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Ovine Johne's disease is a chronic debilitating disease of sheep caused by Mycobacterium avium subsp. paratuberculosis (Mptb) which results in diarrhoea, emaciation and mortalities in infected animals. Vaccination with Gudair® has been a key strategy for controlling the disease in Australia since its approval in 2002. Previous research conducted in Australia has demonstrated that the vaccine is quite effective in reducing sheep mortalities. While some farms have also been successful in reducing the prevalence of the disease in their flocks to undetectable levels, sheep in other flocks continue to shed Mptb in faeces even after an ongoing vaccination program . This study was conducted to investigate management, husbandry and biosecurity factors associated with paratuberculosis infection in Gudair® vaccinated sheep flocks in Australia. We enrolled 64 sheep farmers and interviewed them to obtain information about their management and biosecurity practices. Pooled faecal samples were collected from sheep at each farm and cultured to create two outcome variables: Mptb positive (yes/no) and disease prevalence level (nil, < 1 %, ≥ 1 %). Binary and ordinal logistic regression analyses were conducted to evaluate the association of management, husbandry and biosecurity factors with these outcome variables. Farms were more likely to have Mptb positive sheep and a higher disease prevalence in their flocks if they: (a) provided supplementary feed on the ground (instead of in a trough); (b) had a greater number of neighbours with sheep; and (c) had introduced rams from a greater number of sources. The results suggest the effectiveness of Gudair® vaccination to control OJD can be improved if sheep producers maintain other risk management strategies and biosecurity practices. Extension agencies should advise farmers not to relax their biosecurity practices and to purchase rams from only low-risk sources, even if they are continuing to vaccinate their flocks.

At present diagnosis of true resistance and determination of drug efficacy in Fasciola hepatica infection rely solely on terminal experiments. The coproantigen ELISA (cELISA) has been reported previously as a sensitive and specific tool appropriate to detect treatment failure, and potentially drug resistance. Two studies were conducted to determine whether the cELISA was appropriate for on-farm efficacy and resistance testing in Australian Merino sheep. In Study 1 sheep were infected orally with 50 F. hepatica metacercariae on three occasions, twelve, six and two weeks prior to a single flukicide treatment with triclabendazole, closantel or albendazole. Sheep were sampled weekly for a further seven weeks prior to necropsy. Following effective treatment, no faecal antigen was detected from 1 week. When immature stages (≤6 weeks) survived treatment, coproantigen reappeared from 6 weeks post-treatment. Therefore, cELISA conducted 1-4 weeks after treatment will demonstrate obvious treatment failure against adult F. hepatica, but is not sufficiently sensitive to detect survival of immature fluke until these reach maturity. In study 2, fluke burdens of sheep necropsied 13 weeks post single infection were compared to fecal worm egg counts (FWEC) and cELISA at necropsy. Regression analysis demonstrated that cELISA correlated strongly with fluke burden, whilst FWEC correlated weakly with cELISA. The correlation between FWEC and fluke burden was also weak, although stronger than that of FWEC with cELISA. The cELISA is an appropriate tool for monitoring effectiveness of treatments against Fasciola hepatica if an adult infection is present, however when immature stages of the parasite are present it is not as reliable. Where immature parasites are present it is recommended that initial cELISA be followed with a secondary cELISA at least 6 weeks after treatment to ensure resistance to immature stages is detected. Further testing is justified for monitoring the effectiveness of control programs by detecting adult populations that have survived a treatment regime.

Information on the susceptibility status of Fasciola hepatica isolates is lacking in the literature, even for those isolates considered to be laboratory reference strains. Four controlled efficacy studies were conducted on two Fasciola hepatica isolates from Australia, viz. 'Oberon' and 'Sunny Corner' with treatment at either 2, 6 or 10 weeks post-infection (wpi) as defined in each study. Fluke burdens and examination of livers occurred at necropsy in weeks 12 (Study 1) or 13 (Studies 2, 3 and 4) post-infection. The triclabendazole (TCBZ) resistance status of the Oberon isolate was confirmed in 6 and 10-week old F. hepatica, utilizing the drug alone (Fasinex; 71.5% and 31.1%, respectively) and in combination with oxfendazole (Flukazole C; 79.9% and 0%, respectively). The susceptibility of this isolate to albendazole, as well as salicylanilide and sulphonamide drugs was confirmed. The Sunny Corner isolate was confirmed as susceptible to TCBZ (>99% all stages) and closantel (>90% at ≥6 wpi).

Ovine Johne's disease (OJD) is a degenerative wasting condition of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). Similar to other pathogenic mycobacterial infections it is a slow-progressing chronic disease and subclinically infected individuals can spread the disease while appearing healthy. MAP infects macrophages and the host responds by mounting a cell-mediated immune response. Disease progression is associated with immune dysfunction but the reasons are unknown. Increasing our current knowledge of the immunological mechanisms involved in disease progression, including apoptotic responses, may allow advancements in the area of early diagnosis, identification of resistant animals and disease control. We describe lymphocyte apoptosis in peripheral blood mononuclear cells (PBMC) and lymph node cells from sheep exposed to MAP as well as from healthy non-exposed sheep. Apoptosis in intestinal lymph node cells from MAP-exposed infected sheep, but not in MAP-exposed uninfected sheep, increased in response to MAP antigen. In this first longitudinal study of lymphocyte apoptosis using an experimental infection model of MAP infection, we found that there was a transient increase of ex vivo PBMC apoptosis in MAP-exposed sheep soon after exposure to MAP (4 months post inoculation). MAP antigen-specific apoptosis occurred later, at 12 months post inoculation. The cells involved were mainly γδ and CD4+ T cells. Antigen-mediated lymphocyte apoptosis during mycobacterial disease progression could contribute to the immune dysfunction in Johne's disease.

Haemonchus contortus (Barber’s pole worm or “BPW”) is the nematode “nemesis” of small ruminant production systems in tropical and subtropical regions of the world. Its reputation derives from a combination of high fecundity and a short generational interval that provides an enviable developmental plasticity for adaptation or resistance to control measures. This review critically examines the historical and current literature on the host–parasite–environment interaction for H. contortus, particularly in sheep, to highlight changes in parasite distribution and ecology on pasture, changes to the seasonal inhibition of fourth stage larvae and the most appropriate models to identify protective responses and assess vaccines. The review also proposes pathways to bring host genetics to fruition and avenues where advances in the parasite genome may complement control measures.

