Jacqueline ; Gelfi

Michael Pappalardo

Carine Claverys

Brigitte Peralta

Jean-Luc Guerin
email: jl.guerin@envt.fr

Safety and efficacy of an inactivated Carbopol-adjuvanted Goose haemorrhagic polyomavirus vaccine for domestic geese

Keywords: goose, polyomavirus, vaccine, carbopol

Summary

Hemorrhagic Nephritis Enteritis of the goose (HNEG) is an epizootic viral disease in domestic geese. The causal agent is a polyomavirus, namely Goose haemorrhagic polyomavirus (GHPV). To help control the disease, an inactivated vaccine was developed, based on viral particles produced in goose kidney cells. Viral material was quantified using real-time quantitative PCR, inactivated with β-propiolactone and adjuvanted with Carbopol, an acrylic acid polymer. Carbopol proved to be more immunogenic than aluminum hydroxide and was totally safe when administered to young goslings and breeders alike. Carbopoladjuvanted vaccine induced a high serological response. Moreover, goslings hatched from vaccinated breeders were protected against viral challenge, indicating that maternally-derived neutralizing antibodies (MDA) were efficiently transferred. MDA were still detectable 15 days post-hatch. Clinical trials will be necessary to accurately evaluate a vaccine-based HNEG control strategy under field conditions. [START_REF] Bernath | Investigations for clearing the etiology of the disease appeared among goslings in 1969[END_REF]. It was later reported in France and Germany [START_REF] Schettler | Détection en France de la néphrite hémorragique et entérite de l'oie (NHEO)[END_REF][START_REF] Kisary | Haemorragic Nephritis and Enteritis of Geese[END_REF][START_REF] Guérin | Hemorrhagic Nephritis Enteritis of Geese[END_REF]. The causative agent of HNEG was isolated and identified in 2000 and was the first polyomavirus to be considered as pathogenic in poultry [START_REF] Guérin | A novel polyomavirus (Goose haemorrhagic polyomavirus) is the agent of Hemorrhagic Nephritis Enteritis of Geese[END_REF]. This virus, namely GHPV (Goose haemorrhagic polyomavirus), is clearly divergent from the other polyomaviruses identified so far. As a nonenveloped DNA virus, it is highly resistant to heat, desiccation and lipid solvents (Shah, 1996;[START_REF] Guérin | Hemorrhagic Nephritis Enteritis of Geese[END_REF]. During spontaneous or experimental infections, the clinical picture includes lameness and nervous signs and, at necropsy, lesions due to nephritis and oedema.

Histologically, necrosis of endothelial cells and lymphocyte depletion in the cloacal bursa are a classic picture [START_REF] Lacroux | Pathology of spontaneous and experimental infections by Goose haemorrhagic polyomavirus[END_REF]. Infection seems widespread in the field, affecting fattening goslings as well as breeders; carriage is asymptomatic in birds over 13 weeks of age [START_REF] Palya | Epizootic occurrence of haemorrhagic nephritis enteritis infections of geese[END_REF][START_REF] Guérin | Hemorrhagic Nephritis Enteritis of Geese[END_REF]. Recently, GHPV infection has been described in Muscovy and mule ducks, without apparent association with specific clinical signs [START_REF] Pingret | Goose haemorrhagic polyomavirus (GHPV) infection in ducks[END_REF]. When facing outbreaks of HNEG, biosecurity measures alone are not sufficient to control the disease. Vaccination of geese might be a solution, applied either to growing goslings or their parents. Even if clinical signs occur mostly after 4 weeks of age, infection may actually begin in the very early life of the birds. As the immune status of neonate goslings may be critical in HNEG control, vaccination of breeders may contribute to the protection of their offspring, as shown for Avian Polyomavirus (APV) infections in psittacines [START_REF] Ritchie | An inactivated avian polyomavirus vaccine is safe and immunogenic in various Psittaciformes[END_REF]. Attempts to produce GHPV viral-like particles (VLP) based on the VP1subunit have been described and applied to the production of antigen for [START_REF] Zielonka | Generation of virus-like particles consisting of the major capsid protein VP1 of goose haemorrhagic polyomavirus and their application in serological tests[END_REF] and recently as a subunit vaccine for goslings (Mato et al., 2009). This vaccine was preliminarily assayed in goslings and showed to elicit some protection.

In this article, we describe the production of GHPV antigen, the preparation of a Carbopol-adjuvanted inactivated vaccine and the first safety and immunogenicity trials in geese. We also present the ELISA assay designed to measure serological response. Finally, the efficacy of this vaccine was assessed in day-old goslings hatched from vaccinated geese.

Materials and Methods

Cells and virus.

A stock of Goose haemorrhagic polyomavirus (GHPV), strain Toulouse 2000, was grown in goose kidney (GK) cells, derived from a day-old gosling. This viral strain has been described previously [START_REF] Guérin | A novel polyomavirus (Goose haemorrhagic polyomavirus) is the agent of Hemorrhagic Nephritis Enteritis of Geese[END_REF] and the sequence of its major capsid protein VP1 gene has been deposited in GenBank under the accession number AF226991. These cells were cultivated at 37°C in DMEM with 10% foetal calf serum and were permissive to GHPV infection as previously shown [START_REF] Guérin | A novel polyomavirus (Goose haemorrhagic polyomavirus) is the agent of Hemorrhagic Nephritis Enteritis of Geese[END_REF]. Optimized conditions of viral production were determined: briefly, after 2h adsorption at 39°C, the viral inoculum was removed and infected cells were incubated in DMEM with 10% foetal calf serum for 7 days at 39°C. After 3 freeze-thaw cycles, suspensions were centrifuged to remove cells debris and stored as virus stock at -80°C until use.

Detection and quantification of GHPV DNA using either classical or quantitative PCR.

In all vaccination or challenge experiments, tissues (i.e. spleen and kidney) or cloacal swabs were sampled and stored at -20°C until DNA extraction, using the High pure PCR template Preparation kit (Invitrogen). PCR amplification was performed as previously described, using the set of primers VP1F (5'-GAGGTTGTTGGAGTGACCACAATG-3') and VP1R (5' -ACAACCCTGCAATTCCAAGGGTTC-3') [START_REF] Guérin | A novel polyomavirus (Goose haemorrhagic polyomavirus) is the agent of Hemorrhagic Nephritis Enteritis of Geese[END_REF]. PCR products were analyzed by electrophoresis on a 1.5% agarose gel. A PCR product was cloned into a p-GEMT plasmid vector (Stratagene®), amplified, purified and finally quantified by 260/280 OD spectrophotometry. Serial dilutions of this plasmid were subsequently used as standard for real-time PCR analysis. In order to quantify the viral load in virus stock, a SYBR  Green quantitative PCR (qPCR) assay was performed using the same set of primers as described above. Briefly, reaction mixes were obtained by combining 12.5µl of 2× SYBR  Green MasterMix (Applied Bio Systems) and 2.5µl of each primer, used at a final concentration of 500µM. Real time PCR was performed on a GeneAmp 9700 cycler, using the following programme: 5 min at 95°C, then 40 cycles of 15s at 95°C and 1 min at 60°C. For each reaction, the Cycle threshold (Ct) value, corresponding to the number of cycles required for the fluorescence intensity graph to cross threshold, was determined and quantification was performed by reference to a standard VP1-pGEMT plasmid. In order to assess the specificity of PCR products, a dissociation curve was generated after the last PCR cycle, resulting in an expected melting temperature (Tm) of 78.6°C.

