Parasite Immunology 1992,14,645-655
Vaccination of young Dorset lambs against haemonchosis ANGELA S.TAVERNOR, TREVOR S.SMITH, CORDELIA F.LANGFORD, EDWARD A.MUNN & MARGARET GRAHAM Immunology Department, AFRC Institute of Animal Physiology, Cambridge Research Station, Babraham, Cambridge CB2 4AT, UK Accepted for publication 29 May 1992 Summary Six Dorset Horn lambs were each vaccinated at age 7 weeks and 9 weeks with 50 pg of glycosylated integral membrane proteins, particularly enriched in the protein H11 from the intestinal brush border of adult Haemonchus. At 11 weeks of age the lambs were infected with 10000infective third stage Haemonchus larvae. Compared with the average for the control group the vaccinated group of lambs had a 78% reduction in parasite egg output over the patent period, with four of the six better than 93% protected. At autopsy 35 days post-infection the mean total worm burden of the vaccinated lambs was 83% reduced compared with the controls. The serum antibody titres to H11 correlated with the degree of protection. Keywords: Haemonchus contortus, immune response, vaccination, young lambs
Introduction Previous studies have established that lambs 4 months old or more when vaccinated may be substantially protected against haemonchosis by injection with preparations enriched in the integral membrane protein HI 1 which is expressed only in the intestinal microvilli of parasitic stage Haemonchus contortus (Munn et al. 1992). One of these H1 1-enriched preparations, designated CABP, contains H11 and much smaller amounts of other microvillar integral membrane proteins (designated P1) which bind to Concanavalin A (Con-A) (Smith et al. 1992). When injected in suitable amounts P1 will give protection against haemonchosis, but at the level present it contributes very little to the protective activity of CABP and most of this protective activity is attributable to H11. Based on dose-response studies it was shown that between 10 and 100 pg CABP given in two equal injections protected Clun Forest lambs ten months old (Tavernor et al. 1992). To be of value a vaccine against Haemonchus must be effective in young lambs less than 4 months old as at this age they are particularly susceptible to haemonchosis. Attempts to vaccinate lambs by stimulating natural immune responses by exposure to attenuated larvae, whilst very effective in lambs some six months old or more were not Correspondence: E.A.Munn.
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successful in younger lambs (Jarrett et al. 1959, Mulligan er al. 1961,Urquhart et al. 1962, 1966a,b, Lopez & Urquhart 1967).In the course of natural immune responses to infection with Huemonchus many circulating antibodies to larval and adult antigens are produced. None of these antibodies, however, is specificfor the protein HI 1, or other proteins at the luminal surface of the parasite’s intestine (Munn et al. 1987, 1992). It thus became of interest to determine whether H 1 1 would induce the formation of protective antibodies in young lambs. In the experiment described here lambs were first injected immediately after weaning and infected when 1I weeks old. The H11 preparation CABP was used in two doses of 50 pg. Materials and methods PARASITES A N D IMMUNOGEN PREPARATION
Adult Haemonchus contortus were obtained and extracted as described in Smith et al. (1992). Briefly, adult Huemonchus stored at -20°C in approximately their own weight of phosphate buffered saline containing 0.02% NaN3 (pbs/a) were thawed and then homogenized in ice-cold pbs/a. The homogenate was centrifuged and the pellets were reextracted with pbs/a. The pellets were extracted with 1% Tween 20 and then with 1% Thesit (polyoxyethylene 9 lauryl ether, Boehringer Mannheim GmbH) in pbs/a. The Thesit extract thus obtained was ultracentrifuged and the supernatant liquid contained essentially all of the H 11 protein as judged by sodium dodecylsulphatepolyacrylamidegel electrophoresis (SDS-PAGE). This solution was applied to a Con-A column and bound proteins were eluted with methyl a-D-glucopyranoside. Fractions coinciding with the UV (280 nm) peaks were run on SDS-PAGE and those containing H11 (and P1) were pooled to give CABP (Con A-binding proteins). Approximately 85% of CABP is composed of H1 1; the bulk of the remainder being P1 (Smith et al. 1992). Protein concentrations were determined using the Micro BCA protein assay reagent (Pierce Europe B.V., Oud Beijerland, The Netherlands), avoiding high concentrations of Con-A buffer which cause interference in the estimation. EXPERIMENTAL ANIMALS A N D VACCINATION
