Isolation and degranulation of mucosal mast cells from the small intestine of parasitized sheep.

The isolation of mucosal mast cells and globule leucocytes from the small intestine of sheep immunized with Trichostrongylus colubriformis is described. Sheep mast cell protease was released from these cells in a dose-dependent fashion after incubation with soluble protein from T. colubriformis larvae. Release also occurred with other T. colubriformis antigens whereas non-parasite antigens at comparable protein concentrations evinced only a minimal response. Mucosal mast cells prepared from worm-free sheep also produced a similar minimal response. This is the first report describing the release of sheep mast cell protease from isolated sheep intestinal mucosal mast cells after addition of specific parasite antigens.     

Population dynamics of Trichostrongylus colubriformis and Ostertagia circumcincta in single and concurrent infections.

Twenty-one-week-old worm-free pen-reared lambs were infected weekly with either 10,000 T. colubriformis larvae, 5000 O. circumcincta larvae, or with both species (15,000 larvae per week). Larval establishment and total worm burdens were estimated after 4, 7, 10 and 13 weeks of infection. Faecal egg counts and lamb bodyweights were measured weekly, and numbers of eosinophils in blood were estimated before infection and at weeks 5, 8 and 14. For both species of worms, the dynamics of infection (establishment, worm burdens, egg counts) were not affected by concurrent or pre-existing infection with the other species. Infection with T. colubriformis alone did not protect against O. circumcincta, but infection with O. circumcincta alone provided slight protection against the T. colubriformis larvae. Blood eosinophils increased between 5 and 8 weeks of infection and were similar for the three infections. This corresponded to the reduction in establishment for both species.     

The establishment rate of Ostertagia circumcincta and Trichostrongylus colubriformis in lactating Romney ewes

The ability of lactating Romney ewes to resist establishment of ingested infective-stage larvae (L3) of Ostertagia circumcincta and Trichostrongylus colubriformis was measured in the field. Three groups of seven single-lamb-bearing ewes were selected on the basis of uniformity of lambing date from a large flock held on pasture. Either 2, 4 or 6 weeks after parturition, groups of ewes were dosed with 24000 L3 of known oxfendazole-resistant parasite strains; 12000 of each species. Ten to 14 days later the ewes, along with their lambs, were transferred from the field to indoor pens. Twenty-five days after the challenge dose the ewes were drenched with oxfendazole to remove any field-derived infection and 3 days later slaughtered for worm counts. Mean establishment of the resistant parasites was low at all times, with the highest rate recorded being 6.1% for O. circumcincta 2 weeks after parturition. Establishment of O. circumcincta 4 and 6 weeks after parturition, and of T. colubriformis at all times, never exceeded 2%. By comparison, mean establishment in lambs held indoors and parasite free for 13 weeks prior to infection, was 24.9% and 47.1% for O. circumcincta and T. colubriformis, respectively. These results indicate that the lactating ewes were exhibiting a substantial ability to prevent establishment of ingested larvae. The results of this and other similar studies suggest that the dynamics of parasitism in lactating Coopworth and Romney ewes in New Zealand is substantially different to that in Merino ewes in Australia, and that these differences influence optimal strategies for the management of anthelmintic resistance in the two countries.     

The effects of immunization of sheep with Trichostrongylus colubriformis larvae on worm burdens acquired during grazing

The effects of immunization of sheep with Trichostrongylus colubriformis larvae on worm burdens acquired during grazing. International Journal for Parasitology 19: 177-181. Romney sheep, reared helminth-free in pens to 5 months of age, were immunized against Trichostrongylus colubriformis by giving two doses of 200,000 T. colubriformis infective larvae at 15 day intervals to assess protection from natural challenge during grazing. Five immunized sheep and five unimmunized sheep were grazed on infested pasture for 4 weeks, and were then returned to the pens for 4 weeks before slaughter. Worm burdens, gastrointestinal histology and mucus antiparasite activity were examined at slaughter. Faecal egg counts and haematological examinations were carried out at regular intervals throughout the trial. Significant protection (P less than 0.05) was afforded immunized sheep against adult T. colubriformis (87%), T. axei (67%), Nematodirus spathiger (91%) and Ostertagia spp. (42%). Greater numbers of immature Nematodirus spp. and Ostertagia spp. were present in immunized sheep Overall, a significant (P less than 0.05) 42% reduction in total nematode burdens was afforded by immunization of the sheep with T. colubriformis larvae. Immunized sheep had significantly (P less than 0.05) more globule leukocytes, mast cells and eosinophils in gastrointestinal tissue and significantly (P less than 0.05) higher levels of mucous antiparasite activity than unimmunized sheep. Haematological observations showed some sheep had transient eosinophilia during immunization or grazing. Both immunized and unimmunized sheep showed depressed (P less than 0.05) total leukocyte counts during grazing which returned to pre-grazing levels within 1 week of return of the sheep to the pens. Overall, haematological parameters reflected parasite challenge and were unrelated to worm burdens acquired.     