The capacity of a commercially available gamma interferon (IFNγ) assay to detect infected sheep early in the pathogenesis of Johne's disease enables the removal of such animals from the flock before bacterial shedding and pasture contamination. However, nonspecific IFNγ responses in the assay have meant that to achieve high-test specificity, there has been a reduction in sensitivity. Although the optimal conditions for the use of the assay in cattle have been well documented, there have been few studies optimizing the assay for use in sheep. The current study details the effect of anticoagulant, duration of incubation, cell concentration, blood storage temperature, time of stimulation of cells with antigen relative to time of sample collection, and temperatures during transit on IFNγ synthesis. Maximal IFNγ synthesis occurred with incubation periods of 48 hr in blood collected into heparinized tubes. Decreasing the leukocyte population by diluting the total peripheral blood leukocyte concentration was associated with a decreasing IFNγ response. Conversely, concentrating the peripheral blood concentration 2-fold resulted in an increase in the IFNγ production. In field studies, immediate incubation of blood samples with antigen at 37°C resulted in larger IFNγ responses; however, significantly lower IFNγ values were obtained if the samples were transported at ambient temperature. The results of this study indicate that optimization of the IFNγ assay may enable increased synthesis of IFNγ during the stimulation phase of the assay and that future work may determine whether this translates to increased sensitivity of the assay in detecting early infections in sheep.

To determine whether fistulation and differing strains of Haemonchus contortus complicate genome analysis of the host response to infection, two pilot experiments examined parasite development and gene expression in the abomasal mucosa of parasitised sheep. No significant differentially-expressed genes were detected in a comparison between ivermectin-susceptible McMaster and ivermectin-resistant CAVR strains of H. contortus. This demonstrated that the sheep response was not significantly altered by the ivermectin-resistance status of the parasite. However, sheep infected with McMaster strain had a significantly lower proportion of larvae and a higher mean FEC at post-mortem than sheep infected with CAVR, suggesting that McMaster larvae advance to patency faster than CAVR larvae. Abomasal fistulation resulted in significant upregulation of three genes and significant downregulation of two genes. Fistulated sheep had significantly lower FEC than the other groups but the proportion of larvae at post-mortem was not significantly different to other groups infected with the same strain (CAVR). Hence fistulation does not alter establishment of the CAVR isolate, but may slow its progression to patency. The observation that different H. contortus strains and abomasal fistulation induced minimal changes in mucosal gene expression validated the design of a subsequent experiment (manuscript in preparation) where sequential biopsies taken during infection were analysed by microarray to describe the molecular responses which inhibit larval establishment.

Haemonchus contortus is a blood-feeding nematode which parasitizes the abomasum of sheep and represents a serious constraint to sheep production. Anthelmintics are currently the most common method of worm control but the worldwide development of multiple-drug resistance and issues of residues in the food chain make alternatives to anthelmintics a priority. Biotechnology-driven solutions to parasitism include vaccines and silencing of genes regulating nematode development. To pursue gene targets that may be suitable for parasite control, a two stage differential-display PCR (dd-PCR) approach was developed to observe differential gene expression between Haemonchus from immune and control sheep. Twenty-four reproducible differentially-expressed bands were identified in 60 pairs of dd-PCR comparisons. The first of these cloned and sequenced corresponded to the H. contortus 60S ribosomal protein L35A. The remaining bands are being cloned and validated and may provide new targets for parasite control.

Monoclonal antibodies (MAbs) were developed against recombinant ovine interleukin-5 (IL-5) produced in the baculovirus expression vector system. One MAb, D11 (isotype IgG1), neutralised the activity of both recombinant and native sources of IL-5 in a biological assay (Baf cell assay) but was only weakly reactive in immunocytochemistry. Conversely, a second MAb, A8 (isotype IgA), successfully detected IL-5 in immunocytochemistry but did not display neutralising activity. The development of these MAbs will enable the assay of ovine IL-5 in vitro and permit studies into the role of hypersensitivity reactions in sheep by neutralisation of IL-5 in vivo.

In two trials, lambs were trickle infected with 400 or 1000 infective larvae of Haemonchus contortus (HcL(3)), twice weekly for 4 weeks from the day of birth. Following anthelminthic treatment at 6 weeks (Trial 1) or 7 weeks of age (Trial 2), lambs were challenged 1 week later with a trickle infection totalling 5000 (Trial 1) or 10000 HcL(3) (Trial 2). In both cases, significant protection (P

In order to analyse Th2-type immune responses in sheep by the assay of interleukin (IL)-5 in biological fluids, the ovine IL-5 gene was cloned and expressed in Spodoptera frugiperda Sf9 insect cells using the baculovirus expression vector system. The recombinant product was purified as BAC-OV-IL-5 from the supernatant fluid. The ovine IL-5 was biologically active in a bioassay using IL-5-dependent Baf cells, which have been used previously to specifically detect human IL-5. The specificity of Baf cells for ovine IL-5 was examined by two methods. First, Baf cells only proliferated in response to BAC-OV-IL-5 and did not respond to addition of recombinant ovine cytokines granulocyte-macrophage colony stimulating factor (GM-CSF), IL-1beta, IL-2, IL-3, IL-6, IL-8, stem cell factor (SCF) or IFN-gamma at doses from 0.01 to 1 microg/well. Second, the rat monoclonal antibody to murine IL-5, TRFK-5, neutralized murine, but not ovine, IL-5. However, rabbit antisera to BAC-OV-IL-5 neutralized murine and ovine recombinant IL-5 and abolished responses of Baf cells to IL-5 activity in supernatant fluids from mesenteric lymph node cells (MLNC) of parasitized sheep. The bioassay had a sensitivity to detect 8 ng in a 200 microL assay (40 ng/mL). Thus, the specificity of Baf cells to detect human IL-5 also extends to ovine IL-5 and therefore provides a method for monitoring the production of Th2 immune reactivity in sheep.

The ability of young Merino lambs to achieve protective immunity following vaccination via viable nematode infections was assessed. Lambs were infected from 1 month of age by repeated continuous low dose (trickle) administration of Trichostrongylus colubriformis or Haemonchus contortus infective larvae (L3), or by truncated infections with high doses of viable T. colubriformis L3. After 7 weeks all groups were drenched with anthelmintic and at 3 months of age they were re-infected with the homologous species. Protection was assessed by faecal egg counts at 3, 4, 5, 6 and 7 weeks after challenge, and worm count at 7 weeks after challenge. Young lambs were partially protected by 3 months of age against Trichostrongylus by trickle infection. This protection correlated with local mast cell and T-cell priming, increased numbers of local antigen-presenting cells and T-cells and increased worm-specific antibody titres in the intestine. However, there was no evidence that young lambs were capable of immunologically recognising H. contortus antigens following trickle infection, nor did trickle infection significantly protect young lambs against Haemonchus challenge.

This study examined the role of CD8+ and WC1+ T-cells and of interferon (IFN)-gamma in the development of protective immunity against infection with the enteric nematode parasite Trichostrongylus colubriformis in sheep. Monoclonal antibodies (mAb) were administered during induction of the immune response to deplete or neutralise these components. Protection against the primary and challenge infections was assessed by faecal egg count and total worm count. Prolonged administration of mAb recognising IFN-gamma and CD8 resulted in significantly increased protection during the 6 week primary infection and following challenge. CD8+ cells were depleted from blood but not from intestinal mucosa. After injection of mAb (CC15) recognising the surface antigen WC1, WC1+ and Tcr gamma delta + cells were depleted from blood but not markedly from enteric mucosa, and protection against challenge, although variable, was increased by up to 88%. It appears that CD8+ and WC1+/gamma delta+ cells and IFN-gamma all retard the potential development of naturally acquired immunity against the parasite.

