Gordon Hughes

Claire Lawson

RSCPA and the criminology of social control

Keywords: animal abuse, policing, Royal Society for the Prevention of Cruelty to Animals (RSPCA), welfarism, multi-agency governance

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Introduction

In the emerging subfield of the criminology of animal abuse, much attention has been on the aetiology of the perpetrator and the nature of the dyad between (human) criminal/s and (non-human) victim/s. Much less attention has been paid to the dynamics of control associated with the various agencies, both state-based and voluntary or third sector. In this paper we begin by mapping the major institutional circuits of control, linked variously to detection and enforcement, welfare provision and rights based practices in the multi-agency governance of animal abuse. In order to explore in depth these control processes we then focus on the longest established, yet curiously so often ignored, institutional player in the policing of animal abuse and the protection of animal welfare in Britain over the past two hundred years, namely, the RSPCA. To date there has been no sustained criminological analysis of this key institutional player 1 . The case of the RSPCA is notable given that "on the ground" it is arguably both an unusual historical and contemporary instance of a non-state agency having more importance in law enforcement and regulation than the public police in addressing crimes and related harms with regard to the human ill-treatment of animals in a modern centralised state. Accordingly for nearly two hundred years it has been a powerful, non-state player in one area of the field of crime control and harm reduction in which the state has played a limited "hands-off" role, other than in the creation of laws pertaining to animal abuse and dangerous animals. The RSPCA is also an institution which today is surrounded by controversy in the eyes of animal rights lobbyists due to its welfarist philosophy and its reformist, and in some eyes "conservative", policies on farming and food production and scientific experimentation on animals alongside its role in controlling dangerous and neglected animals and killing unwanted and badly abused animals. The RSPCA is thus an institutional player actively involved in the contested politics and "morality war" with regard to the "appropriate" relationship of the human/non-human animal world. It is ideologically representative of the humanitarian, welfarist animal protection movement (as against the "karmic" animal rights movement). Again such highly contested and unfinished political and normative issues are of central importance to the contemporary debates in criminology regarding environmentalism and the broader study of harms, both human and non-human in nature.

It has been noted by several key contributors to this Special Issue that animal abuse remains both a marginal and marginalised area for criminological investigation. The absence of sustained research and analysis on the causes of animal abuse in its myriad forms has rightly of late gained greater attention not least through the efforts over two decades of Piers Beirne, in particular, across the fluid disciplinary borders of sociology and criminology. Writing in the mid-1990s Beirne [START_REF] Beirne | The use and abuse of animals in criminology: a brief history and current review[END_REF] highlighted the dearth of dedicated texts and research and in the fifteen years since this article there has been but limited growth of criminological research and expert commentary, largely confined to socalled environmental or green criminology [START_REF] Beirne | Animal rights, animal abuse and green criminology[END_REF][START_REF] White | Crimes Against Nature: Environmental criminology and ecological justice[END_REF] but, not, surprisingly, "zemiology" 2 .

A useful working definition of animal abuse is offered by Beirne and Messerschmidt ([5] p.152) as: any act that contributes to the pain, suffering or death of an animal or that otherwise threatens its welfare. Animal abuse may be physical, psychological, or emotional, may involve active maltreatment or passive neglect or omission, and may be direct, or indirect, intentional or unintentional. Some forms of animal abuse are socially acceptable.

Given that the key problematic in contemporary sociological criminology is that crimes are interactional phenomena necessarily conceived as emergent properties and products of the wider human figurations in which they are embedded, it is in turn vital that theory of and research into the crimes (and non-criminalised harms) of animal abuse must in part centre questions of "reaction" or control (alongside action and causation). Once again the silence of criminologists on the policing of animal abuse is striking with a few exceptions (see [START_REF] Beirne | Confronting animal abuse: Law, criminology, and human-animal relationships[END_REF][START_REF] Pierpoint | Animal Abuse[END_REF]) This paper aims to unsettle this situation and contribute to the promotion of a sustained research programme on animal abuse control and animal welfare protection.

The policing of animal abuse: mapping a criminological terra incognita

We offer in this section of the paper an initial mapping of the major institutional players in the circuits of control, linked variously to detection and enforcement, welfare provision, and rights based practices in the multi-agency governance of animal abuse as evident in the contemporary UK. What are the key features to the contemporary "control complex" [START_REF] Garland | The Culture of Control[END_REF] regarding detection, prosecution and prevention of crimes and harms associated with human-animal relations? Pierpoint and Maher ([35] pp.481-5) offer a valuable overview of both the legislative context regarding animal cruelty including the numerous pieces of legislation that have been enacted since the first successful anti-cruelty bill in Britain in 1822 which gave cattle, horses and sheep some limited protection. In particular such laws have created offences of cruelty to animals, and regulations and sanctions to protect wildlife and the welfare of livestock. The Animal Welfare Act (AWA) of 2006 is the most comprehensive piece of primary legislation to date and it now covers most of the issues contained in previous major pieces of legislation. In passing the RSPCA has been the major non-state based "expert" lobbyist in this history of law-making as well as policy implementation.

Despite the plethora of legislation since the nineteenth century, there remains little systematic statistical evidence regarding trends and patterns in recorded animal abuse. As Pierpoint and Maher ([35] pp.485-6) note, the little that is known about the prevalence of reported animal abuse is derived from court records and animal welfare charities. Throughout this period it would appear that the RSPCA has consistently brought the majority of prosecutions to the courts. However, there is a major evidential hole awaiting any attempt to assess systematically the trends in prevalence of animal abuse both over time and cross-sectionally at any given time in Britain. Most significantly, it was accepted by the Secretary of State for Environment, Food and Rural Affairs in the post-legislative assessment of AWA in December 2010 that there was no national enforcement database regarding the enforcement of the Act despite the original intention of this being part of a regulatory impact assessment [START_REF]Memorandum to the Environment, Food and Rural Affairs Committee: Postlegislative Assessment of AWA 2006[END_REF]. Furthermore, animal cruelty offences recorded by the police are not collected by the Home Office -we therefore have little other than anecdotal testimony in the absence of sustained criminological research to rely on in dealing with the seeming growth in the problem, for example, of abuse of dogs and their involvement in crime and anti-social behaviour (see Hughes et al. [START_REF] Hughes | Status dogs', young people and criminalisation: Towards a preventative strategy[END_REF]).

Even given the above caveats regarding the nature of the problem of animal abuse and lack of systematic and coordinated recorded information regarding public efforts to control and regulate it in its myriad forms, it is clear that there are multiple types of responses. In particular, private, interestgroup involvement is well established in this field, again epitomised by the work of the Society for the Prevention of Cruelty to Animals (SPCA) since 18243 but also including smaller charities and pressure groups. This is a field of control characterised by the actions of plural agencies though less often coordinated multi-agency partnership working, the latter being processes increasingly characteristic of crime prevention and community safety practices across many contemporary late modern societies [START_REF] Hughes | The Politics of Crime and Community[END_REF].

RSPCA and plural policing

The field of animal abuse control is characterised by plural policing. In this regime both private and state-based public bodies share the field from initial inquiry to prosecution and back again to prevention. Whilst much current criminological analysis points to the recent, potentially seismic shifts towards governance "beyond" the state [START_REF] Edwards | Comparing safety in Europe: a geo-historical approach[END_REF], little if any attention as yet has been given to the long-term continuity in plural policing and indeed private prosecutions in the control of animal abuse in the UK.

In Private Security and Public Policing, Jones and Newburn ( [START_REF] Jones | Private Security and Public Policing[END_REF] p.18) examined the terminology of policing and declared that it includes "organised forms of order maintenance, peace-keeping, rule or law enforcement, crime investigation and prevention and other forms of investigation and associated information-brokering". Whilst plotting the growth in both private and plural policing, the influential work of Jones and Newburn [START_REF] Jones | Private Security and Public Policing[END_REF][START_REF] Jones | The Transformation of Policing?: Understanding Current Trend in Policing Systems[END_REF] failed to refer to the role of the voluntary sector and in particular to the RSPCA as a private, charitable policing service. In turn Crawford et al. [START_REF] Crawford | Plural Policing: The mixed economy of visible patrols in England and Wales[END_REF] more recently researched the trend in plural policing in terms of "visible patrols". However, they omit any mention of a public-facing body such as the RSPCA and its nationwide uniformed body of inspectors in what is otherwise a comprehensive review of visible policing and its "extended family". Button ([7] p.133) has also provided an extensive discussion of the key trends in private policing but whilst noting that "given the growing concern with the environment and with animal welfare issues, it is surprising the RSPCA has received little attention", he does little to address this. More perturbing perhaps is that Button"s justification for studying the RSPCA is lodged in terms of the current growing public and criminological interest and not in the two centuries of legislation and law enforcement already in existence around animal welfare.

The silence to date of policing researchers regarding the practices of the RSPCA as a charitable organisation taking the lead on much of our society"s "fight" against cruelty to and neglect of animals stretching unbroken from the nineteenth to twenty-first centuries is indicative of the marginality of crimes and violence against animals to the criminological enterprise writ large. Later in the paper we consider the important critiques lodged at the feet of animal welfare organisations such as the RSPCA and the Humane Society of the United States ("HSUS") which have emerged from an admixture of radical moral philosophy and the animal rights movement since the latter decades of the twentieth century. These critiques have informed the emerging "green" criminological thinking on animal abuse and the associated critique of "speciesism" of which this Special Issue is itself largely a product. As we shall see shortly, important connections may be made between anti-speciesist critiques with regard to the "reformist" and "conservative" strategies of large-scale corporate animal protection organisations such as the RSPCA and the grand neo-marxist, structuralist criminological narratives of state and corporate control and punitiveness more broadly. These compelling structuralist-theoretical narratives of control also come at a conceptual price in possibly closing off healthy agonistic dispute over the difficult moral and political questions associated with the competing claims regarding the abolition of animal exploitation and the defence of a broad animal protectionism.

RSPCA, animals and the state in modern Britain

We have been at pains to note the neglect of both animal abuse and those institutions tasked with regulating such human/animal figurations in the criminological canon. Of course Beirne [START_REF] Beirne | The use and abuse of animals in criminology: a brief history and current review[END_REF] is right to note that there has been an often hidden presence of the animal in criminology"s meditations on the crime question over the course of its chequered disciplinary history. What concerns us here is somewhat more limited in scope: namely developing both a conceptual and empirical framework for locating the key control institution of the RSPCA within contemporary criminological thinking on social control more broadly.

