Anders Miki Bojesen

H L Shivaprasad

Genetic diversity of Gallibacterium isolates from California turkeys

Keywords: Gallibacterium, AFLP, Turkey, Genetic diversity

The aim of the present study was to investigate the genetic diversity of Gallibacterium isolates recovered from lesions in turkeys. Gallibacterium has been isolated from various bird species including turkeys, but no large investigations have yet been made to characterise isolates from turkeys genetically. Therefore we genotyped 53 Gallibacterium isolates obtained from turkeys between 1998 and 2004. Fifty isolates originated from 29 different flocks in California and the remaining three came from three German turkey flocks. All were recovered from birds with lesions, mainly in the upper respiratory tract. Five chicken isolates from California and five Gallibacterium reference strains were also included. Amplified fragment length polymorphism analysis demonstrated substantial genetic diversity among the Gallibacterium isolates.

However, we also demonstrated that some Gallibacterium clones were present in consecutive rotations at the same farm during the entire six-year observation period and were present in different flocks from different farms. Similarly, the same clone was identified from two of the three German flocks. Further investigation of the spread of Gallibacterium between turkey flocks, including infections acquired from chickens or wild birds should be carried out.

 [START_REF] Christensen | Genetic relationships among strains of biovars of avian isolates classified as "Pasteurella haemolytica", "Actinobacillus salpingitidis" or Pasteurella anatis with proposal of Gallibacterium anatis gen. nov., sp[END_REF]. The importance of Gallibacterium as a pathogen is not fully understood although a number of reports have indicated a pathogenic potential. Isolates have been recovered from mixed infections and in pure culture from a range of pathological lesions in poultry, including septicaemia, oophoritis, follicle degeneration, salpingitis, peritonitis, enteritis and respiratory tract lesions [START_REF] Kjos-Hanssen | Oviduct peritonitis in hens due to pathogenic "cloacal bacteria[END_REF][START_REF] Kohlert | Untersuchrungen zur Ätiologie der Eileiterentzündung beim Huhn[END_REF][START_REF] Mráz | Actinobacilli in domestic fowl. Zentralblat für Bakteriologie und Hygieine Orig[END_REF][START_REF] Gerlach | Significance of Pasteurella haemolytica in poultry[END_REF][START_REF] Matthes | Experimentelle Untersuchrungen zur Übertragung von Bakterien über das Hühnerei[END_REF][START_REF] Mushin | Pasteurella haemolytica found in respiratory tract of fowl[END_REF][START_REF] Bisgaard | Salpingitis in poultry. II. Prevalence, bacteriology, and possible pathogenesis in egg-laying chickens[END_REF][START_REF] Shaw | Pasteurella haemolytica as a co-pathogen in pullets and laying hens[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]Bojesen et al., 2003a;[START_REF] Jordan | Observations on salpingitis, peritonitis and salpingoperitonitis in a layer breeder flock[END_REF].

Bacteria belonging to the genus Gallibacterium seem to have a wide host spectrum based on isolations from domestic as well as a range of non-domestic birds including chickens, turkeys, geese, ducks, pheasants, partridges, cattle egrets and others [START_REF] Mushin | Pasteurella haemolytica found in respiratory tract of fowl[END_REF][START_REF] Bisgaard | Ecology & significance of Pasteurellaceae in animals[END_REF]. Most studies of this organism have been in chickens and isolates obtained from other hosts have not been characterized to any great extent. A recent study reporting the occurrence of Gallibacterium anatis biovar haemolytica in California diagnostic avian specimens during 1994 to 2003 stated that G. anatis bv. haemolytica was isolated from a total of 152 out of 3609 meat turkeys subjected to post mortem analysis (Zellner et al., 2004). However, little is known about the epidemiological aspects of Gallibacterium infections in turkeys at farm, flock or at host level. Genotypic methods involving characterization of the whole genome such as pulsed field gel electrophoresis (PFGE) and amplified fragment [START_REF] Bojesen | Prevalence and transmission of haemolytic Gallibacterium species in chicken production systems with different biosecurity levels[END_REF][START_REF] Christensen | Genetic relationships among strains of biovars of avian isolates classified as "Pasteurella haemolytica", "Actinobacillus salpingitidis" or Pasteurella anatis with proposal of Gallibacterium anatis gen. nov., sp[END_REF]. AFLP and PFGE represent highly reproducible and discriminatory genotyping methods allowing identification of clonal lineages in bacterial populations (Vos et al., 1995;Spratt & Maiden, 1999).

Consequently, the aim of the present study was to characterize a collection of G. anatis bv. haemolytica isolates from turkeys by AFLP and evaluate population dynamics over time and within natural populations.

Materials and Methods

Bacterial strains. Fifty-three Gallibacterium anatis bv. haemolytica isolates from turkeys were characterized in this study. The California isolates were isolated between 1998 and 2004 from a total of 29 flocks owned by five different operations. An additional five isolates from chickens, also from California, were included for comparison. The chicken isolates originated from five epidemiologically unrelated flocks and were isolated in 2004. All isolates were retrieved from accessions in the California Animal Health and Food Safety Laboratory System. The remaining three isolates were obtained from three German turkey flocks. Five Gallibacterium reference strains (CCM5974, CCM5975, CCM5976, F149 T , 12656-12), including the type strain, were also included.

All the field strains were isolated from lesions, mainly in the upper respiratory tract. Other agents were also identified in most of the cases, the most prevalent being Ornitobacterium rhinotracheale (six cases), Bordetella avium (six cases), Escherichia coli (five cases), Pasteurella multocida (one case), Mycoplasma spp. (one case) or a mixed bacterial population mainly composed of two or more of the above agents (24 cases) (Zellner et al, 2004). Swabs were plated on blood agar base supplemented with 5% bovine blood and the plates incubated overnight at 37 o C in plastic bags. Circular smooth, shiny greyish raised colonies with a diameter of 1.0-2.0 mm after 24 h at 37 o C and appearing strongly β-haemolytic, opaque, with a butyrous consistency were sub-cultured on blood agar plates to obtain pure cultures. Frozen stock cultures were subsequently made from cultures in Heart Infusion Broth (HIB; Difco, Detroit, MI.) incubated overnight at 37 o C. Seven hundred microlitres of HIB culture were mixed with 300 µl sterile 50% (v/v) glycerol and stored at -80 o C. Bacterial identification. All isolates were subjected to phenotypic characterization in accordance with Bisgaard (1982) and [START_REF] Christensen | Genetic relationships among strains of biovars of avian isolates classified as "Pasteurella haemolytica", "Actinobacillus salpingitidis" or Pasteurella anatis with proposal of Gallibacterium anatis gen. nov., sp[END_REF] and genotypic identification by the recently described Gallibacterium-specific PCR (Bojesen et al., in press). This PCR is based on primers targeting the 16S and 23S rRNA genes generating two or three amplicons depending on the number of different ribosomal operons present. An internal amplification control based on the rpoB gene is also a part of the PCR protocol.

Genotyping by AFLP. All isolates were characterized as described by [START_REF] Bojesen | Genetic diversity of Gallibacterium anatis in chicken flocks representing different production systems[END_REF]. Briefly, chromosomal DNA was extracted from cell pellets of overnight cultures in HIB by the use of Easy-DNA kit  (Invitrogen, Carlsbad, CA, USA). The AFLP reactions were based on the non-selective BglII primer (FAM-5´GAGTACACTGTCGATCT 3´) and the non-selective BspDI primer (5´ and 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 program GelCompar II (Applied Maths, Kortrijk, Belgium). Normalised AFLP fingerprints were compared using the Dice similarity coefficient with a 0.5% optimization and 0.1% position tolerance, and clustering analysis was performed by the unweighted pair group method with arithmetic averages (UPGMA).

Results and Discussion

All included turkey isolates were β-haemolytic and classified with Gallibacterium based on the phenotypic characters tested. Similarly, Gallibacterium-specific amplicons at the expected sizes of 780 and 1060 bp were generated by PCR in all but one isolate (F0101711; data not shown). In addition, an intermediate size amplicon of ~900 bp was present in all isolates. Only the small and intermediate sized amplicons were present in F0101711. An amplicon of intermediate size indicating the existence of a third size ribosomal operon has been described previously by [START_REF] Christensen | Genetic relationships among strains of biovars of avian isolates classified as "Pasteurella haemolytica", "Actinobacillus salpingitidis" or Pasteurella anatis with proposal of Gallibacterium anatis gen. nov., sp[END_REF], however based on the previous and present results this operon does not seem to occur very frequently in Gallibacterium (Bojesen et al., in press). The internal amplification control based on the rpoB gene was positive in all samples. The The AFLP procedure was repeated only for those isolates (approximately 5%) that generated fingerprints that did not allow proper analysis in the first run. Repeat reactions were deemed unnecessary for others as our earlier study using the same AFLP protocol had a reproducibility of 97.5%, indicating that it was very robust [START_REF] Bojesen | Genetic diversity of Gallibacterium anatis in chicken flocks representing different production systems[END_REF].

