Lieven Van Waeyenberghe
email: lieven.vanwaeyenberghe@ugent.be

Frank Pasmans

Angèle Lies

Freddy Beernaert

Francis Haesebrouck

Frank Vercammen

Gerry M Verstappen

Dorrestein

H W Corné

An Klaassen

Martel

Lies A Beernaert

Freddy Haesebrouck

Francis Vercammen

Frank Verstappen

Gerry M Dorrestein

Klaassen

An Martel

Microsatellite Typing of Avian Clinical and Environmental Isolates of Aspergillus fumigatus

Keywords: aspergillosis, Microsatellite typing, avian, human Genotyping Aspergillus fumigatus isolates

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Currently it is not known if there is a link between the environmental isolates and/or human isolates of A. fumigatus and those responsible for aspergillosis in birds. Microsatellite typing was used to analyze 65 clinical avian isolates and 23 environmental isolates of Aspergillus fumigatus. The 78 genotypes that were obtained were compared to a database containing genotypes of 2514 isolates from human clinical samples and from the environment. There appeared to be no specific association between the observed genotypes and the origin of the isolates (environment, human or bird). Eight genotypes obtained from isolates of diseased birds were also found in human clinical samples. These results indicate that avian isolates of Aspergillus fumigatus may cause infection in humans.

Introduction

Fungal infections due to Aspergillus species are a major cause of morbidity and mortality among certain species of birds, captive as well as free-ranging, independent of age or status of the immune system [START_REF] Tell | Aspergillosis in mammals and birds: impact on veterinary medicine[END_REF][START_REF] Beernaert | Modelling Aspergillus fumigatus infections in racing pigeons (Columba livia domestica)[END_REF]. Aspergillus fumigatus, a ubiquitous and saprophytic fungus, is the major etiological agent responsible for aspergillosis [START_REF] Tell | Aspergillosis in mammals and birds: impact on veterinary medicine[END_REF].

To investigate the genetic and the epidemiological relationship between environmental and clinical isolates, fingerprinting methods with high discriminatory power must be applied.

Also interlaboratory reproducibility and objective interpretation of the fingerprinting data are highly recommendable (de Valk et al., 2007). All of these characteristics can be found in typing methods based on short tandem repeats (STRs), such as microsatellite length polymorphism (MLP) and STRAf typing [START_REF] Bart-Delabesse | Microsatellite markers for typing Aspergillus fumigatus isolates[END_REF][START_REF] De Valk | Use of a novel panel of nine short tandem repeats for exact and highresolution fingerprinting of Aspergillus fumigatus isolates[END_REF]de Valk et al., 2007;Vanhee et al., 2008a). While the discriminatory power is high in patternbased techniques, such as random amplified polymorphic DNA (RAPD) analysis, restriction fragment length polymorphism (RFLP) analysis, and amplified fragment length polymorphism (AFLP), STRAf typing proved to be more simple and reproducible [START_REF] Bart-Delabesse | Microsatellite markers for typing Aspergillus fumigatus isolates[END_REF][START_REF] De Valk | Use of a novel panel of nine short tandem repeats for exact and highresolution fingerprinting of Aspergillus fumigatus isolates[END_REF]de Valk et al., 2007a;Vanhee et al., 2008a,b). However, any typing method is associated with its specific advantages and disadvantages (de Valk et al., 2007 b) and any technical complications associated with microsatellite typing [START_REF] Pasqualotto | A cautionary tale: lack of consistency in allele sizes between two laboratories for a published multilocus microsatellite typing system[END_REF][START_REF] Alvarez-Perez | Characterization of multiple isolates of Aspergillus fumigatus from patients: genotype, mating type and invasiveness[END_REF] can be properly addressed (de Valk et al., 2009;[START_REF] Klaassen | Molecular typing of Aspergillus fumigatus isolates[END_REF].

Previous molecular typing studies showed that there was a high variability among avian isolates and multiple genotypes were recovered from healthy and diseased birds [START_REF] Lair-Fulleringer | Differentiation between isolates of Aspergillus fumigatus from breeding turkeys and their environment by genotyping with microsatellite markers[END_REF][START_REF] Olias | Microsatellite genotyping and virulence assessment of Aspergillus fumigatus isolates from white stork nestlings and their environment[END_REF][START_REF] Alvarez-Perez | Polyclonal Aspergillus fumigatus infection in captive penguins[END_REF]. However, currently it is not known if there is a link between environmental isolates and/or human isolates of A. fumigatus and those responsible for aspergillosis in birds.

Material and Methods

Isolates. Sixty-five clinical avian and 23 environmental isolates of A. fumigatus were subjected to STRAf typing. The clinical isolates were collected at 6 different institutes, 4 located in Belgium and 2 in The Netherlands and were obtained from birds, belonging to 13 orders, 18 families and 35 species (Table 1), that died from an A. fumigatus infection. To collect the environmental isolates, Sabouraud dextrose agar plates were placed at 20 different locations in the vicinity of Ghent, Belgium. After incubation for three days at room temperature, the plates were placed in an incubator at 37°C. Fast-growing greenish colonies were purified on Sabouraud dextrose agar.

The isolates were identified based on the macro-and micro-morphology of the fungus.

Determination of partial DNA sequences of the beta-tubulin and rodletA genes [START_REF] Alcazar-Fuoli | Aspergillus section fumigati antifungal susceptibility patterns and sequence based identification[END_REF] and the ability to grow at 48°C were used to confirm species identity.

STRAf assay. Fungal DNA was prepared from all isolates as described by [START_REF] Beernaert | Modelling Aspergillus fumigatus infections in racing pigeons (Columba livia domestica)[END_REF]. PCR primers for the STRAf2, -3 and -4 panels and amplification conditions were as

Results

A total of 88 A. fumigatus isolates were analyzed using STRAf typing and a total of 78 genotypes were obtained. Seventy genotypes were found once, 6 genotypes were found twice and 2 genotypes were found three times. From the genotypes observed more than once, no relation could be observed between the species of bird in the clinical isolates and the area of isolation in environmental isolates. There was also no relation between the geographic area of for the clinical avian isolates was calculated to be 0.995.

In the dendrogram (Figure 1), 8 clusters can be identified of related genotypes differing only at a single locus. In one case , an identical genotype was found in a clinical avian isolate and an environmental isolate. Two clusters of genotypes contained clinical and environmental isolates, four clusters only clinical isolates and two clusters only environmental isolates.

Comparing the results of our analysis to a database containing 2514 genotypes from human and environmental isolates revealed that 58 genotypes in our collection of avian and environmental isolates were not observed before. Twenty genotypes were observed before either in human clinical samples or in environmental samples. Twelve genotypes from avian clinical isolates were identical to genotypes found before in human (n=3) or environmental (n=4) isolates, or both (n=5) from the Netherlands, Switzerland, Germany and Spain. Eight environmental genotypes from Belgium were identical to genotypes found in human and/or environmental isolates from the Netherlands, Spain, Germany, the United States of America and Norway.

Discussion

Previous epidemiological studies have investigated the origin of avian aspergillosis in limited geographical areas and in one species of birds [START_REF] Lair-Fulleringer | Differentiation between isolates of Aspergillus fumigatus from breeding turkeys and their environment by genotyping with microsatellite markers[END_REF][START_REF] Olias | Microsatellite genotyping and virulence assessment of Aspergillus fumigatus isolates from white stork nestlings and their environment[END_REF][START_REF] Alvarez-Perez | Polyclonal Aspergillus fumigatus infection in captive penguins[END_REF]. This is the first study determining the genetic diversity among A. genotypes in 114 isolates from 30 healthy and 2 diseased turkeys. The low number of distinct genotypes in relation to the number of isolates in those studies could be explained firstly by the fact that a limited number of animals with aspergillosis were used. Secondly, the animals belonged to closed collections and multiple isolates from each animal were examined. Finally, the genotyping was performed with a method described by [START_REF] Bart-Delabesse | Microsatellite markers for typing Aspergillus fumigatus isolates[END_REF] including only four microsatellite markers being less discriminatory than the panel of 9 microsatellite markers described here. Alternatively, these animals may have been exposed to a common source of material(s) that may have been contaminated with a limited genotypic diversity of A. fumigatus spores such as bird feed.

The environmental and avian isolates are widespread throughout the dendrogram, suggesting that any environmental isolate of A. fumigatus is possibly infectious to birds. This finding is supported by the study of [START_REF] Peden | Pathogenicity differences of multiple isolates of Aspergillus fumigatus in turkeys[END_REF], who inoculated isolates from diverse origins (environmental, mammalian, and avian) in air sacs of turkeys. All isolates were able to induce aspergillosis in these birds.

High reproducibility of the STRAf assay and the ease of interlaboratory exchange of the results allowed comparing the dataset of 88 clinical avian and environmental isolates to a dataset of 2514 genotypes from human and environmental isolates [START_REF] De Valk | Interlaboratory reproducibility of a microsatellite-based typing assay for Aspergillus fumigatus through the use of allelic ladders: proof of concept[END_REF].

There appeared to be no specific association between the observed genotypes and the origin of the isolates (environment, human or bird). This was expected because of the high degree of genetic diversity among A. fumigatus isolates, independent of the species or geographical region [START_REF] Debeaupuis | Genetic diversity among clinical and environmental isolates of Aspergillus fumigatus[END_REF][START_REF] Chazalet | Molecular typing of environmental and patient isolates of Aspergillus fumigatus from various hospital settings[END_REF][START_REF] Rosehart | Microsatellite analysis of environmental and clinical isolates of the opportunist fungal pathogen Aspergillus fumigatus[END_REF][START_REF] Menotti | Epidemiological study of invasive pulmonary aspergillosis in a haematology unit by molecular typing of environmental and patient isolates of Aspergillus fumigatus[END_REF][START_REF] Klaassen | Utility of CSP typing to sub-type clinical Aspergillus fumigatus isolates and proposal for a new CSP type nomenclature[END_REF]. Moreover, 8 genotypes derived from diseased birds were also isolated from human clinical samples indicating that avian isolates of A. fumigatus could be considered infectious to humans.

  of the most common causes of death in captive birds. Aspergillosis in birds is mainly caused by Aspergillus fumigatus, a ubiquitous and opportunistic saprophyte.

  this study STRAf typing was performed on environmental and avian clinical isolates of A. fumigatus and the results were compared with a database containing genotypes from A. fumigatus isolated from clinical human samples and from the environment.

  described by[START_REF] De Valk | Use of a novel panel of nine short tandem repeats for exact and highresolution fingerprinting of Aspergillus fumigatus isolates[END_REF], except that FAM labelling was replaced by VIC labelling, HEX labelling by NED labelling and TET labelling by PET labelling. Allelic ladders were used with the same fluorescent labels as above for each of the three trinucleotide markers in the STRAf3 panel as described by[START_REF] De Valk | Interlaboratory reproducibility of a microsatellite-based typing assay for Aspergillus fumigatus through the use of allelic ladders: proof of concept[END_REF]. The obtained PCR products were diluted 10-fold with distilled water. Two microliters of the diluted PCR products were added of formamide (Amresco Inc, Ohio, USA) and 1 µl of GS 500 LIZ size standard (Applied Biosystems, Halle, Belgium). Following denaturation of the samples for 2 min at 95°C and rapid cooling to 0°C for 30 min, they were injected onto an ABI Prism 3100 Genetic Analyzer (Applied Biosystems, Halle, Belgium) equipped with a 16 capillary array. Genemapper v3.5 (Applied Biosystems, Halle, Belgium) was used to determine the size of each amplified fragment. All results are reported as repeat numbers. Repeat numbers for the markers in the STRAf3 panel were determined by comparison to the allelic ladders. The repeat numbers of the markers in the STRAf2 and -4 panels were determined with the reference size values taken from the original publication and validated using a set of reference isolates with known genotypes[START_REF] De Valk | Use of a novel panel of nine short tandem repeats for exact and highresolution fingerprinting of Aspergillus fumigatus isolates[END_REF]. Data analysis. Typing data was imported into BioNumerics version 6 software (Applied Maths, St-Martens-Latem, Belgium) and analyzed using the categorical multistate similarity coefficient with UPGMA clustering. The obtained genotypes were then compared to a database containing 2514 genotypes from human and environmental A. fumigatus isolates from Europe and the USA.

