Understanding the molecular epidemiology of the footrot pathogen Dichelobacter nodosus to support control and eradication programs.

The Gram-negative anaerobe Dichelobacter nodosus is the primary etiologic agent of ovine footrot. Few studies of the genetic diversity and epidemiology of D. nodosus have been done, despite the economic cost and welfare implications of the disease. This study examined a large collection of Australian isolates; 735 isolates from footrot-infected sheep from 247 farms in Western Australia (WA) were tested by pulsed-field gel electrophoresis (PFGE), and a subset of 616 isolates was tested by infrequent restriction site PCR (IRS-PCR). The genetic diversity of WA isolates was compared to that of 61 isolates from three other Australian states. WA isolates were genetically diverse, with 181 molecular types resolved by PFGE, resulting in a simple diversity ratio (SDR) of 1:4 and a Simpson's index of discrimination value (D) of 0.98. IRS-PCR resolved 77 molecular types (SDR = 1:8 and D = 0.95). The isolates were grouped into 67 clonal groups by PFGE (SDR = 1:11, D = 0.90) and 36 clonal groups by IRS-PCR (SDR = 1:17, D = 0.87). Despite the high genetic diversity, three common clonal groups predominated in WA and were found in other Australian states. On some farms, molecular type was stable over a number of years, whereas on other farms genetically diverse isolates occurred within a flock of sheep or within a hoof. This study provides a large database from which to appropriately interpret molecular types found in epidemiological investigations and to identify common and unknown types that may compromise footrot eradication or control programs.

Effect of the interval between shearing and dipping on the spread of Corynebacterium pseudotuberculosis infection in sheep.

OBJECTIVE: To determine whether the spread of Corynebacterium pseudotuberculosis infection to sheep in dips could be controlled by increasing the time between shearing and dipping. DESIGN: A controlled treatment trial where only the time between shearing and dipping was varied. ANIMALS AND PROCEDURE: One hundred and ninety-five sheep were found to be negative for C. pseudotuberculosis exposure by assay of CLA toxin antibody, were divided into four treatment groups. Each was shorn at either 0, 2, 4 or 8 weeks before dipping in a solution containing C. pseudotuberculosis. Blood samples were taken 6 weeks after dipping and sheep were slaughtered 12 weeks after dipping. A fifth smaller group of 14 sheep shorn 26 weeks before dipping, was also exposed to C. pseudotuberculosis and was slaughtered with the other sheep. RESULTS: The occurrence of caseous lymphadenitis abscesses did not differ between groups or with sheep shorn 26 weeks before dipping. The proportion of sheep that seroconverted to the C. pseudotuberculosis toxin and cell wall ELISA was larger in sheep dipped immediately after shearing than in sheep in the other groups. CONCLUSIONS: Delaying dipping until 8 weeks after shearing did not decrease the C. pseudotuberculosis infection rate due to dipping. Sheep dipped immediately after shearing developed higher concentrations of antibody to C. pseudotuberculosis than sheep when dipping occurred between 2 and 8 weeks and later after shearing.

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.

Antimicrobial susceptibility testing of Serpulina hyodysenteriae.

The macrobroth dilution technique was used to test the in-vitro effectiveness of 4 commonly used antimicrobial agents against 23 Australian isolates and 7 overseas strains of Serpulina hyodysenteriae. Minimum inhibitory concentrations and minimum bactericidal concentrations were determined. The growth of 90% of isolates was inhibited by dimetridazole at a concentration of 4 micrograms/mL, and by tiamulin at 8 micrograms/mL. Australian isolates resistant to both antimicrobial agents were identified. Lincomycin was less effective than these antimicrobial agents, with 90% of isolates requiring a concentration of 128 micrograms/mL for inhibition of growth, and 54% being susceptible at 64 micrograms/mL. Tylosin did not prevent the growth of the majority of S hyodysenteriae isolates tested, and 90% were resistant to concentrations of > or = 128 micrograms/mL. Resistant isolates came from different geographical areas. Resistance was not related to overall genetic background of the spirochaetes, and was not correlated with the presence of plasmids or the serogroup of the isolates.