Characterization of IL-10-producing neutrophils in cattle infected with Ostertagia ostertagi.

IL-10 is a master regulator of immune responses, but its cellular source and function in cattle during the initial phase of immune priming have not been well established. Despite a massive B cell response in the abomasal draining lymph nodes in Ostertagia ostertagi (OO)-infected cattle, protective immunity is slow to develop, and partial protection requires years of repeated exposure. In addressing this problem, our initial hypothesis was that B cells produce IL-10 that downregulates the host protective immune response. However, our results showed that neutrophils made up the majority of IL-10-producing cells in circulation and in secondary lymphoid tissues, particularly the spleen (80%). Conversely, IL-10-producing B cells were rare. In addition, approximately 10% to 20% of the neutrophils in the blood and spleen expressed MHC II and were IL-10 negative, suggesting that neutrophils could also participate in antigen presentation. In vitro investigation of bovine neutrophils revealed that exposure thereof to OO extract increased IL-10 and MHC II expression in these cells in a dose-dependent manner, consistent with IL-10+/MHC II+ neutrophils detected in cattle shortly after experimental OO infection. Co-culture of untreated neutrophils with anti-CD3 antibody (Ab)-stimulated CD4+ T cells led to enhanced T cell activation; also, IL-10 depletion with neutralizing Ab enhanced the stimulatory function of neutrophils. OO extract depressed neutrophil stimulation of CD4+ T cells in the presence of IL-10-neutralizing Ab, suggesting that OO utilizes both IL-10-dependent and independent mechanisms to manipulate the bovine immune response. Finally, contact and viability were required for T cell-stimulatory neutrophil function. This report, to the best of our knowledge, is the first to demonstrate that neutrophil-derived IL-10 is directly involved in T cell regulation in cattle. Our data suggest that neutrophils and neutrophil-derived IL-10 are co-opted by nematode parasites and other pathogens to attenuate host immune responses and facilitate pathogen survival.

Infection of sheep with adult and larval Ostertagia circumcincta: gastrin.

Gastric endocrine cell populations and serum and tissue gastrin have been examined in sheep which were infected either intraruminally by tube with 150,000 Ostertagia circumcincta larvae followed by a trickle infection of 10,000 larvae thrice weekly for 8 weeks or by the transfer of 15,000 adult worms directly into the abomasum and killed 8 days later. Depletion of both antral gastrin and somatostatin was evident in both groups: tissue gastrin concentrations were reduced by 85% in the trickle infection and both G cells (gastrin-containing) and D cells (somatostatin-containing) were pale and fewer after adult worm transfer. The concurrent depletion of antral gastrin and somatostatin supports the contention that the hypergastrinaemia in parasitised sheep is largely secondary to the increase in abomasal pH. Although there was no change in the proportions of G34 and G17 in the tissues, there was an increase in the longer form of gastrin in the circulation of the larval-infected sheep, suggesting that there may be differential secretion of G17 and G34 which may be exaggerated as the rate of secretion increases. Although the fundic mucosa was thicker following trickle infection, there was no evidence of enterochromaffin-like cell hyperplasia in either infected group. It is suggested that hyper-gastrinaemia may be beneficial to the host, as it may allow the abomasum to regain the ability to acidify its contents during continued exposure to the parasites.

Infection of sheep with adult and larval Ostertagia circumcincta: abomasal morphology.

The infection of parasite-naive sheep with approximately 15,000 adult Ostertagia circumcincta via abomasal cannulae resulted in marked changes in the structure and function of the abomasum. The functional changes, which have been characterised previously, included elevated abomasal pH and increased serum concentrations of pepsinogen and gastrin. Eight days after the transplant of adult worms, the abomasa of recipient animals were significantly heavier than those of controls (P < 0.001), the thickness of the fundic mucosa was greater (P < 0.01), there were fewer parietal cells (P < 0.01) and increases in the numbers of mitotic figures and mucus-producing cells. Mucous cell hyperplasia was also evident in the fundic mucosae of sheep receiving a trickle infection of infective, third-stage O. circumcincta larvae and was prominent within nodules associated with larval development. In non-nodular mucosa, there was hyperplasia of mucous cells and changes in the distribution of parietal cells. Decreases in the number of parietal cells at the gland base were offset by increases at a mid-gland level, probably due to chronic hypergastrinaemia, so that, overall, total parietal cell number was unaffected. Mucous cell hyperplasia and the diminution of parietal cell number are seen in a diverse range of disease states and may be mediated by host growth factors such as Transforming growth factor-alpha. Alternatively, the cellular and/or the secretory changes in response to the presence of adult worms are mediated by chemicals that are cytotoxic/inhibitory for parietal cells, and released by the parasites themselves.