  K7 and the environmental isolate O7. The Simpsons index of diversity(D)

  fumigatus isolates obtained from 35 avian species collected at 6 different institutes and environmental isolates collected at 20 different locations in Belgium.The results of this study demonstrate that the genetic diversity among avian clinical isolates is extremely high. The 65 isolates, collected from 65 birds affected with aspergillosis, distinct genotypes. In contrast,[START_REF] Alvarez-Perez | Polyclonal Aspergillus fumigatus infection in captive penguins[END_REF] reported 13 distinct genotypes in 33 isolates from 5 diseased birds andLair-Fullinger et al. (2003) 23 distinct

Figure 1 .

 1 Figure legends

Figure 1 :

 1 Figure 1: Dendrogram generated from genotyping 65 clinical and 23 environmental A. fumigatus isolates. Isolates recovered from clinical samples are denoted as K and strains isolated from the environment are denoted as O. Framed are the genotypes that show microvariation. The scale bars above the dendrogram indicates the percentage identity between the genotypes. 177x279mm (600 x 600 DPI)

Table 1 .

 1 Order, Family and Species of domestic and wild birds from which A. fumigatus isolates were obtained.

	Order	Family	Species	N° of	Designation of
				isolates	isolates
	Accipitriformes	Accipitridae	Geranoaetes	2	K30; K57
			melanoleucus

E-mail: cavanagh@metronet.co.uk URL: http://mc.manuscriptcentral.com/cavp Avian Pathology

Acknowledgements

This work was supported by the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT Vlaanderen), Brussels, Belgium.


Mahesh ; Kumar

H J Geerligs
email: harm.geerligs@pfizer.com

G.-J Boelm

C A M Meinders

B G M Stuurman

J Symons

J Tarres-Call

T Bru

R Vila

M Mombarg

K Karaca

W Wijmenga

EFFICACY AND SAFETY OF ATTENUATED LIVE INFECTIOUS BRONCHITIS VIRUS STRAIN QX-LIKE IN CHICKENS

Keywords: QX-like IB, attenuated, safety, efficacy

The attenuation of infectious bronchitis (IB) QX-like virus strain L1148 is described. The virus was passaged multiple times in embryonated specific pathogen free (SPF) chickens eggs and at different passage levels samples were tested for safety for the respiratory tract and kidneys in one-day-old SPF chickens. There was a clear decrease in pathogenicity for the respiratory tract and kidneys when the virus had undergone a large number of passages.

Passage level 80 was investigated for safety for the reproductive tract in one-day-old and 7 day-old SPF chickens. In one-day-old chickens 12.5% of the vaccinated animals had macroscopical lesions. No lesions were observed if the chickens had been vaccinated at 7 days of age. Passage level 80 was investigated for its ability to spread from vaccinated to nonvaccinated chickens and for dissemination in the tissues. The virus was able to spread from vaccinated chickens to groups of non-vaccinated chickens, and in the vaccinated animals the virus was found frequently in oro-pharyngeal and cloacal swabs. A fragment of the hypervariable region of the S1 protein of passage level 80 was sequenced and there were nucleotide changes resulting in 2 amino acid substitutions. Passage level 80 was given additional passages to levels 82 and 85. Both passage levels were tested for efficacy in SPF chickens and passage level 85 was tested in commercial chickens with MDA against a challenge with QX-like strain IB D388. In both SPF chickens and chickens with MDA the vaccines based on strain IB L1148 were efficacious against challenge.

IB disease signs include respiratory distress, reduced weight, reduced egg production, increased frequency of abnormal eggs and increased rates of mortality [START_REF] Cavanagh | Infectious bronchitis[END_REF]. Several different serotypes and genotypes of IB viruses have been identified and new variants are still emerging. One of these new variants is QX-like IB virus. [START_REF] Wang | Isolation and identification of glandular stomach type IBV (QX IBV) in chickens[END_REF] were the first to isolate IBV QX-like virus from a flock of chickens suffering with proventriculitis in China. Later on the virus was reported more frequently in relation to cases of nephritis and false layer syndrome [START_REF] Benyeda | Comparison of the pathogenicity of QX-like, M41 and 793/B infectious bronchitis strains from different pathological conditions[END_REF]. QX-like IB viruses were detected in East Russia in 2001 and in 2002 in West Russia [START_REF] Bochkov | Molecular epizootiology of avian infectious bronchitis in Russia[END_REF]. From 2003 onwards IB QX-like viruses were detected in many countries in Europe: Germany, Holland, Belgium, France, Italy [START_REF] Worthington | A reverse transcriptase-polymerase chain reaction survey of infectious bronchitis virus genotypes in Western Europe from 2002 to 2006[END_REF][START_REF] Monne | QX genotypes of infectious bronchitis virus circulating in Europe[END_REF], Poland [START_REF] Domanska-Blicharz | New variant of IBV in Poland[END_REF], the UK [START_REF] Gough | Chinese QX strain of infectious bronchitis virus was isolated in the UK. The Veterinary Record, 162, 99-100. with Mycoplasma synoviae and chicken-passaged infectious bronchitis vaccine virus[END_REF] and Spain [START_REF] Dolz | First report of IBV QX-like strains in Spain[END_REF].

Various sources report that IB QX-like viruses are currently present in all European countries.

Clearly, IB QX-like viruses pose a serious threat to the poultry industry. No vaccines specific for IB QX-like viruses are available yet. In this paper the development of a live attenuated IB QX-like vaccine virus based on strain IB L1148 is described and the results of studies on safety and efficacy are discussed. Most of the tests described here were performed in accordance with the requirements of the European Pharmacopoeia (EP) monograph 01/2008:442, to which reference should be made for more detail of specific requirements.

Materials and methods

Viruses. IB QX-like virus strain L1148, which was used for vaccine development, was isolated from broilers with respiratory disease. Allantoic fluid from the 7 th passage of strain IB L1148 in eggs was kindly provided by Mrs. K.J. Worthington Department of Veterinary Pathology, University of Liverpool, UK [START_REF] Worthington | A reverse transcriptase-polymerase chain reaction survey of infectious bronchitis virus genotypes in Western Europe from 2002 to 2006[END_REF].

IB QX-like challenge strain D388 was kindly provided by the Dr. J.J. de Wit of the Animal Health Service, Deventer, The Netherlands (NL) [START_REF] Landman | High incidence of false layers in (re)production hens supposedly attributed to a juvenile infectious bronchitis virus infection[END_REF]. Specific pathogen free (SPF) chickens and chicken eggs. SPF eggs were obtained from Lohmann Tierzucht GmbH, Cuxhaven, Germany, or from SPAFAS, North Franklin, Connecticut, USA. For animal experiments performed at Animal Health Service, Deventer, NL, SPF chickens were used from the in house SPF layer flock. For production of virus and for titrations, eggs were incubated at 37°C in a relative humidity of 55%. For hatching, eggs were incubated under similar conditions but with turning.

Passaging of virus in SPF eggs. Virus passages were made, from passage 8 up to passage 101, by inoculation of 9 to 11 day embryonated SPF chicken eggs into the allantoic cavity, each with a quantity of 0.1 ml of the virus. After 1 days incubation, dead eggs were discarded and after 2 to 3 days incubation, the allantoic fluid was harvested from the surviving eggs and pooled. The pooled allantoic fluid was centrifuged at 1000g to remove debris. The supernatant was filled out in small portions. The portions were partly used for further passages and partly frozen and stored at -70°C for further use. For subsequent passages the virus was diluted 1:1000 in phosphate buffered saline. If considered necessary samples of allantoic fluid were mixed with a stabilizer in a 3:1 allantoic fluid: stabilizer ratio. The stabilizer was an autoclaved solution containing 65g peptone, 68g gelatin, 50g d-mannitol and 50g inositol per litre in distilled water. The mixture was filled in 3ml glass vials, 1 to 1.5ml per vial, and the vials were lyophilized according to standard manufacturing procedures.

Lyophilized samples were stored at -20°C.

Virus titration. Dilutions of the sample to be tested were inoculated into the allantoic cavity of 10-day-old embryonated SPF chicken eggs, 6 eggs per dilution. After 1 days incubation dead embryos were considered nonspecific deaths and discarded. After an incubation period Dead embryos were considered positive for IBV. Live embryos were examined for signs of IBV infection, for example dwarfing, curling and stunting. The titre, expressed as the median embryo infectious dose (EID 50 )/ml, was calculated according to the method of Spearmann-Karber [START_REF] Finney | The Spearman-Kärber method[END_REF].

Identity testing by PCR. In order to distinguish or verify the IB strain, RNA was isolated using the High Pure Viral RNA isolation kit of Roche, according to the manufacturer's instructions.

The RNA was used in an RT-PCR reaction using a one step RT-PCR kit from Invitrogen, in which the RNA underwent a cDNA synthesis step for 30 min at 50°C, followed by an initial denaturation step for 10 min at 95°C. Forty repeat steps of denaturation for 30 sec at 95°C, annealing for 30 sec at 50°C and extension for 45 sec at 72°C were performed. The final extension step had a duration of 7 min at 72°C and the reaction was stopped by incubation of the PCR mixture for 5 min at 4°C. The IB L1148 specific primer sequences used for the test on the presence of the virus were 5'-GCTTATGCAGTAGTCAAT-3' as forward primer and 5'-CACGTGGAATCATGCCTGTTAT-3' as reverse primer. These primers were designed from the nucleotide sequence of the hypervariable region of the S1 protein of strain IB L1148, NCBI Genbank accession nr. DQ431199 [START_REF] Worthington | A reverse transcriptase-polymerase chain reaction survey of infectious bronchitis virus genotypes in Western Europe from 2002 to 2006[END_REF]. The specificity of the primers had been tested in a PCR test in which RNA from IB strains H120, D274 and M41 was tested. No PCR product was formed from the RNA of these IB strains. After completion of the RT-PCR reaction, the size of the PCR product was determined by agarose-gel electrophoresis. The fragments were made visible with Ethidium Bromide. The position of the fragments was shown as orange bands by illumination with UV light. The size of the fragments was calculated by comparing the position of the bands with base pair markers which had also been loaded on the gel.

