IMMUNITY TO EXPERIMENTAL STAPHYLOCOCCAL MASTITIS—COMPARISON OF LIVE AND KILLED VACCINES

SUMMARY Eleven pregnant Merino ewes were immunised with either a killed Staphylococcus aureus cell-toxoid vaccine (intramuscularly) or a living culture of the same organism (subcutaneously). A further 3 animals were used as non-immunised controls. There were no significant differences between the vaccinated groups for agglutinating antibody to staphylococci or for anti-α-haemolysin in either serum or whey. Three weeks after lambing the ewes were challenged by intramammary infusion of virulent staphylococci. All animals developed an acute mastitis with significant decreases in milk yields being recorded 48 hours post-challenge. Seven days after challenge the mean milk production of ewes given the live vaccine had recovered to within 5% of the pre-challenge mean yield. However, milk productions of controls and ewes given the killed vaccine had further decreased and were significantly lower than for animals vaccinated with live staphylococci. There were no significant differences between the two vaccinated groups for numbers of bacteria or leucocytes in milk samples collected after challenge. The ewes were killed 7 days postchallenge and mammary tissues were examined for immunoglobulin-containing cells. Large numbers of IgA-containing cells, and few IgM-containing cells were found, but there were no significant differences between the treatment groups for these parameters.     

Vaccination against staphylococcal mastitis

Extract

Sir:- Immunisation with staphylococcal vaccines is a potential means of increasing the resistance to bacterial invasion of the udder. However, the development of an effective vaccine for control of staphylococcal mastitis in ruminants has proved to be an elusive goal. There have been numerous attempts to develop vaccines against coagulase-positive Staphylococcus aureus mastitis, but very few attempts to develop vaccines using coagulase-negative staphylococci. Development of a vaccine against coagulase-negative staphylococci may help control the sub-clinical mastitis caused by these organisms and assist in the search for an effective vaccine against mastitis caused by the more pathogenic coagulase-positive S. aureus.We describe here an experiment in which ewes were immunised with live coagulase-negative staphylococcal vaccine and challenged during lactation by intramammary infusion of a homologous strain.     

Vaccination against experimental staphylococcal mastitis in ewes

Experiments were carried out in ewes using a new vaccine developed for the prevention of mastitis caused by Staphylococcus aureus. The vaccine comprised three major components: (i) killed S aureus cells which had been cultured to induce synthesis of pseudocapsule; (ii) toxoided staphylococcal beta haemolysin and (iii) the adjuvant dextran sulphate. Ewes systemically vaccinated twice during pregnancy developed significantly elevated circulating levels of IgG1 and IgG2 anti-pseudocapsule antibody, as well as increased serum titres of anti-beta haemolysin. Five different strains of S aureus were used to challenge both vaccinated and control ewes by the intramammary route during the ensuing lactation. The incidence of acute gangrenous mastitis and nonacute, clinical mastitis was significantly lower in vaccinated than in control groups after challenge with each strain. Vaccinated ewes produced significantly more milk than control ewes after challenge with four of the five strains of S aureus.     

Immunophysiological activity of supramammary lymph nodes of the ewe during the very early phase of staphylococcal mastitis

Efferent mammary lymph was collected from lactating ewes which were unimmunised (controls) or immunised during pregnancy with two doses of an attenuated live Staphylococcus aureus vaccine either in the hindlimb ("directly primed' supramammary nodes) or in the brisket ("indirectly primed' supramammary nodes). Mammary lymph was also collected from unimmunised animals in which the supramammary nodes had been extirpated several months before. Ewes in which the supramammary nodes had been directly primed by staphylococcal vaccination before challenge had a significantly greater output of IgM- and IgG2-containing cells in lymph and higher concentrations of IgG1 and IgG2 antibody against S aureus surface antigens than did other groups. Lymphadenectomised ewes had fewer leucocytes in mammary lymph but a much higher proportion of neutrophils than other ewes, indicating that afferent mammary lymph has an unusually high number of neutrophils and most of these cells are filtered out in the supramammary lymph nodes under normal circumstances. The results indicated that most of the leucocytes in efferent lymph were derived from the supramammary nodes. After induction of experimental staphylococcal mastitis there was a rapid drop in leucocyte output in lymph within one to four hours after infection; the data indicated that events within the supramammary nodes were responsible for this phenomenon. The output of immunoglobulin-containing cells was reduced during this phase. No significant increases in output of lymphoblasts, immunoglobulin-containing cells or specific antibody occurred during the six hours immediately following infection.     

