Dale L. Godson

The acute phase response of haptoglobin and serum amyloid A (SAA) in cattle undergoing experimental infection with bovine respiratory syncytial virus.

Abstract The ability of a pure virus infection to induce an acute phase protein response is of interest as viral infections are normally considered to be less efficient in inducing an acute phase protein response than bacterial infections. This was studied in a bovine model for infection with bovine respiratory syncytial virus (BRSV), analysing the induction of the two most dominant bovine acute phase proteins haptoglobin and serum amyloid A (SAA). Strong and reproducible acute phase responses were detected for both proteins, peaking at around 7–8 days after inoculation of BRSV, while no response was seen in mock-inoculated control animals. The serum concentrations reached for SAA and haptoglobin during the BRSV-induced acute phase response were generally the same or higher than previously reported for bacterial infections in calves. The magnitude and the duration of the haptoglobin response was found to correlate well with the severity of clinical signs (fever) and with the extent of lung consolidation while SAA responded most rapidly to infection.

Serum haptoglobin as an indicator of the acute phase response in bovine respiratory disease

Abstract The early stages of the host response to infectious agents include a number of physiologic changes, collectively known as the acute phase response. The acute phase response is comprised of reactions localized at the site of infection, as well as the initiation of systemic responses, which include a rapid increase in the serum concentration of some proteins, known as acute phase proteins (APP). Using polyacrylamide gel electrophoresis, we detected two APP of approximately 22 and 37 kDa molecular weight in sera obtained from cattle with bovine respiratory disease (BRD). Based on their presence in the sera of sick, but not normal animals, the molecular weights, N-terminal amino acid sequence analysis, and the ability to bind hemoglobin, we identified these proteins as the α and β subunits of haptoglobin. The haptoglobin molecule and the α subunit were isolated from serum, purified, and used to produce monoclonal and polyclonal antibodies. With these reagents, an enzyme linked immunosorbent assay was developed to measure the concentration of haptoglobin in bovine serum. Using an experimental model of BRD induced by a sequential challenge of calves with bovine herpesvirus type-1 and Pasteurella haemolytica, we observed a temporal relationship between the increase in haptoglobin concentration in serum and the onset of bacterial infection. The haptoglobin concentration ranged from undetectable in the serum of most calves prior to challenge, to greater than 1 mgml−1 in over one-third of the calves at the height of disease. Furthermore, the concentration of haptoglobin was associated significantly with other measures of the severity of disease. Together, these results indicate that quantification of acute phase proteins in animals with BRD could be a valuable diagnostic and prognostic aid.

Protection of chickens against Escherichia coli infections by DNA containing CpG motifs.

ABSTRACT Synthetic oligodeoxynucleotides (ODN) containing CpG motifs (CpG-ODN) have been shown to be effective immunoprotective agents in murine models for a variety of viral, intracellular bacterial, and protozoan infections. Until now, the use of CpG-ODN to protect against extracellular bacterial infections has not been reported. The objective of this study was to investigate the effect of CpG-ODN against cellulitis and colibacillosis in broiler chickens, using a well-established model. At 22 days of age, birds received CpG-ODN by either the subcutaneous or intramuscular route. Three days later, a virulent isolate of Escherichia coli was applied to a scratch site on the caudal abdominal skin. Birds were examined for 10 days after the E. coli challenge, and pathological and bacteriological assessments were conducted on all birds. The control group of birds receiving no CpG-ODN(2007) had a survival rate of 15%. In contrast, groups that received CpG-ODN(2007), by either subcutaneous or intramuscular injection, had significantly higher survival rates (P < 0.0001). Furthermore, the size of the cellulitis lesion was significantly smaller in groups that received CpG-ODN(2007) by the subcutaneous route (P < 0.01). A dose of as little as 3.16 μg of CpG-ODN(2007), delivered 3 days prior to challenge by either the subcutaneous or intramuscular route, significantly protected birds against E. coli infection (P < 0.01). This study demonstrates that CpG-ODN(2007) has both local and systemic protective effects in broiler chickens. This is the first time that CpG-ODN(2007) has been demonstrated to have an immunoprotective effect against an extracellular bacterial infection in any food animal species.

