Wielerella bovis gen. nov., sp. nov. a member of the family Neisseriaceae associated with bovine endocarditis.

Seven bacterial strains isolated from bovine endocarditis in six animals from different geographic regions were investigated in a polyphasic taxonomic approach. Phylogenetic analysis based on 16S rRNA gene sequences placed all seven isolates on a distinct, monophyletic cluster in the family Neisseriaceae with closest similarity to type strains of Alysiella filiformis (97.06 %) and Kingella kingae (96.34 %). Whole genome sequence analysis of isolates confirmed their species status, with an average nucleotide identity >96 % between isolates and <80 % to other type species of genera of Neisseriaceae while digital DNA-DNA hybridization values were >80 % and<18 %, respectively. The DNA G+C content was 42.5-43.0 mol%. Whole genome sequence based phylogeny showed the isolates being monophyletic and separated from established genera, thereby forming a new genus within the family Neisseriaceae. Similarly, analysis of MALDI-TOF MS reference spectra clustered the isolates close together and clearly separated from other genera, making this the method of choice for identification. Biochemical markers based on classical as well as commercial identification schemes allowed separation from closely related Neisseriaceae genera, even though the new taxon is biochemically not very active. Major fatty acids are C12 : 0, C14 : 0 and C16 : 0. The major quinone is ubiquinone Q-8. In the polar lipid profile, diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and phospholipid were predominant. We propose the novel genus Wielerella with the type species Wielerella bovis gen. nov., sp. nov. The type strain is CCUG 44465T (=DSM 113289T=JF 2483T) isolated post mortem from a cow with endocarditis in Switzerland.

Detection of treponemes in digital dermatitis lesions of captive European bison (Bison bonasus).

A newly-discovered foot disease of unknown origin in captive European Bison (Bison bonasus) was recently detected at Berne Animal Park. Dermatitis of the interdigital cleft of varying degrees of severity was diagnosed in all animals (n = 10). The aim of this study was to describe the gross and histological lesions of the interdigital cleft found in 10 captive European bison and to identify involved potential pathogens in affected feet using molecular-based methods for Treponema spp., Dichelobacter nodosus and Fusobacterium necrophorum. Lesions were scored according to the degree of gross pathology at limb level. In a single animal, the gross lesions were restricted to focal lesions on the dorsal aspect of the digital skin of each foot (score 1), whereas all other animals showed at least one foot with extended lesions including the interdigital cleft (score 2). The presence of viable spirochaetes was observed in all animals using dark field microscopy. Applying fluorescence in situ hybridisation (FISH) on biopsies, Treponema spp. were identified, infiltrating the skin lesions in varying numbers in nine animals. Nested PCRs for Treponema medium, Treponema phagedenis and Treponema pedis of swab samples showed three positive animals out of ten for the latter two, whereas pooled biopsy samples were positive in all ten animals for at least T. phagedenis (9/10) and/or T. pedis (7/10), while all samples were negative for T. medium. However, none of these Treponema species could be isolated and sequence analysis of the amplified products showed 100% match of 365 base pairs (bp) to Treponema phylotype PT3 and almost full match (530 of 532 bp, 99.6%) to Treponema phylotype PT13. The presence of T. phagedenis, PT3 and PT13 phylotypes was confirmed by FISH analyses. The phylotypes of T. phagedenis were present in all hybridized positive biopsies of Treponema spp., and PT13 and PT3 were less abundant. Neither D. nodosus nor F. necrophorum were detected. The histological Treponema score was mostly mild. Digital dermatitis in captive European Bison is contagious and differs from bovine digital dermatitis, concerning associated pathogens as well as gross appearance.

A filter-assisted culture method for isolation of Treponema spp. from bovine digital dermatitis and their identification by MALDI-TOF MS.