Cytokine profile induced by a primary infection with Ostertagia ostertagi in cattle.

Changes that occur in the local draining lymph nodes including, changes in cell surface markers and cytokine gene expression were studied over the first 4 weeks of a primary, Ostertagia ostertagi infection of the abomasum. Cells recovered from the abomasal lymph nodes (ABLN) after infection showed a decrease in the percentage of CD3+ cells, and an increase in the percentage of IgM+ cells and cells bearing the TcR1 marker. These changes were coincident with an increase in the proportion of activated cells (II-2R). Analysis of mitogen-stimulated ABLN cells by RNase protection assay (RPA) showed a dramatic reduction in IL-2 and IFN-gamma transcription after infection. In addition, analysis of unstimulated ABLN cells by competitive RT-PCR showed a similar decrease in demonstrable levels of IL-2 mRNA, but IL-10, IL-4 and IFN-gamma mRNA levels were elevated.

Isolation and phenotypic characterization of abomasal mucosal lymphocytes in the course of a primary Ostertagia ostertagi infection in calves.

Isolation and characterization of surface marker phenotypes of abomasal intraepithelial (IEL), lamina propria (LPL) and abomasal lymph node lymphocytes (ABLN) from uninfected calves were conducted, and the dynamics of change in these populations during the course of a primary Ostertagia ostertagi infection were defined. To obtain viable IEL and LPL from the abomasal mucosa of cattle, a modified isolation method was developed. The phenotypic characterization of abomasal lymphocytes was accomplished by indirect immunofluorescence staining. In uninfected animals, numbers of T cells exceeded the number of immunoglobulin-bearing cells in IEL, LPL and ABLN. The predominant T cell type in IEL and LPL was CD8+ cells, while the CD4+ T cell predominated in ABLN. Levels of activated cells and T cell receptor-1 gamma delta T cells were higher in IEL and LPL compared to ABLN. Within 3 weeks of infection, the number of lymphocytes recovered from the abomasal lamina propira and the mass of the ABLN was dramatically increased when compared to uninfected animals. Laser flow cytometric analysis demonstrated increased levels of immunoglobulin-bearing cells, gamma delta T cells, and activated T cells in IEL, LPL and ABLN in the infected animals. The greatest changes in LPL and ABLN took place during the first days of infection, and these changes were apparent throughout the 28 days covered by the experiment.

Different patterns of faecal egg output following infection of Scottish blackface lambs with Ostertagia circumcincta.

Faecal nematode egg counts were monitored in 184 Scottish Blackface lambs during natural exposure to a mixed, predominantly Ostertagia circumcincta infection and 12 lambs were selected which showed consistently zero (low count group; nine lambs) or consistently positive faecal egg counts (high count group; three lambs). These lambs were then treated with anthelmintic and challenged with 50,000 infective larvae of O. circumcincta and monitored thrice-weekly for 38 weeks; they were then re-challenged with another 50,000 infective larvae of O. circumcincta and monitored for a further 8 weeks. All sheep gave positive egg counts following deliberate infection. However, there was considerable variation among sheep in the size and timing of the peak in egg production. In particular, the pattern of mean values for faecal egg counts was different in the two groups. Egg counts were lower in the later periods of the extended infection in both groups of sheep. During the first half of the extended infection, egg counts were lower in sheep from the low count group, but during the second half of the infection the pattern was reversed and egg counts were lower in sheep from the high count group. There was a weak positive correlation between egg counts following anthelmintic treatment and 28 days exposure to natural infection and egg counts 28 days after a deliberate infection. Egg counts in the later stages of the deliberate infection were strongly but negatively correlated with egg counts following natural infection. The results of these studies show that differences in egg count following natural infection can be reproduced in experimental infections and that there is substantial variation in the pattern of egg production over time in different sheep. They also suggest that naturally resistant lambs are better able to delay worm development than naturally susceptible lambs.

A sequential study of the pathology associated with the infection of sheep with adult and larval Ostertagia circumcincta.

