Fusaro

Le Menach

Garba Maina A 7 , N'

Goran Koffi J-N

Giovanni Cattoli

EMERGENCE OF A NEW GENETIC LINEAGE OF NEWCASTLE DISEASE VIRUS IN WEST AND CENTRAL AFRICA -IMPLICATIONS FOR DIAGNOSIS AND CONTROL

Keywords: Newcastle Disease, poultry, Africa, genetic characterization

Newcastle disease (ND) is an OIE listed disease caused by virulent avian paramyxovirus type 1 (APMV-1) strains, which affect many species of birds and may cause severe economic losses in the poultry sector. The disease has been officially and unofficially reported in many African countries and still remains the main poultry disease in commercial and rural chickens of Africa.

Unfortunately, virological and epidemiological information concerning ND strains circulating in the Western and Central regions of Africa is extremely scarce. In the present study, sequence analysis, pathotyping and detailed genetic characterization of virulent ND strains detected in rural poultry in West and Central Africa revealed the circulation of a new genetic lineage, distinguishable from the lineages described in the Eastern and Southern parts of the continent. Several mismatches were observed in the segment of the matrix gene targeted by the primers and probe designed for the molecular detection of APMV-1, which were responsible for the false negative results in the diagnostic test conducted. Furthermore, deduced amino acid sequences of the two major antigens eliciting a protective immune response (F and HN glycoprotein) revealed protein similarities < 90% if compared to some common vaccine strains. Distinct mutations located in the neutralizing epitopes were revealed, indicating the need for detailed assessment of the efficacy of the current vaccines and vaccination practices in Africa. The present investigation provides important information on the epidemiology, diagnosis and control of NDV in Africa and highlights the importance of supporting surveillance in developing countries for transboundary animal diseases.

subfamily Paramyxovirinae, genus Avulavirus [START_REF] Lamb | The negative sense single stranded RNA viruses[END_REF]. This genus contains the 9 serogroups of avian paramyxoviruses (APMV-1 to -9) described so far. According to their virulence in poultry, APMV-1 isolates can be classified as lentogenic, mesogenic or velogenic (i.e. of low, moderate or high virulence for poultry, respectively). In the present paper the term ND virus (NDV) refers to the virulent APMV-1 strains that cause ND.

The virulence of an APMV-1 isolate can be determined in the laboratory by an in vivo test performed by the experimental inoculation of SPF day-old chicks with the isolate (intracerebral pathogenicity index, ICPI) or it can be predicted molecularly by the analysis of the amino acid sequence at the cleavage site of the fusion glycoprotein (F protein) [START_REF] Alexander | Ecology and epidemiology of Newcastle disease[END_REF]. Both these methods are officially recognized for APMV-1 pathotyping by the World Organisation for Animal Health -OIE - (OIE, 2008). The ICPI is considered the most sensitive "in vivo" test [START_REF] Terregino | Conventional diagnosis of Newcastle disease virus infection[END_REF] and an index value of 0.7 or greater classifies the isolate as virulent (i.e. velogenic or mesogenic) thus, confirming a diagnosis of ND in the infected hosts. Alternatively, the virulence of ND strains can be assessed by the determination of the presence or not of multiple basic amino acids (e.g. lysine -K; or arginine -R) located at the C-terminus of the F1 protein and phenylalanine (F) at the N-terminus of the F2 protein, which correspond to the cleavage site of the precursor F0 glycoprotein (positions 112 to 117) (OIE, 2008).

In addition to the gene encoding the structural glycoprotein F, the APMV-1 genome of about 15 kb is composed of 5 other genes encoding 5 structural proteins (nucleoprotein, NP; matrix protein, M; phosphoprotein, P; RNA polymerase, L and haemagglutin-neuraminidase, HN). Two additional proteins are encoded by the RNA editing of the P protein, namely proteins V and W. The cleavability of protein F is certainly the main determinant for viral virulence, but other proteins such A c c e p t e d M a n u s c r i p t 5 as HN and V are also believed to influence pathogenicity (Huang et al., 2004;[START_REF] De Leeuw | Virulence of Newcastle disease virus is determined by the cleavage site of the fusion protein and by both the[END_REF][START_REF] Mebatsion | A recombinant newcastle disease virus with low-level V protein expression is immunogenic and lacks pathogenicity for chicken embryos[END_REF].

As for many other RNA viruses, genetic variability of APMV-1 is significant. Recent analyses have revealed the existence of two main distinct genetic clades, termed class I and II [START_REF] Czeglédi | Third genome size category of avian paramyxovirus serotype 1 (Newcastle disease virus) and evolutionary implications[END_REF]. Class I includes almost exclusively low virulence strains recovered from live bird markets in US or wild waterfowl worldwide. Class II is comprised of the vast majority of viruses of high and low virulence isolated from poultry and wild birds [START_REF] Czeglédi | Third genome size category of avian paramyxovirus serotype 1 (Newcastle disease virus) and evolutionary implications[END_REF][START_REF] Kim | Phylogenetic diversity among low-virulence newcastle disease viruses from waterfowl and shorebirds and comparison of genotype distributions to those of poultry-origin isolates[END_REF].

Restriction enzyme and partial sequence analysis of the gene encoding for the F protein (F gene) classified class II APMV-1 isolates in eight main distinct groups (I to VIII) with several sublineages within them (Herczeg et al., 1999). In another study, based on the partial F gene sequence of a large number of APMV-1 collected worldwide [START_REF] Aldous | A molecular epidemiological study of avian paramyxovirus type 1 (Newcastle disease virus) isolates by phylogenetic analysis of a partial nucleotide sequence of the fusion protein gene[END_REF], the viruses were grouped in 6 distinct genetic lineages (1 to 6) with several sublineages within them. The majority of the NDV isolates responsible of the recent outbreaks in Europe, Asia and Southern Africa are placed in lineages 3, 4 and 5 [START_REF] Alexander | Antigenic and genetic characterisation of Newcastle disease viruses isolated from outbreaks in domestic fowl and turkeys in Great Britain during 1997[END_REF]Herczeg et al., 1999;Herczeg et al., 2001;[START_REF] Cattoli | Characterization of Newcastle disease viruses isolated in Italy in 2000[END_REF][START_REF] Aldous | A molecular epidemiological study of avian paramyxovirus type 1 (Newcastle disease virus) isolates by phylogenetic analysis of a partial nucleotide sequence of the fusion protein gene[END_REF][START_REF] Abolnik | A phylogenetic study of South African Newcastle disease virus strains isolated between 1990 and 2002 suggests epidemiological origins in the Far East[END_REF]. As an OIE listed disease the notification of ND outbreaks by the OIE member countries is mandatory. APMV-1 is distributed worldwide and ND outbreaks have been notified regularly in all continents since the first description of the disease in 1926 [START_REF] Alexander | Ecology and epidemiology of Newcastle disease[END_REF]. In Africa, outbreaks in poultry caused by NDV have been described in the Eastern and Southern part of the continent (Herczeg et al., 1999;[START_REF] Otim | Molecular characterization and phylogenetic study of Newcastle disease virus isolates from recent outbreaks in eastern Uganda[END_REF]Abolnick et al, 2004). The disease has been officially and unofficially reported in other parts of Africa and, according to the United Nations-Food and Agricultural Organization (UN-FAO), ND still remains the main poultry disease in commercial and rural chickens of West Africa [START_REF] Bebay | Première évaluation de la structure et de l'importance du secteur avicole commercial et familial en Afrique de l'Ouest[END_REF].

Unfortunately, virological and epidemiological information concerning ND strains circulating in the Western and Central regions of Africa is extremely scarce and while historically ND has been reported to be widespread [START_REF] Lancaster | Newcastle disease: Virus and Spread[END_REF] in recent years there has been only a single report [START_REF] Snoeck | Newcastle disease virus in West Africa: new virulent strains identified in non-commercial farms[END_REF]. In that investigation, NDVs were detected by PCR in non-A c c e p t e d M a n u s c r i p t 6 commercial farms in Niger, Nigeria and Burkina Faso but viral isolates were not obtained. For this reason, characterization of the detected ND was restricted to the sequence analysis of partial F gene sequences. The authors suggested that NDVs responsible for the outbreaks in the three countries were phylogenetically distinguishable from NDVs circulating in Eastern and Southern Africa and were tentatively grouped in new sublineages within lineage 5, namely 5e, 5f and 5h [START_REF] Snoeck | Newcastle disease virus in West Africa: new virulent strains identified in non-commercial farms[END_REF].

