Immune Sheep Research Articles

Immunization with a Streptococcus bovis Vaccine Administered by Different Routes Against Lactic Acidosis in Sheep

Streptococcus bovis is an important lactic acid bacterium in the rumen, which contributes to the development of lactic acidosis. This study was designed to test the efficacy of immunization with S. bovis primed either intramuscularly (i.m.) or intraperitoneally (i.p.) against lactic acidosis. Forty-five wethers were allocated to three treatment groups. Two groups were injected with a S. bovis vaccine by either the i.m. or i.p. route for primary immunization; both groups were further immunized by the same route(s) (oral and/or i.m.) for boosters. The third group was not immunized (control).Antibody concentrations were measured in saliva prior to and following animals being fed a grain diet, and also in the rumen fluid, before the animals were suddenly introduced to a grain diet. The average antibody concentration in the animals of the i.m. group was higher than the i.p. group (P< 0.05). The antibody concentration in the rumen fluid of immunized sheep was higher than the control animals (P< 0.01). The difference in the rumen fluid antibody concentration between the i.m. and i.p. groups was not statistically significant (P> 0.05). In the i.m. group, there was a significantly greater feed intake, higher rumen pH, lower diarrhoea scores, and less increase in blood packed cell volume following grain feeding than in the animals of the control group. The severity of diarrhoea and the increase of blood packed cell volume in the animals of the i.p. group were also less than in the animals of the control group. The results suggest that the risk of lactic acidosis can be reduced by immunization against S. bovis, and that the immunization primed i.m. is more effective than the immunization primed i.p.

A Sandwich Enzyme-Linked Immunosorbent Assay for the Detection of Almonds in Foods

An enzyme-linked immunosorbent assay was developed to detect almonds as potential allergenic contaminants in food. Polyclonal antibodies directed against roasted almonds were partially purified from immunized sheep and rabbits and used as capture and secondary antibodies, respectively, in a sandwich-type, 96-well plate format. Food samples and almond-spiked samples were extracted 1:10 in phosphate-buffered saline at 60°C for 2 h, centrifuged, and applied to wells coated with sheep anti-almond antibody. After incubation, washing, and the addition of rabbit anti-almond antibody, the amount of almond present was detected with the subsequent addition of goat anti-rabbit immunoglobulin G–alkaline phosphatase conjugate and p-nitrophenyl phosphate substrate. Plate absorbances were read at 410 nm, and standard curves were developed in all matrices to quantify unknowns. Antibodies developed were specific for almond; however, some cross-reactivity was observed with extracts of some tree nuts and sesame seeds. Sodium dodecylsulfate–polyacrylamide gel electrophoresis and Western immunoblotting indicated that sheep anti-almond antibody recognized proteins extracted from black walnuts, Brazil nuts, cashews, hazelnuts, macadamia nuts, pistachios, and sesame seeds in addition to those from almond. The assay was optimized to detect less than 1 ppm of almond and was used successfully to determine almond residues in cereal and chocolate without cross-reacting interferences. A retail survey of 20 brands of cereal demonstrated that the assay produced statistically consistent results. This assay provides a useful quality control tool for the food industry for the protection of consumers allergic to almonds.

The effect of oxfendazole terminated infections with Haemonchus contortus on the development of immunity in sheep

The relative contribution of third (L3), fourth (L4) or adult stages of Haemonchus contortus to the development of immunity was evaluated in three groups of sheep subjected to infections terminated by oxfendazole treatments at the L3, L4 or adult stage. A control group did not receive immunising infections. All the groups were challenged with 5000 L3, to evaluate the protection provided by the different protocols. All sheep were necropsied at the end of the experiment to count the abomasal worm burdens. A marked reduction in egg counts after challenge infection was only observed in sheep in which the infection was terminated in the adult stage (Group 4). A significant reduction in worm burden was also observed in Group 4. The immunising infections and/or the challenge infection resulted in moderately elevated IgG antibody levels against L3, L4 and adult somatic antigens in all the groups. In contrast, a strong IgG response against H. contortus excretory/secretory (ES) antigens was observed in the groups in which the immunising infection was terminated in the L4 and the adult stage. An elevated lymphocyte proliferation response against Haemonchus ES antigens was found only in the group that had their immunising infection terminated at the adult stage. The combined data suggest that exposure to and elicited immunological responses to ES antigens are important for the development of immunity against H. contortus.

