R Strong

S A La Rocca

G Ibata

T Sandvik
email: t.sandvik@vla.defra.gsi.gov.uk

Antigenic and genetic characterisation of border disease viruses isolated from UK cattle

Keywords: Pestivirus, Border disease virus, Cattle, Genetic typing, Monoclonal antibody

Available empirical data on the natural occurrence of ruminant pestiviruses has shown that bovine viral diarrhoea virus (BVDV) is nearly exclusively found in cattle, whereas both border disease virus (BDV) and BVDV can be isolated from sheep. During routine genetic typing of pestivirus RNA from UK cattle diagnosed as BVDV positive between 2006 and 2008, five samples that were classified as BDV positive yielded positive virus isolates in cell cultures. The samples originated from animals that had shown signs typical for BVD.

Phylogenetic analysis of the bovine BDVs showed that two belonged to the BDV-1a group and three to the BDV-1b group, thereby matching the genetic diversity seen for previously described UK ovine BDVs. Antigenic typing with a set of monoclonal antibodies (MABs) showed that all bovine BDVs lacked one or more epitopes conserved among ovine BDV-1 isolates, and that they had gained reactivity with at least one BVDV-1 specific MAB. Serial passaging of two of the virus isolates in ovine cell cultures did not change the epitope expression pattern. These findings suggest that the presumed natural resistance of cattle against infection with BDV no longer holds. A consequence of this is that BVD diagnostic assays should be checked for their ability to also detect BDV, and also highlights the need for monitoring of the BDV status in sheep that may be in contact with cattle in areas with organised BVD control programmes.

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

Introduction

Pestiviruses cause economically important diseases in domestic ruminants and pigs worldwide. There are currently four recognized members of the genus Pestivirus, family Flaviviridae, three of which infect ruminants naturally; bovine viral diarrhoea virus (BVDV) 1, BVDV-2 and border disease virus (BDV) [START_REF] Thiel | Family Flaviviridae[END_REF]. Acute BVDV infection in cattle can vary from sub-clinical infection to mild clinical disease with inappetence and transient fever, or with more specific signs such as diarrhoea, respiratory or reproductive dysfunction. Vertical transmission of virus in pregnant animals to their unborn young that can result in abortion, mummification of the foetus, congenital defects, stillbirth or the birth of persistently infected calves immunotolerant to BVDV (PI). PI animals have been shown to excrete high levels of infectious virus in the absence of clinical disease. Identification of persistently infected animals within the herd is obviously of great economic importance. The presence of BVDV-1 infection in cattle has been detected worldwide. In contrast BVDV-2 infection has only been seen since the 1990s when it was detected in cattle in North America (Pellerin et al., 2000). More recently, BVDV-2 isolates have been detected in Europe, Japan, Korea and South America [START_REF] Vilcek | Bovine viral diarrhoea virus genotype 1 can be separated into at least eleven genetic groups[END_REF][START_REF] Nagai | Genomic and serological diversity of bovine viral diarrhea virus in Japan[END_REF][START_REF] Flores | Clinical, pathological and antigenic aspects of bovine viral diarrhea virus (BVDV) type 2 isolates identified in Brazil[END_REF].

Border disease is principally a disease of sheep that shares some of the characteristics of BVD in cattle, but with more pronounced emphasis on reproductive disease such as abortion, and birth of stillborn or mummified fetuses. It can also cause the birth of small weak lambs that suffer with "hairy-shaker" syndrome named due to the hairy fleeces and tremors suffered by the lambs [START_REF] Nettleton | The production and survival of lambs persistently infected with border disease virus[END_REF]. Birth of PI animals is common in sheep but more rare in goats.

The Pestivirus genome consists of a single stranded positive-sense RNA of approx 12.3 kb in length, encoding for a single open reading frame that is flanked by a 5" and 3" untranslated regions (UTR). The genome codes for 4 structural proteins, the capsid (C) and three envelope proteins (E rns , E1 and E2), plus several non-structural proteins [START_REF] Thiel | Family Flaviviridae[END_REF].

Phylogenetic studies generally classify virus isolates based on sequences generated from the 5'-UTR or N pro . BDV isolates have been divided into six phylogenetic groups; BDV-1 has been detected in sheep from the USA [START_REF] Sullivan | Genetic characterization of ruminant pestiviruses: sequence analysis of viral genotypes isolated from sheep[END_REF], the UK [START_REF] Vilcek | Molecular characterization of ovine pestiviruses[END_REF], Australia [START_REF] Becher | Molecular characterization of border disease virus, a pestivirus from sheep[END_REF] and New Zealand [START_REF] Vilcek | Genetic typing of pestiviruses from New Zealand[END_REF]; BDV-2 in ruminants in Germany [START_REF] Becher | Genetic and antigenic characterization of novel pestivirus genotypes: implications for classification[END_REF]; BDV-3 in Switzerland [START_REF] Stalder | Genetic heterogeneity of pestiviruses of ruminants in Switzerland[END_REF] and Austria [START_REF] Krametter-Froetscher | Influence of Communal Alpine Pasturing on the Spread of Pestiviruses among Sheep and Goats in Austria: First Identification of Border Disease Virus in Austria[END_REF], BDV-4 in Spain (Valdazo-Gonzalez et al., 2007) and BDV-5 and -6 in France [START_REF] Dubois | Genetic characterization of ovine pestiviruses isolated in France, between 1985 and 2006[END_REF]. Isolates from Turkey form a possible seventh group, BDV-7, that remains to be fully characterised (Oguzoglu et al., 2008).

Historically pestiviruses were named after the animal host species they were isolated from.

However, the ability of interspecies transmissibility has been shown both naturally and experimentally [START_REF] Paton | Identification of herd-specific bovine viral diarrhoea virus isolates from infected cattle and sheep[END_REF]1997). For instance, BVDV also infects sheep, goats, wild ruminants and pigs [START_REF] Becher | Phylogenetic analysis of pestiviruses from domestic and wild ruminants[END_REF]. In sheep, BVDV-1 has been detected in Germany, Sweden, UK and the United States [START_REF] Becher | Molecular characterization of border disease virus, a pestivirus from sheep[END_REF][START_REF] Vilcek | Molecular characterization of ovine pestiviruses[END_REF][START_REF] Willoughby | Development of a real time RT-PCR to detect and type ovine pestiviruses[END_REF] and BVDV-2 in sheep in Germany, UK and the US [START_REF] Becher | Further characterization of border disease virus isolates: evidence for the presence of more than three species within the genus pestivirus[END_REF][START_REF] Sullivan | Genetic characterization of ruminant pestiviruses: sequence analysis of viral genotypes isolated from sheep[END_REF], Vilcek et al., 1997). Conversely, classical swine fever virus (CSFV) has never been isolated from naturally infected cattle, although an experimental study has shown cattle to be susceptible to CSFV [START_REF] Dahle | Transmission of pestiviruses between animal species: experimental infection of swine with the virus of bovine virus diarrhea (BVD) and of cattle with the virus of European swine fever (ESP)[END_REF].

Although BVDV is commonly seen in sheep there is very little evidence of natural infection with BDV in cattle. One exception is the Australian V/TOB isolate, which W.A. [START_REF] Snowdon | Mucosal Disease: its incidence and diagnosis in Australia[END_REF] described as antigenically different from other bovine isolates, and that later was genotyped as BDV by [START_REF] Becher | Phylogenetic analysis of pestiviruses from domestic and wild ruminants[END_REF]. More recently, one case of BDV in cattle was identified during routine testing in Austria as part of their BVDV control program [START_REF] Hornberg | Genetic diversity of pestivirus isolates in cattle from Western Austria[END_REF].

Material and methods

Origin of BDV isolates

Three suspect BVD cases were identified as being caused by BDV by a routine TaqMan RT-PCR genotyping as reported by [START_REF] Cranwell | Detection of Border disease virus in cattle[END_REF]. Three further cases have tested positive for BDV; one case in 2006 by RT-PCR of spleen tissue from an aborted foetus, and in 2008 two more cases initially diagnosed as BVDV positive by antigen ELISA (Serelisa BVD/MD Ag, Synbiotics, France) were typed as BDV positive by the TaqMan RT-PCR (Table 1).

Information on the case histories and potential epidemiological links between cattle and sheep on the farms where the isolates came from was obtained either by farm visits or sought from the private veterinary surgeons attending the cases.

Virus isolation

Either plasma or clarified supernatant from homogenized tissue samples were set up for virus isolation by inoculation onto pestivirus-free foetal bovine turbinate (fBT) cells, maintained in minimum essential medium containing 10% foetal bovine serum proven free from pestiviruses and pestivirus neutralizing antibodies. After a further virus passage on fBT cells, samples were fixed and immunostained using an unconjugated pool of pan-pestivirus reacting monoclonal antibodies (MABs) (WB103/WB105/WB112) and a peroxidase-linked secondary mouse antibody as described by [START_REF] Paton | Border disease virus: delineation by monoclonal antibodies[END_REF].