Eleven 7-week-old, just-weaned Dorset lambs raised helminth-free on the ewe at the AFRC Institute of Animal Health, Compton, were assigned to two groups; group A of 5 animals and group B of 6. The groups were matched for lambs of similar body weight and both groups contained males and females. Group A animals, the controls, were each injected with two doses of 50 pg horse spleen ferritin (Type 1,Sigma Chemical Co, Poole, Dorset, UK), chosen as an unrelated protein. The lambs in group B were each injected with two doses of 50 pg CABP. The first injections were given whilst the lambs were at Compton. The second injections were given 15 days later after the lambs had been transferred to Babraham. Each dose totalled 2 ml and was injected subcutaneouslyat 4 or 5 sites in the groin region. The lambs were housed indoors on straw and maintained on a high plane of nutrition. They were examined daily with the intention that any showing overt clinical symptoms should be removed from the trial. This proved necessary for one of the control animals (see Results).
Vaccination against haemonchosis
647
The antigens in sodium acetate buffer pH 5.2 were mixed with an equal volume of Alhydrogel (Superfos Biosector a/s, Vedbaek, Denmark) and twice their volume of Freund’s complete adjuvant (FCA) (CFA, Difco Labs. W. Molesey, Surrey, UK) for the first injection, or Freund’s incomplete adjuvant (FIA) for the second injection. The mixtures were homogenized using an Ultra-Turrax T25 motorized benchtop homogenizer (Janke & Kunkel GmbH, Staufen, Germany) until they formed a stable emulsion and were used immediately. BLOOD SAMPLES
Four ml of blood for serum production was collected by jugular venepuncture prior to the first injection and then at intervals throughout the course of the experiment. At postmortem larger volumes were collected. The sera were stored at - 20°C. Haematocrit values (packed cell volumes, pcv) were determined from 1 ml heparinized blood samples taken from the jugular vein. ELISAs Serum from each animal was diluted 1 : 1000 in pbs and assayed in triplicate for antibodies against CABP or ferritin by enzyme-linked immunosorbent assay (ELISA) as described by Munn et al. (1992). All reagents were titrated before use to establish optimum conditions for detection of antibodies. The I : 1000 dilution was chosen because it gave a good range of values (OD45o) over the course of the experiment. INFECTIVE L A R V A E
Infective third stage larvae were obtained, as described by Munn et a1 (1987), from the faeces of sheep harbouring pure experimental infections of Haemonchus contortus. The strain used was originally provided by Dr R.Connan (School of Veterinary Medicine, University of Cambridge). The larvae were stored in a shallow layer of glass-distilled water at 4°C for less than six months. The strain of Haemonchus and their conditions of storage were the same as used by Tavernor et al. (1 992). CHALLENGE
Fourteen days after their second injection the lambs were challenged with 10000 infective larvae in 20 ml water administered via a stomach tube directly into the rumen. F A E C A L EGG COUNTS
(FECS)
Rectal faecal samples were collected at one week intervals, beginning one week before larval challenge and continuing until 35 days post challenge when the sheep were killed. Egg counts were carried out using a McMaster counting chamber (Soulsby 1982). The results are presented as eggs per gram (epg) faeces. WORM BURDENS
The total worm burden was determined when the sheep were slaughtered 35 days after
648
A.S.Tavernor et al.
