Serum and abomasal antibody response of sheep to infections with Haemonchus contortus

Serum and abomasal IgA, IgG and IgM antibody response against adult worm, L3 and egg antigens of Haemonchus contortus was monitored by the ELISA technique after one or two infections with this nematode. Following the first infection, antibody levels in serum did not change materially. After administration of a challenge dose of infective larvae, antibodies of the three immunoglobulin classes in infected animals rose slightly, but this rise appeared later than the fall in the faecal egg counts. In contrast, in abomasal mucosa, IgA anti-larval antibody levels, which did not increase materially after the primary infection, rose rapidly after a transient inhibition when sheep were challenged. A close temporal relationship was observed between the rise in local anti-worm IgA antibodies and the self-cure reaction, but antibody levels fell rapidly after worm diminution. The local antibody response was thus considered to be related to immunity of sheep to H. contortus.     

Serum antibody response of Castellana sheep to Haemonchus contortus infection and challenge: relationship to abomasal worm burdens

Primary and secondary serum antibody responses to Haemonchus contortus were studied in Castellana sheep. Ten-month-old sheep were infected (200 L3/kg live weight (lw)) and challenged (400 L3/kg lw) or uninfected and equally challenged with H. contortus. Primary infections induced a partially protective response upon challenge, characterized by higher serum protein levels, longer prepatent periods, lower fecal egg counts, and significant reduction in the establishment rate of the parasite and abomasal adult and L4 worm burdens. The resistant status of the infected and challenged sheep was not clearly related either to the serum specific antibody levels (IgG: IgG1, IgG2; IgM; IgA) estimated by ELISA or to immunodetection patterns in the Western blots.     

Partial protection of lambs against Haemonchus contortus by vaccination with a fractionated preparation of the parasite

Florida Native lambs, less than 6 months of age, were successfully vaccinated against Haemonchus contortus with a high mol. wt fraction (greater than 30,000 daltons) derived from a somatic extract of H. contortus larvae (SEL) and excretions and secretions (ES) of larvae isolated during in vitro development from the infective 3rd to 4th stage. A 59% reduction in adult worm numbers was obtained in vaccinates compared to naive lambs following challenge. The protection in vaccinated lambs was similar to that seen in lambs exposed to a primary infection of H. contortus larvae which had been cleared with anthelmintic prior to the challenge infection. The unfractionated SEL/ES preparation and a low mol. wt fraction gave no significant protection against challenge infection.     

Protection against high-dose homologous infection in calves immunized with intestine or membrane extracts from Haemonchus placei

Control of Haemonchus placei, one of the most important cattle nematodes in Brazil, relies on the use of anthelmintics. However, there is a need for integrated control, which includes active immunization. The aim of this work was to assess the protection afforded to calves by immunization with adult H. placei extracts against a high-dose challenge infection, a condition frequently found in the tropics. Holstein calves aged 8-10 months were immunized four times with intestinal extracts (Group D) or with a Triton X-100-soluble fraction of adult H. placei (Group A), challenge-infected with 120,000 infective larvae and sacrificed 40 days later. Immunized animals had higher IgG titers than the controls against tested fractions after the 2nd immunization, peaking after the 4th. Sera from both immunized groups recognized bands of similar apparent mass in both antigenic preparations, some of which were similar in molecular weight to Haemonchus contortus antigens with known protective effect to sheep. Egg counts were 49% and 57% lower in Groups A and D than in controls, respectively. High levels of protection were observed in two of the four calves in Group D, as evidenced by very low worm numbers recovered at necropsy, absence of eggs in the uteri of the recovered females and reduced worm length. Group D animals also showed milder signs of anemia than the other infected animals. Results demonstrate that protection against homologous high-dose challenge can be achieved by immunizing calves with H. placei gut antigens.     

