Tina Broennum Pedersen

John Elmerdahl Olsen

Magne Bisgaard

Persistence of Salmonella Senftenberg in poultry production environments and investigation of its resistance to desiccation

Keywords: Salmonella, poultry, persistence, desiccation

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Introduction Salmonella enterica subsp. enterica serovar Senftenberg (S. Senftenberg) is frequently isolated from feed raw materials, the processing line and environmental samples from feed factories and from poultry farms [START_REF] Bailey | Sources and movement of Salmonella through integrated poultry operations: a multistate epidemiological investigation[END_REF][START_REF] Nesse | Molecular analyses of Salmonella enterica isolates from fish feed factories and fish feed ingredients[END_REF]Anonymous, 2006a;Broennum et al., 2007b). In addition, S. Senftenberg has been associated with several foodborne outbreaks of salmonellosis in humans [START_REF] Rushdy | National outbreak of Salmonella Senftenberg associated with infant food[END_REF][START_REF] Mohle-Boetani | Escherichia coli O157 and Salmonella infections associated with sprouts in California, 1996-1998[END_REF].

The routes of introduction of Salmonella into a broiler flock include vertical transmission from Salmonella infected parent flocks, persistence in the environment and contaminated feed [START_REF] Bisgaard | Mulige sammenhaenge mellem regelgrundlag, frivillige handlingsplaner og opnåede resultater på fjerkraeområdet. Til "Høring om Salmonella[END_REF]. In 1994 Denmark implemented EU directive 92/117/EEC ("the Zoonosis Directive"), under which all breeding stock are tested for S.

Enteritidis and S. Typhimurium, with elimination of positive flocks. By December 1996, the Danish Salmonella Control Program was extended to include all Salmonella serovars, and eggs from a flock can only be used for production of heated egg products if any Salmonella type is detected. The Danish Poultry Council, in cooperation with feed mills, has run a voluntary HACCP-based hygiene programme that includes heat treatment since 1989 [START_REF] Bisgaard | A voluntary Salmonella control program for the broiler industry, implemented by the Danish Poultry Council[END_REF] and heat treatment of animal feed became compulsory in 1993. These initiatives resulted in a significant decrease in the prevalence of Salmonella positive broiler flocks and table egg flocks, with current annual prevalences of approximately 2% and 1%, respectively (Anonymous, 2006a). Surprisingly, the prevalence of isolation exotic serovars has not decreased, indicating that feed and/or the production environment remain significant sources.

S.

Senftenberg is able to colonize and persist in feed mills and poultry houses [START_REF] Wray | The epidemiology of Salmonella infection of calves: the role of dealers[END_REF]Anonymous, 2006a;[START_REF] Anonymous | The national Salmonella control programme for the production of table eggs and broilers 1996-2002[END_REF][START_REF] Löfström | Improvement and validation of RAPD in combination with PFGE analysis of Salmonella enterica ssp. enterica serovar Senftenberg strains isolated from feed mills[END_REF] and it has been suggested that it is more resistant to stresses such as low pH, heating, desiccation and irradiation than other serovars [START_REF] Liu | Thermal resistance of Salmonella Senftenberg 775W in dry animal feeds[END_REF][START_REF] Mackey | The effect of sublethal injury by heating, freezing, drying and gamma-radiation on the duration of the lag phase of Salmonella Typhimurium[END_REF]. However, understanding of the reasons for its increased resistance and unambiguous demonstration of its persistence, as demonstrated by molecular typing methods, have been lacking. This study was conducted to investigate how long S. Senftenberg might persist in cleaned and disinfected poultry houses and be able to infect new Salmonella-free stock, and to examine whether persistent S. Senftenberg isolates survived desiccation better than other S. Senftenberg isolates.

F

Materials and methods

Bacterial strains. A total of 34 S. Senftenberg strains were investigated for persistence using amplified fragment length polymorphism (AFLP) and identical pulsed field gel electrophoresis (PFGE) analysis. The 34 strains included two isolates from 2002 and a single isolate from 2004 from the same farm (Farm 3), as well as two isolates from Farm 4 from January 2000 and July 2001 (Table 1). After slaughter of the S. Senftenberg positive broiler parent stock (PS) on Farm 3 in February 2003, the farm was cleaned, disinfected and remained free of poultry for 10 months until December 2003. Subsequently, Salmonella free stock for table egg production were introduced. Sampling of the flocks was carried out according to national legislation [START_REF] Anonymous | The national Salmonella control programme for the production of table eggs and broilers 1996-2002[END_REF]. In December 2004, the table-egg producing flock tested positive for S. Senftenberg, 22 months after the farm had been properly cleaned and disinfected and after six consecutive samples were negative for Salmonella. Two pairs of sock samples, obtained by wearing the socks over the boots during a walk through the entire poultry house [START_REF] Skov | Evaluation of sampling methods for the detection of Salmonella in broiler flocks[END_REF], were collected from each house and 60 eggs were tested each time [START_REF] Anonymous | The national Salmonella control programme for the production of table eggs and broilers 1996-2002[END_REF]). Another broiler parent flock (Farm 4) tested positive for S. Senftenberg approximately three months before slaughter after having tested negative on a weekly basis for 27 weeks. Subsequently, the flock tested negative 10 times until processing. After cleaning, disinfection and dry-out, a subsequent flock tested negative for the whole production period (40 times). A subsequent flock tested negative for five weeks, after which the house again tested positive for S. Senftenberg. However, 35 subsequent samplings all tested negative for Salmonella. Full cleaning and disinfection had been carried out in between the flocks. 1) were included to investigate diversity. All the S.

F

Senftenberg isolates had been serotyped using the Kauffmann White Scheme [START_REF] Popoff | Antigenic formulas of the Salmonella serovars, 8 th Ed[END_REF]. Single isolates of Pantoea agglomerans and Enterobacter purinus were used in the desiccation assay as reference strains. These isolates had been found persisting on a poultry feed processing line (Broennum et al., unpublished data) Desiccation assay. Pure cultures of all strains were inoculated into 10 ml sterile Luria Bertani (LB) broth [START_REF] Sambrook | Molecular Cloning: A Laboratory Manual 2[END_REF]) and incubated at 37°C for 24 h. Cells from 1 ml of this overnight (ON) culture were harvested by spinning at 15,000 rpm for 5 min at 4°C. The pellets were suspended in 1 ml MilliQ-water to avoid salt stress during desiccation. Aliquots of 50 µl were added to wells in microtitre trays (Nunc A/S, Roskilde, Denmark). The trays were dried at 37°C in an incubation chamber at approximately 70% relative humidity (RH) for 24 h, after which they were transferred to a desiccator maintained at 38-40% RH and at 37°C for the remainder of the study (34 days). Culturable cell counts were performed on samples subjected to desiccation for 0, 1, 2, 5, 9, 15, 20 and 35 days. The desiccated cells were rehydrated by adding 100 µl physiological saline, incubating for 10 min and then mixing by pipetting before plating. Colony forming units (CFU) per ml were calculated by inoculating 10 µl samples from tenfold dilution series onto LB agar plates [START_REF] Sambrook | Molecular Cloning: A Laboratory Manual 2[END_REF]. AFLP analysis. Strains were grown overnight in LB broth [START_REF] Sambrook | Molecular Cloning: A Laboratory Manual 2[END_REF]) at 37°C and DNA was extracted using the EASY-DNA kit (Invitrogen, Carlsbad, CA, USA). AFLP analysis was performed as described by [START_REF] Torpdahl | Population structure of Salmonella investigated by amplified fragment length polymorphism[END_REF] using BspDI and 6carboxyfluorescein (FAM) labelled BglII primers. GeneScan 3.1 fragment analysis software

Results

Analysis of persistence by molecular typing. A total number of 34 S. Senftenberg isolates were analysed by AFLP. Cluster analysis generated a dendrogram with all similarities above 84% (Figure 1). The AFLP profiles consisted of 65 to 89 fragments between 50 and 500 bp.

Twenty-one isolates made up one large cluster (A) based on an arbitrary threshold point of 98.7% similarity, and this cluster included isolates obtained from both the farms suspected to have had persistent contamination (Farm 3 and 4), isolates from Farm 1, and isolates from feed raw materials, imported turkey and chicken meat, and imported curry. This cluster could be divided into subclusters A1, A2, A3 and A4. All three isolates from Farm 3 belonged to subcluster A1. Two isolates from 2002 and 2004 (21 and 22, respectively) were identical, while a second isolate from 2002 (24) differed from these two by a single band. Two isolates from imported curry and single isolates from imported chicken and turkey meat shared the same AFLP profile and made up subcluster A2. Nine isolates from a broiler farm (Farm 1) and a broiler breeder farm (Farm 4) made up subcluster A3. Isolates from Farm 1 were obtained between February 2004 and May 2005, while isolates from the breeder farm were obtained between January 2000 and July 2001. Subcluster A4 included isolates from soy, process line samples from a feed mill, and one isolate from Farm 1. These isolates differed by four bands.

Cluster B had 98% similarity to cluster A and included isolates from imported turkey meat and exported swine meat. Two isolates from poultry feed processing line samples, which grouped together with one isolate from Farm 2, formed cluster C (Figure 1), although there were 16 band differences between these isolates. Isolates in cluster D had least similarity to the other isolates. This cluster also included a feed mill process line isolate and an isolate from Farm 5. However, there were eight band differences between these isolates. Cluster E included two highly related isolates from imported chicken meat. In addition to these five clusters, there were single isolates with distinct profiles.

