Claes Fellström

Phenotypical characterisation of intestinal spirochaetes isolated from pigs.

A combined evaluation of the phenotypical properties of five Serpulina type or reference strains and 163 Swedish isolates of spirochaetes from pigs and two from birds was made. The porcine isolates were collected from herds with a history of dysentery or severe diarrhoea and from herds chosen at random. On the basis of beta-haemolysis, indole production, hippurate hydrolysis, and alpha-galactosidase, alpha-glucosidase and beta-glucosidase activity, the isolates could be divided into four main groups, I to IV, with three subgroups in group III. Group I included the type strain for Serpulina hyodysenteriae (B78). Group II was differentiated from group I only by weak beta-haemolysis. Group III included the type strain for Serpulina innocens (B256). Group IV included the pathogenic, weakly haemolytic strain P43. Group IV-spirochaetes were characterised by their ability to hydrolyse hippurate and by their lack of beta-glucosidase activity. Group I and II-spirochaetes were isolated only from dysenteric or diarrhoeic pigs. There was a statistical relationship between pigs with diarrhoea and the isolation of group IV spirochaetes but no relationship with group III spirochaetes.

Emended descriptions of indole negative and indole positive isolates of Brachyspira (Serpulina) hyodysenteriae.

Two type/reference strains of Brachyspira (B.) hyodysenteriae, 14 Belgian and German indole negative, and 14 Belgian, German and Swedish indole positive field isolates of strongly beta-haemolytic intestinal spirochaetes were compared by pulsed-field gel electrophoresis (PFGE) patterns, biochemical reaction patterns, 16S rDNA sequences and MIC determinations of six antibacterial substances. Three tests for indole production, including a spot indole test, were compared with congruent results. All field isolates were classified as B. hyodysenteriae due to a high genetic and phenotypic similarity with the type strains. The Belgian and German indole negative isolates had identical and unique PFGE patterns for the tested restriction enzymes MluI and SalI, as well as identical 16S rDNA sequences, and they could not be differentiated by any of the methods used. Seven unique PFGE patterns were achieved from the 14 indole positive field isolates. The patterns were identical and unique for epidemiologically related isolates. Type/reference strains and isolates without known relation to other tested isolates showed unique banding patterns. The MICs of tylosin, tiamulin, erythromycin, clindamycin, carbadox and virginiamycin were determined in broth for all isolates. In contrast to Belgian and German isolates, the majority of the Swedish field isolates were susceptible to tylosin, erythromycin and clindamycin. Probable pathways of infection for some of the Swedish isolates were determined. The PFGE patterns of epidemic clones of B. hyodysenteriae remained stable for a period of up to 8 years. In vivo development of resistance to macrolide and lincosamide antibiotics due to use of tylosin was clearly indicated for two epidemic clones.

Antimicrobial Susceptibility Testing of Porcine Brachyspira (Serpulina) Species Isolates

ABSTRACT No standardized method for susceptibility testing of Brachyspira spp. is currently available. A broth dilution procedure was evaluated and used to test the activities of six antimicrobial agents for 108 isolates of Swedish porcine Brachyspira spp. representing biochemical groups I, II, and III. Group I corresponds to Brachyspira hyodysenteriae, group II corresponds to B. intermedia, and group III corresponds to B. murdochii and B. innocens. A panel was designed with the antimicrobial agents dried in tissue culture trays with wells that allowed a liquid volume of 0.5 ml in each and agitation of the broth when incubated on a shaker. The MICs were determined by using brain heart infusion broth with 10% fetal calf serum. For 10 isolates, the results obtained in broth were compared to the MICs obtained on two different types of agar. Different inoculum densities and incubation times were also compared. The concentrations at which 90% of the B. hyodysenteriae isolates (n = 72) were inhibited in the broth dilution test by tiamulin (0.25 μg/ml), tylosin (>256 μg/ml), erythromycin (>256 μg/ml), clindamycin (>4 μg/ml), virginiamycin (4 μg/ml), and carbadox (0.06 μg/ml) were determined. The MICs tended to be lower in broth than on agar. Differences in inoculum densities and incubation times had little influence on the MICs. The evaluated broth dilution test was simple to perform, the end points were easily read, and the results were reproducible and reliable. No isolates with decreased susceptibility to tiamulin were found among the Swedish isolates tested.

