A purified stage-specific 31 kDa antigen as a potential protective antigen against Ostertagia circumcincta infection in lambs.

The 31 kDa antigen of third-stage (L3) Ostertagia circumcincta larvae was evaluated as a potential prophylactic antigen by an analysis of the protective, humoral and cell-mediated responses of lambs immunized with this antigen. Six lambs were immunized by subcutaneous injection with a total of 400 micrograms of the purified 31 kDa antigen in 250 micrograms ml-1 Quil A adjuvant. Five sheep given identical injections but without the 31 kDa antigen were used as controls. All animals were challenged with 4.2 x 10(4) infective L3 O. circumcincta larvae 1 week after the last booster injection. The protection afforded by the 31 kDa antigen was demonstrated by a significant reduction in faecal egg counts (p less than 0.05) and total worm counts (p less than 0.005) in vaccinated animals. Elevated ELISA antibody levels specific to the 31 kDa antigen were detected in the sera of vaccinated animals as early as 3 weeks after immunization. Specific antibodies were further demonstrated by Western blot 4 days after the first booster immunization at 3 weeks. In control animals no antibodies to the 31 kDa antigen were detected in Western blots throughout the course of the experiment. Immunized lambs had significantly higher in vitro lymphocyte stimulation indices (p less than 0.05) than control animals. In haematoxylin and eosin stained sections taken at necropsy from vaccinated sheep, compared with control lambs, there were more larvae present in the crypts of the abomasal epithelium and these larvae were surrounded by lymphocytes and eosinophils. In vaccinated lambs a marked infiltration of lymphocytes in the lamina propria and oedema in the submucosa were also observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of three gene regions associated with virulence in Dichelobacter nodosus, the causative agent of ovine footrot.

Dichelobacter nodosus (formerly Bacteroides nodosus) is a Gram-negative strict anaerobe and is the primary pathogen involved in ovine footrot. A comparative hybridization strategy was used to isolate recombinant clones which hybridized to DNA from a virulent strain of D. nodosus but not with a benign isolate. Three virulence-associated gene regions were identified and one of these regions was shown to be present in multiple copies in the D. nodosus genome. Hybridization studies on 101 clinical isolates of D. nodosus showed that these strains could be divided into three hybridization categories which could be correlated with the virulence of the isolates. The recombinant clones have considerable potential for the development of a gene-probe-based method for the differential diagnosis of ovine footrot.

The distribution and localization of the stage-specific GP31 antigen from infective Ostertagia circumcincta larvae.

The 31,000 mol. wt glycoprotein (GP31) antigen of infective third-stage (L3) Ostertagia circumcincta larvae was shown, by surface labelling experiments and immunofluorescent antibody staining of whole larvae and larval sections, to be distributed internally. When transverse sections of L3 O. circumcincta, taken from the anterior pharyngeal region, were further examined by electron microscopy, after immunogold staining with rabbit anti-GP31 antiserum, the GP31 antigen was found to be specifically located in 'secretory organelles' within the cells of the oesophageal glands. By in vitro culturing L3 O. circumcincta in medium supplemented with 35S-methionine and then analysing the excretory-secretory material released by the larvae, it was found that the GP31 molecule was one of the major components of the excretory-secretory complex. The purified GP31 molecule had no detectable proteolytic activity in protein degradation assays. On examination of Triton X-100 extracts of infective larvae from other nematode parasite species, a predominant antigen similar to GP31 was found in Trichostrongylus colubriformis and Haemonchus contortus, but in Toxocara canis a minor component corresponding in mol. wt to GP31 was also detected. Based on these results the possible role of GP31 as a candidate antigen for a broad spectrum molecular vaccine against gastrointestinal nematode parasites in sheep is discussed.

Extraction and identification of a 31,000 mol.wt glycoprotein antigen of Ostertagia circumcincta by sera from resistant sheep.

Under similar extraction conditions, Triton X-100 sonicates gave higher yields of protein from third stage larvae and adult O. circumcincta than seven other detergents tested. Using sera from sheep which had been experimentally defined by both immunological and parasitological parameters as being either resistant or susceptible to O. circumcincta, a molecule from Triton X-100 extracts of third stage O. circumcincta larvae was identified which reacted preferentially with sera from resistant sheep. This molecule has a molecular weight of 31,000 and preliminary characterization studies revealed it to be a glycoprotein which was not found in later larval stages or adult worms. Antibodies to this 31,000 mol.wt antigen were present in sera of sheep as early as 3 weeks after experimental infection with O. circumcincta.