Vaccine preparation. After real-time PCR titration, the virus stock was used for vaccine preparation. Inactivation of GHPV was performed according to the procedure described for Avian Polyomavirus [START_REF] Ritchie | An inactivated avian polyomavirus vaccine is safe and immunogenic in various Psittaciformes[END_REF]. Briefly, the virus supernatant was incubated for 12h at room temperature with 0.2% β-propiolactone (Sigma), after which pH was adjusted to 7.0 and β-propiolactone was added to a final concentration of 0.3%. After 2h incubation, the preparation was neutralized with 40 mM Sodium thiosulfate. Complete inactivation was certified by indirect immunofluorescence test 48h post inoculation (p.i.) on a GK monolayer, using a post-infectious anti-GHPV goose serum and a secondary goat anti-duck serum (KPL) [START_REF] Guérin | A novel polyomavirus (Goose haemorrhagic polyomavirus) is the agent of Hemorrhagic Nephritis Enteritis of Geese[END_REF]. PCR detection was also performed to confirm the destruction of GHPV DNA. After inactivation, antigen stock was used for preparation of experimental vaccines, adjuvanted with either Aluminum Hydroxide (Al(OH) 3 ) or a carbomer.

An Al(OH) 3 -adjuvanted vaccine (Called Al-Vac later in the paper) was prepared using a Al(OH) 3 aqueous gel (Alhydrogel 2%, Superfos), gently mixed with the antigen stock at a final load of 12.5 mg/mL. Each dose was adjusted to a final volume of 0.5 mL.

A Carbopol-adjuvanted vaccine (called Carbo-Vac later in this paper) was prepared using 2 mg per dose of Carbopol 934, an acrylic acid polymer (Noveon Pharma) mixed with the antigen stock. Each dose was adjusted to a 0.5 mL final volume.

Vaccination of goslings.

Goslings were supplied from a local hatchery and came from flocks without any history of HNEG during the growing period, and PCR negative for GHPV (data not shown). They were housed in BSL-2 facilities. In a first assay, 3 batches of five goslings each were administered an Al(OH) 3 -adjuvanted vaccine containing respectively 10 9 , 10 10 or 10 11 genomes per dose twice, at 3 and 6 weeks of age. Goslings were bled at weeks 3, 6 and 9 and their sera tested by ELISA for antibodies.

A second trial was performed on 3 separated groups of five birds each: one group was non-vaccinated (control group), the second group received a 0.5 mL dose of Al-Vac twice and a third group received a 0.5 mL dose of Carbo-Vac vaccine twice. At day of vaccination and 2, 4, 6 and 7 weeks post-vaccination, the birds were bled and their sera collected for testing by ELISA.

At the end of the two trials, birds were killed and their kidneys were sampled and submitted to real-time PCR analysis in order to further confirm the complete inactivation of GHPV in the vaccines. Vaccination of breeders. Three hundred future breeder geese, housed in conventional facilities, were shown to be free of GHPV, as well as GHPV-specific antibodies, on the basis of an ELISA and a real time PCR analysis performed on the sera of 30 birds. The birds received a subcutaneous injection of Carbo-Vac, 6 weeks and 2 weeks before the beginning of the laying period. Ten females were identified as non-vaccinated controls and maintained together with the vaccinated birds. All male geese were also vaccinated. At day 56 postvaccination, 20 vaccinated females randomly sampled in the flock and the 10 non-vaccinated control birds were bled and their sera collected for ELISA testing. A cloacal swab was collected from each bird for subsequent PCR detection of GHPV DNA. Eggs laid from either vaccinated or non-vaccinated geese were identified distinctively for subsequent study of their offspring. Throughout all the experiments, vaccinated birds were monitored daily for 50 days for their behaviour, food intake, weight, egg production and hatchability and were also subjected to a complete clinical examination. ELISA method. An ELISA was developed by adapting procedures routinely used in the laboratory [START_REF] Gelfi | Development of an ELISA for detection of myxoma virus-specific rabbit antibodies: test evaluation for diagnostic applications on vaccinated and wild rabbit sera[END_REF]. For this purpose, semi purified GHPV antigen was prepared as follows: monolayers of GK cells were infected with GHPV stock at 39°C for 4 to 6 days in DMEM with 10% foetal calf serum. When cytopathic effect reached 100%, the supernatant was collected and clarified by centrifugation at 3000g to remove cells debris. Virus was then pelleted by ultracentrifugation at 100,000g for 2h. Pellets were suspended in nanopure water with protease inhibitors and the optical density (OD) was read at 260 and 280nm. The purity of the preparation was determined by an OD ratio 260/280 of roughly 1,2. Correlation between OD and qPCR quantification was established through 5 independent assays: 1 OD at 280 nm corresponded to 3x10 10 viral genomes/ml of viral suspension. To perform the ELISA, 96-well microplates were coated overnight at 37°C with semipurified GHPV particles corresponding to 5x10 7 genomes/well, diluted in PBS buffer. The wells were washed 3 times in PBS, blocked by incubation for 1h with 15mg/mL gelatin in PBS buffer and then incubated with serial 2-fold dilutions of the serum sample in duplicate.

Positive and negative standards were included in each plate. Wells were washed 3 times in PBS-0.05% Tween 20 buffer, and then incubated with a 1:100 dilution of goat anti-duck IgY conjugated with alkaline phosphatase (KPL). Finally, after 3 washes in PBS-0.05% Tween 20 and one in PBS, detection was performed using PNPP (P-NitroPhenyl Phosphate) as substrate and absorbance was read in a spectrophotometer (wavelength: 405 nm). To determine cut-off value for positivity, the sera of 10 goslings sampled before vaccination were pooled as standard negative control. Each negative serum was first individually tested at a dilution of 1:20. ELISA titres were expressed as the inverse of the dilution giving an OD value 3 times as great as the OD value of the standard negative control. For the ELISA titration in egg yolk, IgY were purified using a commercial kit "Eggcellent Chicken IgY Purification Kit" (Pierce, Rockford, Illinois, USA). The purified fraction was resuspended in a volume of buffer corresponding to the volume of egg yolk processed for extraction. Aliquots were frozen at -20°C and later processed for ELISA testing as described above.

Viral challenge and serological study on day-old goslings. Fifteen day-old goslings hatched from eggs laid by the experimental breeders flock at day 56 post vaccination, were housed in BSL-2 facilities, according to the guidelines of the European Community on Animal Care (European Council directive 3086/609/ECC, 24 November 1986), in wire-floored cages with infrared lamps for heating, and were provided with food and water ad libitum. These 15 birds were inoculated by the subcutaneous route with 200µl of GHPV inoculum (strain Toulouse 2000), purified from cell culture as previously described [START_REF] Guérin | A novel polyomavirus (Goose haemorrhagic polyomavirus) is the agent of Hemorrhagic Nephritis Enteritis of Geese[END_REF]. This inoculum analysis. Fifteen goslings hatched from a non-vaccinated flock were used as controls, 5 birds were inoculated with 10 7 genome-equivalent GHPV, using the same protocol and 10 goslings were mock-infected. The 3 batches of birds were housed separately. All birds were clinically monitored on a daily basis. At 8 days p.i. all the birds which were still alive were submitted to a cloacal swab sampling and screened for GHPV DNA. Finally, either at spontaneous death during the experiment or when killed at day 20 p.i., all birds were submitted to a complete post mortem examination and sampled for real-time PCR analysis of kidney and spleen tissues.

Fifteen goslings hatched from eggs laid at day 56 post-vaccination by vaccinated breeders and 15 hatched from eggs laid by non-vaccinated breeders, were bled and weighed at 1, 5, 10 and 15 days of age and their serum collected for ELISA testing.

