Impact of urban environment and host phenotype on the epidemiology of Chlamydiaceae in feral pigeons (Columba livia)emi_

Environmental Microbiology (2011) doi:10.1111/j.1462-2920.2011.02575.x Impact of urban environment and host phenotype on the epidemiology of Chlamydiaceae in feral pigeons (Columba livia)emi_2575 1..8 J. Gasparini, 1 * N. Erin, 1,2 C. Bertin, 3 L. Jacquin, 1 F. Vorimore, 3 A. Frantz, 1 P. Lenouvel 1 and K. Laroucau 3 1 Laboratoire Ecologie et Evolution (EcoEvo), CNRS ENS UPMC UMR 7625, Université Pierre et Marie Curie, 7 quai St Bernard, 75252 Paris, France. 2 Department of Evolutionary Ecology, Max-Planck Institute for Evolutionary Biology, August-Thienemann Str. 2, 24306 Ploen, Germany. 3 Bacterial Zoonoses Unit, French Agency for Food, Environmental & Occupational Health & Safety (ANSES), Maisons-Alfort, France. Summary Chlamydiaceae are obligate intracellular Gramnegative bacteria found all over the world and known to cause various forms of disease in animals and humans. Urban pigeons are known to be an important reservoir of Chlamydia psittaci, the agent of human psittacosis. In this study, we examined the influence of pigeon houses used to regulate pigeon populations and of melanin-based coloration on several epidemiological parameters of Chlamydiaceae in 708 urban pigeons in Paris. We also identified species and genotypes of Chlamydiaceae present in Parisian populations. First, our results revealed that pigeons roosting and breeding in pigeon houses were equally infected by Chlamydiaceae as those that did not. Second, we found that dark melanic pigeons excreted more Chlamydiaceae than pale melanic ones. Finally, species and strain diversities were very low: all samples were of C. psittaci genotype B. Nevertheless, two atypical Chlamydiaceae were identified based on 16S rrna and ompa sequences. Our study thus highlights the importance of considering environmental and host phenotype when investigating the epidemiology of infectious diseases. Received 28 January, 2011; accepted 25 July, 2011. *For correspondence. E-mail jgaspari@snv.jussieu.fr; Tel. (+33) (1) 44 27 38 23; Fax (+33) (1) 44 27 35 16. Introduction Chlamydiaceae spp. are obligate intracellular Gramnegative bacteria distributed worldwide, known to cause various forms of disease in animals including humans. According to the most recent taxonomy, the family of Chlamydiaceae with its single genus Chlamydia (C.) contains nine species (Kuo et al., 2010). One of these, C. psittaci can be transmitted from animals to humans, inducing influenza symptoms (pneumonia) that are potentially lethal without antibiotic therapy (Petrovay and Balla, 2008). A recent review outlined the feral pigeon Columba livia as an important reservoir of this zoonosis in urban areas with a seroprevalence of 24 96% in pigeons from different European cities (Magnino et al., 2009). B and E C. psittaci genotypes are commonly detected in urban pigeons (Magnino et al., 2009) but C and D genotypes, which are highly virulent for humans, have also been recently reported in Belgium (Dickx et al., 2010). As they live close to humans, urban pigeons are thus an important reservoir of this zoonotic disease, but factors influencing the transmission risk of this pathogen are still poorly known (Haag-Wackernagel and Moch, 2004). Urban areas provide a particular environment where closely interacting biological and social constraints may either directly or indirectly impact on the presence of Chlamydiaceae in pigeons (e.g. via influences on the pigeon phenotype, which in turn may affect the prevalence of the zoonosis). In this study, we first focused on one particular environmental factor, the presence of pigeon houses. Pigeon houses are artificial breeding facilities tested in different European cities to limit local nuisances and to improve the health of urban pigeons (e.g. Basel, Switzerland