Javier Millán

Nieves Negre

Elena Castellanos

Lucia De Juan

Mateos Ana

Lluis Parpal

Alicia Aranaz

Control, Govern Illes Wildlife Health

Balears Negre

Nieves Castellanos

Elena De Juan

Lucia Ana

Mateos Parpal

Lluis Aranaz

Lucía De Juan

Ana Mateos

Avian mycobacteriosis in free-living raptors in Majorca Island, Spain

Keywords: Raptor, Mycobacterium avium, Tuberculosis, Majorca

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

by PCR and, in all the cases, infection with Mycobacterium avium subsp. avium was confirmed. The observed prevalences are similar to those previously observed in Holland, though the actual prevalence detected in this study is likely to be higher than reported because only birds with gross lesions were subjected to culture. Further molecular characterization with a set of six MIRU-VNTR loci was used to sub-typify the isolates in order to show the existence of possible epidemiological links. Six different genotypes were found, which points to infection from multiple focuses. No temporal or geographical aggregation of the cases was observed associated to the presence of positive birds or to the different VNTR allelic profiles. The most feasible origin might be water or food sources, though the reservoir of mycobacteria remains unknown.

Introduction

Avian mycobacteriosis (AM) is a chronic, infectious disease caused by Mycobacterium avium complex (MAC) organisms [START_REF] Thoen | Tuberculosis[END_REF][START_REF] Thorel | Mycobacterium avium infections in animals. Literature review[END_REF], although other mycobacteria can produce mycobacteriosis in birds [START_REF] Hoop | Etiological agents of mycobacterioses in pet birds between 1986 and 1995[END_REF][START_REF] Tell | Mycobacteriosis in birds[END_REF].

All avian species are susceptible to infection by the MAC, although some bird orders, such as Galliformes, Columbiformes or Gruiformes may be more susceptible to the disease [START_REF] Friend | Tuberculosis (avian)[END_REF]. AM is a relatively common disease in domestic flocks [START_REF] González | Outbreak of avian tuberculosis in 48-week-old commercial layer hen flock[END_REF][START_REF] Bougiouklis | Outbreak of avian mycobacteriosis in a flock of two-year-old domestic pigeons (Columba livia f. domestica)[END_REF], or in captive wild birds [START_REF] Singbeil | Isolation of Mycobacterium avium from ringneck pheasants (Phasianus colchicus)[END_REF][START_REF] Marco | Mycobacterium infection in a captive-reared capercaillie (Tetrao urogallus)[END_REF][START_REF] Dvorska | Avian tuberculosis in naturally infected captive water birds of the Ardeideae and Threskiornithidae families studied by serotyping, IS901 RFLP typing, and virulence for poultry[END_REF]. This is mainly due to the persistence of the organism once established, its contagiousness and its ability to survive in the environment [START_REF] Thorel | Mycobacterium avium infections in animals. Literature review[END_REF]. Nevertheless, reports of AM in free-living wild birds are not uncommon [START_REF] Smit | Avian tuberculosis in wild birds in the Netherlands[END_REF][START_REF] Millán | Mycobacterium avium infection in wild red-legged partridges (Alectoris rufa L.)[END_REF][START_REF] Gerhold | Avian tuberculosis in a wild turkey[END_REF].

Captive raptorial birds are not free of suffering from AM [START_REF] Van Nie | Tuberculosis in raptorial birds. Clinical cases and differential diagnosis[END_REF].

However, in free-living raptors, cases are only sporadically reported. [START_REF] Kaliner | Dual infection of an African fish eagle with acid-fast bacilli and an Aspergillus sp[END_REF] detected a case of AM in an African fish eagle (Haliaeetus vocifer). In North America, single cases of AM were reported in two red-tailed hawks (Buteo jamaicensis) [START_REF] Emerson | Mycobacteriosis in a red-tailed hawk[END_REF][START_REF] Sykes | Tuberculosis in a red-tailed hawk (Buteo jamaicensis)[END_REF] and in two American bald eagles (Haliaeetus leucocephalus) [START_REF] Hoenerhoff | Mycobacteriosis in an American bald eagle (Haliaeetus leucocephalus)[END_REF][START_REF] Heatley | Disseminated mycobacteriosis in a bald eagle (Haliaeetus leucocephalus)[END_REF]. In Europe, reports include those of two common kestrels (Falco tinnunculus) [START_REF] Wilson | Avian tuberculosis, an account of the disease in poultry, captive birds and wild birds[END_REF][START_REF] Bucke | Tuberculosis in a barn owl (Tyto alba)[END_REF]) and a barn owl (Tyto alba) [START_REF] Wilson | Avian tuberculosis, an account of the disease in poultry, captive birds and wild birds[END_REF]. [START_REF] Smit | Avian tuberculosis in wild birds in the Netherlands[END_REF] isolated M. avium from multiple dead-found raptor species, both diurnal and nocturnal. The only reported case in Spain was detected in a Eurasian goshawk (Accipiter gentilis) from southern peninsular Spain [START_REF] Aranaz | Laboratory diagnosis of avian mycobacteriosis[END_REF]. In the present article we report the

Material and Methods

In the period 2004-2007, 589 free-living raptors were submitted for necropsy at the "Cofib" rehabilitation centre at Majorca (Table 1). When gross lesions compatible with mycobacteriosis (e.g. granuloma) were identified, tissue samples of the affected organs were frozen at -20 o C and analyzed for the detection and identification of mycobacterias.

Tissues from each animal were homogenized with sterile distilled water and decontaminated with 0.35% hexadecylpyridinium chloride (Sigma Aldrich Chemie GmbH, Buch, Switzerland) for 30 min (Corner and Trajstman, 1988), centrifuged at 1,068 x g for 30 min and cultured onto Colestos, 0.2% (wt/vol) pyruvate-enriched Löwenstein-Jensen media (bioMèrieux España and Biomedics, Madrid, Spain) and Middlebrook 7H10 (Biocult Laboratorios, Madrid, Spain) at 37ºC. Isolates were identified by staining for acidalcohol fastness, a multiplex PCR amplification targeting the Mycobacterium specific 16S rRNA fragment [START_REF] Boddinghaus | Detection and identification of mycobacteria by amplification of rRNA[END_REF][START_REF] Wilton | Detection and identification of multiple mycobacterial pathogens by DNA amplification in a single tube[END_REF], and PCRs aimed at specific insertion sequences IS901 and IS1245 [START_REF] Kunze | Biologically distinct subtypes of Mycobacterium avium differ in possession of insertion sequence IS901[END_REF][START_REF] Guerrero | A novel insertion element from Mycobacterium avium, IS1245, is a specific target for analysis of strain relatedness[END_REF].

Recent studies have shown the usefulness of tandem repeats or repetitive units to characterize members of MAC [START_REF] Bull | Mycobacterial interspersed repetitive units (MIRU) differentiate Mycobacterium avium subspecies paratuberculosis from other species of the Mycobacterium avium complex[END_REF][START_REF] Thibault | New variable-number tandem-repeat markers for typing Mycobacterium avium subsp. paratuberculosis and M. avium strains: comparison with IS900 and IS1245 restriction fragment length polymorphism typing[END_REF][START_REF] Mobius | High genetic diversity among Mycobacterium avium subsp. paratuberculosis strains from German cattle herds shown by combination of IS900 restriction fragment length polymorphism analysis and mycobacterial interspersed repetitive unit-variable-number tandem-repeat typing[END_REF]. Included within these tandem repeats are the variable number tandem repeats (VNTR), which are repetitive units dispersed throughout the genome with a length between 10-100 bp [START_REF] Supply | Variable human minisatellite-like regions in the Mycobacterium tuberculosis genome[END_REF]; and also the mycobacterial interspersed repetitive units (MIRU), small interspersed repetitive elements described by [START_REF] Supply | Identification of novel intergenic repetitive units in a mycobacterial two-component system operon[END_REF] in with six specific loci previously published: MIRU-1, MIRU-2, MIRU-3, MIRU-4 [START_REF] Bull | Mycobacterial interspersed repetitive units (MIRU) differentiate Mycobacterium avium subspecies paratuberculosis from other species of the Mycobacterium avium complex[END_REF], VNTR-25 and VNTR-32 (Thibault et al. 2007;[START_REF] Mobius | High genetic diversity among Mycobacterium avium subsp. paratuberculosis strains from German cattle herds shown by combination of IS900 restriction fragment length polymorphism analysis and mycobacterial interspersed repetitive unit-variable-number tandem-repeat typing[END_REF], using the same specific primers described (Table 2).

PCR mix contained 5 µl of sample DNA, 1x standard reaction buffer with 1.5 mM MgCl 2 , 200 µM deoxynucleoside triphosphate (Biotools, B&M Labs S.A, Madrid, Spain), forward and reverse primers (Roche Diagnostics S.L., St. Cugat del Vallés, Spain) (0.4 µM), 10% of DMSO (Sigma Aldrich Chemie GmbH) and 2.5 U of Hot-Start-Taq-Polymerase (Qiagen GmbH, Hilden, Germany), in a final volume of 50 µl. For MIRU-3, primer concentrations were three times higher than for the rest of the loci [START_REF] Mobius | High genetic diversity among Mycobacterium avium subsp. paratuberculosis strains from German cattle herds shown by combination of IS900 restriction fragment length polymorphism analysis and mycobacterial interspersed repetitive unit-variable-number tandem-repeat typing[END_REF].

The amplification reactions for these MIRU-VNTR loci were: denaturation at 95°C for 15 min, followed by 40 cycles of denaturation at 95°C for 30 sec, annealing at 58°C for 1 min, extension at 72°C for 2 min, with a final cycle of extension at 72°C for 10 min; conditions reported by Frothingham and Meeker-O'Connell (1998) with slight modifications. In the case of MIRU-3, MIRU-4 and VNTR-32 the same recommended melting temperatures [START_REF] Mobius | High genetic diversity among Mycobacterium avium subsp. paratuberculosis strains from German cattle herds shown by combination of IS900 restriction fragment length polymorphism analysis and mycobacterial interspersed repetitive unit-variable-number tandem-repeat typing[END_REF][START_REF] Thibault | New variable-number tandem-repeat markers for typing Mycobacterium avium subsp. paratuberculosis and M. avium strains: comparison with IS900 and IS1245 restriction fragment length polymorphism typing[END_REF] were used.

