Partial sequencing of sodA gene and its application to identification of Streptococcus dysgalactiae subsp. dysgalactiae isolated from farmed fish

AIMS: To investigate the difference between Lancefield group C Streptococcus dysgalactiae (GCSD) strains isolated from diseased fish and animals by sequencing and phylogenetic analysis of the sodA gene. METHODS AND RESULTS: The sodA gene of Strep. dysgalactiae strains isolated from fish and animals were amplified and its nucleotide sequences were determined. Although 100% sequence identity was observed among fish GCSD strains, the determined sequences from animal isolates showed variations against fish isolate sequences. Thus, all fish GCSD strains were clearly separated from the GCSD strains of other origin by using phylogenetic tree analysis. In addition, the original primer set was designed based on the determined sequences for specifically amplify the sodA gene of fish GCSD strains. The primer set yield amplification products from only fish GCSD strains. CONCLUSIONS: By sequencing analysis of the sodA gene, the genetic divergence between Strep. dysgalactiae strains isolated from fish and mammals was demonstrated. Moreover, an original oligonucletide primer set, which could simply detect the genotype of fish GCSD strains was designed. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that Strep. dysgalactiae isolated from diseased fish could be distinguished from conventional GCSD strains by the difference in the sequence of the sodA gene.     

Diversity and mobility of integrative and conjugative elements in bovine isolates of Streptococcus agalactiae, S. dysgalactiae subsp. dysgalactiae, and S. uberis

Bovine isolates of Streptococcus agalactiae (n = 76), Streptococcus dysgalactiae subsp. dysgalactiae (n = 32), and Streptococcus uberis (n = 101) were analyzed for the presence of different integrative and conjugative elements (ICEs) and their association with macrolide, lincosamide, and tetracycline resistance. The diversity of the isolates included in this study was demonstrated by multilocus sequence typing for S. agalactiae and pulsed-field gel electrophoresis for S. dysgalactiae and S. uberis. Most of the erythromycin-resistant strains carry an ermB gene. Five strains of S. uberis that are resistant to lincomycin but susceptible to erythromycin carry the lin(B) gene, and one has both linB and lnuD genes. In contrast to S. uberis, most of the S. agalactiae and S. dysgalactiae tetracycline-resistant isolates carry a tet(M) gene. A tet(S) gene was also detected in the three species. A Tn916-related element was detected in 30 to 50% of the tetracycline-resistant strains in the three species. Tetracycline resistance was successfully transferred by conjugation to an S. agalactiae strain. Most of the isolates carry an ICE integrated in the rplL gene. In addition, half of the S. agalactiae isolates have an ICE integrated in a tRNA lysine (tRNA(Lys)) gene. Such an element is also present in 20% of the isolates of S. dysgalactiae and S. uberis. A circular form of these ICEs was detected in all of the isolates tested, indicating that these genetic elements are mobile. These ICEs could thus also be a vehicle for horizontal gene transfer between streptococci of animal and/or human origin.     

Cloning and expression of serum opacity factor in fish pathogenic Streptococcus dysgalactiae and its application to discriminate between fish and mammalian isolates

Lancefield group C Streptococcus dysgalactiae (GCSD) is known as a causative agent of bovine mastitis and cardiopulmonary diseases in humans. Recently, GCSD has been isolated from diseased fish in Japan. Almost all culture supernatants and sodium dodecyl sulfate extracts obtained from GCSD isolated from farmed fish possessed serum opacity activity. Serum opacity factor (SOF) is a bifunctional cell-associated protein that causes serum opacification. In this study, a gene coding SOF, which was named sof-FD, was identified from GCSD isolated from fish. The amino acid sequence of sof-FD showed 40.1-46.5% identity to those of other SOFs from mammalian strains of S. dysgalactiae and Streptococcus pyogenes. Repetitive fibronectin binding domains were also observed in sof-FD, the structures of which were similar to those of other SOFs, as previously reported. The amino acid sequence of SOF was identical among fish isolates. A primer set targeting the sof-FD gene was designed and applied to a PCR assay for discriminating fish isolates from mammalian isolates.     

