A.D. Wilson

Detection of IgG and IgE serum antibodies to Culicoides salivary gland antigens in horses with insect dermal hypersensitivity (sweet itch)

We postulated that all horses exposed to the bites of Culcoides (midges) would have an antibody response to the antigen secreted in Culcoides saliva, but that IgE antibody would be restricted to allergic individuals. Using immunohistology on sections of fixed Culicoides, we have demonstrated the presence of antibodies in horse serum which recognise Culicoides salivary glands. Antibodies were detected in the serum of horses with insect dermal hypersensitivity and in the serum of normal horses exposed to Culicoides bites. In contrast, no antibodies were detected in serum from native Icelandic ponies which had not been exposed to Culicoides. Anti-salivary gland IgG antibodies were detected in serum from both allergic and healthy horses exposed to Culicoides. IgE antibodies were only detected in horses with signs of insect dermal hypersensitivity, they were not found in serum of healthy controls nor in the serum of horses with a history of hypersensitivity but in remission at the time of sampling. Using western blotting we confirmed the presence of antibodies to Culicoides antigens and demonstrated that individual horses react to different numbers of antigens. This paper demonstrates the ability of serum from allergic horses to detect Culcoides antigens and will enable further studies to isolate and characterise the allergens.

Report of the 3rd Havemeyer Workshop on allergic diseases of the horse, Hólar, Iceland, June 2007

Allergic diseases occur in most mammals, although some species such as humans, dogs and horses seem to be more prone to develop allergies than others. In horses, insect bite hypersensitivity (IBH), an allergic dermatitis caused by bites of midges, and recurrent airway obstruction (RAO), a hyperreactivity to stable born dust and allergens, are the two most prevalent allergic diseases. Allergic diseases involve the interaction of three major factors: (i) genetic constitution, (ii) exposure to allergens, and (iii) a dysregulation of the immune response determined by (i) and (ii). However, other environmental factors such as infectious diseases, contact with endotoxin and degree of infestation with endoparasites have been shown to influence the prevalence of allergic diseases in humans. How these factors may impact upon allergic disease in the horse is unknown at this time. The 3rd workshop on Allergic Diseases of the Horse, with major sponsorship from the Havemeyer Foundation, was held in Hólar, Iceland, in June 2007 and focussed on immunological and genetic aspects of IBH and RAO. This particular venue was chosen because of the prevalence of IBH in exported Icelandic horses. The incidence of IBH is significantly different between Icelandic horses born in Europe or North America and those born in Iceland and exported as adults. Although the genetic factors and allergens are the same, exported adult horses show a greater incidence of IBH. This suggests that environmental or epigenetic factors may contribute to this response. This report summarizes the present state of knowledge and summarizes important issues discussed at the workshop.

The immune response to dietary antigens and its influence on disease susceptibility in farm animals

Transient hypersensitivity reactions of the intestinal immune system to dietary antigens result in increases in enterocyte turnover and villous atrophy. These changes occur in the intestine of the post weaned piglet and precede the proliferation of E. coli and the development of post-weaning diarrhea. We therefore postulated that a transient cell mediated immune response to dietary antigens may increase susceptibility to disease. The interaction of dietary and microbial antigens upon the gut immune system has been investigated in mice and pigs and it has been shown that both exert powerful regulatory effects upon each other.

Virus-specific cytotoxic T cell responses are associated with immunity of the cottontop tamarin to Epstein-Barr virus (EBV)

