A Spirochaete is suggested as the causative agent of Akoya oyster disease by metagenomic analysis.

Mass mortality that is acompanied by reddish browning of the soft tissues has been occurring in cultured pearl oyster, Pinctada fucata martensii. The disease is called Akoya oyster disease (AOD). Although spreading pattern of the disease and transmission experiments suggest that the disease is infectious, the causative agent has not yet been identified. We used shotgun and 16S rRNA-based metagenomic analysis to identify genes that are present specifically in affected oysters. The genes found only in diseased oysters were mostly bacterial origin, suggesting that the causative agent was a bacterial pathogen. This hypothesis was supported by the inhibition of AOD development in naïve oysters injected with the hemolymph of diseased animals followed immediately with penicillin bath-administration. Further analyses of the hemolymph and mantle specifically and universally detected genes of bacteria that belong to phylum Spirochaetes in diseased pearl oysters but not in healthy oysters. By in situ hybridization or immunostaining, a Brachyspira-like bacterium was observed in the smears of hemolymph from affected oysters, but not from healthy oysters. Phylogenetic analysis using 16S rRNA sequences showed that the presumptive causative bacterium was outside of but most closely related to family Brachyspiraceae. We propose 'Candidatus Maribrachyspira akoyae' gen. nov, sp nov., for this bacterium.

Brachyspira and its role in avian intestinal spirochaetosis.

The fastidious, anaerobic spirochaete Brachyspira is capable of causing enteric disease in avian, porcine and human hosts, amongst others, with a potential for zoonotic transmission. Avian intestinal spirochaetosis (AIS), the resulting disease from colonisation of the caeca and colon of poultry by Brachyspira leads to production losses, with an estimated annual cost of circa £ 18 million to the commercial layer industry in the United Kingdom. Of seven known and several proposed species of Brachyspira, three are currently considered pathogenic to poultry; B. alvinipulli, B. intermedia and B. pilosicoli. Currently, AIS is primarily prevented by strict biosecurity controls and is treated using antimicrobials, including tiamulin. Other treatment strategies have been explored, including vaccination and probiotics, but such developments have been hindered by a limited understanding of the pathobiology of Brachyspira. A lack of knowledge of the metabolic capabilities and little genomic information for Brachyspira has resulted in a limited understanding of the pathobiology. In addition to an emergence of antibiotic resistance amongst Brachyspira, bans on the prophylactic use of antimicrobials in livestock are driving an urgent requirement for alternative treatment strategies for Brachyspira-related diseases, such as AIS. Advances in the molecular biology and genomics of Brachyspira heralds the potential for the development of tools for genetic manipulation to gain an improved understanding of the pathogenesis of Brachyspira.

Brachyspira hyodysenteriae contains eight linked gene copies related to an expressed 39-kDa surface protein.

A tandemly linked set of four open reading frames (ORFs), identified as vspA-D (variable surface protein) had been identified from previous cloning and sequencing of clones from a genomic library constructed from Brachyspira hyodysenteriae strain B204. The predicted translation products of these closely related genes were homologous to (but not identical with) a characterized 39-kDa surface-exposed membrane protein from this animal pathogen. Additional screening of the genomic library has been performed to retrieve what are believed to be additional vsp genes including the one expected to encode this 39-kDa protein. Four new vsp genes have been identified and found to be associated in a second set of four tandemly linked alleles. This new gene cluster of 7481 nucleotides is not adjacent to the original vspA-D gene cluster described but does appear to have arisen from a gene (region) duplication event. The new vsp genes (identified as vspE-H) are oriented parallel to one another and appear to have a set of similar but distinct regulatory elements that may control separate expression of their ORFs. The four adjacent ORFs are of similar size (361-390 codons) and share from 83% to 90% identity in their amino acid sequence. The organization and homologies of these highly conserved multiple gene copies are 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.

Experimental infection of C3H mice with avian, porcine, or human isolates of Serpulina pilosicoli.

C3H/HeJ (lps(d)/lps(d)) and C3H/HeOuJ (lps(n)/lps(n)) mice were infected via gastric intubation with avian, porcine, or human isolates of weakly hemolytic spirochetes classified as Serpulina pilosicoli. Upon histopathological examination of cecal tissue from mice infected with avian or porcine isolates, colonization of spirochetes attached end-on to the apical surface of enterocytes was observed. There were no apparent differences in severity of cecal lesions between the lipopolysaccharide (LPS)-responsive (C3H/HeOuJ) and LPS-hyporesponsive (C3H/HeJ) mouse strains infected with these isolates. Transmission electron microscopy showed spirochetes invaginated into the host cell membrane with resultant effacement of microvilli and loss of the glycocalyx. End-on attachment of the human isolate S. jonesii was not observed in the present studies, although weakly hemolytic spirochetes were reisolated from mice infected with S. jonesii. Moreover, results of Western immunoblot experiments showed mice developed serum antibody responses to the S. pilosicoli isolates examined. Thus, the present results indicate that specific isolates of S. pilosicoli can colonize mice and exhibit end-on attachment to cecal enterocytes.

Comparative analysis of the genomes of intestinal spirochetes of human and animal origin.

The aim of the present work was to compare the genomes of 21 strains of intestinal spirochetes, which were isolated from patients suffering intestinal disorders, with those of Treponema hyodysenteriae (strain P18), the known etiological agent of swine dysentery (bloody scours), and of a nonpathogenic strain (M1) of Treponema innocens. The percent guanine-plus-cytosine value of the 23 DNAs was found to be 25.5 to 30.1, as determined by a double-labeling procedure based on nick-translation by DNA polymerase I. The genome size of two spirochetal strains, of human and porcine origin, was found to be similar (4 x 10(6) base pairs) and close to that of the reference bacterium Escherichia coli (4.2 x 10(6) base pairs). Restriction analysis showed the presence of two modified bases in spirochetal DNA. Methyladenine was present in the GATC sequence of DNA from 15 spirochetes of human origin, and methylcytosine was present in several sequences occurring in all strains. The DNA of T. hyodysenteriae displayed a 30 to 100% homology with respect to that of 21 spirochetes from humans, thus suggesting the occurrence of a genetic heterogeneity in the latter group. These data indicate that the intestinal spirochetes analyzed in the present work are related; hence there is a possibility of domestic animals being reservoirs of microorganisms pathogenic for humans. A classification of intestinal treponemes into subgroups has been proposed on the basis of restriction analysis and hybridization experiments.