Finally, PCR amplicons were separated in a 2.5% agarose gel, stained with SYBR safe (Invitrogen, product no. 531478) and using 100 bp molecular ladder (Biotools B&M Labs, Madrid, Spain) to detect the variation in the number of repeats for each of the locus.

Chi 2 test was used to analyze differences in the observed prevalence among bird species and years. Tissue samples from all the birds with lesions were cultured and then M. avium subsp.

Results

F

avium was identified by PCR amplification of the insertion sequences IS901 and IS1245.

Lesions were observed in three species: 12 of 297 common kestrels (4.0%), one of 58 long-eared owls (Asio otus) (1.7%) and one of 109 scops owls (Otus scops) (0.9%). No lesions were observed in the other species analyzed (Table 1). The kestrels were 9 adult birds (5 males, 4 females) and 3 sub-adults between 1 and 2 years-old (2 males, 1 female); the long-eared owl was an adult female; and the scops owl was a sub-adult female. The affected birds arrived throughout the year, dead or sick, from multiple locations in Majorca (Figure 1). Non-affected raptors also arrived from all around the island, including from the locations were infected birds were found.

Infected birds included road-killed birds (n=5) as well as debilitated birds (n=9). Of the latter, one bird suffered from gunshot wounds while the others presented no traumatic lesions. The latter birds arrived as a rule emaciated but did not present other clinical signs.

With the exception of one kestrel that survived for eight months after arrival, the other affected birds died within few days (usually 1-2 days, mean= 11.8 ±16.9 hours). Another kestrel was euthanatized two months after arrival for humane reasons. On necropsy, all the birds were cachectic and presented white-yellowish nodules from pinpoint size to 1 cm in diameter in diverse organs, chiefly in liver, spleen and intestine (Table 3 andFigure 2 genotypes, where allelic profile number 1 (1-8-1-5-9-5) and 5 (1-8-1-5-7-5) represented 42.86% and 28.57% of the isolates tested respectively (Table 4). The two isolates from the scops owl and the long-eared owl were both clustered into the most common allelic profile no.1, which was shared with four isolates from kestrel.

No differences were observed in the prevalence of AM lesions among years. Prevalence of AM lesions was higher in common kestrel than in barn owl, though differences were only residually significant (Chi 2 =3.13, p=0.07).

Discussion

Other than the single goshawk reported by [START_REF] Aranaz | Laboratory diagnosis of avian mycobacteriosis[END_REF], this is the first report of widespread AM in raptorial birds from Spain. As mentioned above, AM in free-living raptors has rarely been diagnosed. In the Netherlands, [START_REF] Smit | Avian tuberculosis in wild birds in the Netherlands[END_REF] isolated M. avium from, among other raptors, 17/450 (3.8%) of common kestrels, and in 3/313 (0.96%) of long-eared owls. Those prevalences are similar to these reported in the present article. As mentioned before, a case of AM was also reported in one common kestrel from UK [START_REF] Wilson | Avian tuberculosis, an account of the disease in poultry, captive birds and wild birds[END_REF]. As far as we know, AM was not previously diagnosed in a scops owl.

However, it is worth to note that the prevalence of mycobacteriosis was likely to be higher than reported here, because only tissue samples from birds with gross lesions were subjected to culture.

Affected birds were mostly adults. This is in accordance with most of the previous reports of AM in raptors [START_REF] Sykes | Tuberculosis in a red-tailed hawk (Buteo jamaicensis)[END_REF], or in other wild birds [START_REF] Millán | Mycobacterium avium infection in wild red-legged partridges (Alectoris rufa L.)[END_REF] [START_REF] Thoen | Tuberculosis[END_REF]. According to [START_REF] Thorel | Mycobacterium avium infections in animals. Literature review[END_REF], affected birds die within two months or may survive for six months depending on the extent of disease.

On necropsy, macroscopic lesions were seen in the liver, spleen and intestine of all the affected raptors, which is the typical feature of AM in birds (Skyes, 1982;[START_REF] Thorel | Mycobacterium avium infections in animals. Literature review[END_REF][START_REF] Millán | Mycobacterium avium infection in wild red-legged partridges (Alectoris rufa L.)[END_REF][START_REF] Saggese | Mycobacteriosis in naturally infected ring-neck doves (Streptopelia risoria): investigation of the association between feather colour and susceptibility to infection, disease and lesions type[END_REF]. According to the distribution of the lesions, the affected birds probably contracted the infection by ingestion. However, due to the presence of lesion in the lungs in one third of the cases, an inhalation route should not be excluded [START_REF] Kaliner | Dual infection of an African fish eagle with acid-fast bacilli and an Aspergillus sp[END_REF].

The source of the infection remains unknown. The hypothesis of a nosocomial infection can be ruled out due to the affected birds arrived dead or survived for a short period. [START_REF] Smit | Avian tuberculosis in wild birds in the Netherlands[END_REF] proposed that kestrels and other aggressive raptors may contract AM through injuries during fights, and even isolated M. avium from some local injuries.

However, in the present case no infected injuries were observed in any of these birds, though subcutaneous granulomas were detected in seven birds that may be derived from old injuries. In any case, the most feasible origin of the mycobacterias might be water or food sources. Therefore, feeding ecology may explain the differences in prevalence among species. Very little is known about the diet of the three affected species in Majorca. In the Mediterranean part of the peninsular Spain, the common kestrel feeds mainly upon lizards (Lacerta spp.), which are no present on Majorca, passerines, several species of insects and micromammals [START_REF] Valverde | Estructura de una comunidad de vertebrados terrestres[END_REF][START_REF] Veiga | Ecología de las rapaces de un ecosistema mediterráneo de montaña[END_REF]. For the long-eared owl, the most important prey species are Murinae, followed by Arvicolinae (not present in Balearic Islands) and passerine birds [START_REF] Corral | Contribución al estudio de la alimentación de Asio otus en sur de España[END_REF][START_REF] Corral | Contribución al estudio de la alimentación de Asio otus en sur de España[END_REF], although this species depends less on rodents in Mediterranean regions [START_REF] García | Notas sobre la variación estacional y geográfica de la dieta del Búho Chico Asio otus[END_REF]. However, the barn owl also feeds mainly on rodents in Balearic Islands [START_REF] Sommer | Feeding of the barn owl, Tyto alba with first record of the European free-tailed bat, Tadarida teniotis on the island of Ibiza (Spain, Balearics)[END_REF], and no individual out of 75 would indicate a species specific difference in susceptibility, with barn owls being less susceptible. However, several cases of AM have been reported in the species [START_REF] Wilson | Avian tuberculosis, an account of the disease in poultry, captive birds and wild birds[END_REF][START_REF] Bucke | Tuberculosis in a barn owl (Tyto alba)[END_REF]. Thus, differences may be related to prey accessibility more than to susceptibility.

According to [START_REF] Friend | Tuberculosis (avian)[END_REF], AM is most commonly seen in scavenger species.

Thus, an alternative hypothesis of the infections is that the affected birds contracted AM through the scavenging of carcasses of diseased red-legged partridges or pigeons (Columba spp.), which consitute too large a prey for the affected raptors [START_REF] Calderón | El papel de la perdiz roja (Alectoris rufa) en la dieta de los predadores ibéricos[END_REF]. Due to the intense, often illegal predator control carried out in Spanish hunting estates, sick animals are not removed from the environment by carnivores, developing disseminated AM and even dying by the disease, as has been sporadically observed in partridges [START_REF] Millán | Mycobacterium avium infection in wild red-legged partridges (Alectoris rufa L.)[END_REF].

Regarding the molecular characterization by MIRU-VNTR, it discriminated the M. a. avium isolates into six different genotypes. From the loci included for the analysis, MIRU-3 yielded the highest number of allelic variants in comparison with the rest of the loci. MIRU-3 is located between a two-component regulatory system SenX3-RegX3.

Former studies found the presence of a lower number of TRs at this locus in the M. bovis (BCG) and M. avium subspecies paratuberculosis (316F) vaccine strains in comparison to the rest of the strains they tested [START_REF] Bull | Mycobacterial interspersed repetitive units (MIRU) differentiate Mycobacterium avium subspecies paratuberculosis from other species of the Mycobacterium avium complex[END_REF][START_REF] Magdalena | Specific differentiation between Mycobacterium bovis BCG and virulent strains of the Mycobacterium tuberculosis complex[END_REF]). Probably differences in pathogenesis and progression of the disease have a relationship with the variation of TR units at this particular locus. To our knowledge, this is the first analysis of M. a. avium isolates from animals using MIRU-VNTR typing; therefore we cannot further assess our results in comparison to other populations. However, available information from literature shows that the genotypes isolated from raptors described in this report would be 10 related to that described for the M. a. avium reference strain ATCC 25291 (1-7-1-5-5-5) [START_REF] Bull | Mycobacterial interspersed repetitive units (MIRU) differentiate Mycobacterium avium subspecies paratuberculosis from other species of the Mycobacterium avium complex[END_REF][START_REF] Inagaki | Comparison of a variable-number tandem-repeat (VNTR) method for typing Mycobacterium avium with mycobacterial interspersed repetitive-unit-VNTR and IS1245 restriction fragment length polymorphism typing[END_REF]. Further studies concerning this issue would be necessary.

In the light of the cases presented in this article, AM must be considered a factor in the conservation of raptors in Majorca and the rest of the Balearic Islands. An undetected source is acting as a reservoir of mycobacteria that is currently affecting raptors and potentially may affect other protected species, livestock, or humans. As the infected raptors were retrieved in several points of the Majorca Island, and six different genotypes were found, there may be multiple infection focuses. However, though the affected species are territorial, could have become infected in other locations different from where they were found. In addition, some of the affected birds could be migrant individuals and may have become infected in remote locations. Studies to detect the different reservoirs of this infection in Majorca are currently being performed.