Prevalence of a Streptococcal Inhibitor of a Complement-Mediated Cell lysis-like Gene (sicG) in Streptococcus Dysgalactiae subsp. Equisimilis

Streptococcus dysgalactiae subsp. equisimilis isolates (n = 110) were analyzed by PCR to determine whether the gene encoding SICG, a homolog of Streptococcus pyogenes SIC, was present. Nineteen strains (17%) had this gene of which 11 (55%) were isolated from patients with invasive disease. All 19 strains possessed group G carbohydrate. Molecular characterization of emm type revealed that the majority of emm sequences were stG643 and stG2078. Only the N-terminal sequence of SICG was similar to that of SIC in S. pyogenes. Although we found no significant relationship between pathogenic severity and sicG possession, further investigation into the mechanism of SICG may elucidate the virulence in S. dysgalactiae subsp. equisimilis infection.     

Diversity of lactic acid bacteria associated with fish and the fish farm environment, established by amplified rRNA gene restriction analysis

Lactic acid bacteria have become a major source of concern for aquaculture in recent decades. In addition to true pathogenic species of worldwide significance, such as Streptococcus iniae and Lactococcus garvieae, several species have been reported to produce occasional fish mortalities in limited geographic areas, and many unidentifiable or ill-defined isolates are regularly isolated from fish or fish products. To clarify the nature and prevalence of different fish-associated bacteria belonging to the lactic acid bacterium group, a collection of 57 isolates of different origins was studied and compared with a set of 22 type strains, using amplified rRNA gene restriction analysis (ARDRA). Twelve distinct clusters were delineated on the basis of ARDRA profiles and were confirmed by sequencing of sodA and 16S rRNA genes. These clusters included the following: Lactococcus raffinolactis, L. garvieae, Lactococcus l., S. iniae, S. dysgalactiae, S. parauberis, S. agalactiae, Carnobacterium spp., the Enterococcus "faecium" group, a heterogeneous Enterococcus-like cluster comprising indiscernible representatives of Vagococcus fluvialis or the recently recognized V. carniphilus, V. salmoninarum, and Aerococcus spp. Interestingly, the L. lactis and L. raffinolactis clusters appeared to include many commensals of fish, so opportunistic infections caused by these species cannot be disregarded. The significance for fish populations and fish food processing of three or four genetic clusters of uncertain or complex definition, namely, Aerococcus and Enterococcus clusters, should be established more accurately.     

A new simple medium for the detection of Enterococcus faecalis and Enterococcus faecium by measurement of conductance changes

Streptococcus suis, Streptococcus bovis and the mastitis pathogens Streptococcus dysgalactiae and Streptococcus uberis were the most frequently occurring streptococci in tonsils of cattle. Streptococcus suis dominated in samples from calves between 1 month and 1 year of age, but was much less frequent in calves less than 1 month old. The mastitis pathogen Strep, dysgalactiae was found more often in calves than in older animals. Enterococci were relatively rare, except in the youngest age group. Nearly one third of the strains examined could not be identified to known species.     

Streptococcus dysgalactiae-derived mitogen (SDM), a novel bacterial superantigen: characterization of its biological activity and predicted tertiary structure

A mitogenic substance, designated Streptococcus dysgalactiae-derived mitogen (SDM), was purified from S. dysgalactiae culture supernatant, and the gene encoding the mitogen was cloned. Both native and recombinant SDM expressed in Escherichia coli significantly activated human V beta 1+ and V beta 23+ T cells in association with major histocompatibility complex (MHC) class II molecules on accessory cells, indicating that SDM possesses superantigenic properties. The sdm gene consists of two segments encoding a signal peptide and a mature 25 kDa protein composed of 212 amino acids. Three of 34 S. dysgalactiae strains but none of 28 Streptococcus pyogenes strains examined carried sdm. Phylogenetic analysis indicated that SDM belongs to a family distinct from established bacterial superantigens. SDM showed around 30% homology with other superantigens at the amino acid sequence level. The tertiary structure of SDM was predicted by modelling onto streptococcal pyrogenic exotoxin C and streptococcal mitogenic exotoxin Z-2, both of which share highly homologous structure-determining regions. SDM showed overall structural similarity to both these superantigens. This is the first study to characterize fully a bacterial superantigen from S. dysgalactiae.     

