J L Jorda

T K Jondo

R Abraham

M T Cohen-Adad

C Opagiste

M Couach

A Khoder

F Sibieude

Preparation of pure Tl 2 Ba 2 CuO 6 + x : the contribution of phase equilibrium studies

The formation of the pure thallium-based cuprate Tl 2 Ba 2 CuO 6+x (2201) using a convenient process in the ternary TlO 1.5 -BaO-CuO phase diagram is described. This process is based on the congruent formation of the oxide Tl 2 Ba 2 O 5 to which CuO is mixed, constituting a quasi-binary system. In the resulting 2201 compound no trace of the main poisoning magnetic impurity BaCuO 2 could be detected by AC susceptibility measurements. The orthorhombic to tetragonal transformation is asserted to be due to thallium deficiency which occurs during the heat treatments at a temperature depending on the oxygen partial pressure.

I. Introduction

Despite possible problems related to the toxicity of thallium and its compounds, the superconducting properties of the thallium-based cuprates have been extensively studied. The review of Greenblatt et al. [START_REF] Greenbtatl | Studies of High Temperature Superconductors[END_REF] reports more than 25 investigated Tl-based systems. Actually, since the initial work of Sheng and Hermann [START_REF] Sheng | [END_REF] the thallium cuprates have the highest superconducting transition temperature, Tc, recently enhanced up to 130 K for Tl 2 Ba 2 Cu 3 0 10 by Kaneko et al. [3]. The first member of the two Tl0-layers series Tl 2 Ba 2 Ca n-1 Cu n O 2n+4 with n= l, referred to here as "2201", is particularly interesting. First, the compound is reported to undergo a structural transformation from orthorhombic to tetragonal symmetry [START_REF] Greenbtatl | Studies of High Temperature Superconductors[END_REF][4][5][6][7][8] but the nature of the transition remains to be clarified. Relations between the transformation temperature and the oxygen and thallium content are unknown. Very recently Shimakawa et al. [9] suggested that the transition is composition dependent but quantitative effects have not been determined. Secondly, whereas the tetragonal form is known to become superconducting with Tc scaling up to 95 K [5], there are some doubts about the superconductivity of the orthorhombic form. In reality, the difficulty to obtain definitive results is obviously related to the difficulty to reproduce well defined Tl 2 Ba 2 CuO 6+x samples starting with the elemental compounds CuO or Cu 2 0, BaO, BaO 2 or BaCO 3 and Tl 2 0 3 . The difficulties not only concern the thallium and oxygen stoichiometries but also the formation, during the first stages of the sintering process, of impurity phases. BaCuO 2 especially cannot be completely eliminated, as observed in several studies [10,11], without a high temperature treatment. This in turn affects the thallium composition. In most cases, the amount of impurity phases is relatively low (1-3 vol.%) so that it cannot be detected by conventional X-ray diffraction techniques. The inconsistency between the usually claimed purity and the volume of superconducting phase is thus a puzzling problem.

In this paper we report a method to prepare "2201" at atmospheric pressure, which avoids the formation of BaCuO 2 and allows one to obtain reproducible thallium concentrations. The orthorhombic to tetragonal transformation is confirmed to be due to thallium departure. The low temperature AC susceptibility of normal and superconducting Tl 2 Ba 2 Cu0 6+x is used as a purity test.

Experimental

The "2201" samples used in this study have been prepared with Tl 2 Ba 2 0 5 ("2200") precursor. In our previous work on the TlO 1.5 -BaO system [12] we found that Tl 2 Ba 2 0 5 is a congruent melting compound at 1080°C with moderate Tl 2 O 3 vaporization before melting. Thus, the thallium stoichiometry of "2201" may be controlled during the sintering process. Powdered "2200" and a convenient amount of CuO (Rhone-Poulenc, 99.9%) have been mixed and fired at 850°C for 5 min and, after compaction sintered at 630°C for 70 h in flowing oxygen. The resulting weight losses, about 2% for 400-500 mg samples were reduced to 0.4% for samples of 4-5 g. The weight change and phase transition were determined by use of a Setaram TAG24 apparatus allowing one to perform simultaneous differential thermal analysis and thermo-gravimetry (DTA/TG) measurements in controlled atmospheres (He, Ar, 0 2 ). The orthorhombic to tetragonal phase transition was also investigated by high temperature X-ray difffactometry (XRD) in the range 20= 31-34 degrees including the orthorhombic doublet (200)-(020). The room temperature XRDs were recorded with a Siemens D520 powder diffractometer. The composition has been controlled either by grain analysis with an energy dispersive X-ray system attached to a scanning electron microscope (SEM/EDX) or by plasma emission spectroscopy. The thallium content was obtained from chemical analysis using a procedure described by Paranthanam et al. [ 13 ]. The low temperature behaviour of the samples was studied by AC susceptibility. The method is efficient in investigating magnetic properties and thus may be used to detect magnetic impurities at a low level. It is also a powerful tool to study in detail the bulk superconductivity [14,[START_REF] Couach | Magnetic Susceptibility of Superconductors and other spin Systems[END_REF].

Results and discussion

1. Phase formation

As already mentioned T l 2 Ba 2 0 5 is a congruent melting compound at 1080±20)°C. A monoclinic distortion of an orthorhombic type structure has been proposed for the compound by Zhou et al. [START_REF] Zhou | [END_REF] but all the diffraction lines observed in our "2200" master samples could be indexed assuming an orthorhombic symmetry with the lattice parameters a=6.226(4)Å, b=6.047(7)Å and c=17.179(5)Å. In an oxygen atmosphere of 1 bar, the decomposition of CuO occurs at 1120C [17]. The Tl 2 Ba 2 0 5 -CuO system is a quasi-binary system and except along an invariant line, no ternary equilibrium should be observed. The composition must be solely a combination of Tl 2 Ba 2 0 5 and CuO. Figure 1 shows the direct reaction of powdered "2200" and CuO in the DTA cell. The two endotherms at 880°C and 920°C are due to the eutectic plateau and the liquidus respectively, at a composition close to "2201". The formation reaction of the mixed oxide cannot be observed but from isothermal experiments we found that a flash heat treatment at 850°C improved the efficiency of "2201" formation. From the TG curve in fig. 1, it may be seen that the thallium content is not significantly changed before the melting of the sample, allowing one to obtain an accurate control of the composition and a drastic reduction of toxicity problems.

Formation from the liquid state appears in fig. 2 which is the quasi -binary section extracted from the study of the ternary system TlO 1.5 -BaO-CuO [START_REF] Jondo | XVllI6mes JEEP[END_REF]. Tl 2 Ba 2 CuO 6 is shown to be formed by the peritectic reaction: TI 2 Ba 2 O 5 + liquid → Tl 2 Ba 2 CuO z at 930 ± 5°C. DTA/TG of orthorhombic "2201" reveals that in our experimental conditions, in flowing oxygen, the thallium stoichiometry at the peritectic temperature is decreased to 1.9, assuming Tl 2 0 3 departure. At this level of thallium vacancy, no solid-state phase transition was observed, neither by DTA nor by XRD analysis of quenched specimens, indicating that the material remains orthorhombic. The results are consistent with the neutron scattering data of Hewat et al. [6] showing that the orthorhombic "2201" accepts up to 12.5% thallium deficiency. Moreover, they suggest that the transition from orthorhombic to tetragonal-type structure is not a simple temperature dependent transformation.

