Genetic analysis of Clavibacter toxicus, the agent of annual ryegrass toxicity.

Multilocus enzyme electrophoresis was used to examine the relatedness of 52 isolates of Clavibacter toxicus, the agent of annual ryegrass toxicity. These included 37 Western Australian (WA) field isolates sampled in 3 distinct locations over a 2-year period, and 15 isolates sampled from 6 different host plant species in 3 states in Australia over approximately 8 years. Seventeen reference strains for the related genera Curtobacterium, Rhodococcus and Arthrobacter were examined for comparison. The 69 isolates were divided into 29 electrophoretic types (ETs), separated by genetic distances of 0.06 to 0.81. The C. toxicus isolates fell into 12 ETs, 11 of which formed a tightly clustered group separated by a genetic distance of 0.23 or less. Thirty-one of the WA field isolates of C. toxicus fell into a single ET, and four into another ET. Clavibacter toxicus therefore formed a closely related group which was genetically distinct from the other plant pathogenic species, and a dominant widely disseminated strain of the species was identified in WA.

Genetic differences between nitrate-negative and nitrate-positive C. pseudotuberculosis strains using restriction fragment length polymorphisms.

Corynebacterium pseudotuberculosis has been classified into two biotypes according to ability to breakdown nitrate (Biberstein et al., 1971). Restriction enzyme analysis (REA) has shown to reflect this differentiation, but numerous bands generated by this technique make interpretation difficult (Songer et al., 1988). Restriction fragment length polymorphism's (RFLP's) has become an accepted genetic tool and was used in this study to determine if differences in nitrate reduction and other phenotypic characteristics could be identified genetically. Thirteen C. pseudotuberculosis isolates from four species of domestic animals from different parts of the world were investigated for phenotypic and genetic differences. Three closely related bacteria, Corynebacterium ulcerans, Actinomyces pyogenes (previously C. pyogenes),and Rhodococcus equi (previously C. equi) were included in the study to determine if the RFLP bands were unique to C. pseudotuberculosis. All C. pseudotuberculosis isolates were positive for urease production. Some differences in maltose and sucrose fermentation ability and nitrate reduction were recorded. Genetic differences were identified between the nitrate-positive group and the nitrate-negative group using non-radioactive ribosomal RNA (rRNA) probes Southern blotted to restriction digests of ApaI, PstI, and SstI. A small number of bands were seen, with distinct differences between the nitrate-positive and the nitrate-negative strains. No genetic variations were seen between strains which reflected differences in carbohydrate fermentation. Strains isolated from different animal species and from different parts of the world could not be differentiated genetically using these three restriction enzymes.

The spread of Corynebacterium pseudotuberculosis infection to unvaccinated and vaccinated sheep.

The decrease in the prevalence of Corynebacterium pseudotuberculosis after two generations of vaccination against the disease it causes, was used to estimate the rate of control of caseous lymphadenitis (CLA). Three groups of 150 sheep, of which 50 in each group were artificially infected with C pseudotuberculosis and 100 in each group were uninfected sheep, were run separately for 40 months and shorn 5 times to promote the spread of CLA. One lot of 50 infected sheep and 2 lots of 100 uninfected sheep were vaccinated against CLA. The rate of spread of CLA was recorded. Sheep vaccinated against CLA and naturally exposed to infection had a 74% lower infection rate than unvaccinated sheep. Sheep vaccinated against CLA and exposed to only vaccinated infected sheep had a 97% lower infection rate. Unvaccinated sheep had a 76% infection rate, with 77% of the transmission occurring at the 4th and 5th shearings, without any discharging CLA abscesses being observed. This study supports the view that in Australian wool producing flocks, CLA spreads mainly from sheep with discharging lung abscesses to sheep with shearing cuts. Vaccinated sheep infected with CLA have 96% fewer lung abscesses compared with unvaccinated infected sheep and are therefore less likely to spread this disease to other sheep.

Dermatophilus chelonae sp. nov., isolated from chelonids in Australia.

Three isolates of a previously undescribed Dermatophilus sp. obtained from chelonids (two strains obtained from turtles and one strain obtained from a tortoise) were compared with 30 Dermatophilus congolensis isolates obtained from Australian mammals. The microscopic appearance, the colony morphology, and most biochemical test results for the chelonid isolates were characteristic of the genus Dermatophilus. Our isolates differed from the mammalian D. congolensis isolates in a number of cultural characteristics, including faster growth at 27 degrees C than at 37 degrees C, formation of two hemolysis zones around colonies on blood agar at 37 degrees C in the presence of 10% CO2, poor motility, and production of a distinctive odor. The DNA restriction enzyme digestion and protein electrophoresis patterns of our strains were distinct. The electrophoretic mobilities of 11 enzymes differed from the mobilities observed with D. congolensis strains. A monoclonal antibody to a surface antigen of an ovine isolate did not react with zoospores or filaments of the chelonid isolates. Biochemical differences between our isolates and D. congolensis included the ability of the chelonid isolates to reduce nitrate to nitrate and the fact that the chelonid isolates exhibit collagenase activity in vitro. We propose that the chelonid isolates should be placed in a new species, Dermatophilus chelonae. Strain W16, which was isolated from a nose scab on a snapping turtle, is the type strain; a culture of this strain has been deposited in the American Type Culture Collection as strain ATCC 51576.