Accipiter nisus 0 - 1 - 1 - 2 - 4 0 (0-53) Circus pygargus 0 - 0 - 0 - 1 - 1 0 (0-95) Circus aeruginosus 0 - 1 - 1 - 0 - 2 0 (0-77) Pandion haliaetus 0 - 1 - 0 - 0 - 1 0 (0-95) Aegypius monachus 0 - 0 - 1 - 0 - 1 0 (0-

  in free-living raptors that were brought to a wildlife rehabilitation centre at Majorca Island, Spain.

  M. tuberculosis. Isolates of M a. avium were sub-typified by MIRU-VNTR

  ).Lesions did not markedly differ among species. No other signs were observed in the infected birds, with the exception of one kestrel in which oral lesions of trichomoniasis confirmed by microscopy were observed.Regarding MIRU-VNTR typing, only MIRU-3 and MIRU-4 showed allelic variation among the M. a. avium isolates tested with five and two allelic variants

  combination of loci allowed the differentiation into six different

  affected. If rodents were a major source of mycobacteria, this

Figure 1 .

 1 Figure Legends

Figure 2 .F

 2 Figure 2. Avian tuberculosis granulomas in a scops owl (A) and two common kestrels (B,

F

  Number or TRs and allelic variants observed in the Mycobacterium avium subspecies avium isolates with the combination of loci used.Avian tuberculosis granulomas in a scops owl (A) and two common kestrels (B, C). A: in liver (box), lung (arrowhead), spleen (dotted arrow) and intestine (arrow); B: in liver (arrow) and subcutaneous tissue (arrowhead); C: in lung. 82x53mm (300 x 300 DPI)

  . Older animals are more commonly affected by mycobacteriosis because of the long incubation

	period and accumulated risk of exposure
	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	O n l
	y
	8

Table 1 .

 1 Species of raptor necropsied per year and prevalence (in %) of avian mycobacteriosis in a wildlife rehabilitation centre in MajorcaIsland (Spain).

		2004		2005		2006		2007			Total
		n (pos.) prev. n (pos.) prev. n (pos.) prev. n (pos.) prev.	n (pos.)	prev. (95% C.I.)
	Accipitriformes
	Hieraaetus pennatus	2	-	7	-	5	-	0	-	13	0 (0-19)

Table 2 .

 2 List of primers and melting temperatures [Tm ( o C)] for the MIRU-VNTR loci used in this study.

	VNTR-MIRU	Reference	Primer F (5'-3')	Primer R (5'-3')	Tm
	MIRU-1	Bull et al. 2003	cgcggacttgatggtctc	ccgttgtccaggtggagt	58
	MIRU-2	Bull et al. 2003	gaacgaagatcctgggactg	cgacgacgaacacctcaac	58
	MIRU-3	Bull et al. 2003	acattcaccctgtccattcc	cctccttacggagcaggaa	52
	MIRU-4	Bull et al. 2003	cgttcagcctgtgcatgg	caagtcgtcacgggcaac	53
	25	Thibault et al. 2007	gtcaagggatcggcgagg	tggacttgagcacggtcat	58
	32	Thibault et al. 2007	ccacagggtttttggtgaag ggaaatccaacagcaaggac	55

Table 3 .

 3 Locations of the granulomas due to avian mycobacteriosis observed in 14 raptors in Majorca Island(Spain). Some individuals presented lesions in multiple organs.

	Organ / Tissue	n	Locations
	Liver	14
	Spleen	14
	Intestine	14
	Subcutaneous tissue	7	Neck (7), mandible (1)
	Gizzard	7
	Lung	5
	Kidney	4
	Pancreas	1
	Muscle	3	Stifle (2), intercostal (2), pectoral (1), neck (1)
	Substernal serosa	1
	Oral mucosa	1	Sublingual

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

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

Acknowledgements

We wish to thank the team of "Protecció d'especies" (Conselleria de Medi Ambient, Govern de les Illes Balears), Rangers of the Fish and Game Service (Govern de les Illes Balears), members of the SEPRONA (Servicio de Protección de la Naturaleza, Guardia Civil), and all the individual people who brought animals to the rescue centre.

We also thank Devin J. Morey for the careful revision of the manuscript.


De Boever

Kristel Demeyere

Bart Lambrecht

Patrick De Backer

Evelyne Meyer

S De Boever

S Croubels

FLOW CYTOMETRIC DIFFERENTIATION OF AVIAN LEUKOCYTES AND ANALYSIS OF THEIR INTRACELLULAR CYTOKINE EXPRESSION

Keywords: chicken, leukocyte identification, CD45, cytokine expression

A flow cytometric method for the identification of chicken blood leukocyte subpopulations and thrombocytes was developed. An anti-chicken CD45 phycoerythrin-labelled antibody was used to distinguish leukocytes from red blood cell nuclei. Leukocytes and thrombocytes were identified using a combination of their CD45-positivity and their typical side scatter properties. The identity of the CD45-positive cells was confirmed by sorting the subpopulations and subsequent light microscopic evaluation.

In these differentiated cell populations, intracellular expression analysis of the proinflammatory cytokines interleukin-1β and IL-6 was subsequently optimised on whole blood after in vitro stimulation with lipopolysaccharide from Escherichia coli strain O127:B8.

Introduction

Flow cytometry is a preferred method to phenotype ex vivo derived individual leukocytes from various species (Bohls et al., 2006). The flow cytometric differentiation of avian leukocytes has been described for monocytes [START_REF] Mast | Characterization of chicken monocytes, macrophages and interdigitating cells by the monoclonal antibody KUL01[END_REF], lymphocyte subpopulations [START_REF] Fair | Immunophenotyping of chicken peripheral blood lymphocyte subpopulations: Individual variability and repeatability[END_REF] and thrombocytes (Viertlboeck and Göbel, 2007). [START_REF] Uchiyama | Counting absolute number of lymphocytes in quail whole blood by flow cytometry[END_REF] used anon-specific fluorescent lipophilic dye DiOC 6 (3) for differentiation and counting of quail and chicken blood cells.

In mammals, the common leukocyte antigen CD45 is a haemopoietic cell-specific surface glycoprotein with a cytoplasmic tyrosine phosphatase domain that is believed to play a role in T-and B cell receptor signal transduction [START_REF] Tchilian | Altered CD45 expression and disease[END_REF]. Various isoforms are expressed on different types of lymphohematopoietic cells, while it is absent on 3 mammalian platelets and mature erythrocytes. The avian CD45 homolog is expressed on all chicken leukocytes but not on erythroid cells [START_REF] Paramithiotis | High levels of CD45 are co-ordinately expressed with CD4 and CD8 on avian thymocytes[END_REF]. Chicken thrombocytes, which are the equivalent of mammalian platelets, also express the CD45 antigen although at consistently lower levels compared to lymphocytes [START_REF] Horiuchi | A monoclonal antibody against chicken thrombocytes reacts with the cells of thrombocyte lineage[END_REF]Viertlboeck and Göbel, 2007).

Avian heterophils, which are the equivalent of mammalian neutrophils, blood monocytes, and thrombocytes, release a cascade of cytokines upon stimulation with various infectious or non-infectious agents such as lipopolysaccharide (LPS) [START_REF] Rhind | Intracellular monocyte and serum cytokine expression is modulated by exhausting exercise and cold exposure[END_REF][START_REF] Kogut | Expression and function of Toll-like receptors in chicken heterophils[END_REF][START_REF] Scott | Thrombocytes respond to lipopolysaccharide through Tolllike receptor-4, and MAP kinase and NF-κB pathways leading to expression of interleukin 6 and cyclooxygenase-2 with production of prostaglandin E2[END_REF][START_REF] Ferdous | Broiler chick thrombocyte response to lipopolysaccharide[END_REF]. LPS is commonly used to induce an acute phase reaction in humans and animals because of its proinflammatory properties [START_REF] Skarnes | Role of prostaglandin E in the biphasic fever response to endotoxin[END_REF][START_REF] Klosterhalfen | Time course of various inflammatory mediators during recurrent endotoxemia[END_REF]Erroi et al., 1993;[START_REF] Franco | The in vivo kinetics of tissue factor messenger RNA expression during human endotoxemia: relationship with activation of coagulation[END_REF][START_REF] Maxwell | Contrasting the role of costimulation and the natural adjuvant lipopolysaccharide during the induction of T cell immunity[END_REF]De Boever et al., 2008). LPS interacts with the innate immune system through Toll-like receptor 4 (TLR4), a member of the family of cell-surface and endosomally expressed receptors that recognize conserved molecular patterns unique to microorganisms (Palsson-Mcdermott and O'Neill, 2004;[START_REF] Janssens | Role of Toll-like receptor in pathogen recognition[END_REF]. Recognition of microbial components by TLRs triggers a cascade of cellular signals that culminates in the activation of Nuclear Factor -κB which leads to inflammatory gene expression (Verstrepen et al., 2008). Acute endotoxemia represents a tool for gaining insight into inflammatory processes [START_REF] Remick | Evaluation of endotoxin models for the study of sepsis[END_REF]. Furthermore, such a controlled inflammation model is of interest to test the pharmacodynamic properties of anti-inflammatory drugs. To be able to monitor the progress of the inflammatory response and evaluate the immune-modulatory effect of anti-inflammatory drugs, several parameters can be determined including body temperature, blood pressure, cytokine expression and the production of acute phase proteins.

Of relevance in this context is that the cytokines IL-1β and IL-6 have been characterized as major endogenous pyrogens or fever-inducers using several animal models [START_REF] Kluger | Fever: role of pyrogens and cryogens[END_REF] , 1998;[START_REF] Leon | An antipyretic role for interleukin -10 in LPS fever in mice[END_REF]. Flow cytometry is a powerful analytical technique allowing the quantitative assessment of cytokine production at the single cell level.

Using multiple colour-labelling, the simultaneous identification of different selected parameters can be performed (Bueno et al., 2001;[START_REF] Schultz | Influence of specimen age and use of different negative controls in determination of intracytoplasmic levels of cytokines after whole-blood culture assay[END_REF][START_REF] Schuerwegh | Comparison of intracellular cytokine production with extracellular cytokine levels using two flow cytometric techniques[END_REF].