Production and properties of bacteriocin-like inhibitory substances from the swine pathogen Streptococcus suis serotype 2

Streptococcus suis serotype 2 is a major pathogen found in the upper respiratory tract of swine. In this study, isolates of this bacterial species were tested for the production of bacteriocin-like inhibitory substances (BLIS). Of the 38 strains tested, four inhibited the growth of other S. suis isolates according to a deferred-antagonism plate assay. Interestingly, three of the strains were originally isolated from healthy carrier pigs and were considered nonvirulent. Three isolates (94-623, 90-1330, and AAH4) that produced BLIS in liquid broth were selected for further characterization. None of the inhibitory activities was related to the production of either organic acids or hydrogen peroxide. The BLIS produced by these strains were heat stable and proteinase K, pronase, and elastase sensitive but were trypsin and chymotrypsin resistant. They were stable at pH 2 and 12 and had molecular masses in the range of 14 to 30 kDa. Maximum production was observed during the mid-log phase. Following a curing procedure with novobiocin, only 90-1330 lost the ability to produce BLIS, suggesting that the BLIS might be plasmid encoded. Analysis of the inhibitory spectra revealed that the BLIS-producing strains also inhibited the growth of Actinobacillus minor, Actinobacillus porcinus, Enterococcus durans, Micrococcus luteus, Streptococcus agalactiae, Streptococcus dysgalactiae subsp. dysgalactiae, Streptococcus equi subsp. zooepidemicus, and S. dysgalactiae subsp. equisimilis. This study reports for the first time the ability of the swine pathogen S. suis serotype 2 to produce BLIS with the characteristics of classic bacteriocins. Further studies are required to investigate the possibility of using bacteriocin-producing strains to prevent swine infections caused by virulent strains of S. suis serotype 2.     

A genetic comparison of pig, cow and trout isolates of Lactococcus garvieae by PFGE analysis

Aims: Genetic comparison of Lactococcus garvieae isolated from mammals and fish. Methods and Results: One hundred and ninety‐seven L. garvieae isolates obtained from trout (n = 153), cow (n = 7) and pigs (n = 37) were genetically characterized by determining their pulsed‐field gel electrophoresis (PFGE) profiles after macrorestriction with Bsp120I. Overall, L. garvieae isolates from pigs, cow and trout exhibited distinct PFGE patterns, with a low genetic relationship between them. Isolates from trout generated two pulsotypes [Genetic diversity (GD) 0·01] showing that the fish isolates were more genetically homogenous than the others. The L. garvieae isolates from cows displayed five (GD 0·71) different pulsotypes, while the swine isolates displayed 13 different pulsotypes (GD 0·35). Twenty‐one of the 37 swine strains (56·8%) were grouped in a single cluster that included two closely related (93% similarity) pulsotypes. These pulsotypes exhibited a high frequency of isolation from different organs of the animals, and they were also broadly distributed among herds, suggesting a wide distribution across the swine population. This suggests that L. garvieae might be able to colonize different organs of the swine cardio‐respiratory system. Conclusions: Results indicate that most L. garvieae isolates from pigs and trout exhibited a distinct genetic background. Significance and Impact of the Study: The present study describes the isolation of L. garvieae from both diseased and healthy pigs for the first time, and the findings suggest that pigs could be a previously unknown reservoir of this pathogen.     

Phenotypic and Genotypic Changes in the Phytophthora infestans Population in Taiwan – 1991 to 2006

Late blight, caused by Phytophthora infestans , is one of the most destructive diseases of tomato in Taiwan. A total of 655 isolates of P. infestans , including 29 isolates from potato, was collected from major tomato and potato production areas of Taiwan during 1991 to 2006. Isolates were characterized for their pathogenicity, mating type, in vitro metalaxyl sensitivity and molecular genotype (including allozyme pattern, mitochondrial genomic haplotype and DNA fingerprint) to monitor population changes in P. infestans . The population of P. infestans in Taiwan underwent a dramatic genetic shift in the 1997–1998 cool growing season. Isolates collected from tomato before 1997 were aggressive to tomato but not potato; most isolates obtained after 1998, were aggressive to both hosts. Metalaxyl sensitivity of isolates changed from sensitive/intermediate before 1997 to resistant since 1998. Similarly, the isolates obtained before 1997 were all US-1 clonal lineage (including variants US-1.1, US-1.2, US-1.3 and US-1.4). During the 1997–1998 cool growing season, the US-11 clonal lineage and the TW-1 genotype appeared, possibly introduced on imported table potatoes. The US-11 lineage spread rapidly and since 1999 has almost completely displaced the old population in Taiwan. Mating type determined by pairing with A1 and A2 reference isolages of P. parasitica , showed all isolates were of the A1 mating type, suggesting that the A2 mating type had not become established in Taiwan. The increasing percentage (up to 42.3% in 2006) of the US-11 variants (including US-11.l, US-11.2, US-11.3 and US-11.4) implied that genomic diversity of the pathogen is changing quickly. Therefore, it is important to continuously monitor the population changes of P. infestans and develop an integrated management strategy for this disease.     