Enhancement of in vitro phagocytosis of Staphylococcus aureas by polymorphonuclear leucocytes.

Polymorphs collected from ewes which had been infected with live staphylococci and immunised with a killed staphylococcal vacccine, had significantly enhanced capacity for phagocytosis of staphylococci compared with polymorphs from ewes which received only a killed vaccine. The numbers of viable bacteria were greatly reduced when blood serum or whey were incorporated in incubation mixtures compared to incubation mixtures containing only polymorphs and staphylococci. When serum was used in the incubation mixtures, the numbers of staphylococci which survived were significantly less than when colostral or milk whey was used. No significant difference was observed between whey collected from locally immunised mammary glands and that from non-immunised glands within each group.     

Vaccination of pregnant ewes against infection with Salmonella Brandenburg

AIMS: To develop a challenge model for Salmonella Brandenburg infection in pregnant ewes. To compare efficacies of a live attenuated Salmonella Typhimurium mutant, a subunit preparation from a virulent S. Brandenburg isolate, and a commercial multivalent inactivated vaccine in their ability to prevent experimental S. Brandenburg infection. To assess the efficacy of the live attenuated S. Typhimurium mutant against natural S. Brandenburg infection in lambs.

METHODS: Two-year-old ewes were immunised with either a live attenuated vaccine (eye-drop; n=20), a subunit vaccine (n=20) or an inactivated bacterin vaccine (n=20), both administered subcutaneously, or served as unvaccinated controls (n=21). Four weeks later, the sensitising regime was repeated as a booster vaccination, and the ewes were challenged 6 weeks later with a virulent S. Brandenburg isolate, approximately 6 weeks prior to lambing. The presence of clinical signs, abortion or death was noted following challenge. The presence and number of Salmonella spp in faecal samples taken throughout the trial, and in organs collected post mortem, were determined using an enrichment selection procedure, and confirmed by serology and pulsed-field gel electrophoresis (PFGE). Half of the surviving lambs were vaccinated with the live attenuated vaccine and all (n=39) were exposed to natural infection from contaminated pasture.

RESULTS: There was no significant protection against mortality and abortion following vaccination with the live attenuated, subunit and inactivated vaccines following experimental challenge with S. Brandenburg. There was a significant but transient decrease in the number of ewes shedding S. Brandenburg (live attenuated, p=0.05; subunit, p=0.05; inactivated, p=0.01), and in the quantity of these bacteria in the sheep from the vaccinated groups (p<0.05) compared with controls, 6 weeks after challenge. Lambs from the challenged ewes did not shed Salmonella spp after being vaccinated with the live attenuated vaccine, in contrast to some of the controls, when grazed on pasture contaminated with S. Brandenburg.

CONCLUSIONS: The use of live attenuated, subunit and inactivated vaccines did not significantly protect sheep against lethal experimental challenge with S. Brandenburg.     

Immune response of heifers against a Staphylococcus aureus CP5 whole cell and lysate vaccine formulated with ISCOM Matrix adjuvant

Staphylococcus aureus is the most frequently isolated pathogen from bovine intramammary infections worldwide. Commercially available vaccines for mastitis control are composed either of S. aureus lysates or whole-cells formulated with traditional adjuvants. We recently showed the ability of a S. aureus CP5 whole-cell vaccine adjuvanted with ISCOM Matrix to increase specific antibodies production in blood and milk, improving opsonic capacity, compared with the same vaccine formulated with Al(OH)₃. However, there is no information about the use of ISCOM Matrix for the formulation of bacterial lysates. The aim of this study was to characterize the innate and humoral immune responses induced by a S. aureus CP5 whole-cell or lysate vaccine, formulated with ISCOM Matrix after immunization of pregnant heifers. Both immunogens stimulated strong humoral immune responses in blood and milk, raising antibodies that increased opsonic capacity. Lysate formulation generated a higher and longer lasting antibody titer and stimulated a higher expression of regulatory and pro-inflammatory cytokines compared with the whole-cell vaccine.     