Complement fixation test to assess humoral immunity in cattle and sheep vaccinated with Brucella abortus RB51.

The live attenuated Brucella abortus strain RB51 is a rifampin-resistant, lipopolysaccharide (LPS) O-chain-deficient mutant of virulent B. abortus 2308. The reduced O-chain content in RB51 prevents this bacterium from inducing antibodies detectable by the conventional serologic tests for bovine brucellosis diagnosis that mainly identify antibodies to LPS. The absence of available serologic tests for RB51 also complicates the diagnosis of possible RB51 infections in humans exposed to this strain. The purpose of this study was to evaluate the suitability of a complement fixation (CF) test performed with the rough strain B. abortus RB51, previously deprived of anticomplementary activity, in detecting anti-B. abortus RB51 antibodies in cattle and sheep experimentally vaccinated with this strain. The results of this study showed that a CF test with RB51 as the antigen is able to specifically detect antibodies following RB51 vaccination in cattle and sheep. In addition, this method could be a useful tool for detecting B. abortus RB51 infection in humans.

Duration of immunity, efficacy and safety in sheep of a recombinant Taenia ovis vaccine formulated with saponin or selected adjuvants

The efficacy and safety of a recombinant Taenia ovis protein was tested in sheep using 13 different adjuvant formulations, including oil adjuvants, aluminium salts, saponin, Iscoms and DEAE-dextran. The oil adjuvants, saponin and DEAE-dextran gave the highest antibody responses and greatest degree of protection against challenge infection with T. ovis eggs. Duration of immunity studies with a saponin based vaccine showed that highly significant protection (>90% reduction of cyst numbers) was achieved when sheep were challenge infected one month after immunisation. Significant protection (79%) was still present when sheep were challenged 6 months after immunisation. The optimum dose for this batch of saponin was 10 mg, which stimulated a peak antibody titre of 38,400, 4 weeks after immunisation and did not cause injection site reactions. Dialysed saponin was shown to retain its adjuvant properties and allowed an increase in dose to 30 mg without site reaction, resulting in a peak antibody titre of 51,200.

Ileal and jejunal Peyer's patches play distinct roles in mucosal immunity of sheep.

The majority of pathogens enter the body through mucosal surfaces and it is now evident that mucosal immunity can provide effective disease protection. However, the induction of mucosal immunity will require efficient targeting of mucosal vaccines to appropriate mucosa-associated lymphoid tissue. An animal model, based upon the surgical preparation of sterile intestinal 'loops' (blind-ended segments of intestine), was developed to evaluate mucosal and systemic immune responses to enteric vaccines in ruminants. The effectiveness of end-to-end intestinal anastomoses was evaluated and fetal surgery did not disrupt normal intestinal function in lambs up to 6-7 months after birth. The immunological competence of Peyer's patches (PP) within the intestinal 'loops' was evaluated with a human adenovirus 5 vector expressing the gD gene of bovine herpesvirus-1. This vaccine vector induced both mucosal and systemic immune responses when injected into intestinal 'loops' of 5-6-week-old lambs. Antibodies to the gD protein were detected in the lumen of intestinal 'loops' and serum and PP lymphocytes proliferated in response to gD protein. The immune competence of ileal and jejunal PP was compared and these analyses confirmed that jejunal PP are an efficient site for the induction of mucosal immune responses. This was confirmed by the presence of gD-specific antibody-secreting cells in jejunal but not ileal PP. Systemic but not mucosal immune responses were detected when the vaccine vector was delivered to the ileal PP. In conclusion, this model provided an effective means to evaluate the immunogenicity of potential oral vaccines and to assess the immunological competence of ileal and jejunal Peyer's patches.