In the present investigation we perform a full molecular characterization of ND viruses causing ND outbreaks in poultry in 7 Western and Central African countries. We also discuss the diagnostic implications of the genetic variability detected.

Materials and methods

Samples included in this study

Virological investigations using real time RT-PCR (rRT-PCR) for APMV-1 and virus isolation (VI) attempts were performed on 50 samples collected from rural chickens in Nigeria, Niger, Ivory Coast, Burkina Faso, Mauritania, Cameroon andBurundi between 2006 and2008. Samples consisted of internal organs (such as trachea, lungs and intestines) and swabs (cloacal and tracheal) collected during surveillance programs (3,614 samples) or in cases of suspected disease (387 samples) and submitted to the OIE/FAO Reference Laboratory for Newcastle disease and Avian Influenza at the Istituto Zooprofilattico Sperimentale delle Venezie (IZSVe) in Italy within the framework of diagnostic activities and international collaboration projects to support developing countries.

Laboratory testing

For molecular testing, viral RNA was extracted from the samples using the Nucleospin RNA II Kit (Machery-Nagel, Duren, Germany) and the detection of APMV-1 RNA was performed using a rRT-PCR protocol targeting the M gene [START_REF] Wise | Development of a real-time reverse-transcription PCR for detection of Newcastle disease virus RNA in clinical samples[END_REF]. For virus isolation (VI), samples were prepared and inoculated in SPF embryonated chicken eggs according to the international regulations (OIE, 2008). The allantoic fluid of eggs showing embryo mortality was collected and tested for A c c e p t e d M a n u s c r i p t 7 haemagglutinating activity. Haemagglutinating agents were identified by means of haemagglutination inhibition (HI) tests using standard protocols (OIE, 2008). The virulence of the APMV-1 isolates for chickens was assessed by intracerebral pathogenicity index (ICPI) tests in day-old SPF chickens and by genetic sequencing of the fusion protein cleavage site [START_REF] Terregino | Conventional diagnosis of Newcastle disease virus infection[END_REF].

Nucleotide sequencing and genetic pathotyping

Viral RNA was extracted from the allantoic fluid of APMV-1 positive SPF eggs as described above.

For genetic pathotyping a 266-bp hypervariable region of the F gene encompassing the cleavage site (positions 4652 to 4917, with reference to NDV strain La Sota, GenBank accession number AF077761) was RT-PCR targeted [START_REF] Cattoli | Molecular diagnosis of Newcastle disease virus[END_REF]. Briefly, the APMV-1 F gene segment was amplified with primers NOH-For (5' TACACCTCATCCCAGACAGG 3') and NOH-Rev (5' AGTCGGAGGATGTTGGCAGC 3') using the Qiagen one-step RT-PCR kit (Qiagen, Hilden, Germany). Amplicons were sequenced using the Big Dye Terminator v3.1 cycle sequencing kit (Applied Biosystem, Foster City, CA -USA). The products were cleaned using PERFORMA DTR Ultra 96-Well kit (Edge BioSystems, Gaithersburg, MD -USA) and sequenced in a 16-capillary ABI PRISM 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA -USA).

Phylogenetic analysis

To determine the phylogenetic relationships between West and Central Africa viruses and APMV-1 viruses previously isolated in Africa and other parts of the world, the sequences of the 266-bp hypervariable region of the F gene were compared to the corresponding region of 332 representative viruses available in GenBank (http://www.ncbi.nlm.nih.gov/), for which the genotype was known.

Previous studies have demonstrated that sequences of 250 base pairs give meaningful phylogenetic analyses, comparable with those obtained with much longer sequences [START_REF] Seal | Characterization of Newcastle disease virus isolates by reverse transcription PCR coupled to direct nucleotide sequencing and development of sequence database for pathotype prediction and molecular epidemiological analysis[END_REF][START_REF] Lomniczi | Newcastle disease outbreaks in recent years in western Europe were caused by an old (VI) and a novel genotype (VII)[END_REF]Snoeck et al., 2008).

The lineage-based nomenclature illustrated in a previous study [START_REF] Aldous | A molecular epidemiological study of avian paramyxovirus type 1 (Newcastle disease virus) isolates by phylogenetic analysis of a partial nucleotide sequence of the fusion protein gene[END_REF]) is adopted throughout this paper.

A c c e p t e d M a n u s c r i p t 8

To confirm the robustness of the genetic groupings obtained by the phylogenetic analysis of the partial F gene sequences and to detect mutations potentially associated with antigenic and virulence changes, the complete sequences of the fusion (F), haemagglutinin-neuraminidase (HN) and matrix (M) genes of selected African APMV-1 isolates were obtained. For this purpose, RNAs extracted from infective allantoic fluid were reverse transcribed with the SuperScript III Reverse Transcriptase kit (Invitrogen, Carlsbad, CA -USA) and PCR amplification was performed using gene specific primers (Table S1). Using the full length genome positions from the La Sota vaccine strain complete genome (accession number AF077761), the nucleotide regions sequenced were as follows: the complete coding region for the genes F (positions 4544 to 6205; n = 16 isolates), HN (positions 6412 to 8127; n = 17 isolates), M (positions 3290 to 4384; n = 18 isolates). Sequence alignments were constructed and used to develop phylogenetic trees using Bayesian methods available in the computer program MrBayes version 3.1.2 [START_REF] Ronquist | MrBayes 3: Bayesian phylogenetic inference under mixed models[END_REF].

Specifically, we executed a Markov chain Monte Carlo search for one million generations using two runs and four chains (temperature=0.05) and represented the results of the search as a 50% majority rule consensus tree. In each case, the best-fit model of nucleotide substitution was identified using the Akaike information criterion [START_REF] Posada | Model selection and model averaging in phylogenetics: advantages of akaike information criterion and bayesian approaches over likelihood ratio tests[END_REF] as implemented in Modeltest version 3.7 [START_REF] Posada | Selecting the best-fit model of nucleotide substitution[END_REF] (parameter values available from authors on request).

Nucleotide diversity was calculated using the Maximum Composite Likelihood method implemented in the MEGA4 software package [START_REF] Tamura | MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0[END_REF].

Nucleotide sequence accession numbers

GeneBank accession numbers of the sequences analysed in this study are FJ772445 to FJ772495

Results

Detection of APMV-1 isolates in the samples

The rRT-PCR protocol detected the presence of APMV-1 RNA in 43 of 50 of the clinical samples included in the investigation (Table 1). Forty-one of the samples positive by rRT-PCR were also

Phylogenetic analysis of partial F gene sequences

For genetic sequencing, 28 of the 48 APMV-1 isolates were selected, based on the year of isolation, country of origin and outbreak location. The 266 bp region of the fusion gene of these isolates was sequenced and compared phylogenetically to the corresponding region of 332 representative viruses belonging to the six lineages characterized by [START_REF] Aldous | A molecular epidemiological study of avian paramyxovirus type 1 (Newcastle disease virus) isolates by phylogenetic analysis of a partial nucleotide sequence of the fusion protein gene[END_REF]. The resulting phylogenetic tree is presented in figure 1.

From the topology of the Bayesian tree, presented in figure 1, it can be seen that the two NDV isolates from Burundi were placed in sublineage 5b, a heterogeneous sublineage which is composed of isolates mainly originating from Europe, Africa, Asia and America [START_REF] Aldous | A molecular epidemiological study of avian paramyxovirus type 1 (Newcastle disease virus) isolates by phylogenetic analysis of a partial nucleotide sequence of the fusion protein gene[END_REF]. The sequences of these viruses revealed the highest similarity at nucleotide (nt) level (97.6%) with an isolate from South Africa ZA/71/BA/94 (accession number AF532751). In contrast, the other viruses analysed clustered together in a distinct group showing a mean nt similarity of 88.7% with the sequences of lineage 5 viruses and, more specifically, 89.4% with sublineage 5b viruses. Since the mean nt diversity observed between this new group and lineage 5 (11.3%) falls within the range of nt diversity (10.3% -43.2%) calculated for the existing 6 lineages, as shown in table 2, we considered this group represented a new lineage, provisionally named lineage 7. At present 33 isolates make up lineage 7, all originating from West Africa. Twenty-six isolates were sequenced in A c c e p t e d M a n u s c r i p t 10 the present study and the sequences of seven other viruses were obtained in a previous study [START_REF] Snoeck | Newcastle disease virus in West Africa: new virulent strains identified in non-commercial farms[END_REF]. Lineage 7 can be further subdivided into four, statistically supported, sublineages (7a to 7d) with posterior probabilities >91% (figure 2). Sublineage 7a contained 8 isolates, of which 7 were from samples obtained in 2006 in Mauritania and one from a 2008 Ivory Coast sample. The maximum nt divergence within this group was 6.4%. Sublineage 7b was the most heterogeneous sublineage in terms of country of origin of the viruses included in this study. This lineage consisted of 14 viruses originating from four different countries, namely Niger(n=4), Nigeria (n=4), Cameroon (n=2) and Burkina Faso (n=4) obtained from samples collected in 2006 and 2008. The maximum nt divergence within this group was 4.7%. Sublineage 7c consisted exclusively of three isolates from Burkina Faso obtained in 2006, the partial sequences of which were recently published in GenBank [START_REF] Snoeck | Newcastle disease virus in West Africa: new virulent strains identified in non-commercial farms[END_REF]. The maximum divergence within this group was 1.5%.

