Temporal regulation of cytokine mRNA expression in equine recurrent airway obstruction.

Acute and chronic inflammation of the airway remains an important health problem for equids. "Heaves" or recurrent airway obstruction (RAO) remains one of the most commonly diagnosed conditions affecting the lung of older horses in Europe and the United States. The typical clinical signs of RAO include non-productive coughing, serous nasal discharge, labored expiratory effort, and flaring of the nostrils. Auscultation of the lungs of the affected horse often reveals abnormal respiratory sounds, described as crackles and wheezes, throughout the area of the lung field. These clinical signs occur secondary to an inflammatory response that results in bronchospasm, excessive mucus production and airway obstruction. This inflammatory response is characterized by the presence of excessive mucus and inflammatory cells, primarily neutrophils, in the small airways. Most evidence suggests that RAO is the result of a pulmonary hypersensitivity to inhaled antigens. Exposure of affected horses to hay dust, pollens, and mold spores leads to neutrophil accumulation in the lung and bronchospasm. The identification of allergen-specific IgE in bronchoalveolar lavage (BAL) fluid and sera of affected horses supports the involvement of a late phase, IgE-mediated, hypersensitivity reaction in the pathogenesis of equine RAO. The production of IgE antibodies is regulated by the cytokines IL-4 and IL-13. Using a quantitative PCR method we have reported that horses with RAO exhibit a modified Type 2 cytokine response characterized by the production of IL-4 and IL-13 mRNA, but not IL-5 mRNA in BAL cells. Interferon-gamma mRNA was also elevated, suggesting a mixed response. While these results are consistent with equine RAO being the result of an aberrant Type 2 cytokine response to inhaled allergens, others have failed to find any evidence of elevated Type 2 cytokine mRNA in BAL from horses with "heaves". It is likely that these disparate results could be the result of differences in the clinical stage of the affected animals or the timing of sample collection. Here, we report a diverse pattern of cytokine gene expression when sampling a group of affected horses over a period of time.

A novel model for equine recurrent airway obstruction.

Equine recurrent airway obstruction (RAO; a term combining both chronic obstructive pulmonary disease (COPD) and summer pasture associated obstructive pulmonary disease (SPAOPD)) is one of the most common equine respiratory diseases with up to 50% of horses affected worldwide. The etiopathogenesis of RAO is unknown although pulmonary hypersensitivity to inhaled mold antigens may be involved. Recent work in our laboratory demonstrating elevated levels of IL-4 and IL-13 mRNA in the airways and peripheral blood of horses with RAO is consistent with an atopic component to RAO. Little is known regarding the earliest phases of RAO in horses. Here we describe the development of a novel airway model for equine RAO that utilizes ovalbumin-coated polystyrene beads for airway sensitization and challenge. Aerosol challenge of sensitized ponies with OVA-coated microbeads resulted in decreased airway compliance, increased percentage of lymphocytes and neutrophils in the bronchoalveolar lavage fluid, and evidence of a Th2 cytokine response in the bronchoalveolar cells. These results suggest that this approach may be useful in describing the initial stages of RAO development in the horse.

Increased dietary fat prevents sleep deprivation-induced immune suppression in rats.

BACKGROUND AND PURPOSE: Fatty acid composition of rodent diets can affect baseline immune function as measured in vitro and in vivo. Stress, in a variety of forms, can also affect immune function. Possible interaction between diet and other stressors has not been fully explored. We examined the interaction between sleep deprivation stress and dietary fatty acid composition in altering lymphocyte responses to mitogen stimulation. METHODS: Rats were fed diets containing various sources of fatty acids, then were subjected to sleep deprivation. Splenocytes were harvested and assayed for responsiveness to various mitogens, using a 72-h proliferation assay. RESULTS: Rats subjected to sleep deprivation experienced significant suppression of in vitro proliferative response to various mitogens. This immune suppression was dependent on duration of sleep deprivation. Feeding sleep deprived rats a diet enriched in fatty acids abrogated the effect of sleep deprivation. CONCLUSIONS: The fat content of rodent diets can have a marked effect on baseline and stress-modulated immune responses.

Altered immune responses to a heterologous protein in ponies with heavy gastrointestinal parasite burdens.