The effect of two challenge doses of Dictyocaulus filaria on the acquired immunity of sheep vaccinated with "Filiraq".

Twenty six lambs divided into four groups were used to study the effect of experimental infection with two challenge doses of D.filaria ( 100 &amp; 300 larvae / Kg. B. W.) on the aquired immunity after vaccination with irradiated larval vaccine “ Filiraq”. The clinical symptoms , eosinophi lic response, blood picture, faecal larval counts, gross pathological changes &amp; adult worm counts in the lungs were studied ... &#x0D; The results showed that a high dose of challenge (300 LI kg) prevents the development of expected acquired immunity, however the other vaccinated &amp; challenged group ( 100 LI Kg B.W ) showed good level of acquired immunity in comparison with other group.

Immunization of rats and sheep against granulosa cell-inhibitory factor from bovine follicular fluid increases the number of large follicles in rats and the ovulation rate in sheep.

Granulosa cell-inhibitory factor (GCIF), a low molecular weight factor from bovine follicular fluid, inhibits the proliferation of bovine granulosa cells in vitro and the growth of large follicles in rats in vivo. In this study the effects of (1) immunization of rats against GCIF on follicular growth and (2) immunization of sheep against GCIF on ovulation rate were studied. The ability of antiserum from sheep immunized against GCIF to reduce the inhibitory effect of GCIF on bovine granulosa cell proliferation in culture was also examined. Immunization of rats against GCIF increased the number of large follicles (P < 0.001) but decreased the number of small follicles (P < 0.05) per ovary. Ovarian mass (P < 0.05) and uterine wet (P < 0.05) and dry (P < 0.01) masses were increased in immunized rats. Immunization of sheep against GCIF, followed by boosting over two breeding seasons, increased ovulation rate (P < 0.01). Addition of antiserum from sheep immunized against GCIF reduced or abolished the inhibitory effect of GCIF on granulosa cell proliferation (P < 0.01). These data provide further evidence that GCIF has an important role in controlling follicle growth and ovulation in vivo.

Anti-retroviral immunity

The mechanisms of immunity that protect from infection by viruses are being studied to help in vaccine design and immune intervention therapies. Research into retroviruses is often fuelled by the concern about HIV infection. Two papers 1 Hislop A.D. et al. Vaccine induced cytotoxic T lymphocytes protect against retroviral challenge. Nat. Med. 1998; 4: 1193-1196 Crossref PubMed Scopus (38) Google Scholar , 2 Dittmer U. Brooks D.M. Hasenkrug K.J. Requirement for multiple lymphocyte subsets in protection by a live attenuated vaccine against retroviral infection. Nat. Med. 1999; 5: 189-193 Crossref PubMed Scopus (81) Google Scholar have recently been published that show the wide difference in results obtained with two different retrovirus infections. The first study used a peptide of a cytotoxic T lymphocyte (CTL) epitope to immunize sheep against bovine leukemia virus (BLV) 1 Hislop A.D. et al. Vaccine induced cytotoxic T lymphocytes protect against retroviral challenge. Nat. Med. 1998; 4: 1193-1196 Crossref PubMed Scopus (38) Google Scholar . All the immunized sheep showed anti-peptide CTL (CD8+ T-cell) responses, but only ∼50% showed anti-viral CTL responses; these animals were protected from an intravenous challenge with BLV- infected cells. There was no evidence of any other type of immune mechanism (antibody or CD4+ T-cell) involvement in protection. The second study used a murine retrovirus model, Friend virus (which is a mix of two viruses), a replication-competent helper virus and the replication-defective pathogenic virus 2 Dittmer U. Brooks D.M. Hasenkrug K.J. Requirement for multiple lymphocyte subsets in protection by a live attenuated vaccine against retroviral infection. Nat. Med. 1999; 5: 189-193 Crossref PubMed Scopus (81) Google Scholar . Immune spleen cells from mice were immunized with an attenuated vaccine virus, separated into CD4+, CD8+ T cells or CD19+ B cells, and adoptively transferred into naive recipients to see which subsets provided protection against an intravenous virus challenge. Although CD8+ T cells could help to clear infection, they did not prevent establishment of infection and the appearance of persistently infected cells. All three cell types were necessary together to see protection from acute and persistent infection.

