Repeated Oral Vaccination of Cattle with Shiga Toxin-Negative Escherichia coli O157:H7 Reduces Carriage of Wild-Type E. coli O157:H7 after Challenge.

Subcutaneous vaccination of cattle for enterohemorrhagic Escherichia coli O157:H7 reduces the magnitude and duration of fecal shedding, but the often-required, repeated cattle restraint can increase costs, deterring adoption by producers. In contrast, live oral vaccines may be repeatedly administered in feed, without animal restraint. We investigated whether oral immunization with live stx-negative LEE+E. coli O157:H7 reduced rectoanal junction (RAJ) colonization by wild-type (WT) E. coli O157:H7 strains after challenge. Two groups of cattle were orally dosed twice weekly for 6 weeks with 3 × 109 CFU of a pool of three stx-negative LEE+E. coli O157:H7 strains (vaccine group) or three stx-negative LEE- non-O157:H7 E. coli strains (control group). Three weeks following the final oral dose, animals in both groups were orally challenged with a cocktail of four stx+ LEE+E. coli O157:H7 WT strains. Subsequently, WT strains at the RAJ were enumerated weekly for 4 weeks. Serum antibodies against type III secretion protein (TTSP), the translocated intimin receptor (Tir), and EspA were determined by enzyme-linked immunosorbent assay (ELISA) at day 0 (preimmunization), day 61 (postimmunization, prechallenge), and day 89 (postchallenge). Vaccine group cattle had lower numbers of WT strains at the RAJ than control group cattle on postchallenge days 3 and 7 (P ≤ 0.05). Also, vaccine group cattle shed WT strains for a shorter duration than control group cattle. All cattle seroconverted to TTSP, Tir, and EspA, either following immunization (vaccine group) or following challenge (control group). Increased antibody titers against Tir and TTSP postimmunization were associated with decreased numbers of WT E. coli O157:H7 organisms at the RAJ.IMPORTANCE The bacterium E. coli O157:H7 causes foodborne disease in humans that can lead to bloody diarrhea, kidney failure, vascular damage, and death. Healthy cattle are the main source of this human pathogen. Reducing E. coli O157:H7 in cattle will reduce human disease. Using a randomized comparison, a bovine vaccine to reduce carriage of the human pathogen was tested. A detoxified E. coli O157:H7 strain, missing genes that cause disease, was fed to cattle as an oral vaccine to reduce carriage of pathogenic E. coli O157:H7. After vaccination, the cattle were challenged with disease-causing E. coli O157:H7. The vaccinated cattle had decreased E. coli O157:H7 during the first 7 days postchallenge and shed the bacteria for a shorter duration than the nonvaccinated control cattle. The results support optimization of the approach to cattle vaccination that would reduce human disease.

Poor stimulation of bovine dendritic cells by Mycobacterium bovis culture supernatant and surface extract is associated with decreased activation of ERK and NF-κB and higher expression of SOCS1 and 3.

The cell envelope of pathogenic mycobacteria interfaces with the host. As such, the interaction of bacterial products localized at or released from the cell surface with the host's immune system can determine the fate of the bacterium in its host. In this study, the effects of three different types of Mycobacterium bovis cell envelope fractions-purified protein derivative, total cell wall lipids and culture supernatant and surface extract-on bovine dendritic cells were assessed. We found that the culture supernatant and surface extract fraction induced little to no production of the pro-inflammatory cytokines TNF-α and IL-12 in bovine dendritic cells. Moreover, this muted response was associated with poor activation of ERK and NF-κB, both of which are critical for the pro-inflammatory response. Furthermore, culture supernatant and surface extract treatment increased the expression of suppressor of cytokine signaling 1 and 3, both of which are negative regulators of pro-inflammatory signaling, in bovine dendritic cells. These observations taken together suggest the M. bovis culture supernatant and surface extract fraction contain immunomodulatory molecules that may aid in M. bovis pathogenesis.

Draft Genome Sequences of 12 Histophilus somni Strains Isolated from Feedlot Cattle in Alberta, Canada.

Histophilus somni is a Gram-negative opportunistic pathogen associated with respiratory disease in cattle. Here, we report the draft genome sequences of 12 Histophilus somni strains isolated from feedlot cattle in Alberta, Canada, which were diagnosed with respiratory disease.

