The role of pharmacologically-active amines in resistance to Trichostrongylus colubriformis in the guinea-pig

The role of histamine and 5-hydroxytryptamine in resistance to Trichostrongylus colubriformis in the guinea-pig has been investigated by studying the effect of amine antagonists (promethazine, mepyramine and methysergide), inhibitors of amine synthesis (α-hydrazino analogue of histidine and α-methyl dopa), depletion of tissue stores of the amines with reserpine and by attempts to elevate levels of the amines by oral administration of the amines and their immediate metabolic precursors (L-histidine, L-tryptophan and 5-hydroxy-DL-tryptophan).The results show that promethazine suppressed the development of resistance during a primary infection and inhibited expulsion of the parasite in actively and adoptively immunized animals. Mepyramine and the α-hydrazino analogue of histidine inhibited expulsion of the parasite in actively immunized guinea-pigs although methysergide and α-methyl dopa were not effective. Reserpine suppressed rejection of a challenge infection in actively and adoptively immunized animals, and oral administration of the histamine precursor (L-histidine) and 5-hydroxytryptamine increased the resistance which develops during a primary infection. These results show that histamine and 5-hydroxytryptamine play roles in the mechanism of resistance to T. colubriformis in the guinea-pig.It is suggested that the mechanism of resistance to the helminth is biphasic. The first phase is immunologically specific and probably involves interaction between antigens and sensitized lymphocytes, which acts as a trigger for myeloid (eosinophil and basophil) involvement and the release of pharmacologically active amines. The second phase, which is non-specific, appears to be the final effector mechanism, and involves the rejection of the parasites either directly or indirectly by the action of the amines.     

Passive protection with homologous antiserum against Trichostrongylus colubriformis in the guinea-pig

The successful passive inmunization of guinea-pigs against Trichostrongylus colubriformis was achieved with three out of four pools of homologous antiserum. The degree of protection conferred, although significant, was not as strong as that resulting from active immunization.     

Calcium and phosphorus absorption in lambs exposed to Trichostrongylus colubriformis

Ten lambs (29 +/- 1.2 kg) reared parasite-free and prepared with rumen, duodenal and ileal cannulae were paired and one of each pair was given a daily oral dose of 2500 Trichostrongylus colubriformis larvae for 14 weeks. Untreated animals received the amount of ration consumed by their infected pair-mates the previous day. During weeks 6 and 12 of infection, all lambs underwent a 7 day calcium (Ca) and phosphorus (P) balance. During weeks 7 and 13, duodenal and ileal samples were collected to determine the amounts of Ca and P entering and leaving the small intestine. The infection caused varying degrees of feed refusal in all infected animals. As a result, the data on Ca and P in excreta and the amounts of Ca and P entering and leaving the small intestine were regressed against dry matter (DM) intake for each group at each period. There were no between-period differences in these relationships. Calcium absorption and retention were unaffected by the stress of infection. Infection affected several aspects of P metabolism. Blood P concentrations were markedly reduced. Absorption of P from the small intestine was greater (P less than 0.01) in control lambs (at 1 kg DM intake 6.6 g per day) than in infected animals (2.2 g P per day), but there was a greater (P less than 0.05) duodenal flow rate of P in control lambs which suggested much higher rates of salivary secretion of P than in infected animals. Phosphorus flow rates at the ileum were greater (P less than 0.01) in infected lambs, despite the lower duodenal flow rates, which indicated a major abnormality (P less than 0.01) in small intestine absorption of P in infected animals; this may have contributed to the growth check experienced by these lambs.     

In vitro leucocyte proliferative responses and lymphocyte sub-types in guinea pigs with genetically determined high- and low-level responsiveness to Trichostrongylus colubriformis

In vitro leucocyte proliferative responses to parasite antigens and to mitogens as well as lymphocyte sub-types were compared in guinea pigs with genetically determined differences in their ability to express protective immunity against Trichostronylus colubriformis infection. Proliferative responses to parasite antigens were greatest in high-responder (HR) animals, but cells from low-responder (LR) animals were generally more responsive to mitogens. However, HR circulating leucocytes were more responsive to the T-cell-dependent B-cell mitogen pokeweed mitogen (PWM), and the response of HR, but not LR, cells increased during primary infection with T. colubriformis. Flow cytometry revealed significantly greater numbers of circulating B-cells in HR animals and, as observed for responsiveness to PWM, the number of circulating B-cells increased in HR, but not LR, animals during primary infection with this parasite. These findings suggest a larger and more labile population of B-cells in HR guinea pigs. Received: 16 August 1999 / Accepted: 21 October 1999     

