the LPS in the presence of N-acetylmuramic acid and N-acetylglucosamine, as components of PGN, and the competition binding assay to CD14 and LBP was performed. N-acetylmuramic acid inhibited the induction of inflammatory cytokine expression by LPS and the binding of LPS to CD14 or LBP whereas N-acetylglucosamine did not show such effect. Collectively, the results suggest that S. sanguinis PGN inhibited the cytokine expression induced by the LPS of periodontopathogens due to the inhibition of LPS binding to LBP and CD14. N-acetylmuramic acid of PGN may play a role in inhibition of the LPS binding of periodontopathogens to CD14 and LBP.

In silico prediction of Gallibacterium anatis pan-immunogens.

The Gram-negative bacterium Gallibacterium anatis is a major cause of salpingitis and peritonitis in commercial egg-layers, leading to reduced egg production and increased mortality. Unfortunately, widespread multidrug resistance and antigenic diversity makes it difficult to control infections and novel prevention strategies are urgently needed. In this study, a pan-genomic reverse vaccinology (RV) approach was used to identify potential vaccine candidates. Firstly, the genomes of 10 selected Gallibacterium strains were analyzed and proteins selected on the following criteria; predicted surface-exposure or secretion, none or one transmembrane helix (TMH), and presence in six or more of the 10 genomes. In total, 42 proteins were selected. The genes encoding 27 of these proteins were successfully cloned in Escherichia coli and the proteins expressed and purified. To reduce the number of vaccine candidates for in vivo testing, each of the purified recombinant proteins was screened by ELISA for their ability to elicit a significant serological response with serum from chickens that had been infected with G. anatis. Additionally, an in silico prediction of the protective potential was carried out based on a protein property prediction method. Of the 27 proteins, two novel putative immunogens were identified; Gab_1309 and Gab_2312. Moreover, three previously characterized virulence factors; GtxA, FlfA and Gab_2156, were identified. Thus, by combining the pan-genomic RV approach with subsequent in vitro and in silico screening, we have narrowed down the pan-proteome of G. anatis to five vaccine candidates. Importantly, preliminary immunization trials indicated an in vivo protective potential of GtxA-N, FlfA and Gab_1309.

An in vitro method for evaluating endotoxic activity using prostaglandin E(2) induction in bovine peripheral blood.

Severe side effects of veterinary vaccines, in particular Histophilus somni-containing vaccines for cows, have frequently been reported in Japan. These side effects are probably caused by endotoxins. Contamination levels of endotoxins could be monitored using the Limulus amebocyte lysate (LAL) test; however, the LAL test is not completely adequate for evaluation of in vivo endotoxic activities. In this study, we established a method for evaluating endotoxic activities using prostaglandin E2 (PGE2) induction in bovine peripheral blood. Blood and standard endotoxin, derived from Escherichia coli, were mixed and incubated. The concentration of induced PGE2 in the culture supernatant reached a maximum after 24-h incubation. A linear dose-response curve was observed for PGE2 concentration and the logarithmic transformed standard endotoxin concentration (5-5000 ng/ml). The endotoxic activity of H. somni in cows was the highest among those of several tested endotoxins. However, the LAL activities of H. somni were not as high as those of the other tested endotoxins. These results may provide a reason for the many report of side effects of H. somni-containing vaccines. The PGE2 detection assay described here could be a valuable method for evaluating the endotoxic activities of vaccines in cows.

The fimbrial protein FlfA from Gallibacterium anatis is a virulence factor and vaccine candidate.

The Gram-negative bacterium Gallibacterium anatis is a major cause of salpingitis and peritonitis in egg-laying chickens, leading to decreased egg production worldwide. Widespread multidrug resistance largely prevents treatment of this organism using traditional antimicrobial agents, while antigenic diversity hampers disease prevention by classical vaccines. Thus, insight into its pathogenesis and knowledge about important virulence factors is urgently required. A key event during the colonization and invasion of mucosal surfaces is adherence, and recently, at least three F17-like fimbrial gene clusters were identified in the genomes of several G. anatis strains. The objective of this study was to characterize the putative F17-like fimbrial subunit protein FlfA from G. anatis 12656-12 and determine its importance for virulence. In vitro expression and surface exposure of FlfA was demonstrated by flow cytometry and immunofluorescence microscopy. The predicted function of FlfA as a fimbrial subunit protein was confirmed by immunogold electron microscopy. An flfA deletion mutant (ΔflfA) was generated in G. anatis 12656-12, and importantly, this mutant was significantly attenuated in the natural chicken host. Furthermore, protection against G. anatis 12656-12 could be induced by immunizing chickens with recombinant FlfA. Finally, in vitro expression of FlfA homologs was observed in a genetically diverse set of G. anatis strains, suggesting the potential of FlfA as a serotype-independent vaccine candidate This is the first study describing a fimbrial subunit protein of G. anatis with a clear potential as a vaccine antigen.

Effects of vaccination on the acute-phase protein response and measures of performance in growing beef calves.