The isolates in sublineage 7d were grouped in two further distinguishable clusters: the first (group I) contained five isolates exclusively from Niger, collected in 2006 and 2007 and the second cluster (group II) was composed of three viruses obtained in 2006, two from Niger and one from Nigeria.

The maximum divergence within this group was 7.7%.

Pathotyping of APMV-1 isolates

For the APMV-1 isolates selected, the virulence was determined by molecular sequencing and ICPI tests . The deduced amino acids at the F0 protein protease cleavage site revealed that all 28 of the selected isolates had motifs that are typical of velogenic strains (OIE, 2008). Three possessed the motif 112 RRRKR/F 117 and the other 25 had the motif 112 RRQKR/F 117 . In order to reduce the use of experimental animals, ICPI tests were limited to representative isolates for each country that possessed the different deduced amino acid motifs at the cleavage site of the F protein (Table 3). As shown in table 3, the ICPI values were similar for the two cleavage site motifs and confirmed the virulence of these viruses. The lineage 5b isolate from Burundi produced the highest ICPI value of 1.97. Viruses placed in the new lineage 7 gave ICPI values ranging from 1.7 to 1.87.

A c c e p t e d M a n u s c r i p t 11

Phylogenetic analyses of complete F, HN and M gene sequences

For lineage 7, the topology of the phylogenetic tree created using 266-bp fusion gene fragments was confirmed by analyses of the complete coding regions of the F, M and HN genes of 16, 18 and 17 representative viruses, respectively (figure 3). By this analysis it was possible to confirm the results obtained for three of the four sublineages, 7a,7b and 7d. For sublineage 7c containing viruses that were not available for sequencing in the present study, only partial F gene sequences had been deposited in GenBank [START_REF] Snoeck | Newcastle disease virus in West Africa: new virulent strains identified in non-commercial farms[END_REF] and therefore sublineage confirmation was not possible.

Sequences of F, HN and M genes show comparable ratios of non-synonymous to synonymous substitutions per site (K a /K s =0.086, 0.109 and 0.053 respectively) indicating the absence of positive selection.

Complete nucleotide sequences of the HN genes of viruses belonging to lineage 7 showed the highest nucleotide diversity (12%) compared to the complete sequences of F and M genes of the same viruses for which 11.5% and 10% diversity was recorded respectively. However, in the M gene several mismatches were observed in the region targeted by the primers and probe designed for the rRT-PCR detection of APMV-1 (Wise et al; 2004) (Figure 4).

Additional rRT-PCR testing (data not shown) allowed us to establish that the presence of at least 3 mismatches in the probe was presumably responsible for the false negative results in the diagnostic molecular test.

Analysis of amino acid sequences

At the F protein cleavage site, sublineages 7a, 7b and 7d-group I possessed four basic amino acids (RRQKR/F), as do most of the viruses belonging to lineage 5. Sublineages 7c and 7d-group II showed five basic amino acids (RRRKR/F). The amino acid arginine at position 114 was codified by two different codons (CGA for sublineage 7c and CGG for sublineage 7d) suggesting that this point mutation occurred independently in the two groups of viruses.

Potential N-glycosylation sites at positions 85,191,366,447,471,541, characteristic of APMV-1, were conserved in the F glycoprotein of all the NDV isolates belonging to lineage 7. The HN A c c e p t e d M a n u s c r i p t 12 glycoprotein sequence of lineage 7 viruses contained the five potential N-glycosylation sites (residues 119, 341, 433, 481, 508) that are conserved among APMV-1 viruses. One isolate from Nigeria and one from Niger contained an additional site at position 538. However, a previous study has shown that two of these sites (at positions 508 and 538) are not truly glycosylated [START_REF] Mcginnes | The role of individual oligosaccharide chains in the activities of the HN glycoprotein of Newcastle disease virus[END_REF].

Residues within the HN protein, which is essential for receptor binding, neuraminidase (NA) and haemagglutination (HA) activity of APMV-1 viruses, [START_REF] Seal | Nucleotide and predicted amino acid sequence analysis of the fusion protein and hemagglutinin-neuraminidase protein genes among Newcastle disease virus isolates. Phylogenetic relationships among the Paramyxovirinae based on attachment glycoprotein sequences[END_REF] were conserved in all the isolates analysed.

For 13 of the 14 viruses for which the complete HN and F sequences were obtained at least one amino acid substitution was detected within the known virus neutralizing epitopes of the HN and F proteins (table 4). Specifically, mutations E347D, Y350H and D569G were detected in three neutralizing epitopes of the HN protein (Iorio, et al., 1991), while amino acid substitutions K78R, D170S and L343P were detected within three epitopes located in the F protein (Yusoff et al., 1989).

Comparison of the nt and amino acids sequences available for some common vaccine strains, such as La Sota (Genbank accession number AF077761) and V4 (Genbank accession number AY225110), revealed HN protein similarity ranging between 85.5% and 87.7% with the La Sota strain and between 85.1% and 87.4% with the V4 strain . For the F protein, similarity ranged from 86.4% to 88.8% with the La Sota strain and from 86.2% to 88.5% with the V4 strain, while for M protein similarity ranged from 87% to 89.5% with the La Sota strain and from 88.8% to 92.2% with V4 strain.

The HN proteins of each of the analysed viruses had a length of 571 amino acids, which is the length most commonly observed for the virulent NDV isolates and possessed a cysteine at position 123, which is required for the formation of disulphide-linked HN homodimers. A previous study demonstrated that the amino acid cysteine at this position has an influence on both attachment activity and fusion promotion activity and increased the virulence and the pathogenicity of NDV viruses (Romer-Oberdorfer et al., 2006). information is available on the characteristics of the strains circulating in this continent, with the exception of those seen in South Africa (Herczeg et al, 1999;[START_REF] Aldous | A molecular epidemiological study of avian paramyxovirus type 1 (Newcastle disease virus) isolates by phylogenetic analysis of a partial nucleotide sequence of the fusion protein gene[END_REF][START_REF] Abolnik | A phylogenetic study of South African Newcastle disease virus strains isolated between 1990 and 2002 suggests epidemiological origins in the Far East[END_REF] and Uganda [START_REF] Otim | Molecular characterization and phylogenetic study of Newcastle disease virus isolates from recent outbreaks in eastern Uganda[END_REF]. In West and Central Africa, virological data concerning APMV-1 were almost completely absent. Very recently, [START_REF] Snoeck | Newcastle disease virus in West Africa: new virulent strains identified in non-commercial farms[END_REF] detected NDVs belonging to lineage 3 and 4 in Nigeria. But other APMV-1 viruses detected in Nigeria, Burkina Faso and Niger were tentatively placed in lineage 5 by genetic analysis of a partial fragment of the gene F. In this study [START_REF] Snoeck | Newcastle disease virus in West Africa: new virulent strains identified in non-commercial farms[END_REF] suggested these viruses formed novel sublineages, namely 5g, 5f and 5h or even new genetic lineages. However, isolates were not obtained and the conclusions were considered preliminary by the authors since data were obtained on partial sequence (354 nt) of the F gene only.

In our present study the results of the phylogenetic analyses indicate that the majority of the West and Central African strains belong to a novel lineage, here provisionally named lineage 7, which includes the viruses previously assigned to 5g, 5f and 5h sublineages by [START_REF] Snoeck | Newcastle disease virus in West Africa: new virulent strains identified in non-commercial farms[END_REF].

