Martin Ryll

Phenotypic characteristics of Riemerella anatipestifer and similar micro-organisms from various hosts

A total of 199 Riemerella anatipestifer (RA) and RA-like field strains isolated culturally from birds of 12 different species and from pigs were characterized using classical phenotypic and chemotaxonomic tests. The RA reference strain ATCC 11845 was included in the study. On the basis of the classical phenotypic characteristics studied and the numerical analysis of the whole-cell fatty acid patterns, the RA reference strain and 123 field isolates were assigned to the indole negative (IN) variant and 10 isolates to the indole positive (IP) variant of the species RA. The IN strains were isolated not only from poultry and free-living wild ducks, but also from pigs, guillemots and from a budgerigar and a herring gull. All the IP isolates were isolated from domestic ducks. One field strain from a chicken and one from a black-headed gull, which were distinguished from RA mainly by the negative a-glucosidase reaction and production of yellow pigment respectively, showed fatty acid methyl ester profiles chemotaxometrically different from those of RA. Another 64 field strains isolated from domesticated ducks, geese and muscovy ducks with signs and lesions very similar to those caused by RA were phenotypically and chemotaxometrically clearly different from RA and could not be classified to any of the known species. This possible bacterial pathogen is therefore given the preliminary designation of Riemerella-like (RA-L) taxon 1502.

Pathogenesis of Riemerella anatipestifer in turkeys after experimental mono-infection via respiratory routes or dual infection together with the avian metapneumovirus

Riemerella anatipestifer (RA) is the causative agent of septicaemic and exudative diseases in a variety of bird species. Despite numerous outbreaks, little is known about the pathogenicity of RA for turkeys. We investigated the development of RA-induced disease in commercial turkey poults following RA inoculation via different respiratory routes. Inoculation by aerosol or injection into the abdominal air sac led to systemic infection and mild gross lesions, including pericarditis, epicarditis and airsacculitis, which were less pronounced compared with field outbreaks. It was speculated, that viral pathogens, such as the avian metapneumovirus (aMPV), may exacerbate RA pathogenesis under field conditions. We inoculated turkey poults with virulent aMPV. Subsequently, aMPV-infected and virus-free birds were exposed 3 to 5 days later to a high dose of RA by aerosol (>1010 colony-forming units/ml in 8 ml aerosol per 11 or 12 birds) or were inoculated 4 days later with a low RA dose (104.9 colony-forming units per bird) via the intranasal route. Intranasal RA inoculation with the low bacterial dose led to a respiratory and systemic RA infection in aMPV-infected birds, while virus-free birds remained RA-negative. Following exposure to a high RA dose by aerosol, aMPV-infected groups showed slightly enhanced incidences of gross lesions and RA re-isolation. The present study clearly confirms that RA is pathogenic for turkeys after experimental inoculation via respiratory routes, which are speculated to be the natural route of infection. However, experimental models in this study did not reproduce the severity of RA-related disease as observed under field conditions, which emphasizes the importance of other contributing factors. aMPV-induced respiratory lesions may serve as a predisposing factor for the establishment of RA infection, since they favour colonization of the bacterium.

Epidemiological investigations on the possible risk of distribution of zoonotic bacteria through apparently healthy homing pigeons.

Clinically healthy homing pigeons may serve as an unnoticed reservoir for zoonotic bacteria. Hence, healthy pigeons from 172 different racing pigeon lofts were examined for Salmonella serovars, Campylobacter spp. and Chlamydophila (Chlamydia) psittaci. Two samplings were performed during the racing season in summer (1242 adult and 1164 juvenile pigeons) and two during winter (1074 adult pigeons). Each sampling was accompanied by a questionnaire to identify risk factors for positive lofts. Between 0.9 and 3.7%, 13.1 and 23.7%, and 12.8 and 42.6% of lofts were tested positive by cultural methods or polymerase chain reaction for Salmonella Typhimurium var. Copenhagen, Campylobacter jejuni and C. psittaci, respectively. The detection rate of C. psittaci was twice as high in samples from juvenile pigeons (29.1%) compared with samples from adult pigeons (15.0%, P <0.001). No other influence of age or season was detected. For the first time, pigeon-derived C. jejuni isolates (n=15) were characterized for their ability to invade human enterocytes in vitro. All isolates were invasive with an invasion index between 0.4 and 34.1 (human reference strain: average 11.3). Of 50 C. jejuni isolates tested for antimicrobial susceptibility, 46.0% were resistant to ciprofloxacin. All isolates were sensitive to erythromycin and tetracycline. The analysis of risk factors in association with the infection status of lofts for C. jejuni and C. psittaci suggested that biosecurity measures reduce the risk of infection. This study indicated a zoonotic potential of pigeon-derived C. jejuni. However, clinically healthy homing pigeons pose only a low risk for transmission of the investigated pathogens to humans.