Digital dermatitis (DD) is a major infectious foot disease of cattle worldwide. Some DD stages are associated with lameness, and the disease has significant economic and animal welfare consequences. The pathogenesis of the disease is not yet fully understood, but Treponema spp. have been associated consistently with clinical cases. Isolation of these fastidious bacteria is difficult and cumbersome. We describe an improved method enabling the culturing of the 3 Treponema spp. (T. pedis, T. phagedenis, and T. medium) from bovine foot specimens derived from DD lesions, using a combination of membrane filtering and subsequent growth on selective agar media. The entire procedure from sampling to verification of individual Treponema spp. takes up to 24 d. In addition, we established a MALDI-TOF MS-based identification method to be applied for confirmation of the different Treponema spp. This scheme provides an unambiguous, simple, and straightforward identification procedure for DD-associated Treponema spp.

Mannheimia pernigra sp. nov., isolated from bovine respiratory tract.

Over a period of 1 year, 270 isolates identified as Taxon 39 of Bisgaard were obtained from the nasopharynx of veal calves at 11 epidemiologically independent Swiss fattening farms. Two isolates from each farm and the Australian Taxon 39 reference strain BNO311 were further characterized by genetic and phenotypic methods. Phylogenetic analysis of 16S rRNA and recN gene sequences placed the isolates in a single, distinct cluster within the genus Mannheimia. As to the rpoB gene, most isolates clustered together, but four strains formed a separate cluster close to Mannheimia varigena. Genome sequence analysis of isolates from both rpoB clusters confirmed their species status, with an average nucleotide identity (ANI) >98.9 % between isolates and <84 % to the closest species, M. varigena. Based upon whole genome sequences, the G+C content was determined as 39.1 mol%. Similarly, analysis of MALDI-TOF MS reference spectra clustered the isolates clearly separated from the other Mannheimia species, making this the method of choice for identification. In addition, numerous biochemical markers based on classical as well as commercial identification schemes were determined, allowing separation from other Mannheimia species and identification of the new taxon. Major fatty acids for strain 17CN0883T are C14 : 0, C16 : 0, C16 : 1 ω7c and C18 : 1 ω7c. Major respiratory quinones are ubiquinone-7 and ubiquinone-8. We propose the name Mannheimia pernigra sp. nov. for former Taxon 39 of Bisgaard. The type strain is 17CN0883T (=CCUG 74657T=DSM 111153T) isolated from a veal calf in Switzerland.

In vitro and ex vivo testing of alternative disinfectants to currently used more harmful substances in footbaths against Dichelobacter nodosus.

A footbath-based control program for ovine footrot, a contagious disease caused by Dichelobacter nodosus, will be implemented in Switzerland. The currently used footbath disinfectants formaldehyde, zinc sulfate and copper sulfate are carcinogenic or environmental pollutants. Hence, the aim of this study was to identify alternative disinfectants, which are highly effective, non-carcinogenic, environmentally acceptable, inexpensive, available as concentrate and suitable for licensing. The antimicrobial effect of a series of potential chemicals such as lactic acid, propionic acid, hydrogen peroxide, sodium hypochlorite, octenidine dihydrochloride, chlorocresol, Ampholyt 20 and the registered biocide DESINTEC® Hoof Care Special D (Desintec) were investigated by culture based in vitro testing. The microcidal effect of various Desintec concentrations were then compared against routinely used 4% formaldehyde and 10% zinc sulfate in ex vivo assays on sheep feet from slaughter. For this purpose a newly established PMA (propidium monoazid) real-time PCR using the improved dye PMAxx™ was applied that allows discrimination of viable and dead D. nodosus. In the ex vivo experiments, 4% formaldehyde was significantly more effective than 10% zinc sulfate and was chosen as positive control for assessing the new disinfectant. The disinfectant effect of Desintec in a minimal concentration of 6% was equally effective as 4% formaldehyde, meaning that it offers a comparable antimicrobial effect against virulent D. nodosus. In conclusion, Desintec is a promising disinfectant for replacing formaldehyde, copper sulfate and zinc sulfate in footbaths against footrot.

Treponema phagedenis (ex Noguchi 1912) Brumpt 1922 sp. nov., nom. rev., isolated from bovine digital dermatitis.

