A combined Clostridium perfringens/Trueperella pyogenes inactivated vaccine induces complete immunoprotection in a mouse model.

Clostridium perfringens (C. perfringens) and Trueperella pyogenes (T. pyogenes) are two bacterial pathogens frequently associated with wound infections and following lethal complications in livestock. However, prudent use of antimicrobial agents is highly required given the emergence of multidrug-resistant strains of both bacteria and need for food safety. In the current study, a combined vaccine, composed of inactivated C. perfringens and T. pyogenes, was prepared. The amount of formaldehyde being used to inactivate two bacteria was optimized to retain the immunogenicity of antigens. Three adjuvants were tested for their potency in improving specific immune responses against the candidate antigens. Then inactivated combined C. perfringens/T. pyogenes vaccine was prepared using inactive cultures of two organisms. The ratio of inactive cultures of two organisms for preparation of combined vaccine was optimized to gain effective protective immunity against the two pathogens. Results revealed that combined C. perfringens/T. pyogenes inactive vaccine can elicit high level of exotoxins and cell-associated antigen-specific antibodies and induce complete protection against C. perfringens and T. pyogenes infections in mice. The combined vaccine could be used as an alternative of antibiotics for prevention of C. perfringens and T. pyogenes infections in animals.

Fleece rot and dermatophilosis in sheep.

Fleece rot and dermatophilosis reduce health and production of sheep and predispose them to blow fly strike. This paper reviews aetiology, prevalence, pathogenesis, resistance, attempts to develop vaccines and prospects for new control strategies to these important skin diseases. Although the severity of fleece rot is associated with the abundance of Pseudomonas aeruginosa on skin, microbial ecology studies are providing new insights into the contribution of other bacteria to the disease. Wool traits and body conformation traits that predispose sheep to fleece rot and dermatophilosis are heritable and have been used as indirect selection criteria for resistance for many years. Selection against BoLA-DRB3-DQB class II haplotype in cattle can substantially reduce the prevalence of dermatophilosis and holds promise for identification of gene markers for resistance to these bacterial diseases in sheep. Immune responses in skin and systemic antibody responses to bacterial antigens are acquired through natural infection and contribute to resistance; however, prototype antibacterial vaccines have to date failed to provide protection against the diversity of isolates of Dermatophilus congolensis and Pseudomonas species present in the field. Opportunities for future control through breeding for resistance, vaccines and non-vaccine strategies for controlling the microbial ecology of fleece are discussed. In combination, control strategies need to reduce the risk of transmission, minimise exposure of animals to stressors that enhance the risk of infection, and enhance resistance though genetics or vaccines.

The ERK MAP kinase cascade mediates tail swelling and a protective response to rectal infection in C. elegans.

The nematode Caenorhabditis elegans is proving to be an attractive model organism for investigating innate immune responses to infection. Among the known pathogens of C. elegans is the bacterium Microbacterium nematophilum, which adheres to the nematode rectum and postanal cuticle, inducing swelling of the underlying hypodermal tissue and causing mild constipation. We find that on infection by M. nematophilum, an extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase cascade mediates tail swelling and protects C. elegans from severe constipation, which would otherwise arrest development and cause sterility. Involvement in pathogen defense represents a new role for ERK MAP kinase signaling in this organism.

Caenorhabditis elegans as a host for the study of host-pathogen interactions.

Recently, pathogenicity models that involve the killing of the genetically tractable nematode Caenorhabditis elegans by human pathogens have been developed. From the perspective of the pathogen, the advantage of these models is that thousands of mutagenized bacterial clones can be individually screened for avirulent mutants on separate petri plates seeded with C. elegans. The advantages of using C. elegans to study host responses to pathogen attack are the extensive genetic and genomic resources available and the relative ease of identifying C. elegans mutants that exhibit altered susceptibility to pathogen attack. The use of Caenorhabditis elegans as the host for a variety of human pathogens is discussed.

Reticulostimulating properties of killed vaccines of anaerobic coryneforms and other organisms.

