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.     

Proteomic analysis of the abomasal mucosal response following infection by the nematode, Haemonchus contortus, in genetically resistant and susceptible sheep

Sheep have a variable ability to resist gastrointestinal nematode infection, but the key factors mediating this response are poorly defined. Here we report the first large-scale application of quantitative proteomic technologies to define proteins that are differentially abundant between sheep selectively bred to have an enhanced (resistant) or reduced (susceptible) ability to eliminate nematodes. Samples were collected from the abomasal mucosa three days after experimental challenge with the nematode, Haemonchus contortus. This timing reflects the initial interaction of host and parasite, and the tissue represents the immediate interface. We identified and quantified more than 4400 unique proteins, of which 158 proteins showed >1.5 fold difference between the resistant and susceptible sheep. Trefoil factor 2, a member of RAS oncogene family (RAP1A) and ring finger protein 126 were amongst the proteins found to be highly abundant in the abomasal surface of resistant sheep, whereas adenosine deaminase and the gastrokine-3 like precursor were found at higher levels in susceptible sheep. Construction of gut proteome interaction networks identified mitochondrial function and energetic partitioning as important components of an effective nematode eliminating response. The differentially abundant proteins may be useful targets for phenotypic tests that aim to identify sheep with an enhanced ability to resist nematode infection.     

Changes in protein expression in the sheep abomasum following trickle infection with Teladorsagia circumcincta

Continual low-level exposure of sheep to the helminth Teladorsagia circumcincta elicits a temporary protective immunity, where factors in the immune abomasal mucosa prevent penetration of infective larvae, but which is essentially lost within 6 weeks of cessation of parasite challenge. Here, a proteomic approach was used to identify proteins that are differentially regulated in immune compared to na?ve sheep, as potential key mediators of immunity. Six na?ve sheep and 12 sheep trickle-infected with T. circumcincta were treated with anthelmintic, and the na?ve (control) and 6 immune sheep were killed 7 days later. The remaining 6 sheep (immune waning) were killed 42 days after anthelmintic treatment. Abomasal tissue samples were subjected to 2D-gel electrophoresis and densitometric analysis. Selected spots (n=73) were identified by peptide mass fingerprinting and confirmatory Western blotting was carried out for 10 proteins. Spots selectively up-regulated in immune versus control, but not immune waning versus control sheep, included galectin-15 and thioredoxin, which were confirmed by Western blotting. In immune sheep, serum albumin was significantly down-regulated and albumin proteolytic cleavage fragments were increased compared to controls. Unexpectedly, albumin mRNA was relatively highly expressed in control mucosa, down-regulated in immune, and was immunolocalized to mucus-producing epithelial cells. Thus we have identified differential expression of a number of proteins following T. circumcincta trickle infection that may play a role in host protection and inhibition of parasite establishment.     

The key components of resistance to Ostertagia circumcincta in lambs

One of the most remarkable features of parasitic infections in general, and Ostertagia circumcincta infection of sheep in particular, is the extensive variation among hosts in resistance to infection, as assessed by parasite burdens and production of eggs or infective larvae. Here Mike Stear, Michael Park and Stephen Bishop describe the factors that account for the variation among animals within a flock, including dam, sire, sex, date of birth and history of exposure to infection. There are no detectable genetic effects in lambs less than three months old. Genetic control of an acquired response develops in two stages: first, a reduction in the average egg production per worm, which is associated with the development of a parasite-specific local IgA response; and second, control of worm burden, which is associated with the production of globule leukocytes in the abomasal mucosa.     

Isolation and characterization of a novel inducible mammalian galectin

A novel mammalian galectin cDNA (ovgal11) was isolated by representational difference analysis from sheep stomach (abomasal) tissue infected with the nematode parasite, Haemonchus contortus. The mRNA is greatly up-regulated in helminth larval infected gastrointestinal tissue subject to inflammation and eosinophil infiltration. Immunohistological analysis indicates that the protein is localized in the cytoplasm and nucleus of upper epithelial cells of the gastrointestinal tract. The protein is also detected in mucus samples collected from infected abomasum but not from uninfected tissue. The restricted and inducible expression of ovgal11 mRNA and limited secretion of the protein support the hypothesis that OVGAL11 may be involved in gastrointestinal immune/inflammatory responses and possibly protection against infection.     

Effects of a series of infections of Ostertagia circumcincta on gastric secretion of sheep.

