This article has been accepted for publication in Veterinary Record, 2015, following peer review, and the Version of Record can be accessed online at
http://dx.doi.org/10.1136/vr.103218
Chlamydiosis in corvids
A. Di Francesco a*, K. Laroucau b, F. Vorimore b, R. Aaziz b, A. Balboni a, R. Galuppi a, G. Merialdi
c, D. Salvatore a, M. Renzi c
a Department of Veterinary Medical Sciences, University of Bologna, Ozzano Emilia (Bologna), Italy
b Bacterial Zoonoses Unit, French Agency for Food, Environmental & Occupational Health Safety (Anses), Maisons-Alfort, France
c IZSLER Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia-Romagna, Sezione di Bologna, Italy
E-mail for correspondence: antoniet.difrancesco@unibo.it (A. Di Francesco)
Keywords: Chlamydiaceae; Wild birds; Eurasian magpies; Hooded crows; Italy
Abstract
The aim of this study was to investigate the presence of Chlamydiaceae in some corvid birds, such as magpies and hooded crows that in recent years have shown a rapid increase in their populations in urban environment. In total, 78 dead birds, in particular 54 Eurasian magpies (Pica pica) and 24 hooded crows (Corvus cornix) from the outer periphery of Bologna city (Northern Italy), were tested by a 16S rRNA encoding gene nested PCR. A total of 24 out of 78 (31%) samples, 18 from magpies and 6 from carrion crows, were PCR positive. Twenty-one samples were positive for C.
suis, two samples for C. abortus and one sample to C. psittaci. Detection of C. suis in birds is reported here for the first time, at our knowledge, confirming a pathogen transmission from livestock to wildlife
Avian chlamydiosis is primarily caused by the intracellular bacterium Chlamydia psittaci, belonging to the Chlamydiaceae family. Depending on the species and age of the bird and the virulence of the infectious bacterial strain, avian chlamydiosis can be subclinical or characterised by respiratory, digestive, or systemic disorders (Knittler and others 2014).
Seven C. psittaci outer-membrane protein A (ompA) genotypes (A-F and E/B), have been initially detected in birds. All these genotypes can be transmitted to humans by contact with contaminated faeces or feathers or by inhalation of an infectious aerosol, causing a mild flu-like illness or severe atypical pneumonia. Recently, six additional C. psittaci ompA genotypes, all occurring in wildlife birds, have been proposed (Sachse and others 2008).
Recent studies suggested that more chlamydial agents, beyond C. psittaci, can be involved in avian chlamydiosis. In this respect, Chlamydia abortus, Chlamydia pecorum, Chlamydia trachomatis and Chlamydia pneumoniae have been detected in birds (Pantchev and others 2009, Sachse and others 2012, Frutos and others 2015). Recently, two new bacterial species belonging to the Chlamydiaceae family have been described: Chlamydia avium from pigeons and psittacine birds and Chlamydia gallinacea from poultry (Sachse and others 2014). In addition, a novel candidate species, named Chlamydia ibidis, has been proposed (Vorimore and others 2013).
In Italy, the occurrence of avian chlamydiosis in free-living birds in the urban environment has been investigated in columbiform birds, showing a common exposition to C. psittaci (Magnino and others 2009, Donati and others 2015). Recently, C. avium has been reported in pigeons (Sachse and others 2014).
The aim of this study was to investigate the presence and prevalence of Chlamydiaceae species in corvids, since only limited information is available so far for these birds.
From March to June 2014, 76 dead birds, including 52 Eurasian magpies (Pica pica) and 24 hooded crows (Corvus cornix) from the outer periphery of Bologna (northern Italy), were submitted for diagnostic investigation to the IZSLER, province of Bologna. A cloacal swab was taken from each bird and stored at −20°C. DNA was extracted from the swabs using a commercial kit (QIAamp DNA Stool Mini Kit, Qiagen, Germany). The samples were examined by a 16S rRNA-based nested PCR assay performed, as previously described, by using the primers 16SIGF and CL1 for the primary amplification, and CL2 and 16SIGR for the secondary amplification (Di Francesco and others 2013), targeting the Chlamydiales 16S rRNA‘signature sequences’ (Everett and others 1999).
A blank extraction control, a blank PCR control and a positive PCR control (C. psittaci 6BC
reference strain) were included in each reaction. Each sample was examined in duplicate. The CL2/16SIGR PCR amplicons with the expected size (approximately 243 bp) were purified for DNA sequencing using QIAquick PCR purification kit (Qiagen). Both strands of PCR amplicons were sequenced with the respective (CL2 and 16SIGR) PCR primers (Bio-Fab Research, Italy). The nucleotide sequences were compared with those available in GenBank by using the BLAST server from the National Center for Biotechnology Information (http://blast.ncbi.nlm.nih.gov/Blast.cgi).
Twenty-two samples (16 from magpies and six from hooded crows) out of 76 (29 per cent) samples tested, were positive for 16S rRNA-based nested PCR. Sequence from 21 samples, 16 from magpies and five from hooded crows, were identical to C. suis sequences detected in naturally infected piglets (GenBank accession numbers AY661794, AY661797), whereas one sequence from a hooded crow showed 100 per cent identity to C. psittaci sequences detected in birds or mammalians (GenBank accession numbers CP003790, CP003796, JN426966). The C. suis 16S rRNA sequences from a hooded crow and a magpie were submitted to the GenBank database where the following accession numbers have been assigned: KP896507 (C. suis EU01/2014 from hooded crow) and KP896508 (C. suis EU44/2014 from magpie).
Recent decades have seen in Europe an adjustment of wild animal populations to specific conditions of urban environment. Magpies and hooded crows are birds of high adaptability and both have developed a successful adaptation to urban ecological niches (Luniak 2004). In the period 2000–
2013, corvids showed a substantial annual increase in population levels in Italy, with a trend of 2.48 (±0.3) for magpies and 1.51 (±0.2) for hooded crows (Rete Rurale Nazionale & LIPU 2014). Sorace and Gustin (2008) investigated the power of urbanisation on bird communities in Italian towns and showed that magpies and hooded crows were present in most of the tested towns and in each urban sector evaluated. In view of the rapid increase in corvidae population in the urban environment, the presence of agents with zoonotic potential in corvids should be investigated. Few studies focused on the occurrence of Chlamydiaceae species in corvids, until now. A recent investigation reported the isolation of C. psittaci genotype A in a magpie and a carrion crow (Kalmar and others 2014). The results of the present study confirm C. psittaci colonisation in corvids. Detection of C. suis in birds is reported here for the first time, to our knowledge. The birds were from hilly areas where the presence of wild boars was consistently reported. Only a few pig farms were in the vicinity of the sampling area: pigs were intensively reared with no contacts with the outside environment and the waste from the farms was stored in basins for a period of 180 days before removal from farms.
Taking into account the previous studies reporting the presence of C. suis in wild boar populations
in the province of Bologna (Di Francesco and others 2011, 2013), the source of C. suis for corvids might be related to contact with wild boars.
The results suggest a potential receptivity of birds to C. suis. Our molecular data should be confirmed by C. suis isolation from birds. In addition, the molecular comparison among C. suis isolates from birds, wild boars and pigs could be useful to elucidate the source of avian infection.
Zoonotic potential of C. suis has been recently investigated by De Puysseleyr and others (2014).
Although the risk of chlamydial transmission to humans through contact with wild birds has not been widely documented, appropriate control measures in all activities involving contact with wild birds should be recommended.
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