Results

Preparation of GHPV vaccines. GHPV was produced in optimized conditions in GK cells as described above. Virus production was then quantified by real-time PCR and inactivated with β-propiolactone. Virus inactivation was confirmed by three successive passages in GK cells and immunofluorescence assay. Results show a good quantification with a viral suspension of up to 5x10 11 genomes/ml and a complete efficiency of the inactivation procedure.

Carbopol adjuvant is immunogenic in geese. In a first attempt, the antigen load was optimized by means of a comparison of Al(OH) 3 -adjuvanted vaccines containing 10 9 , 10 10 or 10 11 genomes per dose, respectively. The curve measuring serological response showed that the highest antibody titre was obtained with the vaccine containing 10 10 genomes of GHPV (Figure 1a). This dose was therefore used for all the following trials of vaccines, adjuvanted with either Al(OH) 3 or Carbopol.

In a second step, we compared Carbopol and classical Al(OH) 3 as adjuvants. Two groups of 5 goslings each were inoculated with a vaccine adjuvanted with either Al(OH) 3 or Carbopol in accordance with the protocol described above. Based on ELISA assays, the Carbopol-adjuvanted vaccine induced a significantly stronger serological response as early as after the first injection, the difference being 4-fold at day 49 (Figure 1b). This experiment was repeated twice with the same result. Based on all these findings, the subsequent vaccination assays were based on a Carbopol-adjuvanted vaccine containing an antigenic load corresponding to 10 10 genome-equivalent GHPV / GHPV-equivalent genome.

The Carbopol vaccine induces a strong serological response in growing goslings and breeder geese. To evaluate Carbo-Vac safety and immunogenicity, goslings were vaccinated twice with a 3-week time interval between the two injections (Figure 2). Vaccinated goslings showed a rapid seroconversion. In both vaccinated and control birds, cloacal swabs remained PCR negative throughout the study. In particular, no vaccinated bird presented any viral carriage detectable by PCR. The breeders were challenged in order to assess immunogenicity and transmission of maternal antibodies (MDA) to goslings. In a first attempt, a serological analysis was performed at day 56 post vaccination, without follow-up of the serological kinetics. Indeed, a significant amount of specific antibodies was detected in the geese sera, in the vitellus of eggs and in the serum of their offspring (Figure 3). Transmission and decay of maternally-derived antibodies. The objective of the breeders' vaccination scheme is the passive transfer of MDA from vaccinated geese to goslings through the vitellus. To evaluate this transmission, 15 goslings hatched from the vaccinated breeder geese in the previous experiment were monitored for 15 days. These goslings were hatched from eggs laid at day 56, i.e. 5 weeks after the second vaccination. The average antibodies titre in these goslings was 330 at day-old, then decreased rapidly to the level of the controls by 15 days. In the same period, the goslings' weight had increased five fold (Figure 4).

Both Al-Vac and

Goslings hatched form vaccinated breeders are protected against viral challenge.

Viral challenge was performed on 15 one-day-old goslings hatched from vaccinated breeders and 10 day-old goslings hatched from non-vaccinated breeders. Five day-old goslings hatched from non-vaccinated breeders were not challenged as controls. The results of the challenge are shown in Table 1. Goslings hatched from vaccinated breeders showed neither clinical signs suggestive of HNEG, nor detectable GHPV infection after a virulent challenge at oneday-old.

Discussion

Polyomaviruses infect mammals and birds but, to date, avian species are the only host in which polyomaviruses show a significant pathogenicity. Within the family Polyomaviridae, these inflammatory features are highly specific of avian polyomaviruses (APV) [START_REF] Johne | Polyomaviruses of birds: etiologic agents of inflammatory diseases in a tumor virus family[END_REF]. Vaccination was implemented for the control of APV infections in psittacines, with a significant efficacy. By contrast, attempts to vaccinate humans against polyomaviruses infections, such as JC, have been poorly investigated since these viruses affect immunocompromised hosts only.

Propagation of GHPV was successfully performed in GK cells derived from a day-old gosling, which allowed the production of significant amounts of viral particles [START_REF] Guérin | A novel polyomavirus (Goose haemorrhagic polyomavirus) is the agent of Hemorrhagic Nephritis Enteritis of Geese[END_REF]. Culture medium and procedures were optimized to produce a high and reproducible amount of viral material (data not shown). In our hands, these kidney cells allow the production of a significant viral yield on a routine basis, corresponding to up to 10 11 GHPVgenomes/ml of culture supernatant. Intriguingly, GHPV propagation could not be obtained by other groups with primary kidney cells [START_REF] Johne | The genome of Goose haemorrhagic polyomavirus, a new member of the proposed subgenus Avipolyomavirus[END_REF]. This discrepancy may be related to specific properties of the GK cell seed obtained in our laboratory or, alternatively, an adaptation of the GHPV strain Toulouse 2000 to cell culture.

The quantification of viral yield was performed indirectly using real-time PCR, since plaque titration in GK cells seldom gives a clear cytopathic effect. Virus titration on goose embryo was previously described but this method is time consuming and can hardly be applied to routine titration of GHPV (Bernath et al., 2006). We developed a quantitative PCR assay to obtain an indirect quantification of the virus. The correlation between the number of genome copies and the viral particles was described for SV40 (Simian virus 40) and showed to be reliable in this model [START_REF] Shi | A real-time quantitative PCR-based method for the detection and quantification of Simian virus 40[END_REF]. The same procedure was used in all the experiments described in this study.

Alternatively, recombinant VP1 may be used for the purpose of serological assays or vaccination. Indeed, recombinant VP1 was shown to induce a serological response and provide protection to goslings after vaccination once at day-old, or twice by boosting at 18 et al., 2009). These results confirm that VP1 is the major antigenic determinant of polyomaviruses and can be expressed in subunit or vectored vaccines. Furthermore, recombinant proteins can easily be quantified, allowing a control of antigenic load. However, in a field perspective, the marketing of such a recombinant vaccine would be severely restricted by the European licensing regulation applied to biologicals based on genetically modified organisms.

Among the common poultry species, the goose is, by far, the most sensitive to adjuvant-associated general secondary reactions. We therefore had to find an alternative adjuvant to the oil-based ones routinely used in poultry vaccines. Since the Al(OH) 3adjuvanted vaccine offered very poor immunogenicity, Carbopol polymer was assayed, according to previous uses in horses [START_REF] Mumford | Antigenicity and immunogenicity of equine influenza vaccines containing a Carbomer adjuvant[END_REF][START_REF] Minke | Effective priming of foals born to immune dams against influenza by a canarypox-vectored recombinant influenza H3N8 vaccine[END_REF], swine and pigeons [START_REF] Vereecken | The effect of vaccination on the course of an experimental Salmonella Typhimurium infection in racing pigeons[END_REF]. The adjuvant properties of polyacrylic acids, designated by the term carbomers, may vary greatly with the number of carboxyl groups present in the final molecule. Here we have shown that Carbopol 934 is actually immunogenic and may be a relevant alternative to oil in avian species for which safety is a major concern. However, it would be of interest to further study the efficacy of Carbopol 934 in poultry, by comparison with other carbomers and oil emulsions; some trials are under way in our group to address this. The most challenging issue is the duration of the serological response, since the antibody response is well known for being weaker and shorter in waterfowl than in chickens [START_REF] Higgins | Comparative immunology of avian species[END_REF]. This problem recently arose in the vaccination campaign of ducks against highly pathogenic influenza virus H5N1 [START_REF] Steensels | Primeboost vaccination with a fowlpox vector and an inactivated avian influenza vaccine is highly immunogenic in Pekin ducks challenged with Asian H5N1 HPAI[END_REF]. Further trials will be necessary to assess a protective response during a whole laying period of breeder geese, even though in this species the laying period is remarkably short (i.e. less than 6 months), compared to other poultry breeders. The transmission of MDA through the vitellus has been confirmed here.

