Antigenic variability of Vibrio anguillarum serotype O2a: A hurdle for vaccine efficacy against vibriosis in rainbow trout (Oncorhynchus mykiss).

Despite vaccination, outbreaks of vibriosis still occur in sea-reared rainbow trout in Denmark. Vibriosis outbreaks are caused mainly by V. anguillarum serotypes O1 and O2a, and bacterins of both serotypes are included in the commonly used vaccine against this disease in Danish aquaculture. However, while the strains belonging to serotype O1 are genetically similar, the strains belonging to serotype O2a are highly diverse. This work aimed first at examining how the antibody response and protection induced by bacterin-based vaccines were affected by the antigenic variability within V. anguillarum serotype O2a strains. Following vaccination of rainbow trout with either a commercial or an experimental vaccine, specific antibody reactivity in serum from vaccinated fish was examined by ELISA against 23 strains of V. anguillarum serotype O2a (VaO2a). The strains were divided into 4 distinct subgroups according to the observed detection pattern. Seven strains were strongly recognized only by sera from fish vaccinated with the experimental vaccine (EV-I antisera), while 13 other strains were primarily recognized by sera from fish vaccinated with the commercial vaccine (CV antisera). Two strains were recognized by both EV-I and CV antisera, but with intermediate reactivity, while one strain was not recognized at all. A partly similar recognition pattern was observed when purified lipopolysaccharide (LPS) was used as antigen in the examination of antibody reactivity in Western blotting. The level of protection was highly dependent on both the vaccine and the strain used for challenge and showed no consistent correlation with antibody reactivity. Secondly, we attempted to use a bacterin vaccine based on one of the V. anguillarum O2a strains intermediately recognized by both EV-I and CV antisera to investigate whether that could potentially provide protection across strain variability. The immunized fish did mount a cross-reactive antibody response, but protection still varied depending on the strain used for challenge. Interestingly, the grouping of strains according to antibody reactivity correlated not only with genotyping based on single nucleotides polymorphisms analysis (SNP) but also with variability in the accessory genome, indicating that presence or absence of protein antigens or proteins associated with the biosynthesis of antigenic epitopes may explain the observed distinct serological subgrouping within VaO2a strains by trout immune sera. In terms of vaccination against VaO2a, our results demonstrate that it is important to take (local) antigen variations into account when using bacterin-based vaccines but also that alternatives to traditional bacterin-based vaccines might be needed to induce protection against the highly virulent Vibrio anguillarum serotype O2a strains.

Polar bear-adapted Ursidibacter maritimus are remarkably conserved after generations in captivity.

Most species in the bacterial family of Pasteurellaceae colonize one specific host species. Vertebrates of very different evolutionary descent including fish, turtles, marsupials, eutherians and birds are colonized by different members of Pasteurellaceae. This one-to-one microbial-host species partnership makes Pasteurellaceae species valuable candidates to study biodiversity, bacterial-host co-evolution and host adaptation, and their widespread distribution across vertebrates provide the possibility to collect a wide array of data, where wildlife species are essential. However, obtaining samples from wild animals comes with logistic, technical and ethical challenges, and previous microbiota studies have led to the presumption that captive animals are poor models for microbial studies in wildlife. Here, we show that colonization of polar bears by Ursidibacter maritimus is unaffected by factors related to captivity, reflecting a deep symbiotic bond to the host. We argue that the study of ecological and evolutionary principles in captive wildlife is possible for host-adapted taxa such as those in the Pasteurellaceae family. Moreover, studying captive, often trained animals protects wild populations from the stress associated with obtaining samples.

Analysis of 44 Vibrio anguillarum genomes reveals high genetic diversity.

