Remigio Martínez

Alfredo García

Jesús E Blanco

Jorge Blanco

Joaquín Rey

Juan M Alonso

Luis Gómez

Sergio Sánchez

Jesús E Blanco

Jorge Blanco

Occurrence of verocytotoxin-producing in the faeces of free-ranging wild lagomorphs in southwest Spain

Keywords: Verocytotoxin-producing Escherichia coli (VTEC), E, coli O157:H7, Wild lagomorphs, Oryctolagus cuniculus, Lepus granatensis

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

Verocytotoxin-producing Escherichia coli (VTEC) has recently emerged as an important food-borne pathogen. Human diseases ranging from mild diarrhoea to haemorrhagic colitis and the life-threatening haemolytic-uraemic syndrome (HUS) can be caused by VTEC, typically affecting infants, young children and the elderly [START_REF] Paton | Pathogenesis and diagnosis of Shiga toxin-producing Escherichia coli infections[END_REF]. The pathogenic capacity of VTEC resides in a number of virulence factors, including verocytotoxins (VT1 and VT2), intimin, enterohaemolysin and the autoagglutinating adhesin (Saa) [START_REF] Gyles | Shiga toxin-producing Escherichia coli: an overview[END_REF]. Serotype O157:H7 especially represents a major public health concern worldwide; however, non-O157 VTEC should not be overlooked in human disease investigations because non-O157 VTEC strains are more prevalent than E. coli O157:H7 in the faeces of meat-producing animals, indicating that humans are more likely to become exposed to such strains (Blanco et al. 2004a;[START_REF] Djordjevic | Serotypes and virulence gene profiles of Shiga toxin-producing Escherichia coli strains isolated from feces of pasture-fed and lot-fed sheep[END_REF]). Although healthy domestic ruminants (mainly cattle) are the best recognised animal reservoir for VTEC (Blanco et al. 2004b;Sánchez et al. 2010a), VTEC strains have also been isolated from wildlife, especially from deer [START_REF] Renter | Escherichia coli O157:H7 in free-ranging deer in Nebraska[END_REF][START_REF] Sánchez | Detection and characterisation of Shiga toxinproducing Escherichia coli other than Escherichia coli O157:H7 in wild ruminants[END_REF]). The isolation of E. coli O157:H7 and non-O157 VTEC from wild rabbits has been previously reported [START_REF] Pritchard | Wild rabbits-a novel vector for verocytotoxigenic Escherichia coli O157[END_REF][START_REF] Scaife | Wild rabbits (Oryctolagus cuniculus) as potential carriers of verocytotoxin-producing Escherichia coli[END_REF]) and colonisation of laboratory rabbits by VTEC has also been demonstrated in experimental laboratory infections [START_REF] Li | The effect of enterohemorrhagic Escherichia coli O157: H7 on intestinal structure and solute transport in rabbits[END_REF][START_REF] García | The rabbit as a new reservoir host of enterohemorrhagic Escherichia coli[END_REF]. The aim of the current study was to analyse faecal samples from wild rabbits and hares killed by hunters and intended for their own consumption for the presence of E. coli O157:H7 and non-O157 VTEC.

During the 2007-2008 and 2008-2009 hunting seasons (from October to January), collaborating hunters collected faecal samples from wild lagomorphs harvested in different game estates in the Extremadura region in southwest Spain. They were provided with sampling equipment as well as step-by-step instructions for sample collection before the start of every season. When harvesting the animal, the hunters were asked to use a pair of disposable gloves, insert a sterile swab into the rectum of the animal, collect a faecal sample (one per animal) by rapid in-and-out motion and transfer the swab to a tube containing Cary-Blair transport medium (DeltaLab). The samples were transported to the laboratory under refrigeration and placed in culture media within 24 h. Overall, wild rabbit (Oryctolagus cuniculus) provided 124 samples and 117 came from hare (Lepus granatensis). The samples were plated onto both MacConkey (MAC) and cefixime tellurite sorbitol MacConkey (CTSMAC) agars (Oxoid). For isolation of non-O157 VTEC, ten suspect E. coli colonies from MAC plates were tested for the genes encoding VT1 and VT2 toxins (VT1 and VT2 genes) by PCR as previously described [START_REF] Rey | Serotypes, phage types and virulence genes of Shiga-producing Escherichia coli isolated from sheep in Spain[END_REF]. For isolation of E. coli O157:H7, after a selective enrichment step involving immunomagnetic separation, ten non-sorbitolfermenting colonies from the CTSMAC plates were tested for the genes encoding O157 and H7 antigens as previously described [START_REF] García-Sánchez | Presence of Shiga toxinproducing E. coli O157:H7 in a survey of wild artiodactyls[END_REF]). The resulting VTEC isolates were confirmed biochemically as E. coli by the API 20E system (bioMérieux) and tested for additional virulence genes (eae, ehxA and saa). The identification of O and H antigens (serotyping) in VTEC isolates was carried out as described by [START_REF] Guinée | Escherichia coli associated with neonatal diarrhoea in piglets and calves[END_REF] using the full range of O (O1 to O185) and H (H1 to H56) antisera.

Overall, VTEC were detected and isolated in four (1.66%) of the 241 animals sampled. E. coli O157:H7 was isolated only from one of 124 (0.81%) wild rabbit faecal samples while non-O157 VTEC were isolated from two of 124 (1.61%) wild rabbit faecal samples and one of 117 (0.85%) hare faecal samples (Table 1). All isolates obtained in the present study (four in total) were further characterised (Table 1). They belonged to four different O: H serotypes, including two serotypes (O84:H-and O157: H7) previously associated with human infection and in particular with causing HUS (http://www.lugo.usc.es/ecoli; http://www.microbionet.com.au/vtectable.htm). The PCR procedure indicated that one isolate carried the VT1 gene and the rest carried the VT2 gene. The ehxA and eae genes were detected only in the E. coli O157:H7 isolate and none of the isolates contained the saa gene.

The results of the present study suggest a low prevalence of VTEC infection in free-ranging wild lagomorphs in this area of Spain, although it is important to note that this was not a random survey but only biased to areas used by hunters. Prevalence rates of E. coli O157:H7 and non-O157 VTEC obtained in this study are much lower than those currently observed in domestic ruminant species such as cattle, sheep or goats [START_REF] Oporto | Escherichia coli O157:H7 and non-O157 Shiga toxin-producing E. coli in healthy cattle, sheep and swine herds in Northern Spain[END_REF][START_REF] Orden | A longitudinal study of verotoxinproducing Escherichia coli in two dairy goat herds[END_REF]Sánchez et al. 2010a). This is not surprising as livestock are much more intensively reared and the potential for exposure and colonisation is much greater than for most wildlife; however, VTEC strains have also been isolated from other wild animal species, such as deer and wild boars [START_REF] Sánchez | Detection and characterisation of Shiga toxinproducing Escherichia coli other than Escherichia coli O157:H7 in wild ruminants[END_REF](Sánchez et al. , 2010b)). Wild rabbits and hares are commonly found in areas inhabited by human beings and livestock and consequently may share common sources of exposure to VTEC. Indeed, wild rabbits' faeces have been associated with E. coli O157:H7 infection in visitors to a wildlife park in the United Kingdom [START_REF] Pritchard | Wild rabbits-a novel vector for verocytotoxigenic Escherichia coli O157[END_REF]. Furthermore, indistinguishable genotypes of E. coli O157: H7 have been identified (by the pulsed-field gel electrophoresis technique) in cattle and wild rabbits occupying the same range, indicating either a common source of exposure or transmission from one species to the other [START_REF] Pritchard | Wild rabbits-a novel vector for verocytotoxigenic Escherichia coli O157[END_REF]). In the studied areas, wild rabbit and hare populations usually have access to the same pastures as cattle and sheep, although they have no contact with freeranging wild ruminants. Therefore, environmental contamination with VTEC excreted by domestic livestock and indirect transmission to wild lagomorphs from the contaminated environment could explain the occurrence of VTEC in their faeces. In addition, [START_REF] Miko | Assessment of Shiga toxin-producing Escherichia coli isolates from wildlife meat as potential pathogens for humans[END_REF] recently reported the contamination of meat from wild animals (including hare meat) with VTEC and concluded that such strains should be recognised as a public health problem. Consequently, wild lagomorphs such as wild rabbits and hares may play an important role as a source of VTEC in the environment, as a source of exposure to human beings and livestock and as a vehicle for dispersing these pathogens. These findings have implications for the zoonotic risk to hunters, people consuming meat from wild animals and others in contact with wild animal faeces, and also in the development of programmes for controlling VTEC at the farm level.