A genetic model describing the evolution of levamisole resistance in Trichostrongylus colubriformis, a nematode parasite of sheep

Data from 21 generations of selection on a levamisole-resistant strain of Trichostrongylus colubriformis, either exposed to selection with the anthelmintics levamisole (LEV) or thiabendazole (TBZ), or unexposed, were used to fit a genetic model describing the evolution of LEV resistance in this parasite species. A statistical model describing the dose-response relationship for a mixed population of susceptible and resistant parasite eggs exposed to anthelmintic was fitted to egg-hatch assay data for each generation and for each selection regimen. Estimated parameters from the statistical model provided the input for the genetic model from which were obtained estimates of the relative fitness of susceptible and resistant genotypes under each selection regimen. The experimental data and the genetic models both indicated that, in this parasite strain, LEV resistance was determined by a single dominant gene, and that TBZ selects for LEV susceptibility. A variety of drug alternation programmes was simulated for this genetic system. The programme that minimized the development of LEV resistance involved alternating the drugs (LEV and TBZ) between each worm generation.     

The cellular transfer of immunity to Trichostrongylus colubriformis in an isogenic strain of guinea-pig: II. The relative susceptibility of the larval and adult stages of the parasite to immunological attack

Cells obtained from mesenteric lymph nodes of highly inbred guinea-pigs (Heston strain) resistant to Trichostrongylus colubriformis were injected into virgin animals of the same genotype. The adoptively immunized recipients were challenged with 1000 T. colubriformis larvae 4 days after transfer and slaughtered at intervals which correspond to critical times in the development of the parasite. Differential worm counts carried out on specimens of intestine showed that a sharp decline in the number of parasites occurred between days 7 and 9. This period corresponds to the time required for the parasite to develop to the fourth larval stage.

Variation of the time interval between cell transfer and challenge showed that immune cells transferred on the day of challenge and on days 4, 6 and 8 after challenge inhibited the development of infection to patency, while cells injected on day 10 were without effect. This observation confirmed that the fourth larval stage of the parasite is uniquely susceptible to the immunological attack initiated by the transferred cells and showed that these cells are effective within 24–48 hours after injection. This latter finding excludes the possibility of active participation in the response by the recipient.

Resistance can be transferred by spleen cells and by cells obtained from lymph nodes other than the mesenteric nodes which drain the site of infection. However the local nodes are more effective and resistance was regularly transferred with as few as 10 × 10⁶ cells injected intravenously.

     

RNA metabolism in the guinea pig infected with the nematode Trichostrongylus colubriformis

The concentration of RNA/mg DNA fell progressively in skeletal muscle, but was unchanged in the liver during infection of the guinea pig by the nematode Trichostrongylus colubriformis. The rate of RNA turnover was increased in muscle and liver.The specific activity of RNA 24 hr after the injection of ³H-uridine was unchanged in muscle, but increased in liver. While the concentrations of RNA precursors were unchanged in liver. The activity of muscle RNA polymerase was decreased, but that of liver was unchanged by the infection.From these results, it was concluded that the fall of the concentration of muscle RNA was due to both the increased rate of turnover and a decreased rate of synthesis, whereas the increased rate of turnover of liver RNA was approximately equal to the increased rate of synthesis.The significance of these findings is discussed in relation to changes of muscle and liver protein metabolism in this nematode and in bacterial or viral infections, as well as those that occur following reduced feed intake.     

An enhanced role for the recirculating lymphocyte in the neonatal immune system

Lymphocytes continually recirculate between the blood and the tissues via the lymph independent of antigen. A great deal is known regarding both the physiology and the molecular mechanisms responsible for the process in adults. However, relatively little is known regarding the development of the recirculating lymphocyte pool in very young animals or fetuses. We have directly measured the recirculation of lymphocyte subsets in antigen-inexperienced newborn animals, and found extensive recirculation of T cells through both intestinal and subcutaneous lymph nodes. Apparent selective migration of recirculating lymphocytes could be attributed to subset-specific migration of gammadelta-T cells through subcutaneous lymph nodes. This clearly demonstrates that the preference for gammadelta-T cells to recirculate through SCLN is lineage specific, and independent of the presence of antigen. Most surprising was the observation that the recirculating lymphocyte pool was proportionately larger in neonatal animals than in adults, which correlated with the histological appearance of newborn lymph nodes. This data strongly suggests that development of the recirculating lymphocyte pool is inversely correlated with antigen exposure, and decreases in size with age and the acquisition of immunological memory.     

