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Scouring (diarrhoea) is a major concern for sheep producers as the accumulation of faecal material (dags) around the breech pre-disposes sheep to flystrike. Scouring occurs when the consistency of faeces is fluid with a low percentage of dry matter. In temperate areas such as the southern half of Australia, New Zealand and the United Kingdom, scouring is associated with ingestion of parasitic nematode larvae, mainly Teladorsagia circumcincta and Trichostrongylus colubriformis (Larsen et al., 1994). Breeding sheep to be resistant to these nematodes is a sustainable parasite-control strategy due to reduced reliance on chemical treatment. However, in adult sheep, scouring appears equally prevalent in resistant animals and, in some environments, is even more severe than in susceptible sheep (Karlsson et al., 2004). In this experiment, we investigated how faecal dry matter (FDM) in sheep from a flock bred for resistance to parasitic nematodes changed when challenged with infective larvae. We expected that FDM would be lower in challenged sheep compared to unchallenged controls, and FDM would also be lower in sheep with high dag scores compared to sheep with low dag scores.

The cultivation of bone marrow was used to quantitate the levels of eosinophil differentiation factors (EDF) produced in conditioned medium (CM) by incubation of mesenteric lymph node cells (MLNC) with mitogens or specific antigens from the intestinal nematode parasite, Trichostrongylus colubriformis. In liquid cultures with 20 units ml-1 recombinant murine interleukin-5 (IL-5), bone marrow cells (BMC) from either normal or infected donors contained less than 5% eosinophils and differentiated to greater than 50% eosinophils over 2-3 weeks. Conditioned medium from 3-4 week infected donors produced between 20 and 50% eosinophils when donor MLNC were stimulated with the specific antigen preparation SP3, but macrophages predominated when using CM from MLNC incubated with Concanavalin A (ConA). CM from MLNC of challenged donors incubated with SP3 produced 30-70% eosinophils in BMC assays, with highest levels induced by CM from high responder (HR) donors. Marrow from parasitized or normal donors gave rise to comparable proportions of eosinophils. CM was also produced from LNC of donors given protein or parasite antigens in adjuvant where between 28 and 35% eosinophils were produced in culture. There were no differences between activities attributable to the antigen, but Freund's complete adjuvant induced earlier differentiation of BMC than alum-induced CM. The results confirm that high levels of EDF activity are specifically induced by parasitic infection, and can also be produced by intraperitoneal and subcutaneous inoculation of adjuvanted antigens. Consistent with the greater eosinophilia exhibited by HR guinea pigs to infection with T.colubriformis L3, their MLNC also produced the highest levels of EDF activity.

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.

Immune-mediated scouring in sheep is a mucosal hypersensitivity response incited by infective larvae of gastrointestinal nematodes in the southern half of Australia and in New Zealand, regions characterised by a Mediterranean, winter-rainfall climate. To gain insights into the underlying mechanism(s), this study investigated the relationship between nematode infection of sheep in pens, faecal dry matter and release of inflammatory mediators in the intestinal tract. Six worm-free, immune rams received a dose of 3000 Trichostrongylus third stage larvae (L3) and 3000 Ostertagia L3 per week for 6 weeks, after which they were euthanased. Three rams acted as uninfected controls. Faecal dry matter was significantly lower in the infected group from the first week of infection until the end of the trial. Concentrations of cysteinyl leukotrienes and prostaglandin E2 were higher in mucus from both the abomasum and the small intestine of infected rams compared with controls. Within the infected group, concentrations of both mediators were higher in mucus from the small intestine than from the abomasum. There were greater numbers of eosinophilic granulocytes and mucosal mast cells in abomasal and small-intestinal tissue sections from infected rams compared with the controls. The results are consistent with the hypothesis that nematode infection causes an inflammatory response in the gastrointestinal mucosa, characterised by increased fluid secretion.

The IEEE P1003.5 Working Group is developing a standard Ada Binding to POSIX Operating System services. This paper describes the basic approach and some of the techniques used in developing a prototype implementation of this POSIX Ada Binding. The primary goal of the prototype is to provide a “proof of concept” implementation of the Ada binding on top of an existing C binding to POSIX, and to provide a basis for trial use of the Ada binding. In particular, the paper demonstrates a set of techniques for interfacing to existing C software, and discusses issues in building an Ada binding on top of an existing C binding.

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.

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.