The effect of dietary sainfoin ( Onobrychis viciifolia) on local cellular responses to Trichostrongylus colubriformis in sheep

The effect of sainfoin (Onobrychis viciifolia) hay consumption on the pathophysiology and local cellular responses of growing lambs during infection with Trichostrongylus colubriformis was investigated. Thirty-two lambs, 16 weeks of age, were allocated to 1 of 4 treatment groups (n=8) that were offered either grass (G) or sainfoin (S) hay while concurrently either infected (+), or not (-) with 12,000 L3 T. colubriformis larvae per week for 6 weeks. Liveweight gains were affected by diet (P=0.002) and reduced by infection (P<0.05). Faecal egg count was reduced in S+compared to G+ from days 35 to 42 (P=0.001); however, total egg output, worm burdens at day 42 and worm fecundity were similar between diets (P>0.05).Feeding sainfoin appeared to enhance immune cell development with tissue eosinophils, mast cells and pan T cells present in greater concentrations in S+ than in G+ animals. However, further studies are required to determine if the enhanced immune cell development is a consequence of a greater nutrient supply or a direct influence of sainfoin metabolites on local inflammatory responses to the gastrointestinal nematode T. colubriformis.     

Cellular changes in the intestinal lymph of sheep infected with the enteric nematode, Trichostrongylus colubriformis

Some changes produced in the cell populations of intestinal lymph by infection with the enteric nematode, Trichostrongylus colubriformis, were studied in sheep regularly re-infused with all discharged lymph. Lymphocyte traffic through the intestinal lymphatic duct was reduced until day 35 of primary infection, mainly due to the absence of lymphocytes with smaller cell volumes, but was increased two-fold after day 70 and in immune sheep. Antigen-reactive lymphocytes in blood and lymph were assayed by the uptake of 3H-thymidine in cell culture stimulated by extracts from the larvae of T. colubriformis. Cells from the blood and lymph of immune sheep were highly reactive to worm antigen. A relatively smaller reactivity was present in the blood of worm-free sheep and was abolished during the first 12 days of primary infection. Antigen reactive cells were not detected in intestinal lymph until 12 days after primary infection, and in vitro antigen reactivity in intestinal lymph of immune sheep was increased after challenge with infective larvae. Responses to the mitogens, concanvalin A and phytohaemagglutinin, in cultures of cells from both intestinal lymph and blood were depressed on days 7 and 12 of primary infection. It is proposed that the diminished traffic of lymphocytes in intestinal lymph and the reduced numbers of mitogen and nematode antigen-reactive lymphocytes in both blood and intestinal lymph during the early stages of infection with T. colubriformis is closely related to the slow development of protective immunity to this parasite.     

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

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

Temporal effects of protein nutrition on the growth and immunity of lambs infected with Trichostrongylus colubriformis

The aim of this research was to determine whether metabolisable protein supply during the early period of infection with Trichostrongylus colubriformis influenced resilience and the later stages in the development and magnitude of host resistance in previously nematode-na?ve lambs. Eighty TexelxGreyface lambs were fed pelleted feeds calculated to provide grossly different amounts of metabolisable protein. Sixty of the lambs received a trickle infection of T. colubriformis and 20 lambs were kept as uninfected controls. There were four initial groups, namely infected or uninfected and fed either a moderate or a high protein feed. After 5weeks of infection, a further four groups were established by changing the feed of half of the animals fed the moderate protein feed to the high protein feed and of half of the animals fed high protein to the moderate protein feed. Live weight gain and feed conversion ratio were greatest for lambs fed the high protein feed and were reduced by infection. Faecal egg counts, worm burdens and per capita fecundity of adult female nematodes were unaffected by changes to metabolisable protein supply. Decreasing metabolisable protein supply following 5weeks of infection reduced live weight gain without any effect on resistance to T. colubriformis. Haematological variables, indicative of improved resistance, were also largely unaffected by metabolisable protein supply. It is concluded that the requirements of immune function probably had priority over those of growth and that the metabolisable protein supply provided by the moderate protein feed was sufficient to account for the requirements for the expression of immunity. It is probable that the potential for metabolisable protein supply to enhance resistance to infection from T. colubriformis is dependent on the levels and magnitude (i.e. in relation to maintenance requirements) of metabolisable protein supply being compared and the demand of other competing physiological functions.     