Emergency vaccination of sheep against foot-and-mouth disease: protection against disease and reduction in contact transmission

The ability of several emergency FMD vaccine formulations to elicit early protective immunity in sheep was examined. All vaccine formulations were shown to protect sheep against airborne challenge with homologous FMDV within 4 days of vaccination. Protection was associated in part with the induction of serum antibody responses but was also demonstrated in the absence of any detectable antibody response at the time of challenge. Aqueous Al(OH) 3/saponin vaccine formulations and oil emulsion vaccines based on Montanide ISA 206 adjuvant reduced virus replication and the numbers of animals subclinically infected up to 28 days post-challenge, when compared with non-vaccinated animals, consequently limiting transmission of the disease or infection to in-contact susceptible animals.

Studies on the role of mucus and mucosalhypersensitivity reactions during rejection ofTrichostrongylus colubriformis from the intestine ofimmune sheep using an experimental challenge model

Nematode-naive sheep and sheep immunised by truncated infections with Trichostrongylus colubriformis were fitted with intestinal cannulae to allow administration ofchallenge infection and collection of intestinal fluids. Sheep were slaughtered at various timesafter challenge and the distribution of larvae along the small intestine was determined. Resultsshowed that immune sheep had significantly fewer larvae in their intestines and that some sheepcould expel the challenge infection within 2 h. Mucus samples from immune sheep containedincreased parasite-specific antibody, histamine and anti-parasite activity as measured by larvalmigration inhibition assay. Higher levels of antibody and histamine were seen in intestinal fluids ofimmune sheep after challenge. Immunisation of sheep by truncated infections stimulated serumIgE and resulted in significantly higher numbers of IgE-positive cells in gut tissue sections beforechallenge and at 2 h and 24 h after challenge. Immune sheep also had greater numbers ofmucosal mast cells and globule leucocytes after challenge, compared with naive sheep. Whenchallenge larvae were mixed with mucus from immune sheep and infused back into naive recipientsheep, there was a distinct displacement of the larval population towards the distal part of theintestine, compared with the profile of larval establishment after infusion with mucus from naivesheep. These results are further evidence for an immediate hypersensitivity reaction in the intestineof immune sheep, where challenge larvae are expelled within 2 h and confirm the direct anti-larvalproperties of mucus. The cannulated-sheep challenge model described here will be a useful tool tounravel the mechanism of larval rejection from immune sheep and could lead to novel therapies.

Development of a Protective Immunity againstOstertagia leptospicularisin Trickle-infected Sheep and Parallel Changes of Serum Gastrin, Pepsinogen and Antibody Levels

Nine lambs, approximately 9 months of age were allocated to three groups (A, B, C), with three animals in each. Sheep in Groups A and B were trickle-infected with doses of 1000 third-stage larvae (L3) ofOstertagia leptospicularis(five times per week) over periods of 7.5 and 10.5 weeks, respectively, and were subsequently treated with fenbendazole (7.5 mg/kg). Approximately 3 weeks after anthelmintic treatment, all sheep were challenged with a single dose of 100 000 L3, whereas sheep of Group C received the same dose as a primary infection. Sheep of Groups A and B were almost completely refractory against the challenge infection, as indicated by negative faecal egg counts and adult worm burdens. A relatively high infection level was present in the sheep of Group C.The results indicate that a comparatively short immunization period of 7.5 weeks is sufficient to protect lambs against subsequent larval challenge. During immunization, the pepsinogen-, gastrin- and IgA-responses were similar in the individual sheep. In contrast to parasite-specific IgG1and IgG2levels, IgA decreased rapidly after cessation of trickle infection and parallel anthelmintic treatment, and may therefore indicate current exposure to parasite antigen. After challenge, the majority of the immunized sheep exhibited immediate and short-term responses of pepsinogen, gastrin and IgA in the serum. The time course and the level of each of these responses were very similar in the individual sheep, suggesting that the release of pepsinogen, gastrin and IgA into the circulation was influenced by related mechanisms.