SUMMARY Calves were immunised with fractions of Fusobacterium necrophorum incorporated in mineral oil adjuvant and then each foot was experimentally exposed to interdigital necrobacillosis (foot abscess) by subcutaneous injection of homologous organisms through the interdigital skin. The number of cells from an 18 h liquid culture that might be expected to cause 50% of the feet of control calves to develop marked swellings following subcutaneous injection was shown to be approximately 2.2×103 cells. Immunity was shown to be associated with antigens that were located in the supernatant of the culture, and which may be identical with or closely associated with the exotoxins.

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.

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.

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In this article Emery and Enger not only discuss the results of an experimental use of a computer game, Fiscal Policy, but explain the use of the gap-closing score model in evaluating student gains. Readers are urged to pay close attention to the gap-closing score technique as an analytical tool and might consider using it in their own research and evaluation efforts. The authors' use of the TUCE in this study, and their breakdown of the test into simple application, complex application and recognition-understanding components for purposes of this analysis, will also be of interest.

The paper demonstrates that only with some form of reproduction cost valuation for rate base capital is it possible that the regulatory process might encourage electrical utilities to cooperate or acquiesce with a manufacturers' conspiracy to increase equipment prices. Based on the process function for steam-electric generation developed in the paper, a monopolistic utility subject to rate-of-return regulation using original cost valuation will always have a more profitable alternative than paying conspiratorially inflated equipment prices. Empirically, the acquisition prices for steam-electric generating units purchased by utilities were higher during a "conspiracy" period, 1956-1959 than during the adjacent years, 1954-1955, 1960-1961. Although the statistical results were somewhat ambiguous, it could not be concluded that the type of rate-base valuation scheme applicable to each utility made a significant difference either in the level of prices or in the increase in prices for similar generating units.

When cultures of known pathogenic strains of Fusobacterium necrophorum, isolated either from cattle or sheep were injected through the interdigital skin of cattle typical lesions of interdigital necrobacillosis were produced. The inclusion of Bacteroides melaninogenicus in the inoculum did not appear to contribute to the development of lesions.

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.

The original aim of Industrial Therapy Units, which exist in most psychiatric hospitals, was to preserve or re-establish outside interests and standards by providing work of an industrial nature (Wadsworth, 1962), but often they merely offer the patient activity and occupation. The staff of the Industrial Therapy Unit at Clifton Hospital were concerned about some patients who benefited very little from attendance, and they devised a project to improve this. Qualitatively the patients appeared to benefit but the staff were unable to measure this. They therefore approached the Psychology Department, and a structured programme was designed with emphasis on measurement and control of variables. There was very little to be found in the literature on experiments in an Industrial Therapy setting (see references).

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.

© 2015 Hammer et al.; licensee BioMed Central. Background: Theileria are blood-borne intracellular protozoal parasites belonging to the phylum Apicomplexa. Previously considered a benign parasite in Australia, outbreaks of clinical disease resulting from Theileria orientalis genotypes have been reported in Australia since 2006. Since this time, outbreaks have become widespread in south-eastern Australia, resulting in significant adverse impacts on local dairy and beef industries. This paper provides the first investigation into the possible biological and mechanical vectors involved in the rapid spread of the parasite. Methods: To identify possible vectors for disease, ticks, biting flies and mosquitoes were collected within active outbreak regions of Gippsland, Victoria. Ticks were collected from cattle and wildlife, and mosquitoes and biting flies were collected in traps in close proximity to outbreak herds. Ticks were identified via DNA barcoding of the mitochondrial cytochrome oxidase I gene. Barcoded ticks were pooled according to species or phylogenetic group and tested for the presence of T. orientalis and the genotypes Ikeda, Chitose and Buffeli using real-time PCR. Results: DNA barcoding and phylogenetic analysis identified ticks from the following species: Haemaphysalis longicornis, Ixodes holocyclus, Ixodes cornuatus, Ixodes hirsti, and Bothriocroton concolor. Additional Haemaphysalis, Ixodes and Bothriocroton spp. were also identified. Of the ticks investigated, only H. longicornis ticks from cattle carried theilerial DNA, with the genotypes Ikeda, Chitose and Buffeli represented. Mosquitoes collected in close proximity to outbreak herds included; Aedes camptorhynchus, Aedes notoscriptus, Coquillettidia linealis, Culex australicus, and Culex molestus. Low levels of T. orientalis Buffeli genotype were detected in some mosquitoes. The haematophagous flies tested negative. Conclusions: This is the first demonstration of a potential vector for T. orientalis in the current Australasian disease outbreak.