In this report we describe a flow cytometric method for the identification of avian leukocyte subpopulations and thrombocytes based on their combined CD45 positivity and side scatter properties. The major advantage of this technique is the use of a single antibody directed against a common leukocyte antigen for the identification of the leukocyte subpopulations and thrombocytes. Furthermore, intracellular detection of IL-1β and IL-6 was performed in these different leukocyte subpopulations. This new tool allows us to monitor the cytokine expression after in vivo LPS administration in broiler chickens.

Materials and Methods

Blood samples. Blood samples (0.5 ml) were collected from the leg vein from laying hens, kept as blood donors, into heparin containing tubes (Venoject ® , Terumo Corp., Tokyo, Japan). This procedure was approved by the Ethics committee of the Faculty of Veterinary Medicine (EC 2006/035).

Reagents and antibodies. The phycoerythrin (PE) conjugated Cell Lab mouse monoclonal anti-chicken CD45 antibody, isotype IgMκ was purchased from Analis (Gent, Belgium).

Two polyclonal IgG antibodies, both raised in rabbits and directed against chicken IL-1β (AHP941Z) and IL-6 (AHP942Z), respectively, were used as primary antibodies (AbD LPS of Escherichia coli strain O127:B8 was purchased from Sigma-Aldrich (Bornem, Belgium).

Staining procedure and flow cytometric analysis. Chicken leukocytes and thrombocytes were obtained from whole blood after lysis of the erythrocytes. For this purpose, 4.5 ml of BD FACS Lysing solution, 1/10 diluted in distilled water, was added to 0.5 ml of blood in a Falcon tube for 10 minutes (min) at room temperature. After washing andcentrifugation for 5 min at 500 g, the pellet was resuspended in 500 µl RPMI and 50 µl was pipetted into a flow cytometer tube. Following titration, testing 4, 6, 8, 10 and 20 µg/ml as concentrations, the PE-labelled anti-CD45 was added in a final concentration of 6 µg/ml and samples were incubated for 30 min at 4°C.

For the in vitro stimulation with LPS, 0.5 ml of whole blood was incubated with LPS at a final concentration of 10 µg/ml for 3 hours at 37°C in a shaking warm water bath (Memmert,Model WNB 14,Schwabad,Germany). Subsequently, erythrocytes were lysed and leukocytes and thrombocytes were labelled as described above, followed by permeabilization using BD FACS Permeabilizing solution 2, 1/10 diluted with distilled water, again for 10 min at room temperature. As for CD45, the optimal working dilutions for the primary antibodies directed against IL-1β and IL-6 were also optimized by means of titration, testing 1.6, 3.3, 6.6, 10, 12.5, 25, 50 6 50 µg/ml was used and samples were incubated for 30 min at room temperature. Blocking was performed by adding 20% goat serum in RPMI for 30 min at room temperature. Finally, the APC-labelled secondary antibody was added at a concentration of 6 µg/ml for 30 min at 4°C. Cells were resuspended in 450 µl CellFIX, 1/10 diluted with distilled water. The optimal antibody concentrations of the primary and secondary antibodies were also determined by titration.

Samples were analysed using a double laser bench top flow cytometer FACSCanto (Beckton Dickinson Biosciences) and the data were analysed with FACSDiva Software (Becton Dickinson). Excitation was performed with a 488 nm argon-ion laser and emission collected using a 530/30 nm band pass (BP) filter for FITC conjugates, a 585/42 nm BP filter for PE conjugates and a 660/20 nm BP filter for APC conjugates. Two basic parameters, i.e. the side scatter (SSC), which is proportional to the cellular granularity, and the forward scatter (FSC) which is proportional to cell size, were analyzed. For the CD45 expression, the amount of CD45 antigen present per cell (mean fluorescence intensity, MFI) was determined in all populations as presented in Figure 3.

For the cytokine expression, the % of IL-1β and IL-6 positive cells and their MFI were assessed after differentiation based on CD45 positivity and SSC as described above.

Additionally, flow cytometric cell sorting was performed on CD45 + cells (BD FACSAria II, California, USA). The sorted cell populations were stained (Diff-Quick, Acros Organics N.V., Geel, Belgium) after cytocentrifugation (Shandon, UK) and subsequent analysis of the slides by light microscopy (Olympus BX-UCB, Pennsylvania, USA).

The aim of this study was two-fold, i.e. to develop (1) a straightforward method to differentiate avian white blood cells and (2) to enable the subsequent assessment of their individual intracellular cytokine expression after LPS challenge.

Results and Discussion

(1) Flow cytometric identification of avian monocytes [START_REF] Mast | Characterization of chicken monocytes, macrophages and interdigitating cells by the monoclonal antibody KUL01[END_REF], lymphocyte subpopulations (Bohls et al., 2006;[START_REF] Fair | Immunophenotyping of chicken peripheral blood lymphocyte subpopulations: Individual variability and repeatability[END_REF] and thrombocytes (Viertlboeck and Göbel, 2007) has already been performed. The non-specific fluorescent lipophilic dye DiOC 6 (3) used by [START_REF] Uchiyama | Counting absolute number of lymphocytes in quail whole blood by flow cytometry[END_REF] for differentiation, was very useful for counting of quail and chicken blood cells. We succeeded in the development of a straightforward method to differentiate leukocyte subpopulations and thrombocytes using a single antibody directed against the common leukocyte antigen. The anti-CD45 antibody used in the current study allows a better discrimination between the leukocyte subpopulations. This is especially the case for lymphocytes and thrombocytes because of the reduced interference with the red blood cell nuclei. The presence of nucleated erythrocytes and thrombocytes was indeed a challenge as this is a major difference between mammalian and avian blood cells (Campbell and Ellis, 2007). Lysis of the erythrocytes was tested comparing a Tris-aminomethane/NH 4 Cl buffer with the commercial lysing solution, with the latter giving the best results (data not shown). Nevertheless, after lysis, the red blood cell nuclei were still present and since their size is similar to that of a small lymphocyte, the discrimination between both events was not possible based on the commonly used FSC versus SSC dot-plot as illustrated in Figure 1 (A).

However, the combined use of an anti-chicken CD45 antibody and the SSC, enabled the discrimination between lymphocytes and red blood cell nuclei. On the representative dot plot showing CD45 + cells versus SSC, the different subpopulations, i.e. lymphocytes, thrombocytes, monocytes and heterophils, could be gated (Figure 1 B). The position of the different subpopulations on the dot plot is similar to that seen in mammalian species, except for thrombocytes, which are not present in mammalian blood (Figure 1 C). The optimal concentration of the anti-CD45 antibody (6 µg/ml) was determined by titration (data not shown).

To confirm the identity of the gated populations on the anti CD45-PE versus SSC dotplot, additional sorting was performed, followed by light microscopy (Figure 2). Population L was composed of lymphocytes, characterized by a round nucleus and weakly basophilic cytoplasm. Population M, contains monocytes with the hallmarks of being the largest leukocytes and having a large nucleus. The heterophils (population H) are characterized by a lobed nucleus and abundant eosinophilic granules in the cytoplasm. For the heterophils and lymphocytes, a purity of 96% and 89.4% could be calculated, respectively. For the monocytes and thrombocytes no percentages can be provided as only a few cells were present on the slide.

(2) In a second series of experiments the optimized cell differentiation method was combined with intracellular detection of pro-inflammatory cytokines. More specifically, IL-1β and IL-6 were chosen since these cytokines have been implicated as key mediators of fever [START_REF] Kluger | Fever: role of pyrogens and cryogens[END_REF][START_REF] Jakab | The acute phase reaction syndrome: the acute phase reactants[END_REF][START_REF] Leon | An antipyretic role for interleukin -10 in LPS fever in mice[END_REF]. Commercial antibodies directed against chicken IL-1β and IL-6 are of polyclonal origin and have not yet been tested for flow cytometry. In the current experiment, the method was optimized after in vitro stimulation of heparin-treated whole blood samples with LPS, because cell activation has been reported to be better and cytokine expression is more efficient than with other anticoagulants (Coló Brunialti et al., 2002). Both a commercial lysing agent, BD FACS Lysing solution and a trisaminomethane/NH 4 Cl buffer were tested, with the former giving the best results. It should be remarked that we deliberately chose not to use a secretion block in the in vitro experiments, to mimic the in vivo situation as closely as possible. Moreover, the use of a fixation-containing lysing agent in the first step of the protocol, prevents cytokine secretion after sampling. Several amounts of the commercial permeabilizing solution were tested, with 500 µl resulting in the highest percentage of cytokine expressing cells and the highest MFI (data not shown). As for CD45, the optimal working dilutions for the primary antibodies directed against IL-1β and IL-6 were also optimized by means of titration (data not shown). An APClabeled secondary antibody was chosen for intracellular cytokine detection. APC belongs to a family of phycobiliproteins with a broad excitation spectrum and a large Stoke's shift with a high quantum yield fluorescence [START_REF] Jung | A novel and inexpensive source of allophycocyanin for multicolour flow cytometry[END_REF][START_REF] Telford | Cryptomonad algal phycobiliproteins as fluorochromes for extracellular and intracellular antigen detection by flow cytometry[END_REF]. The optimal concentration of the secondary antibody was also determined by titration.

A fluorescein isothiocyanate (FITC) labelled antibody was first tested as secondary antibody, but since preliminary data confirmed that FITC stains the cytoplasmic granules of avian heterophils [START_REF] Rath | Fluorescein isothiocyanate staining and characterization of avian heterophils[END_REF], this fluorochrome was not suited for intracellular cytokine detection nor could FITC-labelled antibodies be used for the identification of the different leukocyte subpopulations.