A c c e p t e d M a n u s c r i p t 6 2.3 Genetic typing RNA was extracted from infected fBT cells using Trizol (Invitrogen, UK) and RNA was eluted in 100 l nuclease-free water. Reverse transcription (RT) was carried out using the Superscript II Reverse Transcriptase kit (Invitrogen, UK) as per the manufacturer"s protocol. A 242-246 nucleotide (nt) fragment of the 5"-UTR was amplified using primers 324/326 [START_REF] Vilcek | Pestiviruses isolated from pigs, cattle and sheep can be allocated into at least three genogroups using polymerase chain reaction and restriction endonuclease analysis[END_REF] and Taq DNA polymerase (Promega, UK). The N pro gene was amplified with primers 320F (5'-GCCTGATAGGGTGYWGCAGAG-3", forward) and 1040R (5'-TTYCCTTTCTTCTTYACCTGGTA-3", reverse). Sequencing reactions based on purified amplicons were set up in both directions using both the PCR primers and the 356F / 1027R pair [START_REF] Valdazo-Gonzalez | Genetic and antigenic typing of border disease virus isolates in sheep from the Iberian Peninsula[END_REF] using a chain termination protocol based on fluorochrome labeled dideoxynucleotides (Cogenics, UK). Assembled sequences were aligned with matching sequences of BDV and BVDV reference strains using Clustal X [START_REF] Thompson | The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools[END_REF]. Transition/transversion ratios were calculated with Puzzle4 [START_REF] Strimmer | Likelihood-mapping: a simple method to visualize phylogenetic content of a sequence alignment[END_REF] and phylograms were calculated using components of the PHYLIP package [START_REF] Felsenstein | Phylip phylogeny inference package (version 3.5c)[END_REF]. Phylogenetic analyses were carried out using the maximum likelihood distance matrix method using 1000 replicates for calculation of bootstrap values. Trees were drawn with TREEVIEW [START_REF] Page | TREEVIEW: An application to display phylogenetic trees on personal computers[END_REF] and edited with Freelance Graphics. Nucleotide sequences of the new BDV strains have been submitted to GenBank; accession numbers are given in Table 1.

Antigenic typing

fBT cells infected with each virus isolate were acetone fixed and immunostained using a selection of monoclonal antibodies (MABs) raised against different pestiviruses (BDV, BVDV-1, BVDV-2 and CSFV) [START_REF] Edwards | The application of monoclonal antibody panels to characterize pestivirus isolates from ruminants in Great Britain[END_REF][START_REF] Edwards | Antigenic comparisons of hog cholera virus isolates from Europe, America and Asia using monoclonal antibodies[END_REF][START_REF] Paton | Border disease virus: delineation by monoclonal antibodies[END_REF]. The staining intensity was graded as negative, weak positive and strong positive with staining of less than 50% of the monolayer also being recorded. Ten serial passages were A c c e p t e d M a n u s c r i p t carried out on two of the isolates using pestivirus-free sheep choroid plexus (SCP) cells maintained in minimum essential medium containing 10% foetal bovine serum proven free from pestiviruses and neutralizing antibodies to pestiviruses. Each passage was fixed, immunostained and results recorded as described for the isolates grown in fBT cells.

Results

Case histories

The available case history information was variable. For the adult animals the presentation was undistinguishable from typical signs seen in animals PI with low-virulent BVDVs (Table 1). Four of the cases came from farms that also kept sheep, but the degree of contact appeared to have been variable. There was no record of recent border disease among the sheep on any of the farms involved, nor any obvious signs of BVD in other cattle.

BDV isolates

A pestivirus was isolated from five of the six cases that had been recorded positive by RT-PCR. The sixth sample remained negative after two virus isolation attempts with spleen samples from the aborted foetus, probably due to the cytotoxicity of the available sample.

When cultured on fBT cells, all five BDV isolates viruses were of the non-cytopathogenic biotype.

Genetic typing

The genetic analysis of partial 5"-UTR sequences showed that all five bovine BDV isolates grouped with other UK ovine BDV-1 sequences. Three isolates clustered within the BDV-1b

viruses, together with isolate K1729/3 from 1987, whilst the other two isolates were allocated to different clusters within the BDV-1a viruses (Fig. 1). Pairwise sequence comparison with the closest matching ovine BDV 5'-UTR sequence showed between two to five nucleotide substitutions, corresponding to sequence identities between 97.9 and 99.2 %. Within the negative case showed greatest similarity to that of isolate 1505744, but with a significantly lower sequence identity (81%).

Phylogenetic analysis based on full length N pro sequences resulted in the same classification of the five bovine BDVs as obtained with the partial 5'-UTR sequences, but with much higher bootstrap values (Fig. 2). Pairwise sequence identities for the group of bovine BDVs ranged from 98.4 to 75.4 %, with the same order of sequence identity as seen for the partial 5'-UTR sequences.

Antigenic typing

The five bovine BDV isolates were most similar to ovine BDVs by immunostaining, although an unusual reactivity pattern was observed compared to the BDV reference strains (Fig. 3).

The otherwise broadly reactive BDV MAB WS381 failed to detect four of the five bovine BDV isolates. Similarly, MAB WB160 did not recognise three of the bovine BDVs. Most of the bovine BDVs were immunostained by MABs WB214, CA1, and CA39, which have been known as BVDV-1 specific. In general, the staining intensity with the anti-BDV MABs was more variable for the bovine BDVs than for the ovine BDVs. The immunostaining pattern of the bovine isolate 1505744 was more similar to BDV reference strains than the other four bovine BDVs.

Two of the bovine BDV isolates, 1062689 and 1118212, were maintained for up to 10 passages on SCP cells to assess any changes in the MAB reactivity pattern. No change in the MAB reactivity pattern was seen for either of these isolates (data not shown).

A c c e p t e d M a n u s c r i p t 9

Discussion

It has been well documented that BVDV not only infects cattle but also other members of the Order Artiodactyla, whilst BDV has mainly only been found in small ruminants. This study describes antigenic and genetic properties of the first BDVs isolated from UK cattle.

Previous in vivo transmission studies of BVDV between cattle and sheep showed host animal dependent reversible changes in the MAB reactivity pattern [START_REF] Paton | Establishment of serial persistent infections with bovine viral diarrhoea virus in cattle and sheep and changes in epitope expression related to host species[END_REF]. To avoid artificial induction of an ovine antigenic profile, the cattle BDV isolates were maintained in bovine cell cultures for the antigenic characterization. Although variable, the MAB reactivity pattern of the cattle isolates revealed an antigenic profile most similar to ovine BDV reference strains. The most striking exception was that nearly all the bovine BDVs expressed the BVDV-1 specific WB214, CA1 and CA39 epitopes, which reside within the major envelope protein E2. Interestingly, these epitopes were the only ones that were expressed exclusively in a bovine environment in the study by [START_REF] Paton | Establishment of serial persistent infections with bovine viral diarrhoea virus in cattle and sheep and changes in epitope expression related to host species[END_REF]. This may indicate that the WB214, CA1 and CA39 epitopes are directed against cell surface receptors important for replication in bovine, but not ovine cells. These changes, along with the general lower reactivity against BDV specific MABs indicate that BDVs benefit from adapting antigenically when shifting to a new host environment. However, this would not explain why antigenic differences in non-structural proteins were seenwhilst the NS3 specific MAB WB160 has been known to recognise most BVDV-1 and BDV isolates, three of the five bovine BDVs were not immunostained with this MAB. The observation that the "bovine MAB reactivity pattern" did not alter after 10 passages in ovine cell cultures is consistent with the previously observed delayed reversion to an ovine antigenic pattern of a bovine BVDV after transfer from cattle to sheep [START_REF] Paton | Establishment of serial persistent infections with bovine viral diarrhoea virus in cattle and sheep and changes in epitope expression related to host species[END_REF]. This indicates that the antigenic adaptation after host animal changes may be a clonal selection process that takes some time, in the absence of immune selective pressure.

A c c e p t e d M a n u s c r i p t 10 Phylogenetic analysis showed that the bovine BDV isolates grouped together with previously described UK ovine BDV-1 isolates [START_REF] Vilcek | Molecular characterization of ovine pestiviruses[END_REF]. This genetic classification is not unlikely considering the expected continued field circulation of UK BDVs since they were isolated 15-30 years ago. Based on the partial 5'-UTR sequences, the best sequence match for the bovine BDVs was for the group of three BDV-1b"s with a Scottish isolate obtained 20 years ago, whereas the remaining two bovine BDVs were allocated to two different groups of BDV-1a"s. The same genotyping pattern was seen in the N pro based phylogram, with sufficient statistical support to verify the 5'-UTR based classification. Direct comparison of N pro sequences of the bovine BDVs with their closest matches in the 5'-UTR phylogram was not possible since the latter N pro sequences were not available from GenBank. Interestingly, the Australian V-TOB BDV isolate, reported to have originated from a case of acute mucosal disease, and being antigenically distinct from contemporary BVDV isolates [START_REF] Snowdon | Mucosal Disease: its incidence and diagnosis in Australia[END_REF], is also a BDV-1a, but genetically different from the bovine BDVs characterised in this paper.

