Léa Bordes

Nicolas Dumont

Anne Lespine

Elise Souil

Jean-François Sutra

Françoise Prévot

Christelle Grisez

Lola Romanos

Aurélie Dailledouze

Philippe Jacquiet
email: philippe.jacquiet@envt.fr

First report of multiple resistance to eprinomectin and benzimidazole in Haemonchus contortus on a dairy goat farm in France

Keywords: Goat, Multiple anthelmintic resistance, Benzimidazole, Eprinomectin, Haemonchus contortus Graphical

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Gastrointestinal nematode (GIN) infection is one of the main health threats for grazing dairy goats. Three GIN species predominate in the digestive helminthofauna of goats in temperate countries of Western Europe: Teladorsagia circumcincta, H. contortus and Trichostrongylus colubriformis. All these species cause growth retardation and milk production losses; however, only H. contortus infections can lead to high mortality rates in kids as well as in adult dairy goats. H. contortus proliferates particularly when climatic conditions are optimal for the development of the free-living stages on pastures (wet and warm periods) and when flock-management practices favor infection (permanent use of small pastures close to the farm). To regulate infection, control measures are thus essential and depend heavily on anthelmintics (AHs) [START_REF] Hoste | Control of gastrointestinal parasitism with nematodes in dairy goats by treating the host category at risk[END_REF]. Since the 1960s, benzimidazole which has a zero-day milk withdrawal period has been used extensively, but given the very high prevalence of benzimidazole resistance in France, the use of this family of AHs is no longer relevant on many dairy sheep or goat farms [START_REF] Chartier | Prevalence of anthelmintic resistant nematodes in sheep and goats in Western France[END_REF][START_REF] Chartier | Prevalence of anthelmintic resistance in gastrointestinal nematodes of dairy goats under extensive management conditions in southwestern France[END_REF][START_REF] Geurden | Anthelmintic resistance and multidrug resistance in sheep gastro-intestinal nematodes in France, Greece and Italy[END_REF]. Levamisole is not approved for use in dairy goats and closantel is tolerated only at the beginning of the dry period, due to the persistence of the drug in milk and milk products [START_REF] Iezzi | Closantel plasma and milk disposition in dairy goats: assessment of drug residues in cheese and ricotta[END_REF]. Oral moxidectin is a macrocyclic lactone approved for use in dairy goats and can be used as an alternative to benzimidazole with a short milk withdrawal period (5 days). However, GIN resistance to moxidectin has begun to appear in France, mainly on meat sheep farms [START_REF] Paraud | Cross-resistance to moxidectin and ivermectin on a meat sheep farm in France[END_REF]. On dairy goat farms, milk is used to make cheese and the goats lactate from 9 to 10 months per year, so the milk residue issue is of major concern and farmers favor the drugs with the shortest milk withdrawal period when treating their animals.

In the nineties, the macrocyclic lactone eprinomectin was developed and approved for use against a broad range of endoparasites (pulmonary and gastro-intestinal nematodes) and ectoparasites (mange, lice and warble fly) in cattle of all ages [START_REF] Shoop | Eprinomectin: a novel avermectin for use as a topical endectocide for cattle[END_REF]. Because of its interestingly low partitioning in milk [START_REF] Alvinerie | Pharmacokinetics of eprinomectin in plasma and milk following topical administration to lactating dairy cattle[END_REF], it is the only endectocide approved for use during lactation with a zero-day milk withdrawal period. Until 2016, eprinomectin was available for dairy cattle as a 0.5 mg/kg topical formulation ("pour-on"), but is now also marketed for subcutaneous administration at 0.2 mg/kg for use in cattle only. Since 2016, pour-on eprinomectin is also marketed for dairy sheep and goats with a zero-day milk withdrawal period [START_REF] Hamel | Eprinomectin pour-on (EPRINEX® Pour-on, Merial): efficacy against gastrointestinal and pulmonary nematodes and pharmacokinetics in sheep[END_REF][START_REF] Hamel | Eprinomectin pour-on: Prevention of gastrointestinal and pulmonary nematode infections in sheep[END_REF]. However, the pour-on formulation has several disadvantages such as low plasma levels and high individual variability, making the therapeutic outcome uncertain. In order to improve the efficacy of eprinomectin, the dose was increased from 0.5 mg/kg to 1 mg/kg in sheep and goats [START_REF] Hamel | Eprinomectin pour-on (EPRINEX® Pour-on, Merial): efficacy against gastrointestinal and pulmonary nematodes and pharmacokinetics in sheep[END_REF][START_REF] Chartier | Activity of eprinomectin in goats against experimental infections with Haemonchus contortus, Teladorsagia circumcincta and Trichostrongylus colubriformis[END_REF]. In addition, on farms, pour-on eprinomectin is sometimes given orally to dairy sheep and goats to improve its efficacy [START_REF] Badie | Kinetics and anthelmintic efficacy of topical eprinomectin when given orally to goats[END_REF]. In parallel, the pharmacokinetics and efficacy of a subcutaneous formulation of eprinomectin (0.2 mg/kg) was evaluated in sheep [START_REF] Termatzidou | Anthelmintic activity of injectable eprinomectin (eprecis® 20 mg/mL) in naturally infected dairy sheep[END_REF] and goats [START_REF] Lespine | Eprinomectin in goat: assessment of subcutaneous administration[END_REF][START_REF] Briqué-Pellet | Pharmacokinetics and anthelmintic efficacy of injectable eprinomectin in goats[END_REF] and displayed high efficacy against GIN. However, this formulation is not yet approved for small ruminants, and if used "off-label" then a mandatory milk withdrawal period of seven days is required. Nevertheless, eprinomectin is now considered as the main AH drug in dairy sheep and goats in France and is widely used during the lactation period.

It is well established that the frequent use of the same AH on a farm favors the selection of resistant GIN populations. Increasing numbers of cases of multiple resistance in GIN of small ruminants are being documented in Europe [START_REF] Ploeger | Alarming levels of anthelmintic resistance against gastrointestinal nematodes in sheep in the Netherlands[END_REF] and France [START_REF] Paraud | Cross-resistance to moxidectin and ivermectin on a meat sheep farm in France[END_REF][START_REF] Cazajous | Multiple-resistance to ivermectin and benzimidazole of a Haemonchus contortus population in a sheep flock from mainland France, first report[END_REF]; however, data regarding eprinomectin resistance in GIN populations are still scarce, and no cases of resistance have been reported in France. In Brazil, some eprinomectin-resistant populations of Cooperia spp., Haemonchus spp., Oesophagostomum spp., Ostertagia spp. and Trichostrongylus spp. have been reported in cattle [START_REF] Ramos | Anthelmintic resistance in gastrointestinal nematodes of beef cattle in the state of Rio Grande do Sul, Brazil[END_REF]. Eprinomectin-resistant H. contortus populations have been recorded in goats from Switzerland and Southern Germany [START_REF] Scheuerle | Anthelminthic resistance of Haemonchus contortus in small ruminants in Switzerland and Southern Germany[END_REF][START_REF] Murri | Frequency of eprinomectin resistance in gastrointestinal nematodes of goats in canton Berne, Switzerland[END_REF]. In northern Italy [START_REF] Zanzani | Gastrointestinal nematodes of dairy goats, anthelmintic resistance and practices of parasite control in Northern Italy[END_REF], eprinomectin resistance was suspected on one farm but was not confirmed at a later date. It is important to note that since plasma concentrations of the drug were not reported in these studies, we cannot exclude that the loss of efficacy was due to low exposure of the worms to the drug, rather than true worm resistance.

In the present study, we report the first case of multiple resistance to eprinomectin and benzimidazole in H. contortus on a dairy goat farm in southwestern France. This study was performed according to the WAAVP guidelines [START_REF] Coles | World Association for the Advancement of Veterinary Parasitology (WAAVP) methods for the detection of anthelmintic resistance in nematodes of veterinary importance[END_REF]. Multiple resistant species were identified both by the morphology of infective larvae [START_REF] Van Wyk | Morphological identification of parasitic nematode infective larvae of small ruminants and cattle: A practical lab guide[END_REF] and using molecular tools [START_REF] Milhes | A real-time PCR approach to identify anthelmintic-resistant nematodes in sheep farms[END_REF].

Plasma concentrations of eprinomectin were measured to determine the level of exposure of the worms to the AH in the treated animals. Finally, the factors that may be involved in the selection of resistant GIN populations on this farm are discussed and recommendations for dairy goat farmers are provided.

Materials and methods

Farm history

This study was performed on a farm comprising 70 adult dairy goats of the Alpine breed, located in the French Pyrénées-Atlantiques département (a département is a French administrative and territorial unit) in southwestern France. This flock was first established in 2012 with the purchase of 30 one-month-old kids from several different flocks. The kids were not infected with gastro-intestinal nematodes because they were born and kept indoors until the date of purchase. Each year since 2012, all does undergo artificial insemination (AI) in September, and non-pregnant does after AI are mated with bucks in October.

The kids, born in February or March, remain indoors until aged 1 year. Lactating goats are kept indoors from September to March but are allowed to graze the rest of the year. More precisely, a first pasture of 4 ha is grazed two to three hours per day from March to June.