These findings are supported by the analysis of the complete sequences of the gene F, M and HN.

Furthermore, our study indicate that viruses of this newly described lineage are circulating in a vast area of Africa, and are probably endemic in the region between Cameroon in the Central part of Africa and Mauritania in the extreme west. Of the samples from West and Central African countries that were examined in the present study only those from Burundi failed to yield virus of lineage 7.

We detected a high degree of genetic variability within lineage 7, which was similar in magnitude to that described for lineage 5 [START_REF] Aldous | A molecular epidemiological study of avian paramyxovirus type 1 (Newcastle disease virus) isolates by phylogenetic analysis of a partial nucleotide sequence of the fusion protein gene[END_REF].

A c c e p t e d M a n u s c r i p t

14

The present study also demonstrated the important impact genetic variability of viruses may have on the diagnosis of this infection, highlighting the limit of the advanced molecular diagnostic tests on viral disease diagnosis. [START_REF] Kim | Phylogenetic diversity among low-virulence newcastle disease viruses from waterfowl and shorebirds and comparison of genotype distributions to those of poultry-origin isolates[END_REF] recently demonstrated that the broad genetic diversity between class I, which includes the genotype 6 described earlier [START_REF] Aldous | A molecular epidemiological study of avian paramyxovirus type 1 (Newcastle disease virus) isolates by phylogenetic analysis of a partial nucleotide sequence of the fusion protein gene[END_REF], and class II APMV-1 may impair the performances of rapid molecular tests, including those having conserved genes as target for PCR amplification. The results presented in our study on the sequence analysis of the matrix gene demonstrated that genetic variability within class II viruses, may be equally responsible for false negative molecular test results. A broader number of strains are necessary to assess whether this problem is restricted to lineage 7 viruses or extends to other lineages.

In a comprehensive study Czeglédy et al., (2006) concluded that the origin of the most recent virulent genotypes was not clear, but it appears that the diversification of the recent epizootic viruses and lineages, has accelerated in the last few decades and suggested that vaccination could have played a role in the generation and spread of the most recent virulent viruses. It has been recently demonstrated [START_REF] Miller | Antigenic differences among Newcastle disease virus strains of different genotypes used in vaccine formulation affect viral shedding after a virulent challenge[END_REF] that genetic diversity in APMV-1 strains influences the efficacy of vaccination control, not necessarily in terms of clinical protection, but mainly in term of virus shedding and subsequent spread of the infection. [START_REF] Miller | Antigenic differences among Newcastle disease virus strains of different genotypes used in vaccine formulation affect viral shedding after a virulent challenge[END_REF] showed that virus shedding in chickens experimentally challenged with ND strains correlated with HN and F protein similarities between the challenge and vaccine strains. HN and F protein similarities less than or equal to 90%, as those observed in our study, were significantly related to higher virus shedding. In addition to this, the distinct mutations detected in the African strains analysed in the present study, located in the neutralizing epitopes of the two major antigens (F and HN glycoprotein) targeted by a protective immune response [START_REF] Seal | The avian response to Newcastle disease virus[END_REF], present the scientific rationale for deeper investigations on the antigenic properties of these isolates and for detailed assessments of the efficacy of the current vaccines and vaccination practices in Africa.

To conclude, the present investigation provides important information on the epidemiology, diagnosis and control of NDV in Africa and highlights the importance of supporting surveillance in

  Reference Laboratory for avian influenza and Newcastle disease, Istituto Zooprofilattico Sperimentale delle Venezie, Viale dell'Università 10, 35020 Legnaro (PD), Italy. Tel +39 0498084389 Fax +39 0498084360 gcattoli@izsvenezie.it A c c e p t e d M a n u s c r i p t Introduction Newcastle disease (ND) is a viral disease affecting many species of birds and causing severe economic losses in the poultry sector. The aetiological agent is a single stranded, non-segmented, negative-sense RNA virus belonging to the order Mononegavirales, family Paramyxoviridae,

  A c c e p t e d M a n u s c r i p t 9 positive by virus isolation. One sample from Niger and one from Nigeria, both collected in 2008 were positive by rRT-PCR but gave a negative result by virus isolation. However, 7/16 of the samples collected in 2006 (3 in Niger and 4 in Mauritania) were negative by the rRT-PCR protocol used but positive by virus isolation in eggs. The M gene rRT-PCR test was carried out on the positive allantoic fluids obtained in the virus isolation tests from these samples in which APMV-1 viruses had been confirmed by HA and HI tests, but the rRT-PCR results were also negative. When the rRT-PCR-negative/HI-positive allantoic fluids were tested by the RT-PCR protocol used for molecular pathotyping targeting the F gene positive results were obtained, thus confirming the HA/HI test results and enabling the pathotyping of the isolates.

  www.oie.int/wahis/public.php. Accessed on 2nd February 2009). Nineteen of the 38 countries are in West and Central Africa. APMV-1 is clearly widespread in Africa but very limited

  transboundary animal diseases. It provides evidence on the need to facilitate data and sequence sharing and to monitor the efficacy of validated diagnostic tests continuously. As demonstrated by the present and previous studies (Xing et al., 2008), neglecting traditional assays such as virus isolation may pose serious risks for the correct diagnosis of contagious diseases. As predicted by Aldous et al. (2003), novel genetic lineages and sublineages are emerging as a consequence of the continuous evolution of APMV-1. However, well defined criteria for lineage or sublineage designation are currently not existing for this virus. This study provide additional genetic data which might be useful for the definition of general criteria for lineage designation, similarly to what has been done for the highly pathogenic avian influenza virus A/H5N1 (WHO/OIE/FAO H5N1 Evolution Working Group, 2008).

Figure 1

 1 Figure 1 -A radial phylogram constructed by Bayesian analysis of 359 NDV isolates belonging to

Figure 2 -

 2 Figure 2 -A radial phylogram constructed by Bayesian analysis of 33 NDV isolates (26 sequenced

Figure 3 -

 3 Figure 3 -Phylogenetic trees constructed by Bayesian analysis of the complete coding region of

Figure 4 -

 4 Figure 4 -Nucleotide sequence alignment of the matrix gene from representative samples tested

  Figure 1

Table 2 -

 2 ). Emerg Infect Dis. [serial on internet]. Available from: http://www.cdc.gov/EID/content/14/7/e1.htm Xing, Z., Cardona, C., Dao, P., Crossley, B., Hietala, S., Boyce, W.; 2008. Inability of real-time reverse transcriptase PCR assay to detect subtype H7 avian influenza viruses isolated from wild The mean number of base substitutions per site for all sequence pairs between lineages is shown . Analyses were conducted using the Maximum Composite Likelihood method in MEGA4 software package.

	birds. J Clin Microbiol. 46(5):1844-6.
	Yusoff, K., Nesbit, M., McCartney, H., Meulemans, G., Alexander, D.J., Collin, M. S., Emmerson
	P. T., Samson, A. C. ;1989. Location of neutralizing epitopes on the fusion protein of Newcastle
	disease virus strain Beaudette C. J Gen Virol. 70 ( Pt 11):3105-9.

Table 3 .

 3 Pathotyping results of representative African APMV-1 isolates

	Isolate	Cleavage site
		amino acid motif

Table 4 -

 4 Amino acid substitutions located within the neutralizing epitopes ofHN and F proteins

	Gene Substitution	Virus
		E347D	NDV_ck_2415_580_Burkina Faso_2008 NDV_ck_2415_577_Burkina Faso_2008
			NDV_ck_3490_147_Cameroon_2008
	HN	Y350H	NDV_ck_2602_605_Niger_2008 NDV_ck_3490_149_Cameroon_2008
			NDV_ck_2602_625_Niger_2008
		D569G	NDV_ck_1377_1_Niger_2006
			NDV_ck_1377_1_Niger_2006
		K78R	NDV_avian_3724_6_Nigeria_2008
	F	D170S	NDV_ck_2601_Ivory Coast_2008 NDV_avian_1532_14_Mauritania_2006 NDV_ck_2601_Ivory Coast_2008
		L343P	NDV_ck_2415_361_Burkina Faso_2008

Acknowledgments

The Food and Agricultural Organization of the United Nations partially funded this study and facilitated the collection and submission of samples (LoA/PR 37772, Project EPIAAF, Project OSRO/RAF/602). The Italian Ministry of Health (RC IZSVe 26/2007) and the European Commission (EPIZONE) also partially funded this study. The Ministry of Agriculture of the Islamic Republic of Mauritania, Niger, Nigeria, Cote d'Ivoire, Burkina Faso, Burundi, Cameroun, are also gratefully acknowledged. We are grateful to Dr G Koch (CVI-Lelystad, NL) for his technical assistance on NDV sequencing and to Dr DJ Alexander for the critical revision of this manuscript.


