Limited performance of MALDI-TOF for identification of fish Aeromonas isolates at species level.

The aim of this study was to evaluate the usefulness of the MALDI-TOF MS to identify 151 isolates of Aeromonas obtained mostly from diseased fish. MALDI-TOF MS correctly identified all isolates to the genus level but important differences in the percentage of isolates correctly identified depending on the species were observed. Considering exclusively the first identification option, Aeromonas bestiarum, Aeromonas hydrophila, Aeromonas salmonicida, Aeromonas veronii and Aeromonas sobria were the best identified with results >95%. However, considering the first and second identification options, the only species that showed values >90% was A. hydrophila. Overall, when the database was supplemented with 14 new spectra, the number of accurate identifications increased (41% vs. 55%) and the number of inconclusive identifications decreased (45% vs. 29%), but great differences in the success of species-level identifications were found. Species-distinctive mass peaks were identified only for A. hydrophila and A. bestiarum (5003 and 7360 m/z in 95.5% and 94.1% of their isolates, respectively). This work demonstrates the utility of MALDI-TOF MS for Aeromonas identification to the genus level, but there is no consistency for the accurate identification of some of the most prevalent species implicated in fish disease.

Coinfection by Streptococcus phocae and cetacean morbillivirus in a short-beaked common dolphin Delphinus delphis.

We describe gross, histopathological, and immunohistochemical features of Streptococcus phocae and cetacean morbillivirus coinfection in a short-beaked common dolphin Delphinus delphis. Major gross findings were cutaneous purulent nodules in the tail fluke, vegetative mitral valve endocarditis, and presumed postpartum pyometra. Histologic examination revealed bacterial septicemia characterized by widespread intravascular coccoid bacterial emboli. These were associated with fibrinonecrotizing to pyogranulomatous dermatitis and panniculitis, embolic pneumonia, neutrophilic and lymphoplasmacytic meningochoroiditis, random neutrophilic hepatitis, lymphoplasmacytic myocarditis and epicarditis, necrotizing adrenalitis, suppurative endometritis, and multicentric reactive lymphadenopathy. Bacteriology and molecular analysis with sequencing of the 16S rRNA gene identified S. phocae from lung, brain, and adrenal gland tissue. Immunohistochemical analysis for morbillivirus detection revealed positive immunolabeling in the epithelium of the choroid plexus of the fourth ventricle. Published reports on S. phocae infection in cetaceans are rare, and pathological details are limited. The present case indicates that S. phocae has potential pathogenic capacity in common dolphins. The pathogenesis is proposed to have involved cutaneous penetration after a skin trauma, leading to initial cutaneous disease and eventual systemic infection.

Streptococcus caprae sp. nov., isolated from Iberian ibex (Capra pyrenaica hispanica).

Biochemical and molecular genetic studies were performed on a novel Gram-stain-positive, catalase-negative, coccus-shaped organism isolated from tonsil samples of two Iberian ibexes. The micro-organism was identified as a streptococcal species based on its cellular, morphological and biochemical characteristics. 16S rRNA gene sequence comparison studies confirmed its identification as a member of the genus Streptococcus, but the organism did not correspond to any species of this genus. The nearest phylogenetic relative of the unknown coccus from ibex was Streptococcus porci 2923-03T (96.6 % 16S rRNA gene sequence similarity). Analysis based on rpoB and sodA gene sequences revealed sequence similarity values lower than 86.0 and 83.8 %, respectively, from the type strains of recognized Streptococcus species. The novel bacterial isolate was distinguished from Streptococcus porci and other Streptococcus species using biochemical tests. Based on both phenotypic and phylogenetic findings, it is proposed that the unknown bacterium be classified as representing a novel species of the genus Streptococcus, for which the name Streptococcus caprae sp. nov. is proposed. The type strain is DICM07-02790-1CT ( = CECT 8872T = CCUG 67170T).

Streptococcus cuniculi sp. nov., isolated from the respiratory tract of wild rabbits.