We have noted that social scientists and in particular criminologists have neglected both issues of animal abuse and the policing of this problem. This stands in sharp relief to the work of moral philosophers, both ancient and modern [START_REF] Beirne | For a Non-Speciesist Criminology: Animal Abuse as an Object of Study[END_REF][START_REF] Garner | Animals, Politics and Morality, Second Edition[END_REF]. It also compares unfavourably with the work of social historians of the emergent industrial capitalist and urbanising society of the nineteenth century [START_REF] Perkin | The Origins of Modern English Society[END_REF][START_REF] Thomas | Man and the Natural World[END_REF].

Foremost among these scholars in contextualising the place of the RSPCA in terms of being a mirror reflecting and refracting key ideological, cultural and political currents and fissures in that society is the research undertaken by Harrison some forty years ago [START_REF] Harrison | Animals and the State in nineteenth-century England[END_REF]. As with all good historiography, Harrison"s 1973 essay Animals and the State in nineteenth century England provides both a window on the particularities of past human collective practices and insights for understanding our history of the present.

In this section of the paper we begin with the lessons offered by social historical research for social scientists today interested in the control and regulation of animal abuse before we turn our attention to contemporary developments, and both continuities and discontinuities with the past, associated with the work of the RSPCA at the beginning of twenty-first century.

Origins of the RSPCA: contemporary criminological lessons from the past?

Harrison ([22] p.786) begins by contrasting the commonsensical claim from judge Lord Devlin in 1968 that "there is hardly anyone nowadays who would not be disgusted by the thought of deliberate cruelty to animals" with the realities of living in a predominantly rural society in 1800 where the dependence on the slaughter of animals was a harsh and mundane fact of life. He contrasts this with the transition wrought by the 1870s where the claims of animals for both kinder treatment and much greater regulation had become institutionalised -albeit in the face of often fierce resistance -and as statistically represented by the registration of one million licensed dogs and over 800,000 licensed horses by this latter decade.

Much of this cultural transformation was in large measure made possible by the work of the SPCA and subsequently RSPCA. It is also evident to a social historian like Harrison that the story of the RSPCA has powerful resonances with socio-political issues in his own contemporary context of the late twentieth century. Noting the pertinence of the RSPCA story to political scientists" contemporary studies of "humanitarian" pressure groups, Harrison provides a powerful, empirically-based argument that it was one of the most continuously influential bodies on nineteenth century opinion but also almost uniquely one of the better known reform groups which consistently "collaborated with the authorities" ( [START_REF] Harrison | Animals and the State in nineteenth-century England[END_REF] p.787). The RSPCA was thus often caught between the opinions of its own members and those of the government department with which it had to work. Robert Garner [START_REF] Francione | The Animal Rights Debate[END_REF] has more recently reaffirmed this strategy of the RSPCA continuing into the twenty-first century as we shall see below. The other key feature of the RSPCA"s "mission" in the nineteenth century was the desire for "moral and religious progress", particularly with regard to "the pleasures and livelihood of the very poor" ( [START_REF] Harrison | Animals and the State in nineteenth-century England[END_REF] p.789), both regarding the cruelty to "beasts of burden" and to animals used in sport and for pleasure. The origins of the RSPCA were certainly of a Christian missionary and civilising zealaccording to its critics, past and present, it was a morally prudish movement and organisation of upperand middle-class mobilisation against the dangerous and uncouth habits of the poor and laboring classes [START_REF] Pemberton | Mad Dogs and Englishmen: Rabies in Britain 1830-2000[END_REF] 4 .

Harrison organises his inquiry into four questions, namely, (i) what is the chronology of legislation, (ii) how was the latter enforced, (iii) what techniques were used in winning support for animal protection, and (iv) how was it defended in argument ( [START_REF] Harrison | Animals and the State in nineteenth-century England[END_REF] p.787). These questions provide a valuable template for the sociological analysis of the RSPCA in our contemporary conjuncture.

Let"s begin with the first area of concern: the legislative landmarks in the nineteenth century.

Animals are considered as property only: to destroy or to abuse them, from malice to the proprietor, or with an intention injurious to his interest in them, is criminal; but the animals themselves are without protection; the law regards them not substantively; they have no rights! (Lord Thomas Erskine, 15th May 1809)

The above quote is from Erskine"s introduction to his Cruelty to Animals Bill in the House of Lords debate -which signified a shift in attitude among some influential sections of "polite" society" regarding the status of animals. Although this Bill and a later version failed, it laid the foundations for legislation that would protect animals for the first time in the UK. Indeed, "by encouraging kindness to animals, the Society hoped eventually to civilise manners, and hence to make the masses more receptive to religious instruction" [START_REF] Harrison | Religion and Recreation in Nineteenth-Century England[END_REF].

Lord Erskine failed on two occasions to pass his Act to Prevent Malicious and Wanton Cruelty

to Animals in 1809 and 1810 and such efforts were greeted with widespread ridicule. The cudgels were then taken up by Richard Martin MP supported by a group of "evangelical humanitarians" who in 1822 were finally successful in securing the first major animal protection legislation in the UK with the Act to Prevent the Cruel and Improper Treatment of Cattle. This was to establish the principle of protecting animals against cruelty by law and through pressure from the privileged supporters of the (R)SPCA further types of cruelty were, once again, to be banned including a range of animal fighting and baiting.

Following the 1822 Act Martin, nicknamed "Humanity Dick" by King George IV, was soon in need of "policing machinery" to enforce the new law ([4] p.11). As Radford ([36] p.40) observes, for legislation "to be effective, it must be enforced" but at this historical juncture of laissez faire capitalism the State undertook but a minimal "nightwatchman state" role. Into this void the RSPCA developed its own "constabulary", later renamed as the "inspectorate". Radford ([36] p.40) recounts that at the time of the creation of the RSPCA there was significant scepticism regarding, and often overt rejection of, the state"s role in prosecutions and enforcement of the criminal law, thus the majority of such legal actions were brought by private individuals. This is perhaps why the Metropolitan Police, the first modern professional police force in Britain, was not created until after the SPCA in 1829. Curiously the Metropolitan Police were invited in 1852 to take over managing the RSPCA"s constables, but a satisfactory agreement was never reached ( [START_REF] Harrison | Animals and the State in nineteenth-century England[END_REF] p.811).

"Policing" was a term in regular use long before the advent of the modern police force. Indeed in the eighteenth century, before the concept of a professional police force, there were alternatives operating in law enforcement, namely the Associations for the Prosecution of Felons which attempted to address the quandary that the prosecution was dependent on the victim discovering, detaining, and funding the prosecution, of the perpetrator ( [START_REF] Sharp | Crime order and historical change[END_REF] p.136). This was the system into which the (R)SPCA was born and its relevance is vital to understanding the Society"s role today. Garland ([18] p.32) has influentially analysed the decline of the private police and prosecution bodies in the nineteenth century but failed to recognise that at least one such body continued and thrived. While, as he describes (ibid), "citizens increasingly orientated their complaints to the state", in terms of animal cruelty and the RSPCA there is no evidence that this was the case.

The traditional "Whiggish" narrative of the onward march of progress and enlightenment tells the story in the following picaresque Dickensian terms. Several leading, religiously motivated figures such as Rev. Arthur Broome and William Wilberforce gathered at the Old Slaughter"s Coffee House in London on June 16th 1824 and formed the Society for the Prevention of Cruelty to Animals. At its inauguration the members of the new SPCA described its purpose as to ensure "the mitigation of animal suffering, and the promotion and expansion of the practice of humanity towards the inferior classes of animated beings" ( [START_REF] Radford | Animal Welfare Law in Britain[END_REF] p.41). This is of course the language of Christian missionary evangelism directed not least also at the "inferior" human classes. Despite the archaic sounding wording, this humanitarian and anthropocentric welfarist disposition remains that of the RSPCA to this day. In the framework provided by the sociology of deviancy we can interpret the nineteenth century history of the RSPCA as being that of a moral and political crusade manufactured by the lobbying of an increasingly influential network of "moral entrepreneurs", successfully mobilising influential opinion by the chronicling of a "moral panic" with its "folk devils" (uncivilised, un-Christian savages) which needed to be subjected to reformation through new governmental modes of discipline and punishment.

We have noted that the RSPCA did not content itself with legislating as an elite pressure group. Rather, it is peculiar in creating agencies which enforced the new legal rules: "a sort of private police force with a strictly limited area of concern" ( [START_REF] Harrison | Animals and the State in nineteenth-century England[END_REF]

p.793). Between 1830 and 1900 Harrison

[ibid] has estimated that its prosecutions doubled in every decade with the 1890s witnessing over 71,000 prosecutions. This successful expansion of the reach of the de facto major institutional expression of formal animal abuse control and repression was linked to the co-operation between the paid constables and unpaid subscribers to the Society. Harrison acknowledges that we have little written evidence of what the "policework" of RSPCA inspectors in this period really looked like. There is no rich evidence of the "crucially important moments when the Society made contact with the culture it despised. Reflective accounts of these incidents would greatly clarify the relationship between nineteenth-century humanitarianism and social class" ( [START_REF] Harrison | Animals and the State in nineteenth-century England[END_REF] p.795). Whilst the RSPCA today certainly highlights the work of its constituent employees and volunteers in its publicity documents and public accounts (see, for example, RSPCA [START_REF]Annual Review 2009[END_REF]), we still lack a rich body of ethnographic evidence on latterday RSPCA policing work on the ground. We lack then this particular ethnography of animal abuse control as "socially situated action" [START_REF] Edwards | Control as socially situated action[END_REF] which could contribute to a wider research-based criminology of social control.

Harrison"s third and fourth lines of inquiry focused on what we term the RSPCA"s "mobilisation strategy" and "ideological defence" which he described as both "prudent" and "professional" and opposed to what a contemporary member described as "any fanatical excesses". This was very evident in the RSPCA"s strategy towards vivisection. Whereas anti-vivisectionists in the later decades of the nineteenth century moved towards abolitionism, the RSPCA "adopted the alternative and characteristic strategy of pressing for stricter regulation" ( [START_REF] Harrison | Animals and the State in nineteenth-century England[END_REF] p.805 emphasis added). Harrison alludes to the institution from its earliest origins to its late twentieth-century incarnation always being identified with the "Establishment". According to Harrison ([22] p.808) this conservative" strategy was to prove successful and may have important lessons for the current debates raging over animal rights and animal protection.