Haag-Wackernagel, 1995). Since 2003, several pigeon houses have been set up in Paris but their effects on pathogen transmission and public health have not yet been investigated. Such regulation strategies can have important impacts on the ecology of pigeons, as suggested by a recent study (Jacquin et al., 2010). Pigeon houses could indeed have two opposite effects on pathogen transmission risk. First, as pigeons roosting in pigeon houses are provided with food and therefore likely to be in better physical condition, they should be less susceptible to pathogens than 2011 Society for Applied Microbiology and Blackwell Publishing Ltd

2 J. Gasparini et al. pigeons roosting in the street. Inversely, high pigeon densities in such facilities could favour pathogen transmission between birds. In this study, we examined the correlation between habitat and C. psittaci excretion and also between habitat and seroprevalence of pigeons, to evaluate the potential impact of pigeon houses on public health. Furthermore, both intrinsic resistance to pathogens and pathogen excretion can be highly variable between individuals and are likely to have a strong impact on the epidemiology of C. psittaci. While seroprevalence and excretion prevalence are indices of epidemiological infection, serotitre of seropositive individuals is a proxy of the intensity of the immune response developed by individuals against this infection, and the level of cloacal excretion informs on the direct risk of infectious Chlamydiaceae present in the environment likely to infect humans. Therefore, we also examined the link between melanin-based coloration, serotitre and chlamydial excretion of individual pigeons. This is because urban pigeons display a melanic colour polymorphism and a recent study reports the existence of a link between melanic coloration and resistance to pathogens for this species (Jacquin et al., 2011). This observation confirms previous results found in other avian species (Gasparini et al., 2009; see Ducrest et al., 2008 for a review) and raises the interesting possibility that, in our case, pigeon phenotype could also be related to host resistance against Chlamydiaceae. To date, there is little information available on the role of urban environment and host phenotype on the presence of this pathogen in urban areas. In this study, we examined correlations between the presence of Chlamydiaceae and both environmental (presence of pigeon houses) and phenotypic factors (body mass and coloration) in urban pigeons. We also identified species and genotypes of Chlamydiaceae circulating in the Parisian pigeon population. This is important from a public health point of view, providing information on whether new potentially virulent species of Chlamydiaceae are circulating in Parisian pigeons. Results Pigeon house, coloration and presence of Chlamydiaceae The seroprevalence and prevalence of chlamydial excretion were respectively 29.3% (range 0 100%) and 17.6% (range 0 43%) (Table 1). There was a significant association between the seroprevalence and the excretion prevalence (n = 376, rj =0.10, c 2 1 = 4.0, P = 0.05) and there was a strong heterogeneity of seroprevalence and excretion prevalence between locations (location effect on seroprevalence: c 2 11 = 43.5, P < 0.0001, on excretion prevalence: c 2 14 = 39.3, P = 0.0003). We found no difference in seroprevalence and excretion prevalence between pigeons roosting in pigeon houses and those roosting in the street (Table 2, Fig. 1). In addition, we found no effect of melanin-based coloration on seroprevalence and excretion prevalence (Table 2). In contrast, for the subset of individuals for which body mass was measured, we found that seropositive individuals were heavier (320 g 3) than seronegative individuals (310 g 2, mixed-model ANCOVA for binomial data, F 1,357 = 5.27, P = 0.02). There was however no effect