A total of 41 individual Gallibacterium clones were identified among the 63 isolates using the 90% Dice similarity cut-off. This cut-off limit has been used in previous studies employing AFLP to assess the genetic diversity of Gallibacterium and other bacteria [START_REF] Janssen | Discrimination of Acinetobacter genomic species by AFLP fingerprinting[END_REF][START_REF] Savelkoul | Amplified-fragment length polymorphism analysis: the state of an art[END_REF][START_REF] Bojesen | Genetic diversity of Gallibacterium anatis in chicken flocks representing different production systems[END_REF].

Of the 41 clones, 33 appeared as single isolates whereas the remaining eight were part of clonal complexes as defined as groups of identical (S D ≥ 90%) bacterial isolates (Figure 1). Three of the complexes contained from three to nine identical isolates.

Interestingly, the same clone was isolated at different time points from operation A over the entire observation period from 1998 to 2004 (Figure 1). A similar picture was seen with the German turkey isolates where two flocks were infected with the same clone.

One of the Californian clones was isolated in one case from turkeys originating from six different flocks owned by two different operations (Figure 1). Strict biosecurity, including hygiene barriers and visitor clearing was practiced in all the operations included in our investigation although none of them operated on an 'all in all out' basis. For example, there were eight to 15 houses on each farm and the individual houses contained birds of different ages. Each farm was serviced by two or three workers, who did not visit flocks on any other farms. However, there was traffic Gallibacterium organisms are not expected to survive well in poultry houses between rotations due to the cleaning and disinfection procedures and their poor viability outside the host [START_REF] Bisgaard | Ecology & significance of Pasteurellaceae in animals[END_REF], but spread of these organisms and other pathogens between flocks and farms, for example by the wind, remains a possibility.

There are other examples of bacterial clones, as in the case of Pasteurella multocida, that have been transmitted between poultry from different production systems and between farmed and wild birds [START_REF] Christensen | Phenotypic and characters of isolates of Pasteurella multocida obtained from backyard poultry and from two outbreaks of avian cholera in avifauna in Denmark[END_REF][START_REF] Pedersen | Pasteurella multocida from outbreaks of avian cholera in wild and captive birds in Denmark[END_REF].

In addition to isolation from various types of poultry Gallibacterium organisms have been isolated from different bird species kept in captivity and from wild birds [START_REF] Bisgaard | Ecology & significance of Pasteurellaceae in animals[END_REF]. However, virtually nothing is known about possible interchange of organisms between these different bird populations. Although the current study was focussed on Gallibacterium isolated from turkeys, a small number of isolates from chickens, also from California, were characterized by AFLP. Interestingly, the California chicken isolates that were included appeared to be related to the turkey isolates and in one case even grouped together with a turkey clone, indicating that there could be a link between the different production systems (Fig. 1).

The five Gallibacterium reference strains were only distantly related to the California isolates, but this was expected due to the large temporal and geographical separation between them. A previous study assessing the genetic diversity among Gallibacterium isolates from chickens found that typically only one clone was present in each flock [START_REF] Bojesen | Genetic diversity of Gallibacterium anatis in chicken flocks representing different production systems[END_REF]. However, isolates from only two flocks were

  organisms previously reported as [Actinobacillus] salpingitidis, avian [Pasteurella] haemolytica or [P.] anatis has recently been reinvestigated leading to formation of a new genus, Gallibacterium

  AFLP) have been used successfully to clarify epidemiological aspects of Gallibacterium infections in chicken flocks but isolates from turkeys have never been investigated on a large scale

  and used to amplify the fragments subsequent to restriction digestion and ligation to their corresponding adaptors. Amplification products were detected on an ABI 377 automated sequencer (PE Biosystems). Each lane included internal-lane size standard labelled with ROX dye (Applied Biosystems)

  results from the current investigation strongly indicate that all of the included isolates have been correctly identified as Gallibacterium.

  trucks carrying feed, live and dead birds. In addition, all farms were located in areas that were densely populated with commercial poultry including chickens and turkeys.

  of isolates from more flocks is probably necessary to substantiate this observation.In conclusion, we have demonstrated a high genetic diversity among Gallibacterium isolates from Californian turkeys. In addition, we have shown that some Gallibacterium clones were present in consecutive flocks on the same farm over six years and in different flocks at different farms. Further studies are required to provide more insight into clonal spread and persistence of Gallibacterium. .G. & Maiden, M.C. (1999). Bacterial population genetics, evolution and epidemiology. Philosophical by the Royal Society of London. Biological Sciences 354, 701-710. Vos, P., Hogers, R., Bleeker, M., Reijans, M., van de Lee, T., Hornes, M., Frijters, A., Pot, J., Peleman, J., Kuiper, M. & Zabeau, M. (1995). AFLP: a new technique for DNA fingerprinting. Nucleic Acids Research, 23, 4407-4414. Zellner, D.E., Charlton, B.R. Cooper G. & Bickford, A.A. (2004). Prevalence of Gallibacterium species in California poultry over 10 years (1994-2003) from diagnostic specimens. In: Proceedings of Annual Conference of the American Association of Avian Pathologists (p. 64). Philadelphia, Pennsylvania.

  Figure Legends

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

Acknowledgement

Vi Phuong Thi Nguyen is thanked for excellent technical assistance.

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Claire Valiente Moro

Philippe Fravalo

Michel Amelot

Claude Chauve

Gilles Salvat

Lionel Zenner

Valiente Moro
email: valienteclaire@yahoo.fr

Lionel Zenneramelot

Colonization and organ invasion in chicks experimentally infected with Dermanyssus gallinae contaminated by Salmonella Enteritidis

Keywords: Salmonella Enteritidis, Dermanyssus gallinae, organ colonization, chicks

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.

The poultry red mite, Dermanyssus gallinae is the most important and common ectoparasite of laying hens in Europe. This haematophagous mite has been experimentally demonstrated to be a vector of Salmonella Enteritidis by acquiring bacteria through the blood meal or cuticular contact. We have evaluated another route of infection by orally inoculating chicks with mites previously infected by S. Enteritidis. Two methods of infecting the mites were tested: mites contaminated by cuticular contact or during the blood meal. After the washing of mites with Paraformaldehyde, groups of 10 Salmonella-contaminated mites were inoculated individually into 1-day-old chicks. The titre of the inoculum suspension was evaluated by crushing mites and followed by bacteriological counting. It was 3x10 4 colony-forming units (CFU) and 2.7x10 6 CFU/chick, respectively, for cuticular contact and orally mediated contamination of mites. Each bird was found to be positive 12 days postinoculation. Salmonella colonized the intestinal tracts and invaded livers and spleens. Caecal content concentration reached a mean number of S. Enteritidis of 8.5x10 4 MPN Salmonella g -1 . This experiment demonstrated the ability of mites to orally infect one-day-old chicks with subsequent colonization and multiplication of Salmonella. Consequently, mites infected by S. Enteritidis constitute potential reservoir hosts of this bacterium, allowing it to persist in the poultry house as a source of infection for newly introduced animals. If contaminated mites are found in poultry facilities, effective red mite control should be performed before new batches are introduced into the facility.

Introduction

Salmonella is associated with many of the most important outbreaks of food-borne diseases worldwide [START_REF] Lacey | Food-borne bacterial infections[END_REF]. Eggs or broiler meat contaminated with S. enterica have been described as sources of infection for consumers. As poultry is one of the most important reservoirs of Salmonella, its transmission to humans through the food chain is a major public health concern. Since the 1980s, the serovar most often responsible for Salmonella intoxication in humans is S. Enteritidis, strongly associated with egg production. S.

Typhimurium human cases are more commonly associated with consumption of contaminated meat (poultry, pig and bovine) [START_REF] Food | The Community Summary Report on Trends and Sources of Zoonoses, Zoonotic Agents and Antimicrobial Resistance in the European Union in[END_REF]. Poultry facilities have identified risk factors associated with primary production contamination. Birds can be infected from contaminated food, water, litter, and rodents and the persistence properties of Salmonella in the birds' environment still represents an important characteristic of Salmonella epidemiology in poultry (Davies & Breslin, 2003). Many investigations have emphasized the role of wildlife species such as litter beetles, nematodes or houseflies in the transmission or the persistence of S. Enteritidis in poultry [START_REF] Noreen | Vector potential of the german cockroach (dictyoptera:blatellidae) in dissemination of Salmonella enteritidis serotype typhimurium[END_REF]Chadfield et al. 2001;Olsen & Hammack, 2000;Rose et al. 2000;Skov et al. 2004). The role of Dermanyssus gallinae as a causative agent in the transmission of Salmonella between laying hens in successive flocks is still under question.