The tetragonal phase formation

In the literature there is a general agreement concerning the formation of a tetragonal Tl 2 Ba 2 CuO z which is obtained, as summarized in table 1, after various heat treatments of the orthorhombic form. However, the transition is not clearly described and has been considered with a microscopic approach. Bordet et al. [20], on the basis of electron diffraction experiments associated the tetragonal symmetry to shortrange ordering whereas Manthiram et al. [21] argued an ordered extrusion of "TlO" units for describing the orthorhombicity of the 2201 compound. In fact the formation of tetragonal 2201 also has to be considered from a thermodynamic viewpoint, on the basis of a phase diagram. We have already mentioned that in samples prepared using Tl 2 Ba 2 0 5 as precursor the thallium stoichiometry may be decreased to 1.9 without affecting the orthorhombicity. From accurate TG measurements we found that the formation of the tetragonal form implies thallium oxide departure which, following the recent work of Walhbeck et al. [22], is here assumed to be Tl 2 0 3 . All the Tl compositions after the various heat treatments hereafter reported have been calculated using this assumption and noting that, for a molecular weight of 843 g of Tl 2 Ba 2 CuO 6 , one TlO 1.5 unit corresponds to 27.1 weight%. The starting master samples with a typical weight of 400 mg were orthorhombic with the lattice parameters: a= 5.432(5) Å, b=5.493(5) Å, c=23.12(1) Å . From plasma emission analysis, the thallium content was 1.9, in agreement with weight loss measurements. In static or flowing oxygen atmospheres the tetragonal symmetry can be obtained by annealing an orthorhombic 2201 specimen above 800°C for times depending on sample size. Alternatively, a deficient Tl material may be directly prepared. Our study on the ternary TlO 1.5 -BaO-CuO system [START_REF] Jondo | XVllI6mes JEEP[END_REF] showed that the 2201 specimens were orthorhombic except for 3(c)). These results contradict the usually accepted [START_REF] Greenbtatl | Studies of High Temperature Superconductors[END_REF] reversibility of the orthorhombic-tetragonal transformation with the temperature. Actually inversibility may be ascertained by taking into account the change in composition. We have found that departure of 0.1 mole of TlO l.5 from the initial Tl 1.9 Ba 2 CuO z promoted tetragonal "2201" which should be described as Tl 1.8 Ba 2 CuO z . Heat treating such tetragonal compound in flowing oxygen did not change the structure up to 915°C, the melting temperature, slightly lower than for orthorhombic "2201" (fig. 4). From 850°C to 910°C the weight losses were reduced to 1.3% i.e. 0.05 TlO 1.5 , the sample remaining tetragonal, suggesting a small homogeneity range in the TlO 1.5 direction which extends to Tl 1.7 Ba 2 CuO z . From that composition, pure orthorhombic "2201" could be recovered solely by addition of Tl 2 0 3 followed by annealing at 650°C thus s h o w i n g t h e inversibility of the reaction. In an oxygen free atmosphere (10 -2 mbar, flowing argon or helium), thallium losses occur at temperatures as low as 500°C. Figure 5 represents the evolution of the XRD pattern in helium of orthorhombic "2201" as the temperature is increased. From room temperature to 500°C, the characteristic diffraction lines (020), (200) of the orthorhombic structure are separated by 2θ = 0.37(2)°. At 630°C, the (200) peak seems to merge into (020) but the (110) diffraction line of the tetragonal structure is apparent, suggesting the existence of a two-phase field between tetragonal and orthorhombic "2201". The reaction is complete at 670°C and simultaneously, BaCuO 2 appears as minor phase, increasing with time and reflecting compositional change. From the (200), ( 020) and (115) diffraction lines of orthorhombic "2201" and the ( 110) and ( 105) lines of the tetragonal form we deduced the thermal expansion of the lattice parameters. Figure 6 shows a linear temperature dependence of the orthorhombic parameters a o (T) and b o (T). The slight difference between the thermal expansion coefficients α a = 14.2 × 10 -6 K -1 and α b =13.5 × 10 -6 K -1 reflects the stability of the Tl-O (3) slab which produces the orthorhombicity, thus extending to high temperatures the results of Parise et al. [23] who studied the low temperature range 12 K< T< 293 K. Note that the thermal expansion does not differ too much from the values for YBa 2 Cu 3 0 7-x , as deduced from the study of Jorgensen et al. [24]. However, in the case of thallium based 2201, the orthorhombic a o and b o do not converge to a "'virtual" a tetra obtained from the real lattice a T by a tetra = € 2a T which seems to follow a similar temperature dependence. Owing to the stability of the two Tl0 layers, relative to thallium vacancies, there is no discontinuity in the c-direction between orthorhombic and tetragonal cells and thus, a linear orthorhombic and tetragonal cells and thus, a linear volumetemperature dependence is observed, with a volume thermal expansivity β= 39.4 × 10 -6 K -1 . We argue that orthorhombic and tetragonal TI 2 Ba 2 CuO 6 are two ordered phases which may be schematically represented in the enlarged phase diagram on fig. 7. In table 2 we report the sample analyses which supports our conclusions. The thallium contents were deduced by the determination of the weight loss and have been confirmed for samples (I) and ( I + II ) by wet chemical analysis and plasma emission spectroscopy which provided the compositions (TlO 1.5 ) 1.91 (BaO) 2 (CuO) 1.12 a n d (TlO 1.5 ) 1.74 (BaO) 2 (CuO) 1.11 respectively. Nevertheless, it should be interesting to complement this thermodynamic approach by careful investigations on the crystallographic structure of the tetragonal phase. We have to note that a study of our samples by high-resolution electron microscopy is in progress [25].

Superconductivity

The magnetic susceptibility of the samples has been measured in the temperature range 1.2-150 K. As a general trend, the absolute value of about 10 -4 SI is in agreement with previous results of Junod et al. [26]. Superconductivity was not observed in samples with the orthorhombic-type structure. However, the latter revealed an interesting low temperature behavior. The magnetic susceptibility of orthorhombic "2201" is reduced remarkably for T< 50 K (fig. 8) and remains unchanged when a static magnetic field (up to 1.5T) is superimposed on the AC field. This last experiment demonstrates that this behavior does not originate in superconductivity but reflects an intrinsic property such as magnetic ordering. In addition, the susceptibility measurement confirms the phase diagram expectation. BaCuO 2 cannot be produced in the quasi-binary Tl 2 Ba 2 O 5 -CuO system. The AC susceptibility of BaCuO 2 , displayed in fig. 9, shows a positive contribution, decreasing with a superimposed DC field. This does not appear, at ppm levels in orthorhombic "2201" samples. However, it has to be mentioned that a small deviation from ideal thallium stoichiometry might produce BaCuO 2 traces (see fig. 7) and thus modify the behavior. There is clear evidence of this during the formation of the tetragonal phase from orthorhombic samples by thallium losses, implying an inhomogeneous composition which may result in the presence of BaCuO 2 as a minor impurity (in the range of 10 -2 -10 -3 vol.%). In fact, for tetragonal "'2201" a positive contribution to the AC susceptibility preceding superconductivity is observed. This is shown in fig. 8 for a sample with a superconducting transition temperature of about 6 K and a very low level of superconductivity which was easily destroyed by an external field of 1T. As expected for the tetragonal symmetry [5], the superconducting transition temperature is strongly dependent on the heat treatment (fig. 10) which affects both the oxygen content and the thallium stoichiometry. The tetragonal sample used for the measurements reported in fig. l 0 was obtained after the heat treatments I + II (see table 2) and was initially not superconducting down to 4.2 K. Annealing in argon for 2 h at 500°C resulted in a weight loss of 0.1%. This is thought to be due to oxygen departure. Tc grew to 53 K (number 9 in fig. 10 Further annealing in vacuum (5 X 10 -2 atm) at 475°C decreased the Tl content to 1.77 without change of T c onset , but here the large transition reflects a composition gradient in the samples (numbers 12 and 17 in fig. 10). Subsequent treatment in an encapsulated silica tube under argon pressure allowed the equilibrium to be established for Tl l.77 Ba 2 CuO 5.603 with a critical temperature of Tc=22 K (number 24 in fig. 10). It is worth noting that despite the large screening level (χ= -1 SI), the grain contribution revealed by the effect of a superimposed DC field on a strong AC field ( 30 Oe ) is of the order of 10% volume ( fig. 11), largely lower than the percolation threshold. This apparent contradiction may result from the grain size of our samples, a few 1000 Å, less than the penetration depth. Metallographic observations support this assumption.

In conclusion we have established a working method to prepare orthorhombic Tl 2 Ba 2 CuO 6 , excluding the formation of BaCuO 2 . At normal pressure, the transition from orthorhombic to tetragonal "2201" implies thallium deficiency and was found to be inversible. Superconductivity could not be found on samples with the orthorhombic-type structure which, however, displayed a magnetic-like ordering at low temperature.

Fig. I .

 I Fig. I. Direct formation in the DTA/TG apparatus of Tl 2 Ba 2 CuO 6 showing the reduced weight losses ( 4% ) preceding the melting of the sample at 930°C.

Fig. 2

 2 Fig. 2 The quasi-binary section TI 2 Ba 2 O 5 -CuO, where (∆) is DTA, (O) single phase by XRD, and (•) two-phase.

t h o s e b e l o n g i n g t o t h e t e r n a r y Tl 2

 2 Ba 2 O 5 +BaCuO 2 +2201 phase field which presented the tetragonal symmetry.

  l.5 ) 0.53 (BaO) 0.43 (CuO) 0.04 in the three phase field Tl 6 Ba 4 O 13 + Tl 2 Ba 2 O 5 + orthorhombic Tl 2 Ba 2 CuO z ( f i g . 3 ( a ) ) , o f (TlO l.5 ) 0.26 (BaO) 0.30 (CuO) 0.44 in the field CuO+BaCuO 2 +orthorhombic Tl 2 Ba 2 CuO z (fig. 3 (b)) and finally (TlO l.5 ) 0.33 (BaO) 0.47 (CUO) 0.20 in the Tl 2 Ba 2 O 5 , BaCuO 2 and tetragonal "2201" field (fig.

Fig. 4 .

 4 Fig. 4. DTA of 2201 samples showing (---) the peritectic plateau at 930°C corresponding to the melting of ortho 2201 containing 1.9 Tl, deduced from the weight change (_._._.). The liquidus is found at 940°C. (__) is the melting of tetragonal 2201 containing, from the TG curve (--), 1.77 Tl at 915°C.

