Eefke Weesendorp
email: eefke.weesendorp@wur.nl

Keywords: Classical swine fever virus; Diagnosis, Organs, Survival, Tissue degradation

A considerable part of tissue samples that are collected for the monitoring of classical swine fever (CSF) from the wild boar population or from domestic pigs are unsuitable for virus detection using the fluorescent antibody test (FAT) or virus isolation (VI), due to tissue degradation. Reverse transcription polymerase chain reaction (RT-PCR) has a higher sensitivity than the FAT and VI, and is supposed to be less sensitive to sample degradation.

Reliable and quantitative information on how long viral RNA and infectious virus can be detected in organs and which organs are most susceptible to degradation is, however, lacking. In the present study we generated survival curves of infectious CSF virus (CSFV) and viral RNA in the tonsil, mesenteric lymph node, spleen and kidney, obtained from twenty-four pigs infected with a moderately virulent CSFV strain. Tissue samples were stored at room temperature and tested by VI and RT-PCR, directly after storage and 1, 2, 3, 4, 7, 14 and 21 days later. It was shown that the RT-PCR is not only more sensitive than VI on fresh tissue samples, but RT-PCR is also less vulnerable to sample degradation. Average half-life values of viral RNA in the tissues ranged from 0.95 to 2.55 days, while half-life values of infectious virus ranged from 0.21 to 0.31 days. The tonsil and spleen are regarded as the most appropriate organs for the detection of infectious virus and viral RNA, not only in fresh samples, but also in samples that suffer from tissue degradation.

Introduction

Classical swine fever (CSF) is a highly contagious viral disease in domestic pigs and wild boar. It is included in the list of diseases notifiable to the OIE. Several large outbreaks of CSF occurred in the European Union in the last twenty years [START_REF] Koenen | Epidemiological characteristics of an outbreak of classical swine fever in an area of high pig density[END_REF][START_REF] Elbers | The classical swine fever epidemic 1997-1998 in the Netherlands: descriptive epidemiology[END_REF][START_REF] Fritzemeier | Epidemiology of classical swine fever in Germany in the 1990s[END_REF][START_REF] Sharpe | Epidemiology of the 2000 CSF outbreak in East Anglia: preliminary findings[END_REF], causing high economic losses, mainly due to restrictions in the trade of animals and animal products [START_REF] Terpstra | The 1997/1998 epizootic of swine fever in the Netherlands: control strategies under a non-vaccination regimen[END_REF]. The main sources of these outbreaks have been illegal swill feeding, contact with contaminated livestock trucks, infected pigs or wild boar [START_REF] Moennig | Introduction to classical swine fever: virus, disease and control policy[END_REF][START_REF] Moennig | Clinical signs and epidemiology of classical swine fever: A review of new knowledge[END_REF]. In Europe, CSFV was reported from the wild-boar population in Bulgaria, Croatia, France, Germany, Hungary, Romania andSlovakia between 2005 and2009. The danger of virus introduction due to (indirect) contact with wild boar and wild boar meat was demonstrated in Germany. Between 1993 and1998, 59% of the primary outbreaks in domestic pigs were due to this route of transmission [START_REF] Fritzemeier | Epidemiology of classical swine fever in Germany in the 1990s[END_REF].

A c c e p t e d M a n u s c r i p t 3 Surveillance in domestic pigs, but also wild boar, is the key to quickly detect classical swine fever upon a new introduction in a country. For early diagnosis of CSF, the detection of the virus is more suitable than the detection of antibodies, as antibodies can be found only in a later stage of the infection [START_REF] Koenen | Epidemiological characteristics of an outbreak of classical swine fever in an area of high pig density[END_REF][START_REF] Dewulf | Analytical performance of several classical swine fever laboratory diagnostic techniques on live animals for detection of infection[END_REF]. Virus can be detected with high titres in blood and several organs like the spleen, bone marrow, lymph nodes, tonsil, kidney, ileum and lung [START_REF] Ressang | Studies on the pathogenesis of hog cholera. I. Demonstration of hog cholera virus subsequent to oral exposure[END_REF][START_REF] Van Oirschot | Classical swine fever and related viral infections[END_REF][START_REF] Kamolsiriprichaiporn | A comparison of the pathogenicity of two strains of hog cholera virus. 2. Virological studies[END_REF].

Samples that are being used for the detection of the virus, or viral genome, are therefore generally blood or tissue samples. In this paper we will focus on the latter.

The traditional techniques to detect virus in tissue samples are virus isolation (VI) and the fluorescent antibody test (FAT). These techniques require good sample quality, however, obtaining fresh tissue samples may be difficult or impossible because of postmortem alterations (haemolysis, putrefaction or virus destruction), that will increase in time. Especially in wild boar that are found dead, with an unknown length of time between death and sampling, this will quickly reduce the suitability of such samples for a reliable diagnosis of CSF. According to the experiences of several research groups, up to 30%, and even exceptionally 50% of field samples from wild boar may be unsuitable for laboratory tests [START_REF] Artois | Classical swine fever (hog cholera) in wild boar in Europe[END_REF][START_REF] Rossi | Long-term monitoring of classical swine fever in wild boar (Sus scrofa sp.) using serological data[END_REF]. The same may apply for samples from domestic pigs. Three percent of all samples (18,000) from domestic pigs taken during the 1997-1998 outbreak in

The Netherlands were deteriorated to such an extent that they were unsuitable for testing in the FAT [START_REF] De Smit | Laboratory decision-making during the classical swine fever epidemic of 1997-1998 in The Netherlands[END_REF]. In a long-running Dutch surveillance program for CSFV, for which tonsils were collected from all domestic pigs that were sent in for a post-mortem examination, approximately 15% of the tonsils were deteriorated to such an extent that they were unsuitable for testing in the FAT (authors' own observation).

More recently developed techniques enabled the detection of the viral genome by reverse transcription polymerase chain reaction (RT-PCR). This method is not only more sensitive than VI and FAT [START_REF] Blome | Assessment of classical swine fever diagnostics and vaccine performance[END_REF][START_REF] Depner | Evaluation of real-time RT-PCR assay for the routine intra vitam diagnosis of classical swine fever[END_REF][START_REF] Greiser-Wilke | Diagnostic methods for detection of Classical swine fever virus-status quo and new developments[END_REF] but may also be less susceptible to the deterioration of tissue and degradation of virus than both conventional techniques. If so, RT-PCR may be more suitable to detect CSFV in deteriorated tissue, therefore allowing for a more reliable surveillance in wild boar and domestic pigs, in case tissue samples are collected for testing.

In this study the effect of tissue degradation on virus infectivity and viral RNA stability in a range of commonly used organs was studied to find out how this affects the possibility to diagnose CSF by VI and RT-PCR.

Materials and Methods

Experimental animals and housing

A c c e p t e d M a n u s c r i p t

Because of animal welfare concerns and the need to limit the number of experimental animals, organs were used from pigs that were included in a transmission study. For that study, twenty-four male pigs, eight weeks of age, were obtained from a conventional, but pestivirus free pig herd in the Netherlands. Pigs were housed in six pens, with four pigs each, within one room of an isolation unit. Two pigs were inoculated at the start of the study (pigs 23 and 24), and within the 31 days the study lasted, the other 22 pigs became infected through contact infections (Table 1). All pigs were euthanized at day 31 post-inoculation (p.i.). The experiment was approved by the Ethics Committee for Animal Experiments of the Animal Sciences Group of Wageningen UR.

Viruses and inoculation of animals

The two pigs (number 23 and 24) were inoculated with the moderately virulent strain Paderborn (genotype 2.1). This strain was isolated in 1997 during the outbreak in the Paderborn area of Germany [START_REF] Greiser-Wilke | Molecular epidemiology of a large classical swine fever epidemic in the European Union in 1997-1998[END_REF]. The virus suspension, with a dose of 10 5 TCID 50 (50% tissue culture infectious dose), was administered intranasally, 1 ml per animal, 0.5 ml per nostril.

Collection and treatment of samples

Oropharyngeal fluid samples were collected from all pigs at days -4, 3, 5, 7, 10, 12, 14, 17, 19, 21, 24, 26, 28 p.i. to determine the approximate moment of infection of each animal (Table 1). Oropharyngeal fluid was obtained with a sterile gauze tampon held by a 30-cm long forceps, which was scrubbed against the dorsal wall of the pharynx behind the soft palate. The oropharyngeal fluid was suspended in 4 ml medium (Eagle minimum essential medium [EMEM], supplemented with 5% fetal bovine serum [FBS] and 10% antibiotics solution ABII [1000 U/ml Penicillin, 1 mg/ml Streptomycin, 20 μg/ml Fungizone, 500 μg/ml; Polymixin B, and 10 mg/ml Kanamycin]).

After centrifugation (1800 g for 15 min) the samples were stored at -70°C until they were analysed by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR).

From each pig, entire tonsils, several mesenteric lymph nodes, and a piece of at least 5 grams of spleen and kidney were collected at day 31 p.i. Tissue samples were stored in petri-dishes at room temperature for 21 days. Directly after collection and at days 1, 2, 3, 4, 7, 14 and 21, a piece of 0.15 g was cut off and added to 0.5 ml of the same medium as described for the oropharyngeal fluid samples. The tissue was homogenized in the MagNa Lyser (Roche Applied Science) for 30 sec at 3500 g. After centrifugation (9500 g for 1 min), an additional one ml of medium was added. Samples were centrifuged again (9500 g for 5 min) and the supernatant was stored at -70 °C until they were analysed by VI and RT-qPCR.

A c c e p t e d M a n u s c r i p t

From the tonsil, mesenteric lymph node, spleen and kidney of six randomly selected pigs, additional samples were taken from fresh organs to test for homogeneity of the virus in the organs. Four samples were taken from different locations of each organ. These samples were analysed by RT-qPCR.

Tests

Virus isolation and titration

Virus titration was performed to detect the presence of infectious virus in the tissue samples. Ten-fold dilutions of the tissue supernatant were prepared. Four replicates of 125 µl of each serial dilution (10 -1 to 10 -6 ) were incubated for four days on a monolayer of SK6 cells in a 24-wells plate (Greiner). After four days, the growth medium was discarded, and the monolayers were washed in a 0.15M NaCl solution, dried for 1 hour at 37°C and frozen for 1 hour at -20°C. The monolayers were fixed with 4% cold (5 C) paraformaldehyde in Phosphate-Buffered Saline (PBS) for 10 min. After fixation, the plates were washed in 0.15 M NaCl. Monolayers were stained by the immunoperoxidase monolayer assay (IPMA) (Wensvoort et al., 1986), using two horseradish peroxidase (HRPO)conjugated CSFV specific MAbs (V3/V4) diluted in PBS (1:4000) with 4% horse serum. Monolayers were stained with 300 l 0.05 M NaAc solution, containing 5% 3-amino-9-ethylcarbazole (AEC) and 0.05% H 2 O 2 , and examined for stained cells. Virus titres were calculated as TCID 50 using the Spearman-Kärber method [START_REF] Finney | Statistical Methods in Biological Assay[END_REF]. The detection limit was 10 2.4 TCID 50 /g of tissue, since tissue samples were tested 100 times diluted in fourfold.

Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR)

The concentration of viral RNA in the tissue samples was analyzed by RT-qPCR. For RNA isolation, 200 µl of the sample were pipetted manually into MagNA Pure sample cartridges (Roche Applied Science, Mannheim, Germany). In each run of thirty-two samples, one negative control sample and five dilutions of a positive control sample (standard curve) were included. The standard curves were constructed as a dilution series in medium with a well known concentration of infectious virus (Paderborn strain). The RNA was extracted with the Total Nucleic Acid Isolation Kit (Roche Applied Science, Mannheim, Germany) according to the manufacturer's instructions, using the automated MagNA Pure LC instrument (Roche Applied Science, Mannheim, Germany). After RNA isolation, the nucleic acids were immediately processed for the RT-qPCR or stored at -70°C in the sample cartridge until the PCR was carried out.

The RT-qPCR was performed with a LightCycler (LC) instrument (Roche Applied Science, Mannheim, Germany), using the RNA Master Hybridization Probes Kit as described by Van Rijn et al. (2004). Analysis was sample could be calculated using the standard curve. It has to be stressed that TCID 50 equivalents are only a relative measure for the amount of RNA and that no correlation exists with the amount of infectious virus.

Statistical analysis

Differences between organs in initial infectious virus and viral RNA titres

Significant differences between organs in initial infectious virus and viral RNA titres were calculated with the non-parametric Wilcoxon matched-pairs test. For the multiple comparisons, Bonferroni's correction was applied.

Virus or viral RNA negative tissue samples were excluded from the calculation. Due to the Bonferroni's correction, a p value less than 0.05/3 indicated a significant relationship.

Homogeneity of viral RNA distribution in the organs

From each of the six tonsils, mesenteric lymph nodes, spleens and kidneys, the average viral RNA titre and standard deviation of the four locations was calculated. Per organ type an average standard deviation was calculated.

Calculation of half-life values

For all tissue samples, the decay of infectious virus (log 10 TCID 50 ) or viral RNA (log 10 TCID 50 equivalents) as a function of time after storage at room temperature was determined using a random coefficient regression model.

The dispersion parameters were estimated by restricted maximum likelihood (REML), employing GenStat (Eleventh edition, copyright 2008) software. The half-life (h) of infectious virus or viral RNA in the kidney, spleen, tonsil and mesenteric lymph nodes was then estimated by: h = -log 10 2 / b. Here, b is the estimated (expected) slope in the regression of log 10 CSFV on time for a random sample of the specific tissue.

Differences in half-life values between organs were statistically analysed using a paired T-test. Mean differences were considered significant when the p value was less than 0.05.

Results

Initial infectious virus and viral RNA titres in fresh organs

Seventy-nine out of the 96 fresh tissue samples tested positive in the VI with initial infectious virus titres ranging from below the detection limit (lower than 10 2.4 TCID 50 /g) to 10 8.9 TCID 50 /g (Table 1). Only one of the pigs A c c e p t e d M a n u s c r i p t was VI-negative in all four organs. This was one of the two inoculated pigs, which by then had fully recovered from the infection. The initial infectious virus titres were on average the highest in the spleen and the tonsil (on average 10 7.0 and 10 6.7 TCID 50 /g). With the exception of the recovered pig, all fresh tonsils were VI-positive. Significantly lower titres than in the tonsil were observed in the mesenteric lymph node (on average 10 6.2 TCID 50 /g) and the lowest titres in the kidney (on average 10 5.6 TCID 50 /g). However, variations in virus titres were large, depending of the estimated time of infection of the pig.

Ninety-five out of the 96 tissue samples tested positive in the RT-qPCR with initial viral RNA titres in the 95 positive samples ranging from 10 2.4 to 10 9.1 TCID 50 equivalents/g (Table 2). The only negative result originated from the kidney sample of the recovered pig that was completely VI-negative. The initial viral RNA titres were highest in the tonsil and the mesenteric lymph node (on average 10 7.1 and 10 6.9 TCID 50 equivalents/g). Significant lower titres were observed in the spleen (on average 10 6.3 TCID 50 equivalents/g) and the lowest titres in the kidney (on average 10 5.1 TCID 50 equivalents/g). As seen with infectious virus titres, variations in viral RNA titres were also large, depending of the estimated time of infection of the pig.

Homogeneity of viral RNA distribution in the organs

The viral RNA was least homogeneously distributed in the kidney, as the viral RNA titres at the different locations of the organ showed an average standard deviation of 0.25 log 10 TCID 50 equivalents/g (minimum 0.09 and maximum 0.53 log 10 TCID 50 equivalents/g). In the tonsil the average standard deviation was 0.15 log 10 TCID 50 equivalents/g (minimum 0.07 and maximum 0.30 log 10 TCID 50 equivalents/g), in the spleen 0.11 log 10 TCID 50 equivalents/g (minimum 0.06 and maximum 0.19 log 10 TCID 50 equivalents/g), and in the mesenteric lymph node 0.10 log 10 TCID 50 equivalents/g (minimum 0.01 and maximum 0.24 log 10 TCID 50 equivalents/g).

Decay of infectious virus and viral RNA in tissue samples

The average half-life values for infectious virus ranged from 0.21 to 0.31 days (Table 3). There were small differences in half-life values between the organs, however, these were not statistically significant. After 7 days of storage at room temperature, virus could still be isolated from only 6 out of the 96 organs. Four of these were spleen samples and two were lymph node samples.

The average half-life values for viral RNA ranged from 0.95 to 2.55 days (Table 4). The shortest average half-life value was observed in the lymph node, while the longest average half-life value was observed in the spleen.

After 7 days of storage at room temperature, viral RNA could still be detected in 74 out of the 96 organs, and even

Discussion

In the present study, survival curves of infectious CSFV and viral RNA in tissue samples from tonsils, mesenteric lymph nodes, spleens and kidneys were generated. These survival curves showed that the RT-PCR is not only more sensitive than VI on fresh tissue samples, but also that viral RNA can be detected much longer in autolytic samples than infectious virus. Average half-life values of viral RNA in the tissues, as detected with RT-qPCR, ranged from 0.95 to 2.55 days, and in 50% of the organs, viral RNA could still be detected after 21 days of storage at room temperature. On the other hand, half-life values of infectious virus, as detected with VI, ranged from 0.21 to 0.31 days, and in only 6% of the cases, infectious virus could still be detected after 7 days of storage at room temperature.

The results of the present study are not unexpected and confirm previous observations that the RT-PCR is more sensitive than virus isolation [START_REF] Dewulf | Analytical performance of several classical swine fever laboratory diagnostic techniques on live animals for detection of infection[END_REF]Van Rijn et al., 2004), and is supposed to be less influenced by tissue degradation. However, half-life values of viral RNA in tissue samples were not known. Information on halflife values of infectious CSFV have been published before, but focused mainly on survival of the virus in medium, faeces and urine [START_REF] Depner | Thermal and pH stability of pestiviruses[END_REF]Weesendorp et al., 2008). In medium, a mean half-life of 2 days at neutral pH and a temperature of 20°C was observed [START_REF] Depner | Thermal and pH stability of pestiviruses[END_REF]. In faeces and urine of pigs infected with the Paderborn strain, half-life values of 0.14 days (faeces) and 0.21 days (urine) were more comparable to the results of the present study (Weesendorp et al., 2008). Differences between the results of the present study and others could be due to differences in condition between tissues and faeces, urine or medium, differences in storage condition, spiking versus using tissues from infected pigs, or differences in virus strain.

The tonsil and the spleen are the organs of preference to detect viral RNA. The spleen has the highest halflife value in deteriorating samples, when the RT-qPCR is used. The tonsil on the other hand, has a significantly higher initial viral RNA titre than the spleen. In both the tonsil and spleen, viral RNA was still detectable in the majority of the samples at the end of the storage period of 21 days. For the detection of infectious virus by VI, the tonsil and spleen are also the preferential organs. From fresh samples, tonsils gave the highest rate of positive VI, with significantly higher initial virus titres than in kidney and mesenteric lymph node samples. Half-life values for infectious virus in deteriorating tissue samples were, however, not significantly different.

A c c e p t e d M a n u s c r i p t

The tissue samples in the present study were stored at room temperature, which is approximately 20-24 degrees Celsius. Average temperatures over a whole day will rarely exceed this in most European countries, even during the summer. It is therefore expected that the deterioration that was simulated in this study will represent the worst case scenario in the field, both for wild boars and domestic pigs. However, during the day in the summer, temperatures may occasionally be higher, which might result in a faster deterioration of tissue and degradation of virus and viral RNA. In these cases the spleen might be somewhat less appropriate than the tonsils as it may be more susceptible to decomposition and contain a higher level of endogeneous RNases, as was suggested by [START_REF] Blacksell | The effect of sample degradation and RNA stabilization on classical swine fever virus RT-PCR and ELISA methods[END_REF]. To extend the duration of virus detectability a stabilization reagent like RNAlater TM or Trizol could be used, as it has been shown that this prevents or reduces the RNA decomposition [START_REF] Blacksell | The effect of sample degradation and RNA stabilization on classical swine fever virus RT-PCR and ELISA methods[END_REF][START_REF] Hofmann | Rescue of infectious classical swine fever and foot-and-mouth disease virus by RNA transfection and virus detection by RT-PCR after extended storage of samples in Trizol®[END_REF]. However, this will only apply for stabilizing samples during transport, and may therefore be of limited value.

In this study, organs were kept at room temperature for up to 21 days. This resulted in severe deterioration of some of the organs, almost liquefying them. In reality, it will be very rare for samples to be subject to such a long period of deterioration before they are being tested. In the field, carcasses of wild boar are quickly consumed by scavengers. Piglets may decompose in less than 48 hours, but this depends on the climatic conditions, body mass and location of animals (Sophie Rossi, personal communication). At that time, viral RNA could still be detected in 94% of the samples used in this study. What is more, wild boar, and also domestic pigs, dying of CSF, and therefore usually showing severe clinical symptoms and fever shortly before death, are likely to have high or very high initial virus titres in their organs Even with the lowest half-life values observed in this study in the spleen and tonsil samples, only an initial viral RNA titre of 10 4.4 TCID 50 equivalents/g is required to detect the virus after 48 hours (with a detection limit of the RT-qPCR of 10 2 TCID 50 equivalents/g).

Depending on the technique of preparation of samples for either VI or RT-PCR, only a very small piece of an organ may be sampled to detect the virus. This is actually a general trend as a result of ongoing robotisation and the use of smaller sample volumes in diagnostic tests. Distribution of the virus in the organ may therefore become crucial to obtain a sufficiently high sensitivity. If the virus is heterogeneously distributed, it would be possible to miss infected animals by simply taking the wrong small piece of tissue. Homogeneity was tested in the fresh organs by taking samples from multiple places. It was shown that the virus was distributed very homogeneously in the tonsils, spleen, and lymph nodes. The viral RNA titres at the different locations of these organs showed an average standard deviation between 0.10 and 0.15 log 10 TCID 50 equivalents/g. The standard deviation of the RT-qPCR itself was between 0.07 and 0.17 log 10 TCID 50 equivalents/g. This was calculated by testing the reproducibility of five Taking one small sample from these organs is therefore likely to give a representative and reproducible test result. A somewhat larger variation between individual samples was observed in the kidney, with an average standard deviation of 0.25 log 10 TCID 50 equivalents/g. It may be concluded that distribution of virus in the kidney may not be as homogeneous, therefore giving an additional argument why kidney samples are less suitable than tonsil and spleen. Whether the homogeneity that was found in fresh organs will last during the deterioration process was not investigated.

