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.     

Parasitological and immunological responses of genetically resistant Merino sheep on pastures contaminated with parasitic nematodes.

One hundred and twenty lambs were grazed continuously from weaning until 9 months of age on 12 plots contaminated with larvae of three nematode species (Haemonchus contortus, Trichostrongylus colubriformis and Ostertagia circumcincta). The lambs were sired by either a genetically resistant ram or susceptible rams (determined by the response of previous progeny to artificial H. contortus infection). Half the resistant and half the susceptible lambs were given strategic anthelmintic treatment and the remainder remained untreated. Faecal egg counts and blood packed cell volume were measured frequently in all animals. One and 5 months after weaning, two lambs from each plot were slaughtered, and worm burdens and larval establishment rates of the three species of nematode were estimated. At the second slaughter, leukotriene levels and larval migration inhibitory (LMI) activity were measured in mucus collected from the small intestine. The dominant species in all faecal samples and the gastrointestinal tract was T. colubriformis. Lambs of the resistant genotype had lower faecal worm egg counts, lower worm burdens and higher levels of resistance to larval establishment. There were no differences in larval migration inhibition (LMI) activity, but resistant lambs had higher levels of the leukotriene LTC4/D4/E4. Further, the resistant genotype, identified on responsiveness to artificial infections with H. contortus, was more resistant to infections of three important species acquired naturally from contaminated pastures. All these genetic differences were maintained while the lambs were subject to strategic anthelmintic treatment.     

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.     

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.     

Interactions of Giardia lamblia with Human Intestinal Mucus: Enhancement of Trophozoite Attachment to Glass1

Giardia lamblia trophozoites frequently are associated with mucus in vivo. We investigated the effects of human intestinal mucus on parasite attachment and survival in vitro. All samples of mucus from the duodenum and ileum (from four humans and two rabbits) enhanced attachment at 100 μm/ml. Attachment increased with mucus concentrations from 1 to 1000 μg/ml but declined toward the unstimulated level at concentrations above 1000 μg/ml. Mucus from the small intestine also promoted the survival of the parasites during the 2-h incubation. In contrast, colonic mucus promoted survival, but inhibited attachment. Fractionation of mucus from the human small intestine by cesium chloride equilibrium density gradient ultracentrifugation revealed that both attachment- and survival-promoting activities were in the low density, protein-rich fraction. The high density fractions containing the mucins were devoid of activity. Thus, a non-mucin fraction of mucus from the human small intestine may promote colonization by G. lamblia.     

Influence of Urea,Hydroxyurea, and Thiourea on Meloidogyne javanica and Infected Excised Tomato Roots in Culture

Urea (U), hydroxyurea (HU), and thiourea (TU), in various concentrations, were added to chemically defined plant tissue culture medium on which Meloidogyne javanica was reared on excised tomato roots. Concentrations as low as 3 ppm HU or 12 ppm TU inhibited nematode maturation by 70-90% 4 weeks after inoculation, and the coenocytes in the parasitized tissue were poorly developed. Gall weight was also inhibited by 50% in cultures treated with 3 and 6 ppm HU. However, exposing juveniles of M. javanica and Tylenchulus semipenetrans or juveniles and adults of Pratylenchus thornei to increasing concentrations of HU or TU, up to 100 ppm, was not lethal. These two urea derivatives still inhibited nematode maturation when the infected region of the root was not in direct contact with the chemicals. Therefore, we suggest that these urea derivatives inhibit nematode development by affecting the plant metabolism essential to coenocyte formation, an occurrence similar to the hypersensitive reaction in a naturally resistant plant.     

Cholecystokinin and anorexia in sheep infected by the intestinal nematode Trichostrongylus colubriformis

Symons L. E. A. and Hennessy D. R. 1981. Cholecystokinin and anorexia in sheep infected by the intestinal nematode Trichostrongylus colubriformis. Internationaljournal for Parasitology11: 55–58. It was postulated that there is a correlation between anorexia in intestinal nematode infection and the plasma concentration of cholecystokinin (CCK). In the first experiment plasma concentration of CCK rose as food consumption fell until, when sheep infected with Trichostrongylus colubriformis were almost completely anorexic, it had increased by 65%. Plasma CCK and food consumption returned to pre-infection levels in from four to six days after administration of an anthelmintic. In the second experiment food consumption by uninfected sheep was reduced in the first ten minutes after intravenous infusion of 150–300 μg of the octapeptide of CCK. It was concluded that anorexia in these infections may be due to or be mediated by higher concentrations of CCK.     