'Treponema phagedenis' was originally described in 1912 by Noguchi but the name was not validly published and no type strain was designated. The taxon was not included in the Approved Lists of Bacterial Names and hence has no standing in nomenclature. Six Treponema strains positive in a 'T. phagedenis' phylogroup-specific PCR test were isolated from digital dermatitis (DD) lesions of cattle and further characterized and compared with the human strain 'T. phagedenis' ATCC 27087. Results of phenotypic and genotypic analyses including API ZYM, VITEK2, MALDI-TOF and electron microscopy, as well as whole genome sequence data, respectively, showed that they form a cluster of species identity. Moreover, this species identity was shared with 'T. phagedenis'-like strains reported in the literature to be regularly isolated from bovine DD. High average nucleotide identity values between the genomes of bovine and human 'T. phagedenis' were observed. Slight genomic as well as phenotypic variations allowed us to differentiate bovine from human isolates, indicating host adaptation. Based on the fact that this species is regularly isolated from bovine DD and that the name is well dispersed in the literature, we propose the species Treponema phagedenis sp. nov., nom. rev. The species can phenotypically and genetically be identified and is clearly separated from other Treponema species. The valid species designation will allow to further explore its role in bovine DD. The type strain for Treponema phagedenis sp. nov., nom. rev. is B43.1T (=DSM 110455T=NCTC 14362T) isolated from a bovine DD lesion in Switzerland.

Differential innate immune responses induced by Mycoplasma hyopneumoniae and Mycoplasma hyorhinis in various types of antigen presenting cells.

Mycoplasma (M.) hyopneumoniae is the etiological agent of enzootic pneumonia in pigs and is closely related to M. hyorhinis, which can be isolated from the healthy mucosal surfaces of the upper respiratory tract. In rare cases it can also cause arthritis and polyserositis. Since the innate immune system is an important first line of defense and promotes adaptive immune responses, we characterized the innate immune response of various antigen presenting cells (APCs) to M. hyopneumoniae and M. hyorhinis, which differ in their pathogenicity in vivo. Porcine peripheral blood mononuclear cells were infected with different multiplicities of infection (MOI) of live and inactivated porcine mycoplasmas. Both Mycoplasma species induced strong tumour necrosis factor (TNF) responses in monocytes, with a stronger activation by M. hyorhinis. This higher stimulatory activity was also confirmed for CD40 upregulation. Conventional and plasmacytoid dendritic cells (cDC and pDC, respectively) did not or poorly respond to mycoplasmas in terms of TNF expression but more efficiently in terms of CD40 upregulation. Again, these responses were generally stronger with M. hyorhinis than with M. hyopneumoniae. Both Mycoplasma species also activated B cells in terms of CD25 upregulation, proliferation, and IgM secretion. Interestingly, while the induction of CD25 and in particular proliferation was higher with M. hyorhinis, the IgM secretion did not differ between the two species with the exception of the highest dose of M. hyopneumoniae,which appeared to suppress IgM responses. Taken together, our results provide a comparative analysis of innate immune response with different porcine APCs and demonstrate Mycoplasma species-dependent differences, which could relate to their different pathogenicity in vivo.

Whole-genome-based revisit of Photorhabdus phylogeny: proposal for the elevation of most Photorhabdus subspecies to the species level and description of one novel species Photorhabdus bodei sp. nov., and one novel subspecies Photorhabdus laumondii subsp. clarkei subsp. nov.