This transmission is supposed to be restricted to the complete forms of IgY, i.e. excluding the [START_REF] Liu | Yolk-sac transmission and post-hatching ontogeny of serum immunoglobulins in the duck (Anas platyrhynchos)[END_REF]. This uncommon mechanism, specific to waterfowl species, may explain the apparent discrepancy between ELISA titres observed in our study in breeders and day-old goslings respectively.

Infection of breeders seems to be highly prevalent in geese flocks [START_REF] Palya | Epizootic occurrence of haemorrhagic nephritis enteritis infections of geese[END_REF].

Within an infected flock, a great variability is classically encountered, inducing heterogeneity in transmission of MDA to their offspring (Guérin et al., unpublished data). These discrepancies may lead to rolling infections in brooding flocks. Beside transmission of MDA, vaccination of breeders induces a strong humoral response, which may prevent an active infection during the laying period and subsequently, spreading of the virus either through faecal contamination of the shell or via a still-to-demonstrate vertical route. In this perspective, vaccination of breeders should contribute to a global control of HNEG in the goose industry. Vaccination of growing goslings will have to be applied in order to supplement the decaying MDA in the case of strong viral challenge. Long-term field applications of GHPV vaccine will be required to accurately evaluate a vaccine-based HNEG control strategy.

  of the goose (HNEG) is a viral infection in domestic geese, first described in 1970 in Hungary

  Carbo-Vac vaccines are totally innocuous in goose. No local inflammatory reaction at the point of injection was noticed during any of the experiments the vaccination assays in both young goslings and future breeders, the health status of the animals never altered and food intake remained in accordance with standards. Vaccination of future breeders had no effect on their laying performance or on the hatchability of embryonated eggs.

  IgY, which are much more abundant in the serum of breeders

Figure 1a .

 1a Figure legends

Figure 1b .

 1b Figure 1b. ELISA antibody titres in goslings after vaccination using either the Aluminium hydroxide or Carbopol-adjuvanted vaccine (5 birds in each group). A group of 5 control, non-vaccinated birds was also included in the assay and did not show any detectable antibody during the experiment. Vertical bars represent standard deviation.

Figure 2 .

 2 Figure 2. ELISA kinetics of GHPV-specific antibodies in groups of 15 growing geese vaccinated at day 0 with a boost at day 28, or non-vaccinated as controls. Vertical bars represent standard deviation. (D= day of age).

Figure 3 .

 3 Figure 3. Comparison of ELISA titres of GHPV specific antibodies at day 56 post vaccination in breeder geese (n=20), vitellus of eggs laid the same day (n=10) and day-old-goslings (n=20) hatched from eggs laid the same day. Vertical bars represent standard deviation.

Figure 4 .

 4 Figure 4. ELISA titre kinetics of maternally derived antibodies in goslings hatched from vaccinated or non-vaccinated breeder geese (n=15 birds in each group). The growth curve of vaccinated and control birds weighed throughout the study is included. Vertical bars represent standard deviation. (D= day of age).

Figure 1a .

 1a Figure 1a. ELISA antibody titres observed in goslings after vaccination with an Aluminium hydroxide-adjuvanted vaccine containing 10 9 ; 10 10 ; 10 11 genomes (gen) per dose (5 birds in each group). Vertical bars represent standard deviation.

Figure 1b .Figure 2 .Figure 3 .Figure 4 .

 1b234 Figure 1b. ELISA antibody titres in goslings after vaccination using either the Aluminium hydroxide or Carbopol-adjuvanted vaccine (5 birds in each group). A group of 5 control, non-vaccinated birds was also included in the assay and did not show any detectable antibody during the experiment. Vertical bars represent standard deviation.

Table 1 .

 1 Virus challenge performed subcutaneously on day-old goslings hatched from either vaccinated or control breeders. Birds were daily monitored for clinical signs and were sampled at day 8 post inoculation for testing by GHPV PCR.

	Group of birds	Virus challenge	Total mortality	GHPV cloacal excretion
		at 1-day-old	20 days post-challenge	8 days post-challenge
	Goslings	10 7 genomes	7/10 a	10/10 b
	hatched from
	non-vaccinated
	breeders (n=10)
	Goslings	10 7 genomes	0/15	0/15
	hatched form
	vaccinated
	breeders (n=15)
	Goslings	None	0/5	0/5
	hatched from
	non-vaccinated
	breeders (n=5)

a Number dead/ number in group. b Number of PCR-positive birds/ number in group.

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

Acknowledgements

We gratefully thank our colleagues Benoit Severac, Stéphane Bertagnoli and Christelle Camus-Bouclainville for their helpful suggestions and careful reading of the manuscript. This work was supported by Régions Aquitaine and Midi-Pyrénées, CIFOG (Comité Interprofessionnel des Palmipèdes à Foie Gras) and OFIVAL (Office Interprofessionnel des Viandes, de l'Elevage et de l'Aviculture).


Ewa Malicka

Tadeusz Groń

Danuta Skrzypek

Andrzej W Pacyna

Dariusz Badurski

Alicja Waśkowska

Slawomir Mazur

Rafal Sitko

Correlation between negative magnetoresistance effect and magnon excitations in

Keywords: electron paramagnetic resonance, magnetoresistance, spinels, thermoelectric power magnetoresistance, spin waves, thermoelectric effects magnetoresistance, spin waves, thermoelectric effects, electron spin resonance .manuscriptcentral.com/pm-pml

teaching and research institutions in France or abroad, or from public or private research centers.

Introduction

Kanamori [1] predicted that 90° ferromagnetic (FM) superexchange occurs between octahedrally coordinated 3d 3 ions. For example, in the p-type FM semiconductor CdCr 2 Se 4 with the Curie temperature T C = 142 K and the Curie-Weiss (CW) temperature θ CW = 190 K the FM superexchange occurs within the Cr sublattice via the 90° Cr-Se-Cr σ/π bonding which links a half-filled t 2g (π) orbital of one of Cr-ions with an empty e g (σ) level on the other Cr [2,3]. In the parent FM CuCr 2 Se 4 being a p-type metallic conductor with T C = 416 K and θ CW = 436 K [4], the chromium spins are coupled ferromagnetically via double exchange interaction involving the electrons jumping between Cr 3+ and Cr 4+ ions [START_REF] Lotgering | Proceedings of the International Conference on Magnetism[END_REF][START_REF] Lotgering | [END_REF]. The X-ray photoelectron spectroscopy and polarized neutron diffraction study for CuCr 2 Se 4 provided a direct evidence for the presence of monovalent copper atoms with a 3d 10 configuration [7,8]. However, it was not possible to measure in CuCr 2 Se 4 the magnetic splitting of the Cr 3s levels, because these levels overlapped with the Cu 3p ones [7]. The second end composition of the CuCr 2-x V x Se 4 system, i.e CuV 2 Se 4 is not known, yet. In the isostructural CuCr 2- x V x S 4 spinel system a rapid decrease of T C from 267 K for x = 0.25 to 8 K for x = 1.75 was observed [9]. This fact has been explained by being weaker Cr-S-V superexchange interactions than the original Cr-S-Cr ones as well by a weakening of the nearest neighbor ferromagnetic Cr-S-Cr interactions due to the initial lattice expansion [9].