Responses of isolated mucosal mast cells (MMC) during infections with either Trichostrongylus colubriformis or Haemonchus contortus were examined by measuring the release of sheep mast cell protease (SMCP) in a degranulation assay. MMC from sheep immune to T. colubriformis released maximal amounts of SMCP and histamine within 0.5h of incubation with larval antigen whereas maximum secretion of leukotrienes occurred 3h after addition of antigen. It was only after 8 weeks of a primary T. colubriformis infection, that MMC released significantly elevated levels of SMCP (23%); this occurred when the worm burden was being rejected. In contrast, the SMCP release from MMC of immune sheep was significantly higher at 40%, and occurred within 1-4 days after challenge (DAC). The SMCP release peaked at 6-8 DAC at 51%, and declined after 56 DAC to < 25%. MMC isolated from the duodenum and mid-small intestine of immune sheep released 2-3 times higher proportion of SMCP than did cells recovered from the terminal ileum. Mast cell numbers were similar in the 3 regions but the quantity of globule leucocytes (GL) was 2.5 times higher in the duodenum. During infections with H. contortus in the abomasum, MMC isolated from the small intestine released greater levels of SMCP when incubated with larval antigens than did abomasal MMC. There was no increased release during the first 12 weeks of a primary infection although the SMCP release (23%) from immune MMC at 7-10 DAC was significantly enhanced. Once again the release from MMC isolated from the three intestinal regions of sheep immune to H. contortus was lowest in the terminal ileum.(ABSTRACT TRUNCATED AT 250 WORDS)

A mAb (IFN-9) which neutralizes biologically active ovine and bovine IFN-gamma was used to deplete levels of the cytokine in vivo in sheep and examine the consequences for immune responses to adjuvanted antigen and skin reactivity to Bacillus-Calmette-Guerin (BCG). Groups of sheep were immunized with ovalbumin in the adjuvants, Quil A or dextran sulphate (MW 500,000; DXS), both of which elicit production of IFN-gamma. MAb anti-IFN-gamma or an isotype control mAb (anti-carbonic anhydrase positive particles) were inoculated i.v. during primary and/or secondary responses. Reactions monitored in efferent prefemoral lymph indicated that anti-IFN-gamma effectively depleted levels of IFN-gamma in lymph but had no effects on the magnitude and kinetics of lymphocyte and lymphoblast traffic, or total or isotypic titres of specific Ig. When incubated in vitro with ovalbumin, antigen-reactive cells from anti-IFN-gamma treated sheep did not produce IFN-gamma, suggesting an on-going modification to cytokine production. In contrast, skin reactions to purified-protein derivative in sheep immunized with BCG were reduced by < 40% by anti-IFN-gamma. The results indicate that IFN-gamma production may not be obligatory for delayed-type hypersensitivity reactions or for the adjuvant action of Quil A or DXS, and that specific mAb can alter the profile of cytokines produced by antigen-reactive cells in sheep.

The effects of protein supplementation and infection with Trichostrongylus colubriformis on production responses and immune function in young sheep and on nematode population dynamics were assessed. Eighty-four 3-month-old Merino wether sheep were housed in individual pens and fed ad libitum chopped oaten hay containing 0.5% urea, together with 50 g day-1 lucerne meal. Fish meal (FM) was given as a source of protected protein at 0, 50 or 100 g day-1 (FM0, FM50, FM100; from Days --28 to 140). From Days 1 to 140, 0 or 1000 T. colubriformis infective larvae were given on Mondays, Wednesdays and Fridays. Infected sheep were slaughtered after 35, 70, 105, or 140 days of infection. Live-weight gain was reduced significantly by infection with T. colubriformis in sheep given FM0, but not in sheep given FM50 or FM100. Greasy wool production and fibre diameter were increased by FM, whereas the effects of infection with T. colubriformis on wool measurements depended on the level of FM given. Worm egg concentrations in faeces were significantly lower for sheep given FM100 than for those given FM0 or FM50 during the last 28 days of infection. Similarly, the apparent rate of worm expulsion was considerably higher in sheep given FM than in those not given FM. The rate of expulsion of T. colubriformis correlated with levels of circulating eosinophils as well as with the concentration of intestinal sheep mast cell proteases. Levels of parasite-specific and non-specific circulating antibodies were either unaffected or reduced as a result of supplementation with FM, although lymphocyte stimulation in vitro in response to T. colubriformis third stage larval antigen was enhanced significantly in infected animals given FM100. It was concluded that supplementary feeding with FM substantially reduced the production losses attributable to infection with T. colubriformis and was associated with enhanced expulsion of the parasite burden.

Helminthologically naive merino sheep were given either a single infection of 30,000 or a trickle infection of 6000 Trichostrongylus colubriformis infective larvae (TcL3) per week. Faecal egg counts started to fall after 8 weeks in the single infection and after 11 weeks in the trickle infection. Small intestinal contents were collected from indwelling intestinal fistulae over the next 14 weeks. Concentrations of sheep mast cell protease (SMCP) in these contents increased to highest levels 9–11 weeks and 6–10 weeks after the single infection and from the start of the trickle infection, respectively. Similarly, peptidyl leukotriene (PLT) concentrations were highest at 6 weeks and at 6–9 and 13 weeks, respectively. Histamine concentrations increased slightly after both infections to peak values at 7 weeks and 9 weeks, respectively. Inhibition of migration of larvae in vitro was increased in contents sampled at 8 weeks after the single infection and after 6–10 weeks of the trickle infection. Another 2 groups of sheep were immunised by repeated infections with TcL3. Gut contents from 1 group sampled immediately before and after challenge with 30,000 TcL3 at 0 and 18 days had increased levels of larval migration inhibitory (LMI) activity throughout the 35 day period, especially 7–14 days after challenge (DAC). The mediators SMCP increased significantly 5–7 DAC while PLT increased 7–14 DAC. In the second group of immunised sheep, levels of SMCP and PLT increased rapidly within 1 DAC and further increased 3–14 DAC. Increased concentrations of platelet activating factor PAF occurred only at 14 DAC, while LMI activity was greatest in mucus collected 5–8 DAC. Since increased concentrations of these mediators occurred around the time when egg counts started to decline in primary infections and were increased after challenge of immune sheep, the results suggest roles for the mediators and/or their cellular source in the expression of immunity.