Bacterial symbionts are crucial for the infectivity and success of entomopathogenic nematodes as biological control agents. The current understanding of the symbiotic relationships is limited by taxonomic uncertainties. Here, we used whole-genome sequencing and traditional techniques to reconstruct the phylogenetic relationships between all described Photorhabdus species and subspecies as well as 11 newly isolated symbiotic bacteria of Heterorhabditis nematodes, including the unreported bacterial partner of H. beicherriana. In silico DNA-DNA hybridization, orthologous average nucleotide identity and nucleotide sequence identity of concatenated housekeeping genes scores were calculated and set into relation with current cut-off values for species delimitation in bacteria. Sequence data were complemented with biochemical and chemotaxonomic markers, and ribosomal protein fingerprinting profiles. This polyphasic approach resolves the ambiguous taxonomy of Photorhabdusand lead to the proposal for the elevation of most of them into a higher taxon and the creation of several new taxa: 15 new species, one of which is newly described: Photorhabdus bodei sp. nov. (type strain LJ24-63T=DSM 105690T=CCOS 1159T) and the other 14 arise through the proposal of elevating already described subspecies to species, and are proposed to be renamed as follows: Photorhabdus asymbioticasubsp. australis as Photorhabdus australis sp. nov., Photorhabdus luminescenssubsp. akhurstii as Photorhabdus akhurstii sp. nov., Photorhabdus luminescenssubsp. caribbeanensis as Photorhabdus caribbeanensis sp. nov., Photorhabdus luminescenssubsp. hainanensis as Photorhabdus hainanensis sp. nov., Photorhabdus luminescenssubsp. kayaii as Photorhabdus kayaii sp. nov., Photorhabdus luminescenssubsp. kleinii as Photorhabdus kleinii sp. nov., Photorhabdus luminescenssubsp. namnaonensis as Photorhabdus namnaonensis sp. nov., Photorhabdus luminescenssubsp. noenieputensis as Photorhabdus noenieputensis sp. nov., Photorhabdus luminescenssubsp.laumondii as Photorhabdus laumondii sp. nov., Photorhabdus temperatasubsp. cinerea as Photorhabdus cinerea sp. nov., Photorhabdus temperatasubsp. khanii as Photorhabdus khanii sp. nov., Photorhabdus temperatasubsp. stackebrandtii as Photorhabdus stackebrandtii sp. nov., Photorhabdus temperatasubsp. tasmaniensis as Photorhabdus tasmaniensis sp. nov., and Photorhabdus temperatasubsp. thracensis as Photorhabdus thracensis sp. nov. In addition, we propose the creation of two new subspecies, one of which arises through the reduction of rank: Photorhabdus laumondii subsp. laumondii comb. nov. (basonym: P. luminescenssubsp. laumondii) and the second one is newly described: Photorhabdus laumondii subsp. clarkei subsp. nov. (type strain BOJ-47T=DSM 105531T=CCOS 1160T). Finally, we propose to emend the description of three species, which results from the proposal of elevating three subspecies to the species status: Photorhabdus asymbiotica, Photorhabdus temperata and Photorhabdus luminescens, formerly classified as Photorhabdus asymbioticasubsp. asymbiotica, Photorhabdus temperatasubsp.temperata and Photorhabdus luminescenssubsp. luminescens, respectively.

Uruburuella testudinis sp. nov., isolated from tortoise (Testudo).

A polyphasic taxonomic analysis was carried out on 11 uncommon Gram-stain-negative, non-motile, catalase- and oxidase-positive, but indole-negative, bacterial strains isolated from tortoises. Phenotypically and genetically they represented a homogeneous group of organisms most closely related to, but distinct from, Uruburuella suis. In a reconstructed 16S rRNA gene tree they clustered on a monophyletic branch next to U. suis with gene similarities between strains of 99.5-100%, and of up to 98.2% with U. suis . DNA-DNA hybridization indicated the organisms represented a novel species with only 40% DNA-DNA similarity with U. suis . Partial sequencing of rpoB resulted in two subclusters confirming the 16S rRNA gene phylogeny; both genes allowed clear separation and identification of the novel species. Furthermore, they could be unambiguously identified by matrix-assisted laser desorption ionization time-of-flight MS, where, again, they formed a highly homogeneous cluster separate from U. suis and other members of the family Neisseriaceae . The major fatty acids were C(16 : 0) and summed feature C(16 : 1)ω7c/iso-C(15 : 0) 2-OH. The DNA G+C content was 54.4 mol%. Based on phenotypic and genetic data we propose classifying these organisms as representatives of a novel species named Uruburuella testudinis sp. nov. The type strain is 07_OD624(T) ( = DSM 26510(T) = CCUG 63373(T)).

Frederiksenia canicola gen. nov., sp. nov. isolated from dogs and human dog-bite wounds.