Panton–Valentine leukocidin and some exotoxins of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> and antimicrobial susceptibility profiles of staphylococci isolated from milks of small ruminants

The aims of this study were to determine the existence of pvl gene, some toxin genes, and mecA gene in Staphylococcus aureus strains isolated from sheep milk and to examine antimicrobial resistance profiles in staphylococci from sheep and goats' milk. The milk samples were collected from 13 different small ruminant farms in Kirikkale province from February to August 2009. A total of 1,604 half-udder milk samples from 857 ewes and 66 half-udder milk samples from 33 goats were collected. Staphylococcus spp. were isolated and identified from the samples. Toxin genes and mecA gene among S. aureus strains were determined by PCR. Antimicrobial susceptibility of staphylococci was examined by the disk diffusion method on Mueller-Hinton agar, and interpreted according to the Clinical Laboratory Standards Institute (CLSI) guidelines. The prevalence of subclinical intramammary infection in both ewes and goats was 5.2%. The most prevalent subclinical mastitis agents were coagulase-negative staphylococci and S. aureus with prevalences 2.8% (n:46) and 1.3% (n = 21), respectively. The prevalence of resistances in isolated Staphylococcus spp. to penicilin G, tetracycline, erythromycin, gentamicin, and enrofloxacin were found as 26.9% (18), 7.5% (5), 6.0% (4), 3.0% (2), and 1.5% (1), respectively. Only 3 of the 21 S. aureus ewe isolates (13.4%) were shown to harbor enterotoxin genes being either seh, sej or sec. However, fourteen (66.6%) of the 21 S. aureus isolates had pvl gene while none of the isolates harbored mecA gene. In conclusion, Staphylococci were shown to be the most prevalent bacteria isolated from subclinical mastitis of ewes and goats and these isolates were susceptible to most of the antibiotics. In addition, S. aureus strains isolated from ewes were harboring few staphylococcal enterotoxin genes. However, Panton–Valentine leukocidin produced by S. aureus could be an important virulence factor and contribute to subclinical mastitis pathogenicity.     

Prevalence of capsular serotypes among Staphylococcus aureus isolates from cows with mastitis in the United States

Development of an appropriate Staphylococcus aureus vaccine for bovine mastitis has eluded researchers for decades. The ability of S. aureus to form a protective exopolysaccharide capsule has posed a major obstacle because of the multiple serotypes and the poor immune response elicited by exopolysaccharides. This study characterized S. aureus serotypes isolated from cases of bovine mastitis obtained from veterinary diagnostic laboratories that service 44% of the dairy cattle in the United States. Major milk producing areas of the northeast, north central, Pacific coast and southwest were proportionately represented. Sub-samples of mastitic milk that contained S. aureus were frozen and sent to our laboratory for strain serotyping. The only other regional serotyping of S. aureus from bovine mastitis to date was done in France. The primary serotypes found were types 5 (51%) and 8 (18%) and 31% were non-typeable. In the current study, serotype 5 accounted for 18% of the isolates and serotype 8 for 23%. More importantly 59% of the isolates were not typeable with either type 5 or 8 antisera. These data indicate that S. aureus vaccines employing serotypes 5 and 8 would only be marginally effective in the United States. These data also suggest that development of a S. aureus vaccine for bovine mastitis should take into account regional variation in S. aureus serotypes.     

The early phase of the immune response to live and killed Staphylococcus aureus vaccines in sheep — Cellular and immunoglobulin parameters

Various cellular and protein parameters in efferent lymph were monitored during early stages of immune responses in the draining popliteal lymph nodes of sheep following injection with either live or killed $\text{Staphylococcus aureus}$ vaccines. Significantly higher outputs of leucocytes and lymphoblasts were measured in lymph from animals injected with the live vaccine (Group 1) compared with animals given the killed vaccine (Group 2) and non-immunised controls (Group 3). At 48 h post-injection the mean output of leucocytes in lymph for Group 1 was 9.5 × 10⁷ cells/h, and for Groups 2 and 3 comparable mean outputs were 5.2 × 10⁷ and 2.2 × 10⁷ respectively.There was a marked increase in IgM-containing cells in Group 1 animals, significant differences between Group 1 and 2 occurring 96 h post-injection. A difference was observed between the two immunised groups in the kinetics of output of cells containing antibody to a staphylococcal antigen extract. There was a slower increase in antibody-containing cells for Group 1 than for Group 2 animals. The mean proportion of antibody-containing cells reached 0.09% (of total leucocytes) at 72 h post-injection for Group 2 and 0.10% at 120 h post-injection for Group 1.Rapid increases in flow rate of lymph were recorded in animals in Group 1. These results were in contrast to lower corresponding values observed in sheep in Groups 2 and 3. There was a twofold increase in the lymph: serum (L:S) concentration ratio for IgG2 in animals in Group 1 and the evidence suggested that this was due to local synthesis of IgG2 by lymphoid cells in the abscess at the site of injection and/or in the draining popliteal lymph node. This change in L:S ratio for IgG2 was not recorded for sheep in Groups 2 and 3.     

