Zsuzsanna Papp

Mucosal immunization of calves with recombinant bovine adenovirus-3: induction of protective immunity to bovine herpesvirus-1

To determine the potential of replication-competent (E3-deleted) bovine adenovirus-3 (BAV-3) as a delivery system for vaccine antigens in calves, we evaluated the ability of recombinant BAV-3 expressing different forms of of bovine herpesvirus-1 (BHV-1) glycoprotein gD to protect against BHV-1 infection in calves that had pre-existing BAV-3 specific antibodies. Three- to four-month-old calves, vaccinated intranasally with recombinant BAV-3 expressing full-length gD (BAV3.E3gD) or a truncated version of gD (gDt) (BAV3.E3gDt), or with E3-deleted BAV-3 (BAV3.E3d; control), were challenged with BHV-1 strain 108. Vaccination with BAV3.E3gD or BAV3.E3gDt induced gD-specific antibody responses in serum and nasal secretions, and primed calves for gD-specific lymphoproliferative responses. In addition, all calves developed complement-independent neutralizing antibodies against BHV-1. Protection against viral challenge was observed in calves vaccinated with recombinant BAV3.E3gD or BAV3.E3gDt as shown by a significant reduction in body temperature and clinical disease, and a partial reduction in the amount and duration of virus excretion in nasal secretions. These results indicate that replication-competent BAV-3-based vectors can induce protective immune responses in calves (the natural host) that have pre-existing BAV-3-specific antibodies.

Ileal and jejunal Peyer’s patches play distinct roles in mucosal immunity of sheep

The majority of pathogens enter the body through mucosal surfaces and it is now evident that mucosal immunity can provide effective disease protection. However, the induction of mucosal immunity will require efficient targeting of mucosal vaccines to appropriate mucosa‐associated lymphoid tissue. An animal model, based upon the surgical preparation of sterile intestinal ‘loops’ (blind‐ended segments of intestine), was developed to evaluate mucosal and systemic immune responses to enteric vaccines in ruminants. The effectiveness of end‐to‐end intestinal anastomoses was evaluated and fetal surgery did not disrupt normal intestinal function in lambs up to 6–7 months after birth. The immunological competence of Peyer’s patches (PP) within the intestinal ‘loops’ was evaluated with a human adenovirus 5 vector expressing the gD gene of bovine herpesvirus‐1. This vaccine vector induced both mucosal and systemic immune responses when injected into intestinal ‘loops’ of 5–6‐week‐old lambs. Antibodies to the gD protein were detected in the lumen of intestinal ‘loops’ and serum and PP lymphocytes proliferated in response to gD protein. The immune competence of ileal and jejunal PP was compared and these analyses confirmed that jejunal PP are an efficient site for the induction of mucosal immune responses. This was confirmed by the presence of gD‐specific antibody‐secreting cells in jejunal but not ileal PP. Systemic but not mucosal immune responses were detected when the vaccine vector was delivered to the ileal PP. In conclusion, this model provided an effective means to evaluate the immunogenicity of potential oral vaccines and to assess the immunological competence of ileal and jejunal Peyer’s patches.

The effect of pre-existing adenovirus-specific immunity on immune responses induced by recombinant adenovirus expressing glycoprotein D of bovine herpesvirus type 1.

We investigated whether pre-existing adenovirus-specific immunity influenced the development of immunity to a foreign antigen expressed by recombinant adenovirus. Active adenovirus-specific immunity was induced in cotton rats by i.n. administration of wild type human adenovirus type 5 (HAd5) two weeks before immunisation with a HAd5 vector expressing the glycoprotein D (gD) of bovine herpesvirus type 1 (gD-dE3 recombinant adenovirus). Active adenovirus-specific immunity inhibited gD-specific immune responses, following either i.n. or gastrointestinal immunisation with gD-dE3. An inhibitory effect was present even if infection with HAd5 and immunisation with gD-dE3 were 13 weeks apart. Passive transfer of adenovirus specific antibodies to cotton rats one day before immunisation, however, did not significantly inhibit gD-specific immune responses induced by i.n. immunisation with gD-dE3. Repeated administration of an adenovirus vector, therefore, may have a limited ability to deliver antigen, while passive immunity to adenovirus may not interfere with the success of immunisation.

Mucosal immunization with recombinant adenoviruses: induction of immunity and protection of cotton rats against respiratory bovine herpesvirus type 1 infection.

