Cellular and chemical mediators of type 1 hypersensitivity in calves infected with Ostertagiaostertagi: Mast cells and eosinophils

Abomasal mucosal mast cell and eosinophil accumulation was morphometrically evaluated in 26 Holstein steers after natural or experimental infection with Ostertagiaostertagi. Results showed that following infection, accumulation of mast cells and eosinophils in abomasal tissue was dependent on infection pattern. Eosinophilia was greater in steers with type 1 ostertagiosis, while mastocytosis was more pronounced in steers with type 2 ostertagiosis.     

Studies of stage-specific immunity against Trichostrongylus colubriformis in sheep: immunization by normal and truncated infections.

Sheep immunized with multiple normal infections of 30,000 Trichostrongylus colubriformis larvae (T.c. L3) suppressed the fecundity, establishment and survival of adoptively transferred adult worms, showing that these parasites were susceptible to the effects of host immunity. When sheep were immunized by four 'truncated' larval infections of 4, 7 or 10 days' (d) duration with 10(5) T.c. L3, animals given 4 x 4d infections were susceptible to challenge, whereas sheep given 4 x 7d and 4 x 10d infections were significantly protected. A serial analysis of the rejection of T. colubriformis from nine sheep given 5 x 7d infections revealed that the challenge larval infection given intraduodenally was expelled within 3 days after challenge (DAC). However, another five of these sheep only rejected around 50% of transferred adult worms by 21 DAC when compared with control animals. The results indicate that stage-specific antigens produced by early L3 and L4 stages of T. colubriformis effectively immunize sheep against a larval challenge but appear less reliably protective against adult worms.     

Reactions of cells of nasal and sinusoidal mucosa of goats and sheep naturally infected by Oestrus ovis Linné 1758 (Diptera: Oestridae)]

OEstrosis is a very common myiasis of sheep and goats in mediterranean and tropical countries. Goats are suitable hosts for OEstrus ovis but the parasitic burden remain lower than in sheep. Cellular responses (mucous and serous mast cells, eosinophis and globules leucocytes) were measured in 30 infected and 30 non infected sheep and in 23 infected and 24 non infected goats. The presence of OEstrus ovis larvae led to an important inflammatory response in sheep and in goats as well. Furthermore, the intensity of the cellular response correlated with the larva burden, specially with globules leucocytes and eosinophils. Nevertheless, huge differences occurred between sheep and goats responses even in similar larval burden range. Infected sheep showed larger counts of mucous mast cells than goats, the differences were smaller in serous mast cells and eosinophils and no difference was detected in globules leucocytes (intraepithelial mast cells) counts between the two hosts species. These results were compared with those obtained in gastro-intestinal strongyles infections.     

Studies on the role of mucus and mucosal hypersensitivity reactions during rejection of Trichostrongylus colubriformis from the intestine of immune sheep using an experimental challenge model.

Nematode-naive sheep and sheep immunised by truncated infections with Trichostrongylus colubriformis were fitted with intestinal cannulae to allow administration of challenge infection and collection of intestinal fluids. Sheep were slaughtered at various times after challenge and the distribution of larvae along the small intestine was determined. Results showed that immune sheep had significantly fewer larvae in their intestines and that some sheep could expel the challenge infection within 2 h. Mucus samples from immune sheep contained increased parasite-specific antibody, histamine and anti-parasite activity as measured by larval migration inhibition assay. Higher levels of antibody and histamine were seen in intestinal fluids of immune sheep after challenge. Immunisation of sheep by truncated infections stimulated serum IgE and resulted in significantly higher numbers of IgE-positive cells in gut tissue sections before challenge and at 2 h and 24 h after challenge. Immune sheep also had greater numbers of mucosal mast cells and globule leucocytes after challenge, compared with naive sheep. When challenge larvae were mixed with mucus from immune sheep and infused back into naive recipient sheep, there was a distinct displacement of the larval population towards the distal part of the intestine, compared with the profile of larval establishment after infusion with mucus from naive sheep. These results are further evidence for an immediate hypersensitivity reaction in the intestine of immune sheep, where challenge larvae are expelled within 2 h and confirm the direct anti-larval properties of mucus. The cannulated-sheep challenge model described here will be a useful tool to unravel the mechanism of larval rejection from immune sheep and could lead to novel therapies.     