Risk factors and impacts of clinical and subclinical mastitis in commercial meat-producing sheep flocks in Quebec, Canada

We conducted a prospective observational study on clinical and subclinical mastitis in 30 commercial meat-producing sheep flocks from 2 regions of the province of Quebec, Canada. A total of 2792 ewes selected in late gestation were followed from lambing to weaning of lambs. The incidence of clinical mastitis for the total lactation period (average of 58 days) ranged among flocks from 0 to 6.6%, with a median of 1.2%. The most frequently isolated bacteria from the cases of clinical mastitis, in pure or mixed culture, were Mannheimia haemolytica (26%), Staphylococcus aureus (23%), and coagulase-negative staphylococci (17%). Incidence of clinical mastitis was higher in ewes that gave birth to 3 or more lambs and from the Estrie region, and was associated with an increase in ewe mortality, an increase in lamb mortality at the litter level, and a decrease in lamb's weaning weight for lambs born in multiple litter size or from ewes ≥4 years old.Among 354 selected ewes with clinically normal udder at the end of lactation, 28.8% had potentially pathogenic bacteria isolated from milk. The most prevalent bacteria were S. aureus (9.3%) and coagulase-negative staphylococci (9.3%). The risk of having a positive culture in at least one half was different between the two regions. Prevalence of ewes (n = 261) with California Mastitis Test (CMT) positive result in at least one half was 24.1 and 14.9% using a cut-off of ≥1+ and ≥2+, respectively. Prevalence of culture-positive udder halves was 11.7% for CMT-negative compared with 53.6% for CMT 3+ halves. CMT status was positively associated with the isolation of coagulase-negative staphylococci, M. haemolytica, S. aureus, and various Streptococcus species, but not with other isolated bacteria. Additionally, prevalence of CMT-positive halves was higher in ewes from the Estrie region, aged of ≥4 years versus 1 year, having clinical mastitis previously detected in the lactation and/or with low body condition score. Lamb weaning weight was associated with CMT status of ewes, while weaning weight was not associated with milk culture results. More research is needed to understand the dynamic of milk SCC and IMI in ewes from meat-producing flocks, its economical impact and best ways to control it.     

Variations in immunoglobulin isotype produced during the antibody response to Brucella abortus and Staphylococcus aureus vaccines in sheep

Experiments in sheep were carried out to examine factors modifying the immunoglobulin (Ig) isotype of the antibody response to Brucella abortus. Live B abortus (S19) stimulated higher titres of agglutinating antibody and IgG1 and IgG2 antibody than did killed B abortus. Live B abortus stimulated a more protracted synthesis of IgG2 antibody during the primary and secondary responses than did the killed S19 vaccine. In a second experiment, the capacity of live and killed Staphylococcus aureus to modify the antibody response to killed B abortus was examined. Both live and killed S aureus enhanced production of anti-brucella antibodies; this response was attributed to the adjuvant properties of S aureus. Killed S aureus enhanced production of anti-brucella antibody to a greater extent than live S aureus. Live S aureus did not preferentially enhance production of IgG2 anti-brucella antibody. The results suggested that the enhanced production of IgG2 antibody induced by live vaccines does not depend solely on a pyogenic lesion at the vaccination site.     

Toxoplasmosis in sheep III. Further evaluation of the ability of a live Toxoplasma gondii vaccine to prevent lamb losses and reduce congenital infection following experimental oral challenge

The aim of this study was to compare the ability of a live incomplete strain (Strain 48) and a live complete strain (Strain 89) of Toxoplasma gondii to protect against abortion and congenital infection following an oral challenge of T. gondii oocysts. Sixty-nine two-tooth ewes were immunised pre-tupping with live Strain 48 of T. gondii tachyzoites and seventy ewes were immunised with Strain 89. Eighty-two serologically negative ewes served as controls. At mid-pregnancy half of the ewes were challenged orally with T. gondii oocysts (2×10⁵/ewe).

