Rebeka Lucijana Berčič

Ivanka Cizelj

Daliborka Dušanić

Mojca Narat

Olga Zorman-Rojs

Peter Dovč

Dušan Benčina
email: dusan.bencina@bf.uni-lj.si

Neuraminidase of Mycoplasma

Keywords: M. synoviae, neuraminidase, chicken glycoproteins, desialylation

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Introduction

Mycoplasma gallisepticum and Mycoplasma synoviae are major poultry pathogens that cause great economic losses in intensively reared chickens and turkeys (Kleven, 2003;[START_REF] Ley | Mycoplasma gallisepticum infection[END_REF].

Although M. gallisepticum and M. synoviae are phylogenetically distant, they share hosts, tissue tropism, ways of transmission, as well as numerous horizontally transferred genes [START_REF] Papazisi | The complete genome: sequence of the avian pathogen Mycoplasma gallisepticum strain R low[END_REF]Vasconcelos et al., 2005). Some of these genes encode proteins, which are important in interactions with host cells and most likely, in adaptation of M. gallisepticum and M. synoviae to the poultry hosts [START_REF] Noormohammadi | Multigene families encoding the major hemagglutinins in phylogenetically distinct mycoplasmas[END_REF][START_REF] Sirand-Pugnet | Evolution of mollicutes: down a bumpy road with twist and turns[END_REF]Vasconcelos et al., 2005). These two species adhere to host cells via receptors containing sialic acid (SA) and share haemagglutinating lipoproteins VlhA involved in their binding to erythrocytes [START_REF] Benčina | Haemagglutinins of pathogenic avian mycoplasmas[END_REF][START_REF] Markham | Molecular cloning of a member of the gene family that encodes pMGA, a hemagglutinin of Mycoplasma gallisepticum[END_REF][START_REF] Noormohammadi | Mycoplasma synoviae has two distinct phase-variable major membrane antigens, one of which is a putative hemagglutinin[END_REF]1998;[START_REF] Razin | Mycoplasma adherence[END_REF]. Sialidases (neuraminidases) are their potential virulence factors. Higher neuraminidase activity has been reported in M. gallisepticum and M. synoviae strains that are more invasive and/or more pathogenic for chickens and turkeys (Berčič et al., 2008b;[START_REF] May | Sialidase activity in Mycoplasma synoviae[END_REF].

Significant differences in NEAC (up to 65-fold) have been reported for M. synoviae strains [START_REF] May | Sialidase activity in Mycoplasma synoviae[END_REF]. Moreover, M. synoviae ULB 925 cultures and one culture of ULB 9122 lacked detectable neuraminidase enzymatic activity (NEAC), but the molecular basis responsible for the absence of NEAC has not been reported (Berčič et al., 2008b).

Because desialylation of host receptors can reduce or abolish cytadherence by both M. gallisepticum and M. synoviae, a hypothesis has been proposed that a balance between variable sialidase activity (i.e. NEAC) and avidity of adherence to sialylated receptors on host cells is predictable (May & Brown, 2008;2009a). However, only for M. gallisepticum strain TT it has been reported that it cleaved sialic acid from human transferrin, human α1- [START_REF] Sethi | Neuraminidase activity in Mycoplasma gallisepticum[END_REF]. Generally, cultures of M. synoviae showed higher levels of NEAC than those of M. gallisepticum (Berčič et al., 2008b), but there was no data about M. synoviae capability to desialylate glycoproteins of its host or other glycoproteins.

In this study, we attempted to address several aspects related to NEAC of M. synoviae strains. With specific antibodies we determined the expression of their NanH neuraminidase.

In M. synoviae cultures without detectable NEAC and NanH protein the nanH gene sequences were determined and this enabled identification of mutations responsible for the absence of NEAC. We also demonstrated for the first time, that M. synoviae desialylates several chicken glycoproteins, including those from the upper trachea. This capability could enable an efficient colonization and long persistence of M. synoviae in the upper respiratory tract of chickens.

Materials and Methods

Mycoplasma synoviae strains and their growth. M. synoviae strains used in this study are shown in Table 1. Except strain ULB 07/P5, they have been characterized in previous studies [START_REF] Narat | Hemagglutination-positive phenotype of Mycoplasma synoviae induces experimental infectious synovitis in chickens with a higher frequency than the hemagglutination-negative phenotype[END_REF][START_REF] Benčina | The characterization of Mycoplasma synoviae EF-Tu protein and proteins involved in hemadherence and their N-terminal amino acid sequences[END_REF][START_REF] Benčina | Molecular basis of the length variation in the N-terminal part of Mycoplasma synoviae hemagglutinin[END_REF]Benčina et al., , 2005;;Berčič et al., 2008 a,b). Cultures of the type strain WVU 1853 were from two Mycoplasma culture collections (Berčič et al., 2008b).

Those used throughout of this study and designated WVU 1853, were from the WVU 1853 culture provided by S. H. Kleven [START_REF] Benčina | Molecular basis of the length variation in the N-terminal part of Mycoplasma synoviae hemagglutinin[END_REF]. The second WVU 1853 culture (designated WVU 1853 Aa), donated by Janet M. Bradbury, was from the collection of FAO/WHO collaborating Center for Animal Mycoplasmas, Aarhus, Denmark. Strains without NEAC (Berčič et al., 2008b) [START_REF] Narat | Hemagglutination-positive phenotype of Mycoplasma synoviae induces experimental infectious synovitis in chickens with a higher frequency than the hemagglutination-negative phenotype[END_REF]. ULB 925 has been isolated from the hock joint of a chicken inoculated with the haemagglutination positive (HA + ) culture of AAY-4 [START_REF] Narat | Hemagglutination-positive phenotype of Mycoplasma synoviae induces experimental infectious synovitis in chickens with a higher frequency than the hemagglutination-negative phenotype[END_REF][START_REF] Benčina | The characterization of Mycoplasma synoviae EF-Tu protein and proteins involved in hemadherence and their N-terminal amino acid sequences[END_REF]. ULB 925 HA + clone KF was cloned to obtain HA + subclone KF9 (and its subclones KF9/3 and KF9/4) and HAnegative (HA -) subclone KF6 [START_REF] Benčina | The characterization of Mycoplasma synoviae EF-Tu protein and proteins involved in hemadherence and their N-terminal amino acid sequences[END_REF][START_REF] Benčina | Molecular basis of the length variation in the N-terminal part of Mycoplasma synoviae hemagglutinin[END_REF]Berčič et al., 2008b). ULB 9122 was isolated in 1991 from the upper trachea of a hen of brown egg-layer of breed RIR in Slovenia [START_REF] Benčina | Molecular basis of the length variation in the N-terminal part of Mycoplasma synoviae hemagglutinin[END_REF](Benčina et al., , 2005)). ULB 9122 culture C1 had NEAC, whereas its another culture (C2) was NEAC-negative (Berčič et al., 2008b). Both cultures, C1 and C2, underwent 20 passages (Fig. 1). ULB 9122 culture C3 (also NEAC-negative) derived from C2 after additional 2 passages. Strain ULB 07/P5 was isolated in 2007 from the choanal cleft of a chicken belonging to the same chicken breed as chicken from which ULB 9122 was recovered 16 years earlier. Sequences of highly polymorphic M. synoviae genes (5'-vlhA, nanH and for a putative cysteine protease MS53_0590) of ULB 9122 and ULB 07/P5 were identical, except a single nucleotide polymorphism (SNP) in the nanH gene (see Results).

M. synoviae cultures were grown in Frey's medium at 37-38 °C and were harvested before pH of the broth reached pH 6.7 (Berčič et al., 2008b;Dušanić et al., 2009). The number of colony forming units (CFU) of their broth cultures was determined as recommended [START_REF] Rodwell | Methods for direct and indirect measurement of mycoplasma growth[END_REF]. The 30-µl PCR mixture contained 3 µl of 10 x PCR buffer (MBI Fermentas, Lithuania), 0.5 U of Taq DNA polymerase (MBI Fermentas), 1.5 µl of each primer at a concentration of 10 pM, 1.5 µl of mixture of all four deoxynucleoside triphosphates at a concentration of 2mM and 2.4 µl of 25 mM MgCl 2 . The program consisted of initial denaturation at 95 °C for 3 min, followed by 35 cycles of denaturation for 1 min at 95 °C, annealing for 30 s (for specific set of primers the annealing temperature is described in Table 2), extension for 150 s at 72 °C, and final extension for 10 min at 72 °C. The PCR products were sent for sequencing to Macrogen DNA Sequencing Service. Sequence analyses were performed by EMBL-EBI Tool ClustalW2 [START_REF] Thompson | Clustal W: improving the sensitivity of progressive multiple sequences aligment through sequence weighting, position-specific gap penalties and weight matrix choce[END_REF], BCM Search Launcher [START_REF] Smith | BCM Search Launcher -an integrated interface to molecular biology data base search and analysis services available on the World Wide Web[END_REF], NCBI BLAST Tools [START_REF] Zhang | PowerBLAST: A new network BLAST application for interactive or automated sequence analysis and annotation[END_REF].

Polymerase chain reaction (PCR

Evaluation of neuraminidase enzymatic activity (NEAC).

M. synoviae cells were harvested from their broth cultures (50 or 100 ml) by centrifugation, washed in sterile phosphate buffered saline (PBS, pH 7.4) and their suspension in PBS were prepared for NEAC assays. Ten µl suspension of M. synoviae cells (~10 8 or 10 9 CFU) was mixed (in small microfuge tubes with caps) with ten µl of the chromogenic substrate containing 5µg of 5bromo-4-chloro-3-indolyl-α-D-N-acetylneuraminic acid (called hereafter BIN; B4666, Sigma-Aldrich, St. Louis, USA).

Samples were incubated at room temperature and times of their positive NEAC reactions, colour change from colourless to indigoblue, were recorded. M. synoviae cultures without NEAC (see Table 1) were also assayed at higher cell-density (10 10 CFU) and longer incubation with BIN (at least 24 hours). In each NEAC assay the positive control was Assays using neuraminidase inhibitor. To confirm that neuraminidase is involved in the NEAC reaction and desialylation of glycoproteins by M. synoviae, the neuraminidase inhibitor 2,3-didehydro-2deoxy-N-acetylneuraminic acid (DANA; D9050, Sigma-Aldrich) was used. Stock solution of DANA (10 mg/ml) was kept at -20 ºC. To demonstrate neuraminidase inhibition of M. synoviae cells (10 8 or 10 9 CFU) their samples were incubated with 1 -10 mg/ml DANA (10 or 100 µg/30 µl sample) and 5 µg of BIN. Parallel NEAC assays were performed with C. perfringens neuraminidase (1 or 10 µg) and 10 or 100 µg of DANA. One µg of C. perfringens neuraminidase had 1.7 mU if the neuraminidase substrate was mucin (data from Sigma).

Antibodies to the NanH neuraminidase and other antibodies. Chicken antibodies specific for the NanH protein of M. synoviae were custom produced by GenWay (San Diego, CA, USA). A synthetic peptide, DKRVNNKDDRGHLEQD, representing theoretically highly immunogenic "epitop" of NanH (residues 464-478) was coupled (via cysteine added to the first residue of this peptide) to keyhole limpets haemocyanin (KHL). Two chickens were immunized, each received ~ 2 mg of the NanH peptide coupled to KLH during the immunization procedure. Immunoglobulin G (IgG) antibodies were purified from their egg yolks. Affinity-purified IgG to NanH were obtained using the NanH peptide as a specific ligand immobilized on CNBr beads (isolated by GenWay). They were received as a solution containing 1 mg of IgG per ml.

