Identification of a novel clade of group A rotaviruses in fatally diseased domestic pigeons in Europe.

Rotaviruses are well-known causative agents of enteric disorders in humans and other mammals, but little is known about their virulence and pathogenic role in pigeons and other birds. Starting in summer 2017, a series of outbreaks of an acute disease with high mortalities was reported in domestic pigeons in Germany, Belgium and Denmark. The clinical picture was characterized by diarrhoea, vomiting, hepatic necrosis and sudden fatalities. From these severe outbreaks, we discovered several previously unknown group A rotavirus (RVA) lineages of genotype G18P[17]-I4-R4-C4-M4-A4-T4-N4-E19-H4, which were closely related but not identical to an RVA variant identified in cases of fatal hepatic necrosis in Australian pigeon lofts in 2016. Retrospective analysis demonstrated that the predecessors of the highly virulent variants have circulated in Europe since at least 2010. Our data indicate that reassortment and intercontinental spread has led to the emergence of novel RVA variants, which may constitute a major threat to animal welfare and health of domestic pigeon populations worldwide.

Colonization patterns of Enterococcus cecorum in two different broiler production cycles detected with a newly developed quantitative real-time PCR.

BACKGROUND: Although Enterococcus cecorum (EC) infection is one of the most important bacterial diseases in modern broiler chickens today, many aspects of epidemiology and pathogenesis are still unknown. There is a need for better detection methods for EC than classical cultivation. In the present study, we describe the validation and application of a newly developed quantitative TaqMan real-time PCR (qPCR) assay based on the 16S-rRNA-gene for the detection of EC. RESULTS: Fifty EC strains isolated from 12 different animal species were detected with the assay, while none of the other 26 examined bacterial species were tested positive during validation procedure. The detection limit of the PCR was 6.25 CFU/ml PBS. The qPCR assay was also considerably more sensitive using intestine and organ samples than the classical cultivation method. Field application of the PCR setup was tested comparing two different broiler production cycles on one farm: in cycle I broilers showed signs of enterococcal spondylitis (ES) from day 24 post hatch onwards while broilers in cycle II developed no ES. Two totally different colonization patterns were found in the two cycles with the qPCR using cloacal swabs. Animals in cycle I showed significantly (P ≤ 0.05) higher detection rates of EC at the day of placement and throughout the cycle than broilers of cycle II. Additionally, significantly higher detection rates were found in the cecum compared to duodenum, jejunum and ileum within one cycle. CONCLUSIONS: The new qPCR for EC is highly specific, more sensitive than classical cultivation and was able to show differences in colonization in a broiler cycle with later EC disease outbreak compared to a healthy cycle. These findings may be explained by infection with different strains, pathogenic EC isolates are probably more effective in colonization than commensal isolates. A high correlation was found between qPCR results from cecum and cloacal swabs in this study, indicating that cloacal swabs can be used to examine intestinal colonization of broilers with EC. The new qPCR significantly improves the diagnostic of EC infections and may help to answer open questions concerning epidemiology and pathogenesis.

Enterococcus cecorum infection in a racing pigeon.

Until now, Enterococcus cecorum (EC) has been known as a pathogen for broilers, broiler breeders, and Pekin ducks. In the present report, we describe a fatal systemic EC infection in a young racing pigeon (Columba livia forma domestica). EC was isolated from the heart, liver, spleen, and intestine of the bird in pure culture. In the pathologic examination, the pigeon showed enteritis and an ulcerative gastritis, which may have been predisposing factors for the development of the generalized EC infection. An accumulation of gram-positive cocci in spleen tissue was found in the histopathologic examination and confirms the presence of a systemic EC infection in the pigeon. Additionally, EC was isolated from cloacal swabs of other pigeons in the same loft, but no additional pigeons were submitted for necropsy. All EC isolates tested were negative by PCR for the enterococcal virulence factors cytolysin, enterococcal surface protein, aggregation substance, hyaluronidase, and gelatinase. Therefore, the reason for the enhanced virulence of the EC isolate remains unknown. Our report confirms EC as a disease-causing agent in pigeons and presents the first data concerning the analysis of EC for virulence factors.

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.

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.