Channel Formation by LktA of Mannheimia (Pasteurella) haemolytica in Lipid Bilayer Membranes and Comparison of Channel Properties with Other RTX-Cytolysins.

Cytolysin LktA is one of the major pathogenicity factors of Mannheimia haemolytica (formerly Pasteurella haemolytica) that is the cause of pasteurellosis, also known as shipping fever pneumonia, causing substantial loss of sheep and cattle during transport. LktA belongs to the family of RTX-toxins (Repeats in ToXins) that are produced as pathogenicity factors by a variety of Gram-negative bacteria. Sublytic concentrations of LktA cause inflammatory responses of ovine leukocytes. Higher concentrations result in formation of transmembrane channels in target cells that may cause cell lysis and apoptosis. In this study we investigated channel formation by LktA in artificial lipid bilayer membranes made of different lipids. LktA purified from culture supernatants by polyethylene glycol 4000 precipitation and lyophilization had to be activated to frequently form channels by solution in 6 M urea. The LktA channels had a single-channel conductance of about 60 pS in 0.1 M KCl, which is about one tenth of the conductance of most RTX-toxins with the exception of adenylate cyclase toxin of Bordetella pertussis. The LktA channels are highly cation-selective caused by negative net charges. The theoretical treatment of the conductance of LktA as a function of the bulk aqueous concentration allowed a rough estimate of the channel diameter, which is around 1.5 nm. The size of the LktA channel is discussed with respect to channels formed by other RTX-toxins. We present here the first investigation of LktA in a reconstituted system.

Multistage vaccines containing outer membrane, type III secretion system and inclusion membrane proteins protects against a Chlamydia genital tract infection and pathology.

Pathogens with a complex lifecycles can effectively evade host immunity in part due to each developmental stage expressing unique sets of antigens. Multisubunit vaccines incorporating signature antigens reflecting distinct developmental stages (multistage vaccines) have proven effective against viral, bacterial and parasitic infection at preventing pathogen evasion of host immunity. Chlamydia trachomatis is characterized by a biphasic extra/intracellular developmental cycle and an acute/persistent (latent) metabolic state; hence a multistage vaccine may prevent immune evasion and enhance clearance. Here we tested the efficacy of a multistage vaccine containing outer membrane (MOMP and PmpG), type three secretion system (T3SS) (CdsF and TC0873) and inclusion membrane proteins (IncA and TC0500) in mice against an intravaginal challenge with Chlamydia muridarum. Comparison of single (eg. MOMP) and double antigen vaccines (eg. MOMP and PmpG), largely targeting the extracellular stage, elicited significant yet comparable protection against vaginal shedding when compared to unimmunized control mice. Utilization of different adjuvants (ISCOMATRIX - IMX, PCEP/polyI:C/IDR1002 - VIDO, CTA1-DD and ADVAX) and numerous immunization routes (subcutaneous - SQ and intranasal - IN) further enhanced protection against infection. However, a multistage vaccine elicited significantly greater protection against vaginal shedding and upper genital tract pathology than vaccines targeting only extra- or intracellular stages. This indicates that protection elicited by a vaccine targeting extracellular chlamydial antigens could be improved by including chlamydial antigen expressed during intracellular phase.

Immune responses to in ovo vaccine formulations containing inactivated fowl adenovirus 8b with poly[di(sodium carboxylatoethylphenoxy)]phosphazene (PCEP) and avian beta defensin as adjuvants in chickens.

Inclusion body hepatitis (IBH) is one of the major viral infections causing substantial economic loss to the global poultry industry. The disease is characterized by a sudden onset of mortality (2-30%) and high morbidity (60-70%). IBH is caused by a number of serotypes of fowl adenovirus with substantially low levels of serotype cross protection. Thus far, there is no effective and safe vaccine commercially available in the North America for the control of IBH in chickens. Poly[di(sodium carboxylatoethylphenoxy)]phosphazene (PCEP) is a high molecular weight, biodegradable water soluble polymer that has been well characterized as a safe and effective adjuvant for a number of experimental veterinary vaccines. Similarly, host defence peptides, including ß-defensins, have also been shown to exhibit strong adjuvant potential. In this study, we evaluated the adjuvant activity of PCEP and avian beta defensin (ABD) in a vaccine formulation containing inactivated fowl adenovirus (FAdV) serotype 8b administered in ovo. Our data showed that a combination of PCEP and inactivated virus is capable of inducing a robust and long lasting antibody response. Moreover, significant enhancement of IFN-γ, IFN-α, IL-12(p40) and IL-6 gene expression under the influence of PCEP suggests that as an in ovo adjuvant PCEP has the ability to activate a substantial balanced immune response in chickens. To our knowledge, these are the first studies in which PCEP and ABD have been characterized as adjuvants for the development of an in ovo poultry vaccine. It is expected that these preliminary studies will be helpful in the development of safer and more effective in ovo vaccine against IBH and other infectious diseases affecting chickens.