Fig. 5 .

 5 Fig. 5. High-temperature X-ray powder diffraction of ortho 2201 in helium: (a) T= 500°C: the (020) and (200) doublet of ortho 2201; (b) T=630°C: growth of tetragonal 2201. One finds evidence that the sample at this temperature is two-phase; (c) T=670°C: tetragonal (110) diffraction line. The formation of BaCuO 2 as impurity may be observed at 2θ= 33.85°.

Fig. 6 .

 6 Fig. 6. The linear temperature dependence of the cell parameters of the ortho and tetra 2201 deduced from hightemperature XRD in He. Note the coexistence of the two phases above 500°C. The low temperature value of a of the tetragonal cell suggests similar expansion as for the ortho parameters. The accuracy is affected by the temperature precision (± 50°C).

Fig. 7 .

 7 Fig. 7. Enlarged phase diagram at the vicinity. of the 2201 phase.

Fig. 8 .

 8 Fig. 8. AC susceptibility of 2201 samples. H AC =35 Oe (--) ortho 2201, The lowering of χ' is not destroyed by a static superimposed field up to 1.5T and thus does not correspond to superconductivity; ( …. ) tetra 2201. The large positive contribution preceding the superconducting transition at about 6 K is due to BaCuO 2 . Superconductivity is destroyed by a superimposed DC field of 1 T.

Fig. 9 .

 9 Fig. 9. AC susceptibility of BaCuO 2 prepared in oxygen flowing at 960°C. H AC = 35 Oe. The parameter is an external static field up to 1.8T.

Fig. 10 .

 10 Fig. 10. The superconducting transition temperature by AC susceptibility of tetragonal 2201 after different heat treatments. H AC =0.05 Oe, H DC =0 T. (9) As prepared followed by 2 hours in argon at 500°C. (12)+1 hour in vacuum (5 X l0 -2 atm) at 475°C. (17) Repeated treatment. (24) Subsequent anneal at 475°C in argon. Encapsulated quartz tubes.

  ) c o r r e s p o n d i n g t o t h e c o m p o u n d Tl 1.83 Ba 2 CuO 5.693 assuming O = 6 initially,.

Fig. 11 .

 11 Fig. 11. AC susceptibility of samples of fig. 10 but under a strong AC field (30 Oe). A superimposed DC field is supposed to reveal the grain contribution but here is interfered with the grain size of our samples, less than the penetration depth.


Christian Ducrot
email: ducrot@clermont.inra.fr

Carole Sala

Giuseppe Ru

Aline De Koeijer

Hazel Sheridan

Claude Saegerman

Thomas Selhorst

Mark Arnold

Miroslaw P Polak

Didier Calavas

M Arnold Vla

Sutton Bonington

Modelling BSE trend over time in Europe, a risk assessment perspective

Keywords: Age period cohort model, Basic reproduction number, Bovine spongiform encephalopathy, Epidemiology, European Union, Risk assessment, Prevention and control cohort model BSE Bovine spongiform encephalopathy EU

BSE is a zoonotic disease that caused the emergence of variant Creuzfeldt-Jakob disease in the mid 1990s. The trend of the BSE epidemic in seven European countries was assessed and compared, using Age-Period-Cohort and Reproduction Ratio modelling applied to surveillance data 2001-2007. A strong decline in BSE risk was observed for all countries that applied control measures during the 1990s, starting at different points in time in the different countries. Results were compared with the type and date of the BSE control measures implemented between 1990 and 2001 in each country. Results show that a ban on the feeding of meat and bone meal (MBM) to cattle alone was not sufficient to eliminate BSE. The fading out of the epidemic started shortly after the complementary measures targeted at controlling the risk in MBM. Given the long incubation period, it is still too early to estimate the additional effect of the ban on the feeding of animal protein to all farm animals that started in 2001. These results provide new insights in the risk assessment of BSE for cattle and Humans, which will especially be useful in the context of possible relaxing BSE surveillance and control measures.

Introduction

The first cases of Bovine spongiform encephalopathy (BSE) were observed in 1985 in the United Kingdom (UK), and evidence of the zoonotic impact of the disease was found in 1996 [START_REF] Will | A new variant of Creutzfeldt-Jakob disease in the UK[END_REF], as the source of variant Creuzfeldt-Jakob disease (vCJD); since then, 216 cases of vCJD were detected in Humans (http://www.cjd.ed.ac.uk/vcjdworld.htm; consulted June 29, 2008). Epidemiological studies on BSE indicated that meat and bone meal (MBM) played a major role in disease transmission [START_REF] Wilesmith | Bovine spongiform encephalopathy: epidemiological studies[END_REF][START_REF] Wilesmith | Bovine spongiform encephalopathy: case-control studies of calf feeding practices and meat and bonemeal inclusion in proprietary concentrates[END_REF]. This led to a ban on the feeding of MBM to ruminants, implemented in 1988 in the UK and subsequently throughout the European Union (EU). Although the ban significantly decreased exposure [START_REF] Heim | Surveillance of BSE[END_REF], by itself it proved insufficient to eliminate disease. Further measures were taken in the mid 1990s by some countries (controls on cross contamination, SRM removal) to eliminate the residual risk posed by MBM. In 2001 the European Union imposed a ban on the feeding of animal protein to all farmed animal species with limited exceptions (so called total feed ban) (http://ec.europa.eu/food/food/biosafety/ bse/chronological_list2008_en.pdf; consulted October 20, 2008). As the various control measures, prior to the total EU ban in 2001 were implemented at different times in different EU Member States, it is expected to see variations in the trend of the BSE epidemic by country connected with the date and type of control measures introduced in each of them.

As the incidence of disease declines, there is a huge pressure from different stakeholders to lift certain BSE control measures. In the TSE roadmap published in 2005 (http://ec.europa.eu/food/food/biosafety/bse/roadmap_en.pdf; consulted 20 October 2008), the European Commission planned a relaxation of aspects of the total feed ban provided certain conditions were met. In part these relaxations are based on risk assessments. For this reason, analyses of the trend of the epidemic and the effect of control measures is of major concern for risk assessments and risk management in the coming years.

A comprehensive surveillance system for BSE has been in place in the EU since 2001. The surveillance system is based on clinical surveillance as well as on systematic rapid testing of risk animals (emergency slaughter and animals that die on farm) over 24 months of age and healthy slaughtered cattle over 30 months of age. Data generated by this surveillance programme has been analyzed in the past and has provided insight into the trend of the epidemic in different countries (Netherlands [START_REF] De Koeijer | Quantifying BSE control by calculating the basic reproduction ratio R 0 for the infection among cattle[END_REF], UK [START_REF] Arnold | Estimation of the age-dependent risk of infection to BSE of dairy cattle in Great Britain[END_REF], Italy [START_REF] Ru | Epidemiological study of the decline of BSE in Italy[END_REF], France [START_REF] Morignat | Prevalence of BSE in cattle found dead euthanased or emergency slaughtered on farms in western France in 2000-2001 and 2002[END_REF][START_REF] Bonnardie | Estimating the trend of the French BSE epidemic over six birth cohorts through the analysis of the abattoir screening in 2001 and 2002[END_REF], Switzerland [START_REF] Schwermer | Cases of bovine spongiform encephalopathy born in Switzerland before and after the ban on the use of bovine specified risk material in feed[END_REF]. The length and variability of the incubation period for BSE [START_REF] Ferguson | The epidemiology of BSE in cattle herds in Great Britain. II. Model construction and analysis of transmission dynamics[END_REF] means that a large percentage of the infected animals are already dead or slaughtered before infection can be detected. This must be taken into account when analyzing the epidemic. Over the years different approaches have been used [START_REF] Ducrot | Review on the epidemiology and dynamics of BSE epidemics[END_REF] varying from relatively simple approaches based on the age of cases at detection [START_REF] Saegerman | Evolution de l'a ˆge moyen lors de la de ´tection des bovins atteints d'ence ´phalopathie spongiforme bovine (ESB): un indicateur utile du stade de la courbe e ´pide ´mique d'un pays[END_REF][START_REF] Saegerman | Trends in age at detection in cases of bovine spongiform encephalopathy in Belgium: an indicator of the epidemic curve[END_REF] to more complicated approaches such as back calculation modelling [START_REF] Arnold | Estimation of the age-dependent risk of infection to BSE of dairy cattle in Great Britain[END_REF][START_REF] Supervie | The unrecognised French BSE epidemic[END_REF] and birth cohort modelling with extrapolation based on the reproduction ratio (called R 0 in the text) [START_REF] De Koeijer | Quantifying BSE control by calculating the basic reproduction ratio R 0 for the infection among cattle[END_REF][START_REF] De Koeijer | Factors that influence the age distribution of BSE cases: potentials for age targetting in surveillance[END_REF][START_REF]Risk for Human and Animal Health related to the revision of the BSE Monitoring regime in some Member States, Scientific Opinion of the Panel on Biological Hazards (Question No EFSA-Q-2008-007)[END_REF]. Regression integrating birth cohort and year of test [START_REF] Morignat | Prevalence of BSE in cattle found dead euthanased or emergency slaughtered on farms in western France in 2000-2001 and 2002[END_REF], and Age-Period-Cohort (APC) [START_REF] Cohen-Sabas | Age-periodcohort analysis of the Bovine spongiform encephalopathy (BSE) epidemic in Switzerland[END_REF] models have also been used to model disease trends.