Sheep which had been either previously infected with 3. circumcincta or maintained worm-free, were surgically prepared with separated fundic pouches and abomasal cannulae and subsequently infected with 20,000 O. circumcincta larvae three times weekly. A reduction in food intake and increases in total acid output from the pouches and plasma pepsinogen levels were evident in both groups of sheep 4 days after repeated infections commenced; effects which increased in severity after 12 or more days. Except for a transient period of slight failure, previously infected sheep retained the capacity to acidify their abomasal contents whereas previously worm-free sheep lost this capacity. These changes were reversed between 2 and 7 days after treatment with thiabendazole (88 mg.kg-1). Secretory capacity of the fundic pouches was tested with histamine (40 mug.kg-1), the histamine antagonist (burimamide 8 mg.kg-1) and atropine (100 mug.kg-1). Ostertagiasis reduced or abolished the stimulatory effects of histamine. An increase in secretion volume and acid output was obtained after food was freshly provided, even though as little as 25 gm was consumed. Atropine and burimamide both caused a profound decrease in pouch secretion and acid output. These data are consistent with the hypothesis previously stated that in ostertagiasis the hypersecretion from fundic pouches is due to increased levels of circulating gastrin.     

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.     

L3 and adult Ostertagia (Teladorsagia) circumcincta exhibit cyanide sensitive oxygen uptake

Oxygen consumption by L3 and adult Ostertagia (Teladorsagia) circumcincta was examined in vitro to determine whether oxygen can be utilised in metabolism. The oxygen concentration in the abomasal fluid of sheep infected with O. circumcincta was also measured. Rates of consumption (in nmol O2/h/1000 worms) were 13+/-1 in sheathed L3, 34+/-6 in ex-sheathed L3, and 1944+/-495 in adult worms. Constant rates of consumption were maintained until media oxygen concentration dropped to between 10 and 20 microM. Consumption was inhibited 95% by cyanide in L3 and 74% in adults. Oxygen concentration in abomasal fluid varied between 10 and 30 microM in both infected and uninfected animals. During infection, oxygen concentration decreased slightly with increased abomasal pH, though the correlation between the two was poor (r=-0.30). In conclusion, O. circumcincta can consume oxygen and oxygen concentration at the infection site is sufficient to support at least some aerobic metabolism.     

Evaluation of reference genes for real-time PCR studies of Brazilian Somalis sheep infected by gastrointestinal nematodes

Precise normalization with reference genes is necessary, in order to obtain reliable relative expression data in response to gastrointestinal nematode infection. By using sheep from temperate regions as models, three reference genes, viz., ribosomal protein LO (RPLO), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and succinate dehydrogenase complex subunit A (SDHA), were investigated in the abomasum, abomasal lymph nodes and small intestine of Brazilian Somalis sheep, either resistant or susceptible to gastrointestinal nematodes infections. Real time PCR was carried out by using SYBR Green I dye, and gene stability was tested by geNorm. RPLO was an ideal reference gene, since its expression was constant across treatments, presented lower variation, and was ranked as the most stable in abomasum and lymph node tissues. On the other hand, SDHA was the most stable in the small intestine followed by RPLO and GAPDH. These findings demonstrate the importance of correctly choosing reference genes prior to relative quantification. In addition, we determined that reference genes used in sheep from temperate regions, when properly tested, can be applied in animals from tropical regions such as the Brazilian Somalis sheep.     

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.     

Gastric dysfunction in sheep infected with Trichostrongylus colubriformis, a nematode inhabiting the small intestine

Barker I. K. and Titchen D. A. 1982. Gastric dysfunction in sheep infected with Trichostrongylus colubriformis, a nematode inhabiting the small intestine. International Journal for Parasitology12: 345–356. Six of 12 lambs infected with Trichostrongylus colubriformis had reduced abomasal acidification (pH 4.0–8.1) in comparison with uninfected pair-fed and replete controls (pH <3.5), though less than 0.8% of the worm burden was in the abomasum. Loss of prominence of parietal cells and encroachment of mucous cells deep into fundic glands was seen by light microscopy. Under the electron microscope, parietal cells had little canalicular or tubulovesicular development, had large vacuoles, many polyribosomes and few mitochondria in comparison with those in controls. In a further 8 sheep prepared with abomasal fistulae and separated fundic pouches and inoculated orally with T. colubriformis, the volume of fundic pouch secretion declined as feed intake dropped and in 7 out of 8 animals H⁺ concentration in fundic pouch secretion also fell. Infection generally reduced volume and acidity of pouch secretion more than did a pre-inoculation fast. In 5 sheep, abomasal content exceeded pH 4. Inoculation of T. colubriformis by enterotomy and Ostertagia circumcincta per os, in a lamb with a separated fundic pouch, caused depression of volume and acidity of pouch secretion characteristic of T. colubriformis infection, rather than the hypersecretion typical of abomasal infection with Ostertagia. Factors inhibitory to parietal cell differentiation and gastric acid secretion may be released from the small intestine of some sheep in response to changes in the gut induced by the presence of T. colubriformis. Abomasal dysfunction is a manifestation of severe intestinal trichostrongylosis.     