Polyphasic analysis was done on 24 strains of Bisgaard taxon 16 from five European countries and mainly isolated from dogs and human dog-bite wounds. The isolates represented a phenotypically and genetically homogenous group within the family Pasteurellaceae. Their phenotypic profile was similar to members of the genus Pasteurella. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry clearly identified taxon 16 and separated it from all other genera of Pasteurellaceae showing a characteristic peak combination. Taxon 16 can be further separated and identified by a RecN protein signature sequence detectable by a specific PCR. In all phylogenetic analyses based on 16S rRNA, rpoB, infB and recN genes, taxon 16 formed a monophyletic branch with intraspecies sequence similarity of at least 99.1, 90.8, 96.8 and 97.2 %, respectively. Taxon 16 showed closest genetic relationship with Bibersteinia trehalosi as to the 16S rRNA gene (95.9 %), the rpoB (89.8 %) and the recN (74.4 %), and with Actinobacillus lignieresii for infB (84.9 %). Predicted genome similarity values based on the recN gene sequences between taxon 16 isolates and the type strains of known genera of Pasteurellaceae were below the genus level. Major whole cell fatty acids for the strain HPA 21(T) are C14:0, C16:0, C18:0 and C16:1 ω7c/C15:0 iso 2OH. Major respiratory quinones are menaquinone-8, ubiquinone-8 and demethylmenaquinone-8. We propose to classify these organisms as a novel genus and species within the family of Pasteurellaceae named Frederiksenia canicola gen. nov., sp. nov. The type strain is HPA 21(T) (= CCUG 62410(T) = DSM 25797(T)).

Evolution of the leukotoxin promoter in genus Mannheimia.

BACKGROUND: The Mannheimia species encompass a wide variety of bacterial lifestyles, including opportunistic pathogens and commensals of the ruminant respiratory tract, commensals of the ovine rumen, and pathogens of the ruminant integument. Here we present a scenario for the evolution of the leukotoxin promoter among representatives of the five species within genus Mannheimia. We also consider how the evolution of the leukotoxin operon fits with the evolution and maintenance of virulence. RESULTS: The alignment of the intergenic regions upstream of the leukotoxin genes showed significant sequence and positional conservation over a 225-bp stretch immediately proximal to the transcriptional start site of the lktC gene among all Mannheimia strains. However, in the course of the Mannheimia genome evolution, the acquisition of individual noncoding regions upstream of the conserved promoter region has occurred. The rate of evolution estimated branch by branch suggests that the conserved promoter may be affected to different extents by the types of natural selection that potentially operate in regulatory regions. Tandem repeats upstream of the core promoter were confined to M. haemolytica with a strong association between the sequence of the repeat units, the number of repeat units per promoter, and the phylogenetic history of this species. CONCLUSION: The mode of evolution of the intergenic regions upstream of the leukotoxin genes appears to be highly dependent on the lifestyle of the bacterium. Transition from avirulence to virulence has occurred at least once in M. haemolytica with some evolutionary success of bovine serotype A1/A6 strains. Our analysis suggests that changes in cis-regulatory systems have contributed to the derived virulence phenotype by allowing phase-variable expression of the leukotoxin protein. We propose models for how phase shifting and the associated virulence could facilitate transmission to the nasopharynx of new hosts.

Hormographiella aspergillata keratomycosis in a dog.

A 4-year-old, female, Border Collie was presented to the University of Bern Veterinary Teaching Hospital, because of a corneal lesion of 10 days duration. The axial cornea presented a whitish fluorescein-positive plaque with irregular margins. A diagnosis of keratomycosis was made based on cytology. Medical therapy with local broad-spectrum antibiotic and fluconazole was instituted. After 1 week of treatment, the improvement was deemed unsatisfactory. Therefore, a lamellar keratectomy and conjunctival pedicle flap were performed. After surgery, the cornea healed uneventfully. Histology confirmed the diagnosis of keratomycosis. The fungus could not be grown in culture and a precise etiological diagnosis could only be obtained with genetic identification of the fungus. A PCR technique was used to amplify the fungal genome from the cornea. Hormographiella aspergillata, the asexual reproductive form of the basidiomycete Coprinopsis cinerea, was identified. As advised in human medicine, we encourage the use of this molecular technique to obtain an early species diagnosis, allowing targeted medical therapy.