From July to September, a second pasture of 1 ha is grazed. As grass availability is not sufficient to cover the nutritional needs of the lactating animals, they are given concentrates two times per day.

Lactating goats were treated regularly with anthelmintics from 2012 to 2017: twice a year with eprinomectin (EPRINEX Multi ® , Boeringher Ingelheim, 1 mg/kg of bodyweight (BW)) in June and August. A third treatment was administered every year in December (SUPAVERM ® , Elanco France, with closantel, 10 mg/kg BW and mebendazole, 15 mg/kg BW) to control gastro-intestinal nematodes and liver fluke.

In 2017, the farm experienced an outbreak of enterotoxemia which was finally contained by vaccination. In summer 2018, lactating goats showed weakness, facial edema and pale ocular mucosae associated with a high mortality rate (17%, whatever the age of adult goats). The flock was treated in June 2018 with EPRINEX Multi ® (1 mg/kg BW), but no improvement in the goats' health was observed. Further fecal examinations were performed on July 20 th and showed substantial levels of GIN egg excretions (composite fecal egg count of 1,650 eggs per gram (EPG)). The goats were immediately treated with EPRECIS ® (CEVA Santé Animale, 0.2 mg/kg BW subcutaneously). However, a second round of fecal examinations fourteen days later did not show any reduction in GIN egg excretion (composite fecal egg count of 2,500 EPG).

At housing (beginning of October), another series of analyses showed high egg excretions in five out of 10 lactating goats (from 1,900 to 11,450 EPG) and poor hematological values (packed cell volumes below 20% in five animals). Subcutaneous eprinomectin (EPRECIS ® 0.2 mg/kg BW) was again administered to these five high egg-shedding goats with poor reduction values again 14 days later (reduction rate: 63%). A fecal egg count reduction test following eprinomectin treatment (both topical and injectable routes) was therefore performed in November 2018. At the request of both the vet practitioner and goat farmer, the efficacies of moxidectin and fenbendazole were also tested on this farm.

Study design, efficacy calculation and evaluation of anthelmintic resistance

According to the guidelines of the WAAVP [START_REF] Coles | World Association for the Advancement of Veterinary Parasitology (WAAVP) methods for the detection of anthelmintic resistance in nematodes of veterinary importance[END_REF], the FECRT was performed on this flock.

Firstly, in November 2018, fecal samples were collected for the entire adult flock (70 goats) to measure individual GIN egg excretion. Animals were ranked according to their pretreatment Fecal Egg Count (FEC). The modified-Mc-Master method [START_REF] Raynaud | Etude de l'efficacité d'une technique de coproscopie quantitative pour le diagnostic de routine et le contrôle des infestations parasitaires des bovins, ovins, équins et porcins[END_REF], with a sensitivity of 15 EPG, was used to determine the individual fecal egg counts. Based on these results, the 50 goats with the highest FECs were allocated to five well-balanced groups of ten animals according to their FEC and age. One group remained untreated (control group), while the other four groups were treated with commercially available anthelmintics: injectable eprinomectin (Inj-EPRI) (EPRECIS ® , CEVA Santé Animale, 0.2 mg/kg BW subcutaneously), topical eprinomectin (pour-on EPRI) (EPRINEX Multi ® , Boeringher Ingelheim, 1 mg/kg BW, along the dorsal line from the withers to the tail head), oral moxidectin (MOX) (CYDECTINE 0.1% ® , ZOETIS, 0.2 mg/kg BW), and oral fenbendazole (FBZ) (PANACUR ® , MSD Animal Health, 5 mg/kg BW). Treatment was initiated (day 0 (D0)) as soon as the FEC results were known (24 hours post-sampling) by the vet practitioner. Young animals (born in 2017) received doses equivalent to 70 kg BW, whereas adult goats received doses equivalent to 80 to 100 kg BW. Based on the goats' body condition scores at the time of the study, these body weights were clearly overestimated. At day 14 (D14), individual fecal samples were collected again. The animals remained indoors during the whole test.

To calculate the percentage of reduction, we used the formula: Efficacy = 100 x (1 -arithmetic mean EPG of the treated group at day 14 / arithmetic mean EPG of the control group at day 14).

WAAVP guidelines state that anthelminthic resistance (AR) occurs when the percentage of reduction in egg counts is less than 95% and when the 95% confidence interval (CI) is less than 90%. If only one of the two criteria was met, the finding should be recorded as suspected AR (SAR) [START_REF] Coles | World Association for the Advancement of Veterinary Parasitology (WAAVP) methods for the detection of anthelmintic resistance in nematodes of veterinary importance[END_REF].

Identification of gastrointestinal nematode species

To identify GIN species in each group, a composite larval culture was made at each date (D0 and D14) and for each group. Each animal within a group contributed more or less equally to the composite larval culture (4 to 5 g of feces) but for some animals, this amount of fecal material was not available. All composite larval cultures were incubated for 12 days at 24 °C ± 1 °C and humidified every two days with tap water. Third stage larvae (L3s) were recovered by filling the beaker with tap water at room temperature (+/-25 °C) and inverting it on a Petri dish [26]. In a first step, L3s were collected in a volume of 40-45 mL of tap water then centrifuged (10 minutes at 4,500 rpm) to obtain a final suspension of 5 mL. These suspensions were stored at 4 °C until the counting and identification step. Morphological identification of the larvae was performed according to the criteria of Van Wyck and Mayhew [START_REF] Van Wyk | Morphological identification of parasitic nematode infective larvae of small ruminants and cattle: A practical lab guide[END_REF]. In addition, molecular identification of GIN species was performed using real-time PCR according to Milhes et al. [START_REF] Milhes | A real-time PCR approach to identify anthelmintic-resistant nematodes in sheep farms[END_REF]. Briefly, genomic DNA was extracted and purified from 500 µL of homogenized larval suspension using the DNeasy PowerSoil kit (QIAGEN). All experiments were based on real-time PCR assays using TaqMan technology in simplex PCR reactions. The primers and probes used are described in detail in Milhes et al. [START_REF] Milhes | A real-time PCR approach to identify anthelmintic-resistant nematodes in sheep farms[END_REF]. Standard curves for larval DNA quantitation were established for each PCR run and for the three species H. contortus, T. circumcincta and T. colubriformis. Additional data are available in Milhes et al. [START_REF] Milhes | A real-time PCR approach to identify anthelmintic-resistant nematodes in sheep farms[END_REF].

Blood sampling and eprinomectin determination

Blood samples were collected on post-treatment days 2 (D2) and 5 (D5) in the two eprinomectin groups. Eprinomectin plasma levels were determined using HPLC according to the previously described method [START_REF] Sutra | Determination of eprinomectin in plasma by high-performance liquid chromatography with automated solid phase extraction and fluorescence detection[END_REF]. The quantification limit of the method was 0.07 ng/mL, and inter-assay coefficients of variation were below 5%. The differences in the plasma levels of eprinomectin between the animals treated with the two formulations were examined using a Kruskal-Wallis non parametric test.

Results

Table A shows FEC and FECRT values at day 0 and day 14. At day 0, although significant between-individual variation was observed for FEC (for example in the control group: 200 to 10,350 EPG), the level of egg excretion was high in most of the goats, revealing substantial GIN infestation. In each treatment group, mean EPG values (from 1,280 to 2,135 EPG) were consistent the values of the control group (2,135 EPG), and the median values were similar in all groups (from 825 to 1,025 EPG).

At post-treatment day 14, mean EPG values remained high after treatment with FBZ (1,045 EPG), injectable EPRI (775 EPG) and pour-on EPRI (1,153 EPG), and in the same range as the untreated group (987 EPG). The median values for these treatment groups (ranging from 275 to 575 EPG) were slightly lower than for the control group (800 EPG). On the other hand, at day 14, no GIN eggs were observed in any of the individual samples from the MOX group, demonstrating the efficacy of this molecule on this farm.

Very poor reduction of egg excretion were calculated for the FBZ (-5.8%), injectable EPRI (21.5%) and pour-on EPRI (-16.7%) groups. Moreover, the lower limit of the 95% CI was -205%, -126% and -237% respectively in the 3 groups and the upper limit was 63%, 73% and 59%, respectively. According to the criteria of the WAAVP guidelines, AR was therefore demonstrated in these groups.

Tables B and C show the morphological and molecular identification results of L3s, respectively, following fecal culture. At day 0, H. contortus was the highly predominant species (93 to 99% of larvae by morphological identification depending on the group), followed by Trichostrongylus/Teladorsagia species. No larvae of Oesophagostomum venulosum or Chabertia ovina species were identified morphologically. This result was confirmed by real-time PCR, which demonstrated the predominance of H. contortus (87% to 98.6% of larvae). The DNA of Trichostrongylus spp. and T. circumcincta were detected in small and very small proportions respectively. Regarding morphological identifications at day 14, H. contortus was the unique species identified in all GIN-positive groups, including the untreated group. However, in the group treated with MOX, no larvae were observed. No 1 mg/kg BW). For injectable eprinomectin, the mean plasma concentrations were 10.2 ± 3.8 ng/mL and 3.6 ± 1.3 ng/mL at days 2 and 5, respectively. The mean eprinomectin plasma concentrations measured after pour-on administration of the drug were 2.91 ± 0.49 ng/ml and 1.77 ± 0.43 ng/ml at days 2 and 5. As expected, drug concentrations in plasma after pour-on administration were significantly lower than those measured after subcutaneous administration (p-value = 0.0002 and p-value = 0.0028 at days 2 and 5, respectively), while pour-on formulation contains five times more active drug. In all cases, there were high individual variations of plasma concentrations as observed.

