R Martin-Iguacel

J M Llibre

H Nielsen

E Heras

L Matas

R Lugo

B Clotet

G Sirera

: E Heras

Lymphogranuloma venereum proctocolitis: a silent endemic disease in men who have sex with men in industrialised countries

Lymphogranuloma venereum proctocolitis: a silent endemic disease in men who have sex with men in industrialised countries

Introduction

Lymphogranuloma venereum (LGV) is a systemic sexually transmitted disease (STD) caused by serovars L1, L2 and L3 of the obligate intracellular bacterium Chlamydia trachomatis. C. trachomatis is classified into 15 serovars based on immunogenic epitope analysis of the major outer membrane protein. Serovars A, B, Ba and C are associated with ocular trachoma and strains D-K with genital tract disease and with inclusion conjunctivitis, and represent the most frequent STD in industrialised countries. Only serovars L1, L2 and L3 cause LGV, probably due to their tropism for the lymphatic system, in contrast to serovars A-K, which affect mucocutaneous tissue. The L2 serovar can be further classified into L2, L', L2a and L2b, and the recently described L2c-L2g according to amino acid differences [START_REF] Gomes | Lymphogranuloma venereum in Portugal: unusual events and new variants during 2007[END_REF][START_REF] Stary | New Chlamydia trachomatis L2 strains identified in a recent outbreak of lymphogranuloma venereum in Vienna, Austria[END_REF].

Endemic in Africa, the Caribbean and many parts of Asia, prior to 2003 LGV was considered rare in industrialised countries and the sporadic cases seen were deemed to be imported from endemic areas. In 2003 a cluster of LGV with a new strain, L2b, was detected in Rotterdam, the Netherlands, among men who have sex with men (MSM) presenting mainly as proctitis [START_REF] Nieuwenhuis | Resurgence of lymphogranuloma venereum in Western Europe: an outbreak of Chlamydia trachomatis serovar l2 proctitis in The Netherlands among men who have sex with men[END_REF]. Since then many countries all across Europe, North America and Australia have reported clusters of cases in sexual networks of MSM with a total of over 1,600 cases of proctitis reported so far [START_REF] Gomes | Lymphogranuloma venereum in Portugal: unusual events and new variants during 2007[END_REF][START_REF] Stary | New Chlamydia trachomatis L2 strains identified in a recent outbreak of lymphogranuloma venereum in Vienna, Austria[END_REF][START_REF] Bremer | Lymphogranuloma venereum emerging in men who have sex with men in Germany[END_REF][START_REF] Macdonald | Initial results of enhanced surveillance for lymphogranuloma venereum (LGV) in England[END_REF][START_REF] Ward | Lymphogranuloma venereum in the United Kingdom[END_REF][START_REF] Jebbari | Update on lymphogranuloma venereum in the United Kingdom[END_REF][START_REF] Gebhardt | Lymphogranuloma venereum (LGV) serotype L2 in Switzerland, 2003-2005[END_REF][START_REF] Goldenberger | Analysis of 721 Chlamydia trachomatis-positive urogenital specimens from men and women using lymphogranuloma venereum L2-specific realtime PCR assay[END_REF][START_REF] Vandenbruaene | Lymphogranuloma venereum outbreak in men who have sex with men (MSM) in Belgium, January 2004 to[END_REF][START_REF] Herida | Rectal lymphogranuloma venereum surveillance in France 2004-2005[END_REF][START_REF] Halioua | Ano-rectal lymphogranuloma venereum: 22 cases reported in a sexually transmitted infections center in Paris[END_REF][START_REF] Cusini | Lymphogranuloma venereum: the Italian experience[END_REF][START_REF] Van De Laar | A slow epidemic of LGV in the Netherlands in 2004 and 2005[END_REF][START_REF] Koedijk | An ongoing outbreak of lymphogranuloma venereum in the Netherlands, 2006-2007[END_REF][START_REF] Velicko | Lymphogranuloma venereum-increased spread in Sweden. A rare Chlamydia subtype which become more and more common among men who have sex with men[END_REF][START_REF] Savage | European Surveillance of Sexually Transmitted Infections n (2009) Lymphogranuloma venereum in Europe, 2003-2008[END_REF][START_REF] Castro | Anorectal lymphogranuloma venereum: the first two confirmed cases in Portugal[END_REF][START_REF] Berglund | Two cases of lymphogranuloma venereum (LGV) in homosexual men in Stockholm[END_REF].

The majority of patients were HIV-infected with high rates of promiscuity and high-risk sexual behaviour, often involving international sexual networks, which correlates with the high rate of concomitant STD observed.

The atypical clinical presentation, the unawareness of physicians and patients with regard to the disease, and the lack of a routine diagnostic test for LGV serovars of C. trachomatis have conditioned a delay in the diagnosis and even misdiagnosis.

The untreated disease is associated with significant sequelae with chronic inflammation of the lymphatic vessels, progressive lymphoedema and sclerosing fibrosis leading to fistula formation and strictures and disfiguring conditions such as genital elephantiasis and esthiomene [START_REF] Mabey | Lymphogranuloma venereum[END_REF].

The current resurgence of LGV represents furthermore a significant public health problem as the ulcerative nature of LGV may facilitate both transmission and acquisition of HIV infection and other STD as well as other blood-borne diseases like hepatitis C [START_REF] Götz | A cluster of acute hepatitis C virus infection among men who have sex with men-results from contact tracing and public health implications[END_REF].

It is therefore critical to increase the awareness of the reemergence of this disease among health care professionals in industrialised countries for prompt diagnosis and appropriate treatment. It is important to highlight that specific diagnostic tests not routinely used are required to allow the diagnosis of LGV infections.

This review addresses the characteristics of the present recurrence of LGV proctocolitis in MSM in high-income, non-tropical settings.

Materials and methods

This review used a systematic search in the literature using PubMed. The incorporating criteria were "Chlamydia trachomatis", "Lymphogranuloma venereum", "homosexual male", "men who have sex with men" and "proctitis". The search was restricted to the period 1990-2009 and only those series reporting more than 5 patients were included in the present review. A total of 25 studies were identified.

Epidemiology

Lymphogranuloma venereum was endemic in Europe and the US in the pre-antibiotic era, but thereafter only sporadic cases were seen in the western world, and all were considered to be imported [START_REF] Koch | Public health aspects of lymphogranuloma venereum[END_REF][START_REF] Geiger | Venereal lymphogranuloma: public health aspects[END_REF]. However, in the 1980s some clusters of LGV in MSM presenting as proctitis were reported in industrialised, non-tropical areas [START_REF] Quinn | The polymicrobial origin of intestinal infections in homosexual men[END_REF][START_REF] Levine | Chronic proctitis in male homosexuals due to lymphogranuloma venereum[END_REF]. The estimates of incidence are difficult to ascertain, as LGV was not a mandatory reportable disease in most countries. It is still currently endemic in parts of Africa, South East Asia, South America and the Caribbean.