In conclusion, this study showed the advantage of the RT-PCR over virus isolation as a diagnostic assay on fresh organs, but also on deteriorated samples. A reliable detection of viral genome can be expected in deteriorated samples within a reasonable period that these samples may be subject to deterioration in the field. The organs of preference to detect CSF infections in dead pigs, being either domestic pigs or wild boar, are the tonsil and the spleen. Wensvoort, G., Terpstra, C., Boonstra, J., Bloemraad, M., Van Zaane, D., 1986. Production of monoclonal antibodies against swine fever virus and their use in laboratory diagnosis. Vet. Microbiol. 12, 101-108.

A c c e p t e d M a n u s c r i p t 3 Max = maximum half-life value observed.

1

  The effect of tissue degradation on detection of infectious virus and viral RNA to diagnose classical

  LC software. The viral RNA concentration (TCID 50 equivalents per gram) of each individual

  , viral RNA could still be detected in 48 of the 96 organs. At 21 days the highest recovery rates were observed in the tonsil and the spleen.

  the standard curve of decreasing viral RNA concentrations, that were included in 36 RT-qPCR-tests performed in this study. Thus, the variation of the viral RNA titres found in the tonsil, spleen and lymph nodes, can be attributed as much to just test variation as it can to a non-homogeneous distribution over the organ.

  Van Rijn, P.A., Wellenberg, G.J., Hakze-van der Honing, R., Jacobs, L., Moonen, P.L., Feitsma, H., 2004. Detection of economically important viruses in boar semen by quantitative RealTime PCR technology. J.Virol. Meth. 120,[151][152][153][154][155][156][157][158][159][160]

Table 3 .

 3 Decay of infectious virus, as determined by virus isolation, in organs from pigs infected with the Paderborn strain

	Organ	Average half-life (days)	se 1	Min 2	Max	3
	Kidney	0.31	0.051	0.10	1.00
	Spleen	0.28	0.042	0.14	2.41
	Tonsil	0.21	0.019	0.11	0.57
	Lymph node	0.29	0.047	0.08	2.41
	1 se = standard error

2 Min = minimum half-life value observed.

Acknowledgements

The authors thank Linda Smits-Mastenbroek for the technical assistance. Bas Engel and Willem Buist are thanked for their valuable help with the statistical analysis.

This study was financially supported by the Dutch Ministry of Agriculture, Nature and Food Quality (WOT-01-003-010-01).

A c c e p t e d M a n u s c r i p t Table 1.Overview of all 24 pigs from which organs were collected at day 31 after inoculation of two of them (pigs 23 and 24). Time of infection will be related 1 to the first day the pigs tested positive in the oropharyngeal fluid (OPF). Infectious virus titres are given for all organs, including the last day after storage at 2 room temperature that the samples were still positive. 1.9 a-c Means within row with no common superscript differ significantly (p< 0.05). b.d.l = below the detection limit of the virus isolation (2.4 log 10 TCID 50 /g). * First 4 day after inoculation of the first two pigs that the oropharyngeal fluid (OPF) was positive in the qRRT-PCR. 5

A c c e p t e d M a n u s c r i p t Table 2.Overview of all 24 pigs from which organs were collected at day 31 after inoculation of two of them (pigs 23 and 24). Time of infection will be related 7 to the first day the pigs tested positive in the oropharyngeal fluid (OPF). Viral RNA titres are given for all organs, including the last day after storage at room 8 temperature that the samples were still positive. 9 3 Max = maximum half-life value observed.

Pig


Philipp Kaysser

Wolf Von Bomhard

Linda Dobrzykowski

Dr Hermann Meyer
email: hermann1meyer@bundeswehr.org

Genetic diversity of feline cowpox virus, Germany 2000 -2008

Keywords: Orthopoxvirus, cowpox virus, cats, Germany, hemagglutinin

Recent cowpox virus (CPXV) infections of humans in Europe transmitted from cats and pet rats have risen public awareness of this rare zoonosis. Based on serosurveys wild rodents are regarded as primary reservoir hosts. Cats can become infected while hunting and could therefore serve as a sentinel for CPXV strains circulating in wild rodents. In a retrospective study we analysed 73 formalin-fixed paraffin-embedded skin samples from cats with a histologically proven CPXV infection. Specimens had been collected in different parts of Germany during 2000 to 2008.

Following DNA extraction part of the hemagglutinin gene was amplified and sequenced from 72 samples. A phylogenetic analysis was inferred resulting in a total of 21 different CPXV genetic variants. The geographic distribution was imposed on a map.

Poxviridae, was associated with localized lesions in cattle. However, during the last 3 decades CPXV has not been isolated from cattle anymore. Instead, domestic cats and zoo animals are currently those animal species most frequently diagnosed with CPXV [START_REF] Bennett | Cowpox virus[END_REF]. Wild rodents seem to be the primary reservoir host, shown by serological transmission studies [START_REF] Hazel | A longitudinal study of an endemic disease in its wildlife reservoir: cowpox and wild rodents[END_REF]. Cats, zoo animals and humans are accidental hosts with humans getting infected by close contact to infected cats or pet rats (Schulze et al, 2006;[START_REF] Campe | Cowpox virus transmission from pet rats to humans, Germany[END_REF][START_REF] Ninove | Cowpox virus transmission from pet rats to humans, France[END_REF]. The fact that most feline CPXV infections occur in the late summer [START_REF] Pfeffer | Retrospective investigation of feline cowpox in Germany[END_REF] where rodent populations usually are at their peak levelunderlines the hypothesis that domestic cats can be regarded as a sentinel for CPXV prevalence among rodents. In general, CPXV infections predominantly manifest as skin lesions which progress from papule, vesicle, pustule, ulcer to scarring. In cats, CPXV infections are often misdiagnosed clinically as pyodermia, eosinophilic granuloma or neoplasm. In most cases these clinical differential diagnoses can be ruled out by histopathological examination and a CPXV infection can be confirmed. Sequence data from sporadic CPXV infections or small outbreaks has been published on a case-by-casebasis in the last years (Schulze et al, 2006;[START_REF] Campe | Cowpox virus transmission from pet rats to humans, Germany[END_REF][START_REF] Ninove | Cowpox virus transmission from pet rats to humans, France[END_REF], some indicate possible chains of infection. However, systematic data on the genetic diversity of CPXV does not exist. Recent rat-to-human infections in Germany and France [START_REF] Campe | Cowpox virus transmission from pet rats to humans, Germany[END_REF][START_REF] Ninove | Cowpox virus transmission from pet rats to humans, France[END_REF] or cat-to-human infections in Italy [START_REF] Carletti | Cat-to-human orthopoxvirus transmission, northeastern Italy[END_REF] have re-raised the question for virus population dynamics and genetic diversity of CPXV in Europe and suggest improved surveillance efforts. To fulfil this need we conducted a retrospective study on formalin-fixed paraffin-embedded tissues from histopathologically confirmed feline cowpox infections aiming to describe the genetic diversity of CPXV in Germany. For amplification 5 µL of template DNA were added. PCR-grade water served as no-templatecontrol (NTC) and vaccinia virus (VACV) strain Lister-Elstree (ATCC VR-1549, LGC Standards GmbH, Wesel, Germany) served as positive control.

The PCR was performed in a touch-down format with a pre-incubation of five minutes at 94°C, its first ten cycles starting at 94°C for one minute, then reducing the annealing temperature from 65°C to 55°C in one-degree steps for one minute and an extension phase for one minute at 72°C. Further amplification was done by 30 cycles each consisting of one minute at 94°C, 55°C and 72°C. After ten minutes at 72°C samples were held at 4°C. 4 µl of the amplicon were electrophorized in a 2 % agarose gel.

Sequencing and analysis. HA-positive samples were purified using the QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. The final elution step was done with 50 µl molecular biology grade water (5 Prime GmbH, Hamburg, Germany) and 3 µl were electrophorized in a 2 % agarose gel to determine the DNA concentration by using a 50 -2000 bp AmpliSize Molecular Ruler (BioRad, Munich, Germany).

DNA was adjusted to give a total yield of about 10 µg/ml and was shipped for commercial sequencing (Eurofins MWG GmbH, Ebersberg, Germany). Contigs were build using BioNumerics 5.1, sequences were aligned using ClustalW in MEGA4 [START_REF] Tamura | MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0[END_REF]. The final phylogenetic analysis was also done using MEGA4, the diagram in Figure 5 was created using Base-by-Base [START_REF] Brodie | Base-By-Base: Single nucleotide-level analysis of whole viral genome alignments[END_REF].

Nucleotide sequence accession numbers. 72 sequences representing part of the HA gene were deposited into GenBank and assigned the numbers FJ944556 to FJ944627.

Results

Samples and DNA extraction. The report search yielded 73 hits for the keywords "pox" or "orthopox" of feline origin. 72 samples were sent in from small animal practices in Germany, one from Luxemburg. In 52 cases preliminary diagnoses were conveyed by the sender, in nine cases an orthopox virus infection was suspected. The other preliminary diagnoses were: neoplasm (n = 15), eosinophilic granuloma ( 14), bacterial infection (9), mycosis (3), autoimmune disease (1) and herpes virus infection (1). The places of origin were distributed all over Germany with a focus on the South and West of Germany (Figure 3).

We were able to amplify part of the HA gene from 72 out of the 73 samples. Despite repeated attempts, one sample did not show an amplicon.

The seasonal distribution of the accumulated case numbers over the period from January 2000 to January 2008 is shown in Figure 1. Almost 72 % (n = 52) of the cases were found in the months August to November with a peak in September (n = 18). Sample numbers ranged from 2 (in 2006) up to 17 (in 2007) with a median of 9 samples per year.

Phylogeny and geographical distribution. After sequencing and eliminating primer-specific sequences the adjusted lengths of the resulting sequences varied from 214 to 229 base pairs (exact sizes were either 214, 217, 223 or 229 bp). All sequences were located within the hemagglutinin gene's reading frame, no frameshifts were observed. BLAST search identified all 72 study sequences as CPXV with query coverages ranging from 98 to 100 % and strain identities from 97 to 100 %. A total of 21 different genetic variants were found, ten of these have not been described in GenBank so far. The specifications of these genetic variants are outlined in Table 1 and Figure 5.

The phylogenetic tree resulting from a Neighbor-Joining analysis with 500 bootstraps is shown in Figure 2. Based on the branching, two major groups A and B with up to three subgroups were A c c e p t e d M a n u s c r i p t 7 defined. To correlate the phylogenetic analysis with the place of origin a map overlay was designed in Google Maps that shows the different subgroups and origin of the samples (Kaysser et al., 2009).