Sequence analysis. The nucleotide sequence coding for a part of the S1 protein of the attenuated IB L1148 strain was determined. RNA was isolated using the High Pure Viral RNA isolation kit of Roche, according to the manufacturer's instructions. The RNA was used in an RT-PCR reaction using a one step RT-PCR kit from Invitrogen, in which the RNA underwent a cDNA synthesis step for 30 min at 50°C, followed by an initial denaturation step for 10 min at 95°C. Forty repeating steps of denaturation for 60 sec at 95°C, annealing for 45 sec at 51.9°C, and extension for 120 sec at 72°C were performed. The final extension step had a duration of 10 min at 72°C, and the reaction was stopped by incubation of the PCR mixture for 5 min at 4°C.

The IB L1148 specific primer sequences used for the test on the presence of the virus were 5'-CTTCAGGGTATGGCTTGGTCT-3' as forward primer and 5'-AATTCTTCTGGCACTATCATCTTT-3' as reverse primer. These primers were designed from the nucleotide sequence flanking the hypervariable region of the S1 protein of strain IB L1148, NCBI Genbank accession nr. DQ431199 [START_REF] Worthington | A reverse transcriptase-polymerase chain reaction survey of infectious bronchitis virus genotypes in Western Europe from 2002 to 2006[END_REF]. The PCR product was purified with the Qiaquick PCR purification kit from Qiagen, according to the instructions of the manufacturer. After completion of the RT-PCR reaction, the size of the PCR product was determined by agarose-gel electrophoresis. The fragments were made visible with Ethidium Bromide. The position of the fragments was shown as orange bands by illumination with UV light. The size of the fragments was calculated by comparing the position of the bands with base pair markers which had also been loaded on the gel. The nucleotide sequence was determined by Baseclear B.V., Leiden, NL. The obtained sequence was compared with sequences of other QX-like IB viruses using BLAST analysis.

Safety testing for respiratory tract and kidneys in chickens. The degree of virulence of virus at the various egg passage levels was determined by administering the virus to chicks, just after hatching, by eye drop at a titre of 10 6.0 EID 50 per dose in 0.1 to 0.2 ml. A dose of 10 6.0 EID 50 was used in the safety tests for the respiratory tract and kidneys, because the EP requires testing 10 times the maximum release titre for safety of the respiratory tract, and testing the maximal release titre in the test for safety of the reproductive tract. It was anticipated that a dose of 10 5.0 EID 50 would be the maximal titre for the vaccine. In experiment A, we tested egg passage levels 8, 25, 40 and 50, and in experiment B we tested egg passage levels 65 and 80. In each test a non-infected control group was included. Five chickens were killed humanely 5 days after inoculation, by inhalation of 100% CO 2 , another 5 at 7 days after inoculation and the remaining 5 at 10 days after inoculation. The ciliostasis test was performed in accordance with the EP. Briefly, the trachea was removed, rinsed and stored in physiological saline at 37°C. Transverse sections of 0.6 mm of the trachea were made using a McIlwain Tissue Chopper (Mickle Laboratory Engineering Co. Ltd., Surrey, U K). The ciliary activity of 3, 4 and 3 sections of respectively, the upper, middle and lower part of the trachea was determined by light microscopy at a magnification of 400x. A score of 0 was given if the cilia in the complete tracheal section showed movement; a score of 1 was given if the cilia of 67% to 100% of the tracheal section showed movement; a score of 2 if the cilia 33% to 67% of the trachea showed movement; a score of 3 if the cilia of less than 33% but not 0% of the tracheal section showed movement; and a score of 4 if the cilia in the complete tracheal section showed no movement at all. For each group the average ciliostasis score was calculated. Gross-pathological examination was done on the kidneys of each chicken used for the ciliostasis test. During the examination, some samples of the kidneys were taken for further histological examination and IHC staining. In some instances it was considered necessary to take additional samples for histology and IHC staining. The samples were preserved by fixation in formalin. Kidney lesions were scored as follows: -no lesions, + some infiltration of lymphocytes locally, ++ strong infiltration of lymphocytes through whole kidney, +++ strong infiltration of lymphocytes, some necrosis of epithelia in tubuli, atrophic foci. Histological examination was performed by the Animal Health Service, Deventer, NL.

Immunohistochemical staining. Sections of the tracheas and kidneys were deparaffinised.

Endogenous peroxidase activity was blocked by incubation with 1% H 2 O 2 containing 0.1% NaN 3 for 20 min at room temperature (RT) and subsequently boiled in Tris (0.01 M) -EDTA (0.001 M) pH 9.0 for 10 min. The binding of Fc-receptors was blocked by incubation with 10% foetal bovine serum for 20 min at RT. Immunohistochemical staining was performed using MAb 48.4 (Prionics, Lelystad, NL) directed against the IBV nucleocapsid protein [START_REF] Koch | Rapid detection of infectious bronchitis virus by nucleoprotein specific antigen capture assay[END_REF] for 30 min at RT (dilution 1:20,000) in Normal Antibody Diluent (Klinipath, Duiven, NL). After 3 subsequent wash steps with PBS containing 0.05 % Tween, the sections were treated with anti-mouse Dako EnVision+ (Dako UK Ltd, Cambridgeshire, UK) for 30 min at RT. Again, sections were washed 3 times with PBS Tween and then treated with diaminobenzidine (Dako UK Ltd, Cambridgeshire, UK) for 5 min at RT. This was followed by counterstaining using haematoxylin. Sections incubated in the absence of primary antibody were included as negative controls. Immunohistochemical (IH) staining was performed by the Animal Health Service, Deventer, NL.

Testing safety for the reproductive tract of chickens. The safety of IB strain L1148 (passage 80) for the reproductive tract was assessed by inoculating groups of 50 female SPF chicks at the day of hatching or at 7 days of age, with 10 5.0 EID 50 per dose by eye drop. This dose was used because the EP requires the maximum release titre of the vaccine to be tested for safety of the reproductive tract and it was anticipated that this would be a titre of 10 5.0 EID 50 . During the study, the birds were observed daily for clinical signs. Eleven weeks after vaccination the birds were killed humanely, the coelomic cavity was opened and the complete oviduct examined macroscopically, externally and internally for the presence of cysts, strictures, deformation or aplasia. This test was performed in accordance with the EP monograph.

Dissemination in the body of chickens and spread of the virus strain. On day of hatching, SPF chickens were vaccinated with 10 5.0 EID 50 of IB L1148 passage 80 in a volume of 0.1 ml by eye-drop; this dose being selected to comply with the EP monograph. Three days after vaccination, 30 of the vaccinated chickens were added to a containment unit in which a group of 35 non-vaccinated chickens of the same age and of the same origin was housed. Seven days after vaccination, a group of 30 non-vaccinated one-day-old chickens was added to this containment unit. Fourteen days after vaccination, 35 non-vaccinated chickens of the same age and origin as the vaccinated chickens were added to the containment unit. At regular Samples of bursa, duodenum, lung, kidney, pancreas and trachea were taken in order to determine the presence of IBV by IHC staining as described above. The cloaca and larynx of these chickens were also swabbed after killing. The presence of IB QX-like virus in the swabs was determined by PCR.

Testing the efficacy of the experimental vaccine strain against IB QX-like challenge in SPF chickens. On the day of hatching, SPF chickens, group housed in isolators, were vaccinated with doses of 10 3.0 or 10 3.3 EID 50 of IB L1148 passage levels 82 and 85, and with a dose of 10 3.3 EID 50 of IB L1148 passage level 101. The samples of the passage levels 82 and 85 were lyophilized; the samples of passage level 101 were frozen allantoic fluid. IB strain L1148 at passage levels 82 and 85 was chosen to test its efficacy as a vaccine against challenge with a virulent IB QX-like virus, because we took into account that passage level 80 would most likely be the passage level of the master seed virus. In order to be able to produce a vaccine from the master seed virus, a few passages are required to scale up the virus. Passage 101 was tested to investigate whether higher passage levels would also be efficacious. Vaccine titres per dose of 10 3.0 and 10 3.3 EID 50 were chosen because it was anticipated that a vaccine based on the attenuated IB L1148 strain would have a minimal titre of 10 3.0 or 10 3.3 EID 50 per dose. The freeze-dried vaccines were reconstituted in distilled water. Reconstituted virus and the allantoic fluid of passage 101 were further diluted to the appropriate titre in distilled water. The chickens were vaccinated by coarse spray using a Hozelock flower sprayer. Generation of pressure and flow had been determined before vaccination began. The sprayer had a flow of approx. 2.5 ml per sec. Before vaccination the exact vaccination time for the applicable number of animals was determined (0.5 ml per animal) and the sprayer was disinfected with 70% ethanol, followed by 3 rinses with distilled water. After preparation, the container with the diluted vaccine was placed in a bath with melting ice. Just before vaccination the vaccine was put into the sprayer and the sprayer was put under pressure by moving the pump up and down 40 times. Prior to vaccination, it was tested for flow. The animals, which had been placed in a small box, were vaccinated by spraying the vaccine from a height of 40 to 50 cm for the predetermined period. After spraying, the animals were kept for at least 15 min inside the box. Subsequently, the animals were released into their housing facilities. A non-vaccinated group served as control. At 21 days of age, 20 chickens taken from each group were challenged with 10 4.0 EID 50 of IB D388 per dose of 0.1 ml. The challenge virus was applied by eye-drop. Of the non-vaccinated group, half was challenged; the other half was not challenged. During the whole study period the chickens were observed daily for clinical signs. Five days after challenge the ciliary activity of tracheal explants was examined in 20 chickens per vaccine group and in 10 chickens per control group. Chickens were killed by injecting sodium pentobarbital (T61) intravenously. Immediately after death the trachea was removed, rinsed with, and stored in, physiological saline at 37°C. Small transverse sections of the trachea were cut by hand.

Ciliary activity of 3 sections of the upper part, 4 sections of the middle part and 3 sections of the lower part of the trachea were examined by light microscopy at a magnification of 400x.

The ciliary activity was scored as described in the EP monograph using the following classification criteria: 1 if >50% of the tracheal section showed ciliary activity, 0 if <50% of the tracheal section showed ciliary activity. For a given tracheal section, ciliary activity was considered as normal when at least 50% (score 0) of the internal ring showed vigorous ciliary movement. A chicken was considered protected if not fewer than 9 out of 10 rings showed normal ciliary activity. Significance of differences between groups was evaluated by Chi-square analysis.