Serine Protease(s) Secreted by the Nematode Trichuris muris Degrade the Mucus Barrier

The polymeric mucin component of the intestinal mucus barrier changes during nematode infection to provide not only physical protection but also to directly affect pathogenic nematodes and aid expulsion. Despite this, the direct interaction of the nematodes with the mucins and the mucus barrier has not previously been addressed. We used the well-established Trichuris muris nematode model to investigate the effect on mucins of the complex mixture of immunogenic proteins secreted by the nematode called excretory/secretory products (ESPs). Different regimes of T. muris infection were used to simulate chronic (low dose) or acute (high dose) infection. Mucus/mucins isolated from mice and from the human intestinal cell line, LS174T, were treated with ESPs. We demonstrate that serine protease(s) secreted by the nematode have the ability to change the properties of the mucus barrier, making it more porous by degrading the mucin component of the mucus gel. Specifically, the serine protease(s) acted on the N-terminal polymerising domain of the major intestinal mucin Muc2, resulting in depolymerisation of Muc2 polymers. Importantly, the respiratory/gastric mucin Muc5ac, which is induced in the intestine and is critical for worm expulsion, was protected from the depolymerising effect exerted by ESPs. Furthermore, serine protease inhibitors (Serpins) which may protect the mucins, in particular Muc2, from depolymerisation, were highly expressed in mice resistant to chronic infection. Thus, we demonstrate that nematodes secrete serine protease(s) to degrade mucins within the mucus barrier, which may modify the niche of the parasite to prevent clearance from the host or facilitate efficient mating and egg laying from the posterior end of the parasite that is in intimate contact with the mucus barrier. However, during a T_H2-mediated worm expulsion response, serpins, Muc5ac and increased levels of Muc2 protect the barrier from degradation by the nematode secreted protease(s).     

Changes in hydrolytic enzyme activities of naïve Atlantic salmon Salmo salar skin mucus due to infection with the salmon louse Lepeophtheirus salmonis and cortisol implantation

The changes in the activities of mucus hydrolytic enzymes and plasma cortisol levels were examined following infection of Atlantic salmon Salmo salar with the salmon louse Lepeophtheirus salmonis and these changes were compared with those resulting from elevated plasma cortisol. Salmon were infected at high (Trial 1; 178 +/- 67) and low (Trial 2; 20 +/- 13) numbers of lice per fish and the activities of proteases, alkaline phosphatase, esterase and lysozyme in the mucus, as well as plasma cortisol levels were determined. At both levels of infection, there were significant increases of protease activity over time (1-way K-WANOVA; Trial 1, p = 0.004; Trial 2, p < 0.001). On several sampling days, generally on later days in the infections, the mucus protease activities of infected fish were significantly higher than control fish (Student's t-tests; p < 0.05). In addition, zymography experiments demonstrated bands of proteases at 17 to 22 kDa in the mucus of infected salmon that were absent in the mucus from non-infected fish and absent in the plasma of salmon. The intensity of these protease bands increased in the mucus over the course of both infections. However, plasma cortisol levels were elevated only in the heavily infected fish from the first trial. At high infection levels (Trial 1), alkaline phosphatase activity was higher in the mucus of infected fish at all days (t-test, p < 0.05). However, at the lower infection level (Trial 2), the mucus alkaline phosphatase activity did not differ significantly between infected and non-infected fish. Esterase and lysozyme activities were very low and did not change with time nor between non-infected and infected salmon in either challenge. Mucus enzyme activities of cortisol-implanted salmon did not change over time, nor were there any differences in activities between cortisol-implanted and control salmon. The present study demonstrates biochemical changes resulting from sea lice infection of Atlantic salmon occurring at the site of host-pathogen interaction, the mucus layer. However, the origin of these enzymes, whether host or pathogen, remains to be determined.     