Ultrastructural aspects of the host cellular immune response to Taenia crassiceps metacestodes.

When three Taenia crassiceps metacestodes were injected intraperitoneally into C3H mice primed by previous subcutaneous inoculation of metacestodes, larvae which were resistant to early immune damage by the humoral response were encapsulated by host cells and rejected. Initially, normal larvae were encapsulated primarily by eosinophils and macrophages. In the early stages of encapsulation, both cell types showed severe degenerative changes and disruption of cell membranes, but there was no evidence of tegumental damage to the encapsulated larvae. Later, mast cells appeared in the capsules surrounding the larvae. After mast cells became common, all of the cell types present were normal, and damage to the larval Tegument became apparent. Ultimately, interaction of eosinophils, mast cells, macrophages, and lymphocytes resulted in death of the encapsulated larvae. These results suggest that larvae may secrete substances toxic to host cells, and that mast cells are necessary for rejection of larvae.     

The isolation and purification of a proteinase with chymotrypsin-like properties from ovine mucosal mast cells

A mast cell granule proteinase was purified from isolated ovine mucosal mast cells by cation exchange chromatography, which defined the conditions for enzyme purification from sheep gastric mucosae. Antibodies raised against the proteinase were used in subsequent purification procedures which yielded 78 micrograms of enzyme per 5 g wet wt of abomasal tissue. Immuno-histochemistry confirmed that mucosal mast cells were the source of the enzyme. The proteinase had chymotrypsin-like esterase activity, with a molecular weight between 19,000 and 25,000.     

IgE-Independent Activation of Human Mast Cells Indicates their Role in the Late Phase Reaction of Allergic Inflammation

Mast cells are tissue dwelling cells that have a clear-cut pathologic role in allergy. Besides that, they participate in several chronic inflammatory conditions, helminitic parasitosis, and in some solid tumor reactions, but also in physiological situations, such as wound healing and innate immunity. Mast cells produce and release various mediators after activation induced by either IgE-dependent or IgE-independent mechanisms. Although much information has been gathered on the immunological (IgE-dependent) mast cell activation both in vivo and in vitro, not much is known about the non-immunological (IgE-independent) activation particularly in human mast cells. Mast cell IgE-independent activation is mediated through Gi3alpha which has been identified in rat mast cells as the pertussis toxin (Ptx)-sensitive heterotrimeric G protein that interacts with cationic secretagogues inducing PLC-independent mast cell exocytosis. Mast cell IgE-independent activation in allergy most likely occurs when mast cells encounter eosinophils, the main inflammatory cells that persist throughout the late and chronic phases of the allergic reaction. This review summarizes the influence of eosinophils on mast cell activation demonstrating that IgE-independent activation has a significant role in pathophysiological processes.     

Ostertagia ostertagi: excretory secretory chemotactic substance from infective larvae as cause of eosinophil locomotion

The infective (third stage) larva of Ostertagia ostertagi produced an excretory secretory substance that is chemotactic for bovine eosinophils. The eosinophil chemotactic substance was present in supernatants of larval cultures within 6 hr of incubation in Eagle's Minimum Essential Medium containing 25 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid. The substance responsible for eosinophil chemotaxis has activity in small amounts, a molecular weight (MW) greater than 2000, and is heat labile at 100 C. Bovine eosinophils appeared to have a receptor for the chemotactic excretory secretory substance which is either identical or structurally similar to the previously described eosinophil chemotactic substance present in soluble third stage larval extracts. The larval substance may cause eosinophil accumulation in the abomasal tissue of cattle with ostertagiasis.     