larval challenge. Great care was taken to ensure that every worm was recovered from each abomasum.The surfaceof the mucosa, to which the bulk of the worms were attached, was washed gently with pbs and the washings and abomasa contents were pooled and made to 150 ml. The attached worms were rubbed from the surface of the mucosa into fresh pbs and the volume made to 700 ml. In both cases the pbs was at less than 20°C in order to minimize the possibility of the worms clumping at this stage. When the number of worms was judged to be small all the worms in these suspensions were counted. When the number of worms was judged to be comparatively large the worms were counted in samples taken immediately the suspensionswere obtained. Each suspension was stirred vigorously and a sample of about 70 ml obtained by plunging a 50 ml beaker into the suspension. Two further samples were taken in the same way and the total volume of the pooled samples was determined accurately. The blotted-dry weight of the worms was also determined. For those worm burdens determined by sampling, the female to male ratio (FMR)was calculated by counting the number of males present in a sample containing 100 females. For each animal the lengths of 3 1 individual worms of each sex in a random sample were determined by the method of Coadwell & Ward (1977). STATISTICS
The data were analysed statistically using the Mann-Whitney U test.
Results HAEMATOCRIT VALUES
The haematocrits of all the lambs in both groups had fallen to between 60-80% of their starting values by 13 days post-infection (Table 1). Those of the control group continued to fall. The haematocrit of one control animal, A4, determined on day 26 p.i. because the lamb seemed listless and off its food, was found to be only 20% of its starting value and this animal was therefore euthanased immediately.On day 35 p.i. the day all animals were slaughtered, two other animals in the control group, A2 and A5, had packed cell volumes less than 20% of their pre-challengevalues. Neither of these animals however, showed any sign of listlessness or inappetence. The haematocrits of the animals vaccinated with HI 1 reached a minimum (mean of 0-18 0-14)on day 21 p.i. and then started to recover so that by day 35 p.i. the average pcv for this group was 68.9% of the pre-infection value with B2, B4 and B5 having the highest values (Table 1). FAECAL EGG COUNTS
The mean values for the faecal egg counts of the vaccinates were significantly less than those of the control group (P
Vaccination of young Dorset lambs against haemonchosis ANGELA S.TAVERNOR, TREVOR S.SMITH, CORDELIA F.LANGFORD, EDWARD A.MUNN & MARGARET GRAHAM Immunology Department, AFRC Institute of Animal Physiology, Cambridge Research Station, Babraham, Cambridge CB2 4AT, UK Accepted for publication 29 May 1992 Summary Six Dorset Horn lambs were each vaccinated at age 7 weeks and 9 weeks with 50 pg of glycosylated integral membrane proteins, particularly enriched in the protein H11 from the intestinal brush border of adult Haemonchus. At 11 weeks of age the lambs were infected with 10000infective third stage Haemonchus larvae. Compared with the average for the control group the vaccinated group of lambs had a 78% reduction in parasite egg output over the patent period, with four of the six better than 93% protected. At autopsy 35 days post-infection the mean total worm burden of the vaccinated lambs was 83% reduced compared with the controls. The serum antibody titres to H11 correlated with the degree of protection. Keywords: Haemonchus contortus, immune response, vaccination, young lambs
Introduction Previous studies have established that lambs 4 months old or more when vaccinated may be substantially protected against haemonchosis by injection with preparations enriched in the integral membrane protein HI 1 which is expressed only in the intestinal microvilli of parasitic stage Haemonchus contortus (Munn et al. 1992). One of these H1 1-enriched preparations, designated CABP, contains H11 and much smaller amounts of other microvillar integral membrane proteins (designated P1) which bind to Concanavalin A (Con-A) (Smith et al. 1992). When injected in suitable amounts P1 will give protection against haemonchosis, but at the level present it contributes very little to the protective activity of CABP and most of this protective activity is attributable to H11. Based on dose-response studies it was shown that between 10 and 100 pg CABP given in two equal injections protected Clun Forest lambs ten months old (Tavernor et al. 1992). To be of value a vaccine against Haemonchus must be effective in young lambs less than 4 months old as at this age they are particularly susceptible to haemonchosis. Attempts to vaccinate lambs by stimulating natural immune responses by exposure to attenuated larvae, whilst very effective in lambs some six months old or more were not Correspondence: E.A.Munn.