Based on the relatively high genetic similarity of the isolates 1062689, 1502304 and 1505744, it is possible that they might have emerged from the same parental virus strain, although the differences in the MAB reactivity patterns did not support this. On the other hand, from the more similar MAB profile of isolates 1118212 and 1376527 one might assume they are more closely related than the bovine BDV-1b isolates, but the genotyping does not support this either. However, it should be noted that the genetic typing is based on non-coding nucleotide sequences, as opposed to antigenic properties that to a large extent reside in structural glycoproteins involved with host cell receptor binding.

Recently a wide genetic diversity of BDV isolates has been described. Although all current BDV reference strains belong to the BDV-1 cluster [START_REF] Thiel | Family Flaviviridae[END_REF], several new genetic groups have now been described, most recently in southern Europe and around the Mediterranean [START_REF] De Mia | Genetic characterization of a caprine pestivirus as the first member of a putative novel pestivirus subgroup[END_REF][START_REF] Thabti | Detection of a novel border disease virus subgroup in Tunisian sheep[END_REF][START_REF] Valdazo-Gonzalez | Genetic and antigenic typing of border disease virus isolates in sheep from the Iberian Peninsula[END_REF] A c c e p t e d M a n u s c r i p t

11 [START_REF] Dubois | Genetic characterization of ovine pestiviruses isolated in France, between 1985 and 2006[END_REF][START_REF] Oguzoglu | Border disease virus (BDV) infections of small ruminants in Turkey: A new BDV subgroup?[END_REF]. To some extent there appears to be a geographical bias in the distribution of BDVs in sheep. In the UK, BDV-1 is the only genetic group that has been found [START_REF] Vilcek | Molecular characterization of ovine pestiviruses[END_REF], but this group has also been identified in Australasia and the Americas. The genetic groups BDV-2 to -6 have only been described regionally in Germany, Switzerland and Austria, Spain and France, respectively [START_REF] Becher | Genetic and antigenic characterization of novel pestivirus genotypes: implications for classification[END_REF][START_REF] Stalder | Genetic heterogeneity of pestiviruses of ruminants in Switzerland[END_REF][START_REF] Krametter-Froetscher | Influence of Communal Alpine Pasturing on the Spread of Pestiviruses among Sheep and Goats in Austria: First Identification of Border Disease Virus in Austria[END_REF][START_REF] Valdazo-Gonzalez | Genetic and antigenic typing of border disease virus isolates in sheep from the Iberian Peninsula[END_REF][START_REF] Dubois | Genetic characterization of ovine pestiviruses isolated in France, between 1985 and 2006[END_REF]. Of these, only BDV-1 and BDV-3 have been isolated from cattle in Australia, the UK, Italy and Austria [START_REF] Snowdon | Mucosal Disease: its incidence and diagnosis in Australia[END_REF][START_REF] Cranwell | Detection of Border disease virus in cattle[END_REF][START_REF] Schirrmeier | Border disease virus infection in cattle: Epidemiological and diagnostic impact[END_REF][START_REF] Hornberg | Genetic diversity of pestivirus isolates in cattle from Western Austria[END_REF], and on each occasion the bovine BDV isolates were of the common local genotype. Thus it seems like the BDV strain is less important than how sheep and cattle are managed. A sufficiently close epidemiological contact between the two animal species appears to be the most important risk factor for interspecies transfer. In the UK, sheep and cattle are kept in relatively close contact on many farms, including the majority of the cases investigated here. It is not unlikely that this also is a contributing factor to the high prevalence of BVDV in sheep in the UK [START_REF] Vilcek | Molecular characterization of ovine pestiviruses[END_REF]; if so, detection of BVDV in sheep in other countries could be an indicator of likely presence of local BDVs in cattle as well.

When a pestivirus is isolated from particularly a novel host animal, it is important to ensure that the virus actually originated from the animal and is not a laboratory contamination. Cell cultures and foetal calf serum used to supplement cell culture medium have frequently been

shown to be contaminated with BVDV [START_REF] Bolin | Survey of cell lines in the American Type Culture Collection for bovine viral diarrhea virus[END_REF][START_REF] Sandvik | Detection and identification of ruminant and porcine pestiviruses by nested amplification of 5' untranslated cDNA regions[END_REF][START_REF] Schirrmeier | Genetic and antigenic characterization of an atypical pestivirus isolate, a putative member of a novel pestivirus species[END_REF]. This may be one explanation of the general high prevelence of pestivirus infections worldwide, and also of some instances where the origin of sets of isolates has been questioned [START_REF] Vilcek | Molecular characterization of ovine pestiviruses[END_REF][START_REF] Vilcek | A RT PCR assay for the rapid recognition of border disease virus[END_REF]. This potential problem can be overcome by detection of pestiviruses by RT-PCR directly in original sample material, and with identity confirmation by partial sequencing of PCR products. Following this approach, Detection of natural infection of cattle with BDV highlights the needs for BVDV diagnostic tests that are able to detect all three ruminant pestiviruses; BVDV-1 and -2 as well as BDV.

The majority of the cases described here were originally diagnosed as BVDV antigen ELISA positive, using an assay specific for the NS2-3 antigen. Being the most conserved of all pestivirus proteins, NS2-3 is the most likely viral antigen to be detected by broadly reactive MABs. Recently BVDV antigen ELISAs targeting the E rns glycoprotein have become more popular. It may be difficult to know to what extent E rns ELISAs will detect bovine BDVs, since the antigenic profile of the latter differed from BDV reference strains. Diagnostic RT-PCR assays generally have a high sensitivity, and most assays targeting the 5"-UTR by use of Pestivirus generic primers should be able to amplify BDV cDNA, but it is important to ensure that the fluorogenic detection probes in use also recognise BDV.

Lastly, from a disease control point of view, it would be of interest to assess to what extent inactivated BVDV vaccines are able to provide immunoprotection against BDVs, of both ovine and bovine origin. Since sheep previously have been known to be infected with BVDV, sheep should not be considered a greater risk for BVD control programmes after natural infection of cattle with BDV has been described, since all domestic inter-ruminant contact should be considered a risk for successful eradication of BVD. ovine BDVs [START_REF] Vilcek | Molecular characterization of ovine pestiviruses[END_REF], and representatives for other BDV genetic groups [START_REF] Valdazo-Gonzalez | Genetic and antigenic typing of border disease virus isolates in sheep from the Iberian Peninsula[END_REF][START_REF] Dubois | Genetic characterization of ovine pestiviruses isolated in France, between 1985 and 2006[END_REF]. BVDV strain NADL was used as outgroup species.

Conclusion

5

  During recent screening for exotic pestiviruses in UK cattle samples positive for BVDV antigen, six cases were found to be BDV positive by TaqMan RT-PCR. Here we describe the genetic and antigenic typing of five bovine BDV isolates from the UK.

  BDVs sequence identities ranged from 97.1 to 88.5 %. A partial 5'-UTR nucleotide sequence (140 nucleotides) derived from a PCR amplicon of the virus isolation

  the five bovine BDVs characterised in this study although genetically distinguishable were allocated to three different phylogenetic clusters, it is highly likely that BDV has jumped host species from sheep to cattle on at least three different occasions in the UK. This route of infection is supported by the findings of[START_REF] Schirrmeier | Border disease virus infection in cattle: Epidemiological and diagnostic impact[END_REF] and[START_REF] Hornberg | Genetic diversity of pestivirus isolates in cattle from Western Austria[END_REF], who both also detected BDV-3 directly in blood samples by RT-PCR and sequencing.

  five BDV-1s from naturally infected cattle has indicated that they are likely to have jumped host species from sheep to cattle within the UK. These bovine BDVs showed an antigenic profile differing from that of ovine BDVs, with lower reactivity to BDV specific MABs, whilst they were expressing epitopes normally only seen in BVDV-1.The previously assumed natural resistance of cattle against infection with BDV therefore no longer holds.

Fig. 1 .

 1 Fig. 1.

Fig. 2 .

 2 Fig. 2.Unrooted phylogenetic tree based on full N pro sequences (504 nucleotides) of five UK bovine

Fig. 3 .

 3 Fig. 3.

  Figure 2

Table 1 .

 1 Case history details of bovine border disease virus isolates characterised in this study.

	Isolate	Year	County	Clinical signs, age 1	Contact with sheep	Sample	GenBank Accession no.
							5'-UTR	N pro
	1062689	2006	West Yorkshire	Diarrhoea / MD, 30	Not on farm of origin	Blood	EU887952	EU887957
	1118212	2006	Clywd	Diarrhoea / wasting, 12	Pet lambs	Blood	EU887953	EU887958
	1376527	2007	Powys	Neonatal death, 0	250 ewes on farm	Thymus	EU887954	EU887959
	1502304	2008	Buckinghamshir	Diarrhoea / wasting, 15	2000 ewes on farm	Blood	EU887955	EU887960
			e
	1505744	2008	Durham	Diarrhoea / wasting, 24	Sheep on farm	Blood	EU887956	EU887961
	1 in months

Acknowledgements

Thanks to the following VLA veterinary investigation officers for following up and obtaining case history data; M.C. Cranwell, C. Featherstone, K.L. Foyle, R. Hogg, P. Holmes, J.P.

Hutchinson, A. Otter and K. Whitaker. Also to J. Errington (VLA-Penrith) for the initial identification of cases, and for providing partial sequence data for the virus isolation negative case, to J. Peake (VLA Weybridge) for assistance with MAB typing, and DEFRA for funding this work under research contract OD0345.