Routine phenotypic identification of bacterial species of the family Pasteurellaceae isolated from animals.

Pasteurellaceae are bacteria with an important role as primary or opportunistic, mainly respiratory, pathogens in domestic and wild animals. Some species of Pasteurellaceae cause severe diseases with high economic losses in commercial animal husbandry and are of great diagnostic concern. Because of new data on the phylogeny of Pasteurellaceae, their taxonomy has recently been revised profoundly, thus requiring an improved phenotypic differentiation procedure to identify the individual species of this family. A new and simplified procedure to identify species of Actinobacillus, Avibacterium, Gallibacterium, Haemophilus, Mannheimia, Nicoletella, and Pasteurella, which are most commonly isolated from clinical samples of diseased animals in veterinary diagnostic laboratories, is presented in the current study. The identification procedure was evaluated with 40 type and reference strains and with 267 strains from routine diagnostic analysis of various animal species, including 28 different bacterial species. Type, reference, and field strains were analyzed by 16S ribosomal RNA (rrs) and rpoB gene sequencing for unambiguous species determination as a basis to evaluate the phenotypic differentiation schema. Primary phenotypic differentiation is based on beta-nicotinamide adenine dinucleotide (beta-NAD) dependence and hemolysis, which are readily determined on the isolation medium. The procedure divides the 28 species into 4 groups for which particular biochemical reactions were chosen to identify the bacterial species. The phenotypic identification procedure allowed researchers to determine the species of 240 out of 267 field strains. The procedure is an easy and cost-effective system for the rapid identification of species of the Pasteurellaceae family isolated from clinical specimens of animals.

Analysis of gene order data supports vertical inheritance of the leukotoxin operon and genome rearrangements in the 5' flanking region in genus Mannheimia.

BACKGROUND: The Mannheimia subclades belong to the same bacterial genus, but have taken divergent paths toward their distinct lifestyles. For example, M. haemolytica + M. glucosida are potential pathogens of the respiratory tract in the mammalian suborder Ruminantia, whereas M. ruminalis, the supposed sister group, lives as a commensal in the ovine rumen. We have tested the hypothesis that vertical inheritance of the leukotoxin (lktCABD) operon has occurred from the last common ancestor of genus Mannheimia to any ancestor of the diverging subclades by exploring gene order data. RESULTS: We examined the gene order in the 5' flanking region of the leukotoxin operon and found that the 5' flanking gene strings, hslVU-lapB-artJ-lktC and xylAB-lktC, are peculiar to M. haemolytica + M. glucosida and M. granulomatis, respectively, whereas the gene string hslVU-lapB-lktC is present in M. ruminalis, the supposed sister group of M. haemolytica + M. glucosida, and in the most ancient subclade M. varigena. In M. granulomatis, we found remnants of the gene string hslVU-lapB-lktC in the xylB-lktC intergenic region. CONCLUSION: These observations indicate that the gene string hslVU-lapB-lktC is more ancient than the hslVU-lapB-artJ-lktC and xylAB-lktC gene strings. The presence of (remnants of) the ancient gene string hslVU-lapB-lktC among any subclades within genus Mannheimia supports that it has been vertically inherited from the last common ancestor of genus Mannheimia to any ancestor of the diverging subclades, thus reaffirming the hypothesis of vertical inheritance of the leukotoxin operon. The presence of individual 5' flanking regions in M. haemolytica + M. glucosida and M. granulomatis reflects later genome rearrangements within each subclade. The evolution of the novel 5' flanking region in M. haemolytica + M. glucosida resulted in transcriptional coupling between the divergently arranged artJ and lkt promoters. We propose that the chimeric promoter have led to high level expression of the leukotoxin operon which could explain the increased potential of certain M. haemolytica + M. glucosida strains to cause a particular type of infection.

Emended description of Actinobacillus capsulatus Arseculeratne 1962, 38AL.