Biochemical and molecular genetic studies were performed on four unknown Gram-stain-positive, catalase-negative, coccus-shaped organisms isolated from tonsils (n = 3) and nasal samples (n = 1) of four wild rabbits. The micro-organism was identified as a streptococcal species based on its cellular morphological and biochemical tests. Comparative 16S rRNA gene sequencing confirmed its identification as a member of the genus Streptococcus, but the organism did not correspond to any recognized species of this genus. The closest phylogenetic relative of the unknown cocci from wild rabbits was Streptococcus acidominimus NCIMB 702025(T) (97.9% 16S rRNA gene sequence similarity). rpoB and sodA sequence analysis of the novel isolate showed interspecies divergence of 16.2% and 20.3%, respectively, from the type strain of its closest 16S rRNA gene phylogenetic relative, S. acidominimus. The novel bacterial isolate could be distinguished from the type strain of S. acidominimus by several biochemical characteristics, such as the production of esterase C4, acid phosphatase and naphthol-AS-BI-phosphohydrolase and acidification of different sugars. Based on both phenotypic and phylogenetic findings, it is proposed that the unknown bacterium be classified as a novel species of the genus Streptococcus, Streptococcus cuniculi sp. nov. The type strain is NED12-00049-6B(T) ( = CECT 8498(T) = CCUG 65085(T)).

Flavobacterium tructae sp. nov. and Flavobacterium piscis sp. nov., isolated from farmed rainbow trout (Oncorhynchus mykiss).

Four Gram-staining-negative, catalase- and oxidase-positive, pale-orange pigmented bacterial strains (435-08(T), 47B-3-09, 412R-09(T) and 60B-3-09) were isolated from diseased rainbow trout. Analysis of their 16S rRNA gene sequences suggested their adscription to the genus Flavobacterium. Strains formed two phylogenetic groups represented by strains 435-08(T) and 47B-3-09 (group A), and strains 412R-09(T) and 60B-3-09 (group B) displaying 16S rRNA sequence similarities greater than 99.8-99.9% within their respective groups. Strain 435-08(T) exhibited the highest levels of similarity with Flavobacterium aquidurense WB-1.1.56(T) (98.6% sequence similarity) and strain 412R-09(T) with Flavobacterium frigidimaris KUC-1(T) and Flavobacterium aquidurense WB-1.1.56(T) (98.9% and 98.6% sequence similarity, respectively). DNA-DNA hybridization studies showed low levels of relatedness between strain 435-08(T) and strain 412R-09(T) and between both strains and the most closely related species of the genus Flavobacterium. The genomic DNA G+C contents of strains 435-08(T) and 412R-09(T) were 36.2 and 34.3 mol%, respectively. The predominant respiratory quinone of both strains was MK-6 and the major fatty acids were iso-C(15 : 0), C(16 : 1)ω7c and C(15 : 0). The two groups of strains could be distinguished from each other and from related species of the genus Flavobacterium by a number of phenotypic properties. Phylogenetic, genotypic and phenotypic evidence indicated that strains of groups A and B represent two novel species of the genus Flavobacterium, for which the names Flavobacterium tructae sp. nov. (type strain 435-08(T) = CECT 7791(T) = CCUG 60100(T)) and Flavobacterium piscis sp. nov. (type strain 412R-09(T) = CECT 7911(T) = CCUG 60099(T)) are proposed.

Meningitis caused by an unusual genotype (ST3) of Streptococcus suis.

We describe a case of meningitis due to Streptococcus suis with the unusual ST3 genotype. The bacterial pathogen was isolated from blood samples. S. suis genotype ST3 was initially isolated from carrier pigs, but it has not been previously associated with invasive human infections. The patient developed serious endogenous bilateral endophthalmitis which resulted in severe visual deficiency.

Chryseobacterium viscerum sp. nov., isolated from diseased fish.

A taxonomic study was carried out on five Gram-staining-negative, catalase- and oxidase-positive, rod-shaped bacteria isolated from the gills and livers of five diseased rainbow trout. The five novel isolates were designated strains 687B-08(T), 445-08, 452-08, 453B-08 and 967B-08. In phylogenetic analyses based on 16S rRNA gene sequences, the five novel strains appeared almost identical (99.0-100 % sequence similarity) and to belong to the genus Chryseobacterium. Strain 687B-08(T) (the strain selected to represent the five novel isolates) was found to be most closely related to Chryseobacterium oncorhynchi 701B-08(T) (98.9% sequence similarity), Chryseobacterium ureilyticum F-Fue-04IIIaaaa(T) (98.6%), Chryseobacterium indologenes ATCC 29897(T) (98.3%), Chryseobacterium jejuense JS17-8(T) (98.1%) and Chryseobacterium gleum ATCC 35910(T) (98.1%). In DNA-DNA hybridizations, DNA-DNA relatedness values of 99-100% were recorded between the five novel strains. Lower DNA-DNA relatedness values (21-57%) were recorded between strain 687B-08(T) and C. oncorhynchi 701B-08(T), C. ureilyticum F-Fue-04IIIaaaa(T) and the type strains of other closely related, established species of the genus Chryseobacterium. The predominant respiratory quinone of strain 687B-08(T) was MK-6 and the major cellular fatty acids were iso-C(15:0), iso-C(17:1)ω9c, iso-C(17:0) 3-OH and C(16:1)ω6c. The G+C content of the genomic DNA of strain 687B-08(T) was 38.6 mol%. Based on the phenotypic and genotypic evidence, the five novel strains isolated from rainbow trout represent a single, novel species of the genus Chryseobacterium, for which the name Chryseobacterium viscerum sp. nov. is proposed. The type strain is 687B-08(T) ( = CECT 7793(T)  = CCUG 60103(T)).