In February 2003 the first case of LGV proctitis in Europe was detected in a recently infected HIV-positive bisexual white man who presented with a painful perianal ulcer, bilateral inguinal lymphadenitis and general malaise in Rotterdam, the Netherlands (NL) [START_REF] Nieuwenhuis | Unusual presentation of early lymphogranuloma venereum in an HIV-1 infected patient: effective treatment with 1 g azithromycin[END_REF]. Subsequently, a cluster of 13 MSM with LGV serovar L2 was detected through contact tracing [START_REF] Nieuwenhuis | Resurgence of lymphogranuloma venereum in Western Europe: an outbreak of Chlamydia trachomatis serovar l2 proctitis in The Netherlands among men who have sex with men[END_REF], most of whom were HIVpositive. They presented with a median duration of 3 months of unspecific intestinal symptoms and 46% had another concomitant STD [START_REF] Gomes | Lymphogranuloma venereum in Portugal: unusual events and new variants during 2007[END_REF][START_REF] Stary | New Chlamydia trachomatis L2 strains identified in a recent outbreak of lymphogranuloma venereum in Vienna, Austria[END_REF][START_REF] Nieuwenhuis | Resurgence of lymphogranuloma venereum in Western Europe: an outbreak of Chlamydia trachomatis serovar l2 proctitis in The Netherlands among men who have sex with men[END_REF][START_REF] Bremer | Lymphogranuloma venereum emerging in men who have sex with men in Germany[END_REF][START_REF] Macdonald | Initial results of enhanced surveillance for lymphogranuloma venereum (LGV) in England[END_REF][START_REF] Ward | Lymphogranuloma venereum in the United Kingdom[END_REF][START_REF] Jebbari | Update on lymphogranuloma venereum in the United Kingdom[END_REF][START_REF] Gebhardt | Lymphogranuloma venereum (LGV) serotype L2 in Switzerland, 2003-2005[END_REF][START_REF] Goldenberger | Analysis of 721 Chlamydia trachomatis-positive urogenital specimens from men and women using lymphogranuloma venereum L2-specific realtime PCR assay[END_REF][START_REF] Vandenbruaene | Lymphogranuloma venereum outbreak in men who have sex with men (MSM) in Belgium, January 2004 to[END_REF]. Several outbreaks in MSM have so far been reported in Western Europe, Australia and the USA, clustering in large cities [START_REF] Stary | New Chlamydia trachomatis L2 strains identified in a recent outbreak of lymphogranuloma venereum in Vienna, Austria[END_REF][START_REF] Bremer | Lymphogranuloma venereum emerging in men who have sex with men in Germany[END_REF][START_REF] Macdonald | Initial results of enhanced surveillance for lymphogranuloma venereum (LGV) in England[END_REF][START_REF] Ward | Lymphogranuloma venereum in the United Kingdom[END_REF][START_REF] Jebbari | Update on lymphogranuloma venereum in the United Kingdom[END_REF][START_REF] Gebhardt | Lymphogranuloma venereum (LGV) serotype L2 in Switzerland, 2003-2005[END_REF][START_REF] Goldenberger | Analysis of 721 Chlamydia trachomatis-positive urogenital specimens from men and women using lymphogranuloma venereum L2-specific realtime PCR assay[END_REF][START_REF] Vandenbruaene | Lymphogranuloma venereum outbreak in men who have sex with men (MSM) in Belgium, January 2004 to[END_REF][START_REF] Herida | Rectal lymphogranuloma venereum surveillance in France 2004-2005[END_REF][START_REF] Halioua | Ano-rectal lymphogranuloma venereum: 22 cases reported in a sexually transmitted infections center in Paris[END_REF][START_REF] Cusini | Lymphogranuloma venereum: the Italian experience[END_REF][START_REF] Van De Laar | A slow epidemic of LGV in the Netherlands in 2004 and 2005[END_REF][START_REF] Koedijk | An ongoing outbreak of lymphogranuloma venereum in the Netherlands, 2006-2007[END_REF][START_REF] Kropp | Emergence of lymphogranuloma venereum in Canada[END_REF][28][START_REF] Lee | Lymphogranuloma venereum becomes an established infection among men who have sex with men in Melbourne[END_REF][START_REF] Stark | Lymphogranuloma venereum in Australia: anorectal Chlamydia trachomatis serovar L2b in men who have sex with men[END_REF][START_REF] Ahdoot | Lymphogranuloma venereum in human immunodeficiency virusinfected individuals in New York City[END_REF][START_REF] Vall Mayans | Outbreak of lymphogranuloma venereum among men who have sex with men in Barcelona 2007/08-an opportunity to debate sexual health at the EuroGames 2008[END_REF].

A total of 232 cases of LGV proctitis had been recorded in the NL by March 2007 [START_REF] Koedijk | An ongoing outbreak of lymphogranuloma venereum in the Netherlands, 2006-2007[END_REF]. Previously, the NL had an incidence of LGV of fewer than 5 cases per year. In the UK a total of 492 cases had been recorded by April 2007 [START_REF] Bremer | Lymphogranuloma venereum emerging in men who have sex with men in Germany[END_REF][START_REF] Jebbari | Update on lymphogranuloma venereum in the United Kingdom[END_REF][START_REF] Velicko | Lymphogranuloma venereum-increased spread in Sweden. A rare Chlamydia subtype which become more and more common among men who have sex with men[END_REF][START_REF] Herida | Rectal lymphogranuloma venereum, France[END_REF]. In Canada there were 88 cases by September 2007 [28]. The first case of LGV in Barcelona was reported in an MSM in 2005 and as of April 2009 a total of 42 confirmed cases had been reported in Catalonia, an autonomous community in the north-east corner of the Iberian Peninsula with a population of around 7 million (Fig. 1).

In all these countries LGV proctitis has been described nearly exclusively in MSM most of whom were HIVinfected, but typically not immunosuppressed, and some had seroconverted to HIV at around the time of the LGV diagnosis [START_REF] Nieuwenhuis | Resurgence of lymphogranuloma venereum in Western Europe: an outbreak of Chlamydia trachomatis serovar l2 proctitis in The Netherlands among men who have sex with men[END_REF][START_REF] Ward | Lymphogranuloma venereum in the United Kingdom[END_REF][START_REF] Herida | Rectal lymphogranuloma venereum, France[END_REF]. The epidemic might be expanding beyond the initial core group of high-risk MSM [START_REF] Gomes | Lymphogranuloma venereum in Portugal: unusual events and new variants during 2007[END_REF][START_REF] De Munain | Two lymphogranuloma venereum cases in a heterosexual couple in Bilbao (Spain)[END_REF], even though the detection of LGV in the general population has not been confirmed in other studies [START_REF] Goldenberger | Analysis of 721 Chlamydia trachomatis-positive urogenital specimens from men and women using lymphogranuloma venereum L2-specific realtime PCR assay[END_REF][START_REF] Herida | First case of urethritis due to Chlamydia trachomatis genovar L2b[END_REF][START_REF] Geisler | Absence of lymphogranuloma venereum strains among rectal Chlamydia trachomatis outer membrane protein A genotypes infecting women and men who have sex with men in Birmingham, Alabama[END_REF]. The majority reported high-risk sexual behaviour including unprotected anal intercourse, fisting, sharing sex toys, often connected to the sex party scene and involving international sexual networks [START_REF] Nieuwenhuis | Resurgence of lymphogranuloma venereum in Western Europe: an outbreak of Chlamydia trachomatis serovar l2 proctitis in The Netherlands among men who have sex with men[END_REF]. In a cross-sectional study the use of enemas was found to be associated with a higher risk of LGV proctitis, probably because such a procedure leads to mucosal damage before receptive anal intercourse favouring the transmission of the disease [START_REF] Vries | Lymphogranuloma venereum proctitis in men who have sex with men is associated with anal enema use and high-risk behavior[END_REF].

Contact tracing was difficult as most cases reported multiple, mainly anonymous sexual contacts.

The HIV-1 infection has been identified as the strongest risk factor for anorectal LGV [START_REF] Nieuwenhuis | Resurgence of lymphogranuloma venereum in Western Europe: an outbreak of Chlamydia trachomatis serovar l2 proctitis in The Netherlands among men who have sex with men[END_REF][START_REF] Van Der Bij | Diagnostic and clinical implications of anorectal lymphogranuloma venereum in men who have sex with men: a retrospective case-control study[END_REF]. MSM have not been confirmed to be a reservoir of LGV and the high rate of HIV co-infection probably reflects the confounding effect of HIV as a surrogate marker of highrisk behaviour; nevertheless, a potential effect of HIV infection increasing the susceptibility to infection or facilitating the expression of the disease cannot be ruled out [START_REF] Stary | New Chlamydia trachomatis L2 strains identified in a recent outbreak of lymphogranuloma venereum in Vienna, Austria[END_REF][START_REF] Klint | Lymphogranuloma venereum prevalence in Sweden among men who have sex with men and characterization of Chlamydia trachomatis ompA genotypes[END_REF][START_REF] Tinmouth | Is there a reservoir of sub-clinical lymphogranuloma venereum and non-LGV Chlamydia trachomatis infection in men who have sex with men?[END_REF][START_REF] Alexander | What is the prevalence of lymphogranuloma venereum in the United Kingdom among men who have sex with men, and is there an asymptomatic reservoir of infection?[END_REF].

Since the introduction of highly active anti-retroviral therapy an increase in unprotected sexual practices has been observed worldwide among both HIV-positive andnegative MSM, which could have contributed to facilitating the spread of LGV in this core group. During the 1980s the Bahamas registered a sequential epidemic of crack cocaine use, genital ulcer-inguinal adenopathy disease (including bubonic LGV) and HIV infection in heterosexuals related to unsafe sexual behaviours [START_REF] Bauwens | Epidemic lymphogranuloma venereum during epidemics of crack cocaine use and HIV infection in the Bahamas[END_REF].