Vaccines prepared from 115 strains of anaerobic coryneforms and other organisms were tested in mice for their reticulostimulating ability as judged by the degree of spleen hypertrophy produced after ip injection. Almost all vaccines caused a statistically significant increase in spleen weight, but the ability to produce spleen ratios (test mean wt:control mean wt) of 4 or more was confined to Propionibacterium acnes and P. avidum strains. P. acnes, type II, gave high spleen ratios more frequently than strains of any other type.

Effects of Corynebacterium granulosum on weight and histology of lymphoid organs, response to mitogens, skin allografts, and a syngeneic fibrosarcoma in mice.

We studied the effect of single and multiple injections of Corynebacterium granulosum on weight and histology of lymph nodes and spleen, on peripheral white blood cell count, response of peripheral blood lymphocytes, lymph node, and spleen cells to phytohemagglutinin and pokeweed mitogen, survival of skin allografts, and lung metastases of a syngeneic fibrosarcoma in C3Hf/Bu mice. Corynebacterium parvum was used in some studies on antitumor activity. The weight of lymph nodes and spleen was markedly increased by single and multiple i.p. injections of C. granulosum, the peak enlargement occurring at Day 7 in lymph nodes and at Day 16 in spleen. Histologically, there was an extensive proliferation of nucleated cells in the enlarged organs. C. granulosum did not change the total white blood cell count but caused a temporary lymphopenia. In general, in vitro response to phytohemagglutinin and pokeweed mitogen of blood lymphocytes and spleen cells was decreased. Lymph node cell response to phytohemagglutinin was increased by small doses (0.025 mg) of C. granulosum, was not altered by a single large dose (0.5 mg), and was decreased by multiple doses. The response of lymph node cells to pokeweed mitogen was increased by all treatments. These changes in response to mitogens were demonstrable for about 2 months after treatment. Treatment i.v. with 0.1 or 0.25 mg of C. granulosum given before but not after grafting significantly prolonged the survival of grafted BALB/c skin. Smaller doses of this bacterium were not effective. Splenectomy of skin graft recipients did not prevent the effect of C. granulosum. Treatment i.p. or i.v. with this bacterium significantly decreased the number of lung metastases from i.v.-injected fibrosarcoma cells, even if the cells were injected 3 to 4 months later. The magnitude of this effect varied with the dose and frequency of injection of C. granulosum and C. parvum.

Serum KL-6 concentrations in dairy farmers.

STUDY OBJECTIVES: Serum KL-6 (Krebs von den Lungen-6) has been recognized to be a marker for the activity of diffuse interstitial lung diseases. The purpose of the study is to evaluate serum KL-6 measurement as a marker for farmer's lung disease (FLD). DESIGN: A cross-sectional survey of a cohort of dairy farmers. Retrospective measurement of KL-6 stored serum samples from those dairy farmers previously screened for FLD. SETTING: University hospital screening project for FLD within a dairy-farming community in Japan. PARTICIPANTS: Four hundred seventy-two dairy farmers were invited to attend a local clinic. MEASUREMENTS AND RESULTS: We examined serum KL-6 concentrations in 272 farmers. Subjects were classified into three groups: (1) 5 farmers with FLD, (2) 30 farmers with positive serum precipitating antibodies to Saccharopolyspora rectivirgula and/or Thermoactinomyces vulgaris without FLD (Ab(+)), and (3) 237 farmers without these antibodies (Ab(-)). Serum KL-6 concentrations in the FLD group were significantly higher than those in the Ab(+) and the Ab(-) groups (1,263 +/- 288 [SEM], 328 +/- 57, and 207 +/- 6 U/mL, respectively, p < 0.001). Serum KL-6 concentrations in those with FLD were significantly higher than KL-6 concentrations from stored screening samples from the same individual when FLD was not diagnosed (1,263 +/- 288 and 419 +/- 209 U/mL, respectively, p < 0.05). Serum KL-6 concentrations of the Ab(+) group were significantly higher than those of the Ab(-) group (p < 0.001). In the Ab(+) group, farmers with high serum KL-6 concentrations had lower permeability coefficients than farmers with normal serum KL-6 concentrations (p < 0.05). These results may suggest that subclinical FLD can be detected in farmers with high KL-6 concentrations and precipitating antibodies. CONCLUSION: Serum KL-6 concentration can be a useful marker for assessing the activity of FLD and may be able to be used to detect subclinical disease.