Vibriosis, a hemorrhagic septicemic disease caused by the bacterium Vibrio anguillarum, is an important bacterial infection in Danish sea-reared rainbow trout. Despite of vaccination, outbreaks still occur, likely because the vaccine is based on V. anguillarum strains from abroad/other hosts than rainbow trout. Information about the genetic diversity of V. anguillarum specifically in Danish rainbow trout, is required to investigate this claim. Consequently, the aim of the present investigation was to sequence and to characterize a collection of 44 V. anguillarum strains obtained primarily from vibriosis outbreaks in Danish rainbow trout. The strains were sequenced, de novo assembled, and the genomes examined for the presence of plasmids, virulence, and acquired antibiotic resistance genes. To investigate the phylogeny, single nucleotide polymorphisms were identified, and the pan-genome was calculated. All strains carried tet(34) encoding tetracycline resistance, and 36 strains also contained qnrVC6 for increased fluoroquinolone/quinolone resistance. But interestingly, all strains were phenotypic sensitive to both oxytetracycline and oxolinic acid. Almost all serotype O1 strains contained a pJM1-like plasmid and nine serotype O2A strains carried the plasmid p15. The distribution of virulence genes was rather similar across the strains, although evident variance among serotypes was observed. Most significant, almost all serotype O2 and O3 strains, as well as the serotype O1 strain without a pJM1-like plasmid, carried genes encoding piscibactin biosynthesis. Hence supporting the hypothesis, that piscibactin plays a crucial role in virulence for pathogenic strains lacking the anguibactin system. The phylogenetic analysis and pan-genome calculations revealed great diversity within V. anguillarum. Serotype O1 strains were in general very similar, whereas considerable variation was found among serotype O2A strains. The great diversity within the V. anguillarum serotype O2A genomes is most likely the reason why vaccines provide good protection from some strains, but not from others. Hopefully, the new genomic data and knowledge provided in this study might help develop an optimized vaccine against V. anguillarum in the future to reduce the use of antibiotics, minimize economic losses and improve the welfare of the fish.

OCCURRENCE OF PASTEURELLACEAE BACTERIA IN THE ORAL CAVITY OF SELECTED MARSUPIAL SPECIES.

Eighty-two Pasteurellaceae isolates from marsupials characterized by phylogenetic analysis of rpoB gene sequences formed five distinct groups. Twenty-one strains from long-nosed potoroos ( Potorous tridactylus apicalis), spotted-tailed quolls ( Dasyurus maculatus), and eastern quolls ( Dasyurus viverrinus) made up group 1, which classified with Frederiksenia canicola. Group 2, 15 strains from Tasmanian devils ( Sarcophilus harrisii), common wombats ( Vombatus ursinus), common ring-tailed possums ( Pseudocheirus peregrinus), and eastern quolls, grouped with Pasteurella multocida. Three strains from koalas ( Phascolarctos cinereus) formed group 3 and clustered with Lonepinella koalarum. Group 4, 13 common wombat strains only distantly related to other Pasteurellaceae, probably represent a new genus. Finally, 29 strains from Tasmanian devils, spotted-tailed quolls and eastern quolls formed group 5 and clustered with 15 previously described Tasmanian devil strains, belonging to a yet unnamed Pasteurellaceae taxon. The results strongly indicate that Pasteurellaceae bacteria represent a part of the normal oral microbiota in marsupials.

Testudinibacter aquarius gen. nov., sp. nov., a member of the family Pasteurellaceae isolated from the oral cavity of freshwater turtles.

A total of 13 Pasteurellaceae isolates from healthy freshwater turtles were characterized by genotypic and phenotypic tests. Phylogenetic analysis of partial 16S rRNA and rpoB gene sequences showed that the isolates investigated formed a monophyletic group. The closest related species based on 16S rRNA gene sequencing was Chelonobacter oris CCUG 55632T with 94.4 % similarity and the closest related species based on rpoB gene sequence comparison was [Pasteurella] testudinis CCUG 19802T with 91.5 % similarity. All the investigated isolates exhibited phenotypic characteristics of the family Pasteurellaceae. However, they could be separated from existing genera of the Pasteurellaceae by the following test results: indole, ornithine decarboxylase and Voges-Proskauer positive; and methyl red, urease and PNPG (α-glucosidase) negative. No X- or V-factor requirement was observed. A zone of ß-haemolysis surrounded the colonies after 24 h of incubation on bovine blood agar at 37 °C. Acid was produced from l-arabinose, dulcitol, d-mannitol, sucrose and trehalose. Representative strain ELNT2xT had a fatty acid profile that was characteristic for members of the Pasteurellaceae. ELNT2xT expressed only one respiratory quinone, ubiquinone-8 (100 %). The DNA G+C content of strain ELNT2xT was 42.8 mol%. On the basis of both phylogenetic and phenotypic evidence, it is proposed that the strains should be classified as representatives of a novel species of a new genus, Testudinibacter aquarius gen. nov., sp. nov. The type strain of Testudinibacter aquarius is ELNT2xT ( = CCUG 65146T = DSM 28140T), which was isolated from the oral cavity of a captive eastern long-necked turtle (Chelodina longicollis) in Denmark in 2012.

