Molecular and serological characterization of Riemerella isolates associated with poultry in Australia.

A total of 62 isolates of Riemerella-like organisms, originally isolated from Australian poultry (10 from chickens, 46 from ducks, five from unknown hosts and one vaccine strain), were included in this study. On the basis of two published polymerase chain reaction (PCR) assays that are reported to be specific for Riemerella anatipestifer, 51 of the isolates were identified as R. anatipestifer. Forty-six of these isolates had a detailed history and were sourced from ducks, while five were of unknown origin. The 11 remaining isolates failed to yield a positive reaction in either PCR with 10 originating from chickens and one from a duck. Amplification and sequencing of the 16S rRNA gene of these isolates identified the duck isolate as Moraxella lacunta. Phylogenetic analysis of the 10 chicken isolates identified one as R. columbina and the remaining nine isolates as Riemerella-like taxon 2. The 51 Australian R. anatipestifer isolates were assigned by gel diffusion test to serovars 1 (26 isolates), 6 (seven isolates), 8 (five isolates), 9 (two isolates), 13 (one isolate) and 14 (one isolate) while nine isolates gave no reaction to any antiserum. A commercial system was used to perform DNA fingerprinting using rep-PCR analysis, which revealed different clusters with a lack of a clear relationship between the clusters and the serovars.

Data on the epidemiology and pathology of anatipestifer disease in Hungary (2010-2014).

Anatipestifer disease is a contagious disease caused by Riemerella anatipestifer, affecting primarily ducks, geese and turkeys, and characterised by listlessness, diarrhoea, sneezing, nasal discharge, and nervous signs. Sporadically, it occurs in a wide range of other domesticated and wild birds as well. The incidence and characteristics of the disease seen in the three main host species are summarised based on birds submitted for routine laboratory investigation in Hungary over the period 2010-2014. The infection was diagnosed in a higher percentage in geese (9.9%) and ducks (7.5%). It occurred in 5-day-old to 17-week-old geese and 3- to 6.5-week-old ducks, respectively. The pathological lesions were comparable in these two species: enlarged spleen, serofibrinous pericarditis, perihepatitis, airsacculitis, catarrhal enteritis, subcutaneous oedema and hyperaemia over the cranium, mucopurulent exudate in the nasal cavity and occasionally pneumonia, conjunctivitis, purulent arthritis and caseous salpingitis. In some cases, R. anatipestifer produced only secondary lesions, which complicated other diseases such as circovirus infection, mycotoxicosis, mycoplasmosis, or Derzsy's disease. In turkeys, the disease occurred rarely (0.5%) and at an older age (12 to 19 weeks). The lesions most frequently seen were purulent osteomyelitis of the cranium and seropurulent meningitis. Purulent osteomyelitis in the cranium caused by R. anatipestifer infection had not been reported in turkeys previously. To various extents, other local lesions such as serofibrinous pericarditis, airsacculitis, arthritis, and in one case septicaemia were also observed. The high incidence of the disease in waterfowl underlines the importance of appropriate treatment and prevention that should be based on accurate diagnosis and antimicrobial susceptibility testing, proper biosecurity and vaccination with regard to the serotype(s) present on the farm.

Antimicrobial susceptibility of Riemerella anatipestifer strains isolated from geese and ducks in Hungary.

Riemerella anatipestifer causes anatipestifer disease in many avian species. A total of 185 R. anatipestifer strains isolated in Hungary between 2000 and 2014 from geese and ducks were tested against 13 antibiotics (ampicillin, doxycycline, enrofloxacin, erythromycin, florfenicol, flumequine, gentamicin, penicillin, spectinomycin, streptomycin, sulphamethoxazole-trimethoprim, sulphonamide compounds, and tetracycline) by the Kirby-Bauer disk diffusion method. The majority of the strains were susceptible to florfenicol (97.9%), ampicillin (95.1%), penicillin (93%), sulphamethoxazole-trimethoprim (92.4%), and spectinomycin (86.5%). The highest resistance rates were observed for flumequine, tetracycline, erythromycin and streptomycin (94%, 91.4%, 75.1% and 71.4% resistance, respectively). The resistance patterns showed some variation depending on the geographical origin of the strains. The average rate of extensive drug resistance was 30.3%, and its proportion tended to increase in the period examined.

Composition and Predicted Metabolic Capacity of Upper and Lower Airway Microbiota of Healthy Dogs in Relation to the Fecal Microbiota.