Two experiments were conducted to evaluate the influence of vaccination on the acute-phase protein (APP) reaction (Exp. 1 and 2) and measures of performance (Exp. 2) in growing beef calves. In Exp. 1, the APP reaction was assessed in newly weaned steers administered 1 of 3 treatments (n = 8 steers/treatment), consisting of 1) Mannheimia haemolytica vaccine (One Shot; Pfizer Inc., New York, NY), 2) Clostridium vaccine (UltraBac 7; Pfizer, Inc.), or 3) saline-injected control. Blood samples for the evaluation of APP concentrations were collected on d 0, 1, 3, 5, 7, 10, and 14 and steer BW measured on d 0 and 21 relative to treatment administration. Plasma concentrations of haptoglobin (Hp) increased (P < 0.05) in vaccinated but not control calves and reached a peak on d 3 and 5 for steers receiving Mannheimia haemolytica and Clostridium vaccine, respectively. Plasma concentrations of ceruloplasmin (Cp) and fibrinogen (Fb) increased (P < 0.05) in all calves after treatment administration and Fb concentrations were greatest (P < 0.01) in calves receiving Mannheimia haemolytica vaccine on d 3 and 5 compared with the other treatments. There were no treatment effects (P = 0.44) on 21-d steer ADG (0.43 kg/d; SEM = 0.082). In Exp. 2, 23 heifers were randomly assigned to 2 treatments: 1) vaccinated (Mannheimia haemolytica vaccine (One Shot; n = 12) and 2) saline control (n = 11). After vaccination, blood samples were collected for determination of APP concentrations on d 0, 3, 6, 9, 12, and 15. During this period, individual heifer DMI was measured using an automated feed intake measuring system (Model 4000E; GrowSafe Systems Ltd., Airdrie, Alberta, Canada). Initial and final shrunk BW did not differ (P > 0.36) among treatments. On d 1, plasma Cp concentrations increased (P < 0.01) sharply in vaccinated heifers but not control heifers and were greater (P < 0.05) in vaccinated vs. control heifers on d 3, 6, 9, and 12 relative to injection. Daily DMI did not differ (P = 0.66) among treatments (average = 9.1 kg/d; SEM = 0.34); however, ADG and G:F were greater (P ≤ 0.05) for control vs. vaccinated heifers (1.14 vs. 0.87 kg/d and 0.13 and 0.10 kg, respectively; SEM = 0.064 and 0.011). These data indicate that within a 2 wk period after vaccination, beef calves experience an acute-phase protein response, which may result in reduced ADG and feed efficiency.

Proteomic analysis and immunogenicity of Mannheimia haemolytica vesicles.

Mannheimia haemolytica, a major causative agent in bovine respiratory disease, inflicts extensive losses each year on cattle producers. Commercially available vaccines are only partially efficacious. Immunity to M. haemolytica requires antibodies to secreted toxins and outer membrane proteins (OMPs) of the bacterium. Gram-negative bacteria produce membrane blebs or vesicles, the membrane components of which are primarily derived from OMPs. Accordingly, vesicles have been used as immunogens with various degrees of success. This study characterized components of M. haemolytica vesicles and determined their immunogenicity in mice and cattle. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of vesicles from this bacterium identified 226 proteins, of which 58 (25.6%) were OMPs and periplasmic and one (0.44%) was extracellular. Vesicles were used to vaccinate dairy calves and BALB/c mice. Analyses of sera from calves and mice by enzyme-linked immunosorbent assay (ELISA) showed that circulating antibodies against M. haemolytica whole cells and leukotoxin were significantly higher on days 21 and 28 (P < 0.05) than on day 0. For control calves and mice, there were no significant differences in serum anti-whole-cell and leukotoxin antibody levels from days 0 and 21 or 28, respectively. Lesion scores of lungs from vaccinated calves (15.95%) were significantly (P < 0.05) lower than those from nonvaccinated calves (42.65%). Sera from mice on day 28 and calves on day 21 showed 100% serum bactericidal activity. Sera from vesicle-vaccinated mice neutralized leukotoxin.

Cellular and mucosal immune responses in the respiratory tract of Nigerian goats following intranasal administration of inactivated Recombinant Mannheimia hemolytica bacterine.

This experiment was conducted to evaluate the cellular and mucosal responses in the respiratory tract of Nigerian goats vaccinated intranasally with recombinant Mannheimia hemolytica bacterine. Twenty one goats were divided into five groups, five goats each in three vaccinated groups while three goats each in two other groups serve as positive and negative control. Group A was vaccinated once; group B was vaccinated twice at one week interval, and group D at twice at two weeks interval. Group C1 were the unvaccinated and challenged, while group C2 were unvaccinated and unchallenged. The bronchoalveolar lavage differential counts and bronchial associated lymphoid tissue (BALT) responses were measured using Giemsa stained thin smear of the cell fraction of the lavage and histomorphometry. ANOVA were employed and significance was at p>0.05. The post-challenge macrophage to neutrophil (M:N) ratio values of group B goats was the highest and the ratio differed from other groups which had much lower M:N values. The exposure in group B resulted in significant increase in number and size of BALTs as well as the number of lymphocytes in BALT than those of the other groups. This study showed that intranasal vaccination of the recombinant Mannheimia hemolytica bacterine twice at a week interval was more efficient in inducing strong mucosal and defensive cellular responses in the respiratory tract.