The intensity of resistance by mature Merino ewes against Haemonchus contortus and Tichostrongylus colubriformis in single-species and combined-species infection

Three-year-old, non-lactating and non-pregnant Merino ewes, raised on pasture under a program of strategic treatment with anthelmintic and found to be extremely resistant to "trickle" infection with Haemonchus contortus, were given single-dose infections with either H. contortus or Trichostrongylus colubriformis or both species together. The purpose was to ascertain the intensity of protective immunity against the 2 parasites in sheep with immunity acquired from a presumably slight exposure to infection. To provide a criterion, some infected ewes were immunosuppressed with corticosteroid, dexamethasone. Untreated ewes were extremely resistant to challenge infection with either 15,000 or 150,000 H. contortus or 15,000 T. colubriformis. Surprisingly, when mixed infection was given, egg counts for H. contortus were significantly elevated compared with infection by that species alone. Antibody to antigens from infective larval and adult H. contortus was measured in serum by enzyme-linked-immunosorbent assay (ELISA) during the course of infection. Serum titres against larval antigens were significantly depressed when infections with either H. contortus or T. colubriformis were permitted by immunosuppression with dexamethasone, whereas those against adult antigen were depressed when infection with T. colubriformis was permitted.     

Serodiagnosis of haemonchosis with a somatic antigen (Hc26) in several breeds of sheep.

Sera from 53 sheep belonging to Castellano, Churro, Manchego, and Merino breeds were analyzed to test the diagnostic value of a 26-kD antigen from adult Haemonchus contortus at prepatency and early and late patency of experimental haemonchosis. Animals that received zero, 1, or 2 infections with the parasite were tested. In addition, sera from 20 experimentally infected and 10 noninfected Texel sheep were used to test the antigen. Sera from 37 infected animals at prepatency as well as at patency in primary and secondary infection were found positive with the 26-kD antigen. However, sera from 10 animals with the lowest worm burdens (second infection) did not recognize the antigen during early patency (day 28 postinfection). IgG1 was the only isotype implicated in antigen recognition because IgG2, IgA, and IgM, in the same sera, showed no reactivity with the peptide. Antigen specificity was confirmed because hyperimmune sera against infective larvae and adult stages of the most common gastrointestinal nematodes found in natural infections in sheep (Trichostrongylus colubriformis and Teladorsagia circumcincta) did not recognize this peptide. The antigen was recognized only by anti-adult H. contortus hyperimmune sera and appeared to be absent in the L3 parasite stage. In addition, the partial N-terminal amino acid sequence of the diagnostic peptide is reported.     

Partial biochemical and serological characterization of exsheathing fluid and somatic extracts of infective larvae and adult females of Haemonchus contortus.

Exsheathing fluid from infective larvae of Haemonchus contortus may contain antigens which induce protective immunity upon infection in sheep. In this study, a rapid method of preparing exsheathing fluid was used to determine its biological activity, purity and antigenic complexity. Protein in exsheathing fluid ranged from 21-59.5 micrograms/mL and no carbohydrate was detected. Gel filtration showed a single broad peak which retained biological activity and polyacrylamide gel electrophoresis demonstrated a single band staining with Coomassie blue. Biological activity of exsheathing fluid was present in extracts of infective larvae but not in extracts of exsheathed larvae or in extracts of adult female H. contortus. Rabbit antiserum against exsheathing fluid proteins cross-reacted with infective larvae extracts but not with adult female worm extracts. Immunoelectrophoresis of exsheathing fluid proteins against this antiserum showed a single polydisperse precipitin line. Thus, exsheathing fluid produced by the rapid method is a pure preparation of a biologically active and antigenically simple material suitable for further studies of protective immunity in sheep to H. contortus.     

Evaluation of immune response to artificial infections of Haemonchus contortus in Gulf Coast Native compared with Suffolk lambs

Due to their critical functions in worm physiology, glutathione peroxidases in Haemonchus contortus are potential candidates for vaccine to control haemonchosis. However, information on the protection potential of these molecules is lacking. In this study, recombinant HC29 antigen was tested for protection against experimental H. contortus infections in goats. Fifteen animals were allocated into three trial groups. The animals of HC29 group was vaccinated with a recombinant HC29 DNA vaccine twice at day 0 and day 14, and then challenged with 5000 infective H. contortus L3 (third larval stage) on the 28th day. An unvaccinated positive control group was challenged with L3 at the same time. An unvaccinated negative control group was not challenged with L3. The results indicated HC29 DNA vaccine was transcribed at local injection sites and expressed in vivo post immunizations, respectively. Following L3 challenge, the mean eggs per gram feces and worm burdens of HC29 group were reduced by 36.1% and 35.6% respectively compared to the positive control group. After immunization with the DNA vaccine, significantly high levels of serum IgG, serum IgA, mucosal IgA and CD4(+) T lymphocytes were detected. These results suggest that recombinant H. contortus HC29 glutathione peroxidase DNA vaccine induced a partial immune response and has protective potentials against caprine haemonchosis.     