The taxonomic position of Actinobacillus capsulatus, a member of the family Pasteurellaceae found in rabbits, hares and hamsters, has been challenged. 16S rRNA gene (rrs) sequence data show the species to be heterogeneous. Using a polyphasic approach, 23 strains that were identified previously as belonging, or closely related, to A. capsulatus were analysed. Eighty characters were included in the phenotypic analysis. Phylogenetic analysis was done based on rrs, rpoB, infB and recN sequences. In addition, the recN sequence similarities were used to calculate the whole-genome sequence relatedness of all strains investigated as well as that with other members of the family Pasteurellaceae. The phenotypic analysis allowed identification of five groups. The major group of 17 strains could be classified as A. capsulatus. Two hamster isolates were closely related to A. capsulatus but differed in a few characters. Single isolates from a rabbit and snowshoe-hare were phenotypically related to Actinobacillus suis. One rabbit isolate was related to the genus Mannheimia, while another isolate could not be classified phenotypically with known taxa. The phylogenetic analysis confirmed the phenotypic grouping. In contrast to the rrs-based tree, the A. capsulatus strains clustered unambiguously with the type species and related species of the genus Actinobacillus in the rpoB-, infB- and recN-based trees. Genome similarity comparison using recN finally confirmed the high genomic relationship of the A. capsulatus strains with the type species and related species of the genus Actinobacillus and allowed a clear assignment of the other unrelated strains to the phenotypic and phylogenetic clusters outlined. The present findings allow the description of A. capsulatus to be emended and separate it more clearly from other species, both phenotypically and genotypically. The type strain of A. capsulatus is CCUG 12396(T) (=Frederiksen 243(T)=ATCC 51571(T)=NCTC 11408(T)=CIP 103283(T)).

Proposed minimal standards for the description of genera, species and subspecies of the Pasteurellaceae.

Principles and guidelines are presented to ensure a solid scientific standard of papers dealing with the taxonomy of taxa of Pasteurellaceae Pohl 1981. The classification of the Pasteurellaceae is in principle based on a polyphasic approach. DNA sequencing of certain genes is very important for defining the borders of a taxon. However, the characteristics that are common to all members of the taxon and which might be helpful for separating it from related taxa must also be identified. Descriptions have to be based on as many strains as possible (inclusion of at least five strains is highly desirable), representing different sources with respect to geography and ecology, to allow proper characterization both phenotypically and genotypically, to establish the extent of diversity of the cluster to be named. A genus must be monophyletic based on 16S rRNA gene sequence-based phylogenetic analysis. Only in very rare cases is it acceptable that monophyly can not be achieved by 16S rRNA gene sequence comparison. Recently, the monophyly of genera has been confirmed by sequence comparison of housekeeping genes. In principle, a new genus should be recognized by a distinct phenotype, and characters that separate the new genus from its neighbours should be given clearly. Due to the overall importance of accurate classification of species, at least two genotypic methods are needed to show coherence and for separation at the species level. The main criterion for the classification of a novel species is that it forms a monophyletic group based on 16S rRNA gene sequence-based phylogenetic analysis. However, some groups might also include closely related species. In these cases, more sensitive tools for genetic recognition of species should be applied, such as DNA-DNA hybridizations. The comparison of housekeeping gene sequences has recently been used for genotypic definition of species. In order to separate species, phenotypic characters must also be identified to recognize them, and at least two phenotypic differences from existing species should be identified if possible. We recommend the use of the subspecies category only for subgroups associated with disease or similar biological characteristics. At the subspecies level, the genotypic groups must always be nested within the boundaries of an existing species. Phenotypic cohesion must be documented at the subspecies level and separation between subspecies and related species must be fully documented, as well as association with particular disease and host. An overview of methods previously used to characterize isolates of the Pasteurellaceae has been given. Genotypic and phenotypic methods are separated in relation to tests for investigating diversity and cohesion and to separate taxa at the level of genus as well as species and subspecies.

Nicoletella semolina gen. nov., sp. nov., a new member of Pasteurellaceae isolated from horses with airway disease.