The poultry red mite, Dermanyssus. gallinae, is a common haematophagous arthropod found in laying hen facilities where it can cause bird anaemia, decreased egg production and in extreme cases, death of its bird hosts [START_REF] Kirkwood | Anaemia in poultry infested with the red mite Dermanyssus gallinae[END_REF]. This mite parasites its host only during blood meals and the rest of time it hides in the darkness to digest and/or reproduce. It can also bite humans and is recognized as a real problem for people working on poultry farms (Chauve, 1998). Moreover, its population can rapidly reach great number of individuals since its life cycle is particularly short (about 10 days), and the current methods of control having for most of them low efficiency, it has become a very difficult pest to eradicate [START_REF] Beugnet | Resistance of the red poultry mite to pyrethroïd in France[END_REF]. D. gallinae is also suspected to be a possible vector of various pathogens (Valiente Moro et al. 2005) and has been demonstrated to be an experimental vector of Salmonella Enteritidis (Valiente Moro et al. 2006). It has been shown that the mite can be infected by S.

Enteritidis after both cuticular contact and during a contaminated blood meal, that the bacteria can multiply, can be transmitted to the next generation and that transtadial transmission from protonymphs to deutonymphs is also possible. Finally, it has been shown that previously infected mites are also able to recontaminate the blood in an in vitro feeding device. Since birds commonly peck and even ingest red mites in breeding hen facilities, the ingestion of contaminated mites by chickens as a potential route for poultry infection is of particular interest and merits further investigation.

Therefore, we have orally contaminated chicks with mites previously infected by S.

Enteritidis. The ability of Salmonella to colonize the intestinal tract of the birds and invade their internal organs after inoculation of mites was then analysed.

Materials and Methods

Mites and bacterial strain. Mites were collected from broiler chickens in the Rhône-Alpes region (Bresse, France) and were manipulated with a paint brush and a suction pump connected to a collector. The absence of Salmonella in the mites was confirmed on 20 groups of 10 mites by culture of crushed mites onto SMID. The Salmonella Enteritidis strain was provided by the "Laboratoire Centrale de Recherche Avicole et Porcine" (AFSSA, Ploufragan, France) which had previously isolated it from a poultry hatchery sample. It was maintained by regular plating on Salmonella Medium Identification (SM ID), (Biomérieux, Craponne, Fance). Chicks. One-day-old Isabrown chicks were used for the experiments. Chicks came from a layer hatchery free of Salmonella. Prior to the test, 10 chicks were confirmed to be S.

Enteritidis-free by analysing their intestinal tract. The bottoms of the boxes in which the chicks had been transported as well as any droppings in the boxes were also checked for Salmonella contamination with negative results. All experimental birds received a commercial broiler finisher diet which was confirmed as S. Enteritidis-free. All experimental procedures were performed according to French law concerning animal experimentation (decree 87-848 modified by 2001-464 of 29/5/2001).

Infection of mites.

As described in our experimental model, two methods of infecting the mites with Salmonella were used (Valiente Moro et al. 2006). The infection during the blood meal consisted of in an in vitro feeding device using chicken skin over a pipette tip used as a blood reservoir. Adult chickens (Gallus gallus) were killed, their blood defibrinized by vigorous shaking and their skin carefully dissected from the corpses and scraped free of subcutaneous tissue. The absence of Salmonella in the blood used was previously checked by culture on specific medium SM ID. Blood was infected with S. Enteritidis at the rate of 10 8 CFU/mL. Using a pipette the mites were then placed in contact with the chicken skin overlying the infected blood reservoir and allowed to feed for 4 hours in an incubator under standardized conditions (temperature: 27°C ± 1°C, relative humidity: 75% and total darkness). This resulted in an average contamination of 2x10 4 Salmonella/mite. Cuticular infection of the mites was achieved by leaving them on a dry Salmonella coating. Briefly, 500µl of a S. Enteritidis concentrated solution in Trypton Salt (TS) (BioMérieux, Craponne, France) was streaked onto SM ID agar and incubated at 37°C for 18-24 h. Sterile Whatman paper was placed in contact with the bacterial coating and then transferred to a sterile petri dish. This allowed the mites to walk on the paper without suffering from the humidity Inoculum preparation. Forty pools of 10 mites were selected by randomly taking engorged mites which had been artificially infected during bloodmeals 6 to 13 days before. The same sampling was performed for cuticle-infected mites which had been infected 6 to 10 days earlier. To ensure that any bacteria subsequently detected were those located inside the mites, we used the external decontamination and washing protocol previously described by Zeman et al., (1982); each set of 10 mites was washed twice in 100 µl of 4% paraformaldehyde for 7 min, once in 100 µl of sterile water followed by a final washing in 100 µl of TS. The final washing solutions were pooled and analysed for the presence of Salmonella to check the efficiency of the washing protocol. Each group of mites was finally suspended in 100 µl of TS and stored at 4°C. These mite suspensions were used as the inoculum for the chicks. Control mites, namely non contaminated mites, were also washed to check that the Paraformaldehyde solution was not toxic for chicks. Six pools of 10 mites were kept for each route of infection to assess the titre of the feeding suspension. Mites were crushed in 200 µl of TS and a ten-fold serial dilution was made. Then, 100 µl of each dilution were streaked onto Rambach agar and incubated at 37°C for 18-24 h. When bacterial levels were undetecttable, the presence of Salmonella in the remaining 80 µl of the initial solution was checked by pre-enrichment in Peptone Buffer Water (PBW), (BioMérieux, Craponne, France) and enrichment on MSRV agar.

Experimental infection of chicks. Ninety eight one-day-old chicks were inoculated orally: 34 received mites contaminated during the blood meal, 34 were infected with mites contaminated by the cuticular route and 30 chicks, the negative control, were inoculated with Fecal samples. At 6 days postinoculation (Figure 1), fecal samples were randomly collected from each incubator (about 25g per incubator). These samples were transferred to 225 ml of BPW (BioMérieux, Craponne, France) and mixed by stomaching for 30 sec. After preenrichment for 16-20 h at 37°C, an MSRV (Modified Semi-solid Rappaport Vassiliadis agar) plate was spot-inoculated with three drops of the pre-enriched medium and incubated at 41.5°C for 24 h and if necessary for 48 h. On MSRV agar, Salmonella strains migrated from the inoculation spot. The margin of this migratory zone was streaked onto a Rambach agar plate. If no migration was noted after 48 h of incubation, the MSRV result was considered as negative. After 24 h incubation of the Rambach agar plate, typical colonies were biochemically confirmed using API20E gallery (BioMérieux, Craponne, France).

Internal organ samples. At 12 days after inoculation in each trial (Figure 1), all chicks were sacrificed to allow bacteriological culture of their internal organs. For chicks inoculated with uncontaminated mites, livers, spleens and caeca were aseptically removed, pooled in groups of 5, transferred to BPW (1/10 dilution w/w) and homogenized for 30 sec in a Stomacher Model. For chicks inoculated with contaminated mites, their livers and spleens were pooled in groups of 5 and mixed by stomaching for 30 sec in BPW. Salmonella detection was performed individually on their caeca. All samples were incubated for 16-20 h at 37°C, and a MSRV agar plate was spot-inoculated and incubated for 24 and 48 h at 41.5°C. Finally, a loopful was streaked onto Rambach and Xylose Lysine Desoxycholate (XLD) agar and incubated for 18-24 h at 37°C. The enumeration of Salmonella was also performed by direct plating of 100 µl from the liver and spleen pool suspensions onto Rambach agar and cultured overnight at 37°C. For caeca individually isolated from contaminated chicks, the Salmonella count was performed using a MPN approach based on miniaturisation of MSRV enrichment.

The method consisted in 3 repeat successive dilutions using the mini MSRV method [START_REF] Fravalo | Convenient method for rapid and quantitative assessment of Salmonella enterica contamination : the mini-MSRV technique[END_REF]. Any presumed colonies of S. Enteritidis were confirmed biochemically and serotyped.

Results

Determination of the feeding suspension titres. The number of Salmonella associated with the pool of 10 Dermanyssus inoculated per bird was determined by crushing mites, homogenizing the suspension and subsequent plating of convenient ten-fold serial dilutions in TS onto Rambach agar. The mean infectious titres were respectively 3.0x10 4 CFU/chick and 2.7x10 6 CFU/chick for birds inoculated with cuticular contaminated mites and those contaminated by blood-engorged mites (Figure 2A et 2B). Before the chicks were inoculated, the final washing solutions of the mites were also controlled. The residual contamination measured for the 4 ml pools of the final washing solutions from the 340 cuticle-contaminated mites was 10 CFU/100 µl and 7.5x10 4 CFU/ 100 µl for the blood-contaminated mites.

Isolation of S.

