J Terraube
email: julien.terraube@uclm.es

B E Arroyo

F Mougeot

T E Katzner

E A Bragin

F J Bairlein

Terraube

Á F Mougeot

Breeding biology of Montagu's Harrier Circus pygargus in north-central Kazakhstan

Keywords: Breeding biology, Steppes, Kazakhstan, Diet, Conservation

The ecology and conservation status of Central Asian populations of Montagu's Harriers Circus pygargus are poorly known. We studied the breeding biology of this species during 3 years in the Naurzum region, north-central Kazakhstan. Most Montagu's Harriers in the study area nested in the forest-steppe transition area, in bushy areas dominated by dogrose Rosa canina, which was apparently the nesting vegetation type providing highest and densest nest cover in the study area. Laying occurred from 26 April to 7 June (average 13 May, n = 49) and, although it varied significantly between years, was earlier than in western European populations of similar latitude. Mean (±SD) clutch size was 4.44 ± 0.86 (range 2-6; n = 50), in the higher range observed for the species. There was no significant interannual variation in clutch size, despite large variations in the abundance of small mammals in the area. Diet was mainly composed of lizards (54.2%, n = 533 identified prey in all 3 years), with small mammals (17.1%), passerine birds (14.3%) and insects (13.6%) also being consumed. Mean brood size at the last visit was 2.55 ± 2.10 (range 0-6; n = 51). Failure rate was relatively high; the main identified cause of nest failure was predation. We compare the data obtained in this population breeding in natural steppes with breeding parameters from the well-studied western European populations, and discuss the implications for the conservation of this species.

Introduction

The Montagu's Harrier Circus pygargus is a migratory raptor species that breeds from north Africa and west and central Europe across to Russia and central Asia [START_REF] Cramp | The birds of the western Palearctic[END_REF]Del Hoyo et al. 1994). In Europe, a decade ago, the species had a conservation status of ''leastconcern'' [START_REF] Tucker | Birds in Europe: their conservation status[END_REF], although this categorisation was mainly based on a very large estimation of the breeding populations in European Russia [START_REF] Tucker | Birds in Europe: their conservation status[END_REF], possibly unreliable due to the few data available on recent population trends of this species in this part of its distribution range. More recently, it is still considered as ''secure'' mainly based on a positive trend in the large Russian population, but which has been insufficiently quantified (Birdlife International 2004). In the core areas of western European populations (France and the Iberian Peninsula), it is considered vulnerable or conservation dependent [START_REF] Millon | Busard Cendre ´. Circus pygargus[END_REF][START_REF] Arroyo | El aguilucho cenizo y el aguilucho pa ´lido en Espan ˜a[END_REF]. There is no information about the conservation status or trends of the Asian populations, which are thought to contain a significant part of the global breeding population (ca. 25,000-30,000 pairs; Ferguson-Lees and Christie 2001), although again these estimates are not very precise.

This ground-nesting species traditionally breeds in open landscapes with dense and tall herbaceous plant communities including marshes, lowland heath, rough grasslands and steppes. However, in the western Palaearctic, it has taken to agricultural areas in recent decades, breeding mainly in winter cereal or alfalfa fields. In the late twentieth century, more than 90% of breeders in the Iberian Peninsula nested within cereal crops; the proportion of birds breeding in cereal crops was 70-80% in France, and 40-50% in other western European countries [START_REF] Arroyo | Land use, agricultural practices and conservation of Montagu's Harrier[END_REF]. The change to agricultural habitats was probably catalysed by degradation or loss of natural breeding habitats, but it is likely that this was not the only reason. Indeed, the change from natural to agricultural areas has had a geographic spread (starting in south-west Europe and progressing east and north), and increased rapidly after the first occurrence in each area, more rapidly than the rate of habitat loss [START_REF] Arroyo | Land use, agricultural practices and conservation of Montagu's Harrier[END_REF].

The Montagu's Harrier is a relatively well-studied species of harrier, with numerous population studies in western Europe in recent decades (review in [START_REF] Arroyo | Circus pygargus Montagu's harrier[END_REF]. Most of those studies, however, considered populations breeding in agricultural areas. In such areas, population sustainability depends mainly on conservation measures that reduce breeding losses due to crop harvesting [START_REF] Arroyo | Conservation of the Montagu's harrier (Circus pygargus) in agricultural areas[END_REF]. In contrast, very few studies have been carried out in Montagu's Harrier populations breeding in natural and unaltered habitats (e.g. [START_REF] Limin ˜ana | Vegetation height selection in Montagu's harriers (Circus pygargus) breeding in a natural habitat[END_REF]. In particular, there is little information on the breeding ecology of Montagu's Harriers in Asian steppes, despite the apparent demographic relevance of these populations [START_REF] Ferguson-Lees | Raptors of the world[END_REF]. A better knowledge of the breeding ecology of Montagu's Harriers in steppe areas may help improving our understanding of the stability of Montagu's Harrier populations at a global scale.

In this paper, we report on a 3-year study of the density, breeding habitat and breeding parameters of Montagu's Harriers in north-central Kazakhstan. This population breeds primarily in natural vegetation, in a steppe ecosystem partly protected from major anthropogenic modifications or used extensively for grazing. We compare our results with those observed in well-studied populations breeding in natural and agricultural habitats in western Europe, and discuss results in terms of conservation for the species.

Methods

Study area and field methods

The study area was located in and around the Naurzum Zapovednik (National Nature Reserve) in the Kostanay Oblast, north-central Kazakhstan (51°N, 64°E). This area is at the juncture of the southern limit of Siberian forests and the northern limit of Eurasian steppes. We regularly checked for breeding harriers along the network of accessible tracks within the area, which totalled 350 km in length, which means, assuming a visibility of 1 km at each side of the transect line, a total surveyed area of ca. 700 km 2 . The habitat included a mosaic of dry steppes with feather grass Stipa spp. and bunch grasses on the poorer soils, low sagebrush Artemisia spp. and other nutrient-rich shrubs and grasses on denser soils. Reeds and phragmites occurred along rivers or in semi-permanent ponds throughout the study area. The area outside the Reserve boundaries was used by humans mainly for sheep, horse and cattle grazing (grazing is not authorised within the Reserve boundaries). Additionally, more than 50% of the steppe outside the Reserve was ploughed for wheat cultivation during the 'virgin lands' program initiated in the 1950s [START_REF] Katzner | Relationships between demographics and diet specificity of imperial eagles Aquila heliaca in Kazakhstan[END_REF], so part of the area was covered by agricultural land (mainly cereal crops). Much of this area turned to fallow in the 1990s, having been neglected since the dissolution of the USSR [START_REF] Katzner | Ecology and behaviour of four coexisting eagle species at Naurzum Zapovednik, Kazakhstan[END_REF], although we observed a trend for agricultural re-intensification between 2000 and 2007. Finally, interspersed within the steppe matrix, there were three distinct woodland patches (all within the Reserve) dominated mainly by Scots pine Pinus sylvestris, birch Betula spp. and aspen Populus spp. The transition areas between forest and steppe ecosystems were characterised by bushy areas mainly composed by dogrose Rosa canina and spirea Spirea spp. bushes. Detailed quantitative data on the surface covered by these different habitat types is currently lacking, but based on semi-quantitative observations along our network of tracks, 20% of the monitored area was agricultural land, 60% was unprotected non-agricultural areas (including steppe, riverine habitats, and bushy areas, the latter not covering more than 5% of the surface), and 20% was protected areas (within Naurzum Reserve, including steppe, forest, riverine habitats, and bushy areas, the latter not covering more than 5% of the surface).

On the study area, voles Microtus ssp exhibit strong annual fluctuations in abundance like in other more studied vole populations [START_REF] Zhang | Extrinsic and intrinsic factors determine the eruptive dynamics of Brandt's vole Microtus brandti in Inner Mongolia, China[END_REF]Lambin et al. 2006). Fieldwork took place in June 2000, June 2006 and between April and July 2007. We quantitatively assessed vole abundance in 2006 and 2007 by using an index based on the presence/absence of fresh vole droppings. Twenty-five quadrats (25 cm 9 25 cm) were randomly located in 11 (2006) and 25 (2007) randomly selected 1 9 1 km 2 areas spread throughout the study site and sampled for the presence (1) or absence (0) of fresh voles faeces (moist and greenish in colour). Presence/absence scores were then summed across the 25 quadrates in each area [START_REF] Madders | Hen harrier Circus cyaneus foraging activity in relation to habitat and prey[END_REF]. These sampling showed a mean score of 13.8. ± 7.6 per area in 2006 (n = 11), versus 0 ± 0 in 2007 (n = 25). We did not quantitatively assess vole abundance in 2000, but, based on data on the breeding success of other raptor species present on the study area and on qualitative observations of voles at night during mammal censuses (Bragin and Katzner, personsal communication;[START_REF] Terraube | Breeding biology of the pallid harrier Circus macrourus in north-central Kazakhstan: implications for the conservation of a near-threatened species[END_REF], we estimated that this year was a high vole abundance year, similar to 2006.