SummaryGenetic resistance to gastrointestinal worms is a complex trait of great importance in both livestock and humans. In order to gain insights into the genetic architecture of this trait, a mixed breed population of sheep was artificially infected withTrichostrongylus colubriformis(n=3326) and thenHaemonchus contortus(n=2669) to measure faecal worm egg count (WEC). The population was genotyped with the Illumina OvineSNP50 BeadChip and 48 640 single nucleotide polymorphism (SNP) markers passed the quality controls. An independent population of 316 sires of mixed breeds with accurate estimated breeding values for WEC were genotyped for the same SNP to assess the results obtained from the first population. We used principal components from the genomic relationship matrix among genotyped individuals to account for population stratification, and a novel approach to directly account for the sampling error associated with each SNP marker regression. The largest marker effects were estimated to explain an average of 0·48% (T. colubriformis) or 0·08% (H. contortus) of the phenotypic variance in WEC. These effects are small but consistent with results from other complex traits. We also demonstrated that methods which use all markers simultaneously can successfully predict genetic merit for resistance to worms, despite the small effects of individual markers. Correlations of genomic predictions with breeding values of the industry sires reached a maximum of 0·32. We estimate that effective across-breed predictions of genetic merit with multi-breed populations will require an average marker spacing of approximately 10 kbp.

Sheep immunized with multiple normal infections of 30,000 Trichostrongylus colubriformis larvae (T.c. L3) suppressed the fecundity, establishment and survival of adoptively transferred adult worms, showing that these parasites were susceptible to the effects of host immunity. When sheep were immunized by four 'truncated' larval infections of 4, 7 or 10 days' (d) duration with 10(5) T.c. L3, animals given 4 x 4d infections were susceptible to challenge, whereas sheep given 4 x 7d and 4 x 10d infections were significantly protected. A serial analysis of the rejection of T. colubriformis from nine sheep given 5 x 7d infections revealed that the challenge larval infection given intraduodenally was expelled within 3 days after challenge (DAC). However, another five of these sheep only rejected around 50% of transferred adult worms by 21 DAC when compared with control animals. The results indicate that stage-specific antigens produced by early L3 and L4 stages of T. colubriformis effectively immunize sheep against a larval challenge but appear less reliably protective against adult worms.

Immune-mediated scouring due to ingested parasite larvae is a major concern for sheep producers in Mediterranean climates. We investigated immune-mediated scouring in parasite-resistant Merino sheep in Australia. Forty-adult, parasite-resistant Merino rams were judged to be either susceptible or non-susceptible to immune-mediated scouring on the basis of dag scores taken under field conditions. We hypothesised that the susceptible rams would have lower faecal dry matter during larval challenge than non-susceptible rams and that, at post-mortem examination, inflammatory mediators and granulocytes would be negatively correlated with both faecal dry matter and worm numbers. In pens, the rams received a dose of 500 Teladorsagia circumcincta L3 and 500 Trichostrongylus colubriformis L3 each day for 6 weeks before euthanasia. Ten rams acted as unchallenged controls. Challenging sheep with larvae reduced faecal dry matter at 2, 3 and 4 weeks after challenge began and the greatest reductions were with the sheep susceptible to scouring. The sheep showed good resistance to the parasite challenge as evidenced by low faecal worm egg counts and low total worm counts at post-mortem, with the numbers of T. colubriformis particularly low. Sheep with low faecal dry matter had significantly higher numbers of eosinophils in small intestine tissue. Sheep with low total worm counts had significantly higher levels of bradykinin in abomasum mucus. Sheep with more granulocytes in tissue and inflammatory mediators in mucus tended to have fewer numbers of T. circumcincta but there was little relationship with numbers of T. colubriformis. Our results show that dag scores are correlated to a reduction in faecal dry matter, which can be attributed to the challenge with infective parasite larvae. Inflammation during worm infection is associated with rejection of the worm challenge and may result in more fluid faeces and consequently diarrhoea. Therefore, sheep breeders should focus on breeding for both low worm egg counts and also low dag scores.

Johne's disease is a severe chronic enteritis of ruminants caused by Mycobacterium avium subspecies paratuberculosis (Map). Repeatable infections of known duration are required for validation of new diagnostic tests, evaluation of pathogenesis and development of improved vaccines. In the first study of its type, a standardised experimental model for Johne's disease was developed based on a lyophilised, low passage, pure culture, seedstock of Map. Experimental inoculations of sheep with accurately enumerated doses of Map resulted in infection outcomes across multiple trials that were modulated by the interval between inoculation and examination. Compared to an inoculum consisting of an intestinal mucosal homogenate from a naturally affected sheep, clinical signs from the pure culture of Map were manifested later, but other measures of infection were similar. Immunological assays showed that most of the inoculated animals were IFN-gamma positive in the early stages of the infection. Over time, an increasing number of sheep became Map-specific antibody positive, developed typical histopathological lesions and shed Map in their faeces. The repeatability and utility of this experimental infection model will enable study of many aspects of Johne's disease. It is the first study to show that models for Johne's disease can be standardised in relevant species using traditional microbiological approaches to production and storage of seedstock. It is recommended that an international bank of master seedstock be established, containing low passage isolates that are representative of the major strains of Map, S and C.

Merino sheep were immunized against the intestinal nematode, T. colubriformis, by repeated infections, and proliferative responses of their peripheral blood lymphocytes (PBL) against parasite extracts and excretory-secretory (ES) antigens were monitored over 130 days. Maximal responses occurred 7–14 days after challenge. The ability of soluble proteins and parasite antigens to induce proliferation was compared with that of antigen-bearing particles obtained after antigen was adsorbed onto nitrocellulose. Blank particles increased c.p.m. two- to three-fold above that obtained in medium alone, and to elicit proliferative responses of comparable magnitude between 10 and 100 times more antigen was required when antigen-bearing particles were used instead of soluble extracts or denned proteins. Blood leucocytes as well as T-cell lines established by stimulation with parasite antigens in vitro reacted to moieties of from 5000 to 38,000 mol. wt in ES antigens on nitrocellulose particles. Direct comparisons of T-lymphocyte responses with antibody responses as assessed by immunoblots revealed different profiles of immunogenicity among ES proteins within individual sheep, but the 10,000, 30,000 and 75,000–90,000 mol. wt proteins were immunodominant. These proteins were also those consistently recognized by T-lymphocytes and sera from sheep immunized with ES proteins in adjuvant. Thus, this technique can be applied to identify parasite material which is immunogenic for T-lymphocytes, but the sensitivity of the procedure in sheep is less than reported in human studies.

Anthelmintics are currently the most common method of worm control. The emergence of worms with multiple-drug resistance and issues of residues in the food chain make alternative parasite control measures a priority. To develop improved and sustainable methods for controlling Haemonchus contortus such as genetic selection of resistant sheep, a better understanding of the host-parasite relationship is required. A trial was undertaken using sheep surgically implanted with abomasal fistulas to enable sequential biopsy of the abomasal mucosa during trickle infection with two strains of H. contortus. These were ivermectin-resistant CAVR and ivermectin-sensitive McMaster. From a gross parasitology perspective, this approach enabled the effect of developing immunity to be observed on both the establishment and maturation of two CAVR doses within and between groups. Since the only difference in parasite treatment between the groups was the staggering of the two CAVR doses, microarray results from biopsies taken on the same day in different groups were combined and compared between different biopsy dates to observe differential gene transcription over time. Differential gene transcription was detected by comparing transcription in our array data between different biopsy dates using a low P value screen (P0.01) and by compiling a list of 82 immunoparasitology-related genes and examining transcription in this list with a higher P value screen (P0.05). Our microarray data were validated in silico by comparison with intelectin 2, trefoil factor 3, calcium activated chloride channel and mucin 5 from other gene transcription studies and with phenotypic data such as the response by gammadelta T cells and immunoglobulins to H. contortus. The first four genes are involved in non-specific responses to infection and mucosal healing. These were upregulated at the early time points and intelectin 2 remained prominent throughout the trial. As the trial progressed, immunoglobulin genes became strongly upregulated. These included IgCgamma IgG2a heavy chain constant region, IGHE immunoglobulin heavy constant epsilon and IGHM immunoglobulin heavy constant mu.