Henrik Hasman

A Moodley

L Guardabassi

M Stegger

R L Skov

F M Aarestrup

spa type distribution in Staphylococcus aureus originating from pigs, cattle and poultry

A c c e p t e d M a n u s c r i p t -2 -Abstract Methicillin resistant S. aureus (MRSA) of clonal complex 398 (CC398) is emerging globally among production animals such as cattle, pigs and poultry as well as among humans. However, little is known about the prevalence of CC398 among methicillin sensitive S. aureus (MSSA) or the relative clonal distribution of S. aureus isolated from these three animal reservoirs. To study this, we have analyzed a random sample of S. aureus consisting of 296 epidemiologically unrelated isolates from infections and colonization of pigs, cattle and poultry. These were examined and compared by spa and multi-locus sequence typing (MLST) and the result was compared to the most common spa types found among human blood isolates. Little overlap in spa types was seen between isolates from the three animal reservoirs or between animals and humans.

Most of the porcine isolates had the spa types t034 (CC398), t1333 (CC30) and t337 (CC9), while the bovine isolates mainly had spa types t518 (CC50), t524 (CC97) and t529 (CC151). None of these spa types are common among human blood isolates in Denmark. Surprisingly, almost all of the poultry isolates (96%) belonged to CC5 (spa types t002 and t306), which is also known to be commonly found among human blood isolates and subsequent pulsed field gel electrophoresis (PFGE) analysis identified indistinguishable PFGE patterns among a poultry isolate and selected human isolates. In conclusion, strains of MSSA CC398 were commonly present in pigs but not present at all in the other reservoirs tested.

A c c e p t e d M a n u s c r i p t Introduction Staphylococcus aureus is an opportunistic pathogen and a frequent coloniser of many animal species, including humans. In addition to colonizing various hosts, it can cause a wide range of different infections ranging from dermatitis, pneumonia, septicaemia to osteomyelitis and meningitis in humans and swine as well as bovine mastitis in cattle and bumblefoot disease in poultry [START_REF] Quinn | Staphylococcus species[END_REF].

It has generally been thought that S. aureus from different reservoirs were host-specific [START_REF] Devriese | Characteristics of Staphylococcus aureus strains isolated from different animal species[END_REF][START_REF] Devriese | A simplified system for biotyping Staphylococcus aureus strains isolated from animal species[END_REF][START_REF] Sung | Staphylococcus aureus host specificity: comparative genomics of human versus animal isolates by multi-strain microarray[END_REF]. However, recently human carriage and infections has increased in especially pig farmers in many countries by a specific lineage (ST398/CC398) of methicillin-resistant S. aureus originating from livestock animals [START_REF] Huijsdens | Community-acquired MRSA and pigfarming[END_REF][START_REF] Lewis | Pigs as source of methicillin-resistant Staphylococcus aureus CC398 infections in humans, Denmark[END_REF][START_REF] Smith | Methicillin-resistant Staphylococcus aureus (MRSA) strain ST398 is present in midwestern U.S. swine and swine workers[END_REF].

A large number of different typing methods, such as MLST, ribotyping, PFGE and Staphylococcal protein A (spa) typing have been used in epidemiological investigations of S. aureus from humans and several animal species [START_REF] Blumberg | Epidemiologic typing of Staphylococcus aureus by DNA restriction fragment length polymorphisms of rRNA genes: elucidation of the clonal nature of a group of bacteriophagenontypeable, ciprofloxacin-resistant, methicillin-susceptible S. aureus isolates[END_REF][START_REF] Melles | Comparison of multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and amplified fragment length polymorphism (AFLP) for genetic typing of Staphylococcus aureus[END_REF][START_REF] Monecke | Microarray based study on virulence-associated genes and resistance determinants of Staphylococcus aureus isolates from cattle[END_REF]. It is generally agreed that MLST today is the method that gives most reliable information about the phylogenetic clustering of S. aureus isolates [START_REF] Feil | How clonal is Staphylococcus aureus?[END_REF][START_REF] Turner | The secret life of the multilocus sequence type[END_REF]. However, the discriminatory power is still relatively low compared to other typing methods such PFGE and spa typing. Therefore, the most widely used method today for first line typing and epidemiological studies is spa typing [START_REF] Frenay | Molecular typing of methicillin-resistant Staphylococcus aureus on the basis of protein A gene polymorphism[END_REF]. This method allows easy and rapid characterisation of isolates and at the same time gives robust evidence for the phylogenetic relationship, since spa types are normally associated to specific MLST types [START_REF] Strommenger | Assignment of Staphylococcus isolates to groups by spa typing, SmaI macrorestriction analysis, and multilocus sequence typing[END_REF].

In this study we investigated the clonal diversity, as determined by spa and supportive MLST typing, of a random sample of 296 epidemiologically unrelated S. aureus isolates from cattle, pigs and poultry in Denmark. Here, we provided evidence at the molecular level of the host-preference of different S. aureus lineages and also showed that MSSA ST398 is the predominant S. aureus

Material and methods

Bacterial isolates

Clinical S. aureus isolates included in this study were randomly sampled from diagnostic submissions sent from all over Denmark for identification and antimicrobial susceptibility testing to either the National Food Institute -DTU or KU-Life between 1993 and 2007. In total 101, 46 and 85 clinical isolates from cattle, pigs and poultry, respectively, were analyzed. Colonizing S. aureus isolates (nasal carriage) were either sampled at individual farms from healthy animals or sampled as part of the DANMAP surveillance program [START_REF] Danmap | DANMAP 2007 -Consumption of antimicrobial agents and occurence of antimicrobial resistance from food animals, food and humans in Denmark[END_REF] in 2007 from healthy animals at the time of slaughter. In total, 11 and 53 isolates from colonization of cattle and pigs, respectively, were included in this study. The criteria used in the strain selection process of both clinical and colonizing isolates excluded duplicate farm isolates in order to minimize sampling bias. All isolates from the National Food Institute -DTU were screened phenotypically for susceptibility towards the 3rd generation cephalosporin ceftiofur as MIC determinations using a commercially prepared, dehydrated panel (Sensititre), while all isolates from KU-LIFE were screened for methicillin resistance using ORSAB screening agar (Oxoid).

In Denmark, human S. aureus bacteremia isolates (MSSA as well as MRSA) identified at any hospital has to be submitted to the National Staphylococcal laboratory at Statens Serum Institute (SSI) for further analysis. In 2007, a total of 1356 isolates were isolated in Denmark and subjected to spa typing as described below. In addition, human MRSA isolates from all hospitals in Denmark have for many years routinely been collected at the National Staphylococcal laboratory at SSI and analyzed by PFGE SmaI digestion.

A c c e p t e d M a n u s c r i p t -6 -spa typing Amplification of the spa repeat region was performed using primers spa 1113f (5'-AAAGACGATCCTTCGGTGAGC-3') and spa 1514r (5'-CAGCAGTAGTGCCGTTTGCTT-3') with assay conditioned as described by SeqNet (www.SeqNet.org) and using 1 unit of VWR Taq DNA polymerase (VWR International) per reaction. The spa types were determined based on the sequencing results using the spa plugin included in the Bionumerics v4.6 software (Applied Math).

MLST typing

MLST was performed by the method previously described by Enright et al. [START_REF] Enright | Multilocus sequence typing for characterization of methicillin-resistant and methicillin-susceptible clones of Staphylococcus aureus[END_REF] on at least one isolate from all spa types with more than two isolates. In case, where isolates with the same spa sequence originated from different animal groups, at least one isolate originating from each animal group were analysed. MLST Sequence Types (ST) were assigned through the MLST database (http://www.mlst.net). The eBURST algorithm was used to assign individual STs to specific CC types (http://eburst.mlst.net).

PFGE

PFGE was performed as described previously [START_REF] Murchan | Harmonization of pulsed-field gel electrophoresis protocols for epidemiological typing of strains of methicillin-resistant Staphylococcus aureus: a single approach developed by consensus in 10 European laboratories and its application for tracing the spread of related strains[END_REF] on selected Danish poultry isolates and Danish human MRSA isolates displaying spa type t002.

PCR

The presence of the mecA gene in suspected methicillin-resistant isolates was tested genotypically using a multiplex PCR directed against the 16S ribosomal RNA, the mecA gene and the nucA gene as described previously [START_REF] Maes | Evaluation of a triplex PCR assay to discriminate Staphylococcus aureus from coagulase-negative Staphylococci and determine methicillin resistance from blood cultures[END_REF].

A c c e p t e d M a n u s c r i p t -7 -

Results

A total of 296 S. aureus isolates originating from cattle, pigs and poultry in Denmark were included in this study. Among these, 48 different spa types were identified. Subsequently, MLST analysis was performed on representative isolates from the predominant (more than two isolates within a spa type) of these spa types. Based on this, sequence types and clonal complexes (CCs) was inferred through the MLST database (www.mlst.net) using the eBURST algorithm. This resulted in grouping the spa types into 12 different clonal complexes (CC's) as well as two singletons (Table 1).

