B.G. Combs

The prevalence of Serpulina pilosicoli in humans and domestic animals in the Eastern Highlands of Papua New Guinea

In a survey of five villages in the Eastern Highlands of Papua New Guinea, Serpulina pilosicoli was isolated from rectal swabs from 113 of 496 individuals (22.8%). Colonization rates ranged from 22.6-30.1% in four of the villages but was only 8.6% in the other village. In comparison colonization was demonstrated in only 5 of 54 indigenous people (9.3%) and none of 76 non-indigenous people living in an urban environment in the same region. Colonization did not relate to reported occurrence of diarrhoea, age, sex, or length of time resident in a village. A second set of 94 faecal specimens was collected from 1 village 6 weeks after the first set. S. pilosicoli was isolated from 27 of 29 individuals (93.1%) who were positive on the first sampling and from 7 of 65 individuals (10.8%) who previously were negative. In this case, isolates were significantly more common in watery stools than in normal stools. The annual incidence of infection in the village was calculated as 93.6%, with an average duration of infection of 117 days. S. pilosicoli could not be isolated from any village pig (n = 126) despite its confirmed presence in 17 of 50 commercial pigs (34.0%) sampled at a local piggery. Four of 76 village dogs (5.3%) and 1 of 2 village ducks were colonized with S. pilosicoli, suggesting the possibility of cross transmission between humans and animals.

Development and evaluation of polymerase chain reaction tests as an aid to diagnosis of swine dysentery and intestinal spirochaetosis

Polymerase chain reaction (PCR) tests were established for detection of Serpulina hyodysenteriae, the agent of swine dysentery, and S. pilosicoli, the agent of intestinal spirochaetosis. Both reactions were specific when tested with DNA from 107 strains of various intestinal spirochaetes. For diagnostic use, faeces were plated to selective medium, and diatomaceous earth extraction used to obtain DNA prior to PCR. This procedure detected 103–104 cells of either organism seeded into 0·2 g of faeces. When applied to 63 samples from pigs of eight piggeries naturally infected with either S. hyodysenteriae or S. pilosicoli, both PCRs were specific, more rapid, and detected more positive samples than did routine faecal culture and isolation.

Multilocus enzyme electrophoresis for identification and typing of Treponema hyodysenteriae and related spirochaetes.

An assessment was made of multilocus enzyme electrophoresis as a means of identifying and typing spirochaetes isolated from pigs. Using five enzyme systems, 36 isolates from Australia, the U.K. and the U.S.A. were divided into 12 electrophoretic types or multilocus genotypes, comprising four major, genetically distinct groups. All 26 isolates of Treponema hyodysenteriae fell into one group, members of which showed relatively little genetic diversity. Ten isolates of non-pathogenic spirochaetes fell into three genetically different groups. Although the technique was capable of typing organisms within the groups, it was not always as discriminatory as DNA-restriction endonuclease analysis. Examination of additional enzyme loci should increase the sensitivity of the method for typing and for overall assessment of genetic relationships between spirochaetes.

Genetic relationships between isolates of Serpulina (Treponema) hyodysenteriae, and comparison of methods for their subspecific differentiation

Multilocus enzyme electrophoresis (MEE) was used to examine the extent of genetic diversity amongst 98 isolates of Serpulina (Treponema) hyodysenteriae. The species contained four major genetic divisions (A, B, C and D) and 29 electrophoretic types (ETs). Division D was relatively distinct, being separated from the other three divisions by fixed allelic differences at an average of 6.6 of 15 enzyme loci. Electrophoretic differences were compared with results of DNA restriction endonuclease analysis (REA) and serological typing of the isolates. Most isolates with the same or similar REA banding patterns shared the same or similar ETs. This demonstrated that both techniques could be used as sensitive and specific methods of identifying closely related isolates. However, using MEE analysis, some isolates that had quite different REA patterns were found to be genetically closely related. Therefore ET designations had an advantage over REA patterns in that they were readily quantifiable as a means of estimating genetic relatedness between isolates. Most isolates that were genetically similar to each other were of the same serological group, but some antigenic types were widely distributed across the genetic divisions.

Typing of Australian isolates of Treponema hyodysenteriae by serology and by DNA restriction endonuclease analysis

A total of 91 isolates of Treponema hyodysenteriae which were obtained from 62 piggeries located around Australia were typed by serology and by DNA restriction endonuclease analysis (REA). The isolates fell into eight serogroups, of which groups B and D were the most common. Isolates with different REA patterns were recognised within serogroups, whilst a few isolates with the same REA pattern were placed into different serogroups. Some of the latter isolates were either from the same piggery or from farms with epidemiological links, thus indicating the bacteria may have altered their antigenic properties. A total of 31 different REA patterns were recognised amongst the Australian isolates. These comprised eight major patterns, with four of these being subdivided on the basis of minor differences in banding. Where a number of isolates were obtained from individual piggeries these all had the same REA pattern, and in one piggery isolates with the same pattern were recovered over a five year period. Plasmid bands were observed in 70 of the Australian isolates (77%), and in six of the seven overseas isolates included in the study for comparison. These plasmids did not affect the REA pattern. Of the States from which substantial numbers of isolates were examined, the greatest number of different strains (12 amongst 19 piggeries) were found from Victoria, and there were 10 REA patterns in strains from 24 piggeries in Queensland. Despite the large total number of different strains of T. hyodysenteriae in Australia, only three were found in more than one State.

Typing of Treponema hyodysenteriae by restriction endonuclease analysis

Restriction endonuclease analysis (REA) was used to type eight well-characterised strains of Treponema hyodysenteriae originating from the U.K., Canada and the U.S.A., and 16 isolates from cases of swine dysentery in Western Australia (W.A.). Several of the W.A. isolates were also serotyped by the method of Baum and Joens (1979), and the two typing techniques were compared. REA typing was more discriminatory than serotyping, being able to distinguish strains within serotypes. The new technique was neither more difficult nor more time-consuming to perform than serotyping. Within the 16 W.A. isolates, three different REA patterns were identified, with common patterns found on different farms. The eight overseas strains had seven different REA patterns, all of which could be distinguished from the patterns of the W.A. isolates.

Use of a whole chromosomal probe for identification of Treponema hyodysenteriae

A whole chromosomal DNA probe labelled with photobiotin was used in a dot blot hybridisation to identify DNA from isolates of Treponema hyodysenteriae, the aetiological agent of swine dysentery. The probe was evaluated using DNA from 13 isolates of T hyodysenteriae and 13 isolates of non-T hyodysenteriae spirochaetes recovered from pigs. The initial test had both a sensitivity and specificity of 92.3 per cent, although when it was repeated the specificity fell to 84.6 per cent. The test was helpful in distinguishing between T hyodysenteriae and other morphologically similar treponemes that are part of the normal flora in the large intestine of pigs. The probe could also be used to detect as little as 10 ng of purified DNA from T hyodysenteriae, or DNA from 2 x 10(6) bacterial cells lysed directly onto nitrocellulose.