Molecular analysis of one of multiple protease-encoding genes from the prototype virulent strain of Bacteroides nodosus.

The aim of these studies was to examine the organization of the Bacteroides nodosus protease-encoding gene(s). The extracellular serine proteases (38 kDa) from the prototype virulent strain of B. nodosus were purified and used to raise a specific antiserum in rabbits. This antiserum was used in a colony immunoassay to screen a genomic DNA library constructed in Escherichia coli using BamHI-digested B. nodosus DNA and the plasmid pBR322. An E. coli clone expressing a 50-kDa immunoreactive polypeptide was identified. No protease activity was detected in the culture media, or in crude soluble and membrane fractions prepared from this clone. Restriction mapping and deletion analysis of the recombinant plasmid, pEKM2, was used to locate the coding region to a 1.4-kb EcoRI-BamHI fragment which was subsequently sequenced. A large open reading frame was found to extend through the BamHI site from a putative start codon just downstream from the EcoRI site, which indicated that the complete gene was not isolated. Southern blotting demonstrated that there were at least three B. nodosus BamHI fragments which were homologous to the 0.4-kb PstI-BamHI fragment of pEKM2. Based on these results the existence of multiple protease genes in B. nodosus was postulated.

A comparison of nematode control programs for cattle in south western Victoria.

Nematodiasis and its subsequent effect on production in Hereford weaner steers in western Victoria was studied during 1983 and 1984. In the first summer, steers were allocated to 2 replicates of 6 treatments--No treatment (Nil); Morantel slow release bolus in March (M1); Morantel bolus in March and June (M2); pour-on levamisole in January, May and July (R3); albendazole in January and July (V2) and albendazole in January, May and July, (V3). In 1984, treatment M2 was discontinued to provide extra replicates for Nil and M1. The replicate paddocks were 5 ha and were stocked with 7 and 8 steers in 1983 and 1984, respectively. Nematode egg counts in faeces, were generally less than 50 epg, indicating low numbers of adult nematodes. Faecal egg counts were highest in autumn and declined during the year. There was a significant (P = 0.02) effect of treatment on mean faecal egg count. Mean egg counts for treatment groups Nil and M1 were 16 and 10 epg above the overall mean (47 epg); those of the other treatments were 6 to 12 epg below the mean. There were no significant (P = 0.8) differences between treatments in the numbers of nematode larvae on pasture, during the experiment. At the end of both years of the experiment most nematodes (92%) were early fourth stage larvae of O. ostertagi. There were no consistent differences in nematode counts between treatments. There were no significant (P = 0.33) differences between treatments in bodyweights at any time during the experiment.(ABSTRACT TRUNCATED AT 250 WORDS)

Anthelmintic resistance and sheep management practices in south western Victoria.

Twenty-eight farms in 7 shires in south western Victoria were selected and tested for presence of benzimidazole-resistant nematodes between November 1979 and June 1981. Mean faecal egg counts of sheep were less than 100 strongyloid eggs/g on 11 farms. Faecal egg count reduction tests were conducted on the remaining 17 farms and thiabendazole was less than 90% efficient in reducing egg counts in sheep from 5 (29%) of these farms. Thiabendazole-resistant Teladorsagia circumcincta were identified at necropsy of experimentally infected treated sheep. In further studies a survey of 104 farms was conducted in the Mount Rouse and Dundas shires of western Victoria in 1981 and 1982 respectively to determine the prevalence of anthelmintic resistance in these shires. Mean faecal egg counts among weaner sheep in the winter-spring of both years were less than 100 eggs/g which indicated low levels of parasitic nematode populations. A faecal egg count reduction test was conducted on 10 farms and thiabendazole was less than 90% efficient on 3; levamisole was greater than 90% efficient in all 10 tests. Most of the surveyed farms carried Merino or Merino crossbred sheep at 10 to 15 dry sheep equivalents per ha and weaners were treated with anthelmintics 3 to 6 times per year. Management procedures based mainly on anthelmintic therapy were effective in controlling nematode populations in weaner sheep, although many producers alternated between different groups of anthelmintics within the same year contrary to current recommendations for long-term preservation of anthelmintic efficacy. It was concluded that anthelmintic resistance was not of practical importance to the majority of sheep producers in the region.