Discussion

Zoonotic diseases originating from wild animal reservoirs such as tularemia, hantavirus or monkeypox virus infections are of growing interest to physicians worldwide. The reservoir host of CPXV has long been unknown, but it is now widely accepted that bank voles (Clethrionomys glareolus) and wood mice (Microtus agrestis) are primary reservoir hosts [START_REF] Chantrey | Cowpox: reservoir hosts and geographic range[END_REF][START_REF] Hazel | A longitudinal study of an endemic disease in its wildlife reservoir: cowpox and wild rodents[END_REF]. A good correlation between population density and seroconversion rates (up to 60 % against OPV) with yearly peak infection levels in the rodent population has been observed in late summer and autumn. A link between feline CPXV infections acquired from hunting CPXV-infected wild rodents was shown by a serologic study in Germany demonstrating a seroconversion rate of 2 % in 2.173 cats investigated [START_REF] Czerny | A monoclonal blocking-ELISA for detection of orthopoxvirus antibodies in feline sera[END_REF]. The prevalence of CPXV in local rodent populations is certainly the cause for sporadic CPXV infections in cats which subsequently can infect humans. In this study the peak month with regard to time of infection is September, which confirms results from a previous study in cats [START_REF] Pfeffer | Retrospective investigation of feline cowpox in Germany[END_REF] and correlates well with data from rodent population studies [START_REF] Chantrey | Cowpox: reservoir hosts and geographic range[END_REF][START_REF] Hazel | A longitudinal study of an endemic disease in its wildlife reservoir: cowpox and wild rodents[END_REF]. The winter 2006/2007 was extremely mild in Germany, whichtogether with other climatic factors -led to a significant rise in vole populations and subsequent hantavirus infections among humans in 2007 [START_REF] Piechotowski | Emergence of hantavirus in South Germany: rodents, climate and human infections[END_REF]. And indeed, the highest number of CPXV infections in cats (n = 17) was observed in 2007 (Figure 1).

Since CPXV has never been isolated from rodent species in Western Europe, the use of molecular techniques for analysing feline CPXV could shed light on a potential genetic diversity of CPXV in the primary rodent host. In the past, open reading frame (ORF) sequences of the A c c e p t e d M a n u s c r i p t 8 hemagglutinin (HA) protein have proven very useful to allocate a given isolate to a distinct OPV species, thereby confirming the current taxonomy of OPV species which is based on phenotypic markers. In this study we attempted to sequence the entire ORF of the HA gene from FFPE specimens. However, pretests in our lab failed to amplify the entire ORF (which accounts for 900 to 951 bp, depending on the strain investigated) in the majority of the 73 FFPE blocks (data not shown). The success rate of this PCR approach largely depends on the fixative itself, the age of the FFPE block and the duration of fixation, since all these factors are likely to promote DNA degradation and making amplification sometimes impossible [START_REF] Johansen | Detection of Mycobacterium tuberculosis complex in formalin-fixed, paraffin-embedded tissue specimens with necrotizing granulomatous inflammation by strand displacement amplification[END_REF]. We therefore chose a second PCR assay amplifying a much shorter sequence within the HA gene (about 270 base pairs). The segment amplified lies on the upstream end of the HA gene (starting at nucleotide 41 of the CPXV type strain Brighton Red). This rather short sequence of the HA gene allowed identification of all 72 amplified samples as CPXV using BLAST search [START_REF] Blast | Basic Local Alignment Search Tool[END_REF]. CPXV always was the top hit, in four cases (CPXV 2007/33255 (FJ944556), CPXV 2005/27380 (FJ944603), CPXV 2006/28727 (FJ944615), CPXV 2004/28150 (FJ944626)) a sequence conserved in both, vaccinia and cowpox virus was found.

ATIs are regarded as a pathognomonic marker for an infection with CPXV. This was confirmed to nearly 100 % in this study by PCR findings. A re-examination of one hematoxylin and eosin (HE)-stained tissue section that did not yield a CPXV amplicon revealed a pattern characteristic of a viral infection. However, inclusions were not clearly located intracytoplasmic. A herpes virus could therefore be the causative agent in this case. The high correlation between PCR and light microscopy supports the key role and the high sensitivity and specificity of histopathology.

Only 17 % of the preliminary diagnoses were correctly made by the sender of the sample. CPXV in cats seems to be "underdiagnosed" by the general practitioner since the reasons for submission of skin specimens to the pathologist were mostly other dermatologic diseases and not a suspected CPXV infection. Both groups overlap to some extent in central Germany.

It is remarkable that subgroup B.2 (red markers) shows close clustering in and around Berlin (including the strain Biber V940/97, described by [START_REF] Hentschke | An outbreak of cowpox in beavers (Castor fiber canadensis) and bearcats (Ailurus fulgens) in Berlin zoo[END_REF]. Strain GRI-90 from Moscow is loosely related to this small cluster. Unfortunately the study lacks further data from Mecklenburg-Western Pomerania (the federal state north of Berlin) and in general from the area of the former German Democratic Republic to draw conclusions on the appearance of further subgroup B.2 isolates. It would also be valuable to add data from Poland and the Czech Republic to the comparison to see if the two-group-distribution extends further to the East of Europe.

Group B.1 is very small and to some extent heterogeneous, in our opinion conclusions should not yet be drawn out of its existence. In this case again, data from Austria and Italy could add valuable information to the map. The reference strains all fit well into the clustering, they were added to illustrate the potential of combined phylogenetic analyses and the use of geographical information systems.

It could be argued that the sequence variation described in this study is merely a clonal selection in the infected cat of highly different CPXVes circulating in the reservoir host. However, OPVs are genetically remarkably stable in vivo and in vitro. This was shown in a recent study of human monkeypox in the Democratic Republic of Congo. Monkeypox-like CPXV has a broad host range and is maintained in a rodent reservoir, however, the HA gene sequence of 50 human monkeypox virus isolates was 100 % identical [START_REF] Rimoin | Endemic human monkeypox, Democratic Republic of Congo, 20012004[END_REF] which argues against a variation in the reservoir host.

Isolates of CPXV do differ in restriction enzyme pattern and generally greater differences occur between geographically distinct isolates. Based on bioinformatic analyses of the genomes A c c e p t e d M a n u s c r i p t 10 sequenced so far it has been proposed that the two CPXV reference strains Brighton (UK) and Grishak (Russia) are quite divergent and should be reclassified as separate OPV species [START_REF] Gubser | Poxvirus genomes: a phylogenetic analysis[END_REF].

Regarding the apparent variation among isolates in central Europe which was also confirmed in this study, CPXV might have originated from central Europe. A geographically and host-related independent evolution might explain the genetic divergence seen today. Isolation of new or a more detailed genomic characterization of existing strains might increase the number of genetic variants considerably. However, due to the shortness of the amplicon the resulting phylogenetic tree in Figure 2 should be interpreted with caution: while the full HA gene has proven to be wellsuited for phylogenetic analyses, this is the first attempt to perform an analysis on such a short segment. It might also be that an even larger genetic variation would have appeared if the full length HA gene would have been amplified, but it is also possible that the segment under investigation shows an unusually high grade of base exchanges. A certain degree of blur in the phylogenetic tree must be attributed to the short amplicon.

A large database is a prerequisite for an adequate prediction of risk to humans, but the proper understanding of animal surveillance data is the key challenge for linking veterinary research to human risk prediction [START_REF] Scotch | Linkages between animal and human health sentinel data[END_REF]. The data generated in this study using cats as a sentinel for CPXV infections might add to the understanding of CPXV distribution in Germany and show the potential of integrating geographical information with sequence data in outbreak investigations and risk prediction. Due to its self-limiting course in humans an its low impact on public health, CPXV infections are not a notifiable disease in Germany, surrogate parameters as presented in this study seem to be the only practical approach to human risk prediction.

Conclusion

A c c e p t e d M a n u s c r i p t 11 CPXV infectionalthough relatively rare in animals and uncommon among humanshas gained some recognition through micro-outbreaks in recent years. The fact that immunity to smallpox infection (with its cross-reactivity to CPXV) in the world population is constantly decreasing due to cessation of mandatory smallpox vaccination nearly 30 years ago, puts more persons to the risk of zoonotic CPXV infections. Since CPXV is not a notifiable disease in Germany and distinct virological diagnosis relies on specialized labs, it is often not included in the differential diagnoses of most general practitioners (veterinary or human). A higher grade of public awareness and a linkage between human and veterinary health authorities through databases will reduce the individual risk of infection. This study can serve as part of this effort by providing data on the geographical distribution of CPXV in Germany. Table 1. Features of 21 cowpox virus genetic variants detected in the study. 2

A c c e p t e d M a n u s c r i p t Figure 2. Evolutionary relationships of 80 CPXV sequences. The evolutionary history was inferred using the Neighbor-Joining method [START_REF] Saitou | The neighbor-joining method: a new method for reconstructing phylogenetic trees[END_REF]. The optimal tree with the sum of branch length = 0,17798791 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) is shown next to the branches [START_REF] Felsenstein | Confidence limits on phylogenies: An approach using the bootstrap[END_REF]. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method [START_REF] Tamura | Prospects for inferring very large phylogenies by using the Neighbor-Joining method[END_REF] and are in the units of the number of base substitutions per site. All positions containing gaps and missing data were eliminated from the dataset (Complete deletion option). There were a total of 214 positions in the final dataset. Phylogenetic analyses were conducted in MEGA4 [START_REF] Tamura | MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0[END_REF]. For reference eight additional CPXV strains were included (by accession number, sequences were truncated for facilitated alignment using the primer pair applied for amplification described above): AF482758 (Brighton Red), AY298785 (Callithrix jacchus), AY902287 (Nancy), AY902260 (Biber V940/97), AY944028 (CPV91ger3), EF612709 (ITA 1 PG), FJ654467 (Shiva), X94355 (GRI-90). Data is presented using virus acronym, sequence identifier, GenBank accession number (study samples) or virus acronym, city of origin, strain, GenBank accession number (reference strains).

  cowpox virus (CPXV), a member of the genus Orthopoxvirus (OPV), family

  Routine tissue specimens received for histopathological examination had been processed in an automatic tissue processor, embedded in paraffin blocks, sectioned at 5-6 µm on a standard rotary microtome, and mounted on glass microscope slides for automated staining with hematoxylin-eosin. Samples with the characteristic histopathologic features of a necrotizing and eosinophilic dermatitis and folliculitis with large intracytoplasmic eosinophilic A-type inclusion (ATI) bodies were considered CPXV positive. Scanning the report database (January 2000 to January 2008) for the keywords "pox", "orthopox" and "cat" resulted in 73 cases. Corresponding formalin-fixed paraffin-embedded (FFPE) blocks were retrieved and stored individually in a plastic box. For every sample the following data from each report were included in a software database (BioNumerics 5.1, Applied Maths NV, Sint-Martens-Latem, Belgium): case number (year/individual code), species, preliminary diagnosis, kind of material, place of origin, postal code, country of origin and month of transmittal. Using the postal code and place of origin coordinates were generated for display in Google Maps (Google Maps, 2009). DNA extraction. To reduce contamination, all initial sample handling and preparation was done in a class 2 biosafety cabinet with single-use scalpels (Aesculap AG, Tuttlingen, Germany). Approximately 3 x 1 x 3 mm tissue sections were removed and transferred into sterile, DNAsefree Eppendorf safe-lock Biopur 1.5 mL test tubes (Eppendorf AG, Hamburg, Germany). The DNA elution was performed with the QIAamp® DNA FFPE Tissue Kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. The final elution step was done in 60 µl of ATE buffer. Orthopox virus-specific PCR. A PCR yielding an approximately 270 bp amplicon within the OPV hemagglutinin (HA) gene was performed on a Perkin Elmer GenAmp® PCR System 2400 (Applied Biosystems, Singapore, Singapore) with the following DNA Master mix: 6 µL of LightCycler® FastStart DNA Master SYBR Green I (Roche Applied Science, Mannheim, each of primer HA fw (5`-TAC TTT TGT TAC TAA TAT CAT TAG-3´) and HA rev (5´-AGC AGT CAA TGA TTT AAT TGT-3´) both at 10 µM (TIB MOLBIOL, Berlin, Germany), 7.2 µL MgCl 2 (final concentration of 4,8 mM MgCl 2 ) and 25.8 µL PCR-grade water.

  in Figure2reveals two major groups (A and B), group B could be further divided into three subgroups based on the clustering. Group A specimens have been sent in from the northern part of Germany, whereas group B specimens have a higher incidence in the south.