We located harrier nests by following males carrying prey and watching where females landed after a food pass. During the first survey (June 2000), we located 34 Montagu's Harrier nests of which 16 were visited. In June 2006, we found and visited 26 Montagu's Harrier nests. During the 2007 field season, 26 nests were also found and visited. Upon the first nest visit, we recorded vegetation type and height at the nest site (in m, averaged from two measures at the edge of the nest). Additionally, vegetation density was categorised as sparse, medium, dense or very dense, by estimating the percentage of plant cover within a 1-m 2 area including the nest. Vegetation height or density were not measured at random points, so it was not possible to evaluate selection of nest sites in relation to vegetation height. However, for comparison, we present data on vegetation heights recorded (with a similar methodology to that described above for Montagu's Harrier nests) at Pallid Harrier Circus macrourus nests across the study area, in order to give an indication of the range of heights of other potentially suitable vegetation available in the area. Pallid Harriers nested in three habitats representative of the main vegetation types in the open habitats of our study area, steppe vegetation (ray-grass or Artemisia), riverine habitats (reeds and phragmites) and agricultural areas (set-asides or cereal fields) [START_REF] Terraube | Breeding biology of the pallid harrier Circus macrourus in north-central Kazakhstan: implications for the conservation of a near-threatened species[END_REF].

For each nest visit, we recorded clutch size or number of nestlings. If the nest contained nestlings, we measured the 8th primary length (in mm) to age nestlings [START_REF] Arroyo | Breeding ecology and nest dispersion of Montagu's harrier Circus pygargus in Central Spain[END_REF]. Laying date was estimated directly if the nest was visited during laying, or by subtracting 30 days from the estimated date of hatching (estimated from nestling age). We calculated relative laying date for each nest as the difference between the laying date for that nest and the average laying date for that year. First year and adult ([1 year old) females were identified from plumage characteristics (see [START_REF] Forsman | The Raptors of Europe and the Middle East. A Handbook of Field Identification[END_REF].

During nest visits, we tried to minimise the length of the stay at the nests and to leave the vegetation as undisturbed as possible, to minimise the risk that human disturbance would make nests more vulnerable to predation. We believe that our nest visits were unlikely to have significantly increased predation rate because of two observations: (1) in 2000, nests monitored at distance without being visited failed at the same rate than visited nests (authors, personal observations); and (2) Pallid Harrier nests on the study area in the same study years (whose nests were visited in the same manner as those of Montagu's Harriers) had very low predation rates (see [START_REF] Terraube | Breeding biology of the pallid harrier Circus macrourus in north-central Kazakhstan: implications for the conservation of a near-threatened species[END_REF].

The location of all nests was marked with a GPS. Between-nest distances were calculated in Arc View 3.2 from GPS locations. We used data from all the nests located, including those that were not visited. We calculated for each nest the number of breeding neighbours present within 1 km using Arcview 3.2.

To characterise diet, we collected pellets each year at nests or perching sites, and also included observations of prey deliveries at the nest, when we could identify the prey type. Pellets were analysed and their contents were classified as ''birds'', ''small mammals'', ''reptiles'' and ''insects''. We could not identify all pellet contents to species, but most (95.3%, n = 37) small mammals identified were voles. We assessed the minimum number of each prey category per pellet. Data from pellets only were too few (particularly in 2006) to allow Chi-square analyses among years, so we have combined pellets and prey observations at nests. Prey biomass was estimated for each prey type following [START_REF] Arroyo | Breeding ecology and nest dispersion of Montagu's harrier Circus pygargus in Central Spain[END_REF] as 20 g for small mammals, 29 g for birds, 10 g for reptiles, 5 g for insects and 15 g for eggs.

Weather data for the 3 years of the study was obtained through the meteorological station of the Naurzum Nature Reserve (E. Bragin, unpublished data). Available data were average daily temperature for 2006 and 2007, and average temperature for each 10-day period each month for 2000. Additionally, we had access to monthly total rainfall each year. Given that daily temperatures were only available for 2006 and 2007, we only performed statistical tests for those 2 years.

Data analysis

We used generalised linear models (GLM) to test for between-year differences in breeding density, laying date or temperature. Inter-nest distance, laying date or temperature were fitted to models using a normal error distribution. Number of neighbours within a 1-km radius was fitted to models using a Poisson distribution.

We also analysed variations in clutch and brood size in relation to breeding phenology and year using GLM, fitting the response variables to a normal distribution. The initial model included ''laying date'', ''year'' and their interactions as explanatory variables. Non-significant terms were removed sequentially until the most parsimonious model was obtained.

Given that in two of the years monitoring occurred only during a limited time span (1 month), we could not calculate breeding success directly by evaluating the proportion of pairs that start breeding that lead to at least one fledging (the usual procedure in raptor studies; [START_REF] Steenhof | Assessing raptor reproductive success and productivity[END_REF]. In contrast, and to avoid underestimations, breeding success (i.e. the proportion of nests in which there was at least one fledging) was estimated by modulating the observed failure rate by the ''exposure time'' (number of days between first and second visit; [START_REF] Mayfield | Suggestions for calculating nest success[END_REF]. Nests visited only once were not included in this analysis. This means there were not sufficient data for 2000 to calculate breeding success, and data are only presented for 2006 and 2007.

All tests are two-tailed and data are presented as means ± SD.

Results

Breeding habitat

Surveys to locate harrier nests were conducted in a large variety of habitat types, including steppe, marshland and agricultural areas (see [START_REF] Terraube | Breeding biology of the pallid harrier Circus macrourus in north-central Kazakhstan: implications for the conservation of a near-threatened species[END_REF]. For the 67 nests monitored during the 3 years, nest vegetation-type ranged from steppe areas with Artemisia patches (5.97%, n = 4), to dense reeds on riverine habitats (2.98%, n = 2), ray grass (1.49%, n = 1), Spirea bushes (1.49%, n = 1), or berry bushes (4.47%, n = 4). However, the majority of nests (83.58%, n = 55) were found in dogrose bushes in the forest-steppe transition area. Vegetation height at the nest site averaged 109 ± 33 cm (range 50-200, n = 64). For comparison, mean vegetation height at Pallid Harrier nests was 53.63 ± 14.9 cm for nests found in steppe vegetation (n = 22), 156.11 ± 62.3 cm for nests found in riverine habitats (n = 36), and 30 ± 18.3 cm for nests in agricultural areas (n = 8). Vegetation density around Montagu's Harrier nests was classified as very dense in 15% of the cases (n = 53), 53% of the nests were situated in dense vegetation, and medium dense in 32% of the cases. No nests were found in sparse vegetation. For comparison, 44% of Pallid Harrier nests appeared in sparse vegetation (mainly in agricultural or steppe vegetation), and 56% in dense vegetation (in riverine habitats) (n = 75).

Breeding density

Distance to nearest breeding neighbour averaged 1,031 ± 2,487 m (range 39-15,733 m; n = 65 active nest-sites). Average breeding density (number of breeding neighbours present within a 1-km circle around each nest) was 2.75 ± 2.14 (range 0-8, n = 65). Average breeding density varied significantly between years (GLM: v 2 2 = 8.46, P = 0.015) with density being significantly higher in 2007 than in 2006 (GLM: v 1 2 = 7.60, P = 0.006). There was also a tendency for density to be higher in 2007 than in 2000 (GLM: v 1 2 = 3.64, P = 0.056).

Breeding phenology

Laying occurred from 26 April to 7 June (n = 49; average: 13 May). Mean laying dates varied significantly between years (GLM; v 2 2 = 23. When taking into account annual differences, laying onset extended over 8 weeks (Fig. 1a). However, most (75.9%) clutches each year were laid within a 3-week period (Fig. 1b).