The porcine isolates (n=99) mainly clustered into three clonal complexes, namely CC398 (39%), CC30 (29%) and CC9 (27%). Of these, only one was found to be mecA positive and this isolate belonged to ST398/CC398. This isolate as well as most of the other CC398 isolates belonged to spa type t034, thus being the most predominant spa type among MSSA isolated from pigs in Denmark. Among the remaining two predominant CC's found among pigs, a single spa type in each CC were again dominating, namely t1333 of CC30 and t337 of CC9. Forty-six percent of the porcine isolates originated from infections and 54% from colonisation of the nostrils. There was no evident difference in the spa type distribution between isolates from infections and colonisation (Table 1).

The bovine isolates (n=112) were all methicillin susceptible, and again three major CC's were observed, but here three different clonal lineages CC50 (31%), CC97 (28%) and CC151 (16%) dominated. As with the porcine isolates, each of the three CC's were mainly associated with a specific spa type, namely the t518 of CC50, t524 of CC97 and t529 of CC151 (Table 1).

Interestingly, several of the remaining isolates belonged to CC9, one of the main porcine CC's.

However, different spa types dominated in the CC9 isolates from the two animal reservoirs i.e. spa A c c e p t e d M a n u s c r i p t -8 -types t1419 and t1430 were most dominant among the bovine CC9 isolates and t337 were the dominant porcine type although there was some overlap (marked in bold in Table 1). In particular, isolates with the spa type t899 was found in both reservoirs with low prevalence. We therefore tested the three t899 isolates by MLST and found these to belong to ST9, which is within the CC9.

Finally, a minor fraction of the bovine isolates belonged to CC131, CC20 and CC8.

The isolates from the poultry infections (n=85), belonged almost exclusively to CC5 (96%) with the t002 as the far most dominant spa type (Table 1). The most frequent spa type in poultry (t002) was the seventh most common human spa type involved in human S. aureus blood infections in Denmark in 2007 (data not shown). To examine the clonal relationship between poultry and human t002 isolates, a subset (n=24) of the poultry isolates were subjected to PFGE using the SmaI enzyme and compared to the six most dominant PFGE types identified among the human MRSA isolates in Denmark. In general, a large diversity was seen among the poultry PFGE types, even though some isolates with indistinguishable SmaI profiles were seen (data not shown).

Interestingly, one of the poultry isolates had a PFGE profile identical to one of the most common human PFGE types, indicating that some poultry and human t002 isolates were in fact closely related.

Discussion

Recently, several studies have examined the prevalence of MRSA in pigs with special attention to the emerging ST398/CC398 clonal linage, which was first detected among pigs and has now been isolated from livestock animals worldwide [START_REF] Armand-Lefevre | Clonal comparison of Staphylococcus aureus isolates from healthy pig farmers, human controls, and pigs[END_REF][START_REF] Huijsdens | Community-acquired MRSA and pigfarming[END_REF]Lewis et al., 2009;[START_REF] Nemati | Antimicrobial resistance of old and recent Staphylococcus aureus isolates from poultry: first detection of livestock-associated methicillin-resistant strain ST398[END_REF]. The initial emergence of this clonal linage in pigs is somewhat of an enigma as this is not one of the traditional lineages associated with MRSA in A c c e p t e d M a n u s c r i p t -9 -human medicine. It could be hypothesized that this lineage was already present among pigs as MSSA and has subsequently emerged as MRSA by acquiring SCCmec cassettes through a yet unknown donor. However, the spa type distribution in a larger collection of epidemiologically independent MSSA porcine isolates has to our knowledge not been studied in detail before. A recent study of the clonal distribution of sequence types of isolates from healthy pig farmers (n=44) and pigs (n=14) from France also identified some porcine isolates belonging to CC398, CC9 and CC30 (ST433) in healthy pigs as well as in pig farmers [START_REF] Armand-Lefevre | Clonal comparison of Staphylococcus aureus isolates from healthy pig farmers, human controls, and pigs[END_REF], but the animal sample size was to small to give an indication of the relative prevalence of the isolates in general. Here we show that ST398/CC398 is a common MLST type in pigs together with two other types. Interestingly, spa type t034 of ST398 seemed to be the most dominant type among pigs in Denmark, which is also the predominant spa type of Danish MRSA isolates isolated from breeding pig as well as from pigs at the time of slaughter (data not shown) and thus supporting the hypothesis for emergence of MRSA ST398. However, this is difficult to prove unless other typing methods with higher discriminating power can be used examine the isolates further. Unfortunately, ST398 is untypable by standard PFGE, which could otherwise have been used. As other CC398-related spa types such as t011 and t108 seems to be dominant in other European countries [START_REF] Witte | Methicillin-resistant Staphylococcus aureus ST398 in Humans and Animals[END_REF]) it would also be interesting to analyse MSSA isolates or porcine origin in these countries to see, if these are correlated with the MRSA spa types found in these countries.

Several studies have examined spa types or MLST types of S. aureus isolates of bovine origin. A intercontinental MLST study of 258 bovine-associated S. aureus isolates from United States, Chile and the United Kingdom identified CC97 to be the far the most dominant clonal complex constituting a total of 87.4% of the tested isolates, however spa types were not analysed [START_REF] Smith | Multilocus sequence typing of intercontinental bovine Staphylococcus aureus isolates[END_REF]. These isolates were sampled from milk, teat skin, milking machines and various other A c c e p t e d M a n u s c r i p t -10 -locations but were not sampled from animals with clinical signs of bovine mastitis. Among our bovine sample material, which mostly originate from animals with clinical signs of mastitis (90%), we found 28% of the isolates to belong to CC97, but also found two other dominant clonal complexes (CC50 and CC151), which were only present among few of the intercontinental isolates. An explanation for the more diverse result obtained with our isolates compared to the intercontinental study could be related to this difference in sample material (healthy vs. mastitis origin) or it could be related to geographical variation of the clonal lineages. In support of the latter, bovine mastitis isolates from countries like United States and United Kingdom traditionally have a high prevalence of reduced susceptibility towards penicillin as opposed to Danish isolates, which have lower prevalence of penicillin resistant S. aureus [START_REF] Vintov | Association between phage types and antimicrobial resistance among bovine Staphylococcus aureus from 10 countries[END_REF] A recent study from Switzerland and Germany on 128 bovine mastitis isolates [START_REF] Monecke | Microarray based study on virulence-associated genes and resistance determinants of Staphylococcus aureus isolates from cattle[END_REF] identified 20 different subgroups based on microarray analysis and found CC97 and CC151 to be the most predominant with other CC's being present in smaller numbers (CC8, CC20, CC398, CC133, CC5 and CC1). Interestingly, most of these CC's were also present among the isolates examined in our study, even though the prevalence of the individual CC's does differ between the two studies.

As only a limited number of the bovine isolates included in our study originated from colonisation of the animal nares, it is difficult to evaluate if the same relative distribution of spa types was found among isolates from infections and colonisation. However, nothing indicates that this should not be the case, as the spa types of isolates associated with colonisations were all found among the mastitis isolates as well (Table 1). Two (MSSA) isolates from pigs and one isolate from cattle had the spa type t899. This spa type has previously been assigned to both ST398 [START_REF] Van Duijkeren | Transmission of methicillin-resistant Staphylococcus aureus strains between different kinds of pig farms[END_REF][START_REF] Wulf | Prevalence of methicillin-resistant Staphylococcus aureus among veterinarians: an international study[END_REF] as well as CC9 [START_REF] Liu | Molecular evidence for spread of two major methicillin-resistant Staphylococcus aureus clones with a unique geographic distribution in Chinese hospitals[END_REF]. Our three isolates was shown here to belong to CC9 as opposed to the MRSA t899 isolates described previously [START_REF] Wulf | Prevalence of methicillin-resistant Staphylococcus aureus among veterinarians: an international study[END_REF]. To verify that isolates with spa type A c c e p t e d M a n u s c r i p t -11 -t899 in fact can belong to both clonal complexes, we repeated the spa and MLST typing of two of the t899 isolates, which were previously reported to belong to ST398 [START_REF] Wulf | Prevalence of methicillin-resistant Staphylococcus aureus among veterinarians: an international study[END_REF]. The tests confirmed that the previous isolates had been typed correctly initially and thus were indeed spa type t899 and positioned in CC398. A closer examination of the spa repeats of spa type t899 (r07r16r23r02r34) would position this type within the CC9 spa types, rather than within the CC398, which bares little similarity to the spa repeats of t899 (see Table 1). Therefore, a likely explanation for some t899 isolates to be located in CC398 is horizontal transfer of the spa gene from a t899 strain into a CC398 strain some point in time rather than horizontal transfer of the seven household genes used to determine the sequence type.

Interestingly the spa types from poultry isolates were far more clonally conserved than the spa types from the two other animal reservoirs. As the poultry isolates used in this study were submitted between 1993 and 2007 from different farms located all across Denmark and thus representing a very diverse sample material, this result indicates that there is a very narrow range of S. aureus isolates, which are involved in poultry infections.