of body mass on excretion level (mixed-model ANCOVA for binomial data, F 1,529 = 0.00, P = 0.98). Focusing on seropositive individuals, we found no relationship between serotitre and body mass (mixed-model ANCOVA, F 1,71 = 0.70, P = 0.41), melanin-based coloration or habitat (roosting in pigeon houses or in the street, Table 2). For positively excreting individuals, there was no relationship between intensity of excretion (log [number of calculated inclusion-forming units per sample]) and body mass (mixed-model ANCOVA, F 1,89 = 0.00, P = 0.99) or habitat (Table 2). In contrast, we found that dark melanic pigeons (morph class 4) excreted significantly more Chlamydiaceae in their faeces than the other morphs (Table 2, Fig. 2). Strain isolation and genotyping All PCR-positive samples (n = 125) were examined by bacterial culture and 32 strains were eventually isolated. Sequencing the partial ompa fragment showed a strong conservation of the ompa sequence among strains isolated from pigeons captured in different geographical areas, which were all identical to the C. psittaci CP3 reference strain (genotype B). All samples that gave a positive signal with the 23S- real-time PCR and that were not cultivated were all re-analysed with a C. psittaci specific real-time PCR. Results are presented in Table 1. Among samples having a Ct value lower than 34 (more than 500 calculated inclusion-forming units/samples) (n = 56) with the Chlamydiaceae real-time PCR, all were positive with the C. psittaci real-time PCR, except for two samples (09-489/LP23 and 09-589/S46). Analysis of ompa sequences directly obtained from the highest-titre C. psittaci real-time PCR-positive samples (n = 22) revealed only genotype B. To establish the identity of the two particular samples (09-489/LP23 and 09-589/S46), which did not react with the C. psittaci real-time PCR, the 16S rrna and ompa genes were partially sequenced directly on biological samples. BLAST analysis revealed that ompa sequences of 09-489/LP23 and 09-589/S46 were different (84% identity) but exhibited moderate similarity to a C. psittaci strain CPX0308 (AB284064) isolated from an oriental white stork (75% identity). To explore the genetic relatedness of

Chlamydiaceae in pigeons 3 Table 1. Summary of results from Chlamydiaceae analyses. ID number Habitat a City (district number) Dates of sample collection Nb of positive samples (% positive) 23S-rtPCR Chlamydiaceae Positive ompa-rtpcr C. psittaci/positive Mean of excretion (calculated IFU) for 23S rtpcr positive samples SE (min max) Chlamydiaceae Isolated strains Nb of seropositive sample (% positive) Mean of serotitre for seropositive samples SE (min max) D-09-295 Out Courbevoie (92) 2/18/2009 15/60 (25.0%) 4.98 10 4 4.95 10 4 (7.62 10 0 7.42 10 5 ) 11/15 6 20/60 (33.3%) 3.3 0.3 (1 5) D-09-589 Out Creil (60) 5/5/2009 16/66 (24.2%) 3.48 10 5 3.13 10 5 (4.29 10 1 5.01 10 6 ) 13/16 3 D-09-295 D-09-711 D-09-0336 D-09-0496 D-09-0592 D-09-0664 Out Gennevilliers (92) 2/18/2009 23/142 (16.2%) 2.18 10 4 1.52 10 5 (3.61 10 0 3.45 10 5 ) 12/23 1 8/52 (15.4%) 2.5 0.6 (1 5) In Paris_Jussieu (V) 28/02/2009 30/03/2009 15/04/2009 02/05/2009 D-09-543 In Fontenay sous bois_la Fontaine (93) 13/69 (18.8%) 1.50 10 4 1.06 10 4 (6.11 10 0 1.39 10 5 ) 7/13 5 18/46 (39.1%) 3.3 0.3 (2 5) 4/10/2009 7/77 (9.1%) 8.16 10 3 5.72 10 3 (1.93 10 1 3.90 10 4 ) 6/7 2 na na D-09-489 In Clamart_La Plaine (92) 3/27/2009 10/29 (34.5%) 5.61 10 4 5.39 10 4 (2.42 10 1 5.41 10 5 ) 8/10 3 4/29 (13.8%) 3.3 0.5 (2 4) D-09-662 In Paris_La Roquette (XI) 4/30/2009 3/11 (27.3%) 4.74 10 1 3.17 10 1 (1.48 10 1 1.11 10 2 ) 1/3 na 4/11 (36.4%) 3.3 0.6 (2 4) D-09-661 In Paris_Lazareff (IV) 4/27/2009 0/14 (0%) na na na 0/14 (0%) D-09-450 In Clamart_Maison Blanche (92) 3/23/2009 9/41 (22.0%) 2.41 10 4 2.19 10 4 (8.98 10-1 1.99 10 5 ) 6/9 5 17/41 (41.5%) 3.1 0.3 (1 5) D-09-588 In Paris_Montreuil (XX) 4/16/2009 