Enteritidis from fecal samples and tissue. At 6 days after inoculation, faecal samples from both sets of infected chicks were positive for Salmonella while the control corresponding to chicks inoculated with uncontaminated mites remained negative for Salmonella. On day 12 post-inoculation, S. Enteritidis was isolated from the caeca of all birds which had received contaminated mites with an average number of S. Enteritidis above 8.5x10 4 MPN Salmonella g -1 (Table 1). Using direct plating, S. Enteritidis was counted from the spleen and the liver of all birds. Chi2 square analysis did not show any significant difference (P<0.5) between the infection routes.

Discussion

It has recently been demonstrated that D. gallinae is an experimental vector of S. Enteritidis (Valiente Moro et al. 2006). It has been shown that the mite can be infected through the blood meal or by cuticular contact. This infection is followed by bacterial survival of up to 14 days inside the mite as well as multiplication, transovarial and transtadial transmission and contamination of host blood after a blood meal. However, the contamination of hens through eating previously infected mites has not yet been demonstrated. Therefore, in the present study, we inoculated chicks with mites experimentally infected by S. Enteritidis in order to assess the potential of D. gallinae to transmit Salmonella in this way.

The mean infectious titre of the inoculum given to chicks was equal to 3x10 4 CFU and 2.7x10 6 CFU for chicks respectively inoculated with cuticule-infected and engorged mites.

Other experimental studies performed with young chicks at 1-day-old or 1-week-old showed that low dosages of S. Enteritidis ranging from less than 10 organims to 5x10 4 CFU could induce infection in chicks (Cooper et al, 1994;Duchet-Suchaux et al 1995;Van Immerseel et al 2004). It is important to note that this dose is dependant on the age of the host, the number of bacteria in the organs and the serotype of Salmonella used. For example, it is about 10 12 CFU for older chickens infected with S. Typhimurium considering the fact that they can trigger their host's defence mechanisms after the colonization of the gastrointestinal tract [START_REF] Muir | Comparison of Salmonella typhimurium challenge models in chickens[END_REF]. In our experiment, mites used as the infecting inoculum contained sufficient infectious levels to cause infection in one-day-old chicks. After the chicks had been orally inoculated with contaminated mites, excretion of S.

Enteritidis was observed from 6 days post-inoculation in both infected groups. When the birds were sacrified on day 12, all those which had received contaminated mites showed increased Salmonella levels in their livers, spleens and caeca (higher than 8,5x10 4 MPN Salmonella g -1 tissue) indicating colonisation and multiplication of the bacteria. The results of our tests on the final washing solutions were respectively 10 CFU/100 µl and 7.5x10 4 CFU/100 µl, respectively, for cuticule-infected and engorged mites. It suggests that the washing procedure was not performed adequatly. However, it would be expected that the mites that were infected by walking on a Salmonella coating would have more Salmonella on the outside that the mites that have been infected by a blood meal. But the results show that the washing solution of the engorged infected mites is more contaminated than the other group. So, another explanation for this contamination could be the accidental crushing of a mite during the washing process. This hypothesis is supported by the fact that the level of contamination in the solution could be compatible with the concentration found in a single infected mite 7 or 14 days after the engorgment considering the fact that bacterial multiplication has been observed in some mites (Valiente Moro et al. 2006).

Previous studies on S. Enteritidis infections in poultry have been primarily carried out by inoculating chickens with a suspension of bacteria to demonstrate the invasive character of these bacteria when administered orally to chickens (Desmidt et al. 1997). In a model using 1week-old chicks, CFU levels were highest during the first week(s) after oral inoculation with 5x10 4 S. Enteritidis and then decreased progressively (Duchet-Suchaux et al. 1995). Another study involving S. Enteritidis as an inoculum of 2x10 2 CFU administrated orally to 1-day-old chicks showed that the clearance of Salmonella was fast with 60% of caecal samples which were found to be S. Enteritidis positive at 18 h pi (Asheg et al 2001). [START_REF] Hinton | Experimental Salmonella enteritidis infection in chicks[END_REF] further demonstrated that birds may readily become infected by eating feed contaminated with In spite of the Salmonella-positive results in the washing solutions, the level of infection obtained in both the infection models tested shows that previously in-and outside infected mites, when eaten by one-day-old chicks, could represent a new source of Salmonella infection. The level of infection obtained in both the infection models tested shows that previously infected mites, when eaten by one-day-old chicks, could represent a source of Salmonella infection. The invasion of organs such as the liver and spleen is an indication of systemic infection. So, the reproductive organs could be also contaminated and it could introduce a risk for humans when eggs are consumed. However, we can not say how many mites are ingested each day by hens and it is difficult to assess the real impact of the mite in breeding facilities. However, we can state that this mode of infection is possible in one-dayold chicks Moreover, the challenge model we have used has been performed with chicks, so, in future investigations, it would be interesting to confirm if such infection takes place in older birds. To give a ruling on the possibility of this type of contamination in the field, it would also necessary to study the levels of Salmonella in facilities which have already had problems with Salmonella contamination or acarian infestation both before and after the arrival of new flocks. Finally, carriage of Salmonella by arthropods has already been reported in previous studies. Most recorded examples refer to litter beetles (Alphitobius diaperinus) or cockroaches but their role in the transmission of infection remains unproven [START_REF] Mcallister | Reservoir competence of the lesser mealworm (Coleopta: tenebrionidae) for Salmonella typhimurium (Eubacterials: Enterobacteriacae)[END_REF]Davies & Wray, 1995;[START_REF] Gray | Reservoir competence of Carcinops pumilio for Salmonella Enteritidis[END_REF]. [START_REF] Ash | Vector potential of the German cockroach (dictyoptera: blatellidae) in dissemination of Salmonella enteritidis serotype typhimurium[END_REF] showed that the German cockroach, Blatella germanica, is an effective mechanical transmitter of S. typhimurium via faeces, although the bacteria were recoverable from its gut for about 10 days longer than from the faeces. In addition, the role of litter beetles as a potential reservoir for Salmonella enterica between broiler flocks has also been observed (Skov et al. 2004). Davies

  agar. They remained in contact with Salmonella for 48 h at room temperature and were finally collected and stored in sterile plastic tubes until inoculation of chicks.

  mites. Using a 200 µl pipette, each chick received orally 10 mites suspended in 100 µl of TS. Each group of birds was housed in separate isolator units and kept 12 days after inoculation.

  Enteritidis. This latter experiment is closer to our study because Salmonella transmitted via feed given to birds, is similar to Salmonella transmitted via mites ingested by birds.

  1993) demonstrated that the feeding of contaminated Lucilia sericata larvae to chickens is a potent means of establishing S. Enteritidis infection.In conclusion, this study has demonstrated an experimental model of S. Enteritidis infection which could occur in poultry farms: natural consumption of contaminated mites leading to a lasting infection. It is the first time that Salmonella infection has been induced via the ingestion of an arthropod vector. Infected D. gallinae could represent a new source of contamination when ingested by chickens.

  ., Permin, A., Nansen, P. & Bisgaard, M.. (2001). Investigation of the parasitic nematode Ascaridia galli (Shrank 1788) as a potential vector for Salmonella enterica dissemination in poultry. Parasitology Research, 87, 317-325. Chauve, C. (1998).The poultry red mite Dermanyssus gallinae (DeGeer, 1778): current situation and future prospects for control. Veterinary Parasitology, 79, 239-245. Cooper, G.L., Venables L.M., Woodward, M.J. & Hormaeche, C.E. Invasiveness and persistence of Salmonella Enteritidis, Salmonella Typhimurium, and a genetically defined S. Enteritidis aroA strain in young chickens. Infection and Immunity, 62, 4739-4746. Davies, R.H. & Breslin, M. (2003). Persistence of Salmonella enteritidis phage type 4 in the environment and arthropod vectors on an empty free-range chicken farm. Environmental Microbiology, 5, 79-84. Davies, R.H. & Wray, C. (1995). The role of the lesser mealworm beetle (Alphitobius diaperinus) in carriage of Salmonella Enteritidis. Veterinary Record, 137, 407-408. Davies, R.H. & Wray, C. (1993). Use of larvae of Lucilia serricata in colonization and invasion studies of Salmonella enteritidis in poultry. Proceedings of the Flair Workshop No. 6, 10-13 December 1992. Southampton., pp. 10-13. Desmidt, M., Ducatelle, R. & Haesebrouck, F. (1997). Pathogenesis of Salmonella enteritdis phage type four after experimental infection of young chickens. Veterinary Microbiology, 5, 99-109. 1997. Duchet-Suchaux, M., Léchopier, P., Marly, J., Bernardet, P., Delaunay, R. & Pardon, P.(1995). Quantification of experimental Salmonella enteritidis carrier state in B13 Leghorn chicks. AvianDiseases, 39,[796][797][798][799][800][801][802][803]