Harrison"s conclusion and guarded celebration of the work of evangelical "do-gooders" sits uneasily with the scepticism of much critical criminology over reforms in justice and punishment in modernity. Such scepticism, which reaches its apogee in the anti-humanist, anti-modernist and dystopian analysis of Michel Foucault [16] on the technologies of penality, is of course an important corrective to the Whiggish historiographies plotting the inevitable and onward march of progress and enlightenment from the great "Victorian" reformers. However, the cost of such grand, dystopian and deterministic narratives from Foucault and his acolytes is the downplaying of the "real stuff of disposition, choice and actionthe stuff of which society and history are actually made" ( [START_REF] Garland | The Culture of Control[END_REF] p.25).

We need to be as wary of assuming the inferred repressive dystopian intentions and consequences of elitist reform movements and control agencies such as the RSPCA as we are regarding the supposed "pathologies" of the poor "deviant" sections of society most often subject to regulation and control from above.

Harrison"s historical monograph represents the most sustained academic research on the RSPCA as a social institution. But the issues Harrison raises are also worthy of detailed attention given their contemporary resonance for understanding the axial role played by the RSPCA as a key agency of animal abuse control in our present times. Let us now map some of the key features of the RSPCA as a late-modern control institution.

What's late-modern about the RSPCA?

Looking across the globe, the RSPCA stands out as one of the giants of the animal protection and welfare "enterprise" [START_REF] Francione | The Animal Rights Debate[END_REF]. The RSPCA is a large-scale corporate body and represents one of the largest charities in the UK today. Of its total annual income over £70 million derives from legacy income built up over the last two centuries from bequests and £35-40 million from donors. Organisationally, the institution also remains reliant on voluntary support, not just from sizeable donations as a charity but also as a result of its 170 branches run by local volunteers (who were termed "subscribers" in the nineteenth century). Its expenditure is also large-scale, amounting to over £100 million per annum, of which £32 million is spent on the inspectorate, £23 million on its establishments, £21 million on the cost of generating funds, and £11 million on prosecutions [39].

The RSPCA is also one of the UK"s most recognised "brand names" (no doubt helped -as well as stigmatised by opponents -by the monarchical moniker "Royal"). It was ranked recently as the fourth most prominent charity in the UK in the Charity Brand Index ( [START_REF] Donovan | Charity Brand Index[END_REF] p.4). Such a status would appear to indicate significant sections of the public"s affection for the organisation given that factors such as reputation and income generated are incorporated in this Index. Such a celebrated identity could perhaps have deterred critical inquiry if there appeared little merit in pursuing a seemingly virtuous organisation or in commonsensical, ideological terms a "national treasure". However, it is perhaps more likely that researchers have misinterpreted either the law enforcement work of the RSPCA or the legal status and mandate it has to enforce such laws.

Paradoxically such a myth that the organisation is a government arm conflicts with the very prominent charity status recorded in the Index above. Further still, if the RSPCA"s status has indeed been construed as quasi-governmental, then the question of why this function has not been the subject of any significant academic scrutiny remains striking 5 .

As noted above, Harrison divides his monograph on the nineteenth century development of the animal protection movement and in particular the axial role of the RSPCA in these processes into four themes covering (1) legislative and legal developments, (2) enforcement of the law(s), (3) mobilisation strategy, and (4) ideological defence. This heuristic categorisation is helpful to our understanding of the institution at the beginning of the twenty-first century.

First the RSPCA continues to influence legal developments. This is most recently evident in its lobbying and expert advice on the most comprehensive, enabling legislation at the time of writing, the Aninal Welfare Act (AWA) of 2006. It has been claimed by leading RSPCA representatives that the most notable shift in the RSPCA"s explicit role in crime prevention emerged when it succeeded in persuading the Government to pass the new AWA given that this legislation contains the ability to proactively prevent the suffering of animals for the first time [START_REF] Robinson | Enforcing Animal Welfare[END_REF]. As a result of this legislation, a duty of care is placed on the owner or keeper of the animal and the needs of the animal must be provided for. There is no need to prove that the animal has suffered -only that it is likely suffer should the situation not improve. Inspectors appointed under the Act within local authorities can now serve statutory improvement notices. The combination of the threat of prosecution and the softer provision of assistance and financial support appear to have enabled individuals to comply with the notices, but more importantly the animals have not gone on to suffer ([37] p.6).

I n turn, the RSPCA continues to offer both expert advice and guarded criticism on current deficiencies in the legislative arsenal of the state, such as the Dangerous Dogs Act of 1991 (amended 1997) and proposals for amendment of this piece of legislation which is widely regarded as being populist, ill-thought through and largely symbolic. The list of interventions in laws could go on and would now include the RSPCA"s international lobbying for legislative changes at the EU level and most recently with the lengthy negotiations with the Chinese state in formulating its first formal enactment of anti-animal cruelty regulations. It is no exaggeration then to view the RSPCA as a global player in animal project debates beyond the UK [START_REF]Supporting the Implementing of OIE Animal Welfare Standards: The role that civil society plays[END_REF].

In terms of enforcement of the law, the RSPCA remains de facto the major investigative, protection and prosecution agency on animal abuse and neglect cases 6 . Annually the organisation"s national control centre receives about 1.3 million calls from the public. In turn 135,000 animals were collected and rescued by the institution [39]. The latest data compiled by the agency for 2009 reveals a count of 141,000 cruelty complaints investigated which resulted in 2,500 known convictions of 1,500 persons. The contemporary RSPCA annual reports present graphic in-depth cases of successfully prosecuted offences in its review of its activities, highlighting for public consumption in no uncertain terms the depths of cruelty perpetrated by its human offenders. In turn it is able to boast of a high success rate (98%) for cases brought to court. The types of offences prosecuted or dealt with less adversarially range in descending order from cruelty (mostly to dogs), welfare offences, adult written cautions, breach of disqualification, offences against wildlife, juvenile-related offences, and animal fighting [40]. In their discussion of the national databases, Pierpoint and Maher [START_REF] Pierpoint | Animal Abuse[END_REF] conclude that the RSPCA prosecutes the majority of cases in comparison to the other two major prosecuting agencies, namely the Crown Prosecution Service, and local authorities. However, they acknowledge the quantitative and qualitative data to substantiate this is lacking.

With regard to front-line "policing" staff, the RSPCA employs 300 uniformed inspectors (including inspectors in the special operations unit often working with the police on the most serious cases of abuse and cruelty). Alongside this most visible outward facing "enforcement" group, the agency has a large body of staff including veterinary and animal care specialists in its hospitals, clinics and branches, which in recent years have treated in excess of 200,000 animals annually. According to its own latest official figures, the RSPCA"s animal centers routinely microchip 73,000 and neuter 87,000 domestic animals per annum. The RSPCA also has four science departments and much of its self-promotion in terms of improving "the lot" of livestock animals of the agri-business complex is associated with the monitoring and registration of its "Freedom Food" label of humane treatment which currently covers 900 million animals in the UK each year [39]. As we shall see below this is an area of work for which the RSPCA has been widely criticised of late.

What, then, are the legal powers of the RSPCA as a prosecuting agency? A degree of misunderstanding or misinformation on this subject has always been prevalent and arguably utilised by the RSPCA itself [START_REF] Lawson | Where is the 'P' in RSPCA: A Criminological Case Study of Crime Prevention Within Animal Cruelty[END_REF]. Scott ([41] pp.62-63) is certainly mistaken in classifying the RSPCA as an example of "delegated regulatory power" and as an institution that possesses a "statutory power to prosecute". In fact the RSPCA is a registered charity, with its law enforcement role operating solely under the legal jurisdiction of England and Wales. This charitable status is an important designation to comprehend for it requires that the purpose of the organisation to be ultimately for public benefit. For the RSPCA, and other animal welfare charities, prevailing legal judgments have established that public benefit to be defined as the "promotion of human morality"; thus animals are only to benefit indirectly ([6] p.58].

It is not technically a formal prosecuting body because, "in pursuing alleged offenders, it is acting entirely as a private prosecutor" ( [START_REF] Radford | Animal Welfare Law in Britain[END_REF] p.380), and thus is not obliged but may have regard for the Crown Prosecution Service Code [START_REF]The Code for Crown Prosecutors[END_REF]. It is the category of the legislation being utilised which determines the RSPCA"s ability to prosecute. The legal theorist Radford ([36] p.380) notes that, without any formal declaration, both the criminal justice authorities and the RSPCA consider their Inspectors as investigating officers under the Police and Criminal Evidence Act. The rules under this legislation bind officers with regard to actions during custody and interrogation. However, RSPCA Inspectors possess no power or authority beyond that of an ordinary citizen, so cannot enter a premises without permission nor arrest or remove evidence; thus police assistance in these matters is paramount ([23] p.220). The government"s own position regarding the RSPCA"s legal status as a prosecuting body has been obscure and until very recently had to be unearthed painstakingly from often obscure guidelines and documents. For example, under "Relations with Other Agencies" in the Domestic and Captive Animals section of the CPS"s website [START_REF]Legal Guidance: Offences involving domestic and captive animals[END_REF], the only note says:

The Royal Society for the Protection of Cruelty to Animals (RSPCA) has a long established expertise in both the investigation and prosecution of cases involving animal welfare and has built up a useful body of precedent and case law.

In December 2010, the RSPCA was acknowledged in the post-legislative assessment of the AWA by DEFRA as "the principal agency responsible for the day-to-day enforcement of the Act" ( [START_REF]Memorandum to the Environment, Food and Rural Affairs Committee: Postlegislative Assessment of AWA 2006[END_REF] p.13).

This remains a rare instance where such formal public recognition has ever been found, which could suggest a reluctance on behalf of the state authorities to fully disclose the reality, or perhaps the ignominy, of the situation [START_REF] Lawson | Where is the 'P' in RSPCA: A Criminological Case Study of Crime Prevention Within Animal Cruelty[END_REF].

Moving on to questions of mobilisation strategy and ideological defence, the historian Harrison ([22] p.805) recounts how from its earliest foundations the RSPCA "found itself in a political situation, where opposition to the powerful on one issue had to be moderated in the hope of attracting support from the powerful on another". To repeat our earlier point, this centrist strategy has consistently been its doctrine into the twentieth and twenty-first centuries. For supporters of this strategy "radical activities have been eschewed in favour of patiently but persistently accumulating a formidable body of protective legislation" ( [START_REF] Harrison | Animals and the State in nineteenth-century England[END_REF] p.808).