To facilitate the evaluation of vaccines against bovine herpesvirus type 1 (BHV-1), a cotton rat model of intranasal (i.n.) BHV-1 infection was established. Cotton rat lung cells were similar to bovine cells in their ability to support BHV-1 replication in vitro. Furthermore, i.n. inoculation of cotton rats with BHV-1 resulted in pulmonary lesions comparable to BHV-1 infection in cattle. Using this model, the potential of i.n. and gastrointestinal (g.i.) immunization was examined with recombinant human adenoviruses expressing glycoprotein D (gD) of BHV-1 to induce protective immunity against BHV-1. The replication-competent virus (gD-dE3) was more efficient than the replication-defective virus (gD-dE1E3) in inducing gD-specific antibody in the serum and in the respiratory tract. Furthermore, i.n. immunization with gD-dE3 stimulated antigen-specific antibody-secreting cells in the lung 12 weeks following immunization. Protection against BHV-1 challenge correlated with gD-specific antibody levels such that i.n. immunization with gD-dE3 conferred complete protection, while g.i. immunization conferred only partial protection of the lungs of most animals against BHV-1 challenge. In comparison, immunization with gD-dE1E3 by either route resulted in only a partial reduction of BHV-1 titre in the respiratory tract. The results obtained demonstrate that mucosal immunization with replication-competent recombinant adenovirus expressing gD of BHV-1 can induce immunity and protection against BHV-1 challenge.

Effects of Exposure to Naphthenic Acids in Tree Swallows (Tachycineta bicolor) on the Athabasca Oil Sands, Alberta, Canada

Naphthenic acids (NAs) are a group of carboxylic acids that are of particular concern to the steadily growing oil sands mining industry of Alberta, Canada, because they become highly concentrated in the water used for oil sands extraction and are toxic to aquatic biota and mammals. Upon mine closure, vast amounts of process-affected water will need to be reclaimed and proven safe for wildlife colonizing reclaimed areas. The effects of exposure to NAs have not been investigated in avian species. To address this void, tree swallow (Tachycineta bicolor) nestlings were dosed with NAs while being reared normally by their free-ranging parents on a site in the vicinity of the oil sands. Nestlings received 1.5 mg NAs/day (approximately 0.075 g/kg body mass) from d 7 to d 13 of age, which represented a 10-fold “worst exposure” scenario. Nestling growth, hematocrit, blood biochemistry, organ weights, and ethoxyresorufin O-deethylase (EROD) activity were unaffected by NAs. The only change detected on histopathological evaluation of major organs was an increase in extramedullary erythropoiesis in the liver. These findings indicate that nestling tree swallows can successfully tolerate short-term exposures to environmentally realistic concentrations of NAs. However, this study did not investigate the chronic or reproductive toxicity of NAs. More research needs to be conducted to complete this initial assessment, to determine environmental risks on reclaimed areas where birds will be breeding and where their exposure to NAs could extend for several weeks.

The immunogenicity and efficacy of replication-defective and replication-competent bovine adenovirus-3 expressing bovine herpesvirus-1 glycoprotein gD in cattle.

Replication-competent and replication-defective bovine adenovirus type 3 recombinants expressing the bovine herpesvirus type 1 (BHV-1) glycoprotein D (gD) were tested for induction of gD specific immune responses in calves using intratracheal (1st and 2nd immunization) and sub-cutaneous (3rd immunization) route of immunization. The replication-defective recombinant BAV501 induced systemic immune responses against gD as low titers of anti gD-IgG were detected in the serum. However, the efficacy of the replication-competent BAV3.E3gD to induce gD-specific antibodies in the serum and the nasal secretions was superior to that of replication-defective BAV501 when both viruses were given at the same dosage. Partial protection from challenge was induced in calves immunized with replication-competent BAV3.E3gD. A dramatic increase in the titers of anti-gD IgG and IgA levels, both in serum and nasal secretions, following BHV-1 challenge (anamnestic response) suggested that the animals immunized with replication-defective BAV501 had been primed for gD-specific antibody responses.

VALIDATION AND NOVEL APPLICATIONS OF THE WHOLE-BLOOD CHEMILUMINESCENCE ASSAY OF INNATE IMMUNE FUNCTION IN WILD VERTEBRATES AND DOMESTIC CHICKENS

The whole-blood chemiluminescence (WBCL) assay is a simple and rapid method of measuring production of reactive oxygen species by circulating leukocytes, particularly heterophils (birds) and neutrophils (other vertebrates). In the interest of substantiating a broadly applicable measure of innate immunity, we investigated the microplate WBCL method for several wildlife species as well as domestic broiler chickens. Lucigenin as a light enhancer was used for all avian blood, wild and domestic, while luminol was used in bear and frog blood. Use of ethylenedinitrilo-tetraacetic acid (EDTA) as the anticoagulant caused hemolysis of frog blood and decreased WBCL responses in all animals tested. Heparin, even in high concentrations, caused modest to no decrease of WBCL responses. The WBCL response correlated highly with heterophil or neutrophil numbers in all species tested. The WBCL response was tested in freshly collected blood as well as in blood one to five days postcollection in order to determine the utility of this assay for field studies when immediate access to laboratory facilities is not possible. One to three days of delay in performing the test after blood collection caused no, or only a slight, decay of the chemiluminescence response in most animals. Using domestic chickens, we tested the sensitivity of the WBCL method to detect differences between treatment groups and looked for loss of chemiluminescence response over several days using the original blood samples. Significant differences in the WBCL response between experimental groups of broiler chicken were detectable in freshly collected blood, as well as one- to four-day-old blood. Our results show that the innate immune response of populations of wild animals may be successfully compared using this assay, even when blood cannot be tested until a few days after collection.

