leptospirosis in free-ranging endangered european mink ... - BioOne

Journal of Wildlife Diseases, 46(4), 2010, pp. 1141–1151 # Wildlife Disease Association 2010

LEPTOSPIROSIS IN FREE-RANGING ENDANGERED EUROPEAN MINK (MUSTELA LUTREOLA) AND OTHER SMALL CARNIVORES (MUSTELIDAE, VIVERRIDAE) FROM SOUTHWESTERN FRANCE Marie Moinet,1,2,13 Christine Fournier-Chambrillon,1,15 Genevie`ve Andre´-Fontaine,2,3 Ste´phane Aulagnier,4 Alain Mesple`de,5 Beátrice Blanchard,6 Ve´ronique Descarsin,7,14 Philippe Dumas,8 Yann Dumas,9 Christophe Coı¨c,10 Laurent Couzi,11 and Pascal Fournier1,12 1

Groupe de Recherche et d’Etude pour la Gestion de l’Environnement, route de Prećhac, 33730 Villandraut, France Ecole Nationale Ve´te´rinaire de Nantes, route de Gachet, 44307 Nantes, France 3 Laboratoire de Bacte´riologie Me´dicale et Molećulaire des Leptospires, route de Gachet, 44307 Nantes, France 4 Comportement et Ecologie de la Faune Sauvage, Institut National de la Recherche Agronomique, BP 52627, 31326 Castanet-Tolosan, France 5 Laboratoire De´partemental des Landes, 1 rue Marcel David, 40004 Mont-de-Marsan, France 6 Adiage`ne, 38 rue de Paris, 22000 Saint-Brieuc, France 7 AES Laboratoire, rue Maryse Bastie´, Ker Lann, CS 17219, 35172 Bruz cedex, France 8 Office National de la Chasse et de la Faune Sauvage, Service De´partemental de Charente, 4 rue de l’e´te´, 16440 Nersac, France 9 Fe´de´ration De´partementale des Chasseurs de Dordogne, boulevard de Saltgourde, BP 232, 24052 Pe´rigueux cedex 9, France 10 Cistude Nature, chemin de Moulinat, 33185 Le Haillan, France 11 Ligue pour la Protection des Oiseaux Aquitaine, 109 quai Wilson, 33130 Be`gles, France 12 Socie´te´ Française pour l’Etude et la Protection des Mammife`res, Museúm National d’Histoire Naturelle, 57 rue Cuvier, 75231 Paris cedex 05, France 13 Current address: Agence Nationale de Sećurite´ Sanitaire, Laboratoire de la Rage et de la Faune Sauvage de Nancy, Technopole Agricole et Ve´te´rinaire, Domaine de Pixerećourt, BP 40009, 54220 Malze´ville, France 14 Current address: Cabinet Ve´te´rinaire, 15 avenue Rioust des Villes Audrain, 56800 Ploermel, France 15 Corresponding author (email: [email protected]) 2

To study the possible role of disease in the decline of endangered European mink (Mustela lutreola), we conducted a survey of antibody prevalence and renal carriage of pathogenic leptospira (Leptospira interrogans sensu lato) using serum and kidney samples collected from 1990 to 2007 from several free-ranging small carnivores and farmed American mink (Mustela vison) in southwestern France. An indirect microscopic agglutination test using a panel of 16 serovars belonging to 6 serogroups (Australis, Autumnalis, Icterohæmorrhagiæ, Grippotyphosa, Panama, Sejroe) revealed antibodies in all species, with significant differences in antibody prevalences: 74% in European mink (n599), 65.4% in European polecats (Mustela putorius, n5133), 86% in American mink (n574), 89% in stone martens (Martes foina, n519), 74% in pine martens (Martes martes, n519), 35% in common genets (Genetta genetta, n579), and 31% in farmed American mink (n551). Serogroups Australis and Icterohæmorragiæ were dominant in most free-ranging species; serogroup Grippotyphosa had high prevalences in European mink. Such high antibody prevalences have never been reported. They are probably related to the large number of known reservoirs, rats (Rattus spp.), muskrat (Ondatra zibethicus), and coypu (Myocastor coypu), in the study area. The polymerase chain reaction test specific for pathogenic leptospiral DNA detected renal carriage in 23% of 34 European mink, 22% of 18 polecats, and 15% of 33 free-ranging American mink, with no significant differences. Renal carriage shows that mustelids may shed leptospira for short periods, but their epidemiologic role is probably limited. High antibody prevalences suggest that the disease is unlikely to be highly pathogenic for these species; however, chronic forms of the disease (abortions, renal lesions) could reduce the reproductive success or life span of infected animals. Further studies on the pathogenicity of leptospirosis in these populations are needed to measure its impact on the population dynamics of these rodent predators. Key words: France, Genetta, Leptospira interrogans sensu lato, Martes, Mustela, renal carriage, serology. ABSTRACT:

INTRODUCTION

The European mink (Mustela lutreola) is one of the five most threatened

carnivores in Europe, and is currently listed as endangered by the International Union for the Conservation of Nature and Natural Resources (Maran et al., 2008).

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This small semiaquatic mustelid has vanished from its former range during the last century and is now distributed in three well-separated populations, one in northern Spain and southwestern France, a second in Romania, and a third larger population in Belarus and Russia (Maran et al., 2008). The populations are still declining in these three areas due to several causes, including anthropogenic pressure (habitat loss and degradation, historic hunting, accidental trapping, predation by dogs, and collisions with motor vehicles), interspecific competition with the alien invasive American mink (Mustela vison), and infectious diseases (Fournier and Maizeret, 2003; Maran, 2007). Moreover, genetic studies clearly illustrate an absence of genetic diversity within the western population (Michaux et al., 2005). Identification of the role of infectious disease is an important concern for conservation strategies, especially because animals with low genetic diversity could be more vulnerable to infectious disease (O’Brien and Evermann, 1988). Recent studies of the western population of European mink revealed the prevalence of Aleutian disease virus and canine distemper virus, whereas exposure to other viral pathogens appeared to be low (Fournier-Chambrillon et al., 2004a; Philippa et al., 2008). Another potential disease is leptospirosis, a worldwide zoonotic disease caused by a pathogenic spirochete, Leptospira interrogans sensu lato (L. interrogans sl), which affects a wide range of vertebrates, including humans. The epidemiology of this disease is complex, with more than 200 pathogenic serovars, and it is possible to be both clinically susceptible to one serovar and a reservoir for another (Levett, 2001). Transmission occurs mainly through direct or indirect contact with the urine of infected animals. Clinical features range from asymptomatic infection for the maintenance host to severe or even lethal hepatic, renal, or cerebral dysfunctions for incidental hosts. For some domestic

species such as horses, cattle, or swine, reproductive problems due to chronic infection are known to have important economic consequences (Andre´-Fontaine, 2004b). Wildlife is considered to be an important reservoir (Levett, 2001), but although the universal role of rodents in the epidemiology of the disease is well documented (Fennestad and Borg-Petersen, 1972; Aviat et al., 2009), little is known about other wild species. Within the superfamily Musteloidea, striped skunks (Mephitis mephitis) and raccoons (Procyon lotor) are considered to be reservoirs for leptospira (Reilly, 1970; Ferguson and Heidt, 1981; Richardson and Gauthier, 2003). No clinical signs of the disease or abortions have been described in these species, but there is evidence of renal carriage, long-lasting leptospiruria, and associated renal lesions (Roth et al., 1963; Crowell et al., 1977; Schowalter et al., 1981). Leptospiral infection has also been reported in sea otters (Enhydra lutris) and North American river otters (Lontra canadensis; Hanni et al., 2003; Gaydos et al., 2007), small Indian mongoose (Herpestes javanicus auropunctatus; Everard et al., 1983), Asian palm civets (Paradoxurus hermaphroditus; Gordon Smith et al., 1961), and genets (Genetta spp.; Sebek et al., 1989). In Europe, some infected mustelids have been reported (Fennestad and Borg-Petersen, 1972; Hathaway et al., 1983), but in most cases, samples were very small, and tests were negative. More recently, Millań et al. (2009) studied leptospirosis in wild and domestic carnivores from Andalusia, Spain, and recorded a 12.5% antibody prevalence in 24 common genets (Genetta genetta). We conducted a similar study within the French range of the European mink in order to identify the potential role of the disease on the decline of the species. First, we investigated the prevalence of antibodies to L. interrogans sl in several freeranging small carnivores and in farmed