The 34 S. Senftenberg isolates were also subjected to PFGE analysis. A subset of 25 isolates is shown in Figure 2. All isolates from Farm 1 had identical profiles. Likewise, isolates 28 and 33 had identical profiles, as well as isolates 18 and 19 (not shown). PFGE separated the isolates into 15 profiles with less than 90% similarity (Figure 2). Six clusters (I-VI) had greater than 90% similarity. Isolates in cluster I had identical profiles and included all the isolates obtained from Farm 3. All isolates from Farm 4 (cluster II) were identical, as were those from Farm 1 (cluster III). Isolates from Farm 4 were similar to isolate 34, which was obtained from a sample of soy. Isolates from imported turkey meat in cluster IV were also similar, as were isolates from imported turkey and pig meat in cluster V, and isolates from imported curry and process line samples in cluster VI.

Resistance to desiccation. The mean concentration of viable cells after overnight culture of all the S. Senftenberg isolates was 9.64 log 10 CFU/ml. After the cells were washed and dispensed into the wells there was a mean loss of viability of approximately 13%, leaving a mean concentration of 9. 57 log 10 CFU/ml for the desiccation trial. Over the first 24 hours the concentration of viable cells decreased by 79% to approximately 8.87 log 10 CFU/ml (Figure 3). After 48 hours there was a further decrease in concentration of only 6%. Over the first 20 days there was an 83% decrease in the concentration of viable cells and a further decrease of 36% between days 20 and 35. Over 35 days of desiccation at 40% RH, there was a 99.8% reduction in the concentration of viable cells, to a final concentration of 6.12 log 10 CFU/ml. The variation between isolates increased with duration of desiccation (Figure 3), and the concentration of the different strains at day 35 ranged from 4.22 to 7.77 log 10 CFU/ml (Figure 3). The loss in viability of the three isolates from Farm 3 was less than the mean of all

Discussion

We investigated the persistence of S. Senftenberg in poultry environments. The three isolates obtained from Farm 3 over a period of 30 months were identical by PFGE analysis and by AFLP analysis the isolates obtained in July 2002 and December 2004 were identical while a single isolate obtained in August 2002 differed from them by only one DNA fragment. These data suggest that S. Senftenberg can persist for very long periods of time. Over this 30 month period, the environment of the poultry house was exposed to detergents, disinfectants and subsequent desiccation. This observation extends those of [START_REF] Davies | Persistence of Salmonella Enteritidis in poultry units and poultry food[END_REF], who were able to isolate Salmonella Enteritidis PT4 from floor sweepings and rehydrated wall fabric junctions in an empty poultry house 22 weeks after cleaning and disinfection. However, their characterization relied on serotyping and phage typing, whereas we used two different genotyping methods and serotyping to demonstrate clonality. The ability of S. Senftenberg to persist in poultry production facilities and infect subsequent stock was also demonstrated by observations on both Farms 1 and 4 in our study.

Despite the demonstration of persistence of S. Senftenberg on Farms 3 and 4, salmonella were not isolated from repeated weekly samples over extended periods. Salmonella were not isolated from the broiler parent flock on Farm 3 during rearing or the first three weeks on the production farm prior to the first positive sample from Houses 1 and 6 in July 2002. The following week, a positive sample was also obtained from House 2, while samples from the other houses were negative. After two weeks of negative samples, a sample from House 1 was again positive, then samples for an additional 12 weeks were negative, followed by a positive sample and a further 13 negative samples. Samples from House 6 were negative every week until the eleventh week after the first positive sample, then this positive sample was followed by 18 negative samples. A total of 55 samples tested negative for Salmonella between these two samplings. Similar observations of samples intermittently positive for S. Enteritidis and/or S. Typhimurium have been reported previously [START_REF] Gradel | A questionnaire-based, retrospective field study of persistence of Salmonella Enteritidis and Salmonella Typhimurium in Danish broiler houses[END_REF][START_REF] Anonymous | The national Salmonella control programme for the production of table eggs and broilers 1996-2002[END_REF]. The intermittently positive samplings might be explained by either a lack of sensitivity of the sampling and test method, combined with a very low infection pressure [START_REF] Skov | Evaluation of sampling methods for the detection of Salmonella in broiler flocks[END_REF]. However, as doses as low as 5 cells [START_REF] Milner | Bacteriologic studies of experimental Salmonella infections in chicks[END_REF] or 10 2 cfu [START_REF] Van Immerseel | Intermittent long-term shedding and introduction of carrier birds after infection of chickens early posthatch with a low or high dose of Salmonella Enteritidis[END_REF]) have shown to be able to colonize chickens, the possibility of persistence in a location to which the chickens are infrequently or indirectly exposed should be considered. Age differences in susceptibility to infection [START_REF] Beal | Age at primary infection with Salmonella enterica serovar Typhimurium in the chicken influences persistence of infection and subsequent immunity to re-challenge[END_REF][START_REF] Van Immerseel | Intermittent long-term shedding and introduction of carrier birds after infection of chickens early posthatch with a low or high dose of Salmonella Enteritidis[END_REF]) might also partially explain the intermittently positive samplings.

Serovars such as S. Senftenberg and S. Infantis appear to persist in poultry houses [START_REF] Gradel | A questionnaire-based, retrospective field study of persistence of Salmonella Enteritidis and Salmonella Typhimurium in Danish broiler houses[END_REF][START_REF] Anonymous | The national Salmonella control programme for the production of table eggs and broilers 1996-2002[END_REF]. These persistent serovars have also been found in raw materials for poultry feed production [START_REF] Bisgaard | Mulige sammenhaenge mellem regelgrundlag, frivillige handlingsplaner og opnåede resultater på fjerkraeområdet. Til "Høring om Salmonella[END_REF][START_REF] Anonymous | The national Salmonella control programme for the production of table eggs and broilers 1996-2002[END_REF]Broennum et al. unpublished data). Although Salmonella were not isolated from any finished poultry feed in Denmark over the period in which these isolates were obtained from the farms [START_REF] Anonymous | The national Salmonella control programme for the production of table eggs and broilers 1996-2002[END_REF]Broennum et al. unpublished data), epidemiological investigations indicate that feed still represents a risk to the poultry industry [START_REF] Davies | Bacteriological and serological investigation of persistent Salmonella Enteritidis infection in an integrated poultry organisation[END_REF][START_REF] Bisgaard | Mulige sammenhaenge mellem regelgrundlag, frivillige handlingsplaner og opnåede resultater på fjerkraeområdet. Til "Høring om Salmonella[END_REF][START_REF] Jensen | Salmonella Control In Primary Breeders[END_REF]Broennum et al. unpublished data). PFGE analysis of isolates from Farms 3 and 4 suggested that they were related to isolates from feed raw materials.

Since the isolates from soy were obtained in July 2002 (Feed Mill A) and November 2003 (Feed Mill C) and the isolates from Farm 4 were from January 2000 and July 2001, there does not appear to be any direct link. However, as Feed Mills A, B and C sometimes share bulk imported raw feed materials and as S. Senftenberg may persist in bulk feed tanks and other parts of the unclean area of the a feed mill [START_REF] Nesse | Molecular analyses of Salmonella enterica isolates from fish feed factories and fish feed ingredients[END_REF][START_REF] Anonymous | The national Salmonella control programme for the production of table eggs and broilers 1996-2002[END_REF] , 2006), the poultry feed mill might have been contaminated with S. Senftenberg from soy products imported prior to 2000. Soy seems to pose a particular risk for Salmonella contamination (Anonymous, 2006a;Broennum et al., unpublished data).

Finished poultry feed and feed raw materials are normally stored at 12-14 % RH to prevent microbial growth. The water activity (a w ) and organic matter in the environment or in feed influence the survival of Salmonella during desiccation [START_REF] Mattick | The survival and filamentation of Salmonella enterica serovar Enteritidis PT4 and Salmonella enterica serovar Typhimurium DT104 at low water activity[END_REF][START_REF] Mattick | Effect of challenge temperature and solute type on heat tolerance of Salmonella serovars at low water activity[END_REF]. Other feed ingredients, such as fat, also influence bacterial survival (Greenwood & Hopper, 1983). [START_REF] Duncan | Effects of a chemical additive and of formaldehyde gas fumigation on Salmonella in poultry feeds[END_REF] suggested that in feeds with similar moisture contents, different ingredients may affect a w values, providing different environments for bacterial survival and growth. As the desiccation assay used in this study was based upon dehydration of the cells after washing in MilliQ-water, enhanced survival due to lipids, proteins and cross-protection by adaptation to salt were excluded. Over 35 days of desiccation at 40% RH, we observed a mean 99.8% reduction in the concentration of viable cells of the 34 S. Senftenberg isolates. [START_REF] Ha | Indigenous feed microflora and Salmonella Typhimurium marker strain survival in poultry mash diets containing varying levels of protein[END_REF] observed a 66% decrease in the concentration of Salmonella after 56 days in poultry mash containing soybean meal and a 71% decrease in mash containing meat and bone meal (a w approx. 0.63%). We found that all the S.

Senftenberg isolates survived better than a Pantoea agglomerans isolate. However, S.

Senftenberg has not previously been found to persist any longer in poultry feed than S.

Enteritidis, S. Typhimurium or S. Mbandaka [START_REF] Davies | Persistence of Salmonella Enteritidis in poultry units and poultry food[END_REF]. The Pantoea agglomerans isolate used in our study has previously been shown to persist over a period of 14 days in the cooler in a processing line producing poultry feed, where the RH of the feed was below 20% (Broennum et al., unpublished data).