645
646
A.S.Tavernor et al.
successful in younger lambs (Jarrett et al. 1959, Mulligan er al. 1961,Urquhart et al. 1962, 1966a,b, Lopez & Urquhart 1967).In the course of natural immune responses to infection with Huemonchus many circulating antibodies to larval and adult antigens are produced. None of these antibodies, however, is specificfor the protein HI 1, or other proteins at the luminal surface of the parasite’s intestine (Munn et al. 1987, 1992). It thus became of interest to determine whether H 1 1 would induce the formation of protective antibodies in young lambs. In the experiment described here lambs were first injected immediately after weaning and infected when 1I weeks old. The H11 preparation CABP was used in two doses of 50 pg. Materials and methods PARASITES A N D IMMUNOGEN PREPARATION
Adult Haemonchus contortus were obtained and extracted as described in Smith et al. (1992). Briefly, adult Huemonchus stored at -20°C in approximately their own weight of phosphate buffered saline containing 0.02% NaN3 (pbs/a) were thawed and then homogenized in ice-cold pbs/a. The homogenate was centrifuged and the pellets were reextracted with pbs/a. The pellets were extracted with 1% Tween 20 and then with 1% Thesit (polyoxyethylene 9 lauryl ether, Boehringer Mannheim GmbH) in pbs/a. The Thesit extract thus obtained was ultracentrifuged and the supernatant liquid contained essentially all of the H 11 protein as judged by sodium dodecylsulphatepolyacrylamidegel electrophoresis (SDS-PAGE). This solution was applied to a Con-A column and bound proteins were eluted with methyl a-D-glucopyranoside. Fractions coinciding with the UV (280 nm) peaks were run on SDS-PAGE and those containing H11 (and P1) were pooled to give CABP (Con A-binding proteins). Approximately 85% of CABP is composed of H1 1; the bulk of the remainder being P1 (Smith et al. 1992). Protein concentrations were determined using the Micro BCA protein assay reagent (Pierce Europe B.V., Oud Beijerland, The Netherlands), avoiding high concentrations of Con-A buffer which cause interference in the estimation. EXPERIMENTAL ANIMALS A N D VACCINATION
Eleven 7-week-old, just-weaned Dorset lambs raised helminth-free on the ewe at the AFRC Institute of Animal Health, Compton, were assigned to two groups; group A of 5 animals and group B of 6. The groups were matched for lambs of similar body weight and both groups contained males and females. Group A animals, the controls, were each injected with two doses of 50 pg horse spleen ferritin (Type 1,Sigma Chemical Co, Poole, Dorset, UK), chosen as an unrelated protein. The lambs in group B were each injected with two doses of 50 pg CABP. The first injections were given whilst the lambs were at Compton. The second injections were given 15 days later after the lambs had been transferred to Babraham. Each dose totalled 2 ml and was injected subcutaneouslyat 4 or 5 sites in the groin region. The lambs were housed indoors on straw and maintained on a high plane of nutrition. They were examined daily with the intention that any showing overt clinical symptoms should be removed from the trial. This proved necessary for one of the control animals (see Results).