CpG-containing oligodeoxynucleotides augment and switch the immune responses of cattle to bovine herpesvirus-1 glycoprotein D☆

The adjuvanticity of a synthetic oligodeoxynucleotide containing unmethylated CpG motifs (CpG ODN) was determined in cattle. Calves were immunized with a truncated secreted version of glycoprotein D (tgD) of bovine herpes virus-1 (BHV-1) formulated with alum, CpG ODN, or a combination of both. BHV-1 tgD formulated with CpG ODN or with alum and CpG ODN induced a stronger and more balanced immune response than tgD in alum. This level of immunity was of sufficient magnitude to minimize weight loss and significantly reduce the duration of virus shedding after intranasal viral challenge. Local tissue reactions generated by CpG ODN were very mild and transient, whereas reactions induced by alum or a combination of CpG ODN and alum were moderate in severity and duration. These data demonstrate that CpG ODN causes minimal injection site reactions and yet acts as an effective adjuvant in cattle.

Bovine and ovine blood mononuclear leukocytes differ markedly in innate immune responses induced by Class A and Class B CpG-oligodeoxynucleotide.

Cytosine-phosphate-guanosine (CpG)-DNA can induce an impressive array of innate immune responses that may directly or indirectly contribute to the clearance of infectious agents. Assays, such as lymphocyte proliferative responses, have been used to demonstrate that the immunostimulatory activity of CpG-DNA is conserved among a broad range of vertebrate species, but no studies have been completed to determine if qualitative differences exist among species for CpG-oligodeoxynucleotide (ODN)-induced innate immune responses. In this study, we assessed the capacity of a Class A (ODN 2216) and a Class B (ODN 2007) CpG-ODN to induce innate immune responses in two closely related species, ovine (n = 28) and bovine (n = 29). The secretion of interferon (IFN)-alpha and IFN-gamma and non-major histocompatability complex (MHC)-restricted cytotoxic activity were assayed with CpG-ODN-stimulated peripheral blood mononuclear cells (PBMC). These investigations revealed significant interspecies and intraspecies variation in the responses. As expected, ODN 2216 was a potent inducer of IFN-alpha secretion by both bovine and ovine PBMC, but ODN 2007 also induced dose-dependent, CpG-specific IFN-alpha secretion by ovine PBMC. In contrast, a significant dose-dependent, CpG-specific IFN-gamma secretion response was only observed following ODN 2216 stimulation of bovine PBMC. Furthermore, both ODN 2216 and ODN 2007 induced CpG-specific non-MHC-restricted cytotoxicity with ovine but not bovine PBMC. Finally, there was not a single assay in which PBMC from all sheep or cattle responded at a detectable level. A striking aspect of these results is that such marked differences in CpG-ODN induced innate responses existed both between and within two closely related species.

Th1/Th2 biasing effects of vaccination in cattle as determined by real-time PCR

Real-time polymerase chain reaction (PCR) is now becoming an accepted tool for measuring gene expression at the transcriptional level. In this study, a direct comparison between real-time PCR, enzyme-linked immunosorbent assay (ELISA) and enzyme-linked immunosorbent spot (ELISPOT) assay was performed. When interferon-gamma (IFN-gamma) gene expression was assessed, both ELISA and ELISPOT data strongly correlated to results obtained by real-time PCR. Real-time PCR was subsequently used to measure bovine IFN-gamma (bIFN-gamma) and bovine interleukin-4 (bIL-4) gene expression by antigen stimulated peripheral blood mononuclear cells (PBMC), isolated from bovine herpesvirus-1 (BHV-1) infected animals. BHV-1-infected animals were either non-vaccinated or vaccinated using one of two adjuvants prior to infection. With non-vaccinated infected animals, a Th1 bias occurred, based on IFN-gamma expression exceeding IL-4 expression. The level of cytokine expression, and the IFN-gamma/IL-4 ratio could be significantly affected, depending on the manner in which animals were vaccinated.