Our desiccation study was performed at 40% RH and 37°C. Thirty-one S. Infantis isolates desiccated under similar conditions had comparable losses of viability and the losses in viability at 37°C were similar to those seen at 20°C over 35 days (Broennum et al., Previous epidemiological studies on the persistence of Salmonella in poultry environments have used serotyping and plasmid profiling [START_REF] Baggesen | Plasmid profiles and phage types of Salmonella Typhimurium isolated from successive flocks of chickens on three parent stock farms[END_REF][START_REF] Brown | Salmonella enterica: infection, cross infection and persistence within the environment of a broiler parent stock unit in Denmark[END_REF]. In our investigation, a combination of fluorescently labelled AFLP and PFGE analysis was used to ensure reliability, as the discriminatory power of typing methods differs between Salmonella serotypes (Liebana et al., 2001a). PFGE of fragments generated by XbaI digestion of genomic DNA has been recognised as a sensitive method for molecular fingerprinting of several Salmonella serotypes [START_REF] Lyytikainen | Molecular epidemiology of an outbreak caused by Salmonella enterica serovar Newport in Finland and the United Kingdom[END_REF][START_REF] Valdezate | Evaluation of phenotypic and genotypic markers for characterization of the emerging gastroenteritis pathogen Salmonella Hadar[END_REF]Liebana et al., 2001b).

AFLP has previously been used for separation of epidemiologically unrelated isolates within serovars of Salmonella enterica with varying results [START_REF] Lindstedt | Fluorescent amplifiedfragment length polymorphism genotyping of Salmonella enterica subsp. enterica serovars and comparison with pulsed-field gel electrophoresis typing[END_REF][START_REF] Torpdahl | Population structure of Salmonella investigated by amplified fragment length polymorphism[END_REF]. A threshold of greater than 90% similarity has been used previously to define genetically similar isolates of S. Typhimurium DT 126 in fluorescent AFLP studies [START_REF] Ross | Use of AFLP and PFGE to discriminate between Salmonella enterica serovar Typhimurium DT126 isolates from separate food-related outbreaks in Australia[END_REF]. However, use of a threshold of 90% similarity for AFLP analysis of S. Senftenberg, did not generate the same clusters as obtained using PFGE, suggesting that the threshold similarity for AFLP analysis must be determined empirically for each organism to enable meaningful biological and epidemiological conclusions to be drawn.

In conclusion, S. Senftenberg was demonstrated to persist in several poultry production environments for periods of up to 30 months. In addition, we demonstrated that S.

  , Naerum, Denmark) was used for fragment size determination and pattern analysis. Only AFLP profiles comprising fragments in the size range of 50-500 bp were considered for numerical analysis. This analysis was performed on data files comprising both bacterial AFLP profiles and internal size standards (ABI GeneScan™-500 ROX, Applied Biosystems, Naerum, Denmark) using the program Gelcompar II version 3.5 (Applied Maths, St. Martens-Latem, Belgium). Normalised AFLP fingerprints were compared using the Dice similarity coefficient and clustering analysis was performed by the unweighted pair group method with arithmetic averages (UPGMA). AFLP fingerprints consisting of approximately 65 to 89 fragments per isolate were compared. The arbitrary similarity (S D ) cut off level for individual clonal types was set at 98%. PFGE analysis. PFGE was performed as described by[START_REF] Christensen | Salmonella enterica serovar Gallinarum biovar gallinarum in layers: Epidemiological investigations of a recent outbreak in Denmark[END_REF], using XbaI to digest chromosomal DNA. DNA fragments were separated in 1% SeaKem GTG agarose (Camprex Bio Science, Copenhagen, Denmark) using the CHEF DRIII system set at 14°C and 5.6 V/cm with pulses ramping from 1 to 35 s over 20 h . The DNA fragments were stained with ethidium bromide and photographed with ultraviolet transillumination using a Geldoc 2000 system (BioRad, Herlev, Denmark) and Quantity One 4.0.3 gel (BioRad, Herlev, Denmark) documentation software. The images of the gels were saved in TIFF format and imported into the program Gelcompar II version 3.5 (Applied Maths BVBA). Cluster analysis was performed using the Dice correlation for band matching with a 1% position tolerance and UPGMA was used to generate a dendrogram describing the relationships between the S.

  Senftenberg isolates (data not shown). However, strains isolated from turkey meat (numbers 11 and 14), a feed mill processing line sample (number 27), and a sample of soy (number 32) showed the greatest resistance to desiccation. There was no significant difference in resistance to desiccation between the isolates from the different sources (farms, meat, spices and feed mills). The resistance of the Enterobacter purinus isolate was similar to that of the S. Senftenberg isolates, while the Pantoea agglomerans isolate lost viability rapidly and the concentration of viable cells was only 1.3 log 10 CFU/ml after 35 days of desiccation (Figure3).

  . Therefore, although our study of resistance of S. Senftenberg to desiccation was performed at 37°C, it may still be indicative of survival in poultry houses, in which ambient temperatures range from 34°C to 18°C between the introduction of day old chicks and the time of slaughter.

  Senftenberg was relatively resistant to 35 days of desiccation at 40% RH. Finally, our study highlights the problems associated with reisolation of Salmonella on farms and underlines the related to the introduction of feed-associated Salmonella serovars such as

Figure 2 .Figure 3 .

 23 Figure 2. Dendrogram showing PFGE fingerprints from 25 isolates of Salmonella Senftenberg representing the total diversity of isolates included in this study. Using the unweighted pair group method with arithmetic averages (UPGMA), cluster analysis was performed with the Dice correlation method. The position tolerance was set to 1% and optimisation was 1%. The broken line indicates the division into clusters made at 90% similarity with the Dice coefficient

Table 1 .

 1 S. Senftenberg isolates analysed by AFLP and PFGE and subjected to desiccationFigure 1. AFLP fingerprints of 34 S. Senftenberg strains. Dendrogram calculated using GelCompare II. The broken line indicates the division into clusters based upon 98% similarity with the Dice coefficient

	Isolate No. Isolation date Origin	Remarks
	1	February 2004 Farm 1	Isolate from sock sample, broiler production
	2	February 2004 Farm 1	Isolate from sock sample, broiler production
	3	March 2004	Farm 1	Isolate from sock sample, broiler production
	4	March 2004	Farm 1	Isolate from sock sample, broiler production
	5	March 2004	Farm 1	Isolate from sock sample, broiler production
	6	November 2004 Farm 1	Isolate from sock sample, broiler production
	7	November 2004 Farm 1	Isolate from sock sample, broiler production
	8	May 2005	Farm 1	Isolate from sock sample, broiler production
	9	May 2005	Farm 1	Isolate from sock sample, broiler production
	10	July 2004	Farm 2	Isolate from sock sample, broiler production
	11	May 2003	Imported meat Turkey meat, import control
	12	October 2003	Imported meat Chicken meat, import control
	13	January 2004	Imported meat Turkey meat, import control
	14	August 2004	Imported meat Turkey meat, import control
	15	December 2004 Imported meat Chicken meat, import control
	16	February 2005 Imported meat Chicken meat, import control
	17	March 2005	Imported meat Turkey meat, import control
	18	March 2003	Imported spices Curry
	19	March 2003	Imported spices Curry
	20	April 2003	Meat	Swine meat, export control
	21	July 2002	Farm 3	Isolate from sock sample, broiler parent production
	22	December 2004 Farm 3	Isolate from sock sample, table egg production
	23	January 2000	Farm 4	Isolate from sock sample, broiler parent production
	24	August 2002	Farm 3	Isolate from sock sample, broiler parent production
	25	January 2002	Farm 5	Isolate from sock sample, broiler parent production
	26	January 2002	Feed Mill A	Process sample
	27	December 2003 Feed Mill B	Process sample
	28	January 2004	Feed Mill C	Process sample
	29	January 2004	Feed Mill C	Process sample
	30	July 2001	Farm 4	Isolate from sock sample , broiler parent production
	31	July 2002	Feed Mill A	Soy, raw feed material sample
	32	July 2002	Feed Mill A	Soy, raw feed material sample
	33	November 2003 Feed Mill C	Process sample
	34	November 2003 Feed Mill C	Soy, raw feed material sample

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


Claudia Neubauer
email: claudia.neubauer@vu-wien.ac.at

Magdalena De Souza-Pilz

Miki Bojesen

Magne Bisgaard

Michael Hess

De Souza-Pilz

A M Bojesen

Tissue distribution of haemolytic Gallibacterium isolates in laying birds showing clinical signs of egg peritonitis

Keywords: Gallibacterium, layers, salpingitis, peritonitis

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 genus Gallibacterium was recently established within the family of Pasteurellaceae Pohl 1981 [START_REF] Christensen | Genetic relationships among avian isolates classified as Pasteurella haemolytica, Actinobacillus salpingitidis or Pasteurella anatis with proposal of Gallibacterium anatis gen. nov., comb. nov. and description of additional genomospecies within Gallibacterium gen[END_REF]. The genus contains avian bacteria formerly known as Pasteurella haemolytica, Actinobacillus salpingitidis or Pasteurella anatis and currently includes the species Gallibacterium anatis and Gallibacterium genomospecies 1 and 2. Two biovars are described within G. anatis, a haemolytic biovar haemolytica and a nonhaemolytic, biovar anatis. Gallibacterium spp. can be isolated from a great variety of birds, such as chickens, turkeys, ducks, geese, psittacine birds, partridges and guinea fowl [START_REF] Harbourne | A haemolytic cocco-bacillus recovered from poultry[END_REF][START_REF] Hacking | Pasteurella hemolytica in pullets and laying hens[END_REF][START_REF] Mushin | Pasteurella haemolytica found in the respiratory tract of fowl[END_REF][START_REF] Addo | Atypical Pasteurella haemolytica type A from poultry[END_REF][START_REF] Bisgaard | Ecology and significance of Pasteurellaceae in animals[END_REF].