Streptococcus porcorum sp. nov., isolated from domestic and wild pigs.

Seven isolates of an unidentified Gram-stain-positive, catalase-negative, coccus-shaped organism isolated from domestic and wild pigs were characterized by phenotypic and molecular-genetic methods. Based on cellular morphology and biochemical criteria, the isolates were tentatively assigned to the genus Streptococcus, although the organisms did not appear to correspond to any recognized species. Comparative 16S rRNA gene sequencing showed that the unknown bacterium was phylogenetically closely related to, but distinct from, Streptococcus suis (97.5 % 16S rRNA gene sequence similarity to the type strain). rpoB and sodA sequence analysis showed minimum interspecies divergence from phylogenetically close 16S rRNA gene sequence-based relatives of 13.8 and 18.6 %, respectively. DNA-DNA hybridization of a strain of the unidentified organism demonstrated 8-18 % reassociation with S. suis NCTC 10234(T). The novel bacterium could be distinguished from S. suis and other Streptococcus species using biochemical tests. On the basis of phenotypic and phylogenetic evidence, it is proposed that the unknown isolates from domestic and wild animals be assigned to a novel species of the genus Streptococcus, Streptococcus porcorum sp. nov. The type strain is 682-03(T) ( = CCUG 58479(T)  = CECT 7593(T)).

Weissella ceti sp. nov., isolated from beaked whales (Mesoplodon bidens).

During an investigation into the microbiota of beaked whales (Mesoplodon bidens), nine isolates were obtained from different organs of four animals. The isolates were Gram-positive-staining, catalase-negative, short rod-shaped or coccoid organisms. A phylogenetic analysis based on 16S rRNA gene sequences of these isolates allocated them to the genus Weissella, showing 96.3 % and 96.0 % 16S rRNA gene sequence similarity with Weissella viridescens NRIC 1536(T)and Weissella minor NRIC 1625(T), respectively. On the basis of phenotypic, physiological and phylogenetic evidence, it is proposed that the new isolates from whales represent a novel species of the genus Weissella, Weissella ceti sp. nov. The type strain of Weissella ceti is 1119-1A-09(T) ( = CECT 7719(T) = CCUG 59653(T)).

Streptococcus rupicaprae sp. nov., isolated from a Pyrenean chamois (Rupicapra pyrenaica).

Biochemical and molecular genetic studies were performed on an unknown Gram-stain-positive, catalase-negative, coccus-shaped organism isolated from clinical samples of a Pyrenean chamois. The micro-organism was identified as a streptococcal species based on its cellular morphological and biochemical tests. 16S rRNA gene sequence comparison studies confirmed its identification as a member of the genus Streptococcus, but the organism did not correspond to any species of this genus. The nearest phylogenetic relative of the unknown coccus from chamois was Streptococcus ovis (95.9 % 16S rRNA gene sequence similarity). The rpoB and sodA sequence analysis showed sequence similarity values of less than 85.7 % and 83.0 %, respectively, with the currently recognized species of the genus Streptococcus. The novel bacterial isolate was distinguished from S. ovis and other species of the genus Streptococcus using biochemical tests. Based on both phenotypic and phylogenetic findings, it is proposed that the unknown bacterium be classified as a novel species of the genus Streptococcus, Streptococcus rupicaprae sp. nov., with the type strain 2777-2-07(T) ( = CECT 7718(T)  = CCUG 59652(T)).

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.

Capsular type diversity of Mannheimia haemolytica determined by multiplex real-time PCR and indirect hemagglutination in clinical isolates from cattle, sheep, and goats in Spain.