This study was performed to test the hypothesis that immunity to heterologous vaccination would improve when the parasites were removed. It was also expected that parasitised ponies would exhibit a biased Th2 cytokine response to KLH immunisation. Helminth parasites are common in horses even in the era of highly effective broad-spectrum antiparasiticides. These parasites have been shown to alter the outcome to heterologous immunisation in a number of host species. The effect of gastrointestinal parasites on heterologous vaccination has not been addressed in equids. In the current study, humoral, lymphoproliferative, and cytokine responses to a single i.m. injection of keyhole limpet haemocyanin (KLH) were compared between groups of ponies with high, medium or low gastrointestinal parasite burdens. Antibody levels determined by ELISA showed that animals with low levels of parasites had a trend toward increased KLH specific total immunoglobulin, IgG(T) and IgA compared to heavily parasitised ponies. Medium and heavily parasitised ponies demonstrated a trend toward reduced lymphoproliferative response to KLH that was not restored after the addition of interleukin-2 (Il-2). Cells from these ponies also produced significantly lower levels of IL-4 compared to lightly parasitised ponies. These data indicate that heavily parasitised ponies have uniformly decreased cellular and humoral immune responses to soluble protein immunisation. The mechanisms involved may have potential deleterious effects on standard vaccine protocols of parasitised equines.

Exercise alters the immune response to equine influenza virus and increases susceptibility to infection.

Equine influenza virus remains a major health concern for the equine industry in spite of ongoing vaccination programmes. Previous work has shown that the immune system of horses can be affected by strenuous exercise. The possible adverse consequence of exercise-induced alterations in lymphocyte responses measured in vitro was unknown. Here we demonstrate that subjecting vaccinated ponies to a 5 day strenuous exercise programme results in a significant suppression of their T cell-mediated immune response to equine influenza virus as measured by decreased lymphoproliferation and gamma interferon production measured in vitro. These same ponies also demonstrated increased susceptibility to influenza disease following a challenge exposure to the same strain of virus. Rested ponies that had received the same vaccine and challenge were completely protected from disease. Our results demonstrate that exercise-induced suppression of the equine immune response to influenza virus can be associated with an increased susceptibility to disease.

Mechanism of exercise-induced augmentation of lymphokine activated killer (LAK) cell activity in the horse.

Intense exercise affects various parameters of the immune system. The overall effect of exercise on immune function is dependent upon the physical condition of the subject, the intensity and duration of the exercise period, and the immune parameter assessed. Unconditioned horses subjected to a single bout of intensive exercise exhibit multiple alterations in immune function, including an augmentation of lymphokine activated killer (LAK) cell function. This increase in LAK cell activity is not due to an increase in circulating LAK precursors. While peripheral blood mononuclear cells from exercising horses exhibit greater responsiveness to IL-2, this is not due to an increase in IL-2 receptor expression. LAK cell generation in vitro is augmented by those catecholamines and neuropeptides which are produced during exercise, suggesting a direct effect of these compounds on LAK cell generation at a step post IL-2 receptor binding.

Induction of lymphokine-activated killer cells of equine origin: specificity for equine target cells.

The in vitro stimulation of peripheral blood mononuclear cells (PBMC) with interleukin 2 (IL-2) results in the development of potent cytotoxic effector cells, referred to as lymphokine-activated killer (LAK) cells. LAK cells are capable of lysing a wide variety of autologous, allogeneic and xenogeneic tumor cells. The exact mechanism of target cell recognition by LAK cells remains unknown. LAK cell activity has been reported for a variety of domesticated species except the horse. We report here that IL-2-stimulated equine PBMC, which fail to lyse either human or murine tumor cell lines, exhibit potent cytolytic activity against an equine tumor cell line, EqT8888. Cytolytic activity against the EqT8888 cells required 3 days of incubation with IL-2, was mediated primarily by T-cells, and was not restricted by major histocompatibility complex antigens. Though LAK activity could only be demonstrated using equine-derived target cells, xenogeneic targets could be lysed in a lectin-mediated cytotoxicity assay. The xenogeneic targets also failed to block LAK cell-killing of the EqT8888 cells in a cold-target competition assay. These results indicate that LAK cells in the horse appear to utilize a species-specific recognition mechanism during target cell lysis.