Members of the genus Gallibacterium have been isolated from birds with various clinical conditions. In fact, Gallibacterium spp. can be isolated from healthy birds and it has been suggested that these bacteria may be part of the normal microbiota in the upper respiratory and the lower genital tracts [START_REF] Bisgaard | Incidence of Pasteurella haemolytica in the respiratory tract of apparently healthy chickens and chickens with infectious bronchitis. Characterisation of 213 strains[END_REF][START_REF] Mushin | Pasteurella haemolytica found in the respiratory tract of fowl[END_REF]. Contrary to this, Gallibacterium spp. have been obtained from layers with lesions affecting the reproductive organs including salpingitis and severe drops in egg production [START_REF] Kohlert | Colonization of Escherichia coli in young turkeys and chickens[END_REF][START_REF] Matthes | Enzootisches Auftreten der Pasteurella haemolytica beim Huhn[END_REF][START_REF] Hacking | Pasteurella hemolytica in pullets and laying hens[END_REF][START_REF] Gerlach | Significance of Pasteurella haemolytica in poultry[END_REF][START_REF] Bisgaard | Salpingitis in poultry. II. Prevalence, bacteriology, and possible pathogenesis in egg-laying chickens[END_REF][START_REF] Mirle | Studies into the incidence of Pasteurella haemolytica infections and their relevance to hens, with particular reference to diseases of the egg-laying apparatus[END_REF].

Furthermore, a mortality of 73% was noted in experimentally immunosuppressed 15-weekold brown layers after intravenous infection, emphasizing the importance of Gallibacterium spp. as a pathogen [START_REF] Bojesen | In vivo studies of Gallibacterium anatis infection in chickens[END_REF]. In a field study performed in Denmark Bojesen et al. (2003b) demonstrated that the biosecurity level influenced the prevalence of Gallibacterium spp. with a lower level of biosecurity resulting in a more frequent detection of these pathogens.

In spite of all the experimental and epidemiological data reported so far, no data have been published on the presence of G. anatis in different organs of laying birds showing reproductive disorders. The present study was designed to investigate the prevalence of haemolytic G. anatis in individual birds kept in alternative husbandry systems and suffering from reproductive disorders. Flocks investigated. In total 31 layer flocks were investigated: 14 organic free range flocks (54 birds), six conventional free range flocks (32 birds) and 11 conventional deep litter flocks (55 birds). Data on the size and the age of these flocks are shown in Table 1. The flocks were selected on the following criteria: a) an increase of mortality reported by the farmer (compared with the normal baseline mortality of the flock), b) a drop in egg production (up to 10% within one week), c) apathy of birds, d) pasting around the vent and e) pathological signs of salpingitis/peritonitis noted during necropsy performed on site. During the site visit the clinical status of the flocks was recorded.

Material and Methods

F

All flocks were vaccinated according to a standard vaccination schedule including vaccinations against coccidia, Salmonella Enteritidis, avian metapneumovirus, infectious bronchitis virus, infectious bursal disease virus and Newcastle disease virus. In addition some flocks were vaccinated against E. coli (eight with a commercially available vaccine, Nobilis® E. coli inac., and two with an autogenous vaccine), Pasteurella multocida (one flock with an autogenous vaccine) and Mycoplasma gallisepticum (one flock with a commercially available vaccine, Poulvac® MG).

Prior to any therapeutic treatment three to eight birds per flock were brought to the clinic and killed for necropsy followed by a very detailed sampling regime. The only exception was a group of birds from a single conventional deep litter flock which were treated with neomycin directly before sampling.

Necropsy findings. The necropsy findings of the birds were recorded according to the following protocol: trachea: haemorrhages; lungs: airsacculitis and oedema/hyperaemia; heart: haemorrhages, hydropericardium and pericarditis; liver: hepatomegaly, haemorrhages, necrosis and perihepatitis; ovary: haemorrhages, atrophy, deformed follicles, broken follicles, regression; oviduct: haemorrhages, non-functional; kidney: renomegaly and haemorrhages; spleen: splenomegaly; abdominal cavity: peritonitis and ascites. Bacteriological investigations. Bacteriological investigations were carried out from the following ten sites: choana, trachea, lung, heart, liver, spleen, ovary, oviduct, duodenum and cloaca. Each sample was plated out directly on a blood agar plate (Columbia agar with 5% sheep blood; BioMeriéux, Vienna, Austria) and a McConkey agar plate (BioMeriéux, Vienna, Austria) for isolation of Gallibacterium spp. and E. coli, respectively. The plates were incubated aerobically at 37°C for 24 h. Haemolytic Gallibacterium isolates were identified by their growth on blood agar within 24 h which is characterized as follows: circular, raised colonies with an entire margin, shiny and semi-transparent with beta-haemolytic zone. Such colonies were regarded as suspicious of Gallibacterium. Suspect colonies were subcultured on blood agar to obtain pure cultures. Gram-staining and cytochrome oxidase tests were performed for all isolates.

Up to five E. coli colonies per plate were tested for avian pathogenic serovars O1:K1, O2:K2 and O78:K80 using slide agglutination (Veterinary Laboratories Agency, Surrey, UK).

Briefly, one drop of the agglutination sera was mixed with one single E. coli colony on an object slide and swirled for 2 min. The absence/appearance of agglutination with each serovar test sera was recorded.

Extraction of DNA and PCR. The QIAGEN DNEasy Tissue Kit (Qiagen, Hilden, Germany) was used for DNA extraction following the manufacturer's protocol. All isolates suggestive of Gallibacterium spp. were investigated according to [START_REF] Bojesen | Specific identification of Gallibacterium by a PCR using primers targeting the 16S rRNA and 23S rRNA genes[END_REF]. Using this PCR, two fragments of approximately 790 bp and 1080 bp were expected for strains of the genus Gallibacterium. All PCR products were separated by electrophoresis on 2% agarose gels following a standard procedure [START_REF] Sambrook | Detection of DNA in Agarose Gels[END_REF].

Amplified Fragment Length Polymorphisms (AFLP).

The genetic diversity of a subset of 83 isolates from 18 flocks was evaluated by AFLP as reported previously [START_REF] Christensen | Genetic relationships among avian isolates classified as Pasteurella haemolytica, Actinobacillus salpingitidis or Pasteurella anatis with proposal of Gallibacterium anatis gen. nov., comb. nov. and description of additional genomospecies within Gallibacterium gen[END_REF]. From each flock at least one isolate from the respiratory tract and one from liver, heart, spleen, ovary, oviduct or intestinal tract was chosen. Briefly, the non-selective BglII primer (FAM-5´GAGTACACTGTCGATCT 3´) and the non-selective BspDI primer (5´ GTGTACTCTAGTCCGAT 3´) were used to amplify the fragments subsequent to restriction digestion and ligation to their corresponding adaptors. All AFLP reactions were repeated at least twice to allow evaluation of the reproducibility of the method. Amplification products were detected on an ABI 377 automated sequencer (PE Biosystems). Each lane included internal-lane size standards labelled with ROX dye (Applied Biosystems). GeneScan 3.1 fragment analysis software (Applied Biosystems) was used for fragment size determination and pattern analysis. AFLP profiles comprising fragments in the size range 50-500 bp were considered for numerical analysis with the software GelCompar II (Applied Maths, Kortrijk, Belgium). Normalised AFLP fingerprints were compared using the Dice similarity coefficient and clustering analysis was performed by the unweighted pair group method with arithmetic averages (UPGMA).

Results

Flocks investigated. In addition to the criteria applied for selecting flocks the most frequent additional findings were lack of uniformity (17/31), cannibalism (13/31), shaking heads (10/31) and nasal discharge (5/31).

Necropsy findings. Three to eight birds per farm were investigated, totalling 141 birds. In addition to peritonitis (21%) the main pathological findings were lesions in the reproductive tract as summarized in Table 2. A high percentage of birds demonstrated regression of the ovaries (40%) and a non-functional oviduct (31%). Furthermore, 28% of the birds had deformed follicles and in 10 birds, severe egg concretions were found within the oviduct.

Fibrinous perihepatitis and pericarditis were found in only three and two birds, respectively. During necropsy gastrointestinal helminths (Ascaridia spp.) were found in the small intestine of birds from three organic free-range flocks. Many birds (36/141) showed signs of fatty liver syndrome. Severe pecking wounds due to cannibalism, mainly localized around the cloaca were observed in 63 birds. No lesions were found in the respiratory tract.

Bacteriological investigations.

A total of 310 bacterial isolates suggestive of haemolytic G. anatis were obtained. All these demonstrated a wide beta-haemolytic zone (1 to 2 mm) as reported by [START_REF] Bisgaard | Isolation and characterization of some previously unreported taxa from poultry with penotypical characters related to Actinbacillus-and Pasteurella species[END_REF] and [START_REF] Christensen | Genetic relationships among avian isolates classified as Pasteurella haemolytica, Actinobacillus salpingitidis or Pasteurella anatis with proposal of Gallibacterium anatis gen. nov., comb. nov. and description of additional genomospecies within Gallibacterium gen[END_REF]. All were Gram-negative rods that were sometimes pleomorphic, and were cytochrome oxidase-positive.