  .R. & Hammack, T. (2000). Isolation of Salmonella spp. from the housefly, Musca domestica, and the dumpfly (Hydrotaea aenescens)(Wiedmann) (Diptera:Muscidae) at caged-layer houses. Journal of Food Protection, 63, 958-960. Rose, N., Beaudeau, F., Drouin, P., Toux, J.X., Rose, V. & P. Colin, P. (2000). Risk factors for Salmonella persistence after cleansing and disinfection in French broiler-chicken houses. Preventive Veterinary Medicine, 44, 9-20. Skov, M.N., Spencer, A.G., Hald, B., Petersen, L., Nauerby, B., Carstensen, B. & Madsen, M. (2004). The role of litter beetles as potential reservoir for Salmonella enterica and thermophilic Campylobacter spp. between broiler flocks. Avian Diseases, 48, 9-18. Valiente Moro, C., Chauve, C. & Zenner, L. (2005). Vectorial role of some Dermanyssoid mites (Acari, Mesostigmata, Dermanyssoidea). Parasite, 12, 99-109. Valiente Moro, C., Fravalo, P., Amelot, M., Chauve, C., Salvat, G. & Zenner, L. Experimental studies on the vector potential of the Red Mite, Dermanyssus gallinae to Salmonella Enteritidis. In Proceedings of the XIIth International Congress of Acarology, Amsterdam, The Netherlands, 21-26 August 2006. Van Immerseel, F., De Buck, J, Pasmans, F;, Bohez, L., Boyen, F., Haesebrouck, F & Ducatelle, R. Intermittent long-term shedding and induction of carrier birds after infection of chickens early posthatch with a low or high dose of Salmonella Enteritidis.PoultryScience, 83, 1911-1916. Zeman, P., Stika, V., Shalka, B., Bartik, M., Dusbabeck, F. & Lavickova, M. (1982).Potential role of Dermanyssus gallinae(DeGeer, 1778) in the circulation of the agent of pullurosis-typhus in hens. FoliaParasitologica, 29,[371][372][373][374]

Figure legends Figure 1 .

 legends1 Figure legends

Figure 2 .

 2 Figure 2. Determination of the feeding suspension titres calculated from cuticular contaminated mites (A) and from blood-meal contaminated mites (B). The number of salmonella associated with the pool of 10 Dermanyssus inoculated per bird was determined by crushing mites, homogenizing the suspension and plating convenient ten-fold serial dilutions in TS onto Rambach agar. The experiment was repeated six times.

Table 1 .

 1 Isolation and enumeration of S. Enteritidis in spleen, liver and caecum on day 12 post-inoculation ( a Percentage of positive birds, b with the number of CFU per gram of tissue, c Caecal walls and contents estimated usingMPN method)

		Spleen	Liver		Caecum
		(CFU/g tissue) (x5)	(CFU/g tissue) (x5)	(CFU/g tissue) (x1)
		%positive a CFU/g b	%positive a CFU/g b	%positive a MPN
					Salmonella/g b
	engorged mites	100 2.7 +/-1.8 x10 3	100 1.9 +/-1,9 x10 2	100	> 8.6x10 4
	cuticular mites	100 3.6 +/-3.5 x10 3	100 1.8 +/-2.9 x 10 3 100	> 8.6x10 4

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

Acknowledgments

We gratefully acknowledge the technical assistance of the service personnel of "Elevage et Expérimentation en Pathologie Aviaire" (Agence Française pour la Sécurité Sanitaire des Aliments, Ploufragan) as well as Wanda Lipsky and Richard Sullivan for English language correction.


Paul G Mbuthia

Lucy W Njagi

Uswege Philip Njeru Nyaga

Mbonile Minga

John Kamundia

Lily C Bebora

John Elmerdahl Olsen

U Minga

Pasteurella multocida in scavenging family chickens and ducks: carrier status, age susceptibility and transmission between species

Keywords: fowl cholera, carrier, age susceptibility, transmission Pasteurella multocida, fowl cholera, carrier, cross-transmission, family poultry, age susceptibility ¶

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Introduction

The Gram negative rod, Pasteurella multocida causes fowl cholera, which is a severe disease of poultry. It is seen either as acute or chronic forms, and the clinical signs vary depending on the form of the disease. Symptoms include depression, ruffled feathers, fever, anorexia, mucous discharge from the mouth, diarrhoea and an increased respiratory rate (Rhoades & Rimler, 1989). Carrier birds play a major role in the transmission of fowl cholera [START_REF] Christensen | Fowl cholera[END_REF].

Free-range chicken are important livestock species for many rural families worldwide. They are traditionally raised in a low input low output production system, where birds obtain feed by scavenging and only rarely are offered supplementary feed.

Birds used in this type of production are of low genetic potential as they are often of undefined indigenous breeds that have not been subjected to a clear selection strategy. In the current publication, the term family chickens/ducks/poultry will be used for such birds. While the disease is well described from intensive production systems (see [START_REF] Christensen | Fowl cholera[END_REF] for a review), little information is available as to the presence of this disease among family chickens. Recent studies have shown family poultry may be carriers of P. multocida [START_REF] Muhairwa | Occurrence of[END_REF], however, clinical cases are not frequently described, and chronic manifestations seem to be the normal clinical picture in this production system.

Family poultry are kept in a production system where different age groups mix freely during scavenging. Knowledge of the most susceptible age group is important, since disease control measures and development of health programs depend on such In chicken raised under industrial conditions, fowl cholera is mainly diagnosed in mature chickens (Salami et al., 1989), and based on challenge of 16 and 45 week-old New Hampshire chickens with two different P. multocida serotypes (Heddleston,1962) it has been generally accepted that mature birds are more susceptible than the young chickens. In support of this, [START_REF] Heddleston | Fowl cholera: Comparison of serologic and immunologic responses of chickens and turkeys[END_REF], showed that 9-16 week-old New Hampshire chickens were less susceptible than 52 week old birds. In such studies, the attack criterion has been mortality, since the P. multocida strains used were lethal to the chickens. However, no studies on age susceptibility have been done in poultry using a less virulent strain causing clinical signs but no mortality, as seems to be the normal form of fowl cholera in family poultry.

Fowl cholera is also a recognized cause of duck mortality [START_REF] Hunter | Pathology of experimental avian cholera in Mallard ducks[END_REF]Pehlivanoglu et al., 1999;[START_REF] Amonsin | DNA finger printing of Pasteurella multocida recovered from avian sources[END_REF]. Fewer studies have been performed with this species. However, by use of 5, 11, 16 and 18 week-old Mallard ducks, it has been demonstrated that birds older than 11 weeks are less vulnerable to P. multocida infection than young ones [START_REF] Hunter | Pathology of experimental avian cholera in Mallard ducks[END_REF]. In many developing countries, chickens and ducks are kept under scavenging conditions along side each other, and the close proximity may sustain infection if transmission can take place between species. Studies of transmission between chicken and ducks (and vice versa) have, however, not been undertaken.

The present study aimed to estimate the prevalence of carriers of P. multocida among healthy looking family chickens and ducks and to determine the most susceptible age group among the family chickens and ducks when challenged with a low virulent strain causing signs of chronic fowl cholera. Further it aimed to evaluate if transmission inoculums. The inoculums were prepared as described by Petersen et al. (2001) and each bird was inoculated intra-tracheal with 0.5 ml culture containing P. multocida in brain heart infusion (BHI) broth. The size of the inoculums was determined by plate spread method and contained 1.2 -2.0 x 10 8 CFU per bird. Control birds were inoculated with 0.5 ml of BHI broth and housed in different houses than the challenged birds.

Materials and Methods

Samples

Animal experimentation. Birds and general management conditions. Family chicks and ducklings, hatched from incubated eggs or bought as one day old birds from farms with indigenous flocks, were brooded and reared in an isolation house, away from other birds up to the required experimental age. They were a mixture of eco-types used for local production and could not be included with any described commercial breed. They were screened for P. multocida before used in experiments, as described under bacteriological examination below.When used for experimentation, groups were separated in different rooms located in different houses and left to acclimatize for 48 hr before challenge. They were kept at ambient temperature on the floor, which allowed all birds to mix freely.

Each room had own utensils and did not communicate with other rooms through ventilation. Birds were fed on commercial chick and grower's feed (Unga Limited, Kenya) and given water ad libitum. Experimental infections were performed with permission according to Kenyan law.

Age susceptibility studies. A total of one hundred and twenty family chickens and 120 family ducks were divided into age groups (4, 8, 12, and 16 weeks) with 30 birds in each group. For each age susceptibility group, the birds were split into two groups of 15 wing was observed between repeats, and the result section presents pooled results from both experiments. Of the 15 birds in each experimental round, 10 were infected with P. multocida while 5 were used as controls. They were challenged with P. multocida strain NCTC 10322 T as described above and 24 hours post challenge, the birds were swabbed on oro-pharynx and cloaca to confirm the establishment or absence of P. multocida. On day 14 post-challenge, all birds (challenged and control) were sacrificed and examined for gross lesions and swabs were taken for bacterial isolation.

Studies of cross transmission between species.