Rapid improvement of immunity to Teladorsagia circumcincta is achieved through a reduction in the demand for protein in lactating ewes

Protein supplementation can improve the resistance to parasites of periparturient ewes, as indicated by reduced nematode egg excretion and worm burdens. However, the rate at which this improvement can occur is largely unknown. We investigated the rate of improvement by assessing temporal changes in faecal egg counts after we experimentally reduced nutrient demand. Three groups of nine pregnant ewes each were trickle infected with Teladorsagia circumcincta from day(-70) to day(16) (parturition is day0). Two groups of twin-rearing ewes were fed at 0.8 (L22) or 1.2 (H22) times their assumed metabolizable protein requirements, and a third group was fed the same daily food allowances as L22 ewes, but one of their lambs was removed on day10 (L21). Ewes were slaughtered on day21 to assess worm burdens, in vitro larval establishment on abomasal explants, and mucosal inflammatory cells. Faecal egg counts of L22 ewes were higher than H22 ewes throughout lactation. After the removal of one lamb, faecal egg counts of L21 ewes decreased within 5 days to levels similar to H22 ewes. Relative to L22 ewes, L21 and H22 ewes had lower worm burdens, parasite per capita fecundity and in vitro establishment rates of both T. circumcincta and Haemonchus contortus. Mucosal mast cell and eosinophil counts were similar for all ewes, but H22 ewes had higher globule leukocyte counts than L22 and L21 ewes. The data suggest that a reduction in protein demand can rapidly improve periparturient immunity to T. circumcincta. This may be associated with increased parasite expulsion, reduced fecundity and non-parasite specific reduction of in vitro larval establishment.     

The effect of vaccinating infection during pregnancy and dietary protein supply on the peri-parturient immune response of sheep to infection with Teladorsagia circumcincta and Trichostrongylus colubriformis larvae

It is well established that dietary protein supply can influence the peri-parturient breakdown of immunity to nematode parasites but there is no information on the importance of exposure to nematode larvae during pregnancy for this response. We investigated this by exposing housed pregnant sheep, scanned as carrying two lambs, to a vaccinating infection with a trickle mixed infection of Teladorsagia circumcincta and Trichostrongylus colubriformis larvae (L3) or to no infection during weeks − 9 to − 4 relative to parturition. At the beginning of week − 3 all sheep were treated with anthelmintic to remove any vaccinating worm burden and from week − 2 to week +6 received a trickle challenge infection with the same nematodes. Within each vaccinating treatment there were two nutritional treatments (no. = 20 per subgroup) designed to provide 1.5 or 1.0 and 1.3 or 0.8 of metabolisable protein (MP) requirement during pregnancy and lactation, respectively. Five ewes were necropsied during weeks +1 and +3 to measure worm burdens and mucosal inflammatory cells and the remainder maintained until week +6. Serum levels of total, IgA and IgE antibodies against L3 antigen of each nematode were measured.Scanning errors and lamb losses resulted in some ewes carrying and/or rearing only one lamb. Numbers of lambs reared was therefore introduced as a treatment effect. Vaccinating infection delayed the peri-parturient rise in faecal egg count (FEC) by an average of 2 weeks but its effect on FEC during the first 6 weeks of lactation was smaller and less persistent than that of dietary MP supply and single- v. twin-suckling.Populations of both nematodes were lower in association with high MP supply, vaccination and single suckling. These changes were associated with increases in numbers of mucosal mast cells (MMC) as a result of both increased MP supply and vaccination. Evidence for a more rapid return of host ability to limit populations of the abdominal nematode T. circumcincta than of the intestinal nematode T. colubriformis was associated with fewer eosinophils and more globule leucocytes (GL) in abomasal than in intestinal tissue.None of the serum antibody isotypes was affected by dietary protein supply. Total and IgA antibodies were maintained by a current larval (vaccinating) intake. IgA titres, however, increased progressively during pregnancy, especially in twin-bearing ewes. IgE titres appeared to be sensitive primarily to the reproductive cycle itself, peaking around parturition.This work supports the conclusion that availability of MP supply influences the recruitment and activity of cells of the immune armoury of the gastro-intestinal tract to nematode parasites. The precise outcome may differ with site and/or nematode species.     

Ileal mucosal mast cell, eosinophil, and goblet cell populations during Hymenolepis diminuta infection of the rat.