Discussion

Our study shows a low fecal egg count reduction (FECR) after treatment with eprinomectin, whatever the route of administration, and with benzimidazole. This clearly indicates the lack of efficacy of these two drugs against H. contortus in the flock studied. Interestingly, this H. contortus population remained highly sensitive to moxidectin. While there is some degree of cross resistance between the avermectin and moxidectin [START_REF] Paraud | Cross-resistance to moxidectin and ivermectin on a meat sheep farm in France[END_REF], it is well documented that moxidectin at the recommended dose can still be highly effective in ivermectin-resistant nematode isolates in sheep or goats [START_REF] Prichard | Moxidectin and the avermectins: Consanguinity but not identity[END_REF]. A similar pattern was recently reported in a H. contortus isolate from a meat sheep farm located in the French Pyrenees [START_REF] Cazajous | Multiple-resistance to ivermectin and benzimidazole of a Haemonchus contortus population in a sheep flock from mainland France, first report[END_REF]. Since the lack of efficacy could be due to suboptimal exposition of the worms to the active compound, we measured the plasma concentrations of eprinomectin in the two eprinomectin-treated groups at days 2 and 5 following drug administration which correspond, respectively, to the peak drug concentration in the plasma and the last elimination phase. The concentrations measured in this study were in the same range as those reported in the literature using similar treatment protocols [START_REF] Briqué-Pellet | Pharmacokinetics and anthelmintic efficacy of injectable eprinomectin in goats[END_REF][START_REF] Rehbein | Pharmacokinetics and anthelmintic efficacy of topical eprinomectin in goats prevented from grooming[END_REF]. As expected, plasma levels were higher at days 2 and 5 in the animals that received subcutaneous eprinomectin compared with pour-on eprinomectin. The animals treated by subcutaneous injection hence displayed higher levels of the drug in their blood, even though they received five times less drug (0.2 mg/kg) than those treated by topical administration (1 mg/kg). These results are in complete agreement with the well-known poor availability when the drug is applied topically when compared with the subcutaneous route.

Nevertheless, most of the eprinomectin concentrations measured in both groups treated with eprinomectin were above 2 ng/mL, which is considered as the minimal active concentration for macrocyclic lactones [START_REF] Hamel | Eprinomectin pour-on (EPRINEX® Pour-on, Merial): efficacy against gastrointestinal and pulmonary nematodes and pharmacokinetics in sheep[END_REF][START_REF] Guillot | Concentration of ivermectin in bovine serum and its effect on the fecundity of psoroptic mange mites[END_REF], indicating that the lack of efficacy was not primarily due to suboptimal exposition of worms to the drug. Therefore, based on drug concentrations, FECRT and the presence of GIN DNA in bulk larval cultures, we conclude that the nematodes infecting the goats on this farm were resistant to eprinomectin. We clearly identified that H. contortus was the resistant species. Indeed, before implementing the FECRT on this dairy goat farm, a high mortality rate was reported with anemia (assessed by pale color of ocular mucosae and decreased packed cell volumes) and heavy egg excretions, which are typical symptoms of H. contortus infection. This was confirmed by the morphological and molecular identification of H. contortus infective larvae obtained after larval cultures in both eprinomectin-treated groups.

We noted some between-group variations of the number of L3/mL after larval culture. As previously stated, each composite larval culture was made with the same amount of individual fecal samples, but the feces of some animals were absent from the larval cultures due to an insufficient quantity of fecal material.

The identification of very few T. colubriformis revealed that multiple resistance appears to be marginal in this species. It is important to note that H. contortus was the only eprinomectinresistant species identified in a previous study [START_REF] Ramos | Anthelmintic resistance in gastrointestinal nematodes of beef cattle in the state of Rio Grande do Sul, Brazil[END_REF] and the main eprinomectin-resistant species in another [START_REF] Scheuerle | Anthelminthic resistance of Haemonchus contortus in small ruminants in Switzerland and Southern Germany[END_REF]. The primary causes of multiple drug resistance in H. contortus isolates are still under debate. The flock described in this study is relatively young. In 2012, the farmer purchased 30 one-month old female kids, which marked the beginning of the farm.

At that time, no quarantine or drench was applied to these young kids because they were kept indoors from their birth to their arrival in the flock. Interestingly, no new kids or bucks have been introduced to the flock since 2012. Consequently, multiple resistance to eprinomectin and benzimidazole probably occurred on this farm over a short period (2012 to 2018) and involved many different factors. Firstly, the animals were treated frequently over a short grazing period that did not exceed six months (from March to September). Three AHs were administered routinely to the adult goats: two eprinomectin treatments in June and August and another treatment that combined mebendazole and closantel in December. From 2012 to 2017, pour-on eprinomectin was used off-label, often at 0.5 mg/kg BW (i.e. the half of the recommended dose for goats), with a fixed milk withdrawal time of seven days. Sometimes the product was administered orally (Dumont, personal communication). Badie et al. [START_REF] Badie | Kinetics and anthelmintic efficacy of topical eprinomectin when given orally to goats[END_REF] reported that the oral administration of topical eprinomectin was 100% efficient against GIN and that maximum concentrations of eprinomectin residues determined in milk after oral treatment were below the Maximum Residue Limits for goat milk (defined by the European Medecines Agency, 27 July 2018, EMA/CVMP/607398/2017). However, changing the recommended route of administration of an AH drug is not allowed. In 2017 and 2018, the number of eprinomectin treatments was increased from two to three per year due to the low response of treated animals in terms of health and milk production recoveries. This probably accelerated the resistance selection process over the last two years. Most of goats were lean in 2017 and 2018, and the extensive loss of fat reserves may have contributed to diminished drug levels in heavily infected animals [START_REF] Lespine | The influence of parasitism on the pharmacokinetics of moxidectin in lambs[END_REF], leading to sub-therapeutic concentrations of the drug, and favoring the selection of drug resistant worms. Because pour-on eprinomectin is active for only a relatively short time (around 15 days) compared with the grazing period [START_REF] Chartier | Duration of activity of topical eprinomectin against experimental infections with Teladorsagia circumcincta and Trichostrongylus colubriformis in goats[END_REF][START_REF] Molina | Eprinomectin "pour-on" and the shedding of Haemonchus contortus eggs in experimentally infected goats[END_REF], goat farmers are advised to treat their animals several times during the grazing period. In the present study, the whole lactating adult flock was treated with eprinomectin each time, without prior FEC assessment. Therefore, almost no refugia were maintained for susceptible worms. Finally, the use of very small pastures (3 ha for 70 adult goats) inevitably led to overgrazing and heavy contamination, and hence repeated treatment. When combined, these factors created an ideal environment for development of multiple drug resistance.

In this context, it is important to propose appropriate recommendations for goat farmers in order to minimize the impact of resistant GINs on animals, reduce the population of resistant GINs and avoid the selecting of drug-resistant worms. Zero-grazing for a period of at least one year (to let multiple-resistant H. contortus infective larvae completely disappear from the pastures) was proposed as an option. The producer refused this option as his business image was based on grazing animals on pasture. Obviously, eprinomectin and benzimidazole cannot be now used on this farm. Since moxidectin inhibited egg excretion in the flock, it still represents a relevant alternative. But it should be used with parsimony to avoid rapid selection of resistance. Closantel was not tested in this study but should be considered as a possibility, and its efficacy on this H. contortus isolate should be tested as soon as possible. The recommended treatment scheme could be the alternation of oral moxidectin during the lactation period (five days of milk withdrawal) and closantel at the beginning of the dry period. However, to avoid the selection of moxidectin resistance, treatment should be targeted and selective after FECs performed on a representative number of animals of the flock. Hoste et al. [START_REF] Hoste | Control of gastrointestinal parasitism with nematodes in dairy goats by treating the host category at risk[END_REF] demonstrated that leaving a part of the flock without treatment during the grazing period is a possible option. Epidemiological observations identified the categories of host populations at risk (high milk producers and young adult goats). Targeted selective treatments on this basis were shown to be relevant in dairy goat farms as they did not compromise the annual amount of milk production on the farm and because this strategy reduces the amount of endectocide residues in feces which affect the non-target fauna [START_REF] Lumaret | Pour-on formulation of eprinomectin for cattle: Fecal elimination profile and effects on the development of the dung-inhabiting Diptera Neomyia cornicina (L.)(Muscidae)[END_REF]. Pasture management including frequent tillage and ray grass seeding could be useful to decrease the contamination of grazed areas with infective larvae between two grazing seasons.