Sebastian Guenther

Matthias Filter

Karsten Tedin

Istvan Szabo

Lothar H Wieler

Karsten Nöckler

Nicole Walk

Peter Schierack
email: peter.schierack@hs-lausitz.de

Enterobacteriaceae populations during experimental Salmonella infection in pigs Running title Enterobacteriaceae in Salmonella-infected pigs

Keywords: pig, Salmonella, E. coli, Enterobacteriaceae, intestine, infection

Salmonella infection might affect other intestinal Enterobacteriaceae populations and possible correlations between single Enterobacteriaceae populations would help to predict subclinical Salmonella infections in pigs. In one experimental setup, weaned piglets (n=40) were infected with Salmonella and sacrificed at 3 h, 24 h, 72 h or 28 days post infection (p.i.). Dilutions of intestinal contents and mucosal tissues were plated on agar plates for determinations of Enterobacteriaceae. In another experimental setup, weaned piglets (n=12) were infected with Salmonella and probed over a period of 28 days p.i., and dilutions of rectal contents were also

tested for Enterobacteriaceae populations. The occurrence of single Enterobacteriaceae populations was correlated with the occurrence of other tested Enterobacteriaceae populations as well as with clinical parameters of the piglets.

Salmonella (infection strain), E. coli (hemolytic and non-hemolytic) and another six non-E. coli/non-Salmonella Enterobacteriaceae (NENSE) genera with eight species were identified.

In general, the absolute numbers of E. coli, Salmonella and NENSE populations decreased with increasing age of the animals. In the jejunum, the numbers of NENSE, E. coli and Salmonella were all highly positively correlated with each other. The occurrence of hemolytic E. coli had no obvious effects on the occurrence of other Enterobacteriaceae. Furthermore, only few associations of Enterobacteriaceae populations with clinical parameters were observed.

In conclusion, we did not observe evidences for either a competition between or benefits for specific Enterobacteriaceae populations during Salmonella infection indicating that changes in the composition of the intestinal Enterobacteriaceae microflora are not useful indicators of subclinical Salmonella infections.

A c c e p t e d M a n u s c r i p t Introduction Salmonella is one of the most important causes of human gastroenteritis worldwide. In Germany, a total of 42,851 human Salmonellosis cases were reported in 2008 (http://www3.rki.de/SurvStat/; Robert Koch Institute, Berlin, Germany). The consumption of contaminated pork and pork products are one of the main sources for Salmonella infections [START_REF] Steinbach | Attempt to estimate the share of human Salmonella infections, which are attributable to Salmonella originating from swine[END_REF]. Pork and pork products are commonly contaminated with Salmonella during processing of meat from Salmonella-infected animals. Salmonella infections have been reported to be widespread in pig farms [START_REF] Berends | Identification and quantification of risk factors in animal management and transport regarding Salmonella spp. in pigs[END_REF], and infected pigs can carry Salmonella without being recognized as carriers harboring the high risk for transmission of Salmonella via the food production chain to humans. Thus, from the vantage point of human nutrition most critical Salmonella infections in pigs are asymptomatic, subclinical infections.

After infection of the host, Salmonella pathogenesis is characterized by three phases: the initial colonization of the intestine, the invasion of enterocytes, and finally the dissemination to lymph nodes and other organs [START_REF] Darwin | Molecular basis of the interaction of Salmonella with the intestinal mucosa[END_REF]. During the initial colonization phase, Salmonella must compete with the autochthonous microflora for nutrients and attachment/adhesion sites [START_REF] Baba | The role of intestinal microflora on the prevention of Salmonella colonization in gnotobiotic chickens[END_REF][START_REF] Snoeyenbos | Protecting chicks and poults from Salmonellae by oral administration of "normal" gut microflora[END_REF]. During the second phase of Salmonella infection, invasion mechanisms and intraepithelial growth might also be affected by other, resident bacteria including other Enterobacteriaceae [START_REF] Hudault | Escherichia coli strains colonising the gastrointestinal tract protect germfree mice against Salmonella typhimurium infection[END_REF][START_REF] Kleta | Detection and distribution of probiotic Escherichia coli Nissle 1917 clones in swine herds in Germany[END_REF]Tsai et al., 2005). It might therefore be expected that there might be correlations between the occurrence and/or persistence of Salmonella in intestinal contents and the mucosa through the presence of other Enterobacteriaceae species.

In this study we characterized populations of Enterobacteriaceae in the porcine intestine. The main focus of the study was to examine interactions between Salmonella and other non-Salmonella Enterobacteriaceae (NSE) during experimental, subclinical Salmonella infections. (LAGetSi), Berlin, Germany (No. G0037/02). Litters were infected subsequently within a one year period. Piglets from five litters (hybrids of Deutsche Landrasse and Duroc) were housed in groups after weaning (28 days post partum (p.p.). Piglets had ad libitum access to liquid feed with the essential components wheat and soybean. No antibiotics were administered to piglets and their sows for at least three months prior to the trial [START_REF] Szabo | Influence of a probiotic strain of Enterococcus faecium on[END_REF].

On day 29 p.p., piglets were sedated by intramuscular application of 1.0 mg/kg azaperon and challenged with Salmonella Typhimurium DT104 intragastrically using a stomach tube. The Salmonella Typhimurium DT104 (BB440) strain used for the infection study was originally isolated from a pig with sepsis. A nalidixic acid-resistant derivative transformed with a GFP-expressing plasmid, pFVP25.1, was used for the infections. The nalidixin acid-resistance simplified the reisolation of Salmonella from fecal and tissue samples. The GFP-expressing plasmid was introduced into the Salmonella strain for tracing Salmonella infection routes in organs (lymph nodes, spleen etc.) of animals by fluorescence analysis in other studies as part of the Deutsche Forschungsgemeinschaft (DFG) Research Unit ''An Integrative Analysis of Mechanisms of Probiotic Action in Pigs'' network. The GFP-expressing plasmid had no impact on the virulence of the strain which was tested in preliminary studies (data not shown). Extensive data from network partners are not included in this manuscript as they are the subject of other publications.

For infection, Salmonella was cultured in Luria-Broth (LB) at 37°C overnight. The following day, 1 ml was inoculated into 100 ml pre-warmed LB and grown at 37°C to an optical density (OD) of 2.0, corresponding to an average final count of approximately 3 x 10 9 /ml. Each pig was infected with 2 ml of this culture mixed with 8 ml Buffered Peptone The study consisted of two parts. In the first experimental part (1), from each of five litters, a total of eight weaned piglets were infected with the Salmonella strain. Two infected piglets from each litter were randomly selected and sacrificed at 3h, 24h, 72h and 28 days post infection (p.i.). These 40 sacrificed animals were tested for Enterobacteriaceae populations of the two intestinal compartments "content" and "mucosa" from the two intestinal sections "jejunum" and the "colon". Mucosa-associated bacteria included adherent as well as intracellular bacteria. Evaluation of the consistency of intestinal contents and additionally of feces (rectum) was measured using a macroscopic score (1: solid granular, 2: firm, 3: soft, 4: pulpy, 5: liquid, 6: watery, [START_REF] Szabo | Influence of a probiotic strain of Enterococcus faecium on[END_REF]).

In the second experimental part (2), twelve animals were randomly selected out of the five litters mentioned above (including six animals from experimental part 1 which were sacrificed at day 28 p.i.) and were tested on day 5 prior to infection (ante infectionem, a.i.), day 0 (the day of infection, samples were taken approximately 3 hours a.i.) and days 1, 2, 3, 6, 9, 15, 21 and 28 p.i. (33 days time series) for rectal Enterobacteriaceae populations. Rectal samples were taken by sterile cotton swabs. During the observation period of experimental part 2, clinical parameters of the 12 animals were monitored for (1) weight (twice weekly), (2) evaluation of general condition and fecal consistency which was measured daily using a macroscopic score from 1 to 6 (see above), and (3) rectal temperature (daily). approach was chosen because several species occurred only very rarely and thus a separate statistical analysis for each species was not useful. However, if a genus/species was isolated and defined more than 5 times in an intestinal section, a separate statistical analysis was additionally included for this genus/species.