The population dynamics in the enteric connective tissues of eosinophils, mucosal mast cells (MMC), and in the mucosal epithelium of goblet cells were examined morphometrically in fixed ileal tissue of outbred Sprague Dawley rats during the first 32 days of infection with the tapeworm Hymenolepis diminuta. MMC and eosinophils were present in the lamina propria and submucosa; however, only eosinophils were also present in the muscularis externa. Eosinophilic infiltrate was first observed in the lamina propria at 15 days postinfection (dpi) and the numbers of eosinophils remained elevated through 32 dpi. Initial mucosal mastocytosis was detected on 6 dpi and MMC numbers continued to rise over the study period without reaching a plateau. Goblet cell hyperplasia occurred only at 32 dpi. In contrast to some intestinal nematode infections where these same 3 cell types are associated with the host's expulsion responses, H. diminuta is not lost by a rapid host response in the outbred Sprague Dawley rat strain used in these experiments. We suggest that either the induction of hyperplasia of these host effector cells in ileum tissue during H. diminuta infection is not capable of triggering parasite rejection mechanisms, or the function of the induced hyperplasia is necessary for some as yet unassociated physiological or tissue architecture change in the host's intestine.     

Experimental skin lesions from larvae of the bot fly Dermatobia hominis

Skin biopsies from larvae of Rattus norvegicus, experimentally infested with Dermatobia hominis (Linnaeus Jr) (Diptera: Cuterebridae), were processed for histopathological studies. Two days after infestation, the first-stage larvae (L1) were located deep in the dermis, surrounded by an inflamed area infiltrated predominantly by neutrophils. On the fourth day a thin necrotic layer could be seen close to the larvae, surrounded by large numbers of neutrophils, lymphocytes, macrophages with a few eosinophils and mast cells. A small warble was formed after the fourth day, increasing in size until the seventh day, when the L1 moulted to the second-stage larva (L2). The inflammatory process continued with increasing numbers of neutrophils, macrophages, lymphocytes, eosinophils and mast cells invading the area, as well as the proliferation of fibroblasts and endothelial cells and the appearance of a few localized haemorrhages. After 18-20 days, the L2 moulted to the third-stage larva (L3), when a few plasma cells could be seen in the inflamed area. At 25-30 days there was a reduction in the necrotic layer, as well as in the number of neutrophils and lymphocytes, although large amounts of eosinophils, plasma cells, and collagen fibres were seen. The L3 usually left the host after 30 days. Two days later, the larval cavity was reduced, mast cells infiltrated the region and collagen fibre production were increased. After 7 days, an intense infiltration of plasma cells and scattered necrotic areas could be seen. A scar formed after 10 days. This study showed the laboratory rat to be a suitable model for studies of D. hominis infestation.     

Studies of stage-specific immunity against Trichostrongylus colubriformis in sheep: immunization with adult parasites.

Merino sheep immunized by the adoptive transfer of adult T. colubriformis for 8 weeks were significantly protected against a challenge infection of 20,000 larvae. Two additional groups of sheep received a primary infection of 9000 adult worms which were allowed to persist for 14 weeks before one group was drenched. When both groups were challenged 10 days later with 30,000 larvae, serial necropsies of these and naive sheep revealed that worm rejection did not occur until 7-10 days after challenge. By comparison with the rapid rejection of larval challenges from sheep immunized with normal primary infections, the results suggest that the antigens which elicited rejection in these experiments are stage-specific and were only present or synthesized in sufficient quantities when parasites had developed for 1 week.     

Local eosinophil- and mast cell-related responses in abomasal nematode infections of lambs