Testing the efficacy of the experimental vaccine strain against IB QX-like challenge in chickens with maternally derived antibodies (MDA). We used commercial layer type chickens on the day of hatching. The parent chickens had been vaccinated against amongst other pathogens, IB at one day of age with a live IB Massachusetts type strain, at 5 weeks of age with a live IBMM vaccine, at 9 weeks of age with a live IB 4/91 and at 16 weeks of age with an inactivated combination vaccine containing IB M41 and IB D274. Five chickens were killed and blood samples were collected to test for the presence of IB MDA. This test was performed using the ELISA test kit, Flock check, cat. no. 04-01103-07, IDEXX Laboratories, Westbrook, Maine, USA. The chickens were housed on wood shavings in containment facilities. Each group was housed in a separate unit. The chickens were vaccinated with 10 3.0 EID 50 or 10 3.3 EID 50 per dose of IB strain L1148 passage level 85 as described above. A third group of MDA-positive chickens was not vaccinated. A group of day-old SPF chickens was vaccinated with 10 3.0 EID 50 per dose. The vaccine virus was reconstituted and further diluted to the appropriate titre in distilled water. The chickens were vaccinated by coarse spray. At 21 days of age, 20 chickens were taken from each group and challenged with 10 4.0 EID 50 of IB D388 per dose of 0.1 ml. The challenge virus was applied by eye-drop. During the whole study period the chickens were observed for clinical signs. Five days after challenge the ciliary activity of tracheal explants was examined in the challenged chickens. All challenged chickens were killed by injecting sodium pentobarbital (T61) intravenously. The ciliostasis test was performed as described above for the SPF chickens.

Results

Passaging of the virus. During passaging allantoic fluid was harvested 2 to 3 days after inoculation. The titres of the allantoic fluids ranged between 10 8.0 and 10 8.8 EID 50 per ml. The virus titres did not change during the passages until passage level 85. After lyophilisation passage 80 had a titre of 10 8.2 EID 50 per vial, containing 1 ml of allantoic fluid plus stabiliser mixture. Lyophilised preparations of passage 82 and passage 85 were also prepared. The titres per ml were 10 8.41 EID 50 and 10 8.20 EID 50 , respectively. Surprisingly, passage 101 had a much higher titre, i.e. 10 9.51 EID 50 per ml. All samples, including passage 101, which were tested for identity, reacted positively in the PCR with the IB L1148 specific primers (data not shown), i.e. a clearly visible band was formed in the agarose gel with the expected size. The nucleotide sequence of a 340BP fragment of the region coding for a portion of hypervariable region of the S1 protein QX-like strain IB D388 was also available from the GenBank (DQ674739) (Figure 1). In comparison to the original IB L1148 strain there was one silent substitution from A to T at position 1485 in this 340 BP fragment.

Safety for respiratory tract and kidneys in chickens.

No clinical signs were observed during the safety tests. Passage 8 of strain IB L1148 had a maximum average ciliostasis score at 5 and 7 days after vaccination, and a score of 38 at 10 days, which gives a total average score of 39 (Table1). There was a strong decrease in average score to18 at passage level 25.

Surprisingly, there were increases in ciliostasis score at passage levels 40 and 50. Passage 50 had an average score of 31. At passage levels 65 and 80 there was a decrease in ciliostasis scores to 21 and 11 respectively. The highest average ciliostasis scores were observed at 7 days after vaccination, with the exception of passage level 25, which had the highest value at day 10. In a number of instances there were great differences between individual values in one group, for example with passage 25 at day 5, 3 chickens had a high ciliostasis score, whereas the other 2 had scores of 0. No ciliostasis at all was observed in the uninoculated groups. The results presented in Table 2 show that in study A in which passage levels 8, 25, L1148 affected the kidneys histologically, but there was a decrease in the severity of the lesions in the kidneys with increasing passage level. Passage levels 8 and 25 caused quite severe lesions; scores being ++ until 10 days after vaccination in both groups, and likewise at 5 days after vaccination with passage level 40. At higher passage levels lesion scores were never higher than +. Lesion scores of +++ were not observed in any group. Passage level 65 hardly caused any effect on the kidneys histologically, and passage level 80 had no effect.

The kidneys in the uninoculated group were normal. In the IHC test IB antigen could be detected frequently in the kidneys from chickens inoculated with lower passage 8, i.e. in total in 9 of the 15 samples tested. In the kidneys from the chickens with the higher passage levels, IB virus could be detected less frequently. In both passages 65 and 80, IB was detected in only 1 sample.

Safety for the reproductive tract in chickens.

No clinical signs were observed during the whole 11 week observation period. In the group vaccinated at day of hatching 2 chickens died because of cannibalism, whereas in the group vaccinated at 7 days one chicken died because it was trapped in the feeding system. When passage 80 was administered at day of hatching, there was a damaging effect on the oviducts in 6 of the 48 chickens. These chickens had cystic oviducts and aplasia of the upper segment. The tubular structure of the oviducts was affected because of the presence of cysts in the interior. These cysts were present especially in the middle section of the oviducts. In the birds vaccinated at 7 days of age no effect on the oviducts was observed. In the control group all oviducts were in good condition.

Dissemination in the body of chickens and spread of the virus strain. The results are

given in Table 3. The vaccine virus was not found in the bursa, duodenum, lung or kidney of any of the chickens, therefore these organs are not included in Table 3. In the inoculated chickens the vaccine virus was detected with IH staining in tracheas from 4 to 11 days and in the pancreas on day 4. The virus was found in one cloacal swab on day 14 and in oropharyngeal swabs from days 2 to 11. In the 3 groups of contact animals, the virus could be detected frequently in oro-pharyngeal swabs, and was found also in cloacal swabs, trachea and once in the pancreas. There were no great differences in susceptibility of the contact chickens for the virus, in the one-day-old chickens introduced 7 days after vaccination, virus was detected in the swabs and in the trachea, but not in the pancreas. Chickens introduced 2 weeks after vaccination became infected, because some swabs from these chickens were also positive.

Efficacy of the experimental vaccine strain against IB QX-like challenge in SPF

chickens. No clinical signs were observed after vaccination. The results of examining tracheas for damage after challenge using the ciliostasis test are given in Table 4. The results show that maximum damage (highest possible ciliostasis score) was seen in the nonvaccinated chickens, whereas the tracheas of the non-vaccinated, non-challenged chickens appeared normal. After challenge the chickens vaccinated with passage levels 82 and 85 of IB L1148 showed hardly any damage in the tracheas. The results complied with the requirements of the EP, which prescribe that at least 80% of the vaccinated chickens should be protected from ciliostasis. Chi-square analysis of the data showed that the differences between the groups vaccinated with passage levels 82 and 85 on one side, and the non-vaccinated controls on the other, were statistically significant (P ≤ 0.05). Passage level 101 also was efficacious and the difference between the vaccinated group and the non-vaccinated control group was statistically significant. Passage level 101 was less efficacious than passages 82 and 85, but the difference in levels of protection by passages 82 and 85 on one side, and passage level 101 on the other, was not statistically significant. Passage level 101 did not comply with the EP requirements.

Efficacy of the experimental vaccine strain against IB QX-like challenge in commercial chickens

with MDA. Blood samples taken from 5 chicks of the MDA positive flock were tested for the level of IB antibodies by ELISA. The chickens had antibody titres of log 2 9.9, 11.5, 11.5, 12.1 and 9.7, respectively. The average titre was log 2 10.9, which is normal for MDA against IB. After vaccination as well as after challenge no clinical signs were observed. The results in Table 5 show that the maximal damage (the highest possible ciliostasis score) to the tracheas after challenge was seen in the non-vaccinated group. In the group vaccinated with 10 3.0 EID 50 of IB L1148, passage level 85, 70% of the birds were protected against tracheal damage after challenge, whereas 80% of the group vaccinated with 10 3.3 EID 50 of IB L1148, passage level 85 was protected. The difference in level of protection observed in the vaccinated group compared to the non-vaccinated controls was statistically significant by Chi-square analysis.

In this study, a group of SPF chickens was included, because this is a recommendation in the EP monograph for efficacy studies using birds with MDA. 95% of the SPF chickens were protected against damage to the tracheal ciliated epithelium after challenge, which is in line with the results of the study in SPF chickens. The protection level observed in the SPF chickens was not significantly different from that in the MDA positive chickens as evaluated by Chi-square analysis.

Discussion

Many poultry vaccines are based on live-attenuated vaccine strains. Examples of live attenuated IB vaccines which are common in Europe are D274, H52, H120, 4/91, MM and CR88. The classical procedure for attenuation of IB viruses is by passage in embryonated eggs (MacDonald & McMartin, 1976;[START_REF] Klieve | Immunity and cross-protection to nephritis produced by Australian infectious bronchitis viruses used as vaccines[END_REF][START_REF] Jackwood | Attenuation, safety, and efficacy of in infectious bronchitis virus GA98 serotype vaccine[END_REF][START_REF] Huang | Development of attenuated vaccines from Taiwanese infectious bronchitis virus strains[END_REF][START_REF] Huang | Sequence changes of infectious bronchitis virus isolates in the 3' 7.3 kb of the genome after attenuating passage in embryonated eggs[END_REF][START_REF] Liu | S1 gene sequence heterogeneity of a pathogenic infectious bronchitis virus strain and its embryo-passaged, attenuated derivatives[END_REF]. In our studies, 80 passages were required to achieve a virus strain which was attenuated sufficiently to comply with safety requirements for live IB vaccines. This is in line with number of passages required to attenuate other IB strains. It is not known what type of mechanism causes a virus to attenuate. [START_REF] Huang | Sequence changes of infectious bronchitis virus isolates in the 3' 7.3 kb of the genome after attenuating passage in embryonated eggs[END_REF] sequenced the 3' 7.3 kb of the genome of an attenuated and parent virus strain from which an attenuated strain was derived. They found that there were amino acid substitutions in the spike 1 and 2 subunits, the small membrane protein and the No substitutions were detected in the nucleoprotein. We sequenced a substantial part of the genomic region coding for the hypervariable region of the S1 protein or our strain [START_REF] Worthington | A reverse transcriptase-polymerase chain reaction survey of infectious bronchitis virus genotypes in Western Europe from 2002 to 2006[END_REF]. In comparison to the parent field isolate there were not more than 4 substitutions in 1424 nucleotides, resulting in 2 amino acid substitutions. The substitutions we detected were different from those found by [START_REF] Huang | Sequence changes of infectious bronchitis virus isolates in the 3' 7.3 kb of the genome after attenuating passage in embryonated eggs[END_REF]. The role of these substitutions remains unclear.