Cultivation of Enterobacteriaceae.

Enterobacteriaceae from sacrificed piglets were isolated from intestinal contents and from mucosal tissues from either the distal jejunum (50 cm proximal to the Plica ileocaecalis) or proximal to the ileo-caecal opening. The distal jejunum was chosen as representative of the small intestine since in previous studies the more proximal jejunum generally contained no intestinal contents and bacteria of distal jejunum are thought to be similar to bacteria of the ileum but distinct from the colon [START_REF] Dixit | Diversity analysis of commensal porcine Escherichia coli -associations between genotypes and habitat in the porcine gastrointestinal tract[END_REF].

The proximal colon was chosen as part of the large intestine as other authors found uniformity of bacterial adhesion at a number of sites of the large intestine indicating that small biopsy samples are likely to be representative of larger areas of the gut epithelium [START_REF] Hartley | Adhesion of commensal bacteria to the large intestine wall in humans[END_REF][START_REF] Swidsinski | Mucosal flora in inflammatory bowel disease[END_REF].

Piglets were euthanized by injection with sodium pentobarbital, and clamped intestinal sections were removed for bacterial isolations from intestinal contents. Intestinal contents were plated by serial dilutions on CHROMagar orientation agar [START_REF] Merlino | Evaluation of CHROMagar Orientation for differentiation and presumptive identification of gram-negative bacilli and Enterococcus species[END_REF], MacConkey agar for lactose fermentation (Oxoid, Hampshire, UK), and Xylose-Lysine-Desoxycholat agar (XLD, Oxoid, Hampshire, UK). Bacteria from the mucosa were isolated from an approximately 2 x 5 cm section of intestinal tissue of each intestinal sample washed twice in phosphate-buffered saline (PBS) to remove visible fecal material. These short washing steps were not expected to affect mucosa-attached bacteria as mucosa-or epithelial A c c e p t e d M a n u s c r i p t 8 cell-associated E. coli remain adherent after up to four to six changes of physiological saline washes [START_REF] Hartley | Adhesion of commensal bacteria to the large intestine wall in humans[END_REF][START_REF] Swidsinski | Mucosal flora in inflammatory bowel disease[END_REF]. Approximately 0.5 g of each mucosal sample was scraped from connective tissue with glass slides. Mucosal samples were transferred to a Dounce-homogenizer, were homogenized in 5 mL 1X PBS and serial dilutions were plated on agar plates. Tubes containing rectal samples (containing approximately 0.1 g rectal content on a cotton swab) from the Experimental part 2 were filled with 3 ml 1X PBS and vortexed for 1min at 3000 rpm. Serial dilutions were plated on agar plates as described above.

Intestinal contents (digesta samples) and mucosal tissues (mucosal samples) were plated in parallel on CHROMagar orientation agar, MacConkey agar and XLD agar plates to obtain an overview of the composition of the total Enterobacteriaceae population. The absolute numbers of coliform bacteria were on average lower on MacConkey and XLD agar plates compared to CHROMagar orientation agar plates (data not shown). For comparability of data from several samples we chose to enumerate E. coli and other non-Salmonella Enterobacteriaceae (NSE) on the basis of the least selective CHROMagar orientation agar plates only.

The determination of populations of E. coli (pink coliform colonies) and for non-E. coli/non-Salmonella Enterobacteriaceae (NENSE, defined as blue to dark violet Enterobacteriaceae-like colonies in this study) was done by using CHROMagar orientation agar plates. These agar plates enable the determination of both E. coli as well as NENSE numbers [START_REF] Alali | Longitudinal study of antimicrobial resistance among Escherichia coli isolates from integrated multisite cohorts of humans and swine[END_REF][START_REF] Merlino | Evaluation of CHROMagar Orientation for differentiation and presumptive identification of gram-negative bacilli and Enterococcus species[END_REF][START_REF] Schierack | Composition of intestinal Enterobacteriaceae populations of healthy domestic pigs[END_REF]. Determinations of Salmonella (pink colonies with a black central spot) were based on XLD agar plates only.

Hemolytic activity of E. coli colonies was tested on sheep blood agar plates. If the NENSE isolates of one intestinal sample comprised more than 5% of NSE, one representative NENSE isolate of one intestinal sample was verified using API 20 E® (Biomerieux, France) tests for A c c e p t e d M a n u s c r i p t 9 further species identification. Our definition of the 5% cut-off point to define a single NENSE species by the API system was based on the practicability of verification due to the common occurrence of NENSE below this cut-off point (see Supplementary Figure S1). However, for calculations of correlations between E. coli, NENSE and Salmonella, exact numbers for all three populations were included. Salmonella-like colonies were additionally examined for their green fluorescence on XLD agar plates.

Data from a mucosal sample (mucosa-associated bacteria) comprised adherent as well as intracellular bacteria. Data from a digesta samples comprised bacteria of the intestinal contents. Data from an intestinal section includes data from both the digesta and the mucosal sample of this section. Data from a rectal sample comprised bacteria of the rectal contents.

Statistical analysis. Statistical analysis was performed using SPSS 12.0.2 (SPSS Inc.). In all cases, non-parametric statistical tests were applied. Associations between ordinally or metrically scaled parameters were tested by application of the Spearman rank correlation test procedure. Differences in central tendencies between two sample groups were analyzed by application of the Mann-Whitney-U test (independent samples) or the Wilcoxon-test (paired samples). In general, the level of significance α was set to 0.05. In Experimental part 2, data correlations were separately analyzed for each time point and were only considered relevant when statistical tests were significant at least half the time. As this study was intended to provide a descriptive analysis of the relationships between microbial parameters, a correction for the effects of multiple testing was not applied. Nevertheless, while results with p-values below 0.01 are indicated as additional information, in general, the results of this study should be considered as descriptive in nature. Hemolytic E. coli were sporadically present in all piglet groups and occurred in colonic and/or jejunal and/or rectal samples. However, the occurrence of hemolytic E. coli had no obvious effects on the occurrence of other E. coli, NENSE or Salmonella. In addition, the isolation frequency of hemolytic E. coli did not change after Salmonella infection.

Experimental part 1. Analysis of Enterobacteriaceae in digesta and mucosal samples of killed pigs. The absolute counts of E. coli numbers in the jejunum and colon, NENSE numbers in the jejunum digesta and the absolute Salmonella counts in the mucosa of the jejunum and colon decreased significantly with the age of the animals (Table 1).

There were in general higher numbers of E. coli in the colon compared to the jejunum (p<0.01), but no differences for NENSE and Salmonella. In addition, there were higher numbers of E. coli and NENSE in the digesta compared to mucosa (jejunum as well as colon, A c c e p t e d M a n u s c r i p t 11 all tests with p-values < 0.01) and higher numbers of Salmonella in the digesta compared to mucosa (p<0.01). Additionally, high numbers of mucosa-adherent bacteria of one population correlated with high numbers of bacteria of this population in the digesta (Fig. 1).

In the jejunum the numbers of isolated E. coli, NENSE and Salmonella were highly positively intercorrelated, "i.e." if a jejunum sample showed a high number of NENSE, there was a higher probability of observing high numbers of E. coli or Salmonella as well. In the colon, samples with this strong intercorrelation pattern were less frequently observed although some correlations were statistically significant (E. coli vs. Salmonella in colon digesta: p<0.05; NENSE vs. Salmonella in colon digesta: p<0.05) (Fig. 2).

The frequency of isolated single NENSE species is shown in Table 2. No single NENSE genus/species was isolated more than 5 times in the colon. Klebsiella was the only genus which was sampled and defined more than 5 times in the jejunum, a separate statistical analysis was performed for this genus. However, the occurrence of Klebsiella did not correlate with E. coli and Salmonella populations of the jejunum.

The occurrence of Salmonella in jejunum as well as in colon samples varied considerably between individual pigs even at 3h p.i. For example, in a single pig (animal No.8 in Fig. 3) we detected no Salmonella in the jejunum or colon (digesta or mucosa) at this time point. In contrast, only two other animals had Salmonella in the jejunum and colon (digesta and mucosa, animals No. 3 and 9 in Fig. 3). The highest number of Salmonella-positive samples was detected at 24 h p.i. (Table 3).