To this day, explicit engagement in what we may call the "morality war" around the seemingly irreconcilable positions of animal exploitation abolitionists and those of reformist animal protection positions (of which the RSPCA is a powerful and brooding "Establishment" representative) is impossible to find in any official RSPCA documents and statements. At best we find briefing notes for teachers on "humans and other animals", "creating an animal welfare charter", "ethics, animal experiments and the law" and such like in its education publications which steadfastly avoid the type of deeply engaged moral and political debate characteristic of animal rights lobbying organisations. The ideology of the RSPCA on the vexed issue of animal rights versus animal welfare remains tellingly under-if not un-stated in any explicit manner of the kind we find in the work of an animal protectionism advocate such as Garner [START_REF] Francione | The Animal Rights Debate[END_REF]. As an Establishment institution "above" politics, it would appear that the RSPCA pragmatically avoids public debate on the politics and ethics of animal rights versus animal welfare. Despite this public silence, the RSPCA is an institution which is surrounded today by controversy in the eyes of animal rights lobbyists and "scholar-activists" ([45] p.1209) and a growing number of proponents of a "non-speciesist" criminology. Much of this controversy is linked to its "humanitarian welfarist" philosophy and its reformist, and in the eyes of some [START_REF] Francione | The Animal Rights Debate[END_REF], "conservative" policies and practices particularly with regard to "humane" farming and animal food production and scientific experimentation on animals 7 . Meanwhile in the eyes of anti-state, libertarian critical criminologists, criticism is also lodged at the RSPCA"s feet for its axial, governmental role in controlling dangerous and neglected animals and killing unwanted, badly abused animals, particularly "dangerous" dogs (Hallsworth, this Issue, Kaspersson, [START_REF] Kaspersson | On Treating the Symptoms and not the Cause. Reflections on the Dangerous Dogs Act[END_REF]). The RSPCA is thus an institutional player which is, willingly or not, actively implicated in a contested politics and "morality war" with regard to the "appropriate" relationship between humans and non-human animals.

What in brief are the features of the "animal welfare" view for which the RSPCA is both globally renowned and increasingly vilified? 8 The animal welfare view is widely regarded as being one in which animals are viewed as being at least "partial members" of the moral community" due to their sentience whilst accepting that humans may use animals for human purposes. This welfarist position also claims that it is morally wrong to inflict unnecessary harm or suffering on animals or to treat them in ways that are not "humane". According to supporters of this avowedly anthropocentric world-view, even the property status of animals is not an inevitable obstacle to them receiving better treatment, as evidenced in the comparative treatment of animals across different cultures today ([12] pp.124-46). 7 Animal rights critics of the RSPCA say little, it would seem, about its routine repression of animal cruelty and regular prosecution of human perpetrators which, as is clear from our previous analysis makes up a very significant area of its work and institutional logic. 8 Of course, we do not come to this debate "value-free" and "politics-free". Our broad normative position is that of humanitarian and anthropocentric welfarism. We remain wary of the more "karmic" direction in debates on human/non human animal interface whereby the human is re-absorbed into natural history ([17] p.5). We broadly concur with the modernist founders of sociology on the uniqueness of the human animal as residing in their "participation in large-scale corporate projects that defy the gene"s eye-view of the world" ([17] p.6), not least at times in reversing or neutralising the effects of "natural selection". This is indeed a feature of both animal protection and animal rights political projects.

What in brief are the counter-claims of abolitionist proponents? Let"s focus on a number of key claims made in Francione"s abolitionist critique of the RSPCA and other globally influential animal welfare institutions. According to Francione ([12] p.51), "Making society feel more comfortable about animal exploitation is more often than not an explicit goal of animal welfare campaigns and organisations". Specifically cited evidence in support of this claim is the labelling schemes for humanely farmed animal food produce and in particular the RSPCA"s Freedom Food label ([12] pp.52-3). In turn, it is suggested that such schemes do not translate into significant welfare benefits for nonhuman animals kept under such "humane" conditions, backed up "empirically" by the citing of notorious examples of badly regulated enterprises. Furthermore, and most crucially, it is contended that the overall consequence of animal welfarism has been to reinforce the "property paradigm" regarding non-human animals and is often based on "increasing the efficiency of exploitation" ( [START_REF] Francione | The Animal Rights Debate[END_REF] p.45).

Radical, if not revolutionary, animal rights "scholar-activists" like Francione conclude by condemning reformist "traditionally conservative groups" like RSPCA and HSUS and newer, supposedly more progressive groups such as People for the Ethical Treatment of Animals (PETA), for in effect perpetuating and propping up the exploitative system. Not unexpectedly we can find no explicit response of the RSPCA to such damning criticisms of its role in both propping up and helping perpetuate "the system" (by "humanising" animal exploitation). Again, this silence speaks to the mobilisation strategy and ideology of this Establishment institution. Those seeking sophisticated normative and ideological rationales of the politics of animal protectionism and the new welfarism have to turn to reformist "scholar-activists" such as Garner [START_REF] Francione | The Animal Rights Debate[END_REF].

Conclusion

Much of this paper"s focus has been on the RSPCA as a key, if also "strange", exemplar of developments in the policing and control of animal abuse. The RSPCA is certainly a key player both in British society and also increasingly across the globe in the politics of animal protection and abuse control. Its "strangeness" may be due both to its charitable status and nineteenth-century Christian missionary roots and its continuing core role in policing animal abuse in a late-modern system of crime control. It is clearly an institution of social control worthy of interest in its own right. By exploring the historical continuities and discontinuities of this original "Victorian" invention over two centuries, we also hope to have shed light on the broader issues raised for the nascent criminology of animal abuse. We await future criminological work which may inform the future practice of animal abuse control and animal welfare protection and which might ambitiously help reconstitute the governmental savoir surrounding this key area of public policy and practice.

Historians have not been so guilty of this neglect, as we shall see below. The silence of most criminologists may reflect both the state-based, criminal justice system focus of modern criminology and also he more widely acknowledged marginality of animal abuse to the sociological study of crime and its control.

The study of social harms, recently badged as "zemiology", would appear an appropriate home for the growing research surrounding harms done to animals, including those in sport, entertainment and farming for they are legal activities that animal welfare and animal rights lobbies have thus far been unable to inveigle governments to criminalise. Whilst it may seem obvious to situate animal abuse within zemiology there is little evidence to demonstrate zemiologists have open arms. For example, Hillyard et al.([24] p.48) catalogue a vast array of social harms at corporate, state and international levels yet animal abuse is overlooked.

The prefix "Royal" was added later as a result of formal royal patronage from Queen Victoria in 1840.

This accusation of an obsession with the dangerous habits of the lower orders or in contemporary argot, the "underclass", has been reaffirmed with regard to the RSPCA"s role in the policing and control of "dangerous" dogs by libertarian journalists (O"Neil,[START_REF] O"neil | In defence of devil dogs[END_REF]) and libertarian criminologists (Hallsworth, this issue, Kaspersson,[START_REF] Kaspersson | On Treating the Symptoms and not the Cause. Reflections on the Dangerous Dogs Act[END_REF]) alike.

Note, however, the brief but undeveloped analysis in Grabosky[START_REF] Grabosky | Using Non-Governmental Resources to Foster Regulatory Compliance[END_REF] and Scott,[START_REF] Scott | Private Regulation of the Public Sector: A Neglected Facet of Contemporary Governance[END_REF].

Despite the inclusion of the term "prevention" in its title, it is the enforcement of the law with which the RSPCA is most associated, both internally (Lawson,[START_REF] Lawson | Where is the 'P' in RSPCA: A Criminological Case Study of Crime Prevention Within Animal Cruelty[END_REF]) and in the broader public perception of its mission. In terms of notions of crime and harm prevention involving an emphasis of proactive actions before crimes or harms ensue, the preventive role of the RSPCA remains underdeveloped (see Hughes et al.[START_REF] Hughes | Status dogs', young people and criminalisation: Towards a preventative strategy[END_REF]) despite the provisions in the AWA noted above. As noted previously, much of the day-to-day work of the RSPCA is filling in an enforcement and prosecutorial "void" and providing a governmental function which the state has not addressed directly. In the words of a leading RSPCA spokesperson at its Status Dogs Summit in 2010, commenting on the policy transfer implications of the "British model" for policing and law enforcement of animal abuse, "An NGO taking the slack is not the best model to start with".


Andrew J Wilson
email: a.j.wilson@leeds.ac.uk

Andrea M Mcghee

Jeffrey P Plante

Colin A Kilner

Solid State Structures of Ureidoimidazoles

Keywords: hydrogen-bonding, linear arrays, self-assembly hydrogen-bonding, linear arrays, self-assembly

NMR Spectra

Introduction

The design and synthesis of hydrogen-bonding motifs exhibiting well-defined supramolecular organization both in solution and the solid state is an ongoing area of interest. [1][2][3][4][5][6][7][8] Hydrogen-bonding motifs are important building blocks for the assembly of supramolecular polymers [8][9][10] and more generally of self-assembling gelators 11,12 which attract attention as tuneable "smart materials". They also play a central role in crystalline assemblies which are significant in a number of areas including polymorphism of active pharmaceutical ingredients 13 and solid-state reactivity. 14 Furthermore, solid state structures can provide valuable information on molecular recognition behaviour in solution.

Our group have initiated an investigation concerned with the development of linear arrays of hydrogen-bonds 15,16 to be used as components of supramolecular polymers. [8][9][10] The current manuscript focuses on the ureidoimidazole donor-donor-acceptor motif previously shown by us to exhibit conformer independent heterodimerisation with the complementary amidoisocytosine acceptor-acceptor-donor array. 16 Although, our prior studies illustrated negligible self-association of the ureidoimidazole motif in solution, the current results reveal an unusual mode of self-assembly in the solid-state for a series of compounds; this is remarkably conserved across the series and subtly modulated through both steric effects and proximal hydrogen-bonding groups.

Results and Discussion

The ureidoimidazole derivatives 1, 2 and 3a-c were synthesized using standard procedures as illustrated in scheme 1.