Development of a serologic panel for the recognition of nocardial infections in a murine model.

A murine model was used to develop a sensitive and specific serologic test for clinical and subclinical infections caused by Nocardia. The following tests were used: (a) enzyme-linked immunosorbent assays (ELISAs) with culture filtrate and cytoplasmic extract antigens from Nocardia asteroides; (b) ELISA with N. asteroides trehalose dimycolate (cord factor); (c) indirect immunofluorescent antibody assay with whole cells of N. asteroides; and (d) Western-blot analysis for the 54 to 55-kD, 36-kD, and 62-kD proteins of N. asteroides. The sera from BALB/c mice, experimentally infected with nonlethal doses of three species of Nocardia, nine species of Mycobacterium, Rhodococcus equi, two species of Actinomadura, and two species of Streptomyces were tested using this panel. The serologic tests did, indeed, identify mice infected with nocardiae and could differentiate them from mice infected with the other actinomycetes, including mycobacteria.

Immunogenic properties of glycolipids of Nocardiopsis dassonvillei.

Two specific glycolipids identified as monomannosyl diglyceride (G1) and monoacylated glucose (G2), isolated from Nocardiopsis dassonvillei strains, were found to be biologically active. They elicited antibody response in rabbits when administered with some carrier components: lecithine, methylated bovine serum albumin (MBSA) and Freund incomplete adjuvant (FIA). Antibodies to the glycolipids were detected in antisera to G1 and in the sera against crude cell antigen of N. dassonvillei (N). The ability of the lipids to induceskin delayed hypersensitivity (SDH) as well as adjuvant and mitogenic effect were also studied. Generally glycolipid G1 seemed to be more active in antibody response whereas glycolipid G2 was a better stimulator of cellular response. Glycolipid G2 appeared to be distinct adjuvant and mitogenic factor.

Isolation and identification of cheese-smear bacteria inhibitory to Listeria spp.

A newly developed hydrophobic grid membrane method was used to rapidly screen 105 traditional French cheeses for surface smear microorganisms inhibitory to Listeria monocytogenes strain V7. Approximately 125,000 colonies comprising a wide variety of bacteria were examined of which less than 0.1% produced visible zones of inhibition. Isolates possessing antilisterial activity consisted of various strains of Enterococcus faecalis, Staphylococcus xylosus, Staphylococcus warneri and coryneform bacteria, including one orange coryneform resembling Brevibacterium linens. All strains of E. faecalis and the orange coryneform that inhibited L. monocytogenes V7 exhibited strong inhibition against a panel of 21 Listeria strains comprised of L. monocytogenes (14 strains), L. innocua (two strains), L. ivanovii (two strains), L. seeligeri (two strains) and L. welshimeri (one strain). The remaining cheese isolates showed moderate to weak inhibition towards the same set of Listeria strains. Inhibitory substances produced by all strains except the orange coryneform were sensitive to one or more of five proteolytic enzymes tested and were therefore classified as bacteriocin-like inhibitory agents.

Inhibition of Dermatophilus congolensis by substances produced by bacteria found on the skin.

Bacteria, isolated from the skins of clinically normal sheep, were tested for inhibitory activity against Dermatophilus congolensis grown in vitro. Out of 85 bacterial isolates, 19, mainly Bacillus spp., showed zones of inhibition when grown together with D. congolensis. The inhibitory activity was shown to be due to the metabolites released by the bacteria.

Serum and skin surface antibody responses in merino sheep given three successive inoculations with Dermatophilus congolensis.

Three antigens prepared from different phases of the life cycle of Dermatophilus congolensis were used in an enzyme-linked immunosorbent assay to measure serum and skin surface antibody responses in sheep after a first, second and third inoculation with D. congolensis. After the first inoculation, a strong antibody response to the flagella, filament and soluble antigens was detected after 7-21 days in the sera from sheep that were regularly biopsied; the antibody response at the skin surface was detected 28-42 days after inoculation, when the lesions were resolving. Strong anamnestic responses were detected in the serum of sheep that were biopsied and some of the nonbiopsied sheep after the second and third inoculations, but the skin surface antibody response at these times was variable.