After stimulation of whole blood with LPS, cytokine production was measured in the leukocyte subpopulations and thrombocytes as illustrated in Figure 3. The MFI of LPSstimulated cells for both cytokines is shown in Table 1. On the dot plot of samples for which only the CD45-PE antibody and the secondary APC-labelled antibody were added (Figure 3 A), a grid was placed to be able to discriminate between specific and non-specific staining of the secondary antibody. As could be expected from literature [START_REF] Schultz | Influence of specimen age and use of different negative controls in determination of intracytoplasmic levels of cytokines after whole-blood culture assay[END_REF][START_REF] Schuerwegh | Comparison of intracellular cytokine production with extracellular cytokine levels using two flow cytometric techniques[END_REF], stimulation with LPS evoked a right-shift of the populations, indicating the intracellular presence of IL-1β and IL-6. Monocytes showed the largest shift, both for IL-1β and IL-6. Thrombocytes are also capable of synthesizing cytokines. This observation is in accordance with Scott et al. (2007) and [START_REF] Ferdous | Broiler chick thrombocyte response to lipopolysaccharide[END_REF] [START_REF] Kogut | Expression and function of Toll-like receptors in chicken heterophils[END_REF]. However, the latter authors mention that mRNA levels do not necessarily equate to bioactive protein. A confocal microscopic image of a lymphocyte expressing IL-6 is shown in Figure 2B.

Finally, it should be remarked that heterophils clearly expressed more CD45 molecules after LPS stimulation (data not shown). This observation corroborates findings in human granulocytes that contain an intracellular pool of CD45 and upon stimulation with formylmethionylleucylphenylalanine, ionophore A23187 or LPS increase the surface expression of CD45 [START_REF] Lacal | Intracellular location of T200 and Mol glycoproteins in human neutrophils[END_REF]Caldwell et al., 1991;[START_REF] Stie | Reorganization of the human neutrophil plasma membrane is associated with functional priming: implications for neutrophil preparations[END_REF].

In conclusion, we developed a straightforward method for the differentiation of the different leukocyte subpopulations in avian blood, using a monoclonal antibody directed against chicken CD45 in combination with SSC properties. Furthermore, intracellular IL-1β and IL-6 expression in these individual populations was assessed. This method can now be applied for the detection of intracellular cytokine expression after intravenous administration of LPS to broiler chickens. (2008). Immunophenotyping of chicken peripheral blood lymphocyte subpopulations: Individual variability and repeatability. Veterinary Immunology and Immunopathology, 125, 286-273. Ferdous, F., Maurice, D., Scott, T. (2008). Broiler chick thrombocyte response to lipopolysaccharide. Poultry Science, 87, 61-63.

Franco, R.F., de Jonge, E., Dekkers, P.E.P., Timmerman, J.J., Spek, C.A., van Deventer, S.J.H., [START_REF] Franco | The in vivo kinetics of tissue factor messenger RNA expression during human endotoxemia: relationship with activation of coagulation[END_REF]. The in vivo kinetics of tissue factor messenger RNA expression during human endotoxemia: relationship with activation of coagulation. Blood, 96, 554-559. Horiuchi, H., Tanaka, K., Shigata, A., Yoshida, K., Kushima, K., Ohta, H., Furusawa, S., Matsuda, H. (2004). A monoclonal antibody against chicken thrombocytes reacts with the cells of thrombocyte lineage. Immunolology, 66, 243-250. [START_REF] Janssens | Role of Toll-like receptor in pathogen recognition[END_REF]. Role of Toll-like receptor in pathogen recognition. Clinical Microbiology Reviews, 16, 637-646. Jakab, L., Kalabay, L. (1998). The acute phase reaction syndrome: the acute phase reactants.

Acta Microbiologica et Immunologica Hungarica, 45, 409-418. Jung, T.M., Dailey, M.O. (1989). A novel and inexpensive source of allophycocyanin for multicolour flow cytometry. Journal of Immunological Methods, 121, 9-18. [START_REF] Klosterhalfen | Time course of various inflammatory mediators during recurrent endotoxemia[END_REF]. Time course of various inflammatory mediators during recurrent endotoxemia. Biochemical Pharmacology, 43, 2103-2109[START_REF] Kluger | Fever: role of pyrogens and cryogens[END_REF]. Fever: role of pyrogens and cryogens. Physiological Reviews, 14, 93-127. 13 [START_REF] Kogut | Expression and function of Toll-like receptors in chicken heterophils[END_REF]. Expression and function of Toll-like receptors in chicken heterophils. Developmental and Comparative Immunology, 29, 791-807. Lacal, P., Pulidos, R., Sànchez-Madrid, F., Mollinedo, F. (1988). Intracellular location of T200 and Mol glycoproteins in human neutrophils. Journal of Biological Chemistry, 20, 9946-9951.

Leon, L.R., Kozak, W., Rudolph, K., [START_REF] Leon | An antipyretic role for interleukin -10 in LPS fever in mice[END_REF]. An antipyretic role for interleukin -10 in LPS fever in mice. American Journal of Physiology, 276, R81-89. Answer to the editor:

We would like to thank the editor and the three reviewers for their constructive remarks, which were of great help to improve this methodological manuscript. Please find below a point-by-point list of answers to the issues raised.

Concerning the specific comments of the editor, following arguments can be provided:

-the main concern raised by reviewers #1 and #2 regarding the need for an appropriate negative control has been addressed by showing that an isotype control (polyclonal antibody, ab) does not cause a positive signal. Nevertheless, based on our arguments outlined in detail to both these reviewers, we propose to include the additional control results for the selective intracellular localization but not those for the isotype control in the revised manuscript, unless decided otherwise by the editor.

-we agree that lymphocytes are not the main producer of IL-1beta and IL-6. However, according to our experimental findings and literature, lymphocytes are indeed capable of producing both these cytokines. Our flow cytrometric observations were supported by confocal microscopy after stimulation of whole blood with LPS. Cell nuclei were stained using propidium iodide (PI), an Alexa 488-labeled secondary ab was used for visualizing IL-1β/-6. Leukocyte differentiation was based on the nuclear morphology and cell size. A representative image (Figure 2B) is now included in the revised manuscript illustrating the intracellular staining of chicken lymphocytes for both cytokines.

-the data in the original Figure 3 were indeed not quantitative. To illustrate that our results

were confirmatory, the % of positive events expressed as mean ± standard deviation (µ ± SE, n = 6) are now provided in each of the upper right quadrants of the dot plots in the revised Figure 3 as requested. Moreover, the adequate reproducibility (n = 6) of our method is further illustrated in table 1 showing the mean fluorescence intensity (MFI) data (µ ± SE) for each of the different cell types, both for IL-1β and IL-6. We preferred this format instead of the suggested bar graph because of space limitations. We would like to thank the reviewer for his/her constructive remarks which were of great help to improve this methodological manuscript.

-We fully agree with the main concern regarding the degree of non-specific binding of our primary polyclonal antibodies (abs). As suggested by the reviewer, two additional negative control experiments were carried out:

o A polyclonal isotype control (Rabbit IgG, EPITOMICS) was tested in the same concentration and under the same conditions as the primary polyclonal rabbit anti- o Staining of the LPS-treated cells without permeabilising solution was performed, but since the FACSlyse (Becton Dickinson, BD) solution used to lyse the red blood cells has already a significant permeabilization effect, these data could not be used as additional negative control. Indeed, the permeabilization step after incubation with the anti-CD45 antibodies is recommended by BD only to be sure that all cells are properly permeabilized. Therefore, an alternative control for the binding of our abs to the cell surface was carried out by confocal microscopy after stimulation of whole blood with LPS. Cell nuclei were stained using propidium iodide (PI), an Alexa 488-labeled secondary ab was used for visualizing IL-1β/-6.

Leukocyte differentiation was based on the nuclear morphology and cell size. A representative image (2B) is now included in the revised manuscript illustrating the intracellular staining of chicken lymphocytes for IL-6.

Although the results from both complementary negative control experiments are satisfactory, we propose to include only the confocal image showing the absence of cell surface binding of the abs in the revised manuscript. The reason not to include the isotype control results is that we are not convinced of the added value of this type of control to prove the absence of non-specific binding for our application. Following arguments motivate our decision: the company of the primary antibodies (Serotec) provided negative advice on the use of a polyclonal isotype ab, while the use of a monoclonal isotype is also We are aware that the high concentrations at which our primary abs were used increase the risk for non-specific binding, but we obtained these after appropriate titration of the abs. Addionally, preliminary results using chicken serum in an additional blocking step before labeling with our abs did not have any influence as shown below.

IL -High levels of LPS cause indeed cell death as correctly mentioned by the reviewer. Since the white blood cells are fixed and already partly permeabilized after lysis of the red blood cells by FACSlyse, these cells are dead and consequently stain positively with PI. Cell death of the white blood cells after contact with LPS in vivo was described in a former paper from our group (De Boever et al., Avian Pathology, in press), using Annexin-V to detect apoptosis.

Reviewer 2

We would like to thank the reviewer for his/her constructive remarks which were of great help to improve this methodological manuscript.

-The reviewer remarked that the CD45 staining shown in Fig. 1 is low given that CD45 is an abundant cell surface antigen. In Fig. 3 the CD45 staining for the different cell types is indicated on the Y axis. This intensity of CD45 staining is in accordance with the findings of [START_REF] Paramithiotis | High levels of CD45 are co-ordinately expressed with CD4 and CD8 on avian thymocytes[END_REF], where the mean fluorescence intensity (MFI) values also vary between 10 2 and 10 3 .

-The data on the %CD45 + cells is indeed not shown, but the MFI can be determined from o Staining of the LPS-treated cells without permeabilising solution was performed, but since the FACSlyse (Becton Dickinson, BD) solution used to lyse the red blood cells has already a significant permeabilization effect, these data could not be used as additional negative control. Indeed, the permeabilization step after incubation with the anti-CD45 antibodies is recommended by BD only to be sure that all cells are properly permeabilized. Therefore, an alternative control for the binding of our abs to the cell surface was carried out by confocal microscopy after stimulation of whole blood with LPS. Cell nuclei were stained using propidium iodide (PI), an Alexa 488-labeled secondary ab was used for visualizing IL-1β/-6.

Leukocyte differentiation was based on the nuclear morphology and cell size. A representative image ( 2B) is now included in the revised manuscript illustrating the intracellular staining of chicken lymphocytes for IL-6.