Breeding parameters

Clutch size averaged 4.44 ± 0.86 (range 2-6, n = 50). There was no significant variation in clutch size among years and this parameter was not significantly related to laying date (GLM; year: v 2 2 = 0.83, P = 0.66; laying date: v 1 2 = 0.44, P = 0.51), or relative laying date (GLM; relative laying date: v 1 2 = 0. 18, P = 0.67). Mean brood size at the time of the last visit was 2.55 ± 2.10 (range 0-6, n = 51). Brood size variation was not significantly explained by year (GLM; v 2 2 = 3.40, P = 0.18) or laying date (GLM; v 2 2 = 0.90, P = 0.34). The proportion of breeding females that were first year birds was 6.3% (n = 16) in 2000, and 11.5% in 2006 and in 2007 (n = 26 for each year). This proportion did not differ significantly between years (v 2 2 = 0.41, = 0.82).

Nest failures

The mean percentage of failed nests over the 3 years of the study was 30%, ranging from 7. Although data for 2000 were insufficient to calculate Mayfield nest success, we know that 7 of the 16 nests visited in 2000 failed, all but one of them in a single colony. In other colonies, the proportion of located nests that were visited was smaller, but we know that they were active at last visit, so overall failure rate may have been lower than that calculated from our own nest visits.

Diet

Combining pellets and observations in all 3 years, lizards formed the main prey category by number (54.18%, n = 537 identified prey in total), whereas the percentage of small mammals was 17.13%, birds represented 14.33% and insects 13.59% of all prey. In terms of biomass, birds were the most important prey (38.70%), followed by small mammals (31.30%) and lizards (27.91%). Comparing pellets with observations at the nest, lizards were more frequent at nests (66.6%, n = 96 identified prey in nest deliveries) than in the pellets (51.47%, n = 441 identified prey in pellets). Insects were never identified at nests, although their frequency in pellets was 16.55%. Percentages of small mammals and birds were similar, regardless of how diet was determined. The overall proportion of each prey type in the diet changed significantly among years (v 6 2 = 37.95, P \ 0.001), with birds being more common, and lizards less common in 2000 than in other years (Fig. 3).

Discussion

The main findings of this study were that Montagu 0 s Harriers in north-Kazakhstan: (1) use dogrose bushes for nesting more frequently than they use other vegetation types;

(2) had a diverse diet based on reptiles, passerines and small mammals; (3) had large clutches, suffered relatively high nest failure rates, but appeared nevertheless to have relatively high productivity; and (4) experienced little interannual variation in breeding parameters.

The regular use of dogrose as a nesting habitat, with most (84%) nests found in that vegetation despite it covering less than 10% of the total surface (see ''Methods''), was probably associated with the height of that vegetation. It is known that Montagu's Harriers select relatively tall vegetation for placing their nests (Arroyo et al. 2004 and references therein). The steppe vegetation is relatively low throughout the whole breeding season (see ''Breeding habitat'' for mean vegetation height around Pallid Harrier nests in steppe vegetation), and the reeds or agricultural vegetation are not grown when Montagu's Harriers return to the breeding grounds (authors, personal observatons). Dogrose bushes may thus be the only vegetation in the area that is tall enough at settlement time to be attractive to Montagu's Harriers. This may mean that, in the natural steppes of Kazakhstan, the suitable habitats available for nesting are limited. The observed high vegetation density at Montagu's Harrier nests (as compared to nests of Pallid Harrier) may be a by-product of Montagu's Harriers nesting in Dogrose, usually denser than alternative vegetation, or may indicate that Montagu's Harriers also prefer dense vegetation as a protection from predators [START_REF] Claro | Ecologia reproductiva do Tartaranhao-cac ¸ador Circus pygargus (L.) na regiao de Evora[END_REF].

Average first egg laying date for the Montagu's Harriers in this population was mid-May. Laying dates are known to vary with latitude [START_REF] Arroyo | Circus pygargus Montagu's harrier[END_REF]. Combining data for the 3 years of the study, laying dates in the Naurzum region were 5 days earlier than expected from the relationship between laying date and latitude in other studied populations (Fig. 2). This difference increased to 15 days if excluding data from 2007, the year when laying was significantly later, most likely because of bad weather conditions (see below). All other studied Montagu's Harrier populations winter in western or central Africa [START_REF] Limin ˜ana | Autumn migration of Montagu's harriers (Circus pygargus) tracked by satellite telemetry[END_REF][START_REF] Trierweiler | Satellite tracking of two Montagu's harriers (Circus pygargus): dual pathways during autumn migration[END_REF]). The observation that Montagu's Harriers breeding in Kazakhstan breed somewhat earlier (relative to latitude) than western European birds may indicate that they winter in areas that are closer to their breeding grounds than their western European counterparts (or that their spring migration starts relatively earlier), or else that weather conditions on arrival to the breeding grounds in Kazakhstan are more benign than those of western Europe at similar latitudes. Indeed, mean temperatures in Naurzum in April were low (Table 1) in comparison with a same latitude western European site (Rotterdam, mean temperature in April: 8.2°C. http://www. worldclimate.com). But mean temperatures in Naurzum increased quickly and reached a mean of 14.2 ± 4.9°C in May, as compared to an average of 12.5°C in May in Rotterdam. This difference could be related to the strong continental climate characteristic of central Asian steppes, which could explain the earlier laying dates observed in our study population. The likely effect of weather on laying dates is also supported by the fact that laying in 2007 was delayed by 2 weeks. Interannual differences in laying date in a given population might be explained by differences in weather conditions (Sergio, 2003a, b;[START_REF] Macdonald | Weather dictates reproductive success and survival in the Australian brown falcon Falco berigora[END_REF]) and/or food abundance [START_REF] Potapov | What determine the population density and reproductive success of the rough-legged buzzard, Buteo lagopus, in the Siberian tundra?[END_REF][START_REF] Steenhof | Interactive effects of prey and weather on golden eagle reproduction[END_REF][START_REF] Salamolard | Strate ´gie d'utilisation des resources chez une espe `ce de rapace semi-colonial, le Busard cendre ´(Circus pygargus). The `se de doctorat[END_REF][START_REF] Nielsen | Effects of food abundance, density and climate change on reproduction in the sparrowhawk Accipiter nisus[END_REF]. It is unlikely that food abundance was the main reason behind the delay in laying observed in 2007, given that clutch sizes were large and similar to those of previous years. On the other hand, the low temperatures observed in 2007 during the pre-laying period might have delayed laying that year.

The mean clutch size in our study population was among the largest found for this species in western Europe (Table 2). Interestingly, we found no significant interannual variations in clutch size or the proportion of breeding first-year females, despite strong variations in the abundance of one of their prey, voles. This is in contrast to that observed in western European vole-eating populations [START_REF] Salamolard | Strate ´gie d'utilisation des resources chez une espe `ce de rapace semi-colonial, le Busard cendre ´(Circus pygargus). The `se de doctorat[END_REF][START_REF] Millon | Comparative biology of hen Harrier and Montagu's harrier: an 8-year study in north-eastern France[END_REF][START_REF] Koks | Do voles make agricultural habitat attractive to Montagu's harrier Circus pygargus[END_REF]). Montagu's Harriers in the study area had a generally diverse diet, and voles were not the most important part of the diet, even in years of high vole abundance (population outbreaks). This may be related to abundance of other prey: although we could not measure those during this study, it was qualitatively apparent that both passerines and lizards were very abundant in the area (authors, personal observations). Observed interannual variations in diet could be linked to variations in the abundance of non-vole prey, or to differences in sampling between years (e.g. low sample size in 2000; Fig. 3). Interannual differences in clutch size have nevertheless been observed in generalist populations in western Europe [START_REF] Arroyo | Diet composition influences annual breeding success of Montagu's harriers (Circus pygargus) feeding on diverse prey[END_REF] or Castan ˜o 1995), so it is possible that the duration of our study (3 years) was not enough to detect interannual variations, which are expected to be lower in amplitude for generalist than for specialist predators.