The ewes vaccinated with Strain 48 were significantly (p<0.05) protected against the effects of experimental challenge and the rate of congenital infection was also significantly (p<0.15) reduced. The ewes vaccinated with Strain 89 were also significantly (p<0.05) protected.

The serological response to challenge as measured by both the Dye test and the Indirect Haemagglutination test varied considerably between the two vaccinated groups.     

Alarelin active immunization influences expression levels of GnRHR,FSHR and LHR proteins in the ovary and enhances follicular development in ewes

We investigated the effects of gonadotropin releasing hormone (GnRH) agonist on expressions of GnRH receptor (GnRHR), follicle‐stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHR) proteins in the ovaries and follicular development in the ewes. Forty‐two pre‐pubertal ewes were assigned to experimental groups 1 to 5 (EG‐I to EG‐V) and control group (CG). Ewes in EG‐I, EG‐II and EG‐III were subcutaneously injected with 200, 300 or 400 μg alarelin antigens twice (on days 0 and 14), respectively. Ewes in EG‐IV and EG‐V were subcutaneously injected with 200 μg and 300 μg alarelin antigen four times (on days 0, 7, 14 and 21). Ewes in CG were subcutaneously injected with a solvent twice (on days 0 and 14). Serum concentrations of GnRH antibody in the EGs increased and were higher than (P < 0.05) that of CG from day 14 to day 60. GnRH antibody concentrations in EG‐IV and EG‐V were higher than that in EG‐I, EG‐II and EG‐III from days 35 to 45. Expressions of GnRHR protein in EG‐IV and EG‐V were lower than that in CG (P < 0. 01). Expressions of FSHR and LHR proteins in EGs increased. Levels of FSHR and LHR proteins in EG‐IV and EG‐V (P < 0.05) were higher than CG. Ovarian weights in EGs increased. Values of follicle vertical diameter, follicle transverse diameter, follicle wall thickness, follicle externatheca thickness and follicle internatheca thickness in EG‐III and EG‐V were greater than other groups. Primordial follicles and primary follicles developed quickly in alarelin‐immunized animals. Secondary follicles and mature follicles became more abundant. Mitochondria, mitochondrial cristaes and cortical granules increased. Serum FSH concentrations of EGs remained higher than that in CG from days 28 to 70 (P < 0.05). Alarelin immunization stimulated GnRH antibody production, suppressed expression of GnRHR protein, enhanced expressions of FSHR and LHR proteins in ovaries, promoted FSH secretion and thereby accelerated the development of ovaries and follicles in ewes.     

Protective immunity following immunisation of pigs with aerosol of Actinobacillus pleuropneumoniae serotype 2

To introduce antigen to the respiratory mucosa, killed Actinobacillus pleuropneumoniae with quil A as adjuvant was administered to pigs as an aerosol. Immunisation by this aerosol induced a marked IgA response in the bronchoalveolar and nasal fluids, and in the serum. Following challenge with live bacteria two weeks after the last exposure to the aerosol, the immunised pigs were protected from the severe pleuropneumonia which developed in non-immunised pigs. The immunised pigs had lower antibody titres in the mucosal fluids and serum after exposure to the challenge. The immune response after experimental infection of non-immunised animals was a weak IgA antibody response in the bronchoalveolar and nasal fluids, whereas the systemic immune response after challenge included both IgA and IgG antibodies.     

Humoral immunity in the ewe. 1. The influence of adjuvancy and immunogenic substitution on immune reactivity following immunisation with protein antigens

The nature of antigen and presence of adjuvant are major factors which influence the level of immune reactivity following immunisation. This study examines the quantity and isotype of antibody produced in ewes immunised with different proteins in combination with different adjuvants. Results using indirect ELISA assays show that animals immunised with keyhole limpet haemocyanin (KLH) in adjuvant produced lower levels (P less than 0.05) of immunoglobulin M (IgM) and had increased levels and persistence of immunoglobulin G1 (IgG1) compared with ewes immunised with antigen in saline (P less than 0.005). The anti-KLH immunoglobulin G2 (IgG2) titre was significantly higher in animals given oil-emulsion adjuvant than all other groups (P less than 0.005). Ewes were also immunised with bovine serum albumin (BSA) or BSA haptenated with trinitrophenyl (TNP) for the primary injection and carrier BSA alone for the secondary inoculation. Chemical haptenation of BSA antigen reduced primary IgM (P less than 0.005), IgG1 (P less than 0.005) and IgG2 (P less than 0.005) levels compared with animals immunised with pure BSA. There was an increased secondary anti-BSA IgM response in all animals first immunised with TNP-BSA (P less than 0.05). The class of anti-hapten antibody produced to TNP determinants was influenced by the degree of TNP haptenation of the carrier BSA. Mid-range molar ratios of TNP produced the strongest IgM, IgG1 and IgG2 anti-TNP responses compared with all other groups (P less than 0.01).     