Antibodies to bacterial glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were raised in mouse. GAPDH of Bacillus (Geobacter) stearotermophillus was purchased (G5892, ). The BALB/c mouse was immunized with GAPDH (total protein ~ 100 µg) using previously described procedure [START_REF] Benčina | Molecular basis of the length variation in the N-terminal part of Mycoplasma synoviae hemagglutinin[END_REF] and finally with the M. synoviae cytoplasmic protein fraction obtained following osmotic lysis of WVU 1853 cells [START_REF] Razin | Cell lysis and isolation of membranes[END_REF]. In this fraction GAPDH was the most abundant protein. A week later the antiserum to GAPDH was collected. In immunoblots it strongly reacted with GAPDH of G. stearotermophillus (at dilution 1:5000), whereas in M. synoviae it reacted only with a protein similar in Mw to M. synoviae GAPDH (~ 36 kDa).

Monoclonal antibody (mAb) 1F5 was obtained after immunization of a mouse with chicken IgG (cIgG, also termed cIgY) purified from egg yolk [START_REF] Narat | Production, characterization and use of monoclonal antibodies recognizing IgY epitopes shared by chicken, turkey, pheasant, peafowl and sparrow[END_REF]. mAb 1F5 recognizes heavy chain (HC) of cIgG (~ 67 -70 kDa), as well as HC of turkey and pheasant IgG [START_REF] Narat | Production, characterization and use of monoclonal antibodies recognizing IgY epitopes shared by chicken, turkey, pheasant, peafowl and sparrow[END_REF].

Analyses of proteins with electrophoresis and immunoblotting. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting of M. synoviae proteins were performed with the PhastSystem and PhastGel Gradient (8-25) gels (Healthcare Bio-Sciences, Sweden) using ~1 µg of protein per lane [START_REF] Benčina | Variable expression of epitopes on the surface of Mycoplasma gallisepticum demonstrated with monoclonal antibodies[END_REF][START_REF] Narat | Hemagglutination-positive phenotype of Mycoplasma synoviae induces experimental infectious synovitis in chickens with a higher frequency than the hemagglutination-negative phenotype[END_REF].

Analyses also were performed in larger gels (10% gel, 12 x 10 cm) using 10 µg of protein per lane (Berčič et al., 2008a). Samples of M. synoviae cells were lysed by SDS (final SDS concentration 2%), heated at 95 ºC for 5 min and usually analyzed under reducing conditions (5% β-ME). For comparison of NanH expression (see Fig. 2 AB) samples were first analyzed by SDS-PAGE and staining by coomassie blue (PhastGel TM BlueR) to determine dilutions at which samples contained similar amount of major proteins like EF-Tu, enolase and DnaK (Berčič et al., 2008a). Following the SDS-PAGE, proteins were transferred to the Immobilon P membrane (Millipore, USA). NanH was detected by affinity-purified antibodies (2 µg/ml) and incubation for 60 min. After washing the membrane was incubated for 45 min with rabbit Desialylation of glycoproteins by M. synoviae. DIG Glycan Differentiation Kit (Roche, Manheim, Germany) was used to determine sialylation of glycoproteins, as well as levels of their desialylation with the M. synoviae neuraminidase. Sialylation with SAα(2-6)galactose linkage was detected by SNA (Sambucus nigra agglutinin), whereas SAα(2-3)gal linkage was detected by MAA (Maackia amurensis agglutinin) using the recommended reagents and procedure (Roche). The same kit included PNA (Peanut agglutinin) and DSA (Datura stramonium agglutinin), used to show cleavage of sialic acid from glycoproteins with distinct galactose and N-acetylglucosamine linkages. Reference glycoproteins (fetuin, asialofetuin, transferrin) from the same kit were used at concentrations from 0.01 to 0.1 mg/ml in PBS (pH 7.4). Ten or 20 µl samples of glycoproteins were mixed with equal volumes of M. synoviae cells (in PBS) in microcentrifuge tubes. Samples of M. synoviae strains (from 10 8 to 10 9 CFU) were from their cultures with determined NEAC (see Table 1). Incubations took place at 37-38 °C for 12-18 hours (overnight) () or longer (24-72 hours). Negative controls, consisting of PBS alone (without M. synoviae) were incubated with appropriate glycoprotein and included in each assay. In addition, C. perfringens neuraminidase (type V, N 2876, Sigma) was used as a positive control (0.1 or 1 µg/sample) in desialylation assays. Following incubation, samples were centrifuged (30 000 x g for 10 min) and supernatants examined for desialylation of glycoproteins. Samples were assayed with the PhastSystem and PhastGel Gradient (8-25) or Desialylation of chicken serum glycoproteins and chicken IgG. Sera of adult specific pathogen-free (SPF) chickens were diluted from 1:60 to 1:100 (final dilution) in PBS pH 7.4. Initial analyses using SNA and MAA showed that serum glycoproteins contained SAα(2-6)gal, rather than SAα(2-3)gal moiety. In desialylation assays samples of diluted sera (10 or 20 µl) were mixed with the tested M. synoviae cells (10 8 or 10 9 CFU in 10 or 20 µl PBS) and incubated at 37-38°C overnight or longer. Assays included NEAC-positive, as well as NEACnegative cultures (shown in Table 1). Positive control was C. perfringens neuraminidase (0.5 -5 µg per sample of serum glycoproteins), whereas the negative control was PBS alone. The neuraminidase inhibitor DANA was used (up to 100 µg DANA per sample) in assays in which we tried to confirm that neuraminidase desialylated chicken serum glycoproteins.

Following incubation, the samples were centrifuged (30 000 x g, 20 min), supernatants collected, their proteins subjected to SDS-PAGE and transferred to Immobilon P as described above. Levels of SAα(2-6)gal moiety on serum glycoproteins were estimated by visual comparison of staining intensities of bands to which SNA bound (Roche). Samples of purified cIgG from egg yolks or from sera of SPF chickens were prepared as described elsewhere [START_REF] Narat | Production, characterization and use of monoclonal antibodies recognizing IgY epitopes shared by chicken, turkey, pheasant, peafowl and sparrow[END_REF]. Samples of cIgG were analyzed by SDS-PAGE and Coomassie blue staining of proteins. They were used in assays only when they contained pure IgG (~ 170 kDa protein). Desialylation assays with cIgG were conducted as described above for chicken serum glycoproteins. Cleavage of SAα(2-6)gal moiety from cIgG was determined by SNA probe. In order to show that the heavy chain (HC) of cIgG was desialylated by M. synoviae and C. perfringens neuraminidases, supernatants of cIgG samples incubated with M. synoviae were reduced by 5% β-ME and heated at 95 ºC for 5 min. Following SDS-PAGE and transfer of cIgG or its HC to Immobilon P, the membrane was probed with SNA to determine levels of sialylation with SAα(2-6)gal moiety. Then, the membrane was incubated with mAb 1F5 (to HC of cIgG) for 60 min. After washing, the membrane was incubated 45 min with goat IgG (to mouse IgG) conjugated with HRP and developed using TrueBlue.

Desialylation of glycoproteins from chicken tracheal mucus. Samples of tracheal mucus

were collected from upper trachea of adult healthy chickens using a syringe and kept at -20 ºC in microfuge tubes. These samples were thawed, diluted 1:10 in PBS (pH 7.4), centrifuged (20 000 x g, 20 min) and supernatant was used (final dilution of mucus 1:50 or 1:100) for analyses of glycoproteins with sialic acid linkages detected by SNA or MAA. Three samples with relatively abundant sialylation of glycoproteins with SAα(2-3)gal moiety, recognized by MAA, were used in desialylation assays.

Assays were performed at 37-38 °C in microfuge tubes with caps. Typically, 10 µl of diluted mucus sample was mixed with suspension of M. synoviae cells (10 8 or 10 9 CFU) in 10 µl PBS, pH 7.4 and incubated overnight or longer (see Table 1 for M. synoviae strains tested).

Analyses of mucus glycoproteins desialylation were performed by the MAA probe using procedures described above for other glycoproteins.

In some assays, additional consecutive reactions were conducted with the lectin DSA which binds to galactose β(1-4)-N-acetylglucosamine to examine if M. synoviae cleaved from mucus glycoproteins only terminal sialic acid linked via α(2-3) to galactose (see Fig. 7 A,B). 1, Fig. 2 and data not shown). In the strain ULB 02/OV6 which expressed 110-kDa protein with the N-terminal amino acid sequence of NanH confirmed by amino acid sequencing (see Berčič et al., 2008a), specific antibodies also recognized 110-kDa NanH. As shown in Fig. 2A, two NEAC-positive cultures of WVU 1853 and that of F10 2AS expressed ~ 110-kDa NanH, whereas NEAC-negative cultures of ULB 925 (KF9/3 and KF9/4) and ULB 9122 (culture C2) did not. However, they expressed the enzyme GAPDH which is encoded by two gene copies of which the first is adjacent to the nanH gene (Fig. 2B). Strains AAY-4, ULB 925, as well as clones and subclones of ULB 925 lacked NEAC and expression of NanH (Table 1 andFig 1). On the other hand, in ULB 9122 its cultures C2 and C3 lacked NEAC and NanH, whereas its culture C1 had NEAC and synthesized NanH. In addition, all examined cultures of the strain ULB 07/P5, which is closely related to ULB 9122, expressed NanH and had NEAC (Table 1 and data not shown).

Deletions in the nanH gene causing premature terminations of translation. To elucidate the molecular basis for the absence of NanH and NEAC in AAY-4, ULB 925 and ULB 9122 cultures C2 and C3, sequences of their nanH gene, its promoter and intergenic region downstream of nanH were analysed (see Table 1). Strains AAY-4 and ULB 925 (its clone KF, and subclones KF6 and KF9) had identical sequences (4051 bp) with the same point mutation in the nanH gene (Fig 3A). In the codon for the K510 (lysine) one adenine was deleted. This would cause a frameshift introducing a stop codon UAA (TAA) six bases downstream and consequently, a premature termination of translation. Theoretically, the 1). Fig. 7 A, B shows an example of a test examining cleavage of SAα(2-3)gal moiety from mucus glycoproteins. ULB 925 KF9 lacking NEAC did not cleave this moiety, whereas WVU 1853 visibly reduced SAα(2-3)gal moiety on ~ 80 kDa glycoprotein. Consecutive probing with the lectin DSA probe revealed that WVU 1853 cleaved from ~ 80-kDa glycoprotein sialic acid, but not galactose β(1-4)-Nacetylglucosamine which bound DSA probe (Fig. 7B).

Discussion

NEAC is associated with virulence of M. gallisepticum and M. synoviae and varies among their strains, as well as among cultures of individual strains [START_REF] May | Sialidase activity in Mycoplasma synoviae[END_REF]May & Brown, 2009a;Berčič et al., 2008b). Among M. synoviae strains compared, WVU 1853 had the highest level of NEAC [START_REF] May | Sialidase activity in Mycoplasma synoviae[END_REF]Berčič et al., 2008b). It seems that NEAC is dependent on the NanH neuraminidase expression, at least in strains AAY-4, ULB 925 and ULB 9122 (cultures C2 and C3) which were without NEAC and NanH expression due to deletions in nanH gene and premature termination of translation into functional NanH polypeptide.

In Mycoplasma species slipped-strand mispairing during chromosomal replication frequently causes insertions or deletions in short polyA tracts and affects gene expression at the level of transcription initiation (mutations in promoters) or at translation due to frameshift and premature termination of translation (Citti et al., 2005). We found deletions in polyadenosine tracts in two different regions of the nanH gene (Fig. 3 A,B). In both cases, strains with such deletions did not express NanH (Table 1 andFig 17 which introduces stop codon TAA in the gene encoding ABC transporter prevented its expression and even a truncated form of this protein was absent [START_REF] Theiss | Localized frameshift mutation generates selective, highfrequency phase variation of a surface lipoprotein encoded by a Mycoplasma ABC transporter operon[END_REF].

