F.N.J. Kooyman

Core α1→3-fucose is a common modification of N-glycans in parasitic helminths and constitutes an important epitope for IgE from Haemonchus contortus infected sheep

Synthesis of parasite specific IgE plays a critical role in the defence against helminth infections. We report here that IgE from serum from Schistosoma mansoni infected mice and Haemonchus contortus infected sheep recognizes complex‐type N‐glycans from Arabidopsis thaliana, which contain R‐GlcNAcβ1→4(Fucα1→3)GlcNAcβ1‐Asn (core α1→3‐Fuc) and Xylβ1→2Manβ1→4GlcNAcβ1‐R (core β1→2‐Xyl) modifications, and honeybee phospholipase A2, which carries N‐glycans that contain the core α1→3‐Fuc epitope. Evidence is presented that core α1→3‐fucosylated N‐glycans bind a substantial part of the parasite specific IgE in serum of H. contortus infected sheep. These results suggest that the core α1→3‐Fuc antigen may contribute to induction of a Th2 response leading to the production of IgE. In addition we show here that N‐glycans carrying core α1→3‐Fuc and β1→2‐Xyl antigens are synthesized by many parasitic helminths and also by the free living nematode Caenorhabditis elegans. Since N‐glycans containing the core α1→3‐Fuc have also been implicated in honeybee and plant induced allergies, this conserved glycan might represent an important common IgE epitope.

The effect of ivermectin treatment against inhibited early third stage, late third stage and fourth stage larvae and adult stages of the cyathostomes in Shetland ponies and spontaneous expulsion of these helminths

A controlled and critical test on the efficacy of ivermectin against larval and adult stages of the cyathostomes was carried out in six yearling castrated male Shetland ponies. The ponies grazed together as one group from 3 May to 4 October 1990, after which they were housed. Three ponies were treated with ivermectin on 29 October while the others served as controls. The shedding of helminths in the faeces was followed in all ponies until necropsy on 14 November. Comparison of worm counts of both groups before and after necropsy showed no evidence for an effect of ivermectin against inhibited early third stage larvae (EL3) and mucosal late third stage (LL3) and fourth stage larvae (L4). However, a high, but not 100%, efficacy was observed against adults and lumenal L4. A remarkable observation was the high incidence of spontaneous expulsion of L4 and adult populations of some species in two of the untreated ponies.

Protection studies with recombinant excretory/secretory proteins of Haemonchus contortus.

The efficacy of two recombinant proteins of Haemonchus contortus was studied in both adult sheep and young lambs. These 15 and 24 kDa excretory/secretory proteins were given combined, either supplemented or not with a glycan‐rich insect cell extract. In 9‐month‐old sheep (trial 1), faecal egg output and worm burden were reduced by 49% and 55%, respectively, after vaccination with rec15/24, and by 46% and 65% after vaccination with rec15/24 and glycan extract. No reduction in egg output or number of worms was found in young lambs using the above recombinant proteins plus glycan‐rich extract (trial 2). When trial 1 was repeated (trial 3), the protection could not be reproduced, possibly due to differences in batches of recombinant proteins. In all sheep, independent of their age, rec15/24‐specific immunoglobulin (Ig)G1 and IgA titres were present, but 9‐month‐old protected sheep had significantly higher IgA titres than the lambs. Addition of glycans resulted in lower rec15/24‐specific IgG1 and IgA in 9‐month‐old sheep after challenge. This did not affect the level of protection. A significant negative correlation was found between IgA and worm numbers in protected sheep immunized with rec15/24 supplemented with glycans. Total IgE and rec15/24 specific IgE titres were low. The number of eosinophils, mast cells, sheep mast cell protease (SMCP)+ cells and IgA+ cells did not differ between the protected and unprotected sheep, but the lambs had significantly fewer mast cells independent of their immunization.

IgE antibody during infection with the ovine abomasal nematode, Teladorsagia circumcincta: primary and secondary responses in serum and gastric lymph of sheep

A monoclonal antibody to ovine IgE was employed in an ELISA to investigate the IgE antibody responses in serum and gastric lymph to a primary infection of Teladorsagia circumcincta, and following challenge in previously infected sheep. During a primary response, IgE antibody to antigens derived from the infective third stage (L3) and adult (L5) worms were negligable, with low levels of IgE antibody detected in serum and lymph. In contrast, there was a pronounced IgE antibody response in 2/4 sheep to L3 antigens during 2–8 days after challenge of previously infected animals but low levels of IgE antibody to L5 antigens. This response was confirmed in a second but similar experiment, where relatively high levels of IgE antibody was detected to antigens from L3. Antibody levels were higher in lymph than in serum from the same animals, and Western blots of L3 antigen following SDS‐PAGE under reducing conditions revealed several bands of MW26–96KD which reacted with the IgE antibody from gastric lymph. Immunohistochemical staining indicated that these IgE antibodies may be reacting with allergens associated with the surface cuticle of the worms.