The upper and lower airways of healthy humans are reported to harbor stable and consistent bacterial populations, and the composition of these communities is altered in individuals affected with several respiratory diseases. Data regarding the presence of airway microbiota in other animals are scant and a better understanding of the composition and metabolic function of such bacterial populations is essential for the development of novel therapeutic and diagnostic modalities for use in both veterinary and human medicine. Based on targeted next-generation sequencing of feces and samples collected at multiple levels of the airways from 16 healthy female dogs, we demonstrate that canine airways harbor a topographically continuous microbiota with increasing relative abundance of proteobacterial species from the upper to lower airways. The lung-associated microbiota, as assessed via bronchoalveolar lavage fluid (BALF), was the most consistent between dogs and was dominated by three distinct taxa, two of which were resolved to the species level and one to the level of family. The gene content of the nasal, oropharyngeal, and lung-associated microbiota, predicted using the Phylogenetic Investigations into Communities by Reconstruction of Unobserved States (PICRUSt) software, provided information regarding the glyoxylate and citrate cycle metabolic pathways utilized by these bacterial populations to colonize such nutrient-poor, low-throughput environments. These data generated in healthy subjects provide context for future analysis of diseased canine airways. Moreover, as dogs have similar respiratory anatomy, physiology, and immune systems as humans, are exposed to many of the same environmental stimuli, and spontaneously develop similar respiratory diseases, these data support the use of dogs as a model species for prospective studies of the airway microbiota, with findings translatable to the human condition.

The siderophore-interacting protein is involved in iron acquisition and virulence of Riemerella anatipestifer strain CH3.

Riemerella anatipestifer causes epizootic infectious disease in poultry and serious economic losses especially to the duck industry. However, little is known regarding the molecular basis of its pathogenesis. The ability to acquire iron under low-iron conditions is related to the virulence of a variety of bacterial pathogens. In this study, a sip (Riean_1281) deletion mutant CH3Δsip was constructed and characterized for iron-limited growth, biofilm formation, and pathogenicity to ducklings. Results showed that siderophore-interacting protein (SIP) was involved in iron utilization and the sip deletion significantly reduced biofilm formation and adherence to and invasion of Vero cells. In addition, the sip gene was absent in 1 of 24 (4.17%) virulent strains and 2 of 3 (66.7%) avirulent strains of R. anatipestifer, and the sip gene from six R. anatipestifer strains, which belong to serotypes 1, 2, and 10, respectively, shared 100% amino acid identities to those of R. anatipestifer strains DSM15868 and RA-GD. These results suggested that siderophore-mediated iron acquisition may be an important iron-uptake pathway in R. anatipestifer. Animal experiments indicated that the median lethal dose of the CH3Δsip mutant in ducklings was about 35-fold higher than that of the wild-type CH3 strain. Thus, our results demonstrated that R. anatipestifer SIP was involved in iron acquisition and necessary for its optimal virulence.

Sequence-based genotyping methods to assess the genetic diversity of Riemerella anatipestifer isolates from ducklings with tremor.

The aim of this study was to investigate sequence-based genotyping methods to distinguish 27 Riemerella anatipestifer isolates from ducklings in South Korea. The 16S rRNA sequences of the 27 R. anatipestifer isolates showed 99�100% similarities to each other and to reference sequences from Genbank (AY871822.2, AY871834.2, CP002562.1, EU715016.1, EU016548.1, EU715000.1, EU715008.1 and EU715011.1). In addition, the ompA gene sequences of 25 of the 27 R. anatipestifer isolates were 100% identical to each other, and these sequences were also 100% identical to reference sequences (CP002562.1, GQ415419.1, DQ059079, FJ765034.1, AY606207.1, AF104937.1, and FJ765033.1). Alternatively, four housekeeping genes (mdh, gdh, pgi, and rpoB) and three virulence-associated genes (prtC, hagA, and sspA) were used for a multi-locus sequence typing (MLST) and a single-locus sequence typing (SLST) among R. anatipestifer isolates. Compared to 16S rRNA and the ompA gene, seven genes showed higher genetic divergence patterns, and the isolates were separated into three distinct groups in phylogenetic trees.

Development and evaluation of a trivalent Riemerella anatipestifer-inactivated vaccine.