Experimental part 1. Associations between numbers of Enterobacteriaceae in digesta

and mucosal samples of killed pigs and disease-related parameters. There were no significant dependencies detectable between the numbers of E. coli or NENSE in digesta or mucosal samples and the body temperatures of animals. However, lower numbers of A c c e p t e d M a n u s c r i p t 12 Salmonella in colonic digesta samples were associated with elevated body temperatures of the animals (p<0.01). This correlation remained significant even after correcting for the influence of the animal age by application of the partial correlation analysis algorithm on Salmonella non-negative samples.

Fecal consistencies were significantly positively associated with E. coli counts from the colon digesta and mucosal samples and colon digesta consistencies were significantly associated with E. coli counts from the colon digesta samples, "i.e." the more E. coli were detected the more solid the digesta and feces were (all p<0.05). In contrast, where NENSE were detectable in jejunal digesta samples, high NENSE counts were negatively associated with digesta consistencies, "i.e." the more NENSE were detected the more liquid the digesta was (p=0.074 for the samples from digesta, p<0.05 for samples from the mucosa). The number of Salmonella was not significantly associated with digesta consistencies in the colon or jejunum, but Salmonella from colon digesta and jejunum mucosa showed a strong positive correlation with solid feces, as was observed for E. coli counts (p<0.01). As Klebsiella was the only genus which was sampled and defined more than 5 times in the jejunum, a separate statistical analysis was performed for this genus. However, the occurrence of Klebsiella did not correlate with digesta consistencies or body temperatures of animals.

Experimental part 2. Analysis of Enterobacteriaceae in rectal samples of live piglets

during a 33 days time series. The absolute E. coli counts of rectal samples decreased significantly with the age of the animals (p<0.01), whereas for the absolute NENSE counts such time-dependency could not be verified due to the high percentage of NENSE-negative samples. When analyzing the intercorrelations between E. coli, Salmonella and NENSE numbers separately at each time point of this time series, the occurrence of high numbers of isolates of a single Enterobacteriaceae-population was not correlated to high numbers of The occurrence of Salmonella in rectal samples varied considerably between individual pigs. An apparent increase of Salmonella shedding was observed between 3 h and 24 h p.i. followed by a decrease of Salmonella shedding after 24 h until day 28 p.i. (p<0.01). The level of Salmonella shedding in feces during the three days following the infection (24 h to 72 h p.i.) varied between 3 to 8 log units (log(cfu/g)).

Experimental part 2. Associations between numbers of Enterobacteriaceae in rectal

samples and disease-related parameters during a 33 day time series. During the observation period of 28 days post infection, the weight of animals increased continuously, corresponding to the normal development of body weight in healthy, weaning pigs. Elevated body temperature over 40.0°C was observed in 33.3% (4/12) of the animals during the four week observation period. The fecal consistency of all samples before infection was soft to solid (scores 2-3). Diarrhea was observed in 16.7% (2/12) of animals within the first three days p.i. and in 41.6% (5/12) of animals within the four weeks observation period (score 5=liquid feces). However, the bacterial counts taken during this time showed no significant dependencies between the numbers of E. coli or NENSE or Salmonella in rectal samples and either the fecal consistencies or body temperatures of animals. Individual NENSE species could not be correlated with disease-related parameters due to low abundances of these species.

A c c e p t e d M a n u s c r i p t 14

Discussion

With the exception of E. coli, the composition of the autochthone porcine intestinal Enterobacteriaceae microflora has been only partially studied [START_REF] Schierack | Composition of intestinal Enterobacteriaceae populations of healthy domestic pigs[END_REF], and changes during a Salmonella infection remained unknown. In this study, we determined the dynamics of the autochthone Enterobacteriacea microflora and investigated possible relationships between the occurrence of intestinal Salmonella and the occurrence of other Enterobacteriaceae during an experimental Salmonella infection. The animal infections were performed with Salmonella Typhimurium phagetype DT104, one of the most frequently isolated Salmonella serovar in pork, and a common Salmonella serovar isolated from a wide range of other animals and humans and of particular concern because of its acquisition of multiple antibiotic resistances [START_REF] Baggesen | Characterisation of recently emerged multiple antibiotic-resistant Salmonella enterica serovar typhimurium DT104 and other multiresistant phage types from Danish pig herds[END_REF][START_REF] Poppe | Diversity in antimicrobial resistance and other characteristics among Salmonella typhimurium DT104 isolates[END_REF]Threlfall et al., 2000). Our goal was to monitor infection-dependent changes and possible protective functions of ratios of members of the authochtone Enterobacteriaceae microflora against a Salmonella infection. We also performed correlation analyses for the occurrences of E. coli, non-E. coli/non-Salmonella Enterobacteriaceae (NENSE) and Salmonella with alterations in clinical parameters which were also monitored during an experimental subclinical to mild Salmonellosis.

In our study, a specific ratio between the E. coli and the total NENSE population as well as the occurrence of single NENSE genera/species were not correlated with low numbers of Salmonella and thus appeared to have no protective function against the Salmonella infection.

In contrast, high Salmonella counts were accompanied by high numbers of non-Salmonella Enterobacteriaceae (NSE) and high numbers of mucosa-associated Salmonella were also correlated with high numbers of mucosa-attached E. coli. Such a rather proportional than competing effect was also seen comparing the E. coli and the total NENSE population as high numbers of NENSE were also markedly correlated with high numbers of E. coli. These results During the course of the infection study, the absolute numbers of E. coli, NENSE and Salmonella continuously decreased in rectal samples. This decrease was associated with a decrease of these Enterobacteriaceae populations in both the jejunum as well as in the colon.

Comparison of the data from this study with a recent study of non-infected piglets of the same pig population [START_REF] Scharek | Influence of a probiotic Enterococcus faecium strain on development of the immune system of sows and piglets[END_REF], indicated that the decrease of coliform bacteria after Salmonella infection was no more prominent than the decrease of coliform bacteria in noninfected animals. Such an age-dependent decrease of coliform bacteria seems to be common in the fast developing intestines of piglets [START_REF] Katouli | The effect of zinc oxide supplementation on the stability of the intestinal flora with special reference to composition of coliforms in weaned pigs[END_REF]. In summary, the Salmonella infection did not appear to result in a marked reduction or increase of NSE. However, a decrease in the rectal populations might reflect a decrease of the jejunal or colonic Enterobacteriaceae populations.

We observed a correlation between numbers of bacteria from an Enterobacteriaceae population and the consistency of the digesta samples. The colon digesta and feces consistencies were associated with E. coli counts from the colon, "i.e." the more E. coli were detectable the more solid the digesta and feces were. A similar correlation was found for Salmonella in the jejunum and colon and the consistency of animal feces. In contrast, the more NENSE detectable in the jejunum, the more liquid the digesta was. These correlations might be explained by two possibilities: (1) The changes in fecal/digesta consistency may have been due to changes of the intestinal milieu which in turn reflected changes of the reabsorption/excretion capacity of the mucosa. The Enterobacteriaceae populations might be expected to adapt to such intestinal alterations with increased/decreased numbers of an Enterobacteriaceae population reflecting the consequences of the changes of the intestinal Changes in the intestinal milieu including weaning, changes in feeding regime and intestinal diseases (e.g. viral diseases) are thought to support the emergence or dominance of pathogenic hemolytic E. coli [START_REF] Franklin | Characterization of microbial populations and volatile fatty acid concentrations in the jejunum, ileum, and cecum of pigs weaned at 17 vs 24 days of age[END_REF][START_REF] Hampson | Coliform numbers in the stomach and small intestine of healthy pigs following weaning at three weeks of age[END_REF][START_REF] Lecce | Rotavirus and hemolytic enteropathogenic Escherichia coli in weanling diarrhea of pigs[END_REF][START_REF] Pluske | Feeding different types of cooked white rice to piglets after weaning influences starch digestion, digesta and fermentation characteristics and the faecal shedding of betahaemolytic Escherichia coli[END_REF]. Although Salmonella infection led to mild diarrhea in several animals in our study and hemolytic E. coli were present in all piglet groups, an increase or a dominance of hemolytic E. coli were not detectable. We conclude that hemolytic E. coli already present in the intestinal microflora did not exploit or reinforce the intestinal disturbances caused by the Salmonella infections [START_REF] Szabo | Influence of a probiotic strain of Enterococcus faecium on[END_REF]. In this study we have made a number of additional general observations which may or may not be independent of a Salmonella infection. Here we report for the first time the isolation of several Enterobacteriaceae species from the pig (Enterobacter sakazakii, Klebsiella ornithinolytica, Klebsiella oxytoca, Rahnella aquatilis and Serratia marcescens). This confirmed our previous assumptions that the porcine intestine is a natural habitat for a A c c e p t e d M a n u s c r i p t 17 broad variety of different Enterobacteriaceae species [START_REF] Schierack | Composition of intestinal Enterobacteriaceae populations of healthy domestic pigs[END_REF]. However, also in this study, E. coli was the clearly most dominant Enterobacteriaceae species in the jejunum, the colon as well as in the rectum [START_REF] Schierack | Composition of intestinal Enterobacteriaceae populations of healthy domestic pigs[END_REF].

