Chlamydia psittaci in Eurasian Collared Doves (Streptopelia decaocto) in Italy Author(s): Manuela Donati, Karine Laroucau, Mauro Delogu, Fabien Vorimore, Rachid Aaziz, Eleonora Cremonini, Roberta Biondi, Claudia Cotti, Raffaella Baldelli, and Antonietta Di Francesco Source: Journal of Wildlife Diseases, 51(1):214-217. Published By: Wildlife Disease Association URL:

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DOI: 10.7589/2014-01-010

Journal of Wildlife Diseases, 51(1), 2015, pp. 214–217 # Wildlife Disease Association 2015

Chlamydia psittaci in Eurasian Collared Doves (Streptopelia decaocto) in Italy Manuela Donati, 1 Karine Laroucau, 2 Mauro Delogu, 3 Fabien Vorimore, 2 Rachid Aaziz, 2 Eleonora Cremonini, 1 Roberta Biondi,1 Claudia Cotti,3 Raffaella Baldelli, 3 and Antonietta Di Francesco3,4 1Section of Microbiology DIMES, S. Orsola Hospital, University of Bologna, Via Massarenti 9, 40138, Bologna, Italy; 2Bacterial Zoonoses Unit, French Agency for Food, Environmental & Occupational Health Safety (Anses), 4 rue Pierre et Marie Curie, 94701 Maisons-Alfort, France; 3Department of Veterinary Medical Sciences, University of Bologna, Via Tolara di Sopra 50, 40064 Ozzano Emilia (Bologna), Italy; 4Corresponding author (email: [email protected])

of an infectious aerosol or direct contact with contaminated feces or feathers, particularly in high-risk individuals such as veterinarians, bird breeders, and petshop or slaughterhouse workers. In view of the zoonotic potential, several epidemiologic studies have been performed on C. psittaci circulation in birds in contact with humans. Since the 1980s, C. psittaci has been repeatedly detected in Eurasian Collared Doves (Streptopelia decaocto) (De Gruchy 1983; Gough and Bevan 1983). The Eurasian Collared Dove is a Columbiformes species with a range of colonization, extending from India to the Middle East, Western Europe, and, more recently, Mediterranean countries including Italy and Spain. The collared dove lives in rural as well as suburban or urban areas, sharing some habitat with feral pigeons (Columba livia). A recent study on the avifauna of 26 Italian towns showed the presence of S. decaocto in each urban sector tested (center, inner periphery, outer periphery), with a frequency slightly lower than that of the urban pigeon (Sorace and Gustin 2008). A seroepidemiologic survey conducted in Great Britain showed higher antibody titers and prevalence of antibody to C. psittaci in collared doves than in the pigeons, even if the number of samples tested was relatively small (Bracewell and Bevan 1986). Serotyping (Duan et al. 1999; Andersen 2005) and genotyping (Laroucau et al. 2008) studies of C. psittaci isolates from Eurasian Collared Doves identified serotype/ genotype B. To date, no investigations

We investigated the Chlamydia spp. occurrence in Eurasian Collared Doves (Streptopelia decaocto) from urban and suburban areas in northern Italy. Among 76 doves screened, prevalence of Chlamydia spp. was 61%. Chlamydia psittaci genotype E was identified in 33 of the 46 positive samples. The multilocus sequence typing pattern of one highly positive sample showed a new allelic combination. The same molecular features were observed in a C. psittaci strain subsequently isolated from a live dove. Our results reveal a high C. psittaci prevalence in S. decaocto. The spread of this zoonotic pathogen from collared doves to other birds or humans seems to be a potential risk. Key words: Chlamydiaceae, Chlamydia psittaci, Eurasian Collared Dove, MLST, MLVA, real-time PCR, Streptopelia decaocto. ABSTRACT:

Avian chlamydiosis is a zoonotic disease caused by an obligate intracellular bacterium, Chlamydia psittaci, belonging to the family Chlamydiaceae. Depending on the virulence of the infectious chlamydial strain and the avian host, chlamydiosis can be subclinical or characterized by mild to severe respiratory signs and can also induce digestive symptoms (Andersen 1997). Seven genotypes (A to F and E/B) have been described in birds on the basis of the outer membrane protein A (ompA) gene. All these genotypes can be transmitted to humans, causing a mild flu-like illness or severe atypical pneumonia with dyspnea; genotypes A and D are more virulent (Vanrompay et al. 1994; Gaede et al. 2008). Recently, six additional C. psittaci ompA genotypes were proposed, all occurring in wild birds (Sachse et al. 2008). Zoonotic transmission occurs by inhalation 214