Table 1

 1 Results of faecal culture for VTEC, serotypes and virulence genes of VTEC isolates recovered from wild lagomorphs VT2 genes encoding VT1 and VT2 toxins, eae gene encoding intimin, ehxA gene encoding enterohaemolysin, HNT H antigen non-typeable

	Animal	Number cultured	E. coli O157:H7		Non-O157 VTEC
			Number positive (%)	Virulence genes	Number positive (%)	Serotype	Virulence genes
	Wild rabbit	124	1 (0.81)	VT2 eae ehxA	2 (1.61)	O22:HNT	VT2
						O117:H8/H21 a	VT2
	Hare	117	0		1 (0.85)	O84:H-b	VT1
	VT1/

a The isolate cross-reacted with the respective H antisera b Serotype previously associated with human infection and in particular with causing HUS

J. E. Blanco : J. Blanco Laboratorio de Referencia de E. coli (LREC), Departamento de Microbiología y Parasitología, Universidad de Santiago de Compostela, 27002 Lugo, Spain L. Gómez Histología y Anatomía Patológica, Departamento de Medicina Animal, Facultad de Veterinaria, Universidad de Extremadura, 10071 Cáceres, Spain Present Address: S. Sánchez (*) Instituto de Investigación en Recursos Cinegéticos (IREC), CSIC-UCLM-JCCM, Ronda de Toledo s/n, 13071 Ciudad Real, Spain e-mail: sergio.sanchezprieto@uclm.es Eur J Wildl Res (2011) 57:187-189 DOI 10.1007/s10344-010-0431-8

Eur J Wildl Res (2011) 57:187-189

Acknowledgements The authors thank hunters for indicating a willingness to participate in sample collection. The authors also thank R. Rubio for skilful technical assistance. This study was partially supported by grants from the Junta de Extremadura and FEDER (grants 3PR05A009-III Plan Regional de Investigación and PRI09A022). R. Martínez acknowledges the Junta de Extremadura for his research fellowship (PRE06053). S. Sánchez acknowledges the Consejería de Educación y Ciencia de la Junta de Comunidades de Castilla-La Mancha and Fondo Social Europeo for his research fellowship (09/02-C).


Roland Kozdrowski
email: roland.kozdrowski@up.wroc.pl

Michał Dzięcioł

Ewa Stańczyk

Małgorzata Ochota

Piotr Sławuta

Marta Jolanta Siemieniuch

: E Stańczyk

Artificial induction of superfetation in the European hare (Lepus europaeus)

Keywords: Lagomorpha, Artificial insemination, Superfetation, Pregnancy, GnRH

The aim of this study was to induce superfetation in hares. On day 37 or 39 of pregnancy, female hares were treated with GnRH analogue and artificially inseminated with fresh semen. Ultrasonographic examination showed that pregnant females had follicles on the ovary on 37 days of pregnancy. After a few days from insemination performed during pregnancy, all females gave birth to healthy young, and one of the females inseminated on day 37 of pregnancy gave birth to two healthy young 42 days after insemination performed during pregnancy (39 days post first delivery). The same female in the next year gave birth to two healthy young 43 days after second insemination that was performed on the day 37 during pregnancy. The obtained results confirm that superfetation is possible to occur in hares.

Introduction

European hare reproduction strategy is based on a relatively long breeding season (January-August). During this period, a female hare can give birth to four litters and an average litter size is 2-4 [START_REF] Raczyński | Studies on the European hare. V. Reproduction[END_REF]). However, European hares inhabiting Mediterranean ecosystems can reproduce all over the year [START_REF] Antoniou | Evidence of autumn reproduction in female European hares (Lepus europaeus) from southern Europe[END_REF]. Additionally, a phenomenon of superfetation (conception by an already pregnant female) may occur in a hare, which, according to [START_REF] Raczyński | Studies on the European hare. V. Reproduction[END_REF], occurs very occasionally in the natural environment and does not have a greater influence on the effectiveness of the European hare reproduction. In general, superfetation occurs very occasionally in other animal species and is recognized as pathology [START_REF] Gee | A case of superfetation in the cow[END_REF], whereas it is considered to be normal in the hare. [START_REF] Bloch | Beobachtungen zur Superfetation beim Feldhasen (Lepus europaeus)[END_REF] described presence of a foetus, nearly ready to be delivered, and fertilized ova (divided into four cells) in the uterus of a pregnant hare. [START_REF] Martinet | Oestrus behavior, follicular growth and ovulation during pregnancy in the hare (Lepus europaeus)[END_REF] showed that copulation occurred frequently in the hare from days 34 to 40 of pregnancy (the peak of sexual activity occurred on days 37 and 38 of pregnancy, i.e. 3-4 days before parturition), and copulation at this time induced ovulation resulting in the superfetation [START_REF] Caillol | Preliminary results on plasma progesterone levels during pregnancy and superfetation in the hare, Lepus europaeus[END_REF]. [START_REF] Martinet | Oestrus behavior, follicular growth and ovulation during pregnancy in the hare (Lepus europaeus)[END_REF] also showed that mating or treatment of hares with hCG during pregnancy induced ovulation. On the other hand, [START_REF] Stavy | Interbirth interval and duration of pregnancy in hares[END_REF] maintained a breeding colony of paired hares in Israel for over 10 years, but no cases of superfetation were observed. [START_REF] Lincoln | Reproduction and "March madness" in the brown hare, Lepus europaeus[END_REF] observed ovulation immediately following parturition or sometimes a short before it, but no females ovulated during early and middle phase of pregnancy and no case of superfetation was found. As can be seen, the data on superfetation in hares are very different, and this subject has been recently reviewed in detail by Roellig et al. (2010b).

Therefore, the aim of this study was to induce the superfetation experimentally in hares by induction of ovulation in pregnant females with the use of GnRH analogue and insemination with fresh semen. In order to explain conditions that enable the induction of superfetation in the European hares, we used ultrasonographic examination of reproductive tract in female hares with the special attention on the ovary, i.e. if follicles are presented on the ovary during the late stage of pregnancy.

Materials and methods

Eleven adult females (body weight from 3.5 to 4.1 kg) and two adult males (body weight of 3.8 and 4.2 kg) were used in this experiment. During the experiment, the hares were housed in individual cages located in Wrocław (south-west Poland, 57°07′ N, 17°02′ E) and were exposed to natural daylight and ambient temperature. The hares were watered and fed ad libitum with a diet composed of hay, oat and a full-component mixture for hare. Additionally, branches from fruit and willow trees were given. In the middle part of February, all females were artificially inseminated. Just before artificial insemination (AI), the females were treated with a mixture of xylasin (Sedazin; Biowet Puławy, Poland) and ketamin (Bioketan; Biowet Puławy, Poland) (4 and 12 mg, respectively, per kilogramme of body weight, intramuscular injection). A dozen or so of minutes after the hares were immobilized, each female was then treated with a GnRH analogue (Receptal; Intervet International GmbH, 0.00168 mg of buserelin, intramuscular injection) for induction of ovulation as described before [START_REF] Kozdrowski | The effect of trehalose on post-thaw viability and fertility of European brown hare (Lepus europaeus Pallas, 1778) spermatozoa[END_REF]Kozdrowski et al. 2009;[START_REF] Kozdrowski | Inducing ovulation and artificial insemination in the European brown hare (Lepus europaeus Pallas, 1778)[END_REF].

The semen for AI was collected by electroejaculation [START_REF] Kozdrowski | The effect of trehalose on post-thaw viability and fertility of European brown hare (Lepus europaeus Pallas, 1778) spermatozoa[END_REF]Kozdrowski et al. 2009). Males, before semen collection, were subjected to 12-h starvation and were then treated with a mixture of xylasin (Sedazin; Biowet Puławy, Poland) and ketamin (Bioketan; Biowet Puławy, Poland) (6 and 15 mg, respectively, per kilogramme of body weight, intramuscular injection). A few minutes after achieving full anaesthesia, semen was collected. After initial evaluation, the semen was diluted with the extender of the following composition: tris, 313.79 mM; citric acid, 103.07 mM and glucose, 33.3 mM [START_REF] Roca | Viability and fertility of rabbit spermatozoa diluted in Tris-buffer extenders and stored at 15°C[END_REF]. The diluted semen was used for AI within 1 h after dilution and each female received 1 ml of the diluted semen containing 30×10 6 spermatozoa with spermatozoa motility above 80% [START_REF] Kozdrowski | Inducing ovulation and artificial insemination in the European brown hare (Lepus europaeus Pallas, 1778)[END_REF]. The diluted semen was slowly deposited with a plastic pipette introduced into the vagina as deep as it was possible (8-10 cm).

The inseminated females were divided into two groups. Six females included in group A were again anaesthetized with the use of the above-mentioned protocol on day 37 after AI (AI day 0). At least 12 min after the hares were immobilized, palpable examination for detection of pregnancy were performed. Immediately after the examination, the pregnant females were treated with the same dose of GnRH analogue as mentioned above and were inseminated again (second AI) with the same dose of fresh semen as mentioned above. Five females included in group B underwent the same procedures 2 days later, i.e. on day 39 after the first AI (AI day 0). Next, no procedures were performed on the females from groups A and B until delivery and for 7 weeks following the delivery. Every day in the morning (during feeding), the cages were checked for newborn hares. The young were separated from their mothers 4 weeks post-partum.