Figure captions Figure 1 .

 captions1 Figure captions

Figure 3 .

 3 Figure 3. Geographical distribution of 77 feline specimens which proved CPXV positive. Group

Figure 4 .

 4 Figure 4. Paw pad of a twelve year old female cat: necrotizing dermatitis with abundant brightly

Figure 5 .

 5 Figure 5. Graphical overview of 21 genetic variants of cowpox virus detected in the study.

  Figure 1

  Figure 2

  Figure 3

  Figure 4

Sequence number Accession number Sequence previously described Group according to Figure 2

	CPXV 2000/12671	FJ944598	yes	A
	CPXV 2000/13381	FJ944584	no	A
	CPXV 2001/27888	FJ944589	no	A
	CPXV 2001/3718	FJ944621	yes	A
	CPXV 2002/28462	FJ944624	yes	A
	CPXV 2005/23475	FJ944599	yes	A
	CPXV 2005/24618	FJ944557	yes	A
	CPXV 2005/36869	FJ944602	yes	A
	CPXV 2005/40050	FJ944619	yes	A
	CPXV 2007/33816	FJ944573	no	A
	CPXV 2000/20780	FJ944572	no	B.1
	CPXV 2007/26903	FJ944563	no	B.1
	CPXV 2007/37472	FJ944574	no	B.1
	CPXV 2004/28150	FJ944626	no	B.2
	CPXV 2005/27380	FJ944603	yes	B.2
	CPXV 2000/19404	FJ944608	yes	B.3
	CPXV 2000/33736	FJ944627	yes	B.3
	CPXV 2001/27666	FJ944577	yes	B.3
	CPXV 2001/33149	FJ944566	no	B.3
	CPXV 2004/30583	FJ944580	no	B.3
	CPXV 2005/18989	FJ944581	no	B.3
	* Identical to strain Brighton Red AF482758.

Table

Acknowledgements

We thank Jutta Brohl, Anke Stark and Antal Lodri for excellent technical support throughout the study.

Conflict of interest statement 1

The authors declare that they have no conflict of interest. 2


Delphine Sylvie

Anne Beeckman
email: delphine.beeckman@ugent.be

Daisy C G Vanrompay

Biology and intracellular pathogenesis of high or low virulent Chlamydophila psittaci strains in chicken macrophages

Keywords: Chlamydophila psittaci, Type III secretion, macrophages, mitochondria, actin

Within a few days post infection of SPF turkeys, highly pathogenic Chlamydophila (Cp.) psittaci genotype A and D strains can be found in blood monocytes/macrophages, while this effect is less pronounced for infection with a milder genotype B strain. To elucidate on the observed difference, we studied the developmental cycle of avian Cp. psittaci strains of varying virulence in a matched avian monocyte/macrophage cell line (HD11) by electron microscopy and immunofluorescence and determined the gene transcription of 26 Type III secretion related genes and six control genes upon infection of HD11 cells. The genotype A (84/55) and D (92/1293) strains 1) clearly induced actin recruitment to the site of entry, 2) initiated host cell degeneration at earlier time points, and 3) survived and proliferated better when compared to the milder CP3 strain. Strain 84/2334, genetically intermediate between Cp. psittaci and Cp. abortus, did not induce actin recruitment. Limited mRNA transcripts for the cell division genes ftsW and ftsK were in agreement with the observed low replication of Cp. psittaci in these host cells. The results also indicated that genes coding for the structural components of the Type III secretion system were transcribed earlier compared to an infection in epithelial cells.

Based on the presented results, we postulate that upon infection of blood monocytes/macrophages, Cp. psittaci deliberately limits its replication and immediately arms itself to infect other cells elsewhere in the host, whilst using the monocytes/macrophages as a quick transport vehicle.

A c c e p t e d M a n u s c r i p t

INTRODUCTION

The obligate intracellular pathogen Chlamydophila (Cp.) psittaci can infect both mammalian and avian hosts, with Psittacidae and Columbiformes among the most frequently infected avian orders [START_REF] Kaleta | Avian host range of Chlamydophila spp. based on isolation, antigen detection and serology[END_REF]. Survival and replication within a membrane bound vacuole in the host cell is essential to chlamydial pathogenesis. During their unique biphasic life cycle, Chlamydiaceae are able to transfer effector molecules in the host cell by means of a Type III secretion system (T3SS). Genes encoding the T3SS and its effectors seem to be temporally transcribed as early (1.5 to 8 h p.i.), middle (12 to 18 h) or late (by 24 h) in the developmental cycle [START_REF] Slepenkin | Temporal expression of type III secretion genes of Chlamydia pneumoniae[END_REF], in agreement with the transition from elementary body (EB) to reticulate body (RB), RB multiplication and RB to EB redifferentiation.

Serovars largely correspond to the currently accepted classification in ompA (coding for MOMP) genotypes defined by sequencing [START_REF] Everett | Emended description of the order Chlamydiales, proposal of Parachlamydiaceae fam. nov. and Simkaniaceae fam. nov., each containing one monotypic genus, revised taxonomy of the family Chlamydiaceae, including a new genus and five new species, and standards for the identification of organisms[END_REF][START_REF] Geens | Sequencing of the Chlamydophila psittaci ompA gene reveals a new genotype, E/B, and the need for a rapid discriminatory genotyping method[END_REF]. Infection with genotypes A and D results in a more elevated pathogenicity for turkeys than genotype B: for genotype B, the incubation period is longer, maximal replication is delayed, the period during which bacteria are observed in the same tissue is shorter and tissue tropism seems to be less extensive [START_REF] Vanrompay | Chlamydia psittaci in turkeys: pathogenesis of infections in avian serovars A, B and D[END_REF]. During the initial replication in epithelial cells from the turkey respiratory system, macrophages are attracted, normally acting as a first line of (innate) defense against infection (Vanrompay et al., 1994a) and Cp. psittaci has already been detected in monocytes/macrophages recognized by the specific marker KUL04 [START_REF] Vanrompay | Chlamydia psittaci in turkeys: pathogenesis of infections in avian serovars A, B and D[END_REF]. The present study therefore aims to further shed light on the complex interaction between Cp. psittaci and its avian host by studying the biology and intracellular pathogenesis of high and low virulent Cp. psittaci strains in the chicken monocyte/macrophage cell line HD11.

A c c e p t e d M a n u s c r i p t 4

MATERIALS AND METHODS

Organism and cell culture

The HD11 chicken monocyte/macrophage cell line [START_REF] Beug | Chicken hematopoietic cells transformed by seven strains of defective avian leukemia viruses display three distinct phenotypes of differentiation[END_REF] was cultured in Dulbecco's modified Eagle's minimal essential medium (DMEM) supplemented with 5% heat-inactivated fetal calf serum, 1% sodium pyruvate, 1% L-glutamine and 0.5% gentamicin (all products from Invitrogen, Merelbeke, Belgium), further referred to as HD11 culture medium. Cultures were incubated at 5% CO 2 and 37 °C. Four different Cp. psittaci strains were used in this study: 84/55 [isolated from the lung of a budgerigar, ompA genotype A [START_REF] Vanrompay | Ultrastructural changes in avian Chlamydia psittaci serovar A-, B-, and D-infected Buffalo Green Monkey cells[END_REF]], CP3 [isolated from a pigeon, genotype B [START_REF] Page | Investigation of a recent ornithosis epornitic in Californian turkeys[END_REF]], 92/1293 [isolated from a pooled homogenate of lungs, cloaca and spleen of diseased turkeys, genotype D [START_REF] Vanrompay | Serotyping of European isolates of Chlamydia psittaci from poultry and other birds[END_REF]] and 84/2334, an intermediate strain in the evolution of Cp. abortus from Cp. psittaci, isolated from a yellow-headed Amazon parrot [START_REF] Van Loock | Missing links in the divergence of Chlamydophila abortus from Chlamydophila psittaci[END_REF].

All strains were cultured and titrated as described before [START_REF] Vanrompay | Ultrastructural changes in avian Chlamydia psittaci serovar A-, B-, and D-infected Buffalo Green Monkey cells[END_REF].

Transmission electron microscopy

HD11 cells were seeded on Cytodex 3 microcarriers (GE Healthcare, Diegem, Belgium) essentially as described before [START_REF] Vanrompay | Ultrastructural changes in avian Chlamydia psittaci serovar A-, B-, and D-infected Buffalo Green Monkey cells[END_REF]. Briefly, 60 mg of microcarriers were hydrated and autoclaved according to the manufacturer's instructions, and subsequently resuspended in HD11 culture medium. Before seeding on the microcarriers, HD11 cells were grown for 72 h with 10% FCS to obtain a large fraction of adherent macrophages. The adherent cells were trypisinized, pelleted and 2 x 10 7 cells were added to the microcarriers in a Petri dish. After incubation for 5 h at 37 °C and 5% CO 2 , the non-adherent cells were washed away with sterile PBS and A c c e p t e d M a n u s c r i p t 5 cells and microcarriers were resuspended in HD11 medium with 10% FCS. The microcarrier cultures were incubated overnight on a rotating platform, distributed into six new Petri dishes with fresh HD11 culture medium to maintain optimal growth conditions and further incubated on the rotating platform for 24 h. Contents of each Petri dish were centrifuged for 4 min at 135 x g and pellets were resuspended in 20 ml HD11 medium supplemented with 5.5 mg/ml glucose (Sigma, Antwerp, Belgium) and each of the four Cp. psittaci strains under study at a multiplicity of infection (MOI) of 100. Infected microcarrier cultures were incubated at 37 °C and 5% CO 2 for 3 h whereafter the inoculum was removed by centrifugation and a washing step with sterile PBS. Cultures were then resuspended in HD11 medium supplemented with glucose and further incubated on a rotating platform until sample taking at 0, 1, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 21, 26, 30, 36, 42, 45, 50, 52 and 72 h post inoculation (p.i.). For the negative control, the medium was replaced with HD11 medium with glucose. Infection was checked on dried and acetone fixed spotted microcarriers on a glass slide using a direct immunofluorescence staining at 30 and 50 h (IMAGEN Chlamydia, Oxoid, Drongen, Belgium) as previously described (Vanrompay et al., 1994b). At each time point 100 µl of microcarrier culture was added to 1 ml of Karnovsky fixative [START_REF] Karnovsky | Formaldehyde-glutaraldehyde fixation of high osmolality for use in electron microscopy[END_REF] and fixed overnight at 4 °C.

Afterwards, the fixative was replaced with sodium cacodylate buffer (0.1 M in BiDest, pH 7.4, Sigma) until further processing. Post-fixation was performed using 4% osmiumtetroxide and samples were dehydrated in a step gradient of ethanol at room temperature. The samples were transferred to 100% alcohol/Spurr's resin (1:1) at 4 °C overnight, brought to 100% alcohol/Spurr's resin (1:2) for 8 h (4 °C), and transferred to 100% Spurr's resin and left overnight at 4 °C. Polymerization was performed at 70 °C for 16 h. Sections (70 nm thick) were made using a Reichert Sections were post-stained with a Leica EM stain for 30 min in uranyl acetate at 40 °C and 10 min in lead citrate stain at 20 °C. The grids were examined with a JEM 1010 Jeol electron microscope equipped with imaging plates which were scanned digitally (Ditabis, Pforzheim, Germany).