Preparation and use of hyperimmune serum for prophylaxis and therapy of Ebola virus infections.

To obtain hyperimmune serum appropriate for the treatment of filovirus infection, methods were developed to immunize nonsusceptible animals with live Ebola (EBO) virus preparations. Immune plasma with high ELISA and neutralization-specific antibody titers was obtained by multiple immunization of sheep and goats with preparations of live EBO virus. Goat immunoglobulin was prepared by Cohn's method and tested on guinea pigs, using an EBO virus strain that is highly pathogenic for guinea pigs. Prophylaxis with these immunoglobulins within 48 h after infection was effective in challenge experiments, with a log10 prophylaxis index as high as 1.92+/-0.52. Other studies have shown that equine anti-EBO virus immunoglobulins worked well in baboons. The goat immunoglobulins were also tested in preclinical trials on laboratory animals; after being positively evaluated, they were administered to volunteers in clinical trials for biologic safety and reactivity, and they were administered to researchers suspected of becoming infected with EBO during their experimental work. These immunoglobulins may be useful for the emergency treatment of persons accidentally infected with EBO.

Vaccination against the gastrointestinal nematode, Haemonchus contortus, using a purified larval surface antigen

Sheep were immunized with a purified antigen ( Hc-sL3) expressed on the surface of L3 larvae of the gastro-intestinal parasite, Haemonchus contortus, using different adjuvant and immunization routes. In the first experiment, intradermal immunization of sheep with Hc-sL3 and QuilA did not result in reductions in faecal egg counts after subsequent challenge infection while significant reductions were obtained when aluminium hydroxide (AH) was used as the adjuvant. Significant protection with Hc-sL3 absorbed on AH was confirmed in a second experiment and this protection was maintained when dextran sulphate was added to the Hc-sL3/AH mixture while the addition of pertussis toxin abrogated the protective effect. Significant levels of protection, as determined by reductions in both faecal egg counts and worm burdens, were also obtained when the Hc-sL3/AH mixture was injected into the rectal mucosa or the Hc-sL3 antigen was deposited on the surface of the rectal mucosa with cholera toxin. No correlations with antibody levels or isotype and protection were observed.

Characterization of immune responses caused by bovine leukemia virus envelope peptides in sheep.

To study the immunomodulative activity caused by bovine leukemia virus envelope (BLV Env) peptide, sheep were immunized with two kinds of Th-epitope peptides, peptide 98 (BLV Env 98-117), and 61 (BLV Env 61-78). Four of eight immunized sheep showed specific proliferative responses against both of the peptide stimulations. To characterize the cells responding to the peptides, peptide-specific cells were established from the responding sheep by the continuous stimulation of peripheral blood mononuclear cells (PBMCs) with either peptide 98 or 61 in vitro. The peptide 98-specific cells consisted of CD4-positive cells, whereas the peptide 61-specific cells consisted of CD8-positive cells and MHC class II-positive cells. In addition, cytokine profile analysis indicated that the peptide 98-stimulated cells expressed IFN-gamma but not IL-10, although the peptide 61-stimulated cells expressed IL-10 but not IFN-gamma. These results show that BLV envelope peptides 98 and 61 can modulate immune responses of sheep lymphocytes in different ways and may contribute to the pathogenesis of BLV infection.

Passive immunisation of sheep against adipose tissue: effects on metabolism, growth and body composition