A total of 21 wing-tagged chicken (12 week old) and 21 wing-tagged ducks (6 week old) were used for study of cross transmission between species. In experiment number one, 6 ducks were infected intratracheal with P. multocida NCTC 10322 T as described above (seeder birds), while 15 chickens (sentinel birds) were mixed with them under conditions that allow free mixing six hours post inoculation for contact cross infection. In the second experiment, 6 chickens were infected intra-tracheal (seeder birds), while 15 ducks (sentinel birds) were mixed with them under conditions that allowed free mixing six hours post inoculation.

Five sentinel birds in each experiment were individually swabbed on the oropharynx and cloaca daily. The remaining 10 sentinel birds were randomly paired and sacrificed on days 1, 3, 5, 7, and 10 after mixing for post mortem examination. On day 14 post-infection the sentinel birds that were regularly swabbed and the seeder birds were sacrificed and examined for gross lesions and swabs taken for bacterial isolation. Clinical observations. Daily observations started with birds in the control house before proceeding to the infected house. Clinical sign observations (See Table 1 for a list of signs noted), temperature and weight measurements were done once daily, by the same person throughout the study period. The observer took 30 minutes per day per group at the same time each day for the measurements. Each day, observations were first made without disturbing the birds and later a close examination and cloacal temperature measurement were carried out while holding each bird; and its weighing was done and recorded. Observed signs were graded as severe, moderate, or mild. A sign was mild if it was observed 1-3 times during the 30 min observation period, moderate if it was observed 4-6 times and severe if it was observed 7 or more times on an individual bird during the 30 minutes of observations. Depression was mild if there was only dullness; moderate if accompanied by wing drooping; and severe when there is addition sign of dosing and/or tucked head under the wings. Ruffled feathers around the head and neck was taken as mild; when general body was affected it was taken as moderate; while with drooping wings it was regarded as severe. Cloacal temperature below 41. (2001), to improve the bacterial recovery rate. Inoculated mice were sacrificed after 48 hours, however, severely affected mice were sacrificed on a running basis. Culture was performed from aseptically removed liver and spleen.

Bacterial colonies from initial swabs and mice organs morphologically resembling those of P. multocida were sub-cultured on BA and differentiated following the procedures described elsewhere [START_REF] Bisgaard | Characterization of some previously unclassified Pasteurella spp. obtained from the oral cavity of dogs and cats and description of a new species tentatively classified with the family Pasteurellaceae Pohl 1981 and provisionally called Taxon 16[END_REF]. The biochemical reactions of the isolates were compared with those of P. multocida strain NCTC 10322 T and strains were assigned to sub-species according to Mutters et al. (1985).

Post mortem procedure. Post mortem examination was done as described by [START_REF] Bermudez | Disease prevention and Diagnosis[END_REF]. The sacrificed birds were opened aseptically and post mortem examination done, gross lesions noted and swabs taken from the oro-pharynx, cloacae, lungs, liver, spleen, caecal tonsils, and uropygial gland for bacterial examination.

Statistical analysis. The number of clinical signs in different age groups and the proportion of infected birds at farm and at marked were compared by the chi-square method. A p-value of 0.05 was taken as significant.

Results

Formatted: Indent: First line: 36 pt Seventeen isolates were obtained from swabbing of oropharynx and seven from cloacae.

Four isolations were made by mouse passage only, while in the remaining 20 cases, isolation was done both from swabs and mice. Seven isolates were characterized as P. multocida subsp. multocida, 12 isolates as P. multocida subsp. gallicida while 5 isolates were characterized as P. multocida subsp. septica. Of the 162 chickens sampled, 10 (6.2 %) yielded P. multocida. The proportion of infected birds at market was 9/74 (12.1 %) was significantly higher than that of birds sampled on farm (1/88 (1.1 %; p<0.05)). Of the 54 family ducks screened, 14 (25.9%) yielded P. multocida. The proportion of infected market birds, 4/7 (57.1%) was significantly higher than that of birds sampled on farm (10/47 (21.3%; p<0.05)).

Age susceptibility in family chickens. Clinical signs in chicken in relation to age group.

No birds died during the experiments although all chickens but two (16 week old) expressed clinical signs of fowl cholera at some point during the 14 days observation.

The frequency of the individual clinical signs was significantly different between groups (p<0.05) except for diarrhea, mouth discharges and cyanosis (Table 1). Control birds in all groups did not show any clinical sign throughout the observation period.

Gross lesions and P. multocida re-isolation from chickens. On post mortem the 4 weekold chickens had no visible gross lesions, while three birds in the 8 week-old group had fibrin remnants on the airsacs and thickened airsacs, one bird had fibrotic lung and another had splenomegally. Five from the 12 week-old ones had remnants of fibrin and fibrosis of the lungs and airsacs, one had necrotic liver lesions, and another one had splenomegally, while three of the 16 week-old ones had fibrosis on the lungs and airsacs.

P. multocida was isolated from oropharynx of three of the 12 week-old and two of the 16 week-old chickens, 24 hours post-infection but not from the 4 and 8 week-old chickens.

There were no gross pathological lesions or P. multocida recovered from the control birds from all the chicken age groups.

Weight changes between control and P. multocida infected chicken. The infected birds in all the age groups gained significantly (P<0.05) less weight than the control birds (Table 2).

Age susceptibility in family ducks. Clinical signs in ducks in relation to age group.

All infected ducks except three (one 12 week old and two 16 week old) expressed clinical signs of fowl cholera at some point during the 14 days observation. No mortalities were recorded. The frequency of all signs, except diarrhea and fever, varied significantly between age groups (Table 1).

The eight week old ducks expressed significantly more clinical signs (188) compared to the other age groups. This was also the case for the four week old ducks (117). The susceptibility among the 12 (80) and 16 (83) weeks old ducks was equal.

Moreover, the number of severe signs was highest among the 8 week old birds (Table 2) Control birds of all ages did not show any clinical sign throughout the observation period.

Gross lesions and P. multocida re-isolation from ducks. On post mortem examination two 4 week-old ducks had remnants of fibrin and fibrous strands on the air sacs. One of these had pericarditis and perihepatitis. Eight of the 8 week-old ducks had remnants of fibrin and fibrous strands on the air sacs, two had fibrosis on the lungs and another two had necrotic spots on spleen and another on the liver. Two of the 12 week-old ducks had remnants of fibrin and fibrous strands on the air sacs; while four of the 16 week-old ducks had remnants of fibrin on the air sacs; one of these had necrotic spots on the liver. P. multocida was isolated from 4 of the 8 week-old and 2 of the 16 week-old ducks and one each from the 4 and 12 week-old ducks, respectively, 24 hours post infection. There were no gross pathological lesions or P. multocida recovered from the control birds from all the age groups. Weight changes between controls and P. multocida infected ducks. The infected birds in all the age groups gained less weight than the infected birds. However, statistically the difference was only significant in the 8, 12 and 16 week-groups (Table 2).

Transmission of P. multocida from seeder to sentinel chicken and ducks. The demonstration of P. multocida in both oropharyngeal and cloacal swabs among carrier animals is an indication that birds sharing drinking water and feed can easily transmit bacteria to each other. Therefore we decided to investigate the possibility of contact transmission between chicken and ducks (and vice versa) kept together, a conditions often seen in developing countries.

P. multocida was re-isolated on day one post infections from 5 out of the 6 birds of each species that were inoculated (seeder birds). This was taken as an indication that the experimental infection was successful and that they could shed the P. multocida organisms. Table 3 summarizes the daily P. multocida recoveries from swabs collected from sentinel birds after mixing with the seeder birds. Within 24 hours 4/5 chickens and 2/5 ducks had P. multocida organisms recovered from their swabs. By the 3 rd day, all the five sentinel chickens examined and 3/5 sentinel ducks examined had P. multocida organisms. All the five sentinel ducks were infected by the 6 th day after mixing the sentinel and seeder. The number of infected sentinel chicken declined up to day 14. The chickens appeared to clear the P. multocida organisms from their oropharynx and cloacae faster than the ducks. expressed clinical signs and pathological lesions of fowl cholera at one or more point during the study. The gross lesions observed were fibrino-purulent necrotic lesions on lungs and airsacs from day-three after mixing. Thereafter, the lungs and airsacs had fibrotic spots and remnants of insissipated fibrin on their abdominal airsacs appearing up to day 7. Duck had more severe lesion than chicken. No bacterial isolates were recovered from internal organs of these birds.