Another interesting result was the absence of seasonal decline in clutch size, as reported in many other studies on Montagu's Harrier [START_REF] Castan ˜o | Ecologı ´a reproductiva del aguilucho cenizo Circus pygargus en el campo de Montiel. Un estudio aplicado a la conservacio[END_REF][START_REF] Salamolard | Strate ´gie d'utilisation des resources chez une espe `ce de rapace semi-colonial, le Busard cendre ´(Circus pygargus). The `se de doctorat[END_REF][START_REF] Millon | Comparative biology of hen Harrier and Montagu's harrier: an 8-year study in north-eastern France[END_REF][START_REF] Koks | Do voles make agricultural habitat attractive to Montagu's harrier Circus pygargus[END_REF], other raptors [START_REF] Newton | Seasonal trend in the breeding performance of sparrowhawks[END_REF][START_REF] Korpima ¨ki | Clutch size of kestrels: seasonal decline and experimental evidence for food limitation under fluctuation food conditions[END_REF][START_REF] Mougeot | Breeding biology of the red kite Milvus milvus in Corsica[END_REF]) and more generally in bird species [START_REF] Arnold | Seasonal declines in reproductive success of the common tern Sterna hirundo: timing or parental quality?[END_REF]. A seasonal decline in clutch size is usually related to a difference in the quality of individuals breeding earlier versus later in the season, or to a degradation of environmental conditions, with poorer breeding conditions later in the season (decreased food resources and/or more frequent adverse climatic events). Only one previous study on this species did not find any relationship between laying date and clutch size [START_REF] Corbacho | Clutch size and egg size in the breeding strategy of Montagu's harrier Circus pygargus in a Mediterranean area[END_REF]. In our study population, this absence of seasonal clutch size decline could be explained by the marked laying synchrony observed in the 3 years of the study, when at least 63.2% of the monitored pairs started laying within a 2-week period (see Fig. 1b). A similar pattern has been observed by [START_REF] Arroyo | Breeding ecology and nest dispersion of Montagu's harrier Circus pygargus in Central Spain[END_REF] in a Spanish population of Montagu's Harriers during years of high breeding synchrony.

Estimates of nest failure rates were higher than in western European populations, when excluding failures (2000, 2006 and 2007). Sample sizes (above columns) refer to the number of identified prey items due to harvesting activities (Arroyo et al. 2004 and references therein). The most important identified cause of failure was predation. A high predation rate in our study population may be expected, since the predator community of north-central Kazakhstan steppes is very rich. However, it is difficult to discriminate between ultimate and proximate causes of failure, because in most cases no remains were found (so the cause of failure is unknown), and if broken eggshells appeared it was difficult to differentiate whether the nest had been predated, or the eggs had been abandoned and subsequently eaten by scavengers (such as corvids). A combination of poor food supply to incubating females, inclement weather and predation may be involved.

Overall, in any case, the Montagu's Harrier population in the Naurzum area appeared to have high nest failure rates, with predation being a non-negligible cause of failure.

Predation rates could also be high after fledging, because the Naurzum area supports an extremely high density of eastern Imperial Eagles Aquila heliaca that regularly prey on harriers [START_REF] Katzner | Spatial structure in the diet of imperial eagles Aquila heliaca in Kazakhstan[END_REF]. The role of predation on the breeding success of this population needs further research.

Despite relatively high failure rates, it appeared that the Montagu's Harrier population had a relatively high productivity. Unfortunately, fieldwork was only undertaken during a short period during the first 2 years of the study, and there was thus not enough information to evaluate fledging success and productivity for those years. Nevertheless, there were no differences in brood size among years, including 2007 when monitoring was more intense, and the mean brood size at the last visit was in the top range of those observed in western European populations nesting in natural vegetation or when conservation measures to protect nestlings from harvesting activities are in place (2.74 in Castello ´n, Spain, Limin ˜ana et al. 2006;1.80 ± 0.21 in Groningen, Netherlands, Koks et al. 2001;1.83 ± 1.33 in western France, Salamolard et al. 2000;2.50 ± 1.52 in Extremadura, Spain, Corbacho et al. 1997;1.90 ± 1.57 in central Spain, Arroyo 1995).

In conclusion, Montagu's Harrier populations breeding in natural steppes of northern Kazakhstan appear to be relatively productive despite high failure rates, which suggest that they may have sustainable dynamics. However, they seem dependent on a relatively scarce habitat (dogrose bushy areas), which may be further depleted if steppe areas are turned into alternative land uses, such as agriculture, or could be sensitive to overgrazing. One of the main threats for steppe habitats in north Kazakhstan is the likely expansion of cereal agriculture in the near future. In our study area, good meteorological conditions and the overall economic growth in recent years has allowed the expansion of the agricultural areas onto adjacent steppe biotopes [START_REF] Bragin | Osobennosti rasprostraneniya, chislennosti I nekotorye cherty ekologii stepnogo lunya (Circus macrourus) v Kustanaiskoi oblasti. Distribution, number and some features of ecology of the pallid harrier (Circus macrourus) in Kustanai region[END_REF]. It is relevant that the most preserved shrubland areas (holding the main breeding colonies of Montagu's Harriers) in our study area were inside a National Nature Reserve, whereas this type of habitat appeared to be more fragmented in grazed and farmed steppe areas. Additionally, climate changes through an increase in fire frequency (as it seem to have been the case in recent years) could further reduce the availability of Montagu's Harrier breeding habitats in our study area. It would be important to determine whether this is the trend in central Asia at large.

It would also be interesting to evaluate the population implications of breeding in agricultural versus natural habitats for the species. Agricultural habitats are widely available, but published data show that at present 60% of nestlings in the Iberian Peninsula and France would die because of harvesting activities if unprotected by conservation measures, and that such a reduction in productivity would make harrier populations unsustainable in the absence of conservation measures [START_REF] Arroyo | Conservation of the Montagu's harrier (Circus pygargus) in agricultural areas[END_REF]. Additionally, the current intensification rate of western Europe agricultural areas has led to important reductions in food abundance (voles and passerine birds; [START_REF] Butet | Effects of agriculture development on vole dynamics and conservation of Montagu's harrier in western French wetlands[END_REF][START_REF] Donald | Agricultural intensification and the collapse of Europe's farmland bird populations Faralli U (1994) Breeding biology, habitat selection and conservation of Montagu's harrier Circus pygargus I the northern Apeninnes, Italy[END_REF], so food availability might be more limiting in western European areas than in the preserved steppes of Central Asia. Breeding in natural vegetation may lead to higher productivity (e.g. [START_REF] Limin ˜ana | Vegetation height selection in Montagu's harriers (Circus pygargus) breeding in a natural habitat[END_REF]), but these populations may be more threatened by habitat disappearance and, as predator communities are more diverse, may suffer higher predation rates during the breeding period. Further research is needed to better understand the dynamics of these different populations and the interplay between breeding habitat selection and availability, breeding success, food availability and predation levels.

Zusammenfassung

Brutbiologie der Wiesenweihe (Circus pygargus) in Nord-Zentralkasachstan U ¨ber die O ¨kologie und den Bedrohungsstatus zentralasiatischer Populationen der Wiesenweihe Circus pygargus ist nur wenig bekannt. Wir haben die Brutbiologie dieser Art in der Naurzum-Region in Nord-Zentralkasachstan u ¨ber einen Zeitraum von drei Jahren untersucht. Die meisten Wiesenweihen im Untersuchungsgebiet bru ¨teten in der U ¨berganszone zwischen Wald und Steppe in Buschbereichen, die von der Hunds-Rose Rosa canina dominiert wurden; dieser Vegetationstyp bot im Untersuchungsgebiet anscheinend die ho ¨chste und dichteste Deckung fu ¨r die Nester. Die Eiablage erfolgte vom 26. April bis zum 7. Juni (Mittelwert 13. Mai, n = 49) und erfolgte fru ¨her als in westeuropa ¨ischen Populationen vergleichbarer geographischer Breite, wenn der Termin auch signifikant zwischen den Jahren schwankte. Die mittlere (±SD) Gelegegro ¨ße betrug 4,44 ± 0,86 (Spannweite 2-6; n = 50), was am oberen Rand des fu ¨r diese Art beobachteten Bereichs liegt. Es gab keine signifikante Variation in der Gelegegro ¨ße zwischen den Jahren, obwohl die Abundanz kleiner Sa ¨ugetiere in der Region stark schwankte. Die Nahrung bestand hauptsa ¨chlich aus Eidechsen (54,2%, n = 533 identifizierte Beutestu ¨cke in allen drei Jahren), doch auch kleine Sa ¨ugetiere (17,1%), Sperlingsvo ¨gel (14,3%) und Insekten (13,6%) wurden gefressen. Die mittlere Brutgro ¨ße bei der letzten Kontrolle betrug 2,55 ± 2,10 (Spannweite 0-6; n = 51). Die Verlustrate war relativ hoch; der ermittelte Hauptgrund fu ¨r Nestverluste war Pra ¨dation. Wir vergleichen die Daten, die in dieser in natu ¨rlicher Steppe bru ¨tenden Population gewonnen wurden, mit Brutparametern aus gut untersuchten westeuropa ¨ischen Populationen und diskutieren die Folgen fu ¨r den Schutz dieser Art.