Haemonchus contortus (Nematoda: Trichostrongylidae) infection in lambs elicits an unequivocal Th2 immune response

Selection of resistant animals and host immunization have been proposed as alternative methods for the control of gastrointestinal nematode parasites. However, a better knowledge of the mechanisms involved in protective immunity against these parasites is required for the development of optimal strategies. In this study, 3 month old INRA 401 lambs (n = 81) were allocated into three groups: uninfected control, challenged either once (primary-infected animals) or twice (previously-infected animals) with 10,000 Haemonchus contortus L3. Uninfected control and challenged animals were sequentially sacrificed at 3, 7, 15 and 28 days post challenge. In both challenged groups, a clear Th2-oriented immune response was observed in the abomasal lymph node and mucosa. IL-4 and IL-13 mRNA over-expression, recruitment of eosinophils, mast cells and globule leukocytes and production of specific systemic IgG and mucosal IgA were observed earlier in previously-infected animals than in primary-infected ones. At 28 days post infection, no differences between intensities of these responses were observed between the challenged groups. Worm establishment rates were similar in previously-infected and primary-infected lambs. However, reductions of worm development, female fecundity and fecal egg output were observed in previously-infected sheep. We conclude that H. contortus infection in young INRA 401 lambs induced an unequivocal Th2 immune response resulting in the regulation of worm egg production without affecting their establishment.     

Serum IgG response of Manchego lambs to infections with Haemonchus contortus and preliminary characterization of adult antigens

Manchego lambs (16-18 weeks old) were infected with 2500 infective larvae (L3) of Haemonchus contortus and challenged 2 months later with 5000 L3. The serum IgG anti-Haemonchus response was estimated by enzyme-linked immunosorbent assay (ELISA) using soluble proteins from adults and L3. Previously infected Manchego lambs failed to mount a protective immune response against challenge, at least as assessed by faecal egg counts and pre-patency periods. Primary infection did not provoke any rise in specific anti-parasite serum antibodies, whereas a weak but significant rise was observed in challenged 6.5-month-old lambs which was very similar in both infected and non-infected lambs. The serum IgG anti-parasite response was higher against larval antigens than adult soluble proteins. Preliminary characterization of adult and larval soluble proteins by electrophoresis under reducing and denaturing conditions and Western blotting showed high cross-reactivity of both extracts. Immunoblots of adult H. contortus probed with infected and challenged lambs' sera did not yield conclusive results, although some low molecular weight peptides were recognized.     

Expression of acquired immunity to a local isolate of Haemonchus contortus by the Nigerian West African Dwarf goat.

The capacity of young Nigerian West African Dwarf (WAD) goats to express good acquired immunity to their native geographic strain of Haemonchus contortus and the correlates of this responsiveness were studied in a laboratory experiment involving forty 7-8 month old kids. A primary immunising infection with 2000 L3 (equivalent to 260-450 L3/kg body weight) with or without challenge on D42 with 2000 L3 resulted in a mild chronic infection with a pre-patent period of 18-20 days and little or no reduction in worm burden between D14 and D56. In contrast, another group (D) of kids, whose immunising infection had been truncated with fenbendazole on D35 and later received similar challenge infection, developed good protection against challenge. Thus, worm burdens were largest in group E (challenge control), larger in group C (primary+challenge) and least in group D. Of the measures of infection used, namely faecal worm egg counts (FECs), circulating eosinophil (EOS) responses, packed cell volume (PCV) and body weight, FEC and EOS responses exhibited marked individual variability, but only FEC (geometric mean of transformed counts) and PCV showed strong correlation with worm burden. There was also a significant negative correlation between FEC and PCV. The size of inoculum used was well tolerated by the kids, as it induced only mild changes in PCV in some goats and no effect at all on body weights. This suggests that the WAD goat may possess a good measure of resistance to the pathogenic effects of its native strain of H. contortus. The wide individual variability in FEC and its strong relationships to worm burden and PCV are pointers to its likely genetic basis. There are, therefore, good prospects for further studies to identify H. contortus resistant genotypes among the WAD goat population.     