However, M. synoviae strains lacking NanH expressed GAPDH (one copy of its gene is adjacent to nanH) like WVU 1853 and other M. synoviae strains (Berčič et al., unpublished data). Interestingly, ULB 925, its clones and their subclones that all lacked NEAC, had either HA + or HA -phenotype like clone KF6 [START_REF] Benčina | The characterization of Mycoplasma synoviae EF-Tu protein and proteins involved in hemadherence and their N-terminal amino acid sequences[END_REF] showing that there was no association of NEAC with ability to agglutinate chicken erythrocytes (Fig. 1).

Although AAY-4 lacks NEAC, its culture, with the HA + phenotype, induced acute synovitis in chickens [START_REF] Narat | Hemagglutination-positive phenotype of Mycoplasma synoviae induces experimental infectious synovitis in chickens with a higher frequency than the hemagglutination-negative phenotype[END_REF]. It replicated in hock joints and was recovered as an isolate ULB 925 which cultures lack NEAC, as well [START_REF] Benčina | The characterization of Mycoplasma synoviae EF-Tu protein and proteins involved in hemadherence and their N-terminal amino acid sequences[END_REF]Berčič et al., 2008b). On the other hand, AAY-4 did not colonize tracheas of chicken when it was inoculated into their upper respiratory tract (Benčina, unpublished data). This may be due to its incapability to desialylate glycoproteins present in the respiratory mucus, which represents antiadhesive barrier, particularly for pathogens like M. synoviae and M. gallisepticum that target and bind to sialic acid moieties of receptors on epithelial cells. This is consistent with a recent report that inactivation of the neuraminidase (i.e. sialidase) gene abolished in M. gallisepticum strain R LOW its NEAC (sialidase activity) and its ability to colonize tracheas of chickens (May et al., 2008). (Hunt & Brown, 2007). For comparison, sialidase (neuraminidase) activity (enzymatic units per CFU) of M. synoviae (WVU 1853) is 100-fold stronger as compared to M. alligatoris (May & Brown, 2009b).

Several lines of evidence indicate that neuraminidase

M. synoviae and M. gallisepticum bind to sialic acid-receptors of hosts cells and must overcome various obstacles, including mucins displaying sialylated glycoproteins, for efficient colonization of host's mucosa. Desialylation of host proteins by the NanA neuraminidase of Streptococcus pneumoniae is important for its survival in humans (King et al., 2004). Such events may be relevant to survival and persistence of M. synoviae in chickens. We postulate that desialylation of hosts glycoproteins by M. synoviae enables colonization of the respiratory tract, as well as its spreading to other tissues and organs. This is consistent with observations that strains of M. synoviae and M. gallisepticum with high levels of NEAC were more invasive than their strains with weak NEAC (Berčič et al., 2008b;[START_REF] May | Sialidase activity in Mycoplasma synoviae[END_REF]Dušanić et al., 2009). M. synoviae also is capable of cleaving sialic acid α(2-6) linked to galactose from various chicken serum glycoproteins, including from HC of IgG. This may contribute to its survival in the bloodstream and consequently, to its spreading to various organs including joints. Indeed, WVU 1853 having the strongest NEAC among M. synoviae strains examined [START_REF] May | Sialidase activity in Mycoplasma synoviae[END_REF]Berčič et al., 2008b and this study) was recovered from the blood of chickens up to day 54 after infection (Kerr & Olson, 1970).

Notably, WVU 1853 showed in vitro the highest relative invasion frequency of chicken erythrocytes (Dušanić et al., 2009). was incubated with mAb 1F5 to HC of cIgG [START_REF] Narat | Production, characterization and use of monoclonal antibodies recognizing IgY epitopes shared by chicken, turkey, pheasant, peafowl and sparrow[END_REF] The neuraminidase gene was amplified using PCR primers designed to anneal in the flanking nanA (NEUI 1 F) and gapA (NEU R, GAP 3 F/R) genes as well as inside the neuraminidase gene (NEUI 1 R, NEUI 2 F/R, NEUI 3 F/R, NEU F) open reading frame of Mycoplasma synoviae strain 53 (NC 007294).

  and closely related strains are shown in Fig. 1. AAY-4 has been infectious synovitis in chickens

  ) and sequencing. Purified DNA of M. synoviae strains was obtained by RTP Spin Bacteria DNA Mini Kit according to the manufacturers' instruction (Invitek). The oligonucleotide primers used for PCR are presented in Table2. The published M. synoviae genome sequence was used to design primers enabling amplification and sequencing the region from the 3'-end of nanA gene, the entire nanH (neuraminidase) 5'-end of the gapDH gene i.e. region from nt 221086 to 225216(Vasconcelos et al., 2005, GenBank accession number AE017245).

  Clostridium perfringens (type V, N2876, Sigma-Aldrich), whereas the negative control was PBS.

  cIgG) conjugated with horseradish peroxidase (HRP) (A9046, Sigma). The chromogenic substrate used was either diaminobenzidine tetrahydrochloride (DAB; D5905, Sigma) or TrueBlue TM (Kirkegaard & Perry Laboratories, USA). Mouse antiserum to bacterial GAPDH was used at a dilution of 1:500 for 60 min. As secondary antibodies, goat Ig (to mouse IgG) labeled with HRP (A4416, Sigma) were used at a dilution of 1:2000 for 45 min and then, staining was developed by TrueBlue.

  (12 x 10 cm). The proteins resolved in gels were transferred to Immobilon P and probed by the DIG glycan differentiation kit according to the manufacturer's instructions (Roche).

  cultures lacking NEAC did not express NanH neuraminidase. Immunoblots with antibodies to NanH showed that NEAC-positive M. synoviae strains expressed NanH of approximately 110 kDa (Table

  would have only 512 instead 938 residues and would lack domains that are critical for the neuraminidase activity. However, a truncated NanH was not detected by specific antibodies, although they recognize an epitope (residues 464 -478) within the molecule predicted for such truncated NanH. Cultures of ULB 9122 had identical sequences except a deletion in the polyA sequence of the nanH gene in cultures C2 and C3 lacking NanH. In ULB 9122 C2 and C3 a deletion of one adenine (change 6A to 5A) caused frameshift and introduced stop codon TAA eight bases downstream (Fig. 3B). The predicted NanH polypeptide of C2 and C3 cultures (832 residues) would lack 106 amino acids and would have Mw approximately 97 kDa. Specific antibodies did not detect in their cultures NanH of any size, whereas culture C1 (without deletion in nanH) expressed 110-kDa NanH. Strain ULB 07/P5, isolated 16 years after the isolation of ULB 9122, had sequence (comparison of 4063 bp) almost identical to that of ULB 9122, culture C1. In the nanH gene of ULB 07/P5 a single nucleotide polymorphism was observed in the codon for residue 740 (change CTT → CCT). Thus, at this position ULB 9122 has leucine, whereas ULB 07/P5 has proline. Neuraminidase of M. synoviae desialylates fetuin and transferring. M. synoviae strains cultures expressing NanH cleaved sialic acid from SAα(2-6)gal moiety recognized by SNA on reference transferrin (

  lectin probe, that binds only to asialofetuin confirmed cleavage of sialic acid from fetuin in consecutive tests (not shown). M. synoviae neuraminidase cleaves sialic acid SAα(2-6)gal moiety of chicken serum glycoproteins. This showed that M. synoviae can cleave sialic acid from SAα(2-6)gal moiety of various chicken serum proteins. Overnight incubation with WVU 1853 cells (~10 8 CFU) reduced the quantity of SAα(2-6)gal moiety detected by SNA on serum glycoproteins with Mw ranging from ~ 60 to ~ 200 kDa (Fig. 4, lane 4). Longer incubation with WVU 1853 resulted with the absence of SAα(2-6)gal moiety (Fig. 4, lane 6). However, intensely sialylated serum glycoproteins, of ~ 170 kDa (presumably IgG) and of ~ 80 kDa, lost all detectable SAα(2-6)gal moiety after 15 hours of incubation with ~ 10 9 CFU of WVU 1853 (Fig. 5, lane 2). Strains lacking NEAC, like ULB 925/KF9, did not cause desialylation of serum glycoproteins (Fig. 4, lane 5). Other M. synoviae strains expressing NanH and NEAC, including F10 2AS, ULB 07/P5 and ULB 9122 (culture C1), also cleaved SAα(2-6)gal moiety from serum glycoproteins. To confirm that neuraminidase desialylates chicken serum glycoproteins, the neuraminidase inhibitor DANA was used in assays. DANA obviously inhibited NEAC of WVU 1853 (measured by BIN), but high concentrations of DANA (> 50 µg of DANA per sample with 10 9 CFU of WVU 1853) were needed to prevent reaction of BIN after incubation of 15 hours. Therefore 100 µg of DANA was used in assays of desialylation of serum glycoproteins with WVU 1853. As shown in Fig. 5, DANA prevented cleavage of SAα(2-6)gal moiety from serum glycoproteins incubated with WVU 1853 for 40 hours. Complete cleavage of this moiety from glycoproteins of ~ 170 and ~ 80 kDa was observed already after 15-hour incubation with WVU 1853 (Fig. 5, lane 2).

  desialylation was observed with 0.5 µg of C. perfringens neuraminidase, but DANA (100 µg/sample) also prevented its neuraminidase activity (Fig.5, lanes 6 and 7).M. synoviae cleavesSAα(2-6)gal moiety from heavy chain of chicken IgG. Assays with serum glycoproteins indicated that M. synoviae cleaved SAα(2-6)gal moiety from chicken IgG of ~ 170 kDa and IgG purified from chicken serum or egg yolk. M. synoviae cultures having NEAC and expressing NanH, were capable of cleaving SAα(2-6)gal moiety from chicken IgG, whereas those without NEAC and NanH were not. This moiety is in the Nglycosylation site (N 308) of the CH2 domain of heavy chain of chicken IgG (Suzuki & Lee, 2004). As shown in Fig. 6, after reduction of IgG (~ 170 kDa) SNA probe bound to ~ 70-kDa heavy chain. Incubation of IgG with neuraminidase of C. perfringens or M. synoviae strains with NEAC caused cleavage of SAα(2-6)gal moiety from heavy chain (Fig. 6A). Consecutive reaction with mAb 1F5 to heavy chain of chicken IgG revealed the position of desialylated heavy chain (Fig. 6B, lanes 4 and 6). M. synoviae cultures lacking NEAC and NanH (ULB 925/KF9 and ULB 9122/C2) did not cleave SAα(2-6)gal moiety from cIgG heavy chain. M. synoviae cleaves sialic acid from SAα(2-3)gal moiety of mucus glycoproteins. Tests performed with tracheal mucus samples from different chickens showed considerable differences concerning molecular weight of glycoproteins containing sialic acid and level of their sialylation. However, they contained SAα(2-3)gal moieties detected by MAA, rather than SAα(2-6)gal moiety being predominant in serum glycoproteins. WVU 1853 was able to cleave SAα(2-3)gal moiety of ~ 40 kDa mucus glycoprotein and to reduce this moiety of ~ 80-and 110-kDa glycoproteins after incubation of 15 hours. Several other strains also cleaved sialic acid from mucus glycoproteins, including ULB 9122 (culture C1), whereas those NanH expression did not (Table