Passive immunisation of sheep with anti-ovine adipocyte membrane serum was examined. Antibodies were raised in donkeys by primary immunisation with sheep adipocyte plasma membrane protein in Freund's complete adjuvant followed by booster injections at 3, 5 and 15 weeks and subsequently at 4-month intervals with Freund's incomplete adjuvant. Finnish Landrace crossbred sheep were injected with 50 ml of donkey serum on three consecutive days. A control group ( n=16) received serum intravenously from a donkey which had not been immunised, while the other sheep were treated subcutaneously (SC, n=16) or intravenously (IV, n=10) with anti-ovine adipocyte membrane serum. Eight SC and eight control sheep were slaughtered after 5 weeks and the remainder after 12 weeks. Immunisation against adipocyte plasma membranes decreased ( P<0.001) dry matter intake in the first week after treatment but overall growth and feed conversion efficiency were not affected. The omentum fat depot was decreased ( P<0.05) 12 weeks after SC immunisation but not after 5 weeks. There were no other immunisation×time of slaughter interactions. Immunisation (both SC and IV) decreased ( P<0.05) the kidney/pelvic fat depot, while SC immunisation also decreased ( P<0.05) the omentum fat depot. The depth of fat covering the loin joint was similarly decreased ( P<0.05) by SC and IV immunisation, while SC immunisation decreased ( P<0.05) the fat proportion and increased ( P<0.05) the lean proportion of the loin joint. These data demonstrate the potential of this approach as a means of decreasing adiposity in farm animals. However, to be commercially viable, this approach must be further developed so that decreases in the fat content of the total carcass can be achieved.

Immunization of Sheep Against GNRH Early in Life: Effects on Gonadotropins, Follicular Growth and Responsiveness of Granulosa Cells To FSH and IGF-I in Two Breeds of Sheep With Different Prolificacy (Romanov and ILE-DE-France)

The prolific Romanov (R, ovulation rate = 3) and non-prolific Ile-de-France (IF, ovulation rate = 1) breeds were compared for their ovarian sensitivity to gonadotropins and IGF-I before puberty. For this purpose, the effects of in vivo immunization against GnRH on populations of ovarian follicles and in vitro sensitivity of granulosa cells to FSH and IGF-I were studied in prepuberal lambs from both breeds. Seventeen prepuberal lambs of each breed were actively immunized against GnRH between 3 wk and 6 mo of age. Relative to untreated lambs, FSH levels at 4, 5, and 6 mo of age were (respectively) 41%, 25%, and 29% for IF, and 43%, 24%, and 36% for R lambs. In a first experiment, histological analysis of ovaries was performed. Immunization treatment decreased the number of small (100–390 μm in diameter) and large size follicles (<1500 μm) in both breeds at 6 mo of age. In both breeds, gonadotropin (FSH - LH -hCG) treatment increased the number of large size follicles (<1500 μm in diameter) and induced the formation of preovulatory follicles in immunized as well as untreated lambs. The ovulation rate was less in immunized animals, but it was not different between breeds. In a second experiment, the effects of FSH and IGF-I were studied on granulosa cells from follicles between 1000 and 2000 μm in diameter. In both breeds, IGF-I increased granulosa cell proliferation, but enhanced progesterone secretion was observed only in R lambs after FSH and IGF-I stimulation. Granulosa cell response to FSH treatment was lost by immunization, whereas response to IGF-I remained unchanged in both breeds. These results indicate that long-term immunization of prepuberal lambs against GnRH reduced systemic concentrations of FSH, follicular development, and response to gonadotropins in vivo, similarly in the prolific R and the non-prolific IF breed. However, granulosa cells from R lambs had higher steroidogenic capacities and were more responsive to FSH. In addition, these results suggest that IGF-I could play an important role in regulating growth of small follicles both in immunized and non-immunized lambs.

Active immunization against somatostatin alters regulation of gastrin in response to gastric acid secretagogues.