Glyceradehyde-3-phosphate dehydrogenase as a suitable vaccine candidate for protection against bacterial and parasitic diseases.

The enzyme glyceraldehyde-3-P-dehydrogenase (GAPDH) has been identified as having other properties in addition to its key role in glycolysis. The ability of GAPDH to bind to numerous extracellular matrices, modulation of host-immune responses, a role in virulence and surface location has prompted numerous investigators to postulate that GAPDH may be a good vaccine candidate for protection against numerous pathogens. Although immune responses against GAPDH have been described for many microorganisms, vaccines containing GAPDH have been successfully tested in few cases including those against the trematode-Schistosoma mansoni, the helminth-Enchinococcus multilocularis; the nematode filaria- Litomosoides sigmodontis; fish pathogens such as Aeromonas spp., Vibrio spp., Edwarsiella spp., and Streptococcus iniae; and environmental streptococci, namely, Streptococcus uberis and Streptococcus dysgalactiae. Before GAPDH-based vaccines are considered viable options for protection against numerous pathogens, we need to take into account the homology between the host and pathogen GAPDH proteins to prevent potential autoimmune reactions, thus protective GAPDH epitopes unique to the pathogen protein must be identified.

Antibodies Directed against Shiga-Toxin Producing Escherichia coli Serotype O103 Type III Secreted Proteins Block Adherence of Heterologous STEC Serotypes to HEp-2 Cells.

Shiga toxin-producing Escherichia coli (STEC) serotype O103 is a zoonotic pathogen that is capable of causing hemorrhagic colitis and hemolytic uremic syndrome (HUS) in humans. The main animal reservoir for STEC is ruminants and hence reducing the levels of this pathogen in cattle could ultimately lower the risk of STEC infection in humans. During the process of infection, STECO103 uses a Type III Secretion System (T3SS) to secrete effector proteins (T3SPs) that result in the formation of attaching and effacing (A/E) lesions. Vaccination of cattle with STEC serotype O157 T3SPs has previously been shown to be effective in reducing shedding of STECO157 in a serotype-specific manner. In this study, we tested the ability of rabbit polyclonal sera against individual STECO103 T3SPs to block adherence of the organism to HEp-2 cells. Our results demonstrate that pooled sera against EspA, EspB, EspF, NleA and Tir significantly lowered the adherence of STECO103 relative to pre-immune sera. Likewise, pooled anti-STECO103 sera were also able to block adherence by STECO157. Vaccination of mice with STECO103 recombinant proteins induced strong IgG antibody responses against EspA, EspB, NleA and Tir but not against EspF. However, the vaccine did not affect fecal shedding of STECO103 compared to the PBS vaccinated group over the duration of the experiment. Cross reactivity studies using sera against STECO103 recombinant proteins revealed a high degree of cross reactivity with STECO26 and STECO111 proteins implying that sera against STECO103 proteins could potentially provide neutralization of attachment to epithelial cells by heterologous STEC serotypes.

Large animal models for vaccine development and testing.