Abstract Eosinophil numbers in peripheral blood and eosinophil potentiating activity(EPA) and sheep mast cell protease (SMCP) in efferent gastric lymph were monitored in lambs during infections with Ostertagia circumcincta . Worm burdens, eosinophil numbers in bone marrow, abomasal mucosa and gastric lymph node, as well as mast cell numbers and SMCP concentrations in mucosa and mucus, were determined in post mortem samples. In naive lambs, high and relatively uniform worm burdens were present 10 days after primary infection and these were associated with only mild blood and tissue eosinophilia. By day 21 worm burdens were markedly lower and more variable. There was more evidence of eosinophil and mast cell accumulation in mucosa, and numbers in bone marrow were also higher than on day 10. However, neither EPA nor SMCP were detectable in lymph. By contrast, EPA and SMCP were present in substantial amounts in draining lymph within 48 h of challenge (secondary) infection of previously exposed lambs. EPA was inversely related to worm burdens recovered on day 10, as were abomasal mucosal and mucus SMCP concentrations. Elevated eosinophil numbers were also consistently detected in blood, bone marrow, mucosa and gastric lymph node. The results suggest that host immune defence against secondary, but not primary, exposure to O. circumcincta involves a rapidly mobilised local inflammatory component.     

Secretion of anti-parasite substances and leukotrienes from ovine gastrointestinal tissues and isolated mucosal mast cells

The presence of larval migration inhibitory (LMI) compounds in the gastrointestinal mucus of nematode resistant sheep has been shown previously to be associated with increased numbers of gastrointestinal mucosal mast cells (MMC) and globule leukocytes (GL). This experiment was designed to determine if LMI compounds were secreted by MMC/GL in response to nematode antigenic challenge and if so, could secretion account for levels observed in mucus. Rommey sheep were immunized by repeated cycles of infection with Trichostrongylus colubriformis or Haemonchus contortus larvae and anthelmintic treatment. After slaughter, gastrointestinal tissue was taken for examination of histology and mucus anti-parasite activity. Segments of small intestine were ligatured to form sacs which were incubated with exsheathed nematode larvae or larval excretory/secretory antigens. Tissue slices from small intestine or abomasum were also incubated with nematode larvae or antigens. After homologous challenge, levels of leukotrienes secreted into small intestinal tissue sacs were significantly higher than levels in heterologously challenged sacs or unimmunized sheep intestinal sacs challenged with larvae of any nematode species (279.4±33.7, 141.0±27.8 and 39.5±15.2 ng h⁻¹ respectively). Tissue slices gave a similar pattern of leukotriene secretion. LMI activity was also significantly elevated in intestinal sacs from immunized sheep challenged homologously with nematode larvae or antigen (64±10 and 68±14% respectively cf. heterologous challenge 32±10% and unimmunized sheep sacs 15±6%). Histological examination of abomasal and small intestinal sections showed that immunized sheep had significantly greater numbers of MMC/GL than unimmunized sheep. MMC/GL isolated and purified from immunized sheep secreted leukotrienes and compounds having LMI activity when cultured with homologous nematode larvae or antigens. Secretion of leukotrienes and molecules having LMI activity from MMC/GL could account for the levels of these substances observed in small intestinal mucus.     

IL-6 is required for protective immune responses against early filarial infection

IL-6 has a wide range of biological activities that includes anti- and pro-inflammatory aspects. In this study, we investigated the role of IL-6 in immune responses to the rodent filarial nematode Litomosoides sigmodontis, a model for human filarial infections. IL-6^?/? mice had a significantly increased worm burden after natural infection compared with wild type controls at early time points p.i. Given that the worm burden in IL-6^?/? mice was already increased at the time point the infective larvae reached the pleural cavity, immune responses that may facilitate the migration from the site of infection (skin) via the lymphatics to the pleural cavity were analysed. Increased vascular permeability may facilitate larval migration, but blocking of histamine receptors had no effect on worm burden and vascular permeability was similar between IL-6^?/? mice and wild type controls. In contrast, blocking mast cell degranulation reduced the worm burden in IL-6^?/? mice partially, suggesting that release of mast cell-derived mediators improves larval migration to some degree. Protective immune responses within the skin were involved, as bypassing the skin barrier by inoculating infective L3s subcutaneously resulted in a comparable worm recovery in both mouse strains. Analysis of the cellular composition by flow cytometry and PCR array in the skin after exposure to filarial extract or L3s, respectively, indicate that the absence of IL-6 results in a delayed recruitment of neutrophils and macrophages to the site of initial infection. These results demonstrate that IL-6 is essentially involved in protective immune responses within the skin that impair migration of infective L3s.     

Hypergastrinaemia, abomasal bacterial population densities and pH in sheep infected with Ostertagia circumcincta.