To our knowledge this is the first time, poultry MSSA isolates has been analysed by spa and MLST typing. A recent study of poultry isolates from Belgium [START_REF] Nemati | Antimicrobial resistance of old and recent Staphylococcus aureus isolates from poultry: first detection of livestock-associated methicillin-resistant strain ST398[END_REF] found a relatively large proportion (12%) of Belgian poultry isolates to be MRSA ST398 (spa types t011 and t567).

These were isolated at five out of 39 tested poultry farmers in 2006 whereas no MRSA ST398 was found among similar isolates obtained between 1970 and 1972. However, as only the MRSA isolates were subjected to spa typing, this study did not give any information on the spa type distribution among the remaining MSSA isolates. Therefore this result can not be used to estimate the clonal diversity of the poultry isolates studied per se, but does rather emphasize the recent spread of MRSA ST398 in other livestock reservoirs than pigs.

Few studies have investigated the clonal diversity of S. aureus among the three animal groups, which we have examined in our study, or compared them to S. aureus isolated from humans, but A c c e p t e d M a n u s c r i p t -12 -based on the antigenic pattern differences between animal and human isolates, it has previously been suggested that animal and human S. aureus isolates does appear to be antigenically different [START_REF] Oeding | Antigenic properties of Staphylococcus aureus[END_REF]. Similar results have been observed, based on phage typing of mastitis isolates [START_REF] Vintov | Association between phage types and antimicrobial resistance among bovine Staphylococcus aureus from 10 countries[END_REF]. In Denmark, all S. aureus isolates (MSSA and MRSA) involved in human blood infections at Danish hospitals collected in 2007 were spa typed at the Statens Serum Institute.

Only one of the most predominant spa types (t034) found among Danish bovine and porcine isolates are located among the top 50 human spa types from 2007 (number 46 on the list, consisting of 4 isolates out of 1356 isolates tested), indicating very little overlap between the spa types found in humans and in Danish pigs and cattle. In fact, subsequent analysis of these four t034 isolates have revealed that these were isolated from patients with close association with animal production, such as pig farmers and slaughterhouse workers [START_REF] Lewis | Pigs as source of methicillin-resistant Staphylococcus aureus CC398 infections in humans, Denmark[END_REF] An explanation for the clonal organization of S. aureus in the three animal reservoirs is not deducible from the data as it is not known whether it is caused by the fact that there is only limited contact between the different animal species in modern farming or if the different S. aureus clonal lineages have specific preferences to each of these three animal species. In the present study, however, at least one clonal lineage seems to contradict this clonal organization, as isolates belonging to CC9 (ST9) were found among both bovine and porcine isolates, even though the relative prevalence of spa types within the CC9 isolates varied between the two animal reservoirs. Also, the MRSA CC398 seems to be able to spread rapidly between different animal reservoirs.

However, based on the fact that MRSA CC398 was first isolated from pigs as well as the data presented here, where MSSA CC398 are only identified among pig isolates, it could be suggested that the MRSA CC398 initially emerged within the porcine reservoir. As CC398 does not seem to be prevalent among bovine and poultry isolates, the current spread to these animal reservoirs could be driven by a selective force. A likely candidate for this selective force would be one or more antimicrobial agents used in all three reservoirs; however, the exact antimicrobial agent has not A c c e p t e d M a n u s c r i p t -13 -been identified yet and will await further investigations. Tetracycline has been suggested previously as most CC398 MRSA isolates appears to be resistant to tetracycline, however, tetracycline susceptibility testing of the MSSA CC398 isolates indentified in this study showed that they were also resistant to tetracycline (data not shown). Therefore this particular antimicrobial agent would not give the MRSA CC398's a selective advantage compared to the other MSSA CC398 isolates present on the pigs.

Finally, an explanation for the low level of variation among the spa types found in poultry isolates compared to isolates from the two other animal reservoirs could be attributed to the fact that the number of hatcheries in Europe is very limited and therefore most poultry chicks originate from the same hatcheries. The link observed between poultry and human clinical isolates belonging to S. aureus lineage CC5 t002 will require further investigation to assess possible zoonotic risks associated with poultry farming.

  the pig population, but was absent among isolates of bovine and poultry origin.

Table 1 :

 1 Prevalence data of the spa and MLST types among the 296 isolates of porcine, bovine and poultry origin included in this study. Spa types, which are present in more than one animal group has been marked in bold.

	388
	Source	spa type	Spa repeats		Number (i/c) 1	MLST Sequence type	MLST Clonal complex
	Pigs	t034	r08r16r02r25r02r25r34r 24r25	32 (14/18)	ST398	CC398
		t571	r08r16r02r25r02r25r34	r25	2 (1/1)	ST398	CC398
		t011	r08r16r02r25	r34 r24r25	1 (1/0)	ST398*	CC398
		t108	r08r16r02r25	r24r25	1 (0/1)	ST398*	CC398
		t1255	r08r16	r34r24r25	1 (0/1)	ST398	CC398
	Cattle	t1793 t2876 t1333 t318 t631 t2840 t337 t1334 t2462 t899 t1430 t2498 t2839 t127 t1491 t230 t2678 r03r16r12r21r17r23r13r17r17r17r23r24 r08r16r02r25r02r25r34r24r24r25 r08r16r02r25r02r25r 24r25 r15r12r16r34r 02r25r17r24 r15r12r16r16r02r16r02r25r17r24 r15r12r16r 17r24 r15r12r16r34r02r25r17r24r17r24 r07r16r23 r23r02r12r23r02r34 r07r16r23 r23r02r34 r07 r23 r23r02r34 r07r16r 23r02r34 r07r16r23 r02r12r23r02r34 r07r16r16r23r23r02r12r23r02r34 r07r16r23 r23r23r02r34 r07r23r21 r16r34r33r13 r07r23r21r17r13r34r34r16r34r33r13 r08r16r02r16r34 a n u s c r i p t 1 (0/1) ST398 CC398 1 (1/0) na na 26 (13/13) ST433 CC30 1 (0/1) ST30* CC30 1 (0/1) na na 1 (1/0) na na 16 (9/7) ST9 CC9 3 (0/3) ST9 CC9 3 (0/3) ST9 CC9 2 (0/2) ST9 CC9 1 (0/1) ST9 CC9 1 (1/0) na na 1 (1/0) ST9 CC9 1 (1/0) ST1* CC5 1 (1/0) ST1* CC5 1 (1/0) ST45* CC45 1 (1/0) na na t518 r04r20r17r23r24 r20r17r25 25 (25/0) ST50 CC50 t519 r04 r20r17r25 8 (8/0) ST50 CC50 M t3463 r04r20r17r23r31r24r20r17r25 1 (1/0) na na
		t3465	r04r20r17r17r17r16r20r17r25	1 (1/0)	na	na
	t524 t529 t528 t1403 t2873 t527 r07r23r12r21r17r34r34r34r34r34r33r34 r04r17 r04r34 r04 g r03r23r24 r04r20r17r31r24 t267 r07r23r12r21r17r34r 34r34r33r34 t521 r07r23r12r21r17r34r34 r34r34r33r34 t1419 r07 r23r02r12r23r02r34 t1430 r07r16r23r02r12r23r02r34 t3464 r07r23r02r12r23r02r02r34 t899 r07 r16r23r02r34 t2839 r07r16r23r 23r23r02r34 t2875 r07r16r06r20r12r06r02r34 t008 r11r19r12r21r17r34r24r34r22r25 t731 r07r06r17r21r34r22r34 A c c e p t e d t3046 r03r16r12r21r17r23r13r17r17r17r23r25	25 (24/1) 17 (15/2) 1 (1/0) 7 (6/1) 5 (4/1) 4 (3/1) 1 (1/0) 1 (1/0) 3 (3/0) 3 (2/1) 2 (1/1) 1 (1/0) 1 (1/0) 1 (1/0) 2 (2/0) 1 (1/0) 1 (1/0)	ST71 ST151 ST151 ST133 ST479 ST97 ST97 ST97 ST9 ST9 na ST9 na ST9 ST8 ST513 na	CC97 CC151 CC151 CC133 Singleton CC97 CC97 CC97 CC9 CC9 na CC9 na CC9 CC8 CC20 na
		t3830	r14r34r17r34r50r17		1 (1/0)	na	na
	Poultry t002	r26r23r17r34r17r20r17r12r17 r16	66 (66/0)	ST5	CC5
		t306	r26r23r17r34r17r20r17r12r17r17r16	13 (13/0)	ST5	CC5
		t688	r26r23r17r34r17		r16	1 (1/0)	ST5	CC5
		t2049	r26r23r17		r17r16	1 (1/0)	na	na
		t2308	r26r23r17r34r17r17r20r17r12r17r16	1 (1/0)	na	na
		t213	r07r23r12r21r24r33r22r17	2 (2/0)	ST385	Singleton
		t304	r11r10r21r17r34r24r34r22r25	1 (1/0)	ST8*	CC8
				-19 -

. (i/c) = (infections/colonization), na = not available, * = ST type retrieved from the Rindom 389 spaServer.

Acknowledgements

The authors wish to thank our technicians for their excellent help. This study was funded by a grant 274-05-0117 from the Danish Research Agency.

Conflict of interest

None to declare

Role of the funding source

The grant sponsor had no involvement in the any step of this manuscript.