Recently, numerous efforts have been made to find a correlation between the thermoelectric power and the double exchange and superexchange interactions in the CdCr 2-x Ga x Se 4 , Zn x Cu y Cr z Se 4 , Cu x Ga y Cr z Se 4 and Cu x Co y Cr z Se 4 spinel series [10][11][12][13][14]. These studies have shown that the strong ferromagnetic coupling connected mainly with the double-exchange mechanism, makes easier both the magnon excitations and a transfer of the phonon momentum to the electron gas. The opposite behavior for the ferrimagnetic and antiferromagnetic spin arrangements was observed.

It seems thus natural to expect a coupling between the magnon and electron systems leading to the negative magnetoresistance effect in single-crystalline CuCr 1.6 V 0.4 Se 4 spinel. For that the Bloembergen and Wang model of paramagnetic relaxation processes [15], the high-temperature expansion of the magnetic susceptibility and the thermoelectric power analysis were used.

Experiment

Preparation

The single crystal of CuCr 1.6 V 0.4 Se 4 spinel was obtained by the chemical vapour transport method [16] in a closed quartz ampoule containing binary selenide CuSe, and stoichiometric amounts of high purity (99.99%) elements: vanadium and selenium. Anhydrous chromium chloride CrCl 3 was used as the chemical transport agent. The ampoule was heated in a horizontal zone furnace for four weeks. The temperature of the solution and crystallization zone was 1273 K and 1223 K, respectively. The resulting single crystal was of octahedral shape with regular {111} faces, the edge length being 4 mm.

For the crystal structure characterization, describing the cation distribution over the tetrahedral and octahedral sites formed by the selenium sublattice, a good quality sample was selected for the intensity data collection with a single crystal X-ray diffraction technique. The intensities were measured with a KM-4/CCD (Oxford Diffraction) instrument, operating in κ geometry and using graphite monochromated MoKα radiation (λ=0.71073 Å). The ω-scan technique was used for data collection. Integration of the intensity data and corrections for Lorentz -polarization effects and absorption were made using CrysAlis software [17]. The absorption correction was applied with the Gaussian face-indexed numerical routine [START_REF] Sheldrick | SHELXL-99, Program for the Refinement of Crystal Structures[END_REF]. The structure calculations were performed using the SHELXL-97 program system [START_REF] Sheldrick | SHELXL-99, Program for the Refinement of Crystal Structures[END_REF].

The chemical composition of the CuCr 1.6 V 0.4 Se 4 single crystal was determined by the energy-dispersive X-ray spectrometry (EDXRF). The sample was excited by the air-cooled side-window Rh target X-ray tube of 125 µm thickness Be window and ca. 100 µm nominal focal spot size (XTF 5011/75, Oxford Instruments, USA). The X-ray tube was supplied with the XLG high- the resolution of 145 eV at 5.9 keV. The Si-PIN detector was coupled to a multichannel analyzer (PX4 Amptek, Bedford, MA, USA). In the constructed spectrometer, the incidence and take-off angles were 45°. The position of the sample was moved using the X-Y stage and was monitored by CCD camera and two laser pointers. The weight composition of the single crystal under study is as follows: Cu -13.1 ± 0.2, Cr -17.3 ± 0.3, V -4.26 ± 0.08, Se -65.4 ± 0.8. These values have been converted into percent of the atomic content.

Electrical and magnetic measurements

The mass magnetization was measured at 4.6 K and in external magnetic fields up to 60 kOe while the static (dc) mass susceptibility measurements were performed in the temperature range 4.2-350 K and in applied external magnetic field of 900 Oe using a Faraday type Cahn RG automatic electrobalance.

The electrical measurements have been done in the temperature range 7-300 K. The electrical resistivity ρ has been measured with the 4-point dc method using the apparatus with Keithley K181 digital multimeters. The maximal error δρ/ρ was less than ±1 %. The magnetoresistance measurements were performed in the magnetic fields up to 8 T at 4.3, 10, 15, 20 and 40 K and at fixed magnetic field 8 T in the temperature range 7-110 K. The direction of magnetic field was perpendicular to the current sample. The thermoelectric power was measured with a differential method using the temperature gradient ∆T of about 2 K. The accuracy of the value of thermopower was estimated to be better than 3 µV/K.

The electrical and thermal contacts between the single crystal and the copper rods were maintained with a silver lacquer mixture (Degussa Leitsilber 200).

The thermoelectric power and the electrical resistivity of the same sample were measured along the <001> direction. The crystal orientation about the <001> crystallographic zone axis, was based on 12-18 centered X-ray diffraction reflections with the Bragg angles in the range 107 ≤ 2θ ≤ 144º (MoKα radiation). The checking electrical measurements in the <111> direction, performed for comparison, showed unimportant difference with those along <001>.

ESR measurements

The electron spin resonance (ESR) spectra were measured in the temperature range 77-275 K using a 100-kHz field-modulated spectrometer operated at 9 GHz, so that the derivatives of the absorption signals were

Results

Crystallographic properties

Crystal data, experimental details and structure refinement results for the single-crystal of CuCr 1.6 V 0.4 Se 4 spinel are collected in Table 1. They are showing that the compound crystallizes in a cubic structure with space group m 3 Fd (#227) and the unit cell of dimension a = 1034.99 (12) pm, being slightly elongated in comparison with a = 1033.5(3) pm of the parent CuCr 2 Se 4 [START_REF] Lotgering | [END_REF]. The structure model with the diamagnetic Cu ions occupying the tetrahedral 8a positions and the magnetic Cr and V-ions sharing the octahedral 16d sites, converged satisfactorily. The site occupancy factors treated as free variables in the least-squares structure refinement, confirmed the sample composition and the cation distribution according to the spinel formula: Cu[Cr 1.6 V 0.4 ]Se 4 .

Electrical and magnetic properties

The electrical resistivity ρ(T) and thermoelectric power S(T) measurements depicted in Fig. 1 reveal a p-type semiconducting behaviour of the CuCr 1.6 V 0.4 Se 4 single crystal. The values of ρ(T) and S(T) are close to those for CuCr 2 Se 4 [19]. The variation of ρ(T) does not follow a simple exponential behaviour, because a gradual decrease of resistivity and strong slope at T C with increasing temperature are observed. The S(T) shows a broad hump at 100 K and reaches a minimum at T C from one side and goes to (0, 0) from the other. The ρ(T) dependence decreases with increasing magnetic field, revealing the negative magnetoresistance effect shown in Fig. 2. Moreover, the lower temperature, the larger negative magnetoresistance, ∆ρ/ρ 0 (H). However, in the temperature range 10-20 K the negative values of ∆ρ/ρ 0 (H) increase insignificantly as temperature decreases (Fig. 2).

The results of the magnetic measurements are depicted in Figs. 3 and4 the Curie constant C σ = 8.873⋅10 -3 K⋅cm 3 /g. This value is somewhat higher than the spin-only value of 5.22 µ B /f.u. for 3d 3 Cr 3+ and 3d 2 V 3+ ions as well as of 5.50 µ B /f.u. for 3d 9 Cu 2+ , 3d 3 Cr 3+ and 3d 2 V 3+ ions. It means that the chromium ions may form a mixed valence band. In Fig. 4 a shape of magnetic isotherm, σ(H), measured up to 60 kOe and at 4.6 K exhibits a saturation magnetization of 53.84 Oe•cm 3 /g (4.66 µ B ) at 4 kOe already. In the inset of Fig. 4 a linear increase of magnetization with increasing magnetic field, indicates a lack of spontaneous magnetization below 4 kOe.