Scheme 1

Single crystals of each compound suitable for a diffraction study were obtained as described in the experimental section and subjected to crystallographic structure determination. The crystal structure of compound 1 was previously been disclosed by us. 16 Compound 1 crystallizes in the monoclinic P2 1 /n space group, with one molecule present in the asymmetric unit. Of the two conformational possibilities: intramolecular urea NH to imidazole N hydrogen-bonded and intramolecular urea carbonyl to imidazole NH hydrogen-bonded only the latter is observed (Figure 1 a). The packing of compound 1 is mediated by intermolecular hydrogen-bonds, which, based on the intermolecular heteroatom hydrogen distances, would be described as weak hydrogen-bonds. Bifurcated hydrogenbonding between urea NH's and the imidazole nitrogen is observed with each monomer perpendicular to two others, whilst the backface of the molecule interacts through carbonyl to imidazole NH hydrogen-bonding creating an infinite two dimensional structure (Figure 1b). Van der Waals packing is observed between sheets (Figure 1c). At a similar time to our original report, 16 Barboiu reported a structure of compound 1 that is virtually identical to the one we reported. 17 2a). As for compound 1 weak bifurcated hydrogen-bonding between urea NH's and the imidazole nitrogen is observed with each monomer perpendicular to two others, however no hydrogen-bonds are observed involving the carbonyl and imidazole NH's on the opposite side of the molecule to the DDA array (Figure 2b,c). Therefore infinite one dimensional chains are observed that pack in the second and third dimensions through aromatic π-π edge to face (ArC-H-π interaction), π-π face to face and Van der Waals interactions (Figure 2c, d ande).

Figure 2

Compound 3a crystallizes in the monoclinic P2 1 /n space group, with two molecules and two solvent methanol molecules present in the asymmetric unit. The conformations of each molecule of 3a are only subtly different. As for the first two examples only intramolecular urea carbonyl to imidazole NH hydrogen-bonding is observed, however, the packing structure is significantly different in the presence of solvent (Figure 3a). The same core mode of dimerisation that is observed for 1 and 2 is observed for 3a in that urea NH bifurcated hydrogen-bonding to the imidazole is observed, however in this instance the solvent molecule acts as a bridge -the interactions are therefore significantly stronger as evidenced by the shorter intermolecular distances -and a coplanar rather than perpendicular arrangement of 3a is observed i.e. a self-complementary dimer is observed bridged by two methanol molecules (Figure 3b,c). The infinite one dimensional polymer is propagated through self-complementary hydrogen-bonds involving the carbonyl and imidazole NH's on the opposite side of the molecule to the DDA array as is the case for compound 1. Packing in the second and third dimensions is mediated by Van der Waals interactions (Figure 3d). Compound 3b crystallizes in the triclinic P1 space group, with two molecules and three solvent methanol molecules present in the unit cell. One of the methanol molecules is disordered over two equally occupied positions. There are only minor differences between the structures of the individual monomer units. Again the conformation of 3b is determined by intramolecular urea carbonyl to imidazole NH hydrogen-bonding and the DDA present in the structure is the same as the other compounds (Figure 4a). The solid state assembly is similar in nature to 3a. The principle difference is that the infinite one dimensional polymer is propagated through self-complementary hydrogen-bonds involving the carbonyl of the ester (as opposed to the urea) and imidazole NH -an additional hydrogen bond between from the urea carbonyl to a solvent CH is also observed (Figure 4b,c). Packing in the second and third dimensions is mediated by Van der Waals interactions (Figure 4d).

Figure 4

Compound 3c crystallizes in the triclinic P1 space group, with two molecules and two solvent methanol molecules present in the asymmetric unit. Each molecule of 3c is only subtly different.

Like all the other examples, a single defined conformation is observed that is dictated by the intramolecular hydrogen-bonding (Figure 5a). The packing structure is very similar to compound 3a and all of the same hydrogen-bonding features are present within the observed infinite one dimensional polymer with Van der Waals interactions mediating the packing in the second and third dimensions (Figure 5b-d). Overall, the salient solid state assembly properties of the ureidiomidazole series are summarised in Fig. 6. Each structure adopts a common conformation mediated by intramolecular hydrogenbonding between the imidazole NH and the carbonyl functionality of the urea motif to present a donor-donor-acceptor array. In the absence of solvent, direct interaction of each urea group to the acceptor on the imidazole of an adjacent molecule is observed. The perpendicular orientation (which presumably results from steric constraints imposed by R 1 ) results in an infinite one dimensional chain as is observed for 1 and 2 (Fig 6a). Assembly for this series can be propagated in a second dimension through self-complementary hydrogen-bonding between the rear faces as for 1.

This feature is "switched-off" with sterically bulky uredioimidazoles as for 2 -the R 2 group in Fig 6a is clearly positioned to influence packing of adjacent molecules. In the presence of methanol, stronger hydrogen-bonding is observed and the solvent acts as a bridge between the urea NH's and imidazole Ns on two coplanar ureidoimidazole motifs as is observed for 3a-c (Fig 6b). The infinite one dimensional chain is propagated through self-complementary hydrogen-bonding between the rear faces. This interaction is the same as that which is observed to mediate propagation in the second dimension when solvent is absent (i.e. compound 1), although it should be noted that the presence of additional hydrogen-bonding functionality on the ureidoimidazole can play a role as is the case where for 3b where Y = CO 2 Et (the size of R 2 is also anticipated to influence assembly).

Figure 6 Conclusions

We have reported the synthesis and solid-state structure of a series of ureidoimidazole motifs. In solution (chloroform), the ureidoimidazole motif has been shown by us to participate in selfassociation to a negligible extent (< 10 M -1 ) 16 however, hydrogen-bonding plays a significant role in determining the solid-state structures that are observed. In the absence of a bridging solvent, the reinforces the solution observations. The urea motif has a strong tendency to engage in bifurcated hydrogen-bonding involving both its NH's, 2 and this seems to play a prominent role here in controlling the solid-state assembly. These observations will be used in our ongoing efforts to design and synthesize novel linear arrays of hydrogen-bonding groups for supramolecular assembly.

Experimental

All reagents were purchased from Aldrich or Alfa-Aesar and used without further purification unless otherwise stated. Where anhydrous solvents were required, THF was freshly distilled from sodium benzophenone ketyl radical, CH 2 Cl 2 was freshly distilled from calcium hydride and CHCl 3 was freshly distilled from calcium chloride under a nitrogen atmosphere. Anhydrous DMF was obtained 'sure-sealed' from Sigma-Aldrich. Triethylamine was distilled from calcium hydride and stored, under nitrogen, over potassium hydroxide pellets. All non-aqueous reactions were carried out under a nitrogen atmosphere. Analytical thin layer chromatography (TLC) was conducted using Merck Kieslegel 0.25mm silica gel pre-coated aluminium plates with fluorescent indicator active at UV 245 . Purification by column chromatography was carried out using Merck Kieselgel 60 silica gel.

NMR spectra were obtained using Bruker DMX500 or Bruker AMD300 spectrometers operating at 500MHz or 300MHz for 1 H spectra and 125 MHz or 75 MHz for 13 C spectra as stated. Proton spectra are referenced to TMS at 0.00 ppm, and carbon spectra to CDCl 3 at 77.4 ppm, unless otherwise stated. Melting points were determined using a Griffin D5 variable temperature apparatus and are uncorrected. IR spectra were obtained using Perkin-Elmer FTIR spectrometer.

Microanalysis was carried out on a Carlo Erba Elemental Analyser MOD 1106 instrument. High

Resolution Mass Spectra (HRMS) were recorded on a Micromass GCT Premier using electron impact ionisation (EI) or a Bruker Daltonics micrOTOF using electro spray ionisation (ESI). The title compound was prepared as described previously 16 2-tert-Butoxyamido-4-tert-butylimidazole 16 The title compound was prepared as described previously 16

N-Phenyl-N'-(imidazo-2-yl)urea 1

The title compound was prepared as described previously 16

1-(1H-benzo[d]imidazol-2-yl)-3-phenylurea 2

A stirred solution of 2-aminobenzimidazole (0.500 g, 3.75 mmol) in anhydrous pyridine under nitrogen was brought to 100°C. Phenylisocyanate (0.387 mL, 0.4245 g, 3.57 mmol) was then added and stirring continued. After twenty minutes a precipitate began to form and after one hour the reaction was cooled and filtered to give a white solid (0.340 g). The filtrate was then poured into water to give a precipitate (0.366 g) which was filtered and dried. Both samples were found to be identical giving a total yield of product (0.706 g, 87%) as a white solid which was not purified

General Procedure: Synthesis of ureido-4-tert-butylimidazoles

2-tert-Butoxyamido-4-tert-butylimidazole (1.0 equiv.) was stirred in 20% HCl/EtOH (100 mL per 1.0 g of starting material) for 1 h. The solution was concentrated and the residue dried under vacuum for 1 h. The resulting material was dissolved in dry THF (40 mL per 1.0 g of material) at room temperature under N 2 and triethylamine (1.5 equiv.) added. The reaction was heated to reflux and the appropriate isocyanate (1.0 equiv., 1.0 M in THF) was added dropwise. The reaction was stirred at reflux for 16 h. After cooling to room temperature the volatiles were removed under 1-(4-Tert-butyl-1H-imidazol-2-yl)-3-phenylurea 3a 16 Adopting minor modifications to our published method, 16 the general procedure D, using

Ethyl 4-(3-(4-tert-butyl-1H-imidazol-2-yl)ureido)benzoate 3b

The general procedure D, using 2-tert-butoxyamido-4-tert-butylimidazole (820 mg, 3.

1-(4-Tert-butyl-1H-imidazol-2-yl)-3-(4-(trifluoromethyl)phenyl)urea 3c

The general procedure D, using 2-tert-butoxyamido-4-tert-butylimidazole (842 mg, 3.50 mmol) and

Crystal Structure Determination for 1

The single crystal diffraction study for compound 1 was reported previously. 16 We note that, since the publication of our paper, a further report of the structure of 1 has appeared in the literature. 17

Crystal Structure Determination for 2

Single crystals were grown by the slow evaporation of a solution of 2 in chloroform/ methanol. Xray diffraction data were collected at the University of Leeds using a Bruker APEX2 instrument. CCDC 795262 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.