Lymphocyte phenotypes in the intestinal mucosa of sheep infected with Trichostrongylus colubriformis

Monoclonal antibodies to ovine lymphocyte surface antigens were used in an immunohistochemical study of the intestine of sheep. In the epithelium CD8+ cells predominated whereas the majority of lamina propria T lymphocytes were CD4+. Infection of sheep with the parasitic nematode Trichostrongylus colubriformis including sufficiently large numbers of parasites to induce protective immunity did not alter the number of CD4+ or CD8+ lymphocytes in the intestinal mucosa. In contrast, exposure of naive sheep to a single large infection of T. colubriformis resulted in a substantial decrease in number of CD8+ cells and moderate decreases in number of CD4+ cells in the duodenal but not the jejunal mucosa. MHC class II antigens were not detected either in or on epithelial cells of the sheep small intestine.     

The contribution of a partial tricarboxylic acid cycle to volatile end-products in thiabendazole-resistant and susceptible Trichostrongylus colubriformis

Acetate, propionate, ethanol and propanol were the predominant end-products released during incubation of a thiabendazole resistant and a susceptible strain of Trichostrongylus colubriformis. The parasites in all the incubations appeared to be deficient in reducing equivalents if the end-products arose from the classical catabolic pathway through fumarate reductase (EC 1.3.1.6). Possible alternative pathways for accounting for redox balance, including beta-oxidation, the pentose phosphate pathway and amino acid metabolism were investigated. Palmitate was oxidised aerobically. Radiolabelled tricarboxylic acid cycle intermediates, citrate and alpha-ketoglutarate, were decarboxylated to 14CO2 indicating that at least a partial tricarboxylic acid cycle to succinyl-CoA via alpha-ketoglutarate operates both anaerobically and aerobically in T. colubriformis. These data and the pattern of end-products suggest the presence of two pathways to propanol and propionate either through fumarate reduction or alpha-ketoglutarate oxidation. T. colubriformis may apportion carbon flow through these pathways to maintain a stable redox ratio. Similar calculations on previously reported data indicate that both pathways may also operate in Haemonchus contortus. Exposure of resistant T. colubriformis to thiabendazole under anaerobic conditions caused an increased accumulation of end-products, especially propanol, in the incubation medium. The alpha-ketoglutarate pathway may lower the dependence of the parasite on the fumarate reductase route which is sensitive to thiabendazole. The operation of the alpha-ketoglutarate pathway, with propanol as an end-product, may provide a mechanism for regulating redox balance in trichostrongylidae.     

Plasma zinc and inappetence in sheep infected with Trichostrongylus colubriformis

The concentration of zinc in plasma before and after the occurrence of inappetence was measured in sheep infected with Trichostrongylus colubriformis and in uninfected animals fed freely or pair-fed with the infected group. Infection reduced mean food consumption to about one-third of that of the sheep fed ad libitum, half becoming completely anorexic. Plasma zinc concentrations in the infected sheep were reduced by 17 per cent whereas they were unchanged in the uninfected groups.     

The cellular transfer of immunity to Trichostrongylus colubriformis in an isogenic strain of guinea-pig: IV. The localization of immune lymphocytes in small intestine in infected and non-infected guinea-pigs

Immune and non-immune mesenteric lymph node and Peyer's patch cells were labelled in vitro with ⁵¹Cr and injected intravenously into infected and non-infected syngeneic guinea-pigs (Heston strain). From the distribution of the radioactive label in the small intestine (the site of infection of T. colubriformis) it was concluded that immune lymphocytes: (1) preferentially localize or `home in' on the infection, (2) come into intimate contact with the parasite in the epithelium, and (3) rapidly undergo `allergic death' or lysis at the site of infection.     

Anthelmintic effect of heather in goats experimentally infected with Trichostrongylus colubriformis