Fig. 1

 1 Fig. 1 Frequency distribution of laying dates (a) and relative laying dates (b) in Montagu's Harrier Circus pygargus in north-central Kazakhstan for the 3 years of the study. In Fig. 1b, week 0 is the week of the average lay date in each year

Fig. 2

 2 Fig.2Relationship between mean laying date and latitude in 11 populations of Montagu's Harrier (western Europe black dots, Kazakhstan white triangle) (data fromArroyo et al. 2004 and this study)

Fig. 3

 3 Fig. 3 Diet composition (percentages based on prey numbers) of Montagu's Harriers in the Naurzum region during the study period(2000, 2006 and 2007). Sample sizes (above columns) refer to the number of identified prey items

  25, P \ 0.0001), with laying being almost 2 weeks later in 2007 than in the other years studied (2000: 6 May ± 13 days, n = 13; 2006: 7 May ± 8 days, n = 19; 2007: 22 May ± 9 days, n = 17; Fig. 1a). Mean April temperatures were significantly lower in 2007 than in 2006 (v 2 2 = 7.52, P = 0.0061). More precisely, temperatures of the first and second decades of April were significantly lower in 2007 than in 2006 (v 2 2 = 12.66; P = 0.0004 and v 2 2 = 16.02; P \ 0.001, respectively), and also much lower than in 2000 (Table1).

Table 1

 1 Mean temperatures and precipitation levels of the three spring months of the study period(2000, 2006 and 2007)

		Year	2000		2006		2007
			Mean	Precipitation	Mean	Precipitation	Mean	Precipitation
			temperatures	(mm)	temperatures	(mm)	temperatures	(mm)
		April 1 a	4.5		5.0 ± 5.3		-2.5 ± 2.7
		April 2	11.6		11.4 ± 4.1		4.2 ± 2.6
		April 3	14		8.0 ± 5.4		10.5 ± 2.1
		April	10 ± 4.9	20.7	8.1 ± 5.2	13.9	4.0 ± 5.9	24.3
		May 1	9.9		9.4 ± 4.1		7.0 ± 8.1
		May 2	9.9		15.3 ± 2.4		15.3 ± 3.7
		May 3	14.2		19.4 ± 3.6		20.7 ± 2.7
		May	11.3 ± 2.4	36.8	14.8 ± 5.3	29.6	14.5 ± 7.7	56.4
		June 1	17.8		21.8 ± 3.6		12.3 ± 4.5
		June 2	20		25.5 ± 4.5		19.5 ± 5.4
	a April 1, 2, 3 refer to the first,	June 3	25.5		20.0 ± 0.9		24.7 ± 4.8
	second and third decades of	June	21.1 ± 3.9	32.9	22.4 ± 4.0	12.1	18.8 ± 7.0	27.9
	April, respectively (same for May and June)	Vole index No data		13.8 ± 7.6		0 ± 0

Table 2

 2 Variations of clutch size of Montagu's Harriers in different parts of Europe and Central Asia (modified from[START_REF] Arroyo | Circus pygargus Montagu's harrier[END_REF]

	Location		Clutch size		n	Range	References
	The Netherlands		4.20 ± 0.80		112	2-7	Bijlsma et al. (1993)
	Poland		3.90-4.40		125	2-5	Krogulec and Leroux (1994)
	England		4.02 ± 0.07		227	2-10	Underhill-Day (1990)
	France, Morbihan		4.27 ± 0.14		29	3-6	Hays (1971)
	France, Maine et Loire		3.69 ± 0.19		26	2-5	Cormier (1985)
	France, Vende ´e		3.61 ± 0.17		23	2-5	Cormier (1985)
	France, Vende ´e		3.75 ± 0.80		101	2-6	Arroyo et al. (1998)
	France, Charente-Maritime	2.80 ± 0.40-4.39 ± 0.15	200	1-6	Butet and Leroux (2001); Arroyo et al. (1998)
	France, Deux Sevres		4.28 ± 1.09		143	2-8	Arroyo et al. (1998)
	France, Champagne-Ardennes	3.65 ± 1.01		111	1-6	Millon et al. (2002)
	Italy, N. Appenines		3.32		31	-	Faralli (1994)
	Italy, Marche		3.8		37	-	Pandolfi and Giacchini (1991)
	Italy, Emilia-Romana		3.70 ± 0.80		61	2-5	Martelli and Sandri (1991)
	Spain, Madrid		3.93 ± 0.84		215	2-6	Arroyo et al. (1998)
	Spain, Ciudad Real		4.30 ± 1.00		120	2-9	Castan ˜o (1997)
	Spain, Badajoz		3.48 ± 0.99		98	1-5	Corbacho et al. (1997)
	Spain, Castello ´n		4.34 ± 0.84		266	2-8	Limin ˜ana et al. (2006)
	Spain, Ca ´ceres		3.31 ± 0.65		19	2-5	Pe ´rez Chiscano and Ferna ´ndez Cruz (1971)
	Portugal, Evora		3.97 ± 1.04		36	2-6	Claro (2000)
	Portugal, Castro Verde		2.82 ± 0.40		11	2-3	Onofre (1995)
	North-central Kazakhstan	4.44 ± 0.86		50	2-6	This study
		(39)		(69)	(429)
		100%
	percentages based on numbers	20% 40% 60% 80%
		0%
		2000		2006	2007
		eggs	insects	reptiles birds small mammals

J Ornithol (2010) 151:713-722

Acknowledgments We thank Mike Madders, Jeff Watson, Becky Wills, Ainur Akhmetova, Robert Stakim, Alexey Timoshenko and Maxim Glushkov for help with data collection, and Natural Research for funding this work. Becky Wills also acted as a translator in 2000, and Ainur Akmetova helped with translations and logistics in 2006. We also thank the current director of the Reserve, Muttakov Bagitjan Zhakezhanovich, the deputy director, Zeinelova Maria Alexandrova, and all the staff of the Naurzum Nature Reserve for help in setting up our fieldwork in the Naurzum region.


Michelle Jayne Cole
email: michelle.cole@hpa.org.uk

Stephanie Angela Chisholm

Catherine Ann Ison

Catherine M Lowndes

Steen Hoffmann

Angelika Stary

European Surveillance

Reinhild Strauss

Tania Crucitti

Susan Cowan

Véronique Goulet

Sednaoui

Peter Kohl

Vasileia Konte

Guðrún Sigmundsdóttir

Paola Stefanelli

Judite Pirsko

Elvira Lavrinovica

Christopher Barbara

St Luke's Hospital

Jackie Maistre Melillo

Ineke Linde

Marianne Van Der Sande

Hilde Klovstad

General Jacinta Azevedo

Maria-José Borrego

Jan Mikas

Irena Klavs

Julio Vazquez

Mercedes Diez

Anders Blaxhult

Inga Velicko

Hans Fredlund

Magnus Unemo

Peyman Altan

Mike Catchpole

Gwenda Hughes

Health Emma Savage

European Surveillance of antimicrobial resistance in Neisseria gonorrhoeae

Keywords: Neisseria gonorrhoeae, gonorrhoea, antimicrobial resistance, surveillance 2

la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Neisseria gonorrhoeae is the causative agent of gonorrhoea, a sexually transmitted infection (STI) which continues to present a significant public health problem across Europe and Worldwide, with approximately 62 million new cases diagnosed globally each year. [START_REF]Sexually Transmitted Diseases: Gonorrhoea[END_REF] Gonorrhoea primarily presents as a mucosal infection of the lower genital tract, which is predominantly symptomatic in men presenting as urethritis but is thought to be asymptomatic in about 50% of women. N. gonorrhoeae can ascend to the upper genital tract, if undiagnosed or inadequately treated, leading to complications such as infertility which occur more frequently in women or can invade the blood causing disseminated infections (e.g. septic arthritis, meningitis, endocarditis). Gonorrhoea is associated with an increase in the acquisition and transmission of HIV. [START_REF] Fleming | From epidemiological synergy to public health policy and practice: the contribution of other sexually transmitted diseases to sexual transmission of HIV infection[END_REF] Treatment of gonorrhoea, therefore, is essential not only for individual patient management but also to intercept transmission chains and so reduce the overall disease burden. However, successful treatment of gonorrhoea is severely compromised by the propensity of N. gonorrhoeae to develop resistance to antimicrobials used for therapy, and the subsequent worldwide spread of resistant gonococci is well documented. [START_REF] Who Western | Surveillance of antibiotic resistance in Neisseria gonorrhoeae in the WHO Western Pacific Region, 2006[END_REF][START_REF]Sexually Transmitted Disease Surveillance 2007 Supplement, Gonococcal Isolate Surveillance Project (GISP) Annual Report[END_REF][START_REF] Dillon | Challenges in the control of gonorrhea in South America and the Caribbean: monitoring the development of resistance to antibiotics[END_REF][START_REF]GRASP 2008 Report:Trends in Antimicrobial Resistant Gonorrhoea[END_REF][START_REF] Kubanova | National surveillance of antimicrobial susceptibility in Neisseria gonorrhoeae in 2005-2006 and recommendations of first-line antimicrobial drugs for gonorrhoea treatment in Russia[END_REF][START_REF] Tapsall | Annual report of the Australian Gonococcal Surveillance Programme[END_REF] Therefore, surveillance of antimicrobial resistance (AMR) for N. gonorrhoeae is of paramount importance to inform prescribing policy to ensure patients receive appropriate therapy.