Riemerella anatipestifer infections cause major economic losses in the duck industry. In this study, a trivalent inactivated vaccine of R. anatipestifer, including strains CH3 (serotype 1), NJ3 (serotype 2), and HXb2 (serotype 10), was developed. Animal experiments showed that the ducks that received two immunizations with the vaccine were 100% protected from challenge with strains from any of the three serotypes (1, 2, or 10). No death or clinical signs of diarrhea, tremors, or limb swelling were shown in the protected ducks. Also, no R. anatipestifer bacteria were isolated from the livers or brains of the protected ducks. Furthermore, no histopathological changes were observed in the liver, spleen, or brain samples from the protected ducks during histological examination. The ducks that received two immunizations with the vaccine generated high antibody titers of 1:3,200 to 1:6,400 against the three serotypes of strains. The vaccine significantly enhanced the production of gamma interferon (IFN-γ) and interleukin 2 (IL-2) after one immunization and enhanced the production of IL-4 and IL-10 after two immunizations. In addition, real-time PCR indicated that the expression of major histocompatibility complex I (MHC-I), as well as that of CD40 and CD154 molecules, was significantly increased after one immunization, and the expressions of both MHC-I and MHC-II molecules were increased after two immunizations. Our study indicates that the vaccine can induce both humoral and cellular immunities in ducks and offer effective protection against R. anatipestifer infection.

Evaluation of different diagnostic tools for the detection and identification of Riemerella anatipestifer.

Riemerella anatipestifer (RA) is an important avian pathogen with considerable impact on poultry production worldwide. However, the diagnosis of RA infections may be difficult, mainly due to problems with unequivocal differentiation of RA from other Flavobacteriaceae and a lack of standardized methods and reagents. The aim of the present study was therefore to complement the routine diagnostic strategies for RA by design and evaluation of alternative diagnostic tools. We designed and validated a new RA-specific polymerase chain reaction assay, which proved to be a valuable tool for the identification of RA isolates as well as for rapid and sensitive RA detection directly from diagnostic samples. Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry fingerprinting of whole bacterial cells was also demonstrated to identify RA isolates efficiently. Furthermore, this method may also provide opportunities for RA subtyping. In our study, a stable subcluster was formed by the mass spectroscopy profiles of a group of RA isolates originating from turkey flocks in northern Germany, suggesting an epidemiological relationship of these isolates. Serotyping is a further important measure to characterize RA isolates. We tested a set of commercially available anti-RA sera with RA serotype reference strains and field isolates to allow comparison between these sera and reference sera. In summary, this report contributes to the improvement of present microbiological and molecular strategies for the diagnosis of RA infections by providing new tools as well as enhanced knowledge on existing methods.

Description of Riemerella columbipharyngis sp. nov., isolated from the pharynx of healthy domestic pigeons (Columba livia f. domestica), and emended descriptions of the genus Riemerella, Riemerella anatipestifer and Riemerella columbina.

A group of 11 bacterial strains was isolated during microbiological investigations of pharyngeal swabs collected from domestic pigeons (Columba livia f. domestica). Phenotypic properties of the isolates closely resembled those of members of the genus Riemerella within the family Flavobacteriaceae. The genus presently contains two species, Riemerella anatipestifer and Riemerella columbina. The pigeon isolates differed from R. columbina by their lack of pigment production and negative CAMP co-haemolysis reaction. They grew more slowly at 37 °C under microaerobic conditions and showed reduced viability during storage under aerobic conditions at different temperatures, compared with both Riemerella species. Comparisons of protein profiles with matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS analysis allowed differentiation between the new pigeon isolates and both R. anatipestifer and R. columbina. Phylogenetic analysis based on 16S rRNA gene and rpoB gene (encoding RNA polymerase beta subunit) sequences supported the affiliation of the 11 strains to a novel species within the genus Riemerella, for which we propose the name Riemerella columbipharyngis sp. nov. The type strain is 8151(T) (=DSM 24015(T) = LMG 26094(T)). Emended descriptions of the genus Riemerella and of its species Riemerella anatipestifer and Riemerella columbina are also presented.

Development and application of specific polymerase chain reaction assay targeting the gyrB gene for rapid detection of Riemerella anatipestifer.

A pair of PCR primers was designed and synthesized to amplify a gyrB gene sequence from Riemerella anatipestifer (RA). A fragment of 194 bp was detected in RA-positive isolates, whereas other isolates were negative, which confirmed the high specificity of the primers and PCR conditions. As little as 1.6 × 10(4) cfu/mL of cultural liquid was required by this method. We compared a 16S rRNA sequence-based PCR method and a Biolog bacterial identification system used in the detection and identification of suspicious isolates of RA in clinical tests. The results showed that the gyrB-based PCR was consistent with the results of the Biolog identification system and was more specific. By applying the gyrB-PCR to detect RA strains in 56 duck livers, a positive rate of 46% (26/56) was observed, whereas the positive rate of 85 throat swabs from clinically healthy ducks was 11%. Thus, this method could be used for the epidemiological investigation and preliminary isolate identification of RA.