We found that there was a clear effect of increased egg passage level on the results of ciliostasis scores. The effect was not gradual. There was a clear decrease in ciliostasis scores after 25 passages, followed by an increase until passage level 50. After 50 passages ciliostasis scores decreased again. To our knowledge this effect has not been reported before. At passage level 80 a ciliostasis score of 11 was determined, which is much lower than the maximum score of 40, often found with virulent IB strains, therefore the virulence of the virus strain for the respiratory tract has been decreased significantly. There is no threshold defined to distinguish safe IB strains from unsafe one. In the EP monograph an average value of 25 has been indicated for many years as the threshold, but nowadays the requirement is that a risk benefit analysis should be made, taking into account the average ciliostasis score and the benefits expected from the use of the vaccine. The average ciliostasis score for passage level 80 was well below the threshold of 25. Kidney lesions also decreased to very low levels at passage 80, and virus could be detected in the kidneys in only one occasion. These results clearly demonstrate that the virus was attenuated during passaging and the data for passage level 80 can be considered in compliance with requirements in the EP, with respect to safety for the respiratory tract and kidneys.

Following vaccination of female chickens on the day of hatching with passage 80, 12.5% developed lesions in the oviducts. IB has been described as an agent causing such lesions [START_REF] Crinion | Pathogenicity of two embryo-passage levels of avian infectious bronchitis virus for the oviduct of young chickens of various ages[END_REF]. They found that at egg passage level 55, IBV caused lesions only in chickens vaccinated at one day of age, but no lesions were found in chickens vaccinated at 8 days of age. These findings are in line with our results. IBV replicates at many epithelial surfaces. [START_REF] Cavanagh | Coronaviruses in poultry and other birds[END_REF] mentioned Harderian gland, trachea, oesophagus, lung, kidney, oviduct, duodenum, caecum, rectum, ileum and bursa as potential sites of replication of IBV. We recovered the virus from pancreas and trachea, and from cloacal and oro-pharyngeal swabs. In the contact birds which were infected by spreading of the virus from the vaccinated chickens, we found that oro-pharyngeal swabs were often virus-positive, whereas in the tracheal tissues the virus could no longer be detected. This may be because the cells producing the virus had been destroyed, whereas the virus which was produced from these cells was still present. It is possible also that the PCR test was more sensitive than the IHC staining method. Furthermore, it has to be said that the PCR test detects genomic material of the virus, i.e. a part of the genomic region coding for the hypervariable region of the S1 protein, whereas the IHC staining method is based on the reactivity of a monoclonal antibody with the nucleoprotein of the virus. The observed presence of the virus in trachea and pancreas suggest that this attenuated IB L1148 strain at passage level 80 has the trachea and the alimentary tract as main sites of replication.

Replication of IBV in the trachea is a common finding and replication in the alimentary tract has been described also [START_REF] Wang | Isolation and identification of glandular stomach type IBV (QX IBV) in chickens[END_REF], but this is not the case for replication of IBV in pancreatic tissue. [START_REF] Qian | Isolation and characterization of a coronavirus from pigeons with pancreatitis[END_REF] isolated an IB virus from sick pigeons and when the isolated virus was inoculated into SPF chickens pancreatitis developed. We did not observe any abnormalities in the pancreatic tissue in the dissemination study. In the other safety studies likewise we did not observe any abnormalities which could be attributed to IBV infection in the pancreas. Thus, there is no evidence that the attenuated IB L1148 strain has a damaging effect on the pancreas. It has to be mentioned that the IHC test detects the presence of epitope 48.4 of the nucleoprotein of the IB virus [START_REF] Koch | Rapid detection of infectious bronchitis virus by nucleoprotein specific antigen capture assay[END_REF]. More research will need to be done to confirm that the virus replicates in the pancreas.

In the dissemination study we introduced 3 groups of non-vaccinated contact chickens to the vaccinated chickens. Two groups were chickens of the same age as the vaccinated chickens; the other group consisted of 1-day-old chickens. In these younger chickens the vaccine virus was present in the same tissues as in the chickens of the same age as the vaccinated chickens. No signs of disease were observed in the contact chickens of the same age as the vaccinated chickens or in the younger contact chickens. It is common knowledge that IB vaccine viruses can spread from vaccinated to non-vaccinated chickens (Hopkins & Yoder, 1983), so the results described in this paper are not surprising. Just as for other IB vaccine viruses, it is strongly recommended to ensure that if a flock is vaccinated; all birds are vaccinated directly to avoid rolling reactions.

Our results show that chickens with MDA and vaccinated with IB strain L1148 passage level 85 were protected against challenge with IB D388. The degree of protection in the SPF chickens was higher than that in the MDA positive chickens. This can be a consequence of the presence of MDA which may have had a negative impact on vaccine efficacy. However, a direct comparison between the SPF chickens and the MDA positive chickens is difficult, because genetic differences between the chicken lines may also have had an effect on the susceptibility of the chickens to the IB challenge [START_REF] Ignjatovic | Susceptibility of three genetic lines of chicks to infection with a nephropathogenic T strain of avian infectious bronchitis virus[END_REF]. The MDA will have been directed against the Massachusetts, 4/91 and D274 types of IB, because the parent animals had been vaccinated with these IB vaccines, but it can not be excluded that the MDA was directed against IB QX-like viruses also, because of possible field infections. It has to be said that the MDA-positive chickens came from France, where IB QX-like viruses are prevalent [START_REF] Worthington | A reverse transcriptase-polymerase chain reaction survey of infectious bronchitis virus genotypes in Western Europe from 2002 to 2006[END_REF].

A vaccine titre of 10 3 EID 50 per dose was sufficient to generate protection in SPF chickens as well as chickens with MDA. If it is taken into account that virus titres of higher than 10 8 EID 50 per ml could be produced easily in SPF chicken eggs, it is obvious that a commercial vaccine based on IB L1148 passage level 85 is achievable.

Vaccines based on QX like strains do not yet exist. In order to find ways to protect poultry against IB QX like viruses, combinations of existing vaccine strains have been tested. IB 4/91 conferred protection against IB QX like virus challenge. It is not unusual for chickens to be vaccinated with different combinations of vaccines to generate broad protection. A consequence of the use of combinations of vaccine viruses, not related to the virus which has to be controlled, may result in a situation in which new virus strains, i.e. the vaccine strains, are introduced in the environment. This is not the case if a vaccine derived directly from the field virus is used. In this respect the use of such vaccine virus strains is advantageous.

However, other aspects, such as degree of attenuation and interactions and compatibilities with other vaccines also should be taken into account. A None 0, 0, 0, 0, 0 0 0, 0, 0, 0, 0 0 0, 0, 0, 0, 0 0 0 65 0, 1, 40, 40, 2 17 38, 40, 39, 40, 38 39 31, 0, 0, 1, 0 6 21 80 0, 0, 0, 0, 0 0 39, 40, 1, 0, 38 24 35, 0, 2, 3, 0 8 11 B None 0, 0, 0, 0, 0 0 0, 0, 0, 0, 0 0 0, 0, 0, 0, 0 0 0 a Ten tracheal rings were examined/ bird as detail in the Methods section. Each was scored as follows. 0: cilia in whole section showed movement; 1: 67% to 100% of the cilia showed movement; 2: 33% to 67% of the cilia showed movement; 3: less than 33% but more than0% of the cilia showed movement; 4: no ciliary movement. For each group the average ciliostasis score was calculated.

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--c +-d -- -- -- -- -- IHC a Trachea -- ++ e +- +- +- -- -- Cloacal swabs -- -- -- -- -- +- -- -- -- Vaccinated chickens (vaccine group) PCR b Oro-pharyngeal swabs ++ ++ ++ -- +- -- -- -- -- Pancreas -- -- -- +- -- -- IHC Trachea -- -- +- - -- -- Cloacal swabs -- -- -- -- +- -- +- -f
Non-vaccinated chickens of the same age in contact with vaccinated chickens 3 days after vaccination PCR Oro-pharyngeal swabs -- -----------------------------------------------------------------------------------------------------------DQ341199 392 GTGATCATATTCGTATTTCTGCAATGAAAAATGGTTCTTTATTTTATAATTTAACAGTTAGCGTATCTAAATACCCTAATTTTAAATCTTTTCAATGTGTTAACAAC IB L1148 P80 -----------------------------------------------------------------------------------------------------------DQ341199 499 TTCACATCTGTTTATTTAAATGGTGATCTTGTTTTTACTTCCAATAAAACTACTGATGTTACGTCAGCAGGTGTGTATTTTAAAGCAGGTGGACCTGTAAATTATAG IB L1148 P80 -----------------------------------------------------------------------------------------------------------DQ341199 606 TATTATGAAAGAATTTAAGGTTCTTGCTTACTTTGTTAATGGTACAGCACAAGATGTAGTTTTGTGCGACAATTCCCCCAAGGGTTTGCTAGCTTGTCAATATAACA IB L1148 P80 -----------------------------------------------------------------------------------------------------------DQ341199

+- +- -- -- -- +- -f Pancreas -- IHC Trachea -- Cloacal swabs -- -- -- -- -- Non-vaccinated
713 CTGGCAATTTTTCAGATGGCTTTTATCCTTTTACTAATAGTACTTTGGTTAGGGAAAAGTTCATCGTCTATCGCGAAAGTAGTGTTAATACTACTCTGGCGTTAACT IB L1148 P80 ---------------------------------------------------------------------------------------------T-------------DQ674739

---------------------------------------------------------------------------DQ341199 820 AATTTCACTTTTACTAATGTAAGTAATGCACAGCCTAATAGTGGTGGTGTTAATACTTTTCATTTATACCAAACACAAACAGCTCAGAGTGGTTATTATAATTTTAA IB L1148 P80 -----------------------------------------------------------------------------------------------------------DQ674739

-----------------------------------------------------------------------------------------------------------DQ341199 927 TTTGTCATTTCTGAGTCAGTTTGTGTATAAGGCAAGTGATTTTATGTATGGGTCCTACCACCCTAGTTGTTCTTTTAGACCAGAAACCATTAATAGTGGTTTATGGT IB L1148 P80 -----------------------------------------------------------------------------------------------------------DQ674739 -----------------------------------------------------------------------------------------------------------DQ341199

1034 TTAATTCCTTGTCAGTTTCTCTTACTTATGGACCCCTACAGGGAGGGTGTAAGCAATCTGTTTTTAGTGGTAAGGCAACGTGTTGTTACGCCTACTCTTATAAAGGC IB L1148 P80 -----------------------------------------------------------------------------------------------------------DQ674739 -------------------------------------------T-------DQ341199

1141 CCAATGGCATGTAAAGGTGTTTATTCAGGTGAATTAAGCACGAATTTTGAATGTGGATTGCTGGTTTATGTTACTAAGAGTGATGGCTCTCGTATACAGACTAGAAC IB L1148 P80 ---------------------------TT-C----------------------------------------------------------------------------DQ341199

1248 AGAGCCCTTAGTATTAACGCAATACAATTATAATAATATTACTTTAGATAAGTGTGTTGCCTATAATATATATGGCAGAGTAGGCCAAGGTTTTATTACTAATGTGA IB L1148 P80 -----------------------------------------------------------------------------------------------------------DQ341199

1355 CTGATTCTGCTGCTAATTTTAGTTATTTAGCAGATGGTGGGTTAGCTATTTTAGATACGTCGGGTGCCATAGATGTTTTTGTTGTACAGGGCATCTATGGTCTTAAT IB L1148 P80 -----------------------------------------------------------------------------------------------------------DQ341199

1462 TATTACAAGGTTAATCCTTGTGAAGATGTTAATCAACAATTTGTAGTGTCTGGTGGCAATATAGTTGGCATTCTTACTTCTAGAAATGAAACAGGTTCTGAACAGGT IB L1148 P80 -----------------------------------------------------------------------------------------------------------DQ341199

1569 TGAGAACCAGTTTTATGTTAAGTTAACCAATAGCTCACATCGTCGTAGGCGTTCTATTGGCCAAAATGTAACAAGTTGTCCTTATGTTAGTTATGGCAGATTTTGTA IB L1148 P80 -----------------------------------------------------------------------------------------------------------DQ341199

1676 TTGAACCAGATGGTTCGTTAAAGATGATAGTGCCAGAAGAATTGAAACAGTTTGTGGCACCTTTACTTAATATTACTGAAAGTGTACTCATACCTAACAGTTTTAAC IB L1148 P80 ---------------------------------

  IB) virus is a coronavirus which causes respiratory disease in chickens.

  the embryos were examined for the presence of specific lesions caused by the virus.

  of each group were removed and killed by inhalation of 100% CO 2.