This study compares the utility of a commercially available multiplex q-PCR assay for serotyping A1, A2, and A6 M. haemolytica serotypes with indirect hemagglutination, for determining the relative distribution of M. haemolytica capsular types associated with respiratory disorders in cattle, sheep, and goats. For the 129 isolates analyzed, both q-PCR and IHA assays exhibited nearly complete agreement for capsular types A1 (k = 0.965) and A2 (k = 0.888) and substantial agreement for A6 (k = 0.801). Despite the overall good performance of the commercial q-PCR, its effectiveness differed between the host origin of the isolates. The serotype was identified by q-PCR in 83.3 % of cattle, 77.8 % of goat, and 53.8 % of sheep isolates. Combining the results of both methods, A1 was the most prevalent in cattle and sheep (55.6 % and 22.25 %, respectively) but was not detected in goats, A2 was the most prevalent in goats (61.1 %) and the second most prevalent in cattle (16.7 %) and sheep (20.5 %). The prevalence of A6 was 7.4 %, 5.1 %, and 16.7 % in cattle, sheep, and goats, respectively. Other capsular types determined exclusively by IHA were A16 in cattle, A9 in goats, and A7, A8, A9, and A13 in sheep. Capsular type diversity was greater in sheep (H = 0.601) than in cattle (H = 0.408) and goat (H = 0.330) isolates. The commercial multiplex q-PCR is a valuable tool, alternative to IHA, for identifying isolates of capsular types A1, A2, and A6, the most frequent serotypes of M. haemolytica associated with respiratory disease in ruminants. However, when testing sheep isolates it should be complemented with immunological assays due to the wider range of serotypes implicated.

Moraxella porci sp. nov., isolated from pigs.

Nine Gram-negative, catalase- and oxidase-positive, coccus-shaped bacteria were isolated from pigs affected by different pathological processes. Phenotypic and genotypic methods were adopted to determine the relationships of these new isolates to recognized species of the genus Moraxella. Analysis of the 16S rRNA gene sequences demonstrated that the clinical isolates represented a new lineage within the genus Moraxella. The isolates were closely related to Moraxella cuniculi and Moraxella pluranimalium with 16S rRNA gene sequence similarities of 98.1 % and 99.1 %, respectively. The isolates displayed DNA-DNA relative binding ratios of 74 % to each other, but distinctly lower levels of DNA-DNA hybridization were observed with phylogenetically closely related moraxellae (<32 %). The new isolates could be distinguished from all other recognized species of the genus Moraxella by physiological and biochemical tests. On the basis of the phenotypic and molecular data, the nine new isolates from pigs represent a novel species within the genus Moraxella, for which the name Moraxella porci sp. nov. is proposed. The type strain is SN9-4M(T) (=CECT 7294(T)=CCUG 54912(T)).

Strength of association between isolation of Pasteurella multocida and consolidation lesions in ovine pneumonic pasteurellosis.

This study investigated the association of Pasteurella multocida isolation and the molecular characteristics of the isolates with the presence of pneumonic lesions in lambs at slaughter to assess its importance as a causative agent of pneumonic pasteurellosis compared with Mannheimia haemolytica. P. multocida was isolated from the 13.9% and 2.7%, and M. haemolytica from the 36.4% and 26.8%, of lungs with and without lesions, respectively (P < 0.05). Both microorganisms were frequently coisolated (23.2% and 12.5% from lungs with and without lesions, respectively). Isolation of P. multocida alone exhibited greater strength of association with pneumonic lesions (OR 11.4; 95% CI 3.2-40.6) than that exhibited by M. haemolytica alone (OR 3.0; 95% CI 1.6-5.4). Cluster analysis grouped the lungs into four clusters characterized by the isolation of M. haemolytica or P. multocida alone (clusters 1 and 4), coisolation of both microorganisms (cluster 3), and isolation of neither (cluster 2). Cluster 4 lungs exhibited higher frequencies of pneumonic lesions (87.5%) and severe (20.8%) and moderate (25.0%) lesions. Lungs coinfected with both pathogens (cluster 3) did not exhibit a higher frequency of severe and moderate consolidation lesions (6.1% and 14.3%, respectively), suggesting that P. multocida and M. haemolytica do not act synergically to cause more severe pneumonic infections. The greater strength of association of P. multocida isolation with pneumonic lesions together with the higher severity of the lesions caused could indicate a greater role played by this pathogen in the aetiopathogenesis of pneumonic pasteurellosis in sheep than is commonly assumed.

Nervous signs associated with otitis and cranial osteomyelitis and with Ornithobacterium rhinotracheale infection in red-legged partridges (Alectoris rufa).