Fibrinogen-degrading proteins from Haemonchus contortus used to vaccinate sheep.

Sixteen nonsibling sheep, approximately 12 months old, that were raised in a helminth-free environment, were used for 2 protection studies 6 months apart. Sheep were vaccinated weekly for 5 weeks by IM injection of fibrinogen-degrading proteins derived from the intestinal tract of adult Haemonchus contortus. Ten days after the last vaccination, sheep were given 2,500 infective H contortus larvae by intraruminal injection. Vaccinated sheep produced specific antibodies, and were protected from the worm challenge. Significant differences in mean fecal worm egg counts for 56 days after worm challenge, in mean numbers of H contortus worms, and female fecundity ratios at necropsy were detected in vaccinated sheep, compared with those in control sheep. These data suggest that the fibrinogen-degrading proteins have a protective role in vaccination of sheep against H contortus.     

Haemonchus contortus infection in sheep: parasite fecundity correlates with worm size and host lymphocyte counts

Two experiments were conducted to elucidate the timing and nature of the sheep immune response to Haemonchus contortus (Barber's pole worm). The first experiment examined the establishment of H. contortus populations and the immune response by comparing a bolus infection of third-stage larvae in na?ve sheep with a group previously primed by a trickle infection. The second experiment used staggered doses of ivermectin-resistant larvae to compare the development of adult worms during different durations of trickle infection with ivermectin-sensitive larvae. Infections successfully generated pathological signs of haemonchosis such as anaemia. Image analysis software was used to measure the area and perimeter of worms collected at post-mortem, and the number of eggs present in individual adult females (fecundity) was significantly correlated with worm size. A significant inverse correlation was found between blood lymphocyte counts and worm fecundity. The absence of correlation between worm fecundity and other leukocyte and erythrocyte counts highlighted the specificity of the lymphocyte response. This is the first report of a link between haematology profiles and worm fecundity in haemonchosis. The correlation observed between adult worm size and egg content leads to the hypothesis that egg production in H. contortus is limited by immune regulation of worm size and presumably growth. Mean worm size and fecundity declined as sheep received more prolonged trickle infections before necropsy, confirming previous reports that immune responses to adult worms are enhanced by ongoing larval challenge. Immunohistochemical results showed trends consistent with a Th2 (humoral) immune response which has been implicated in reducing nematode burdens in several species.     

Studies on Haemonchus contortus. IX. The effect of Trichostrongylus axei in Merinos on natural pasture

Four groups of 6-7-month-old Merino lambs were each dosed with 40 000 infective larvae of Trichostrongylus axei on 2 November 1976 and subsequently exposed to challenge with Haemonchus contortus on natural grazing at the University of Pretoria's Experimental Farm in the eastern suburbs of Pretoria. One of these groups and one group of controls were killed every 8 weeks from the end of December 1976-June 1977. Predosing with T. axei was greater than 50% effective against 5th stage and adult H. contortus in greater than 50% of sheep for 164 days (Class C), improving to greater than 60% in greater than 60% of sheep (Class B) 220 days after dosing T. axei. The numbers of retarded 4th stage larvae (L4) of H. contortus in the undosed controls as well as in the sheep predosed with T. axei rose from a low level in summer (December) to a peak in late Autumn (June).     

Fecundity in adult Haemonchus contortus parasites is correlated with abomasal tissue eosinophils and γδ T cells in resistant Canaria Hair Breed sheep

Canaria Hair Breed (CHB) sheep are more resistant than Canaria sheep (CS) to experimental Haemonchus contortus infection. Protective responses appear effective against the adult stage of the parasite, not as commonly reported in other breeds against the larval stages. In this study we have quantified several abomasal immune cells and correlated these with parasitological variables for each breed. A significant negative correlation between CD4+ T cell numbers and worm burden or length at 28 dpi was seen only in CS sheep. Significant negative correlations for both abomasal eosinophils and γδ/WC1+ T cells, and fecundity of the adult worms were observed only in the resistant CHB sheep breed. Tissue eosinophils and γδ/WC1+ T cells were positively correlated in CHB sheep. We suggest that the two sheep breeds have disparate immune responses following infection with the parasite and that γδ+ T cells in association with eosinophils may play a hitherto unrecognised role in modulating fecundity in H. contortus adult female parasites.     