have been undertaken on large sampling of Eurasian Collared Dove populations. We assessed the circulation of Chlamydia spp. in a relatively large population of Eurasian Collared Doves in northern Italy, providing species and genotype determination. We tested 86 collared doves (46 males, 40 females) for Chlamydiaceae. From 2010 to 2013 we collected cloacal swabs from 76 dead collared doves from suburban areas in Emilia-Romagna region (40 from Bologna, 44u299N, 11u209E; 36 from Faenza, 44u179N, 11u539E). In October 2013, we sampled cloacal swabs from 10 live collared doves housed in a wildlife center in Bologna. We extracted DNA from the cloacal swabs of the 76 dead doves using the QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. A Chlamydiaceaespecific real-time (rt) PCR targeting the 23S ribosomal RNA gene (Ehricht et al. 2006) was used to screen the samples. The quantitative 23S-rtPCR also allowed us to compute the excretion level. The number of corresponding inclusion-forming units (IFU) per sample was calculated from the cycle threshold (Ct) value on the basis of the calibration curve from serial 10-fold dilutions of a titrated DNA (kindly provided by Dr. K. Sachse, Friedrich Loeffler Institute, Jena, Germany). Samples with Ct values of ,40 were considered positive. All positive samples were reanalyzed by rtPCR assays specific for C. psittaci (Me´nard et al. 2006), as well as for Chlamydia gallinacea (Zocevic et al. 2012) and Chlamydia avium (Zocevic et al. 2013), two new species detected in birds (Sachse et al. 2014). Samples positive for C. psittaci with Ct values ,35 were genotyped by a multilocus variable-number tandem-repeat analysis (MLVA) typing system (Laroucau et al. 2008). Multilocus sequence typing (MLST) (Pannekoek et al. 2010) was also performed on the highly rtPCR C. psittaci-positive samples. Samples positive for C. psittaci with Ct values of .35 were tested by an


ompA nested PCR (Heddema et al. 2006) and sequenced. Cloacal swabs collected from live doves were placed into sucrose phosphate transport medium, transported at 4 C, and processed immediately. Specimens were vortexed, then inoculated in duplicate onto Macaca mulatta kidney (LLC-MK2) cells seeded in individual vials containing a glass coverslip at the bottom. After centrifugation at 800 3 G for 3 hr, the infected cell monolayers were incubated at 37 C for 48 hr and fixed in methanol before detection of intracellular chlamydial inclusions by immunofluorescence (Donati et al. 2002). The DNA was extracted from the positive cell cultures and tested by ompA nested PCR and MLST, as described earlier. In vitro sensitivity of the isolates to tetracycline was determined according to Donati et al. (2010). Forty-six (23 males and 23 females) of 76 (61%) samples tested were positive for Chlamydiaceae by 23S-rtPCR (mean Ct value532). Twenty-two positive doves were from Bologna and 24 from Faenza areas. When retested by a C. psittacispecific rtPCR, 33 of 46 samples were positive; none was positive for C. gallinacea or C. avium. Twenty-two of the C. psittaci-positive samples with higher DNA contents (Ct values between 23 and 33 corresponding to a concentration between 53103 and 53106 IFUs/mL) exhibited a MLVA pattern [1] already observed in two Italian genotyped E strains isolated from feral pigeons (Laroucau et al. 2008). The MLST pattern was determined for one of the highly C. psittaci-positive samples and a new allelic combination was observed (gatA_11, oppA_14, hflX_11, gidA_14, enoA_13, hemN_11, fumC_13) (data not shown). Closely related MLST types were already detected in seven previously typed isolates, including four Italian strains from feral pigeons (genotype B or E) (Pannekoek et al. 2010). Sequencing of the ompA nested PCR amplicons identified genotype E in the remaining 11 C. psittaci-positive



samples that could not be typed by MLVA. Finally, the identity of the species could not be determined for 13 cases (28% of all Chlamydia spp.-positive samples) because of a low DNA content. Of the 10 live doves tested by cell culture, five were positive for Chlamydia spp. One isolate (AR13) that grew better than the others was molecularly typed and the same new MLST pattern described earlier was obtained. The isolate was sensitive to doxycycline, showing a minimum inhibitory concentration of 0.06 mg/ mL and a minimum bactericidal concentration of 0.125 mg/mL. We document a higher Chlamydia spp. prevalence (61%) in S. decaocto compared with that (14–42%) described in feral pigeons in Italy (reviewed by Magnino et al. 2009), to date. The Chlamydia spp. prevalence was calculated without considering the data obtained from live doves. Considering that the five positive live birds were housed in the same cage, the Chlamydia spp. prevalence in this group could be artificially influenced. Interestingly, AR13 isolate obtained from a collared dove from Bologna showed the same particular MLST pattern as detected in a dove from Faenza. The high Chlamydia spp. prevalence detected in collared doves, as well as the finding of the same Chlamydia spp. genotype in birds collected from different geographic areas in different periods, suggest that S. decaocto represents an effective reservoir for C. psittaci. The spread of Chlamydia infection from Eurasian Collared Doves to others birds and humans seems to be a potential risk. Kalmar et al. (2014) reported the occurrence of C. psittaci infection in wild birds (including Eurasian Collared Doves) sampled in a wild bird refuge center, resulting in the infection of the staff members. Our findings suggest that workers in wildlife centers and people who may have professional or other contact with Eurasian Collared Doves should be informed about the risk of contracting

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Chlamydia psittaci in Eurasian Collared Doves (Streptopelia decaocto) in Italy.

We investigated the Chlamydia spp. occurrence in Eurasian Collared Doves (Streptopelia decaocto) from urban and suburban areas in northern Italy. Amon...
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