9/21 (42.9%) 2.63 10 2 2.62 10 2 (7.13 10 0 1.86 10 3 ) 4/9 3 11/21 (52.4%) 3.2 0.4 (1 5) D-09-542 In Pantin (93) 4/1/2009 4/71 (5.6%) 2.16 10 4 1.74 10 4 (2.44 10 1 7.29 10 4 ) 4/4 1 19/47 (40.4%) na D-09-726 Out Paris_St Denis (X) 5/18/2009 2/36 (5.6% 1.88 10 0 1.89 10-1 (1.69 10 0 2.07 10 0 ) 1/2 na 3/3 (100%) na D-09-727 Out Paris_Sacré Cœur (XVIII) 5/18/2009 5/19 (26.3%) 1.00 10 6 9.54 10 5 (5.15 10 1 4.81 10 6 ) 3/5 na na na D-09-487 In Clamart_Trivaux (92) 3/25/2009 5/25 (20.0%) 2.87 10 5 2.78 10 5 (1.28 10 1 1.40 10 6 ) 5/5 2 2/25 (8.0%) 4.0 1 (3 5) D-09-544 In Paris_Vanves (XIV) 4/9/2009 4/27 (14.8%) 8.78 10 5 8.78 10 5 (1.38 10 1 3.51 10 6 ) 4/4 1 4/27 (14.8%) na Total 125/708 (17.6%) 1.43 10 5 6.29 10 4 (8.98 10-1 5.01 10 6 ) 85/125 (68.0%) 32 110/376 (29.3%) 3.2 0.1 (1 5) a. Roosting in or out pigeon houses. IFU, inclusion-forming units.

4 J. Gasparini et al. Table 2. Mixed-model ANOVA with excretion prevalence, intensity of excretion, seroprevalence and serotitre for Chlamydia as the dependent variable in separate models, habitat (roosting or not roosting in pigeon houses) and melanic coloration morphs as cofactors. Excretion prevalence Excretion intensity Seroprevalence Serotitre F df P F df P F df P F df P Habitat 0.17 1,13 0.68 0.09 1,12 0.77 1.31 1,10 0.28 0.04 1,7 0.85 Morph 0.24 4,689 0.92 3.79 4,107 0.006 1.12 4,360 0.35 0.67 4,72 0.61 We included the location as a random effect nested within habitat, as individuals sharing the same location were not statistically independent. F-values, degrees of freedom (df) and P-values (P) are given for fixed effects. Interactions between morph and habitat were all non-significant (P > 0.26) and were then removed from the final model. these two samples, 16S rrna sequences were aligned with corresponding sequences of strains representing the established species of the genus Chlamydia and a corresponding sequence of an atypical Chlamydiaceae (08-1274_Flock21) recently isolated from chickens (Laroucau et al., 2009). A phylogenetic tree (Fig. 3) indicates that the two identical 16S rrna sequences are closely related to those of the novel atypical one isolated from chickens. Discussion Prevalence and seroprevalence of Chlamydiaceae Fig. 1. Excretion and seroprevalence of Chlamydia SE in pigeons roosting or not roosting in pigeon houses. Differences between prevalence of pigeons roosting or not roosting in pigeon houses were not significant (Table 1). The seroprevalence and prevalence of excretion of Chlamydiaceae were relatively low in pigeon populations of Paris during spring 2009 (respectively 30% and 18%) compared with previous studies conducted in different European countries (Magnino et al., 2009). A recent study reports a prevalence of excretion of 53% in Madrid, Spain (Vázquez et al., 2010). In contrast, the prevalence of excretion was very low (8%) in Amsterdam, Netherlands (Heddema et al., 2006). In Paris, previous studies performed in 1999 and in 2003 showed mean seroprevalences of respectively 48% and 50% (Laroucau et al., 3 2 1 0 C.suis_R22 (U68420) C.trachomatis_A/Har-13 (NC_007429) C.muridarum_MoPn (D85718) C.pneumoniae_TW183 (AE009440) C.pecorum_E58 (NR_027576) 08-1274_Flock21 (GQ398029) 09-489/LP23 (JF756077) 09-589/S46 (JF756078) C.abortus_B577 (AB001783) C.psittaci_6BC (AB001778) C.caviae_GPIC (D85708) C.felis_FE145 (AB001785) Fig. 2. Mean of log [level of chlamydial excretion (calculated inclusion-forming units per sample)] SE in relation to melanic coloration morphs (visual score). Post-hoc tests revealed that dark morph (4) excreted significantly more than others. Numbers above bars indicate sample size of each morph. Fig. 3. Phylogenetic tree of partial 16S rrna (about 1350 bp) from samples 09-489/LP23 and 09-589/S46, from the type strains of nine Chlamydiaceae and from the recently described atypical Chlamydiaceae sp. 08-1274_Flock21 strain. Tree was constructed using the neighbour-joining method with Jukes-Cantor distance corrections. Horizontal distances correspond to the percentage of substitution per site.