  . 2). A similar event has been observed in Mycoplasma fermentans. A deletion in a polyA tract causing frameshift

  NanH of M. synoviae desialylates chicken glycoproteins. M. synoviae cultures having NEAC and expressing NanH desialylated glycoproteins, whereas M. synoviae cultures lacking NEAC and NanH did not. The neuraminidase inhibitor DANA prevented desialylation of chicken serum glycoproteins with M. synoviae WVU 1853 (which caused desialylation in less than 15 hours) for 40 hours. We used relatively high DANA concentrations (up to 100 µg DANA per sample). However, in experiments with the neuraminidase (sialidase) of Mycoplasma alligatoris degradation of

  In conclusion, NEAC of M. synoviae depends on the expression of the NanH neuraminidase. Mutations in the nanH gene causing frameshifts and premature termination of translation eliminate NanH expression and NEAC. M. synoviae strains are capable of cleaving sialic acid α(2-6) linked to galactose from various chicken serum glycoproteins, including IgG CH 2 domains). M. synoviae neuraminidase also can cleave sialic acid α(2-3) linked to galactose from glycoproteins of chicken tracheal mucus. Such cleavage could be a factor facilitating colonization of the respiratory tract, as well as spreading of M. synoviae to other host organs. ., Narat, M., Bidovec, A. & Zorman-Rojs, O. (2005). Transfer of maternal immunoglobulins and antibodies to Mycoplasma gallisepticum and Mycoplasma synoviae to the allantoic and amniotic fluid of chicken embryos. Avian Pathology, 34, 463-472. Berčič, R.L., Slavec, B., Lavrič, M., Narat, M., Bidovec, A., Dovč, P. & Benčina, D. (2008a). Identification of major immunogenic proteins of Mycoplasma synoviae isolates. Veterinary Microbiology, 127, 147-154. Berčič, R.L., Slavec, B., Lavrič, M., Narat, M., Zorman-Rojs, O., Dovč, P. & Benčina, D. (2008b). A survey of avian Mycoplasma species for neuraminidase enzymatic activity. Veterinary Microbiology, 130, 391-397. Citti, C., Browning, G.F. & Rosengarten, R. (2005). Phenotypic diversity and cell invasion in host subversion by pathogenic mycoplasmas. In Blanchard, A. & Browning, G. (Eds.), Mycoplasmas: Molecular biology, pathogenicity and strategies for control (pp.439-483). Norfolk: Horizon Bioscience, The Cromwell Press. Dušanić, D., Berčič, R.L., Cizelj, I., Salmič, S., Narat, M. & Benčina D. (2009). Mycoplasma synoviae invades non-phagocytic chicken cells in vitro. Veterinary Microbiology, 138, 114-119. Hunt, M.E. & Brown D.R. (2007). Role of sialidase in Mycoplasma alligatoris-induced pulmonary fibroblast apoptosis. Veterinary Microbiology, 121,73-82. Kerr, K.M. & Olson, N.O. (1970). Pathology of chickens inoculated experimentally or contact-infected with Mycoplasma synoviae. Avian Diseases, 14, 291-320. King, S.J., Hippe, K.R., Gould, J.M., Bae, D., Peterson, S., Cline, R.T., Fasching, C., Janoff, E.N. & Weiser, J.N. (2004). Phase variable desialylation of host proteins that bind to Streptococcus pneumoniae in vivo and protect the airway. Molecular Microbiology, 54, 159-171.

Fig. 1 .

 1 Fig. 1. Schematic presentation of M. synoviae cultures, clones and subclones derived

Fig. 2 A

 2 Fig. 2 A,B: Immunoblot analysis of the NanH neuraminidase and GAPDH expression in

Fig. 3 :

 3 Fig. 3: Mutations in the nanH gene associated with the absence of NEAC and NanH

Fig. 4 :

 4 Fig. 4: M. synoviae cleaves sialic acid from SAα(2-6)gal linkage of glycoproteins. SAα(2-

Fig. 5 :

 5 Fig. 5: Neuraminidase inhibitor DANA inhibited desialylation of SAα(2-6)gal moiety of

Fig. 6 A

 6 Fig. 6 A,B: Neuraminidase of M. synoviae and C. perfringens cleave SAα(2-3)gal moiety

  , HRP conjugate and TrueBlue to show the position of HC. Note that HC desialylated by C. perfringens and WVU 1853 (lanes 4 and 6) reacted by mAb 1F5.

Fig. 7 :

 7 Fig. 7: M. synoviae neuraminidase cleaves sialic acid from SAα(2-3)gal moiety of chicken

Fig. 1 .Fig. 3 :Fig. 5 :Fig. 7 :

 1357 Fig. 1. Schematic presentation of M. synoviae cultures, clones and subclones derived from strain AAY-4 (the left side) or ULB 9122 (the right side). HA denotes haemagglutination; HA + -positive; HA --negative; NEAC -neuraminidase enzymatic activity; NEAC + -positive; NEAC --negative. 379x258mm (72 x 72 DPI)

Table 1 )

 1 . M. synoviae cultures lacking NEAC and NanH expression did not desialylate transferrin (Table1).M. synoviae cultures having NEAC and expressing NanH cleaved from fetuin sialic acid from SAα(2-6)gal moiety detected by SNA (Fig.4, Table1and data not shown). M. synoviae cultures lacking NEAC and NanH did not cleave sialic acid from fetuin (Fig.4,

Table 1 )

 1 or transferrin in 72 hours. On the other hand, WVU 1853 cleaved in 24 hours from those glycoproteins all sialic acid moieties detectable by SNA and/or MAA. A strong reaction

Table 1 .

 1 M. synoviae strains, cultures, NEAC, NanH synthesis, ability to desialylate chicken glycoproteins and nanH sequences a Fet/Tra, reference fetuin and transferrin; cIgG, chicken IgG; TM, glycoproteins of mucus from chicken tracheas; + denotes positive; -denotes negative; ND, not determined. b Accession numbers of sequences in GenBank; sequences in parentheses are identical to those with the same designation but were not deposited. c Except deletion show in Fig.2identical to EU 245027. d These sequences were included in comparison in a study reporting diversifying selection of sialidase (neuraminidase) in M. synoviae(May & Brown, 2009a -Suppl. Table1). e It was donated by S.H. Kleven. Another culture of the type strain, designated WVU 1853 Aa, had the nanH sequence identical to EU245026.

				Synthesis of		Desialylation of a
	Strain	Culture	NEAC	NanH	Fet	Tra	cIgG	TM nanH sequence b	Reference
	WVU 1853 S e KD2	+	+	+	+	+	+	EU 245026	Berčič et al., 2008b
	F10 2AS	C1	+	+	+	ND	+	ND (EU245026)	Benčina et al., 2001
	K1968	ZC3	+	+	ND	ND	+	ND EU 532615 d	Benčina et al., 2001
	K 4463B	C5	+	ND	+	ND	+	ND EU245028 d	Benčina et al., 2001
	K 1723	C23	+	+	ND	+	+	ND not deposited	Benčina et al., 2001
	K 2581	C2	+	ND	ND	ND	+	ND EU 245025	Benčina et al., 2001
	K 2426D	AA3	+	+	+	ND	+	ND EU 015400 d	Benčina et al., 2001
	ULB 02/P4	K4	+	+	+	ND	+	+	EU 245029 d	Lavrič et al., 2007
	ULB 02/T6	KA1	+	+	ND	+	+	ND (EU 245029)	Berčič et al., 2008a
	ULB 02/OV6 C4	+	+	+	+	+	ND (EU 245029)	Berčič et al., 2008a
	ULB 07/P5	C5	+	+	+	+	+	+	EU 532616 d	This study
	ULB 9122	C1	+	+	+	+	+	+	not deposited c	Benčina et al., 2005
	ULB 9122	C2 and C3	-	-	-	-	-	-	EU 245027	Berčič et al., 2008b
	AAY 4	K 1/3	-	-	-	ND	ND	ND (EU 532617)	Narat et al., 1998
	ULB 925	KF	-	-	-	-	-	-	EU 532617	Benčina et al., 1999; 2001
	ULB 925	KF6	-	-	-	-	-	ND (EU 532617)	Benčina et al., 1999
	ULB 925	KF9	-	-	-	-	-	-	(EU 532617)	Benčina et al., 1999

Table 2 :

 2 Oligonucleotide primer pairs and their annealing temperatures used in polymerase chain reaction (PCR).

	Primer pairs Forward (F)/sequence 5'-3'	Reverse (R)/sequence 5'-3'	Annealing T
	NEUI 1	TGCGTCTTCATGGAGTAGATG	CCGAGCTTGACGAAGCTAAT	57°C
	NEUI 2	AGGCTCACCGAAAGTGCTAA	GGAGCTGTAGTTACGCTAAGCAA	57°C
	NEUI 3	CATCAACATCACTGTATAAACCTGAA AACCTTCCAGAGTTTGTTAATGC	57°C
	NEU	CTGAAGCTTGAACATAAACTCA	AAATTTGAAACCGATGAAATAG	50°C
	GAP 3	CCGTCTTTTGATTCAACAAAGT	GAAACAAGCCTTACAGACAAAG	54°C

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. This work was supported by grant P4-0220 from the Slovenian Research Agency. We thank Dr. Brigita Slavec and Ana Jakopič for technical assistance.


with AIDS-related illness among older informal carers in two slums in Nairobi, Kenya

Keywords: caregivers, health impact, older people, urban, sub-Saharan Africa

Gender differentials on the health consequences of care-giving to people

Introduction

Long-term care for people with AIDS (PWA), the leading cause of illness and death among adults in sub-Saharan Africa, [START_REF] Who | Projections of mortality and burden of disease to 2030[END_REF] is largely in the hands of informal caregivers who bear a great burden resulting in direct and indirect effects on their mental and physical health. Older people in sub-Saharan Africa provide the bulk of the care for PWA [START_REF] Ntozi | AIDS morbidity and the role of the family in patient care in Uganda[END_REF]. Women in particular have largely been portrayed as central in the care and support for PWA [START_REF] Moller | AIDS: The `grandmothers' disease' in Southern Africa[END_REF][START_REF] Baylies | HIV/AIDS and older women in Zambia: concern for self, worry over daughters, towers of strength[END_REF][START_REF] Nyambedha | Retirement Lost"-The new role of the elderly as caretakers for orphans in Western Kenya[END_REF] and the role of older men as caregivers has received relatively little attention in the literature. This paper aims to fill this gap in the literature by highlighting the role of men and women in care-giving to people with AIDS-related illness. The paper also investigates the association between care-giving and poor health outcomes by comparing self-reported health of non-caregivers with caregivers to people with AIDS-related illness and whether this varies by gender.

Studies which have compared health outcomes between men and women caregivers report mixed findings with more studies pointing to worse health outcomes and perceived higher levels of burden for women compared with men [START_REF] Kiecolt-Glaser | Spousal caregivers of dementia victims: longitudinal changes in immunity and health[END_REF][START_REF] Miller | Gender differences in caregiving: fact or artifact?[END_REF]. These gender differences have however been challenged as 'a research artefact rather than a fact' [START_REF] Miller | Gender differences in caregiving: fact or artifact?[END_REF][START_REF] Kramer | Men as caregivers: an overview[END_REF] especially in self-reported health outcomes. Men and women tend to report feelings of distress differently, with women more likely to report higher levels of distress or health outcomes compared with men [START_REF] Stoller | Theoretical perspective on caregiving men[END_REF][START_REF] Pinquart | Gender differences in caregiver stressors, social resources, and health: An updated meta-analysis[END_REF]. Between-gender comparison would therefore often conclude that women have more distress or poorer health outcomes compared with men even though men and women may undergo the same level of stress or health outcome ( ). Previous analysis from the study (Reference withheld, 2009) found women affected by HIV and AIDS through care-giving and loss of support reported significantly poorer health compared to men and no attempt was made to investigate gender differentials per se. In the current paper, we conduct within-gender comparison by contrasting women affected by HIV and AIDS with women who have not been affected, and conduct similar analysis with the men. This acknowledges inherent gender differences and takes into consideration gender-based attributes in self-reported health where women tend to report poorer outcomes compared with men.