Population dynamics of Trichostrongylus colubriformis in sheep: the effect of infection rate on the establishment of infective larvae and parasite fecundity

The establishment of Trichostrongylus colubriformis was estimated in helminthologically naive 20-week-old Merino sheep given third stage infective larvae (L3) at rates of 2000, 632 or 200 L3 per day, 5 days per week. After varying periods of continuous L3 intake, a levamisole-susceptible strain of T. colubriformis was replaced with a highly resistant strain for 1 week. The animals were then treated with levamisole to remove the susceptible population, and establishment of the cohort of resistant worms was estimated. In previously uninfected sheep, approximately 65% of the L3 given in the first week became established as adults. This fell to low levels (less than 5%) after 7, 10 and 14 weeks of continuous L3 intake for the high, medium and low infection rates, respectively. At the low infection rate, establishment remained at maximum levels for the first 4 weeks, but then fell at a rate similar to that observed for the higher infection rates. This implied that a threshold of worm exposure was required before resistance to establishment developed. Parasite egg production, expressed as eggs per gram of faeces, was proportional to infection rate and is explained by higher worm burdens occurring at high infection rates. However, estimates of fecundity in eggs per female per day showed the opposite relationship with rate of infection. Fecundity stayed high (approximately 600) for 5 weeks at the low infection rate but only maintained this level for 3 weeks and 1 week at the medium and high rates, respectively. This suggests that fecundity, like establishment, was similarly affected at threshold levels of immunological recognition.     

The responses of a tropical breed of domestic rabbit, Oryctolagus cuniculus, to experimental infection with Trichostrongylus colubriformis

Clinical, parasitological and pathological responses of a tropical out-bred domestic rabbit to experimental Trichostrongylus colubriformis infection were used to evaluate its suitability as a laboratory host and model for studying the host-parasite relationships of T. colubriformis. In the first experiment, three groups each of 16, predominantly juvenile male, 8- to 10-week-old rabbits were given a single pulse infection with 500, 5000 or 25000 infective larvae (L3) of T. colubriformis, to represent low, medium and high levels of infection, respectively. A fourth group of 16 rabbits of similar age formed the uninfected controls. In the second experiment, two groups of 10 juvenile (8- to 10-week-old) and 10 adult (8- to 10-month-old) rabbits were similarly infected with 20000 L3, with appropriate naive controls. Prepatency was 14 and 16 days and peak faecal egg counts occurred on days 24 and 20 after infection in young and adult rabbits respectively. Peak worm counts occurred on day 14 in both age groups and at all levels of infection. Subsequently, parasite burdens declined in a highly significantly dose- and age-dependent manner. At low and moderate levels of infection, approximately 83-98% of worms were recovered from the first 60 cm of the small intestine. Worm fecundity was also significantly influenced by host age and larval dose. Host age also had a significant effect on worm length. Infections with T. colubriformis were associated with a highly significant loss of body weight, accompanied by anorexia, diarrhoea and 25% mortality at high dose levels during the patent period of infection. There were no significant changes in packed cell volume and eosinophil counts at all ages and levels of infection but significant lymphocytosis occurred at the high dose level between days 7 and 21. Parasite-specific serum IgG responses were not related to worm burden. Overall, data showed that this miniature, docile and relatively inexpensive breed of rabbit is a potentially valuable laboratory host for studying T. colubriformis infections. The larval dose, duration of infection and host age were major determinants of host responsiveness to primary infections in this rabbit genotype.     