Chlamydiaceae in pigeons 5 2005; Magnino et al., 2009). This spatio-temporal variation could not be explained by differences in methods used to estimate the seroprevalence, as the same method was used by the same laboratory. Therefore, our results suggest that the infection rate of pigeons by Chlamydiaceae decreased between 2003 and 2009 in Paris. Since 2003, several pigeon houses have been set up in Paris to limit local nuisance and to improve the health of urban pigeons. We therefore hypothesized that this decline in Chlamydiaceae seroprevalence could be due to the new introduction of such pigeon houses. This hypothesis was tested by a correlative approach comparing seroprevalence and prevalence of excretion for pigeons roosting in pigeon houses and those roosting in the street. Strong heterogeneity of infection by Chlamydiaceae among locations in Paris was found, but we did not find any difference of prevalence between pigeons roosting in pigeon houses or not (Fig. 1). Our results also suggest that higher density in pigeon houses does not increase prevalence of Chlamydiaceae. The decline in prevalence of Chlamydiaceae observed in Paris may therefore be due either to other factors of the urban environment that have changed during the past decade or to an adaptation of pigeons becoming less susceptible to this bacterium (Frank, 2002). Additional studies in urban areas are now required to understand why infection with this zoonotic disease has decreased during the past decade in pigeons living in Paris. Intensity of immune response to Chlamydiaceae We found that the intensity of the immune response (measured by serotitre) was related to body mass and was not related to melanin-based coloration or to habitat (roosting or not roosting in pigeon houses). This correlative result suggests that individuals in good condition are better able to mount a strong immune response and is in agreement with ecological immunology theories (Sorci et al., 2009). This result highlights that it might be possible to increase immune resistance of urban pigeons to Chlamydiaceae by increasing their nutritional status. Currently, pigeons roosting in pigeon houses are fed every week by local authorities. However, we failed to find differences of body mass between pigeons roosting or not roosting in pigeon houses (mixed-model ANOVA, F 1,11 = 3.50, P = 0.09) suggesting that the food provided every week is not enough to increase their nutritional status, health and immune resistance against Chlamydiaceae. Cloacal excretion of Chlamydiaceae and melanin-based coloration The level of cloacal excretion is an important parameter for public health because it informs us about the direct risk of infectious Chlamydiaceae transmission to humans. Indeed, humans can even be infected by inhalation of aerosols contaminated with faecal dust from infected birds (Magnino et al., 2009). First, we found that the level of cloacal excretion was not related to body mass or habitat (roosting or not roosting in pigeon houses). This result suggests that the risk for humans is not modified by the use of pigeon houses set up by local authorities. Second, we found a link between the level of cloacal excretion and melanin-based coloration. Indeed, post-hoc tests (Fig. 2) revealed that darker individuals (morph class 4) excreted significantly more Chlamydiaceae than the others (morph classes 0, 1, 2 and 3). In disagreement with this, a recent study showed that differently coloured pigeons were similarly exposed to blood parasites but that darker melanic individuals were more able to control the infection (Jacquin et al., 2011; see also Roulin et al., 2001 for experimental support in another avian species). Our results, though