Chlamydia spp. are obligate intracellular bacterial pathogens that cause a number of important diseases in animals and humans. Novel members of the group have been discovered in the last few years [Reference Sachse1–Reference Taylor-Brown, Bachmann, Borel and Polkinghorne5] including Chlamydia gallinacea, which was found primarily in chickens, but was also shown to infect cattle [Reference Hulin6–Reference Li8]. Experimentally infected chickens exhibit no clinical disease symptoms but grow more slowly and have significantly reduced gains in body weight (6·5–11·4%) [Reference Guo7]. Importantly, French slaughterhouse workers exposed to C. gallinacea-infected chickens developed respiratory clinical symptoms, suggesting that C. gallinacea may also infect humans and may represent a zoonotic threat [Reference Hulin6]. The organism appears to be widely distributed and has been reported in four European countries as well as in Argentina and China [Reference Sachse1,Reference Hulin6–Reference Li8]. To determine if this emerging agent might also be present in North America, we used PCR to determine if backyard poultry from Alabama is infected with C. gallinacea.

All samples used in this study were collected for the avian influenza and avian paramyxovirus type-1 surveillance program in avian species organized by the US Department of Agriculture (USDA). Sample collections followed the recommendation for collecting swab specimens from poultry for viral diagnostic testing (USDA SOP WI-AV-0020). In total, 1929 oral–pharyngeal swabs from apparently healthy poultry were collected into 531 tubes (1–5 swabs from the same bird and same owner/per tube) containing 3 ml brain–heart infusion (BHI) medium and transported on ice to the Alabama State Diagnostic Laboratory. For DNA extraction, BHI for each tube was thawed at room temperature and a 50 µl aliquot processed using the MagMax 96 Viral RNA isolation kit (ThermoFisher Scientific, Waltham, MA, USA). The extracted DNA was collected in 50 µl elution buffer and tested for Chlamydia spp. using a previously described Chlamydia spp. real-time FRET-PCR [Reference Guo7]. This PCR amplifies a 168-bp fragment of the 23S rRNA gene of Chlamydia spp. and can detect all 11 Chlamydia species with a detection sensitivity of a single copy/reaction. High-resolution melting curve analysis enables differentiation of the Chlamydia spp. detected [Reference Guo7].

To further characterize the organisms present, we analyzed high-copy C. gallinacea-positive specimens by standard PCRs targeting a variable region of the 16S rRNA (301 bp) of the recognized 11 Chlamydia spp. and a highly variable domain (VD) of the 23S rRNA (329 bp) [Reference Li8,Reference Frutos9]. In addition, C. gallinacea-specific PCRs targeting the ompA VDs 1–2 (478 bp) and VD 3–4 (311 bp) [Reference Li8] were used to investigate the polymorphism of C. gallinacea in the backyard poultry studied (Fig. 1). All PCR products were verified by gel electrophoresis and automated DNA sequencing (Elim Biopharmaceuticals, Inc., Hayward, California, USA).

Fig. 1. Phylogenetic comparison of Chlamydia gallinacea from backyard poultry in this study. The 16S rRNA (301 bp) and 23S rRNA (329 bp) gene sequences (in red font and accession number) found in the current study are compared with those of other representative Chlamydia spp. (in black font with GenBank accession number) using the Clustalx 1.83 alignment software. In addition, the ompA VD 1–2 (478 bp) and ompA VD 3–4 (311 bp) of C. gallinacea strains identified in in this study are compared with other C. gallinacea from poultry in European countries and China. Based on these alignments, phylogenetic trees were constructed by the neighbor-joining method using the Kimura 2-parameter model with MEGA 6.0. Branch lengths are measured in nucleotide substitutions and numbers show branching percentages in bootstrap replicates. Scale bar represents the percent sequence diversity.

Altogether 12·4% (66/531) of the tubes containing the 1929 oral–pharyngeal samples that we collected from backyard poultry in Alabama were positive in the Chlamydia FRET-PCR. The positives comprised 64 of the 472 tubes containing swabs from chickens (13·6%) and two of the six tubes containing swabs from guinea fowl (33·3%). None of the tubes with swabs from turkeys, geese, ducks, quail, pheasants, peafowl, and pigeons were positive. The 16S rRNA and 23S rRNA gene sequences showed that only C. gallinacea was detected, and the strains found in this study were identical to those from Europe and China (Fig. 1a and b ). Sixteen samples positive in the genus-specific Chlamydia-FRET-PCR with over 1000 copies of the 23S rRNA/reaction were further tested with standard PCRs targeting the ompA VD 1–2 and ompA 3–4 domains of Chlamydia spp. [Reference Guo7,Reference Li8]. Phylogenetic comparisons showed that 16 sequenced samples represented 14 biotypes (Fig. 1c and d ), and the sequences were deposited in GenBank (KY206892, KY206893, KY206894-KY206907, and KY206908-KY206920) (Fig. 1a – d ). The value of phylogenetic comparisons based on partial gene sequences of ompA is limited. Research is in progress to isolate C. gallinacea from swabs of backyard poultry, which allows amplification of the whole ompA sequences for complete phylogenetic comparisons.

Avian chlamydiosis is an important infectious disease of domestic poultry as well as companion and wild birds that can be associated with acute, subacute, or chronic respiratory, digestive, or systemic signs. Although C. psittaci was considered to be the sole causative agent of avian chlamydiosis, recent studies indicated that prevalence of C. gallinacea may be higher than that of C. psittaci [Reference Guo7,Reference Sachse, Laroucau and Vanrompay10]. A survey conducted over 1 year in a French abattoir demonstrated 80·0% of the Chlamydiaceae-positive samples contained C. gallinacea [Reference Hulin6]. Our study in the USA adds to the growing evidence that C. gallinacea is a common and widespread chlamydial agent in poultry.

In conclusion, we report for the first time the presence of C. gallinacea in North America. The findings from our study should alert the poultry industry in North America to the possibility of a newly emerging pathogen, C. gallinacea, in their flocks. Further studies are indicated to more definitively establish the organism's pathogenicity, hosts, transmission, and zoonotic potential. Such studies will be aided by Chlamydia species-specific peptide ELISAs that have recently become available [Reference Rahman11].