MOINET ET AL.—LEPTOSPIROSIS IN FREE-RANGING CARNIVORES

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American mink to measure exposure to this pathogen; second, we determined the prevalence of renal carriage of spirochetes in some species in order to better evaluate their potential epidemiologic role. MATERIALS AND METHODS Study area and samples

Sera were obtained from 449 banked serum samples collected from 423 animals: 99 European mink, 133 polecats (Mustela putorius), 74 American mink, 19 stone martens (Martes foina), 19 pine martens (Martes martes), and 79 genets trapped in eight de´partements of southwestern France (42u479–46u229N, 0u549–4u79W; Fig. 1) between 1998 and 2003. Trapping and handling procedures are described in previous papers (Fournier-Chambrillon et al., 2004a; Philippa et al., 2008). Individual animals were sometimes caught several times. In addition, blood samples were collected in 2003 from 51 farmed American mink belonging to three farms in three de´partements in the study area. Blood was centrifuged for 5 min at 3,000 3 G on the same or next day after sampling, and serum samples were stored at 220 C. Kidneys were obtained from banked kidney samples collected during standardized necropsies (Fournier-Chambrillon et al., 2004b) of 34 European mink, 18 polecats, and 33 American mink in the same study area between 1990 and 2007. Kidney samples were stored at 220 C before analysis. Serologic studies

Fifty-microliter serum aliquots were examined for leptospiral agglutinins. The indirect microscopic agglutination test (MAT) was performed according to Faine (1982) using a panel of 16 serovars (reference strains from Institut Pasteur, Paris, France, and local isolates indicated by *) belonging to six serogroups: Australis (serovars: Australis, Bratislava, and Mu¨nchen strain ENVN 372*), Autumnalis (serovars: Autumnalis and Autumnalis strain ENVN 32*), Icterohæmorrhagiæ (serovars: Copenhageni, Icterohæmorrhagiæ, and Icterohæmorrhagiæ strain ENVN 19*), Grippotyphosa (serovars: Grippotyphosa and Vanderhœdoni strain ENVN 35*), Panama (serovar: Panama strain ENVN 374*), and Sejroe (serovars: Sejroe, Hardjo, Wolffi, Saxkœbing, and Saxkœbing strain ENVN 296*). Titers $80 for at least one serovar per serogroup were considered positive.

FIGURE 1. Geographic distribution of 423 freeranging small carnivores tested for Leptospira interrogans sensu lato in southwestern France using a microagglutination test. Each symbol represents the unique sample of each animal included in the analysis. Antibody-negative animals are in gray; antibody-positive animals are in black. PCR analysis

DNA from 30 mg of each kidney was purified with QIAamp DNA mini kitH (Qiagen, Courtaboeuf, France). Real-time polymerase chain reaction (PCR) amplification using the ADIAVETH Lepto real-time PCR kit (Adiage`ne, Saint Brieuc, France) and targeting the hap1 gene specific of pathogenic Leptospira (Branger et al., 2005) was performed on each DNA extract. Two microliters of DNA extract were mixed with 48 ml of a PCR reagent (ADIAVETH) and analyzed following the manufacturer’s recommendations. Twenty-four animals (two European mink, two polecats, and 20 American mink) had been blood-sampled shortly before (0– 6 days before) and were tested using both serologic and PCR methods. Data and statistical analysis

Eleven European mink, four polecats, one American mink, and one genet were sampled twice, three European mink were sampled three times and one European mink was

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TABLE 1. Antibody prevalence (%) to Leptospira interrogans sensu lato in free-ranging small carnivores and farmed American mink in southwestern France using the microagglutination test. CI 5 confidence interval.