significant, are based on a small sample size (only eight pigeons from morph class 4) and therefore need to be confirmed by increasing sample size. This result may however have important implications for characterizing the risk of transmission of Chlamydiaceae to humans. Indeed, it shows that melanin-based coloration could be a key factor affecting transmission risk. Since the level of cloacal excretion is higher in dark melanic pigeons, the risk could be directly linked to the proportion of dark pigeons in the population. If this proportion is high, the risk would be relatively higher. In Paris, dark pigeons (morph class 4) represent only 10% of the population (L. Jacquin, unpubl. data) suggesting that the risk could be fairly low. Species and strain diversity of Chlamydiaceae in Parisian pigeons Genotyping analyses did not reveal strong diversity in species or in strains infecting pigeons in Paris, as we mainly found the C. psittaci species. Contrary to a recent study performed on homing pigeons in Ghent, Belgium, where Dickx and colleagues (2010) discovered three genotypes of C. psittaci (genotypes B, C and D) among 32 pigeons, we only detected genotype B in this study of more than 700 pigeons. This genotype is the most prevalent in pigeons (Heddema et al., 2006; Magnino et al., 2009). This is a reassuring result for public health as this genotype is less virulent for humans than genotypes A and D (Beeckman and Vanrompay, 2010), even if human cases due to genotype B are also reported. However, two atypical Chlamydiaceae were detected from two individuals captured in two different geographical areas (see Fig. 3). Identical 16S rrna and similar ompa sequences were previously obtained from Chlamydiaceae isolated from Italian pigeons (Vicari et al., 2009).

6 J. Gasparini et al. Phylogenetic comparison of their 16S rrna sequences to corresponding reference sequences, including a representative of an atypical Chlamydiaceae recently isolated from chickens (Laroucau et al., 2009), suggests that these two atypical Chlamydiaceae detected from two French pigeons are closely related to the new ones isolated from chickens. Their detection was possible because of the use of real-time PCR targeting all Chlamydiaceae, which is now strongly recommended for laboratory diagnosis of chlamydial infection. Indeed, the spectrum of Chlamydiaceae in Aves is broader than initially suspected, and not restricted to the C. psittaci species (Herrmann et al., 2000; Gaede et al., 2008; Laroucau et al., 2009; Vicari et al., 2009; Lemus et al., 2010). It is therefore important to take into account the presence of these atypical strains of Chlamydiaceae and to estimate their prevalence in pigeon populations. For that purpose, a specific tool is now needed. More generally, our study highlights the importance of taking into account environmental and host phenotype factors when investigating the epidemiology of infectious diseases. Experimental procedure Sample collection Samples were collected from 708 pigeons in 15 different locations in the Parisian agglomeration during spring 2009 (from February to May) either in pigeon houses or in the street in collaboration with different local associations. Table 1 summarizes the information about sample collection. When authorized by local authorities, we collected 1 ml of blood from the ulnar vein using a sterile syringe and performed cloacal swabbing. Blood samples were kept at 10 C until centrifugation during the evening following the capture. The blood serum was then isolated and kept frozen until serological analysis. One panel of swabs was stored in SPG conservation buffer (Spencer and Johnson, 1983) at -80 C until inoculated into chicken eggs. The other panel was stored dry at -80 C until subjected to DNA extraction. During the capture, pigeons were also weighed to the nearest g using Pesola and coloration was