It seems unlikely that LGV constitutes an opportunistic infection involving immunosuppressed HIV-infected patients, and studies have failed to detect a significant asymptomatic reservoir in the general population [START_REF] Goldenberger | Analysis of 721 Chlamydia trachomatis-positive urogenital specimens from men and women using lymphogranuloma venereum L2-specific realtime PCR assay[END_REF][START_REF] Herida | First case of urethritis due to Chlamydia trachomatis genovar L2b[END_REF][START_REF] Geisler | Absence of lymphogranuloma venereum strains among rectal Chlamydia trachomatis outer membrane protein A genotypes infecting women and men who have sex with men in Birmingham, Alabama[END_REF].

On the other hand, the ulcerative nature of LGV could enhance transmission and acquisition of HIV and other STD, as well as other blood-borne diseases like hepatitis C as indicated by the high co-prevalence of STD observed in these patients. Although hepatitis C is not usually considered a STD it has been consistently reported to be sexually transmitted among MSM, especially with high-risk sexual practices that lead to mucosal damage [START_REF] Götz | A cluster of acute hepatitis C virus infection among men who have sex with men-results from contact tracing and public health implications[END_REF].

Almost all patients presented with long-lasting gastrointestinal symptoms and were initially misdiagnosed, which increased the risk of transmission. In some series there was a significant group with no or few symptoms, which could suggest that the number of cases reported might underestimate the real disease occurrence [START_REF] Vries | Lymphogranuloma venereum proctitis in men who have sex with men is associated with anal enema use and high-risk behavior[END_REF][START_REF] Van Der Bij | Diagnostic and clinical implications of anorectal lymphogranuloma venereum in men who have sex with men: a retrospective case-control study[END_REF].

A previously non-described L2b Chlamydia variant has been identified as the main causative agent [START_REF] Herida | Rectal lymphogranuloma venereum surveillance in France 2004-2005[END_REF][START_REF] Kropp | Emergence of lymphogranuloma venereum in Canada[END_REF][START_REF] Stark | Lymphogranuloma venereum in Australia: anorectal Chlamydia trachomatis serovar L2b in men who have sex with men[END_REF][START_REF] Spaargaren | New lymphogranuloma venereum Chlamydia trachomatis variant, Amsterdam[END_REF][START_REF] Meyer | Repeated detection of lymphogranuloma venereum caused by Chlamydia trachomatis L2 in homosexual men in Hamburg[END_REF]. Nevertheless, this variant seems to have been circulating in the NL since 2000 (no earlier samples were available) and has also been detected retrospectively in rectal samples from symptomatic MSM collected from 1979 to 1985 in San Francisco (LGV represented 13% of the collected samples and 67.3% of the C. trachomatispositive samples) [START_REF] Schachter | Lymphogranuloma venereum: how to turn an endemic disease into an outbreak of a new disease? Start looking[END_REF]. This fact could support the hypothesis proposed by some authors of the existence of an ongoing unrecognised endemic disease among MSM for at least 20 years rather than a new outbreak [START_REF] Schachter | Lymphogranuloma venereum: how to turn an endemic disease into an outbreak of a new disease? Start looking[END_REF][START_REF] Spaargaren | Slow epidemic of lymphogranuloma venereum L2b strain[END_REF]. However, other countries have not confirmed the retrospective detection of undiagnosed cases in stored samples [START_REF] Gebhardt | Lymphogranuloma venereum (LGV) serotype L2 in Switzerland, 2003-2005[END_REF][START_REF] Klint | Lymphogranuloma venereum prevalence in Sweden among men who have sex with men and characterization of Chlamydia trachomatis ompA genotypes[END_REF][START_REF] Lister | Variability of the Chlamydia trachomatis omp1 gene detected in samples from men tested in male-only saunas in Melbourne, Australia[END_REF].

The existence of an unrecognised endemic disease could also be supported by the fact that before 2003 no systematic surveillance of LGV was available and that the diagnostic tests necessary to find LGV serovars of C. trachomatis were not routinely available, and anal swabs positive for Chlamydia were recorded as Chlamydia proctitis with no additional testing for LGV being performed. On the other hand, there are some characteristics suggesting an ongoing outbreak like a sudden increase in the number of cases potentially linked and the presence of geographical clustering. The increase in high-risk sexual practices, such as serosorting, among HIV-positive MSM after the introduction of highly active anti-retroviral therapy (HAART) could have contributed to the spread of the disease in the last years in this specific group, as observed for other STD such as syphilis and gonorrhea [START_REF] Fenton | Increasing rates of sexually transmitted diseases in homosexual men in Western Europe and the United States: why?[END_REF]. A selection bias (testing only HIV-infected patients and HIV-infected persons being more aware) cannot be excluded [START_REF] Goldenberger | Analysis of 721 Chlamydia trachomatis-positive urogenital specimens from men and women using lymphogranuloma venereum L2-specific realtime PCR assay[END_REF]. Further investigations are needed to shed light on this issue.

Clinical syndrome

Lymphogranuloma venereum is caused by Chlamydia trachomatis serovars L1-L3. Serovars B and D-K are responsible for the syndromes of non-gonococcal urethritis and cervicitis and can also cause a mild inflammation of the rectal mucosa. It is an invasive disease and has tropism for the lymphatic system, causing severe inflammation, often with systemic implication in contrast to serovars A-K, which are associated with mild or asymptomatic systemic implications.

LGV can be transmitted through vaginal, anal or oral sexual contact. The clinical characteristics depend on the site of inoculation.

The classical description of LGV consists of a primary lesion at the site of inoculation after an incubation period of 3-30 days involving a small, painless genital papula, which may ulcerate, heal spontaneously and leaving no scarring. This lesion frequently passes unnoticed. The initial infection can also be intraurethral, producing an asymptomatic urethritis, cervical, producing cervicitis, or in the rectum, producing proctitis. It was well known that LGV serovars were associated with more severe forms of proctitis, especially seen in the collective of MSM [START_REF] Quinn | The polymicrobial origin of intestinal infections in homosexual men[END_REF][START_REF] Levine | Chronic proctitis in male homosexuals due to lymphogranuloma venereum[END_REF], but this is probably more common than previously presumed. This primary stage is followed after days to weeks by a secondary stage characterised by lymphatic involvement of those lymph nodes that drain the area of the primary lesion. In the classical form tender unilateral or bilateral inguinal and/or femoral ("groove" sign) adenopathies appear. When the primary infection occurs in the rectum the deep iliac lymph nodes are affected, but remain unnoticed. This can also be observed in women due to the drainage of the cervical or upper vaginal area to the perirectal lymph nodes. The lymph nodes become inflamed and painful and may coalesce to form a "bubo" or abscesses that may rupture spontaneously with the development of fistulas or sinus tracts. This stage may be associated with systemic features such as malaise, fever and headaches.

If untreated, a proportion of patients will progress to a chronic granulomatous inflammatory process (tertiary stage) with lymphatic obstruction leading to fistula formation and strictures and disfiguring conditions such as genital elephantiasis and esthiomene (Greek, "eating away") which refers to hypertrophic enlargement with ulceration of the external genitalia.

In the present outbreak of LGV among MSM the majority of patients were HIV-positive with preserved CD4 cell counts. The role of the CD4 cell count in modulating the clinical picture of LGV proctocolitis, as occurs with almost all opportunistic infections associated with HIV, is yet to be elucidated [START_REF] Vries | Lymphogranuloma venereum proctitis in men who have sex with men is associated with anal enema use and high-risk behavior[END_REF]. In a retrospective study no differences in the clinical picture of proctitis investigated by proctoscopy were noted among HIVpositive patients who did or did not receive HAART [START_REF] Van Der Bij | Diagnostic and clinical implications of anorectal lymphogranuloma venereum in men who have sex with men: a retrospective case-control study[END_REF]. However, more than the use of HAART, it would be interesting to examine the clinical presentation stratified by the CD4 count.

The immune restoration inflammatory syndrome (IRIS) seen with many opportunistic infections after the initiation of HAART seems unlikely to modulate the clinical presentation of LGV proctocolitis, since most patients had been on anti-retroviral therapy (ART) for several years before the LGV diagnosis and no signs of IRIS were registered [START_REF] Vries | Lymphogranuloma venereum proctitis in men who have sex with men is associated with anal enema use and high-risk behavior[END_REF][START_REF] Van Der Bij | Diagnostic and clinical implications of anorectal lymphogranuloma venereum in men who have sex with men: a retrospective case-control study[END_REF].