Disturbances in the physiology of the abomasa of sheep infected with either adult Ostertagia circumcincta given via abomasal cannulae, or larvae (L3) given intraruminally were matched by pathological changes in tissues collected by repeated mucosal biopsy. Within 2-3 days of the transplant of adult worms, abomasal pH had increased markedly in five out of six animals, and there also had been rapid increases in serum gastrin and pepsinogen concentrations in all animals. Reductions in parietal cell number were recorded as early as 1 day after the transplant of adults and were associated with the rapid accumulation of many neutrophils and eosinophils. Mucosal hyperplasia, with increased numbers of cells closer in appearance to mucous/mucous neck cells, was a relatively late development, being most pronounced in the latter part of the infection. In sheep given larvae, changes in secretory physiology were again matched by a concurrent fall in parietal cell number and by the accumulation of inflammatory cells. Changes became maximal when most worms could be expected to be present as adults, confirming the role of adults in the natural disease. Some abnormalities were detected in biopsies collected from animals maintained free of parasites and, although milder in degree, there were similarities to those observed in parasitised tissues, there being fewer parietal cells, a modest degree of mucous cell hyperplasia and inflammatory infiltrates of predominantly neutrophils. These changes were the likely result of trauma to the tissues in the immediate vicinity of the cannula, due either to the presence of the cannula itself or to the frequent collection of biopsy material from areas close to it.

An attempt to produce Ostertagia circumcincta infections in Mongolian gerbils.

Adult infections of Ostertagia circumcincta were successfully established in gerbils, although worm growth was retarded compared with infections in sheep. Chronic administration of beta-methasone to the host enhanced development of the parasite reproductive system but did not increase infection rate or prevent worm rejection. Infection rates achieved with single infections were not increased by using daily or weekly trickle infection regimes with or without prior exsheathment of larvae. Examination of stomachs from infected gerbils demonstrated pathological changes in the pyloric region which was the predilection site for O. circumcincta. This is in contrast to infections in sheep in which the fundic region of the abomasum is the preferred site.

Sequential histopathologic changes of type I, pre-type II and type II ostertagiasis in cattle.

Yearling cattle in Louisiana were examined at monthly intervals for abomasal nematode burdens and histological lesions over a year. Tracer calves were grazed for 3 to 4 weeks and removed from pasture for 2 to 3 weeks, then slaughtered; a few animals were killed in extremis shortly after removal from pasture. Histological changes were correlated with worm burdens and characterized according to the type of Ostertagia ostertagi infection present. In cattle with acute Type I ostertagiasis, changes varied from eosinophil infiltration to glandular dilation and slight mucous cell hyperplasia with submucosal edema. During the summer months the cattle had worm burdens that were primarily early 4th stage larvae (EL4), with changes characterized by minimal glandular dilation and mucous cell metaplasia and moderate lymphoid cell proliferation and with intramucosal migration of EL4. In the autumn, the maturation of EL4 produced the Type II syndrome with severe glandular changes, prominent mucosal hyperplasia and marked lymphoid cell accumulation. With increased duration of the pre-Type II interval, there was greater development of the subepithelial lymphoid tissue and increased frequency of epithelial globule leukocytes. The lymphoproliferation which occurred during the prolonged pre-Type II interval appeared to be related to the increased severity and mortality seen with the Type II ostertagiasis syndrome.

The sequential development of type I and type II ostertagiasis in young cattle with special reference to biochemical and serological changes.

The sequential development of Type I and Type II ostertagiasis over a 2-year period in the same naturally infected cattle is described for the first time. Particular reference is made to biochemical and serological changes. Positive relationships were demonstrated between the clinical signs of both Type I and Type II disease, and marked increases in the levels of plasma pepsinogen, plasma gastrin and antibody titres to adult Ostertagia antigen. At necropsy, there were significant relationships between the combined total of adult and developing 5th stage larvae of Ostertagia spp. and the levels of both plasma pepsinogen and gastrin. By the end of the second grazing season the cattle had acquired an immunity to infection with Ostertagia spp. and had very low burdens of this parasite at necropsy. However some of these cattle maintained elevated plasma pepsinogen levels when under natural challenge by Ostertagia spp. larvae and the aetiology of these changes and the problems of diagnosis using this parameter are discussed. Similar trends of infection were observed for Cooperia oncophora, although resistance to the parasite developed more rapidly.

Notes on necropsy and herbage processing techniques for gastrointestinal nematodes of ruminants.

