Veterinary Immunology and Immunopathology 164 (2015) 30–39

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Research paper

Host–pathogen interactions in specific pathogen-free chickens following aerogenous infection with Chlamydia psittaci and Chlamydia abortus Isabelle Kalmar a,1 , Angela Berndt b,1 , Lizi Yin a , Koen Chiers c , Konrad Sachse b,∗,2 , Daisy Vanrompay a,2 a b c

Department of Molecular Biotechnology and Immunology, Ghent University, Belgium Institute of Molecular Pathogenesis, Friedrich-Loeffler-Institut (Federal Research Institute for Animal Health), Jena, Germany Department of Pathology, Bacteriology and Avian Diseases, Ghent University, Belgium

a r t i c l e

i n f o

Article history: Received 6 October 2014 Received in revised form 5 December 2014 Accepted 31 December 2014 Keywords: Avian chlamydiosis Pathogenesis Experimental infection Host immune response Chlamydia psittaci Chlamydia abortus

a b s t r a c t Although Chlamydia (C.) psittaci infections are recognized as an important factor causing economic losses and impairing animal welfare in poultry production, the specific mechanisms leading to severe clinical outcomes are poorly understood. In the present study, we comparatively investigated pathology and host immune response, as well as systemic dissemination and expression of essential chlamydial genes in the course of experimental aerogeneous infection with C. psittaci and the closely related C. abortus, respectively, in specific pathogen-free chicks. Clinical signs appeared sooner and were more severe in the C. psittaci-infected group. Compared to C. abortus infection, more intense systemic dissemination of C. psittaci correlated with higher and faster infiltration of immune cells, as well as more macroscopic lesions and epithelial pathology, such as hyperplasia and erosion. In thoracic air sac tissue, mRNA expression of immunologically relevant factors, such as IFN-␥, IL-1␤, IL-6, IL-17, IL-22, LITAF and iNOS was significantly stronger up-regulated in C. psittaci- than in C. abortus-infected birds between 3 and 14 days post-infection. Likewise, transcription rates of the chlamydial genes groEL, cpaf and ftsW were consistently higher in C. psittaci during the acute phase. These findings illustrate that the stronger replication of C. psittaci in its natural host also evoked a more intense immune response than in the case of C. abortus infection. © 2015 Elsevier B.V. All rights reserved.

1. Introduction The zoonotic and obligate intracellular bacteria Chlamydia (C.) psittaci and C. abortus are genetically closely related,

∗ Corresponding author at: Friedrich-Loeffler-Institut (Federal Research Institute for Animal Health), Naumburger Str. 96a, 07743 Jena, Germany. Tel.: +49 3641 8042334; fax: +49 3641 8042482. E-mail address: konrad.sachse@fli.bund.de (K. Sachse). 1 Equal contribution. 2 Shared senior authorship. http://dx.doi.org/10.1016/j.vetimm.2014.12.014 0165-2427/© 2015 Elsevier B.V. All rights reserved.

but display marked differences in host preference and clinical manifestation of the ensuing diseases. While the former is endemic in birds and primarily causes respiratory disease, the latter is a major abortifacient agent in small ruminants. Cases of zoonotic transmission to humans are well documented (Gaede et al., 2008; Harkinezhad et al., 2009) and can be severe, particularly when early medication is not available. Due to the peculiarities of Chlamydia spp., which include a biphasic obligate intracellular developmental cycle and, therefore, dependence on host cells for in vitro