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 naive 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.

The J558L cell line, previously transfected with the ovine Cepsilon gene, was induced to secrete a chimeric IgE protein composed of the ovine heavy chain and a mouse light chain with MW of approximately 80 and 26 kDa, respectively. After purification, the chimeric protein was used to immunise BALB-c mice and monoclonal antibodies (mAbs) were generated. The mAb 2F1, which had greatest anti-IgE activity in preliminary screens, was chosen for further characterisation and an examination of systemic and local IgE responses to the intestinal nematode, Trichostrongylus colubriformis. The chimeric IgE protein was not recognised in enzyme linked immunosorbent assay (ELISA) by mAbs raised against ovine IgG1, IgG2, IgA or IgM. However, 2F1 was highly specific to the chimeric IgE protein, and did not cross-react with ovine IgG1, IgG2 or IgA. Western blot analysis also showed that 2F1 and secretory IgA (sIgA) did not cross-react, and that 2F1 and the anti-IgA mAb identified different MW bands from colostrum (approximately 200 and 400 kDa, respectively). 2F1 bound to mucosal mast cells (MMC) isolated from the intestines of lambs infected with T. colubriformis, but cultured bone marrow-derived mast cells (BMMC) required prior incubation with the chimeric IgE protein for this binding to occur. Distinctive staining of plasma cells and putative mast cells were observed using 2F1 on immunohistological sections of mesenteric lymph node and jejunum.ELISA incorporating 2F1 was able to detect >0.4 ng chimeric protein. Total IgE in ovine colostrum and intestinal homogenates was quantified using a capture ELISA, with known amounts of chimeric protein used to produce a standard curve. Colostrum from outbred Merino ewes had 0.55-11.05 ng ml(-1) total IgE, and their lambs, at necropsy after infection with a total of 18,000 T. colubriformis infective larvae over a 9-week period, had 45-620 ng g(-1) total IgE in intestinal tissue. Compared to genetically susceptible lambs, antigen-specific levels of IgE were significantly higher in genetically resistant lambs after infection with 4500 T. colubriformis infective larvae (TcL3) per week for 9 weeks (161.4 versus 44.8 geometric mean titres; P=0.043). In western blots, distinctive bands (19-21 and 27 kDa) from T. colubriformis larval antigen were differentially recognised by IgE, as identified by 2F1, in intestinal homogenates from genetically resistant animals. These results have demonstrated the value of 2F1 for quantification of IgE responses in samples derived from ovine fluids and tissues using ELISA, western blots and immunohistology. In this respect, it recognises native ovine IgE and does not require pre-treatment of the sample with denaturing agents or ammonium sulphate.

Wagland B. M. , Jones W. O. , Hribar L. , Bendixsen T. and Emery D. L. 1992. A new simplified assay for larval migration inhibition. International Journal for Parasitology 22 : 1183–1185. A simple method is described for the in vitro detection of substances that impair the motility of third-stage larvae of gastro-intestinal nematodes. The test is based on the ability of larvae to freely migrate through selected mesh sizes of nylon sieves and the reduced ability of larvae to migrate after preincubation with, and in the presence of, substances that inhibit or reduce larval motility.

Following challenge with Trichosirongylus colubrifonizis, increased numbers of T-cells and immunoglobulin responses are seen in the intestine of sheep immunised by repeated infection with live worms. IL-6 mRNA expression in the small intestine from T. colubriformis-immunised and naive sheep was determined by in situ hybridisation, whereas CD4(+), IgA(+), IgG(+) cells in the gut were evaluated by immunohistochemistry. There was constitutive expression of IL-6 mRNA by cells in the naive gut, and the number of these cells was increased by parasite challenge. There were corresponding increases in numbers of CD4(+) and TCR gamma/delta(+) T-cells and IgG(+) B-cells. Our data are consistent with a role for IL-6, perhaps produced by CD4(+) and/or TCR gamma/delta(+) T-cells or B-cells, in B-cell terminal differentiation. Infiltration of B-cells, particularly IgG(+) B-cells, may reflect parasite immunity in the host.

This study examined whether infective larvae (L 3 ) of Trichostrongylus colubriformis could establish throughout the small intestine and were not restricted to the anterior duodenum in susceptible and resistant sheep. The location of worms was similar in susceptible animals given doses of T. colubriformis between 10,000 and 80,000 T. colubriformis larvae, with 90% of worms located in the proximal 3 m of the small intestine. Those worms recovered from resistant sheep were also found in the first 9 m of the intestine. However, worms recovered from immune sheep were significantly ( P=0.0074 ) relocated posteriorly from the first 3 m into the next 6 m of the intestine. By the surgical introduction of worms, it was found that T. colubriformis could establish at any site in the small intestine and to some extent in the caecum. Immunity was generated principally in the site of predilection in the anterior 3 m of the small intestine and effectively expelled challenges given at distal sites and the caecum, indicating dissemination of immunity throughout the gastrointestinal tract. Moreover, the rejection episode had removed worms from the entire small intestine within 2 h of introduction through the pylorus.

Progress towards effective vaccines to control internal parasites, especially those affecting mucosal compartments, has been inhibited by the combined problems of the antigenic complexity of parasites and the lack of understanding of the host response. However, the accumulation of information regarding regulation of mucosal immunity has enabled a reappraisal of vaccination options to provide appropriate mucosal effector responses. The pivotal role of T cell influences, and in particular the contribution of cytokine signals, has been clearly established from in vitro studies, but data emerging from our laboratories provide evidence for these effects in vivo. We have demonstrated the role of T cells in determining the outcome of an intestinal response and propose a role for local Th2 cytokine production in this regard. To support this proposition, the distribution of cytokine mRNA has been determined by in situ hybridisation techniques in normal and parasitised animals. Further, we have shown that in the absence of Th2 cytokines (using gene knockout animals) mucosal responses are grossly deficient; we have also shown that this defect can be overcome by vector-directed gene therapy. These studies have indicated that new mucosal immunisation opportunities exist by combining traditional immunisation approaches with strategies to upregulate local cytokine production. However, the success of these new strategies will depend on selection of highly immunogenic subunit antigens, coupled with techniques for cytokine manipulation and delivery with appropriate adjuvant/vehicle formulations. This paper reviews delivery technologies available to chaperone labile antigenic and genetic material to appropriate sites for mucosal stimulation after systemic or oral administration.