The cytotoxic responses of peripheral blood lymphocytes from cottontop tamarins to in vitro re‐stimulation with autologous lymphoblastoid cell lines (LCL) were assayed. Lymphocytes from immune tamarins that had recovered from EBV challenge developed potent cytotoxicity for natural killer (NK) cell targets and for autologous LCL. The cytotoxicity for LCL targets was EBV‐specific, as B cell blasts uninfected with EBV were not killed. The cell lines could be maintained by repeated stimulation with LCL and the addition of IL‐2. Flow cytometry showed that they were T cell lines expressing CD2, CD3, CD4, CD8 and CD25. Dual‐colour flow cytometry revealed two subpopulations, one CD4+ CD8+ population and the other CD4− CD8+. After separation by magnetic cell sorting both subpopulations were shown to be cytotoxic and the CD4+ CD8+ fraction was also shown to be MHC class II‐restricted; the MHC restriction of the CD8+ subpopulation could not be determined. The unseparated T cells and both the subpopulations were able to inhibit LCL outgrowth in vitro. In contrast, PBL from naive tamarins stimulated by autologous LCL developed less NK cell cytotoxicity and little cytotoxicity for LCL. The cytotoxic response was enhanced at higher levels of LCL stimulation, but the cells were unable to inhibit LCL outgrowth in vitro. We conclude that cytotoxic responses capable of inhibiting LCL growth in vitro correlate with in vivo immunity in the tamarin model and provide a basis for understanding the mechanism of vaccine‐induced immune protection.

Depressed potential for interleukin-2 production following early weaning of piglets.

Spleen cells, but not mesenteric lymph node cells, from 3-week-old piglets abruptly weaned onto a soya-based diet, produced less interleukin-2 (IL-2) following non-specific activation with concanavalin A (Con A) than did cells from age- and litter-matched, unweaned controls. In contrast, the ability to express receptors for IL-2 was only marginally reduced. The effect on IL-2 production was most marked in animals weaned for as little as 24-48 h. Variation within groups increased with time after weaning, indicating differences between individuals in the longer-term effects of weaning. This finding may be due to endogenous production of steroids resulting in generalised impaired immune function or to retention of cells within intestinal sites owing to an active local immune response.

Immunology of the porcine gastrointestinal tract

The gastrointestinal immune system is presented with a contrasting array of antigens, ranging from harmless dietary components to highly pathogenic microorganisms. The mucosal immune system has the ability to recognise different groups of antigens and has evolved a battery of responses from which an appropriate response may be orchestrated. The question as to how the mucosal immune system categories antigens and selects a particular response is central to this process but it remains largely unanswered. The solution to this question is likely to hold the key to the development of safe and effective mucosal vaccines as well as suggesting methods for the prevention and control of allergic responses. Enteric diseases resulting from antigens (microbial and dietary) presented via the gastrointestinal tract are a major cause of morbidity and mortality. In addition to being of substantial economic importance, growing public awareness on animal welfare and food quality confirms the urgent need for new methods of disease control.

Increased IL-4 and decreased regulatory cytokine production following relocation of Icelandic horses from a high to low endoparasite environment

Insect bite hypersensitivity (IBH) is an IgE-mediated dermatitis of horses caused by bites of Culicoides spp. IBH does not occur in Iceland where Culicoides are absent. However, following importation into continental Europe where Culicoides are present, >or=50% of Icelandic horses (1st generation) develop IBH but <or=10% of their offspring born in Europe (2nd generation) do so. Recently, we showed that PBMC from 1st generation horses produce more IL-4 than 2nd generation horses. Since helminths and allergens induce Th2 responses, we investigated whether horses domiciled in Iceland are Th2-biased, and whether this is determined by helminth infection. We compared the parasite burden and T cell responses between Icelandic horses living in Iceland or Switzerland. Horses in Iceland have higher faecal egg counts, higher tapeworm-specific IgG(T) levels and higher total serum IgE levels than horses in Switzerland. Nevertheless, horses in Iceland displayed a low proportion of IL-4-producing cells in PBMC cultures after polyclonal or parasite extracts stimulation. No IL-4-producing cells were found in PBMC from horses after stimulation by Culicoides extract. Addition of anti-IL-10 and anti-TGF-beta1 to PBMC cultures of horses in Iceland increased the proportion of IL-4-producing cells after polyclonal or parasite antigens stimulation but not stimulation with Culicoides extract. This paralleled the high levels of IL-10 and TGF-beta1 found in supernatants from PBMC cultures of horses in Iceland. Collectively, horses living in Iceland have a high parasite burden but low IL-4 production. This supports the hypothesis that heavy helminth infections have a suppressive effect on IL-4 production mediated by IL-10 and TGF-beta1.