Serum gastrin and pepsinogen concentrations, food intake, abomasal pH and abomasal aerotolerant and anaerobic bacterial populations were measured in sheep infected with Ostertagia circumcincta to search for links between hypergastrinaemia, food intake and changes in the abomasal environment. Abomasal pH and serum gastrin and pepsinogen concentrations were elevated in each of five sheep infected via abomasal cannulae with 150000 exsheathed larval stage three, followed 11 days later by 100000 sheathed larvae given intraruminally. Unparasitised abomasa contained aerotolerant bacterial population densities of between 10(3) and 10(6) cells ml(-1) and these did not change significantly following parasitism. In contrast, anaerobic bacterial population densities increased markedly by about 10(4)-fold following parasitism. Anaerobic numbers changed rapidly when abomasal pH increased from 2.5 to 3.5. At pH 4 and above, anaerobic bacterial numbers approached levels expected in rumen contents but parameters other than pH did not relate to bacterial numbers. Brief periods when serum gastrin was lower than expected, coinciding with raised abomasal pH, were not explicable by increased bacterial numbers. Food intake, which decreased for a variable period from around Day 5 p.i., correlated poorly with serum gastrin concentration, suggesting hypergastrinaemia is not the sole cause of anorexia in parasitised animals. The survival of substantial numbers of rumen bacteria in the abomasum at only slightly raised pH may significantly lower the bacterial protein available to the sheep.     

Three surface antigens dominate the mucosal antibody response to gastrointestinal L3-stage strongylid nematodes in field immune sheep

Although gastrointestinal nematode parasites are a major human and veterinary health problem, little is known about how the host is sometimes able to mount an effective immune rejection response. In previous work, we identified a carbohydrate larval surface antigen (CarLA) as the target of mucosal antibodies that can elicit rejection of Trichostrongylus colubriformis L3s in sheep. Here we characterise the natural mucosal antibody responses to L3s from three major strongylid gastrointestinal parasites of sheep, Trichostrongylus colubriformis, Haemonchus contortus and Teladorsagia circumcincta. The mucosal antibody repertoire of naturally field-immune sheep was displayed on bacteriophage as single-chain antibodies (scFvs) and phage were selected for the ability to bind to the surface of living L3s of the three nematode species. All nematode-binding scFvs were found to recognize one of three different antigen classes that are each found in the three strongylid species. These three antigen classes appear to represent all of the major antigens recognized on Western blots by pooled mucosal antibodies from field-immune sheep. One of the antigen classes is a heterogeneous, high molecular weight molecule that is protease-sensitive. The scFvs recognizing this surface antigen also recognize a similar antigen in all strongylids tested. A second antigen class is a protease-insensitive, low molecular weight antigen found only in sheaths and scFvs recognizing this antigen cross-react with a similar molecule found in all strongylids tested. The third surface antigen class is CarLA and all of the anti-CarLA scFvs obtained from the field-immune sheep repertoire were specific to L3s of only one species and often recognized only a subset of the worms. Thus three different L3-stage surface antigens, two that lack a protein component, dominate the natural mucosal antibody response to L3-stage gastrointestinal strongylid nematodes in sheep.     

Digital gene expression analysis of gastrointestinal helminth resistance in Scottish blackface lambs

Digital gene expression (DGE) analysis offers a route to gene discovery which by-passes the need to develop bespoke arrays for nonmodel species, and is therefore a potentially valuable tool for molecular ecologists. Scottish blackface sheep, which vary in resistance to the common abomasal parasitic nematode Teladorsagia circumcincta, were trickle-infected with L3 larvae over 3 months to mimic the natural progression of infection. DGE was performed on abomasal lymph node tissue after the resolution of infection in resistant animals. Susceptible (low resistance) animals showed a large number of differentially expressed genes associated with inflammation and cell activation, but generally few differentially regulated genes in either the susceptible or the resistant group were directly involved in the adaptive immune function. Our results are consistent with the hypothesis that both resistance and susceptibility are active responses to infection and that susceptibility is associated with dysfunction in T cell differentiation and regulation.