Interleukin (IL)-5 is produced by variety of cell types and contributes to both lymphocyte development an eosinophil terminal differentiation in vitro. The coincidence of worm expulsion and eosinophilia in sheep infected with the gastrointestinal nematode Trichostrongylus colubriformis suggest that eosinophils may be involved as effector cells in host immunity against parasite infection. The role of IL-5 in this process was investigated by observing the distribution of IL-5 mRNA+ cells in the small intestine, mesenteric lymph nodes (MLN) and Peyer's patches (PP) by an in situ hybridization technique using a murine IL-5 riboprobe. IL-5 mRNA+ cells were distributed throughout the lamina propria (LP) of the small intestine from the tips of the villi to the muscularis mucosae and in the parafollicular areas of MLN and PP in both naive and immune sheep. The phenotypes of IL-5 mRNA+ cells was explored by simultaneous eosin and immunohistochemical staining using a monoclonal antibody recognizing the T19 marker, which identifies a major subset of gamma/delta TCR+ cells in sheep. In immune sheep, there was about a five-fold increase in the number of eosinophils and IL-5 cell mRNA+ cells in the LP, but there was no significant change in numbers of T19+ cells. Most of the IL-5 mRNA cells in the LP were eosinophils, but many of the T19+ cells also expressed IL-5 mRNA. In contrast, there were fewer eosinophils than T19+ cells in MLN of immune sheep and, compared to controls, a three-fold increase in T19+ cells and a five-fold increase in T19+/IL-5 mRNA+ double positive cells observed in immune sheep. In PP, there were very few eosinophils but substantial numbers of T19+ cells; however, no significant differences in numbers of eosinophils, T19+ or IL-5 mRNA+ cells were observed between control and immune sheep. These results indicate that in sheep, both eosinophils and gamma/delta T cells are capable of IL-5 expression and suggest that IL-5 is an important regulatory factor in autocrine and paracrine activation of effector cells involved in parasite immune expulsion.

In order to examine the role of CD8+ and WCI+ T cells and of IFN-gamma in the development of protective immunity against infection with the enteric nematode parasite Trichostrongylus colubriformis in sheep, mAb were administered during induction of the immune response to deplete or neutralize these components. Protection against the primary and challenge infections were assessed by faecal egg count and total worm count. Prolonged administration of mAb recognizing IFN-gamma and CD8+ resulted in significantly increased protection during the 6-week primary infection. CD8+ cells were depleted from blood but not intestinal mucosa. After injection of mAb (CC15) recognizing the surface antigen WCI, WCI+ and T cell receptor (Tcr) gamma delta+ cells were depleted from blood but not from enteric mucosa, and protection against challenge, although variable, was increased by up to 88%. It appears that CD8+ and WCI+/gamma delta+ cells and IFN-gamma all retard the potential development of naturally-acquired immunity against the parasite.

A total of 8 drugs were examined for their ability to suppress the rejection of Trichostrongylus colubriformis infective larvae (L3) from immune sheep. Specific antagonists of leukotrienes (plroxicam), prostaglandin (indomethacin) and the immunosuppressant cyclosporin A were given orally, while injectable preparations of dexamethasone, chlorpheniramine, BN 52051, WEB 2086 (anti-platelet-activating factor) and theophylline were administered as directed. The drugs were given for 5 days prior to and 4 days after challenge with 20,000 L3, when worm counts were done. Corticosteroids inhibited rejection by around 70% in two experiments, and none of the remaining compounds were effective. In the third study, six of the drugs were given to susceptible sheep and did not affect the establishment of a primary infection with T. colubriformis.

Sheep immunized by truncated larval infections or by the adoptive transfer of adult Trichostrongylus colubriformis were subsequently challenged with single infections of T. colubriformis, Nematodirus spathiger, Haemonchus contortus or Ostertagia circumcincta or combinations of the parasites. Sheep vaccinated with larval infections were >90% protected by 4 days after challenge (DAC) against T. colubriformis L 3 given in a single or combined infection, whereas no significant protection was exhibited against a single-species infection with the unrelated nematodes N. spathiger or O. circumcincta . Sheep challenged with T. colubriformis together with N. spathiger or O. circumcincta were equally protected against both intestinal nematodes, but O. circumcincta was not affected. Sheep immunized with adult T. colubriformis and challenged with a combined infection of T. colubriformis, N. spathiger and H. contortus expelled around 40 and 80% of the intestinal parasites by 4 and 11 DAC, respectively, but showed no protection against the abomasal parasite, H. contortus . The results confirm the previous findings on ‘selfcure’ and the non-specific rejection of unrelated parasites living in the same or downstream niches in the gut when the nematode used to induce immunity is included in the challenge infection. The results also indicate that even though L3 antigens effectively elicited the non-specific rejection, antigens produced by L 4 and later stages could also induce rejection of unrelated worms in the second week after infection.

Merino sheep immunized by the adoptive transfer of adult T. colubriformis for 8 weeks were significantly protected against a challenge infection of 20,000 larvae. Two additional groups of sheep received a primary infection of 9000 adult worms which were allowed to persist for 14 weeks before one group was drenched. When both groups were challenged 10 days later with 30,000 larvae, serial necropsies of these and naive sheep revealed that worm rejection did not occur until 7–10 days after challenge. By comparison with the rapid rejection of larval challenges from sheep immunized with normal primary infections, the results suggest that the antigens which elicited rejection in these experiments are stage-specific and were only present or synthesized in sufficient quantities when parasites had developed for 1 week.

The levels of inflammatory mediators in the intestinal contents of sheep immunized with Trichostrongylus colubriformis larvae increased in the first 6 days after challenge. These mediators were histamine, leukotriene C 4 , 6-keto-prostaglandin F 1α (from prostacyclin) and thromboxane B 2 . Leukotriene C 4 was released in the greatest quantities. Leukotriene B 4 was present but its concentration remained unchanged after challenge. The presence of these particular mediators in the intestinal contents after challenge is consistent with antigeninduced mediator release from the mucosal mast cells found in immune sheep undergoing challenge infection. This is the first sequential analysis of mediator release in sheep that also demonstrates the release of prostacyclin and thromboxane into the intestine during expulsion of a nematode infection.

Peripheral blood leucocytes (PBL) from sheep immunized with pilus protein purified from Bacteroides nodosus serogroup A were cultivated in vitro and cloned in the presence of the specific antigen and autologous antigen-presenting cells (APC). The efficiency of cloning was enhanced by high proliferative responses to pili during the initial week of cultivation, and the provision of recombinant human interleukin-2 (rec-IL-2). After three passages at weekly intervals, bulk cultures of PBL and cloned T-lymphocytes were >99% CD4 + , CD8 − , sIg − , i.e. the characteristic phenotype of helper T-lymphocytes. Cloned T-lymphocytes were devoid of allo-reactivity, and were restricted by class II antigens of the major histocompatibility complex (MHC). Both bulk PBL and cloned T-lymphocytes exhibited similar patterns of reactivity against pili from different serogroups of B. nodosus and the T-lymphocytes reacted to three of six peptides synthesized from the amino-acid sequence of pilus from serogroup A. Although clones of T-lymphocytes could retain antigen specificity for up to 2 months of cultivation, several attempts to recover clones with specific reactivity after storage in liquid nitrogen were unsuccessful.