Differentiation of human and animal strains of Streptococcus dysgalactiae by pulsed-field gel electrophoresis

The genetic diversity among 54 human isolates and 33 animal isolates belonging to the species Streptococcus dysgalactiae (20 α-haemolytic Streptococcus dysgalactiae, 23 Streptococcus equisimilis, 43 group G streptococci and one group L streptococcus) was evaluated by macrorestriction analysis of chromosomal DNA with SmaI and resolution by pulsed-field gel electrophoresis. This technique revealed a high degree of intraspecies polymorphism, leading to the differentiation of 80 distinct banding patterns, and identified the presence of two major clusters, one containing isolates of human origin and the other isolates of animal origin. These results suggest that human and animal isolates of S. dysgalactiae are genetically distinct, and support the recent proposal of the subspecies S. dysgalactiae subsp. equisimilis for human isolates. The heterogeneity revealed within isolates from the same host type indicates that pulsed-field gel electrophoresis is a powerful epidemiological tool for studying S. dysgalactiae infections.     

Genotypic and Phenotypic Profiles of Enterotoxigenic <Emphasis Type="Italic">Escherichia coli</Emphasis> Associated with Acute Diarrhea in Tunis,Tunisia

No past studies of acute diarrhea in Tunisia have examined the phenotypic and genotypic profiles of enterotoxigenic Escherichia coli (ETEC) isolates. We determined 65 ETEC isolates derived from a total of 327 E. coli isolates collected from a previous study (acute diarrheal and healthy persons, children and adults n = 214) and 32 E. coli isolates derived from an acute diarrheal outbreak in Kabaria-Ennour city, Tunis. All E. coli isolates were screened by polymerase chain reaction (PCR) for ETEC virulence genes: sta (heat-stable toxin gene) and elt (heat-labile toxin gene). Seventy-two percent (47 of 65) of ETEC strains expressed the sta gene only, 21.5% (14 of 65) expressed the elt gene and 6.1% (4 of 65) expressed both genes. For the outbreak isolates, the elt gene was predominant (10 isolates out of 14). Ganylioside GM1 enzyme-linked immunosorbent assay (GM1-ELISA) was used to validate the PCR results and this was confirmed by dot blot assay. The same results were obtained. The most common colonization factors (CFs) were CFA/I (44.6%) and coli surface antigen 6 (CS6) (11%), and 44.6% of the isolates showed no association with either CFAs. Resistance of ETEC isolates to tetracycline (38.5%), streptomycin (26%), and beta-lactam agents (ticarcillin 26%, amoxicillin 24.6%, cephalotin 21.5%) was common. Regarding serotypes, the majority of ETEC isolates serotyped as O86:H(-) (n = 16), O128:H2 (n = 11), and O127:H21 (n = 10). Other serotypes found were O111:H(-) (n = 6) and O126: H(-) (n = 5). DNA macrorestriction fragment analysis by pulsed-field gel electrophoresis (PFGE) using the XbaI enzyme was conducted to investigate the epidemiological clonal relationship among ETEC isolates. Major patterns were identified among which some of outbreak ETEC isolates belonged. These data suggest that a proportion of acute diarrhea in Tunis represents the confluence of small epidemics by clonality-related ETEC isolates that are transiently introduced or that persist in our community.     

Application of Congo Red agar for detection of Streptococcus dysgalactiae isolated from diseased fish

The strong clinical similarity between Lancefield group C Streptococcus dysgalactiae subsp. dysgalactiae (GCSD) and Lactococcus garvieae infections, characterized by development of necrotic lesions in the caudal peduncle of infected fish, has hindered differentiation of these two strains, making rapid and accurate diagnosis of diseased fish in fish farms difficult. GCSD from diseased fish were presumptively identified and isolated using Todd-Hewitt agar containing 30 μg ml⁻¹ of Congo Red dye (TH-CR). TH-CR agar was also used to detect and presumptively identify the GCSD obtained from artificially or naturally infected fish. Orange GCSD colonies distinct from the L. garvieae colonies were observed on the TH-CR agar; thus, TH-CR agar can be used to detect and identify GCSD isolated from infected fish.     