Diarrhoea in the growing pig - a comparison of clinical, morphological and microbial findings between animals from good and poor performance herds.

Abstract Diarrhoea among growing pigs (8–13 weeks old) is a significant problem in many herds. Nine herds with poor performance and diarrhoea among growing pigs were selected on the basis of their piglet mean age at a body weight of 25kg, compared to the overall mean age in Swedish herds. In addition, four herds with good average performance and no problems with diarrhoea were selected. Pigs were necropsied and samples for histology and microbiology were collected. Based on the necropsy findings, the pigs from the good performing herds were all judged to be healthy. The presence of Brachyspira pilosicoli and Lawsonia intracellularis was significantly correlated to poor performing herds and the results indicate that these microbes are main pathogens involved in enteric diseases among Swedish grower pigs. In addition, concomitant infections with other presumptive pathogens were commonly found.

Phylogeny of Serpulina based on sequence analyses of the 16S rRNA gene and comparison with a scheme involving biochemical classification

Twenty-one putative Serpulina strains, representing six proposed biochemical groups, were selected for phylogenetic studies based on 16S rRNA sequencing. The biochemical groups were distinguished by the degree of beta-haemolysis, indole production, hippurate hydrolysis and alpha-galactosidase-, and beta-glucosidase activity. The 16S rRNA sequences of the U2 to U5 region, including three evolutionarily variable regions, from representatives of each biochemical group were determined by automated solid phase DNA sequencing after in vitro amplification by the polymerase chain reaction (PCR). The sequences generated were 532 nucleotides in length. Sequence alignments showed that all the strains were closely related, with six informative positions in the region sequenced. A dendrogram was constructed from these data and compared with the tentative biochemical classification. The results support the proposed biochemical classification and indicate that at least five genetic variants of the genus Serpulina can be identified.

The use of culture, pooled samples and PCR for identification of herds infected with Brachyspira hyodysenteriae

Abstract The sensitivity of culturing Brachyspira hyodysenteriae was determined after sampling with swabs from porcine fecal specimens inoculated with tenfold dilutions of a field strain of these microbes. After storage of swabs, Brachyspira hyodysenteriaewas recovered throughout the first 3 weeks after inoculation from feces with more than 140 cells/g. Viable spirochetes could still be recovered after up to 83 days of storage from feces, with 1.4 × 106 cells or more per gram. Culture for Brachyspira spp. was performed on 285 rectal swabs, which were pooled in batches of five. The number of pooled samples positive for B. hyodysenteriae corresponded with the sum results of individual analysis of the corresponding collections of five samples. A PCR system based on the tlyA gene of B. hyodysenteriae was developed and tested on primary cultures of pooled samples. The results of the PCR assay showed a 97% correlation with the culture results. The prevalence of Brachyspira spp. was determined in five swine herds and found to be highest among breeding gilts and boars aged 13–16 weeks and among 6–12-week-old weaned pigs. In contrast, Brachyspiraspp. were only rarely found in sows, which may reflect the development of immunity by adult pigs to all species of the genus.

Porcine proliferative enteropathy: an important disease with questions remaining to be solved.

Proliferative enteropathy caused by the intracellular bacterium Lawsonia intracellularis is an endemic disease with high herd prevalences reported worldwide. The infection has a considerable impact on pig production and herd economics and, with the development of new diagnostic techniques, L. intracellularis is being identified in an increasing number of pig herds and a wider range of species. This paper reviews current knowledge of the disease, with a focus on the epidemiology in pigs. The prevalence of infection, transmission, predisposing factors, microbial features, pathogenesis, clinical signs, diagnosis, treatment and control are discussed. The disease is mainly controlled by antibiotic treatment and vaccination at herd level. In the development of effective measures to prevent the spread of the infection, increased knowledge of the transmission and persistence of the microorganism are crucial.

