Mark J. Redmond

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.

Immunomodulatory Effects of Oat β-Glucan Administered Intragastrically or Parenterally on Mice Infected with Eimeria vermiformis

The immunostimulatory effect of intragastrically or parenterally administered β‐(1→3; 1→4) glucan, extracted from oats (OβG), on disease resistance to Eimeria vermiformis was studied in C57BL/6 mice. Multiple administrations of OβG by intragastric or subcutaneous routes reduced fecal oocyst shedding compared to the non‐treated control group. The administration of OβG by subcutaneous route resulted in higher levels of total serum immunoglobulins and antigen (sporozoite and merozoite)‐specific immunoglobulins as compared with the non‐treated group. To evaluate the effect of a single subcutaneous dose, groups of mice were treated with OβG 2 days before E. vermiformis infection, at the time of infection and at 2 or 6 days after infection. From day 11 post‐infection the oocyst discharge was significantly diminished (P< 0.05‐0.01) in the OβG‐treated groups, except in those treated 6 days after infection, as compared to the non‐treated control group. The proliferative responses to E. vermiformis sporozoite antigen of lymphocytes isolated from the spleen were significantly increased (P < 0.05) when OβG was administered 2 days before or at the time of E. vermiformis infection. Lymphocyte proliferative responses to merozoite antigen were not influenced by treatment. In conclusion, OβG appeared to up‐regulate immune mechanisms and provide enhanced resistance against eimerian coccidiosis in mice.

β-(1→3, 1→4) Oat glucan enhances resistance to Eimeria vermiformis infection in immunosuppressed mice

The effect of intragastrically or parenterally administered beta-glucan, extracted from oats, on the enhancement of disease resistance to Eimeria vermiformis was studied in C57BL/6 mice. Groups of mice were immunosuppressed with dexamethasone (DXM), infected with oocysts of E. vermiformis and treated with oat beta-glucan by the intragastric (i.g.) or subcutaneous (s.c.) routes. Faecal oocyst shedding was reduced in the beta-glucan-treated groups compared to the non-treated group. Immunosuppressed mice which received no beta-glucan treatment showed more severe clinical signs of the disease and a 50% mortality, while minimal clinical signs and no mortality were recorded in the beta-glucan-treated groups. Total IgG, IgG1, IgG2a, IgM and IgA immunoglobulins in the serum of beta-glucan-treated groups were overall higher than those in the non-treated group. Specific IgG anti-sporozoite and merozoite immunoglobulins in serum were significantly higher in the beta-glucan-treated groups than in the non-treated animals. No significant differences were found in the levels of intestinal IgA anti-sporozoite and anti-merozoite immunoglobulins. IFN-gamma- and IL-4-secreting cells, in response to sporozoite antigen, were detected in the spleen and mesenteric lymph nodes of the beta-glucan-treated groups only. In conclusion, the i.g. and s.c. oat beta-glucan treatment increased the resistance to E. vermiformis infection in immunosuppressed mice.

Induction of Systemic and Mucosal Immune Responses Following Immunization with Somatostatin-Avidin Complexes Incorporated Into Iscoms

Synthetic, biotinylated somatostatin-14 (Somatotropin Release-Inhibiting Factor; SRIF) was conjugated to avidin, and the resulting complex incorporated into immune-stimulating complexes (ISCOMS). The ISCOMS were used to study the systemic and mucosal immune responses induced by parenteral and gastrointestinal vaccination. Mice were immunized by intraperitoneal (IP) and intragastric (IG) routes and subsequently by either IP or IG secondary immunizations (groups-IP/IP; IP/IG; IG/IG). Antigen specific IgG and IgA antibody secreting cells (ASC) from the spleen, mesenteric lymph nodes (MLN) and Peyers patches (PPs) were studied by an enzyme-linked immunospot assay (ELISPOT). Specific proliferative responses of spleen cells to avidin and to SRIF were measured. Immunization IP/IP evoked the highest serum IgG levels to avidin and to SRIF as well as the highest numbers of splenic IgG isotype ASC. The greatest IgA response in MLN and PPs was induced by IP/IG immunization. Only marginal mucosal immunity and no splenic cell specific proliferative responses were found by IG/IG immunization. These results indicate that ISCOMS are an effective delivery system for protein-peptide antigens. The ISCOMS system described elicited systemic and mucosal antibody immune responses, and primed specific proliferative response when administered IP/IG. This offers another approach for the design and delivery of mucosally administered peptide vaccines.

Passive immunization against somatostatin increases resistance to Eimeria vermiformis infection in susceptible mice

The effect of in vivo immunoneutralization of somatostatin (SRIF) on Eimeria vermiformis intestinal infection was studied in resistant (BALB/c), and susceptible (C57BL/6) mouse strains. An anti-SRIF monoclonal antibody (MAb-SRIF) was used to passively immunize the mice by intraperitoneal injection. The animals were subsequently orally infected with oocysts of E. vermiformis. Individual fecal samples were collected daily for 21 days to monitor the kinetics of oocyst shedding. The fecal oocyst shedding was significantly higher in the C57BL/6 strain than in the BALB/c strain (P < 0.01). Passive immunization with MAb-SRIF in the C57BL/6 mice significantly reduced the number of oocysts in feces (P < 0.05), when compared to the infected non-immunized mice of the same strain. Infected BALB/c mice showed no difference in oocyst shedding in response to the passive immunoneutralization with MAb-SRIF. In conclusion, passive immunization with MAb-SRIF increased resistance to E. vermiformis-infection in the susceptible C57BL/6 mice, but not in the resistant BALB/c mice. This suggests that SRIF modulates gut immune function in parasitic infection.