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cultivation, the tools for genetic manipulation of strains are still in their infancy (Wang et al., 2011). In this situation, the reasons for the unequal host ranges and aetiopathologies of these two chlamydial species have yet to be identified. For instance, factors and mechanisms determining host specificity of Chlamydia spp. are still largely unknown. In a recent study, Braukmann and co-workers (Braukmann et al., 2012) used a chicken embryo model to compare C. psittaci and C. abortus for their invasiveness, virulence, and capability of eliciting an immune response in the avian host. While both species caused an increase in expression of the pro-inflammatory mediators IL-1␤, IL-6, IL-8, IL-17, lipopolysaccharide-induced tumour necrosis factor alpha factor (LITAF), and inducible nitric oxide synthase (iNOS), as well as IFN-␥, IL-12, IL-18, and toll-like receptor 4 (TLR4), it was also shown that C. psittaci exhibited a significantly better capability of disseminating in host organs and triggered higher macrophage numbers, as well as higher embryo mortality rates. We hypothesized that the more robust and competent behaviour of C. psittaci in the face of the embryo’s emerging response could be due to its capability of significantly up-regulating essential chlamydial genes, such as incA, groEL, cpaf and ftsW, which C. abortus failed to accomplish. Given our observations in chicken embryos, the question arose whether they could be reproduced in young chicks, where the immune system is becoming more mature. In fact, chicks start developing their own defence mechanisms during embryonic life, but immunocompetence triggered by the adaptive system emerges only a few days post-hatch (Mast and Goddeeris, 1999). Thus, an effective humoral immune response with specific antibodies cannot be generated earlier than one week after hatch (Mast and Goddeeris, 1999). For cell-mediated immunity, TCR␣␤ V␤1 (TCR2+ ) T cells migrate to peripheral organs in three discrete waves, with the first influx in spleen and intestine not before day 19 of embryonic life (Dunon et al., 1994). After hatch, two further waves of T cell colonization, around 5 and 9 days after hatch, provide the prerequisite for an effective immune response against various pathogens (Dunon et al., 1994). While the avian lung is the primary target of infection by Chlamydia spp., little is known about the immunological maturation and functional relevance of the respiratory immune system. Immunohistochemical staining proved the occurrence of diffusely distributed leukocytes, mainly monocytes and macrophages at least 5 days after hatch

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(Jeurissen et al., 1989a, 1989b). The development of bronchus-associated lymphoid tissue (BALT) structures was shown to be dependent not only on age, but also environmental stimuli (Jeurissen et al., 1994), so that infections with pathogenic microorganisms cause a significant increase in the number of BALT nodules. In the present study, we investigated pathology and host immune response, as well as systemic dissemination and expression of essential chlamydial genes in specific pathogen-free (SPF) chickens following experimental infection with C. psittaci and C. abortus. 2. Materials and methods 2.1. Bacterial strains and cell culture C. psittaci strain DC15 isolated from an aborted calf foetus in Germany in 2002 (Goellner et al., 2006) and C. abortus strain S26/3 isolated from a case of ovine abortion (McClenaghan et al., 1984) were used in this study. The bacteria were grown in Buffalo Green Monkey (BGM) cells using standard techniques (Vanrompay et al., 1992). The 50% Tissue Culture Infective Dose (TCID50 ) was determined by the method of Spearman and Kaerber (Mayr et al., 1974). 2.2. Experimental infection The experimental setup is presented in Table 1. Three groups of 20 one-day-old SPF chickens (VALO BioMedia, GmbH, Osterholz-Scharmbeck, Germany) were individually tagged and housed in separate negative pressure isolation units (IM 1500, Montair, Sevenum, The Netherlands). At the age of 7 days, chickens were exposed for 1 h to an aerosol (5 ␮m droplets; CirrusTM nebulizer; Lameris, Aartselaar, Belgium) of 106 TCID50 C. psittaci DC15 suspended in phosphate-buffered saline (PBS) (group 1) or 106 TCID50 C. abortus S26/3 suspended in PBS (group 2). The third group received an aerosol of PBS and served as non-infected control. The experimental design was evaluated and approved by the Ethical Commission for Animal Experiments of Ghent University (EC 2010/054). 2.3. Clinical signs and macroscopic lesions Clinical signs were daily scored until 14 days post infection (dpi). Clinical score 0 indicated no clinical signs; score 1: conjunctivitis; score 2: rhinitis; score 3: conjunctivitis

Table 1 Experimental setup. Group

1 (n = 20) 2 (n = 20) 3 (n = 20) a b c d

7 days of age inoculation with

C. psittaci DC15 C. abortus S26/3 PBS

Chlamydia culture. Antibody determination (ELISA). Histopathology. T cell proliferation assay.