  Sequence analysis. The nucleotide sequence of a 1456 BP fragment, between nucleotide position 262 and 1718, of the region coding for the hypervariable region of the S1 protein of the vaccine virus was determined, and the results are given in Figure1. The nucleotide sequence of the original isolate was available from the GenBank (DQ431199). Of the overlapping 1424 BP fragment, there were substitutions at 4 positions, i.e. positions 913, 1275, 1276 and 1278. The substitution of C by G is silent. The substitutions at the other positions result in substitutions of Glycine and Glutamate into Phenylalanine and Glutamine.

  40 and 50 were tested, some kidneys were affected macroscopically; one at 5 days after vaccination with passage level 50 and several at day 10 in the groups inoculated with each virus. The kidneys were swollen and pale. No gross pathological lesions were recorded in study B in which passage levels 65 and 80 were tested. Passage levels 8, 25, 40 and 50 of IB

  For example,[START_REF] Terregino | Pathogenicity of a QX strain of infectious bronchitis virus in specific pathogen free and commercial boiler chickens, and evaluation of protection induced by a vaccination program based on the Ma5 and 4/91 serotypes[END_REF] vaccinated chickens with the IB Ma5 vaccine strain at 1 day of age and with IB 4/91 vaccine strain at 14 days of age. At 5 weeks of age the chickens were challenged with an IB QX like virus, isolated in Northern Italy in 2005 (IBV QX-like virus 1997/V05). The results of that study show that combined vaccination with IB Ma5 and

  ND ND ND ND ND ND ND ND ND ND ND ND a Immunohistochemistry.b Kidney lesions were scored as follows: -no lesions, + some infiltration of lymphocytes locally, ++ strong infiltration of lymphocytes through whole kidney. c More than 5 samples tested. d One of 5 samples was lost. e ND, not done.

F

F

Figure 1 .

 1 Figure 1. Nucleotide sequence of the genomic fragment coding for the hypervariable sequence of the S1 protein of the master seed virus of IB L1148 at passage level 80; nucleotide positions 285 to 1708. DQ341199 is GenBank reference for low egg passage IB strain L-1148A. DQ674739 is GenBank reference for IB strain D388. IB L1148 p80 is the attenuated QX-like IB virus at passage level 80.

  and Prof. R.C. Jones,

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Table 1 .

 1 Results of safety tests with different passage levels of IB L1148 at a dose of 10 6.0 EID 50 for the respiratory tract of SPF chickens.

	Study	Passage			Ciliostasis scores at 5, 7, 10 days post vaccination
		level	Day 5		Day 7		Day 10		Total
		inoculated	Individual	Mean	Individual	Mean	Individual	Mean	mean
		8	40 a , 40, 40, 40, 40	40	40, 40, 40, 40, 40	40	40, 40, 31, 38, 39	38	39
		25	0, 38, 30, 0, 39	21	0, 40, 0, 0, 0	8	40, 0, 37, 3, 37	23	18
		40	30, 0, 0, 40, 40	22	40, 40, 40, 40, 39	40	0, 0, 0, 0, 0	0	21
		50	0, 39, 40, 40, 39	32	40, 40, 40, 40, 40	40	40, 3, 27, 0, 36	21	31

Table 2 .

 2 Results of safety tests with different passages of IB L1148 at a dose of 10 6.0 EID 50 for the kidneys of SPF chickens.

		Passage	Macroscopic examination			Histological examination				IHC staining for IB
		level	Day 5	Day 7	Day 10		Day 5			Day 7			Day 10		Day 5	Day 7	Day 10
	Study	inoculated -b	+	-	+	-	+	-	+	++	-	+	++	-	+	++	-	+	-	+	-	+
		8

Table 3 .

 3 Results of study on dissemination of IB virus in the chicken and spread of the vaccine strain.

	Days after vaccination of the vaccinated group

Table 4 .

 4 Results of cilia movement test following challenge of SPF chickens vaccinated at day of hatching with different doses of IB L1148 at different passage levels.

	Vaccinated with Passage 101	Titre per dose (EID 50 ) 10 3.3	Challenge at day 21 Yes	Protected a / total (%) 13/20 (65)
	Passage 85 Passage 85	10 3.0 10 3.3	Yes Yes	17/20 (85) 17/20 (85)
	Passage 82 Passage 82	10 3.0 10 3.3	Yes Yes	18/19 (95) 18/20 (90)
	None	NA b	Yes	0/20 (0)
	None	NA	No	20/20 (100)

a A chicken was considered protected if not fewer than 9 out of 10 trachea rings/bird showed normal ciliary activity. b Not applicable.

E-mail: cavanagh@metronet.co.uk URL: http://mc.manuscriptcentral.com/cavp Avian Pathology

Acknowledgement.

The efficacy tests were performed by Dr. J.J. de Wit, Dr. W. Landman, Dr. A. Feberwee, H. van de Sande and coworkers at the Animal Health Service, Deventer, NL. The description of the immunohistochemistry was kindly provided by Dr. J.J. de Wit.


Frédéric Wang

Ruan Yuan

Han Studies

Song Studies

Prof Daeyeol Kim

Prof Young Carl

Prof Catherine Jami

Zhang Xuecheng

See D Nivison

) and the Transmission of Classical Texts between the 18 th and 19 th Centuries

Ruan Yuan, a Chinese contemporary of Tasan, was the last of the high officials who advocated Han studies in the Qianlong and Jiaqing periods. As a patron of scholarship at his time, he went beyond his own intellectual propensities and did not set aside all partisans of Song studies. He thus contributed to the harmonisation of Song and Han studies.

Tasan 茶山 (1762-1836), is not an easy task because there is a large number of great scholars who lived between the 18 th and 19 th centuries. Even looking only at the scholars that Tasan's friend Park Che-ga 朴齊家 (1750-1805) met in Beijing between 1790 and 1801, among them were Ji Yun 紀昀 (1724-1805), Li Dingyuan 李鼎元 (jinshi 1778), Zhu Delin 祝德麟 (1742-1798), Tang Leyu 唐樂宇 (circa 1740-1791), Peng Yuanrui 彭元瑞 (1731-1803), Weng Fanggang 翁方綱 (1733-1818), Ruan Yuan 阮元 (1764-1849), Jiang Deliang 江德 量 (1752-1793), Tie Bao 鐡保 (1752-1824), Luo Pin 羅聘 (1733-1799), Sun Xingyan 孫 星衍 (1753-1818), and Xiong Fangshou 熊方受 (jinshi 1790). 1 Living in the Qianlong (1736-1796) and Jiaqing (1796-1821) periods, most of them were expert philologists and heirs to Hui Dong 惠棟 (1697-1758) and Dai Zhen 戴震 (1724-1777). The list becomes longer if we add other erudite scholars such as the historian Zhang Xuecheng 章學誠 (1738-1801), [START_REF]阮元對漢宋學術之平議 (Ruan Yuan's Discussion on the Relation of Han and Sung Studies)[END_REF] and the philosopher Fang Dongshu 方東樹 (1772-1851), a faithful follower of Zhu Xi's 朱熹 (1130-1200) school. The reason for choosing Ruan Yuan is that he was an immediate contemporary of Tasan, who quoted him, and that Kim Chông-hui 金正喜 (1786-1856), a disciple of Park Che-ga, met him in 1809. Even if Ruan Yuan's career was filled with success in comparison to Tasan's more tortuous one, the two thinkers are marked by Han studies (hanxue 漢學), all the while inspiring themselves from the heritage of Song Neo-Confucianism. 3 Some consider that Ruan was one of the most influential Chinese thinkers in Korea in this period. 4 He displayed openness not only towards Korea, but also towards Japan. He referred to Shichikei Mōshi kōbun hoi 七經孟子考文補遺 (Supplementary Thoughts on the Critical Study of the Seven Classics and Mencius) by the Japanese thinker Yamanoi Konron 山井崑崙 (1690-1728) in his new critical edition of the Shisanjing zhushu 十三經 註疏 (Notes and Commentaries on the Thirteen Classics).

The contemporary historian Qian Mu (Ch'en Mu 錢穆, 1895-1990) states concerning this great scholar of the 18 th and 19 th centuries that he was the last of the high officials who advocated Han studies in the Qianlong and Jiaqing periods. 5 Even if he gave greater importance to the philological aspects of classical studies, as his predecessors and contemporaries such as Dai Zhen and Qiao Xun 焦循 (1763-1820) did, he adopted a more open attitude towards the philosophical interpretation of the Neo-Confucians. 6 Who is this individual? What was his role in the transmission of classical texts in the turn of the 18 th and 19 th centuries? What are the principal ideas or rather the original contributions of this thinker? All of these questions are deserving of consideration. References to Ruan Yuan in this paper will be essentially based on his Yanjing shi ji 揅經室集 (Collection from the Studio of the Study of the Classics).

Since Ruan Yuan's biography has already been the subject of an important monograph by Betty Peh-T'i Wei, 7 I will content myself with a few lines to bring out the principal events in Ruan's life according to his biography in the Qingshi gao 清史稿 (Draft History of the Qing Dynasty). 8 Born in Yizheng 儀征 in Jiangsu, Ruan Yuan, zi Boyuan 伯元, grew up and lived out his retirement until his death in Yangzhou. Awarded the jinshi degree in 1789, or in other words the 54 th year of Qianlong's reign, Ruan Yuan had a brilliant official career, as much in the provinces as in the capital. After successfully passing his doctoral examinations, he was elected chief among the compilers at the Hanlin Academy and became a reviser. In the 58 th year of Qianlong's reign (1793), he was appointed director of studies (xuezheng 學政) in Shandong before being transferred to Zhejiang in 1795. From 1798, he was successively director at the Ministries of the Army, Rites, and Revenue. Between 1799 and 1805, and thereafter between 1807 and 1810, he was appointed twice as an itinerant governor (xunfu 巡 撫) in Zhejiang. It is there that he directed actions against pirates. In the 19 th year of the Jiaqing period (1814), he became itinerant governor in Jiangxi and then in Henan. From 1817, he was appointed governor-general (zongdu 總督) of Huguang, and then of Liang Guang (Guangdong and Guangxi) and Yungui (Yunnan and Guizhou). Near the end of his life, he returned to the capital and assumed the duties of president of the Censorate (1836). He retired in 1838 and received the honorific title of Grand Preceptor to the Crown Prince (taizi taibao 太子太保).