Ethics

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


Sylvia S Reemers

Edwin J A Veldhuizen

Cherina Fleming

Daphne A Van Haarlem

Henk Haagsman

Lonneke Vervelde
email: l.vervelde@uu.nl

Transcriptional expression levels of chicken collectins are affected by avian influenza A virus inoculation

Keywords: avian influenza virus, immune response, C-type lectin, innate immunity

Mammalian collectins have been found to play an important role in the defense against influenza A virus H9N2 inoculation, but for chicken collectins this has not yet been clarified. The aim of this study was to determine the effect of avian influenza A virus (AIV) inoculation on collectin gene expression in the respiratory tract of chickens and whether this was affected by age. For this purpose 1-and 4-week-old chickens were inoculated intratracheally with PBS or H9N2 AIV.

Chickens were killed at 0, 8, 16 and 24 h post inoculation and trachea and lung were harvested for analysis. Viral RNA expression and mRNA expression of chicken collectins 1 and 2 (cCL-1 and cCL-2), chicken lung lectin (cLL) and chicken surfactant protein A (cSP-A) were determined using real-time quantitative RT-PCR. In lung, a decrease in mRNA expression of cCL-2, cLL and cSP-A after inoculation with H9N2 was seen in both 1-and 4-week-old birds, although at different time points, while in trachea changes were only seen in 4-week-old birds and expression was increased. Moreover, collectin expression correlated with viral RNA expression in lung of 1week-old birds. These results suggest that both age and location in the respiratory tract affect changes in collectin mRNA expression after inoculation with H9N2 and indicate a possible role for collectins in the host response to AIV in the respiratory tract of chickens.

Introduction

The first line of defense against invading pathogens in the respiratory tract is provided by the innate immune system. This phylogenetically ancient system does not require previous exposure to a pathogen and acts within hours after the first encounter. Especially in the early A c c e p t e d M a n u s c r i p t 3 stages of life, the host is highly dependent on innate immunity for protection because the adaptive immune system is not fully developed yet. An important group of effector molecules of the innate immune system comprises collectins, proteins characterized by a collagen-and a C-type lectin domain. These proteins are pattern recognition molecules that can bind and neutralize a wide array of pathogens including bacteria and viruses (reviewed by [START_REF] Hogenkamp | Collectins -Interactions with pathogens[END_REF].

For mammalian collectins, several in vitro studies have described a strong neutralizing activity against influenza A virus [START_REF] Hartshorn | Mechanisms of antiinfluenza activity of surfactant proteins A and D: comparison with serum collectins[END_REF]Van Eijk et al., 2003;[START_REF] Hogenkamp | Chicken Lung Lectin is a functional C-type lectin and inhibits haemagglutination by Influenza A Virus[END_REF]. Collectins are thought to bind carbohydrate residues present on influenza hemagglutinin and neuraminidase, thereby aggregating viral particles and preventing invasion of host cells [START_REF] Reading | Glycosylation as a target for recognition of influenza viruses by the innate immune system[END_REF]. In addition, a highly reduced clearance of influenza virus was observed in mice lacking either surfactant protein A or surfactant protein D, implicating the importance of these mammalian collectins in defense against influenza virus inoculations in vivo [START_REF] Sano | The lung collectins, SP-A and SP-D, modulate pulmonary innate immunity[END_REF]. In chicken, five collectins have been described thus far, which are mannan binding lectin [START_REF] Laursen | Cloning and sequencing of a cDNA encoding chicken mannan-binding lectin (MBL) and comparison with mammalian analogues[END_REF], surfactant protein A (cSP-A) and chicken collectins 1, 2 and 3 (cCL-1-3) [START_REF] Hogenkamp | Characterization and expression sites of newly identified chicken collectins[END_REF]. In addition, a chicken lung lectin (cLL) with high sequence homology to cSP-A was reported. Whether chicken collectins can protect against influenza A virus inoculation has not been clarified, but cLL has been shown to possess in vitro hemagglutination inhibiting activity [START_REF] Hogenkamp | Chicken Lung Lectin is a functional C-type lectin and inhibits haemagglutination by Influenza A Virus[END_REF].

Avian influenza virus (AIV) enters the respiratory epithelium using sialic acid receptors linked to galactose by an α-2,3 linkage, although the pattern of receptor distribution in chickens is less defined than in mammals (Wan and Perez, 2006). H9N2 AIV has been shown to have a preference for infecting the upper part of the respiratory tract [START_REF] Nili | Natural cases and an experimental study of H9N2 avian influenza in commercial broiler chickens of Iran[END_REF]. After spray inoculation, viral replication was detected at 24 hours post inoculation in both trachea and lung of chickens. Virus was first detected in the epithelium of the trachea, intrapulmonary bronchus and A c c e p t e d M a n u s c r i p t 4 in adjacent parabronchi, and thereafter spread to other parabronchi deeper in the lung [START_REF] Reemers | Differential gene expression and host response profiles against avian influenza virus within the chicken lung due to anatomy and airflow[END_REF].

The aim of the current work was to determine changes in gene expression of chicken collectins upon AIV inoculation in the respiratory tract of 1-and 4-week-old chickens and the effect of age on collectin gene expression after AIV inoculation.

Materials and methods

Inoculation model

AIV, subtype H9N2, isolate A/Chicken/United Arab Emirates/99 was kindly provided by Intervet Schering-Plough Animal Health, Boxmeer, The Netherlands. The experiment was carried out according to protocols approved by the Animal Experiment Committee of Utrecht University (The Netherlands). One-week and four-week-old Lohmann Brown layer chickens were inoculated intratracheally with either 0.1 ml PBS or 10 7.7 EID 50 H9N2. Before inoculation 5 control chickens per age were killed and at 8, 16 and 24 h post inoculation (p.i.) 5 control and 5 H9N2 inoculated chickens per age were killed. The cranial part of the lung containing the intrapulmonary bronchus and the upper trachea were used for analysis.

Real-time quantitative RT-PCR (qRT-PCR)

Lung and trachea were homogenized and total RNA was isolated using the RNeasy Mini Kit and DNase treated (Qiagen Benelux B.V., Venlo, The Netherlands) as described previously [START_REF] Reemers | Differential gene expression and host response profiles against avian influenza virus within the chicken lung due to anatomy and airflow[END_REF]. cDNA was generated with reverse transcription using iScript cDNA Synthesis Kit (Biorad Laboratories B.V., The Netherlands).

Detection of GAPDH, H9 hemagglutinin (HA) and 28S products was performed as described by [START_REF] Reemers | Differential gene expression and host response profiles against avian influenza virus within the chicken lung due to anatomy and airflow[END_REF]. Real-time qRT-PCR was performed for cCL-1, cCL-2, cLL and cSP-A A c c e p t e d M a n u s c r i p t 5 using primers and probes depicted in Table 1 according to the following cycle protocol: 2 min at 50°C, 10 min at 95°C (denaturation); 40 cycles: 15 s at 95°C and 60 s at 60°C. GAPDH was used as a reference gene for correction of viral HA RNA expression and 28S as reference gene for collectin mRNA expression. Corrections for variation in RNA preparation and sampling were performed as previously described [START_REF] Reemers | Differential gene expression and host response profiles against avian influenza virus within the chicken lung due to anatomy and airflow[END_REF]. Results are expressed in terms of the threshold cycle value (Ct) and given as corrected 40-Ct values.

Statistical analysis

Significance between H9N2 inoculated and control samples and between age groups within a time point was determined with an ANOVA. Significance between time points within age groups and treatment groups was determined with an ANOVA and Tukey post-hoc test. Correlation between viral RNA and collectin mRNA expression was based on the Pearson correlation coefficient (r) and determined using SPSS 15.0 software. Data were expressed as means with standard error of the mean (SEM). A p-value < 0.05 was considered significant.

Results

Viral RNA expression in lung and trachea

Viral RNA expression was detected in lung and trachea of all H9N2 inoculated birds (Fig. 1).

There was no significant difference in viral RNA levels in lung between 1-and 4-week-old birds within a time point or between time points within an age group. In trachea there was no significant difference between 1-week and 4-week-old birds within a time point. Only the decline of viral RNA expression in trachea of 1-week-old birds at 24 h p.i. was significantly different from the expression at 16 h p.i. Expression of cCL-1, cCL-2, cLL and cSP-A mRNA was determined in individual birds at all time points in both lung and trachea (Fig. 2 and3). cCL-2, cLL and cSP-A mRNA levels in lung of H9N2 inoculated birds were generally lower compared to control birds. In 1-week-old birds, cCL-2, cLL and cSP-A mRNA levels were significantly decreased after H9N2 inoculation at 16 h p.i. In 4-week-old birds, there was a significant decrease in mRNA levels of cCL-2 at 8 h, cSP-A at 16 h and cLL at 24 h p.i. between lung of H9N2 inoculated and control birds.

Although expression in lung was mostly decreased after H9N2 inoculation, collectin mRNA expression in trachea was mostly increased in H9N2 inoculated birds. The cCL-1 mRNA level was significantly higher at 16 h p.i. in H9N2 inoculated birds compared to control birds in trachea of both 1-and 4-week-old birds. In 4-week-old birds, cCL-2, cLL and cSP-A mRNA levels were significantly increased at 8 h p.i. after H9N2 inoculation. All observed changes were between 1 and 2 Ct values corresponding to a 2-4 fold change in mRNA levels.