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.     

Recombinant expression of a galectin from the sheep gastrointestinal parasite Teladorsagia circumcincta: its use in isolating galectin-glycoconjugates.

Galectins, or beta-galactoside-binding lectins, are a family of proteins that have been described in vertebrates and, more recently, invertebrates, including nematode parasites. A tandem repeat-type galectin from the sheep gastrointestinal parasite, Teladorsagia circumcincta, has previously been isolated and the cDNA cloned. This molecule and each of its domains were expressed as fusion proteins with glutathione S-transferase (GST). The full-length molecule and the C-terminal domain were expressed in soluble form and the purified fusion proteins demonstrated a capacity to bind beta-galactoside sugars, with the greatest preference for lactose. The full-length fusion protein was used to successfully isolate potential galectin-glycoconjugates from within the parasite and from sheep serum.     

The distribution and localization of the stage-specific GP31 antigen from infective Ostertagia circumcincta larvae.

The 31,000 mol. wt glycoprotein (GP31) antigen of infective third-stage (L3) Ostertagia circumcincta larvae was shown, by surface labelling experiments and immunofluorescent antibody staining of whole larvae and larval sections, to be distributed internally. When transverse sections of L3 O. circumcincta, taken from the anterior pharyngeal region, were further examined by electron microscopy, after immunogold staining with rabbit anti-GP31 antiserum, the GP31 antigen was found to be specifically located in 'secretory organelles' within the cells of the oesophageal glands. By in vitro culturing L3 O. circumcincta in medium supplemented with 35S-methionine and then analysing the excretory-secretory material released by the larvae, it was found that the GP31 molecule was one of the major components of the excretory-secretory complex. The purified GP31 molecule had no detectable proteolytic activity in protein degradation assays. On examination of Triton X-100 extracts of infective larvae from other nematode parasite species, a predominant antigen similar to GP31 was found in Trichostrongylus colubriformis and Haemonchus contortus, but in Toxocara canis a minor component corresponding in mol. wt to GP31 was also detected. Based on these results the possible role of GP31 as a candidate antigen for a broad spectrum molecular vaccine against gastrointestinal nematode parasites in sheep is discussed.     

A mechanistic model of developing immunity to Teladorsagia circumcincta infection in lambs

Acquired immunity influences the severity of parasitic disease, but modelling the effects of acquired immunity in helminth infections has proved challenging. This may be due to a lack of suitable immunological data, or to the perceived complexity of modelling the immune response. We have developed a model of T. circumcincta infection in domestic sheep that incorporates the effects of acquired immunity on parasite establishment and fecundity. A large data set from commercially managed populations of Scottish Blackface sheep was used, which included relationships between IgA activity and worm length, and between worm length and fecundity. Use was also made of a recently published meta-analysis of parasite establishment rates. This realistic but simple model of nematode infection emulates observed patterns of faecal egg counts. The end-of-season faecal egg counts are remarkably robust to perturbations in the majority of the parameters, possibly because of priming of the immune system early in the season, reducing parasite establishment and growth and, therefore, faecal egg counts. Lowering the amount of early infection leads to higher end-of-season egg counts. The periparturient rise in egg counts in ewes appears to have an important role in supplying infection for the priming of the immune response. This feedback in the immune priming suggests that nematode infections may be difficult to eliminate.     

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.     

Somatostatin cells in the gastric mucosa of small ruminants. Immunohistochemical study

The distribution of somatostatin cells was investigated immunohistochemically in the epithelium of the abomasal mucous membrane of sheep and goats. Tissue samples were taken from different areas at the lesser and greater curvature of the glandular stomach. More somatostatin cells per 0.5 mm2 were found in sheep than in goats, predominantly in the pyloric gland region. In the pyloric area of both species the mucosa in the greater curvature contained more immunoreactive cells than the one in the lesser curvature. A few somatostatin cells with basal cytoplasmic processes of the 'open type' were observed over the entire abomasal mucosa.