Although the results from both complementary negative control experiments are satisfactory, we propose to include only the confocal image showing the absence of cell surface binding of the abs in the revised manuscript. The reason not to include the isotype control results is that we are not convinced of the added value of this type of control to prove the absence of non-specific binding for our application. Following arguments motivate our decision: the provider of the primary antibodies (Serotec) provided negative advice on the use of a polyclonal isotype ab, while the use of a monoclonal isotype is also not relevant; in general, the use of isotype controls remains to date highly controversial in the field of flow cytometry and recommended typically to test for non-specific binding of monoclonal abs in specific cell types (O'Gorman and Thomas, 1999;Hulspas et al., 2009). To test our argumentation, we evaluated a second polyclonal isotype ab from the same class of immunoglobulin (i.e. IgG) and at the same concentration but with only minor variations in characteristics compared to the first isotype in manufacturing process and formulation (data not shown). This second isotype yielded much much less satisfactory results. We are aware that the high concentrations at which our primary abs were used increase the risk for non-specific binding, but we obtained these after appropriate titration of the abs. Addionally, preliminary results using chicken serum in an additional blocking step before labeling with our abs did not have any influence as shown below. Reviewer 3

We would like to thank the reviewer for his/her constructive remarks and suggestions which helped us to improve this methodological manuscript.

-The polyclonal antibodies for cytokine detection were purchased at Serotec and their reference numbers have been included in the revised manuscript.

-We agree that the correlation with real-time RT-PCR data is an added value and performed mRNA expression analysis on the same time points as the flow cytometric method in an in vivo study carried out after the current in vitro work (De Boever et al., Avian Pathology, in press). In this latter paper, mRNA levels of IL-1β, IL-6 and TL1A

(the chicken homologue of mammalian TNF-alpha) were slightly elevated 1 h after LPS administration. However, care should be taken when comparing the results obtained with both complementary techniques. Because mRNA is extracted from all white blood cells and not from the separate cell types, correlation between mRNA and protein levels is not straightforward.

1. Page 3, line 19: "in mammalia" was changed to "in mammals" A flow cytometric method for the identification of chicken blood leukocyte subpopulations and thrombocytes was developed. An anti-chicken CD45 phycoerythrin-labelled antibody was used to separate leukocytes from red blood cell nuclei. Leukocytes and thrombocytes were identified using a combination of their CD45-positivity and their typical side scatter properties. The identity of the CD45-positive cells was confirmed by sorting the subpopulations and subsequent light microscopic evaluation.

In these differentiated cell populations, intracellular expression analysis of the proinflammatory cytokines interleukin-1β and IL-6 was subsequently optimised on whole blood after in vitro stimulation with lipopolysaccharide from Escherichia coli strain O127:B8.

Keywords: chicken, leukocyte identification, CD45, cytokine expression Flow cytometry is a preferred method to phenotype ex vivo derived individual leukocytes from various species (Bohls et al., 2006). The flow cytometric differentiation of avian leukocytes has been described for monocytes [START_REF] Mast | Characterization of chicken monocytes, macrophages and interdigitating cells by the monoclonal antibody KUL01[END_REF], lymphocyte subpopulations [START_REF] Fair | Immunophenotyping of chicken peripheral blood lymphocyte subpopulations: Individual variability and repeatability[END_REF] and thrombocytes (Viertlboeck and Göbel, 2007). [START_REF] Uchiyama | Counting absolute number of lymphocytes in quail whole blood by flow cytometry[END_REF] used an aspecific fluorescent lipophilic dye DiOC 6 (3) for differentiation and counting of quail and chicken blood cells.

In mammals, the common leukocyte antigen CD45 is a haemopoietic cell-specific surface glycoprotein with a cytoplasmic tyrosine phosphatase domain that is believed to play a role in T-and B cell antigen receptor signal transduction [START_REF] Tchilian | Altered CD45 expression and disease[END_REF]. Various isoforms are expressed on different types of lymphohematopoietic cells, while it is absent on mammalian platelets and mature erythrocytes. The avian CD45 homolog is expressed on all chicken leukocytes but not on erythroid cells [START_REF] Paramithiotis | High levels of CD45 are co-ordinately expressed with CD4 and CD8 on avian thymocytes[END_REF]. Chicken thrombocytes, which are the equivalent of mammalian platelets, also express the CD45 antigen although at consistently lower levels in comparison to lymphocytes [START_REF] Horiuchi | A monoclonal antibody against chicken thrombocytes reacts with the cells of thrombocyte lineage[END_REF]Viertlboeck and Göbel, 2007).

Avian heterophils, which are the equivalent of mammalian neutrophils, blood monocytes, and thrombocytes, release a cascade of cytokines upon stimulation with various infectious or noninfectious agents such as lipopolysaccharide (LPS) [START_REF] Rhind | Intracellular monocyte and serum cytokine expression is modulated by exhausting exercise and cold exposure[END_REF][START_REF] Kogut | Expression and function of Toll-like receptors in chicken heterophils[END_REF][START_REF] Scott | Thrombocytes respond to lipopolysaccharide through Tolllike receptor-4, and MAP kinase and NF-κB pathways leading to expression of interleukin 6 and cyclooxygenase-2 with production of prostaglandin E2[END_REF][START_REF] Ferdous | Broiler chick thrombocyte response to lipopolysaccharide[END_REF]. LPS is commonly used to induce an acute phase reaction in humans and animals because of its proinflammatory properties [START_REF] Skarnes | Role of prostaglandin E in the biphasic fever response to endotoxin[END_REF][START_REF] Klosterhalfen | Time course of various inflammatory mediators during recurrent endotoxemia[END_REF]Erroi et al., 1993;[START_REF] Franco | The in vivo kinetics of tissue factor messenger RNA expression during human endotoxemia: relationship with activation of coagulation[END_REF][START_REF] Maxwell | Contrasting the role of costimulation and the natural adjuvant lipopolysaccharide during the induction of T cell immunity[END_REF]De Boever et al., 2008). LPS interacts with the innate immune system through Toll-like receptor 4 (TLR4), a member of the family of cell-surface and endosomally expressed receptors that recognize conserved molecular patterns unique to microorganisms (Palsson- 'Neill, 2004;[START_REF] Janssens | Role of Toll-like receptor in pathogen recognition[END_REF]. Recognition of microbial components by TLRs triggers a cascade of cellular signals that culminates in the activation of Nuclear Factor -κB which leads to inflammatory gene expression (Verstrepen et al., 2008).

Acute endotoxemia represents an adequate tool for gaining insight in inflammatory processes [START_REF] Remick | Evaluation of endotoxin models for the study of sepsis[END_REF]. Furthermore, such a controlled inflammation model is of interest to test the pharmacodynamic properties of anti-inflammatory drugs. To be able to monitor the progress of the inflammatory response and evaluate the immune-modulatory effect of antiinflammatory drugs, several parameters can be determined including body temperature, blood pressure, cytokine expression and the production of acute phase proteins. Of relevance in this context is that the cytokines IL-1β and IL-6 have been characterized as major endogenous pyrogens or fever-inducers using several animal models [START_REF] Kluger | Fever: role of pyrogens and cryogens[END_REF][START_REF] Jakab | The acute phase reaction syndrome: the acute phase reactants[END_REF][START_REF] Leon | An antipyretic role for interleukin -10 in LPS fever in mice[END_REF]. Flow cytometry is a powerful analytical technique allowing the quantitative assessment of cytokine production at the single cell level. Using multiple colourlabelling, the simultaneous identification of different selected parameters can be performed (Bueno et al., 2001;[START_REF] Schultz | Influence of specimen age and use of different negative controls in determination of intracytoplasmic levels of cytokines after whole-blood culture assay[END_REF][START_REF] Schuerwegh | Comparison of intracellular cytokine production with extracellular cytokine levels using two flow cytometric techniques[END_REF].

In this paper we describe a flow cytometric method for the identification of avian leukocyte subpopulations and thrombocytes based on their combined CD45 positivity and side scatter properties. The major advantage of this technique is the use of a single antibody directed against a common leukocyte antigen for the identification of the leukocyte subpopulations and thrombocytes. Furthermore, intracellular detection of IL-1β and IL-6 was performed in these different leukocyte subpopulations. This new tool allows us to monitor the cytokine expression after in vivo LPS administration in broiler chickens.

MATERIALS AND METHODS

Blood samples Blood samples (0.5 ml) were collected from the leg vein from laying hens, kept as blood donors, into heparin containing tubes (Venoject ® , Terumo Corp., Tokyo, Japan).

This procedure was approved by the Ethics committee of the Faculty of Veterinary Medicine (EC 2006/035).

Reagents and antibodies

The phycoerythrin (PE) conjugated Cell Lab mouse monoclonal anti-chicken CD45 antibody, isotype IgMκ was purchased at Analis (Gent, Belgium).

Two polyclonal IgG antibodies, both raised in rabbits and directed against chicken IL-1β

(AHP941Z) and IL-6 (AHP942Z), respectively, were used as primary antibodies (AbD Serotec, Cergy Saint-Christophe, France). An allophycocyanin (APC)-conjugated fragment F(ab') 2 of a goat antibody to rabbit immunoglobulin (Santa Cruz biotechnology Inc., Heidelberg, Germany) was used as secondary antibody.

Goat serum, RPMI 1640 and bovine serum albumin (BSA) were purchased at Invitrogen (Merelbeke, Belgium). BD FACS Lysing solution, BD FACS Permeabilizing solution 2 and BD CellFIX were purchased from BD Biosciences (Erembodegem, Belgium).

LPS of Escherichia coli strain O127:B8 was purchased at Sigma-Aldrich (Bornem, Belgium).