Variable

Positive/ tested Prevalence 95% CI a

Mustela lutreola

Mustela putorius

Mustela vison

Martes foina

Martes martes

Genetta genetta

Farmed American mink

73/99

87/133

64/74

17/19

14/19

28/79

16/51

74a,b 63.9–82.1

65.4c,d,e 57.7–73.9

35a,d,f,h 25.1–47.1

31b,e,g,i,j 19.2–46.0

86c,f,g 89h,i 73j 76.5–93.3 66.9–98.7 48.8–90.9

Values with the same superscript are significantly different among species (P#0.05).

sampled four times, with a mean interval of 45 wk (6–112 wk). To determine antibody prevalence and for all statistical tests, resampled animals were represented once (the first positive sample, or when more than one sample was positive, the sample positive for most serogroups). To test the influence of sex and species on antibody prevalence, logistic regression was computed using Minitab software (Minitab Inc., State College, Pennsylvania, USA), with species and sex as predictor variables. For the purpose of the analysis, free-ranging and farmed American mink were considered separate species groups. When a variable was significant, antibody prevalences were compared using a chi-square test (or Fischer’s exact test when expected frequencies were insufficient), followed by a multiple comparisons test (Sokal and Rohlf, 1995). The five native species were split into two geographic groups: group 1 for animals trapped in the feral American mink range and group 2 for animals trapped outside this area. To test the prevalence of leptospirosis in animals living in the vicinity of American mink, a second logistic regression was computed with species and geographic group as predictor variables. When a variable was significant, antibody prevalences were compared as described previously. Differences in antibody prevalence between species within each serogroup and differences in antibody prevalence between serogroups within species were tested using a chi-square test followed by a multiple comparisons test (or Fischer’s exact test when expected frequencies were insufficient). The influence of sex and species on the renal carriage rate was tested using logistic regression with species and sex as predictor variables, followed by a chi-square test and a multiple comparison test for the significant variables. For all statistical tests, P#0.05 was considered statistically significant.

RESULTS

Positive reactions to antibodies to L. interrogans sl were recorded for all species (Table 1) and across the entire study area (Fig. 1); prevalences ranged from 31% to 89% depending on species. Most animals were positive for one (n5117) or two serogroups (n595), but some reacted to three (n552), four (n522), or five serogroups (n513). Both regressions (species and sex, species and geographic group) revealed that species was the only significant variable (P,0.001 and P50.007, respectively), and the chi-square test was highly significant (x2577.1, df56, P,0.001). Multiple comparison tests showed that positive animals were significantly less numerous among farmed American mink than all free-ranging species (P#0.002) except the common genet. The occurrence of positive animals was also significantly lower among common genets than all freeranging species (P,0.001) except the pine marten. Moreover, the occurrence of positive animals was significantly lower among polecats than the free-ranging American mink (P50.001). All six serogroups were detected in all species except Panama (Pan), which was not detected in farmed American mink, and Autumnalis (Aut), which was only detected in the mink species (Table 2). Antibody prevalences differed significantly among species within each serogroup (x2.23.0, df56, P,0.001 for all groups). The highest prevalences ($50%) were for Icterohæmorrhagiæ (Ih) in free-ranging

8/51 16 QQ

5/79 6v

4/19 21

5/19 26

13/74 18 V,Y,AA

10/133 7.5 u,M,Q,T

38/99 38 u,v,E,J

Panama

0/51 0 y,aa,NN,OO,PP,QQ,RR

1/79 1 w,z,LL

3/19 16

3/19 16 HH

3/74 4 x,W,BB,DD

24/133 18.0 z,aa,N,R

20/99 20 w,x,y,B,F,H

Same superscripts in capitals within lines indicate significant differences among serogroups within species (P#0.05).

11/51 22 q,s,PP

9/79 11 m,p,r,t,MM

9/19 47 t,JJ

13/19 68 o,r,s,FF,HH

48/74 65 l,n,p,q,X,AA,BB,CC

25/133 18.8 k,n,o,L,P

41/99 41 k,l,m,D,H,I

Grippotyphosa

Values with the same superscript in lowercase letters within columns are significantly different among species within serogroups (P#0.05).