Multiple resistance to anthelmintics by Haemonchus contortus and Trichostrongylus colubriformis in sheep in Brazil

The objective of this study was to determine the level of resistance of Haemonchus contortus and Trichostrongylus colubriformis in sheep to levamisole, albendazole, ivermectin, moxidectin, closantel and trichlorfon. The parasites were isolated from sheep naturally infected by gastrointestinal nematodes and were then kept in monospecifically-infected lambs for production of infective larvae (L3) of both species. Forty-two lambs, at three months of age, were simultaneously artificially infected with 4000 L3 of H. contortus and 4000 L3 of T. colubriformis. The animals were allocated into seven groups with six animals each that received one of the following treatments: Group 1--control, no treatment; Group 2--moxidectin (0.2mg/kg body weight (BW)); Group 3--closantel (10mg/kg BW); Group 4--trichlorfon (100mg/kg BW); Group 5--levamisole phosphate (4.7 mg/kg BW); Group 6--albendazole (5.0mg/kg BW); and Group 7--ivermectin (0.2mg/kg BW). Nematode fecal egg counts (FEC) were carried out on the day of treatment and again at 3, 7, 10 and 14 days post-treatment. On the same occasions, composite fecal cultures were prepared for each group for production of L3, which were identified into genus. The animals were sacrificed for worm counts at 14 days after treatment. The efficacy of each treatment was calculated from the arithmetic mean of the FEC or worm burden of the treated group, compared with the values of the control group. Only trichlorfon and moxidectin treatments resulted in a significant reduction of H. contortus recorded at necropsy (73% and 45% respectively). Moxidectin reduced T. colubriformis worm burdens by 82% and albendazole by 19%. All other anthelmintics resulted in no significant reduction in the numbers of worms found at necropsy. In conclusion, the isolates of H. contortus and T. colubriformis showed multiple resistance to all groups of anthelmintics tested. This is the first report, based on the controlled efficacy test, to show resistance of T. colubriformis to macrocyclic lactones in Brazil.     

Expression of cell surface adhesion molecules by peripheral blood eosinophils during Trichostrongylus colubriformis infection in sheep

The effect of infection of sheep with the gastrointestinal nematode parasite Trichostrongylus colubriformis on expression of adhesion molecules CD11a, CD11b, CD11c, CD18, CD44, CD49d and CD62L by peripheral blood eosinophils was examined by flow cytometry. Initially, to establish the sensitivity of adhesion molecules to inflammatory signals, eosinophil-rich exudates were elicited in non-lactating mammary glands of immune sheep by infusion of 50 microg of soluble antigen extract from T. colubriformis third stage larvae. Eosinophils comprised 40.8% of mammary leucocytes and 4.5% of peripheral blood leucocytes. In comparison with blood, the percentage of eosinophils expressing CD18 increased and the percentage expressing CD62L decreased in exudates and the mean fluorescent intensity, an indicator of receptor number per cell, for CD11a and CD49d also decreased on exudate eosinophils. Peripheral blood eosinophils were examined over 8 weeks during trickle infection of immune sheep with infective or irradiated third stage larvae of T. colubriformis. During the last 3 weeks of infection, CD11a staining decreased in infected sheep and CD44 staining decreased in sheep receiving either infective or irradiated larvae. Other surface markers did not change. The results indicate that systemic changes in expression of adhesion molecules by eosinophils occur during T. colubriformis infection in sheep.     

Population dynamics of Trichostrongylus colubriformis in sheep: the effect of infection rate on loss of adult parasites

Rejection of adult T. colubriformis was examined in lambs given 2000, 1124, 632 or 200 L3 day-1, 5 days per week for up to 20 weeks. Rejection of adults began at approximately the same time for the first three infection rates, and took about 9 weeks to complete. Approximately 20% of adults were rejected by week 10 of infection at the higher infection rates and it was estimated that rejection commenced at about week 7. This coincided with a decline in establishment of larvae to about 1%. For the low infection rate, there was a delay of about 5 weeks before adult worm rejection commenced and reached equivalent levels to those observed at the higher rates. This delay also coincided with an estimated 5-week delay in larval establishment declining to 1% at the low rate.     