Staining procedure and flow cytometric analysis Chicken leukocytes and thrombocytes were obtained from whole blood after lysis of the erythrocytes. For this purpose, 4.5 ml of BD FACS Lysing solution, 1/10 diluted in distilled water, was added to 0.5 ml of blood in a falcon tube for 10 minutes (min) at room temperature. After washing, centrifugation for 5 min at 500 g, the pellet was resuspended in 500 µl RPMI and 50 µl was pipetted into a flow cytometer tube. Following titration, testing 4, 6, 8, 10 and 20 µg/ml as concentrations, For the in vitro stimulation with LPS, 0.5 ml of whole blood was incubated with LPS at a final concentration of 10 µg/ml for 3 hours at 37°C in a shaking warm water bath (Memmert, Model WNB 14,Schwabad,Germany). Subsequently, erythrocytes were lysed and leukocytes and thrombocytes were labelled as described above, followed by permeabilisation using BD FACS Permeabilizing solution 2, 1/10 diluted with distilled water, again for 10 min at room temperature. As for CD45, the optimal working dilutions for the primary antibodies directed against IL-1β and IL-6 were also optimized by means of titration, testing 1.6, 3.3, 6.6, 10, 12.5, 25, 50 and 100 µg/ml as concentrations. A final concentration of 50 µg/ml was used and samples were incubated for 30 min at room temperature. Blocking was performed by adding 20% goat serum in RPMI for 30 min at room temperature. Finally, the APC-labelled secondary antibody was added at a concentration of 6 µg/ml for 30 min at 4°C. Cells were resuspended in 450 µl CellFIX, 1/10 diluted with distilled water. The optimal antibody concentration of the primary and secondary antibody were also determined by titration.

Samples were measured using a double laser bench top flow cytometer FACSCanto (Beckton Dickinson Biosciences) and analysed with FACSDiva Software (Becton Dickinson).

Excitation was performed with a 488 nm argon-ion laser and emission collected using a 530/30 nm band pass (BP) filter for FITC conjugates, a 585/42 nm BP filter for PE conjugates and a 660/20 nm BP filter for APC conjugates. Two basic parameters, i.e. the side scatter (SSC), which is proportional to the cellular granularity, and the forward scatter (FSC) which is proportional to cell size, were analyzed. For the CD45 expression, the amount of CD45 antigen present per cell (mean fluorescence intensity, MFI) was determined in all populations as can be seen in Figure 3. Additionally, flow cytometric cell sorting was performed on CD45 + cells (BD FACSAria II, California, USA). The sorted cell populations were stained (Diff-Quick, Acros Organics N.V., Geel, Belgium) after cytocentrifugation (Shandon, UK) and subsequent analysis of the slides by light microscopy (Olympus BX-UCB, Pennsylvania, USA).

RESULTS AND DISCUSSION

The aim of this study was two-fold, i.e. to develop (1) a straightforward method to differentiate avian white blood cells and (2) to enable the subsequent assessment of their individual intracellular cytokine expression after LPS challenge.

(1) Flow cytometric identification of avian monocytes [START_REF] Mast | Characterization of chicken monocytes, macrophages and interdigitating cells by the monoclonal antibody KUL01[END_REF], lymphocyte subpopulations (Bohls et al., 2006;Fair et al., and thrombocytes (Viertlboeck and Göbel, 2007) has already been performed. The aspecific fluorescent lipophilic dye DiOC 6 (3) used by [START_REF] Uchiyama | Counting absolute number of lymphocytes in quail whole blood by flow cytometry[END_REF] for differentiation, was very useful for counting of quail and chicken blood cells. We succeeded in the development of a straightforward method to differentiate leukocyte subpopulations and thrombocytes using a single antibody directed against the common leukocyte antigen. The CD45 antibody used in the current study allows a better discrimination between the leukocyte subpopulations. This is especially the case for the lymphocytes and thrombocytes because of the reduced interference with the red blood cell nuclei. The presence of nucleated erythrocytes and thrombocytes was indeed a challenge as this is a major difference between mammalian and avian hematology (Campbell and Ellis, 2007). Lysis of the erythrocytes was tested comparing a Tris-aminomethane/NH 4 Cl buffer with the commercial lysing solution, with the latter giving the best results (data not shown).

Nevertheless, after lysis, the red blood cell nuclei were still present and since their size is similar to that of a small lymphocyte, the discrimination between both events was not possible based on the commonly used FSC versus SSC dot-plot as illustrated in Figure 1 (A).

However, the combined use of an anti-chicken CD45 antibody and the SSC, enabled the discrimination between lymphocytes and red blood cell nuclei. On the representative dot plot showing CD45 + cells versus SSC, the different subpopulations, i.e. lymphocytes, thrombocytes, monocytes and heterophils, could be gated (Figure 1 B). The position of the different subpopulations on the dot plot is similar to mammalian species, except for the thrombocytes, which are not present in mammalian blood (Figure 1 C).

The optimal concentration of the anti-CD45 antibody (6 µg/ml) was determined by titration (data not shown).

To confirm the identity of the gated populations on the anti CD45-PE versus SSC dot-plot, additional sorting was performed, followed by light microscopy (Figure 2). Population L was composed of lymphocytes, characterized by a round nucleus and weakly basophilic cytoplasm. Population M, contains the monocytes with the hallmarks being the largest leukocytes and having a large nucleus. The heterophils (population H) are characterized by a lobed nucleus and abundant eosinophilic granules in the cytoplasm. For the heterophils and lymphocytes, a purity of 96% and 89.4% could be calculated, respectively. For the monocytes and thrombocytes no percentages can be provided as only few cells were present on the slide.

(2) In a second series of experiments the optimized cell differentiation method was combined with intracellular detection of pro-inflammatory cytokines. More specifically, IL-1β and IL-6 were chosen since these cytokines have been implicated as key mediators of fever [START_REF] Kluger | Fever: role of pyrogens and cryogens[END_REF][START_REF] Jakab | The acute phase reaction syndrome: the acute phase reactants[END_REF][START_REF] Leon | An antipyretic role for interleukin -10 in LPS fever in mice[END_REF]. Commercial antibodies directed against chicken IL-1β and IL-6 are of polyclonal origin and have not yet been tested for flow cytometry. In the current experiment, the method was optimized after in vitro stimulation of heparin-treated whole blood samples with LPS, because cell activation has been reported to Several amounts of the commercial permeabilizing solution were tested, with 500 µl resulting in the highest percentage of cytokine expressing cells and the highest MFI (data not shown).

As for CD45, the optimal working dilutions for the primary antibodies directed against IL-1β and IL-6 were also optimized by means of titration (data not shown). An APC-labeled secondary antibody was chosen for intracellular cytokine detection. APC belongs to a family of phycobiliproteins with a broad excitation spectrum and a large Stoke's shift with a high quantum yield fluorescence [START_REF] Jung | A novel and inexpensive source of allophycocyanin for multicolour flow cytometry[END_REF][START_REF] Telford | Cryptomonad algal phycobiliproteins as fluorochromes for extracellular and intracellular antigen detection by flow cytometry[END_REF]. The optimal concentration of the secondary antibody was also determined by titration.

A fluorescein isothiocyanate (FITC) labelled antibody was first tested as secondary antibody, but since preliminary data confirmed that FITC stains the cytoplasmic granules of avian heterophils [START_REF] Rath | Fluorescein isothiocyanate staining and characterization of avian heterophils[END_REF], this fluorochrome was not suited for intracellular cytokine detection nor could FITC-labelled antibodies be used for the identification of the different leukocyte subpopulations.

After stimulation of whole blood with LPS, cytokine production was measured in the leukocyte subpopulations and the thrombocytes as illustrated in Figure 3. The MFI of LPS stimulated cells for both cytokines is shown in Table 1. On the dot plot of samples to which only the CD45-PE antibody and the secondary APC-labelled antibody was added (Figure 3 A), a grid was placed to be able to discriminate between specific and nonspecific staining of the secondary antibody. As could be expected from literature [START_REF] Schultz | Influence of specimen age and use of different negative controls in determination of intracytoplasmic levels of cytokines after whole-blood culture assay[END_REF] Formatted: Subscript et al., 2003), stimulation with LPS evoked a right-shift of the populations, indicating the intracellular presence of IL-1β and IL-6. Monocytes showed the largest shift, both for IL-1β and IL-6. Thrombocytes are also capable of synthesizing cytokines. This observation is in accordance with Scott et al. (2007) and [START_REF] Ferdous | Broiler chick thrombocyte response to lipopolysaccharide[END_REF], although in those experiments IL-1β and IL-6 expression was measured on the mRNA level. The heterophils expressed the lowest level of cytokines, although mRNA expression after LPS stimulation is reported [START_REF] Kogut | Expression and function of Toll-like receptors in chicken heterophils[END_REF]. However, the latter authors mention that mRNA levels do not necessarily equate to bioactive protein. A confocal microscopic image of a lymphocyte expressing IL-6 is shown in Figure 2B.

Finally, it should be remarked that the heterophils clearly expressed more CD45 molecules after LPS stimulation (data not shown). This observation corroborates findings in human granulocytes that contain an intracellular pool of CD45 and upon stimulation with formylmethionylleucylphenylalanine, ionophore A23187 or LPS increase the surface expression of CD45 [START_REF] Lacal | Intracellular location of T200 and Mol glycoproteins in human neutrophils[END_REF]Caldwell et al., 1991;[START_REF] Stie | Reorganization of the human neutrophil plasma membrane is associated with functional priming: implications for neutrophil preparations[END_REF].

In conclusion, we developed a straightforward method for the differentiation of the different leukocyte subpopulations in avian blood, using a monoclonal antibody directed against chicken CD45 in combination with SSC properties. Furthermore, intracellular IL-1β and IL-6 expression in these individual populations was assessed. This method can now be applied for the detection of intracellular cytokine expression after intravenous administration of LPS to broiler chickens.

  Saint-Christophe, France). An allophycocyanin (APC)-conjugated fragment F(ab') 2 of a goat antibody to rabbit immunoglobulin (Santa Cruz biotechnology Inc., Heidelberg, Germany) was used as secondary antibody.Goat serum, RPMI 1640 and bovine serum albumin (BSA) were purchased from Invitrogen (Merelbeke, Belgium). BD FACS Lysing solution, BD FACS Permeabilizing solution 2 and BD CellFIX were purchased from BD Biosciences (Erembodegem, Belgium).

  and 100 µg/ml as concentrations. A final concentration of

  , although in those experiments IL-1β and IL-6 expression was measured at the mRNA level. Heterophils expressed the lowest level of cytokines, although mRNA expression of cytokines by these stimulation has been reported

  .M., Taylor-McCabe, K.J., Shou, Y., Marrone, B.L.