High rates of co-infection with other STD and hepatitis C have been observed [START_REF] Nieuwenhuis | Resurgence of lymphogranuloma venereum in Western Europe: an outbreak of Chlamydia trachomatis serovar l2 proctitis in The Netherlands among men who have sex with men[END_REF][START_REF] Ward | Lymphogranuloma venereum in the United Kingdom[END_REF][START_REF] Götz | A cluster of acute hepatitis C virus infection among men who have sex with men-results from contact tracing and public health implications[END_REF]. Tables 1 and2 summarise the main clinical characteristics of different series reported so far, where wide variations in clinical symptoms can be observed. They presented typically with a rectal syndrome (>90%) with moderate to severe ulcerative proctitis or proctocolitis with mucopurulent discharge, rectal pain, bloody rectal discharge, constipation and tenesmus. Systemic symptoms like fever and malaise and weight loss were relatively common. Paradoxically, simultaneous penile lesions and/or inguinofemoral lymphadenopathy have been reported rarely [START_REF] Sethi | Lymphogranuloma venereum presenting as genital ulceration and inguinal syndrome in men who have sex with men in London, United Kingdom[END_REF]. Despite the fact that most patients had reported unprotected insertive and receptive anal sexual intercourse, only few cases of urethritis due to LGV serovars have been reported in the present outbreak [START_REF] Herida | First case of urethritis due to Chlamydia trachomatis genovar L2b[END_REF][START_REF] Ward | The prevalence of lymphogranuloma venereum infection in men who have sex with men: results of a multicentre case finding study[END_REF]. The reason why the bubonic form or the urethral infection is not more commonly reported remains unclear. Some reports have suggested transmission through sex toys, but further research will be required to reveal the exact mode of transmission. Some patients were pauci-symptomatic or asymptomatic, up to 40% in the Dutch series, but other studies have failed to demonstrate a significant reservoir of asymptomatic or urethral infection in MSM or in the general population [START_REF] Stary | New Chlamydia trachomatis L2 strains identified in a recent outbreak of lymphogranuloma venereum in Vienna, Austria[END_REF][START_REF] Klint | Lymphogranuloma venereum prevalence in Sweden among men who have sex with men and characterization of Chlamydia trachomatis ompA genotypes[END_REF][START_REF] Tinmouth | Is there a reservoir of sub-clinical lymphogranuloma venereum and non-LGV Chlamydia trachomatis infection in men who have sex with men?[END_REF][START_REF] Ward | The prevalence of lymphogranuloma venereum infection in men who have sex with men: results of a multicentre case finding study[END_REF][START_REF] Annan | Rectal chlamydia-a reservoir of undiagnosed infection in men who have sex with men[END_REF]. On the other hand, the absence of the classical clinical features of LGV have also conditioned a delay in the diagnosis and treatment in the current outbreak as evidenced by the large number of cases diagnosed retrospectively, allowing ongoing transmission [START_REF] Macdonald | Initial results of enhanced surveillance for lymphogranuloma venereum (LGV) in England[END_REF][START_REF] Gebhardt | Lymphogranuloma venereum (LGV) serotype L2 in Switzerland, 2003-2005[END_REF][START_REF] Herida | Rectal lymphogranuloma venereum surveillance in France 2004-2005[END_REF][START_REF] Spaargaren | New lymphogranuloma venereum Chlamydia trachomatis variant, Amsterdam[END_REF][START_REF] Spaargaren | Slow epidemic of lymphogranuloma venereum L2b strain[END_REF].

The endoscopic findings included mucopurulent exudate with a hyperaemic and friable mucosa, easily bleeding, with multiple ulcers and erosions being replaced by granulomatous tissue and tumorous inflammatory masses. However, in some reports up to 40% of men with LGV proctitis showed no anoscopic abnormalities [START_REF] Vries | Lymphogranuloma venereum proctitis in men who have sex with men is associated with anal enema use and high-risk behavior[END_REF][START_REF] Van Der Bij | Diagnostic and clinical implications of anorectal lymphogranuloma venereum in men who have sex with men: a retrospective case-control study[END_REF], Even these endoscopic findings are unspecific and the disease can be misdiagnosed. The pathology shows typically only nonspecific inflammatory features with an intense chronic inflammatory process. It is crucial to remember that the histology is non-specific and can be easily misinterpreted, even by trained pathologists [START_REF] Nieuwenhuis | Resurgence of lymphogranuloma venereum in Western Europe: an outbreak of Chlamydia trachomatis serovar l2 proctitis in The Netherlands among men who have sex with men[END_REF].

Clinical proctitis is a common problem in MSM and clinicians should suspect LGV in MSM who present with colorectal gastrointestinal symptoms. Diagnosis may be hampered because rectal LGV may clinically and histologically mimic other conditions such as inflammatory bowel disease, colorectal cancer and lymphoma [START_REF] Quinn | The polymicrobial origin of intestinal infections in homosexual men[END_REF], which is why they should be included in the differential diagnosis along with other ulcerative rectal STD such as syphilis and herpetic or cytomegalovirus proctitis. The case of lymphoma is particularly relevant, because the tissue infiltration by lymphocytes and the immunohistochemical markers for the Epstein-Barr virus can be extremely confusing for the pathologist [START_REF] Van Hal | Lymphogranuloma venereum: an emerging anorectal disease in Australia[END_REF]. e HIV status was available for 96 patients, of which 82 were known to be HIV-positive f HIV status was available for 52 patients, of which 50 were known to be HIV-positive

Diagnosis

First of all, it is of paramount importance to bear in mind that LGV proctocolitis will not be diagnosed if specific tests are not ordered, as routine smears and cultures performed in urethral or rectal samples do not identify this pathogen.

Correct diagnosis is essential as a prolonged treatment of 3 weeks is recommended for LGV infection, in contrast to infection with other Chlamydia serovars, where only 1 week (or a single azithromycin dose) is required.

Lymphogranuloma venereum was classically diagnosed by the Frei intradermal skin test, based on the delayed hypersensitivity reactions to chlamydial antigens, but this is no longer in use because of its low sensitivity and specificity. Direct identification of C. trachomatis from clinical specimens is the current standard. Before 2003 we did not have a routine test for diagnosing LGV since the tests that were available did not distinguish between the LGV and non-LGV serovars.

Initially, the laboratory diagnosis of LGV was based in either culture (which was rarely available in routine practice, because it was slow and less sensitive), or nucleic acid amplification testing (NAAT) followed by genotyping of positive samples with PCR-based restriction fragment length polymorphism (RFLP) analysis and/or sequencing of the omp1 gene encoding the major outer membrane protein. All this required a sophisticated laboratory setting and was time consuming. Since 2005, several fast molecular biological diagnostic tests that have a higher sensitivity have been widely available. In 2005, the first RT-PCR technique was developed to detect LGV serovars in a C. trachomatis PCR-positive sample using the polymorphic membrane protein H gene (pmp gene) as a PCR target because of its unique gap in LGV strains, making it highly specific [START_REF] Morré | Lymphogranuloma venereum diagnostics: from culture to realtime quadriplex polymerase chain reaction[END_REF]. This technique can detect the LGV serovars, but it cannot distinguish between them. A multiplex RT-PCR specific to serovar L2, and a quadriplex RT-PCR that simultaneously detects LGV and non-LGV serovars and mixed infections have been developed more recently [START_REF] Chen | A real-time quadriplex PCR assay for the diagnosis of rectal lymphogranuloma venereum and non-lymphogranuloma venereum Chlamydia trachomatis infections[END_REF][START_REF] Chen | The molecular diagnosis of lymphogranuloma venereum: evaluation of a real-time multiplex polymerase chain reaction test using rectal and urethral specimens[END_REF].