The European Surveillance of Sexually Transmitted Infections (ESSTI) project was established in 2002 to develop and coordinate epidemiological and laboratory surveillance of sexually transmitted infections (STIs) in the European region in order to better inform about STI prevention, care and control. Surveillance data collected by ESSTI demonstrated that the number of cases of gonorrhoea, from 1998 to 2007, rose in Western Europe, remained steady in Central Europe and declined in the two Eastern European countries that supplied data. [START_REF]Sexually Transmitted Infections in Europe[END_REF] However there was wide variation in the rate of gonorrhoea diagnoses in individual countries, for example, 0.3/100,000 in Italy to 30.8/100,000 in the United Kingdom in 2007. [START_REF]Sexually Transmitted Infections in Europe[END_REF] Although this may be due to the heterogeneity in the different surveillance and laboratory systems. [START_REF]Sexually Transmitted Infections in Europe[END_REF] Euro-GASP (European Gonococcal Antimicrobial Surveillance Programme), the laboratory component of ESSTI, performed the first European sentinel surveillance study for AMR in N. gonorrhoeae in 2004. [START_REF] Martin | European Surveillance of Sexually Transmitted Infections (ESSTI): the first combined antimicrobial susceptibility data for Neisseria gonorrhoeae in Western Europe[END_REF] This initial study revealed high resistance rates to ciprofloxacin (31%) which was used by most countries for treatment at that time, and tetracycline (60%) and penicillin (21%), along with azithromycin resistance of over 5% observed for the first time. Surveillance studies are essential to demonstrate when resistance rates to antimicrobials exceed 5%, the level at which a therapeutic agent should no longer be recommended as first line therapy. [11] In addition, it is of particular importance to monitor emerging resistance to the current recommended therapy across Europe, the third generation cephalosporins. [START_REF] Bignell | European (IUSTI/WHO) guideline on the diagnosis and treatment of gonorrhoea in adults[END_REF] Euro-GASP has subsequently performed sentinel surveillance studies in 2006 to 2008 to continue to monitor the prevalence of antimicrobial-resistant N. gonorrhoeae across Europe.

METHODS

Participating laboratories

Seventeen N. gonorrhoeae reference or specialist laboratories participated in the ESSTI N. gonorrhoeae AMR surveillance study as previously described. [START_REF]Sexually Transmitted Infections in Europe[END_REF] Three participating laboratories had complete coverage of all diagnosed gonorrhoea in the country (Denmark, Scotland and Sweden). Eight laboratories had good national coverage, defined as representations from all areas of the country but not every case of diagnosed gonorrhoea (Belgium, England and Wales, France, Greece, Latvia, Malta, Portugal and Spain). Six laboratories had good coverage of a particular region of the country but not national coverage (Austria, Germany, Ireland, Italy, The Netherlands and Slovenia).

Collection of N. gonorrhoeae isolates

The aim of the isolate collection was to obtain a minimum of 1000 isolates across Europe, where possible, to maximise the ability to detect changes over time. Each country was asked to contribute 100 isolates from 1 st June each year. Some laboratories (Austria, Portugal, Slovenia, Germany, Greece, Malta, Italy and Latvia) collected isolates prior to the start date due to a low collection rate. Laboratories were requested to collect one isolate from each patient. For each isolate the following data where available was collected: Date specimen obtained, sex and age.

Antimicrobial Susceptibility testing

Isolates were retrieved on GC agar (Becton, Dickinson and Company, Le Pont de Claix, France) with 1% Vitox (Oxoid, Basingstoke, UK) and incubated for 18 -24 hours at 36 o C in 5% CO 2 . Isolates were confirmed to be N. gonorrhoeae by Gram stain, oxidase test and either the Gonogen II test (Bioconnections, Leeds, UK) or the Microtrak test (Trinity Biotech, Wicklow, Ireland) according to manufacter's instructions. Susceptibility testing was performed at the three centres using the agar dilution breakpoint technique for ciprofloxacin, azithromycin, penicillin and tetracycline. In addition, spectinomycin was tested for the 2008 isolates.

The isolates were sub-cultured once before susceptibility testing was performed.

Suspensions of cultures aged 18 -24 hours were prepared equivalent to

McFarland's standard 0.5 (approximately 10 4 cfu/µl) in saline. Using the Multipointelite multipoint inoculator (Mast Group Ltd, Bootle, UK), suspensions were inoculated onto GC agar plates with 1% Vitox, containing a panel of antimicrobials at the following breakpoint concentrations; penicillin (1 mg/L), ciprofloxacin (0.5 mg/L), tetracycline (1 and 8 mg/L), azithromycin (0.5 mg/L) and spectinomycin (64 mg/L).

Plates were incubated for 18 -24 hours at 36 o C in 5% CO 2 and the presence of bacterial growth was recorded for each isolate. All isolates were tested for penicillinase production using the chromogenic reagent, Nitrocefin (Oxoid), following manufacturer's instructions. The ceftriaxone MIC of isolates was determined using Etests (AB bioMérieux, Solna, Sweden) according to the manufacturer's instructions.

Azithromycin MICs were determined by Etest for isolates displaying resistance by the agar dilution breakpoint technique.

The Clinical and Laboratory Standards Institute (CLSI) breakpoints for susceptibility or resistance [START_REF]Performance standards for antimicrobial susceptibility testing[END_REF], as widely used in European countries, were used for ciprofloxacin, penicillin, tetracycline and spectinomycin. The European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints [START_REF]European Committee on Antimicrobial Susceptibility Testing -EUCAST[END_REF] were followed for azithromycin and ceftriaxone, as CLSI have not yet recommended a breakpoint for azithromycin and the lower EUCAST breakpoints for ceftriaxone ensure levels of decreased susceptibility are not under estimated. In summary, strains were categorised according to the following definitions: Ciprofloxacin resistant (MICs ≥ 1 mg/L); penicillinase producing N. gonorrhoeae (PPNG) (β-lactamase positive); tetracycline resistant N. gonorrhoeae (TRNG) (MICs ≥ 16 mg/L); chromosomally resistant N. gonorrhoeae (CMRNG) (penicillin MICs ≥ 2 mg/L and β-lactamase negative and tetracycline MICs 2 -8 mg/L); azithromycin resistant (MICs ≥ 1 mg/L); spectinomycin resistant (MICs ≥ 128 mg/L) and ceftriaxone decreased-susceptibility (MICs > 0.125 mg/L).

Statistical analysis

Fisher's exact test was used to assess the significance of difference between the resistance rates from 2006 to 2008. The significance of difference between all countries and countries that participated in all of the three years was assessed using Fisher's exact test. The differences between the resistance rates of each antimicrobial between all participating countries in each year was assessed using the chi-squared test for penicillin, tetracycline and ciprofloxacin, and the Fisher's exact test was used for azithromycin and fully susceptible strains. P values less than 0.05 were categorised as significant.

Quality assurance and control

Laboratory personnel from each of the three testing centres were trained in a single centre (the HPA) to ensure consistency of technique. Quality control strains (WHO A (spectinomycin resistant), WHO G (penicillin and tetracycline resistant), WHO J (PPNG, ciprofloxacin and tetracycline resistant), and G06-1153 (azithromycin resistant clinical strain)) were included in each susceptibility testing run. To ensure consistent quality assurance (QA), two different panels of 10 strains were tested by the three centres each year using all methods and the results from all three centres were compared.

RESULTS

Isolate and patient data

A total of 3645 isolates were collected over the three year period (Table 1) and after the removal of duplicate specimens 3528 isolates were retrieved and confirmed to be N. gonorrhoeae. The overall retrieval rate was 97% (3528/3645). The number of isolates tested from each country varied from 12 (Germany 2008) to 114 (Spain 2006) (Table 2). Of the 3528 isolates tested, further data was available for 3494 isolates. The majority of gonococci (80%; 2798/3494) were collected from men whereas 19% (675/3494) were from women. Gender was unknown for the remaining 21 isolates (Table 1). The age range of the patients was 6 to 81 years, with a mode and median age of 24 and 31 years respectively.