Cryptosporidiosis associated with immunosuppression attributable to distemper in a pup.

Cryptosporidiosis, giardiasis, trichomoniasis, and distemper were diagnosed in a 6-month-old female Siberian Husky pup. Poor growth rate, mucopurulent ocular and nasal discharges, and diarrhea were observed. Results of immunologic studies revealed decreased serum IgG concentration and undetectable serum IgA concentration. Cultured lymphocytes yielded a less-than-adequate response to mitogen stimulation. The serum also contained a factor that suppressed mitogen stimulation in control cultured lymphocytes.

Effect of serum on lymphocyte blastogenesis. 2. Characterization of disease-induced immunosuppressive factors by chromatography and molecular weight determination.

Numerous infectious and noninfectious diseases are associated with the appearance of suppressive serum lymphocyte immunoregulatory factors (SLIFs). The suppressive SLIFs in sera from clinically healthy dogs and from dogs with bacterial (staphylococcal, brucellar) or mycotic (blastomycotic) infections were further characterized by dialysis, fractionation by ultrafiltrations and HPLC (high performance liquid chromatography) sieving, by affinity chromatography on protein A-Sepharose columns, and by DEAE-cellulose ion exchange chromatography. Factors of various molecular weights and of various elution patterns from DEAE-cellulose and affinity chromatography columns were taking part in the suppressive action of the whole serum. The 'common' inhibitors present in all sera were in the molecular weight range of 28 to 35 Kd, whereas the disease-induced suppressive SLIFs were present in various molecular weight categories. 'Common' suppressor SLIFs and some SLIFs from dogs with staphylococcal infections were partially dialysable; suppressive SLIFs induced in dogs with generalized brucellosis and blastomycosis were not dialysable. Protein A bound suppressive SLIFs from two of three dogs with staphylococcal pyodermas. DEAE-cellulose chromatography gave variable elution patterns with different animal sera. It is concluded that various suppressive SLIFs contribute to the immunosuppressive effect of the whole serum and no disease-specific suppressive SLIF could be identified.

Blastogenesis-suppressing serum factors, decreased immunoglobulin A, and increased beta 1-globulins in Airedale Terriers with diskospondylitis.

In Airedale Terriers with diskospondylitis, immunologic tests revealed heat-stable blastogenesis-suppressing serum factors affecting primarily the effector (phytohemagglutinin-sensitive) lymphocytes; decreased serum concentrations of immunoglobulin A; and increased serum concentrations of undetermined beta 1-globulins. Data indicated that the dogs had decreased immunoglobulin A production and were immunosuppressed, which probably contributed to penetration of bacteria into the body and subsequent spreading to the disks. Eosinophilia and basophilia were also detected, which indicated a potential for a type-I hypersensitivity reaction that may have ameliorated the inflammatory reaction.

Lymphocyte transformation and humoral immune factors in Basenji dogs with immunoproliferative small intestinal disease.

Serum protein concentrations and 4 immunologic factors were determined in 5 Basenji dogs with immunoproliferative small intestinal disease. There was no correlation between the total serum proteins, immunoglobulin G and immunoglobulin M concentrations, and physical health status of the animals. The severity of clinical signs correlated roughly with decrease in albumin and increase in globulin concentrations. The main changes were detected in beta- and fast gamma-globulins. The total hemolytic complement levels were decreased in the 2 most severely affected animals below the minimal laboratory values observed in healthy animals. Alteration in the intrinsic responsiveness of lymphocytes to various mitogens did not correlate with progression in severity of the disease. Correlation between the appearance of blastogenesis-suppressing substances in serum and the severity of the disease was only partial: Sera (at 20% concentration) from the 2 most severely affected dogs completely suppressed blastogenesis induced by all 3 mitogens. The sera of 3 other dogs either did not suppress or suppressed only concanavalin A-induced mitogenesis and to a lesser extent phytohemagglutinin-induced mitogenesis without correlations to the overall clinical status. The disturbances of immunologic mechanisms were detected after the appearance of clinical disease, were not considered the cause of immunoproliferative small intestinal disease, may represent a manifestation of the secondary infection, and may contribute to aggravation of the clinical course.

Counterimmunoelectrophoresis (immunoelectroosmosis) and serum electrophoretic pattern in serologic diagnosis of canine blastomycosis.