Chlamydia nucleic acids can be detected in urethral, endocervical, vaginal, pharyngeal or rectal swab specimens The Netherlands [START_REF] Spaargaren | New lymphogranuloma venereum Chlamydia trachomatis variant, Amsterdam[END_REF] 33.3 13.3 6.6 The Netherlands [START_REF] Nieuwenhuis | Resurgence of lymphogranuloma venereum in Western Europe: an outbreak of Chlamydia trachomatis serovar l2 proctitis in The Netherlands among men who have sex with men[END_REF] 7 7 7 7 6 1 1 5 1 5 0 France [START_REF] Herida | Rectal lymphogranuloma venereum surveillance in France 2004-2005[END_REF] UK [START_REF] Macdonald | Initial results of enhanced surveillance for lymphogranuloma venereum (LGV) in England[END_REF] 36.8 10.5 The Netherlands [START_REF] Van De Laar | A slow epidemic of LGV in the Netherlands in 2004 and 2005[END_REF] 74 85 65 41 24 Belgium [START_REF] Vandenbruaene | Lymphogranuloma venereum outbreak in men who have sex with men (MSM) in Belgium, January 2004 to[END_REF] Switzerland [START_REF] Gebhardt | Lymphogranuloma venereum (LGV) serotype L2 in Switzerland, 2003-2005[END_REF] 0 The Netherlands [START_REF] Vries | Lymphogranuloma venereum proctitis in men who have sex with men is associated with anal enema use and high-risk behavior[END_REF] 4 4 1 3 UK [START_REF] Ward | Lymphogranuloma venereum in the United Kingdom[END_REF] 6 4 7 2 2 3 5 3 2 5 2 9 8 4 UK [START_REF] Jebbari | Update on lymphogranuloma venereum in the United Kingdom[END_REF] 15-24 b The Netherlands [START_REF] Koedijk | An ongoing outbreak of lymphogranuloma venereum in the Netherlands, 2006-2007[END_REF] 66 78 89 25 Spain Australia [START_REF] Lee | Lymphogranuloma venereum becomes an established infection among men who have sex with men in Melbourne[END_REF] 9 Canada [28] Sweden [START_REF] Velicko | Lymphogranuloma venereum-increased spread in Sweden. A rare Chlamydia subtype which become more and more common among men who have sex with men[END_REF] 13 13 Germany [START_REF] Bremer | Lymphogranuloma venereum emerging in men who have sex with men in Germany[END_REF] 13 Italy [START_REF] Cusini | Lymphogranuloma venereum: the Italian experience[END_REF] 23 France [START_REF] Halioua | Ano-rectal lymphogranuloma venereum: 22 cases reported in a sexually transmitted infections center in Paris[END_REF] Austria [START_REF] Stary | New Chlamydia trachomatis L2 strains identified in a recent outbreak of lymphogranuloma venereum in Vienna, Austria[END_REF] 13. and urine samples. However, the use of rectal swabs for nucleic acid testing is not cleared by the US Food and Drug Administration and in consequence is not recommended by the CDC yet [START_REF] Van Der Bij | Diagnostic and clinical implications of anorectal lymphogranuloma venereum in men who have sex with men: a retrospective case-control study[END_REF], although initial data support its validity [START_REF] Alexander | Confirming the Chlamydia trachomatis status of referred rectal specimens[END_REF].

Serology has been replaced by direct detection tests. However, it could still be useful in selected cases. A high titre of antibody (with microimmunofluorescence or with complement fixation) in a symptomatic patient is strongly suggestive of LGV, but cannot distinguish between current or previous infection. Comparative studies between different serological tests are lacking, and there is no consensus regarding their interpretation.

Investigation of proctitis, with or without inguinal lymphadenopathy and genital ulcer, especially in the MSM group, should make the physician suspect LGV. Clinicians should not count on patient complaints and/or proctoscopic findings alone to identify LGV proctitis. Serovar typing is recommended in MSM with a PCRpositive anal swab for Chlamydia infection. If routine LGV serovar typing is not available or until definitive diagnosis, syndromic LGV management is proposed by some authors using clinical predictors of LGV proctitis in MSM with anorectal Chlamydia with either proctitis detected by proctoscopic examination, > 10 WBC detected on Gram stain smears from anal mucosa or presence of HIV infection [START_REF] Van Der Bij | Diagnostic and clinical implications of anorectal lymphogranuloma venereum in men who have sex with men: a retrospective case-control study[END_REF].

Management

According to the current European and US STD guidelines the recommended treatment for LGV is doxycycline (100 mg orally twice a day for 21 days or as long as anorectal symptoms persist). An alternative regimen is erythromycin (500 mg orally four times daily for 21 days). However, this recommendation is not supported by randomised evidence in clinical trials, but by empirical data. Azithromycin (1.0 g orally once weekly for 3 weeks) has also been suggested as an alternative, but clinical data are lacking, as well as data regarding the new fluoroquinolones [START_REF] Mcmillan | The 2007 European Guideline (International Union against Sexually Transmitted Infections/World Health Organization) on the management of proctitis, proctocolitis and enteritis caused by sexually transmissible pathogens[END_REF][START_REF] Mclean | Treatment of lymphogranuloma venereum[END_REF][START_REF] Vries | Delayed microbial cure of lymphogranuloma venereum proctitis with doxycycline treatment[END_REF][START_REF] Centers For | Sexually transmitted diseases treatment guidelines, 2006[END_REF].

Patients should be followed up until symptoms have disappeared, and clinically reviewed at 3-6 weeks.

In uncomplicated chlamydial infections with non-LGV strains a single oral dose of 1 g azithromycin is enough or a shorter (1-week) course of doxycycline. In HIV-infected patients the recommended treatment is the same, even though randomised studies are lacking [START_REF] Mclean | Treatment of lymphogranuloma venereum[END_REF]. A delay in resolution may be observed in immunosuppressed persons, and prolonged therapy may be necessary. Sexual partners who have had unprotected contact with the patient within the previous 60 days of onset of clinical symptoms should be screened or empirically treated with an LGV regimen [START_REF] Mclean | Treatment of lymphogranuloma venereum[END_REF][START_REF] Centers For | Sexually transmitted diseases treatment guidelines, 2006[END_REF]. Aspiration of bubos may be necessary as well as surgical management of anorectal fistulas and strictures in advanced cases.

Patients should receive risk education counselling and be routinely screened for other STD, especially including HIV and hepatitis B and C.

Lymphogranuloma venereum should be a mandatory reportable disease in all countries, and contact tracing is of major importance to prevent further spread within the community.

Clinical implications

Clinicians should suspect LGV proctocolitis, especially in MSM presenting with colorectal symptoms like rectal pain, mucopurulent or bloody discharge, cramping abdominal pain, constipation, tenesmus leading to specific molecular biological diagnostic testing based on RT-PCR of a rectal swab with serovar typing. Systemic symptoms like fever, malaise and weight loss are relatively common. Simultaneous penile lesions and/or inguinofemoral lymphadenopathy are observed rarely in the present outbreak.

Early diagnosis and management are pivotal and furthermore have important public health implications, since the ulcerative character of the disease may facilitate transmission and acquisition of HIV and other STD, including hepatitis C. Screening for HIV, other STD and hepatitis C is mandatory since many of the described cases had concomitant infections. Contact tracing should be performed but it may be difficult given the anonymous nature of many sexual contacts in this core group.

Future challenges

There are still some pivotal questions to be answered concerning LGV proctitis. Diagnostic testing for C. trachomatis with LGV typing of C. trachomatis-positive samples in symptomatic men or contacts of men with

LGV is currently recommended. The role of systematic screening for rectal LGV in stopping the transmission of LGV in HIV-infected MSM needs to be defined. Also, the usefulness of a shorter treatment with tetracyclines, macrolides or quinolones would be of great interest and may also have public health implications in reducing transmission. Moreover, there is a strong need for simpler and more affordable diagnostic methods, particularly those quick on-the-spot results, to help physicians with earlier diagnosis. It would be of great interest to perform prevalence studies among both HIV-positive and HIVnegative MSM.

Last but not least, there is an urgent need to implement regular and coordinated national and international surveillance programs in many countries in order to monitor the disease occurrence, to better identify patterns of disease and possible risk factors and to implement health service needs. This implementation would be of utmost urgency.