A comparative study of helminth parasites from the fish Tilapia zillii and Oreochromis leucostictus in Lake Naivasha and Oloidien Bay,Kenya

The parasitic fauna of two fish species, namely gill-netted samples of 652 Oreochromis leucostictus and 448 Tilapia zillii from Lake Naivasha and Oloidien Bay was investigated during the period from the end of October 1995 to September 1996. Five larval helminth parasites were recovered including the nematode, Contracaecum sp., the acanthocephalan Polyacanthorhynchus kenyensis, the digenetic trematode, Clinostomum sp. and two cestodes, Amirthalingamia sp. and Cyclustera sp. Both prevalence and intensity of the infection of these helminths increased in larger sized fish, whereas male fish were more heavily infected than females. No seasonality in infection level were observed. The health status of both fish species remained unaffected, although O. leucostictus from Oloidien Bay which harboured heavy infections of Contracaecum exhibited stuntedness and the lack of fatty deposits around the digestive caecum.     

Interactions of Giardia lamblia with human intestinal mucus: enhancement of trophozoite attachment to glass

Giardia lamblia trophozoites frequently are associated with mucus in vivo. We investigated the effects of human intestinal mucus on parasite attachment and survival in vitro. All samples of mucus from the duodenum and ileum (from four humans and two rabbits) enhanced attachment at 100 micrograms/ml. Attachment increased with mucus concentrations from 1 to 1000 micrograms/ml but declined toward the unstimulated level at concentrations above 1000 micrograms/ml. Mucus from the small intestine also promoted the survival of the parasites during the 2-h incubation. In contrast, colonic mucus promoted survival, but inhibited attachment. Fractionation of mucus from the human small intestine by cesium chloride equilibrium density gradient ultracentrifugation revealed that both attachment- and survival-promoting activities were in the low density, protein-rich fraction. The high density fractions containing the mucins were devoid of activity. Thus, a non-mucin fraction of mucus from the human small intestine may promote colonization by G. lamblia.     

Changes in blood leukocytes,bone marrow and lymphoid organs in sheep infected with Haemonchus contortus

Haemonchus contortus infection, which causes a blood loss anaemia in sheep, depleted leukocytes and produced leukopenia and a mild lymphopenia. Thymus atrophy, a decreased size of spleen and enlargement of adrenal glands occurred concomitantly in infected sheep which suggested that they were caused by the stress of infection. The overwhelming change in bone marrow of infected sheep was a 4-fold increase in erythroid series cells.Primary immunization with rat erythrocytes produced similar haemagglutinating antibody responses in infected sheep and in sheep allowed to recover from infection after treatment with thiabendazole. This suggests that extant infection with H. contortus was not associated with a secondary immunodeficiency. Blastogenic responses of blood lymphocytes from infected sheep to larval antigen correlated with faecal egg counts but not with haematocrit or leukocyte values. These results suggest that the unresponsiveness of lymphocytes to worm antigen which occur during infection with H. contortus is more closely related to contact with the parasite than to the pathological effects of infection.     

Reduced three-dimensional motility in dehydrated airway mucus prevents neutrophil capture and killing bacteria on airway epithelial surfaces

Cystic fibrosis (CF) lung disease is characterized by persistent lung infection. Thickened (concentrated) mucus in the CF lung impairs airway mucus clearance, which initiates bacterial infection. However, airways have other mechanisms to prevent bacterial infection, including neutrophil-mediated killing. Therefore, we examined whether neutrophil motility and bacterial capture and killing functions are impaired in thickened mucus. Mucus of three concentrations, representative of the range of normal (1.5 and 2.5% dry weight) and CF-like thickened (6.5%) mucus, was obtained from well-differentiated human bronchial epithelial cultures and prepared for three-dimensional studies of neutrophil migration. Neutrophil chemotaxis in the direction of gravity was optimal in 1.5% mucus, whereas 2.5% mucus best supported neutrophil chemotaxis against gravity. Lateral chemokinetic movement was fastest on airway epithelial surfaces covered with 1.5% mucus. In contrast, neutrophils exhibited little motility in any direction in thickened (6.5%) mucus. In in vivo models of airway mucus plugs, neutrophil migration was inhibited by thickened mucus (CF model) but not by normal concentrations of mucus ("normal" model). Paralleling the decreased neutrophil motility in thickened mucus, bacterial capture and killing capacity were decreased in CF-like thickened mucus. Similar results with each mucus concentration were obtained with mucus from CF cultures, indicating that inhibition of neutrophil functions was mucus concentration dependent not CF source dependent. We conclude that concentrated ("thick") mucus inhibits neutrophil migration and killing and is a key component in the failure of defense against chronic airways infection in CF.     