Vaccination against ovine dermatophilosis.

Zoospore, filamentous and soluble antigens were prepared from Dermatophilus congolensis and examined for their ability to protect sheep from challenge with D. congolensis zoospores. In 1 experiment, sheep were vaccinated with Antigens A, B and C. The number of sheep protected in the group vaccinated with Antigen B was greater (P less than 0.05) than that in the unvaccinated group after challenge. The group vaccinated with Antigen B had a higher antibody response (P less than 0.05) to Antigen B than to Antigen A or C. In a second experiment, 2 groups of sheep were vaccinated with Antigen B. All sheep in this study developed lesions after challenge, but those on the vaccinated sheep were less severe (P less than 0.05) than those on the unvaccinated sheep. The antibody response to Antigen A, 28 days after vaccination, was higher (P less than 0.05) than the response to Antigen B.

Ecology of Rhodococcus equi.

A selective broth enrichment technique was used to study the distribution of Rhodococcus equi in soil and grazing animals. Rhodococcus equi was isolated from 54% of soils examined and from the gut contents, rectal faeces and dung of all grazing herbivorous species examined. Rhodococcus equi was not isolated from the faeces or dung of penned animals which did not have access to grazing. The isolation rate from dung was much higher than from other samples and this was found to be due to the ability of R. equi to multiply more readily in dung. Delayed hypersensitivity tests were carried out on horses, sheep and cattle, but only horses reacted significantly. The physiological characteristics of R. equi and the nature of its distribution in the environment suggested that R. equi is a soil organism.

Relationship between chronic ovine dermatophilosis and levels of T6 lymphocyte antigen staining in peripheral blood mononuclear cells.

Quantification of a T6-lymphocyte antigen in peripheral blood mononuclear cells of sheep was used to select 15 from 48 one year old Merino ewes not previously exposed to Dermatophilus congolensis infection. These sheep were compared in response to challenge with D. congolensis zoospores and levels of T-6 lymphocyte antigen in peripheral blood mononuclear cells with 15 Merino ewes of similar age and strain from a different site that had been treated and recovered from chronic dermatophilosis. The T-6 lymphocyte antigen levels were significantly lower in the chronic dermatophilosis sheep and they developed significantly more severe lesions than the selected, previously unexposed sheep despite the former sheep having high serum antibody levels to D. congolensis. Measurement of the fleece characteristics, wax and suint concentration showed no differences between the groups that might have explained the considerable differences in their susceptibility to dermatophilosis.

Cellular immune responses of the rat to experimental infection with Dermatophilus congolensis.

The host cell-mediated immune response was examined following experimentally-induced infection of rats with Dermatophilus congolensis, the causal agent of the skin disease dermatophilosis. Mononuclear cells (MC) isolated from Wistar rats 10 days following the induction of a third infection underwent a strong and specific proliferative response, as assessed by a [3H]thymidine incorporation assay, when cultured with various concentrations of inactivated D. congolensis cocci. Using specific monoclonal antibodies in an indirect fluorescent antibody test, this in vitro response was found to be characterised by a large expansion of the W3/25 (T-helper phenotype) population to form 56% of the total. Finally, the primed and stimulated MC were assessed for their ability to produce factors capable of inhibiting macrophage migration. The culture supernatants of D. congolensis-stimulated MC from infected rats caused significant migration inhibition of normal rat peritoneal exudate cells, whilst the supernatants of similarly-stimulated MC from naive rats failed to cause significant inhibition. The results show that a MC subpopulation becomes primed following experimentally-induced infection with D. congolensis and becomes activated after subsequent, in vitro, exposure.

Evaluation of vaccines against ovine dermatophilosis.