Fig. 1

 1 Fig. 1 Distribution of confirmed cases of lymphogranuloma venereum (LGV) by date of consultation, Catalonia (Spain), January 2007 to April 2009 (Source: CEEISCAT)

g

  Concomitant sexually transmitted disease (STD) h Proctitis syndrome includes, unless otherwise stated, variable degrees of loose, bloody stools associated with the passage of mucus, cramping abdominal pain, rectal tenesmus and low-grade fever. Periods of constipation are also common I Proctitis syndrome was present in 90% of the individuals in the period of 2004-2005 and in 77% in 2006-2007; asymptomatic patients accounted for 5% in 2004-2005 and 18% in 2006-2007 j Asymptomatic patients k 40% were pauci-symptomatic l Signs of proctitis observed in proctoscopy/sigmoidoscopy

Table 1

 1 Main characteristics of lymphogranuloma venereum cases in men who have sex with men (MSM) reported between 1990 and 2009

	Country	Period	Cases	Median	HIV	STD g	Hep C	HAART	Proctitis h	Asymp j	Proc l
		of study	(number)	age (year)	(%)	(%)	(%)	(%)	(%)	(%)	(%)
	The Netherlands	2002-2003	87	37.6	60	21		54			47
	[38]
	The Netherlands	2002-2003	45	35.8	69
	[43]
	The Netherlands	2004	13	39	85	46	8	36	92.3		100
	[3]
	France [11]	2002-2005	244	39	85 e				Most patients
	UK [5]	2004	24	40	90	42	21		94.7
	The Netherlands	2004-2005	114	40	67	Frequent	10		91	18
	[14]
	Belgium [10]	2004-2005	13		92				100
	Switzerland [8]	1999-2005	10		70				100
	The Netherlands	2004-2006	32	39.6	78	19	13			40 k	60
	[37]
	UK [6]	2004-2006	327	38	76	39	19	45	96	13
	UK [7]	2004-2007	492	40	74	48	14		90-77 I	5-18 I
	The Netherlands	2006-2007	57	45	77	Frequent			Most patients
	[15]
	Spain a	2007-2009	42	39.4	91	50			Most patients
	Australia [29]	2005-2008	11	47	82				81.8
	Canada [28]	2001-2007	88	37.5	78	Frequent	6
	Sweden [16]	2007	15	38	73	40		33	80	6.6
	Germany [4]	2004-2005	78	39	96 f				75
	Italy [13]	2006-2007	13		62	38		46	77
	France [12]	2002-2004	22	39.2	57				100
	Austria [2] b	2005-2006	15	41.3	85	53		13	86.7
	France [33]	2003-2004	38 c	40	57	64			100
	Canada [27]	2004-2005	22 d	38	73		25		78
	Portugal [1]	2007	8	36	25	38			12.5	75

a Data from CEEISCAT in Catalonia (Jordi Casabona, personal communication) b Mean CD4 count 469 (±180.4) cells/mL. No specification of CD4 count was available in the other studies c Complete surveillance data were available for 14 of the 38 patients d Surveillance data were available for 19 of the 22 cases, though not complete for all cases

Table 2

 2 Main characteristics of reported lymphogranuloma venereum cases inMSM between 1990 and 2009 (continued)

	Country	Rectal pain	Anal	Constipation	Blood loss	Tenesmus	Systemic a	Inguinal	Anogenital
		(%)	discharge (%)	(%)	(%)	(%)	(%)	nodes (%)	ulcer (%)
	The Netherlands [38]	2	2					24	9

Eur J Clin Microbiol Infect Dis (2010) 29:917-925

Eur J Clin Microbiol Infect Dis (2010) 29:917-925


Juan G Navedo
email: jgnavedo@unex.es

Germán Orizaola

José A Masero

Otto Overdijk

Juan M Sánchez-Guzmán

Long-distance travellers stopover

Keywords: Experience, intraspecific competition, migratory strategies, spoonbill, stopover duration, timing

Long-distance migration is widespread among birds, connecting breeding and wintering areas through a set of stopover localities where individuals refuel and/or rest. The extent of the stopover is critical in determining the migratory strategy of a bird. Here, we examined the relationship between minimum length of stay of PVC-ringed birds in a major stopover site and the remaining flight distance to overwintering area in the Eurasian spoonbill (Platalea l. leucorodia) during four consecutive autumn migrations. We also analysed the potential effect of timing (arrival date), as well as the role of experience in explaining stopover duration of spoonbills. Overall, birds wintering in Africa, and facing a long-distance travel from the stopover site (ca.

3,000 km), stay for longer (2.7 ± 0.4 days) than Iberian winterers (1.5 ± 0.2 days) that perform a much shorter migration (ca. 800 km). These differences were consistent between years. Stopover duration was not significantly affected by the age of the bird. However there is a significant reduction as migration advanced.

Our results suggest that spoonbills develop different stopover strategies depending on the expected distance to the wintering grounds. Adults, especially long-distance migratory ones, could reduce the potential negative effects of density-dependence processes by avoiding stopover at the end of the migration period. These findings are of significant relevance for understanding differences in migratory behaviour within single populations, especially for declining waterbirds, as well as stress the relevance of preserving stopover localities for the conservation of intraspecific diversity in migratory species.

Introduction

Many animals undertake seasonal movements from the breeding to the wintering grounds, which are regarded primarily as an adaptation for exploiting the best conditions in food availability throughout the year [START_REF] Alerstam | Long-distance migration: evolution and determinants[END_REF][START_REF] Newton | The migration ecology of birds[END_REF]). Long-distance migration is a costly process that consists in sequential movements interrupted by stopovers for resting and/or refuelling, and is widespread in many animal groups such as mammals, birds, fish and insects [START_REF] Alerstam | Long-distance migration: evolution and determinants[END_REF].

Migrating birds spend most of the time at stopover sites [START_REF] Schaub | Migrating birds stop over longer than usually thought: an improved capture-recapture analysis[END_REF], that are usually discrete and far apart for many long-distance migratory species, especially waterbirds [START_REF] Pennycuick | Burning the engine: a time-marching computation of fat and protein consumption in a 5420-km non-stop flight by great knots, Calidris tenuirostris[END_REF][START_REF] Battley | How do red knots Calidris canutus leave Northwest Australia in May and reach the breeding grounds in June? Predictions of stopover times, fuelling rates and prey quality in the Yellow Sea[END_REF][START_REF] Gill | Crossing the ultimate ecological barrier: Evidence for a 11000-km-long nonstop flight form Alaska to New Zealand and eastern Australia by Bar-tailed Godwits[END_REF][START_REF] Piersma | Fuel storage rates before northward flights in Red Knots worldwide: facing the severest constraint in tropical intertidal environments?[END_REF]. Stopover duration is the most important factor determining the total length of a migratory journey and the total amount of energy load [START_REF] Alerstam | Long-distance migration: evolution and determinants[END_REF].

Considering the energy-optimisation theory, individuals should stay in a stopover until enough fuel is stored to reach the next stopover site, independently of the rate of fuel deposition [START_REF] Alerstam | Optimal bird migration: the relative importance of time, energy and safety[END_REF][START_REF] Hedenström | Optimum fuel loads in migratory birds: distinguishing between time and energy minimization[END_REF]. On the other hand, in the context of the time-minimisation theory, individuals should be sensitive to the rate of fuel deposition, adjusting their departure fuel load to their fuel deposition rate [START_REF] Alerstam | Optimal bird migration: the relative importance of time, energy and safety[END_REF][START_REF] Hedenström | Optimum fuel loads in migratory birds: distinguishing between time and energy minimization[END_REF]. In terms of energy turnover rate, associated costs may appear in the short-and/or long-term [START_REF] Schmidt-Wellenburg | Costs of migration: short-and long-term consequences of avian endurance flight[END_REF]. Therefore, timing and conditions experienced at stopover sites may affect not only the success of the migration, but also the future individual reproduction and survival, with potential consequences for population dynamics [START_REF] Newton | Can conditions experienced during migration limit the population levels of birds?[END_REF].