Antimicrobial susceptibility

Data from all countries for each year was combined to determine the overall resistance rates (Figure 1). The list of participating countries varied slightly for each year (Table 2). However, there were small, non-significant differences (Fisher's exact test p > 0.05) when the resistance rates from the countries that participated in all of the three years was compared to all countries in each of the three years (Tables 2 and3). For each antimicrobial in each year there was considerable variation in the resistance rates between the countries (chi-squared and Fisher's exact test p ≤ 0.01) (Tables 2 and3).

The overall level of ciprofloxacin resistance significantly increased from 42% in 2006 to 51% (Fishers exact test p < 0.001). All countries displayed more than 5% resistance to ciprofloxacin in each year tested (Table 2). No apparent trend for azithromycin resistance rates was evident (Table 2). The modal MIC of azithromycin resistant isolates in all three years was 1.0 mg/L. Four isolates in 2007 from Scotland and one from Ireland in 2008 were highly resistant to azithromycin (>256mg/L). High level resistance to penicillin (PPNG) and tetracycline (TRNG) remained fairly constant over the study period (Table 3 -Please put as web only) and there was no apparent trend from 2006 to 2008 for chromosomally mediated resistance to both penicillin and tetracycline (CMRNG).

No isolates demonstrated decreased-susceptibility to ceftriaxone. The modal MIC of isolates to ceftriaxone was 0.008 mg/L in 2006 and 2007 and 0.004 mg/L in 2008 (Figure 2).

Quality assurance

The overall concordance for the agar dilution breakpoint technique was 94% (94/100). For the agar dilution technique there were six occasions when the same category of resistance was not achieved for penicillin (n=4), ciprofloxacin (n=1) and azithromycin (n=1) for each QA isolate. However all of these six strains had a MIC on a breakpoint. There was 100% concordance for 10 strains tested by azithromycin Etest. Eighty-five percent (17/20) concordance was achieved for the 20 strains tested by ceftriaxone Etest, where the MICs differed by less than two doubling dilutions. The internal quality control strains results were within one dilution of the expected ranges. [START_REF] Martin | European Surveillance of Sexually Transmitted Infections (ESSTI): the first combined antimicrobial susceptibility data for Neisseria gonorrhoeae in Western Europe[END_REF] and by Etest in the current study.

While these two methods have been shown to be comparable in our laboratory (data not shown), the possibility that differences in modal MIC are partially due to differences in methodologies cannot be fully excluded. However a similar drift in the population susceptibility to ceftriaxone has been documented recently in England and Wales, [START_REF]GRASP 2008 Report:Trends in Antimicrobial Resistant Gonorrhoea[END_REF] and the current study may indicate an emerging problem across Europe, which raises the possibility that MICs may reach a level which leads to treatment failure. The data from this study shows that ceftriaxone is still an appropriate treatment for gonorrhoea in Europe, and as yet treatment failure of urogenital gonorrhoea has not been documented although treatment failure for pharyngeal infection has been recorded. [START_REF] Tapsall | Two cases of failed ceftriaxone treatment in pharyngeal gonorrhoea verified by molecular microbiological methods[END_REF] Ceftriaxone is administered parenterally and this has the advantage of ensuring patient compliance and preventing misuse of the agent, which may delay emerging resistance. In contrast, third generations cephalosporins administered orally, such as cefixime, have already resulted in suspected treatment failures [23;24] The current study did not examine resistance to cefixime, but a review of the susceptibility testing panel for European surveillance of gonococcal antimicrobial resistance in 2009 has lead to the amendment of the testing strategy to include cefixime to ensure all therapeutically relevant agents are monitored in future years.

The threat of emerging resistance to third generation cephalosporins highlights the need to consider future therapeutic options. Azithromycin is not currently a recommended treatment for gonorrhoea [START_REF] Bignell | European (IUSTI/WHO) guideline on the diagnosis and treatment of gonorrhoea in adults[END_REF] but it is proposed that it could be effective as part of a dual therapy in the treatment of pharyngeal gonorrhoea. [25;26] It is therefore important to monitor azithromycin resistance, particularly as the gonococcal population is frequently exposed to this drug as the recommended therapy for treating Chlamydia trachomatis co-infection. The azithromycin resistance rates fluctuated over the study period which is consistent with surveillance studies from the United Kingdom. [6;19] The modal MIC of resistant isolates to azithromycin was 1 mg/L, which is a proposed breakpoint for categorising resistance. [START_REF]European Committee on Antimicrobial Susceptibility Testing -EUCAST[END_REF] Isolates with an MIC on the breakpoint are just one doubling dilution from giving a susceptible category, which is still technically valid, but may explain the inconsistent resistance rates from 2006 to 2008. The numbers however are small and more longitudinal data is required to establish a trend. Of great concern were the five isolates from Scotland and Ireland exhibiting high-level resistance to azithromycin (>256 mg/L).

This resistance phenotype in N. gonorrhoeae was first detected in Scotland in 2004, [START_REF] Palmer | Emergence and spread of azithromycin-resistant Neisseria gonorrhoeae in Scotland[END_REF] and has since been reported from England and Wales, [START_REF] Chisholm | Emergence of high-level azithromycin resistance in Neisseria gonorrhoeae in England and Wales[END_REF] Italy [START_REF] Starnino | Azithromycin-resistant Neisseria gonorrhoeae strains recently isolated in Italy[END_REF] and

Argentina. [START_REF] Galarza | Emergence of high level azithromycin-resistant Neisseria gonorrhoeae strain isolated in Argentina[END_REF] Widespread dissemination of high-level azithromycin resistant strains would eliminate azithromycin as a future treatment option. While no resistance to spectinomycin, another therapeutic option, was observed in the current study, problems with availability of this drug in many European countries limits the utility of this as a therapeutic option.

Development and continuation of a European scheme for surveillance of gonococcal antimicrobial susceptibility is a significant achievement which has clear public health benefits. This is now an ongoing programme and the focus for the future will be to include more European countries and to overcome some of the acknowledged limitations of the current study such as the variability in numbers submitted from each country annually and differences in the representativeness of the selected isolates for each country. In the current study, there was no difference when the rates of resistance from the countries that participated in all of the three years were compared to all countries in each of the three years. However the overall trends may be biased by the variable number of isolates a country submits in each of the three years and the level of representativeness from each country. Further development of this programme will also focus on increasing sampling numbers from countries with high rates of gonorrhoea and on increasing the frequency of testing so changes in gonococci antimicrobial susceptibilities can be detected more rapidly. In addition, future European surveillance work will focus on ensuring the panel of antimicrobials are therapeutically relevant and have the potential for use in the future. Continued efforts to reliably link epidemiological data to the antimicrobial susceptibility data will be critical in establishing which core groups carry the burden of resistance to help focus intervention strategies.

This study has identified continued high rates of ciprofloxacin resistance, high-level azithromycin resistance and an upward ceftriaxone MIC drift in the European gonococcal population. This demonstrates that N. gonorrhoeae AMR surveillance studies are essential to inform treatment guidelines thereby preventing onward transmission and reducing patient morbidity. These studies should continue to monitor for increasing, emerging and high-level resistance, and also to help assist in the decision making process about future antimicrobials options.

KEY MESSAGES

Longitudinal surveillance of antimicrobial resistance in gonorrhoea showed ciprofloxacin is not suitable for treatment in Europe because of continued high levels of resistance.

The emergence of high-level azithromycin resistance is of great concern as widespread dissemination of these strains would eliminate azithromycin as a future treatment option.

Ceftriaxone is an appropriate treatment for gonorrhoea in Europe as all isolates tested were susceptible.

An upward ceftriaxone MIC drift was detected in the European gonococcal population, demonstrating that this situation needs to be monitored carefully.

Pure cultures, 18 -

 18 24 hours old, were saved on Microbank beads (Pro-lab Diagnostics, Richmond Hill, Canada) and stored at -70 o C. The isolates were then sent frozen on dry ice to one of three laboratories for susceptibility testing (Health Protection Agency (HPA), London, UK; Statens Serum Institut, Copenhagen, Denmark and Outpatients' Centre for Diagnosis of Infectious Venero-Dermatological Diseases, Vienna, Austria).

Figure 1 . 3 )Figure 2 .

 132 Figure 1. Overall incidence of resistant and fully susceptible N. gonorrhoeae (arrows

Word Count: 2827 "Figure 1 .Figure 2 .