Intradermal vaccination of live crude filaments (vaccine A) was compared with a vaccine (vaccine B) consisting of a 45 kD zoospore protein and mucoid material coating filaments in its ability to protect sheep from experimental Dermatophilus congolensis infection. Fourteen and 21 days after challenge, vaccine A sheep had fewer lesions (P less than 0.001) than the vaccine B sheep. The lesions on the vaccine A sheep were also less severe 14 and 21 days after challenge (P less than 0.05, P less than 0.01 respectively). In a second study, vaccine A was assessed for its ability to protect against natural challenge. Ten weeks after contact with sheep with active and generalised dermatophilosis no difference was found between the number of lesions present on the vaccine A and unvaccinated sheep and no differences were found in the number of sheep in each group with active lesions.

Strain variation in Dermatophilus congolensis demonstrated by cross-protection studies.

Cross-protection studies were conducted with vaccines prepared from two isolates of Dermatophilus congolensis (designated strain 1 and strain 2). The vaccines were prepared as either heat-inactivated, washed, formalized filamentous phase bacterium, mixed with alum as an adjuvant, and inoculated intramuscularly (type A vaccine) or sedimented live filaments inoculated intradermally (type B vaccine). The vaccinated sheep were challenged with D. congolensis zoospores of one or other strain. Challenge sites were observed for the presence and severity of lesions. Serum antibody levels to D. congolensis were monitored after vaccination and challenge. Type A and B vaccines from both strains produced some reduction in the severity of lesions when sheep were challenged with strain 1 but not with strain 2. Unvaccinated control sheep developed more severe and persistent lesions when challenged with strain 2 than controls challenged with strain 1. Serum antibody levels to the type B vaccine prepared from strain 1 were significantly higher (P less than 0.05) than antibody levels to type B vaccine from strain 2. These findings showed there was significant variation in virulence and antigenicity between these two isolates of D. congolensis.

Immunodominant antigens of zoospores from ovine isolates of Dermatophilus congolensis.

Zoospores of Dermatophilus congolensis were analysed by SDS-PAGE and western blotting. The electrophoretic profiles of zoospores from 13 isolates of D. congolensis were similar but not identical when stained with Coomassie blue or silver. Immunodominant polypeptides with apparent molecular masses of 76 and 31 kDa were identified in western blots of 13 of 13 and 12 of 13 isolates respectively of D. congolensis reacted with hyperimmune, ovine, antizoospore sera. Identical immunodominant polypeptides were observed in western blots reacted with sera obtained from naturally infected sheep. Initial characterisation of the 76 and 31 kDa polypeptides indicated that they were probably surface exposed because (i) antibodies eluted from the surface of live zoospores after adsorption of hyperimmune antizoospore serum, reacted principally against the 76 and 31 kDa subunit polypeptides in western blots, (ii) adsorption of hyperimmune antizoospore serum with live zoospores resulted in significant diminution of reactivity against both the 76 and 31 kDa polypeptides in western blots, (iii) indirect fluorescent immunostaining of zoospores with antiserum prepared against gel-purified 76 kDa polypeptide, resulted in intense staining of the zoospore outer coat. Immuno-gold electron microscopy of negatively stained zoospores with antiserum prepared against gel-purified 31 kDa polypeptide identified this antigen as a flagella subunit.

Cellular responses in the skin of merino sheep to repeated inoculation with Dermatophilus congolensis.

The cellular response in the skin of Merino sheep was examined after three successive inoculations with Dermatophilus congolensis. There was a massive neutrophil influx into the infected epidermis and underlying dermis at 4-10 days after the first inoculation. A lymphocyte-macrophage response occurred at 10-12 days, followed by a plasma cell response at 14-38 days. Resolution of skin lesions after the first inoculation corresponded to the time when the plasma cell response in the skin was most intense. A second inoculation with D. congolensis, 70 days after the first, failed to produce skin lesions typical of dermatophilosis. Typical lesions of dermatophilosis did develop after a third inoculation of the same sheep 140 days after the first inoculation, but the lesions resolved in most sheep within 13 days. Dermatophilosis did not develop in some of these sheep at sites inoculated with 100-1000-fold lower infective doses of D. congolensis, whereas control sheep did develop lesions.