Data for bacteriological isolation are summarized in Table 3. Most of the isolates (161/310) were obtained from the respiratory tract. From heart, liver, spleen, intestine and reproductive tract 16, 14, 19, 63 and 37 isolates were found, respectively. In addition to the intestinal tract (244/847) E. coli was obtained mainly from the respiratory tract (274/844) and the reproductive tract (167/844). From heart, liver and spleen 55, 53, and 54 isolates were recovered, respectively. Almost half of the isolates belonged to avian pathogenic E. coli (405/847). In nine flocks E. coli strains with the antigenic profile O1:K1 were observed while O2:K1 was found in flock numbers 14 and 24 and O78:K80 was found only once in flock number 10. Furthermore, mixed infections with E. coli O1:K1 and O2:K1 were noted in two flocks and mixed infection of O1:K1 and O78:K80 and one further flock. In reproductive tract co-infections of haemolytic G. anatis and E. coli were found in 18 birds from 14 flocks. Haemolytic G. anatis isolates were obtained from eight birds of six flocks and infection of the reproductive tract with only E. coli was seen in 39 birds from 10 flocks. Both E. coli and haemolytic G. anatis were isolated from birds expressing lesions in either the ovaries or the oviduct. However, these microorganisms were also found in birds without lesions in the reproductive tract.

Identification of Gallibacterium by PCR. All 310 isolates that were tentatively identified as Gallibacterium were tested with a Gallibacterium specific PCR, and from all isolates amplicons of approximately 790 bp and 1080 bp were obtained as exemplified in Figure 1.

AFLP. Eighty-three isolates from 18 flocks were investigated. Using a cut-off level of 90% to define a genotype (clone), 34 individual clones were identified. Generally, a high genetic similarity was demonstrated both between and within flocks. In 11 flocks, only one clone was present, this includes the three flocks (6, 7, 8), where only one isolate was obtained. In four flocks two clones were demonstrated. One flock (flock number 4) had three and two flocks (flock numbers 16 and 19) had six clones, respectively (Figure 2). One clone was present in four different flocks.

Discussion

The present study aimed to investigate the association between the presence of haemolytic G. anatis and reproductive disorders in laying birds kept in alternative husbandry systems as birds kept in these systems are more likely to be infected with G. anatis (Bojesen et al., 2003b).

E. coli is considered to be the most common bacterium associated with reproductive disorders in chickens [START_REF] Bisgaard | Salpingitis in poultry. II. Prevalence, bacteriology, and possible pathogenesis in egg-laying chickens[END_REF][START_REF] Jones | Reproductive tract lesions of the laying fowl with particular reference to bacterial infection[END_REF][START_REF] Jordan | Observations on salpingitis, peritonitis and salpingoperitonitis in a layer breeder flock[END_REF].

However, other reports suggest that Gallibacterium spp. may cause these disorders and their importance is likely to be underestimated due to overgrowth by other bacterial species and the difficulties of identification [START_REF] Mirle | Studies into the incidence of Pasteurella haemolytica infections and their relevance to hens, with particular reference to diseases of the egg-laying apparatus[END_REF]Bojesen et al., 2003a).

Based on specific signs in the flocks, birds were selected for further investigations in accordance with [START_REF] Vanderkerchove | Colibacillosis in caged layer hens: characteristics of the disease and the aetiological agent[END_REF]. Drop in egg production, apathy of birds, pasting around the vent and increased mortality were all signs used by the farmer to initiate further investigations as reported in the present study. The results of the post mortem investigations performed on the farms and the notification of salpingitis/peritonitis were additional criteria to include the farm in the study. In total, 141 birds were killed and the samples were taken from fresh carcasses. Except the five birds from flock no. 15, all birds (136/141) were investigated prior to any antibiotic treatment to increase the recovery of bacteria from the different organs.

In the present study most of the haemolytic G. anatis isolates were obtained from the respiratory tract, especially from the choana and trachea. This is not surprising considering previous reports by [START_REF] Bisgaard | Incidence of Pasteurella haemolytica in the respiratory tract of apparently healthy chickens and chickens with infectious bronchitis. Characterisation of 213 strains[END_REF], [START_REF] Mushin | Pasteurella haemolytica found in the respiratory tract of fowl[END_REF] and Bojesen et al. (2003b) indicating that Gallibacterium spp. constitute part of the normal tracheal microbiota in chickens and that under certain conditions they might act as opportunists and cause respiratory disease. In the case of E. coli, the natural route of infection is generally thought to be via the respiratory tract [START_REF] Gross | The development of "air sac disease[END_REF][START_REF] Carlson | Coliform bacteria in chicken broiler house dust and their possible relationship to coli-septicemia[END_REF]) and a similar route might exist for Gallibacterium. The present investigation supports this assumption for both bacteria, as 52% of the G. anatis and 32% of the E. coli isolates were recovered either from the choana, trachea or the lungs of the birds. Penetration of Gallibacterium organisms from the mucosa of the respiratory tract into the systemic circulation may be due to the influence of an impaired host immunity, co-infections and environmental factors [START_REF] Gerlach | Significance of Pasteurella haemolytica in poultry[END_REF][START_REF] Matthes | Beitrag zur Kinetik bakterieller Infektionen beim Huhn[END_REF][START_REF] Nagaraja | Effect of ammonia on the quantitative clearance of Escherichia coli from lungs, air sacs, and livers of turkeys aerosol vaccinated against Escherichia coli[END_REF][START_REF] Shaw | Pasteurella haemolytica as a co-pathogen in pullets and laying hens[END_REF][START_REF] Kohlert | Colonization of Escherichia coli in young turkeys and chickens[END_REF].

From the case history of the flocks it was known that cannibalism occurred in nearly half of the flocks (13/31) and that numerous birds showed extensive pecking wounds around the cloaca. This is in contrast to the findings reported by [START_REF] Jones | Reproductive tract lesions of the laying fowl with particular reference to bacterial infection[END_REF] and [START_REF] Jordan | Observations on salpingitis, peritonitis and salpingoperitonitis in a layer breeder flock[END_REF] who observed only a few cases of vent cannibalism in birds suffering from salpingitis/peritonitis. However, [START_REF] Cumming | The aetiology and importance of salpingitis in laying hens[END_REF] attributed considerable importance to such injuries from which the bacteria may spread into the blood stream resulting in bacteraemia with bacteria to be isolated from the heart, liver, spleen and the reproductive tract. In the present investigation ascending infections may have contributed to the finding of G. anatis and E. coli in the reproductive tract, in addition to the heart, liver and spleen. Bojesen et al. in the ovaries or the oviduct E. coli was isolated more frequently. This might have been because G. anatis was overgrown in such cases as already mentioned above. E. coli are present in the normal microbiota of the intestinal tract in poultry, but certain strains of avian pathogenic E. coli (APEC) possess specific virulence factors and are able to cause severe infections. The APEC serogroups O1, O2 and O78 are most frequently recovered from infected chickens [START_REF] Sojka | Escherichia coli infection in poultry[END_REF][START_REF] Glantz | Escherichia coli serotypes isolated from salpingitis and chronic respiratory disease in poultry[END_REF][START_REF] Cloud | In vitro and in vivo characterization of avian Escherichia coli. I. Serotypes, metabolic activity, and antibiotic sensitivity[END_REF][START_REF] Dozois | Pap-and pil-related DNA sequences and other virulence determinants associated with Escherichia coli isolated from septicemic chickens and turkeys[END_REF]. In the present investigation E. coli was isolated from birds of each flock and APECs (O1:K1, O2:K1 and O78:K80) were found in more than 50% of the flocks. The bacteria were isolated from the respiratory tract (274/844) and the reproductive tract (166/844). [START_REF] Bisgaard | Salpingitis in poultry. II. Prevalence, bacteriology, and possible pathogenesis in egg-laying chickens[END_REF] investigated 150 salpinx samples from layer carcasses condemned at slaughterhouses due to salpingitis and found that E. coli was isolated most frequently. Their findings were confirmed by [START_REF] Jones | Reproductive tract lesions of the laying fowl with particular reference to bacterial infection[END_REF] investigating 292 birds in which salpingitis/peritonitis was diagnosed over a period of two years. Furthermore, these authors also found mixed infections of E. coli and haemolytic Gallibacterium spp., as shown in nearly half of the cases in the present study.

According to the existing literature detection of members of Pasteurellaceae by traditional methods often results in difficulties at isolation, cultivation and identification. The weak and unreliable reactions observed for some of the phenotypic identification tests add to these uncertainties [START_REF] Christensen | Genetic relationships among avian isolates classified as Pasteurella haemolytica, Actinobacillus salpingitidis or Pasteurella anatis with proposal of Gallibacterium anatis gen. nov., comb. nov. and description of additional genomospecies within Gallibacterium gen[END_REF]. Diagnostic PCR assays were therefore developed to complement these diagnostic methods [START_REF] Bojesen | Specific identification of Gallibacterium by a PCR using primers targeting the 16S rRNA and 23S rRNA genes[END_REF] and were found very useful in confirming the classification of the isolates. The genetic diversity assessed by AFLP analysis of 83 isolates confirmed previous indications of high genetic relatedness among isolates from the same flock (Bojesen et al., 2003a). Furthermore, the fact that the same clone could be isolated from the mucosal lining of the upper respiratory tract and from lesions in the reproductive tract, heart, liver and spleen, suggests that, given the right opportunity, Gallibacterium isolates residing in the upper respiratory or lower genital tract may gain access to the systemic circulation and/or the upper reproductive tract and cause disease,.