ESR spectra

The resonance spectra for CuCr

Discussion

An explanation of the negative magnetoresistance effect in singlecrystalline CuCr 1.6 V 0.4 Se 4 spinel is considered within a framework of spin-spin and spin-phonon couplings and also as a coupling between magnon and electron systems. For that a complex model of the paramagnetic relaxation processes and the thermoelectric power analysis are discussed in detail.

ESR studies

The linewidth value of CuCr 1.6 V 0.4 Se 4 at room temperature is about 200 mT.

To understand the origin of the observed linewidth we have to estimate the relaxation contributions of the relevant spin-spin and spin-phonon interactions.

In the chromium spinels, the Cr 3+ ions always occupy the B-site of the spinel structure. The local symmetry at the octahedral site leads to the nondegenerate orbital ground state with S = 3/2. On the other hand, for the 3d 2 configuration of V 3+ (S = 1), the 3 F free ion ground state is split by the octahedral field leaving an orbital triplet (T 2 ) lowest. Because of that, Cr 3+ is the ion weakly coupled to the lattice in comparison with the fast-relaxing V 3+ ions [START_REF] Bates | [END_REF].

The ESR linewidth, ∆B, is related to the relaxation of the spin system. For individual spins ∆B ∼ 1/τ, where τ is the spin relaxation time. In a dense magnetic material, this relationship is modified since the magnetization relaxes towards an effective field instead of the external field. In the examined compound it can be expected that the presence of V 3+ ions essentially changes the resonance linewidth. In paramagnetic state (PM) (i.e. for T > 212 K) the linewidth can be described by the following expression:

∆B = ∆B ss + ∆B s-ph (1)
where ∆B ss is described by the exchange narrowing theory [START_REF] Bencini | Electron Paramagnetic Resonance of Exchange Coupled Systems[END_REF]:

∆B ss = [(∆B dd ) 2 ] / B ex , (2)
(i.e. is proportional to the square of the dipolar produced linewidth ∆B dd divided by the rate of exchange B ex ) and ∆B s-ph represents the contributions of the spinphonon interaction.

The complex model of paramagnetic relaxation processes, illustrated in Fig. 6, can be derived by analogy with the model of Bloembergen and Wang [15,[START_REF] Abragam | Electron Paramagnetic Resonance of Transition Ions[END_REF]. The Zeeman subsystem contains Cr 3+ and V 3+ ions and the vanadium is directly coupled to the lattice. The exchange subsystem includes the exchangecoupled Cr-Cr, Cr-V, V-V pairs. In the parent CuCr 2 Se 4 spinel, which is a well studied metallic ferromagnet with T C = 416 K [4], the high value of T C is related to the exchange through the current of the hole carriers [START_REF] Lotgering | Proceedings of the International Conference on Magnetism[END_REF][START_REF] Lotgering | [END_REF] including also the relaxation includes both the interaction between the conduction electron spins and magnetic spins (Korringa effect) and also the vibrational modulation of exchange interactions. According to the Huber's and Seehra's theory for magnetically concentrated systems [23,24], the relationship between temperature and resonance linewidth can be explained by the Korringa mechanism [START_REF] Barnes | [END_REF] and spin-phonon relaxation processes in which are participating broad bands of phonons.

The temperature dependence of ∆B and B r in the temperature range 212 -193 K (Fig. 5(a) and (b)) indicates that the magnetic short-range order develops.

In this case, the spin correlations act as an effective internal field and therefore cause a shift of the resonance field B r from that at high temperatures, where no magnetic short-range order exists. Simultaneously, the linewidth ∆B as T → T C from the high temperature side can be written as follows:

∆B = ∆B noncrit (T) + ∆B crit (T), (3)
where: ∆B noncrit and ∆B crit factors represent the noncritical and critical contributions to the linewidth, respectively, and ∆B noncrit is given by Eq. ( 1). The FMR can be adequately described in terms of the Landau-Lifshitz-Gilbert equation [25] for the effective internal magnetic fields, which are attributed to demagnetization and magnetocrystalline anisotropy fields. However, as in our resonance measurements the sample in powdered form was examined, the shape of FMR spectrum is the envelop of several components. As can be seen from Fig. 5(a) and (b), the position and linewidth of this spectrum depends monotonously on the temperature.

Thermoelectric power analysis

In this section we consider a coupling between the magnon and electron systems based on the thermoelectric power analysis. Generally, in the magnetic materials the magnetic component of thermopower (MCTP) may result from a coupling of the phonon system to the electron gas via the spin lattice (magnon gas), which is known as the Kasuya model [26], or from the transfer of the magnon momentum to the electron gas (magnon drag) [27]. This magnetic contribution is described by the Bloch law [START_REF] Kittel | Introduction to solid state physics[END_REF], i.e. the spontaneous magnetization decreases with temperature at T 3/2 when T is small [START_REF] Callaway | Quantum theory of the solid state[END_REF].

Analogous temperature dependence of MCTP has been directly derived from a theoretical model of magnon-assisted transport in a mesoscopic tunnel junction between a ferromagnetic metal and a normal (nonmagnetic) metal [30] for a magnetic component of thermoelectric power.

The experimental curve shape of thermopower S(T) of the spinel single crystal under study (Fig. 1) can be easily fitted by the modified Matoba, Anzai and Fujimori semiempirical formula [31] including the magnetic contribution [13] as follows:

2 / 5 2 / 3 2 / 1 4 3 3 ) ( ) ( ) ( ) ( ) ( aa eff aa eff D D J T J T T T K I T H G F T E T D T S + ⋅ + ⋅ + + ⋅ + ⋅ + ⋅ = θ θ (4)
where θ D is the Debye temperature (see Appendix A), and aa eff J is the effective exchange integral for the first coordination sphere (see Appendix B). First and second terms in Eq. ( 4) is the diffusion component where the small correction E is the temperature dependence of D [32], third term in Eq. ( 4) is the phonon drag component, fourth term in Eq. ( 4) is the impurity component, and the last term in Eq. ( 4) is the magnon drag component. D, E, F, G, H, I and K in Eq. ( 4) (listed in Table II) are the curve-fitting parameters. The agreement index R between the experimental (S exp ) and the theoretical (S theor ) thermopowers is high (over 99 %, see Table 2).

The results of thermopower analysis of the single-crystalline CuCr 1.6 V 0.4 Se 4 depicted in Fig. 7 show that the intensity of the magnon drag component (S mag ) is the largest and reaches a maximum of 7.86 µV/K at 33 K. It means that the magnon excitations, usually driven by the double exchange mechanism, are observed only at low temperatures, and the maximum transfer of the magnon momentum to the electron gas may be a main reason for the observed the negative magnetoresistance effect. This effect disappears at higher temperatures.

The contribution to the total thermopower originates then mainly from the phonon component (S ph ) which reveals a maximum of 7.1 µV/K at 100 K, i.e. exactly in the theoretical temperature range θ D /10 -θ D /2 predicted by the Debye theory [27], where in our case θ D = 279.7 K (see Table 2). At high temperatures the diffusion component (S diff ) dominates, and according to the Mott formula [START_REF] Blatt | Thermoelectric power of metals[END_REF] it is proportional to temperature. The S diff = 5.6 µV/K at 300 K. The socalled impurity component of the thermopower (S imp ) described by the T 1/2 law [START_REF] Mott | Conduction in non-crystalline materials[END_REF] generates a maximum 1.51 µV/K at 300 K.