 282712 Figure 1. Overall incidence of resistant and fully susceptible N. gonorrhoeae (arrows indicate the lowest and highest percentage from participating countries (see Tables2 and 3)

Table 1 .

 1 Isolate and patient characteristics

		Year
	2006	2007	2008	All years

Table 2 .

 2 Resistance rates by ciprofloxacin and azithromycin antimicrobials and percentage of strains that were fully susceptible in each participating country

						% Resistance rates			% Fully
		No. of isolates							susceptible
			tested		Ciprofloxacin	Azithromycin		strains
	Country	2006 2007 2008 2006 2007 2008 2006 2007 2008 2006 2007 2008
	Austria	19	107 110 36.8 64.5 80.9	0	1.9	0.9 26.3 11.2 9.1
	Belgium	109 107 108 58.7 62.6 56.5 1.8 12.1	0	9.2 14.0 10.2
	Denmark	111 110 110 45.9 67.3 70.0 8.1 13.6 6.4	1.8	7.3	0
	England & Wales	110 112 112 15.5 25.9 30.4	0	4.5	0	14.5 16.1 5.4
	France	ND	110 107	ND 44.5 43.9 ND	4.5	0	ND	7.3 13.1
	Germany	ND	84	12	ND 59.5 58.3 ND	2.4	8.3	ND	7.1	8.3
	Greece	ND	86	110	ND 55.8 63.6 ND 27.9 3.6	ND	0	0.9
	Ireland	ND	ND	92	ND	ND 29.3 ND	ND	2.2	ND	ND	4.3
	Italy	ND	92	ND	ND 59.8 ND	ND	1.1	ND	ND	5.4	ND
	Latvia	ND	33	ND	ND 21.2 ND	ND	3.0	ND	ND 57.6 ND
	Malta	ND	29	24	ND 89.7 83.3 ND	0	0	ND	0	0
	Netherlands	108 107 106 38.0 36.4 50.0 1.9	2.8	0	2.8	4.7	8.5
	Portugal	29	72	54	48.3 47.2 29.6 6.9	8.3	0	0	0	3.7
	Scotland	110 110 110 28.2 31.8 31.8 2.7	9.1	0.9 10.9 13.6 0.9
	Slovenia	26	28	27	23.1 32.1 29.6 11.5	0	3.7	0	10.7 3.7
	Spain	114 110 103 50.9 60.9 51.5 3.5	1.8	1.0	5.3	6.4	8.7
	Sweden	100 110 100 62.0 66.4 61.0 2.0	9.1	6.0	5.0	1.8	0
	All countries	836 1407 1285 42	52	51.2 3.2	7	1.9	7.1	8.7	5.4
	Consistent participants*	836 973 940	42	51	51.8 3.2	6.8	1.8	7.1	8.7	5.2
	*Countries that participated in all three years; Austria, Belgium, Denmark, England and Wales,
	The Netherlands, Portugal, Scotland, Slovenia, Spain and Sweden.
	ND -No data available
	Bold text = highest and lowest % resistance

Table 3 .

 3 Resistance rates for penicillin and tetracycline antimicrobials in each participating country This high level of ciprofloxacin resistance clearly indicates that this antimicrobial is not an appropriate choice for use in any of the countries which participated in this study, unless isolates are known to be susceptible or local resistance rates are known to be less than 5%.It was encouraging to note that not only were there no examples of decreasedsusceptibility to ceftriaxone over the sampling period, but there was also little change in the ceftriaxone modal MICs from 2006 to 2008. However the observation that the modal MICs of ≤ 0.002 mg/L documented in 2004[10] had increased to 0.008 mg/L in 2006 and 2007, and 0.004 mg/L in 2008 may indicate drift in the susceptibility of the gonococcal population in Europe. It should be noted that ceftriaxone MICs were determined by agar dilution in the 2004 study

		No. of isolates				% Resistance rates
			tested			PPNG			TRNG		CMRNG
	Country	2006 2007 2008 2006 2007 2008 2006 2007 2008 2006 2007 2008
	Austria	19	107 110	5.3 15.0 10.9 26.3 16.8 5.5	0	36.4 33.6
	Belgium	109 107 108 11.0 9.3 16.7 14.7 20.6 22.2 39.4 38.3 25.0
	Denmark	111 110 110 20.7 19.1 11.8 24.3 24.5 14.5 20.7 42.7 39.1
	England & Wales	110 112 112	3.6	8.0 10.7 6.4	8.9	7.1 12.7 18.8 9.8
	France	ND	110 107	ND 10.9 13.1 ND 18.2 19.6 ND 25.5 25.2
	Germany	ND	84	12	ND	8.3 25.0 ND 15.5 16.7 ND 23.8 33.3
	Greece	ND	86	110	ND	4.7	3.6	ND 11.6 8.2	ND 46.5 46.4
	Ireland	ND	ND	92	ND	ND	8.7	ND	ND 14.1 ND	ND 17.4
	Italy	ND	92	ND	ND	9.8	ND	ND 10.9 ND	ND 27.2 ND
	Latvia	ND	33	ND	ND	9.1	ND	ND 12.1 ND	ND 12.1 ND
	Malta	ND	29	24	ND	0	4.2	ND 10.3 4.2	ND 75.9 70.8
	Netherlands	108 107 106	7.4	8.4	9.4 10.2 9.3 14.2 33.3 21.5 29.2
	Portugal	29	72	54	6.9	8.3	9.3	3.4 15.3 24.1 41.4 48.6 14.8
	Scotland	110 110 110	9.1	8.2	8.2 10.0 7.3 12.7 16.4 14.5 17.3
	Slovenia	26	28	27	11.5 7.1	7.4	3.8	0	7.4	7.7 25.0 22.2
	Spain	114 110 103 11.4 19.1 15.5 14.0 20.0 24.3 36.0 31.8 23.3
	Sweden	100 110 100 35.0 30.0 23.0 32.0 40.0 36.0 15.0 30.9 28.0
	All countries	836 1407 1285 13.3 12.2 11.7 15.2 16.5	16	24.4 31.1 27.2
	Consistent participants*	836 973 940 13.3	14	12.8 15.2 17.7 16.9 24.4 30.6 24.9
	*Countries that participated in all three years; Austria, Belgium, Denmark, England and Wales,
	The Netherlands, Portugal, Scotland, Slovenia, Spain and Sweden.
	ND -No data available
	Bold text = highest or lowest % in that year

Ciprofloxacin resistance has increased dramatically since the first European N. gonorrhoeae AMR sentinel study was performed in 2004

[START_REF] Martin | European Surveillance of Sexually Transmitted Infections (ESSTI): the first combined antimicrobial susceptibility data for Neisseria gonorrhoeae in Western Europe[END_REF]

(31% to 51% in 2008) in spite of the fact that ciprofloxacin is no longer recommended for therapy in Europe.

[START_REF] Bignell | European (IUSTI/WHO) guideline on the diagnosis and treatment of gonorrhoea in adults[END_REF]

The rates are above the recommended 5% cut-off

[11]

in all participating countries. Similarly high rates have been demonstrated in individual European countries conducting national surveillance of antimicrobial resistance.

[6;15-21]

It is evident that ciprofloxacin resistance in N. gonorrhoeae confers no selective disadvantage to the organism as resistance has persisted in countries where relatively little ciprofloxacin is now prescribed for treatment of gonorrhoea.

[START_REF]GRASP 2008 Report:Trends in Antimicrobial Resistant Gonorrhoea[END_REF]

ACKNOWLEDGMENTS

We thank all the ESSTI collaborators for their contribution to the ESSTI project (www.essti.org). A special thanks to John Anderson, Lene Berthelsen and Maria Haller for performing the laboratory work and Tom Nichols for his statistical advice.

FUNDING

Financial support for this study was provided by the European Commission (DG SANCO), Agreement No. 2004210: ESSTI European Surveillance of Sexually Transmitted Infections.

Competing interests: None

CONTRIBUTORS

MC contributed to the design of the study, performed some of the laboratory work, analysed the data and prepared the manuscript. SC contributed to the data analysis, the creation of the manuscript and helped supervise the project.

SH and AS contributed to the design of the study, contributed to the testing of isolates and gave project support throughout the study.

CL was the surveillance lead for ESSTI and was involved in the creation of the ESSTI network and contributed to the design of the study. CI was the project and microbiology lead for ESSTI and was involved in the creation of the ESSTI network, contributed to the design of the study and supervised the project.

All authors were part of the ESSTI network, participated in project meetings and reviewed the final draft of the manuscript.