The goal of this study was to compare the trend of the BSE epidemic in several European countries using two different standardized methods and the surveillance data collected between 2001 and 2007. In addition the study examined whether the overall trend was towards eradication and if so at what point in time did this decline start. The results were discussed in comparison with the date and type of control measures introduced. This up to date analysis provides precise risk estimates for birth cohorts born in the second half of the 1990s; it gives new insight into the effect of measures introduced before the total feed ban in 2001, and provides key aspects in a risk assessment perspective for Human Health in the context of relaxing BSE surveillance and control measures.

Materials and methods

Data

Seven EU Member States (Table 1) were involved in the study. BSE cases identified between 2001 and 2007 in all three BSE surveillance streams (clinical surveillance, risk animals and healthy slaughter animals) were used. All positives were confirmed at the Member States' National Reference Laboratories using validated tests (OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals Chapter 2.4.6) [START_REF] Gavier-Wide ´n | Diagnosis of transmissible spongiform encephalopathies in animals: a review[END_REF][START_REF] Grassi | Progress and limits of TSE diagnostic tools[END_REF]. Data from the three surveillance streams were merged to facilitate comparison between countries based on the assumption that the surveillance streams varied little among countries (with the exception of the UK with mainly clinical surveillance) and that rapid tests for BSE are detecting infected animals close to onset of clinical disease.

As animals below 2 years of age are not required to be tested under European legislation, these animals were excluded from the study (http://ec.europa.eu/food/food/ biosafety/bse/chronological_list2008_en.pdf, consulted 20 October 2008). Moreover, imported cases were excluded when such information was available. Atypical cases were included although they have different epidemiological characteristics as not all countries are capable of identifying them and they represented only a small proportion of all BSE cases [START_REF] Biacabe | Atypical bovine spongiform encephalopathies, France, 2001-2007[END_REF]. From each participating country, raw surveillance data has been obtained: in particular data on tested animals, broken down by year of birth (or age in years), year of diagnosis and testing result (negative animals vs positive confirmed cases) were available for statistical analysis and allowed computing stratum (i.e., age, birth cohort and period) specific prevalence rates.

For the calculation of the R 0 an estimation of the adult cattle population of each country was used as the background population. The APC model used the number of animals tested in all three surveillance streams. BSE cases and the tested population were classified by age and by birth cohort. The age was calculated in complete years by subtracting the date of birth from the date of diagnosis. The birth cohort was defined as the calendar year of birth. In the R 0 method, the year of detection was the calendar year of detection while in the APC method the year of detection, called the period, was calculated from the age and birth cohort.

The country specific type and year of implementation of the main BSE control measures prior to 2001 for each country included in the study are detailed in Fig. 1 and summarized in Fig. 2. Initially, the primary BSE control measure was a ban on the feeding of MBM to cattle (or ruminants in some countries). This was followed by complementary measures involving the removal of specified risk material (SRM), controls on cross contamination and the sterilization of MBM at 133°under 3 bar pressure and for 20 min (with size particles less than 5 cm). In January 2001, a total ban on the feeding of animal protein to all farmed animal species with certain limited exceptions has been enforced. Finally in May 2001, control measures were harmonized throughout the EU with the introduction of a comprehensive TSE Regulation (Regulation (EC) 999/ 2001).

Methods

Two complementary methods were used to analyze the data.

Age period cohort analysis (APC)

A logistic regression model was used to model the respective effects, expressed as odds ratios, of the age at diagnosis (age), birth cohort (cohort) and period of detection (period) on BSE prevalence, expressed as cases per Animals born before 1990 were not considered f Animals 13 years old and over (whose three cases) were removed from the analysis because only pooling data for these animals was available (class: ''?12 years'') g One case excluded because secondary and two more cases excluded because the number of tested animals for the same age and period was not estimable h Estimate i Animals 9 years old and over were removed from the analysis because only pooling data for these animals was available (class: ''?9 years'') j Nine cases for which the date of birth was unknown were removed from the analysis k Animals for which the date of birth was unknown and/or under 2 years old were excluded from the analysis 0,00 0,01 0,10 10,000 cows tested. For the cohort, the reference group chosen in each country was the birth cohort in which the highest crude prevalence was observed.

For each country, before fitting the full age-period-cohort (APC) model, we used a stepwise procedure recommended by Clayton and Schiffler [START_REF] Clayton | Models for temporal variation in cancer rates. I: Age-period and age-cohort models[END_REF], and run successively four sub models, including, respectively age, age and drift, age and period, age and cohort. The drift is the global trend, due to the undistinguished linear effects of the period and cohort, that we tested (model 2) before adding the non-linear components of the period (model 3) or cohort (model 4) effects. The drift cannot be attributed specifically to cohort or period, because of the inter-relation between age, period and cohort. Identifying the complete APC model required a minimal additional constraint; this was done by equalizing the effect of two successive periods as we expected little, if any, period effect due to the stability in the surveillance systems during the studied period of time [START_REF] Mason | Some methodological issues in cohort analysis of archival data[END_REF][START_REF] Barrett | The redundant factor method and bladder cancer mortality[END_REF]. The significance of each variable was assessed by a log-likelihood ratio test. Goodness of fit was evaluated by the examination of the residual deviance of the models, which approximates a Chi-square distribution [START_REF] Mccullagh | Generalized linear models[END_REF] (Table 2).

Calculation of the reproduction ratio R 0 per cohort

The reproduction ratio (R 0 ) [START_REF] De Koeijer | Quantifying BSE control by calculating the basic reproduction ratio R 0 for the infection among cattle[END_REF] provides an estimate of the expected number of newly infected animals resulting from an initial infection. When R 0 is above 1, more than one new infection is expected from each infectious animal, so the epidemic increases; similarly, a R 0 smaller than 1 is linked to a decreasing epidemic. A cohort model with yearly increments was applied on the BSE case data that were categorized by age (2-12 years) and year of birth (1991)(1992)(1993)(1994)(1995)(1996)(1997)(1998)(1999)(2000)(2001)(2002). The method is described in [START_REF]Risk for Human and Animal Health related to the revision of the BSE Monitoring regime in some Member States, Scientific Opinion of the Panel on Biological Hazards (Question No EFSA-Q-2008-007)[END_REF][START_REF] De Koeijer | Analyzing BSE transmission to quantify regional risk[END_REF]. We determined the test window of age for each birth cohort which had been tested between January 2001 and December 2007. Next we calculated the expected number of BSE cases in the full cohort using a distribution for the age at onset, derived from the EU15 data [START_REF]Risk for Human and Animal Health related to the revision of the BSE Monitoring regime in some Member States, Scientific Opinion of the Panel on Biological Hazards (Question No EFSA-Q-2008-007)[END_REF]; it was applied to all the evaluated countries without further adjustment to the national age distribution of The reproduction ratio was calculated as the ratio of the expected number of cases estimated to appear in a birth cohort and the expected number of cases in that test year. For estimates before 1997, this required an estimate of the number of cases found in cohorts born before 1991, which was not available in the data. We assumed that no BSE case was found in animals born before 1991; this led to an increasing overestimation of the R 0 , the further we go back from 1997. Confidence intervals (95%) for the reproduction ratio were computed using the method described in [START_REF] De Koeijer | Analyzing BSE transmission to quantify regional risk[END_REF]. The model was based on three major assumptions. Firstly, all infections occurred at a young age (first year of life); secondly, all imports of infectious animals or material are ignored; and thirdly the age at clinical onset distribution was assumed to be the same for all countries, without further adjustment to the country cattle age distribution or the different exposure pattern because of the difficulty in obtaining country specific information.

Results

The results of APC models and R 0 estimates are presented in Table 2 and Fig. 1. The cohort effect from the APC model was expressed as an Odds Ratio (OR) and plotted on a log scale. As BSE is a disease with a low prevalence the OR can be assumed to be equivalent to the Relative Risk (RR); it provides the risk of an animal of a specific cohort to be detected as a BSE case compared to the risk for an animal of the reference cohort at the same age and observation period. The results of the APC model showed that in the UK, the BSE risk decreased significantly from the 1988 birth cohort onwards. In the other countries a significant decrease started later; from the 1995 cohort onwards in France, from the 1996 cohort onwards in Ireland, from the 1997 cohort onwards in Italy and Germany and the from the 1998 cohort onwards in the Netherlands. Birth cohort was not a significant variable in the Polish model pointing to the lack of a specific temporal trend in BSE risk in Poland in the late nineties. A small increase in risk was observed in Germany, Italy and Ireland in 1999 and in the Netherlands in 2000.