Identical experimental

As shown in a previous study, there were higher numbers of Enterobacteriaceae in the colon compared to the jejunum and higher numbers of Enterobacteriaceae in the digesta compared to the mucosa samples [START_REF] Schierack | Composition of intestinal Enterobacteriaceae populations of healthy domestic pigs[END_REF]. However, as also noted in this previous study, there were also determinations showing no Enterobacteriaceae in the jejunum.

For the determination of Enterobacteriaceae numbers we compared plating results on different agar plates. Absolute NSE numbers were in general lower on MacConkey, XLD agar and also on the initially used Gassner agar plates compared to CHROMagar orientation agar plates. However, differences in absolute numbers differed from sample to sample. We therefore chose to only use absolute numbers of E. coli and non-E. coli/non-Salmonella Enterobacteriaceae as determined from CHROMagar orientation agar plates (the less restrictive agar plate). CHROMagar orientation agar plates identify by 99.3% E. coli isolates due to their pink/red colony colors and are generally accepted to be the only plate type sufficiently capable of differentiating E. coli from other Enterobacteriaceae [START_REF] Alali | Longitudinal study of antimicrobial resistance among Escherichia coli isolates from integrated multisite cohorts of humans and swine[END_REF][START_REF] Merlino | Evaluation of CHROMagar Orientation for differentiation and presumptive identification of gram-negative bacilli and Enterococcus species[END_REF][START_REF] Schierack | ExPEC-typical virulence-associated genes correlate with successful colonization by intestinal E. coli in a small piglet group[END_REF][START_REF] Schierack | Composition of intestinal Enterobacteriaceae populations of healthy domestic pigs[END_REF]. CHROMagar orientation agar plates were also the basis for our definition of NENSE. Due to practical reasons here we focused on blue/violet coliform colonies. A broad spectrum of Enterobacteriaceae has been shown to form blue/violet coliform colonies on CHROMagar orientation agar plates with different shades of color: several species of the genera Buttiauxella, Citrobacter, Enterobacter, Hafnia, Klebsiella, Leclercia, Pantoea, Raoultella, Serratia, Yersinia (Merlino et al., 1996;Schierack et al., 2007 and unpublished results). However, we are aware of that other members of the Enterobacteriaceae population might be excluded from our analysis as there might have been Enterobacteriaceae genera/species which do not form pink/red or A c c e p t e d M a n u s c r i p t 18 blue/violet colonies on CHROMagar orientation agar plates. As we also plated intestinal samples to MacConkey and XLD plates (and initially Gassner agar plates), we had the possibility to observe colony differences between NENSE isolates (color, size, surface etc.). However, NENSE isolates of a given sample were always very similar on agar plates. We conclude that the dominant fraction of the NENSE population of one intestinal sample consists largely of one species, as suggested in a prior study [START_REF] Schierack | Composition of intestinal Enterobacteriaceae populations of healthy domestic pigs[END_REF].

Based on the results of this study, we find no correlations or changes in the ratios of single or groups of Enterobacteriaceae populations which would provide predictive or diagnostic markers based on the composition of the Enterobacteriaceae population for asymptomatic or subclinical Salmonella infections. Rather, we observed a similar trend for the endogenous populations concerning absolute numbers of E. coli and NENSE following experimental introduction of Salmonella into the intestinal microflora. In addition, endogenous hemolytic E. coli did not appear to play a role during an experimental Salmonella infection. Clearly the interplay between intestinal microflora populations is complex, but it would appear that these populations are also rather stable, since introduction of a diarrheagenic intestinal pathogen such as Salmonella did not result in perturbations resulting in significant shifts in populations or even single genera. Threlfall, E.J., Ward, L.R., Frost, J.A., Willshaw, G.A., 2000. The emergence and spread of antibiotic resistance in food-borne bacteria. Int. J. Food Microbiol. 62, 1-5. Tsai, C.C., Hsih, H.Y., Chiu, H.H., Lai, Y.Y., Liu, J.H., Yu, B., Tsen, H.Y., 2005. Antagonistic activity against Salmonella infection in vitro and in vivo for two Lactobacillus strains from swine and poultry. Int. J. Food Microbiol. 102,[185][186][187][188][189][190][191][192][193][194].

  . The animal study was approved by the local animal welfare committee of the Landesamt für Arbeitsschutz, Gesundheitsschutz und technische Sicherheit

  , Merck, Darmstadt, Germany). The infection dose of approximately 6 x 10 9 bacteria/ml was chosen to cause only subclinical or mild Salmonellosis.

  Definitions of Enterobacteriaceae populations. Four groups of Enterobacteriaceae were distinguished for clear representation of data: (1) the total E. coli population, (2) the Salmonella population, (3) total non-Salmonella Enterobacteriaceae (NSE) including all Enterobacteriaceae other than Salmonella, and (4) non-E. coli/non-Salmonella A c c e p t e d M a n u s c r i p t 7 Enterobacteriaceae (NENSE) including all Enterobacteriaceae other than E. coli and Salmonella. Although the NENSE population consisted of many different species, this

  comments. NSE were detected in all colonic and rectal samples. In contrast, in several jejunal samples (digesta (n=1/40) or mucosa (n=5/40)) NSE were not detectable. If NSE were isolated from an intestinal section, E. coli were always present. However, there were two jejunal mucosa and one jejunal digesta samples with only Klebsiella pneumoniae or Klebsiella spp. or Enterobacter cloaceae, but with E. coli in the other compartment of a given section.The following NENSE (non-E. coli/ non-Salmonella Enterobacteriaceae) species and genera were isolated from jejunal and/or colonic and/or rectal samples: Citrobacter freundii (up to 90.7% of the total NSE population of a single sample), Enterobacter cloaceae (100%), Enterobacter sakazakii (93%), Klebsiella ornithinolytica (36%), Klebsiella pneumoniae (100%), Klebsiella oxytoca (10.9%), Klebsiella spp. (100%), Pantoea spp. (68.1%), Rahnella aquatilis (15.4%), Serratia marcescens (84.9%) and Serratia spp. (91.3%).

  and single NENSE species could not be correlated with other Enterobacteriaceae populations due to low abundances of these species.

  each Enterobacteriaceae population supported the occurrence of other Enterobacteriaceae or (2), the intestinal milieu affected the intraintestinal growth of all Enterobacteriaceae populations to a similar extent.

  Alternatively, certain Enterobacteriaceae populations may increase/decrease in the intestine in response to intestinal disorders. These increased/decreased numbers of bacterial population(s) may affect the intestinal mucosa and cause the changes in fecal/digesta consistency. Whether changes in the autochthone Enterobacteriaceae populations are the cause or result from changes in intestinal physiology and/or disorders remains speculative.

  Salmonella infection regimes resulted in often very different infection success as determined by intestinal counts in different animals at different time points. Thus a successful Salmonella infection appears to be strongly dependent on the individual status of a given animal. During intestinal Salmonella infection experiments in swine, the numbers of Salmonella in the intestine therefore have to be constantly monitored as a reference point to evaluate individual effects on other microbial, biochemical or immunological parameters.

  serovar Typhimurium DT104 infection in a porcine animal infection model. Appl. Environ. Microbiol. 75, 2621-2628.

  Figure captions

Fig. 2 .

 2 Fig. 2. Correlations between numbers of E. coli and NENSE (A), E. coli and Salmonella (B)

Fig. 3 .

 3 Fig. 3. Individuality of Salmonella numbers in the jejunum and colon of killed piglets (n=10)

  Fig. 1.

  Fig. 3.

Acknowledgements

This work was supported by grant FOR 438/1-1 from the Deutsche Forschungsgemeinschaft.

Nicole Walk was supported by the Schaumann Stiftung. Furthmore, we thank Dr. Wolff and his team from the Center for Animal Experiments of the Federal Institute for Risk Assessment (BfR, Berlin, Germany) for their indispensible support.

Abbreviations