Discussion

This study has confirmed that family chickens and duck kept under scavenging conditions, which is very common production system in developing countries, may be healthy carriers of P. multocida. The isolates obtained were characterized as P. multocida subsp. multocida, P. multocida subsp. gallicida and P. multocida subsp. septica. Contrary to this, [START_REF] Muhairwa | Occurrence of[END_REF] reported only P. multocida subsp. multocida from healthy chickens. The organisms were recovered from both the oropharyngeal and cloacal swabs, but more frequently from the oropharyngeal, as reported by others [START_REF] Lee | The demonstration of P. multocida in the alimentary tract of chickens after experimental oral infection[END_REF]. Systematic investigation on the occurrence of P. multocida has previously been carried out on healthy village chickens [START_REF] Curtis | Investigation to determine whether healthy chicken and turkeys are oral carriers of Pasteurella multocida[END_REF][START_REF] Muhairwa | Occurrence of[END_REF] but not on traded (slaughter and live market) family chickens and ducks. In this study, the isolation rate was higher from both family chickens and ducks than previously reported [START_REF] Muhairwa | Occurrence of[END_REF], especially, the isolation rate was high among birds The degree of P. multocida susceptibility is variable among different types of birds and different age groups within a type (Heddleston and Wato 1965;Rhoades and Rimler, 1989). Challenge of chicken using 16 and 45 week-old New Hampshire chickens and 2 different P. multocida serotypes showed that mature chickens were more susceptible than the young chickens [START_REF] Heddleston | Studies on pasteurellosis. V. Two immunogenic types of Pasteurella multocida associated with fowl cholera[END_REF]. Similarly, an unintentional challenge of large numbers of birds with a highly virulent strain of P. multocida via a contaminated vaccine demonstrated no mortalities in chicken of 9-16 weeks of age while older birds died due to fowl cholera [START_REF] Hungerford | A clinical note on avian cholera. The effect of age on the susceptibility of fowls[END_REF]). Taken together this has lead to the general acceptance that chicks and young growers are less susceptible to fowl cholera than older birds. The birds used in the present study are of not well defined types and cannot be referred to any of the known breeds. [START_REF] Msoffe | Genetic structure among the local chicken ecotypes of Tanzania based on microsatellite DNA typing[END_REF] have characterized indigenous chickens from Tanzania by microsatellite DNA typing. They were divided into distinct genotypes, which correlated with ecotype. We assume the types used in the current study are of a similar diverse population structure, and hence genetic differences may have existed between the different groups. No genotyping was performed on birds used in the present study. Birds were selected to represent the diversity in the study area and although grouping was done in a randomized way, this did not ensure an equal distribution of ecotypes in all experiments. This must be taken into account when evaluating the results of the study. The attack criterion used in the studies mentioned above was mortality, as the P. multocida strains were lethal to the chicken. No studies have been done using a less virulent strain, expressing clinical signs but no mortality, and no observation have been done on the undefined breeds of birds kept in less intensive production systems in developing countries. In the present study, age susceptibility in chicken with respect to P. multocida infection was based on the number and severity of clinical signs. Twelve week-old family chickens had more clinical signs that were more severe than those of the 8, 16 and 4 week-old chickens. The study confirms that different age groups have variable susceptibility to P. multocida as previously reported [START_REF] Heddleston | Studies on pasteurellosis. V. Two immunogenic types of Pasteurella multocida associated with fowl cholera[END_REF][START_REF] Heddleston | Fowl cholera: Comparison of serologic and immunologic responses of chickens and turkeys[END_REF]Rhoades and Rimler, 1989), however, unlike those studies, age susceptible peaked in birds at 12 week. This does not rule out that even older birds, such as 35 week old ones, may be more susceptible than the age groups tested in the present study, however, it seems unlikely since susceptibility declined from 12 to 16 weeks. Further studies are needed to elucidate this. Clinically it was possible to pick manifestation of fowl cholera in family chicken mainly between day one to day five post infection with signs that were similar to those reported by others for intermediate to chronic fowl cholera [START_REF] Gooderham | Avian Pasteurellosis and Pasteurella like organisms[END_REF][START_REF] Christensen | Fowl cholera[END_REF][START_REF] Glisson | Fowl cholera[END_REF].

The difference between this study and previous reports could be due to both the strain of P. multocida used and the breed of chickens. Mortality is not a typical outcome of fowl cholera in family chicken, and we purposely looked for a strain that produced clinical and pathological signs of chronic fowl cholera. The type strain P. multocida NCTC 10322 T was originally isolated from a pig, but, as documented in the present The findings of age susceptibility in ducks agree with the results of previous studies [START_REF] Hunter | Pathology of experimental avian cholera in Mallard ducks[END_REF], who demonstrated that birds younger than 11 weeks were more prone to infection. We demonstrated that 8 week-old village ducks had more severe clinical signs, followed by 4 week-old ducks in susceptibility to P. multocida. This means that age susceptibility most likely peaks somewhere between 4 and 11 weeks based on all available information. As in chicken this may depend on both breed and strain used for challenge, and further studies are needed to give the full picture. Clinically fowl cholera could be picked between day one to five in ducks with signs similar to those mentioned for chicken above.

One of the effects of fowl cholera is decrease in the feed efficiency as previously reported among affected turkeys [START_REF] Morris | Characteristics of fowl cholera out breaks in turkeys in Georgia in 1986[END_REF] and ducks [START_REF] Faddoul | Pasteurellosis in wild birds in Massachusetts[END_REF]. Also older chickens sacrificed at 19 days after exposure to P. multocida were found to be emaciated [START_REF] Heddleston | Fowl cholera: Comparison of serologic and immunologic responses of chickens and turkeys[END_REF]. In the current study, all infected chickens and duck groups, except the 4 week old ducks, had significant weight losses compared to the control groups of their corresponding age. The weight loss was more pronounce in the elder than the younger groups, despite less clinical symptoms, possibly reflecting a higher growth rate at these ages.

Chicken and ducks infected with the low virulent strain of P. multocida passed the infection on to non-infected sentinel birds. The chickens appeared to clear the P. 19 multocida organisms from their oropharynx and cloacae faster than the ducks, but the reason for this is not obvious. The intra-tracheal inoculation of chickens and ducks with P. multocida, although being artificial compared to natural infections, ensured that seeder birds were infected with the bacteria. Having the ducks and chickens sharing the same feed and water simulated the field set ups under scavenging conditions, and ensured contact between the birds. On mixing, chickens were found to peck on the bills of ducks and other chickens in an attempt to remove feed stuck around mouth and head areas. This behavior may tend to facilitate fast transmission of bacteria and other infections located in the upper respiratory tract, especially those that could be emitted through eye, nasal and mouth discharges, like P. multocida. The clinical signs and gross lesions observed in this study are similar to those reported by others [START_REF] Christensen | Fowl cholera[END_REF][START_REF] Glisson | Fowl cholera[END_REF]. This, together with the frequency of isolation of P. multocida from ducks and chickens in contact with the infected chickens or ducks, confirms that this bacterium was successfully transmitted from chickens to ducks. Similar scenario may be happening in market places and at farm level during scavenging, and disease control strategies should target both species of birds in field settings to be effective. The crosstransmission may play a role in the maintenance and propagation of P. multocida organisms at village level.

Despite the diversity and magnitude of clinical signs observed in both chicken and ducks in the different studies, very few pathological lesions were observed at post mortem, after the study period, except remnants of fibrin, and fibrous strands in form of fibrotic scars on the lungs and airsacs indicating chronic process. This indicates that the birds controls the low virulent strain of P. multocida early in the infection process. Bacterial isolates were recovered only in a few animals from their oropharynx shortly after infection, however, one must bare in mind, that especially in the age susceptibility study, bacterial isolation was only attempted to prove that challenge had worked and then from internal organs after 14 days. Isolation of P. multocida is difficult. The rate of isolation can be improved by passing of extracts of clinical samples through mice (Muhaiewa et al., 2001). This also proved the case in our study of carrier animals.

However, mice challenge was not attempted in the study of age susceptibility and cross transmission, and we may have overlooked bacteria in low concentration in the internal organs.

In conclusion, this study has documented the occurrence of P. multocida among healthy-appearing family poultry in tropical setting, and demonstrated that age susceptibility is highest in 12 week old family chicken and 8 week old family ducks when challenges with a low virulent strain of P. multocida. It has further demonstrated that cross transmission of fowl cholera may happen between family ducks and chickens and vice versa.

  in 12 week old family chicken and 8 week old family ducks when challenged with a low virulent strain of P. multocida. It has further demonstrated that cross transmission of fowl cholera may happen between family ducks and chickens and vice versa.

Formatted

  multocida occurs from infected ducks to non-infected chickens and vice versa through contact. Challenge was done using the type strain of P. multocida (NCTC 10322 T -Carter serogroup A). While originally isolated from pigs, this strain causes clinical and pathological signs of chronic fowl cholera.