OCCURRENCE OF PASTEURELLACEAE BACTERIA IN THE ORAL CAVITY OF THE TASMANIAN DEVIL (SARCOPHILUS HARRISII).

The occurrence of bacteria belonging to the family Pasteurellaceae in the oral cavity of captive Tasmanian devils (Sarcophilus harrisii) was investigated using phenotypic and subsequent genotypic characterization and phylogenetic analyses. A total of 62 bacterial isolates obtained from Tasmanian devils, tentatively classified with the family Pasteurellaceae, were further characterized by phylogenetic analysis of rpoB gene sequence similarity, which showed that the isolates investigated formed five distinct groups. A total of 15 strains formed a novel genus-like group within Pasteurellaceae. Thirty-six strains grouped with the type strain of Frederiksenia canicola. Five strains clustered with the type strain of Pasteurella multocida . Interestingly, four of the P. multocida-like strains were ß-hemolytic when incubated on blood agar, which is atypical for this genus. Five strains grouped with a 100% rpoB similarity with Pasteurella dagmatis. Finally, a single strain showed 97.1% resemblance to Haemophilus haemoglobinophilus. The results demonstrate that Tasmanian devils are hosting a variety of bacterial taxa affiliated with the family of Pasteurellaceae as part of their oral microflora.

Bisgaardia miroungae sp. nov., a new member of the family Pasteurellaceae isolated from the oral cavity of northern elephant seals (Mirounga angustirostris), and emended description of the genus Bisgaardia.

A total of 17 bacterial isolates from northern elephant seals, tentatively classified within the family Pasteurellaceae, were further characterized by genotypic and phenotypic tests. Phylogenetic analysis of partial 16S rRNA and rpoB gene sequences showed that the isolates investigated formed a monophyletic group, closely related to the genus Bisgaardia within the family Pasteurellaceae. The rpoB gene sequence similarity was 97.2-100 % within the group and 16S rRNA gene sequence comparisons showed 99.2-99.8 % similarity within the group. According to 16S rRNA gene sequence analysis, the most closely related species with a validly published name was Bisgaardia hudsonensis with 96.9 % similarity and the most closely related species based on rpoB sequence comparison was Bisgaardia genomospecies 1 with an rpoB sequence similarity of 90.9 %. All the isolates investigated exhibited the phenotypic characteristics of the family Pasteurellaceae. However, these isolates could be separated from existing species of the genus Bisgaardia by the following characteristics: ability to grow at 42 °C, and acid production from lactose, melibiose, raffinose and l-rhamnose, but not from d-mannitol or trehalose. On the basis of both phylogenetic and phenotypic evidence, it is proposed that the strains should be classified as representatives of a novel species within the genus Bisgaardia: Bisgaardia miroungae sp. nov. The type strain, Wildatric(T) ( = CCUG 65148(T) = DSM 28141(T)), was isolated from the oral cavity of a wild northern elephant seal at The Marine Mammal Center, California, USA in 2011. To include the novel species, the description of the genus Bisgaardia has been emended.

Draft Genome Sequence of Chelonobacter oris Strain 1662T, Associated with Respiratory Disease in Hermann's Tortoises.

Chelonobacter oris 1662(T) is a type strain of the recently described species of the Pasteurellaceae family. The strain was isolated from the choanae of a captive tortoise with signs of respiratory tract infection. The genome reported here is approximately 2.6 Mb in size and has a G+C content of 47.1%.

Otariodibacter oris and Bisgaardia genomospecies 1 isolated from infections in pinnipeds.