eye scored. Colour variation in urban pigeons (black, red or brown) is due to the deposition of two different types of melanin pigments in the feathers: yellow to red phaeomelanins and black eumelanins (Haase et al., 1992). In this study we focused on eumelanic black coloration because it is the most widespread coloration in urban populations. Pigeons display a continuous variation in this eumelanin-based coloration from white to black (Johnston and Janiga, 1995), which can be easily scored by human eye into five main groups (Johnson and Johnston, 1989; Johnston and Janiga, 1995; Jacquin et al., 2011): (0) White or almost white individuals; (1) Blue bar (gray mantle with two dark wing bars); (2) Checker (a checked mantle with moderate dark spots); (3) T-pattern (a dark mantle with small gray marks); and (4) Spread (a completely melanic plumage). We therefore visually scored all individuals captured. These patterns are mainly genetically determined (Johnston and Janiga, 1995) and differ by the surface of dark area on the wings that corresponds to different levels of melanin deposition in feathers (Haase et al., 1992). Serological analyses A microtitre complement fixation test for chlamydial antibodies was performed according to the national French standard (Collective, 2000). Sera were treated at 59 C for 30 min before testing. Guinea pig complement was used at 2 UH100, and a commercial ornithosis antigen (Dade Behring/ Siemens, Marburg, Germany) was used at the recommended dilution. The presence of 50% of haemolysis starting from the 1/8 dilution of sera was considered positive and enabled us to measure the seroprevalence. To estimate the serotitre, analyses were re-run on positive sera at different dilutions: 1/8, 1/16, 1/32, 1/64, 1/128 and 1/256. The resulting serotitre values are therefore a semi-quantitative measure of anti- Chlamydiae antibody level in the blood. It was estimated as the negative log2 of the last serum dilution exhibiting 50% of haemolysis, i.e. dilution 1/8 is a score of 3, 1/16 of 4, 1/32 of 5, 1/64 of 6, 1/128 of 7 and 1/256 of 8. Direct detection of Chlamydiaceae from cloacal swabs The dry panel of cloacal swabs was subjected to DNA extraction using a QIAamp DNA Mini Kit, following the buccal swab protocol (Qiagen, Courtaboeuf, France). DNA was eluted with 150 ml of AE buffer and stored at -20 C before analysis. A Chlamydiaceae-specific real-time PCR targeting the 23S rrna gene was used in this study (Ehricht et al., 2006). The protocol included primers Ch23S-F (5 -CTGAAACCAGT AGCTTATAAGCGGT-3 ) and Ch23S-R (5 -ACCTCGCCG TTTAACTTAACTCC-3 ), and probe Ch23S-p (FAM-5 - CTCATCATGCAAAAGGCACGCCG-3 -TAMRA). Each reaction mix contained 2 ml of sample DNA template, 10 ml of Universal Mastermix 2X (Applied Biosystems), 0.5 ml of each primer (25 mm) and 2 ml of the probe (1 mm), with 5 ml of deionized water. The temperature time profile was 95 C 10 min, 45 cycles of 95 C 15 s, 60 C 60 s. The Chlamydiaceae-specific real-time PCR (23S-rtPCR Chlamydiaceae) enabled us to compute the prevalence of pigeons excreting Chlamydiaceae (cloacal prevalence) and their excretion level. The number of inclusion-forming units per sample was calculated from the Ct value on the basis of the calibration curve from serial 10-fold dilutions of a titrated DNA (kindly provided by Dr K. Sachse, FLI, Jena). Strain isolation Samples that were diagnosed positive by PCR were tested by culture. Suspensions of cloacal swabs stored in conservation buffer at -80 C were thawed, transferred into sterile Eppendorf tubes and centrifuged at 10 000 r.p.m. for 5 min. Supernatant was transferred into a new sterile tube. Antibiotic solution containing 0.1 mg of vancomycine, 0.1 mg of streptomycin, 0.1 mg of kanamycin and 100 U of nystatin was added to the supernatant and the pellet suspensions, which were then incubated at 37 C for 2 h before inoculation into eggs. Yolk sacs of 7-day-old embryonated eggs were inocu-

