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Abstract

Objective—To assess binding of IgE to native, whole hydrolyzed, and separated hydrolyzed fractions of soy protein in serum obtained from dogs with experimentally induced soy protein hypersensitivity.

Animals—8 naïve Beagles (6 experimentally sensitized to native soy protein and 2 control dogs).

Procedures—6 dogs were sensitized against soy protein by administration of allergens during a 90-day period. After the sensitization protocol was completed, serum concentrations of soy-specific IgE were measured and intradermal skin tests were performed in all 6 dogs to confirm that the dogs were sensitized against soy protein. Serum samples from each sensitized and control dog underwent western blot analysis to assess the molecular mass band pattern of the different allergenic soy fractions and evaluate reactivities to native and hydrolyzed soy protein.

Results—In sera from sensitized dogs, a characteristic band pattern with 2 major bands (approx 75 and 50 kd) and 2 minor bands (approx 31 and 20 kd) was detected, whereas only a diffuse band pattern associated with whole hydrolyzed soy protein was detected in the most reactive dog. Reactivity was evident only for the higher molecular mass peptide fraction. In control dogs, no IgE reaction to native or hydrolyzed soy protein was detected.

Conclusions and Clinical Relevance—Data suggest that the binding of soy-specific IgE to the hydrolyzed soy protein used in the study was significantly reduced, compared with binding of soy-specific IgE to the native soy protein, in dogs with experimentally induced soy hypersensitivity.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To determine the efficacy of a modified-live virus vaccine containing bovine herpes virus 1 (BHV-1), bovine respiratory syncytial virus (BRSV), parainfluenza virus 3, and bovine viral diarrhea virus (BVDV) types 1 and 2 to induce neutralizing antibodies and cell-mediated immunity in naïve cattle and protect against BHV-1 challenge.

Animals—17 calves.

Procedures—8 calves were mock-vaccinated with saline (0.9% NaCl) solution (control calves), and 9 calves were vaccinated at 15 to 16 weeks of age. All calves were challenged with BHV-1 25 weeks after vaccination. Neutralizing antibodies and T-cell responsiveness were tested on the day of vaccination and periodically after vaccination and BHV-1 challenge. Specific T-cell responses were evaluated by comparing CD25 upregulation and intracellular interferon-γ expression by 5-color flow cytometry. Titration of BHV-1 in nasal secretions was performed daily after challenge.

Results—Vaccinated calves seroconverted by week 4 after vaccination. Antigen-specific cell-mediated immune responses, by CD25 expression index, were significantly higher in vaccinated calves than control calves. Compared with control calves, antigen-specific interferon-γ expression was significantly higher in calves during weeks 4 to 8 after vaccination, declining by week 24. After BHV-1 challenge, both neutralizing antibodies and T-cell responses of vaccinated calves had anamnestic responses to BHV-1. Vaccinated calves shed virus in nasal secretions at significantly lower titers for a shorter period and had significantly lower rectal temperatures than control calves.

Conclusion and Clinical Relevance—A single dose of vaccine effectively induced humoral and cellular immune responses against BHV-1, BRSV, and BVDV types 1 and 2 and protected calves after BHV-1 challenge for 6 months after vaccination.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To assess the biological response to recombinant feline interferon-omega (rFeIFN-ω) following ocular or oral administration in cats via estimation of Mx protein expression in conjunctival cells (CCs) and WBCs.

Animals—10 specific pathogen–free cats.

Procedures—In multiple single-dose drug experiments, each cat received various concentrations of rFeIFN-ω administered topically into both eyes (50 to 10,000 U/eye) and orally (200 to 20,000 units). The same cats received saline (0.9% NaCl) solution topically and orally as control treatments. The CCs and WBCs were collected prior to treatment (day 0), on day 1, and every third or seventh day thereafter until samples yielded negative results for Mx protein. Samples were examined for Mx protein expression via immunohistochemistry and immunoblotting procedures involving murine anti-Mx protein monoclonal antibody M143.

Results—After topical application of 10,000 U of rFeIFN-ω/eye, CCs stained for Mx protein for a minimum of 7 days, whereas WBCs were positive for Mx protein for a minimum of 31 days. After topical application of lower concentrations, CCs did not express Mx protein, in contrast to WBCs, which stained for Mx protein at 1,000 units for at least 1 day. Following oral administration, Mx protein was expressed in WBCs at rFeIFN-ω concentrations as low as 200 units, whereas CCs did not stain for Mx protein at any concentration.