Observations, manipulations, sampling Clinical signs all birds individually

Pharyngeal + cloacal swab of all birds [dpi]a

Blood all birds [dpi]b

Euthanasia 5 birds [dpi]c

½ of the spleen 5 birds [dpi]d

Daily Daily Daily

0, 7, 14 0, 7, 14 0, 7, 14

0, 7, 14 0, 7, 14 0, 7, 14

1, 3, 7, 14 1, 3, 7, 14 1, 3, 7, 14

7, 14 7, 14 7, 14

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and rhinitis; score 4: dyspnoea; score 5: conjunctivitis, rhinitis and dyspnoea; score 6: green watery droppings, conjunctivitis, rhinitis and dyspnoea. Five chickens per group were euthanized at 1, 3, 7 and 14 dpi for detailed examination. Macroscopic lesions were graded [minimal pathological change (1), slight (2), moderate (3), marked (4) or severe (5)]. Macroscopic lesion scores were determined by multiplying the pathology grade assigned by the veterinary pathologist for a specific lesion by a weighting factor. This factor (Germolec et al., 2004) was derived from the frequency (number) of animals within a group with a specific grade. For instance, if 2 of 4 animals (50%), in a group were given a grade of 2, and the remaining 50% had a grade of 3, the score for that group was equal to (2 × 2) + (2 × 3). 2.4. Chlamydia excretion and culture scoring Chlamydial excretion prior to infection (day 0; n = 20/group) and at 7 (n = 10/group) and 14 (n = 5/group) dpi was determined on pharyngeal and cloacal rayontipped, aluminium-shafted swabs (Colpan; Fiers, Kuurne, Belgium) immersed in Chlamydia transport medium (Vanrompay et al., 1992). Swabs were stored at −80 ◦ C until processed. Swabs were shaken for 1 h at 4 ◦ C, centrifuged and Chlamydia excretion was monitored using standard procedures for culture and bacterial identification (IMAGENTM immunofluorescence staining kit; Thermo Fisher, Aalst, Belgium). The presence of Chlamydia spp. was enumerated in five randomly selected microscopic fields (600×, BX41 Olympus, Aartselaar, Belgium) and results were scored from 0 to 5. Score 0 indicates that no Chlamydia spp. was present. Score 1 was given when a mean of 1–5 elementary bodies were counted, and scores 2, 3, 4 and 5 stand for a mean of 1–5, 6–10, 11–20 and >20 inclusion-positive cells identified, respectively. 2.5. Chlamydia replication in tissues and histopathology During necropsy of 5 chickens per group at 1, 3, 7 and 14 dpi, tissue samples of the conchae, lungs, abdominal and thoracic airsacs and liver were immersed in methocel (Methocel MC, Sigma), snap frozen in liquid nitrogen and stored at −80 ◦ C until processed. Cryostat (Leica CM1950; Leica Microsystems, Diegem, Belgium) tissue sections (5 ␮m) were examined for the presence of chlamydial bodies by the IMAGENTM immunofluorescence staining (Thermo Fisher). The presence of Chlamydia spp. was enumerated as for chlamydial excretion. During necropsy at 1, 7 and 14 dpi, tissue samples of the conchae, lungs, abdominal and thoracic airsacs and liver were also fixed in 10% phosphate-buffered formalin, processed, embedded in paraffin, sectioned at 5 ␮m, and stained with haematoxylin and eosin. All slides were examined microscopically (Leitz, New York, USA). Microscopic findings were graded [minimal histological change (1), slight (2), moderate (3), marked (4) or severe (5)]. Histopathological scores were determined by multiplying the histological grade assigned by the veterinary pathologist for a specific histological observation by a weighting factor (Germolec et al., 2004) representing number of

animals and severity of a certain lesion in these animals as described above. 2.6. Quantitative real-time PCR (qrtPCR) Dissemination of the challenge strains in bird tissue was monitored using qrtPCR targeting the 23S rRNA gene of Chlamydiaceae as described previously (Ehricht et al., 2006). Quantitation was based on a decimal dilution series of a DNA extract from cell cultures containing a known number of inclusion-forming units (ifu) of C. psittaci DC 15 and C. abortus S26/3, respectively, which was included in each run. 2.7. Quantitative real-time reverse transcriptase PCR (qRT-PCR) Tissue samples from thoracic air sacs collected at 1, 3, 7, and 14 dpi were submerged in RNAlater (QIAGEN, Hilden, Germany) for 24 h and kept frozen at −20 ◦ C. Total RNA was extracted using the RNeasy Mini Kit and residual DNA was digested using the RNase-free DNase set (QIAGEN). The purified RNA was eluted using 50 ␮l RNase-free water (QIAGEN) and stored at −80 ◦ C. All mRNA samples were checked for the quality criteria of A260 /A280 = 1.8–2.2 and A260 /A230 > 2 using spectrophotometry (BioPhotometer, Eppendorf, Hamburg, Germany). Avian-specific primers for IL-1␤, IL-6, IL-12␤ (p40), IL15, and IL-17␣ IL-18, LITAF, iNOS, MIP-1␤, IFN-␥, IL-8, lymphotactin, TLR , 4 and IL-4, as well as primers for the chlamydial genes 16S rRNA, groEL, incA, cpaf, and ftsW of C. psittaci and C. abortus, were validated previously (Braukmann et al., 2012). Determination of mRNA expression rates using the QuantiFAST SYBR Green real-time one-step RT-PCR Kit (QIAGEN) was described previously (Berndt et al., 2007; Braukmann et al., 2012). The threshold method was used for relative quantification of mRNA expression level (Pfaffl, 2001). Glycerinaldehyde-3-phosphate (GAPDH) was included as endogenous standard for normalization of chicken genes, and chlamydial 16S rRNA for chlamydial genes. Results were expressed as fold change (2[−Ct] ) (Pfaffl, 2001) relative to the amount of transcripts detected in mock infection or in the chlamydial inocula prior to infection, respectively. 2.8. Serological analysis of chicken sera Blood (V. ulnaris) was collected at 0, 7 and 14 dpi. Sera for ELISA were pretreated with kaolin to reduce background activity (Novak et al., 1993). Serum antibody titres were determined using urografin density gradient-purified C. psittaci DC15 or C. abortus S26/3 as antigen. Otherwise, the assay was conducted as described (Verminnen et al., 2006). Positive and negative control sera originated from a former experimental infection (Yin et al., 2013). 2.9. Lymphocyte proliferative responses Spleen cells were isolated during autopsy at 7 (n = 5) and 14 dpi (n = 5). Lymphocyte proliferative tests were