In the next year, seven adult females (all were used in the previous breeding season) constituted group C and one adult male (this male was also used in the previous breeding season) housed and fed in the same way as described above were included for observations. Females and semen for AI were prepared in the same way as described above, and first insemination was also performed in the middle part of February. In this group, all females were introduced for ultrasonographic examination on day 37 after first AI. Up to this time, only few papers described ultrasonographic examinations of reproductive tract in European hare females, and their authors focused on foetal growth [START_REF] Hackländer | The assessment of female reproduction and gestational age in European hares (Lepus europaeus) using ultrasonography[END_REF][START_REF] Hildebrandt | Artificial insemination of captive European brown hares (Lepus europaeus PALLAS, 1778) with fresh and cryopreserved semen derived from free-ranging males[END_REF]Roellig et al. 2010a) and also described ultrasonographic findings on the ovary during pregnancy and AI [START_REF] Hildebrandt | Artificial insemination of captive European brown hares (Lepus europaeus PALLAS, 1778) with fresh and cryopreserved semen derived from free-ranging males[END_REF]Roellig et al. 2010a). Hares for USG examinations were positioned in dorsal recumbency; abdomen was shaved and covered with alcohol and gel. Transabdominal ultrasonographic examination was performed with the Ultrasonographic System Mindray M5 (Mindray Medical International Limited, China) equipped with a 8-12-MHz linear transducer. The ovaries were identified behind the caudal pole of the ipsilateral kidney. Just after examination, all pregnant females were again inseminated (second AI) with the same way as described above, and no procedures were performed on those females until delivery and for 7 weeks following the delivery. Every day in the morning (during feeding), the cages were checked for newborn hares. The young were separated from their mothers 4 weeks post-partum.

Results

On day 37 after the first AI, four females from group A were pregnant (including three females with single preg-nancy and one female with twin pregnancy). On day 39, four female hares from group B were also pregnant (including two females with single pregnancy and two females with twin pregnancy). A pregnancy rate for the two groups together amounted to 73%.

Within group A, the female hares with single pregnancy gave birth to healthy young after 40, 41 and 42 days from the first AI (i.e. after 3, 4 and 5 days from the second AI, respectively). The female with twin pregnancy gave birth to healthy litter after 41 days from the first AI (i.e. 4 days from the second AI). Within group B, the female hares with single pregnancy gave birth to healthy litter after 40 and 43 days from the first AI (i.e. 1 and 4 days after the second AI), whereas the females with twin pregnancy gave birth to healthy litter after 41 and 42 days from the first AI (i.e. 2 and 3 days after the second AI).

On day 39 after the first delivery (42 days after the second AI), a female from group A gave birth to two healthy young (the first pregnancy of this female lasted for 40 days and the female gave birth to one healthy young). The remaining females from group A and group B did not give birth after the first delivery (after second AI).

On the day 37 after the first AI, four females from group C were pregnant (57% pregnancy rate). Ultrasonographic examination showed that all pregnant females had follicles on the ovary at this time (Fig. 1). All females from this group gave birth to one healthy young on 41 days after first AI. On day 39 after the first delivery (43 days after the second AI), one female from this group gave birth to two healthy young (second case of superfetation). However, it was the same female, which had successfully induced superfetation in the previous year. The remaining females from this group did not give birth after the first delivery (after second AI).

Discussion

During the breeding season, administration of hCG and GnRH analogues induce ovulation in hares [START_REF] Caillol | Seasonal variations in the pituitary response to LHRH in brown hare (Lepus europaeus)[END_REF][START_REF] Hildebrandt | Artificial insemination of captive European brown hares (Lepus europaeus PALLAS, 1778) with fresh and cryopreserved semen derived from free-ranging males[END_REF][START_REF] Kozdrowski | The effect of trehalose on post-thaw viability and fertility of European brown hare (Lepus europaeus Pallas, 1778) spermatozoa[END_REF]Kozdrowski et al. 2009;[START_REF] Kozdrowski | Inducing ovulation and artificial insemination in the European brown hare (Lepus europaeus Pallas, 1778)[END_REF][START_REF] Stavy | Interbirth interval and duration of pregnancy in hares[END_REF]. After the hormonal stimulation ovulation appears, the corpora lutea are formed and concentration of progesterone increased for about 2 weeks in those female hares that have not been mated or artificially inseminated (pseudopregnancy) [START_REF] Caillol | Seasonal variations in the pituitary response to LHRH in brown hare (Lepus europaeus)[END_REF][START_REF] Stavy | Plasma progesterone levels during pregnancy and pseudopregnancy in the hare (Lepus europaeus syriacus)[END_REF]. It was shown that after administration of GnRH analogue or hCG and insemination with the spermatozoa collected from epididymis or insemination with fresh or frozen/thawed semen, a percentage of pregnant females ranged between 50% and 80% [START_REF] Hildebrandt | Artificial insemination of captive European brown hares (Lepus europaeus PALLAS, 1778) with fresh and cryopreserved semen derived from free-ranging males[END_REF][START_REF] Kozdrowski | The effect of trehalose on post-thaw viability and fertility of European brown hare (Lepus europaeus Pallas, 1778) spermatozoa[END_REF]Kozdrowski et al. 2009;[START_REF] Kozdrowski | Inducing ovulation and artificial insemination in the European brown hare (Lepus europaeus Pallas, 1778)[END_REF][START_REF] Stavy | Interbirth interval and duration of pregnancy in hares[END_REF]. Similarly, in this study, the percentage of pregnant females after the first insemination was 73% for group A and B and 57% for group C in the next breeding season. Out of 12 pregnancies found there, nine were single and three twin pregnancies, which also correspond to the numbers of young in early year pregnancies mentioned by most of the authors [START_REF] Hackländer | The assessment of female reproduction and gestational age in European hares (Lepus europaeus) using ultrasonography[END_REF][START_REF] Hildebrandt | Artificial insemination of captive European brown hares (Lepus europaeus PALLAS, 1778) with fresh and cryopreserved semen derived from free-ranging males[END_REF][START_REF] Kozdrowski | The effect of trehalose on post-thaw viability and fertility of European brown hare (Lepus europaeus Pallas, 1778) spermatozoa[END_REF]Kozdrowski et al. 2009;[START_REF] Kozdrowski | Inducing ovulation and artificial insemination in the European brown hare (Lepus europaeus Pallas, 1778)[END_REF][START_REF] Raczyński | Studies on the European hare. V. Reproduction[END_REF].

We decided to carry out the next induction of ovulation and insemination on day 37 or 39 of pregnancy as, at this stage of pregnancy, the frequency of copulation between the hares considerably increases leading to ovulation and the next gestation (superfetation) [START_REF] Caillol | Preliminary results on plasma progesterone levels during pregnancy and superfetation in the hare, Lepus europaeus[END_REF][START_REF] Martinet | Oestrus behavior, follicular growth and ovulation during pregnancy in the hare (Lepus europaeus)[END_REF]. [START_REF] Stavy | Interbirth interval and duration of pregnancy in hares[END_REF], inducing superfetation by AI with spermatozoa collected from epididymis on day 38 of pregnancy, found abortion in only one case 22 days after insemination (19 days post-delivery). In our experiment, one female inseminated on day 37 of pregnancy gave birth to one healthy young after 3 days from the second insemination and the next two healthy young after the following 39 days (42 days from the second insemination). To the authors' knowledge, it is the first case of the induction of superfetation described.

In this study, the administration of GnRH analogue to the pregnant female hares and the AI procedure did not have a negative impact on the course of pregnancy as all young were born alive and were raised by their mothers until day 28 of their lives. [START_REF] Stavy | Interbirth interval and duration of pregnancy in hares[END_REF] stated, in turn, that the mean gestation period is shortened from 45 to 41 days in those females for which an attempt to induce superfetation was made on day 38 of pregnancy, which resulted in a lower birth weight of the young and, in natural environment, a shortened period of gestation, resulting in lower weight at birth, might be a disadvantage to the offspring. However, the length of gestation in our study lasted from 40 to 43 days (AI day 0), which is consistent with the earlier observations carried out in the same conditions on the female hares in which superfetation was not induced (the length of pregnancy was from 38 to 44 days) [START_REF] Kozdrowski | The effect of trehalose on post-thaw viability and fertility of European brown hare (Lepus europaeus Pallas, 1778) spermatozoa[END_REF]Kozdrowski et al. 2009;[START_REF] Kozdrowski | Inducing ovulation and artificial insemination in the European brown hare (Lepus europaeus Pallas, 1778)[END_REF] and is consistent with the observations in another breeding centre in the Central Europe [START_REF] Hildebrandt | Artificial insemination of captive European brown hares (Lepus europaeus PALLAS, 1778) with fresh and cryopreserved semen derived from free-ranging males[END_REF]). However, Stavy and Terkel ' s (1992) study was carried out under different geographical conditions and in another hare, and perhaps the attempt to induce superfetation on day 38 of pregnancy in the hare population with an average length of gestation amounting to 45 days might have been made too early. In addition, the procedures (insemination, hCG administration and vaginal stimulation) performed with the attempt to induce superfetation resulted in shortening of the gestation period from 45 to 41 days, leading to early delivery [START_REF] Stavy | Interbirth interval and duration of pregnancy in hares[END_REF]. On the other hand, [START_REF] Martinet | Oestrus behavior, follicular growth and ovulation during pregnancy in the hare (Lepus europaeus)[END_REF] stated that injection of hCG in hares carried out between days 14 and 28 of pregnancy caused abortion and after day 32 did not influence the length of pregnancy. On the basis of our observation, one can state that neither the AI nor the injection of GnRH analogue on the days 37 or 39 of pregnancy has a negative impact on the course of gestation, delivery and raising the offspring.