Conclusions and Clinical Relevance—Results indicate that Mx protein expression (a marker of the biological response to rFeIFN-ω) in CCs and WBCs of rFeIFN-ω–treated cats depends on the dose of rFeIFN-ω, site of administration, and cell type.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To determine the effect of fetal bovine serum (FBS) and heat-inactivated FBS (HI-FBS) on lipopolysaccharide (LPS)- and zymosan-induced procoagulant activity of equine and canine mononuclear cells.

Sample Population—Mononuclear cells from 18 horses and 3 dogs.

Procedures—Cells were incubated with various concentrations of FBS, HI-FBS, LPS, zymosan, polymyxin B, and anti–LPS-binding protein monoclonal antibody or combinations of these constituents. A 1stage recalcification assay was used to determine procoagulant activity.

Results—Addition of FBS to media significantly increased procoagulant activity; equine and canine cells were stimulated by 1% and 10% FBS, respectively. Coincubation of cells with FBS and polymyxin B did not reduce this effect, suggesting that the response was not attributable to LPS contamination. Addition of HI-FBS to media did not stimulate procoagulant activity of equine or canine cells, and the sensitivity of the equine cells to LPS was significantly increased by HI-FBS. This increased LPS sensitivity was reduced 40% with monoclonal antibody directed against human recombinant LPSbinding protein. Increasing concentrations of HIFBS significantly increased LPS- and zymosaninduced procoagulant activity of canine cells.

Conclusion and Clinical Relevance—Procoagulant activity production in equine and canine mononuclear cells was significantly increased by addition of FBS, whereas heat inactivation of FBS eliminated this effect. Heat inactivation did not eliminate the function of serum proteins involved in enhancement of LPSand zymosan-induced procoagulant activity. Results suggest that HI-FBS can be used as a source of serum proteins that increase the sensitivity of mononuclear cells to bacterial and yeast cell wall components.

Full access
in American Journal of Veterinary Research

Abstract

Objective—To assess whether dogs with experimentally induced type I hypersensitivity against soy protein would respond to soy hydrolysate and develop cutaneous or gastrointestinal tract reactions after intradermal and oral challenge exposure.

Animals—12 naïve Beagle pups (9 sensitized and 3 control dogs).

Procedure—9 dogs were sensitized against soy protein by administration of allergens during a 90-day period. After the sensitization period, serum concentrations of soy-specific IgE were determined and an intradermal test was performed to confirm the dogs were sensitized against soy protein. An intradermal challenge test and an oral challenge test with native and hydrolyzed soy protein were conducted on 6 sensitized and 2 control dogs.

Results—High serum concentrations of soy-specific IgE and positive results for the intradermal test were observed for the 9 sensitized dogs after completion of the sesitization process. Sensitized dogs challenge exposed with hydrolyzed soy protein had a reduced inflammatory response after intradermal injection and no clinical response after an oral challenge exposure, compared with responses after intradermal and oral challenge exposure with native soy protein.

Conclusions and Clinical Relevance—Soy-sensitized dogs did not respond to oral administration of hydrolyzed soy protein. Thus, hydrolyzed soy protein may be useful in diets formulated for the management of dogs with adverse reactions to food.

Full access
in American Journal of Veterinary Research

Abstract

Objective

To evaluate interactions of human intravenous immunoglobulin (IVIG) with canine lymphocytes and monocytes.

Sample Population

Heparinized blood samples from 4 clinically normal Beagles.

Procedure

Binding ability of IVIG to canine lymphocytes and monocytes was measured by flow cytometry and an indirect immunofluorescent assay. Dualstaining fluorescent assays were done to determine lymphocyte subsets that bind IVIG. Competitive assays were done, using intact canine IgG and Fc fragments, and inhibition of binding was compared with that of F(ab)2 fragments. Ability of IVIG to inhibit phagocytosis of antibody-coated canine RBC also was determined, using a canine mononuclear cell phagocytic assay.