We document the first associations of two recently described species of Pasteurellaceae bacteria with lesions in wild pinnipeds in rehabilitation. Samples were collected from nine lesions in four California sea lions (Zalophus californianus) and two Pacific harbor seals (Phoca vitulina) during necropsy or admission examinations at a rehabilitation facility in northern California. Seven Pasteurellaceae isolates were identified using phenotypic tests and partial rpoB gene sequencing. Six strains of Otariodibacter oris were isolated from California sea lions. Otariodibacter oris was isolated in pure culture from four abscesses, an affected lymph node, and a bone lesion consistent with osteomyelitis. Otariodibacter oris was also cultured with Arcanobacterium phocae and ß-hemolytic streptococci. A pure culture of Bisgaardia genomospecies 1 was obtained from an abscess in a harbor seal. This is the first time, to our knowledge, that O. oris has been associated with infection. Isolation of these bacteria in pure culture from abscesses and osteomyelitis strongly indicates a pathogenic potential of this organism. Likewise, the isolation of Bisgaardia genomospecies 1 in pure culture from an abscess in a harbor seal implies causality.

Occurrence of Pasteurellaceae bacteria in the oral cavity of selected marine mammal species.

The occurrence of bacteria belonging to Pasteurellaceae in the oral cavity of captive marine mammals was investigated using culture and subsequent geno- and phenotypic characterization and phylogenetic analyses. A total of 89 bacterial isolates from pinnipeds tentatively classified with the family Pasteurellaceae were further characterized by phylogenetic analysis of rpoB gene sequences, which showed that the isolates investigated formed five distinct groups. Four strains from California sea lions (Zalophus californianus) made up group I, which was classified with Pasteurella canis. Group II comprised four strains from harbor seals (Phoca vitulina) and grey seals (Halichoerus grypus) classified with Pasteurella stomatis. Group III consisted of 28 strains, isolated from harbor and gray seals and represented Bisgaardia genomospecies 1. Two strains from a harbor and a grey seal, group IV, were classified with Bisgaardia hudsonensis. Fifty-two strains from northern fur seals (Callorhinus ursinus), walruses (Odobenus rosmarus), and California and Steller sea lions (Eumetopias jubatus) formed group V and represented Otariodibacter oris. No Pasteurellaceae isolates were obtained from cetaceans, but Pasteurellaceae were isolated from all sampled pinnipeds. On the basis of these results, it is very likely that Pasteurellaceae bacteria represent a part of the normal oral flora in pinnipeds.

Otariodibacter oris gen. nov., sp. nov., a member of the family Pasteurellaceae isolated from the oral cavity of pinnipeds.

A total of 27 bacterial isolates from California sea lions and a walrus tentatively classified within the family Pasteurellaceae was further characterized by genotypic and phenotypic tests. Phylogenetic analysis of partial 16S rRNA and rpoB gene sequences showed that the isolates investigated formed a monophyletic group, tentatively designated Bisgaard taxon 57. According to 16S rRNA gene sequences, the most closely related species with a validly published name was Bisgaardia hudsonensis and the most closely related species based on rpoB sequence comparison was Pasteurella multocida subsp. multocida; highest similarities between the isolates and the type strains of B. hudsonensis and P. multocida subsp. multocida were 95.0 and 88.2%. respectively. All isolates of Bisgaard taxon 57 exhibit the phenotypic characters of the family Pasteurellaceae. Members of Bisgaard taxon 57 can be separated from existing genera of the Pasteurellaceae by the following tests: positive reactions for catalase, oxidase, Voges-Proskauer and indole; no X- or V-factor dependency; and acid production from L-arabinose (slow), L-fucose, maltose and trehalose, but not from dulcitol, D-mannitol, D-mannose or sucrose. The main fatty acids of Bisgaard taxon 57 (CCUG 59994(T)) are C(14:0), C(16:0), C(16:1)ω7c and the summed feature C(14:0) 3-OH/iso-C(16:1) I. This fatty acid profile is characteristic of members of the Pasteurellaceae. The quinone profile of Bisgaard taxon 57 (DSM 23800(T)) was similar to that of other genera in the Pasteurellaceae. The DNA G+C content of strain Baika1(T) is 36.2 mol%, which is at the lower end of the range for members of the family Pasteurellaceae. On the basis of both phylogenetic and phenotypic evidence, it is proposed that members of Bisgaard taxon 57 should be classified as representatives of a novel species in a new genus, Otariodibacter oris gen. nov., sp. nov. The type strain of Otariodibacter oris is Baika1(T) (=CCUG 59994(T)=DSM 23800(T)), which was isolated from the oral cavity of a healthy California sea lion in Copenhagen Zoo, Denmark, in 2007.