Necropsy techniques for the digestive tract of ruminants used at the University of Utrecht differ from those used elsewhere in three respects: (1) the abomasum is opened immediately after slaughter and the "contents" are treated separately from the "washings"; (2) the first 10 m of the small intestine are treated separately from the remainder of the intestine; (3) the aliquots are coloured with iodine before being examined for worms. The majority of the worms are found in the washings of the abomasum and the first part of the small intestine, whereas the contents of the abomasum and the remainder of the small intestine contain the bulk of the digesta. Because inhibited stages of Ostertagia, Haemonchus and, particularly, the very small third stage larvae (L3) of Trichostrongylus can be overlooked easily in digesta, these methods imply a more rapid and accurate enumeration of worms. This is more important in small ruminants than in cattle because a much higher proportion of the inhibited larvae will be washed out of the mucosa and because Trichostrongylus is more important in small ruminants. Herbage sampling methods for monitoring gastrointestinal nematode infections on cattle pastures in northwest Europe should also be suitable for lungworm. The agar-bile technique of Jørgensen is an elegant method, but disadvantages are that many gastrointestinal nematode larvae exsheath, resulting in identification difficulties, and recovery of lungworm larvae decreases as a result of ageing. A simple sucrose flotation method, based on the principle that a sucrose solution does not mix easily with water containing nematodes, has been tested at our laboratory.(ABSTRACT TRUNCATED AT 250 WORDS)

Globule leukocytes in the lumen of the small intestine and the resistance status of sheep infected with parasitic nematodes.

The presence of globule leukocytes in the lumen (LuGLs) of the small intestine was studied in Romney sheep reared parasite free and then experimentally infected or immunized with Trichostrongylus colubriformis infective larvae, or naturally infected when gazed on pasture. It was discovered that high numbers of LuGLs were associated with both parasitic infection and the sheep protective immune response. A high positive correlation was observed between LuGLs and tissue globule leukocytes. The highest correlation (r = 0.92) observed was between larval migration inhibition (LMI) and numbers of LuGLs. This was the only correlation between LMI and other parameters studied. Significant positive correlations were also observed between LuGLs and IgG1 and eosinophils. There was a negative correlation between LuGLs and the number of parasites in the intestine and parasite egg production. Progeny of genetically resistant and susceptible sires had significantly different abilities to produce LuGLs.

Local immunity and Ostertagia circumcincta: changes in the gastric lymph of sheep after a primary infection.

Some aspects of the kinetics of the primary local immune response to O. circumcincta were studied by monitoring the flow and composition of gastric lymph from 4 sheep for 3 weeks after infection with 50 000 larvae. A significant increase in the frequency of lymphoblasts in the lymph showed that all the sheep were responding immunologically to the parasites from day 8 onwards. However, considerable individual variation in degree and timing was evident in other aspects of the immune response; 2 animals responded with large increases in lymphocyte output which reached a peak on day 5 or day 10, whereas the main response in the other 2 sheep appeared to be inflammatory in nature, with large increases in lymph flow and eosinophil output which occurred during the third week of infection. No significant changes in the content of IgA or IgA anti-worm antibody were detected in the lymph, but pepsinogen concentrations were significantly raised from day 12 onwards.

Osteopenia and reduced serum alkaline phosphatase activity in grazing lambs naturally infected with gastrointestinal nematodes.

The effect of gastrointestinal nematode infections on bone development was investigated in growing sheep on pasture. Forty-five weaned lambs from six groups in a two-factorial design incorporating stocking rate (SR; low, medium and high) and presence or absence of infection on pasture were sampled in the late grazing season. Worm counts were performed at slaughter, and the left metacarpal bones were excised for bone assessment. Faecal egg counts and worm burdens, primarily of Ostertagia circumcincta and Trichostrongylus vitrinus, were considerably higher in the high SR infected group ("I-High") than in comparable animals at low or medium SRs, whereas uninfected groups showed negligible egg excretion. Clinical biochemistry revealed significantly reduced serum concentrations of albumin, calcium and alkaline phosphatase in infected lambs. Nematode infections were associated with significant reductions in bone mineral density (30% at high SR), measured by dual energy X-ray absorptiometry, and in bone size (9%). Histomorphometry indicated thinning of the trabecular structure and reduced bone formation in the infected groups, particularly the I-High group. Bone mineral density, bone tissue volume and structural changes were strongly associated with log-transformed worm counts. The study showed that lambs suffering from moderate to heavy degrees of naturally acquired gastrointestinal nematode infection developed marked osteopenia after weaning, i.eduring the later part of the grazing season.

Gastrin and gastrin-related responses to infection with Ostertagia ostertagi in the calf.

The effects of a single challenge with 60,000 infective Ostertagia ostertagi larvae on blood and gastrointestinal mucosal gastrin concentrations, gastrin-producing G-cell numbers in the pyloric mucosa and growth of different parts of the gut were investigated in 16, two-and-a-half-month-old calves. Infected calves exhibited a rise in abomasal pH which was accompanied by a 145 per cent increase in wet weight of the fundic mucosa (P < 0.05) and a significant rise in blood total gastrin concentrations (P < 0.01). Circulating little gastrin (G-17) was unaffected. Pyloric mucosal total gastrin concentrations remained unaltered in the infected calves until day 28 when levels fell to 36.9 per cent of control group values (P < 0.01). Pyloric mucosal G-cell numbers declined during the experiment in the infected group. It is suggested that release of previously stored tissue gastrin and not a change in G-cell numbers contributes to the hypergastrinaemia associated with ostertagia infection in the calf.