Immunofluorescence assays

We tested whether Cp. psittaci strains of different virulence are all able to induce actin polymerization at the site of attachment and entry and whether mitochondria are being attracted to the inclusions. HD11 cells (3 x 10 5 / ml) were seeded on sterile glass coverslips (13 mm) at the bottom of Chlamydia Trac Bottles (Bibby Sterilin Ltd., Stone, UK) for 24 h at 37 °C and 5% CO 2 and subsequently inoculated at an MOI of 100, performing a centrifugation step (1 000 g) at 4 °C for 15 min to synchronize irreversible attachment and entry. One ml of HD11 culture medium with glucose was added and cells were further incubated at 37 °C and 5% CO 2 until PFA fixation and permeabilization with saponin at 15 min, 8 h, 20 h or 28 h p.i. as described before [START_REF] Beeckman | Effect of ovotransferrin and lactoferrins on Chlamydophila psittaci adhesion and invasion in HD11 chicken macrophages[END_REF].

Colocalization between Cp. psittaci and actin polymerization was assessed using

AlexaFluor 488 coupled phalloidin (Molecular Probes, Invitrogen) and an indirect staining for Cp. psittaci [START_REF] Beeckman | Effect of ovotransferrin and lactoferrins on Chlamydophila psittaci adhesion and invasion in HD11 chicken macrophages[END_REF]. Distribution of the mitochondria was visualized by use of a cross-reacting monoclonal antibody against turkey sperm mitochondria [START_REF] Korn | Production and characterization of a turkey sperm mitochondrial monoclonal antibody and its usefulness for assessment of sperm integrity[END_REF] (kindly provided by Nancy Korn, Clemson University, Dept. of Animal and Veterinary Sciences, South Carolina). Briefly, cell layers were first incubated with 25 µl of reconstituted hybridoma supernatant for 1 h at 37 °C, rinsed and then incubated with 25 µl of an All dilutions and washing steps (three) were performed with 1% BSA (Sigma) in PBS. After a final rinsing with PBS, nuclei were visualized in all samples with DAPI (100 µl at a concentration of 100 ng/ml in BiDest for 5 min at room temperature). A final rinsing with BiDest was followed by mounting of the coverslips using Mowiol (Calbiochem, VWR, Haasrode, Belgium) with 0.01% PPD (Sigma) [START_REF] Valnes | Retardation of immunofluorescence fading during microscopy[END_REF]. Images were acquired using a Radiance 2000 confocal laser scanning microscope (Bio-Rad, Nazareth, Belgium, 600 x). Chlamydial replication at 8 h, 20 h and 28 h was assessed using a scoring system from 0 to 5 as previously described [START_REF] Van Loock | Exacerbation of Chlamydophila psittaci pathogenicity in turkeys superinfected by Escherichia coli[END_REF].

Transcription analysis of control genes and T3S genes

As T3S plays an important role in different phases of the chlamydial life cycle, we studied the gene transcription profile of a panel of T3S related genes (26 in total) for different Cp. psittaci strains in HD11 cells in order to determine whether virulence could be linked to differences in T3S gene transcription profiles. Total RNA was extracted from 1.5 x 10 6 cells infected at an MOI of 100 (time points 1, 2, 4, 8, 12, 18, 24, 36 and 48 h p.i.) and checked for DNA contamination. All the experiments were performed twice. Instead of 0.5 µg, one microgram of total RNA (spiked with 10 ng of coliphage control RNA) was reverse transcribed using selective hexamers (5'-TTANNN-3' and 5'-CTANNN-3') and qPCR using SYBRGreen technology was performed in duplo on each sample. Reverse transcription, primer sequences, cycling A c c e p t e d M a n u s c r i p t 8 and normalization were exactly as performed earlier [START_REF] Beeckman | Identification and characterization of a type III secretion system in Chlamydophila psittaci[END_REF]. In addition, primers were developed to determine the transcription of groEL-1, ompA and omcB as markers of the early, middle and late phase of the chlamydial developmental cycle, respectively [START_REF] Shaw | Three temporal classes of gene expression during the Chlamydia trachomatis developmental cycle[END_REF][START_REF] Slepenkin | Temporal expression of type III secretion genes of Chlamydia pneumoniae[END_REF], the cell division related genes ftsW and ftsK [START_REF] Gerard | Expression of Chlamydia trachomatis genes encoding products required for DNA synthesis and cell division during active versus persistent infection[END_REF], as well as dnaK (hsp70) [START_REF] Cross | Antimicrobial susceptibility testing of Chlamydia trachomatis using a reverse transcriptase PCR-based method[END_REF]. An overview of the RT-qPCR primers, designed based on the currently unpublished genome of Cp. psittaci 6BC (P. Bavoil & G. Myers, personal communication), can be found in Table 1.

To compare the two independent samples (namely gene transcription ratios of 32 genes from two independent infection experiments per strain, each 16S rRNA normalized) at the same time point, a Paired Student's t-test was used (SPSS Inc., Chicago, Illinois, US). Secondly, an analysis of variance (ANOVA, SPSS Inc.) with post-hoc analysis (both Tukey HSD and Tukey-b) was performed along the time axis to determine significant upregulation time points for each gene for each of the four Cp. psittaci strains under study.

2.5.Titration of infectious progeny on BGM cells

To quantify chlamydial replication in HD11 cells, tenfold dilution series from supernatant from cells infected for 48 h with either of the four strains was inoculated onto BGM cells as described before [START_REF] Vanrompay | Diagnosis of avian chlamydiosis: specificity of the modified Gimenez staining on smears and comparison of the sensitivity of isolation in eggs and three different cell cultures[END_REF]. Cells were fixed at 48 h p.i. with methanol and stained with IMAGEN Chlamydia (Oxoid). The number of inclusion forming units (IFU/ml) was determined by the method of Spearman & Kaerber [START_REF] Mayr | Quantitative Bestimmung der Virusinfektiosität (Virustitration)[END_REF].

RESULTS

A c c e p t e d

M a n u s c r i p t 9

Transmission electron microscopy

Predominantly monocytes were present in the early infection stages, differentiating into inflammatory macrophages later on in the course of the infection. For all Cp. psittaci strains, internalization was often accompanied by an invagination of the HD11 host cell membrane (Figure 1B) or the presence of clathrin-coated pits (except for 84/2334, Table 2). Subsequently, the chlamydial organisms pursued their way to the nucleus and from 6 h p.i. on (8 h for strain 84/55), Cp. psittaci EBs or IBs could be observed in the vicinity of the host cell nuclear membrane (Figure 1D). However, not all internalized bacteria arrived at the nucleus, since fusion of the nascent inclusion with lysosomes was detected for all strains (Table 2, Figure 1C), the earliest (2 h p.i.) for the low pathogenic strain CP3. These phagolysosomes then further evolved to the typical morphology given in Figure 1E. For inclusions efficiently escaping the phagolysosomal fusion, the inclusion membrane faded or opened up to the host cell cytoplasm, thus releasing the chlamydiae. As such, a direct interaction between free RBs and the nuclear envelope could sometimes be observed (Figure 1F). However, intact inclusions were also present, containing a limited number of odd shaped RBs (Figure 1G) and at 45 h p.i. phagocytosis of cell fragments could often be observed.

The high virulent strains already induced host cell degeneration from 6 h p.i. on, while for the milder strain CP3 this could be observed six hours later (Table 2). This was characterized by a marked degeneration of the mitochondria (enlarged and cristae more open), dilatation of the endoplasmatic reticulum and nuclear condensation and fragmentation (Figure 1I), which is indicative for apoptosis.

Noteworthy is the bridging between extracellular Cp. psittaci 84/2334 and the host cell membrane at 8 h p.i. (Figure 1H).

Immunofluorescence assays

In order to investigate the host cell reaction to infection with Cp. psittaci strains of different virulence, HD11 cells were stained for host cell actin (15 min p.i.) or mitochondria (8, 20 and 28 h p.i.), bacteria and the nucleus (all time points). At 15 min p.i., strains 92/1293 and 84/55 had attached greatly to the cells, thereby inducing strong host cell actin polymerization at the site of attachment (Figure 2: A andC). Both the level of attachment and the accompanying actin polymerization were less pronounced for inoculation with the less virulent strain CP3 (Figure 2B). Attachment of Cp. psittaci 84/2334 was higher than observed for CP3, but lower compared to the two highly virulent strains 92/1293 and 84/55. However, no colocalization of actin polymerization and the bacteria could be observed for this strain (Figure 2D). No significant differences could be observed in the staining pattern for the mitochondria or the nuclei at 8 h p.i. with the four Cp. psittaci strains (Figure 3, first column). As could be expected from the lower attachment of CP3 at 15 min p.i., fewer bacteria had entered the host cells compared to the other strains. The highest amount of bacteria at this time point could be observed for 92/1293, while comparable amounts of 84/55 and 84/2334 had entered the cells. As confirmed by the electron microscopy experiments, bacteria could already be observed in the vicinity of the nucleus for all strains. Twelve hours later (20 h p.i.), bacteria had further developed, but no dense uniformly stained inclusions could be observed yet, except for the miniinclusions observed for CP3 (Figure 3, second column). For 84/55, an association between the inclusions and host mitochondria could be observed, although not for every inclusion, and heavily infected host cells showed clear signs of degeneration. 2). At 28 h p.i., bacterial development had proceeded for all strains (Figure 3, third column), but no large, dense, uniformly stained inclusions could be visualized. In concordance with earlier time points, strains 92/1293 and 84/55 had proliferated the most, while growth of CP3 was still very limited. Typically, plenty, clearly separate, mini-inclusions could be observed for 84/2334. Mitochondria were no longer stained in all host cells, especially for 92/1293 or 84/55 inoculated cells, possible as a result of the more pronounced host cell degeneration (Figure 1, Table 2). Additionally, inoculation with Cp. psittaci strains of different virulence had an effect on the size of the host cell nuclei, as visualized by DAPI. Condensed nuclei could be observed in heavily infected cells, whereas the surrounding not or only moderately infected cells had enlarged nuclei when compared to the negative controls. For strain 84/2334, both infected and uninfected cells in the same sample had enlarged nuclei, comparable in size to those of the uninfected host cells in 92/1293 or 84/55 inoculated samples. These effects could already be observed at 20 h p.i., but were more pronounced at 28 h p.i. and were not present in CP3 inoculated samples.

Assessing bacterial proliferation at 48 h p.i., immunofluorescence staining of Cp. psittaci 84/55 or 92/1293 infected monocytes/macrophages revealed a significant bacterial load (most for 92/1293). Multiple inclusions could be observed per cell and host cells were degenerating. For inoculation with CP3, results were in concordance

Transcription analysis of control genes and T3S genes

Firstly, the transcription pattern of a number of control genes (groEL-1, ompA, omcB, ftsW, ftsK and dnaK) was determined. All newly developed primer couples yielded an amplicon of the expected size when tested on Cp. psittaci 92/1293 genomic DNA.

However, for groEL-1, no amplicon could be obtained when tested on copy DNA, despite several PCR optimization steps. An overview of the upregulation time points is given in Table 3.