Transtracheal Administration of Interleukin-12 Induces Neutrophil Responses in the Murine Lung

Although the roles of interleukin-12 (IL-12) in the immunomodulation of antigen-specific responses are well characterized, the effects of IL-12 on the respiratory tract following mucosal administration are not well defined. Therefore, we investigated changes in the murine lung shortly after intranasal (i.n.) administration of murine IL-12. We showed that IL-12 induced neutrophil influx to the murine lung in both C57BL/6 and BALB/c mice. Histologic examination revealed that intranasal administration of IL-12 with liposomes induced focal neutrophil infiltration into the alveoli and a significant increase in neutrophils in bronchoalveolar lavage fluids when compared with administration of liposomes alone. In vitro chemotaxis assays indicated that the observed pulmonary neutrophil response induced by IL-12 could have been due in part to the direct chemotactic activity of IL-12 for murine neutrophils.

The ecology of avian influenza viruses in wild dabbling ducks ( Anas spp.) in Canada

Avian influenza virus (AIV) occurrence and transmission remain important wildlife and human health issues in much of the world, including in North America. Through Canada’s Inter-Agency Wild Bird Influenza Survey, close to 20,000 apparently healthy, wild dabbling ducks (of seven species) were tested for AIV between 2005 and 2011. We used these data to identify and evaluate ecological and demographic correlates of infection with low pathogenic AIVs in wild dabbling ducks (Anas spp.) across Canada. Generalized linear mixed effects model analyses revealed that risk of AIV infection was higher in hatch-year birds compared to adults, and was positively associated with a high proportion of hatch-year birds in the population. Males were more likely to be infected than females in British Columbia and in Eastern Provinces of Canada, but more complex relationships among age and sex cohorts were found in the Prairie Provinces. A species effect was apparent in Eastern Canada and British Columbia, where teal (A. discors and/or A. carolinensis) were less likely to be infected than mallards (A. platyrhynchos). Risk of AIV infection increased with the density of the breeding population, in both Eastern Canada and the Prairie Provinces, and lower temperatures preceding sampling were associated with a higher probability of AIV infection in Eastern Canada. Our results provide new insights into the ecological and demographic factors associated with AIV infection in waterfowl.

ECOLOGICAL DETERMINANTS OF AVIAN INFLUENZA VIRUS, WEST NILE VIRUS, AND AVIAN PARAMYXOVIRUS INFECTION AND ANTIBODY STATUS IN BLUE-WINGED TEAL (ANAS DISCORS) IN THE CANADIAN PRAIRIES

Abstract The Canadian prairies are one of the most important breeding and staging areas for migratory waterfowl in North America. Hundreds of thousands of waterfowl of numerous species from multiple flyways converge in and disperse from this region annually; therefore this region may be a key area for potential intra- and interspecific spread of infectious pathogens among migratory waterfowl in the Americas. Using Blue-winged Teal (Anas discors, BWTE), which have the most extensive migratory range among waterfowl species, we investigated ecologic risk factors for infection and antibody status to avian influenza virus (AIV), West Nile virus (WNV), and avian paramyxovirus-1 (APMV-1) in the three prairie provinces (Alberta, Saskatchewan, and Manitoba) prior to fall migration. We used generalized linear models to examine infection or evidence of exposure in relation to host (age, sex, body condition, exposure to other infections), spatiotemporal (year, province), population-level (local population densities of BWTE, total waterfowl densities), and environmental (local pond densities) factors. The probability of AIV infection in BWTE was associated with host factors (e.g., age and antibody status), population-level factors (e.g., local BWTE population density), and year. An interaction between age and AIV antibody status showed that hatch year birds with antibodies to AIV were more likely to be infected, suggesting an antibody response to an active infection. Infection with AIV was positively associated with local BWTE density, supporting the hypothesis of density-dependent transmission. The presence of antibodies to WNV and APMV-1 was positively associated with age and varied among years. Furthermore, the probability of being WNV antibody positive was positively associated with pond density rather than host population density, likely because ponds provide suitable breeding habitat for mosquitoes, the primary vectors for transmission. Our findings highlight the importance of spatiotemporal, environmental, and host factors at the individual and population levels, all of which may influence dynamics of these and other viruses in wild waterfowl populations.