New infection with Corynebacterium pseudotuberculosis reduces wool production.

The effect of natural Corynebacterium pseudotuberculosis infection on wool production and quality in sheep was examined in light of evidence that artificial C pseudotuberculosis infection causes wool production loss. A toxin ELISA was used to identify sheep that had been infected with C pseudotuberculosis. Greasy and clean fleece weights and fibre diameter were compared in infected and uninfected sheep. C pseudotuberculosis infection caused a 3.8 to 4.8% decrease in greasy wool production and a 4.1 to 6.6% decrease in clean wool production. C pseudotuberculosis infection did not affect fibre diameter. The effects of caseous lymphadenitis (the disease caused by C pseudotuberculosis infection) cause an annual loss of about $17 million in wool production to the Australian wool industry.

Variation in cultural, morphological, biochemical properties and infectivity of Australian isolates of Dermatophilus congolensis.

Recent vaccination studies with Dermatophilus congolensis showed that variation of challenge strains had a considerable influence on protection afforded by the vaccines. In this study cultural, morphological and biochemical properties of 30 D. congolensis isolates from throughout Australian were investigated. The infective dose required to produce lesions of equivalent severity by these isolates for sheep, rabbits and guinea pigs was also examined and the isolates were grouped into four clusters of similar infectivity ranking. Analysis of the relationship between cultural, morphological and biochemical characteristics and infectivity rankings of clusters was undertaken to determine if certain properties were linked to infectivity. Considerable variability was found in haemolytic activity on blood agar, mucoid nature of colonies, motility, flagella density and polarity, capsule width, restriction enzyme profiles of bacterial DNA, protein electropherotype, carbohydrate content, and enzymic activity against proteins, maltose, chondroitin-4-sulphate, phospholipids and lipids. Of these properties haemolytic activity and enzyme activity against casein, chondroitin-4-sulphate and lipids showed some link with infectivity ranking for these isolates.

Blood T6-lymphocyte antigen levels as a poor resistance marker in ovine dermatophilosis.

Merino ewes lambs from a flock expected to have a high prevalence of dermatophilosis were examined for lesions at marking, weaning and prior to first shearing. Blood was collected at marking and prior to shearing to enable quantification of T6-lymphocyte antigen levels in peripheral blood mononuclear cells (PBM) using monoclonal antibody SBU-T6 20-27. A group of the lambs that had persistent and/or severe dermatophilosis had significantly lower (P < 0.05) mean levels of T6-lymphocyte staining in PBM than those with minimal or no dermatophilosis in blood samples collected prior to shearing. However, the wide variability of this parameter in resistant and susceptible lambs indicated that this would not be a satisfactory procedure to rank sheep for resistance to dermatophilosis.

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.

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.

The effect of antibody to caseous lymphadenitis in ewes on the efficacy of vaccination in lambs.

The effect of maternal antibody to the toxin of Corynebacterium pseudotuberculosis, produced by caseous lymphadenitis (CLA) in ewes or by vaccinating ewes before lambing, on the efficacy of vaccination against CLA in their lambs was examined. Lambs were allocated to treatments according to either the vaccination history of their dam or level of CLA toxin antibody of their dam. They were vaccinated twice using 2 different inoculation regimes and then artificially infected with CLA organisms. The number of lambs with CLA lesions was assessed at slaughter. In one experiment high levels of CLA toxin antibody activity in ewes were associated with decreased efficacy of CLA vaccination in their lambs, when lambs were vaccinated at 2 and 8 weeks or 8 and 14 weeks of age. In a second experiment the efficacy of lamb vaccination at 8 and 12 weeks, but not at 14 and 18 weeks of age, was decreased. In sheep flocks with a high prevalence of CLA, vaccinating lambs against CLA at less than 10 weeks of age may not produce optimum protection against CLA in lambs. There was no difference in infection rate between lambs from vaccinated and unvaccinated ewes. However, vaccination of lambs at 2 and 8 wks was less effective that vaccination at 8 and 14 weeks, probably due to reduced immunocompetence in young lambs. In sheep flocks where significant numbers of lambs receive their primary vaccination at less than 3 weeks of age vaccination programmes to control CLA in lambs may be less effective.

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.

Inflammatory cell and immune function in Merino sheep with chronic dermatophilosis.