Estimates of R 0 and the upper confidence limit showed that the epidemic was consistently fading out (i.e., estimated upper confidence limit of R 0 below 1) in all countries except Poland. This trend could be observed from the 1996 cohort onwards in the UK, from the 1997 cohort onwards for France and Ireland, from the 1998 cohort onwards for Italy, from the 1999 cohort onwards for the Netherlands and from the 2000 cohort onwards for Germany. In the case of Poland although R 0 was consistently very close to 1, it did not show a clear tendency to decrease below that level. A decline in the Polish BSE epidemic would need to be confirmed using later cohort data that is not yet available.

Figure 2 summarizes conjointly the results of the models and the date of the implementation of control measures per country.

Discussion

Temporal trend of BSE An important result of the study is the clearly observable decline of the BSE epidemic in all studied countries except Poland. This represents a true decline as the study focused on the period 2001-2007, a period during which intensive and exhaustive surveillance with little bias or uncertainty was carried out for BSE in the EU. In addition both methods (APC and R 0 ) produced similar results and indicated similar trends. The decrease of the number of cases detected between 2001 and 2007 already gave the idea of a decline of the epidemic. However, our models accounted for the main factors influencing the prevalence, in particular the length and variability of the incubation time, the age at which the animals are tested, and they provided an adjusted estimate of the year when the decrease started and the slope of it.

The study focused on birth cohorts as previous analyses of data from the UK BSE epidemic have shown that the age at infection is mostly likely to be early in life [START_REF] Arnold | Estimation of the age-dependent risk of infection to BSE of dairy cattle in Great Britain[END_REF], with more than 90% of case animals infected before 12 months of age. Both methods were used in a complementary way. APC modelling was based on an analysis of tested animals and provides an exact calculation of the evolution of BSE risk over successive birth cohorts. R0 modelling provides an estimate of the prevalence per birth cohort, similar to the evolution of risk derived from the APC model, but in addition can provide a measure for the transmission of infection. An assessment of the efficacy of the various control measures can be derived from the results of both models. Unlike the APC model, which looks for statistical correlations in the data, the R 0 model is mechanistic. It assumes that there is a cohort effect because cattle become infected at a young age. It also assumes that there is no period effect, which is likely because there was no change to surveillance requirements during the period of interest. Both of these assumptions are supported by the results of the APC modelling.

The difference in time (a few years) between the first cohort to exhibit decreased risk and the year where R 0 first declined below 1 can be explained by the wave effect [START_REF]Risk for Human and Animal Health related to the revision of the BSE Monitoring regime in some Member States, Scientific Opinion of the Panel on Biological Hazards (Question No EFSA-Q-2008-007)[END_REF]. The first cohort with decreased risk results from a combination of exposure (i.e., infection pressure in the past) and transmission level (i.e., R 0 ). If there was a steeply increasing epidemic in the past, then even if R 0 remains above 1, a temporary decrease in risk for the cohort may be observed. If there are no further changes in the control measures, this would then be followed by an increase in successive birth cohorts, because the exposure from the past is still increasing although the transmission level remains the same. This pattern is typically observed in epidemics when the transmission level (R 0 ) declines more slowly than the exposure level increases. The theoretical background for this is discussed in [START_REF] De Koeijer | Factors that influence the age distribution of BSE cases: potentials for age targetting in surveillance[END_REF], although the development of second waves is not specifically addressed there.

The results produced by the APC model and the R 0 model are in agreement with the evolution of the average age of the cases, as suggested previously by Saegerman et al. [START_REF] Saegerman | Evolution de l'a ˆge moyen lors de la de ´tection des bovins atteints d'ence ´phalopathie spongiforme bovine (ESB): un indicateur utile du stade de la courbe e ´pide ´mique d'un pays[END_REF][START_REF] Saegerman | Trends in age at detection in cases of bovine spongiform encephalopathy in Belgium: an indicator of the epidemic curve[END_REF]. Although not reported in this paper an increase in the mean age of the cases was detected for the period 2001-2007 coinciding with a strong decline in cohort risk and R 0 [START_REF] Ducrot | Review on the epidemiology and dynamics of BSE epidemics[END_REF][START_REF]Risk for Human and Animal Health related to the revision of the BSE Monitoring regime in some Member States, Scientific Opinion of the Panel on Biological Hazards (Question No EFSA-Q-2008-007)[END_REF].

Finally, it can be observed from results of the APC model (Fig. 1) that the shape of the decline in risk between cohorts differed between countries varying from sharp for Ireland through moderate for Germany. This shape results from the combined effect of the force of the epidemic in the years preceding the decline and the effectiveness of control measures introduced. Furthermore, this decline started at different times in different countries; R 0 was already below 1 in the UK in 1996 though it only declined below 1 in the year 2000 in Germany. It is interesting to note the small increase in risk for animals born in 1999 and 2000 in the case of Germany, Ireland, Italy and the Netherlands. This finding has also been observed in other countries such as Japan, Canada and countries that joined EU recently (Poland, Czech Republic, Slovak Republic, Republic of Slovenia) (Ru, personal communication). It would be worth investigating this further to determine whether there is an identifiable source of these miniature increases in prevalence or whether they are just statistical fluctuations caused by low prevalence.

Control measures

The risk of infecting cattle with the BSE agent is for a given birth cohort influenced by three main parameters. The first one is the level of infectivity in cattle in fallen stock and abattoir in that period; it is the direct consequence of the infection rate about 5-7 years earlier due to incubation period and can be viewed as a wave effect [START_REF]Risk for Human and Animal Health related to the revision of the BSE Monitoring regime in some Member States, Scientific Opinion of the Panel on Biological Hazards (Question No EFSA-Q-2008-007)[END_REF]. The second parameter is of course the existence and quality of control measures, which restricts recycling of the BSE agent. The last one is the risk of BSE in other countries in case of imports of feed or living animals. In our study we were interested in analyzing the pattern of the BSE trend in connection with the control measures. This attempt at interpreting the evolution of the BSE trend in terms of efficiency of control measures is possible if we make the assumption that the two other aspects did not have a great impact on the time differences in BSE trend between countries. In fact, the wave effect was almost synchronized between countries, due to the major impact of the UK BSE epidemic on all European countries, with the main peak in 1988 [START_REF] Arnold | Estimation of the age-dependent risk of infection to BSE of dairy cattle in Great Britain[END_REF] and the closest peak wave on birth cohort 1995 for France [START_REF] Supervie | The unrecognised French BSE epidemic[END_REF]. The analysis by reproduction ratio can distinguish this effect from the control measures although this ignores imports of live animals and feed. The third aspect, infections from imported feed might only attenuate the differences in the observed trend of BSE between countries, and might be marginal in most countries with an endemic BSE. For these reasons, we were confident that we can interpret the trend of BSE in relationship with control measures.

Some points arose from analysing the trend of BSE in comparison with the type and date of the introduction of control measures. The first point is that the ban of MBM for cattle alone was not sufficient to control the risk, at least in the Netherlands, France, Italy and Ireland, since it was not followed by a strong decrease of the epidemic and the R 0 remained above 1. This does not mean that in theory the feed ban is not sufficient to control the risk of BSE; it only means that the way it was implemented in various countries did not fully or immediately stop the transmission process. This resulted from the effect of cross-contaminations between feed for monogastrics and feed for cattle, which were observed in different countries and analyzed in detail in different epidemiological studies [START_REF] Ducrot | Review on the epidemiology and dynamics of BSE epidemics[END_REF]. The second point of interest is that in almost all the countries studied, such as Ireland, Italy, France and the Netherlands, we observed that the risk decreased significantly and the R0 was below 1 shortly after removal of SRM from MBM and treatment of MBM at 133°/3bars/20'/particles \ 5 cm were applied. These two measures were often introduced within a short period of time and it is therefore difficult to separate their respective effects. Also, in some countries such as France [START_REF] Supervie | The unrecognised French BSE epidemic[END_REF], the wave effect could have coincided with the effect of a given control measure such as SRM removal, these two effects being difficult to disentangle. It could be argued that since these complementary measures were implemented shortly after the announcement on March 1996 of the zoonotic consequences of BSE, this announcement might have prompted countries and feed industries to step up compliance with previous regulation measures, such as the ban of MBM for cattle. This could have resulted in an overlap between implementation of complementary actions and increased compliance with previous measures. We believe strongly that the announcement of the zoonotic feature of BSE cannot explain alone the decrease trend observed because the starting date of the decrease varied largely between countries, from birth cohort 1995 to birth cohort 1998. We definitely believe that the complementary measures had a significant impact on the observed decrease, specifically because we observed similar timing between the time of implementation of the complementary measures and the time of decrease in the countries studied (Fig. 2).