The development of human vaccines continues to rely on the use of animals for research. Regulatory authorities require novel vaccine candidates to undergo preclinical assessment in animal models before being permitted to enter the clinical phase in human subjects. Substantial progress has been made in recent years in reducing and replacing the number of animals used for preclinical vaccine research through the use of bioinformatics and computational biology to design new vaccine candidates. However, the ultimate goal of a new vaccine is to instruct the immune system to elicit an effective immune response against the pathogen of interest, and no alternatives to live animal use currently exist for evaluation of this response. Studies identifying the mechanisms of immune protection; determining the optimal route and formulation of vaccines; establishing the duration and onset of immunity, as well as the safety and efficacy of new vaccines, must be performed in a living system. Importantly, no single animal model provides all the information required for advancing a new vaccine through the preclinical stage, and research over the last two decades has highlighted that large animals more accurately predict vaccine outcome in humans than do other models. Here we review the advantages and disadvantages of large animal models for human vaccine development and demonstrate that much of the success in bringing a new vaccine to market depends on choosing the most appropriate animal model for preclinical testing.

Role of motAB in adherence and internalization in polarized Caco-2 cells and in cecal colonization of Campylobacter jejuni.

Campylobacter jejuni, a gram-negative motile bacterium commonly found in the chicken gastrointestinal tract, is one of the leading causes of bacterial gastroenteritis in humans worldwide. An intact and functional flagellum is important for C. jejuni virulence and colonization. To understand the role of C. jejuni motility in adherence and internalization in polarized Caco-2 cells and in cecal colonization of chickens we constructed a C. jejuni NCTC11168 V1 deltamotAB mutant. The motAB genes code for the flagellar motor, which enables the rotation of the flagellum. The nonmotile deltamotAB mutant expressed a full-length flagellum, which allowed us to differentiate between the roles of full-length flagella and motility in the ability of C. jejuni to colonize. To study the adherence and invasion abilities of the C. jejuni deltamotAB mutant we chose to use polarized Caco-2 cells, which are thought to be more representative of in vivo intestinal cell architecture and function. Although the C. jejuni deltamotAB mutant adhered significantly better than the wild type to the Caco-2 cells, we observed a significant reduction in the ability to invade the cells. In this study we obtained evidence that the flagellar rotation triggers C. jejuni invasion into polarized Caco-2 cells and we believe that C. jejuni is propelled into the cell with a drill-like rotation. The deltamotAB mutant was also tested for its colonization potential in a 1-day-old chicken model. The nonmotile C. jejuni deltamotAB mutant was not able to colonize any birds at days 3 and 7, suggesting that motility is essential for C. jejuni colonization.

Novel vaccine formulations against pertussis offer earlier onset of immunity and provide protection in the presence of maternal antibodies.

Whooping cough is a respiratory illness most severe in infants and young children. While the introduction of whole-cell (wP) and acellular pertussis (aP) vaccines has greatly reduced the burden of the disease, pertussis remains a problem in neonates and adolescents. New vaccines are needed that can provide early life and long-lasting protection of infants. Vaccination at an early age, however, is problematic due to the interference with maternally derived antibodies (MatAbs) and the bias towards Th2-type responses following vaccination. Here we report the development of a novel vaccine formulation against pertussis that is highly protective in the presence of MatAbs. We co-formulated pertussis toxoid (PTd) and filamentous hemagglutinin (FHA) with cytosine-phosphate-guanosine oligodeoxynucleotides (CpG ODN), cationic innate defense regulator (IDR) peptide and polyphosphazene (PP) into microparticle and soluble vaccine formulations and tested them in murine and porcine models in the presence and absence of passive immunity. Vaccines composed of the new adjuvant formulations induced an earlier onset of immunity, higher anti-pertussis IgG2a and IgA titers, and a balanced Th1/Th2-type responses when compared to immunization with Quadracel(®), one of the commercially available vaccines for pertussis. Most importantly, the vaccines offered protection against challenge infection in the presence of passively transferred MatAbs.

Carrier molecules for use in veterinary vaccines.

The practice of immunization of animals and humans has been carried out for centuries and is generally accepted as the most cost effective and sustainable method of infectious disease control. Over the past 20 years there have been significant changes in our ability to produce antigens by conventional extraction and purification, recombinant DNA and synthesis. However, many of these products need to be combined with carrier molecules to generate optimal immune responses. This review covers selected topics in the development of carrier technologies for use in the veterinary vaccine field, including glycoconjugate and peptide vaccines, microparticle and nanoparticle formulations, and finally virus-like particles.

Salmonella vaccines in poultry: past, present and future.