In order to identify those bacterial antigens which might be involved in immunity against ovine footrot, antisera were raised in sheep to 6 proteins in the outer membrane complex (OMC) of one strain of Bacteroides nodosus. Examination of the specificity of these antisera by Western blotting, crossed immunoelectrophoresis (XIEP) and IEP, revealed that they recognized the homologous OMC protein, but did not precipitate either undenatured pili or OMC, nor could they agglutinate the homologous bacteria. In contrast, anti-OMC and anti-pili sera could precipitate OMC or pili respectively, and agglutinate whole bacteria. Subsequent analysis of these sera against 5 strains of B. nodosus from different serogroups revealed that Proteins 1, 3 and 4 had a similar antigenic structure in all strains examined. The reactivity of anti-pili sera was restricted to homologous bacteria whereas anti-pilin sera (raised against denatured pili) also reacted with pilin from 2 of 3 heterologous strains. However, none of the patterns of staining or absorption of any of these sera matched the spectrum of cross-protection afforded by vaccination of sheep with B. nodosus strain 198 cells. The results question the role of individual OMC proteins in cross-protective immunity and may imply that interactions between several bacterial components are involved in the phenomenon.

SUMMARY The relationship between the cultural and biochemical characteristics of 22 strains of Bacteroides nodosus and their virulence for sheep was examined. Virulent, intermediate and benign strains were recognised. Although there was some relationship between virulence and colony morphology on hoof medium with 4% agar, colonies of one virulent and 4 intermediate strains resembled those of benign strains. However, on hoof medium with 2% agar and on blood Euonagar, colonies of this virulent and one intermediate strain differed from each other and the other 3 intermediate strains, which in turn differed from the benign. The degree of piliation, as assessed by electron microscopy, was not a reliable indicator of virulence in strains not possessing a beaded colony type. Together, the results of colony morphology and proteolytic tests such as zymogram, degrading proteinase and elastin-agar tests allowed better discrimination of virulent and benign strains. Intermediate strains generally possessed virulent protease activity. In strains with benign zymogram patterns, activity bands 2 and 3 were more labile than in strains with virulent patterns. The addition of CaCl2 to the culture medium resulted in greater stability of proteolytic activity, particularly with benign strains, and prevented the disappearance of protease activity in the band 5 position in virulent, intermediate and benign strains during prolonged incubation. There were slight differences in the sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) patterns of outer membrane proteins of some benign strains but those of intermediate category resembled virulent strains. There was some relationship between the apparent Mr, of the pilin monomer on SDS-PAGE gels and serogroup specificity.

Phagocytosis of Bacteroides nodosus by ovine peripheral blood leucocytes (PBL) was examined after organisms had been opsonized in sera from normal sheep, or from animals immune to, or infected with ovine footrot . Ingestion of bacteria, as assessed microscopically or by counting isotopically-labelled organisms spectrometrically was effected in suspensions by polymorphonuclear leucocytes (PMN). Opsonization of bacteria in immune serum, particularly its IgG2 isotype, enhanced the rate of phagocytosis by PMN compared with that promoted by normal serum or medium alone. Whereas IgG2 from immune serum also increased the rate of ingestion of B. nodosus by adherent PMN, IgM and IgG1 from immune serum also initiated phagocytosis of bacteria by adherent ovine monocytes. Leucocytes from normal, immune or infected sheep of different breeds ingested B. nodosus with equal facility.

The gene for pilin, the monomeric protein subunit from which the pilus of Bacteroides nodosus is constructed, has been isolated. Isolation was achieved by cloning the fragmented genome of B. nodosus in Escherichia coli RR1 using the plasmid vector pBR322. Pilin-producing colonies were identified by screening with a colony immunoassay using antiserum from a sheep immunized against purified pili from B. nodosus strain 198, and were further characterized by immunoblot analysis. Final confirmation of the presence of the pilin gene was by nucleotide sequence data which translated to the known pilin amino acid sequence.

Examination by SDS-PAGE of lithium acetate extracts of several strains of depiliated Bacteroides nodosus revealed 6 major outer membrane proteins (including pilin). The 5 membrane proteins exhibited approximate molecular weights of 75000, 50000, 38000, 34500 and 26500 whereas pilin had a MW of 17500 for the majority of strains. All proteins were accessible to lactoperoxidase-catalysed iodination and proteins 1, 2 and 5 were shown to be glycoproteins. Several attempts to isolate individual OMC proteins in pure form by selective solubilization and gel filtration were unsuccessful, but electroelution of individual outer membrane complex proteins resolved by SDS-PAGE provided sufficient quantities of antigen for immunization of sheep and for immunochemical analysis.

SUMMARY The effects of vaccination of Merino sheep with the purified pili or the whole cells of Bacteroides nodosus strain 198, either in oil or alum-oil adjuvant, on the severity of footrot induced with the homologous strain (198) and a heterologous strain (217) were determined in a field experiment, on flood irrigated pasture. The efficacy of the whole call vaccines was comparable to that of purified pili vaccines, against homologous challenge, when both had a similar content of pilus antigen although the purified pili vaccines induced significantly greater homologous pilus agglutinating antibody titres than the whole cell vaccines. However, against heterologous challenge, the whole cell vaccines in oil (CO) or alum-oil (CAO) provided significantly greater protection than a purified pili-in-oil (PPO) vaccine, the number of severely affected feet in sheep vaccinated with PPO being similar to that of the unvaccinated group. The group vaccinated with purified pili In alum-oil (PPAO) was intermediate between these two extremes. The superior performance of the PPAO in comparison to the PPO vaccine, against heterologous challenge, was associated with significantly higher mean ELISA titres to the outer membrane complex. Western blot analyses implicated a role in cross-protection for outer membrane proteins, in particular a protein M, 78,000. The PPO vaccine produced fewer, smaller and less persistent vaccination reactions at the inoculation sites than did the other vaccines. Bodyweight gains in the period prior to challenge were much lower for the groups vaccinated with CO and CAO than for the controls and those vaccinated with purified pili, due presumably to the larger vaccination reactions. During the foot-rot transmission period bodyweight gains were greater for all vaccinated groups than for the controls for those sheep challenged with the homologous strain. However, no such differences were observed between the groups challenged with the heterologous strain.