The third point of interest is that in all studied countries apart from Germany and Poland, the R 0 was already below 1 (progressive fading out of the epidemic) before the implementation of the ban on the feeding of animal protein to all farm animals in 2001. This does not mean that this total feed ban did not have any effect. In the case of a disease with long incubation time, the fading out of the epidemic can be very slow even with the R 0 below 1. However, it is still a bit too early to quantify the precise effect of this total feed ban in these countries.

In this study, we analyzed the data separately for each country. However, the situation of BSE in a given country is linked to other countries because of the high level of trade; without trade, most of these countries would not have had a BSE epidemic in the first place. Therefore, the control measures decided at the EU level (http://ec.europa. eu/food/food/biosafety/bse/chronological_list2008_en.pdf; consulted October 20, 2008) such as ban on the use of proteins derived from mammalian tissues for feeding ruminants in 1994, pressure cooking system for processing mammalian waste into MBM in 1996, prohibition of the use of SRM in 2000 (application of decision 96/534 was postponed until October 2000 with Decision 2000/412), total ban on the feeding of MBM to all farm animals in 2001, might have had a stronger impact on the international BSE situation than those taken at the country level. However, implementation of these measures was not always at the same time so we specifically looked at the real date of implementation of the measures in our analysis (Figs. 1,2).

Conclusion

The modelling of the BSE epidemic in seven EU countries using two different methods has shown very coherent results concerning the effect of various types of control measures as they were applied in these countries. We observed a strong decline of the risk of BSE for countries that applied control measures in the nineties. The results show that the implementation of the ban on feeding MBM to cattle alone was not followed by a decrease of the epidemic, which can be linked with the quality of implementation of this measure in the field. We did observe fading out of the epidemic following the introduction of complementary measures which were targeted at controlling the risk in MBM (SRM removal and MBM treatment). It is still too early to quantify the additional effect of the total feed ban of MBM for farm animals implemented in 2001. These comparative results add a new insight in the risk assessment of BSE for cattle and Humans, which will especially be useful in the context of the feasibility to possible lifting control measures in the future.

Fig. 1

 1 Fig.1Plot (log scale) of the reproduction ratio (R 0 ) and Odds ratio (compared to a reference cohort defined per country (OR = 1)), per birth cohort per country; type and year of control measures are

Fig. 2

 2 Fig. 2 Schematic time schedule of the main control measures and trend of the BSE epidemic per country

Table 1

 1 Data per country included for APC analysis and R 0 estimation Only indigenous animals over 30 months and tested since July 1st, 2001 were included b About 89 cases detected before 2001 and four BSE cases with incomplete or incorrect data were removed from the analysis c 3282 animals tested before 2001 were excluded d About 17 cases born before 1990 and 67 cases without date of birth were excluded

		APC modelling		R 0 estimation		Period of interest
		Nb BSE cases	Nb tested animals	Nb BSE cases	Adult cattle population
					(million heads 2005)
	France	647	17,248,284 a	716	10.4	2001-2007
	Germany	317 b	13,879,451 c	400	5.9	2001-2007
	Ireland	657 d	3,594,378 e	972	3.1	2001-2007
	Italy	135 e	4,506,951 e	137	2.9	2001-2007
	Poland	56 g	2,968,541 h	57	3.0	2002-2007
	The Netherlands	70 i	2,815,671 i	74	1.7	2001-2007
	United Kingdom	1795 j	2,964,963 k	3326	4.9	2001-2007

a e Number of tested animals derived from the Department of Agriculture, Fisheries and Food Computerised Monitoring and Movement system.

Birth cohort Odds ratio & R0 (log scale) France

	Odds ratio & R0 (log scale)	1,00 10,00	1986 United Kingdom 1988 1990 1992 MBM ban cattle SRM removal	1994	1996 total ban MBM	1998	2000	2002	Odds ratio & R0 (log scale)	0,01 0,10 1,00 10,00	1986 Italy 1988	1990	1992 MBM ban 1994 cattle	1996 partial SRM removal	1998 SRM treatment	2000 SRM removal total ban 2002 MBM
														0,00
						Birth cohort									Birth cohort
	Odds ratio & R0 (log scale)	0,01 0,10 1,00 10,00	1986 Ireland 1988	1990 MBM ban cattle	1992	1994		1996 SRM removal SRM 1998 treatment	2000 total ban MBM	2002	Odds ratio & R0 (log scale)	0,01 0,10 1,00 10,00	1986 The Netherlands 1988 1990 1992 MBM ban cattle	1994 MBM ban ruminants	1996 removal 1998 treatment SRM SRM	2000 total ban MBM	2002
		0,00				Birth cohort					0,00				Birth cohort
		10,00			cattle MBM ban		MBM ban ruminants	removal SRM SRM	MBM total ban			10,00
									treatment
		1,00												1,00
			1986	1988	1990	1992	1994		1996	1998	2000	2002			1986	1988	1990	1992	1994	1996	1998	2000	2002
		0,10												0,10
		0,01												0,01
		0,00												0,00

Birth cohort Odds ratio & R0 (Log scale) Germany

	MBM ban		total ban
	cattle	SRM treatment	SRM removal	MBM

Table 2

 2 Best APC model per country, effect of adding each variable to the model and goodness of fit From these initial steps, we obtained the estimated number of BSE cases in all age groups and for every cohort from 1991 to 2003, including the age groups that were culled before 2001 and the ones still alive in 2008. From these estimates we calculated the expected number of cases in each age group by test year.

		Effect of the variables (P-value*)	Best model	Goodness of fit of the best model
		Age	Cohort	Period		Residual deviance	Df b	P-value**
	France	0.00	0.00	0.06	Age-cohort	77.57	167	1.00
	Germany	0.00	0.00	0.19	Age-cohort	45.81	70	0.99
	Ireland a	0.00	0.00	0.88	Age-cohort	49.75	46	0.33
	Italy	0.00	0.00	0.02	Age-period-cohort	48.61	64	0.98
	Poland a	0.00	0.45	0.03	Age-period	52.41	50	0.38
	The Netherlands	0.00	0.00	0.12	Age-cohort	29.95	30	0.47
	UK	0.00	0.00	0.13	Age-cohort	113.39	152	1.00

a For Poland and Ireland, because the structure of the tested population by age and cohort was not available, the period was defined as the calendar year of detection and the birth cohort was calculated by subtracting the age from the year of detection b Df degree of freedom the standing cattle population.

Acknowledgments This work was based on data collected through active surveillance programmes on BSE organized at the EU level. The study was conducted and funded within the framework of Neuroprion NoE (FOOD-CT-2004-506578), risk and control group, coordinated by Thomas Hagenaars.


Kaspar E Vogt

Stephan Gerharz

Jeremy Graham

Marco Canepari
email: marco.canepari@ujf-grenoble.fr

High-resolution simultaneous voltage and Ca 2+ imaging

Keywords: Voltage imaging, Calcium imaging, neurons, cerebellum, hippocampus Simultaneous voltage and Ca 2+ imaging CF, climbing fibre, EPSP, excitatory synaptic potential, PF, parallel fibre, PN, cerebellar Purkinje neuron, S/N, signal-to-noise ratio, ∆V m , membrane potential changes

Combining voltage and Ca 2+ imaging allows the correlation of electrical and chemical activity at subcellular level. Here we describe a novel apparatus designed to obtain simultaneous voltage and Ca 2+ measurements with single-trial resolution from sites as small as a few microns. These measurements can be obtained with negligible optical cross-talk between the two signals and negligible photo-damage of the preparation. The capability of the technique was assessed recording either from individual neurons in brain slices or from networks of cultured neurons. The present achievements open the gate to many novel physiological investigations requiring simultaneous measurement of voltage and Ca 2+ signals.

Introduction

The spatial and temporal correlation of membrane potential changes (∆V m ) and Ca 2+ signals is critical to understanding many physiological processes. Simultaneous ∆V m and Ca 2+ optical measurements were initially achieved using a combination of an absorbance voltage sensitive dye and of a fluorescent Ca 2+ indicator [START_REF] Sabatini | Timing of neurotransmission at fast synapses in the mammalian brain[END_REF], or of fluorescent voltage and Ca 2+ sensitive dyes excited by the same wavelength [START_REF] Sinha | Simultaneous optical recording of evoked and spontaneous transients of membrane potential and intracellular calcium concentration with high spatio-temporal resolution[END_REF][START_REF] Bullen | Indicators and optical configuration for simultaneous high-resolution recording of membrane potential and intracellular calcium using laser scanning microscopy[END_REF]. Whereas absorbance measurements lack singlecell resolution, several limitations affect fluorescence measurements of ∆V m and Ca 2+ signals excited at the same wavelength. In particular, the optical cross-talk of the two dyes contaminates both signals while using the same excitation intensity and exposure time is not ideal for both dyes. These limitations can be minimised by combining a styryl voltage sensitive dye such as di-2-ANEPEQ (JPW1114) with Fura Ca 2+ indicators, separating both excitation and emission (Canepari et al. 2008). Such a combination was used to investigate ∆V m and Ca 2+ signals in different preparations [START_REF] Canepari | Dendritic signals from rat hippocampal CA1 pyramidal neurons during coincident pre-and post-synaptic activity: a combined voltage-and calcium imaging study[END_REF]Canepari & Vogt, 2008;Milojkovic et al. 2008). This approach, however, utilised sequential repetitive recordings of ∆V m and Ca 2+ signals. Sequential measurements (see also [START_REF] Berger | Combined voltage and calcium epifluorescence imaging in vitro and in vivo reveals subthreshold and suprathreshold dynamics of mouse barrel cortex[END_REF] give valid results if identical responses are evoked in repetitive measurements. Many physiological processes, however, are stochastic, showing large trial-to-trial variability. In these cases, the study of the time variability contains important biological information and simultaneous measurement of ∆V m and Ca 2+ signals with single-trial resolution is required.