Salmonella species are important zoonotic pathogens that cause gastrointestinal disease in humans and animals. Poultry products contaminated with these pathogens are one of the major sources of human Salmonella infections. Vaccination of chickens, along with other intervention measures, is an important strategy that is currently being used to reduce the levels of Salmonella in poultry flocks, which will ultimately lead to lower rates of human Salmonella infections. However, despite numerous studies that have been performed, there is still a need for safer, well-defined Salmonella vaccines. This review examines the different classes of Salmonella vaccines that have been tested, highlighting the merits and problems of each, and provides an insight into the future of Salmonella vaccines and the platforms that can be used for delivery.

Radiological pitfalls with laryngopharyngeal injectables.

The aim of this report was to describe the computed tomography features of injected carboxymethylcellulose and how it can be mistaken for tumor recurrence. Carboxymethylcellulose is a low-density injectable indicated for short-term vocal fold augmentation. When used in patients with head and neck cancer, differentiating this agent from a neoplastic recurrence can be difficult on imaging. The imaging findings of five patients who received carboxymethylcellulose augmentation are presented to illustrate a potential pitfall of image interpretation. When injectable carboxymethylcellulose is used in cancer patients, knowledge of the injection sites, material used, and procedural history can avoid misinterpretation of monitory imaging.

Interferon-gamma and B-cell Activating Factor (BAFF) promote bovine B cell activation independent of TLR9 and T-cell signaling.

We previously reported that CD21(+) B cells purified from bovine blood do not respond to CpG-ODN stimulation unless either CD14(+) monocytes or B-cell Activating Factor (BAFF), a cytokine produced by activated monocytes, are present. In this report, we present evidence that CD14(+) monocytes are critical for CpG-specific lymphocyte proliferation within the peripheral blood mononuclear cell (PBMC) population but that this response is not mediated by soluble factors produced by CpG-activated monocytes. We further determine that bovine monocytes stimulated with IFN-γ induce expression of the BAFF gene and that recombinant IFN-γ and BAFF induced robust B cell activation when cultured in the absence of CpG ODN. These data suggest that CpG-stimulated monocytes may indirectly promote B cell activation by promoting release of cytokines and/or other soluble factors from accessory cells which in turn act on CpG-stimulated B cells to promote antigen-independent and T cell independent B cell activation. Understanding the T cell independent signals that induce B cell activation has important implications for understanding B cell development in locations where T cells are limited and in understanding polyclonal B cell activation that may contribute to autoimmune diseases.

Role of GapC in the pathogenesis of Staphylococcus aureus.

Staphylococcus aureus is recognized worldwide as a major pathogen causing clinical or subclinical intramammary infections in lactating cows, sheep and goats. S. aureus produces a wide arsenal of cell surface and extracellular proteins involved in virulence. Among these are two conserved proteins with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity named glyceraldehyde-3-phosphate dehydrogenase-B (GapB) and -C (GapC). In this study, we used the S. aureus wild type strain RN6390 and its isogenic gapC mutant H330 in in vitro and in vivo studies and determined that the S. aureus GapC protein plays a role on adherence to and internalization into bovine mammary epithelial (MAC-T) cells. In addition, we found that S. aureus H330 did not caused mastitis after an experimental infection of ovine mammary glands. Together, these results show that GapC is important in the pathogenesis of S. aureus mastitis.

Influence of maternal antibodies on active pertussis toxoid immunization of neonatal mice and piglets.

Whooping cough caused by infection with Bordetella pertussis, is a serious illness in infants and young children. Mortality due to whooping cough is being reported in infants too young to be immunized as well as those who have not completed their series of vaccinations. One of the major factors that interferes with successful active immunization in early life is the presence of maternal antibodies (MatAbs). Using the mouse and pig models, we evaluated the effect of maternal antibodies on active immunization with pertussis toxoid (PTd) and explored strategies to overcome this interference. Our results indicate that passively transferred maternal antibodies interfered with active immunization using pertussis toxoid. The level of passively transferred antibodies directly correlated with the level of interference observed. However, this interference could be overcome by using a second booster immunization or by co-formulating the toxoid with novel adjuvants. These results support the need for novel vaccine formulations that are optimized for the neonate and that can be used not only to modulate the inherently biased neonatal immune system but also to prime the response in the presence of passively transferred maternal antibodies.