Correlations between viral RNA and collectin mRNA expression in lung and trachea

First the correlation between viral RNA and collectin mRNA expression was determined per age group for all collectins in lung and trachea over time. If a correlation is found this means that viral RNA expression and collectin mRNA expression correlate at every time point. There was no significant correlation between viral RNA expression and mRNA expression of any of the collectins in lung and trachea of 4-week-old birds over time. No significant correlation was seen in trachea of 1-week-old birds for any of the collectins, however in lung there was a significant strong negative correlation over time between viral RNA expression and mRNA expression of cCL-2 (r=-0.668, p=0.007), cLL (r=-0.655, p=0.008) and cSP-A (r=-0.657, p=0.008).

A c c e p t e d M a n u s c r i p t 7 Although no correlation was found between viral RNA and collectin mRNA expression in trachea of 4-week-old birds over time, there was a significant difference in collectin mRNA expression after H9N2 inoculation at certain time points. Only cCL-2 mRNA expression correlated with viral RNA expression at 8 h p.i..

Discussion

The aim of this study was to determine changes in chicken collectin gene expression due to AIV inoculation in the chicken respiratory tract and whether this was affected by age. Collectins have only relatively recently been described in the chicken and their role in the innate immune response is largely unknown. Based on sequence homology and gene expression profiles, two chicken homologues for SP-A were found: cSP-A and cLL. However, the chicken genome did not seem to contain a homologue for the second pulmonary collectin SP-D. In addition, chicken collectins 1, 2 and 3 were found which resemble the mammalian collectin liver 1 (CL-L1) [START_REF] Ohtani | Molecular cloning of a novel human collectin from liver (CL-L1)[END_REF], collectin kidney 1 (CL-K1) [START_REF] Keshi | Identification and characterization of a novel human collectin CL-K1[END_REF] and collectin placenta 1 (CL-P1) [START_REF] Ohtani | The membrane-type collectin CL-P1 is a scavenger receptor on vascular endothelial cells[END_REF]. The biological roles of CL-L1 and CL-K1 have not been determined yet, leaving no indication for a possible role of chicken collectins cCL-1 and cCL-2. However, both cCL-1 and cCL-2 are expressed in the chicken respiratory tract [START_REF] Hogenkamp | Collectins -Interactions with pathogens[END_REF]. The cCL-3 homologue CL-P1 is different from other collectins in the way that it is a type II membrane protein. It acts as a scavenger receptor on endothelial cells, while all other collectins are soluble effector molecules involved in direct neutralization of pathogens. A role for cCL-3 in direct neutralization of viruses in the lung is therefore unlikely.

Based on this similarity to mammalian collectins and the location of expression in the chicken we investigated the changes in mRNA expression of cSP-A, cLL, cCL-1 and cCL-2 after an H9N2 AIV inoculation in the chicken respiratory tract. In the lung of 1 week-old birds, a down A c c e p t e d M a n u s c r i p t 8 regulation was observed for cLL, cSP-A and cCL-2 mRNA, at 16 h p.i. This time point significantly correlated to the observed peak in viral load in the lung. In addition the expression of cCL-2 and cSP-A mRNA was down regulated in 4-week-old birds at 8 and 16 h p.i., but no correlation to viral RNA expression was found. This indicates that expression of cSP-A, cLL and cCL-2 mRNA expression is affected by H9N2 inoculation early after inoculation and in 1-weekold birds is directly related to viral RNA expression in lung. Interestingly, in the trachea, all significant changes due to H9N2 inoculation were related to up regulation of collectin mRNA expression, suggesting host collectin responses after H9N2 inoculation are site specific. cCL-1 was up regulated at 16 h p.i. in trachea of both 1-and 4-week-old birds, while cCL-1 mRNA expression was not affected by H9N2 inoculation in lung. This suggests that changes of cCL-1 mRNA expression after H9N2 inoculation are dependent on the location in the respiratory tract. cCL-2 expression was affected by H9N2 inoculation in 4-week-old birds at 8 h p.i. in both lung and trachea, although down regulated in lung and up regulated in trachea, indicating that cCL-2 mRNA expression changes time specifically at 8 h p.i. after H9N2 inoculation. In lung, changes in mRNA expression of cCL-2, cLL and cSP-A after H9N2 inoculation were seen in both 1-and 4-week-old birds, although at different time points, while in trachea changes were only seen in 4week-old birds. This suggests that both age and location in the respiratory tract affect changes in collectin mRNA expression after H9N2 inoculation, though one has to keep in mind that changes found at early time points are caused by the virus inoculum whereas at 24 h.p.i. viral replication might affect the outcome. In mammals site specific expression and secretion of collectins in the airways has been reported [START_REF] Coalson | Site specificity of surfactant protein expression in airways of baboons during gestation[END_REF][START_REF] Rooney | Regulation of surfactant secretion[END_REF]. Furthermore, down regulation of SP-A gene expression through p38 MAPK and PI-3 kinase pathways have been described in lung epithelial cells (Miakotina et al., 2002a;Miakotina et al., 2002b). Epithelial cells in the lung differ from tracheal ciliated epithelial cells supporting the possibility of site specific collectin Without knowing the exact function of chicken (col)lectins in innate defense , it is difficult to relate the observed changes in gene expression to a biological effect. In mammals, an up regulation of collectins has frequently been observed in both bacterial and viral infections [START_REF] Murray | Expression of surfactant protein D in the human gastric mucosa and during Helicobacter pylori infection[END_REF][START_REF] Grubor | Enhanced surfactant protein and defensin mRNA levels and reduced viral replication during parainfluenza virus type 3 pneumonia in neonatal lambs[END_REF], which is usually interpreted as a direct response of the host to increase its defense against the incoming pathogens. A down regulation of collectins upon infection is less frequently reported, but could reflect a survival strategy of the pathogen to overcome the host's hostile environment, as has been described for other innate immune proteins such as Toll like receptors and defensins (Wang et al., 2000;[START_REF] Shin | Escape from immune surveillance by Capnocytophaga canimorsus[END_REF]. However, whether this applies to AIV in the chicken lung and why the altered expression of chicken (col)lectins is tissue specific is not clear at this moment and requires further investigation.

In conclusion, the results obtained in this study show that mRNA expression of chicken collectins cCL-1, cCL-2, and cSP-A and cLL are affected by H9N2 AIV inoculation. The effect is tissue specific, showing up regulated mRNA expression in the trachea and down regulation in the lung. White, M., Kingma, P., Tecle, T., Kacak, N., Linders, B., Heuser, J., Crouch, E., Hartshorn, K., 2008. Multimerization of surfactant protein D, but not its collagen domain, is required for antiviral and opsonic activities related to influenza virus. J. Immunol. 181, 7936-7943.

  Collectin mRNA expression in lung and trachea

  inoculation in the chicken. Whether similar signalling pathways are involved in regulation of chicken collectins needs to be addressed to clarify our observed tissue specific changes of these collectins.

  Furthermore, changes in collectin mRNA expression are age specific, showing differential gene expression in 1-and 4-week old birds. These observed changes in collectin mRNA expression implicate that chicken collectins can play an important role in innate defense against viral infection, especially in neonates. More research is needed to relate the changes in collectin mRNA expression after H9N2 AIV inoculation to a biological function and clarify the function of collectins within the innate response to infection.

  M., White, M.R., Crouch, E.C., Batenburg, J.J., Vaandrager, A.B., Van Golde, L.M.,Haagsman, H.P., Hartshorn, K.L., 2003. Porcine pulmonary collectins show distinct interactions with influenza A viruses: role of the N-linked oligosaccharides in the carbohydrate recognition domain. J. Immunol. 171, 1431-1440. Wan, H., Perez, D.R., 2006. Quail carry sialic acid receptors compatible with binding of avian and human influenza viruses. Virology 346, 278-286. Wang, T., Lafuse, W.P., Zwilling, B.S., 2000. Regulation of toll-like receptor 2 expression by macrophages following Mycobacterium avium infection. J. Immunol. 165, 6308-6313.

  Figure captions

Fig. 2 .

 2 Fig.2. cCL-1, cCL-2, cLL and cSP-A mRNA expression in lung of 1-and 4-week-old birds at 0,

Fig. 3 .

 3 Fig. 3. cCL-1, cCL-2, cLL and cSP-A mRNA expression in trachea of 1-and 4-week-old birds at

  Fig.1

Figure 1 A

 1 Figure1

Table captions Table 1

 captions1 Real-time qRT-PCR primers and probes.

Acknowledgements

We thank Astrid Hogenkamp from the Department of Pharmaceutical Sciences for stimulating discussions. This study is financially supported by a BSIK VIRGO consortium grant (Grant no. 03012), the EU sixth framework program Flupath (Grant no. 044220) and the Dutch consortium Veterinary Network Avian Influenza (FES).

A c c e p t e d M a n u s c r i p t 13 Table 1 Real-time qRT-PCR primers and probes.

Target

Probe or primer a sequence (5

AF411083 a F, forward; R, reverse.

b Genomic DNA sequence.

Table