Exfoliation rate of mammary epithelial cells in milk on bovine mastitis caused by Staphylococcus aureus is associated with bacterial load

The exfoliation rate of mammary epithelial cells (MECs) in milk is affected by physiological, breeding and environmental factors. Little is known about the relationship between the MEC exfoliation into milk and mammary‐infected Staphylococcus aureus (S. aureus) load on bovine mastitis caused by S. aureus. The aim of this study was to investigate the relationship between S. aureus load and the proportion of MEC exfoliation in milk using five substantial bovine mastitis models. In 64 randomly extracted milk samples from udders at 3–21 days after S. aureus infusion, there were various samples with different numbers of S. aureus counts and somatic cell counts. No significant correlations were found between the S. aureus counts and somatic cell count (r = 0.338). In contrast, a significant correlation was noted between S. aureus counts and the proportion of cytokeratin‐positive cells in the milk from the infused udders (r = 0.734, P < 0.01). In conclusion, the increasing MEC exfoliation rate in milk from mastitis udders caused by S. aureus may contribute to reduced milk yield.     

Antibody Responses in Sheep Vaccinated against Staphylococcus aureus Mastitis: A Comparison of Two Experimental Vaccines Containing Different Adjuvants

A double-blind, placebo-controlled study was conducted to compare two vaccines using different adjuvants with regard to their ability to stimulate antibody production against the - and -toxins and the exopolysaccharide of Staphylococcus aureus. The vaccines contained identical antigens, consisting of inactivated whole bacteria of two strains of S. aureus in addition to - and -toxoid. One vaccine contained mineral oil, while the other used a water-soluble acrylic acid polymer resin (Carbopol) as adjuvant. Saline served as the placebo. One hundred and forty ewes were vaccinated twice before lambing, by subcutaneous injection with vaccine or placebo in the region of the supramammary lymph node, and were observed and sampled over a period of 6 months. The vaccine containing mineral oil as adjuvant induced significantly greater immune responses to the - and -toxins than did the vaccine containing Carbopol. The latter vaccine induced higher levels of antibodies to exopolysaccharide. The degree of local adverse reactions did not differ between the two groups. The results indicate differences between the oil-adjuvanted and Carbopol-adjuvanted vaccines with regard to their ability to stimulate antibody production against S. aureus protein antigens in sheep.     

Combined vaccination of live 1B Chlamydophila abortus and killed phase I Coxiella burnetii vaccine does not destroy protection against chlamydiosis in a mouse model

Q fever and chlamydiosis often affect ovine and caprine flocks simultaneously or successively. Combination vaccines effective against these 2 diseases would be of great value in veterinary medicine. Unfortunately, the current effective vaccines are a live vaccine for chlamydiosis and killed vaccine for Q fever. Vaccination of mice with live chlamydiosis vaccine 1B and killed phase I vaccine against Q fever at 2 points on the back at the same time produced good protection against chlamydial abortion. This suggests that it may be possible to vaccinate ewes and goats against chlamydiosis and Q fever simultaneously.     

Susceptibility of the pre-parturient ewe to infection with Trichostrongylus vitrinus and Ostertagia circumcincta

During two successive years, groups of pregnant ewes and non-pregnant worm-free sheep were given anthelmintic, challenged one week later with 10,000 infective larvae of either Trichostrongylus vitrinus or Ostertagia circumcincta and killed two, four and six weeks after challenge. All of the ewes were killed within three days of their predicted lambing date. There was no annual variation in the response of the ewes to infection with either species. Compared to adult naive animals, ewes expressed resistance to infection with T vitrinus at all times, with significantly lower worm burdens composed largely of inhibited third stage larvae. Ewes challenged with O circumcincta were fully as susceptible as naive animals with regard to the size and stage of development of their worm populations. Mechanisms regulating the numbers of adult O circumcincta were operative in the adult naive animals but not in the ewes.