The effects of heather (composed primarily of Calluna vulgaris with a smaller content of Erica umbellata and Erica cinerea) consumption on the establishment of incoming infective larvae (experiment 1, preventive treatment) and an adult worm population (experiment 2, curative treatment) were investigated in Cashmere goats experimentally infected with Trichostrongylus colubriformis. In experiment 1, 12 castrated male goats were divided into two groups: heather-supplemented vs. non-supplemented animals. After 2 weeks of adaptation to the diet, all goats were experimentally infected per os with 6,000 T. colubriformis third-stage larvae. Three weeks post-infection, the goats were slaughtered, and worm counts as well as female worm fecundity and development were determined. Heather consumption was associated with a close to significant (P?=?0.092) reduction (mean 14 %) in larvae establishment. No effect on fecundity was observed, but the length of female worms in supplemented goats was greater (P?<?0.001). In experiment 2, 15 non-lactating does were experimentally infected with 6,000?T. colubriformis third-stage larvae. At 6 weeks post-infection, three groups were established: control, heather-supplemented and heather-supplemented with polyethylene glycol. Individual faecal nematode egg output was measured twice weekly to assess gastrointestinal nematode egg excretion. The goats were slaughtered 5 weeks after heather administration (11 weeks post-infection), and worm counts as well as female worm fecundity and development were subsequently determined. Heather administration was associated with a significant (P?<?0.001) decrease (between 47 and 66 % compared with control group) in egg excretion from 45 to 76 days post-infection. Although worm counts and female fecundity were lower in supplemented goats, no significant differences were observed. Overall, the results showed a reduction in T. colubriformis larvae establishment and a decrease in nematode egg excretion when heather was administered in experimentally infected goats. The heather plus polyethylene glycol treatment reduced nematode egg excretion levels at the same proportion as heather, thereby suggesting that the threshold of tannins required for an anthelmintic effect is most likely quite low.     

Studies on the pathogenesis of Trichostrongylus colubriformis in Mongolian gerbils (Meriones unguiculatus)

Sixty Mongolian gerbils were infected with 1500 T. colubriformis larvae. During the experiment, the animals were weighed regularly and faeces were examined for parasite eggs. Groups of 5 gerbils were necropsied at intervals throughout the experiment (60 days) and worm burdens, serum protein concentrations and packed cell volumes were ascertained. It was found that gerbils infected with T. colubriformis showed inappetence and loss in body weight. A modest hypoalbuminaemia also developed but there was no evidence of alterations in packed cell volumes in the infected animals. The results indicated that the pathogenic effects of T. colubriformis in the gerbil were similar to those observed in sheep, one of the natural hosts for this parasite. The conclusion was drawn that the Mongolian gerbil is a suitable laboratory host for T. colubriformis and is superior to other small animal hosts previously described.     

Characterization of cDNA clones coding for muscle tropomyosin of the nematode Trichostrongylus colubriformis

The host-protective antigen from detergent-solubilised extracts of the sheep intestinal helminth Trichostrongylus colubriformis has been identified as tropomyosin. Complementary DNA clones coding for T. colubriformis muscle tropomyosin have been isolated and characterised as the first step in obtaining recombinant protein to carry out more extensive vaccination trials. The clones represent an mRNA of 1544 bases, including a relatively long 5' untranslated sequence of 307 bases and a 3' non-coding region of 344 bases. The mRNA codes for a highly alpha-helical protein of 284 residues with a molecular weight of 33,000; characteristics typically observed for the muscle tropomyosins of higher organisms. The T. colubriformis protein has 58% sequence identity with rabbit and Drosophila melanogaster muscle tropomyosins, and the differences in the protein sequence are randomly distributed throughout the molecule. There is complete identity between the three sequences for the N-terminal 9 residues, the region believed to be essential for the polymerisation of tropomyosin molecules and for binding to actin and troponin.     

The cellular transfer of immunity to Trichostrongylus colubriformis in an isogenic strain of guinea-pig: III. The localization and functional activity of immune lymph node cells following syngeneic and allogeneic transfer

Mesenteric lymph node cells obtained from guinea-pigs immunized with Trichostrongylus colubriformis have been transferred allogeneically (McMaster outbred to Heston inbred) and syngeneically (Heston to Heston inbred) by intravenous injection. The immune cells were transferred on days 6, 7 and 8 of infection when the parasite was in the susceptible fourth stage of development. The syngeneically transferred cells caused rejection of the parasite but the allogeneically transferred cells were not effective.

Immune mesenteric lymph node cells were labelled in vitro with ⁵¹Cr before transfer to infected recipients. The percentage of the intravenously injected cells which localized in infected small intestine has been calculated from γ-counts recorded at 1, 6, 16, 24 and 48 hours after injection of the labelled cells. The mean percentage of cell dose per 12 in. of small intestine recorded following syngeneic transfer was 1·29 and following allogeneic transfer 0·68. A significant difference in the localization of allogeneic and syngeneic cells in the small intestine was already apparent at 6 hours following intravenous injection.

     

The effect of antiserum to eosinophils and susceptibility and acquired immunity of the guinea-pig to trichostronglyus colubriformis.