The study aimed to show whether it was possible to induce superfetation in hares, and this aim was achieved; however, we cannot say anything about the frequency of superfetation occurrence and its significance for the hare reproduction. Based on results obtained in this work, it seems that this phenomenon is rather rare because superfetation was successfully induced only two times and in the same female. It is also hard to say unambiguously what conditions must occur for superfetation to take place. However, it was shown that on 37 days of pregnancy, follicles are presented on the ovary. This study showed that single pregnancy is a factor that predisposes to the occurrence of superfetation and that the second AI should be performed 3 or 4 days before delivery. It can also be assumed that multiple pregnancy may also create favourable conditions for the occurrence of superfetation provided that foetuses are situated only on one side of the uterus. The obtained results confirm that superfetation is possible to occur in hares.

Fig. 1

 1 Fig. 1 Ultrasonography of the ovary with antral follicle (narrow arrow) and part of the foetus (wide arrow)

Eur J Wildl Res (2011) 57:333-336

Acknowledgements This study was supported by the grant of the Polish Ministry of Sciences and High Education, R 12 070 03.

Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.


PharmD Sander D Borgsteede

MD Christiaan A Rhodius

Peter A G M Smet

H Roeline

W Pasman

PhD Bregje D Onwuteaka-Philipsen

PhD Mette L Rurup

PharmD P A G M De Smet

PhD H R W Pasman

The use of

Keywords: opioids, pain management, pharmacists, knowledge, education, multidisciplinary cooperation

HAL is

Introduction

Pain is one of the symptoms most frequently encountered at the end of life [1;2], although it can be controlled satisfactorily in most cases if health care professionals have adequate knowledge. [3] Possibilities to manage pain in patients at the end of life have increased in the past decade. These possibilities have been incorporated in handbooks and practice guidelines that were developed to support professionals in evidence-based practice [4][5][6].

However, the existence of professional guidelines does not guarantee that this knowledge is optimally used in daily practice. Several barriers have been identified concerning the prescribing and use of opioids that hamper the quality of pain management. First, there is still lack of theoretical knowledge about opioids among practicing physicians and nurses. [4;7-9] A systematic review of barriers hindering cancer pain management found that inadequate knowledge was one of the most reported barriers among physicians for adequate pain management. [10] The most reported misconceptions were the likelihood of opioid addiction and the belief that morphine dosage had an upper limit. Lack of theoretical knowledge also includes rare effects of opioids such as opioid induced hyperalgesia. [11] Secondly, when health care professionals have sufficient theoretical knowledge about pain management guidelines, this does not necessary lead to adherence to these guidelines in daily practice. An example is that patients who clearly need opioids for pain frequently receive dosages that are too low. [12;13] Another example is prescribing laxatives concomitantly with opioids, a clinical rule that has been recommended in the WHO pain ladder [6] and almost all guidelines about pain management. [4;5;14] In daily practice, only about half of the patients are prescribed a laxative concomitantly. [15][16][17] Nearing end-of-life constipation can still be a problem, although the patient's deteriorating functional status can mean management of constipation has a lower priority. In the last days of life when patients are no longer able to receive oral treatment and consciousness diminishes, oral laxatives should be discontinued. [18] Compared to physicians and nurses, surprisingly few studies have been aimed at the pharmacists' knowledge and experiences concerning pain management and opioid use. Among countries, pharmacists have different responsibilities in pharmaceutical care and hence their role in pain management might differ. In contrast with other countries [19], pharmacists are not allowed to prescribe in the Netherlands. Their primary task is to control the quality of prescribing, check for potential interactions and support prescribers and patients with optimal medication use. [20] Dutch hospital pharmacists frequently contact and advise clinicians, and an increasing number is present at wards for consultation and patient education. [21] In community pharmacy, patient education is common and there are regular pharmacotherapeutic audit meetings between pharmacists and general practitioners. [22] Characteristic for the Netherlands is that euthanasia and palliative sedation are legal options when suffering is unbearable and hopeless [23], and most pharmacies are prepared to fill prescriptions for euthanasia. [24] Although not in the Netherlands, some studies investigated pharmacists' knowledge and attitude. They identified a general need for more knowledge and education in pain management. [25;26] Misconceptions were not only prevalent among physicians [10], but also among pharmacists. [27] Risk of addiction was no barrier to most pharmacists to dispense opioids, but conservative physicians and nurses, and concerns of pharmacists for adverse effects were experienced as barriers for opioid dispensing. [28] Hence, the few studies among pharmacists do not support the possible assumption that pharmacists' knowledge is sufficient for achieving optimal pharmacotherapy at the end of life. On the contrary, they suggest that the pharmacist might be a limiting factor in achieving optimal pharmacotherapy at the end of life. However, there have not been surveys performed among pharmacists recently, and the previous studies were all performed in the United States and had relatively low response rates. [27;28] In end-of-life care, interdisciplinary cooperation among professionals is essential [29], and within this team approach the pharmacist has a responsibility in guarding and optimizing the quality of pharmacotherapy.

Hence, it is important that the pharmacist has adequate and up-to-date knowledge of pain management and the medication involved. We aimed to study the level of knowledge of pharmacists in the Netherlands concerning pain management and the use of opioids at the end of life, how they cooperate with physicians and what barriers to good end-of-life care pharmacists experience. The questionnaire was sent in January 2009 followed by two reminders. With the second reminder a non-response questionnaire was enclosed for use in the non-response analysis. Respondents could complete the questionnaire on paper or online. The written questionnaire was anonymous. A separate answer card with the personal data of the respondent could be mailed to prevent further reminders. The online questionnaire was not anonymous, because it would otherwise not be possible to exclude the possibility that people who were not in the sample would complete the questionnaire. When the database was closed the questionnaires were anonymized.

In total 412 pharmacists were sent a questionnaire, 7 envelopes were returned to sender (respondent had moved in case of private address, or was no longer working at the institution in case of work address). Of the 322 community pharmacists, 147 completed the full questionnaire (response rate 46%). Of the 83 hospital pharmacists, 35 completed the full questionnaire (response rate 42%). The total response rate was 45%. Of the completed questionnaires, 70% were completed in writing and 30% were completed online. The non-response questionnaire was completed by 35 community pharmacists and seven hospital pharmacists.

After the closing of the data collection, a document with the correct answers to the knowledge statements was sent by e-mail to those who had indicated they were interested in receiving this.

Developing the questionnaire and the pilot study

The questionnaire was developed by the authors who have expertise in end-of-life care, pharmacy, medicine, epidemiology and the development of questionnaires. We used previous studies of the knowledge or attitude concerning opioids and pain management at the end of life. [8;30] Questions were adapted and added to be able to answer the research questions, and to fit the specific situation in the Netherlands. For this purpose we used inter alia Dutch guidelines, original articles and review articles. [8;11;31-37] The questionnaire was piloted among GPs in training, elderly care physicians in training, and pharmacists. Opinions differed on the difficulty level of the knowledge statements in the questionnaire, but most respondents did not think the statements were unclear or too difficult; they sometimes even thought they were (too) easy.

The questionnaire consisted of three types of questions: 1) knowledge statements with three answering options: "true", "false" and "don't know" 2) experience and attitude questions with five answering options: "completely agree", "agree more than disagree", "neutral", "disagree more than agree" and "completely disagree" and 3) other questions, partly structured with four answering options: "often", "sometimes", "seldom" and "never" and partly semi-structured. Furthermore several background characteristics were asked, inter alia to grade one's own knowledge about opioids and pain management on a score of 1 to 10.

Analysis

We calculated descriptive statistics for the comparisons between community pharmacists and hospital pharmacists. Chi-square tests were used for comparing proportions between respondents and non-respondents, using linear by linear association for ordinal variables. Student´s t-tests were used for comparing means. For all tests p-values are presented. In Table 3, the categories "completely agree" and "agree more than disagree" were merged to the category "agree" versus the categories "neutral", and "(completely) disagree".

Results

Background characteristics

In Table 1 the background characteristics of the respondents are shown per specialty. Hospital pharmacists were responsible for more patients to whom opioids were dispensed compared to community pharmacists. They also graded their own knowledge concerning opioids and pain management higher.