9/51 18 h,i,j,OO

0/79 0 j,KK,MM

0/19 0 II,JJ

0/19 0 EE,FF,GG

2/74 3 U,X,Y,Z

0/133 0 i,K,P,Q,R,S

1/99 1 h,A,D,E,F,G

Icterohæmorrhagiæ

A

11/51 22 b,d,f,NN

17/79 21 a,c,e,g,KK,LL

10/19 53II

11/19 58 g,EE

40/74 54 e,f,U,V,W

71/133 53.4 c,d,K,L,M,N,O

56/99 56 a,b,A,B,C

Autumnalis

a

Positive/tested Prevalence

Farmed American mink


Genetta genetta

Martes martes Positive/tested Prevalence


Martes foina


Mustela vison


Mustela putorius


Mustela lutreola

Australis

11/51 22 RR

6/79 8 bb,cc

2/19 10

8/19 42 cc,GG

24/74 32 bb,Z,CC,DD

29/133 21.8O,S,T

15/99 15 C,G,I,J

Sejroe

TABLE 2. Antibody prevalence (%) to the six serogroups of Leptospira interrogans sensu lato tested in free-ranging small carnivores and farmed American mink in southwestern France using the microagglutination test.

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American mink and stone martens, and for Australis (Aus) in all other free-ranging mustelids. Antibody prevalences were also relatively high for Ih and Grippotyphosa (Grip) in European mink (41% and 38%, respectively), Ih in pine martens (47%), and Sejroe (Sj) in stone martens (42%). Significant differences among species within serogroups are given in Table 2. Serologic profiles varied among species. Within each species, antibody prevalences differed among serogroups (x 2513.1, df55, P,0.05 for farmed American mink; x2.22, df55, P,0.005 for all free-ranging species), and significant differences are given in Table 2. In European mink, dominant serogroups were Aus (with a large percentage of high titers), Grip, and Ih (Fig. 2); in European polecats and common genets, the dominant serogroup was Aus (with a large percentage of high titers); in American mink, stone martens, and pine martens, dominant serogroups were Ih and Aus (with a large percentage of high titers), and Sj also had a large percentage of high titers in stone martens. In farmed American mink, all serogroups but Pan were equally represented, and the percentage of high titers was high in Ih, Aut, and Grip. Pathogenic leptospiral DNA was detected in the kidneys of all the Mustela species, with a mean renal carriage of 20% that was not significantly different according to sex or species (Table 3). Among the 24 animals tested with both methods (antibody prevalence and DNA detection), three of four antibody-negative animals also had a negative result for PCR, whereas only three of 20 antibody-positive animals had a positive result for PCR (Table 3). All animals positive for PCR were American mink, but the samples of European mink and polecats were too small to be representative. DISCUSSION

Our results demonstrate high antibody prevalence for L. interrogans sl in small

FIGURE 2. Percentages of low and high titers for each of the six serogroups among the total number of positive reactions to Leptospira interrogans sensu lato for six free-ranging carnivores and farmed American mink in southwestern France using a microagglutination test. (Aus 5 Australis, Aut 5 Autumnalis, Gri 5 Grippotyphosa, Ih 5 Icterohæmorrhagiæ, Pan 5 Panama, Sj 5 Sejroe).

carnivores of southwestern France, particularly free-ranging mustelids, and we report the first serologic evidence of leptospiral infection in the European mink. To our knowledge, such high antibody prevalences in free-ranging mustelids and viverrids of Europe have never been reported in the literature, suggesting a high exposure of these species to leptospira in our study area. The source of infection is usually either direct or indirect contact with an infected animal or its urine and the risk of exposure to leptospirosis has been linked to aquatic or humid environments (Andre´-Fontaine


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TABLE 3. Renal carriage prevalence (%) and 95% confidence intervals (CI) for pathogenic Leptospira interrogans sensu lato in free-ranging mustelids from southwestern France using ADIAVETH Lepto real-time PCR kit, and number of microagglutination test (MAT) antibody-positive and antibody-negative polymerase chain reaction (PCR)–positive animals. No significant differences in renal carriage prevalence were observed among species. Number of MAT antibody-positive and antibody-negative PCR-positive animals