Vaccination against haemonchosis
647
The antigens in sodium acetate buffer pH 5.2 were mixed with an equal volume of Alhydrogel (Superfos Biosector a/s, Vedbaek, Denmark) and twice their volume of Freund’s complete adjuvant (FCA) (CFA, Difco Labs. W. Molesey, Surrey, UK) for the first injection, or Freund’s incomplete adjuvant (FIA) for the second injection. The mixtures were homogenized using an Ultra-Turrax T25 motorized benchtop homogenizer (Janke & Kunkel GmbH, Staufen, Germany) until they formed a stable emulsion and were used immediately. BLOOD SAMPLES
Four ml of blood for serum production was collected by jugular venepuncture prior to the first injection and then at intervals throughout the course of the experiment. At postmortem larger volumes were collected. The sera were stored at - 20°C. Haematocrit values (packed cell volumes, pcv) were determined from 1 ml heparinized blood samples taken from the jugular vein. ELISAs Serum from each animal was diluted 1 : 1000 in pbs and assayed in triplicate for antibodies against CABP or ferritin by enzyme-linked immunosorbent assay (ELISA) as described by Munn et al. (1992). All reagents were titrated before use to establish optimum conditions for detection of antibodies. The I : 1000 dilution was chosen because it gave a good range of values (OD45o) over the course of the experiment. INFECTIVE L A R V A E
Infective third stage larvae were obtained, as described by Munn et a1 (1987), from the faeces of sheep harbouring pure experimental infections of Haemonchus contortus. The strain used was originally provided by Dr R.Connan (School of Veterinary Medicine, University of Cambridge). The larvae were stored in a shallow layer of glass-distilled water at 4°C for less than six months. The strain of Haemonchus and their conditions of storage were the same as used by Tavernor et al. (1 992). CHALLENGE
Fourteen days after their second injection the lambs were challenged with 10000 infective larvae in 20 ml water administered via a stomach tube directly into the rumen. F A E C A L EGG COUNTS
(FECS)
Rectal faecal samples were collected at one week intervals, beginning one week before larval challenge and continuing until 35 days post challenge when the sheep were killed. Egg counts were carried out using a McMaster counting chamber (Soulsby 1982). The results are presented as eggs per gram (epg) faeces. WORM BURDENS
The total worm burden was determined when the sheep were slaughtered 35 days after
648
A.S.Tavernor et al.
larval challenge. Great care was taken to ensure that every worm was recovered from each abomasum.The surfaceof the mucosa, to which the bulk of the worms were attached, was washed gently with pbs and the washings and abomasa contents were pooled and made to 150 ml. The attached worms were rubbed from the surface of the mucosa into fresh pbs and the volume made to 700 ml. In both cases the pbs was at less than 20°C in order to minimize the possibility of the worms clumping at this stage. When the number of worms was judged to be small all the worms in these suspensions were counted. When the number of worms was judged to be comparatively large the worms were counted in samples taken immediately the suspensionswere obtained. Each suspension was stirred vigorously and a sample of about 70 ml obtained by plunging a 50 ml beaker into the suspension. Two further samples were taken in the same way and the total volume of the pooled samples was determined accurately. The blotted-dry weight of the worms was also determined. For those worm burdens determined by sampling, the female to male ratio (FMR)was calculated by counting the number of males present in a sample containing 100 females. For each animal the lengths of 3 1 individual worms of each sex in a random sample were determined by the method of Coadwell & Ward (1977). STATISTICS
The data were analysed statistically using the Mann-Whitney U test.
Results HAEMATOCRIT VALUES
The haematocrits of all the lambs in both groups had fallen to between 60-80% of their starting values by 13 days post-infection (Table 1). Those of the control group continued to fall. The haematocrit of one control animal, A4, determined on day 26 p.i. because the lamb seemed listless and off its food, was found to be only 20% of its starting value and this animal was therefore euthanased immediately.On day 35 p.i. the day all animals were slaughtered, two other animals in the control group, A2 and A5, had packed cell volumes less than 20% of their pre-challengevalues. Neither of these animals however, showed any sign of listlessness or inappetence. The haematocrits of the animals vaccinated with HI 1 reached a minimum (mean of 0-18 0-14)on day 21 p.i. and then started to recover so that by day 35 p.i. the average pcv for this group was 68.9% of the pre-infection value with B2, B4 and B5 having the highest values (Table 1). FAECAL EGG COUNTS
The mean values for the faecal egg counts of the vaccinates were significantly less than those of the control group (P