Seasonally inhibited development of cyathostomine nematodes in Shetland ponies in The Netherlands.

Two groups of three yearling Shetland ponies were used in 1988 to study the epidemiological significance of inhibited development of the Cyathostomine nematodes. In Group 1, acquisition of infections was prevented throughout the grazing season whereas in Group 2 strongylid infections were acquired from the beginning of July until the end of September. Worm counts showed that the Cyathostomine nematode populations of Group 1 ponies mainly consisted of adult worms and those of Group 2 ponies of inhibited early third stage larvae (L3). These results indicate that a large proportion of Cyathostomine larvae which establish between the beginning of July and the end of September inhibit their development. The results also suggest that the pool of inhibited larvae which has overwintered in young ponies will be depleted in autumn.

Cross-sectional serological survey on gastrointestinal and lung nematode infections in first and second-year replacement stock in the netherlands: relation with management practices and use of anthelmintics.

A cross-sectional survey was carried out on 86 farms randomly distributed in The Netherlands. After housing following the first and the second grazing season (FGS and SGS) serum samples were collected to determine IgG levels against Cooperia oncophora and Dictyocaulus viviparus, and the pepsinogen content. A questionnaire was used to inquire on grazing management practices and the use of anthelmintic drugs. On 80.7 and 60.2% of the farms FGS and SGS animals, respectively, were treated at least once with an anthelmintic drug. The percentage for the SGS animals indicates that the use of anthelmintic drugs in those animals has increased enormously over the last 10-15 years. Generally, parasitic nematode control in the FGS is good on most farms, but it can be characterised as being overprotective. There is a tendency that if anthelmintic drugs are used in the FGS they also are used more often in the SGS. On 12 farms (14%), no anthelmintic drugs were given in the FGS and the SGS. These farms did not differ from the others with respect to management practices in any obvious way. The serological results were in general very low, indicating low levels of exposure to gastrointestinal nematode infection in both FGS and SGS animals. This was not surprising in view of the good to high level of nematode control practices reported by the farmers. Although not statistically significant, a consistent result was that serological results for the SGS animals were more often positive or on average higher on those farms where FGS parasite control tended to be excessive. For D. viviparus, a prevalence rate of 41% positive farms was found. Following comparison with previous data, it is speculated that lungworm (sero-)prevalence in replacement stock may be declining as a result of continuing high levels of parasite control in replacement stock. It is concluded that the results confirm previous surveys, lending support to the conclusion that parasitic nematode control on Dutch dairy farms, certainly in FGS calves, is good but tends to be overprotective.

Emergence from inhibited development of cyathostome larvae in ponies following failure to remove them by repeated treatments with benzimidazole compounds

The effect of three albendazole treatments at 5-week intervals, beginning at turnout in April, on cyathostome infections in Shetland ponies was compared with the effect of sequential treatments with albendazole, oxfendazole and oxibendazole. The results showed a substantial reduction in faecal egg output after the first albendazole treatment. Since faecal egg counts remained very low, no estimation of the effect of the second treatment was possible. The third treatment with albendazole and oxibendazole was followed by an increase in faecal egg counts to values of greater than 100 eggs g-1 within 4 weeks. A final albendazole treatment in December, 1 week before necropsy, failed to reduce faecal egg counts. These results suggest resistance to albendazole and oxibendazole in the cyathostome populations of the ponies. The increase in faecal egg counts after the third anthelmintic treatment in July occurred, although overwintered pasture infectivity was very low. The most likely explanation for this increase is resumption of the development of worms which overwintered as inhibited larvae in the host.

Studies on the immunoglobulin E responses to Teladorsagia circumcincta in sheep: purification of a major high molecular weight allergen.