In conclusion, we have shown that haemolytic G. anatis isolates are prevalent in layers with reproductive disorders. The results from the current study provide strong support for the theory that Gallibacterium plays a role in reproductive disorders. Furthermore, we have demonstrated that Gallibacterium isolates from different locations i.e. heart, spleen, upper respiratory and lower genital tract were highly similar, indicating that isolates residing in their

. Agarose gel electrophoresis of amplification products from haemolytic

Gallibacterium anatis field isolates. Lane M, 100 bp DNA ladder (Invitrogen); lanes 1 and 10, isolates from cloaca; lanes 2, 6, 11 and 12, isolates from choana; lanes 3 and 8, isolates from intestine; lanes 4 and 7, isolates from trachea; lane 5, isolate from ovary; lanes 9 and 13, isolates from lungs; lanes 14 and 16, negative controls; lane 15, positive control.

0 0 0 0 0 0 1 3 2 0 1 0 3 0 3 0 0 0 0 3 cfr 2/4 O1:K1 1 0 0 0 0 0 1 3 4 0 2 0 1 2 0 4 3 0 3 0 4 ofr 1/3 O2:K1 1 0 2 0 1 0 2 3 3 1 0 0 2 2 2 3 1 2 1 2 5 ofr 1/3 E. coli 0 0 0 0 0 0 1 0 1 1 0 0 0 3 1 2 1 1 2 1 6 ofr 3/3 O1:K1 0 0 0 0 0 2 0 2 0 3 1 3 0 3 0 3 0 3 0 3 7 ofr 1/3 O1:K1 0 3 0 1 0 0 1 1 0 3 0 3 0 3 0 3 0 3 0 3 8 ofr 0/3 O1:K1 0 0 0 0 0 0 1 3 0 0 0 3 0 3 0 3 0 3 0 0 9 ofr 3/3 E. coli 0 0 0 0 2 2 1 2 1 0 0 3 1 3 2 3 0 3 0 2 10 ofr 3/3 O78:K80 0 0 0 0 0 0 2 1 1 1 0 3 0 3 0 3 0 3 0 3 11 cdl 2/5 E. coli 0 0 0 0 0 0 4 5 2 0 2 0 2 5
0/3 0/3 0/3 3/3 1/3 0/3 2/3 3/3 2/3 2/3 5 E. coli 0/3 0/3 0/3 0/3 1/3 0/3 3/3 2/3 1/3 1/3 6 O1:K1 0/3 0/3 2/3 2/3 3/3 3/3 3/3 3/3 3/3 3/3 7 O1:K1 3/3 1/3 0/3 1/3 3/3 3/3 3/3 3/3 3/3 3/3 8 O1:K1 0/3 0/3 0/3 3/3 0/3 3/3 3/3 3/3 3/3 0/3 9 E. coli 0/3 0/3 2/3 2/3 0/3 3/3 3/3 3/3 3/3 2/3 10 O78:K80 0/3 0/3 0/3 1/3 1/3 3/3 3/3 3/3 3/3 3/3 11 E. coli 0/

(

  2004) used fluorescence in situ hybridisation to demonstrate bacteria in the spleen and liver following intraperitoneal infection of chickens with Gallibacterium, indicating that ascending infections might be of relevance.The gross pathological lesions of reproductive disorders due to Gallibacterium infection cannot be distinguished from infections with E. coli and for this reason all samples were also investigated for E. coli. With an increase of birds/flock showing pathological signs

  Figure legends

Figure 2 .

 2 Figure2. Dendrogram (unweighted pair group method using arithmetic averages) of AFLP similarities of 83 Gallibacterium isolates. SD, band-based Dice similarity coefficient (%). Flocks, the flock number from which the isolates originate. Isolates grouping at a 90% similarity level or higher were pooled as indicated by grey triangles. The dotted line indicates the clonal cut-off level at 90% similarity.

F

Figure 1 :

 1 Figure1: Agarose gel electrophoresis of amplification products from haemolytic Gallibacterium anatis field isolates Lane M: 100 bp DNA ladder (Invitrogen); lanes 1,10: isolates from cloaca; lanes 2,6,11,12: isolates from choana; lanes 3,8: isolates from intestine; lanes 4,7: isolates from trachea; lane 5: isolate from ovary; lanes 9,13: isolates from lungs; lanes 14,16: negative control; lanes 15: positive control

Table 1 .

 1 Size and age of the flocks investigated

	Size	<1000	1000-	2000-	>3000
			2000	3000
	Age (weeks)	<30	30-40	>40	<30	30-	>40 <30 30-	>40 <30 30-	>40
						40			40			40
	Organic/free-range	1		1	1	1	3	1	1	3	1	1
	Conventional/free-		2				2		1			1
	range
	Conventional/deep			1			1	1	1	1	1	4	1
	litter

Table 2 .

 2 Main necropsy findings

	Lesion	Organic/free-	Conventional/free-	Conventional/deep	N=141
		range	range	litter
		(54 birds/14 flocks)	(32 birds/6 flocks)	(55 birds/11 flocks)
	Haemorrhages in	10	13	3	26
	ovary				(18%)
	Atrophy of ovary	25	12	21	58
					(40%)
	Deformed follicles	16	13	10	39
					(28%)
	Broken follicles	10	8	4	22
					(16%)
	Haemorrhages in	11	5	1	17
	oviduct				(12%)
	Non-functional	17	8	19	44
	oviduct				(31%)
	Peritonitis	15	9	6	30
					(21%)

Table 3 .

 3 Isolation of haemolytic G. anatis and E. coli from different organs displayed in connection with the housing system and the number of birds expressing lesions in either the ovaries and/or the oviduct

	Flock no.	Housing system a	N b	Sero-typing	G. a. c	Heart	E. c. d	Liver G. a. E. c. G. a. Spleen E. c. G. a. Choana E. c. G. a. Trachea E. c. G. a. Lung E. c. G. a. Duodenum E. c. G. a. Cloaca E. c. G. a. Ovary E. c. G. a. Oviduct E. c.
	1	cfr	5/8	O1:K1	0		0	1	0	0	0	3	3	8	6	3	2	3	5	1	7	1	0	0	0
	2	ofr	2/3	O1:K1	0

Table 4 :

 4 Escherichia coli obtained from different organs

	No.	E. coli	Heart Liver Spleen Choana Trachea Lung Duod. Cloaca Ovary Oviduct
		Typing
	1	O1:K1	0 a) /8 b) 0/8	0/8	3/8	6/8	2/8	5/8	7/8	0/8	0/8
	2	O1:K1	0/3	0/3	0/3	1/3	2/3	1/3	3/3	3/3	0/3	0/3
	3	O1:K1	0/4	0/4	0/4	3/4	0/4	0/4	2/4	4/4	0/4	0/4
	4	O2:K1

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

Acknowledgement

The authors would like to thank Boehringer Ingelheim Vetmedica for co-operation and financial support of the project.


Lise ; Hedegaard

Henrik Christensen

Mark Simon Chadfield

Jens Peter Christensen

Magne Bisgaard

M. S J P Chadfield

M Christensen

Bisgaard

Association of Streptococcus pluranimalium with valvular endocarditis and septicaemia in adult broiler parents

Keywords: streptococcus, endocarditis, layer, PFGE Times New Roman, Not Italic, Complex

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faecalis were transferred to the new genus Enterococcus [START_REF] Schleifer | Transfer of Streptococcus faecalis and Streptococcus faecium to the genus Enterococcus nom. rev. as Enterococcus faecalis comb. nov. and Enterococcus faecium comb[END_REF]. A few years later the genus Streptococcus was divided into three genera, Streptococcus, Lactococcus and Enterococcus [START_REF] Schleifer | Transfer of Streptococcus lactis and related streptococci to the genus Lactococcus gen[END_REF]. Since then many new species have been defined and today the genus Streptococcus contains more than 60 species [START_REF] Euzéby | List of bacterial names with standing in nomenclature: a folder available on the Internet[END_REF][START_REF] Kilian | Topley and Wilson's Microbiology and Microbial Infections[END_REF][START_REF] Facklam | What happened to the streptococci: overview of taxonomic and nomenclature changes[END_REF][START_REF] Hoshino | Use of phylogenetic and phenotypic analyses to identify nonhemolytic streptococci isolated from bacteremic patients[END_REF].

In animals the main habitats of streptococci are the skin and mucous membranes of the respiratory, gastrointestinal and urogenital tracts [START_REF] Ruoff | The genus Streptococcus[END_REF][START_REF] Hirsh | Streptococcus and Enterococcus[END_REF]. Many species are commensals with pathogenic potential, while others are true pathogens in the sense that they spread from animal to animal and cause infection in nonimmune individuals [START_REF] Kilian | Topley and Wilson's Microbiology and Microbial Infections[END_REF]. In poultry, Streptococcus species have been associated with local and systemic infections, including septicaemia and endocarditis [START_REF] Wages | Diseases of Poultry[END_REF].

Species causing septicaemia include S. dysgalactiae [START_REF] Messier | Focal dermatitis and cellulitis in broiler chickens: bacteriological and pathological findings[END_REF], S. equi subspecies zooepidemicus [START_REF] Peckham | An outbreak of streptococcosis (apoplectiform septicemia) in White Rock chickens[END_REF], S. gallinaceus [START_REF] Chadfield | Characterization of streptococci and enterococci associated with septicaemia in broiler parents with a high prevalence of endocarditis[END_REF], S. gallolyticus [START_REF] Chadfield | Geno-and phenotypic diversity of avian isolates of Streptococcus gallolyticus subsp. gallolyticus (Streptococcus bovis) and associated diagnostic problems[END_REF], S. mutans [START_REF] Ivanics | Streptococcus mutans infection in geese[END_REF] and S. suis [START_REF] Devriese | Streptococcus suis infections in birds[END_REF].