STAT6 Expression and IL-13 Production in Association with Goblet Cell Hyperplasia and Worm Expulsion of Gymnophalloides seoi from C57BL/6 Mice

In intestinal helminth infections, Th2 immune respones are generally associated with mucin secretion for worm expulsion from the host intestine. In particular, IL-4 and IL-13 are the important cytokines related with intestinal mucus production via STAT6 signalling in nematode infections. However, this perspective has never been studied in Gymnophalloides seoi infection. The present study aimed to observe the STAT6 signalling and cytokine responses in C57BL/6 mice, a mouse strain resistant to infection with this trematode. The results showed that worm expulsion occurred actively during days 1-2 post-infection (PI), when goblet cells began to proliferate in the small intestine. The STAT6 gene expression in the mouse spleen became remarkable from day 2 PI. Moreover, G. seoi infection induced a significant increase of IL-13 from day 4 PI in the spleen of infected mice. Our results suggested that goblet cell hyperplasia and worm expulsion in G. seoi-infected mice should be induced by STAT6 signalling, in which IL-13 may be involved as a dominant triggering cytokine.     

Helminth infections of some invertebrates of the Georgia Bight

Dominant macroinvertebrates of the Georgia Bight were studied to evaluate the incidence of helminth infection and pathologies produced. Twenty-one stations along seven transects were sampled in February, May, August, and November 1977. Sicyonia brevirostris (rock shrimp) were heavily infected (83%) with cestode cysticercoids in the hepatopancreas and nerve tracts. Loligo pealeii (common squid) were lightly infected with cestodes (6%) throughout the year and with trematodes (<5%) during most of the year, but in the fall 33% were parasitized with trematode metacercarial. This study reemphasizes the high frequency of infection of marine invertebrates over large areas, the considerable pathology produced as well as the remarkable variability in infection patterns in local areas and with season.     

Changes in body tissues and hemolymph composition of Culex pipiens in response to infection by Romanomermis culicivorax

Hemolymph composition of fourth instar larvae of an autogenous strain of Culex pipiens was examined to determine the effects of parasitism by a mermithid nematode, Romanomermis culicivorax. Mosquitoes were reared under two different pH regimens: 4.5 and 7.3. Wet and dry weight of infected mosquitoes reared at either pH were significantly lower than controls. The effects of parasitism in the development of C. pipiens were evaluated from paraffin sections of mosquito larvae 2, 4, and 6 days postinfection. At 2 days postinfection, the infected larvae showed no apparent effects of parasitism; at day 4, the fat body tissue was reduced and imaginal disc development was retarded; and at day 6, parasitized mosquitoes were smaller in cross section, fat body tissue was found only in isolated clumps, and there was a complete absence of imaginal discs. Concentrations of total carbohydrates in hemolymph from infected fourth instar mosquitoes reared at pH 7.3 were reduced. Trehalose and glucose were each reduced by more than half. Total α-amino nitrogen was significantly lower in infected mosquitoes reared at pH 7.3. However, total amino acid concentrations for hemolymph from control and infected larvae reared at pH 7.3 were the same. Methionine sulfoxide decreased 63% and proline increased 2.5 times in infected mosquitoes. Hemolymph protein concentrations were reduced 80% in infected mosquitoes reared at both pHs. The number of hemolymph proteins also declined from 35 to 22 during infection. Two host proteins, 82,000 and 158,000 daltons, remained prominent throughout the mermithid infection.     