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Table 2 Macroscopic lesions in SPF chickens infected with C. psittaci or C. abortus. Macroscopic lesions* in

C. psittacia

C. abortusa

3 dpi

7 dpi

14 dpi

3 dpi

7 dpi

14 dpi

Lungb Congestion (slight/severe) Areas of consolidation surrounded by a hyperaemic zone Petechiae

5 – –

4 4 1

5 3 –

5 – –

4 – –

5 2 1

Thoracic airsacsb Diffuse opacity (slight/severe) Focal fibrinous airsacculitis Diffuse fibrinous airsaccultitis

2 – –

– 5 –

2 2 1

– – –

2 – –

4 1 –

Abdominal airsacsb Diffuse opacity (slight/severe) Diffuse fibrinous airsacculitis

– –

5 –

1 4

– –

– ––

4 –

Liverb Congested (slight/severe) Enlarged (moderately/severely) Petechiaec

– – –

– – –

3 3 –

– – –

4 – 4

3 5 3

Pericardb Fibrinous pericarditis

–

–

1

–

–

–

* a b c

Scoring as described in Materials and Methods. 5 chickens euthanised at each dpi. No lesions observed at 1 dpi and no lesions observed in the control group. One single location on the liver with few petechial haemorrhages of 0.5 mm.

performed as previously described (Vanrompay et al., 1999). The stimulation index (SI) was defined as the ratio of the mean counts per minute (cpm) of stimulated spleen cells versus the mean cpm of the negative control. 2.10. Statistical analysis Statistical analyses were performed using SPSS 22 and results for all groups were compared by use of the nonparametric Mann–Whitney U test. For qrtPCR and qRT-PCR tests, Student’s t test was used for comparison of two independent samples to evaluate significant differences between C. psittaci and C. abortus infection. Results were considered significantly different if p ≤ 0.05. 3. Results 3.1. Clinical signs and macroscopic lesions Both chlamydial species elicited lethargy, rhinitis and dyspnoea in all animals of the group, but symptoms were more severe and started sooner (6 dpi) following C. psittaci infection, as compared to the C. abortus-infected group (7 dpi). At 14 dpi, all C. psittaci-infected chickens lay down and showed severe dyspnoea (breathing with open beaks), while only 2 of 5 C. abortus-infected chickens showed slight dyspnoea. No deaths occurred. Non-infected controls remained healthy throughout the experiment. C. psittaci-infected birds showed severe congestion of the lungs and areas of consolidation surrounded by a hyperaemic zone (pneumonia). Focal fibrinous inflammation of the thoracic air sacs and diffuse opacity of the abdominal air sacs were observed in all animals from 7 dpi onwards (Table 2). At 14 dpi, 4 of 5 (80%) chickens showed diffuse fibrinous inflammation of the abdominal air sacs and 1 of