A case of nervous signs in red-legged partridges (Alectoris rufa) associated with a severe otitis and osteomyelitis is reported. The outbreak was characterized by abnormal head position, torticollis and difficulty in standing, walking and flying. Pathological, microbiological and molecular genetic data supported an association with Ornithobacterium rhinotracheale (ORT) infection. Clinical signs persisted for several days and were accompanied by weight loss leading to death. Morbidity was approximately 20% and most birds died if untreated. Lesions were mainly characterized by a severe osteomyelitis of the cranial bones and purulent inflammation of the external, middle and inner ears. O. rhinotracheale was isolated from ear samples, skull and brain stem in pure culture. Genetic characterization by pulsed-field gel electrophoresis of the clinical isolates showed that the outbreak was caused by a single strain of ORT. This appears to be the first report of otitis associated with ORT in an avian species.

Pasteurella multocida isolates associated with ovine pneumonia are toxigenic.

The P. multocida toxin (PMT), a dermonecrotic protein encoded by the toxA gene, is the major virulence factor of capsular type D P. multocida strains causing progressive atrophic rhinitis (PAR) in pigs. A high frequency of P. multocida isolates harboring the toxA gene has been found among ovine pneumonic isolates, although the ability of these isolates to express PMT has never been examined. In this study we have investigated the ability of ovine toxA+ P. multocida isolates (n = 57) to express a functional toxin by detection of PMT toxin antigen using an ELISA test and its cytopathic effect in a Vero cell assay. PMT antigen was expressed in the great majority (54/57; 94.7%) of toxA+ isolates. Moreover, the 100% toxA+ ovine isolates analyzed produced a cytopathic effect in Vero cells within 24-48 h post-inoculation, identical to that described for porcine toxigenic P. multocida isolates. These results show for the first time that, in addition to isolates associated with PAR, isolates of P. multocida associated with pneumonia in sheep are also toxigenic. In addition, we found a total agreement (Kappa = 1; C.I. 0.75-1.25) between the detection of the toxA gene and the toxigenic capability of P. multocida isolates, indicating the PCR detection of toxA would be a suitable predictive marker of the toxigenic fitness of P. multocida.

Data supporting the morphological/topographical properties and the degradability on PET/PLA and PET/chitosan blends.

These data display evidence of the fracture through the morphologies and the topographical features as well as roughness data of different ratios of R(recycled)-PET/PLA, PET(virgin)/PLA, PET(virgin)/Chitosan and R(recycled)-PET/chitosan. Also, data of the morphologies after degradation under accelerated weathering test and degradation mechanisms are revealed. The data supplement the article "Comparative assessment of miscibility and degradability on PET/PLA and PET/chitosan blends".

Bergeyella porcorum sp. nov., isolated from pigs.

Four Gram-stain-negative, catalase- and oxidase-positive, bacillus-shaped bacterial isolates were recovered from the lungs and tonsils of four pigs. Based on cellular morphology and biochemical criteria the isolates were tentatively assigned to the genus Bergeyella, although the organisms did not appear to correspond with Bergeyella zoohelcum, the only validly named species of this genus. 16S rRNA gene sequencing demonstrated that isolates represented a distinct subline within the genus Bergeyella with <97%. 16S rRNA gene sequence similarity with B. zoohelcum ATCC 43767(T). The predominant cellular fatty acids of strain 1350-03(T) were iso-C15:0 and iso-C17:0 3-OH and the major quinone was MK-6. The DNA G+C content of strain 1350-03(T) was 37.7mol%. The novel isolates can be phenotypically distinguished from B. zoohelcum based on physiological traits. On the basis of both phenotypic and phylogenetic findings, we describe a new species of the genus Bergeyella for which we propose the name of Bergeyella porcorum sp. nov. (1350-03(T)=CCUG 67887(T)=CECT 9006(T)).

Antimicrobial susceptibility of Listeria monocytogenes isolated from meningoencephalitis in sheep.

The antimicrobial susceptibility to different antimicrobial agents of 41 Listeria monocytogenes strains isolated from sheep with meningoencephalitis and from feedstuff was tested by both microdilution and disk diffusion methods. Both sets of isolates of L. monocytogenes were susceptible to penicillin G, amoxicillin, cephalothin, erythromycin, vancomycin, rifampicin, gentamicin, kanamycin, trimethoprim, sulfisoxazole, chloramphenicol and ciprofloxacin, but resistant to tetracycline and doxycycline (7.3 and 4.9%, respectively). Tetracycline was the most frequent resistance trait in L. monocytogenes strains of animal origin. Four strains (9.8%) also exhibited reduced susceptibility (MIC 4 mg/l) to doxycycline suggesting the need of surveillance studies to monitor the antimicrobial resistance of Listeria strains of animal origin.