Crystal Structure Determination for 3c

Single crystals were grown by the slow evaporation of a solution of 3c in moist acetonitrile/ methanol. X-ray diffraction data were collected at the University of Leeds using a Bruker APEX2

1 Solid

 1 (300 MHz, CDCl 3 ) Ethyl 4-(3-(4-tert-butyl-1H-imidazol-2yl)ureido)benzoate 3b 13 C NMR (75 MHz, CDCl 3 ) Ethyl 4-(3-(4-tert-butyl-1H-imidazol-2yl)ureido)benzoate 3b 4-Tert-butyl-1H-imidazol-2-yl)-3-(4-(trifluoromethyl)phenyl)urea 3c State Structures of Ureidoimidazoles Andrea M. McGhee, a Jeffrey P. Plante, a Colin A. Kilner, a Andrew J. Wilson a,b * a School of Chemistry, University of Leeds, Woodhouse Lane, Leeds LS2 9JT, UK. b Astbury Centre for Structural Molecular Biology, University of Leeds, Woodhouse Lane, Leeds LS2 9JT, UK. Fax: +44(0)113-3436565 Tel: +44(0)113-3431409; E-mail: A.J.Wilson@leeds.ac.uk

Figure

  Figure 1

Figure 3 F

 3 Figure 3

Figure 5 F

 5 Figure 5

  these interactions indicate theses interactions are weak which

  the residue partitioned between EtOAc and water. The layers were separated and the aqueous phase extracted with EtOAc. The combined organics were washed with aqueous saturated NH 4 Cl, aqueous saturated NaHCO 3 and brine, dried (Na 2 SO 4 ) and concentrated under reduced pressure before purification by column chromatography.

  Crystal data: C 14 H 12 N 4 O, M = 252.27, crystal size 0.40 x 0.40 x 0.10 mm, Monoclinic, a = 24.2248(6), b = 6.9079(2), c = 16.2372(5) Å, β = 119.482(2) º, U = 2365.33(12) Å 3 , T = 150(2) K, C2/c, Z = 6, µ = 0.108 mm -1 , λ = 0.71073 Å [Mo-Kα], 21752 reflections measured, 3739 unique (R int = 0.0287), observed 2957 (I>2σ(I)). The final R 1 was 0.0418 (observed reflections 0.0580) and wR(F 2 ) was 0.1144 (all data 0.1333) for 175 parameters. CCDC 795260 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif. Single crystals were grown by the slow evaporation of a solution of 3a in moist acetonitrile/ methanol. X-ray diffraction data were collected at the University of Leeds using a Bruker APEX2 instrument. Crystal data: C 15 H 22 N 4 O 2 , M = 290.37, crystal size 0.33 x 0.15 x 0.05 mm, Monoclinic, a = 12.0588(9), b = 9.5465(7), c = 29.231(2) Å, β = 96.888(4)º, U = 3340.8(4) Å 3 , T = 150(2) K, P2 1 /n, Z = 8, µ = 0.079 mm -1 , λ = 0.71073 Å [Mo-Kα], 58508 reflections measured, 8178 unique (R int = 0.042), observed 5905 (I>2σ(I)). The final R 1 was 0.0562 (observed reflections 0.0839) and wR(F 2 ) was 0.1398 (all data 0.1523) for 413 parameters. CCDC 795261 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge fromThe Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.Crystal Structure Determination for 3bSingle crystals were grown by the slow evaporation of a solution of 3b in moist acetonitrile/ methanol. X-ray diffraction data were collected at the University of Leeds using a Bruker APEX2 instrument. Crystal data: C 37 H 56 N 8 O 9 , M = 756.9, crystal size 0.35 x 0.22 x 0.04 mm, Triclinic, a = 8.0694(11), b = 9.3523(13), c = 14.6137(18) Å, α = 99.815(5)°, β = 103.015(5)°, γ = 102.771(5)°, U = 1019.4(2) Å 3 , T = 150(2) K, P1, Z = 1, µ = 0.089 mm -1 , λ = 0.71073 Å [Mo-Kα], 19367reflections measured, 5107 unique (R int = 0.0272), observed 3904 (I>2σ(I)). The final R 1 was 0.0587 (observed reflections 0.0587) and wR(F 2 ) was 0.1664 (all data 0.1798) for 247 parameters.

Figure and Scheme Captions Scheme 1 Figure 1

 11 Figure and Scheme Captions

Figure 2

 2 Figure 2 (a) X-ray crystal structure of compound 2 (b) structure packing diagram of compound 2

Figure 3 ( 13 (

 313 Figure 3 (a) X-ray crystal structure of compound 3a (b) structure packing diagram of compound 3a

Figure 4

 4 Figure 4 (a) X-ray crystal structure of compound 3b (b) structure packing diagram of compound 3b

Figure 5 Figure 6

 56 Figure 5 (a) X-ray crystal structure of compound 3c (b) structure packing diagram of compound 3c

1

  Compound 2 crystallizes in the monoclinic C2/c space group, with one molecule present in the asymmetric unit. Again, only intramolecular urea carbonyl to imidazole NH hydrogen-bonding is

Page 10 of 26 URL: http:/mc.manuscriptcentral.com/tandf/gsch Email: suprachem@mail.cm.utexas.edu

	Supramolecular Chemistry
	observed (Figure
	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	O n l
	y
	4

  , C 17 H 23 N 4 O 3 requires 331.1765.

	F o r
	P
	e
	e r
	R
	e
	v i e
	w	4 mmol) and
	4-ethoxycarbonylphenylisocyanate (721 mg, 3.8 mmol) provided a pale yellow solid which was purified by column chromatography (1:1 EtOAc-hexane) to give the title compound (804 mg, 65%) O n l as a cream coloured solid; R f 0.17 (EtOAc); δ H (500 MHz, CDCl 3 ); 7.95 (2H, d, J = 8.7 Hz, ArCH), y
	7.47 (2H, d, J = 8.7 Hz, ArCH), 6.31 (1H, s, CHN), 4.35 (2H, q, J = 7.2 Hz, CH 3 CH 2 ), 1.38 (3H, t,
	J = 7.2 Hz, CH 3 CH 2 ), 1.29 (9H, s, t Bu); δ C (75 MHz, DMSO-d 6 ); 165.8, 154.8, 144.9, 140.0, 130.7,
	122.9, 117.7, 107.4, 60.6, 31.0, 29.9, 14.6; ν max /cm -1 3312, 2963, 1696, 1591, 1541, 1273, 1172, 1105

cm -1 ; ESI-HRMS found m/z 331.1760 [M + H] +

URL: http:/mc.manuscriptcentral.com/tandf/gsch Email: suprachem@mail.cm.utexas.edu Supramolecular Chemistry

Acknowledgements

This work was supported by the EPSRC [EP/C007638/1]


Krzysztof Śmietanka

Anna Pikula

Zenon Minta

Wlodzimierz Meissner

A Pikuła

Evidence of persistence and multiple genetic modifications of LPAI H7N7 in wild mallards in Poland is supported by phylogenetic studies

Keywords: avian influenza virus, H7N7, mallards, epidemiology

almost exactly one year apartand that points to resident population of mallards (and other resident waterfowl) as responsible for the perpetuation of AIV in the given area between successive winters. On the other hand, the ornithological data discussed in detail in the paper strongly suggest that the virus transmission between close sites but located in different urban areas is most likely achieved by migratory birds, a fact additionally supported by a close relatedness between different gene segments of Polish H7N7 and AIV detected in wild birds in Europe.A high heterogeneity of the gene pool found in the study is indicative of frequent reassortment events.Additionally, two H7N7isolateswere shown to possess selected genes closely related to AIV detected in domestic poultry in Italy and Czech Republic. This study corroborates the importance of active surveillance in wild birds as a valuable tool of early warning of AI in poultry. (Suarez, 2008). The current classification into subtypes is based on the surface glycoproteins: haemagglutinin (HA) and neuraminidase (NA) and so far 16 HA subtypes and 9 NA subtypes have been described for which wild aquatic birds are considered the natural reservoir (Webster et al., 1992;Swayne & Suarez, 2000;[START_REF] Fouchier | Characterization of a novel influenza A virus hemagglutinin subtype (H16) obtained from black-headed gulls[END_REF].In wild birds AI viruses usually persist in the low pathogenic form but some of the H5 and H7 subtypes have the capacity to become highly pathogenic (HP).The emergence of HP from LP AIV is believed to take place in poultry through a number of processes of which acquisition of multiple amino acids at HA2/HA1 cleavage site plays a crucial role (Swayne &Suarez, 2000) In recent years HP outbreaks caused by viruses of H7 subtypes, even thoughovershadowed by H5N1 epidemic (Sims& Brown, 2008), have also had devastating effects on the poultry industry, e.g. resulting in 13 million of birds dead or culled in Italy in 1999-2000 (HPAI H7N1) and 25.6 million birds stamped outin the Netherlands in 2003(HPAI H7N7) [START_REF] Capua | H7N1 avian influenza in Italy (1999 to 2000) in intensively reared chickens and turkeys[END_REF][START_REF] Alexander | Highly pathogenic avian influenza outbreaks in Europe, Asia, and Africa since 1959, excluding the Asian H5N1 virus outbreaks[END_REF].In 2008 and 2009 outbreaks of HPAI H7N7 were recorded in the UK and Spain but they were contained rapidly and did not spread (OIE online reports,2010).

Despite the great importance, there are few reports describing phylogenetic analysis of H7N7 strains isolated from their natural reservoir [START_REF] Campitelli | Molecular analysis of avian H7 influenza viruses circulating in Eurasia in 1999-2005: detection of multiple reassortant virus genotypes[END_REF]Jahangiret al., 2010;[START_REF] Metreveli | Phylogenetic analysis of the hemagglutinin gene of low pathogenic avian influenza virus H7N7 strains in mallards in Northern Europe[END_REF]. Furthermore, there is still little information dealing with virushost ecology.

