Oral Uptake of Chlamydia psittaci by Ducklings Results in Systemic Dissemination

RESEARCH ARTICLE Oral Uptake of Chlamydia psittaci by Ducklings Results in Systemic Dissemination Simon Thierry1☯, Fabien Vorimore1☯, Christelle Rossignol2,3, Sabine Scharf4, Konrad Sachse4¤, Patricia Berthon2,3, Benoit Durand5, Isabelle Virlogeux-Payant2,3, Nicole Borel6, Karine Laroucau1* 1 ANSES, Animal Health Laboratory, Bacterial Zoonoses Unit, Maisons-Alfort, France, 2 INRA, UMR1282 Infectiology and Public Health, Nouzilly, France, 3 UMR1282 Infectiology and Public Health, François Rabelais University, Tours, France, 4 Friedrich-Loeffler-Institut (Federal Research Institute for Animal Health), OIE Reference Laboratory for Chlamydiosis, Jena, Germany, 5 ANSES, Animal Health Laboratory, Epidemiology Unit, Maisons-Alfort, France, 6 Institute of Veterinary Pathology, University of ZurichVetsuisse, Zurich, Switzerland ☯ These authors contributed equally to this work. ¤ Current address: Dept. of Bioinformatics, Friedrich-Schiller-Universität, Jena, Germany * a11111 Abstract OPEN ACCESS Citation: Thierry S, Vorimore F, Rossignol C, Scharf S, Sachse K, Berthon P, et al. (2016) Oral Uptake of Chlamydia psittaci by Ducklings Results in Systemic Dissemination. PLoS ONE 11(5): e0154860. doi:10.1371/journal.pone.0154860 Editor: Guangming Zhong, Univ. of Texas Health Science Center at San Antonio, UNITED STATES Received: November 19, 2015 Accepted: April 20, 2016 Published: May 11, 2016 Copyright: © 2016 Thierry et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper. Funding: The authors received no specific funding for this work. Competing Interests: The authors have declared that no competing interests exist. Enteric infections caused by Chlamydia (C.) psittaci are frequent in ducks, but mostly remain subclinical under field conditions. To emulate natural infection, we investigated the pathogenic potential of a C. psittaci field strain in orally inoculated 4-day-old ducklings. Three different challenge doses were tested and seven contact animals were also mock-inoculated with buffer in each group. Over the course of ten days, the birds were monitored for clinical symptoms and chlamydial dissemination before final examination of tissues using histopathology and immunohistochemistry. While the challenge strain disseminated systemically to all internal organs, mild signs of diarrhea were confined to ducklings inoculated with the highest dose (4.3 x 108 IFU/mL, Group 1). No other clinical symptoms or histopathological lesions were seen. The chlamydial load in internal organs as measured by PCR depended on the challenge dose and was unevenly distributed, i.e. high loads in spleen, liver, and distal small and large intestinal tract (ileum, cecum and rectum) vs. ten times lower values in lungs and proximal small intestinal tract (duodenum and jejunum). Notably, the C. psittaci infection of contact birds became evident on day 10 post-infection, with bacterial loads comparable to those of experimentally-infected animals, thus suggesting rapid bird-to-bird transmission of the challenge strain. Introduction The family Chlamydiaceae comprises a group of obligate intracellular Gram-negative bacteria that are widely distributed throughout the world, causing a wide range of diseases in humans and animals [1]. It is composed of a single genus, Chlamydia, known to contain 11 species: C. abortus, C. avium, C. caviae, C. felis, C. gallinacea, C. muridarum, C. pecorum, C. pneumoniae, C. psittaci, C. suis, and C. trachomatis [2,3]. PLOS ONE | DOI:10.1371/journal.pone.0154860 May 11, 2016 1 / 12 Oral Uptake of Chlamydia psittaci by Ducklings Avian chlamydiosis due to C. psittaci is a major factor of economic loss within the poultry industry, and a permanent risk for zoonotic transmission to humans [4,5]. Infection was observed in more than 467 species of birds belonging to 30 different orders [6], including poultry. In birds, symptoms depend on the virulence of the chlamydial strain and sensitivity of the host [7]. In general, transmission occurs through inhalation of aerosolized respiratory secretions or ingestion of contaminated dust. In turkeys and sometimes in chickens, C. psittaci infections can lead to respiratory symptoms, a drop in daily weight gains and death, all of which impair the farm’s economic performance [7,8]. To investigate the underlying processes, various experimental models of respiratory infections in turkeys or chickens have been developed [9–11]. In contrast, enteric chlamydial infections due to C. psittaci occur frequently in ducks, but mostly remain subclinical [12–15], making this infection primarily a public health issue. The initial factors and clinical course of duck infections under field conditions are poorly characterized. The interaction of horizontal and vertical transmission pathways in the infectious process is unknown, but the massive C. psittaci fecal shedding detected in adult animals on grassland [14,15] strongly suggests indirect transmission through environmental contamination, mainly by the fecal-oral route. To assess the virulence of C. psittaci in ducks in light of the known zoonotic potential for humans, infection models emulating natural transmission pathways can be very helpful. For this purpose, an experimental study on ducklings using the oral inoculation route was conducted in order to mimic field conditions and, in particular, investigate asymptomatic carriage and possible inter-animal transmission of C. psittaci. Materials and Methods Inoculum C. psittaci strain 06–859 (genotype E/B), isolated from a cloacal swab of an asymptomatic mule duck [16], was used as an inoculum. The strain was first passaged four times by injection into the yolk sac of 7-day-old embryonated specific-pathogen-free (SPF) chicken embryos. Once the embryos had died, the vitelline membranes from ten eggs were harvested, ground with sterile glass beads in 2SP medium (sucrose phosphate 2X with 0.1 mg/mL of vancomycin, 0.01 mg/ mL of gentamycin, 0.025 mg/mL of amphotericin B and 10% fetal bovine serum) and then centrifuged at 3000 rpm for 10 min. The supernatant was homogenized and stored at -80°C until inoculation. The titer of the inoculum was determined in Buffalo Green Monkey (BGM) cell culture as follows: BGM cells were seeded on round coverslips and cultured in growth medium consisting of minimal essential medium (Sigma-Aldrich, France) supplemented with 5% fetal calf serum (FCS). Tenfold dilutions of the inoculum were centrifuged at 3400 g for 1 h at 37°C and incubated for 2 h at 37°C. The growth medium was subsequently replaced by UltraMDCK serum-free medium (Ozyme, France). After 48 h of incubation, the cells were fixed in absolute methanol for 10 min. Chlamydial inclusions were detected by direct imm (...truncated)