There was one significant difference in background characteristics between pharmacists who completed the non-response questionnaire (n=44) and pharmacists who completed the full questionnaire (n=182). Nonresponders were more often fulltime employed (93%) than responders (74%, not in Table ). The most important reasons for non-response were no time, too high workload and being understaffed.

Knowledge statements

In Table 2 the answers to the knowledge statements are shown per specialty. Of the 14 presented statements, community pharmacists gave on average between 9 and 10 correct answers. Hospital pharmacists answered more statements correctly (between 10 and 11). Knowledge questions with a low score of correct answers were 'opioids may cause or worsen pain' (statement is true, 11% correct answers), 'drug management of nausea in treatment with opioids is evidence-based' (statement is false, 19% correct answers), 'simultaneous prescription of a weak opioid (e.g. tramadol) and a strong opioid (e.g. morphine) is contra-indicated' (statement is true, 49% correct answers), 'opioids are the favoured drugs for palliative sedation' (statement is false, 61% correct answers) and 'life-threatening respiratory depression is a real danger when titrating morphine to control pain' (statement is false, 71% correct answers). Of the pharmacists, 74% knew that decreased renal function could raise plasma concentration of morphine and its active metabolite (morphine 6-glucuronide), and 82% calculated an example of opioid rotation correctly.

Attitudes, experiences and barriers concerning pain management

Pharmacists did not think that patients always indicate their pain to their physician (Table 3). The majority of the pharmacists (81%) stated that an opioid prescription should consist of both maintenance and as needed medication. Experienced barriers were opioid fear by the patient (79%) and family (72%), and underreporting of pain by patients (77%) and insufficient information about the pain (60%). Practical/logistic issues such as problems with manufacturing/availability, and problems with reimbursement were not often experienced as a barrier.

Attitudes and experiences concerning opioid rotation, tolerance, addiction, and checking prescriptions

Table 4 shows that almost 39% of the pharmacists found calculating of opioid dosages in case of opioid rotation difficult. Fear of addiction was seen as limiting factor for opioid use in daily practice (78%). The majority of the community pharmacists (94%) controlled opioid prescriptions most often after dispensing, the majority of the hospital pharmacists (68%) did not check opioid prescriptions personally. Community pharmacists were more frequently personally involved compared to their clinical colleagues;15% of the community pharmacists, and none of the hospital pharmacists reported to often have personal contact with the patient/caregiver.

Cooperation with physicians by working agreements, advice and contact about prescriptions

Table 5 shows cooperation between pharmacists and physicians. Working arrangements with physicians concerning euthanasia were more frequently reported by hospital pharmacists (97%) than by community pharmacists (77%). Working agreements concerning palliative sedation and opioid usage were less frequent.

Community pharmacist were more often consulted by general practitioners, and hospital pharmacists by nursing home physicians and clinical specialists. Most pharmacists (93%) reported that physicians were open to unsolicited advice concerning the opioids prescribed by the physician. Almost all pharmacists (97%) would contact the physician if there was something wrong with the prescription. Most reported reasons to contact a physician in practice were laxatives not being prescribed (77%), administrative incompleteness (53%) and drugdrug interactions (52%). Most pharmacists contacted the physician when an opioid was prescribed without a laxative (63%). Reasons for not contacting the physician were that he/she might have had a good reason for not prescribing a laxative or that a laxative was not needed as the patient was terminal or being sedated. Other pharmacists dispensed a laxative to the patient according to working agreements, and some did not contact physicians about laxatives. Most pharmacist (96%) did not contact the physician when an opioid was prescribed without prescribing an anti-emetic drug. About three quarters of the pharmacists (76%) indicated that they had enough options for additional education in subjects related to opioids and pain and symptom management at the end of life (not in Table ). Others were very adamant that there were not enough options -neither in their region, nor non-commercial -or concerning information they could use in their daily practice to support physicians. Subjects that were most often mentioned in which further education was wanted were opioid rotation (59%), pain and symptom management in general (52%), and palliative sedation (49%). The preferred methods to receive further education were literature in Dutch journals (65%), conferences (47%), courses on the internet (44%), and literature in international journals (41%).

Additional education

Discussion

The knowledge of the majority of the respondents was adequate on many points. We identified gaps in knowledge about some aspects of opioid use in a considerable part of the pharmacists. Pharmacists answered 71% of the knowledge questions correctly (10 out of 14) . Physicians scored between 31% and 68% correct answers in various studies [38][39][40], and pharmacists scored 46% correct answers in a single previous study. [25] The overall scores in different studies cannot be compared as such because other statements were used.

A point of concern was that only one in nine pharmacists was aware that opioids may cause or worsen pain. This is a rare effect and has not been known for a long time, so it is understandable that many pharmacists are unaware of this fact. However, the clinical effects can be significant if the dose of opioids is increased instead of decreased when a patient suffers from opioid-induced pain sensitivity. [11;41;42] Although the majority of the pharmacists stated that life-threatening respiratory depression is no real danger of opioids used for pain [36] control, 25% thought it was a real danger. Many studies have shown that this is not the case when titrated [35], even in patients with end-of-life COPD. [43] Pharmacists could support physicians in preventing opioid over dosage in patients with renal failure, if they know that half-life of morphine itself and its active morphine 6-glucuronide is increased in the elderly and in patients with renal dysfunction. [44] However, only 74% answered this statement correctly, and for those who did, it is not known if they actually used this knowledge for prevention in practice. Pharmacists could also have a role in supporting physicians with opioid rotation. Opioid rotation is a complex aspect where the use of equianalgesic tables should always be accompanied by clinical judgement. [45] Half of the pharmacists are sometimes or often consulted by physicians with questions with regard to opioid rotation, and the majority of pharmacists sometimes or often advice physicians to rotate when pain control is inadequate or in case of side effects. However, 40% of the pharmacists indicated they found calculating the correct dosage difficult, and 18%

were not able to calculate the correct dosage when asked to do so. Opioid rotation was also the subject that was most often mentioned by pharmacists as a subject in which they wanted further education (59%).

It is crucial that pharmacists involved in end-of-life care are aware that opioids are not the drug of choice for palliative sedation. About one in three pharmacists thought that opioids are the favoured drugs for palliative sedation. According to Dutch guidelines for palliative sedation, sedation with opioids should be considered malpractice. [31] Sedation was mentioned by half of the pharmacists as a subject in which they would want further education. Co-prescribing of laxatives is the most reported reason in this study for physician-pharmacist contacts, and 63% of the pharmacists reports that not prescribing a laxative when an opioid is prescribed, is a reason for contact. Although laxatives should not be prescribed in the last days of life, it is important that physicians should combine opioids with laxatives in most patients receiving palliative care. [18] Studies have found that many patients who are being prescribed opioids are not prescribed laxatives simultaneously [15;16] Although more evidence for the most effective laxative therapy in end-of-life care is needed [46], checking on concomitant use of laxatives can improve quality of pain management. Pharmacists in our study do seldom contact physicians for co-prescribing of anti-emetics for treatment or prevention of nausea during opioid therapy. Although nausea occurs in 15-30% of the patients receiving morphine for cancer, this is understandable as it is difficult to give advice when studies showing superiority of one anti-emetic drug over another are lacking. [32] As nausea is common at the start of opioid therapy [47], pharmacist-physician contact about nausea is most relevant at the initiation of opioid therapy.

Studies have shown that patients are concerned about using opioids for pain relief, and that these concerns may interfere with adherence to analgesic regimens. [48] Pharmacists in our study experience opioid fear of the patient as a barrier to good pharmacotherapy, and also identify this fear under family members of patients prescribed opioids. With most people with an incurable disease preferring to remain at home during the last part of their lives [49], education about pain management should not only be communicated to the patient, but also towards family caregivers. [50] Financial issues were seldom seen as a barrier, although they are acknowledged to be important at the end-of-life in a previous study in different Western countries. [51] With respect to medication this is not an issue as the costs of opioids are fully reimbursed in the Netherlands by the health care system. With a rather good coverage of pharmacies, availability and logistics are seldom experienced as problematic.

Most pharmacists had working agreements with their physicians about euthanasia (81%); the number of 97% of hospital pharmacists was substantially higher than 71% in 1994 [52], reflecting euthanasia is becoming more incorporated in practice in the Netherlands. Working agreements were less common for palliative sedation (46%) and/or opioid prescribing (25%), and were more common in hospital pharmacy than in community pharmacy. In hospital as well as community settings, specific interventions of pharmacists have shown to contribute to the quality of care of patients at the end-of-their life. [53][54][55] It is usual in the Netherlands that community pharmacist checks all prescriptions after dispensing, whereas hospital pharmacist often only check prescriptions 'at risk', e.g. in case of drug-drug interactions. Our results show, however, that one might question whether current hospital practice is sufficient given the number of issues that warrant attention.