  [START_REF] Mast | Characterization of chicken monocytes, macrophages and interdigitating cells by the monoclonal antibody KUL01[END_REF]. Characterization of chicken monocytes, macrophages and interdigitating cells by the monoclonal antibody KUL01. Veterinary Immunology and Immunopathology, 61, 343-357. Maxwell, J.R., Ruby, C., Kerkvliet, N.I., Vella, A.T. (2002). Contrasting the role of costimulation and the natural adjuvant lipopolysaccharide during the induction of T cell immunity. Journal of Immunology, 168, 4372-4381. Pålsson-McDermott, E. & O'Neill, L. (2004). Signal transduction by the lipopolysaccharide receptor, Toll-like receptor-4. Immunology, 113, 153-162. Paramithiotis, E., Tkalec, L., Ratcliffe, J.H. (1991). High levels of CD45 are co-ordinately expressed with CD4 and CD8 on avian thymocytes. Journal of Immunology, 11, 3710-3717. Rath, N.C., Huff, G.R., Balog, J.M., Huff, W.E. (1998). Fluorescein isothiocyanate staining and characterization of avian heterophils. Veterinary Immunology and Immunopathology, 64, 83-95. Remick, D.G., Ward, P.A. (2005). Evaluation of endotoxin models for the study of sepsis.Shock, 24,[7][8][9][10][11]

  .G., Castellani, J.W., Brenner, I.K.M., Shephard, R.J., Zamecnik, J., Montain, S.J., Young, A.J., Shek, P.N.(2001). Intracellular monocyte and serum cytokine expression is modulated by exhausting exercise and cold exposure. American Journal of Physiology Regulatory, Integrative and Comparative Physiology, 281, R66-R75.[START_REF] Schuerwegh | Comparison of intracellular cytokine production with extracellular cytokine levels using two flow cytometric techniques[END_REF]. Comparison of intracellular cytokine production with extracellular cytokine levels using two flow cytometric techniques. Cytometry Part B, 55B, 52-58. Schultz, C., Rott, C., Temming, P., von Puttkammer, J., Bucsky, P. (2002). Influence of specimen age and use of different negative controls in determination of intracytoplasmic levels of cytokines after whole-blood culture assay. Clinical Diagnostic Laboratory Immunology, 9, 295-298. Scott, T., Owens, M.D. (2008). Thrombocytes respond to lipopolysaccharide through Tolllike receptor-4, and MAP kinase and NF-κB pathways leading to expression of interleukin 6 and cyclooxygenase-2 with production of prostaglandin E2. Molecular Immunology, 45, 1001-1008. Skarnes, R.C., Brown, S.K., Hull, S.S., McCracken, J.A. (1981). Role of prostaglandin E in the biphasic fever response to endotoxin. Journal of Experimental Medecine, 154, 1212-1224. Stie, J., Jesaitis, A.J. (2007). Reorganization of the human neutrophil plasma membrane is associated with functional priming: implications for neutrophil preparations. Journal of Leukocyte Biology, 81, 672-685.Tchilian, E.Z.,[START_REF] Tchilian | Altered CD45 expression and disease[END_REF]. Altered CD45 expression and disease. TRENDS inImmunology, 27,[146][147][148][149][150][151][152][153]

  .G., Moss, M.W., Morseman, J.P.,[START_REF] Telford | Cryptomonad algal phycobiliproteins as fluorochromes for extracellular and intracellular antigen detection by flow cytometry[END_REF].Cryptomonad algal phycobiliproteins as fluorochromes for extracellular and intracellular antigen detection by flow cytometry. Cytometry, 44, 16-23. Uchiyama, R., Morimoto, T., Kai, O., Uwatoko, K., Inoue, Y., Nakanishi, T. (2005). Counting absolute number of lymphocytes in quail whole blood by flow cytometry. Avian Pathology, 67, 441-444. Verstrepen, L., Bekaert, T., Chau, T., Tavernier, J., Chariot, A. & Beyaert, R. (2008). TLR-4, IL-1R, TNF-R signalling to NF-kappaB: variations on a common theme. Cellular and Molecular Life Science, 65, 2965-2978. Viertlboeck, B.C., Göbel, T.W. (2007). Chicken thrombocytes express the CD51/CD61 integrin. Veterinary Immunology and Immunopathology, 119, 137-141.

Figure 1 :

 1 Figure Legends:

Figure 3 :

 3 Figure 3: Representative flow cytometrical dot-plots of in vitro stimulated heterophil (H), monocyte (M), thrombocyte (TH) and lymphocyte (L) populations (A) after addition of anti-CD45 PE and of the secondary APC-labelled antibody without the primary IL antibodies; (B)addition of anti-CD45 PE, the primary antibody directed against IL-1β and the secondary APC-labelled antibody; (C) addition of anti-CD45 PE labelled antibody, the primary antibody directed against IL-6 and the secondary APC-labelled antibody. Autofluorescence was consistently lower than 10 2 for all samples. The mean % of positive events ± standard error of the mean (n = 6) is indicated in the upper right quadrants.

Figure 1 :Figure 2 Figure 3 :

 123 Figure 1: (A) Representative flow cytometrical SSC versus FSC dot plot of chicken blood

  chicken abs against IL-1β and IL-6. This isotype gave a low percentage of positive cells indicating the absence of non-specific binding, as shown in the dot-plots of LPS treated cells below.

  not relevant; in general, the use of isotype controls remains to date highly controversial in the field of flow cytometry and recommended typically to test for non-specific binding of monoclonal abs in specific cell types (O'Gorman and Thomas, 1999, Isotype controls -Time to let go? Cytometry; Hulspas et al., 2009, Considerations for the control of background fluorescence in clinical flow cytometry, Cytometry Part B: Clinical cytometry). To test our argumentation, we evaluated a second polyclonal isotype ab from the same class of immunoglobulin (i.e. IgG) and at the same concentration but with only minor variations in characteristics compared to the first isotype in purification process and formulation. This second isotype yielded much much less satisfactory results (data not shown).

Fig. 3 .

 3 Fig. 3. The sentence was rephrased in the text.

-

  We fully agree with the reviewer's remark regarding the technically demanding aspect of intracellular cytokine staining. As requested, the degree of non-specific binding of our primary polyclonal antibodies (abs) was evaluated by carrying out additional negative control experiments: o A polyclonal isotype control (Rabbit IgG, EPITOMICS) was tested in the same concentration and under the same conditions as the primary polyclonal rabbit antichicken abs against IL-1β and IL-6. This isotype gave a low percentage of positive cells indicating the absence of non-specific binding, as shown in the dot-plots of LPS treated cells below.

2.

  Page 3, line 15: a tris-aminomethane/ammoniumchloride buffer was also tested and this is now mentioned in the revised manuscript 3. Page 9, line 15: incubation with 500 µl of the permeabilization agent resulted in the highest MFI and % cytokine+ cells and was therefore used in all subsequent experiments.

  labelled anti-CD45 was added in a final concentration of 6 µg/ml and samples were incubated for 30 min at 4°C.

  expression, the % of IL-1β and IL-6 positive cells and their MFI were assessed after differentiation based on CD45 positivity and SSC as described above.

Formatted

Figure 1 :

 1 Figure captions:

Figure 3 :

 3 Figure 3: Representative flow cytometrical dot-plots of in vitro stimulated heterophil (H), monocyte (M), thrombocyte (TH) and lymphocyte (L) populations (A) after addition of anti-CD45 PE and of the secondary APC-labelled antibody without the primary IL antibodies; (B)addition of anti-CD45 PE, the primary antibody directed against IL-1β and the secondary APC-labelled antibody; (C) addition of anti-CD45 PE labelled antibody, the primary antibody directed against IL-6 and the secondary APC-labelled antibody. Autofluorescence was consistently lower than 10 2 for all samples. The mean % of positive events ± standard error of the mean (n = 6) is mentioned in the upper right quandrants.

Table 1 :

 1 Mean Fluorescence Intensity (MFI) (± standard error of the mean) for the different cell types stained for IL-1β and IL6 (n = 6)* The MFI values of the heterophils should be interpreted with caution due to the low % of cytokine expressing cells as shown in Figure3.

	IL-1β	IL-6

Table 1 :

 1 Mean Fluorescence Intensity (MFI) (± standard error of the mean) for the different cell types for IL-1β and IL6 (n = 6) * The MFI values of the heterophils should be interpreted with caution due to the low % of cytokine expressing cells as shown in Figure 3.

	IL-1β	IL-6

  Also, autofluorescence and secondary ab controls were systematically used throughout the study. Finally, our in vivo study(De Boever et al., Avian Pathology, in press) shows a consistent increase for both cytokines in LPS-versus sham-inoculated animals in MFI but not in %+ cells of heterophils which are typical candidates for non-specific binding after stimulation.

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  Also, autofluorescence and secondary ab controls were systematically used throughout the study. Finally, our in vivo study(De Boever et al., Avian Pathology, in press) shows a consistent increase for both cytokines in LPS-versus sham-inoculated animals in MFI but not in %+ cells of heterophils which are typical candidates for non-specific binding after stimulation.

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  cytokine expression is more efficient than with other anticoagulants(Coló Brunialti et al., 2002). Both a commercial lysing agent, BD FACS Lysing solution and a trisaminomethane/NH 4 Cl buffer were tested, with the former giving the best results. It should be remarked that we deliberately chose not to use a secretion block in the in vitro experiments, to mimic the in vivo situation as closely as possible. Moreover, the use of a fixation-containing lysing agent in the first step of the protocol, prevents cytokine secretion after sampling.

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

Acknowledgements

The authors would like to thank E. Neirinckx, D. De Clercq and K. Jonckheere for the help with the animal experiment. Deleted: is Deleted: the Deleted: n Deleted: ies ACKNOWLEDGEMENTS The authors would like to thank E. Neirinckx, D. De Clercq and K. Jonckheere for the help with the animal experiment. Deleted: ies