The bacterial cell division genes ftsW and ftsK were only poorly transcribed and transcription dropped below the detection limit after 12 h p.i, except for 92/1293 (no transcription after 24 h). For ompA, a typical mid-cycle gene, transcription was predominantly observed from 8 to 18 h p.i., except for 84/55, where limited transcription was already observed from 1 h p.i. on. The typical late-cycle gene omcB was transcribed from 1 h p.i. on for all four strains, although very limited, and reached its peak transcription at 8 h p.i., followed by a secondary upregulation of transcription late in the infection cycle.

As for the transcription of T3S related genes, incB and copB2 were excluded from further analysis, because no working primers could be developed. None of the strains under study transcribed the sctS or ca530 genes to a detectable level. An overview of significant gene transcription upregulation time points along the time axis, as A c c e p t e d M a n u s c r i p t 13 determined by ANOVA, is given in Table 4 and Table 5 and the most remarkable results will be summarized hereafter.

For Cp. psittaci 84/55 (Table 4, column 3-5), no transcription could be observed for sctU (cluster 1), copB1 (cluster 2.2), sctN (cluster 3) or cap1 (non-clustered) and both incA and incC (non-clustered) were shown to be transcribed from 1 h p.i. on.

Transcription results of the both translocon-coding gene clusters revealed that cluster 4 (scc3, copB2, copD2) was generally spoken transcribed later than the scc2-copB1-copD1 cluster.

Striking gene transcription results for CP3 infection in chicken macrophages (Table 4, column 6-8) include the late transcription of the non-clustered genes incA (24 h) and cap1 (12 h). Transcription of copB1 was strongly delayed when compared to the other genes of cluster 2.2 as was also the case for sctC compared to the other genes in cluster 3.

Transcription results for a Cp. psittaci 92/1293 infection of HD11 cells (Table 5, column 3-5) include upregulation of sctT (cluster 2.1) transcription at the 8 h p.i. time point only, but not further on in the developmental cycle. In addition, transcription of incC (cluster 3) occurred early in the infection while the non-clustered incA gene did not seem to be transcribed at all. Transcription of the non-clustered tarp was upregulated already at 4 h p.i., whereas cadd transcription could be detected from 2 h p.i. on. The cap1 gene was upregulated from 8 h pi on, with the highest transcription observed at 36 h p.i.

Noteworthy for 84/2334 (Table 5, column 6-8) is that this bacterium transcribes the pkn5 gene (cluster 3), while none of the other strains do so, and that pkn5 and ca037 share the same transcription upregulation profile. No transcription could be observed for the genes of cluster 2.2 (scc2, copB1, copD1).

A c c e p t e d M a n u s c r i p t 14

3.4.Titration of infectious progeny on BGM cells

A titration was performed on the supernatant of infected HD11 cells at 48 h p.i. through inoculation of dilution series of this supernatant on BGM cells. In concordance with immunofluorescence stainings, TCID 50 /ml for 92/1293 progeny was the highest (2.5 x 10 8 / ml), closely followed by 84/55 (2 x 10 8 / ml). CP3 had proliferated the least in the HD11 cells (TCID 50 5 x 10 7 /ml) while an 84/2334 infection generated an intermediate value of 1 x 10 8 / ml.

DISCUSSION

Considerable evidence exists indicating that Chlamydophila psittaci is nearly endemic in the European turkey industry. Previous research has shown that the virulence of Cp.

psittaci strains for turkeys depends on the ompA ('outer membrane protein A') genotype of the infecting strain: genotypes A and D result in a severe systemic infection of the animals, while genotype B is less invasive [START_REF] Vanrompay | Chlamydia psittaci in turkeys: pathogenesis of infections in avian serovars A, B and D[END_REF].

As a consequence, the poultry industry suffers predominantly from genotype A and D infections. In experimentally infected turkeys, the infection starts with chlamydial replication in epithelial cells and macrophages of the respiratory tract, thereby inducing severe inflammation. Subsequently, chlamydiae are found in blood monocytes/macrophages recognized by the specific marker KUL04 [START_REF] Vanrompay | Chlamydia psittaci in turkeys: pathogenesis of infections in avian serovars A, B and D[END_REF] and the infection spreads to various organs. We hypothesize that the described differences in virulence are correlated with the ability of the infecting strain to invade blood monocytes/macrophages, thereby using them as a transport vehicle to reach other tissues in the host. To elucidate on the complex interaction between Cp. psittaci and its avian host, we characterized the Cp. psittaci 6BC and human monocytes or macrophages have been performed in the past, revealing that both cell types support intracellular replication of Cp. psittaci [START_REF] Rothermel | Oxygen-independent inhibition of intracellular Chlamydia psittaci growth by human monocytes and interferon-gamma-activated macrophages[END_REF][START_REF] Carlin | Potentiation of interferon-mediated inhibition of Chlamydia infection by interleukin-1 in human macrophage cultures[END_REF]. However, [START_REF] Roshick | Comparison of gamma interferon-mediated antichlamydial defense mechanisms in human and mouse cells[END_REF] recently demonstrated the importance of performing chlamydial research in the matched host cell system, hence our choice for the avian monocyte/macrophage cell line HD11. Two high virulent (84/55 and 92/1293) and one low virulent (CP3) Cp. psittaci strains were included in the study, in addition to 84/2334, formerly classified as serovar A but genetically an intermediate between avian Cp. psittaci and mammalian Cp. abortus [START_REF] Van Loock | Missing links in the divergence of Chlamydophila abortus from Chlamydophila psittaci[END_REF].

Within 15 minutes upon attachment to the host cell, Cp. psittaci 92/1293 induces recruitment of actin to the site of invasion, as earlier demonstrated by [START_REF] Beeckman | Effect of ovotransferrin and lactoferrins on Chlamydophila psittaci adhesion and invasion in HD11 chicken macrophages[END_REF]. To determine whether this phenomenon contributes to virulence, we assessed the induction of actin recruitment by the four strains under study. Both high and low virulent strains recruited actin filaments to the site of infection and this effect was more pronounced for the higher virulent strains (Figure 2). This could be linked to a higher transcription of the tarp gene in the more virulent strains. Remarkably, no colocalization of Cp. psittaci and actin polymerization could be observed for an infection of HD11 cells with strain 84/2334, although the Tarp encoding gene can definitely be transcribed by this strain (Table 5). Similarly, at all time points p.i., no association with clathrin-coated pits could be observed for an 84/2334 infection ( Next, we characterized the intracellular development and host cell interaction of the four Cp. psittaci strains. While fusion of internalized CP3 organisms with lysosomes was already observed at 2 h p.i., this was only the case at 8 h the earliest for the other three strains. As a result, fewer and smaller phagolysosomes were present in CP3 infected cells. Combined with the lower attachment and recruitment of actin, this could account for the smaller inclusion size observed at 20 h and 28 h p.i. (Figure 3) and lower infectious progeny at 48 h p.i. (see section 3.4). Contrary to an infection in epithelial cells [START_REF] Vanrompay | Ultrastructural changes in avian Chlamydia psittaci serovar A-, B-, and D-infected Buffalo Green Monkey cells[END_REF], none of the strains completely occupied the host cell cytoplasm at the later time points of the experiment. The limited survival and replication of Cp. psittaci in avian monocytes/macrophages was also reflected by the low gene transcription levels of ftsW and ftsK, encoding proteins required for cell division [START_REF] Gerard | Expression of Chlamydia trachomatis genes encoding products required for DNA synthesis and cell division during active versus persistent infection[END_REF]. Possible explanations for the limited proliferation of Cp. psittaci in monocytes/macrophages compared to an infection in epithelial cells could thus include the fact that not all bacteria in the inoculum effectively attach to and enter the host cells (especially for the low virulent strain CP3) and a less efficient escape of phagolysosomal fusion. On the other hand, the chlamydial organisms can only benefit from this limited proliferation, as chlamydial replication would otherwise further activate these essentially antigen presenting cells (APCs). Indeed, higher replication within the inclusion would involve more secretion of chlamydial proteins, which, just as proteins from bacteria replicating in the cytoplasm, could be processed by the proteasome into peptide fragments. These peptides would then be presented in combination with MHC I at the cell surface to CD8 + T-cells, initiating their We have demonstrated that only a limited proliferation, or barely survival, of Cp. psittaci is possible within avian monocytes/macrophages. One could therefore wonder why chlamydiae infect these cells in the first place. It could of course be that the monocytes/macrophages simply phagocytose the chlamydial bodies. However, the induction of actin recruitment to the entry site actively mediating internalization would, as least partially, contradict this. The answer could come from former studies in Cp. pneumoniae suggesting that blood monocytes/macrophages act as vectors that systemically disseminate Cp. pneumoniae by hematogenous and lymphatic routes [START_REF] Moazed | Evidence of systemic dissemination of Chlamydia pneumoniae via macrophages in the mouse[END_REF][START_REF] Gieffers | Phagocytes transmit Chlamydia pneumoniae from the lungs to the vasculature[END_REF] and Cp. psittaci, revealing presence of the bacteria in monocytes/macrophages recognized by the specific marker KUL04 [START_REF] Vanrompay | Chlamydia psittaci in turkeys: pathogenesis of infections in avian serovars A, B and D[END_REF]. Moreover, heterophils have very recently been shown to function as a transport vehicle for Cp. psittaci in infected birds [START_REF] Johns | What is your diagnosis? Blood smear from an injured red-tailed hawk[END_REF].

The question which then remains is: how do these infected macrophages halt to cross the blood/tissue barrier? Referring to research performed on Cp. pneumoniae, May et al. report that Cp. pneumoniae induces rolling and adhesion of infected macrophages to non-inflamed vessel walls. More specific, Cp. pneumoniae infected monocytic cells attach to the endothelium through upregulation or activation of VLA-4 (very late antigen-4), LFA-1 (lymphocyte function-associated antigen-1) and Mac-1 (macrophage antigen-1) [START_REF] May | Recruitment of Chlamydia pneumoniae-infected macrophages to the carotid artery wall in noninfected, nonatherosclerotic mice[END_REF]. At least one of these integrins, LFA-1, can bind ICAM-2 (Intercellular adhesion molecule 2) which is expressed on resting A c c e p t e d M a n u s c r i p t 18 endothelium [START_REF] De Fougerolles | Characterization of ICAM-2 and evidence for a third counter-receptor for LFA-1[END_REF]. In a similar way Cp. psittaci infected macrophages could adhere to endothelial cells at locations where macrophages can easily escape from the blood vessel (extravasation), e.g. spleen and high endothelial venules, to invade these tissues. This hypothesis is strengthened by the fact that in diseased birds, Cp. psittaci is often found in macrophages and in the spleen [START_REF] Vanrompay | Chlamydia psittaci in turkeys: pathogenesis of infections in avian serovars A, B and D[END_REF].

Remarkably, none of the four strains in this study transcribed the groEL-1 gene upon infection of HD11 cells. Primer incompatibility can be ruled out because the primers were tested on genomic DNA, generating an amplicon of the expected size. Although transcription of the late-cycle omcB gene was generally spoken delayed compared to the mid-cycle ompA gene transcription for all strains (Table 3), transcripts could be detected from 1 h p.i. on, indicating that, contrary to other chlamydial species, Cp. psittaci omcB gene transcription in monocytes/macrophages is not restricted to the later stages of the infectious cycle.

