Date Published: March 28, 2017
Publisher: Public Library of Science
Author(s): Monika Szymańska-Czerwińska, Agata Mitura, Krzysztof Niemczuk, Kinga Zaręba, Agnieszka Jodełko, Aneta Pluta, Sabine Scharf, Bailey Vitek, Rachid Aaziz, Fabien Vorimore, Karine Laroucau, Christiane Schnee, Garry Stewart Anthony Myers.
Wild birds are considered as a reservoir for avian chlamydiosis posing a potential infectious threat to domestic poultry and humans. Analysis of 894 cloacal or fecal swabs from free-living birds in Poland revealed an overall Chlamydiaceae prevalence of 14.8% (n = 132) with the highest prevalence noted in Anatidae (19.7%) and Corvidae (13.4%). Further testing conducted with species-specific real-time PCR showed that 65 samples (49.2%) were positive for C. psittaci whereas only one was positive for C. avium. To classify the non-identified chlamydial agents and to genotype the C. psittaci and C. avium-positive samples, specimens were subjected to ompA-PCR and sequencing (n = 83). The ompA-based NJ dendrogram revealed that only 23 out of 83 sequences were assigned to C. psittaci, in particular to four clades representing the previously described C. psittaci genotypes B, C, Mat116 and 1V. Whereas the 59 remaining sequences were assigned to two new clades named G1 and G2, each one including sequences recently obtained from chlamydiae detected in Swedish wetland birds. G1 (18 samples from Anatidae and Rallidae) grouped closely together with genotype 1V and in relative proximity to several C. abortus isolates, and G2 (41 samples from Anatidae and Corvidae) grouped closely to C. psittaci strains of the classical ABE cluster, Matt116 and M56. Finally, deep molecular analysis of four representative isolates of genotypes 1V, G1 and G2 based on 16S rRNA, IGS and partial 23S rRNA sequences as well as MLST clearly classify these isolates within the C. abortus species. Consequently, we propose an expansion of the C. abortus species to include not only the classical isolates of mammalian origin, but also avian isolates so far referred to as atypical C. psittaci or C. psittaci/C. abortus intermediates.
The family Chlamydiaceae comprises a group of obligatory intracellular bacteria within the single genus Chlamydia (C.) which includes eleven species  and two candidate species [2–3]. Chlamydiae are widely distributed throughout the world, causing a variety of diseases both in humans and animals, including zoonotic infections. Avian chlamydiosis due to Chlamydia psittaci was reported for the first time at the end of the 19th century and became of world concern in 1930 after the large epidemic involving psittacine birds and affecting 750–800 individuals in America and Europe . In birds, the disease is characterized by respiratory, ocular and enteric symptoms occasionally with fatal outcome, but asymptomatic, latent infections are also common. Shedding of the pathogens through feces or ocular and respiratory secretions occurs intermittently in both diseased birds and asymptomatic carriers, thus representing a reservoir of infection for birds and humans . Based on the ompA gene which encodes the major immunogenic protein of chlamydiae, avian C. psittaci has been classified into fifteen genotypes, each one more or less closely associated with certain bird species. Seven of these genotypes (A-F, E/B) are predominant whereas the other eight genotypes (1V, 6N, Mat116, R54, YP84, CPX0308, I and J) were described as provisional [6–10]. Whereas C. psittaci had been considered for a long time to be the sole species hosted by birds, recent evidence suggested that other chlamydial species, such as C. abortus, C. pecorum, C. trachomatis, C. suis and C. muridarum [11–13], can also be harboured by birds as well as the recently described avian species C. gallinacea and C. avium .
In recent years wild animals including birds such as corvids or swans have successfully adapted to live in the specific conditions of an urban environment. Therefore, investigations into the range of prevalence of zoonotic agents like Chlamydia spp. are important from a public health point of view . Prevalence of Chlamydiaceae shedders among free-living birds in Poland was 14.8% based on the results of the present study (n = 894). Previous data published by Krawiec et al.  reported a prevalence of 7.3%, however it should be noted that our survey included a higher number of samples from birds belonging to 16 families. Cloacal shedding of Chlamydiaceae was detected in eight families: Anatidae, Apodidae, Ciconidae, Columbidae, Corvidae, Laridae, Falconidae and Rallidae. The detection rate of Chlamydiaceae is in agreement with the rates reported by other researchers from Europe, although published data have shown high variability. In our study the highest number of Chlamydiaceae shedders was observed in the families Anatidae (19.7%) and Corvidae (13.4%). Special attention should be paid to swans belonging to the Anatidae family, as no detailed data on chlamydiosis in this species was available in the literature, while prevalence of Chlamydiaceae in our study was unexpectedly high with 37.1%. It can be assumed that a possible cause of the high excretion level of Chlamydiaceae in these birds is related to their behaviour. During winter swans dwell in the vicinity of collectors or other non-freezing water reservoirs with poor quality and feed incorrectly, e.g. on bread, which causes malnutrition and loss of fitness. A resulting weakened immunity might, in turn, lead to an increase in Chlamydiaceae infections. A high percentage of positive samples (33.3%) was also obtained for magpies (Corvidae family). A similar prevalence in corvids was reported by Di Francesco et al. . Surprisingly, the percentage of positive sampled pigeons was significantly lower (4.7%) compared to previous studies conducted in other European countries. For example, about 53% of Columba livia tested in Madrid, Spain  were shedders of the pathogen. Our results are closer to data from Amsterdam in the Netherlands, where positively-sampled pigeons accounted for 7.9% . Chlamydiaceae are shed intermittently and this may result in a high variability between values in individual European countries.
Chlamydiaceae seem to be common in Polish wild birds, such as swans and mallards, which often live close to humans so that zoonotic implications have to be assumed. Molecular characterisation of isolates from wild birds provides a unique opportunity to understand the diversity of avian chlamydia agents. Our analyses classified the majority of chlamydiae detected in this study as new genotypes located in close phylogenetic proximity to C. abortus strains. Consequently, we propose an expansion of the C. abortus species to include not only the classical isolates of mammalian origin, but also avian isolates so far referred to as atypical C. psittaci or C. psittaci/C. abortus intermediates. Isolation of representatives of the new genotypes will enable further investigations into epidemiology, host preference, pathogenicity and zoonotic potential of these agents. Moreover, it will be necessary to adapt current molecular diagnostic tools to cover the detection and differentiation of C. abortus and C. psittaci strains according to the proposed taxonomy.