Study context, data, and methods

Study design and data

The study was carried out in two slum areas of Nairobi, Kenya. Urban areas of Kenya have a higher HIV and AIDS prevalence rate compared with the rural populations (NASCOP & Ministry of Health, 2008) with slum areas exhibiting higher prevalence compared to the city as a whole (Ziraba et al., 2010). Stigma and discrimination is still widespread in large parts of Africa, including the slums hindering access to care and support including access to antiretroviral therapy [START_REF] Merten | Patient-reported barriers and drivers of adherence to antiretrovirals in sub-Saharan Africa: a meta-ethnography[END_REF]. 2006). A total of 1,429 older people interviewed in the survey had matching socioeconomic and demographic information.

Health measures

Health status was measured using two indicators: (1). the WHO Disability Assessment Score (WHODAS-12); and (2). presence of a severe illness or morbidity in the 3-month period prior to the interview.

The WHODAS-12 is a multidimensional measure that assesses day-to-day functionality or disability at the physical, personal and social levels. WHODAS was designed as a generic disability measure and not tailored for a specific disease or age [START_REF] Who | Projections of mortality and burden of disease to 2030[END_REF]. WHODAS is based on six different domains: understanding and communication (cognition); getting around (mobility); self care (hygiene, dressing, eating alone); interpersonal interactions (getting along); life activities (domestic responsibilities and work); and participation in society (community activities). The participants were asked to assess 12 functionality items on a five-point Likert scale and the question were phrased in the manner "In the last 30 days, how much difficulty/problems did you have with…". A score for each of the domains was generated by recoding the 12 items with a weight of either 2 or 4 as the maximum possible score which is then summed and converted to a percentage to allow comparison across domains. The resulting score therefore range from zero to 100 with higher scores indicating more functional impairment. The overall disability score is an aggregate of the six domains thus combines both physical and psychosocial dimensions of disability. This paper unpacks the composite score to assess if there are differences in the direction of effect on the individual components that make up the overall disability score by recognising the multifaceted nature of disability. Caring for someone with a chronic condition can be a risk factor as it can impact on functioning or lead to an acceleration of decline in functionality [START_REF] Vitaliano | Is caregiving a risk factor for illness?[END_REF].

To assess the presence of disease or general poor health the participants were asked the following question "what do you consider to be the most severe health problem you have currently?" If the respondents mentioned more than one health problem, they were asked to state one they considered to be the most severe.

Data analysis

The WHODAS disability scores were treated as continuous variables and summary statistics (mean and standard deviations) are presented comparing caregivers with non-caregivers. To investigate differences taking into account other explanatory factors, multiple OLS linear regression models were fitted for each of the six functionality and disability domains, in addition to the overall disability score. The second explanatory variable, presence of severe illness or health problem, is a binary variable indicating whether or not a person reported having what they considered as a severe health problem. A multiple logistic regression was fitted and the odds ratios of reporting a severe health problem are presented. Factors that are known to influence health (age, marital status, education level, income, and wealth status) are controlled for in the models. Separate models were fitted for females and males and the significance level was defined as p < .05. A caregiver was classified as someone who, at the time of the interview, was providing financial, instrumental, psychosocial, or nursing care to a person with a chronic illness (defined as any illness that had persisted for 3 months or longer) or someone who had provided care to a person with a chronic illness at any other time during the three years preceding the survey. In order to isolate care recipients who had an AIDS-related illness, an indirect approach was used. This approach is an adaptation of the WHO's AIDS case definition which categorises a person as having developed AIDS if he or she presents with at least two of the major signs of an AIDS case in combination with at least one of the minor signs. Overall, the WHO definition has very high specificity but low sensitivity. The presence of these major conditions or illness usually indicates advanced progression from asymptomatic HIV stage to full-blown AIDS [START_REF] Grant | ABC of AIDS: HIV infection and AIDS in the developing world[END_REF]. This study categorised care recipients who were reported to be ailing from at least one of the major signs associated with an AIDS case as having an AIDS-related illness.

Definition of a caregiver

Results

Socio-demographic characteristics of study participants

Table 1 presents the socio-demographic characteristics comparing AIDS caregivers, other caregivers and non-caregivers. The median age for the overall population is 56 years, however, the men are relatively younger compared to the women by three years. Among the women, AIDS-caregivers and other caregivers have very similar mean age (56.3 and 56.6 respectively) which is significantly different from noncaregivers with a mean age of about 62 years. The three groups were also significantly different in terms of the highest education level attained. For instance, a higher proportion of AIDS-caregivers (18%) had secondary education or higher, compared with only four percent of other caregivers and about five percent of non- There was no difference between the comparison groups of women with regards to marital status. The men's groups, however, were not significantly different in age and marital status. Unlike the women, where a higher proportion of AIDScaregivers had secondary education or higher, almost similar proportions of men who were AIDS-caregivers (18%), other caregivers (17%) and non-caregivers (18%) had secondary education or higher.

<<TABLE 1 ABOUT HERE>>

Characteristics of the care episode for AIDS-caregivers

About 10 percent (143) of older people reported to be providing or to have provided care to someone with a chronic illness. Out of the 143 carers, 47% (67) were classified as caring for someone with an AIDS-related illness (AIDS-caregivers). The average duration of care-giving for the AIDS-caregivers was 30 months with men generally providing care for longer durations compared with women (38 months vs. 29 months respectively). This arose largely due to gender differences in care-giving roles with women mainly providing nursing care and assistance with instrumental care while men mainly provided financial care. Demand for financial care may begin at the onset of the illness due, for instance, to medical expenses and the need for support towards living expenses during bouts of illness unlike nursing care which is needed at the symptomatic or AIDS stage of the infection [START_REF] Knodel | The economic consequences for parents of losing an adult child to AIDS: Evidence from Thailand[END_REF].

The different forms of care notwithstanding, all forms of care can be a source of burden and stress on the care-giver hence a cause for negative health outcomes.

Functionality/disability scores

Table 2 shows the summary statistics for the disability domains scores and the overall score for women and men by their care-giving experience. Overall, men reported was reported in the mobility domain for both men and women (22.0 ± 23.3 for women and 9.7 ± 16.7 for men) followed by life activities (15.6 ± 23.3 for women and 8.4 ± 17.1 for men) and cognition domains (10.2 ± 15.6 for women and 5.7 ± 11.8 for men) respectively. The lowest disability was reported for self care (4.2 ± 12.9 for women and 1.9 ± 7.8 for men).

<<TABLE 2 ABOUT HERE>> Men who were AIDS-caregivers reported the highest mean disability scores across all the domains (except participation in society) compared with other caregivers and noncaregivers. For instance the AIDS-caregivers recorded a mean disability score of 18.5 in the mobility domain compared with 14.0 among other caregivers and 9.0 for noncaregivers. Similarly, very high disability scores were reported by AIDS-caregivers in the life activities domain which was about twice (16.0) the score reported by other caregivers (8.1) as well as the non-caregivers (8.2). Among the women however, there is no consistent pattern emerging across the domains. Evidently, AIDS-caregivers relative to other caregivers and non-caregivers reported the highest disability scores in mobility and interpersonal domains but lowest in cognition, self care and life activities.

Table 3 presents the results of the multiple regression, modelling the effects of providing care on the different functionality domains and the overall disability score.

Different models are presented for women and men respectively. Older people who were not providing care or had not provided care over the reference period (noncaregivers) are the reference category. The regression results for women show no significant differences between AIDScaregivers and non-caregivers in reporting disability across all the domains after adjusting for known covariates (age and socio-economic indicators). For the men however, AIDS-caregivers had significantly higher disability scores for the mobility (p<0.01), interpersonal interaction (p<0.05), life activities (p<0.001) and the overall disability score (p<0.01) compared with non-caregivers. The direction of association between HIV care-giving and the health domains is not consistent across the domains for men as well as women. For instance, women caregivers were less likely to report disability with all the health domains except for mobility whereas the men were more likely to report disability with cognition, mobility, self-care and life activities and less likely to report disability with inter-personal and participation in society. Both men and women AIDS-caregivers reported higher disability scores with mobility and the overall disability score.

<<TABLE 3 ABOUT HERE>>

Differentials in illness and morbidity

A higher proportion of AIDS-caregivers (84%) reported having a severe health problem at the time of the interview compared with other caregivers (69%) and less than half (43%) of those who were not providing care. Results of the multiple logistic regression models for women and men to predict the odds of reporting a severe health problem are presented in Table 4. Similar to the disability domains, men who were AIDS-caregivers had significantly higher odds of reporting a severe health problem compared with non-caregivers after adjusting for age and other known confounders.

Men who were AIDS-caregivers were almost eight times (CI: 2.95 -21.53) more

Discussion and conclusion

The care for PWA in sub-Saharan Africa is provided largely by informal caregivers and older people provide the bulk of the care and support [START_REF] Ntozi | AIDS morbidity and the role of the family in patient care in Uganda[END_REF]. Care-giving to a person with a terminal illness apart from being physically demanding on the caregiver, can lead to negative health consequences [START_REF] Vitaliano | Is caregiving hazardous to one's physical health? A meta-analysis[END_REF]. In this study, the health of men and women who were providing care to someone with an AIDS-related illness, those providing care to people with other chronic ailments, and non-caregivers are compared. The assumption was that caregivers would present with poorer health outcomes compared with non-caregivers. In particular, it was assumed that AIDS-caregivers would be worse off due to factors such as stigma towards people with AIDS coupled with the long debilitating nature of the disease.

Gender and care-giving

A previous study found that older people who had been affected by HIV and AIDS either indirectly or directly reported poorer health than those not affected. Women also reported worse health compared to men (reference withheld, 2009). This paper examined the association between care-giving and health by conducting withingender comparison of self-reported health among men and women who had provided care to someone with an AIDS-related illness with other caregivers and noncaregivers. Differential gender effect is envisaged due to variation in care-giving perception, attitude and roles between men and women. The study found that women AIDS-caregivers did not differ significantly from other caregivers or non-caregivers in terms of the specific health domains, the overall disability score or in reporting having a severe health problem. On the contrary, men who were AIDS-caregivers were significantly more likely to report poorer health compared with other caregivers and non-caregivers.