Strengths and limitations of this study

A strength of this study is that it is one of the first studies which maps the knowledge level of pharmacists about the use of opioids and pain management at the end of life. Studies like this are necessary to map strengths and weaknesses in the knowledge levels and experiences, and subsequently improve the (additional) education of pharmacotherapy at the end of life. A limitation of this study is that only a limited number of themes were assessed in which pharmacists should have adequate knowledge, and within those themes, not all aspects were studied. Another limitation is the relatively poor response rate of 45%, with other pharmacy studies reporting higher response rates. [56;57] Our initial sample was representative for pharmacists in the Netherlands, and the characteristics of responders and non-responders were comparable, except for full-time employment. This might have biased our results: full time pharmacists might have more experience and knowledge as they work more hours, and the actual knowledge in the population might be higher.

Another limitation is that some knowledge statements and their answer categories (true or false) leave room for interpretation. In retrospect, the statement that 'simultaneous prescription of a weak opioid (e.g. tramadol) and a strong opioid (e.g. morphine) is contra-indicated' may have been more ambiguous than we intended it to be. Technically speaking, this is not a true contra-indication as it is not a medical condition increasing the risk of a serious adverse drug reaction. However, the addition of tramadol to morphine is not a preferable treatment option, since it does not reduce pain and increases side effects. [58] Open interpretability also concerns the question whether management of nausea in treatment with opioids is evidence based. Antiemetics are not licensed for opioid-induced nausea, and there is lack of knowledge about efficacy in end-oflife [59] , but there are best practices for opioid-induced nausea. [60] We do not know how pharmacists interpreted 'evidence based'. Although respondents of the pilot study did not make remarks with respect to interpretation, open interpretability is relevant to all questions -maybe most to those with a low score.

Conclusion

Although the effects of a gap in the knowledge about opioids in clinical practice differ per subject, we can conclude that most of the knowledge questioned in our study should be part of the basic knowledge pharmacists should have, and that a gap in this knowledge can be a barrier to adequate care at the end of life. One in three pharmacists did not think they had sufficient knowledge to advise about pain control, but most were willing to follow additional education. Physicians were open to unsolicited advice from the pharmacist concerning opioid prescriptions. If education is improved, pharmacists could claim a more active role in communication with patients, caregivers and physicians to improve the quality in end-of-life pharmacotherapy. * The following definition of opioid rotation was given in the questionnaire: "With the term "opioid rotation" we mean the replacing of one opioid by another opioid." ** The following definition of tolerance was given in the questionnaire: "By tolerance for a drug we mean that a patient needs a higher dose to reach the same pain relief while the pain stimulus remains the same. Tolerance has proven to be difficult to measure in practice, we are interested in your personal experience." Purpose: What is the level of knowledge of pharmacists concerning pain management and the use of opioids at the end of life, and how do they cooperate with physicians?

Methods: A written questionnaire was sent to a sample of community and hospital pharmacists in the Netherlands. The questionnaire was completed by 182 pharmacists (response rate 45%).

Results:

Pharmacists were aware of the most basic knowledge about opioids. Twenty nine percent of the pharmacists erroneously thought that life-threatening respiratory depression was a danger with pain control and 38% erroneously thought that opioids were the preferred drug for palliative sedation. One in three pharmacists did not think their theoretical knowledge was sufficient to advice about pain control. Most pharmacists had working agreements about euthanasia (81%), but fewer for palliative sedation (46%) or opioid therapy (25%). In the experience of most pharmacists (93%) physicians were open to unsolicited advice concerning opioid prescriptions. The majority of the community pharmacists (94%) checked opioid prescriptions most often only after dispensing, the majority of the hospital pharmacists (68%) most often did not check the prescriptions at all.

Conclusions:

Although the basic knowledge of most pharmacists was adequate, there seemed to be a lack of knowledge in several areas, which may hamper them to improve the quality of care by giving advice to physicians and preventing or correcting mistakes if necessary. If education is improved, a more active role of the pharmacist might stimulate the quality of end-of-life pharmacotherapy.

Introduction

Pain is one of the symptoms most frequently encountered at the end of life [1;2], although it can be controlled satisfactorily in most cases if health care professionals have adequate knowledge. [3] Possibilities to manage pain in patients at the end of life have increased in the past decade. These possibilities have been incorporated in handbooks and practice guidelines that were developed to support professionals in evidence-based practice [4][5][6].

However, the existence of professional guidelines does not guarantee that this knowledge is optimally used in daily practice. Several barriers have been identified concerning the prescribing and use of opioids that hamper the quality of pain management. First, there is still lack of theoretical knowledge about opioids among practicing physicians and nurses. [4;7-9] A systematic review of barriers hindering cancer pain management found that inadequate knowledge was one of the most reported barriers among physicians for adequate pain management. [10] The most reported misconceptions were the likelihood of opioid addiction and the belief that morphine dosage had an upper limit. Lack of theoretical knowledge also includes rare effects of opioids such as opioid induced hyperalgesia. [11] Secondly, when health care professionals have sufficient theoretical knowledge about pain management guidelines, this does not necessary lead to adherence to these guidelines in daily practice. An example is that patients who clearly need opioids for pain frequently receive dosages that are too low. [12;13] Another example is prescribing laxatives concomitantly with opioids, a clinical rule that has been recommended in the WHO pain ladder [6] and almost all guidelines about pain management. [4;5;14] In daily practice, only about half of the patients are prescribed a laxative concomitantly. [15][16][17] Nearing end-of-life constipation can still be a problem, although the patient's deteriorating functional status can mean management of constipation has a lower priority. In the last days of life when patients are no longer able to receive oral treatment and consciousness diminishes, oral laxatives should be discontinued. [18] Compared to physicians and nurses, surprisingly few studies have been aimed at the pharmacists' knowledge and experiences concerning pain management and opioid use. Among countries, pharmacists have different responsibilities in pharmaceutical care and hence their role in pain management might differ. In contrast with other countries [19], pharmacists are not allowed to prescribe in the Netherlands. Their primary task is to control the quality of prescribing, check for potential interactions and support prescribers and patients with optimal medication use. [20] Dutch hospital pharmacists frequently contact and advise clinicians, and an increasing number is present at wards for consultation and patient education. [21] In community pharmacy, patient education is common and there are regular pharmacotherapeutic audit meetings between pharmacists and general practitioners. [22] Characteristic for the Netherlands is that euthanasia and palliative sedation are legal options when suffering is unbearable and hopeless [23], and most pharmacies are prepared to fill prescriptions for euthanasia. [24] Although not in the Netherlands, some studies investigated pharmacists' knowledge and attitude. They identified a general need for more knowledge and education in pain management. [25;26] Misconceptions were not only prevalent among physicians [10], but also among pharmacists. [27] Risk of addiction was no barrier to most pharmacists to dispense opioids, but conservative physicians and nurses, and concerns of pharmacists for adverse effects were experienced as barriers for opioid dispensing. [28] Hence, the few studies among pharmacists do not support the possible assumption that pharmacists' knowledge is sufficient for achieving optimal pharmacotherapy at the end of life. On the contrary, they suggest that the pharmacist might be a limiting factor in achieving optimal pharmacotherapy at the end of life. However, there have not been surveys performed among pharmacists recently, and the previous studies were all performed in the United States and had relatively low response rates. [27;28] In end-of-life care, interdisciplinary cooperation among professionals is essential [29], and within this team approach the pharmacist has a responsibility in guarding and optimizing the quality of pharmacotherapy.

Hence, it is important that the pharmacist has adequate and up-to-date knowledge of pain management and the medication involved. We aimed to study the level of knowledge of pharmacists in the Netherlands concerning pain management and the use of opioids at the end of life, how they cooperate with physicians and what barriers to good end-of-life care pharmacists experience. The questionnaire was sent in January 2009 followed by two reminders. With the second reminder a non-response questionnaire was enclosed for use in the non-response analysis. Respondents could complete the questionnaire on paper or online. The written questionnaire was anonymous. A separate answer card with the personal data of the respondent could be mailed to prevent further reminders. The online questionnaire was not anonymous, because it would otherwise not be possible to exclude the possibility that people who were not in the sample would complete the questionnaire. When the database was closed the questionnaires were anonymized.

In total 412 pharmacists were sent a questionnaire, 7 envelopes were returned to sender (respondent had moved in case of private address, or was no longer working at the institution in case of work address). Of the 322 community pharmacists, 147 completed the full questionnaire (response rate 46%). Of the 83 hospital pharmacists, 35 completed the full questionnaire (response rate 42%). The total response rate was 45%. Of the completed questionnaires, 70% were completed in writing and 30% were completed online. The non-response questionnaire was completed by 35 community pharmacists and seven hospital pharmacists.

After the closing of the data collection, a document with the correct answers to the knowledge statements was sent by e-mail to those who had indicated they were interested in receiving this. The questionnaire was developed by the authors who have expertise in end-of-life care, pharmacy, medicine, epidemiology and the development of questionnaires. We used previous studies of the knowledge or attitude concerning opioids and pain management at the end of life. [8;30] Questions were adapted and added to be able to answer the research questions, and to fit the specific situation in the Netherlands. For this purpose we used inter alia Dutch guidelines, original articles and review articles. [8;11;31-37] The questionnaire was piloted among GPs in training, elderly care physicians in training, and pharmacists. Opinions differed on the difficulty level of the knowledge statements in the questionnaire, but most respondents did not think the statements were unclear or too difficult; they sometimes even thought they were (too) easy.