From the results of the magnetic considerations presented in Table 3 for the first two coordination spheres are positive and negative, respectively, and the absolute value of aa eff J is more than five times larger than ab eff J . The calculated magnetic parameters above mentioned well correlate with semiconducting properties and strongly reduced both the long and short range FM interactions of the CuCr 1.6 V 0.4 Se 4 spinel. For comparison, the relevant calculated magnetic parameters [13] of the parent CuCr 2 Se 4 metallic ferromagnet [START_REF] Lotgering | Proceedings of the International Conference on Magnetism[END_REF] are collected in Table 3.

Conclusions

We have shown that in ferromagnetic semiconductor CuCr 1.6 V 0.4 Se 4 , exhibiting high electrical conductivity the magnon excitations are connected with a double exchange mechanism existing apart from the superexchange interaction, like it was observed in the ferromagnetic spinel systems: Zn x Cu y Cr z Se 4 for y ≥ 0.8 [11], Cu x Ga y Cr z Se 4 for 0.036 ≤ y ≤ 0.394 [12], Cu x Co y Cr z Se 4 for 0.06 ≤ y ≤ 0.11 [13], and indicated by de Gennes in manganites [START_REF] De Gennes | [END_REF]. For comparison, in the CdCr 2-x Ga x Se 4 spinel system [10], the ferromagnetic coupling, being caused only by the superexchange mechanism, does not contribute to the spin wave excitations. The big value of ESR linewidth of CuCr 1.6 V 0.4 Se 4 and its strong temperature increase in PM state suggest the weakly exchange-coupled Cr-Cr pairs to the lattice in comparison with the fastrelaxing V 3+ ions. The spin-phonon coupling is explained as the several-stage relaxation process in which the V 3+ ions, the exchange subsystem and the calculated value of θ D for this spinel is of the same order as for the CuCr 2 Se 4 one (see Table 2).

Appendix B

To determine the exchange constants of the superexchange and double exchange interactions an estimation of the Cr 3+ and Cr 4+ portions is necessary. Portions x 3 and x 4 of the chromium ions: Cr 3+ and Cr 4+ , respectively, were calculated from the saturation magnetization M s :

M s = 2(g 3 S 3 x 3 + g 4 S 4 x 4 + g 4 S 4 x) (B1)
taking into account the following normalization condition: x 3 + x 4 + x =1, where M s = 4.66 µ B is the experimental value of the saturation magnetization, S 3 = 3/2 is the spin of Cr 3+ ion, S 4 = 1 is the spin of Cr 4+ and V 3+ ions, g 3 = 2.0 and g 4 = 1.86 are the Landé factors for Cr 3+ , Cr 4+ and V 3+ ions, respectively, and x = 0.2 is the normalized content of V 3+ ions.

The exchange constants of superexchange and double exchange interactions between the first two coordination spheres were calculated using the exchange Hamiltonian as follows:

H = H se + H de (B2)
where:

∑ ≠ ⋅ - = j i j i j i ij se S S J H , r r , ( B3
)
represents the superexchange Cr n+ -Se-Cr m+ and Cr m+ -Se-Se-Cr n+ interactions [4] with the integrals J ij and c aa = 6 and c ab = 12 numbers of nearest neighbours in

∑ ≠ ⋅ - = j i j i j i ij de S S b x x H , 4 3 6 1 r r , (B4)
represents the double exchange interactions [4] with x 3 and x 4 being the concentration of Cr 3+ and Cr 4+ ions, which is normalized in such way that x 3 + x 4 + x = 1 as from the ESR studies it follows that V 3+ ions are directly coupled to the lattice, and b ij is the hopping integral between the atomic t 2g states |i〉 and |j〉 for neighbours 〈i,j〉. We take the band factor as ∼x 3 x 4 corresponding to probability of finding one atom in the state Cr 3+ and the neighbouring one in the state Cr 4+ . The number 1/6 in the double exchange Hamiltonian (B4) is a constant of a bilinear term

j i S S r r ⋅
in the case of spin 1 [38]. Additionally, we take the hopping electron between two Cr ions on the same footing as the remaining ones. Because all t 2g electrons are quantum mechanically indistinguishable, the Cr cations supply both localized and itinerant 3d moments when Cr 4+ cations are present [4].

According to the Hamiltonian (B2) the effective exchange constants [4] for the first two coordination spheres are: (B6)

The J aa and b aa and J ab and b ab integrals for the first two coordination spheres, respectively, are calculated from the high temperature expansion of the magnetic susceptibility [4]. These calculations are based on the equations describing the follows [39]:

θ CW = θ se + θ de (B7)
where θ CW is the paramagnetic Curie-Weiss temperature (from experiment), θ se is the superexchange contribution to θ CW and θ de is the double exchange contribution to θ CW . The above contributions [4,39] can be read: Eq. (B11) allows to estimate the mixed valence (Cr 3+ ,Cr 4+ ) bandwidth, W d , of the 3d t 2g band from the relation [4]:

∑ = p p p se c J X
W d = 2B. (B14)
Using the Holland and Brown equations [40] we can calculate the superexchange integrals J aa and J ab for the first two coordination spheres, taking into account the estimated values of θ se from Eq. (B12) and the experimental values of the Curie temperature T C .

θ se = 5(3J aa + 18J ab ), (B15)

T C = 5(J aa -2J ab ). (B16)
The double exchange b aa and b ab integrals 4 can be calculated from the equation:

B = 6b aa + 36b ab , ( B17
)
where b aa stands for the contribution from the 6 nearest neighbours and b ab stands for the contributions from the 36 second nearest neighbours and from the Lotgering [START_REF] Lotgering | Proceedings of the International Conference on Magnetism[END_REF] considerations:

b aa = 5b ab (B18)
The calculated magnetic parameters: θ se , θ de , B, W d , J aa , J ab , J ab , b aa , b ab , aa eff J , ab eff J are collected in Table 3. Oe⋅cm 3 /g and µ s = 4.66 µ B are the saturation magnetisation and saturation magnetic moment, respectively. The saturation state is marked by dashed line.

F

Inset: the onset of the σ (H) dependence up to 1.0 kOe.

  Spellman, USA). The tungsten pinhole collimator was used to reduce the size of analyzed area. For the collimators of the size holes of 100, 200, 400, 1000 and 2000 µm, the following focal spot sizes are obtained: 169, 280, 581, 1469, 2888 µm. The X-ray spectra of the samples were collected by thermoelectrically cooled Si-PIN detector (XR-100CR Amptek, Bedford, MA, USA) of 6 mm 2 active area, 500 µm crystal thickness and 12.5 µm Be window thickness. The Si-PIN detector cooled to the temperature of ca. -55°C reaches

  recorded. A continuous nitrogen flow cryostat was used to study temperature dependence. For ESR measurements the obtained crystals were crushed into powder and diluted in the paraffin to reduce the skin effect. The shape and area spectra were analyzed by numerical method.

.