The presence of a H. contortus isolate with multiple resistance to eprinomectin and benzimidazole is alarming in southwestern France. In this region, there are more than 2,500 dairy sheep farms (representing more than 500,000 dairy ewes) and rely exclusively on eprinomectin during lactation. Eprinomectin resistance is probably currently being selected on many of these farms, but data are not yet available. However, we cannot exclude the risk of multiple-resistant H. contortus dissemination [START_REF] Winter | Transmission of Helminths between Species of Ruminants in Austria Appears More Likely to Occur than Generally Assumed[END_REF] by wild animals (in particular roe deer) that could transport resistant worms from one farm to another. This threat should be taken into account and regular evaluations of anthelmintic efficacy should be proposed to sheep and goat farmers.

Conclusion

The multiple-resistant H. contortus isolate described in this study highlights the alarming situation regarding the control of gastrointestinal nematodes on livestock farms, mainly due to the low number of anthelmintic alternatives currently available for this farmer and the high pathogenicity of H. contortus. It is therefore important to establish a new therapeutic scheme to prevent the further spread of resistance. Strategies including maintaining refugia, alternation of molecules and targeted selective treatment have to be put into practice and appropriate GIN control recommendations should be communicated to all farmers and veterinarians in this region.

  GIN DNA was detected by real-time PCR in the larval culture of the MOX group. All other groups demonstrated a high proportion of H. contortus (89.3% to 98.7%) and a small proportion of Trichostrongylus spp. (1.3 to 10.7%). Individual plasma concentrations of eprinomectin at post-treatment days 2 and 5 are shown in

Figure A. 1 (

 1 Figure A.1 (injectable eprinomectin, 0.2 mg/kg BW) and Figure A.2 (pour-on eprinomectin,

Figure A :

 A Figure A: Eprinomectin concentrations in plasma at day 2 and day 5 post-treatment in goats treated (1) with injectable eprinomectin (Eprecis ND; 0.2 mg/kg BW), or (2) pour-on eprinomectin (Eprinex Multi ND; 1 mg/kg BW). Day 2 (blue) and day 5 (red).

Table A :

 A Fecal egg counts (mean, SD, median, minimum and maximum values) at day 0 and day 14 after treatment, number of positive animals at day 14 and results of fecal egg count reduction tests FECRT (% of reduction and 95% confidence interval, CI) within each group.

Table B :

 B Morphological identification of infective larvae obtained from bulk cultures in each group at day 0 and day14 post-treatment.

Table C :

 C Gastrointestinal nematode species in the different groups at day 0 and day 14 based on the molecular identification of L3 larvae.

Table B :

 B Morphological identification of infective larvae (according to Van Wyk and Mayhew[START_REF] Van Wyk | Morphological identification of parasitic nematode infective larvae of small ruminants and cattle: A practical lab guide[END_REF])

	499
			Total number of
	Group	Date	L3/ mL estimated by counting larvae in 80 microliters	Number of H. contortus larvae	Number of Teladorsagia/ Trichostrongylus larvae	Number of Oesophagostomum/ Chabertia larvae
	Control	D0	5,400	99	1	0
	Injectable eprinomectin	D0	3,250	95	4	0
	Pour-on eprinomectin	D0	2,625	93	5	0
	Oral moxidectin	D0	2,850	97	2	0
	Fenbendazole	D0	4,425	97	3	0
	Control	D14	337	43	0	0
	Injectable eprinomectin	D14	760	100	0	0
	Pour-on eprinomectin	D14	5,000	100	0	0
	Oral moxidectin	D14	0	0	0	0
	Fenbendazole	D14	337	47	0	0
	500
						20

Table C :

 C Real-time PCR according to Milhes et al.[START_REF] Milhes | A real-time PCR approach to identify anthelmintic-resistant nematodes in sheep farms[END_REF])

	501 503 505	Figure A.1: Figure A.2
		Group Control Injectable eprinomectin A.1: injectable formulation (Eprecis ND; 0.2 mg/kg BW) Date Total number of Molecular identification of infective larvae A.2: topical formulation (Eprinex Multi ND; 1 mg/kg L3/ mL estimated by H. contortus larvae Teladorsagia circumcincta larvae Trichostrongylus spp. larvae D0 29,486 96.7 0.1 3.2 D0 12,594 91.9 0.2 7.9 Pour-on eprinomectin D0 7,764 98.6 0.6 0.8 Oral moxidectin D0 11,466 87 0.8 12.2 Fenbendazole D0 15,696 91 0.4 8.6 Control D14 1,388 89.3 0 10.7 Injectable eprinomectin D14 6,214 98.7 0 1.3 Pour-on D14 35,882 97.4 0 2.6 Plasmatic concentration of Eprinomectin (ng/ml) 20047 20040 71035 20046 71027 40026 71031 60147 50041 60027 Plasmatic concentration of Eprinomectin (ng/ml) Real-Time PCR (% of % of % of BW)
	506	eprinomectin Oral moxidectin D14 Fenbendazole D14 71025 60144 71030 50043 20053 50055 60133 40027 40022 71028 0 0 0 728 97 0 3 0 Individual Result for each Goat
	504 502	Individual Result for each Goat
		21 22

Acknowledgements

The participation of the goat farmer in the study is greatly acknowledged.

Funding sources

This study was self-funded by the UMT Santé des Troupeaux des Petits Ruminants without any funding from private companies. This research did not receive any specific grants from funding agencies in the public, commercial, or not-for-profit sectors.

Conflict of interest

The authors declare that there were no conflicts of interest at any point of time.


Sunil Kumar

Damien Lenoble

Francis Maury

Naoufal Bahlawane
email: naoufal.bahlawane@list.lu

Synthesis of vanadium oxide films with controlled morphologies: Impact on the metal-insulator transition behaviour

Keywords: chemical vapour deposition, morphology, thin films, vanadium oxide

Precise control over the growth of VO 2 films with different morphologies is achieved by varying the deposition parameters in the DLI-MOCVD process such as temperature, pressure, concentration of precursor and time of deposition. In this study, thin films of VO 2 with wide range of morphologies having Metal to Insulator Transition (MIT) temperature of (t c ) $ 52 8C were deposited. Adjusting the process parameters has allowed the growth of highly porous nanocrystalline films and dense microcrystalline films with controlled crystallite size up to several hundred nanometres. Vanadium (V) oxy tri-isopropoxide was used in this study as a single source precursor. Porous films lead to a diffuse change in resistivity across the transition temperature while the crystalline films have sharp and high resistivity drop (Dr). This enabled a qualitative study of the MIT behaviour with respect to the microstructure of the films and correlates the effect of deposition conditions to the obtained morphologies. Fine control over the morphology without additional doping or post deposition process provides the ability to tailor VO 2 thin films for their respective applications. Scanning electron microscopy, Raman spectroscopy and X-ray diffraction were used to characterize the microstructure of the films while electrical resistivity measurements were carried out to observe the MIT behaviour of the films.

1 Introduction Vanadium (IV) oxide (VO 2 ) undergoes a reversible phase transition from an insulating monoclinic to a conducting rutile structure around the transition temperature (t c ) 68-70 8C [1]. Intrinsic material properties like electrical resistivity, optical transmission and mechanical strain undergo abrupt changes occurring at MIT [2]. This remarkable behaviour of the material has sparked immense interest and has been actively researched for potential applications ranging from ultra-fast nanoelectronic switches, transistors, thermoelectric devices and thermochromic smart windows [3][4][5][6].

In most cases, however, the focus is always mainly on reducing the t c closer to room temperature. Doping with impurities is one way to reduce the t c but resulted in sacrificing the sharpness of the phase transition [7][8][9][10]. Thin undoped nanocrystalline VO 2 films were reported exhibiting the MIT at t c as low as 55 8C. The growth of single phase VO 2 remains a challenge due to the fact that a number of stable phases of vanadium oxides could form in a narrow range of composition, thus, requiring a strict control on growth conditions for obtaining a single pure phase of VO 2 [11]. Various types of thin film deposition techniques have been employed for VO 2 film synthesis like pulsed laser deposition (PLD) [5,12], molecular beam epitaxy (MBE) [3], magnetron sputtering [13], chemical vapour deposition (CVD) [14][15][16][17], sol-gel processing [18,19] and atomic layer deposition [20][21][START_REF] Rampelberg | Low temperature plasma-enhanced ALD of vanadium nitride as copper diffusion barrier[END_REF]. In this study, we use direct liquid injection-metal organic chemical vapour deposition (DLI-MOCVD) and a single source precursor to grow VO 2 films of varying thickness and microstructure.

Several authors have reported results correlating the film thickness, grain size and crystallinity with the MIT [START_REF] Suh | [END_REF][24][25][26] and provided rather conflicting views relating film morphology and MIT behaviour as each case has only one or limited types of morphologies being studied. Therefore, it is important to properly understand this property with respect to various microstructures and their influence on the grain boundary density and crystallinity. In this study, we grow single phase VO 2 films of various morphologies and crystallinities by varying the deposition parameters to compare the MIT behaviour, hysteresis width (DT) and Dr corresponding to each unique microstructure.

2 Experimental 2.1 Deposition of VO 2 Thin films of VO 2 were deposited on silicon substrates by DLI-MOCVD in a warm wall vertical stagnation point flow reactor. Low concentration (5 Â 10 À3 M) ethanol solutions of 99.9% pure V oxy-tri-isopropoxide ([VO(O i Pr) 3 ] were used as the precursor. Liquid injection was performed in an evaporation tube maintained at 200 8C to secure an instantaneous vapourization of the precursor solution.