Gram-negative, nonmotile bacteria that are catalase, oxidase, and urease positive are regularly isolated from the airways of horses with clinical signs of respiratory disease. On the basis of the findings by a polyphasic approach, we propose that these strains be classified as Nicoletella semolina gen. nov, sp. nov., a new member of the family Pasteurellaceae. N. semolina reduces nitrate to nitrite but is otherwise biochemically inert; this includes the lack of an ability to ferment glucose and other sugars. Growth is fastidious, and the isolates have a distinctive colony morphology, with the colonies being dry and waxy and looking like a semolina particle that can be moved around on an agar plate without losing their shape. DNA-DNA hybridization data and multilocus phylogenetic analysis, including 16S rRNA gene (rDNA), rpoB, and infB sequencing, clearly placed N. semolina as a new genus in the family Pasteurellaceae. In all the phylogenetic trees constructed, N. semolina is on a distinct branch displaying approximately 5% 16S rDNA, approximately 16% rpoB, and approximately 20% infB sequence divergence from its nearest relative within the family Pasteurellaceae. High degrees of conservation of the 16S rDNA (99.8%), rpoB (99.6%), and infB (99.7%) sequences exist within the species, indicating that N. semolina isolates not only are phenotypically homogeneous but also are genetically homogeneous. The type strain of N. semolina is CCUG43639(T) (DSM16380(T)).

Comparative phylogenies of the housekeeping genes atpD, infB and rpoB and the 16S rRNA gene within the Pasteurellaceae.

Phylogenies of housekeeping gene and 16S rRNA gene sequences were compared to improve the classification of the bacterial family Pasteurellaceae and knowledge of the evolutionary relationships of its members. Deduced partial protein sequences of the housekeeping genes atpD, infB and rpoB were compared in 28, 36 and 28 representative taxa of the Pasteurellaceae, respectively. The monophyly of representatives of the genus Gallibacterium was recognized by analysis of all housekeeping genes, while members of Mannheimia, Actinobacillus sensu stricto and the core group of Pasteurella sensu stricto formed monophyletic groups with two out of three housekeeping genes. Representatives of Mannheimia, Actinobacillus sensu stricto, [Haemophilus] ducreyi and [Pasteurella] trehalosi formed a monophyletic unit by analysis of all three housekeeping genes, which was in contrast to the 16S rRNA gene-derived phylogeny, where these taxa occurred at separate positions in the phylogenetic tree. Representatives of the Rodent, Avian and Aphrophilus-Haemophilus 16S rRNA gene groups were weakly supported by phylogenetic analysis of housekeeping genes. Phylogenies derived by comparison of the housekeeping genes diverged significantly from the 16S rRNA gene-derived phylogeny as evaluated by the likelihood ratio test. A low degree of congruence was also observed between the individual housekeeping gene-derived phylogenies. Estimates on speciation derived from 16S rRNA and housekeeping gene sequence comparisons resulted in quite different evolutionary scenarios for members of the Pasteurellaceae. The phylogeny based on the housekeeping genes supported observed host associations between Mannheimia, Actinobacillus sensu stricto and [Pasteurella] trehalosi and animals with paired hooves.

Laribacter hongkongensis: a potential cause of infectious diarrhea.

In this study, we describe the isolation of Laribacter hongkongensis, a recently described genus and species of bacterium, in pure culture on charcoal cefoperazone deoxycholate agar from the stool of six patients with diarrhea. Three patients were residents of Hong Kong, and three of Switzerland. In none of the stool samples obtained from these six patients was Salmonella, Shigella, enterohemorrhagic Escherichia coli, Vibrio, Aeromonas, Plesiomonas, or Campylobacter recovered. Rotavirus antigen detection, electron microscopic examination for viruses, and microscopic examinations for ova and cysts were all negative for the stool samples obtained from the three patients in Hong Kong. Enterotoxigenic E. coli was recovered from one of the patients in Hong Kong. Unlike L. hongkongensis type strain HKU1, all the six strains were motile with bipolar flagellae. Sequencing of the 16S ribosomal RNA genes of the six strains showed that they all had sequences with only 0-2 base differences to that of the type strain. Pulsed field gel electrophoresis of the SpeI digested genomic DNA of the six isolates and that of the type strain revealed that the seven isolates were genotypically unrelated strains. More extensive epidemiologic studies should be carried out to ascertain the causative association between L. hongkongensis and diarrhea and to define the reservoir and modes of transmission of L. hongkongensis.