Formatted

  of birds from farms and markets to determine carrier status. Eighty eight family chickens were sampled from 8 farms in three districts of Kenya. The flock sizes ranged from 20 to 74 birds per farm. Forty seven family ducks were sampled from six farms from two districts in Kenya with a population that ranged from 18 to 85 birds per farm. Similarly, a total of 74 chickens and seven ducks were sampled from slaughterhouses and open air markets in Nairobi. The markets only receive birds from non-industrial production and upon sampling birds were ensured to originate from scavenging conditions. The birds originated from seven districts. Pre-wetted oropharyngeal and cloacal swabs were taken and transported directly to the laboratory in transport containers kept at 4º C. Bacterial strain for experimental infections, preparation of inoculums and challenge. The type strain of P. multocida (NCTC 10322 T -Carter serogroup A, originally isolated from a pig) maintained on Dorset egg agar was spread onto blood agar (BA, Oxoid Ltd., CM55, Basingstoke, Hampshire, England) with 5 % citrated calf blood, incubated aerobically at 37 o C, for 24 hours to check for purity prior to preparation of the Formatted: Normal, Line spacing: single

  each, allowing the experiment to be replicated. No statistical difference

Formatted

  5 o C was taken as normal, 41.6 o C as mild, around 42.0 o C as moderate, and that of over 42.0 o C was taken as severe fever.Bacterial detection and identification. Swabs were individually placed in two ml of sterile Phosphate Buffered Saline (PBS) and transported in a cool box to the laboratory, where it was thoroughly vortexed and 0.1 ml was streaked on blood agar (BA) (Oxoid Ltd., CM55, Basingstoke, Hampshire, England) and incubated aerobically at 37 o C for 24 hours for initial culture. Point-one ml of PBS was further inoculated into Pasteurella-free Balb/C mice by intra-peritoneal route, as described byMuhairwa et al.

Formatted

  healthy looking family chickens and ducks. Oropharyngeal and cloacal swab samples from 162 family chickens and 54 ducks were screened for P. multocida on both culture and mouse passage. They yielded 24 positive isolations.

Formatted

Formatted:

  Indent: First line: 36 pt Deleted: ¶ Deleted: The 8 week old ducks expressed significantly more clinical signs (188) than the other age groups followed by the 4 week old ducks (117).

Formatted

  and slaughterhouses. This is an indication that transport and handling in relation to marketing or slaughter may cause a P. multocida to increase in numbers in low level carrier birds. When evaluating this results, however, one should bare in mind that only seven ducks were tested a market/slaughter.

  produces clinical signs in chicken. It remains to be shown whether a more virulent strain may have different age susceptibility in the family chickens and similarly whether a less virulent strain results in different age susceptibility in commercial chicken than a highly virulent strain.

  constructive discussion of the work described in this paper. The study was financed by Danish International Development Agency (DANIDA) through the ENRECA program "Improving the Health and productivity of the Rural Poultry in Africa (IHEPRUCA)"(DANIDA grant 104.Dan.8.f and University of Nairobi grant no. 500-661-092).

  Pasteurella multocida and related species in village free ranging chickens and their animal contacts in Tanzania. Veterinary Microbiology, 78, 139-153. Mutters, R., Ihm, P., Pohl, S., Frederiksen W. & Mannheim, W. (1985). Reclassification of the genus Pasteurella Trevisan 1887 on the basis of deoxyribonucleic acid homology, with

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  new species Pasteurella dagmatis, Pasteurella canis, Pasteurella stomatis, Pasteuella anatis, and Pasteurella langaa. International Journal of Systematic Bacteriology, 35, 309-322. Petersen, K.D., Christensen, J.P., Permin, A. & Bisgaard, M. (2001). Avirulence of P. multocida subsp. multocida isolated from outbreaks of fowl cholera in wild birds for domestic poultry and game birds. Avian Pathology, 30, 27-31. Pehlivanoglu, F., Morishita, T.Y., Aye, P.P, Porter Jr., R.E.. Angrick, E.J., Harr, B.S. &.Nersessian, B. (1999). The effect of route of inoculation on the virulence of raptorial Pasteurella multocida isolates in Pekin ducks (Anas platyrhyrchos). Avian Diseases, 43, 116-121. Rhoades, K.R. & Rimler,R.B. (1989). Fowl cholera. p. 95-113. In, C. Adlam & J.M. Rutter (1989), Pasteurella and pasteurellosis (pp. 95-113), London: Academic press. Salami, J.O., Egbulem, B.N., Kwaga, J.K.P., Yusufu, H.I. & Abdu, P.A.(1989). Diseases diagnosed in poultry inKaduna, Nigeria (1981 to 1985). Bulletin of Animal Health and Production inAfrica, 37,[109][110][111][112][113][114]

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  The number of clinical signs observed in each age group is listed in Table 2. The 12 week old age group expressed significantly more clinical signs (372) than the other age groups. The age susceptibility of the other age groups declined in the order 8 weeks (272 signs; p<0.05 compared to 4 and 16 weeks) compared to the 4 and 16 week old almost similar number of clinical signs (173 and 187). Moreover, severe clinical signs were more often observed in the 12 week-old birds (Table 2).

	12
		Deleted: ¶
	chickens that expressed	¶
	F o
	r
	P
	e e r
	R e v i e w	Deleted: ¶ ¶ Deleted: ¶
	O n
	l y

Table 1 :

 1 Comparison of clinical signs in chickens and ducks of different age groups infected with P. multocida. Number of Clinical Observations per age group # The frequency of clinical signs varied significantly (p<0.05) between age groups of chicken, except for mouth discharges, diarrhea and cyanosis. The frequency of clinical signs varied significantly (p<0.05) between age groups of ducks except for diarrhea and fever.

	Page 25 of 27
	Age group of chicken (weeks) 8 12 16 63 79 42 3 13 21 61 74 27 27 36 20 6 14 4 6 34 1 17 16 13 3 6 2 5 14 9 0 3 1 24 8 2 53 60 38 4 15 7 0 0 0 0 0 0 # Formatted: Font: Bold Age group of ducks (weeks) Clinical Signs 4 4 8 12 16 Depression 55 15 13 6 4 Nervous tics 0 0 0 0 0 Ruffled feathers 36 15 13 6 4 Sneezing 13 3 25 16 14 Ataxia 1 0 8 0 0 Nasal discharges 3 16 58 32 42 Dyspnoea 4 22 26 1 3 Mouth discharges 0 3 10 2 1 Diarrhoea 10 2 1 0 0 Cyanosis 0 Rales 22 15 2 0 0 Fever 29 26 23 15 14 Head scratching 0 0 0 1 0 Coughing 0 0 7 1 1 Eye discharges 0 0 2 0 0 Formatted: Font: Italic Formatted Table

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Table 2 :

 2 Number of clinical signs observed during 14 days in different age groups of chicken and ducks infected with P. multocida. Groups of chicken that show significantly different number of clinical signs are indicated with different letters (a,b,c), while statistically significant groups of ducks are indicated by letters d,e,f. # Severity of signs are listed in the order: mild/moderate/severe signs (see material and

									Formatted: Font: Bold
									Formatted: Font: Italic
	Age group	Number of signs at days post	Total	Severity	Average weight	Formatted Table
			infection		number	of signs #	loss compared to
						of		control group of
						Signs †		same age (g)
		1-3	4-6	7-9	10-14
	Chicks -4 41	42	44	46	173 a	82/63/28	14.1 *
	Ducks -4 51	39	12	16	117 e	72/22/23	7.5
	Chicks -8 51	69	63	89	272 b	143/70/59 20.0 *
	Ducks -8 68	53	37	30	188 f	93/46/49	12.3 *
	Chicks -	97	93	81	101	372 c	217/90/65 25.7 *
	12
	Ducks-12 25	28	14	13	80 d	50/12/18	28.3 *
	Chicks -16 49	45	37	56	187 a	116/47/24 166.9 *
	Ducks -	27	20	17	19	83 d	41/20/22	28.8 *
	16
	Control all	0	0	0	0	0	0/0/0	NA
	ages
	† methods)
	* p< 0.05 compared to control groups
	NA: non applicable

Table 3 :

 3 Daily isolation of P. multocida organisms from sentinel birds mixed

				Formatted: Font: Bold
				Formatted: Font: Italic
	with infected birds			Formatted: Font: Italic
	Days post	Number of P. multocida positive sentinel birds (n=5)	Formatted: Font: Not Bold
	contact infection	Chicken to ducks	Ducks to chickens
	1	2	4
	2	3	4
	3	3	5
	4	3	3
	5	3	3
	6	5	4
	7	4	4
	8	3	3
	9	3	2
	10	3	2
	11	4	2
	12	3	2
	13	2	0
	14	3	1
	N / 70 possible	44	39
				Deleted: ¶
				¶
				¶
				¶
				¶
				¶
				¶
				¶

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

Acknowledgements

The Chairman of Department of Veterinary Pathology, Microbiology and Parasitology is thanked for allowing us to use the laboratory facilities and the staff, especially Zackary M. Wamuri, Justus N. Matata, Henry M. Kinyua, Mary Wanjiru , John Mungai, Patrick Wahome and Samuel Indeche are thanked for technical help. The late Professor Mugera Formatted: Indent: First line: 36 pt Deleted: ¶ Deleted: ACKNOWLEDGEMENTS