A novel IS-like element frequently inserted in a putative virulence regulator in bovine mastitis isolates of Streptococcus dysgalactiae

Streptococcus dysgalactiae, a Lancefield group C streptococcus, is commonly isolated from bovine mastitis. We recently identified a putative regulon in two S. dysgalactiae strains, 8215 and Epi9, consisting of two consecutive genes, dmg and dem, coding for a possible regulatory protein and an M-like protein with fibrinogen- and IgG-binding-properties, respectively. During these studies a short sequence homologous to an IS element was found to be inserted in the dmg gene of strain 8215. The present investigation describes the complete sequence of this IS-like element, named ISSdy1, which consists of 1218 bp and contains two ORFs, flanked by imperfect repeats. The nucleotide sequence of the IS-like element shows 82% identity to the previously reported sequence of IS199 from Streptococcus mutans V403. The deduced amino acid sequences of the ORFs also revealed high homology to transposases from IS elements in Enterococcus faecium, Escherichia coli, and Shigella dysenteriae, all belonging to the IS3 family. We studied the distribution of ISSdy1 in 57 S. dysgalactiae isolates using PCR analysis with specific primers derived from the IS element. Ninety-eight percent of the isolates contained the ISSdy1 element. Surprisingly, in the majority of studied strains a copy of the IS-like element was found to be inserted in the dmg gene, a putative virulence regulator.     

Genotyping of Edwardsiella ictaluri isolates in Japan using amplified-fragment length polymorphism analysis

Aim:  The major objective of the present study was to clarify genetic relationship of isolates of Edwardsiella ictaluri in Japan, which was first found from ayu Plecoglossus altivelis in Japanese rivers in 2007.
Methods and Results:  Ten isolates of Edw. ictaluri in 2007–2008 from ayu and the 1 isolate from bagrid catfish Pelteobagrus nudiceps in Japan were subjected to amplified-fragment length polymorphism (AFLP) analysis. The strains isolated from catfish in United States (ATCC strains) or Indonesia were used as reference strains. The AFLP profiles were all the same among the isolates from Japan, while the polymorphic DNA bands were observed among the strains from United States or Indonesia. The isolates from Japan and Indonesia constituted a genogroup different from the ATCC strains on a dendrogram constructed from the AFLP profiles.
Conclusion:  No DNA polymorphisms were found among Japanese Edw. ictaluri isolates.
Significance and Impact of the Study:  A single clonality of the Edw. ictaluri isolates in Japan suggests the single source of the organism, and the infection in ayu is in the early stage of epidemics.     

Serotypes of Vibrio parahaemolyticus from clinical and environmental sources in Togo (West Africa).

Serological analysis of O and K antigens was performed on 343 strains of Vibro parahaemolyticus isolated from clinical and environmental sources in Togo. Only two strains were not typable by the available O antisera. K untypable strains were found in 4.8% of isolates from gastroenteritis patients, in 11% from healthy carriers, and in 47% and 46% of isolates, respectively, from water and fish samples. Thirteen serotypes identified in Togo are not considered in the Japanese antigenic scheme. The suitability of the Japanese typing scheme for geographic areas outside of Japan is discussed and its extension is suggested.     

Identification of lactoferrin-binding proteins in Streptococcus dysgalactiae subsp. dysgalactiae and Streptococcus agalactiae isolated from cows with mastitis

Three strains of Streptococcus dysgalactiae subsp. dysgalactiae (S. dysgalactiae) and five strains of Streptococcus agalactiae were used to identify lactoferrin-binding proteins (LBPs). LBPs from extracted surface proteins were detected by polyacrylamide gel electrophoresis and Western blotting. All strains of S. dysgalactiae evaluated had 52- and 74-kDa protein bands. All strains of S. agalactiae evaluated had 52-, 70- and 110-kDa protein bands. In addition, a 45-kDa band was detected in two of five S. agalactiae strains evaluated. This study demonstrated that S. dysgalactiae and S. agalactiae of bovine origin contain two and three major LBPs, respectively.     