5 (20%) presented fibrinous pericarditis. At that time, 3 of 5 (60%) chickens showed congestion and severe enlargement of the liver. The severity of macroscopic lesions in the respiratory tract was much lower and the onset was slower in C. abortus infection. C. abortus-infected chickens showed slight diffuse opacity in thoracic and abdominal air sacs, while focal fibrinous airsacculitis was only observed in 1 of 5 (20%) animals. Areas of consolidation surrounded by a hyperaemic zone were only present in lungs of 2 of 5 (40%) chickens. However, most C. abortus-infected chickens presented petechiae on the liver at 7 and 14 dpi, whereas none of the C. psittaci-infected chickens did. 3.2. Chlamydia excretion Pharyngeal and cloacal shedding was most pronounced in chickens infected with C. psittaci (Table 3). Non-infected controls did not shed chlamydiae. 3.3. Chlamydia replication in tissues and histopathology Both chlamydial species infected tissues of upper and lower respiratory tracts from 1 dpi onwards. Using culture scoring, the highest mean scores of chlamydial replication were observed in the air sacs at 7 dpi, with maximal mean scores of 5.0 and 1.4 in C. psittaci- and C. abortusinfected chickens, respectively (Table 4). Examination of thoracic air sac tissue using qrtPCR led to detection of chlamydial 16S rDNA in C. psittaci-infected birds at 3, 7 and 14 dpi, but only at 3 dpi in the C. abortus group (Fig. 4). A plateau of C. psittaci dissemination was reached between 7 and 14 dpi, where chlamydial copy numbers (DNA equivalents to ifu) were in the range of 40–60 per 1000 host cells (Fig. 1). Thoracic air sacs of the control chickens remained negative. In addition,

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Table 3 Mean culture scores* (mean ± SD) for pharyngeal and cloacal excretion of C. psittaci or C. abortus in the course of infection. dpi

N

0 7 14

20 10 5

*

C. psittaci

C. abortus

Pharyngeal

Cloacal

Pharyngeal

Cloacal

0±0 2.4 ± 0.52a 2.4 ± 0.56c

0±0 2.2 ± 0.75b 2.4 ± 0.56d

0±0 1.0 ± 0a 0.5 ± 0c

0±0 0 ± 0b 1.0 ± 0d

Scoring scheme see Materials and Methods. indicates that values are statistically different (at least p ≤ 0.05).

a,b,c,d

Table 4 Culture scores* (mean ± SD, n = 5) for the presence of Chlamydia spp. in tissues of euthanised chickens infected with C. psittaci strain DC15 or C. abortus strain S26/3. C. psittaci 1 dpi Conchae Lung Liver Abd AS Th AS

1.0 0.8 0.0 1.0 1.0

± ± ± ± ±

C. abortus 3 dpi

0.0 0.4 0.0 0.0 0.0

1.2 1.8 0.0 2.4 2.6

± ± ± ± ±

7 dpi 0.4 0.8a 0.0 0.9a 0.5a

2.0 3.6 1.0 5.0 5.0

± ± ± ± ±

14 dpi a

0.0 0.5b 0.0 0.0b 0.0b

2.8 2.6 2.0 5.0 3.8

± ± ± ± ±

1 dpi b

0.8 0.5c 0.7a 0.0c 1.3c

1.0 0.4 0.0 0.4 0.6

± ± ± ± ±

3 dpi 0.0 0.5 0.0 0.5 0.5

0.8 0.2 0.0 0.2 0.2

± ± ± ± ±

7 dpi 0.4 0.4a 0.0 0.5a 0.5a

0.4 0.8 0.6 1.4 1.0

± ± ± ± ±

14 dpi a

0.5 0.4b 0.9 0.9b 0.7b

0.4 0.6 0.4 1.2 0.8

± ± ± ± ±

0.4b 0.5c 0.5a 1.1c 0.8c

* Scoring scheme see Materials and Methods. Tissue scores of control animals were 0. Abd AS: abdominal air sac, Th AS: thoracic air sac. Numbers within a row with the same superscript are significantly different (p ≤ 0.05).

systemic spread as demonstrated by chlamydial replication in hepatic tissue started from 7 dpi onwards, and was most pronounced in C. psittaci-infected chickens (Table 4). Histological examination revealed epithelial hyperplasia and especially epithelial erosions, as well as infiltration of macrophages, lymphocytes and heterophils in abdominal (Fig. 2) and thoracic air sacs of all C. psittaci-infected chickens from 7 dpi onwards till the end of the experiment (Table 5). In lung tissue, the main lesions were found at 7 dpi, showing hyperplasia of BALT and infiltration of heterophils in particular, but also of macrophages and lymphocytes. In the liver, portal inflammatory infiltrate was observed at 7 and 14 dpi. Parenchymatous infiltration of macrophages and heterophils was only observed at 14 dpi. One chicken showed focal necrosis. In the C. abortus-infected group, histology did not reveal notable pathological lesions in examined tissues, except for lymphocyte infiltration in the lungs of 3 of 10 (30%) animals and combined lymphocyte and macrophage infiltration in