  The dc mass susceptibility vs. temperature, χ σ (T), shows a ferromagnetic order with the Curie temperature T C = 193.2 K. Above 200 K, behavior with a positive CW temperature θ CW = 207 K and an effective moment 85 µ B /f.u., where the molar mass M ≈ 482.96 g/mol and

  1.6 V 0.4 Se 4 consist of a single line in the range from room temperature to T 1 = 200 K. At room temperature the intensity line is very weak. Below T 1 the spectra were simulated with two profiles. The Figures 5(a), (b) and (c) show the temperature dependence of the ESR parameters: ∆B -linewidth, B r -resonance field and DI -intensity of the spectrum. The corresponding values of the ∆B and B r were obtained as the fitting parameters and the DI values were calculated as double integration of the spectrum. Starting from the T = 272 K (∆B = 159.2 mT) the linewidth values rapidly decrease as the temperature is lowered to T 1 where ∆B = 27.8 mT. Next, the linewidth passes through a minimum and with decreasing temperature shows a continuous broadening. The line-shape analysis shows that the ESR line remains Lorentzian down to T = 212 K, and below this temperature a deviation towards an asymmetrical line occurs. The shift of the resonance field (B r ) from the high temperature value is observed below T = 212 K. In the temperature range from T = 193 K to T = 85 K this effect is very significant. In Fig. 5(c) the temperature evolution of the intensity of the ESR spectrum is shown with the values reduced to T = 272 K. In the temperature range from T = 225 K to T = 183 K, the intensity rapidly increases. The intensity of ESR spectrum defined as DI (double integration of the spectrum) should be proportional to the spin susceptibility of the sample. The Curie temperature estimated from the DI(T) dependence equals 193.8 K. The agreement with the susceptibility measurements is satisfactory thus the ESR data appear to be consistent with the magnetic ones.

  subsystem of conduction electrons. That model explains also a big ESR linewidth for CuCr 1.6 V 0.4 Se 4 and its temperature dependence in the PM state (Fig.5(a)). The parameter of the thermal broadening linewidth b = d(∆B)/dT = 1.9mT/K is rather high and means a fast energy flow from the spin subsystem to the CuCr 1.6 V 0.4 Se 4 lattice[START_REF] Barnes | [END_REF]. We suppose that the process of spin-lattice

13 =

 13 growth of linewidth (∆B crit ) reflects the increase in the lifetime and the correlation length of the critical fluctuations. The appearance of the short-range magnetic ordering is confirmed by the temperature dependence of the spectrum intensity (Fig.5(c)). As the temperature is reduced from about T = 225 K, the values for intensity rapidly increase. The ferromagnetic ordering temperature T C 193.8 K can be inferred from the location of coincident anomalies in the ESR data: ∆B, B r , DI (Fig.5). The ESR spectrum of CuCr 1.6 V 0.4 Se 4 observed below T = 193 K undergoes qualitative changes and transforms from paramagnetic (EPR) into ferromagnetic (FMR) resonance. At low temperatures the experimental spectrum was fitted by two profiles shown in Fig.5(a) and (b). It is known, that

  (see Appendix B) it follows that with increasing vanadium content x: (1) contribution of the superexchange interaction to the Curie-Weiss temperature θ CW suddenly increases from 90 K for CuCr 2 Se 4 to 163 K for CuCr 1.6 V 0.4 Se 4 , (2) contribution of the double exchange interaction to the Curie-Weiss temperature θ CW and the total hopping integral B rapidly decrease, and (3) the W d bandwidth of the 3d t 2g band due to Cr 3+ and Cr 4+ ions decreases from 0.76 eV for CuCr 2 Se 4 to 0.11 eV for CuCr 1.6 V 0.4 Se 4 . In the studied single crystal the superexchange J aa and J ab integrals for the first two coordination spheres are positive and negative, respectively, the absolute value of J aa is over nine times larger than J ab , the double exchange b aa and b ab integrals for the first two coordination spheres are positive and b aa is five times larger than b ab , and the effective exchange

  act as the intermediate reservoirs.Our results mean that in the mixed Cr valence band with a narrow bandwidth of 0.11 eV a coherent transfer of the magnon momentum to the electron gas results in the negative magnetoresistance effect in the single crystal under study.

  second Cr-Cr coordination spheres, respectively. The spins i the quasiclassical vectors.

  paramagnetic Curie-Weiss temperature resulting from the separate mechanisms of the superexchange and double exchange interactions as

θ

  was taken from Ref.4 and equal to 90 K for the CuCr 2 Se 4 metallic ferromagnet, which means only the pure superexchange contribution, hopping integral for the first and second coordination spheres and X is a mixture of spins due to the presence of Cr 3+ , Cr 4+ and V 3+ ions. A summation over p means the summation over the consecutive coordination spheres, each of them containing c p neighbours of a given central atom. We should set: X = x 3 S 3 (S 3 + 1) + x 4 S 4 (S 4 + 1) + xS 4 (S 4 + 1).(B10) Finally, Eq. (B7) can be written: the following superexchange and double exchange contributions to θ CW , respectively:

Figure 1 .

 1 Figure 1. Electrical resistivity ρ and thermoelectric power S vs. temperature

Figure 2 .

 2 Figure 2. Magnetoresistance ∆ρ/ρ 0 vs. magnetic induction B at 4.3, 10, 15, 20

Figure 3 .

 3 Figure 3. Temperature dependences of mass susceptibility χ σ and reciprocal

Figure 4 .

 4 Figure 4. Mass magnetisation σ vs. magnetic field H at 4.6 K. σ s = 53.84

Figure 5 .

 5 Figure 5. The temperature dependence of the ESR parameters: (a) the resonance

29 Figure 6 .

 296 Figure 6. Model of paramagnetic relaxation processes of the CuCr 1.6 V 0.4 Se 4

Figure 7 .

 7 Figure 7. The thermopower analysis of the CuCr 1.6 V 0.4 Se 4 spinel where S exp is

Fig. 2

 2 Fig.2

  Fig.3

Fig. 4

 4 Fig.4

  Fig.5

  Fig.6

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Table 1 .

 1 Crystal data, experimental details and structure refinement results for the CuCr 1.6 V 0.4 Se 4 single crystal.

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Table 2 .

 2 The fitting parameters: D, E, F, G, H, I and K in Eq. (4) of the thermoelectric power analysis of the CuCr 1.6 V 0.4 Se 4 single crystal.R is the agreement index. The Debye temperature θ D has been calculated from Eq. (A1).

	D	E	F	G	H	I	K	R	θ D
	(µV/K 2 )	(µV/K 4 )	(µV/K)	(10 -3 )	(µV/K 1.5 )	(µV/K)	(10 -3 )	%	(K)
	0.0227 1.3⋅10 -15 3.473	0.0054 0.0276	2.169	0.0071	99.3 279.7

Table 3 .

 3 The calculated magnetic parameters: X, θ se , θ de , B, W d , J aa , J ab , b aa , b ab ,

	aa eff J and ab eff J of the CuCr 2-2x V 2x Se 4 spinel system.
	x	x 3	x 4	X	θ se	θ de	B	W d	J aa	J ab	b aa	b ab	J	aa eff	J	ab eff
	(V 3+ )	(Cr 3+ )	(Cr 4+ )		(K)	(K)	(K)	(eV)	(K)	(K)	(K)	(K)	(K)	(K)
	0	0.512 0.488 2.896 90	340 4391 0.76 63.9	-9.65 332.65 65.53 466.51	-83.05
	0.2	0.41	0.39	2.718 163 44	607 0.11 31.7	-3.47 45.98 9.2	197.55	-38.7
	The values for x = 0.0 are taken from Ref. 13 for comparison.

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Acknowledgments

This work was partly supported by Ministry of Science (Poland) and funded from science resources for years 2007-2009 as a research project (Project No. N N204 178433). We are indebted to the European Community for the UPGOW fellowship awarded to S. Mazur for the year 2008-09.

Appendix A

The Debye temperature [36], θ D , has been estimated from the following formula:

where h is the Planck constant, k is the Boltzmann constant, N = 56 is the number of atoms in the spinel unit cell, V (= a 3 , where a is the lattice parameter) is the volume of the spinel unit cell and v = 809 m/s is the sound speed in the CuCr 2 Se 4 spinel calculated from Eq. (A1) and based on the value of θ D = 280 K estimated from the heat capacity measurements and the Grüneisen constant. 37 We assumed the same value of the sound speed in the CuCr 1.6 V 0.4 Se 4 spinel and