Components of inflammatory and immunological responses were compared in 17 Merino sheep with chronic dermatophilosis (Group 1) and 15 Merino sheep that had recovered from the disease (Group 2). The functions studied included: (i) total and differential white cell counts; (ii) phagocytic function and intracellular killing by neutrophils; (iii) humoral immune response to T-dependent and T-independent antigens and to Dermatophilus congolensis. (iv) lymphocyte blastogenic responses to phytohaemagglutinin; (v) bovine serum albumen and D. congolensis antigens; (vi) quantification of T-lymphocyte subsets in skin lesions resulting after re-infection with D. congolensis zoospores. After all lesions were treated and the sheep were shorn, both groups of sheep were re-infected with D. congolensis. Both groups had similar infection rate, severity of lesions and rate of resolution after re-infection. The Group 2 sheep had significantly higher primary and secondary antibody responses to killed Brucella abortus cells than Group 1 sheep, but Group 1 sheep had higher levels of specific D. congolensis antibody throughout the trial. Neutrophils from Group 1 sheep showed a higher phagocytic rate for D. congolensis zoospores than Group 2 sheep when the zoospores were opsonised by sera from the Group 1 sheep, but there was no difference in their ability to kill ingested zoospores. Although there were some differences between the groups in the proportion of lymphocytes in lesions that reacted with monoclonal antibodies to T4, T8 and T19-19 lymphocyte markers at various times after re-infection, the sheep in Group 2 consistently had higher levels of lymphocytes reacting to a monoclonal antibody for the T6 lymphocyte antigen in skin biopsies collected 9, 15 and 21 days post-inoculation (p.i.) than did sheep in Group 1. Group 2 sheep also had higher levels of epidermal cells with immunohistochemical properties of Langerhans cells at lesion sites 15 and 21 days p.i.

Comparison of the exotoxins of four strains of Corynebacterium pseudotuberculosis.

The activity of exotoxins produced by four strains of Corynebacterium pseudotuberculosis were compared by their ability to kill white mice, their haemolytic activity, staphylococcal haemolysin-inhibiting effect and activity in an enzyme-linked immunosorbent assay (ELISA). Exotoxins with a haemolytic titre of 1 in 256 or more killed all mice and had the most inhibitory effect (1 in 64 or more) on staphylococcal haemolysin. The haemolytic test and staphylococcal haemolysin inhibitory test of exotoxin activities were highly correlated (P less than 0.001) with the mouse toxicity of the four exotoxins examined. No significant correlation was evident between the ELISA and relative toxicity of the exotoxins.

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.

The effect of selenium supplementation on immunity, and the establishment of an experimental Haemonchus contortus infection, in weaner merino sheep fed a low selenium diet.

Immunity in 12 weaner Merino sheep fed a low selenium (Se) diet (low Se sheep) was compared with that in 10 matching sheep fed the same diet but each given an intraruminal Se pellet (high Se sheep), while the sheep were housed in individual, sheltered pens. All sheep were challenged with killed Brucella abortus cells (days 0 and 28), rabbit red blood cells (days 0, 7 and 28) and corynebacterium pseudotuberculosis toxoid (days 0 and 28), and serum antibody titres were measured weekly for 8 weeks from day 0. The sheep were then experimentally infected with Haemonchus contortus, and slaughtered 8 weeks later. The mean antibody titre to B. abortus, measured by 4 different tests, was significantly higher in the high Se sheep on occasions during the primary immune response phase (Rose Bengal test - day 21 (p less than 0.05), day 28 (p less than 0.025); complement fixation - day 7 (p less than 0.05); enzyme-linked immunosorbent assay - day 14 (p less than 0.01); serum agglutination - no differences), but not during the secondary phase. The mean antibody titre to rabbit red blood cells, measured by haemagglutination test, was marginally higher in the high Se sheep on day 49 (p = 0.049). The mean antibody titre to C. pseudotuberculosis, measured by enzyme-linked immunosorbent assay, was not significantly different between the groups at any time during the trial. In addition, the mean in-vitro responsiveness of peripheral blood lymphocytes to stimulation with phytohaemagglutinin in the high Se sheep was significantly greater than that in 10 sheep from the low Se group on day 22 (p less than 0.01), but not day 50.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of caseous lymphadenitis on wool production and bodyweight in young sheep.

Two hundred Merino wether hoggets were used to examine the effect of Corynebacterium pseudotuberculosis infection (caseous lymphadenitis) on wool production and bodyweight. Sheep which were challenged with C. pseudotuberculosis (artificially infected) and not vaccinated against this disease produced 0.20 kg less clean wool than unchallenged controls during the following 12 months. The incidence of sheep with lesions in the group that was vaccinated prior to challenge was 55% lower than in unvaccinated challenged sheep but their wool production was not significantly different from either the controls or the unvaccinated challenged sheep. Vaccinated sheep were also heavier than unvaccinated sheep 12 months after challenge. These results indicate that caseous lymphadenitis infection may reduce wool production.

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.