There is much experimental evidence to suggest that eosinophils are important in resistance to parasitic infection. We tested this hypothesis by treating guinea-pigs with rabbit anti-eosinophil serum (AES) and determining the effect of treatment on susceptibility and acquired immunity to Trichostrongylus colubriformis. The treatment markedly reduced the number of eosinophils in peripheral blood and in the small intestine. The number of T. colubriformis present after initial infection and after a second infection was determined in animals treated with AES and in control animals. Administration of AES to guinea-pigs significantly increased the susceptibility of the animals to initial infection with T. colubriformis larvae; the number of worms recovered was nearly doubled. Similarly- administration of AES resulted in a significant diminution of acquired immunity to a secondary infection. These results are consistent with the view that eoxinophils are important in susceptibility and acquired immunity to infection with T. colubriformis in the guinea-pig. Because T. colubriformis infection is confined to the intestinal tract, our results also suggest that eosinophils may be involved in resistance to parasites at the level of the intestinal mucosa.     

Susceptibility of high and low producer dairy goats to single experimental infection with Trichostrongylus colubriformis

Culled dry dairy goats, which differed in their level of production in previous lactations, received a single infection with Trichostrongylus colubriformis. The objectives of the study were twofold. First, the study aimed at examining the ability of dairy goats to develop an immune response to trichostrongyle infection and the associated cellular changes developing within the intestinal mucosa. Second, a comparison between animals differing in their level of production was assessed, in order to determine whether these differed in their susceptibility to infection. No difference occurred in egg excretion, worm burden and local inflammatory cell responses between high (HP) and low (LP) producer dairy goats, in contrast to observations in previous studies. Because their nutrition was controlled and milk production absent in the goats employed in the present study, these results suggest that any genetic component associated with the selection of HP and LP goats had little influence on the development of acquired resistance to T. colubriformis. The analysis of the relationship between different cell types in the mucosa and some characteristics of the worm population show that eosinophils are negatively related to worm burden. Also, a role is suggested for mast cells and globule leukocytes in the modulation of egg excretion. Received: 4 April 2000 / Accepted: 16 June 2000     

Rapid turnover of the recirculating lymphocyte pool in vivo

Lymphocytes are unique among blood cells in their capacity tocontinually recirculate between blood and the tissues via thelymph. Previous estimates of lymphocyte lifespan in vivo andthe turnover of the recirculating lymphocyte pool have beendeduced from indirect labeling techniques. Using the fluorescentdye PKH-26, individual labeled cells have been tracked in sheepfor periods >2 months. By direct measurement their lifespanwas calculated. This label was found to be stable in vivo, allowinglong-term analysis of the characteristics of the recirculatinglymphocyte pool. It Is possible to calculate the rate of turnoverof cells of the recirculating pool based on the rate at whichlabeled cells disappear from the lymphatic circulation. Therecirculating lymphocyte pool was found to repopulate itselfevery 16.5 ± 3.0 days. Using this label, it was estimatedthat recirculating lymphocytes divide on average once every29.8 ± 6.8 days. Labeled erythrocytes were also examinedand found to have an average lifespan of 153 days, demonstratingno dye loss over the 2 month period of observation. These datasuggest that the recirculating lymphocyte pool is a highly dynamiccompartment, with a high rate of turnover and peripheral celldivision in vivo. This is the first report of the direct measurementof the in vivo turnover of recirculating lymphocyte pools, andthis method may now be used to further analyze the lifespanof individual lymphocyte subsets and the in vivo lifespan ofother cell types in vivo.     

Immunological properties of murine thymus-dependent lymphocyte surface glycoproteins.

The immunological properties of two murine thymus-dependent (T) lymphocyte surface glycoproteins, T200 and T25, were investigated. T200 is a lymphocyte-specific antigen with a high degree of species specificity. It shares antigenic determinants with molecules present on thymus-independent (B) lymphocytes. T25 has antigenic determinants which cross-react with antigens on mouse brain, rat thymocytes and rat brain. An antiserum against a purified rat brain glycoprotein which carries Thy-1.1 reacts with T25. Absorption of this antiserum with BALB/c thymocytes or brain homogenate produces a Thy-1.1 specific serum which reacts with T25 from AKR/J thymocytes but not with T25 from AKR/Cum thymocytes. These results confirm that T25 is the molecule on the surface of mouse T cells which carries the Thy-1 antigen. T25 also carries antigenic determinants, recognized by anti-thymocyte serum (ATS), which were found on secondary mouse embryo fibroblasts and untransformed fibroblast cell lines but which were not detected on fibroblast cell lines transformed with murine sarcoma virus (MSV) or with Simian virus 40 (SV40).