A novel method for rapid production and purification of exfoliative toxin A of Staphylococcus aureus

Three strains of Streptococcus dysgalactiae subsp. dysgalactiae (UT516, UT519, ATCC 27957) were used to determine if bovine lactoferrin (Lf) binds to bacterial cells by biotin avidin binding assay (BABA), enzyme-linked immunosorbent assay (ELISA), and binding inhibition assay. Binding assays revealed that all strains of S. dysgalactiae subsp. dysgalactiae (S. dysgalactiae) evaluated in this study bound to Lf. However, differences in Lf binding capability among strains and between methods used were detected. Binding of Lf was not inhibited by transferrin (Tf) and Lf moiety molecules (mannose, galactose, and lactose) but by Lf. This study demonstrates that S. dysgalactiae bound to bovine Lf in a specific manner.     

Diversities and similarities in PFGE profiles of Campylobacter jejuni isolated from migrating birds and humans

AIMS: To genetically sub-type Campylobacter jejuni strains isolated from migratory birds, and to compare these with clinical strains collected in the same area and corresponding time period, with the aim to increase our knowledge on sub-types occurring among wild birds and their possible impact on human disease. METHODS AND RESULTS: We sub-typed C. jejuni strains from migrating birds (n = 89) and humans (n = 47), using macrorestriction profiling by pulsed-field gel electrophoresis. Isolates from migrant birds often exhibited sub-types with higher levels of similarity to isolates from birds of the same species or feeding guild, than to isolates from other groups of birds. Likewise, could the vast majority of sub-types found among the migrant bird isolates not be identified among sub-types from human cases. Only two bird strains, one from a starling (Sturnus vulgaris) and one from a blackbird (Turdus merula), had sub-types that were similar to some of the human strain sub-types. CONCLUSIONS: Isolates from one bird species, or feeding guild, often exhibited high similarities, indicating a common transmission source for individuals, or an association between certain sub-types of C. jejuni and certain ecological guilds or phylogenetic groups of birds. Sub-types occurring among wild birds were in general distinctively different from those observed in patients. The two bird isolates that were similar to human strains were isolated from bird species that often live in close associations with human settlements. SIGNIFICANCE AND IMPACT OF STUDY: Wild birds have often been mentioned as a potential route for transmission of C. jejuni to humans. Our study demonstrates that strains isolated from birds most often are different from clinical strains, but that some strain similarities occur, notably in birds strongly associated with human activities.     

Intestinal spirochetes isolated from wild-living jackdaws, hooded crows and rooks (genus Corvus): provisionally designated "Brachyspira corvi" sp. nov

Intestinal spirochetes of genus Brachyspira are commonly isolated from mammalian and avian hosts, and several species have been reported to cause enteric disease in pigs and birds. Except for a previous publication on three isolates from corvid birds (order Passeriformes, family Corvidae, genus Corvus), of which two are further studied in this paper, no other reports exist on Brachyspira spp. of passerine birds. In this study, cloacal and intestinal swabs of small and large intestines were collected from 116 corvid birds of three species, i.e. jackdaws (Corvus monedula), hooded crows (Corvus corone cornix) and rooks (Corvus frugilegus), from four separate geographical locations in Sweden. Isolates were obtained by selective culture from 43 of 116 birds. All isolates were weakly hemolytic, indole-negative and lacked hippurate cleavage capacity. Examination by light microscopy did not indicate association with enteric disease in necropsied birds. Pure spirochete cultures were obtained by serial dilution and subculture, and selected isolates were analyzed by PCR (n=14), randomly amplified polymorphic DNA (RAPD) (n=14), and sequencing of the almost complete 16S rRNA (n=14), and partial nox genes (n=4). Positive reactions were noticed by PCR targeting a hexa-T segment of the 16S rRNA gene, which has been previously reported as a signature characteristic of Brachyspira pilosicoli. By 16S rRNA gene sequencing, the isolates formed a separate cluster related to genus Brachyspira, but not consistent with any presently recognized or proposed Brachyspira sp. The sequence similarity of the 16S rRNA gene among the isolates from corvid birds was 99.7-100%. Compared to 16S rRNA gene sequence data from all presently recognized and several proposed Brachyspira spp. the sequence similarity of the isolates from corvid birds varied between 94.1 and 96.5%. In a radial tree based on nox gene sequences, all four analyzed isolates from corvid birds formed a separate cluster. By RAPD analysis, the banding patterns of the isolates differed from all type strains of Brachyspira spp. Based on the results presented in this paper, we propose that the described isolates from corvid birds belong to a novel species within genus Brachyspira, with the provisional name "Brachyspira corvi" (cor'vi. L gen. n. corvi, of a crow).