Isolation of spirochetes of genus Treponema from pigs with ear necrosis

Various ear lesions, often caused by ear biting, are common in pigs. Some herds have a high frequency of ear necrosis, a syndrome characterized by necrotic lesions along the rim of the pinna, often bilateral and sometimes resulting in loss of the entire ear. In samples from such lesions spirochetes have been observed microscopically but never isolated or identified. In this study two herds with periodic outbreaks of ear necrosis among weaners were investigated. Samples were collected from ear lesions and from the gingiva of the pigs. Spirochetes were observed in silver stained histological sections and by phase contrast microscope in scrapings from the necrotic lesions. From an ear lesion a pure spirochete isolate was obtained and identified as a yet unnamed species of genus Treponema, closely related to spirochetes found in digital dermatitis in cattle. From the oral samples two pure isolates were obtained. One of these isolates was identified as the same species as in the ear lesion and one as Treponema socranskii. Species identification was based on 16S rRNA gene sequences.

Brachyspira spp. ( Serpulina spp.) in birds: a review and results from a study of Swedish game birds

Abstract Only limited data concerning the prevalence of intestinal spirochetes are available in game birds. This paper describes the prevalence and biochemical reactions of spirochetes isolated from 25 common partridges, 7 pheasants and 16 mallards originating from nine Swedish game-bird farms. The birds, which had been submitted for post-mortem examination due to various problems, showed a variety of underlying diseases. Additionally, fecal droppings from 22 common partridges, 20 pheasants and 20 mallards obtained at one of the farms were included in the study. Intestinal spirochetes were isolated from 85.4% of the game birds and from 71% of the fecal droppings. Seven biochemical types were identified. Seventeen per cent of all isolates were classified as Brachyspira pilosicoli and 3% as B. intermedia. One isolate showed strong -hemolysis and a positive indole reaction that is consistent with B. hyodysenteriae. In addition, three previously unknown biochemical types were found. Unclassified spirochetes in presumed mixed cultures were observed in 21% of all samples of fecal droppings. Histologic examination of spirochete-positive birds revealed numerous spirochetes in the lumen and crypts of the cecum, in some cases densely adhered by one end to the luminal surface. The significance of the findings is discussed.

Phylogenetic evidence for novel and genetically different intestinal spirochetes resembling Brachyspira aalborgi in the mucosa of the human colon as revealed by 16S rDNA analysis.

Intestinal spirochetes (Brachyspira spp.) are causative agents of intestinal disorders in animals and humans. Phylogenetic analysis of cloned 16S rRNA genes from biopsies of the intestinal mucosa of the colon from two Swedish 60-years old adults without clinical symptoms revealed the presence of intestinal spirochetes. Seventeen clones from two individuals and 11 reference strains were analyzed and the intestinal spirochetes could be divided into two lineages, the Brachyspira aalborgi and the Brachyspira hyodysenteriae lineages. All of the clones grouped in the B. aalborgi lineage. Moreover, the B. aalborgi lineage could be divided into three distinct phylogenetic clusters as confirmed by bootstrap and signature nucleotide analysis. The first cluster comprised 6 clones and the type strain B. aalborgi NCTC 11492T. The cluster 1 showed a 16S rRNA gene similarity of 99.4-99.9%. This cluster also harbored the only other strain of B. aalborgi isolated so far, namely strain W1, which was subjected to phylogenetic analysis in this work. The second cluster harbored 9 clones with a 98.7 to 99.5% range of 16S rDNA similarity to the B. aalborgi cluster 1. Two clones branched distinct and early of the B. aalborgi line forming the third cluster and was found to be 98.7% similar to cluster 1 and 98.3-99.1% to cluster 2. Interestingly, this shows that considerable variation of intestinal spirochetes can be found as constituents of the colonic microbiota in humans, genetically resembling B. aalborgi. The presented data aid significantly to the diagnostic and taxonomic work on these organisms.