A review of the literature to understand the gender differences observed in this study highlights the fact that men are known to downplay their health problems and hence under-report symptoms or distress especially in interview situations. Men are also socialised to be self-reliant, in control, courageous, and to portray self-confidence when faced with challenges or complex situations. Consequently, they are expected not to show feelings such as fear, anxiety, grief, or frustration and should be able to endure stress and bear pain and hence tackle care-giving tasks with control or stoically. Therefore, comparing women and men caregivers as opposed to caregivers and non-caregivers of the same gender, will point to worse health outcomes for women hence as an artefact of differential reporting [START_REF] Benyamini | Gender differences in the self-rated health-mortality association: Is it poor self-rated health that predicts mortality or excellent self-rated health that predicts survival?[END_REF][START_REF] Spiers | Are gender differences in the relationship between self-rated health and mortality enduring? Results from three birth cohorts in Melton Mowbray, United Kingdom[END_REF][START_REF] Idler | Discussion: Gender differences in self-rated health, in mortality, and in the relationship between the two[END_REF]. In this regard, there is little evidence that men are less emotionally sensitive to care-giving and related burden or stress. The true level of health problems for men compared with women is therefore rarely measured [START_REF] Mullan | Aging and informal caregiving to people with HIV/AIDS[END_REF][START_REF] Brown | Help-seeking by older husbands caring for wives with dementia[END_REF]. The other explanation of the differentials between men and women may arise from gender variation in gender-roles and coping strategies. Cultural expectations where men are expected to be in control and to show more endurance may lead to elevated levels of stress among men caregivers leading to poorer health outcomes [START_REF] Kramer | Men as caregivers: an overview[END_REF][START_REF] Stoller | Theoretical perspective on caregiving men[END_REF].Similarly, unlike women, men do not utilise or seek informal or difficulties relating to care-giving [START_REF] Ashley | Gender differences in coping strategies of spousal dementia caregivers[END_REF]. Informal support has been found to have a mediating effect in caregiver burden and strain [START_REF] Fuller-Jonap | Mental and physical health of male caregivers of a spouse with Alzheimer's disease[END_REF][START_REF] Mullan | Aging and informal caregiving to people with HIV/AIDS[END_REF].

Implications for HIV and AIDS health policies and programmes

Informal care-giving for people with AIDS will continue to play a very crucial role in filling the void brought about by the inability of the formal healthcare systems to meet the needs of people with HIV and AIDS. The findings from this study have highlighted the role of older men as caregivers and the gender-variation that exists among caregivers in the extent to which care-giving is associated with poorer health outcomes. Older women have largely been portrayed as the face of HIV and AIDS care and support, with several studies pointing at them as disproportionately affected.

There is however a need to recognize the role of older men as active and not passive caregivers at both the policy and program level. Interventions targeted at people affected by HIV and AIDS should include older men. This may demand having gender-neutral or gender-specific interventions focusing on men and women.

The second implication of this study relates to the healthcare needs for AIDScaregivers. While the current HIV and AIDS policy in Kenya seeks to improve the quality of life for people with HIV and AIDS through strengthening availability and access to treatment and care (National AIDS Control Council, 2005), the policy is silent on the healthcare needs for caregivers. One strategy would be to provide healthcare for a person with AIDS and their caregiver as dyads. The impact of care-giving manifests itself over a long period and the full extent of the effects can only be measured if individuals are followed up over a longer period of time. Other factors that act to mediate the effects of care-giving were not exhausted fully in the paper although attempts were made to control for confounders during the analysis. The cross-sectional nature of the study cannot adequately measure causality between care-giving to person with AIDS-related illness with health, so, the objective of this paper was to assess if there are any significant differences between the cases and the two comparison groups.

Limitations

The study could not take into account heterogeneity among caregivers due to the small sample size of HIV caregivers. Another limitation is the inability to detect whether the comparison groups have not been affected in any way through spill-over effect thus potentially under-estimating or over-estimating if positively affected the impact on the comparison groups [START_REF] Beegle | Methodological Issues in the study of the socioeconomic consequences of HIV/AIDS World Bank[END_REF].

F

  Data are drawn from a population-based study that seeks to investigate the wellbeing of older people living in the slums. The study, Survey on Social, Health and Overall Wellbeing of Older People, is nested within a demographic surveillance system (DSS) which follows up the population of usual residents of the two slum areas. One of the specific objectives of the study relevant to this paper was to look at possible mechanisms through which HIV and AIDS affects older people. All people 50 years and older who were resident in the DSS during the round of update preceding the for interview. The response rate was 79 percent. The survey was conducted between November 2006 and January 2007 using interviewer-administered questionnaires. The survey data are combined with routine socio-economic and demographic information collected over the same period (September -December

  for all the domains compared to women. The highest disability

  a severe health problem compared with non-caregivers. On the contrary, differences among women were not statistically significant. <<TABLE 4 ABOUT HERE>>

  This deters them from seeking support or discussing their problems

Table 1 : Socio-demographic characteristics of HIV-caregivers, other caregivers and non- caregivers HIV- caregivers Other caregivers

 1

	o r
	P
	e
	e r
	R
	e
	v i e
	w
	O n l
	y

for between care-giving differences and F-test significance levels *** <0.001; **<0.01 *<0.05 Source: Authors' analysis of NUHDSS Individual Data File, 2006

	F o
	r
	P
	e e r
	R e v i e w
	O n
	l y
	2

Table 2 : Mean and standard deviation of disability domain scores by caregiving experience for women and men HIV caregivers Other caregivers

 2

				Non-	Total
				caregivers
		Mean (SD)	Mean (SD)	Mean (SD)	Mean (SD)
		Women
	Mobility	26.6 (25.7)	24.7 (26.2)	21.6 (23.0)	22.0 (23.3)
	Cognition	8.6 (14.3)	9.2 (16.4)	10.3	10.2 (15.6)
	Self care	2.2 (7.9)	3.1 (12.2)	4.4 (13.2)	4.2 (12.9)
	Inter-personal	11.8 (13.0)	8.2 (11.7)	9.9 (14.8)	9.9 (14.5)
	Life activities	14.5 (20.7)	18.4 (27.7)	15.5 (23.1)	15.6 (23.3)
	Participation in society	6.6 (17.1)	5.3 (15.6)	9.3 (22.7)	8.9 (22.1)
	Overall disability	12.8 (11.3)	12.2 (14.1)	12.3 (13.5)	12.3 (13.4)
		Men
	Mobility	18.5 (21.3)	14.0 (19.5)	9.0 (16.1)	9.7 (16.7)
	Cognition	10.0 (16.6)	6.0 (11.0)	5.5 (11.5)	5.7 (11.8)
	Self care	2.3 (8.3)	1.3 (5.1)	2.0 (8.0)	1.9 (7.8)
	Inter-personal	8.3 (14.9)	4.0 (8.3)	5.2 (10.6)	5.2 (10.7)
	Life activities	16.0 (23.0)	8.1 (14.9)	8.2 (17.0)	8.4 (17.1)
	Participation in society	2.0 (9.9)	1.1 (7.4)	4.3 (15.4)	4.0 (14.8)
	Overall disability	10.4 (11.8)	6.5 (7.9)	5.9 (9.3)	6.1 (9.4)
	Standard deviation shown in parenthesis

Table 3 : Results of multiple linear regression models of disability domains for women and men 1

 3

		Women		Men
		Coefficient	P-value	Coefficient	P-value
		(standard errors)	sign	(standard errors)	sign
	Model 1: Mobility
	Non-caregivers (Ref)
	Other caregivers	8.686 (4.515)	NS	6.256 (2.204)	**
	HIV-caregivers	6.806 (3.948)	NS	8.407 (2.661)	**
	Constant	-25.986 (10.861)		-19.987 (6.446)
	R 2	11.4		11.8
	Model 2: Cognition
	Non-caregivers (Ref)
	Other caregivers	0.971 (3.013)	NS	2.165 (1.632)	NS
	HIV-caregivers	-1.028 (2.634)	NS	3.102 (1.971)	NS
	Constant	-7.655 (7.248)		0.885 (4.774)
	R 2	8.7		4.2
	Model 3: Self-care
	Non-caregivers (Ref)
	Other caregivers	2.234 (2.523)	NS	-0.019 (1.007)	NS
	HIV-caregivers	-0.415 (2.191)	NS	1.025 (1.229)	NS
	Constant	-2.848 (5.995)		-4.200 (2.925)
	R 2	6.1		4.3
	Model 4: Interpersonal
	interaction
	Non-caregivers (Ref)
	Other caregivers	0.917 (2.762)	NS	-0.490 (1.439)	NS
	HIV-caregivers	3.326 (2.415)	NS	4.056 (1.738)	*
	Constant	-6.305 (6.645)		-12.911 (4.210)
	R 2	9		7.7
	Model 5: Life activities
	Non-caregivers (Ref)
	Other caregivers	9.094 (4.423)	*	-0.861 (2.278)	NS
	HIV-caregivers	2.232 (3.867)	NS	9.773 (2.751)	***
	Constant	-22.357 (10.640)		-12.879 (6.665)
	R 2	8.4		9.4
	Model 6: Participation in
	society
	Non-caregivers (Ref)
	Other caregivers	1.166 (4.368)	NS	-3.459 (2.113)	NS
	HIV-caregivers	0.279 (3.820)	NS	-1.366 (2.551)	NS
	Constant	-6.996 (10.508)		-6.056 (6.181)
	R 2	6.9		2.2

NS-Not significant 1 Age, education level, per capita income and wealth status added as control variables Source: Authors analysis of Survey on Social, Health and Overall Wellbeing of Older People data, 2006/7

	F o
	r
	P
	e e r
	R e v i e w
	O n
	l y
	5

Table 4 : Logistic regression models on reporting a severe health problem for women and men

 4

	1
		Women	Men
		Odds ratio Confidence	Odds	Confidence
		intervals	ratio	intervals
	Non-caregivers	1.000	1.000
	(Ref)
	Other caregivers	0.748 (0.29 -1.88) NS	3.512 (1.87 -6.58)	***
	HIV-caregivers	1.885 (0.81 -4.39) NS	7.983 (2.95 -21.53) ***
	Model χ 2	32.4	60.0
	Degrees of freedom	7	7
	Log likelihood	-328.5	-565.3
	R 2	7.1	5.8
	Significance levels ***<0.001; **<0.05; NS Not significant
	1 Age,

education level, per capita income and wealth status added as control variables

  Source: Authors analysis of Survey on Social, Health and Overall Wellbeing of Older People data 2006/7


Benjamin William Strugnell
email: b.strugnell@vla.defra.gsi.gov.uk

Mark P Dagleish

Colin W Bayne

Mike Browne

Heather Ainsworth

J Nicholas

Alisdair Wood

Chris Hodgson

Benjamin ; Strugnell

Nicholas

Ben Strugnell

R A J Nicholas

J C Hodgson

Investigations into an outbreak of corvid respiratory disease associated with Pasteurella multocida

Keywords: corvid, pasteurella multocida, respiratory disease, UK

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.

Introduction

The Gram-negative bacterium Pasteurella multocida comprises 5 capsule serogroups (A, B, D, E and F) and 16 somatic serotypes. Serogroup A is isolated most frequently in fowl cholera, a disease of major economic importance in commercially reared birds and outbreaks in England are associated primarily with somatic serotypes 1,3 and 3,4 [START_REF] Rhoades | Somatic serotypes of Pasteurella multocida strain isolated from avian hosts (1976-1988)[END_REF]. Molecular typing has shown that wild birds, and possibly mammals, may be the source of infection in some disease outbreaks in commercial poultry [START_REF] Christensen | Fowl Cholera[END_REF]. The species and age of affected birds, their environment and the strain of P. multocida involved influence the severity and incidence of disease and mortality varies from a few to 100 percent [START_REF] Rimler | Fowl cholera[END_REF].

Corvids are intelligent, highly adaptable birds which have frequently capitalised on changes in agriculture and land use resulting in thriving populations, often in close proximity to farmed livestock and crops. Outdoor pig units are a particular niche to which corvids have become adapted [START_REF] Marchant | Number of nesting rooks Corvus frugilegus in the United Kingdom in 1996[END_REF]. However, there is little information on the susceptibility of corvids to, or their ability to transmit, bacterial disease such as fowl cholera.