Renal carriage prevalence

Mustela lutreola Mustela putorius Mustela vison

Positive/tested

Prevalence (95% CI)

PCR-positive/ antibody-positive

PCR-positive/ antibody-negative

8/34 4/18 5/33

23 (11.0–41.8) 22 (6.4–47.6) 15 (5.2–32.3)

0/1 0/1 3/18

0/1 0/1 1/2

et al., 1992). Free-ranging carnivores, particularly semiaquatic species, may be infected through contaminated water or prey. Enteric leptospiral infection has been described in carnivores (Reilly et al., 1970), as well as bites or urine projection during predation. Brown rats (Rattus norvegicus), which are excellent disseminators of leptospira, and invasive alien species like muskrat (Ondatra zibethicus) or coypu (Myocastor coypu) are known reservoirs and potential prey in the study area (Faine, 1982; Michel et al., 2001; Aviat et al., 2009). In our study, antibody prevalence was particularly high in European and American mink, which rarely venture from riverbeds and display a strong preference for flooded habitats and aquatic prey. Prevalence was lower in polecats, which move between terrestrial and aquatic habitats (Fournier et al., 2007, 2008). High antibody prevalence was also recorded in martens, though they cannot be extrapolated to the whole population due to the small sample size. Because these species prefer dry terrestrial habitats, the risk of leptospirosis exposure could be related more to their diet than their habitat. Significantly lower prevalence was observed in common genets, which are usually found in rocky or wooded habitats with thick vegetation (Jennings and Veron, 2009). This species could be less susceptible to infection than mustelids; the serovars could be less adapted to

this species, which is phylogenetically closer to felines than canines (Wilson and Reeder, 2005; Andre´-Fontaine, 2006). The significantly lower antibody prevalences in farmed American mink can be explained by industrial feed and farming conditions in individual aboveground cages, which limit contact with rodents and contaminated water as well as intraspecific contamination. We found antibodies against several serogroups in the same serum, a result that raises two main hypotheses: (1) Successive infections could have occurred during the life of the animal, relatively close in time, so that the animal produced the same titer of antibodies for both serogroups; (2) since cross-reaction frequently occurs between different serogroups, especially in acute-phase samples (Levett, 2001), serogroups responsible for infection are not necessarily those evidenced by MAT. Due to our sampling method, no culture could be done to distinguish coagglutination from multiinfection and confirm the infective strain. Nevertheless, the MAT data reveal the serogroups present in the population (Levett, 2001). Australis and Icterohæmorrhagiæ were the most frequent serogroups detected in free-ranging species, and this is consistent with data available in France on feral rodents (Aviat et al., 2009) and domestic animals (Lefur et al., 2007). High titers frequently found for Australis in our study could be explained by a high

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infection pressure in nature. Animals may be repeatedly infected and present high antibody titers because their immune system is regularly solicited. European mink was the only free-ranging species showing a high antibody prevalence for Grippotyphosa, which can be related to either a higher exposure level or higher receptivity for this serogroup. In farmed American mink, serologic profiles differed strongly from free-ranging species (absence of serogroup Panama, higher seroprevalence of serogroup Autumnalis), confirming that the sources of contamination are different from those of freeranging species. The role of free-ranging carnivores in the epidemiology of leptospirosis has to be considered, because all species infected by leptospira are potential excretors and then amplifier hosts. The relative epidemiologic efficiency must be assessed by the leptospiral emission rate, which depends on receptivity to infection, renal carriage rate, excretion level and duration, demographic importance, quantity of urine produced (relative to the size of the animal), and behavior (Andre´-Fontaine, 2004a). The renal carriage rate assessed in our study for free-ranging minks and polecats, though possibly underestimated due to the lower PCR sensitivity in frozen samples (Branger et al., 2005), shows that they may excrete leptospira after infection. However, the fact that most MAT-positive animals were PCR negative suggests a short urinary excretion, and their role as reservoir is unlikely. Rodents outnumber their predators considerably (up to 10 muskrats and 1000 brown rats per hectare [Le Louarn and Que´re´, 2003] vs. one polecat per km2 [Larivie`re and Jennings, 2009]), and unlike small carnivores, their social behavior increases the risk of intraspecific infection. While other leptospira emission rate factors could not be investigated in this study, we suggest that the epidemiologic role of American mink, polecat, and a fortiori endangered European mink is negligible compared to that