We have examined the coupling between somatostatin, gastrin, and gastric acidity, using sheep chronically immunized against somatostatin. All immunized sheep had high-titer (3.2 x 10(5) +/- 1.1 x 10(4) M), high-affinity (1.5 x 10(11) +/- 1.2 x 10(10) l/mol) antibodies. However, basal gastrin and gastric acidity were similar to those in control animals, indicating that an inhibitory somatostatin tone was not required for the maintenance of normal basal gastrin and gastric acidity. Omeprazole (a proton pump inhibitor) increased gastric pH to a similar extent in both the control and immunized groups but resulted in a smaller increase in plasma gastrin in the immunized sheep, thus calling into question the assumption that hypergastrinemia associated with hypochlorhydria is the result of somatostatin withdrawal. Pentagastrin- or histamine-stimulated somatostatin secretion reversed or attenuated the omeprazole-induced hypergastrinemia in control but not immunized sheep, demonstrating a functional role for somatostatin and the biological efficacy of the somatostatin immunization. In a separate series of omeprazole-treated sheep, restoration of an acidic gastric pH with intragastric HCl reversed the hypergastrinemia in both control and immunized animals. We conclude that somatostatin is not essential for the acid-mediated regulation of gastrin. The use of a chronically immunized model as opposed to the acute administration of somatostatin antibodies has important advantages in determining the steady-state regulatory role of somatostatin.

Effects of active immunization of sheep against an amino terminal peptide of the inhibin alpha C subunit on intrafollicular levels of activin A, inhibin A and follistatin.

Active immunization of ewes against inhibin (IMM) consistently increases ovulation rate but this response is not always accompanied by the expected rise in plasma FSH. Inhibin-related molecules also have local auto/paracrine effects within the ovary and the ovulatory response to IMM could be due to neutralization of one of these effects, independent of changing FSH levels. To investigate this, ovaries were collected from long-term IMM (n = 6) and control (CON; n = 8) ewes killed 48 h after progestagen withdrawal (late follicular phase) and all follicles > or = 3 mm were recovered to determine intrafollicular levels of inhibin A, activin A and follistatin by specific two-site immunoassay and oestradiol and testosterone by radioimmunoassay. Blood samples were collected to assess plasma FSH, oestradiol and inhibin antibody titres. Although plasma FSH levels were similar in IMM and CON ewes, IMM ewes had approximately 3-fold more follicles > or = 3 mm (P < 0.0001) and approximately 3-fold more oestrogenic follicle (P < 0.001) than CON ewes. Compared with CON ewes, follicles from IMM ewes had much higher concentrations of activin A (approximately 6-fold; P < 0.001) and inhibin A (approximately 3-fold; P < 0.001) but only slightly more follistatin (approximately 1.4-fold; not significant). The activin A:follistatin ratio in follicles from IMM ewes (approximately 1:1) was significantly higher (P < 0.001) than in follicles from CON ewes (approximately 0.3:1). Levels of inhibin antibody measured in follicular fluid (FF) from IMM ewes were similar to plasma levels. Given that activin A has been shown previously to up-regulate FSH receptors and aromatase activity in rat granulosa cells, the increase in intrafollicular activin A, unaccompanied by a rise in the concentration of its binding protein (follistatin), could explain how long-term IMM enhances follicle development and ovulation rate without necessarily promoting a sustained increase in FSH secretion.

Eosinophil-mediated killing of Haemonchus contortus larvae: effect of eosinophil activation and role of antibody, complement and interleukin-5.

Eosinophils have previously been shown to accumulate around the tissue invasive (L3) stage of sheep gastrointestinal parasites in vivo. In this study, eosinophils obtained from mammary washes of sheep, were shown to immobilize and kill H. contortus larvae in vitro in the presence of antibody specific against a defined L3 surface antigen. Eosinophils obtained from sheep primed by repeated infusion of H. contortus larvae were more effective than eosinophils obtained after a single infusion of parasite extract in Fasciola hepatica infected ewes suggesting the former were activated in vivo. The level of larval immobilization in the presence of antibody was significantly increased when complement was added to cultures containing activated eosinophils. The addition of interleukin-5 to larval cultures containing antibody and complement resulted in a significant increase in larval immobilization with unactivated eosinophils suggesting that eosinophil effector function is enhanced following priming with this cytokine. Ultrastructural analysis of the eosinophil/larvae interaction at 6 h of incubation revealed degranulation of adhering eosinophils onto the surface of larvae. By 24 h of incubation, many larvae showed signs of damage and most eosinophils had degenerated. These results suggest that eosinophil-mediated killing may be an effector mechanism for the elimination of L3 H. contortus larvae in immune sheep.