The aim of this study was to investigate the aetiology of disease among corvids found either dead or displaying clinical signs of respiratory disease. Similar outbreaks of disease have been reported previously in the UK, sometimes referred to as corvid respiratory disease, without a definitive cause being determined (Pennycott 2005). Background, case definition, inclusion criteria and clinical signs. In August 2007, the carcases of seven rooks (Corvus frugilegus, birds 1-7) were submitted to the Veterinary Laboratories Agency (VLA) Thirsk regional laboratory for post-mortem examination under the UK Department of Environment, Food and Rural Affairs (Defra) Wildlife Incident Investigation Scheme (WIIS) and the VLA Diseases of Wildlife Scheme (VLADoWS). They represented an estimated 30 birds found dead in a 1.62 hectare wood comprised of deciduous trees, mainly ash, beech and oak, adjacent to a 1000-sow outdoor weaner producer pig unit near York, UK. Over the next six months, amid continuing mortality in this population of corvids, there were a further eight submissions (Table 1), comprising: three carrion crows (birds 9, 11 &14) and two rooks (birds 8 & 12) caught live showing clinical signs of respiratory disease; one live carrion crow (with a humeral fracture) showing no clinical signs of respiratory disease (bird 15) and five carrion crows found dead (birds 10, 13, 16, 17 &18).

Materials & Methods

F

Live-caught birds were held in a large crow trap (for at most 8 hours) and were delivered alive to VLA Thirsk except bird 12 which died in the trap. Clinical signs included moderate to severe dyspnoea, a reduced ability or inability to fly, a snicking cough and weakness (Table 1). Disease was considered to have been progressive, culminating in birds that were either easy to catch or found dead. birds. Live birds were euthanased with an intravenous overdose of pentobarbitone (Euthatal, Merial Animal Health) via a brachial vein. Samples of lung from at least one bird from all submissions (except submission 6), and other tissues abnormal on gross examination (Table 1), were placed into 10% formal saline for histopathology. Where indicated (Table 1) samples for bacteriological, virological and toxicological examination were collected from each bird using aseptic (seared surface) techniques. Selection of tissues for bacteriology was on the basis of abnormal appearance as determined by the pathologist performing the postmortem examination (BS) and samples plated out immediately (see below). A different range of tissues was therefore taken from each bird. Tissue samples for virological examination were stored at -80°C (maximum of 3 weeks) prior to investigation and those for toxicology stored at -80°C until required.

Ornithological

Microbiology.

Each sample was concomitantly cultured on sheep blood agar (SBA, Oxoid, Basingstoke, UK), SBA with a 'nurse streak' of Staphylococcus intermedius, SBA cultured in 5% CO 2 or MacConkey's agar (Oxoid). Direct and enrichment cultures for Salmonella using brilliant green agar (Oxoid) and Selenite broth ((Oxoid) were performed on pooled large intestinal contents from the first four submissions (birds 1-7 in 2 pools; 8-11 individually).

Fungal enrichment cultures were performed on all samples of lung and/or air sac which were selected for bacterial cultures using Sabourard's agar (SAB, Oxoid) alone or containing penicillin and streptomycin (SABA, Oxoid). Bacteria were identified using standard laboratory techniques. P. multocida was identified by characteristic smell and colonial morphology, Gram staining and microscopic appearance, lack of growth on MacConkey's agar, a positive indole reaction and a negative urease reaction [START_REF] Blackall | Pasteurellaceae-the view from the diagnostic laboratory[END_REF][START_REF] Dziva | Diagnostic and typing options for investigating diseases associated with Pasteurella multocida[END_REF]. Samples of lung, airsac and/or sinus at the discretion of the pathologist from all but the first seven birds (omitted due to autolysis) were placed in Friss pre-enrichment broth [START_REF] Bradbury | Recovery of mycoplasmas from birds[END_REF] and checked for Mycoplasma growth at 24 and 48hrs, then at weekly intervals for 4 weeks. Positive cultures were identified using denaturing gradient gel electrophoresis [START_REF] Mcauliffe | 16S rDNA and DGGE: a single generic test for detecting and differentiating Mycoplasma species[END_REF].

Bacterial DNA extraction. Isolated bacteria were cultured overnight in brain heart infusion (BHI) broth (Oxoid, UK) at 37°C and DNA extracted from 1ml of suspension using a DNeasy kit (Qiagen Ltd., Crawley, UK) according to manufacturer's instructions and quantified (NanoDrop Spectrophotometer, Nanodrop Products, Wilmington, USA). Samples were stored at -20°C until required.

Determination of capsule type, serotype and presence of virulence associated genes.

Isolates of P. multocida were analysed by PCR for the presence of species-specific sequence KMT1 [START_REF] Townsend | Development of PCR assays for species-and type-specific identification of Pasteurella multocida isolates[END_REF], capsule biosynthesis genes capA, B, D, E & F [START_REF] Townsend | Genetic organization of Pasteurella multocida cap loci and development of a multiplex capsular pcr typing system[END_REF] and the virulence associated genes pfhA, hgbB [START_REF] Ewers | Virulence genotype of Pasteurella multocida strains isolated from different hosts with various disease status[END_REF], tbpA [START_REF] Ogunnariwo | Characterization of a Novel Transferrin Receptor in Bovine Strains of Pasteurella multocida[END_REF] and toxA [START_REF] Lichtensteiger | Direct PCR Analysis for Toxigenic Pasteurella multocida[END_REF], using amplification conditions and primer sequences described by these authors. Primers were tested against positive control isolates which had been typed by conventional methods to confirm capsule type. For all PCR reactions 50 ng of extracted DNA were taken as template and added to the reaction mixture (50 µl) containing 0.2 µM of each primer (Eurofins MWG Operon, Wolverhampton, UK), 0.2 mM of each of the four deoxynucleotide triphosphatases (Invitrogen Ltd., Paisley, UK), 1 × PCR buffer, 1.5 mM MgCl 2 (except for capA PCR reactions which contained 2 mM MgCl 2 ) and 1 unit of Platinum Taq (Invitrogen). Samples were subjected to 35 cycles of amplification in a Lehne Thermocycler TC-3000 Techne Thermal cycler (Bibby Scientific Ltd., Staffordshire, UK). Amplified products were analysed Virology. A real time PCR for the detection of the matrix gene of influenza A virus [START_REF] Anon | Diagnostic manual for Avian Influenza, Ch. VI, Molecular tests and evaluation of results[END_REF] was performed separately on two sample sets from the first 8 birds examined (birds 1-8, submissions 1 and 2) comprising pooled bulk viscera; brain, liver, spleen, lung, kidney, trachea and intestine. For birds 1-11 samples of brain and kidney were tested for West Nile virus (WNV) by reverse transcription-polymerase chain reaction employing specific primers directed at the C-terminal of the C gene and the N-terminal of the prM gene and via 20% (w/v) suspensions of the same samples of brain and kidney after two passages in Vero C1008 (ATCC) cells [START_REF] Gough | Routine virus isolation or detection in the diagnosis of diseases of birds[END_REF][START_REF] Phipps | Detection of West Nile Virus in tissues of specific pathogen free chickens and serological response to laboratory infection: a comparative study[END_REF]. Scanning electron microscopy was performed on selected tissues from bird 8 (pooled bursa and spleen, and a pool of kidney and lung) to investigate the presence of other viruses. 3 mm Copper/Rhodium (100 µm mesh) support grids were used for the analyses. Each grid was pre-treated by immersing into 0.4% formvar in chloroform (w/v), followed by carbon coating, to provide a stable sample platform. Treatment was then completed, by subjecting each grid to plasma glow discharge, to ensure the grids were highly hydrophilic. Each sample analysed (approx. 0.25 cm 3 ) was ground in 2 cm 3 of 0.1 M Sorenson's phosphate buffer (pH 6.6), to form a suspension. An aliquot of each sample suspension (50 µl) was pipetted onto a piece of dental wax and a support grid was then placed copper side upwards onto the aliquot for 30 seconds and excess sample removed by wicking dry. Each grid was then placed as before onto a drop of 2% phosphotungstic acid (w/v) (pH 6.6), for 10 seconds to counter-stain the grid and again excess stain removed by wicking dry. Analysis of each sample was undertaken on a Phillips 2) were processed routinely to wax blocks within 72 hours of collection. Sections (5 µm) were mounted on glass microscope slides and stained with haematoxylin and eosin or subjected to Gram's stain (ProLab Diagnostics, Neston, UK) for bacteria or Grocott Gomori methenamine silver (GGMS) for fungal hyphae.

Further sections, including duplicates for negative control preparations, were mounted on Superfrost™ slides (Menzel-Gläser, Braunschweig, Germany) and subjected to immunohistochemistry (IHC) with polyclonal antiserum raised in a rabbit against P. multocida A:3 as described previously [START_REF] Dagleish | Characterisation and time course of pulmonary lesions in calves after intratracheal challenge with Pasteurella multocida A:3[END_REF]. A positive control sample consisting of a section of lung from a calf infected experimentally with P. multocida A:3 and containing typical lesions of suppurative broncho-pneumonia was included for comparison of the pattern of immunolabelling with that of P. multocida serotype F isolated from crows in this study. All slides were blind-coded and examined for morphological lesions and the presence and distribution of immunolabelling relative to histological lesions.

Toxicology. Screens for alpha chloralose and rodenticides were performed under the Wildlife Incident Investigation Scheme at the Central Science Laboratories, York, UK, using the methods decribed by [START_REF] Brown | Analytical methods used in the United Kingdom Wildlife Incident Scheme for the detection of animal poisoning by pesticides[END_REF], on pooled kidneys and livers respectively from birds 1, 3 & 4 (the least autolysed in submission 1).

Results

Ornithological investigations and observations. The corvid population at the site was estimated at 200-300 birds comprising 65% carrion crows, 25% rooks and 10% jackdaws (Corvus monedula). The nesting capacity of the wood could not accommodate this number of birds suggesting that a large proportion travelled daily some considerable distance to the farm. Approximately 60% of the birds found dead were in and around the wood, the remainder on grass pastures and nearby pig paddocks where it was common for large flocks of all 3 species to forage together.

Gross post-mortem findings. The commonest lesion, present in all 17 birds with lesions suggestive of infectious disease (Table 1), was fibrinous proliferation on at least one serosal surface (pericarditis, perihepatitis or airsacculitis). No gross lesions were present in the viscera of bird 15, live-caught due to a simple fracture of the left humerus with no clinical signs of respiratory disease. Airsacculitis (Figure 1) was present in 12 (70%) of the 17 affected birds. Necrotising pneumonia was seen in 3 (19%) of the 17 diseased birds, including bird 12 (Figure 2) which died awaiting collection. In most cases the gizzards were empty or contained little food and in 3 cases (birds 5, 8 and 14) ascarid worms were present.

Ectoparasites (lice) were noted on live-caught birds 8, 9 and 11. Cestodes were a common finding in the small and large intestines (6 (66%) of 9 rooks; birds 2, 4, 5, 7, 8 and 12 and 3 (33%) of 9 carrion crows; birds 9, 14 and 11). The findings suggested a spectrum of disease from relatively acute (e.g. bird 17) to chronic (e.g. bird 13) pneumonia and polyserositis. No gross lesions suggestive of haemorrhagic meningitis, congestion of meningeal vessels or brain oedema were present in any of the birds examined. Microbiological findings. P. multocida was isolated from at least one of either the lung, airsac, sinus, heart blood, liver or pericardium in all five affected live-caught birds (birds 8, 9, 11, 12 and 14) (Table 1). In bird 9 pure growths of P. multocida were recovered from the lung, air sac, liver and heart blood indicative of a septicaemic distribution. Salmonella spp.

were not isolated from large intestinal contents from birds 1-11 using direct or enrichment culture techniques and no further Salmonella cultures were performed. Escherichia coli, Proteus spp. and other saprophytic bacteria were recovered only from birds found dead (Table 1). Fungal cultures were negative from all tissues examined. Capsular type, virulence associated genes and serotype. Four P. multocida isolates (from birds 8, 9, 12 &14) were available for further testing All four demonstrated the presence of capF capsule gene only, the species specific gene sequence KMT1 and virulence associated genes haemoglobin binding protein (hgbB) and the filamentous haemagglutinin (pfhA).