During migration, many bird species or populations with similar migratory patterns and/or trophic requirements may use the same stopover sites to refuel and/or rest [START_REF] Newton | Population limitation in migrants[END_REF]. Density-dependent processes may thus be promoted at stopovers, and interspecific and/or intraspecific competition could increase (Alerstam andHedenström 1998, Newton 2006). In this context, early arrival should be under high positive selection since feeding resources may be fewer, or even depleted, when later birds arrive at a single stopover [START_REF] Newton | Can conditions experienced during migration limit the population levels of birds?[END_REF][START_REF] Nolet | Prediction of bird-day carrying capacity on a staging site: a test of depletion models[END_REF]. Therefore, experience accumulated along years/migrations could play a significant role to achieve an optimal migration [START_REF] Hedenström | Adaptations to migration in birds: behavioural strategies, morphology and scaling effects[END_REF], and allows the development of different stopover strategies within a population [START_REF] Scheiffarth | Spring migration strategies of two populations of bartailed godwits, Limosa lapponica, in the Wadden Sea: time minimizers or energy minimizers?[END_REF][START_REF] Drent | Pay-offs and penalties of competing migratory schedules[END_REF]. For long-lived migratory birds, such as cranes and storks, experience (i.e. age of the birds) could be a sign of consistent migration success [START_REF] Berthold | Birds in Europe: population estimates, trends and conservation status[END_REF]. Accumulated experience may be crucial to achieve a "dominant competitive status" to be able to outcompete conespecifics at stopover and/or wintering localities [START_REF] Alerstam | Long-distance migration: evolution and determinants[END_REF], especially in species without apparent sexual dimorphism. Experienced birds should be able to reach required fuel-loads faster than inexperienced ones, and thus reducing stopover duration [START_REF] Newton | The migration ecology of birds[END_REF]. Nevertheless, to date, empirical data about ecological factors affecting stopover duration are scarce, and most frequently involves a reduced number of individuals [START_REF] Schaub | Migrating birds stop over longer than usually thought: an improved capture-recapture analysis[END_REF][START_REF] Salewski | Stopover duration of Palearctic passerine migrants in the western Saharaindependent of fat stores?[END_REF].

Decisions on the necessity for stopover and its duration may depend on the expected length of the migratory bout [START_REF] Schaub | Migrating birds stop over longer than usually thought: an improved capture-recapture analysis[END_REF]), but to the best of our knowledge no empirical studies have corroborated this hypothesis. A previous study addressed this question, but looking at two different subspecies of the Northern wheatear Oenanthe oenanthe [START_REF] Dierschke | Stopover behaviour and departure decision of northern wheatears, Oenanthe oenanthe, facing different onward non-stop flight distances[END_REF] and potentially including some confounding factors (i.e. differential ecological/energetic requirements between subspecies). Therefore, the remaining distance to the wintering area has not been fully considered yet as a main factor explaining stopover duration in birds. In this study, we examined the relationship between the length of stay at a major stopover site in Northern Iberia and the overwintering area (Africa or South Iberia) in migrating Eurasian spoonbills (Platalea l. leucorodia). We predicted that adult spoonbills travelling to overwinter in Africa will stopover for longer in North Iberia than birds overwintering in South Iberia, and that experience (age) of the birds will play a significant role in explaining stopover duration.

Methods

The Eurasian spoonbill (Platalea l. leucorodia) maintains a small breeding population in Western Europe (4,800 pairs; [START_REF] Triplet | International Single Species Action Plan for the Conservation of the Eurasian Spoonbill Platalea leucorodia[END_REF]), catalogued as "Rare" (BirdLife 2004). Less than 2,000 pairs breed in northern latitudes, the so-called North-Atlantic population, most (1,600 pairs) in the Netherlands [START_REF] Triplet | International Single Species Action Plan for the Conservation of the Eurasian Spoonbill Platalea leucorodia[END_REF]; see Figure 1). These birds spend the winter in two distant geographic areas: about 20% of the birds in the Iberian Peninsula, most of them in the Southwest [START_REF] Lorenzo | Spoonbill winter population in the Iberian Peninsula and the Balearic islands[END_REF], and the rest of the population in North-Western African wetlands, mainly Mauritania and Senegal [START_REF] Bauchau | Survival of Spoonbills on Wadden Sea islands[END_REF]; Figure 1). Around 35-40% of the North-Atlantic spoonbill population make a strategic stopover in the Santoña Marshes Natural Park (43°30"N 3°30"W; Fig. 1) during autumn migration (Navedo 2006a), where ca. 80% of these birds came from the Wadden Sea islands, 15% from inland Netherlands, and 5% from France (Navedo 2006a). On average, 90% of the spoonbills using Santoña make their stopover during September (Navedo 2006a).

Santoña marshes is an estuarine area located in the northern coast of the Iberian Peninsula with semidiurnal tides exposing the intertidal zone (roughly 1,200 ha.) daily for 5.5--6.5 h. [START_REF] Navedo | Measuring potential negative effects of traditional harvesting practices on waterbirds: a case study with migrating curlews[END_REF]. This estuary is strategically located within the route of the North-Atlantic spoonbill population (Figure 1), just before the geographical barrier of the Cantabrian Mountains, and about 800 km far away from the next main suitable stopover areas at the southern coasts of Iberia (Algarve, Odiel and Doñana). Spoonbill habitat use is invariably driven by the tides, feeding in the intertidal channels during the low tide period and resting at high tide in a supratidal area (Navedo 2006a). Tidal amplitude at Santoña ranges from ca. 2.5 m during neap tides up to ca. 4.5 m during spring ones (mean 3.5 m; [START_REF] Navedo | Efectos del marisqueo tradicional sobre la alimentación de las aves limícolas en áreas intermareales: aportaciones para la gestión de los estuarios cantábricos[END_REF], so it potentially has a great influence in available foraging possibilities, with periods of spring tides significantly enlarging potential spatial and time foraging budget.

We have estimated stopover duration of spoonbills in Santoña during September in four consecutive years (2002)(2003)(2004)(2005) on the base of PVC-ringed birds. Every day from 1st to 28th of September, and throughout diurnal low tide period, two experienced observers carefully checked foraging flocks from a boat looking for PVC-ringed birds. Observers were connected by walkie-talkies with people in two little hills overlooking the estuarine area, which informed them about the localisation of all foraging spoonbills in the intertidal channels.

Thus, we assumed that almost all the spoonbills using the estuary (mean daily number at low tide was 69.4 birds; J.G. Navedo unpubl. data) were checked on a daily basis. When a PVC-ringed bird was located, the boat was slightly beached to identify the code of the PVC-ring with the help of a 20x-60x telescope (mean observation distance: 120 m.). Approximately 15% of the north-atlantic spoonbill population was currently marked with individual PVC ring codes (O. Overdijk, unpubl. data).

Minimum stopover duration was defined as the time elapsed between first location and last relocation of a bird in the estuary. Since resighting probability of birds that stopover three days or more (n=74) was 0.89 ± 0.01, and it was not significantly different between years (F 3,70 = 0.81; p = 0.5), it approaches real stopover duration. Overall, we read 974 PVC-rings during the study (277, 209, 312 and 176; in 2002, 2003, 2004, and 2005, respectively), comprising 263 accurately identified PVC-ringed spoonbills [69 yearlings (1st year-cal); 39 immatures (2nd and 3rd year-cal) and 155 adult birds (4th year-cal or more)]. Of these birds, 38 were located during more than one autumn at Santoña, corresponding to 34 adults and 4 immatures, all of them 3rd year-cal at the time of their first observation (Dutch Spoonbill Working Group Database). Despite most PVCringed yearlings were not resighted during its first wintering season, many PVC-ringed birds identified in Santoña were resighted in its major wintering areas a few months later (data obtained from the Dutch Spoonbill Working Group database). Nonetheless, as this population shows high fidelity to wintering area [START_REF] Voogd | Methods to sex juvenile Spoonbills and Sex-specific migration in Spoonbills[END_REF], data was used even if wintering area was not known for the specific year for which stopover duration was recorded. In these cases, only birds for which wintering area is known, and remained constant for three years during 2002-2008, were included in the analyses. Under these premises, we analysed information about stopover duration and wintering area for a total of 127 birds (17 yearlings, 17 immatures and 93 adults) Spoonbills resighted during November and December at Algarve (Portugal), Odiel and Doñana (Spain) were grouped into the Iberian-wintering class (43% of the PVC-ringed birds), whereas spoonbills resighted at that time at Banc d´Arguin (Mauritania) and Senegal Delta (Senegal) were grouped into the African-wintering class (57% of the PVC-ringed birds).

Statistical analysis

Some spoonbills (n = 38) were resighted on more than one migration at Santoña. Therefore, effects of year and wintering area on minimum stopover duration of spoonbills were analysed by fitting a Linear Mixed Model with individual (ring code) as random effect. Wintering area and year were respectively included into the model as two-level (Iberia/Africa) and four-level (2002/2003/2004/2005) fixed factors, respectively.