Material and methods

Viruses. In the study four avian influenza H7N7 viruses isolated from cloacal swabs collected from wild mallards (Anas platyrhynchos) were used(Table 1 al., 1998)were also found in all of the isolates. The H7 HA amino acid sequence revealed five possible glycosylation sequons but notat positions123 or 149 (H7 numbering, a feature shared by H7 strains isolated from poultry) [START_REF] Banks | Phylogenetic analysis of H7 haemagglutinin subtype influenza A viruses[END_REF]Banks et al.2001). The PB2 E627K mutation, related to an increased adaptation to mammals (Subbaraoet al., 1993;[START_REF] Shinya | PB2 amino acid at position 627 affects replicative efficiency, but not cell tropism, of Hong Kong H5N1 influenza A viruses in mice[END_REF] was not observed. The S31N amino acid substitution in the M2 protein, responsible for conferring resistance to amantadanes [START_REF] Scholtissek | On the origin of the human influenza viruses subtypes H2N2 and H3N2[END_REF], was not detected in any of the tested H7N7.The NS1 protein did not show any deletion and the D92E substitution, associated with viral resistance to cytokines in pigs [START_REF] Seo | Lethal H5N1 influenza viruses escape host anti-viral cytokine responses[END_REF], was not observed.

Phylogenetic analysis.

All of the gene segments of the viruses analyzed in this study were of avian origin and clearly belonged to Eurasian lineage (Fig. 2-5 and Supplementary Fig. S1-S4). With regard to the HA gene, all of the Polish H7 isolates fell into the same major clade of phylogenetic tree (Fig. 2) with the sequence homology 96.2 to 99.3%, and the highest similarity was observed between with similarity of sequences 98.5%.

In the case of the M gene, 97.0 -98.8 % of nucleotide sequence identity was found but none of the Polish isolates were directly related to one another. On the other hand, a high degree of relationship was observed between A/mallard/Poland/01/08 and A/turnstone/Netherlands/1/2007 (99.5% nucleotide sequence similarity, Fig. 5).

The analysis of NS gene demonstrated that Polish H7N7 belonged to allele A [START_REF] Ludwig | Phylogenetic relationship of the nonstructural (NS) genes of influenza A viruses[END_REF]Zohari et al., 2008)

Discussion

Mallards constitute an important host for AIV. According to [START_REF] Munster | Spatial, temporal, and species variation in prevalence of influenza A viruses in wild migratory birds[END_REF], an average prevalence of AIV infections in this species established in a large-scalesurveillance study was 7.3%. However, the frequency of AIV isolations in mallards is dependant on season and geographical location.At the sampling sites in NorthernEuropeAIV isolationwasabout 3-fold higher as compared to wintering sites in the West, with peak in the early fall migration [START_REF] Munster | Spatial, temporal, and species variation in prevalence of influenza A viruses in wild migratory birds[END_REF]. Among mallards breeding in Poland, like in other European countries and in North America, two groups can be distinguished [START_REF] Cramp | The Birds of the Western Palearctic[END_REF][START_REF] Heusmann | Movements and survival rates of park mallards[END_REF] [START_REF] Cramp | The Birds of the Western Palearctic[END_REF]. Females reveal very high philopatry to the breeding grounds, higher than males,and in spring males follow females to the breeding sites [START_REF] Cramp | The Birds of the Western Palearctic[END_REF][START_REF] Rohwer | Female-biased philopatry, monogamy, and the timing of pair formation in migratory waterfowl[END_REF][START_REF] Evrard | Mallard and Blue-winged Teal philopatry in Northwest Wisconsin[END_REF].Therefore some males of the sedentary populations may join migratory populations whereas some migratory males can stay put on the wintering ground. Such behavioral features create a perfect environment for the transmission of AIV between birds from different populations. Due to the fact that mallards are the most numerous waterfowl in Europe (population estimated at ~7.5 million, Wetlands International, 2006), this species may play a crucial role in the long-lasting perpetuation of AIV, as suggested by [START_REF] Munster | Avian influenza virus: Of virus and bird ecology[END_REF].

In our study we analyzed molecular and phylogenetic characteristics of four H7N7 strains isolated from wild mallards in Northern and North-eastern Poland in 3 successive winters (2007/2008, 2008/2009, and 2009/2010).Even though the molecular markers revealed a profile typical of AIV of wild bird origin (low pathogenicity, no markers related to increased adaptation to poultry and mammals or resistance to some antiviral drugs), these featuresneeded to be analyzed even in AIV isolated from wild birds.There have beensporadic cases of HPAI H7 in wild birds (Swayne, 2008).Moreover, some amino acid substitutions in the binding receptor site of HA and mutations associated with resistance to amantadine in a mallard-derived H7 in Italy have also been found [START_REF] Campitelli | Molecular analysis of avian H7 influenza viruses circulating in Eurasia in 1999-2005: detection of multiple reassortant virus genotypes[END_REF].

The phylogenetic study demonstrateda high degree of inter-relationship between Polish H7N7 but only in relation to selected genes. The major conclusion is that the isolate to our studies, on one occasion the virus was detected twice at one site, two months apart. A phylogenetic study of H7 and N7 genes revealed that these isolates were very similar or virtually identical [START_REF] Jahangir | Haemagglutinin and neuraminidase characterization of low pathogenic H5 and H7 avian influenza viruses isolated from Northern pintails (Anas acuta) in Japan, with special reference to genomic and biogeographical aspects[END_REF].However, the high frequency of H7N7 isolation in that region was noted exclusively in one wintering season as opposed to our study in which we demonstrated the presence of AIV with genetically related genes between two consecutive winter seasons in the same area.

On the other hand, the introduction of new AI viruses and their efflux from the area is caused by migratory waterfowl, a fact supported by ornithological observations and our phylogenetic studies in which we found a high degree of relationship between some of the genome segments of the Polish H7N7 and AIV detected in wild birds in Europe.Despite a very close relationship between HA and PB1 of AIV H7N7 isolated from mallards at close locations in Gdańsk (A/mallard/Poland/446/09) and Słupsk (A/mallard/Poland/41/09), i.e. 102 km away from each other, it is the most likely that the precursor viruses bearing these gene segments were transmittedbetween these sites via migratory birds since urban mallards show a very high fidelity to their whereabouts and usually move within very restricted area of the city in which they stay [START_REF] Heusmann | Movements and survival rates of park mallards[END_REF]; W. Meissner -unpublished data from studies conducted in three Polish cities).

The AI virus undergoes frequent reassortment events: the H7N7 isolates detected in Poland never shared the same constellation of all genome segments and the close relatedness (>98.5%) was only found in relation to single genes, indicatingin most cases different studies have also proved that reassortment events between AIV of various originare frequent [START_REF] Hatchette | Influenza A viruses in feral Canadian ducks: extensive reassortment in nature[END_REF][START_REF] Macken | Genotype turnover by reassortment of replication complex genes from avian influenza A virus[END_REF][START_REF] Campitelli | Molecular analysis of avian H7 influenza viruses circulating in Eurasia in 1999-2005: detection of multiple reassortant virus genotypes[END_REF]Briand et al., 2010).

Interestingly, unlike other genes, H7 gene seems to have established a relatively conserved sublineage for wild mallards in Poland.However, the assumption that this is a result of an adaptive process to this species of birds requires more evidence due to alimited number of available H7 isolates. The fragmentary data of virological surveillance in Polandis on one hand caused by a number and species of birds tested (approximately 3,000 a year, mostly mallards), and on the other hand, possibleinsufficient sampling and storage conditions of swabs and faeces,whichmay have also contributed to the reduction of an overall AIV detection rate (<2% a year). Consequently, it cannot be excluded that there may have been other undetected AIV H7 strains of the same and different sublineagescirculating in mallards and other species of waterfowl. This issue will be raised in future after more intensive and targeted surveillance in places with previous records of AIV H7.

Another noteworthy aspect of the phylogenetic studies is a close relationship between 2 genes (HA & NP) of A/mallard/Poland/16/09 and AIV H7N9isolated from a flock of domestic geese in the Czech Republic. On the other hand, as mentioned above, a high relatedness of its NA & NS to A/mallard/Poland/446/09 was shown. The isolate A/mallard/Poland/01/08 was also found to have an NP gene most closely related toH7N3 viruses detected in poultry in Italy in 2003. There is also strong evidence from other studies that low pathogenic AIV spill over to poultry as phylogenetic studies have shown a directrelationship between AIV genes from wild and domestic birds, even from HPAI outbreaks [START_REF] Campitelli | Interspecies transmission of an H7N3 influenza virus from wild birds to intensively reared domestic poultry in Italy[END_REF][START_REF] Fouchier | Characterization of a novel influenza A virus hemagglutinin subtype (H16) obtained from black-headed gulls[END_REF]Terregino et al., 2007 al., 2008;Briand et al., 2010;[START_REF] Metreveli | Phylogenetic analysis of the hemagglutinin gene of low pathogenic avian influenza virus H7N7 strains in mallards in Northern Europe[END_REF]. This study corroborates the importance of active surveillance in free living birds as a valuable tool of an early warningsystem ofLPAI and HPAI outbreaks. It is very important in relation to H7N7viruses, for which the number of available sequences is still very limited. We also suggest that the survey programmes for AIV should not only be targeted at migratory population at major flyways but that resident waterfowl should also be considered.

F

  virus (AIV) is an orthomyxovirus with a genome consisting of eight single-stranded RNA segments encoding for at least 10 different viral proteins: PB2, PB1, PA, HA, NP, NA, M1, M2, NS1, and NS2

  In the present study we have analyzed the molecular features and phylogenetic relationship of AIV H7N7 isolated from wild mallards in Poland in 2007-2009 based on partial or full-length sequencing of all 8 segments of the viral genome and compared the results to AIV sequencesavailable in the public domain.

  Of special significance is the fact that despite A/mallard/Poland/41/09 and A/mallard/Poland/446/09 sharing a very closely related HA gene (>99% nucleotide identity) their NA genes were completely different (~90% nucleotide similarity). Other

F

Figure 1 .Figure 2 .Figure 3 .Figure 4 .Figure 5 .