However, transferrin binding protein gene (tbpA) and dermonecrotoxin gene toxA were not detected. Somatic serotypes were 4 (bird 8), 3,4,7 (bird 9) and 3 (bird 12). The isolate from bird 14 failed to type. WNV ceased after the first 11 birds proved negative on the basis of a low risk during the winter months (little mosquito activity) and a lack of consistent gross and histopathological findings. The EU target species list for HPAI surveillance (Anon 2010a) does not feature corvids so this testing was also restricted to the first two submissions on the basis of low risk and likelihood. Furthermore, histopathological lesions did not suggest avian influenza.

Mycoplasma enrichment cultures. Four different

Toxicological findings. Samples of kidney for alpha chloralose and liver for rodenticides were all negative. Further samples for toxicology were retained but not tested on the basis that the lesions were more typical of an infectious process.

Histopathology and Immunohistochemistry. Pneumonia and/or air sacculitis was the most consistent lesion in the birds found dead or live-caught with clinical signs of respiratory disease. The character of the pneumonia varied between granulocytic and fibrinogranulocytic both with and without areas of necrosis (Figure 3). Positive labelling for P. multocida was present in the lungs (Figures 4a &b) and/or air sacs of all but one of the affected birds examined by specific IHC (Table 2). Bird 17 did not have pneumonia or air sacculitis but a small focus of necrosis present in the lung was positive for P. multocida by IHC. Gram-negative coccobacillary bacterial colonies present extra-cellularly and also within the foamy cytoplasm of macrophages within the lungs and air sacs of some birds were positive for P. multocida by IHC. Bird 15 had a mild cholangiohepatitis. However, no significant lesions were found in the lung, brain or spleen and all tissues were devoid of immunolabelling in this bird. All negative control preparations were devoid of labelling.

Nematode parasite profiles were present in the respiratory systems of three birds and a trematode in one (Table 2). No fungal hyphae or other fungal forms were present in any of

Discussion

There are early reports of disease in wild corvids from the UK [START_REF] Keymer | A survey and review of the causes of mortality in British birds and the significance of wild birds as disseminators of disease[END_REF]McDairmid, 1965) but detailed and recent descriptions are lacking. A syndrome of 'corvid respiratory disease' featuring varying degrees of fibrinous airsacculitis, perihepatitis, pericarditis and pneumonia has been reported (Pennycott, 2005) but most cases were submitted dead and no consistent aetiology was identified.

In this study P. multocida was isolated from the five corvids displaying clinical signs of respiratory disease which were submitted live. All had gross lesions in the respiratory tract.

In all but two of the birds submitted dead, Gram-negative coccobaccilli bacteria were associated intimately with the pulmonary histological lesions and showed positive labelling for P. multocida by IHC. No gross or histological pulmonary lesions, immunolabelling or isolation of P. multocida were observed in a live-submitted bird that did not have clinical signs of respiratory disease. Samples from further birds unaffected by respiratory disease was unfortunately not available. Taken together, these results suggest that P. multocida was the cause of the pathology.

It was unusual for P. multocida to type only as serogroup F, as avian isolates analysed by traditional methods are more usually reported as serogroup A [START_REF] Christensen | Fowl Cholera[END_REF]. However, a comparison of PCR and conventional methods for typing avian isolates of P. multocida, including serogroup F, showed good agreement [START_REF] Shivachandra | Identification of avian strains of Pasteurella multocida in India by conventional and PCR assays[END_REF] and our findings may reflect the greater discrimination between serogroups possible with PCR, [START_REF] Townsend | Genetic organization of Pasteurella multocida cap loci and development of a multiplex capsular pcr typing system[END_REF].

The failure to isolate P. multocida from birds found dead was surprising as others have isolated it readily from carcases of birds which have succumbed to fowl cholera [START_REF] Glisson | Fowl Cholera[END_REF][START_REF] Dziva | Diagnostic and typing options for investigating diseases associated with Pasteurella multocida[END_REF]. It may have been that the strain of P. multocida involved was sensitive to changes in temperature, pH and/or other factors as well as competition from other species of bacteria proliferating post-mortem, especially if bacterial loads were low in this chronic manifestation of disease (see below). Future studies of similar disease outbreaks may benefit from using PCR techniques in addition to bacterial culture to detect P. multocida within tissue samples although the technique does not allow the specific localisation within lesions obtainable with IHC.

Corvids have been reported as becoming infected with P. multocida after feeding on the carcases of birds which have recently succumbed to fowl cholera and in these reports, the disease course in corvids is longer than in waterfowl. [START_REF] Zinkl | An epornitic of avian cholera in waterfowl and common crows in Phelps county, Nebraska, in the spring, 1975[END_REF][START_REF] Taylor | Avian cholera in common crows, Corvus brachyrhynchos, from the central Texas panhandle[END_REF]. This observation is consistent with our findings where sometimes progressive clinical signs were seen in affected corvids, and histopathological findings showed a chronic disease course. The feeding adaptability and ubiquity of scavenging birds such as corvids makes contact between them and domestic species likely and creates a risk of disease transmission from diseased or sub-clinically affected birds. There is experimental and epidemiological evidence to suggest that in some outbreaks of disease in domestic poultry wild birds were responsible for introducing infection [START_REF] Snipes | Pasteurella multocida in wild mammals and birds in California: prevalence and virulence for turkeys[END_REF][START_REF] Petersen | Virulence of Pasteurella multocida subsp. Multocida from outbreaks of fowl cholera in wild birds for domestic poultry and game birds[END_REF], and risks may be increased if disease (and shedding) is chronic. Challenge studies using the P. multocida F isolates from corvids in this series would establish their potential pathogenicity to domestic poultry and therefore the risk infected corvids may pose. However, this work was 14 out-with the scope of this study. In the USA corvids are considered important in the epidemiology of WNV as they are highly susceptible, developing a substantial viraemia prior to death [START_REF] Reisen | Role of corvids in Epidemiology of West Nile Virus in Southern California[END_REF][START_REF] Murray | West Nile Virus and its emergence in the United Ststes of America[END_REF]. In Western Europe reports of human fatalities due to encephalitis caused by this disease have been recentlly reported [START_REF] Papa | Ongoing outbreak of West Nile virus infections in humans in Greece, July-August 2010[END_REF], and surveillance for this agent is important [START_REF] Zoller | West Nile Virus: The need to strengthen preparedness in Europe[END_REF]. However, to date WNV has not been detected in the UK and all tests in this series were negative.

Although several of the birds were parasitised by lice, ascarid nematodes and cestodes, burdens were low and not considered out-with normal limits for wild birds. It is possible that the empty gizzards were a result of the respiratroy disease rather than a cause of it. There was histological evidence of infestation with Syngamus trachea in one bird (bird 10, see table 2), diagnosed with caseous airsacculitis. Parasitic bronchitis may have predisposed to opportunistic bacterial infection (E. coli, not P. multocida, was isolated) in this bird.

However, in general, parasitism was not considered a major factor in this disease outbreak.

Of the Mycoplasma species isolated only M. sturni has been reported to be of pathological significance, being associated initially with conjunctivitis in songbirds in the USA [START_REF] Ley | Mycoplasma sturni from Blue Jays and Northern Mockingbirds with conjunctivitis in Florida[END_REF]. In Europe it is associated with a range of clinical signs, though not conjunctivitis [START_REF] Pennycott | Mycoplasma sturni and Mycoplasma gallisepticum in wild birds in Scotland[END_REF]. In common with the latter study, M. sturni-positive birds in the present report were not suffering from conjunctivitis, and the significance of mycoplasma carriage remains unclear.

The pig unit was closed down during the submission period and before samples could be obtained, consequently it was not possible to investigate whether commensal carriage of P. multocida by pigs on this unit contributed to the epidemiology of the disease. However, serogroup F is rarely isolated in this species [START_REF] Rimler | Pasteurella multocida isolated from rabbits and swine: Serologic types and toxin production[END_REF][START_REF] Pijoan | Pneumonic pasteurellosis[END_REF], making a link with the observed disease in corvids unlikely. On the basis of the findings reported here, P. multocida infection should be considered as a major differential diagnosis for respiratory disease in wild corvids, the diagnostic rate will be enhanced by the use of IHC for P. multocida in such investigations, especially in those birds submitted dead. Additionally, corvids may act as a reservoir of P. multocida for infections in domestic poultry.

F

  and natural history investigations. The site was visited on six occasions at regular intervals during the period from September 2007 until June 2008, in collaboration with the British Trust for Ornithology (BTO), to determine the feeding, roosting and other behaviours of corvids and other wild birds. Estimates of the sizes of corvid populations, population dynamics and interactions were performed subjectively by an experienced local ornithologist (M. Browne).

  Gross post-mortem examination. Post-mortem examinations were performed on all 18

  photographed. Serotypes of P. multocida isolates from birds 8, 9, 12 and 14 were determined

  × 34, 000 magnification, at 80 kV. Analysis time was standardised to 20 minutes viewing of the sample grid or assessment of 25 grid squares for the presence of virus particles, whichever was the shortest Histopathology and Immunohistochemistry. Tissues for histopathology from 17 birds (Table

Virology.

  All samples examined for WNV and High Pathogenicity Avian Influenza (HPAI) were negative and no other viruses were identified in the cell cultures performed to detect WNV. No virus particles were seen on electron microscopy where performed. Testing for

  using special stains (GGMS) nor any structures suggestive of viral inclusions in the bursas or spleens that were available for examination.

  study when a proportion of avian P. multocida isolates characterised previously by traditional methods as serogroup A were shown by PCR to belong to the closely related serogroup F

(Figure 1 .

 1 Figure Legends

Figure 2 .

 2 Figure 2. Severe necrotic pneumonia, confirmed histologically, in an ex situ lung sample from bird 12,

Figure 3 .

 3 Figure 3. Histological preparation of lung (bird 12) showing severe fibrino-granulocytic pneumonia with

Figure 4a .

 4a Figure 4a. Immunohistochemical preparation labelling Pasteurella multocida (brown pigment) in lung

Figure 4b .

 4b Figure 4b. Higher power image showing immunolabelling (brown pigment) of P. multocida within the

  Fig 1

  Mycoplasma species (M. gallopavonis, M. gallinarum, M. colomborale, and M. sturni, Table 1) were identified. Neither M. gallisepticum nor M. synoviae were isolated from any sample.

Table 1 .

 1 Summary of clinical signs, gross pathology and bacteriological findings from this case series. CC= Corvus corone; CF= Corvus frugilegus;

	o r
	P
	e
	e r
	R
	e
	v i e
	w
	O n l
	y

Table 2 :

 2 Histological lesions present and immunohistochemistry results in birds available for examination

Acknowledgments

The authors thank Steven Newlove (owner of the outdoor pig unit) for his diligence and co-operation and Clare Underwood and Jeanie Finlayson (MRI) for expert preparation of histological and immunohistochemical sections. MPD, CWB and JCH are funded by the Scottish Government. The Wildlife Incident Investigation Scheme (WIIS) is funded by Defra. Some work was funded by the Scanning Surveillance VLA Diseases in Wildlife Scheme (ED1600), funded by Defra's food and farming group (FFG).