Arrival date, defined as days elapsed from August 31 st , was introduced as a potential predictive covariable since timing could significantly affect migratory behaviour [START_REF] Newton | The migration ecology of birds[END_REF]. Similarly, age as well as withinsite experience (i.e. number of previous years registered at the area during the study period) were both included as potential predictive variables into the model. Tidal amplitude at arrival, defined as a coefficient ranging from 20 to 120, a measure directly correlated with the height of the tide, was also examined as a predictive covariable due to its potential influence in migratory decisions [START_REF] Piersma | Behavioural aspects of the departure of waders before longdistance flights: flocking, vocalizations, flight paths and diurnal timing[END_REF]). As arrival date and tidal amplitude were correlated we have tested their effects separately (not in the same model). However, tidal amplitude was not finally included into the model since it was not correlated with the dependent variable (Spearman r = -0.06; p > 0.05).

Statistical analyses were performed using STATISTICA v6.1 (StatSoft Inc. 2002). In all cases we used a 0.05 level of significance. Values are presented as means ± SE.

Results

Individuals wintering in Africa spent more time (2.7 ± 0.4 days; n = 72) at Santoña marshes during the autumn migration than those wintering in south Iberia (1.5 ± 0.2 days; n = 55) (Table 1; Figure 2). Minimum stopover duration was similar between years , and longer stays for birds wintering in Africa were always recorded even during the year with the shortest length of stay (2004, Figure 2). As migration period progresses minimum stopover duration decreased significantly (Table 1; Figure 3). However, nor age neither within-site experience did have a significant effect in explaining stopover duration of spoonbills.

Discussion

This study empirically shows that stopover duration in a long-distance migratory bird as the Eurasian spoonbill was influenced by the remaining flight distance to the overwintering area. According to our prediction, spoonbills wintering in Africa, and consequently facing a long-distance travel from a crucial stopover site, stay for longer than those birds wintering in south Iberia that performed a shorter migration.

Although there are alternative reasons to stay at a stopover site than refuelling (e.g. resting, foraging for maintenance or waiting for favorable winds to continue migration, e.g. Weber et al. 1998), spoonbills were foraging most of the available time during stopover (J.G.Navedo pers obs). Therefore, longer stopover duration would reflect higher energy-loads for spoonbills staying at the study area.

Birds wintering in south Iberia seem to minimise the time spent at the stopover locality, flying after a short refuelling. Without large energy requirements, the need to arrive to the best foraging grounds in these short-distance wintering areas ahead of other conspecifics may be the driving force explaining the fast stopover of Iberian-wintering spoonbills. Moreover, as birds wintering in Southern Iberia are close to their destination, they could be less dependent on the foraging conditions at this stopover to successfully complete their migration. Inversely, birds wintering in Africa need to adjust their stopover duration at Santoña Marshes to the requirements of a high energy-demanding flight and the prospects for a longer migratory trip [START_REF] Alerstam | Optimal bird migration: the relative importance of time, energy and safety[END_REF], thus refuelling for longer. Furthermore, at least some birds might take advantage of the high energy load to "skip" those south Iberian wetlands [START_REF] Piersma | Hop, skip or jump? Constraints on migration of arctic waders by feeding, fattening and flight speed[END_REF], where their conspecifics overwinter, to perform a non-stop bout from Santoña to north western African wetlands.

Eurasian spoonbills consistently stopover for less time as migration period progressed. Prey consumption at stopover areas would reduce food availability for migratory birds and even depleted them (Gudmundsson et al. 1991;[START_REF] Prop | Travel schedules to the high arctic: barnacle geese trade-off the timing of migration with accumulation of fat deposits[END_REF][START_REF] Nolet | Prediction of bird-day carrying capacity on a staging site: a test of depletion models[END_REF]). Although we have not measured food availability in our study area (main prey for spoonbills are little fishes and shrimps; J.G. Navedo unpubl. data), a significant decrease of food resources for waterbirds occurs in estuaries around north Iberian coasts during autumn migration, and extending to the winter season [START_REF] Boileau | Ecologie alimentaire de la spatule blanche Platalea leucorodia en migration postnuptiale dans le marais de Brouage (Charente-Maritime)[END_REF]Viegas et al. 2007). If food is limited, an increase in intraspecific competition resulting from a particular good breeding season (i.e. high number of breeding pairs and productivity) may have measurable consequences in the migratory performance within a given population [START_REF] Alerstam | Long-distance migration: evolution and determinants[END_REF], specially for experienced adults [START_REF] Berthold | Birds in Europe: population estimates, trends and conservation status[END_REF]). The reduced stopover duration observed during 2004 could have been affected by the highest population size during the study period reached in that year (a high number of breeding pairs achieving a high reproductive success; O. Overdijk unpubl. data), which also coincided with the maximum number of spoonbills that stopover at Santoña marshes during the study period (Navedo 2006a).

Age-related differences in timing of migration at stopover sites are common in many bird species (e.g.

Restani 2000; [START_REF] Ueta | Difference in migration pattern between adult and immature birds using satellites[END_REF][START_REF] Hake | Age-dependent migration strategy in honey buzzards Pernis apivorus tracked by satellite[END_REF][START_REF] Meissner | Stopover strategy of adult and juvenile Red Knots Calidris c. canutus in the Puck Bay, southern Baltic[END_REF][START_REF] Lee | Age-specific stopover ecology of Black Brant at Humboldt Bay, California[END_REF]). In our case, a preliminary study addressed that the majority of adult spoonbills (ca. 70%) had currently used the area before the arrival of the majority of yearlings (ca. 60%) at the last week of September [START_REF] Navedo | Different stopover timing in juvenile and adult spoonbills during southward migration[END_REF], when overall stopover duration has markedly decreased. Therefore, yearlings that stopover at the last part of the migration period without knowledge about remaining distance to wintering areas, associated energy demands, and/or suitability of stopover areas, may reduce their probability of survive during a long migration [START_REF] Newton | Can conditions experienced during migration limit the population levels of birds?[END_REF], which could be viewed as an evolutionary issue regulating long-distance migratory bird populations [START_REF] Schekkerman | Conservation of long-distance migratory wader populations: reproductive consequences of events occurring in distant staging sites[END_REF]. Inversely, adults could minimise the effects derived from density-dependent processes by avoiding stopover at the end of the migration period, in particular African wintering birds dealing with a fairly narrow time window to satisfy their higher energy demands [START_REF] Skagen | Migrating shorebirds and habitat dynamics at a prairie wetland complex[END_REF], that overall arrive a few days earlier (mode: 13 th September) than Iberian ones (mode: 17 th September).

Notwithstanding the lack of prey surveys and fuelling rates [START_REF] Hedenström | Optimum fuel loads in migratory birds: distinguishing between time and energy minimization[END_REF], our findings suggest that spoonbills may experience a high intraspecific competition during stopover that could result in the different strategies observed [START_REF] Drent | Pay-offs and penalties of competing migratory schedules[END_REF]. However, further studies are needed to clarify this point and to evaluate potential differences between sexes [START_REF] Cristol | Differential migration revisited: Latitudinal segregation by age and sex-class[END_REF], as well as the role of experience (age) within sexes in these long-lived bird [START_REF] Berthold | Birds in Europe: population estimates, trends and conservation status[END_REF], especially for African-wintering spoonbills that refuel for longer.

To sum up, this study provides empirical evidence of different stopover duration in a natural bird population in function of the remaining migratory distance to overwintering area, and remarks the relevance of stopover places for determining migratory route and pace in long-distance migratory birds.

Zusammenfassung

Figure 1

 1 Figure 1 Navedo et al.

Figure 2

 2 Figure 2 Navedo et al.

Table 1

 1 Results of a Linear Mixed Model procedure evaluating the effect wintering area and arrival date on minimum stopover duration (log transformed) of spoonbills (random effect) at Santoña Marshes Natural Park during autumn migration(2002)(2003)(2004)(2005). Non-significant interactions were sequentially removed from the model.

	Source	Effect	d.f.	F	p
	arrival date	Fixed	1	5.80	< 0.02
	wintering area	Fixed	1	9.03	< 0.004
	ring code	Random	125	1.42	ns

Acknowledgements

We thank Alejandro García-Herrera, Álvaro Bustamante, Máximo Sánchez-Cobo, Manuel A. Alcántara, Juan José Aja, Virginia Iturriaga and Ana Casero in addition to all the volunteers from the Spoonbill Migration Monitoring Program (SEO/BirdLife) for field assistance. Emma Dahl, Fernando Arce, Joaquín Bedia, Dra. Auxiliadora Villegas, Dr. Manuel Mota, Dr. Manuel Medina and an anonymous reviewer, helped to improve an earlier version of this manuscript. The Spanish Ornithological Society (SEO/BirdLife) and the Spanish National Parks Service provided us with logistic and financial support during