Isolation and characterization of atypical Riemerella columbina strains from pigeons and their differentiation from Riemerella anatipestifer

Riemerella columbina (RC) and Riemerella anatipestifer (RA) belong to the genus Riemerella within the family Flavobacteriaceae. While RA is a well-described pathogen of waterfowl and other avian species, only little is known about RC. Previous work reporting the isolation of RC from internal organs of clinically diseased pigeons suggested a potential pathogenic role in this avian species. In this study we examined pharyngeal swabs collected from pigeons and found RC to be widely distributed also among healthy birds. Further characterization of 81 RC-isolates revealed several atypical strains, which differed from all previously described RC-isolates by the lack of aesculin-hydrolysis activity (17 isolates) or by expression of yellow or orange pigmentation (6 isolates). Sequence analysis of the 16S rRNA and outer membrane protein A (ompA) gene supported the affiliation of these strains to the species RC. Aesculin-hydrolysis negative isolates were found to be biochemically indistinguishable from RA. We demonstrated that bacterial fingerprinting using matrix assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF MS) analysis is useful for the identification and differentiation of RC and RA.

Experiences with multispecies polymerase chain reaction and specific oligonucleotide probes for the detection of Mycoplasma gallisepticum and Mycoplasma synoviae

Amplified fragments of the rDNA coding for 16S rRNA of Mycoplasma gallisepticum (MG) and Mycoplasma synoviae (MS) were blotted on nylon membranes, followed by dot-blot detection with two species-specific digoxigenin-(DIG)-labeled oligonucleotide probes. The sensitivity and specifity of the tests were determined in titration studies with purified homologous and heterologous DNA. With the detection protocol used, the MSYV8/31 probe showed 100% specifity for MS, while both MG and the related species Mycoplasma imitans were recognized by the MGAV8/31 probe. Both DIG-labeled oligonucleotides gave positive results in the colorimetric assay with 10 to 100 ng homologous non-amplified DNA and polymerase chain reaction (PCR) amplificates of 100 fg homologous template DNA. There was no reaction with heterologous strains when amplificates starting with a 106-fold amount of template DNA (100 ng) were tested in dot-blots. The suitability for field samples was demonstrated with tracheal swabs from turkeys and chickens, and the results were compared with mycoplasma growth in cultures of the same swabs. Both tests had an accuracy of over 95%, a high sensitivity and specificity, and high predictive values of positive or negative results. There was no significant difference between the results obtained by the two methods. PCR in combination with dot-blotting is a relatively simple method for the detection of mycoplasma infections, and a valuable extension of current diagnostic tools.

Epidemiological investigations on the role of clinically healthy racing pigeons as a reservoir for avian paramyxovirus-1 and avian influenza virus

Clinically healthy racing pigeons may harbour notifiable pathogens and serve as an unnoticed reservoir. Thus, 3480 healthy racing pigeons from 172 different lofts were monitored over a period of 2 years for the presence of avian influenza virus (AIV) and avian paramyxovirus-1 (APMV-1). Pharyngeal and cloacal swabs as well as blood samples were collected from juvenile and adult pigeons. Pools of five pharyngeal swabs per loft and age group were initially screened by real-time reverse transcriptase-polymerase chain reaction (rRT-PCR). Pharyngeal and cloacal samples from lofts that were positive or suspect in the AIV rRT-PCR or the APMV-1 rRT-PCR were inoculated into embryonated chicken eggs for virus isolation. In addition, sera were examined for antibodies against AIV by enzyme-linked immunosorbent assay. The antibody levels after vaccination against APMV-1 were determined by haemagglutination inhibition assay. Of the investigated lofts, 0.0 to 1.4% were positive by rRT-PCR for APMV-1 and 0.0 to 6.7% for AIV during this 2-year period with a total of four samplings. No sample yielded replicating virus in egg culture. No antibodies against AIV were detected. Haemagglutination inhibition test of vaccinated racing pigeons indicated age-dependent APMV-1 titres. The results suggest that the examined racing pigeons may have had contact with AIV, but virus replication may have been too low to induce detectable circulating antibody levels. Only a low percentage of samples were positive for APMV-1, but two outbreaks were observed in monitored flocks, indicating ongoing circulation of APMV-1 in the racing pigeon population. These observations highlight the relevance of APMV-1 vaccination and indicate the importance of flock immunity.

Mycoplasma gallisepticum modifies the pathogenesis of influenza A virus in the avian tracheal epithelium

Multiple respiratory infections have a significant impact on health and economy. Pathogenesis of co-infecting viruses and bacteria and their interaction with mucosal surfaces are poorly characterized. In this study we established a co-infection model based on pre-incubation of tracheal organ cultures (TOC) with Mycoplasma (M.) gallisepticum and a subsequent infection with avian influenza virus (AIV). Mycoplasma gallisepticum modified the pathogenesis of AIV as demonstrated in TOC of two different avian species (chickens and turkeys). Co-infection promoted bacterial growth in tracheal epithelium. Depending on the interaction time of M. gallisepticum with the host cells, AIV replication was either promoted or suppressed. M. gallisepticum inhibited the antiviral gene expression and affected AIV attachment to the host cell by desialylation of α-2,3 linked sialic acids. Ultrastructural analysis of co-infected TOC suggests that both pathogens may attach to and possibly infect the same epithelial cell. The obtained results contribute to better understanding of the interaction dynamics between M. gallisepticum and AIV. They highlight the importance of the time interval between infections as well as the biological properties of the involved pathogens as influencing factors in the outcome of respiratory infections.

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.