For simultaneous voltage and Ca 2+ imaging using commercially available styryl voltage sensitive dyes and Fura Ca 2+ indicators, a system must be designed to illuminate the preparation at two wavelengths and record ∆V m and Ca 2+ signals separately. Here we describe a system developed for this application. We used different protocols to characterise the signal-to-noise ratio (S/N) and the spatial and temporal resolution.

We demonstrate that ∆V m and Ca 2+ signals associated with excitation can be resolved in a single trial from sites of a few microns.

Methods

Preparations, solutions, electrophysiology and analysis

Experiments, approved by Basel cantonal authorities, were performed in 250µm thick cerebellar sagittal slices or hippocampal transversal slices from 27-35 days old mice (C57BL/6) prepared as previously described (Canepari & Vogt, 2008;[START_REF] Canepari | Imaging inhibitory synaptic potentials using voltage sensitive dyes[END_REF]. Embryonic (E18) hippocampal cultures were prepared by Dr. Alexander Kriz as described in [START_REF] Reiterer | Sec24and ARFGAP1-dependent trafficking of GABA transporter-1 is a prerequisite for correct axonal targeting[END_REF] (Molecular Devices, Sunnyvale, CA) and stimulation was carried out with pipettes filled with extracellular solution. Individual neurons in slices were loaded with JPW1114 (0.2-0.5mg/mL) and with either 1 mM Fura-FF (Purkinje neurons) or with 0.5mM BisFura-2 (pyramidal neurons). Intracellular staining was accomplished as previously described (Canepari et al. 2008). Cultures were stained with di-8-ANEPPS (0.01 mg/mL) and Fura2-AM (5 µM) for 30 minutes and recordings were done after wash. Temperature was 32º-34º in slice experiments and 22º-24º in culture experiments. Recordings were analysed using Matlab (The MathWorks Inc., Natick, MA). Non-calibrated optical signals were reported as fractional changes in fluorescence (∆F/F). Anatomical reconstruction of neurons was made from two-photon images as described in [START_REF] Canepari | Imaging inhibitory synaptic potentials using voltage sensitive dyes[END_REF].

Apparatus design and data collection

The system in Fig. 1A, used and described in Canepari et al. (2008), was based on a single light source (Xenon Lamp) and a single camera (NeuroCCD-SM, RedShirtImaging LLC, Decatur, GA). Voltage and Ca 2+ imaging was carried out by manually switching between two filter cubes in the microscope. To achieve simultaneous voltage and Ca 2+ imaging we modified the system as shown in Fig. 1B. The epifluorescence port of an Olympus BX51 microscope was used for Ca 2+ fluorescence excitation with a 385 nm OptoLED (CAIRN Research Ltd., Faversham, UK), filtered at 387±6 nm and directed to a Nikon 60X/1.0 NA objective using a dichroic mirror FF506 (Semrock, Rochester, NY; spectrum shown in Fig. 1B). To excite voltage fluorescence, a custom-made unit was designed permitting whole-field illumination of the preparation from above the objective using a 100 mW 543 nm solid state laser (model MLL543; CNI, Changchun, China). To reflect 543 nm and transmit voltage and Ca 2+ fluorescence we used a dualband dichroic mirror FF493_574 (Semrock; spectrum shown in Fig. 1B). The image of the preparation was demagnified by a 0.5X projection lense in both voltage and Ca 2+ channels and separated using another custom-made unit and a 565 nm long-pass dichroic mirror (565DCXR, Chroma, Bellows Falls, VT).

Voltage fluorescence was long-pass filtered at 610 nm (em1 in Fig. 1B), the image was demagnified by 0.38X and acquired by a NeuroCCD-SM camera. Ca 2+ fluorescence was filtered at 510±42 nm (em2 in Fig. 1B), the image was demagnified by 0.38X and acquired by a back-illuminated Ixon+ model 860 EMCCD camera (Andor, Belfast, UK), controlled by the Redshirt system A/D board. The image on the 80X80 pixel chip of the NeuroCCD camera could be matched and aligned with the image projected to a sub-region of 78X78 pixels of the Ixon+ camera, corresponding to ~160X160 µm area in the object plane as shown in

S/N and optical cross-talk

Fig. 1C shows single-trial sequential recordings from a region of ~12.5X12.5 µm obtained with the system in Fig. 1A. The signals are associated with a climbing fibre (CF) excitatory postsynaptic potential (EPSP) from a cerebellar Purkinje neuron (PN). In Canepari & Vogt (2008) CF-EPSP signals could be resolved from regions of that size by averaging 4 trials (bottom traces in Fig. 1C). Fig. 1D shows singletrial simultaneous recordings from a region of ~12X12 µm obtained with the system in Fig. 1B. The improvement in voltage sensitivity using a laser at the edge of the absorption spectrum (see also [START_REF] Holthoff | Rapid time-course of action potentials in spines and remote dendrites of mouse visual cortex neurons[END_REF][START_REF] Canepari | Imaging inhibitory synaptic potentials using voltage sensitive dyes[END_REF]) and the higher UV intensity of the OptoLED resulted in the S/N increase necessary for single-trial resolution. Whereas negligible cross-talk is observed by the Ixon+ camera, the simultaneous UV illumination generated an increase in the recorded light by the NeuroCCD camera. This spurious fluorescence, originating both from the tail UV excitation of the voltage sensitive dye and from the tail red emission of the Fura indicator, could vary between 3% and 10% at different sites of the preparation (Fig. 1E, left traces) using the optimal intensities of UV and green excitation. Nevertheless, the ∆V m signal was the same whether or not measured with concomitant UV illumination (Fig. 1E, right traces), indicating that optical cross-talk was negligible in these measurements.

Results

We assessed the spatial resolution of ∆V m and Ca 2+ signals in individual PNs (Canepari & Vogt, 2008).

In this preparation, ∆V m signals can be calibrated and, using 1 mM of the low-affinity indicator Fura-FF, Ca 2+ signals can be related to intracellular free Ca 2+ concentration changes (∆[Ca 2+ ] i ) (Canepari et al. 2008).

Thus, a CF-EPSP producing a depolarization of similar size over the whole dendrite was used to assess and compare the sensitivity from different sites. In contrast, a parallel fibre (PF) EPSPs train, exciting only a small area, was used to assess to what extent correlated and localized ∆V m and Ca 2+ signals could be resolved. Fig. 2A-C shows an example of CF-EPSP single-trial recording. The S/N of both signals was variable. Fig. 2B shows the ∆V m and ∆[Ca 2+ ] i signals from three regions of 16X16 pixels (~32X32 µm) in different parts of the dendrite. Signals could be resolved with excellent S/N from all the three regions and from regions of the same size in all the cells tested (N=12). Signals with higher spatial resolution could also be obtained from several areas of each neuron. Fig. 2C shows three sub-regions, from region 3 in panel B, covered by 1 pixel (~2X2 µm), 3X3 pixels (~6X6 µm) and 5X5 pixels (~10X10 µm). Both ∆V m and ∆[Ca 2+ ] i signals could be detected from the single pixel and the S/N increased in the two larger regions. In summary, in all cells tested, ∆V m and ∆[Ca 2+ ] i signals could be resolved from regions <20 µm 2 .