lungs and thoracic air sacs of another three animals. As for C. psittaci-infected chickens, portal inflammatory infiltrate was observed at 7 and 14 dpi. 3.4. Serology C. psittaci- and C. abortus-infected chickens were all seronegative at 7 dpi. At 14 dpi, all C. psittaci-infected chickens had developed a primary antibody response [mean antibody titre ± standard deviation (SD); 77 ± 48], while only 2 of 5 (40%) C. abortus-infected chickens had antibodies at that time [mean antibody titre ± SD; 38 ± 57.2]. Controls remained seronegative throughout the experiment. 3.5. Regulation of immune-related host genes Quantitative real-time RT-PCR revealed chlamydial species-dependent differences of transcription levels of a selection of immune-related genes in thoracic air sac tissue (Fig. 3). Generally, up-regulation of mRNA expression of IL-1␤, IL-6, IL-17, and IL-22 was significantly stronger in C. psittaci- than in C. abortus-infected birds between 3 and 14 dpi. In the case of iNOS and IFN-␥, the same distinction was observed at 14 dpi. TLR4 and LITAF mRNAs were expressed at significantly higher levels in C. psittaci than in C. abortus at 7 and 14 dpi. 3.6. Regulation of selected Chlamydia genes

Fig. 1. Copy number of C. psittaci () and C. abortus () per 1000 cells of thoracic air sac tissue from infected chickens between 1 and 14 dpi. Significant differences between groups are shown as *p ≤ 0.05.

Using qRT-PCR, chlamydial 16S mRNA was detected in thoracic air sac tissue of C. psittaci-infected animals at 3 and 7 dpi, but only at 3 dpi of C. abortus-infected birds (Fig. 4). Therefore, transcription of virulence-related chlamydial proteins could be monitored only on these two days. At 3 dpi, normalized mRNA expression ratios of the ftsW, cpaf and groEL genes were significantly higher in C.

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Fig. 2. Haematoxylin and eosin staining of the thoracic airsac of an SPF chicken infected with C. psittaci strain DC15. Massive infiltration of lymphocytes (a) and heterophils (b). Discrete infiltration of macrophages (c) and epithelial erosion (oval).

psittaci- than in C. abortus-inoculated animals. In the C. psittaci group, transcription of groEL was up-regulated from 3 to 7 dpi. In samples of control chicks, no chlamydial mRNA transcription was seen.

3.7. Lymphocyte proliferation assay At 14 dpi, proliferative responses in C. psittaci-infected chickens were significantly higher than in C. abortus

Table 5 Scores* for microscopic lesions in the respiratory tract and liver of SPF chickens infected with C. psittaci or C. abortus. Lesion

Histopathological scores in C. psittaci-infected chickens (n = 5/day) Conchae

Th air saca

Lung

Abd air sacb

Liver

7 dpic

14 dpi

7 dpi

14 dpi

7 dpi

14 dpi

7 dpi

14 dpi

7 dpi

14 dpi

Infiltration of lymphocytes Infiltration of macrophages Infiltration of heterophils Epithelial hyperplasia Epithelial erosions BALT hyperplasia Portal inflammatory infiltratee Necrosis Total score per tissue

5 0 1 0 0 NA NA 0 6

3 0 0 0 0 NA NA 0 3

1 3 6 0 0 11 NA 0 10

1 0 0 0 0 1 NA 0 1

11 11 9 2 11 NA NA 0 44

9 12 6 1 8 NA NA 0 36

5 13 14 3 11 NA NA 0 46

6 12 4 1 7 NA NA 0 30

0 0 0 NAd NA NA 2 0 2

0 1 1 NA NA NA 1 1 4

Lesion

Histopathological scores in C. abortus-infected chickens (n = 5/day) Conchae

Infiltration of lymphocytes Infiltration of macrophages Infiltration of heterophils Epithelial hyperplasia Epithelial erosions BALT hyperplasia Portal inflammatory infiltratee Total score per tissue * a b c d e

Th air saca

Lung

Abd air sacb

Liver

7 dpi

14 dpi

7 dpi

14 dpi

7 dpi

14 dpi

7 dpi

14 dpi

7 dpi

14 dpi

0 0 0 0 0 NA NA 0

0 0 0 0 0 NA NA 0

1 0 0 0 0 0 NA 1

2 0 0 0 0 0 NA 2

3 2 0 0 0 NA NA 5

2 2 0 0 0 NA NA 4

0 0 0 0 0 NA NA 0

0 0 0 0 0 NA NA 0

0 0 0 NA NA NA 1 1

0 0 0 NA NA NA 2 2

Scoring scheme see Materials and Methods. Th air sac: thoracic air sac. Abd air sac: abdominal air sac. dpi: days post infection. NA: not applicable. Lymphocytes, heterophils and macrophages.