 12345 Figure 1.Geographical location of the LPAIV H7N7 cases in wild mallards: A -A/mallard/Poland/01/08, B -A/mallard/Poland/16/09, C -A/mallard/Poland/41/09, D -A/mallard/Poland/446/09; isolates B&D detected at the same sampling location

Molecular characterization of AIV H7N7.All of

  ). The viruses were isolated in 3

	IZSVe, Legnaro, Italy, available upon request).The complete coding sequences or large the investigated isolates had the monobasic
	portions of all 8 genes were generated using BigDye Terminator v3.1 (Applied Biosystems). amino-acid cleavage site motif at the HA cleavage site: PEIPKGR-GLF (3 isolates)
	The products of the sequencing reactions were sequenced in the 3730xl DNA Analyzer andPELPKGR-GLF (1 isolate) indicating low pathogenicity (Tab.1). The amino-acid residues
	(Applied Biosystems) in Genomed, Warsaw. 226Q and 228G (H3amino acid numbering) at the receptor binding sites, associated with
	preferential binding to α(2,3)-linked sialic acid (Vineset
	Phylogenetic analyses. The nucleotide regions investigated in the phylogenetic analyses were
	F o r as follows:PB2: 27-1203, PB1: 1-1920, PA: 983-2135, HA: 26-1637, NA: 20-1264, NP: 46-1442, M: 1-937, NS: 1-812. For sequence editing and analysis, modules SeqMan (assembly of F o r
	P e the consensus sequence from electrophoregrams) and EditSeq (translation from nucleotide P into amino acid sequences) of DNAStar LaserGene (version 8.1.3) were used. Sequences e
	successive winters: 2007/2008 (isolate A/mallard/Poland/01/08), 2008/2009 (isolates A/mallard/Poland/16/09 &A/mallard/Poland/41/09), and 2009/2010 (isolate e r were compared with published sequences of AIV of Eurasian and North American lineages using BLAST local alignement search tool available from the National Center for e r
	Biotechnology	Information,	R R	Bethesdda,	Maryland,	USA
	1.Noteworthy is (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Multiple sequence alignements were established and the fact that the isolates A/mallard/Poland/16/09 and e e
	v i e w phylogenies reconstructed with MEGA 4 software (Tamura et al., 2007) with the use of v i e Clustal W and neighbor-joining tree inference method with the Kimura two-parameter substitution model with 1000 bootstrap replicates. Potential N-glycosylation sites were w
	O n l y NetNGlyc 1.0 (http://www.cbs.dtu.dk/services/NetNGlyc/). Gene sequences were submitted to the EpiFLU predicted with the use of the server O n l nucleotide sequence database (platform.gisaid.org)with the following isolate ID: y
	EPI_ISL_26339, EPI_ISL_60707, EPI_ISL_60708, EPI_ISL_74018.
	Results

A/mallard/Poland/446/09). The geographical location of sampled mallards is presented in Fig.

A/mallard/Poland/446/09 were detected on the same pond (~3500 m 2 ) almost exactly one year apart

(January 2009-December 2009)

. All the viruses were isolated in embryonated chicken specific pathogen free (SPF) eggs followed by identification in haemagglutination inhibition test according to standard procedures

(OIE, 2008)

. Allantoic fluids from inoculated eggs of the 1 st passage exhibiting haemagglutinating activity were used directly for RNA isolation or stored at -80°C until use.

RNA extraction, RT-PCR and gene sequencing.The RNA was extracted from infected allantoic fluid using Qiagen RNeasy Mini Kit according to manufacturer's instruction.

Reverse transcription and PCR reaction were performed in one tube using Qiagen RT-PCR kit using specific primers (sequences and cycling conditions kindly provided by Dr. W. Dundon,

  Interestingly, in contrast to HA gene analysis, the nucleotide homology percentage of the NA gene of A/mallard/Poland/41/09 and A/mallard/Poland/446/09 was very low (90.3%) and both isolates were located on two distant branches of the phylogenetic tree.In reference to the PB2 gene, two Polish isolates A/mallard/Poland/01/08 and A/mallard/Poland/41/09 clustered in the same subgroup (sequence homology 97.3%), A/mallard/Poland/16/09 was located within a sister subgroup of the same group with up to96.0% of sequence similarity. TheA/mallard/Poland/446/09 showed no significant similarity in relation to the other Polish H7N7 (93.5 to 94.2%) or sequences available at GenBank (Fig.

	sequence similarity (99.4%) was found between A/mallard/Poland/16/09 and A/goose/Czech
	Republic/1848/2009(H7N9).The A/mallard/Poland/01/08 grouped closely to AIV H7 detected
	in poultry in Italy: A/turkey/Italy/2685/2003(H7N3) and A/turkey/Italy/220185/2003(H7N3),
	F o r F o r
	P P
	e e
	e r e r
	R R
	e e
		v i e v i e
	w As for the PA gene, A high diversity were found among Polish isolates,which w
	O n l y clustered within 3 different clades with sequence similarity ranging from 94.0 to 96.6% (Supplementary Fig. S2).Some of them, however, showed a very high degree of relationship O n l with PA gene detected in wild waterfowl in Europe, eg. A/mallard/Poland/01/08 to y
	A/mallard/Netherlands/22/2007(H7N1)	(99.2%),A/mallard/Italy/299/05(H7N7)(99.0%)or
	A/common teal/Netherlands/1/2005(H8N4) (99.0%).
	A high heterogeneity was also found in relation to NP gene because all of the H7N7

A/mallard/Poland/41/09 and A/mallard/Poland/446/09 (99.3%). The A/mallard/Poland/16/09 was most closely related to A/goose/Czech Republic/1848/2009(H7N9) (99.8%), isolated from poultry.

The NA genes of the tested strains were 90.3% to 98.7% homologous to each other, and A/mallard/Poland/16/09 and A/mallard/Poland/446/09 (isolated at the same sampling site almost exactly 1 year apart) were the most closely related (Fig.

3

). Both isolates were also related to A/mute swan/Hungary/5973/2007(H7N7) with sequence identity up to 97.9%.

4).

Regarding the PB1 gene, H7N7 viruses isolated in Poland clustered in 3 different subgroups of the phylogenetic tree. The phylogenic positionof A/mallard/Poland/41/09 and A/mallard/Poland/446/09on the same branch (sequence homology 99.1%) resembled theirlocation on the phylogenetic tree constructed for the HA gene.Both isolates were similar to A/mute swan/Hungary/5973/2007(H7N7) (nucleotide sequence similarity up to 98.0%,SupplementaryFig. S1). The A/mallard/Poland/01/08 shared a very similar PB1 gene with A/turnstone/Netherlands/1/2007 (nucleotide identity 99.1%).

detected in Poland clustered into 4 different subgroups (homology of nucleotidesequences ranged from 93.9 to 96.0%, Supplementary Fig.

S3

). Similarly to the HA analysis, the highest

  . Mallards resident in urban areas do not migrate while most of the birds breeding outside of urban regions show regular movements between breeding and wintering sites. However, in mild climate areas of Western Europe breeding populations of this species are sedentary making only local movements during severe weather conditions[START_REF] Scott | Atlas of Anatidae Populations in Africa and Western Eurasia[END_REF]. Mallards ringed in Poland during breeding seasonwere recovered in Western and South-Western Europe. On the other hand, Poland is the wintering site for mallards breeding in Northern and North-Eastern Europe (e.g. Scandinavia, Russia, Baltic states)(Fransson & Petterson, 2001; unpublished data of the Polish Ringing Center).According toStallknecht & Brown (2008) the avian influenza virus maintenance cycle is based on virus transmission on the breeding and wintering grounds between migratory and resident birds but the mechanism

is still poorly understood. It is known that in urban areas in Poland both sedentary and migratory mallards form mixed populations in winter.Pair formation in mallards take place in autumn and winter

  The bird was ringed and sampled on a pond in Gdansk on the 27 th of December 2009 and it was recovered at the same site on the 23 rd of February, 1 st of May and early June of 2010.Additionally, it was seen on the 7 th of January and 7 th of March in the area within a radius of 2 /mallard/Poland/16/09 & A/mallard/Poland/446/09 detected in mallards at the same sampling site 1 year apart were derived from avian influenza virus precursor strain(s) that persisted in the Gdańsk area for a long period of time and emerged in January 2009 and thenin December 2009.Interestingly,[START_REF] Jahangir | Avian influenza and Newcastle disease viruses from northern pintail in Japan: isolation, characterization and inter-annual comparisons during 2006-2008[END_REF] found a high isolation rate of AIV H7N7 in northern pintails (Anas acuta) in wintering season 2007/2008 in Japan,and similarly

	F o r
	P
	e
	e r
	R
	e
	v i e
	w
	O n l
	y
	km from the sampling site (observation by W. Meissner). These findings confirm that
	anindividual carrying A/mallard/Poland/446/09 strain was a resident bird and it reinforces our

A/mallard/Poland/446/09 is a reassortant virus that shares a very recent common ancestor with A/mallard/Poland/41/09 (in relation to HA&PB1 gene), A/mallard/Poland/16/09 (with respect toNA & NS gene) and A/mallard/Poland/01/08 (as regardsNS gene).The spatial and temporal distribution of the cases was significant: the sampling site of birds carrying A/mallard/Poland/16/09 &A/mallard/Poland/446/09 isolates was located on the same pond in

Gdańsk (January-December 2009), approximately 100 km away from the city of Słupsk, in which A/mallard/Poland/41/09 had been detected in March 2009. Both sites were located 330-430 km from the sampling location of the mallard from which A/mallard/Poland/01/08 had been recovered at the end of 2007. The ornithological observations resulted inseveral resightings of the male (ring number SN 11463), the host ofA/mallard/Poland/446/09 isolate.

hypothesis thatresident mallards contribute to the long-term circulation of AIV in the given area between successive winters. We suggest that the NA and NS segments of A

Table 1Molecular

 1Molecular characteristics of the H7N7 Polish isolates in this study

	Isolate	Location	Sample	HA	PB2	NS1	M2
			collection	cleavage site	sequence at	sequence at	sequence at
			date(ring	(pathogenicity)	amino acid 627	amino acid 92	amino acid 31
			number of bird)
	A/mallard/Poland/01/08	Białystok	28.12.2007	PEIPKGR-GLF	E	D	S
			(SN8884)	(low
				pathogenic)
	A/mallard/Poland/16/09	Gdańsk	12.01.2009	PELPKGR-	E	D	S
			(SN 10243)	GLF
				(low
				pathogenic)
	A/mallard/Poland/41/09	Słupsk	16.02.2009	PEIPKGR-GLF	E	D	S
			(SN 10266)	(low
				pathogenic)
	A/mallard/Poland/446/09	Gdańsk	27.12.2009	PEIPKGR-GLF	E	D	S
			(SN 11463)	(low
				pathogenic)

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

Acknowledgements

This work was supported by European Network of Excellence (EPIZONE) WP6.2 "Molecular epidemiology and surveillance of AI and APMV" and EU 6 th framework programmes INN-FLU (SSPE-CT-2006-44372) and NEW-FLUBIRD (044490).