FIRST DETECTION OF CHLAMYDOPHILA PECORUM IN CAPTIVE BIRDS IN ARGENTINA

FRUTOS, M; IVGAULLET, ML; MONETI, M; VENEZUELA, F; KIGUEN, X; RE, V; FERMEPIN M; CUFFINI, C

Holanda

Encuentro; 7th Meeting of the European Society for Chlamydia Research.; 2012

European Society for Chlamydia Research

Bacteria belonging to the family Chlamydiaceae cause a broad spectrum of diseases in a wide range of hosts, including humans, other mammals, and birds. The Chlamydiaceae are antigenically and genetically diverse, belonging to two genera and nine species (3). Inclusions formed in host cells by these bacteria are recognized, but not necessarily distinguished from one another, by microscopy and staining methods. Occasionally, however, specimens that could not be confirmed by techniques positively specific for the Chlamydiaceae were cultured or isolated. PCR and other DNA-based tests for chlamydiae have tended to be specific for groups within the Chlamydiaceae. Tests targeting the ompA gene have shown some promise as tools for identification of all Chlamydiaceae (8, 10). It was previously believed that specific Chlamydiaceae species were likely to be responsible for disease in specific hosts. However, studies have shown that many of the Chlamydiaceae species have a number of different hosts (1; 2; 5). With the emergence of advanced diagnostic technology, new Chlamydiaceae species have been found in animal species, which otherwise are not a major focus for disease investigation. Real-time PCR based on targeting 23S rDNA, was highly sensitive and rapid for detection and quantification of Chlamydiaceae from cloacal samples. These tests are so specific for the Chlamydiaceae that they may be useful for the screening of field specimens. Nucleotide sequencing of the ompA gene can also be used as a tool for identifying Chlamydiaceae species. Chlamydophila pecorum strains have been isolated from ruminants, swine and koalas in several countries. C. pecorum is associated with conjunctivitis, encephalomyelitis, enteritis, pneumonia, polyarthritis, abortion, and reproductive and urinary tract diseases (4; 6; 8). In a work carried out in free healthy pigeons in Japan, three fecal samples were found to be C. pecorum-positive by PCR (9). The epidemiology of Chlamydia infection in animals in Argentina is unknown. Thus, the aim of the present study was to detect Chlamydia in birds in illegal captivity from Córdoba city, Argentina. Cloacal swabs were collected from 28 birds confiscated by the Provincial Secretary for the Environment in September 2010. DNA extract was tested with the TaqMan test previously described by Ehricht et al. 2006. The DNA extract the positive sample was used to amplify a fragment of 576 pb of the variable domains II, III and IV of the omp A gene of Chlamydophila, as described by Sachse and Hotzel (7). By using the TaqMan test, five samples were positive and can be detected as few as 1 IFU of Chlamydiae in 5 ml template. Nested-PCR showed that none of the samples was positive for Cp. psittaci and that five were positive for Cp. pecorum. The latter were confirmed by sequencing. For sequence analysis, the nested-PCR products were purified and and subjected to automated sequencing. The omp A sequences obtained were submitted to GenBank and relatedness of newly characterized sequences was assessed by analysis with 2.2.19 Basic Local Alignment Search Tool (BLAST). Phylogenetic analyses were constructed with Mega software version 4, using the Neighbor-Joining method and their reliability was assessed by bootstraps (2,000 replicates). Phylogenetic analysis showed that the five sequences clustered with Cp. pecorum. The omp A sequence of the Cp. pecorum strains was highly homologous and shared more than 98% similarity with each other. Blast searches revealed that all novel omp A gene sequences shared high homology with the omp A from the iC2, iC3, iC4, 3638/3 and 4283/3 strains. All local strains were characterized by a thymine at position 132, resulting in Asn instead of Lys in the VD IV of gene omp A. The discrepancy found in this position could be defined as a marker of local strains. In 98% of the cases, the bootstrap values in the omp A phylogenetic tree supported the conclusion that all local strains belong to the same group. This chlamydia has generally been associated with the detection in ruminants (11), however, Tanaka et al. has been detected in birds like the ones found in this study. Cp. pecorum infection did not appear to be associated with any clinical signs of these birds. These birds could be either asymptomatic reservoirs or subclinical carriers of Cp. pecorum. The epidemiological significance of Cp. pecorum infection is not clear to us at this stage. Further studies to estimate the zoonotic role of this pathogen are needed.