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Fig. 3. Relative quantification of mRNA expression levels of immune-related proteins in thoracic air sacs of chickens challenged with C. psittaci () and C. abortus () compared to mock-infected controls () between 1 and 14 dpi (n = 5). Significant differences between inoculated groups are shown as *p ≤ 0.05.

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Fig. 4. Relative mRNA expression levels of virulence-associated genes and Ct values of the 16S rRNA of C. psittaci () and C. abortus () and mock-infected controls () in thoracic air sacs compared to chlamydial strains prior to inoculation between 1 and 14 dpi. Significant differences between inoculated groups are shown as *p ≤ 0.05.

counterparts (Table 6). Spleen cells of non-infected controls revealed no proliferative responses against chlamydial antigens. 4. Discussion The present model of aerogenous infection of the avian lung was conceived to reproduce the natural and most common route of chlamydial infections in birds and other animals. Although both anatomy and function of the avian and mammalian lung systems show a number of substantial differences, parts of the respiratory tracts in both animal classes act as a barrier between the huge amount of different pathogens from outside and the interior of the organism. Our infection experiment demonstrated that C. Table 6 Proliferative responses of spleen cells to the homologous chlamydial antigens. Infection

N

C. psittaci C. abortus p value

5 5

Stimulation index (mean ± SD) 7 dpi

14 dpi

2.57 ± 1.83 1.49 ± 0.85 0.266

4.41 ± 1.90* 2.22 ± 0.53* 0.038

Spleen cells of non-infected controls (N = 5) displayed no proliferative responses. * Significantly different.

psittaci was able to elicit more severe illness and stronger lesions than C. abortus in seven-day-old chicks. Pneumonia, i.e. areas of consolidation surrounded by a hyperaemic zone, and airsacculitis appeared sooner and became more severe in C. psittaci-infected chickens. Macroscopic lesions in the liver appeared more severe in the C. abortus-infected group (Table 2), but the observation has not been confirmed by histopathology, as parenchymatous infiltration of inflammatory cells was only seen in livers of C. psittaciinfected chickens. These central findings from histopathology correlated with all other data collected in this study, such as propagation and dissemination of the challenge strains, chlamydial gene transcription and cytokine expression in lung tissue. Braukmann and co-workers (Braukmann et al., 2012) came to similar insights by examining chicken embryos after inoculation of the chorioallantoic membrane (CAM) with the same Chlamydia strains. Comparing the in ovo and in vivo models, the infectious potential of C. abortus in chicken embryos appeared somewhat higher and minor differences in immune responses were observed. But these differences can be attributed to different infection doses and inoculation routes, as well as to the superiority of the more mature and highly specialized pulmonary immune system over that of the CAM. The outcome of an infection depends on both the pathogen’s virulence properties and the effectiveness of the host immune response. At the genomic level, C. psittaci and