Susceptibility of rainbow trout Oncorhynchus mykiss,Atlantic salmon Salmo salar and coho salmon Oncorhynchus kisutch to experimental infection with sea lice Lepeophtheirus salmonis

Physiological, immunological and biochemical parameters of blood and mucus, as well as skin histology, were compared in 3 salmonid species (rainbow trout Oncorhynchus mykiss, Atlantic salmon Salmo salar and coho salmon O. kisutch) following experimental infection with sea lice Lepeophtheirus salmonis. The 3 salmonid species were cohabited in order to standardize initial infection conditions. Lice density was significantly reduced on coho salmon within 7 to 14 d, while lice persisted in higher numbers on rainbow trout and Atlantic salmon. Lice matured more slowly on coho salmon than on the other 2 species, and maturation was slightly slower on rainbow trout than on Atlantic salmon. Head kidney macrophages from infected Atlantic salmon had diminished respiratory burst and phagocytic capacity at 14 and 21 d post-infection (dpi), while infected rainbow trout macrophages had reduced respiratory burst and phagocytic capacities at 21 dpi, compared to controls. The slower development of lice, coupled with delayed suppression of immune parameters, suggests that rainbow trout are slightly more resistant to lice than Atlantic salmon. Infected rainbow trout and Atlantic salmon showed increases in mucus lysozyme activities at 1 dpi, which decreased over the rest of the study. Mucus lysozyme activities of infected rainbow trout, however, remained higher than controls over the entire period. Coho salmon lysozyme activities did not increase in infected fish until 21 dpi. Mucus alkaline phosphatase levels were also higher in infected Atlantic salmon compared to controls at 3 and 21 dpi. Low molecular weight (LMW) proteases increased in infected rainbow trout and Atlantic salmon between 14 and 21 dpi. Histological analysis of the outer epithelium revealed mucus cell hypertrophy in rainbow trout and Atlantic salmon following infection. Plasma cortisol, glucose, electrolyte and protein concentrations and hematocrit all remained within physiological limits for each species, with no differences occurring between infected and control fish. Our results demonstrate that significant differences in mucus biochemistry and numbers of L. salmonis occur between these species.     

Differentially expressed genes in cotton plant genotypes infected with Meloidogyne incognita

Meloidogyne incognita is a nematode responsible for huge losses of economically important crops. The control of this pathogen is heavily centered on chemical nematicides, which are toxic to humans and environment, besides being very expensive. Alternatively, resistant varieties of cotton generated from conventional breeding programs represent an attractive strategy for the control of M. incognita. In this context, the goal of the work reported here was to analyze the gene expression profile of one resistant and one susceptible cotton genotype infected with M. incognita aiming to understand the mechanisms involved in resistance. EST libraries of cotton in both resistant and susceptible to infection by M. incognita were constructed and sequenced, generating 2261 sequences that were assembled into 233 contigs and 1593 singlets. Genes differentially expressed were observed in both resistant and susceptible cotton. Twenty genes were found to be expressed exclusively in the resistant cotton genotype, with functions related to pathogen recognition, signal transduction, defense mechanisms and protein synthesis transport and activation. The coordinated action of these genes suggests the existence of a complex defense pathway towards nematode attack in cotton. Our data indicate some candidate genes for validation and use through transformation in other agronomically important plants.     

Characterization of Anionic Peroxidases in Tomato Isolines Infected by Meloidogyne incognita

Changes in peroxidase activity during nematode infection were studied using root extracts of tomato near-isogenic lines differing in resistance to Meloidogyne incognita. Total peroxidase activity increased slightly in crude extracts of four susceptible isolines but doubled in two resistant lines, Monita and Motaci. Nematode infection enhanced levels of both p-phenylenediamine-pyrocatechol oxidase and syringaldazine oxidase 7 days after inoculation, especially in resistant lines. This elevated peroxidase activity in resistant isolines was caused by an increase in anionic peroxidase activity. These enzymes, which likely are involved in lignification, were isolated and purified from tomato isolines by ammonium sulfate precipitation, high performance ion-exchange chromatography, and gel electrophoresis. The purified anionic peroxidase extracts contained an electrophoretic band with Rf 0.51 that was present in extracts of infected but not uninfected roots.