Substrates were maintained at a constant temperature within the 400-600 8C range during deposition. The precursor injection was maintained at 4 Hz with 2 ms opening time resulting in a liquid flow rate of 2.5 ml min À1 . Nitrogen was used as the carrier gas with a flow rate, 40 sccm and the chamber pressure being adjusted in the 3-9 mbar range.

Time of deposition was adjusted from 15 to 180 min allowing the growth of different film thicknesses. Substrates were allowed to cool to room temperature in nitrogen atmosphere under vacuum before withdrawing from the chamber and no post deposition annealing was performed. All the samples were handled in air after deposition.

Characterization

Film thickness was measured using an Alpha step d-500 Profilometer from KLA-Tencor and FEI Helios Nanolab 650 TM scanning electron microscope (SEM). Surface and cross-sectional morphologies were characterized by SEM at a working distance of 4 mm with 25 kV voltage.

X-ray diffraction (XRD) was used to characterize the films using the Bruker D8, with CuK a as the X-ray source. Data were collected in the u-2u (locked couple) mode from 2u of 20 to 608 with a step size of 0.058.

Raman spectroscopy was performed using an InVia Raman spectrometer from Renishaw with a 532 nm laser at low power of 0.2 mW to avoid non-controlled surface heating and the resulting phase transition upon laser irradiation. The resistivity of the films was measured using a Hall effect measurement system HMS-3000 in van der Pauw configuration. Temperature-dependent resistivity measurements were performed by placing the sample on a heating stage, while a thermocouple was placed on the sample to measure the surface temperature.

The characterization techniques employed in this study provide complementary insights to understand the interplay between the growth control parameters, film characteristics and the corresponding MIT behaviour.

3 Results and discussion CVD enables a convenient number of parameters of deposition namely (i) temperature of deposition, (ii) chamber pressure, (iii) concentration of the precursor and (iv) time of deposition. The main objective of this study is to obtain different morphologies of VO 2 by varying the growth parameters and to investigate their impact on MIT behaviour.

3.1 Deposition temperature An increase in grain size and crystalline morphology is observed when the temperature of deposition was increased from 450 to 600 8C. Figure 1 shows SEM micrographs of films deposited at various temperatures. It depicts a transition of morphology from a porous nanocrystalline to a compact microcrystalline to well-formed large crystallites of VO 2 . Thickness of the resulting films was measured at 1.4 mm, 800 and 200 nm for the deposition temperatures of 450, 500 and 600 8C, respectively.

X-ray diffractograms and Raman spectra, shown in Fig. 2, reveal single phase growth of monoclinic VO 2 in this temperature range, Raman vibrational peaks observed at 141,192, 222, 310, 387, 496 and 613 all correspond to Ag mode VO 2 . No significant change was observed in the Raman spectra when the deposition temperature was increased from 450 to 600 8C.

Precursor concentration

Film thickness and microstructure depend on precursor concentration for MOCVD process, a 2.5 and 5 mM of V oxy tri-isopropoxide in absolute ethanol solution was used as the precursor.

At 500 8C, VO 2 films with high porosity and thickness of 2.2 mm were grown with a 5 mM concentrated precursor solution which contrasts the films grown at 2.5 mM featuring a smooth surface comprising individual large crystallites of VO 2 of thickness 190 nm forming across the surface as shown in Fig. 3.

Deposition time

Highly crystalline wellordered VO 2 films were grown with 2.5 mM precursor concentration. Shown in Fig. 4 are cross-section SEM micrographs of films deposited at 575 8C for 15, 30 and 120 min, revealing film thicknesses of 90, 150 and 720 nm, respectively. The film thickness at different times corresponds to a constant growth rate (5.6 AE 0.6) nm min À1 without an incubation time.

The crystals grew anisotropically while retaining the single layer morphology. Hence, the increase in the film thickness is due to the growth and the merging of individual grains rather than a formation of multi-layered film composed of several microcrystalline grains. This structure reveals the high surface diffusion of the deposition species.

3.4 Chamber pressure With increasing chamber pressure, the films exhibit an increase in the surface roughness as shown in Fig. 5. The two-dimensional morphology at low pressure is partially replaced by a three-dimensional flaky sheet. Although cross-section inspection shows that early stage of deposition yields a similar morphology in the 3-9 mbar pressure range.

It is evident from Fig. 5e and f that formation of these flakes is favoured at higher chamber pressures, while simultaneously resulting in films with increased porosity and surface roughness. On close inspection of the flakes shown in Fig. 6, it was revealed that they are in fact formed by the aggregation of nanocrystalline grains of VO 2 similar to the film shown in Fig. 1a. These small nanocrystalline grains grew upwards at higher pressure and form a microstructure similar to petals in a rose flower. This type of morphology can be very well suited to gas sensing or absorbing applications where high porosity combined with high and accessible surface area play a key role in determining the effectiveness of the device.

MIT behaviour of different morphologies

It is important to note that due to seemingly numerous ways of synthesising VO 2 films exhibiting MIT behaviour and equally varied number if not more ways of measuring this transition, a wide variety of measurement procedures have been reported like resistivity, transmittance, Raman spectra across the t c . To be able to compare the DT and t c for different morphologies, we chose a simple four-point probe resistivity measurement system, which sounds as the most prevalent method depicting the quality of films while still obtaining enough information about the films characteristics.

Electrical resistivity of the films was measured during heating and cooling of the sample in the 20-90 8C temperature window. This offers a convenient way to measure DT which does not extend beyond this temperature interval.

It is widely reported that t c of VO 2 bulk crystals is between 68-72 8C [1] with the ratio of resistivity in insulating (monoclinic) phase to the metallic (rutile) phase of the order of 10 5 . Epitaxial films were reported to exhibit similar properties seen in single-bulk crystals [12,32]. Whereas polycrystalline films have the tendency to have a smaller-resistivity drop and broader-hysteresis curve [5,33,34].

The top and bottom hysteresis plots shown in Fig. 7 correspond to the films depicted in Fig. 4a andb, respectively, which have similar morphology. The films are composed of a single layer of well-formed grains. VO 2 film in Fig. 4a resembles an epitaxial film in appearance with all grains having the same size and shape resulting in higher resistivity drop (Dr) and a narrow DT of 7 8C. While the film in Fig. 4b has a larger grain size but lack uniform size distribution as observed in thinner films, resulting in a broader DT of 12 8C. Films with higher density of grain boundaries and smaller grain size exhibit a diffuse MIT behaviour with a broad DT. The t c for both film morphologies is same at $52 8C in the heating phase. In Fig. 8, the top and bottom hysteresis curves correspond to the morphologies as shown in Fig. 1a (porous) and b (compact), respectively.

Due to the smaller grain size and the porosity of the film in Fig. 1a, Dr is diminished and the DT is broadened. Films with a compact microstructure, as shown in Fig. 1b, exhibit a narrower DT and sharper Dr compared to a porous film. It is, however, interesting to note that the MIT temperature remains relatively unchanged at $52 8C during the heating phase irrespective of the microstructure. On comparing the hysteresis curves obtained from the morphologies of Figs. 4a and 1b shown as the bottom curves of Figs. 7 and8; we observe a higher Dr and lower DT for films with better crystallinity.

4 Conclusions Thin films of VO 2 with different microstructures and morphologies were grown by MOCVD starting from [VO(O i Pr) 3 ]. This process was used to grow films of thickness ranging from 90 nm to 3 mm with fine control over porosity, crystallinity and grain size. This study reveals that morphology of VO 2 plays a pivotal role on the MIT behaviour. Film thickness, grain size, porosity, crystallinity and shape of the crystallites all have an impact on the DT, Dr and t c . Having achieved a precise control over the growth of the films and the resulting microstructure, we have the freedom and ability to fine tune the film growth and MIT as per the desired application.

Figure 1

 1 Figure 1 SEM micrographs of VO 2 films (a), (b) and (c) deposited at 450, 500 and 600 8C, respectively (chamber pressure 6 mbar). The average grain size is (a) 14.8, (b) 15 and (c) 130 nm.

Figure 2 X

 2 Figure2X-ray diffraction (a) and Raman spectra (b) of films deposited at 450, 500 and 600 8C, respectively. The peaks indicated for XRD correspond to PDF data card no. 00-009-0142[8,24,27] and Raman peaks observed also indicate the formation of monoclinic VO 2[27][28][29][30][31].

Figure 3

 3 Figure 3 (a, c) Surface and (b, d) cross-section SEM micrographs of VO 2 films grown at 500 8C with 5 (a, b) and 2.5 mM (c, d) precursor concentration at total pressure 9 mbar.

Figure 4

 4 Figure 4 SEM cross-section micrographs of VO 2 films deposited at 575 8C for the duration of (a) 15, (b) 30 and (c) 120 min.

Figure 5

 5 Figure 5 (a) (b) (c) SEM surface micrographs and (d) (e) (f) are the cross-section of VO 2 films deposited at chamber pressure 3, 6 and 9 mbar, respectively (temperature 450 8C, concentration 2.5 mM).