of rodents. This hypothesis is supported by our finding that the antibody prevalence in animals living in the vicinity of feral American mink was not greater. Mathematic models of bacterial or viral diseases (McCallum and Dobson, 1995) suggest that a disease of high prevalence is unlikely to be a major problem to an endangered species, but these models are single-host species models and assume that the disease primarily increases mortality. However, if the main effect on the host is a decrease in fecundity, highprevalence diseases may have a major impact (McCallum and Dobson, 1995), especially when reservoir species are locally abundant (McCallum and Dobson, 2002). The impact of chronic leptospirosis is often underestimated, even in domestic species, and the reproductive problems recorded in cattle could also harm domestic carnivores (Luciani, 2004; Andre´-Fontaine, 2006). Therefore, we can hypothesize that leptospirosis could cause abortions or decrease in fertility in mustelids as well, and could be one of the factors explaining the smaller litters recorded in the western population of European mink (Fournier-Chambrillon et al., 2010). Insofar as no histopathologic study has been conducted, chronic renal lesions (interstitial nephritis, glomerulonephritis) similar to those observed in evolutionary close species like skunks (Crowell et al., 1977) are likely and could reduce fitness and life span. In the endangered European mink, these demographic consequences could be dramatic. It is practically impossible to protect European mink from leptospirosis in the wild. Remembering that vaccinating an endangered species is always hazardous (Woodroffe, 2001), in addition to the risk of undesirable side effects, the only vaccine available in France, which protects only against the Canicola and Icterohæmorrhagiæ serovars, is of low efficacy against chronic forms of the disease and urine shedding (Andre´-Fontaine et al., 2003). The main action in the wild should


be the strict disinfection of mustelid and rodent traps, along with heightened trapper awareness. Because breeding programs have been initiated elsewhere in Europe and are under consideration in France, strict sanitary controls should be applied to prevent contamination. Breeder enclosures should be impenetrable to rodents, and, on the assumption that leptospirosis can reduce fertility, animals should receive antibiotic preventive treatment at the beginning of the reproductive period. In conclusion, we have shown that freeranging carnivores of southwestern France, particularly mustelids, are largely exposed to pathogenic leptospira. Further studies should be focused on leptospirosal pathogenicity among these populations to improve our knowledge of the impact of this disease on these species, particularly the endangered European mink. ACKNOWLEDGMENTS

This study was funded by the Conseil Geńe´ral des Landes, Conseil Re´gional d’Aquitaine, Groupe de Recherche et d’Etude pour la Gestion de l’Environnement, Laboratoire de Bacte´riologie Me´dicale et Molećulaire des Leptospires, and Ministe`re de l’Ecologie et du De´veloppement Durable. We thank E. Faure, M. Liabeuf, E. Mazzola-Rossi and A. Perrot for their contribution to the handling of the animals, T. Agrafel, S. Goujon and A. Granel who allowed us to sample American mink in their farms, B. Bedel for its contribution to the data capture, and E. Mathieu from Pfizer Sante´ Animale who kindly provided DomitorH and AntisedanH for the anesthesia of the animals. Special acknowledgement goes to all members of the Re´seau Vison d’Europe, the large trapping network which captured the animals. LITERATURE CITED G. 2004a. Connaıˆtre les risques de transmission de la leptospirose a` l’homme par les animaux de compagnie. Le Nouveau Praticien Ve´te´rinaire juin/juillet 2004: 37–40. ———. 2004b. Leptospiroses animales. Bulletin Epide´miologique de l’AFSSA 12: 1–3. ———. 2006. Canine leptospirosis—Do we have a problem? Veterinary Microbiology 117: 19–24. ———, X. PESLERBE, AND J. P. GANIERE. 1992.

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