Feasibility of genus-specific real-time PCR for the differentiation of larvae from gastrointestinal nematodes of naturally infected sheep

Results of real-time PCR analysis of coproculture third stage larvae (L3) using genus specific TaqMan minor groove binder probes were compared with the results of morphological differentiation of L3 after coprocultured and direct morphological worm differentiation from gastrointestinal samples of eight sheep with naturally acquired nematodes infections. Faecal egg counts prior to postmortem confirmed infections with trichostrongyles with a geometric mean count of 4828 eggs per gram for all sheep. Individual egg counts correlated positively with total worm counts (correlation coefficient 0.794). Five different nematode species and one genus were found in the abomasi and small intestines: Cooperia curticei, Haemonchus contortus, Nematodirus spp., Teladorsagia (Ostertagia) circumcincta, Trichostrongylus axei and Trichostrongylus colubriformis. Coproculture of faecal eggs yielded five of these, Cooperia spp., Haemonchus spp., Ostertagia/Teladorsagia spp. and Trichostrongylus spp. Comparison between morphological L3 and worm differentiation data showed high congruence (94%). The agreement between PCR analysis of L3 after coproculture and direct morphological worm differentiation was 84%. Thus, real-time PCR was found to be suitable as a speedy and reliable diagnostic tool for the assessment of gastrointestinal nematode infections of ruminants in the field.     

The response of Awassi and Merino sheep to primary infection with Haemonchus contortus

The possible existence of breed differences in the response of sheep to primary infection with Haemonchus contortus was examined by comparing worm establishment and pathogenic effects of the parasite in Awassi and Merino sheep of haemoglobin type B infected with 500 third stage H. contortus larvae per kg body weight. The results showed that the Merino sheep had lower faecal egg counts and worm burdens and suffered less severe clinical disturbances than sheep of the Awassi breed. This suggests that genetic resistance operates primarily at the level of worm establishment.     

Genetic analysis of the potential role of IgA and IgE responses against Haemonchus contortus in parasite resistance of Creole goats

Small ruminants are affected by gastrointestinal nematode (GIN) infection. A promising alternative strategy for control of GIN infection is to increase the level of resistance in the population by taking advantage of the host's immune response. Immunoglobulin A (IgA) and E (IgE) are known to be involved in immune response to GIN. The aim of this study was thus to investigate genetic parameters of IgA and IgE responses against Haemonchus contortus in Creole kids naturally challenged at pasture and to determine the relationship with other resistance criteria such as faecal egg counts, packed-cell volume, eosinophil counts and bodyweight. Variance and covariance components for genetic and residuals effects for each trait were estimated on 3862 males at 11 months of age. Heritability estimates for IgA and IgE ranged between 0.15 and 0.57. Strong positive genetic correlations were observed between either IgE or IgA responses against L3 and adult excretory/secretory products (ESP) antigens of H. contortus, suggesting that the humoral immune response is not specific to the life cycle stage of the parasite suggesting that there is substantial cross recognition between the different parasite antigens. Heritability estimates for faecal egg count (FEC), packed-cell volume (PCV) and bodyweight (BW) were in accordance with previous results in Creole kids. Blood eosinophil counts were found moderately heritable and negatively correlated with FEC, suggesting that this cell population plays a role in resistance to nematode parasite infection in Creole goats. IgA response was positively correlated to FEC, in contrast with the negative correlation between IgE against L3 of H. contortus and FEC. In Creole goats, IgA response against L3 or ESP of H. contortus would rather be associated with the worm burden than an immune protective response. The immune response involving activity of IgE against L3 of H. contortus may be one important pathway for development of resistance to gastrointestinal nematode infections in Creole goats.