The questionnaire consisted of three types of questions: 1) knowledge statements with three answering options: "true", "false" and "don't know" 2) experience and attitude questions with five answering options: "completely agree", "agree more than disagree", "neutral", "disagree more than agree" and "completely disagree" and 3) other questions, partly structured with four answering options: "often", "sometimes", "seldom" and "never" and partly semi-structured. Furthermore several background characteristics were asked, inter alia to grade one's own knowledge about opioids and pain management on a score of 1 to 10.

Analysis

We calculated descriptive statistics for the comparisons between community pharmacists and hospital pharmacists. Chi-square tests were used for comparing proportions between respondents and non-respondents, using linear by linear association for ordinal variables. Student´s t-tests were used for comparing means. For all tests p-values are presented. In Table 3, the categories "completely agree" and "agree more than disagree" were merged to the category "agree" versus the categories "neutral", and "(completely) disagree".

Results

Background characteristics

In Table 1 the background characteristics of the respondents are shown per specialty. Hospital pharmacists were responsible for more patients to whom opioids were dispensed compared to community pharmacists. They also graded their own knowledge concerning opioids and pain management higher.

There was one significant difference in background characteristics between pharmacists who completed the non-response questionnaire (n=44) and pharmacists who completed the full questionnaire (n=182). Nonresponders were more often fulltime employed (93%) than responders (74%, not in Table ). The most important reasons for non-response were no time, too high workload and being understaffed.

Knowledge statements

In Table 2 the answers to the knowledge statements are shown per specialty. Of the 14 presented statements, community pharmacists gave on average between 9 and 10 correct answers. Hospital pharmacists answered more statements correctly (between 10 and 11). Knowledge questions with a low score of correct answers were 'opioids may cause or worsen pain' (statement is true, 11% correct answers), 'drug management of nausea in treatment with opioids is evidence-based' (statement is false, 19% correct answers), 'simultaneous prescription of a weak opioid (e.g. tramadol) and a strong opioid (e.g. morphine) is contra-indicated' (statement is true, 49% correct answers), 'opioids are the favoured drugs for palliative sedation' (statement is false, 61% correct answers) and 'life-threatening respiratory depression is a real danger when titrating morphine to control pain' (statement is false, 71% correct answers). Of the pharmacists, 74% knew that decreased renal function could raise plasma concentration of morphine and its active metabolite (morphine 6-glucuronide), and 82% calculated an example of opioid rotation correctly.

Attitudes, experiences and barriers concerning pain management

Pharmacists did not think that patients always indicate their pain to their physician (Table 3). The majority of the pharmacists (81%) stated that an opioid prescription should consist of both maintenance and as needed medication. Experienced barriers were opioid fear by the patient (79%) and family (72%), and underreporting of pain by patients (77%) and insufficient information about the pain (60%). Practical/logistic issues such as problems with manufacturing/availability, and problems with reimbursement were not often experienced as a barrier.

Attitudes and experiences concerning opioid rotation, tolerance, addiction, and checking prescriptions

Table 4 shows that almost 39% of the pharmacists found calculating of opioid dosages in case of opioid rotation difficult. Fear of addiction was seen as limiting factor for opioid use in daily practice (78%). The majority of the community pharmacists (94%) controlled opioid prescriptions most often after dispensing, the majority of the hospital pharmacists (68%) did not check opioid prescriptions personally. Community pharmacists were more frequently personally involved compared to their clinical colleagues;15% of the community pharmacists, and none of the hospital pharmacists reported to often have personal contact with the patient/caregiver.

Cooperation with physicians by working agreements, advice and contact about prescriptions

Table 5 shows cooperation between pharmacists and physicians. Working arrangements with physicians concerning euthanasia were more frequently reported by hospital pharmacists (97%) than by community pharmacists (77%). Working agreements concerning palliative sedation and opioid usage were less frequent.

Community pharmacist were more often consulted by general practitioners, and hospital pharmacists by nursing home physicians and clinical specialists. Most pharmacists (93%) reported that physicians were open to unsolicited advice concerning the opioids prescribed by the physician. Almost all pharmacists (97%) would contact the physician if there was something wrong with the prescription. Most reported reasons to contact a physician in practice were laxatives not being prescribed (77%), administrative incompleteness (53%) and drugdrug interactions (52%). Most pharmacists contacted the physician when an opioid was prescribed without a laxative (63%). Reasons for not contacting the physician were that he/she might have had a good reason for not prescribing a laxative or that a laxative was not needed as the patient was terminal or being sedated. Other pharmacists dispensed a laxative to the patient according to working agreements, and some did not contact physicians about laxatives. Most pharmacist (96%) did not contact the physician when an opioid was prescribed without prescribing an anti-emetic drug. About three quarters of the pharmacists (76%) indicated that they had enough options for additional education in subjects related to opioids and pain and symptom management at the end of life (not in Table ). Others were very adamant that there were not enough options -neither in their region, nor non-commercial -or concerning information they could use in their daily practice to support physicians.

Additional

Subjects that were most often mentioned in which further education was wanted were opioid rotation (59%), pain and symptom management in general (52%), and palliative sedation (49%). The preferred methods to receive further education were literature in Dutch journals (65%), conferences (47%), courses on the internet (44%), and literature in international journals (41%).

Discussion

The knowledge of the majority of the respondents was adequate on many points. We identified gaps in knowledge about some aspects of opioid use in a considerable part of the pharmacists. Pharmacists answered 71% of the knowledge questions correctly (10 out of 14) . Physicians scored between 31% and 68% correct answers in various studies [38][39][40], and pharmacists scored 46% correct answers in a single previous study. [25] The overall scores in different studies cannot be compared as such because other statements were used.

A point of concern was that only one in nine pharmacists was aware that opioids may cause or worsen pain. This is a rare effect and has not been known for a long time, so it is understandable that many pharmacists are unaware of this fact. However, the clinical effects can be significant if the dose of opioids is increased instead of decreased when a patient suffers from opioid-induced pain sensitivity. [11;41;42] Although the majority of the pharmacists stated that life-threatening respiratory depression is no real danger of opioids used for pain [36] control, 25% thought it was a real danger. Many studies have shown that this is not the case when titrated [35], even in patients with end-of-life COPD. [43] Pharmacists could support physicians in preventing opioid over dosage in patients with renal failure, if they know that half-life of morphine itself and its active morphine 6-glucuronide is increased in the elderly and in patients with renal dysfunction. [44] However, only 74% answered this statement correctly, and for those who did, it is not known if they actually used this knowledge for prevention in practice. Pharmacists could also have a role in supporting physicians with opioid rotation. Opioid rotation is a complex aspect where the use of equianalgesic tables should always be accompanied by clinical judgement. [45] Half of the pharmacists are sometimes or often consulted by physicians with questions with regard to opioid rotation, and the majority of pharmacists sometimes or often advice physicians to rotate when pain control is inadequate or in case of side effects. However, 40% of the pharmacists indicated they found calculating the correct dosage difficult, and 18%

were not able to calculate the correct dosage when asked to do so. Opioid rotation was also the subject that was most often mentioned by pharmacists as a subject in which they wanted further education (59%).

It is crucial that pharmacists involved in end-of-life care are aware that opioids are not the drug of choice for palliative sedation. About one in three pharmacists thought that opioids are the favoured drugs for palliative sedation. According to Dutch guidelines for palliative sedation, sedation with opioids should be considered malpractice. [31] Sedation was mentioned by half of the pharmacists as a subject in which they would want further education.

Co-prescribing of laxatives is the most reported reason in this study for physician-pharmacist contacts, and 63% of the pharmacists reports that not prescribing a laxative when an opioid is prescribed, is a reason for contact. Although laxatives should not be prescribed in the last days of life, it is important that physicians should combine opioids with laxatives in most patients receiving palliative care. [18] Studies have found that many patients who are being prescribed opioids are not prescribed laxatives simultaneously [15;16] Although more evidence for the most effective laxative therapy in end-of-life care is needed [46], checking on concomitant use of laxatives can improve quality of pain management. Pharmacists in our study do seldom contact physicians for co-prescribing of anti-emetics for treatment or prevention of nausea during opioid therapy. Although nausea occurs in 15-30% of the patients receiving morphine for cancer, this is understandable as it is difficult to give advice when studies showing superiority of one anti-emetic drug over another are lacking. [32] As nausea is common at the start of opioid therapy [47], pharmacist-physician contact about nausea is most relevant at the initiation of opioid therapy.