Multiple administration with interleukin-2 potentiates antigen-specific responses to subunit vaccination with bovine herpesvirus-1 glycoprotein IV.

Interleukin-2 has been described as an effective adjuvant for a number of antigens in different host species. Previously, we demonstrated the adjuvant activity of recombinant bovine IL-2 with a glycoprotein IV (gIV) subunit vaccine from bovine herpesvirus type-1 (BHV-1). In the present study, primary antibody responses were assessed in cattle immunized with either 2 or 50 micrograms of gIV, and treated with multiple doses of IL-2 or combinations of IL-2 and IFN-alpha or IL-2 and IFN-gamma. IL-2 was able to augment significantly antibody responses detected by either ELISA or virus neutralization. More significantly, IL-2 was able to enhance antibody titres in animals immunized with only 2 micrograms gIV to levels similar to those immunized with 50 micrograms gIV in the absence of IL-2. For optimal stimulation, multiple injections of IL-2 and Avridine had to be used in the formulation; other oil adjuvants or IL-2 alone could not induce a primary serum antibody response. Addition of IFN-alpha or IFN-gamma to the IL-2/gIV/Avridine formulation did not affect any of the immune parameters tested. As IFN-alpha is an effective immunoprophylactic agent for infectious bovine rhinotracheitis (IBR), combination vaccine-immunoprophylaxis may become feasible using IL-2 as a co-adjuvant. Thus, extremely low doses of antigen and only one immunization may be an effective vaccine given in combination with interferon prophylactic treatment.

Innate immune responses induced by CpG oligodeoxyribonucleotide stimulation of ovine blood mononuclear cells.

Examples exist in the literature that demonstrate that treatment with immunostimulatory cytosine–phosphate–guanosine (CpG)‐DNA can protect mice against infection by intracellular pathogens. There are, however, few studies reporting that CpG‐DNA offers similar disease protection in other species. In this study, we assessed the potential of a class A and class B CpG oligodeoxynucleotide (ODN) to induce innate immune responses in sheep, an outbred species. Using peripheral blood mononuclear cells, we have for the first time demonstrated CpG‐ODN‐induced innate immune responses, including natural‐killer‐like activity [non‐major histocompatibility complex (MHC)‐restricted cytotoxicity], interferon‐α secretion and 2′‐5′A oligoadenylate synthetase activity, that could contribute to immune protection in sheep. The type and magnitude of these responses were dependent on ODN class and non‐MHC‐restricted killing was not associated with interferon‐γ production. The latter observation is in contrast with observations reported for mice and humans. These observations support the conclusion that differences in CpG‐ODN‐induced responses exist among species and that specific ODN sequences can significantly influence innate immune responses.

Capture immunoassay for ruminant tumor necrosis factor-α: comparison with bioassay

Abstract Monoclonal antibodies and IgG purified from rabbit polyclonal antiserum, raised against recombinant bovine tumor necrosis factor-alpha (TNF-α), have been employed in ELISA procedures to quantitate bovine TNF-α. These antibodies were potent in neutralizing the biological activity of recombinant as well as natural bovine TNF-α. The monoclonal antibodies were used as capture antibodies and were either passively adsorbed or covalently linked to ELISA plates. Polyclonal rabbit anti-TNF IgG was used as the detecting antibody in combination with a biotinylated anti-rabbit serum and a streptavidin-horseradish peroxidase conjugate. The detection limit for recombinant TNF-α medium was 10 pg ml−1 and in bovine or ovine serum was 35 pg ml−1. A good correlation was found between the ELISA and the WEHI-164 Clone 13 biologic assay when TNF-α was measured in medium containing serum or in serum. This capture ELISA was also capable of detecting ovine, but not porcine. TNF in supernatants from cultures of lipopolysaccharide-stimulated pulmonary alveolar macrophages.

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.