Chlamydiaceae in pigeons 7 lated with 0.2 ml per egg. For each set of inoculation, three eggs were inoculated with C. psittaci Loth strain as positive controls, and three other eggs were kept separately as noninfected controls. Eggs were incubated at 38 C and observed daily. Vitellus membranes were collected, then analysed by PCR. Sample and isolate genotyping All samples that gave a positive signal with the 23S-rtPCR were re-analysed with an ompa-based real-time PCR assay specific for C. psittaci as recently described (Pantchev et al., 2009). For all C. psittaci PCR-positive samples, partial ompa gene fragments were amplified from C. psittaci isolates or directly from biological samples as described previously using 3GPF (5 -ACG CAT GCA AGA CAC TCC TCA AAG CC-3 ) and 5GPB (5 -ACG AAT TCC TAG GTT CTG ATA GCG GGA c -3 ) primers (Kaltenboeck et al., 1993). For samples that were Chlamydiaceae PCR-positive but C. psittaci PCR-negative, partial ompa gene fragments were amplified from biological samples using CTU (5 -ATG AAA AAA CTC TTG AAA TCG G-3 ) and ompa-rev (5 -TCC TTA GAA TCT GAA TTG AGC-3 ) primers, and partial 16S rrna fragments were amplified using 16S1 (5 -CGG ATC CTG AGA ATT TGA TC-3 ) (Pudjatmoko et al., 1997) and rp2 (5 - CTA CCT TGT TAC GAC TTC AT-3 ) (Thomas et al., 2006) primers. The DNA sequencing of these products was carried out at MWG (Biotech France, Roissy, France). Nucleotide sequences (partial ompa and 16S rrna genes) of the two atypical Chlamydiaceae are available from GenBank (HQ917531, HQ917532 and JF756077, JF756078 respectively). Sequence data were analysed using the Bionumerics software package version 4.6 (Applied-Maths, Saint- Martens-Latem, Belgium) as a character dataset. A 16S rrna-based phylogenetic tree was constructed by the neighbour-joining method using the Jukes-Cantor model. Statistical analysis We investigated the effect of habitat (pigeon house vs. street) and melanin-based coloration of pigeons (visual coloration score) on cloacal prevalence, intensity of cloacal excretion, seroprevalence and serotitre of Chlamydiaceae. We used mixed-model ANOVAs with cloacal prevalence, intensity of cloacal excretion, seroprevalence and serotitre of Chlamydiaceae as dependent variables and habitat (roosting or not roosting in pigeon houses) and coloration score as cofactors. We controlled for the non-independence of data collected for pigeons captured on the same location by incorporating location (nested within habitat) as a random factor in the mixed models. Non-significant interactions were removed step by step and statistical analyses were performed using the SAS system (version 9.1: SAS Institute, Cary, NC, USA). Levels of excretion (number of calculated inclusion-forming units per sample) were log transformed to normalize the data. (Log [number of calculated inclusion-forming units per sample].) Binomial distributions were used for mixed-model ANOVAs involving prevalence as dependent variable (Proc GLIMMIX). Means are quoted SE, statistical tests are two-tailed and P-values less than 0.05 are considered significant. Acknowledgements All experiments were carried out with the authorization of the Direction Départementale des Services Vétérinaires de Seine-et-Marne (authorization no. 77 05). We thank A.C. Prévot-Julliard and the scientific network Le pigeon en ville for help at different stages of the manuscript. We thank the AERHO association (C. Dehay), Espace association (Y. Fradin), Le jardin solidaire (I. Trinité) and the municipal authorities of Paris (T. Charachon) for providing us logistic support during the field work. We wish to thank two anonymous reviewers for comments on earlier version of the manuscript. We also thank John Kerr and Dr Mahesh Panchal for language correction. 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