To address the contribution of the T3SS during the chlamydial development in avian monocytes/macrophages, the mRNA levels of a set of T3S related genes were determined from 1 to 48 h p.i. of chicken HD11 cells. Transcription results for strain 92/1293 strongly differed for several genes with former results obtained in BGM cells [START_REF] Beeckman | Identification and characterization of a type III secretion system in Chlamydophila psittaci[END_REF]. For example, for a Cp. psittaci 92/1293 infection of HD11 cells, sctT was now shown to be transcribed. Overall, genes encoding structural components of the T3SS seem to be transcribed earlier for a Cp. psittaci infection in monocytes/macrophages compared to a similar infection in epithelial cells as described in [START_REF] Beeckman | Identification and characterization of a type III secretion system in Chlamydophila psittaci[END_REF]. Taking into account the limited proliferation of Cp. psittaci within monocytes/macrophages, we propose that the bacteria use their A c c e p t e d M a n u s c r i p t 19 T3SS to enter the cells, allow for a limited proliferation and directly arm themselves again to infect new (epithelial) host cells elsewhere in the body, after attachment of the infected mononuclear cell to the blood vessel wall (see above). The observation that the inclusion membrane often disappears could be important as contact with the inclusion membrane through the T3SS is a prerequisite for RB replication (Wilson et al., 2006).

When turning to the transcription of T3S effector encoding genes, we found that the Cp. psittaci CP3 cap1 gene was transcribed remarkably late (12 h p.i.) compared to the transcription pattern for 92/1293 in BGM-cells [1 h p.i. [START_REF] Beeckman | Identification and characterization of a type III secretion system in Chlamydophila psittaci[END_REF]].

The same was observed for 92/1293 (8 h) and 84/2334 (12 h) and no transcription of cap1 could be observed in 84/55. For an infection of Chlamydia (C.) trachomatis in McCoy or HeLa cells, early transcription of this gene was observed [START_REF] Belland | Transcriptome analysis of chlamydial growth during IFN-gamma-mediated persistence and reactivation[END_REF][START_REF] Balsara | Developmental regulation of Chlamydia trachomatis class I accessible protein-1, a CD8+ T cell antigen[END_REF], as also for Cp. psittaci 92/1293 in BGM cells [1 h [START_REF] Beeckman | Identification and characterization of a type III secretion system in Chlamydophila psittaci[END_REF]]. The corresponding Cap1 protein is known to stimulate protective CD8 + T-cells (peptides are presented in association with MHC class I) to transform to CTLs, thus reinforcing the cellular immune response. The fact that the expression of Cap1 by Cp. psittaci in HD11 cells is delayed may therefore indicate that Cp. psittaci is trying to avoid recognition of the infected macrophages by CTLs in order to use the macrophage as a transport vehicle throughout the body for as long as required. Transcription of the non-clustered tarp and cadd genes was noticed much earlier (1-4 h and 1-2 h p.i., repectively) when compared to an infection in BGM cells [12 h and 10 h p.i., repectively [START_REF] Beeckman | Identification and characterization of a type III secretion system in Chlamydophila psittaci[END_REF]]. The gene encoding the serine-threonine kinase Pkn5 is not transcribed for an infection of HD11 cells with Cp. psittaci 84/55, CP3 or 92/1293, although the immediately adjacent sctC gene is.

Cp. psittaci 84/2334 is the only strain transcribing pkn5. In addition, sctC and sctQ A c c e p t e d M a n u s c r i p t 20 have the same upregulation pattern, just as is the case for pkn5 and ca037 (Table 5).

These results are in agreement with [START_REF] Hefty | Chlamydial type III secretion system is encoded on ten operons preceded by sigma 70-like promoter elements[END_REF] stating that sctC and pkn5 do not belong to the same operon. Taking into account the transcription profile of sctD, this would seem to suggest that in the 84/2334 genome sequence the gene order has switched from ca037-sctQ-pkn5-sctC to ca037-pkn5-sctQ-sctC. Genome sequencing could provide a definitive answer.

No transcription could be observed for the translocon components CopB1 and CopD1, neither for their chaperone Scc2 after an 84/2334 infection. Primer incompatibility could be the cause, but it could also be that gene cluster 2.2 is not present in the Cp. psittaci 84/2334 genome. However, as these genes are present in all other Chlamydiaceae, this option is less likely. Again, genome sequencing could shed light on this issue.

CONCLUSION

The present study revealed a limited proliferation of high and low virulent avian Cp. psittaci strains in the matched avian monocyte/macrophage cell line HD11. In concordance with its observed lower virulence for turkeys, Cp. psittaci CP3 attached to and invaded the host cells less efficiently, the infection was less productive and induced less cell degeneration compared to the other strains. Infection with the abortus-like 84/2334 strain was characterized by the absence of actin recruitment at the site of entry and a remarkable T3S gene transcription profile. Further experiments, including genome sequencing, will provide more information on the specific characteristics of this strain. We also proposed that Cp. psittaci uses blood monocytes/macrophages as a vehicle to spread to other organs and provided possible mechanisms deployed by the bacteria to reach this goal. Table 3 Gene transcription patterns of control genes for different Cp. psittaci strains in HD11 cells. Total RNA from 1.5 x 10 6 HD11 cells inoculated with Cp. psittaci 92/1293 at an MOI of 13 was isolated at 1, 2, 4, 8, 12, 18, 24, 36 and 48 h p.i. The experiments were performed twice and all samples were tested in duplicate. For each gene the time point at which a specific PCR product was first detected is provided (time point at which the absolute copy number without 16S rRNA normalization was higher than 100). The time point where maximal significant upregulation of the genes was observed is given in the third column, as determined by an ANOVA with post-hoc analysis along the time axis (both Tukey HSD and Tukey-b test, P < 0.01). The fourth column describes further significant upregulation time points after 16S rRNA normalization, in order of their importance.

Table 4 T3S gene transcription patterns for Cp. psittaci 84/55 and CP3 in HD11 cells. In order to describe the gene transcription profile for each of the genes under study, the time point where maximal significant upregulation of the genes was observed is given in a second column per strain. The third column describes other significant upregulation time points after 16S rRNA normalization, in order of their importance. cells. In order to describe the gene transcription profile for each of the genes under study, the time point where maximal significant upregulation of the genes was observed is given in a second column per strain. The third column describes other significant upregulation time points after 16S rRNA normalization, in order of their importance. A c c e p t e d M a n u s c r i p t

  . Formvar-coated single slot copper grids were used.

  diluted AlexaFluor 546 goat-anti-mouse secondary antibody (Molecular Probes), again for 1 h at 37 °C. Subsequent rinsing was followed by detection of the chlamydial organisms by a direct immunofluorescence staining (IMAGEN Chlamydia, Oxoid).

  A c c e p t e d M a n u s c r i p t 11 Similar effects could be observed for the genotype D strain (92/1293), and more scattered green fluorescent staining in the host cells was observed. For 84/2334, more bacteria could be visualized compared to an infection with CP3. Host cells had degenerated less when compared to an inoculation with 92/1293 or 84/55, while for CP3 no degeneration of the HD11 cells could be observed. No clear association between CP3 or 84/2334 and mitochondria could be noticed at this time point (Table

  points: CP3 proliferation was limited, giving rise to multiple miniinclusions per cell. As observed at earlier time points, multiple 84/2334 miniinclusions could be observed per cell, as well as a few larger inclusions (data not shown).

  its interaction with the host cell in the chicken monocyte/macrophage cell line HD11. Studies investigating the interaction between

  Cp. psittaci 84/2334 and the host cell membrane at 8 h p.i. (Figure1H) could represent such a mechanism and requires further investigation.

  s c r i p t differentiation into cytotoxic T-lymphocytes (CTLs). In addition, activated macrophages would more frequently/efficiently phagocytose extracellular organisms or engulf other infected cells, followed by a presentation of chlamydial antigens in association with MHC II to CD4 + T-cells. The subsequent IFN-γ production by CD4 + Th1 cells would then result in further activation of CTLs.

Figure 1

 1 FIGURE LEGENDS

Figure 2

 2 Figure 2 Actin skeleton distribution at 15 min post inoculation with different Cp.

Figure 3

 3 Figure 3 Distribution of mitochondria in HD11 cells after inoculation with different

  Figure 1

  Figure 3

Table 2 )

 2 . It could be that Cp. psittaci 84/2334 induces actin recruitment later on, and/or that other, not yet identified, entry mechanisms are deployed. The observed

TABLE LEGENDS Table 1

 LEGENDS1 Oligonucleotide primers used for RT-qPCR analysis. Legend: Ta, annealing temperature.

Table 2

 2 Overview of specific characteristics observed at different time points p.i. with the four Cp. psittaci strains under study.

Table 5

 5 T3S gene transcription patterns for Cp. psittaci 92/1293 and 84/2334 in HD11

Table 1

 1

	Gene		Primer sequence	Ta	Size (bp)
	ftsW	F a 5'-ATGGAGCAAAGCGATGGTTAG -3'	60 °C	300
		R b 5'-CGCCCACAACAAGAATACATAA -3'
	ftsK	F	5'-ACCACCAACTCCCATTGTCTC -3'	60 °C	323
		R	5'-CACGCAAGGATTCAGGTTTAG -3'
	dnaK	F	5'-GCATTCAAAGGAAACGAAACC -3'	55 °C	308
		R	5'-AAGTAGGCAGGAACCGTAATCA -3'
	groEL-1 F	5'-CGAAGACAAACACGAGAACAT -3'	55 °C	305
		R	5'-CGGCGATGAGATTACCAA -3'
	ompA	F	5'-TATGGGAATGTGGTTGTGC -3'	60 °C	290
		R	5'-TGCTCTTGACCAGTTTACGC -3'
	omcB	F	5'-CTGCGACAACACCAACCTC -3'	60 °C	287
		R	5'-GACGACAACATTACGGGCTAT -3'

a Forward primers (F). b Reverse primers (R).

Table 1

 1

Table 3

 3

	Cluster	Gene	First detection	Major upregulation timepoint	Secondary timepoints of upregulation
	84/55	ftsW ftsK	2h / a	4h c 8h c	2-12h c
		dnaK groEL-1 b	4h	4h	8h
		ompA	1h	8h	12h
		omcB	1h	8-12h	36h
	CP3	ftsW ftsK	1h 4h	12h 12h c	2-8h c 4h c
		dnaK	4h	8h	4-18h
		groEL-1
		ompA	8h	12h	8h
		omcB	1h	8-12h	36h
	92/1293	ftsW ftsK	/ /	8-12h c 24h c	4-18h c
		dnaK	8h	8h	12h & 48h
		groEL-1
		ompA	8h	8h	12-18h
		omcB	1h	48h	8h & 18-24h
	84/2334	ftsW ftsK	/ /	/ 4h c	/
		dnaK	/	/	/
		groEL-1
		ompA	8h	8h
		omcB	1h	8h	24-48h
	a No signal observed b An amplicon could be obtained when using genomic DNA but not cDNA c Faint signal

Table 3

 3

ACKNOWLEDGEMENTS

Delphine S.A. Beeckman is a Post-doctoral fellow of the Research Foundation -Flanders (FWO -Vlaanderen). This study was sponsored by a GOA research project from Ghent University (Grant 01G00805). Simon Brabant and Myriam Claeys are acknowledged for technical assistance. The authors thank Nancy Korn (Clemson University, Dept. of Animal and Veterinary Sciences, South Carolina) for her kind gift of the antibody against turkey sperm mitochondria.

A c c e p t e d M a n u s c r i p t