Identification and characterisation of an aspartyl protease inhibitor homologue as a major allergen of Trichostrongylus colubriformis

Allergens were identified from the gastrointestinal nematode of sheep, Trichostrongylus colubriformis, by probing Western blots of infective larvae (third stage) somatic antigen with IgE purified from the serum of sheep grazed on worm contaminated pasture. A 31 kDa allergen was frequently recognised by sera from immune sheep, particularly those deriving from a line that has been genetically selected over 23 years for parasite resistance. Using a proteomic approach, the 31 kDa allergen was identified as an aspartyl protease inhibitor homologue. The entire coding sequence of T. colubriformis aspartyl protease inhibitor (Tco-api-1) was obtained and the mature protein expressed in Escherichia coli. Anti-Tco-API-1 antibodies revealed that a commonly observed 21 kDa T. colubriformis allergen species is a truncated form of Tco-API-1. Specific IgE responses to T. colubriformis aspartyl protease inhibitor were significantly correlated with the degree of resistance to nematode infection as measured by faecal egg count in sheep. Surprisingly, IgE responses to Tco-API-1 were not correlated with breech soiling (dag score), which is thought to be caused, in part, by allergic hypersensitivity to worms. Therefore, a specific IgE response to this allergen may be a suitable marker for identifying lambs at an early age that will develop strong immunity to gastrointestinal nematodes.     

Effect of rearing density on pecking behaviour and plumage condition of laying hens in two types of aviary

The objective of this experiment was to investigate the effect of rearing density on pecking behaviour and plumage during rearing and throughout the laying period in aviaries. Chicks were reared on sand at high (H; 13 m⁻²) or low (L; 6.5 m⁻²) density, in four rearing pens of 390 chicks and eight pens of 195 chicks, respectively, each pen measuring 30 m². Proportions of chicks per pen performing various types of pecking behaviour were recorded by scan sampling during 16 observation bouts in each rearing pen at 6 weeks of age and during 24 observation bouts at 12 weeks. Individual body weights and plumage condition were recorded. Later, these pullets were housed at 17 hens m⁻² in Tiered Wire Floor (TWF; 3 H and 3 L pens of 275 hens) and Laco-Volétage (2 H and 2 L pens of 275 hens) aviaries. At 35 weeks, two samples of eight hens from each aviary pen were observed for pecking behaviour in a test pen. Throughout the laying period, additional records were collected on pecking behaviour, body weight, plumage condition, egg production, and mortality. The L birds had better plumage condition at 6 weeks of age and throughout the laying period. These birds also ground pecked more frequently than H birds during rearing and the laying period. At 12 weeks, L birds feather pecked less than H birds, but no relationship was found between rearing density and feather-pecking behaviour during the laying period. Although TWF hens feather pecked more frequently than Volétage hens, there was no interaction between rearing density and type of aviary for the various pecking behaviours.     

The effects of concurrent experimental infections of sheep with Trichostrongylus colubriformis and T. vitrinus on nematode distributions, numbers and on pathological changes

Simultaneous infections of Trichostrongylus colubriformis and T. vitrinus in the small intestine of the sheep were examined by comparing the numbers of worms which established and their distribution within the intestine in both monospecific infections and mixed infections. The results differed depending upon the species and number of parasites. The establishment of T. colubriformis was reduced and the distribution of the nematode population was displaced posteriorly within the intestine when 30,000 larvae of both species were administered, compared with pure infections of T. colubriformis. The reduced establishment was less marked with infections of 15,000 larvae of both species and there was only a slight posterior displacement of T. colubriformis. Neither effect was evident with infections of 7,500 larvae of both species. The rate of establishment and distribution of T. vitrinus were unaffected by the presence of T. colubriformis at all three rates of infection. Atrophy of villi and hypertrophy of crypts occurred at the main site of infection in the anterior duodenum. The severity of villus atrophy was related to the number of infective larvae administered and/or the worm burden. In the ileum, beyond the main site of infection, hypertrophy of villi was only found in sheep receiving the greatest number of infective larvae.     