Counterimmunoelectrophoresis (CIEP) with blastomyces and histoplasma antigens was used in a serologic study of 181 dogs clinically suspected of having blastomycosis and of 8 dogs with confirmed blastomycosis or histoplasmosis. Thirteen of the 181 dogs, positive by CIEP, were euthanatized, and the diagnosis was confirmed by cultivation and/or microscopic detection of Blastomyces dermatitidis. Additional CIEP-positive dogs were confirmed by staining of aspirates collected in vivo. Radiographic support for the diagnosis was reported in 4 other dogs in which histoplasmosis was excluded by a negative CIEP with histoplasma antigen. The precipitating antibody may disappear during the course of the disease, as it did in 1 dog treated with amphotericin B, but not cured. This dog reverted from CIEP-positive to CIEP-negative within 17 months of treatment (with a weak reaction after 10 months of treatment). The CIEP-detectable antibody was present only in 1 dog without a confirmation by histopathologic findings or cultivation among 24 well-documented cases and 181 total tested sera. The CIEP was more sensitive and specific than was the gel-diffusion precipitin test, eliminated the problems of anticomplementarity that often affected the results of complement-fixation tests with canine sera, and served well in detecting dogs with blastomycosis. Electrophoretic pattern of sera from CIEP-positive dogs with blastomycosis showed a decrease in albumin and an increase in alpha 2- and often in beta- and gamma-globulins, with a substantial decrease of the albumin/globulin ratio.

Serum proteins in healthy Basenjis and Basenjis with chronic diarrhea.

A serologic survey was conducted to characterize the electrophoretic patterns of serum proteins in healthy Basenji dogs (n = 137) and Basenjis with chronic diarrhea (n = 32). Serum protein electrophoresis values for Basenjis were similar to previously reported values from dogs of other breeds. Dogs with histologically confirmed lymphocytic-plasmacytic enteritis had a statistically significant (P less than 0.05) decrease in total protein, albumin, and albumin/globulin ratio. alpha 2-Globulin and gamma-globulin values were significantly increased in old dogs (9 years of age or older) with lymphocytic-plasmacytic enteritis. Although not statistically significant, gamma-globulin values were generally increased in Basenjis with lymphocytic-plasmacytic enteritis when compared with the values in age-matched clinically healthy Basenjis.

Frequency and duration of paratyphoid organism shedding by experimentally infected bobwhite quail (Colinus virginianus).

Four-week old bobwhite quail (Colinus virginianus) were experimentally infected with Salmonella urbana, S. infantis, S. newport, S. gaminara, S. braenderup, and S. litchfield. Rates of mortality varied from 0 to 50%. The rate of shedding of paratyphoid organisms varied from 14 to 100% for 18 or more days after infection. The maximum duration of shedding was 53 days by 12% of the quail infected with S. braenderup and the minimum duration was 18 days by 14% of the quail infected with S. litchfield.

Evaluation of secondary enrichment for detecting Salmonellae in bobwhite quail.

Secondary enrichment of cultures in tetrathionate-brilliant-green broth substantially increased Salmonella recovery over that achieved with primary tetrathionate-brilliant-green broth or primary selenite-cystine broth.

Infectious bronchitis and mixed infections of Mycoplasma synoviae and Escherichia coli in gnotobiotic chickens. I. Synergistic role in the airsacculitis syndrome.

The synergistic role of infectious bronchitis virus (IBV) and mixed infections of Mycoplasma synoviae (MS) and Escherichia coli (EC) in the airsacculitis syndrome was evaluated in gnotobiotic chickens. Relative air sac lesion score indexes, in descending order of severity, from various combinations of organisms were: 9.5-IBV, MS, EC; 6.8-IBV, EC; 4.5-IBV, MS; 2.7-IBV; and 0.5-MS, EC. Infectious bronchitis virus caused a mild fibrinous inflammation. M. synoviae combined with IBV increased heterophilic and follicular lymphoid infiltration and mortality. E. coli combined with IBV increased exudation and prolonged airsacculitis. Concentrations of fibrinogen, gamma globulin, and total plasma proteins were elevated significantly by combined infections of IBV, MS, and EC (P < 0.01).