Results

IVIG concentrations (10, 1, 0.1, and 0.01 mg/ml) bound to (mean ± SD) 99.6 ± 0.4, 92.4 ± 6.1, 20.4 ± 24.6 and 2.0 ± 5.1% of canine lymphocytes, respectively, Dual staining analyses with IVIG and canine lymphocyte markers indicated that IVIG bound to CD4, CD8, and B lymphocytes. The aforementioned 4 IVIG concentrations bound to 98.0 ±2.1, 85.5 ± 13.5, 64.7 ± 32.8, and 26.5 ± 17.1% of monocytes, respectively. Inhibition of IVIG (0.01 mg/ml) binding to monocytes was significant (P < 0.05) in the presence of 1 and 10 mg of canine IgG/ml and 1 mg of canine Fc fragments/ml. In the presence of F(ab')2 fragments of canine IgG, inhibition was not significant, suggesting that binding is Fc mediated. Co-culturing of monocytes, opsonized RBC, and the 4 concentrations of IVIG and no IVIG resulted in 11.8 ± 5.1, 27.7 ± 12.3, 31.8 ± 15.1, 53.8 ± 6.7, and 45 ± 12% of the monocytes containing RBC, respectively. Phagocytosis inhibition was significant (P < 0.05) at an IVIG concentration of 10 mg/ml.

Conclusions

IVIG binds to canine lymphocytes and monocytes; binding to the latter is Fc mediated. IVIG also inhibits Fc-mediated phagocytosis of antibody-coated RBC.

Clinical Relevance

Owing to its ability to inhibit Fc-mediated phagocytosis of antibody-coated RBC, IVIG may be an effective short-term treatment for dogs with immune-mediated hemolytic anemia. (Am J Vet Res 1998;59:1568-1574)

Free access
in American Journal of Veterinary Research

Abstract

Objective

To determine whether an established bovine mammary epithelial cell line expresses interleukin 8 (IL-8) mRNA and synthesizes antigenic IL-8 in response to lipopolysaccharide (LPS) stimulation.

Sample Population

A bovine mammary epithelial cell line (MAC-T).

Procedure

mRNA was isolated from cells stimulated with graded concentrations of LPS. The first strand of IL-8 cDNA was synthesized, using a reverse transcriptase (RT) reaction with a specific oligonucleotide. Amplification of IL-8 cDNA was obtained by use of polymerase chain reaction (PCR). The MAC-T-derived antigenic IL-8 was quantified by use of a commercial anti-human IL-8 kit in a sandwich ELISA.

Results

RT-PCR revealed expression of MAC-T-derived mRNA within the first hour after stimulation with LPS. Expression of IL-8 mRNA was correlated to production of IL-8 protein detected in medium by use of the sandwich ELISA. Amounts of antigenic IL-8 increased in a dose- and time-dependent manner, and were maximal (57 pg/ml) at 48 hours after stimulation with 20 μg of LPS/ml.

Conclusions

MAC-T cells secrete IL-8 in response to stimulation with LPS in a dose- and time-dependent manner. The results were consistent with our hypothesis that mammary gland epithelial cells can be a source of IL-8 during the early stage of mastitis. Therefore, IL-8 may have a pivotal role in resolving bacterial infections. (Am J Vet Res 1998;59:1563-1567)

Free access
in American Journal of Veterinary Research

Abstract

Objective

To determine functional responses of neonatal chicken and turkey heterophils to various inflammatory agonists.

Animals

100 one-day-old chickens and turkeys.

Procedure

Blood heterophils were isolated and stimulated for 30 minutes at 39 C with ionomycin, phorbol myristate acetate (PMA), opsonized zymosan (OZ), or formyl-methionyl-leucyl-phenylalanine (FMLP). Functional responses (shape change, adherence, phagocytosis, influx of intracellular calcium, and oxidative burst) of stimulated heterophils were measured and compared with responses of unstimulated (control) heterophils.

Results

Turkey and chicken heterophils did not respond to FMLP stimulation. Stimulation of chicken and turkey heterophils with ionomycin resulted in significant increases in adherence, percentage of cells with a shape change, phagocytosis, intracellular calcium concentration, and oxidative burst. Turkey heterophils did not respond to PMA stimulation, whereas stimulation of chicken heterophils with PMA resulted in significant increases in adherence, percentage of cells with a shape change, phagocytosis, and oxidative burst but not intracellular calcium concentration. Stimulation of chicken and turkey heterophils with OZ resulted in significant increases in oxidative burst.