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C. abortus are largely similar, but different in the plasticity zone. Notably, the “6BC clade” of C. psittaci, of which strain DC15 is a member, possesses an intact large cytotoxin (tox) gene similar to the EHEC adherence factor and the three-gene guaAB-add cluster, which is involved in purine nucleotide interconversion and is necessary for bacterial growth. These two elements are absent in C. abortus (Read et al., 2013; Voigt et al., 2012). Another important distinction between the two species is located in the highly variable locus of the pmp gene family, which encodes a number of membrane proteins assumed to mediate escape mechanisms from the systemic host response or adaptation to multiple host environments (Tan et al., 2006; Voigt et al., 2012). In future studies, it will be interesting to explore whether these genomic distinctions account for any species-specific behaviour discussed here. Braukmann and co-workers (Braukmann et al., 2012) postulated that C. psittaci could cope far better than C. abortus with the chicken embryo’s immune response by upregulating essential genes. In the present study, monitoring of mRNA expression of chlamydial genes was generally hampered due to very low RNA yields recovered from organ tissue. Consistent up-regulation has been seen for groEL at 3 and 7 dpi after C. psittaci exposure and, to a lesser extent, for cpaf. Transcription of ftsW was slightly down-regulated at 3 dpi, but expression rates in C. psittaci were still significantly higher than in C. abortus (Fig. 4). The fact that regulation patterns of these chlamydial genes appeared somewhat different from those in the chicken embryo infection indicates that the immunologically more mature chicks probably employ additional mechanisms for protection against chlamydial infection, for instance the response of alveolar macrophages and epithelial or interstitial cells in the mature lung. The possibility of immunological parameters affecting severity and outcome of a chlamydial infection was already shown by other researchers. Huang and co-workers (Huang et al., 1999) reported that administration of IL-12 provided immediate protection against lethality associated with pulmonary C. psittaci infection of mice. In the present work, the increase of IL-12 expression levels remained rather moderate and without significant differences between the challenge strains. In contrast, we found very high transcription levels of IL-17 in C. psittaci-infected birds, with a significant difference to the C. abortus group. It is interesting to note that recent papers singled out a particular role for IL-17 in generating chemokine responses, inducing antimicrobial proteins and recruiting neutrophils for control of extracellular (reviewed in Khader et al., 2009) and intracellular infections (Khader and Gopal, 2010; Wu et al., 2007; Zhang et al., 2009), including those by chlamydiae (Bai et al., 2009). The immune mediator IFN-␥ can be regarded as another key player in anti-bacterial defence as it regulates the expression of many factors involved in the eradication of intracellular pathogens. The elevated mRNA expression level of IFN-␥ in C. psittaci compared to C. abortus infection in our study underlines the relevance of this cytokine in chlamydial lung infections of chickens. A previous study similarly revealed higher IFN-␥ expression in internal organs of C. psittaci-inoculated chicken embryos

compared to C. abortus-infected counterparts (Braukmann et al., 2012). To what extent the transcribed IFN-␥ could have contributed to an effective immune response against chlamydial infection in the present study remains open. However, the production of the enzyme iNOS by avian macrophages is stimulated by IFN-␥ (Kaspers et al., 1997). The function of iNOS in host immunity consists in antimicrobial action via NO release as part of the oxidative burst of macrophages. In our study, iNOS expression was detectable at higher levels in C. psittaci- than in C. abortusinfected birds at 14 dpi. This result together with the relatively high expression of iNOS in C. abortus- infected birds at 3 dpi may indicate a role of IFN-␥ and NO for the much faster eradication of C. abortus by alveolar or interstitial macrophages of the avian lung. In conclusion, our findings on aetiopathology of C. abortus or C. psittaci and local tissue response indicate the presence of a relatively developed pulmonary immune defence system in seven-day-old chicks. Although the birds showed signs of illness after inoculation with the challenge strains, the extent of pathogen dissemination was limited, with a peak at 7 dpi (C. psittaci) and subsequent decline. In particular, the rather suppressed capability of chlamydial strains to transcribe virulence-related proteins and, in contrast to that, the strong expression of many immunologically important mediators emphasizes the competence of the young chickens to cope with these pathogens in the lung. Nevertheless, C. psittaci seems to have available some specific mechanisms that enable it to tackle the avian host immune defence more efficiently than C. abortus. Conflict of interest statement The authors declare no conflict of interest. Acknowledgements The study was funded by Ghent University (grant IOF10/STEP/002). Lizi Yin has a PhD fellowship from the China Scholarship Council (CSC grant; 01SC2812) and from the Special Research Fund of Ghent University (co-funding of the CSC grant). We gratefully thank A. Dumont (Department of Molecular Biotechnology) for technical assistance. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.vetimm.2014.12.014. References Bai, H., Cheng, J., Gao, X., Joyee, A.G., Fan, Y., Wang, S., Jiao, L., Yao, Z., Yang, X., 2009. IL-17/Th17 promotes type 1T cell immunity against pulmonary intracellular bacterial infection through modulating dendritic cell function. J. Immunol. 183, 5886–5895. Berndt, A., Wilhelm, A., Jugert, C., Pieper, J., Sachse, K., Methner, U., 2007. Chicken cecum immune response to Salmonella enterica serovars of different levels of invasiveness. Infect. Immun. 75, 5993–6007. Braukmann, M., Sachse, K., Jacobsen, I.D., Westermann, M., Menge, C., Saluz, H.P., Berndt, A., 2012. Distinct intensity of host-pathogen interactions in Chlamydia psittaci- and Chlamydia abortus-infected chicken embryos. Infect. Immun. 80, 2976–2988.

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