Wherever he was posted, Ruan Yuan always exhorted the population to study. He was the founder of the Gujing jingshe 詁經精舍 (Academy of the Exegesis of the Classics) at Hangzhou when he was itinerant governor in Zhejiang (1800), and of the Xuehai tang 學海 堂 (Academy of the Ocean of Erudition) at Canton in 1820. 9 These two institutions were the bastions of the specialists of Han studies in Zhejiang and Canton, which were regions that played an important role in the academic sphere in this period. In Zhejiang, he directed the compilation of the Jingji zuangu 經籍籑詁 (Exegetical Dictionary of the Classics) in 106 juan, highly praised by the celebrated scholar Wang Yinzhi 王引之 (1766-1834), son of Wang Niansun 王念孫 (1744-1832). 10 In Jiangxi, he reedited the Song version of the Shisanjing zhushu for which he wrote a critical note (jiaokan ji 校勘記) for the editing of each of the Classics. 11 In Canton, he edited the Xuehai tang jingjie 學 海 堂 經 解 (Interpretations of the Classics of the Xuehai Tang), better known under the name Huang Qing jing jie 皇清經解 (Interpretations of the Classics of the Great Qing). This compilation in 1400 juan comprises 188 exegetical works by 73 authors of the 17 th and 18 th centuries. The publication of the Shisanjing zhushu, Jingji zuangu and Huang Qing jingjie brought together a synthesis of Han studies centred on the study of the Classics from the Qianlong and Jiaqing eras. As to his own writings, they are essentially contained in the Yanjing shi ji, which was compiled in the 3rd year of the Daoguang reign (1823) for his 60 th birthday. Ruan Yuan's intellectual training was intimately linked to the school of Dai Zhen. In his own words, he benefited when he arrived in Beijing around 1786 from the teaching of three of Dai Zhen's disciples: Wang Niansun, zi Huaizu 懷祖, Ren Dachun 任大椿 (1738-1789), zi Zitian 子田 et Shao Jinhan 邵晉涵 (1743-1796), zi Eryun 二雲. He became friends with Qian Daxin 錢大昕(1728-1804), Jiao Xun et Ling Tingkan 淩廷堪 (1755-1809). Ruan Yuan touched almost all the realms of knowledge and Gong Zizhen 龔 自 珍 (1792-1841) summarises ten of them : textual criticism, exegesis, bibliographical science, historiography, the study of institutional systems, studies of bronze inscriptions, mathematics, stylistics, philosophy, and astrology. 12 It is important to remember that he is the supervisor of the compilation of the Chouren zhuan 醻 人 傳 (Biographies of Astronomers and Mathematicians). 13 However, the foundational discipline for him is exegesis (xungu 訓詁), since this was the starting point of philosophy. On this point, he remains faithful to Dai Zhen and Qian Daxin, whose work Ruan Yuan edited at his death. He stated: 9 On the relations between the Xuehai tang and the texts of the Classics in "modern writing", see Benjamin A. Elman, "The Hsüeh-hai T'ang and the Rise of New Text Scholarship in Canton", Ch'ing-shih wen-t'i (Late Imperial China), 4, 2 (1979), pp. 51-82. A Chinese version (Xuehai tang yu jinwen jingxue zai Guangzhou de xingqi 學海堂與今文經學在廣州的興起), translated by Che Xingjian 車行健, is published in Hunan daxue xuebao (Shehui kexue ban) 湖南大學學報(社會科學版), vol. 20, 2006, no. 2, pp. 13-20. 10 Wang Yinzhi states in the preface of the Jingji zuangu : "In a single rhyme are gathered all the characters concerned; each one of them conserves all the etymological explanations ; these allow for the understanding of the works" (展一韻而眾字畢備,檢一字而諸訓皆存,尋一訓而原書可識). In sum, the Dao of the saints is the same as a house. The study of words and their origins is the way to accede to it. If you pass beside this, all your steps will turn away from it. How does one rise to the great hall and enter into study? He that searches for the Way too high up and who has but disdain for phrase by phrase exegesis is like a bird that would project itself into the sky from the rooftop. He will certainly go high but will be unable to see what is between the door and corners of the house. For others, who content themselves with classifying names and things without discussing the Dao of the saints, everything happens as if their whole life flowed between the portal and the entryway, without thinking for an instant to explore the great hall and study. 14

聖人之道譬若宮墻,文字訓詁其門逕也。門逕苟誤,跬步皆岐,安能升堂入室乎?學 人求道太高,卑視章句,譬猶天際之翔出於豊屋之上。高則高矣,戶奧之間,未實窺 也。或者但求名物,不論聖道,又若終年寢饋於門廡之間,無復知有堂室矣。 15
The study of the Dao here is that of the Neo-Confucianism of the Song and Ming, or Song studies, whereas the studies of terms and their origins, xungu or exegesis, is more specific to the Confucians of the Han period, hence the name "Han studies". Ruan Yuan is certainly a great heir of this latter tradition. However, he keeps a balanced vision of the two:

The teaching of the two Han excels in the study of the Confucian Classics. The reading of the Song and the Ming brings out the benefits of the Way of the Masters. With regard to the Way of the Duke of Zhou and Confucius, each one has obtained a part. It is not suitable to privilege one and mock the other. The successive sovereigns of our dynasty, endowed with a pure Way and a perfect virtue, inherit all that comes from the ancients. They advocate the Way of human nature in Song studies, but they bring it to concrete expression through the meaning of the Classics of the Han Confucians. 兩漢名教,得儒經之功;宋明講學,得師道之益;皆於周孔之道,得其分合,未可偏 譏而互誚也。我朝列聖,道德純備,包涵前古,崇宋學之性道,而以漢儒經義實之。 second author, on the contrary, judges that the scholars of Han studies "stray in the irrational and in calumnies, reject entirely the teaching of the sages consisting of personal vigilance in conduct, the search for human meaning, self-cultivation, the ordering of the household, the management of the state and the governance of the empire". For him, the advocates of Han studies "in reality introduce disorder into the Classics under the pretext of studying them, setting up obstacles in the Dao all the while pretending to defend it". He goes all the way to saying that the ill effects of Han studies are "a hundred times more important than that of Chan and intuitionism (xinxue 心學) with regard to the teaching of the world and academic work". 19 Jiang Fan was a compatriot of Ruan Yuan who prefaced his work. As well, during his writing, Jiang was Ruan Yuan's counsellor in Canton. At the same time, Fang Dongshu was also a client of Ruan to whom he dedicated his work. Zhu Weizheng 朱維諍 indicates that it is Ruan Yuan's name which appears most frequently in this work. 20 This confirms his primary importance in the camp of Han studies. However, Ruan goes beyond his own intellectual propensity and does not set aside all the partisans of Song studies. Fang Dongshu was employed by him as a teacher at the Xuehai tang 學海堂 which he created. His leadership role surpassed academic cleavages. For Ruan Yuan, knowledge of ancient commentaries serves as a way to access the study of the Dao, whose goal is the understanding of principle, human nature, and the Dao. One can say that Ruan Yuan, the last great representative of Han studies in the Qianlong and Jiaqing periods, prepared their harmonisation with Song studies that was later taken up by such people as Liu Shipei 劉師培 (1884-1919), Zeng Guofan 曾 國 藩 (1811-1872), and Zhang Zhidong 張 之 洞 (1837-1909). [START_REF]On this aspect, see Guo Mingdao 郭明道, « Ruan Yuan yu Qingdai xuefen » 阮元與清代學風 (Ruan Yuan and the academic trends of the Qing)[END_REF] Ruan Yuan never ceases to say that his explanation of the Classics is aimed at "bringing to light the ancient interpretations and searching out for truth in reality" (推明古訓,實事求 是). [START_REF] Yuan | Auto-preface[END_REF] For him, "not only can the deep ideas of the saints and sages not be understood without exegesis, but also their ideas that are apparently simple to access" (聖賢之言,不但深遠者 非詁不明,即淺近者亦非詁不明也). [START_REF] Yuan | Lunyu yiguan shuo" 論語一貫說 (Explanation of the Expression yiguan in the Analects)[END_REF] He says elsewhere that their Dao is preserved in the Classics which cannot be clarified without exegesis (聖賢之道存于經,經非詁不明). [START_REF] Yuan | Xihu Gujing jingshe ji" 西湖詁經精舍記 (Note on the Guijing Academy on the Western Lake)[END_REF] The more distant the period, the closer to the intentions of the sages are the sayings of the commentators. This is why the model needs to be that of the Han interpreters because they lived in a period that was relatively not too distant from the pre-imperial era, and one where the influence of Daoism and Buddhism was still restricted. Among the Han thinkers, it is Zheng Xuan 鄭玄 (127-200) and Xu Shen 許慎 (58-147) who are most particularly highlighted by him. On the request of Sun Xingyan, at that time a teacher at the Jinggu academy, Ruan Yuan decided to venerate these two representatives of the Classics in the Han "ancient writing" (guwen 古文) at the sacrificial altar of the Jinggu jingshe.

The Han studies put forward by Ruan Yuan did not only include guwen, but also those of the Gongyang 公羊 school of jinwen 今文 (modern writing) represented by Dong Zhongshu 董仲舒 (179-104 BCE) and He Xiu 何休 (129-182). 25 Moreover, Ruan Yuan accords a great importance to Mao Qiling 毛奇齡 (1623-1716), an author neglected by Dai Zhen and other Han studies specialists in favour of Gu Yanwu 顧炎武 (1613-1682). He states concerning this great scholar in his postscript to the Work of Mao Qiling that "he took up the responsibility of the study of the Classics by shouting high and strong. Therefore, practical studies were raised in a short lapse of time" (以經學為自任,大聲疾呼,一時之實學頓 起). 26 His sympathy cut short the doubts that certain scholars had about Mao Qiling, the author of Sishu gaicuo 四書改錯 (Corrections of the Errors of the[Commentaries of the] Four Books) that takes direct aim at the Sishu jizhu 四書集注 (General Survey of the Commentaries of the Four Books) by Zhu Xi (1130-1200). It is important to note that Tasan was also aware of Mao Qiling's work.