In addition, Streptococcus species have been associated with infections causing growth depression and increased mortality without obvious clinical signs [START_REF] Chadfield | Characterization of streptococci and enterococci associated with septicaemia in broiler parents with a high prevalence of endocarditis[END_REF]. According to [START_REF] Smyth | Staphylococci, streptococci and enterococci[END_REF], the Streptococcus species most commonly associated with disease in poultry is S. equi ssp. zooepidemicus. Reclassification of old species and recognition of many new species, combined with the lack of unambiguous characters for identification, has made classification and identification of streptococci challenging. In addition, many species seem to share several hosts. However, major genetic differences seem to exist between species, and a combination of phenotypic and molecular methods is likely to enable more certain diagnosis [START_REF] Hoshino | Use of phylogenetic and phenotypic analyses to identify nonhemolytic streptococci isolated from bacteremic patients[END_REF].

During investigations of the aetiology of increased mortality in broiler parent stock at the end of production, pure cultures of streptococcal-like organisms that could not be classified among the six species regarded as pathogens of chickens were obtained from cases of septicaemia or valvular endocarditis and septicaemia. The aim of this study was to

Materials and Methods

Flock Data. During 2004 and[START_REF] Hoshino | Use of phylogenetic and phenotypic analyses to identify nonhemolytic streptococci isolated from bacteremic patients[END_REF], longitudinal studies were carried out on two consecutive broiler parent flocks on a farm in which endocarditis had been seen previously. The flocks contained approximately 42,000 hens and 3,400 roosters [START_REF] Chadfield | Characterization of streptococci and enterococci associated with septicaemia in broiler parents with a high prevalence of endocarditis[END_REF]. A total of 122 dead hens from the first flock, between 44 and 61 weeks old, were randomly selected from the four houses. In the subsequent flock a further 228 dead birds between 46 and 60 weeks of age were randomly selected from all four houses. In addition to this farm, reference strains were included from three other farms (Table 1). All birds were subjected to a comprehensive post mortem examination.

Bacteriology. Pure cultures of streptococcal-like organisms were obtained from cases with septicaemia, valvular endocarditis, and salpingitis, peritonitis, valvular endocarditis and septicaemia. Isolates were initially identified using standard methods [START_REF] Wages | Streptococcosis[END_REF]. Isolates kept at -80ºC were subsequently plated on blood agar base (CM0055, Oxoid) containing 5% sterile bovine blood (BA) and incubated overnight in sealed plastic bags at 37ºC. Initial identification of pure cultures used assessment of colony morphology, haemolysis, Gram staining, catalase and oxidase tests, motility at 37ºC, aerobic growth, catabolism of glucose in Hugh & Leifson medium and pyrrolidonyl-arylaminidase activity (PYR) (Rosco, Denmark). Enzymatic reactions and fermentation of carbohydrates were determined using the API 20 STREP system according to the manufacturer's instructions (bioMérieux, La Balme les Grottes, France).

Pulsed Field Gel Electrophoresis (PFGE)

. Strains kept at -80ºC were plated on BA and incubated overnight at 37ºC. A single typical colony was sub-cultured overnight in 10 ml Brain Heart Infusion (BHI) broth (Oxoid) at 37ºC with shaking. Some strains needed addition of 0.25% glucose and 10% chicken serum to the 10 ml BHI broth for growth. To measure and standardize cell density, the optical density (OD) of a solution containing 800 200 µl of culture in BHI broth was determined at 578 nm. Cells therein a 3 ml sample of an overnight culture in BHI broth were harvested by centrifugation for 10 min at 4ºC. The supernatant was discarded and the pellet resuspended in 4.5 ml SE buffer. After further centrifugation, the supernatant was discarded and the pellet resuspended in sufficient SE buffer to obtain the same OD as was obtained previously. Five hundred microlitres of the suspension was mixed with 500 µl agarose solution (0.02 g Megabase agarose [Bio-Rad], 1 ml SE-buffer) and dispensed into disposable plug molds (Bio-Rad). To release DNA from the cells the blocks were treated overnight at 37ºC in 950 µl lysozyme solution at pH 7.0 containing 2.5 mg hen egg white lysozyme (Roche), 1 ml phosphate buffer (0.1 M Na 2 HPO 4 , 2H 2 O [Merck]) and 20% sucrose (J. T. Baker). After incubation, the blocks were washed for 30 minutes in 1 ml 10:10 TE buffer (10 mM Tris, 10 mM EDTA, pH 7.4) and incubated overnight at 56ºC in 950 µl proteinase K solution (0.5 mg proteinase K [Roche], 1 ml ES buffer (1% N-laurylsarcosine [Sigma-Aldrich], 0.5 M EDTA, pH 9.5). The blocks were then washed in cold TE buffer and incubated for 1 hour at room temperature. This was repeated 3 times. The blocks of purified chromosomal DNA were stored at 4ºC in TE buffer until further use. PFGE was carried out as described by [START_REF] Chadfield | Geno-and phenotypic diversity of avian isolates of Streptococcus gallolyticus subsp. gallolyticus (Streptococcus bovis) and associated diagnostic problems[END_REF] using SmaI for digestion of the DNA, an initial switch time of 1 sec, a final switch time of 15 sec, a potential difference of 5.6 V cm -1 and an included angle of 120ºC. The current was varied between 108 mA and 177 mA for the different gels. The gel was stained in a solution of ethidium bromide (Sigma) in deionised water (10 mg ethidium bromide ml -1 ) for 15 minutes and afterwards de-stained in deionised water for 15 minutes, then photographed using ultraviolet light transillumination. A Low Range PFG Marker (N0350S, BioLabs, New England) was placed in the first, middle and last wells of the gel. The PFGE profiles produced from the clinical isolates were analysed using the unweighted pair-group method with arithmetic averages based on Dice similarity coefficients calculated using GelCompar II (Applied Maths). [START_REF] Chadfield | Geno-and phenotypic diversity of avian isolates of Streptococcus gallolyticus subsp. gallolyticus (Streptococcus bovis) and associated diagnostic problems[END_REF]. The 16S rRNA gene sequences of two isolates from cases of septicaemia in layer hens (178 and C19672L28) and two isolates from epidemiologically unrelated cases in broiler parents (191 and 219) were included. Searches in GenBank [START_REF] Benson | Genbank[END_REF] were performed using BLAST [START_REF] Altschul | Gapped BLAST and PSI-BLAST: a new generation of protein database search programs[END_REF]. Pairwise comparisons for sequence similarity were performed using WATER within the EMBOSS package [START_REF] Rice | EMBOSS: The European Molecular Biology Open Software Suite[END_REF]. Multiple alignments were performed using CLUSTALX [START_REF] Thompson | The CLUSTRAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools[END_REF]. The region corresponding to that between bases 71 and 1447 of the E. coli rrnB gene was examined.

Sequencing of 16S

Maximum-likelihood analysis, including bootstrap analysis, was performed using fastDNAml [START_REF] Olsen | Fast DNAml: a tool for construction of phylogenetic trees of DNA sequences using maximum likelihood[END_REF]. The GenBank accession numbers for the sequences are shown in Figure 2. In addition, strains 21129H3HJ3, 21192H3L2 and 20326H4L1 were deposited under accession numbers EU391532, EU391533 and EU391534, respectively.

Results

Bacteriology. Streptococcal-like organisms were isolated from cases of septicaemia [START_REF] Facklam | What happened to the streptococci: overview of taxonomic and nomenclature changes[END_REF], valvular endocarditis and septicaemia [START_REF] Smyth | Staphylococci, streptococci and enterococci[END_REF], and salpingitis, peritonitis, valvular endocarditis and septicaemia (1). Only four isolates were obtained from the first flock, with the remaining isolates obtained from the second flock. In addition, four isolates from other farms were included (Table 1).

PFGE Analysis. Cluster analysis of PFGE profiles revealed two major clusters (A and B)

with about 50% similarity (Figure 1). Twelve of 15 isolates formed one sub-cluster (A1) with 98% similarity, constituting a single clone according to [START_REF] London | Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel-electrophoresis -criteria for bacterial strain typing[END_REF]. The remaining three isolates, identified as E. avium (A2) and E. faecium (A3), respectively, by phenotypic testing, formed a cluster with 73% similarity to each other. These three isolates had 66% similarity with subcluster A1, which was subsequently identified as S. pluranimalium by 16S rRNA sequencing. Cluster B included nine isolates within several subclusters (B1-4). Five of these isolates with 90% similarity constituted a second clone of S. pluranimalium (B1). This clone had 70% similarity to two other isolates (B2), both of which were subsequently classified as S. pluranimalium by 16S rRNA sequencing. Two were different from those from flock 2 (Table 1, Figure 1). Phenotypic Analysis. Twenty-four isolates tentatively classified as streptococcal-like organisms were characterized. Phenotypic characterization based upon the API 20 STREP system identified the isolates as Aerococcus viridans (10), A. urinae (2), Leuconostoc sp. (4), S. salivarius (2), S. bovis II 3 (1), Enterococcus avium (3), E. faecium (1) or Gemella morbillorum (1) (Table 1). This identification was not supported by PFGE or 16S rRNA sequence analyses. Phenotypic characteristics are shown in Table 1. Phenotypic characteristics, including those in the API20 STREP system that are useful for identification of S. pluranimalium, E. avium and E. faecium are shown in Table 2. Differences in pyrrolidonyl arylamidase and Voges-Proskaur reactions separate the streptococci and enterococci investigated. In addition, differences in hippurate hydrolysis, α-galactosidase and arginine dihydrolase production, and acid formation from D-mannitol, D-sorbitol, inulin, D-raffinose and starch separate E. avium from E. faecium. Isolates within PFGE subclusters A1, B1 and B2 could not be separated. All isolates identified as S. pluranimalium were strongly α-haemolytic on bovine blood agar plates, while isolates of E. avium and E. faecium produced weaker and greener α-haemolysis (Table 2).