Observations on the infectivity and pathogenicity of three isolates of Ostertagia spp sensu lato in calves.

After experimental infection of three groups of calves with three different defined isolates of Ostertagia spp sensu lato a marked variation in the infectivity and degree of pathological change was noted. The most highly infective and pathogenic isolate contained approximately 30 per cent O leptospicularis in addition to O ostertagi (70 per cent) and a negligible number of Skrjabinagia lyrata. The other two isolates consisted solely of O ostertagi (approximately 90 per cent) and S lyrata (approximately 10 per cent) and showed comparable infectivity and pathogenicity to that commonly recorded. The greater pathogenicity of the isolate containing O leptospicularis was attributed to the higher infectivity and the greater damage caused to the gastric mucosa by individual parasites when compared with O ostertagi.

Experimental concurrent infections with Ostertagia ostertagi and Cooperia oncophora in the calf.

An experiment was conducted to investigate the effects of experimental concurrent infection with Ostertagia ostertagia and Cooperia oncophora compared with the effects of monospecific infections with O ostertagi under conditions designed to mimic natural conditions on pasture during a grazing season. The present experiment was not able to demonstrate any significant effect of C oncophora on the course of an O ostertagi infection. Neither did C oncophora influence the build up of immunity to O ostertagi as demonstrated by post mortem worm count results from challenge infections with hypobiosis-prone larvae.

Effect of concurrent infection with Ostertagia circumcincta and Trichostrongylus vitrinus on the performance of lambs.

The interaction between Ostertagia circumcincta and Trichostrongylus vitrinus was investigated in lambs continuously infected over 12 weeks. Four groups of seven lambs were given either zero, 1000 T vitrinus larvae per day, 2500 O circumcincta larvae per day or infected concurrently. Overall liveweight gain was reduced by 17, 20 and 30 per cent in T vitrinus, O circumcincta and concurrent infection, respectively. T vitrinus infection significantly lowered serum phosphorus concentrations by week 4. In the concurrent infection the decline in phosphorus concentration was more gradual and only differed significantly from the controls during the final four weeks of the trial. There were no significant differences between the total numbers of T vitrinus or O circumcincta recovered from single or combined infections. The lack of an additive effect on performance may be due to a delayed establishment of T vitrinus in the presence of O circumcincta.

Concurrent daily infection of calves with Fasciola hepatica and Ostertagia ostertagi.

Three groups of calves were infected daily with either 1500 Ostertagia ostertagi larvae, 20 Fasciola hepatica metacercariae, or 1500 O ostertagi plus 20 F hepatica metacercariae. Weekly measurements were taken of calf weight, faecal egg output, plasma concentrations of albumin, plasma activities of sorbitol dehydrogenase, gamma glutamyl transpeptidase and pepsinogen and standard haematological indices. Calves were killed either 10 or 21 weeks after daily infections began. F hepatica infection had little influence on the size and structure of the O ostertagi worm population or vice versa. Mean worm burdens found at 20 weeks in those animals infected with both F hepatica and O ostertagi were 293 flukes and 20,641 nematodes. While this level of infection is similar to that seen in the disease complex in the field, there was no evidence of clinical disease or any difference in weight gain between the groups in this experiment. Factors other than additive worm burdens are obviously important for the expression of disease under field conditions.

Pathophysiological and parasitological studies on Ostertagia ostertagi infections in calves.

Friesian calves given a low level infection of the abomasal parasite Ostertagia ostertagi over a six week period displayed a mild diarrhoea with high faecal egg counts and elevated plasma pepsinogen values. At necropsy on day 23 abomasal lesions characteristic of ostertagiasis were widespread. At 42 and 84 days oedema and congestion were also prominent. Total worm burdens on days 23 and 42 were similar but a marked decrease had occurred by day 84. Feed digestibility and nitrogen economy were not markedly affected but radioisotopic measurements demonstrated an increase in albumin disappearance and catabolic rates, and plasma faecal clearance during the course of the infection. Prior administration of a morantel sustained release bolus to a group of similarly infected calves reduced the total worm burdens to less than 50 per cent of those recorded in the infected calves on days 23 and 42 and this fell to 3 per cent on day 84. Abomasal damage and the adverse pathophysiological changes associated with infection were prevented in this group.