Among the Confucian Classics, Ruan Yuan insists on the role of the Odes (Shi 詩) and the Documents (Shu 書), considering them as constituting the basis of the study of Confucius and Mencius. However, he also pays particular attention to the Xiaojing 孝經, the Classic of Filial Piety. In an esoteric commentary of this Classic, there is a phrase that claims to take up again what Confucius said: "My ambition lies in the Chunqiu (The Spring and Autumn Annals) and my conduct in the Xiaojing (Classic of Filial Piety)." (吾志在《春秋》,行在 《孝經》). Ruan Yuan considers that this phrase could not in any way have been invented by the apocryphal authors of the Han, but was really from Confucius himself. He pursues his argument in this way:

The two-character title Xiaojing was given by Confucius himself. It is because of this that he said: "My conduct can be read in the Classic of Filial Piety." It is said in the Shiji (The Histories): "Confucius found that Zengzi was able to follow the Way of filial piety. This is why he transmitted it to him by composing the Xiaojing." It is equally stated in the "Bibliographies" chapter of the Book of the Han: "Filial piety constitutes the framework of Heaven, the meaning of Earth and the conduct of the people. To express its basis, there was then the Classic of Filial Piety." From these ancient works we know that the term jing begins to exist from this Classic, from where come the names of the Five Classics, Six Classics, Nine Classics, and Thirteen Classics. If Buddhism and Daoism have attributed titles of works with the character jing, it is that they also inherited it from this. It is true that among the classical texts from the pre-imperial period, only the Xiaojing includes in the title itself the character jing which was added to the others from Han times. The relation of the Classics is not a resolved question even today. Most researchers consider the Xiaojing to have come after Confucius from the fact that the terms Zi yue 子曰 (Confucius said) appear frequently. However this does not pose a problem for Ruan Yuan since the Lunyu (The Analects) and the Mencius only take up or transmit the ideas of the Xiaojing. It is said in the Lunyu: "A gentleman occupies himself with the essential. When the essential is established, the Way emerges. Filial piety and brotherly love are the foundation of the sense of humanity" (Chapter 2). In the Analects, these remarks are attributed to Youzi, Confucius' disciple. However, Ruan Yuan considers them to be originally those of the Master's. For Ruan, filial piety is the primordial aspect of ren (sense of humanity) which he interprets very differently from that of the Song Neo-Confucians.

《孝經》二字標題,乃孔子所自名。故孔子曰:"吾行在《孝經》"。《史記》:"孔 子以曾子為能通孝道,故授之以業,作《孝經》。"《漢書•藝文志》曰:"夫孝,天 之經,地之義,民之行也。擧大者言,故曰《孝經》。"據此諸古籍,知"經"之一 字,始于此書。自此之後,五經、六經、九經、十三經之名,皆出于此。釋、道之名 其書曰"經",亦始襲取于此。 27
Almost all the contemporary commentators of Ruan Yuan's philosophy have remarked on his "Lunyu lun ren lun" 論語論仁論 (On the Sense of Humanity as Discussed in the Analects) included in the Yanjing shi ji. In this text, Ruan Yuan refers to the Da Dai Liji 大戴 禮記 (Records of Rites by Dai Senior) and the Zhongyong 中庸 (Doctrine of the Mean). The first work states that man cannot succeed alone without others just as a horse cannot draw a wagon without forming a group of four or as a drop of water does not flow. As for him, Zheng Xuan comments on the identification of ren with man in the Zhongyong through the expression « xiang ren'ou » 相人偶 which should mean mutual help or relation between people. Ruan Yuan retains this exegesis, which is in accord with that of the Shuowen jiezi 說 文解字 (Etymological Dictionary) of Xu Shen, and which states that « ren'ou 人偶 in its ancient sense, was a term that designated the friendship between you and me » (古所謂"人 偶",猶言爾我親愛之辭). 28 In this text, Ruan Yuan reproaches Zigong, Confucius' disciple, for elevating ren too much, situating it on the same level as holiness (sheng 聖). This criticism is also applicable to the Song Neo-Confucians, who associate it to Heavenly Principle (tianli 天理). In Ruan Yuan's eyes, practising ren is helping them, respecting them… In this way, instead of defining ren as « the virtue of the mind, the principle of love » (心之德,愛之理) after the fashion of Zhu Xi, Ruan Yuan endows it with a more practical dimension, which is for him inherent in the Way of wisdom. He states:

Ren only appears through the application of experience, on the condition that this is performed in the presence of a third person. By shutting himself up at home to fast or crouch down in calm with closed eyes, a man, even if he is internally endowed with virtue and reason, cannot obtain that which the saints call "the sense of humanity".

凡仁,必於身所行者驗之而始見,亦必有二人而仁乃見。若一人閉戶齋居,瞑目靜 坐,雖有德理在心,終不得指為聖門所謂之仁矣。 29

This practical aspect also characterises his explanation of other philosophical terms such as xing 性 (human nature) and xin 心 (mind-heart); he always verifies their etymology and as much as possible, strips them of their metaphysical interpretation. It is perhaps this point as well that differentiates him from Dai Zhen, who more consciously constructed a philosophical system from a critique of Song and Ming Neo-Confucianism 30 . Ruan says : "the semantic category of the term xing is xin, that is to say blood, vital energy, the heart and intelligence" ("性"字從心,即血氣心知也). He criticises what Li Ao 李翺 (circa 772-836) calls "the return to human nature" (fu xing 復性) but puts forward the notion "measure of human nature" (jie xing 節性). 31 This is because Li Ao distinguishes human nature (xing) and the emotions (qing 情) (joy, anger, sadness, fear, love, aversion, and desire). For Ruan Yuan, human nature is an integral part of human emotions. It is sufficient to take proper measures so that desires do not overflow. This is done through the study of the rites, the importance of which was explicitly noted by his friend Ling Tingkan 淩廷堪. 32 Possessing an encyclopaedic spirit, Ruan Yuan played the role of intellectual leader of his time. He gathered around him scholars from all horizons (Han and Song studies, guwen and jinwen study of the Classics) and directed the most important compilations of classical texts under the Qing. His own hermeneutics were founded essentially on previous linguistic work, from which emerges the importance in his work of the search for the etymological meaning of philosophical terms. He did not set out to invent a new philosophy but rather to give a more authentic perspective by respecting the interpretation of the Classics made by the Han commentators and introducing a more practical and less metaphysical dimension. The two academies (Gujing jingshe and Xuehai tang) that he created in Zhejiang and Canton became models of the training of intellectuals who were often endowed with an acute sense of China's new reality. From the Xuehai tang emerged scholars such as Chen Feng 陳灃 (1810-1882), who further developed the harmonisation of Han and Song studies put forward by Ruan Yuan, and Liang Qichao 梁啟超 (1873-1929), who was also much influenced in his youth by Ruan Yuan. Ruan lived all the way to the Opium War and sensed the tension between Chinese and Western knowledge, of which he was completely aware. However, the theorisation of this complex relationship would be the object of his successors such as Zhang Zhitong, Kang Youwei 康有爲 (1858-1927), Liang Qichao and Yan Fu 嚴復 (1854-1921).

25

  Chen Juyuan 陳居淵, "Lun Ruan Yuan de jingxue sixiang" 論阮元的經學思想 (Ruan Yuan's Thoughts on the Study of the Classics), Zhongguo zhexueshi 中國哲學思想史, 2004, no. 1, pp. 90-96. On the quarrels between the jinwen and the guwen under the Han, see Anne Cheng, Étude sur le confucianisme Han : l'élaboration d'une tradition exégétique sur les Classiques (Study on Han Confucianism: the Elaboration of an Exegetical Tradition on the Classics), Paris: Collège de France, IHEC, 1985. 26 Ruan Yuan, "Mao Xihe Jiantao quanji houxu" 毛西河檢討全集後序 (Postscript to the Work of Mao Qiling), Yanjing shi ji, Book 2, juan 7, op. cit., p. 501. 27 "Xiaojing jie" 孝經解, in Yanjing shi ji, Book 1, juan 2, op. cit., p. 42.

and

  Practices of the Han-Learning School during the Qianlong and Jiaqing Periods : Dai Zhen and Ruan Yuan), Qingshi yanjiu 清史研究, no. 3, 2002, pp. 91-98. 31 Ruan Yuan, "Xingming gu xun" 性命古訓, Yanjing shi ji, Book 1, juan 10, pp. 191-214. 32 On Ling Tingkan, see Chang So-an's 張壽安 work: Yi li dai li: Ling Tingkan yu Qing zhongye ruxue sixiang zhi zhuanbian 以禮代理:淩廷堪與清中葉儒學思想之轉變 (Replacement of Principle by Rites: Ling Tingkan and the Transformation of Scholarly Thought in the mid-Qing), Taipei: Zhongyang yanjiuyuan jindaishi yanjiusuo, 1994 ; Shiba shiji li xue kaozheng de sixiang huoli: lijiao lunzheng yu lizhi chong xing 十八世紀禮 學考證的思想活力:禮教論爭與禮秩重省 (The Liveliness of Spirit in the Philological Tradition of the Study of the Rites in the XVIII th Century : The Quarrels concerning the Teaching of the Rites and the New Exam of the Order of Rites), Taipei: Zhongyang yanjiuyuan jindaishi yanjiusuo, 2001.

  11 Certain contemporary researchers attribute this compilation to Duan Yucai 段玉裁 (1735-1815); see the article by Wang Shaoying 汪紹楹, "Ruanshi chongke Songben Shisanjing zhushu kao" 阮氏重刻宋本十三經 註疏考 (Investigation on Ruan Yuan's edition of a Song version of the Shisanjing zhushu), Wenshi 文史, no. 3, 1963, pp. 25-60. 12 Zhang Jian 張鑑, Ruan Yuan nianpu 阮元年譜 (Chronological Table of Ruan Yuan, year by year),Peking: Zhonghua shuju, 1995, appendix 3, pp. 273-274. 13 Catherine Jami tells us that the Chouren zhuan is nowadays believed to have been written mainly by Li Rui 李銳 (1769-1817) who was a client of Ruan Yuan. See also Jean-Claude Martzloff, A History of Chinese Mathematics, trans. Stephen S. Wilson, Berlin / Heidelberg: Springer-Verlag, 2006 (1997), pp. 166-173.

Fang Dongshu, "Preface" to the Hanxue shangdui 漢學商兌, Taipei: Guangwen chubanshe, 1963, p. 2.

Zhu Weizheng, "Hanxue yu fan Hanxue", p. 29; Sun Huajing 孫華璟, "Ruan Yuan dui Han Song xueshu zhi

Ruan Yuan, "Lunyu lun ren lun" 論語論仁論, Yanjing shi ji, Book 1, juan 8, p. 159.

Ibid., p. 157.

Huang Aiping 黃愛平, "Qian Jia Hanxue zhixue zongzhi jiqi xueshu shijian tanxi: yi Dai Zhen Ruan Yuan wei zhongxin" 乾嘉漢學治學宗旨及其學術實踐探析--以戴震、阮元為中心 (The Academic Principle