IgE, IgGa, IgGb and IgG(T) serum antibody levels in offspring of two sires affected with equine recurrent airway obstruction.

Equine recurrent airway obstruction (RAO) is a chronic lower airway disease of the horse caused by hypersensitivity reactions to inhaled stable dust, including mould spores such as Aspergillus fumigatus. The goals of this study were to investigate whether total serum IgE levels and allergen-specific IgE and IgG subclasses are influenced by genetic factors and/or RAO and whether quantitative trait loci (QTL) could be identified for these parameters. The offspring of two RAO-affected sires (S1: n=56 and S2: n=65) were grouped by stallion and disease status, and total serum IgE levels and specific IgE, IgGa, IgGb and IgG(T) levels against recombinant Aspergillus fumigatus 7 (rAspf7) were measured by ELISA. A panel of 315 microsatellite markers covering the 31 equine autosomes were used to genotype the stallions and their offspring. A whole-genome scan using half-sib regression interval mapping was performed for each of the IgG and IgE subclasses. There was no significant effect of disease status or sire on total IgE levels, but there was a significant effect of gender and age. rAspf7-specific IgGa levels were significantly higher in RAO-affected than in healthy horses. The offspring of S1 had significantly higher rAspf7-specific IgGa and IgE levels than those of S2. Five QTLs were significant chromosome-wide (P<0.01). QTLs for rAspf7-specific IgGa and IgE were identified on ECA 1, for rAspf7-specific IgGa and IgGb on ECA 24 and for rAspf7 IgGa on ECA 26. These results provide evidence for effects of disease status and genetics on allergen-specific IgGa and IgE.

Maternal transfer of IgE and subsequent development of IgE responses in the horse (Equus callabus)

Immunoglobulin E (IgE) mediates the immune response to parasites, but can also cause allergies. In humans maternal IgE is not transferred to cord blood and high levels of cord blood IgE are associated with subsequent allergy. In horses, both maternal IgG and IgE are transferred via colostrum; the IgE levels in the mares serum, the colostrum and the foals serum are correlated but the consequences of IgE transfer to foals are not known. By about 6 weeks of age the levels of IgE in foal serum have dropped to a nadir, at 6 months of age the level of IgE has risen only very slightly and is no longer correlated with the levels seen at birth, IgE(+) B-cells could be detected in lymphoid follicles of some foals at this age. Surprisingly, the levels of total IgE detected in a foals serum at 6 months of age are significantly correlated with the level in its serum at 1, 2 and even 3 years of age suggesting that by 6 months of age the foals are synthesizing IgE and that a pattern of relatively higher or lower total serum IgE has been established. The neonatal intestinal mucosa contained connective tissue mast cells which stained for bound IgE in foals up to 9 weeks of age but not mucosal mast cells, thereafter, the intestinal mast cells were IgE negative until 6 months of age. IgE antibodies to Culicoides nubeculosus salivary antigens were detected in Swiss born foals from imported Icelandic mares allergic to Culicoides spp. yet the foals showed no signs of skin sensitization and such second generation foals are known not to have an increased risk of developing allergy to Culicoides. Overall this evidence suggests there is a minimal effector role of maternal IgE also that maternal IgE has waned prior to the onset of IgE synthesis in foals and does not support maternal priming of IgE responses in foals. Furthermore the total levels of IgE in any given foal are seen to be relatively high or low from soon after the onset of IgE synthesis, and most likely they are determined by genetic factors.

PROBIT: weighted probit regression analysis for estimation of biological activity.

PROBIT estimates biologic activity by weighted probit regression analysis and comparison with a known standard. The program is intended to speed up calculation of the concentration of cytokines in large numbers of cell supernatants. Data are input from either sequential or free-form text files created with a spreadsheet or text editor. This allows transfer of data from a beta-counter equipped with a suitable terminal without retyping. Results are displayed and/or printed as relative potency (% of standard) and 50% effective dose (ED50).