Conclusions

Mechanisms regulating initiation of heterophil activation in neonatal chicken and turkey heterophils are consistent with those described for heterophils isolated from mature birds. The biochemical and cytoskeletal systems of neonatal avian heterophils undergo functional alterations following stimulation with inflammatory agonists.

Clinical Relevance

Understanding heterophil activation and regulation should eventually lead to methods for controlling bacterial diseases in poultry. (Am J Vet Res 1998;59:1404–1408)

Free access
in American Journal of Veterinary Research

Abstract

Objectives

To assess effects of vaccination against fescue toxicosis on weight gain, serum prolactin and cholesterol concentrations, and alkaline phosphatase (ALP) activity in mice fed an endophyte-infected (EI) or endophyte-free (EF) fescue diet.

Animals

50 six-week-old male BALB/c mice.

Procedure

Mice were randomly allocated to the following 5 groups: 1, vaccinated intraperitoneally with a bovine serum albumin-ergotamine (EG) conjugate and fed an EI fescue diet; 2, orally vaccinated with cholera toxin (CT) subunit B-EG conjugate mixed with free CT and fed an EI fescue diet; 3, not vaccinated and fed an EI fescue diet; 4, passively vaccinated with monoclonal antibodies specific for ergovaline (EV) and fed an EI fescue diet; and 5, not vaccinated and fed an EF fescue diet.

Results

Antibodies against EG and EV were in serum of mice of groups 1 and 4, respectively. Secretory IgA and IgG coproantibodies against EG were induced in mice of group 2. Weight increased in groups 1 and 2 and tended to be increased in group 4 versus group 3. Prolactin concentration was similar in all groups; cholesterol concentration was decreased in groups 1, 3, and 4, compared with group 5. Compared with that in group 5, serum ALP activity decreased in groups 1 and 4 and was further decreased in group 1, compared with that in groups 2 and 3; it was negatively correlated with anti-EG titer.

Conclusions and Clinical Relevance

Induction of anti-EG antibodies and administration of EV monoclonal antibodies tended to increase short-term weight gain in this murine model of fescue toxicosis. However, systemic IgG antibodies against EG or EV antibodies were not protective against decreases in serum ALP activity and cholesterol concentrations. Clinical significance of decreased ALP activity associated with vaccination is unknown, but represents a worsening of a response often associated with fescue toxicosis in cattle. (Am J Vet Res 1998;59:1258–1262)

Free access
in American Journal of Veterinary Research

Abstract

Objective

To clone and characterize the cDNA encoding feline interleukin-5 (IL-5) cDNA and the 170 basepairs (bp) of the 5' flanking region of the feline IL-5 gene.

Sample Population

Blood mononuclear cells from a healthy cat.

Procedures

Cells were cultured, stimulated for 48 hours with concanavalin A, and harvested for RNA and DNA isolation. Recovered RNA was used in northern blot and reverse transcription-polymerase chain reaction analyses. Resulting cDNA was used for rapid amplification of 3' cDNA ends, dideoxy chain termination sequencing, and primer extension analysis.

Results

Full length cDNA was 838 bp, including a 402-bp open reading frame that encoded a precursor protein of 134 amino acids including a putative peptide signal of 19 residues. Homologies of the nucleotide and derived protein sequences between feline and human IL-5 cDNA were 72 and 71%, respectively. There also was homology between the human and predicted feline cytokines at amino acid positions that are critical for IL-5 receptor binding and signal transduction. The 5' flanking region of the feline gene was homologous to corresponding regions of the human (88%) and murine (72%) genes, and included putative transcriptional regulatory elements.

Conclusions and Clinical Relevance

Identification of feline IL-5 cDNA is an important step toward a detailed, fully comprehensive characterization of the mechanisms that may be operative in the pathogenesis of eosinophilic disorders in cats. The striking homology between the human and feline IL-5 genes suggests that cats can be used as animal models for human diseases characterized by eosinophil infiltration of tissues. (Am J Vet Res 1998;59:1263–1269)

Free access
in American Journal of Veterinary Research