Figure 6

 6 Figure 6 Surface micrographs of VO 2 film grown at 9 mbar shown at different magnifications (temperature 450 8C, concentration 2.5 mM). The average grain size is $16.45 nm.

Figure 7

 7 Figure 7 Comparison of the MIT behaviour of VO 2 film of thickness 150 and 90 nm for the top and bottom plot, respectively.

Figure 8

 8 Figure 8 Comparison of the MIT behaviour of VO 2 with porous (top) and compact (bottom) films with the thickness of 1.4 mm each.

Acknowledgement The authors would like to acknowledge Luxembourg Institute of Science and technology (LIST) for providing the financial support for this work conducted at its Materials Research and Technology Laboratory.


Florence Vanni

Me ´lanie Montaigu

Brigitte Caussat
email: brigitte.caussat@ensiacet.fr

Carine Ablitzer

Xavie ` Re Iltis

Me ´ryl Brothier

Prof Brigitte Caussat

Fluidized-Bed Chemical Vapor Deposition of Silicon on Very Dense Tungsten Powder

Keywords: Chemical vapor deposition, Fluidization, Silicon coating, Silane, Very dense powder

A very dense tungsten powder was coated by silicon from silane using the fluidized-bed chemical vapor deposition process. A reactor of reduced diameter was developed in order to decrease the weight of powders treated. Results show that the fluidization of this powder is possible in this reactor, but with non-optimal gas solid contact induced by the high powder density. Important disturbances of the fluidized-bed temperatures appeared in the presence of silane, due to a partial bed defluidization related to the cohesive nature of the deposit. These disturbances are clearly exacerbated by the exceptional density of the particles. This is probably why a powder agglomeration has been unavoidable when the bed temperature was too low and/or the silane inlet concentration too high. However, a set of operating parameters could be found, allowing a uniform and continuous coating of the whole powder surface.

Introduction

Fluidized-bed chemical vapor deposition (FBCVD) is an efficient process to uniformly coat powders by a great variety of materials [1]. FB reactors have excellent heat and mass transfer capacities, high throughput rates, and can operate continuously, thus reducing operating costs [START_REF] Ydstie | Solar Cells -Silicon Wafer-Based Technologies[END_REF][START_REF] Liu | [END_REF]. Silicon from silane (SiH 4 ) precursor is certainly the most studied coating material using this process because of its development for producing solar-grade silicon to replace the Siemens process [START_REF] Liu | [END_REF][4][5][6][7][8][9][10][11]. It is worth noting that the literature works have most often been conducted on easy-to-fluidize Geldart's group B powders [12], and employing FB reactors of internal diameters between 5 and 20 cm. The particles coated by Si FBCVD from silane are generally silicon seeds for the photovoltaic industry [START_REF] Liu | [END_REF][4][5][6][7][8][9][10][11].

New nuclear fuels with limited enrichment in 235 U are under development for research nuclear reactors. Powdered U(Mo) metallic fuels dispersed in an aluminum-base matrix appear among the most promising materials. However, interfacial interactions between the fuel and its matrix can occur under irradiation, leading to a huge swelling of the fuel [13]. A solution could be to coat the fuel powder by a barrier before its dispersion into the matrix. Silicon seems to be a good candidate [14]. Some promising irradiation tests have been performed with U(Mo) coated with Si by physical vapor deposition (PVD) [START_REF] Van Den Berghe | Proc. of Research Reactor Fuel Management (RRFM)[END_REF][START_REF] Van Den Berghe | Proc. of Research Reactor Fuel Management (RRFM)[END_REF]. FBCVD could be another way to provide U(Mo) coated with Si, but its evaluation remains to be done.

Galerie et al. [START_REF] Galerie | [END_REF] have analyzed the deposition kinetics during silicon FBCVD from silane on iron particles at temperatures lower than 400 °C without giving information on the process. To the best of our knowledge, no other study in literature deals with FBCVD of Si from silane on metallic particles.

The metallic fuel U(Mo) powder has a very high density of about 17 500 kg m -3 [START_REF] Ryu | European Research Reactor Conference[END_REF] close to that of tungsten (19 300 kg m -3 ). This explains why a tungsten powder of mean diameter close to that of the fuel powder has been used to analyze the feasibility to deposit silicon by FBCVD from silane. To the best of our knowledge, this process has never been used to coat such dense powders.

In a previous work of our group, the ability to fluidize this powder has been demonstrated in a reactor of 5 cm internal diameter [START_REF] Rodriguez | [END_REF]. However, the application requires minimizing the weight of powder used for each coating experiment. Consequently, in the present study, a FBCVD reactor of reduced diameter (3.8 cm) has been tested.

First, experimental results about the fluidization hydrodynamics of the tungsten powder in this reactor will be presented prior to detail the thermal behavior of the FBCVD process and then the results obtained about silicon deposition from silane.

Experimental

The FBCVD reactor for this study consists of a vertical cylindrical column of stainless steel, with 3.8 cm internal diameter and 1 m height. It is externally heated by a two-zone electrical furnace and the wall temperatures are monitored by two thermocouples. Five thermocouples are also bundled into a 6 mm diameter stainless-steel tube, placed inside the reactor in order to measure the bed temperatures at various heights. A sixth thermocouple is placed under the stainless-steel porous plate used for gas distribution. The inlet flow rates of silane (SiH 4 ; Air Liquide, N50) diluted in argon (Air Liquide, Alpha1) are controlled by a ball rotameter and a mass flow controller, respectively. A differential fast response pressure sensor with taps under the distributor and top of the column measures the total pressure drop across the bed. Moreover, an absolute pressure sensor allows monitoring the total pressure below the distributor. A DasyLab system enables the online acquisition of differential pressure, total pressure, and axial profile of bed temperatures.

The FBCVD reactor was also used to perform fluidization tests at room temperature and around 600 °C with pure argon as carrier gas. Some fluidization tests at room temperature were also carried out in a glass fluidization column with an internal diameter of 0.05 m and a height of 1 m, equipped with the same porous plate distributor. All experiments were performed under atmospheric pressure.

The tungsten powder was supplied by NEYCO (CERAC, Inc. T-1220). Its grain density is equal to 19 300 kg m -3 . The particle size distribution values measured by laser granulometry analysis on a Malvern MasterSizer Sirocco 2000 in dry mode as illustrated in Fig. 1 a indicated that the distribution of the particle diameters D 10 /D 90 is 50 μm/105 mm with a Sauter diameter D 3.2 of 70 mm.

Fluidization hydrodynamics was studied by plotting the bed pressure drop versus increasing and decreasing gas superficial velocity. A normalized bed pressure drop ΔP* was calculated by dividing the bed pressure drop measured experimentally by the theoretical bed pressure drop, equal to the bed weight per column surface area. A normalized bed expansion H* was also measured as the ratio between the expanded bed height and the initial fixed bed height.

The morphology of the initial powder and of the coated particles was observed by scanning electron microscopy (SEM; Philips XL 30 FEG and LEO 435 VP). SEM observations highlighted that tungsten particles are non-spherical and faceted, as demonstrated in Fig. 1 b. X-Ray diffraction (XRD) analyses were performed on the compacted powders before and after deposition on a Seifert-3000TT equipment.

Results and Discussion

All the results below were obtained without premixing of the powder once placed in the columns. Fig. 2 presents the normalized bed pressure drop curves obtained in the glass column of 5 cm and in the FBCVD reactor of 3.8 cm at ambient temperature, for 1.3 kg of powder. In both curves, a fluidization horizontal plateau is present for ΔP* close or equal to 1, proving that a complete fluidization of particles was achieved. In the glass column, a small part of powders remained stuck on the walls due to electrostatic effects, which could explain that the plateau is not exactly observed at ΔP* = 1 and that an hysteresis appears between increasing and decreasing curves.

The minimum fluidization velocity U mf was classically determined by the method of Davidson and Harrison [START_REF] Davidson | Fluidised Particles[END_REF] at decreasing gas velocity. A similar value of U mf was found in the two reactors, near to 2.7 cm s -1 . This value is very close to that previously found in the 5-cm stainless-steel FBCVD reactor using pure argon [START_REF] Rodriguez | [END_REF]. According to Liu et al. [22] and to Guo et al. [23], when the reduction of the reactor diameter leads to modifications of the FB hydrodynamics, the U mf value increases and a pressure overshoot appears on the pressure drop curves, due to wall effects. In our case, it can then be concluded that the FB hydrodynamics is not markedly modified by the reactor diameter reduction. Liu et al. found that wall effects only appear for reactors of diameter below 2 cm, called micro FB, which is coherent with our own results, even if the density of the powders is very different. The bed expansions generally vary between 1.2 and 2 for conventional fluidized beds [24,25]. In our case (Fig. 2 c), the bed expansions do not exceed 1.4, even for a fluidization ratio U/U mf of 6. This can be explained by the fact that in a fluidized bed the particles are submitted to three different major forces, the gravitational force, i.e., the weight of particles, the gas-particle interaction drag force, and the particle-particle or particle-wall interaction force. According to Wang et al. [26], when very dense particles are fluidized, the gravitational force dominates the others. As a consequence, the bed expansion is low meaning that the number and size of gas bubbles into the bed are low, too. Even if bubbles can have negative impacts such as gas bypassing through bubbles, it is clear that bubble motion and gas circulation around the bubbles contribute to good gas and solids mixing.