Immunization with PCEP microparticles containing pertussis toxoid, CpG ODN and a synthetic innate defense regulator peptide induces protective immunity against pertussis.

We investigated the efficacy of a novel microparticle (MP) based vaccine formulation consisting of pertussis toxoid (PTd), polyphosphazene (PCEP), CpG ODN 10101 and synthetic cationic innate defense regulator peptide 1002 (IDR) against Bordetella pertussis in mice. We studied whether encapsulation of these IDR-CpG ODN complexes into polyphosphazene-based microparticles further enhanced their immunomodulatory activity compared to soluble formulations containing PCEP (SOL), or without PCEP (AQ). In vitro stimulation of murine macrophages showed MP induced significantly higher levels of pro-inflammatory cytokines. When assessed in a B. pertussis infection challenge model, a single immunization with MP formulation led to significantly lower bacterial loads compared to other formulations and non-vaccinated animals. ELISPOT of splenocytes showed that MP group mice had significantly higher number of antigen-specific IL-17 secreting cells. The cytokine profile in lung homogenates of MP group mice after challenge showed significantly higher amounts of MCP-1, TNF-α, IFN-γ, IL-12 and IL-17 and significantly lowered IL-10 levels suggesting a strong Th1 shift. Protection was observed against challenge infection with B. pertussis. On the other hand protective immune responses elicited in Quadracel(®) immunized mice were Th2 skewed. Hence, we conclude that formulation of PTd, PCEP, CpG ODN and IDR into MP generates a protective immune response in mice against pertussis emphasizing the potential of MP as a delivery vehicle for the potential development of single-shot vaccines.

Vaccination with type III secreted proteins leads to decreased shedding in calves after experimental infection with Escherichia coli O157.

Escherichia coli O157:H7 remains a threat to humans via cattle-derived fecal contamination of food and water. Preharvest intervention strategies represent a means of reducing the pathogen burden before harvest. In this study, the efficacy of a commercially produced type III secreted protein (TTSP) vaccine was evaluated with the use of a commingled experimental calf infection model (30 placebo-treated animals and 30 vaccinates). The calves were vaccinated on days 0, 21, and 42 and infected with 10(9) colony-forming units (CFU) of E. coli O157 by oral-gastric intubation on day 56. Fecal shedding was monitored daily for 14 d. Serologic assessment revealed a robust immune response to vaccination; the serum titers of antibodies against EspA, Tir, and total TTSPs were significantly higher in the vaccinates than in the placebo-treated animals on days 21, 42, 56, and 70. Significantly less (P = 0.011) of the challenge organism was shed by the vaccinates than by the placebo-treated animals on days 3 to 10. Peak shedding occurred in both groups on days 3 to 6; during this period the vaccinates showed a mean log reduction of 1.4 (P = 0.002) and a mitigated fraction of 51%. The number of animals shedding was significantly lower among the vaccinates compared with the placebo group on days 3 to 6 (P ≤ 0.05), with a mean prevented fraction of 21%. No differences in the duration of shedding were observed. Owing to the low challenge shedding in both groups on days 11 to 14 (mean CFU/g < 10; median = 0), no significant differences were observed. These data indicate that TTSP vaccination had protective effects through significant reductions in the number of animals shedding and the number of challenge organisms shed per animal and provides evidence that TTSP vaccination is an effective preharvest intervention strategy against E. coli O157.

B-cell activating factor (BAFF) promotes CpG ODN-induced B cell activation and proliferation.

It is controversial whether naïve B cells are directly activated in response to TLR9 ligand, CpG ODN. Although bovine blood-derived CD21(+) B cells express TLR9 and proliferate in response to CpG in mixed-cell populations, purified bovine B cells do not proliferate significantly in response to CpG ODN, even when the B cell receptor is engaged. When co-cultured with CD14(+) myeloid cells and/or B-cell activating factor (BAFF), a cytokine produced by activated myeloid cells, there was a significant increase in CpG-specific B cell proliferation, and the number of large B cells in general or positive for CD25, all of which are markers for B cell activation. These data suggest that activated myeloid cells and BAFF prime B cells for significant CpG-specific activation. Understanding the signals required to mediate efficient CpG-induced, antigen-independent and T-cell independent activation of B cells has implications for polyclonal B cell activation and the development of autoimmune diseases.