16S rRNA Sequencing.

A single isolate (20978H4L6) from the clonal PFGE subcluster A1 had 99.8% 16S rRNA gene sequence similarity with S. pluranimalium strain LMG14257, Acc. No. Y18026). However, two other isolates, 21147H3L6 and 20326H1L1, from PFGE subcluster B1 also had 99.8% similarity with strain LMG14257. Isolate 20315H1HJ4 from subcluster B2 had 99.0% 16S rRNA gene sequence similarity with S. pluranimalium and 16S rRNA gene sequence identity with an isolate (C19672L28) from a septicaemic layer hen (Table 1, Figure 2). Isolates 20978H4L6 (A1) and 20315H1HJ4 (B2) had 99.9% 16S rRNA gene sequence similarity. Isolate 20363H2HJ3 (B2) had 98.9% sequence similarity with S. pluranimalium and 99.9% similarity with 21147H4L2 (B1), and was identical to an epidemiologically unrelated isolate (178) from a layer (Table 1). Isolate 20326H4L1 from PFGE subcluster B4, had 16S rRNA gene sequence similarities of 99.6 and 99.5% with the type strains of Lactobacillus johnsonii and L. gasseri, respectively (Acc. No. AJ002515 and AF519171). Two isolates, 21192H3L2 (A2) and 21129H3HJ3 (B3), which were classified as The close relationships between the ten isolates of S. pluranimalium compared by 16S rRNA gene sequencing were reflected in the inferred phylogeny (Figure 2). The monophyletic group including all these isolates and the reference strain of S. pluranimalium was supported by a high bootstrap value of 99%. S. pluranimalium was found to be closely related to the species S. thoraltensis, S. gallinaceus, S. suis and S. gallolyticus.

There was no correlation between the lesions and the PFGE subclones and there was no obvious difference between the lesions caused by S. pluranimalium, E. faecium or E. avium.

Discussion

S. pluranimalium was first described by [START_REF] Devriese | Streptococcus pluranimalium sp. nov., from cattle and other animals[END_REF]. Based on sequencing of the 16S rRNA gene it is closely related to S. thoraltensis from pigs, S. gallinaceus from poultry and S. suis from pigs and birds (Figure 2). Whole cell protein analysis has confirmed that S. pluranimalium differs from closely related species of streptococci [START_REF] Devriese | Streptococcus pluranimalium sp. nov., from cattle and other animals[END_REF], but phenotypic identification is complicated by variations in biochemical reactions.

Phenotypically, S. pluranimalium resembles S. thoraltensis, S. hyovaginalis, S. suis and S. acidominimus. In addition, S. pluranimalium may be confused with organisms in the S. bovis group. Colonies grown on blood agar plates are surrounded by a broad greenish zone of αhaemolysis, which makes distinction from E. avium and E. faecium difficult. S. pluranimalium has been isolated from several animal species and from different sites, including the genital tract (cervix and vaginal mucus), the mammary glands of cows and from the tonsils of cattle, a cat and goats. In avian species, it has been isolated from the lungs and crop of canaries [START_REF] Devriese | Streptococcus pluranimalium sp. nov., from cattle and other animals[END_REF]. Of these species, only S. suis [START_REF] Devriese | Streptococcus suis infections in birds[END_REF] and E. avium [START_REF] Devriese | Composition of the enterococcal and streptococcal intestinal flora of poultry[END_REF] have been isolated from birds previously.

In the study reported here S. pluranimalium isolated from adult broiler parents had growth characteristics and colonial morphology similar to those reported by [START_REF] Devriese | Streptococcus pluranimalium sp. nov., from cattle and other animals[END_REF], while some variations were seen in biochemical activity, including hydrolysis of hippurate but only two of our eighteen isolates hydrolysed hippurate. However, the bovine isolates varied in a number of phenotypic characteristics, highlighting the difficulties associated with phenotypic identification of this species [START_REF] Devriese | Streptococcus pluranimalium sp. nov., from cattle and other animals[END_REF].

Recent evaluations of phenotypic and molecular methods for identification of nonhaemolytic streptococci have indicated that identifications by commercial kits are correct in fewer than 50% of cases, but that this varies significantly between species [START_REF] Hoshino | Use of phylogenetic and phenotypic analyses to identify nonhemolytic streptococci isolated from bacteremic patients[END_REF]. In our study, correct identification of S. pluranimalium based upon classical phenotypic methods, including the API 20 STREP system, was not possible. Phenotypic characterization identified the 12 isolates from a single clone (A1) as Aerococcus viridans, Leuconostoc sp., S. salivarius and S. bovis II. Other isolates from clusters B1 and B2 were identified as A. viridans, A. urinae, Leuconostoc sp. and S. salivarius (Table 1).

Phenotypically, both E. avium and E. faecium were weakly α-haemolytic, making them difficult to distinguish from S. pluranimalium. However, E. avium and E. faecium could be distinguished from S. pluranimalium by other characteristics listed in Table 1.

Analysis of PFGE patterns showed that E. avium and E. faecium were distinct from S. pluranimalium, with a similarity coefficient of less than 66%. PFGE demonstrated the existence of two clones, in addition to two other distinct isolates of S. pluranimalium, with an overall similarity of 60%.

Birds received for post mortem examination were between 44 and 61 weeks and age might be one of the predisposing factors for valvular endocarditis, since 12 of 19 birds infected with S. pluranimalium suffered from this condition. The bedding quality for the second flock was poor and distinctly different from that for the first flock. Poor bedding quality is a factor that may contribute to the prevalence of some bacterial diseases in poultry, but is difficult to quantitate.

Isolates of S. pluranimalium were obtained from both flocks and from all four houses. Given the high level of biosecurity at parent stock level, introduction of three different clones at the same time, or successively, would only be likely during placement of day-old birds at the rearing farm. Therefore preventive measures should address egg and hatchery hygiene. A number of virulence factors have been identified in streptococci associated with septicaemia and endocarditis [START_REF] Kilian | Topley and Wilson's Microbiology and Microbial Infections[END_REF]. Further investigations on the

  molecular techniques in bacteriology has resulted in numerous revisions of the genus Streptococcus over the last 25 years. In 1984, Streptococcus faecium and S.

  further to obtain an unambiguous diagnosis and possibly elucidate the epidemiology of disease caused by these organisms.

  (75 mM NaCl, 25 mM ethylene-diamine-tetraacetic acid [EDTA], pH 7.4) and

  rRNA genes. Sequencing of 16S rRNA genes of nine strains (20315H1HJ4, 20326H1L1, 20326H4L1, 20363H2HJ3, 20978H4L6, 21129H3HJ3, 21147H3L6, 21147H4L2 and 21192H3L2) representing the major clusters defined using Deleted: of a three Deleted: gel Deleted: with Deleted: and consisting Deleted: of Deleted: , Deleted: unweight Deleted: th Deleted: ed pair-group method, and comparisons made using as described previously by

  distinct profiles (B3 and B4). The PFGE types of the isolates from flock 1

Formatted

  by phenotyping, had 100% and 99.9% 16S rRNA gene sequence similarity, respectively, with the type strain (ATCC14025 T ) of E. avium (Acc. No. DQ411811).

  of pyrrolidonyl arylamidase, β-glucuronidase, alkaline phosphatase and arginine dihydrolase. All the strains characterized byDevriese et al. (1999) hydrolysed

Figure 1 .

 1 Figure Legends

Figure 2 .

 2 Figure 2. Phylogenic relationships between isolates of S. pluranimalium from broiler parents and related species of Streptococcus based on maximum likelihood analysis of 16S rRNA gene sequences. The percentage support for monophyletic groups by bootstrapanalysis are indicated at specific branches. The strains in bold were sequenced in our investigation and strains in parentheses have identical sequences. The scale bar represents branch length based on the maximum likelihood analysis.

Formatted

a

  Fig.2

Table 2 .

 2 Phenotypic characterization of isolates of Streptococcus pluranimalium, Enterococcus avium and E. faecium.

	Characteristics	Streptococcus a	Enterococcus avium a	E. faecium a
		pluranimalium
	Gram positive cocci in chains	+ b	-	(+)
	α-haemolysis on bovine blood agar	+	w	+
	Motility, 37 ºC	-	-	-
	Catalase	-	-	-
	Oxidase, TMPD	-	-	-
	Aerobic growth	+	+	+
	Fermentative, Hugh & Leifson with glucose	+	+	+
	Voges-Proskauer reaction	-	+	+
	Hippurate hydrolysis	d (0.11)	-	+
	Aesculine hydrolysis	+ (0.95)	+	+
	Pyrrolidonyl arylamidase (PYR)	-	+	w
	α-galactosidase	d (0.84)	-	+
	β-glucuronidase	-	-	-
	β-galactosidase	d (0.11)*	-	w
	Alkaline phosphatase	d (0.17

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