Studies on the immunoglobulin (Ig)E immune responses to the gastric nematode, Teladorsagia circumcincta, have demonstrated a major high molecular weight allergen (HMWTc). Cross reactive allergens of similar MW were demonstrated for Trichostrongylus colubriformis and Cooperia curticei, but not for Haemonchus contortus. Purification of HMWTc was achieved by gel‐filtration chromatography, and nonreducing SDS‐PAGE and Western blot analysis revealed two closely associated bands with a molecular weight of approximately 140–150 kDa. Reduction showed four IgE reactive bands of 120, 50, 45 and 30 kDa, and deglycosylation abrogated the immunoreactivity of the 120 and 30 kDa bands. Ultrastructural immunolocalization by electron microscopy revealed that the IgE reactivity was confined to the cuticular surface of the infective (L3) larvae. ELISA studies to determine the IgE anti‐HMWTc responses in lambs during their first grazing season, demonstrated significantly higher IgE antibody in lambs with low accumulative faecal egg count (FEC) compared to animals with high accumulative FEC. These studies provide evidence for a protective function of IgE antibody in Teladorsagia infections in lambs.

The effect of repeated moves to clean pasture on the build up of gastrointestinal nematode infections in calves

The build up of gastrointestinal nematode infections was followed in two grazing experiments. Both experiments included four groups of six calves, a permanently housed non-infected control group and three groups which were grazed from May to October. One of these was moved to aftermath in the beginning of July, the second in the beginning of July and August and the third in the beginning of July, August and September. The build up of gastrointestinal nematode infections was followed by performing faecal egg counts, differentiation of faecal larval cultures, pasture larval counts, serum pepsinogen values, serum antibodies against Cooperia oncophora, weight gain and worm counts. In the second experiment four of the principal trial animals of each group were treated with oxfendazole and subsequently challenged with 100,000 larvae of Ostertagia ostertagi to examine development of immunity against O. ostertagi. The faecal egg counts and the worm counts of the sentinels necropsied in July indicated low initial infections in both experiments. Infection levels in experiment 1 remained low in each group until the beginning of September. However, during the last month, moderate to high infections were acquired by the groups which were moved once or twice. In contrast, low to moderate infections were maintained in the group moved three times. In the second experiment moderate C. oncophora burdens were already observed in the sentinels grazed until the beginning of August. Tracers grazing in August-September with the group moved once acquired high O. ostertagi and C. oncophora infections, whereas those grazed with both other groups acquired moderate infections. In October high infections with both species occurred in the groups moved once and twice, whereas low to moderate infections were observed in the group moved three times. The challenge infection demonstrated a reduction of establishment of O. ostertagi of approximately 70% in all three groups on pasture. The results demonstrate that moving calves at monthly intervals to clean pasture can be an effective method for the control of parasitic gastroenteritis. In addition, the data indicate that it is essential that the last move does not occur more than 1 month before the end of the grazing season.

Serum immunoglobulin E response in calves infected with the lungworm Dictyocaulus viviparus and its correlation with protection

Protection of a primary Dictyocaulus viviparus infection was measured against a homologueous challenge infection in two independent experiments and this was correlated with serum immunoglobulin (Ig)E responses. A primary infection of 30 third stage larvae (L3) of D. viviparus on day 0 protects calves for 70% against a challenge infection of 2000 L3 on day 35 compared to calves with no primary infection. The variation in post mortem worm counts within this group (n = 6) was very large with mean worm counts of 145 (range 3–446) lungworms. Parasite specific IgA, IgE, IgG1 and IgG2 and total IgE levels in serum were measured by ELISA. Parasite specific IgA, IgG1 and IgG2 were elevated after infection, but correlation with protection was only found with IgG1 levels on day 42 and with IgG2 levels on day 70. IgE was measured in a sandwich ELISA using antisheep IgE that cross‐reacts with cattle IgE. No parasite specific IgE could be detected. However, total serum IgE was elevated after infection and total serum IgE levels before and on the day of challenge correlated with protection (P < 0·05). Total serum IgE also correlates with peripheral eosinophil counts between days 14 and 28 after primary infection. Western blots with three different parasite antigen preparations, L1, excretory/secretory products and crude worm adult antigens, were used to detect parasite specific IgE in sera depleted of IgG and IgM. These depleted sera from protected calves contained parasite specific IgE, while sera from nonprotected calves were negative. A band of approximately 100 kDa was recognized in all three antigens. In a second experiment, primary doses of 30, 60, 120, 240, 480 and 960 L3 of D. viviparus were used and necropsy was 11 days after challenge. This experiment confirmed the correlation between protection and total IgE levels before and on the day of challenge. The rapid and strong IgE responses in protected animals after such a low infection might be caused by the specific characteristics of the lungworm antigens or by the somatic migration of the worm and might be involved in the rapid development of protection against lungworm reinfections in cattle.