We next performed a series of experiments (N=4 cells) in which dendritic signals were elicited by trains of PF-EPSPs. In the example of Fig. 2D-F influx during the spikes (Canepari & Vogt, 2008), it is expected to observe a qualitative spatial correlation between the peak ∆V m and ∆[Ca 2+ ] i signals. Thus, in Fig. 2G, we compared the ∆V m and ∆[Ca 2+ ] i signals in the 3X3 pixels maximal region and in the progressively more distal 5X3 pixels, 7X3 pixels and 9X3 pixels regions. Both the ∆V m and ∆[Ca 2+ ] i decreased with distance from the 3X3 pixels maximal region, a result confirmed in the 4 cells (Fig. 2H). This result indicates that single-trial measurements were able to identify sites with correlated ∆V m and ∆[Ca 2+ ] i signals with a spatial resolution of ~6 µm. applying tetanic stimulation (10 pulsed at 100 Hz) to the CA3 region (Fig. 3A). Under control conditions, shown in Fig. 3A-B, stimulation elicited three action potentials that back-propagated in the apical dendrite with random timing. We then applied 50 µM of the GABA A receptor antagonist picrotoxin to block synaptic inhibition and monitored the evoked activity every three minutes. The number and frequency of action potentials increased with time (see also SupplementalMovie1). The capability of the technique to resolve ∆V m and ∆[Ca 2+ ] i signals in single trials was critical in this experiment.

We finally tested whether our system can be used in experiments from cell cultures labelled by bulk application of the dyes. Fig. 3D shows three images from a culture dish obtained by di-8-ANEPPS fluorescence (vf), Fura-2 fluorescence (cf) and transmitted light (tr). Individual cells were clearly visible.

Single-trial recordings shown in Fig. 3E show that ∆V m and ∆[Ca 2+ ] i signals associated with one or four evoked action potentials could be measured exclusively from the stimulated cell 1 (see also SupplementalMovie2).

Discussion

In this report we describe how to achieve simultaneous voltage and Ca 2+ imaging with S/N permitting single-trial resolution. Signals of ~1% change of fluorescence could be discriminated from regions of a few microns. In voltage imaging, 1% signal typically corresponds to ∆V m ~10-20mV in individually stained cells or in cultured cells after bulk staining. Thus, activity could be monitored from small sub-cellular compartments. For Ca 2+ imaging, the size of the optical signal corresponding to a given Ca 2+ signal depends on the affinity and concentration of the dye. However, we showed that Ca 2+ signals associated with individual action potentials could be resolved both with low and high affinity dyes. Two concerns that limit the applicability of the technique are photo-toxicity of the voltage sensitive dye and optical cross-talk.

Full illumination with a 100 mW 543 nm laser is equivalent, in terms of dye absorption, to ~25% illumination with a 300 mW 532 nm laser. In this study, using full-light illumination at 543 nm, we did not detect any photo-damage for at least 20 sequential measurements of 100 ms duration, separated by 30 s. This result is consistent with our previous tests at 532 nm [START_REF] Canepari | Imaging inhibitory synaptic potentials using voltage sensitive dyes[END_REF]. In voltage fluorescence measurement, UV illumination generates a fluorescence increase that depends on the concentrations of the two dyes (Fig. 1E). This spurious fluorescence, however, didn't affect the ∆V m signal.

The present results open the gate to novel investigations requiring simultaneous measurement of ∆V m and Ca 2+ signals, particularly in neuro-physiology and cardio-physiology. The apparatus is based on commercially available equipment that can be adapted to conventional microscopes.

Figure Legend

  and used after 10-14 days in vitro. The extracellular solution contained (mM): 125 NaCl, 26 NaHCO 3 , 20 glucose, 3 KCl, 1 NaH 2 PO 4 , 2 CaCl 2 and 1 MgCl 2 , bubbled with 95% O 2 and 5% CO 2 . The intracellullar solution contained (mM): 125 KMeSO 4 , 5 KCl, 8 MgSO 4 , 5 Na 2 -ATP, 0.3 Tris-GTP, 12 Tris-Phosphocreatine, 20 HEPES, adjusted to pH 7.35 with KOH. Patch-clamp recordings were made using a Multiclamp amplifier 700A

Fig. 1B .

 1B Fig. 1B. Measurements reported in this article were taken at 2000 Hz with the NeuroCCD (voltage indicator fluorescence) and 751.88 Hz (slices) or 200 Hz (cultures) with the Ixon+ (Ca 2+ indicator fluorescence). The time information of simultaneous voltage and Ca 2+ imaging measurements is limited by the sampling theorem as discussed in the Supplementary Information.

  , the ∆V m and ∆[Ca 2+ ] i signals associated with one CF-EPSP and a train of 5 PF-EPSPs at 100 Hz were compared in two 8X8 pixels regions. ∆V m and ∆[Ca 2+ ] i signals associated with large depolarization could be observed in both regions for the CF-EPSP but only in region 1, near the PF stimulating electrode, for the PF-EPSP train. Because the ∆[Ca 2+ ] i originates from Ca 2+

Fig

  Fig. 3A-C illustrates the more common scenario in which ∆V m and ∆[Ca 2+ ] i signals vary in time, as recorded from individual CA1 hippocampal pyramidal neurons in brain slices. The signals were evoked by

Figure 1 .

 1 Figure 1. Imaging apparatus and sensitivity A, Schematic drawing of the imaging apparatus used in Canepari & Vogt (2008); combined voltage and Ca 2+ imaging obtained sequentially by switching between two filter cubes. B, Schematic drawing of the apparatus for simultaneous voltage and Ca 2+ imaging; 385 nm OptoLED (CAIRN) illumination via the epifluorescence port of the microscope reflected by a dichroic mirror FF506 (Semrock, transmission curve shown on the right); 543 nm laser illumination via the top of the microscope is reflected by a dual-band dichroic mirror FF493_574 (Semrock, transmission curve shown on the right); the image in the V m and Ca 2+ channels are demagnified by 0.5X projection lens, separated by a 565 nm dichroic mirror (dic, 565DCXR, Chroma) and filtered by a 610 nm long-pass filter (em1, RG610, Schott) and a 510±42 nm band-pass filter (em2, FF01-510/84, Semrock); the two images were further demagnified by 0.38X projection lens before being acquired by a NeuroCCD camera (Redshirt) and Ixon+ EMCCD camera (Andor); two aligned images of a micrometer scale are shown. C, V m (red traces) and Ca 2+ (blue traces) signals associated with a CF-EPSP from the region (~12.5X12.5 µm) indicated below obtained with the apparatus in A: single trials (top) and averages (bottom). D, simultaneous V m and Ca 2+ signals associated with a CF-EPSP from the region (~12X12 µm) indicated below obtained with the apparatus in B: single trials (top) and averages (bottom). Notice the S/N increase permitting single-trial resolution. E, V m signals associated with a CF-EPSP from the 3 regions indicated on the left with (red traces) and without (gray traces) concomitant UV illumination; superimposed traces (right) show no signal change with UV illumination.

Figure 2 .

 2 Figure 2. Simultaneous V m and Ca 2+ measurements from cerebellar Purkinje neurons A, reconstruction of a PN; the dendritic area in recording position is outlined. B, left: fluorescence image with 3 selected regions of interest (16X16 pixels; ~32X32 µm) outlined; right: ∆V m (red traces) and ∆[Ca 2+ ] i (blue traces) signals associated with a CF-EPSP (single trial) from the three regions of interest; somatic electrical recording shown below. C, left: region of interest 3 from panel B, 3 subregions of 1 (2 µm), 3X3 (6 µm) and 5X5 pixels (10 µm); right ∆V m and ∆[Ca 2+ ] i signals from the three subregions. D, reconstruction of another PN with the dendritic area in recording position outlined. E, fluorescence image with three selected regions of interest (8X8 pixels; ~16X16 µm) outlined. F, ∆V m (red traces) and ∆[Ca 2+ ] i (blue traces) signals associated with a CF-EPSP (left) or with a train of 5 PF-EPSPs at 100 Hz (right) from

Figure 3 .

 3 Figure 3. Simultaneous V m and Ca 2+ measurements from hippocampal neurons A, left: schematic drawing of the hippocampus with stimulating electrode in the CA3 region and the recording site in the CA1 region of a pyramidal neuron; right-reconstruction of a neuron (top) and the recording area outlines (bottom); B, ∆V m (red traces) and ∆[Ca 2+ ] i (blue traces) single-trial signals from the region of interest in A associated with randomly occurring action potentials evoked by CA3 tetanic stimulation (10 pulses at 100 Hz) in control conditions; recordings were done every minute. C, same as B after addition of 50 µM picrotoxin; recordings were done every 3 minutes; animation of this experiment in SupplementalMovie1. D, images from hippocampal cultured cells stained with di-8-ANEPPS and Fura-2-AM; from left to right: voltage sensitive dye fluorescence image (vf), Ca 2+ indicator fluorescence image (cf) and image under transmitted light; regions of interest (1-3) from three identified cells are indicated. E, ∆V m (red traces) and ∆[Ca 2+ ] i (blue traces) single-trial recordings from the regions of interest in D associated with 1 (left) or 4 (right) action potentials evoked in cell 1. Signals were localised; animation in SupplementalMovie2.

  FIGURE 1

Acknowledgements

We thank Dr. Alexander Kriz for preparing the cultures. We thank Dr. Dejan Zecevic for helping us in setting up laser illumination at the early stage of the project and for comments on the manuscript. This work was supported by the SNSF grants 3100A0_122000 (M.C.).

Author contribution