Studies have shown that patients are concerned about using opioids for pain relief, and that these concerns may interfere with adherence to analgesic regimens. [48] Pharmacists in our study experience opioid fear of the patient as a barrier to good pharmacotherapy, and also identify this fear under family members of patients prescribed opioids. With most people with an incurable disease preferring to remain at home during the last part of their lives [49], education about pain management should not only be communicated to the patient, but also towards family caregivers. [50] Financial issues were seldom seen as a barrier, although they are acknowledged to be important at the end-of-life in a previous study in different Western countries. [51] With respect to medication this is not an issue as the costs of opioids are fully reimbursed in the Netherlands by the health care system. With a rather good coverage of pharmacies, availability and logistics are seldom experienced as problematic.

Most pharmacists had working agreements with their physicians about euthanasia (81%); the number of 97% of hospital pharmacists was substantially higher than 71% in 1994 [52], reflecting euthanasia is becoming more incorporated in practice in the Netherlands. Working agreements were less common for palliative sedation (46%) and/or opioid prescribing (25%), and were more common in hospital pharmacy than in community It is usual in the Netherlands that community pharmacist checks all prescriptions after dispensing, whereas hospital pharmacist often only check prescriptions 'at risk', e.g. in case of drug-drug interactions. Our results

show, however, that one might question whether current hospital practice is sufficient given the number of issues that warrant attention.

Strengths and limitations of this study

A strength of this study is that it is one of the first studies which maps the knowledge level of pharmacists about the use of opioids and pain management at the end of life. Studies like this are necessary to map strengths and weaknesses in the knowledge levels and experiences, and subsequently improve the (additional) education of pharmacotherapy at the end of life. A limitation of this study is that only a limited number of themes were assessed in which pharmacists should have adequate knowledge, and within those themes, not all aspects were studied. Another limitation is the relatively poor response rate of 45%, with other pharmacy studies reporting higher response rates. [56;57] Our initial sample was representative for pharmacists in the Netherlands, and the characteristics of responders and non-responders were comparable, except for full-time employment. This might have biased our results: full time pharmacists might have more experience and knowledge as they work more hours, and the actual knowledge in the population might be higher.

Another limitation is that some knowledge statements and their answer categories (true or false) leave room for interpretation. In retrospect, the statement that 'simultaneous prescription of a weak opioid (e.g. tramadol) and a strong opioid (e.g. morphine) is contra-indicated' may have been more ambiguous than we intended it to be. Technically speaking, this is not a true contra-indication as it is not a medical condition increasing the risk of a serious adverse drug reaction. However, the addition of tramadol to morphine is not a preferable treatment option, since it does not reduce pain and increases side effects. [58] Open interpretability also concerns the question whether management of nausea in treatment with opioids is evidence based. Antiemetics are not licensed for opioid-induced nausea, and there is lack of knowledge about efficacy in end-oflife [59] , but there are best practices for opioid-induced nausea. [60] We do not know how pharmacists interpreted 'evidence based'. Although respondents of the pilot study did not make remarks with respect to interpretation, open interpretability is relevant to all questions -maybe most to those with a low score.

  Pharmacists working as health care provider in the Netherlands can specialize in community pharmacy or hospital pharmacy. Both community pharmacists and hospital pharmacists were approached to participate in this study, samples were taken from a national medical address guide. The medical address guide (BSL) is publicly available and holds addresses and basic characteristics of professionals working in Dutch health care. It is updated on a yearly basis. The random sample was taken per specialism: community pharmacists and hospital pharmacists. Inclusion criteria were: working in the Netherlands as a community or hospital pharmacist, not being in training. First all the names were ordered following postal code (region). Next every Nth case was sampled, N being the number in the sample frame divided by the desired number in the sample. The samples were representative for the Netherlands.

  As physicians were generally open to receive unsolicited advice from the pharmacist concerning opioid prescribing (93%), specific interventions can in the Netherlands. The open attitude of physicians might also be a start to develop local working agreements between pharmacists and physicians for palliative sedation and pain management.

  questions have not been validated and this leaves room open to interpretation. However, we believe these results give a good indication of the knowledge level of pharmacists.

  Pharmacists working as health care provider in the Netherlands can specialize in community pharmacy or hospital pharmacy. Both community pharmacists and hospital pharmacists were approached to participate in this study, samples were taken from a national medical address guide. The medical address guide (BSL) is publicly available and holds addresses and basic characteristics of professionals working in Dutch health care. It is updated on a yearly basis. The random sample was taken per specialism: community pharmacists and hospital pharmacists. Inclusion criteria were: working in the Netherlands as a community or hospital pharmacist, not being in training. First all the names were ordered following postal code (region). Next every Nth case was sampled, N being the number in the sample frame divided by the desired number in the sample. The samples were representative for the Netherlands.

  pharmacy. In hospital as well as community settings, specific interventions of pharmacists have shown to contribute to the quality of care of patients at the end-of-their life.[53-55] As physicians were generally open to receive unsolicited advice from the pharmacist concerning opioid prescribing (93%), specific interventions can in the Netherlands. The open attitude of physicians might also be a start to develop local working agreements between pharmacists and physicians for palliative sedation and pain management.

  questions have not been validated and this leaves room open to interpretation. However, we believe these results give a good indication of the knowledge level of pharmacists.ConclusionAlthough the effects of a gap in the knowledge about opioids in clinical practice differ per subject, we can conclude that most of the knowledge questioned in our study should be part of the basic knowledge pharmacists should have, and that a gap in this knowledge can be a barrier to adequate care at the end of life. One in three pharmacists did not think they had sufficient knowledge to advise about pain control, but most were willing to follow additional education. Physicians were open to unsolicited advice from the pharmacist concerning opioid prescriptions. If education is improved, pharmacists could claim a more active role in communication with patients, caregivers and physicians to improve the quality in end-of-life pharmacotherapy.

Table 1 Background characteristics of respondents (n=182).

 1

	Community	Hospital	Total
	pharmacists	pharmacists
	n=147	n=35	n=182	p
	%	%	%

1 Χ-square. 2 Χ-square for trend. Linear by linear association.

3

Student's t.

Table 2 Answers to the knowledge statements per specialty 1 Community pharmacists Hospital pharmacists

 2

	Total	p

% 25 µg p/h 4 % 50 µg p/h 3

	F o r
	P
	e
	e r
		R
		e
	Sedation and shortening of life by opioids	% false % don't know	43 37 11 9 v i e w 65 24 3 9	47 34 9 9	0.183
	12. Opioids titrated against pain, shorten life % true	1	6	2	0.685
		% false	97	94	95
		% don't know	3	0	3
	13. Opioids are the favoured drugs for	% true	36	23	34	0.087
	palliative sedation	% false	59	74	61
		% don't know	5	3	4
	14. Opioids are appropriate drugs to perform	% true	4	0	3	0.078
	euthanasia	% false	94	100	95
		% don't know	1	0	2

1

correct answer is printed in bold .

2

a simultaneous prescription of a weak and a strong opioid is not a contra-indication in the true sense of the word. It is, however, for pharmacodynamic reasons in general not a sensible combination. This is why it is not advocated in the available guidelines for treatment of pain.

3

respondents could circle the following options 12/25/50/75/100/125/150 µg per hour or "don't know". With this question 2 answers were considered correct, because different guidelines give different conversions, which leads to two different answers.

Table 3 Attitudes, experiences and barriers concerning pain management Community pharmacists Hospital pharmacists

 3

	Total	P

Table 4 Attitudes and experiences concerning opioid rotation, tolerance**, addiction, working arrangements with physicians and controlling prescriptions Community pharmacists Hospital pharmacists

 4

	Total	P

Table 5 Cooperation with physicians by working arrangements, advice and contact about prescriptions Community pharmacists Hospital pharmacists

 5

	Total	P

The use of opioids at the end of life: knowledge level of pharmacists and cooperation with physicians

	Journal: Eur J Clin Pharmacol
	Abstract (241 words)	Deleted: 599
	Word count: 3600
	S.D. Borgsteede, PharmD, PhD 1,2 Tables: 5
	C.A. Rhodius, MD 3,4 References: 60
	P.A.G.M. De Smet, PharmD, PhD 5 Running title: opioids and pain management
	H.R.W. Pasman, PhD 3,6 Key words (max 10): opioids, pain management, pharmacists, knowledge, education, multidisciplinary
	B.D. Onwuteaka-Philipsen, PhD 3,6 cooperation
	M.L. Rurup, PhD 3,6
	F o r F o r
	P P
	e e
	e r e r
	R R
	e e
	v i e v i e
	w w
	2 3

European Journal of Clinical Pharmacology

Often/sometimes vs. seldom/never

Acknowledgements

This study was supported by grants from the Platform Palliative Care from the Dutch ministry of Health, the Comprehensive Cancer Centre Amsterdam (IKA) and the Arijan Porsius Fund

Acknowledgements

This study was supported by grants from the Platform Palliative Care from the Dutch ministry of Health, the Comprehensive Cancer Centre Amsterdam (IKA) and the Arijan Porsius Fund

Conflict of interest

The authors declare they have no conflict of interest.

Conflict of interest

The authors declare they have no conflict of interest.