One of the most famous empirical correlations predicting the fluidized-bed expansion, especially for Geldart's group A particles, is that of Richardson and Zaki [27]. This correlation indicates that bed expansion decreases if the particle density increases, meaning that for very dense powders the gas-solid contact is of lower quality. A hysteresis exists between the increasing and decreasing expansion curves at low gas velocity. This means that the powder is aeratable as Geldart's group A particles, probably due to its quite low mean diameter [28]. So, even if non-optimal, the FB hydrodynamics seems convenient to ensure good thermal and mass transfers conditions necessary to perform CVD in this reactor with reduced diameter.

The operating conditions tested by FBCVD and the average bed temperatures before, during, and after each deposition experiment are detailed in Tab. 1. The operating parameters were chosen on the basis of previous results of our group [6,29], in order to not agglomerate the bed and to form silicon deposits of at least 0.5 mm thickness for an easier characterization. For all runs, the fluidization ratio U/U mf was fixed to 5.3 and the weight of powders was 1.5 kg corresponding to a ratio between the fixed bed height and the reactor diameter of 3.4.

The experimental results are summarized in Tab. 2. The silane conversion rates and weight percentages of silicon deposited on powders were deduced from bed weighing before and after deposition. A theoretical thickness of silicon was also calculated from the mass of deposited silicon on the bed, assuming that the deposit is uniform on the whole powder surface. The real thickness of the coatings was determined from SEM analyses in a cross section after mechanical polishing of embedded powders in epoxy or after crushing the coated particles.

As detailed in Tab. diameter of powders is slightly higher than the initial one, indicating that no bed agglomeration occurred, except for runs W5 and W7. The deposit thicknesses could not be deduced from these results because they are in the measurement uncertainty of the method.

Fig. 3 presents the thermal profiles obtained during runs W6, W5, and W7. They are characteristic of the whole thermal profiles obtained without and with bed agglomeration phenomena. The heating step duration was around 70 min. Once stable thermal profiles were obtained, the FB temperature gradient (thermocouples TC1 to TC5) before silane injection was lower than 10 °C. This confirms that the bed of powders was conveniently fluidized at high temperature. The temperature TC6 measured under the distributor was kept below 200 °C in order to not decompose silane before its entrance into the reactor and to not plug the distributor by silicon deposition.

It is worth noting that for experiments without any agglomeration phenomenon like run W6 (Fig. 3 a), as soon as silane is injected into the bed, a thermal gradient systematically appears, the bottom part of the bed being gradually colder. This gradient was on average close to 70 °C and it systematically decreases a few minutes after the end of silane injection, probably due to a partial and reversible defluidization of the bed.

For a deeper analysis of this phenomenon, Tab. 1 provides the average bed temperatures measured before, during, and after silane injection for each run. It appears that the mean temperatures in the presence of silane are always higher than those before silane injection, from 2 °C to 12 °C depending on the conditions tested. This could be due to an overheating of the furnace to meet its target wall temperature when the bed is partly defluidized, since the gas-solid heat transfers are certainly lower. The temperatures corresponding to the period of 15 min after deposition are also often higher than those before silane injection, certainly due to the thermal inertia of the reactor. The bed pressure drop (not shown) was close to the apparent bed weight before silane injection, slightly increased during deposition by 10 mbar on average, and recovered its initial value after deposition. It is well-known that silicon CVD from silane can involve some reversible disturbances of the bed thermal profile and pressure drop due to a partial defluidization of the bed, probably related to the appearance of short-lived agglomerates. Indeed, silicon dangling bonds are probably formed on the surface of each particle during deposition, acting as a glue for the surrounding particles [5,6].

Fig. 3 b presents the bed thermal profile during run W5, in which an irreversible agglomeration of powders occurred a few minutes after silane injection. A sharp increase of the bed pressure drop (not shown) and a marked thermal gradient of more than 200 °C were rapidly observed along the bed, leading to the immediate stop of silane feeding.

Run W7 began with 4 vol % of silane but since strong disturbances of temperatures and of bed pressure drop rapidly appeared, the silane molar fraction was reduced to 2.9 %, thus leading to more stable process conditions (Fig. 3 c). But the thermal gradient remained higher than for runs performed at lower inlet silane concentration. a) These runs have led to irreversible bed agglomeration.

In the case of silicon FBCVD on powders of conventional density, most often of Geldart's group B, and with reactors of larger diameter [START_REF] Liu | [END_REF][4][5][6][7][8][9][10][11], the bed thermal disturbances are clearly lower than in our case even for much higher silane inlet molar fractions. This means that neither the variations of the fluidization gas properties nor the reaction enthalpy can explain the bed pressure drop and thermal profiles disturbances we observed. The latter are clearly linked to the very high density of the tungsten particles.

As previously explained, fluidized particles are submitted to three different major forces, namely, the weight of particles, the gas-particle interaction drag force, and the particle-particle or particle-wall interaction force. Different particle-particle forces can coexist, which are either repulsive like the solid collision forces or attractive like the Van der Waals or the electrostatic forces. In the case of silicon deposition from silane, a specific attractive force appears, related to the deposition chemistry on the particle surface. As long as the repulsive forces are higher than the attractive ones, for worse only a partial defluidization is observed, leading to higher bed pressure drop and thermal profiles. When this is not the case, an irreversible agglomeration of particles occurs. In the case of the very dense tungsten particles, we have previously explained that the dominant force into the bed without silane is the weight of particles, meaning that the repulsive force linked to particle collisions is lower than for powders of more classical density. So, the silane concentrations for which agglomeration happens are lower than for conventional powders.

Agglomeration seems to be exalted if the silane concentration is increased (runs W5 and W7 compared to run W3) and/or if the bed temperature is decreased (run W5 compared to run W4). It is likely that the number of Si dangling bonds per unit surface area of powder increases with silane concentration. The chemistry and kinetics of silane pyrolysis and of silicon deposition are probably slightly different at 600 °C and at 620 °C, leading to more numerous dangling bonds at 600 °C.

As desired, for the runs without agglomeration, the silane conversion rate was always close to 100 % as detailed in Tab. 2. These good conversion rates are due to the combination of low inlet silane concentrations, sufficiently high bed temperatures, and large enough residence times of the reactive gas into the bed [29]. The theoretical thicknesses deduced from bed weighing before and after deposition were always larger than 0.5 mm as initially planned, except for the two peculiar runs W5 and W7.

SEM views of the powder surface before and after silicon deposition are displayed in Fig. 4. The difference between coated and uncoated tungsten surface is obvious: the nontreated surface of particles is relatively smooth, whereas the silicon deposit forms a nodular film. Such morphology is characteristic of silicon CVD films from silane for this range of temperatures under atmospheric pressure [5,6]. This is due to the fact that sili- con deposition in an FB reactor not only involves heterogeneous deposition mechanisms from silane, but also homogeneous formation of silicon nuclei in the gas phase, which can be scavenged by the bed particles [11]. It is worth noting that for all experiments the top part of the reactor was covered by a very thin brown layer probably formed of these homogeneously born silicon nuclei. No clear trend appears for the influence of bed temperature or of silane inlet concentration on the coating morphology, probably because the ranges of variation are too small. Fig. 5 presents SEM views of crushed powders after runs W4 and W6, on which the uniformity and continuity of the deposit on the tungsten surface clearly appears as its nodular morphology. On these SEM micrographs, it is not possible to distinguish an intermediate layer between the substrate and the Si layer that could indicate the formation of a silicide layer as reported by Yoon et al. [30] in a study about planar CVD of Si on W at higher temperatures of 1000 °C and more. It is worth noting that the deposit thickness can be estimated on these micrographs. For the whole runs without bed agglomeration, the measured thicknesses are close to those deduced from bed weighing as detailed in Tab. 2, thus proving the uniformity of the deposit over the whole powder surface. This is a major result since it indicates that even if the bed was partly defluidized, the mixing of the powders by the gas was intense enough to ensure uniform deposition conditions. Some XRD measurements were performed before and after experiments as illustrated in Fig. 6, revealing that for all the conditions tested, the silicon deposit is composed of amorphous and polycrystalline phases, with {111} and {220} preferential orientations. Such mixed product is a classical result for the range of temperatures tested [11]. These crystalline preferential orientations of silicon were also obtained in another work on planar substrates in the same range of temperatures [31]. No influence of the temperature appears on the XRD diffractometers, certainly due to the fact that the tested temperature range is quite small.

Conclusions

Very dense tungsten powders simulating nuclear fuel particles were coated by silicon from silane (SiH 4 ) using the fluidizedbed chemical vapor deposition process. A reactor of reduced diameter in comparison with previous works was selected in order to decrease the weight of powder.

First, it could be verified that the decrease of reactor diameter from 5 to 3.8 cm does not modify the fluidization behavior of the tungsten powder. These particles can fluidize with characteristics of Geldart's group A powders due to their small mean diameter, but with non-optimal gas solid contact certainly induced by their exceptional density.