Peripartal Excretion of Eimeria Oocyst by Cows on Swedish Dairy Farms and the Age of Calves at First Excretion

Faecal samples were collected 3 times a week for 6 weeks from 22 peripartal cows and for up to 15 weeks after birth from 27 calves in 3 herds, to determine the numbers of Eimeria oocysts excreted and the age at which the calves first excreted oocysts. Only low numbers of oocysts were excreted by the cows and no oocysts were detected in 93% of the samples. However, half the cows excreted oocysts at least once. The age at which the calves first excreted oocysts ranged from 2.5 to at least 15 weeks, and there was a significant difference between the herds in their mean age at first excretion. Oocysts of Eimeria alabamensis, E. auburnensis, E. bovis and E. ellipsoidalis were found in numbers ranging from 7 to 8450 oocysts per gram faeces. About 50% of the calves excreted oocysts before they were transferred to group pens. The primary source of infection of the calves was probably their penmates or the previous occupants of the pens, and the cows probably played a subsidiary role.     

Induction of protective immunity to Trichostrongylus colubriformis in neonatal merino lambs.

The premise that any bias of immune reactivity in neonatal lambs towards T-helper (TH)2 responses could benefit the induction of protection against gastrointestinal nematodes was investigated. In two trials, lambs were either trickle-immunised with 2000 infective larvae of Trichostrongylus colubriformis (TcL3), 3 times weekly from the day of birth for 6 weeks or inoculated with a recombinant T. colubriformis 17 kDa antigen in incomplete Freund's adjuvant (IFA). In trial 1, trickle immunised and control neonates challenged at 7 weeks of age had similar worm counts 10 days after challenge, but from 25 days, significant reductions (P<0.01) in mean faecal egg count and worm count in excess of 75% were displayed by the immunised lambs. The results of a second, similar trial, gave 85-91% reductions in parasitism in trickle immunised neonates (P<0.001) and around 50% protection in neonates vaccinated with recombinant 17 kDa antigen. Parasitism in immunised neonates in Trial 2 was significantly reduced (P<0.001) compared to that in 4-month-old animals. Antibody responses in trickle-immunised (protected) and challenge control (infected) neonates were almost exclusively of the IgG1 isotype compared to vaccinated animals which exhibited increased levels of anti-17kD IgG2. Trichostrongylus colubriformis infection, but not specific vaccination, induced interleukin-5 production by mesenteric lymph node cells. The results offer the tantalising prospect of generating protective immunity to gastrointestinal parasites prior to weaning in sheep; this was most effectively generated by viable parasites in this investigation.     

The migration and development of Parascaris equorum in the horse.

Eight worm-free pony foals, aged 2–4 weeks received a single infection of 8000 Parascaris equorum eggs. The foals were killed at intervals to investigate the migration and subsequent development of P. equorum. Signs of larval invasion were found in the liver within 48 h after infection. Larvae migrated from the liver to the lungs from 7 to 14 days after infection and the majority had returned to the small intestine via the tracheooesophageal route by day 23.After completion of their tissue migration, the parasites grew rapidly in size in the lumen of the small intestine, but after day 37 the total number of worms present in this site decreased. Patent infections developed on days 101–104.     

Studies on the resistance to tolerance induction against human IgG in DDD mice. III. Development of the resistance with age and cellular events.

Three-week-old DDD mice were easily rendered tolerant to human IgG while 12-week-old mice were tolerized only partially. Mechanisms of the development of the resistance with age were investigated. It was shown by the cell transfer experiments that spleen T cells, purified on a Tetron wool column, from older mice were responsible for the resistance, which was not associated with the loss of suppressor cells with age. To elucidate the possibility of whether tolerogen-sensitive spleen T cells differentiate into resistant ones, cell transfer experiments were carried out in which thymectomized, lethally irradiated mice were reconstituted with spleen cells from 3-week-old mice and then treated with the tolerogen on various days afterward. The results indicated that tolerance was inducible in these hosts to the same degree, irrespective of the timing of the tolerogen injection, while age-matched intact mice gradually acquired the resistance. Then the possibility of whether age of thymus affected tolerance inducibility of the hosts or not was examined. The tolerogen was injected into irradiated, bone-marrow-reconstituted mice which bore either 4- or 7-week-old thymus. It was shown that helper T cells newly generated under younger thymus acquired higher susceptibility to the tolerogen. There was no difference in tolerance inducibility irrespective as to whether bone marrow cells were prepared from younger or older mice. From these observations it was suggested that the resistance to tolerance induction in DDD mice is acquired through the appearance of resistant T cells which are generated from T-cell precursors in bone marrow under the influence of a radioresistant thymic constitution and predominantly located in the spleen.