A soluble exotoxin (a leucocidin) which was lethal to peripheral blood leucocytes from cattle, sheep, rabbits and man (in order of decreasing sensitivity) was elaborated by a variety of isolates of Fusobacterium necrophorum when the majority of organisms were present as filaments in liquid culture. Maximum production of the leucocidin was achieved by concentrations of bacteria equivalent to between 4 X 10(7) and 4 X 10(8) short cells per ml of culture above which no further increase in titre was observed. The ability of different batches of medium to support production of leucocidin was reflected in their capacity to enable F. necrophorum to grow to this range of concentration. Prolonged culture of the organism, resulting in a decline to below 6 in the pH of the medium was associated with a depression in the titre of leucocidin, presumably due to its inactivation under these conditions. The leucocidin was stable at 4 degrees C for at least 30 days, to extremes of pH (4 to 9) for 1 h at room temperature, and showed maximum activity in assays conducted at 37 degrees C at pH 7 to 8. The exotoxin was inactivated by heating at 56 degrees C for 30 min and possessed a molecular weight around 250,000 to 300,000 as determined by gel filtration and membrane partition chromatography.

Five breeds of sheep, Romney Marsh, Dorset Horn, Border Leicester, Peppin Merinos and Saxon Merinos were examined for their susceptibility to foot-rot by exposure to natural transmission of infection on irrigated pasture or by the application of pure cultures of Bacteroides nodosus to each foot in a pen experiment. On pasture, the sheep encountered a moderate challenge and the British breeds were more resistant than Merinos to the development of severe foot-rot. Resistance was manifested by a rapid resolution of relatively benign lesions in the interdigital skin, rather than a reduction in the number of feet affected. However, under more severe challenges with foot-rot in pens, all breeds were equally susceptible. There was little difference between resistant and susceptible sheep in the kinetics and magnitude of their antibacterial immune responses indicating that resistance did not depend on pre-existing antibody or a more rapid induction of antibody production. In each experiment, humoral immune responses against B. nodosus were not greatly elevated until under-running lesions of the hoof developed.

Bacterial plasmids that direct the expression in Escherichia coli of the pilin of Bacteroides nodosus were constructed. The quantity of pilin produced was greater than that of the pilin synthesized by B. nodosus, but no surface structural pili were present; pilin was found associated with the inner membrane of E. coli. Vaccination of sheep with E. coli containing pilin elicited increases in agglutinating and enzyme-linked immunosorbent assay antibody titers, which in turn were lower than the titers in sheep immunized with pilin from B. nodosus. The E. coli-produced pilin vaccine initially appeared to delay the progression of infection in immunized sheep after a challenge with virulent homologous B. nodosus, but at a later time the severity of foot rot was similar to that in sheep vaccinated with a placebo.

Summary In a field experiment on irrigated pasture, sheep of several breeds were vaccinated twice, subcutaneously, in the upper neck, with Bacteroides nodosus vaccine containing either depiliated cells (DC vaccine), or whole, piliated cells (WC vaccine) and the responses were measured over the following 14 weeks. DC vaccine was as effective as WC vaccine in protecting against the development of foot-rot under conditions of moderate challenge, although the WC vaccine induced significantly higher pilus agglutinating antibody titres. Foot-rot developed in significantly more vaccinated Merinos (Peppin and Saxon strains) than in Romney Marsh, Dorset Horn or Border Leicester breeds. Agglutinating antibody titres after WC vaccination were significantly lower in the Peppin Merino than in the other sheep for the first 6 weeks, while after DC vaccination the titres remained elevated longer in the Border Leicester and Saxon Merino and were significantly higher from 6 weeks onwards. Reactions at the inoculation sites were generally larger in the British breeds than in the Merinos and among the former the reactions were largest, most numerous and most frequently discharged their contents in the Dorset Horn. Bodyweight gains in all vaccinated sheep were initially reduced, compared with controls, but the differences were no longer significant after the eighth week.

SUMMARY A highly purified pilus vaccine prepared from cells of Bacteroides nodosus strain 198 provided a high level of protection against homologous challenge and small, not statistically significant, levels of protection against challenge with 4 other strains each from different serogroups. In a second experiment, a partially purified pilus vaccine from strain 198 induced significant immunity to 1 of 4 heterologous strains which were different from those used in the first experiment. In a third experiment a strain 198 whole cell vaccine produced significant immunity against 3 of 6 heterologous strains used in the first 2 experiments. There was no obvious relationship between the colony type, degree of piliation and level of cross-protection obtained against a particular strain. The results provide further evidence that immunogens associated with, but distinct from, the pilus are involved in cross-protection and that cross-protective antigens are common to some, but not all, strains.

Sixty-one isolates of Fusobacterium necrophorum were recovered for study. Thirty-one were obtained from lesions of foot abscess in cattle (25) and sheep (6), 28 were from interdigital lesions in cattle and 2 were from the normal interdigital skin of cattle. The majority of isolates from lesions of foot abscess were virulent, belonged to biotype AB (Fievez 1963), produced flat, irregular shaped, greyish colonies and haemolysis on blood agar, and grew as turbid filamentous suspensions in liquid media. They produced a soluble exotoxin, a leucocidin, and were pathogenic for cattle and mice. Virulent isolates also produced a haemolysin which most readily lysed bovine, equine and chicken erythrocytes; those from sheep were less susceptible while those of rabbit and pig were the most resistant. Isolates recovered from lesions of the feet not classified as foot abscess and from clinically normal feet were predominantly of the B biotype and caused few experimental lesions, produced convex, round, yellow colonies, flocculated and sedimented while growing in liquid medium and produced little or no haemolysin or leucocidin. Routine differentiation between virulent and non-virulent bovine isolates of F. necrophorum could be achieved by assessing the colour, morphology, and degree of haemolytic activity of colonies grown on blood agar.

Monoclonal antibodies (MAbs) reacting with bovine (2) ovine (3), murine (1) or human (1) Class II MHC antigens were examined for reactivity with bovine peripheral blood leucocytes (PBL) and lymph node cells (LNC) by immunoflourescence, immunoprecipitation and the capacity to inhibit mixed lymphocyte responses (MLR), lectin- and antigen-induced blastogenesis. The 6 MAbs identified comparable percentages of Class II positive lymphocytes in PBL (40.8 to 54.2%) and LNC (6 to 11.5%) regardless of BoLA-A phenotype. Immunohistological staining of Class II MAb was localized principally to the lymphoid follicles in lymph nodes and to isolated epithelial reticular cells in the thymus. The anti-Class II MAb immunoprecipitated α- and β- chains of 26–29K and 32–34K, respectively. These MAb inhibited proliferative responses in the MLR by between 25 and 74%, and diminished blastogenesis induced by specific antigens (purified protein derivative + PPD and ovalbumin) and B-lymphocyte mitogens (PPD, lipopoly-saccharide and dextran sulphate) by between 45 and 75%, regardless of BoLA-A phenotype. In contrast, proliferation in response to concavavalin A and phytohaemagglutinin were unaffected by the anti- Class II MAb. Similarly these MAb did not affect lysis by cytotoxic T-lymphocytes, the activity of which was depressed by anti-Class I MAbs and monospecific alloantisera.