Then, operating conditions involving inlet molar fractions of silane between 2.6 to 5.2 % in argon and bed temperatures between 590 °C and 625 °C were determined, allowing to uniformly deposit continuous silicon films of at least 0.5 mm thickness on the whole surface of the powder. The coatings are of nodular morphology and appear as mixed-phase, amorphous, and polycrystalline. When the inlet molar fraction of silane was too high and/or the bed temperature too low, an irreversible bed agglomeration occurred. Moreover, bed thermal gradients and pressure drop disturbances classically associated to this process were more intense than in previous works involving easy-to-fluidize powders and larger reactor diameters. This could be due mainly to the high density of the tungsten powder and also to the decrease of reactor diameter, both reducing the intensity of the gas-solid mixing and thus favoring defluidization phenomena induced by the cohesive nature of the silicon deposit.

These results prove that the FBCVD process is an appropriate technology to deposit silicon coatings on very dense particles, even in a reactor of reduced diameter. New experiments are in progress in order to study higher deposition temperatures to better meet barrier requirements for nuclear fuel applications.

Figure 1 .

 1 Figure 1. (a) Volume diameter distribution, (b) SEM micrograph of the tungsten powder.

Figure 2 .

 2 Figure 2. (a) Normalized bed pressure drop in the glass column with 5 cm diameter and (b) in the FBCVD reactor with 3.8 cm diameter, (c) normalized bed expansion in the glass column vs. increasing and decreasing argon velocities at ambient temperature.

Figure 3 .

 3 Figure 3. Thermal profiles in the bed (TC1 to TC5) and below the distributor (TC6) during runs (a) W6, (b) W5, (c) W7. Heights above the distributor for thermocouple TC1: 1 cm, TC2: 12 cm, TC3: 7 cm, TC4: 5 cm, TC5: 2.5 cm.

Figure 4 .Figure 5 .

 45 Figure 4. SEM micrographs of the tungsten powder (a) before and (b) after deposition.

Figure 6 .

 6 Figure 6. XRD diffractomers of the powder after (a) run W2quater and (b) run W6.

Table 1 .

 1 Operating conditions tested and measured average bed temperatures.

	Run	Silane inlet vol. [%]	Run duration [min]	Average bed T before deposition [°C]	Average bed T during deposition [°C]	Average bed T after deposition [°C]
	W2quater	2.6	120	587	597	604
	W3	2.6	120	606	608	609
	W4	5.2	60	621	624	620
	W5 a	5.2	15	605	621	580
	W6	2.6	120	618	625	621
	W7 a)	4-2.9	60	604	602	603
	a) These runs have led to irreversible bed agglomeration.

Table 2 .

 2 Experimental results.

	Run	Silane conversion rate [%]	Silicon [wt %] deposited on powders	Thickness deduced from bed weighing [mm]	Thickness deduced from SEM [mm]	Sauter diameter of powders [mm]
	W2quater	100	0.83	0.76	0.75	70.9
	W3	100	0.96	0.87	0.72	71.3
	W4	100	0.78	0.71	0.53	70.9
	W5 a)	-	-	0.27	-	-
	W6	100	0.97	0.88	0.8	72.4
	W7 a)	93.4	0.4	0.37	0.37	69.5

Acknowledgment

The authors would like to thank Michel Molinier, Etienne Prevot, and Marie Line de Solan from LGC as well as Nicolas Tarisien from CEA for technical support.

The authors have declared no conflict of interest.


Paul Vulto
email: pvulto@deis.unibo.it

Nicolas Glade

Luigi Altomare

Jacqueline Bablet

Gianni Medoro

A Leonardi

Aldo Romani

Isabelle Chartier

Nicolo Manaresi

Marco Tartagni

R Guerrieri

Dry film resist for fast fluidic prototyping

Keywords: Dry film resist, microfluidics, prototyping, hybrid chips, packaging

Introduction

Dry film photoresists (DFR) were initially developed for fabrication of printed circuit boards. Application in MEMS technology is scarcely reported and is restricted to fabrication of electroplating moulds and masks for powderblasting of microstructures [1,2]. DFRs have several advantages over liquid resists such as good planarity of the resist, no liquid handling required, good adhesion to almost any substrate and simple fabrication process. In this paper dry film resist is demonstrated applicable for creating microfluidic structures. For this purpose Ordyl SY300 and SY550 (Elga Europe, Italy) is used, a resist that can be transferred into a rigid structure by a postbake. The resist acts as a fluidic spacer offering the possibility to integrate active elements in both bottom and top substrates. Since the simple fabrication does not necessarily require a dedicated cleanroom environment, the technique is extremely suitable for low-cost prototyping. Good compatibility with standard lithography processes and minimal number of process steps on the other hand make the technique suitable for industrial applications as well.

Fabrication

Photoresist (Ordyl SY300, 30 micron thickness, or SY550, 50 micron thickness) is laminated on a substrate using an office laminator (GBC 3500 pro series). Bare glass is used as a substrate, but also coated glass, silicon and a printed circuit board were tested. After removal of the first protective film, the resist is exposed for one minute with a low cost UVlamp (TR1000, Radio Ricambi, Italy) using a photolithographic film (Tecnografika, Italy, max resolution 20 micron) as a mask. The second protective film is removed and the resist is developed and rinsed in commercially available BMR developer and rinser (Elga Europe). Increased resist thickness is realized by laminating multiple layers. A second substrate, in our case a glass lid with powderblasted holes, is bonded on top of the resist under a pressure of 650kPa and a temperature of 70 o C. The temperature is ramped up in steps of 20 o C per 20 min. to 150 o C. A one hour postbake at 150 o C transforms the resist into a rigid structure. A post-exposure bake (PEB) of 85 o C for 5 min. could be inserted after exposure in order to improve the definition of the structures.

Results

Using the low-cost microfabrication set-up, free-standing structures of about 100μm can be achieved and channels of about 130μm for a resist thickness of 90μm (three layers of resist). A good definition of channels is more difficult than for freestanding structures due to scattering of light in the resist during UV exposure. In a cleanroom setting 20μm structures as well as 40μm channels for a resist thickness of 50μm have been achieved (details not shown).

Double bonding is easy to achieve depending on the substrate planarity, surface roughness, and PEB settings. When omitting the PEB a bonding percentage between 95 and 100% could be achieved on glass substrates (Knittel Gläser microscope slides). Inserting a PEB step puts higher constraints to the surface roughness and the planarity of the substrate. An increased resist thickness facilitates the double bonding. Figure 2 shows an ITO/gold patterned glass chip, a printed circuit board and a CMOS chip with a fluidic gasket of photoresist on top and closed by an ITO coated glass lid. The devices are meant for dielectrophoretic manipulation of biological cells and require electrodes on the bottom as well as on the top substrate for establishing the required field gradient [START_REF] Fuchs | Proc. of MicroTas[END_REF][START_REF] Medoro | [END_REF]. The devices were successfully applied for manipulation and selective recovery of cells and beads (data not shown). Due to inexpensive and fast mask fabrication, the devices such as the ones shown can be packaged in no more than two days, including design, mask fabrication and assembly of the microfluidics.

Figure 3 shows pictures of a two level lithography process on a full silicon wafer. Since alignment of the second mask is difficult when using the low-cost UV lamp, a standard mask aligner is used (Karl Suss MA750). A PEB (85 o C, 5 min) is added to improve structure definition. The same structure has been reported feasible using SU8 photoresist [START_REF] Chartier | Proceedings of SPIE[END_REF]. Biocompatibility of the resist was proven by culturing human K562 cells for 5 days on top of a piece of processed and autoclaved resist on glass. The growth curve of the sample was compared with the growth curve of a population cultured on bare glass. The resist, both postbaked and non-postbaked, did not seem to have inhibiting effects on the cell proliferation.

Conclusions

A fast and inexpensive prototyping technology for microfluidic structures has been developed using dry film resist as a fluidic spacer. The resist allowed good adhesion to many substrates, simple double bonding without the use of additional adhesives and multiple lithography steps. It also has proven biocompatible. The fast and simple fabrication process makes the technique not only suitable for prototyping, but makes it interesting for industrial applications as well.

Figure 1 .

 1 Figure 1. Three lane flow profile in a fluidic network sandwiched between a microscope glass slide and a lid with powderblasted holes.

Figure 2 .

 2 Figure 2. Series of three dielectrophoresis chips with a fluidic structure in dry film resist. All chips are closed with an ITO (Indium Tin Oxide) covered lid that is electrically connected with the bottom substrate via a conductive grease and with powderblasted holes for the fluidic interface. a) Gold and ITO patterned glass chip, wire-bonded to a PCB, b) CMOS chip, also wire bonded to a PCB, c) PCB with bare, gold plated electrodes

Figure 3 .

 3 Figure 3. Two-level resist structure on a silicon substrate. a) SEM picture, b) Detail of the 50μm wide opening

Acknowledgements

This project is funded by the European Union Commission (MeDICS IST -2001-32437). Authors would like to thank Matthieu Kipp and Cecile Bory (CEA-LETI) for advice and SEM pictures, prof. Berti (Univ. of Bologna) for his hospitality and Elga Europe for samples and advise.