PREVALENCE OF ANTIBODY TO ALEUTIAN MINK DISEASE VIRUS IN EUROPEAN MINK (MUSTELA LUTREOLA) AND AMERICAN MINK (NEOVISON VISON) IN SPAIN

The European mink (Mustela lutreola) is a riparian mustelid species that has suffered a dramatic population decline during the 20th century (Youngman 1982; Maran 2007). This native species is considered one of the most endangered mammals and is listed as critically endangered in the International Union for Conservation of Nature Red List of Threatened Species (Maran et al. 2011). The species survives in less than 15% of its original range in three isolated populations: NW Russia, Romania/Ukraine, and France/Spain (Maran et al. 2011). The Spanish population was estimated at <500 adults (Palazón and Ceña 2007).

The American mink (Neovison vison) is a semiaquatic mustelid native to North America, with a similar body size. In Europe, the species became established and widespread during the 20th century after accidental and deliberate releases from fur farms (Dunstone 1993; Bonesi and Palazón 2007). The introduction and expansion of the feral North American mink has had negative effects on the European mink and other native animals (Maran et al. 1998; Sidorovich 2001; Macdonald and Harrington 2003; Poˇdra et al. 2013).

American mink were first recorded as a feral species in Spain in 1978 in Segovia, central Spain (M. Delibes and F. Amores, unpubl. data). Now there are five large, widely distributed populations in Spain, all of which are expanding (Ruiz-Olmo et al. 1997; MAGRAMA 2014). As an invasive species, American mink are subject to routine culling in an attempt to control their population (Bonesi and Palazón 2007).

The expansion of American mink into the range of the native species is considered the main threat for the endangered European mink (Maran and Henttonen 1995; Sidorovich 2001; Maran 2007; Poˇdra et al. 2013). Long-term sympatric coexistence of both mink species has not been documented (Maran et al. 2011). However, this co-occurrence of European and North American mink alone is not sufficient to explain the decline of the European mink because, in many areas, the decline of the native mink was evident prior to the arrival of the exotic species (Lodé et al. 2001; Maran 2007). Others have suggested that the decline of the European mink might be attributed to habitat loss, over-hunting, or pollution (Youngman 1982; Lodé et al. 2001; Maran 2007). Aleutian mink disease (AMD) and other viral infections have also been suggested as factors in the decline of the European mink population (Mañas et al. 2001; Skumatov 2001; Philippa et al. 2008).

Infection with Aleutian mink disease virus (AMDV; Parvoviridae) is widespread and AMDV is the most significant infectious agent affecting farmed American mink worldwide (Hossain-Farid et al. 2010; Jahns et al. 2010; Nituch et al. 2011; Sang et al. 2012). Due to the severe economic losses to the fur industry in all mink-breeding countries, there have been attempts to reduce AMDV prevalence in mink farms by implementing serologic screening programs and culling positive animals (Jensen et al. 2011; Espregueira-Themudo et al. 2012). In spite of these efforts, AMDV continues to be a significant problem. Several studies have been conducted to determine the prevalence of AMDV in free-ranging American mink within its original North American range and the introduced distribution area (Table 1).

Infection with AMDV causes acute or chronic disease in farmed American mink and other mustelids, depending on host factors such as age, genotype, and the virulence of the viral strain (Bloom et al. 1980, 1994). These syndromes span from acute progressing interstitial pneumonia with high mortality in newborn mink kits, which are not protected by maternal antibodies (Larsen et al. 1984; Alexandersen et al. 1994), to a chronic, usually persistent, immune complex-mediated disease with hypergammaglobulinemia, infiltration of tissues with lymphocytes and plasma cells, glomerulonephritis, nonsuppurative meningoencephalitis, decreased fertility/abortion, and severe chronic immune dysfunction with increasing susceptibility to other diseases (Ingram and Cho 1974; Bloom et al. 1980; Hansen and Lund 1988; Bloom et al. 1994; Broll and Alexandersen 1996).

Aleutian mink disease virus is highly stable and persistent in the environment. Transmission can be demonstrated both horizontally by blood, urine, feces, saliva, and insect vectors and vertically by the infection of kits by infected dams during the perinatal period (Kenyon et al. 1963; Gorham et al. 1964; Ingram and Cho 1974; Porter and Larsen 1974; Hansen 1985; Keven-Jackson et al. 1996a, b).

Since the first detection of AMDV in European mink in 2001 (Mañas et al. 2001), disease monitoring in this endangered species has been considered a priority in the conservation effort. Routine serologic tests have been included in all field studies where animals are captured. In France, an AMDV antibody prevalence of 12% (n=99) has been reported (Fournier-Chambrillon et al. 2004), and no antibody-positive animals were detected in 84 samples from Navarra (Spain) (Sánchez-Migallón et al. 2008). Our objective in this study was to describe the role of AMDV infection in both mink species (native and introduced) in their distribution areas in Spain over a 16-yr period.

Blood samples from native European mink were obtained by: a) surveys of European mink in the Spanish distribution areas, b) periodic population studies conducted in specific river drainages, c) campaigns to capture founders for the European mink breeding program in Spain (2004) (MAGRAMA 2009), and d) animals captured accidentally during the culling and trapping of feral American mink (MAGRAMA 2014). The management protocol for all captured European mink included blood extraction for AMDV antibody tests (MAGRAMA 2005).

Samples from feral American mink were collected during the population control operations (MAGRAMA 2014), which were conducted by several governmental authorities and performed by rangers and biologists acting as trappers. The captured animals were transported to an official wildlife center for clinical examination and euthanasia following the standards of animal welfare and legal procedures (MAGRAMA 2014). Both species were captured in single entry 15×15×60-cm homemade wire cage traps.

Captured European mink were anesthetized intramuscularly with a combination of 5 mg/kg ketamine hydrochloride (Imalgène 1000, Merial, Lyon, France) and 0.10 mg/kg medetomidine hydrochloride (Domtor, Orion Corporation, Espoo, Finland). Atipamezole (Antisedans, Orion Corporation, Espoo, Finland) was used for reversal at five times the medetomidine dose. All European mink were clinically examined and bled by jugular puncture; sex and weight were recorded, and they were marked with subcutaneous passive transponder tags for identification. After recovery from anesthesia, they were released at their capture locations. Sera were stored at −20 C until tested.

American mink were also anesthetized, and blood samples were collected from the jugular vein or by cardiac puncture. After data collection and while still under anesthesia, animals were sacrificed following the welfare legal standards and submitted for postmortem examination (MAGRAMA 2014). Necropsies included reproductive examination and extraction of a canine tooth for age determination. Only 2% of these samples were obtained at sites within a 50-km radius of active fur farms. Figure 1 shows the American mink distribution in Spain (Bravo 2007) with the six populations included in this study.

Testing for AMDV antibody was performed in serum samples by counter-current immunoelectrophoresis (CIEP: Bloom et al. 1975; Oie et al. 1996) using the same procedure performed in mink farms for serologic screening (Jensen et al. 2011; Espregueira-Themudo et al. 2012).

To avoid disturbance during the reproductive period, trapping was halted between May and August in the European mink distribution area. Animals captured between January and April were assigned to the prereproductive period, and those captured between September and December were assigned to the postreproductive period. Only animals captured during the prereproductive period were included in the weight analysis to avoid the smaller size of subadult animals and difficulties in separating subadult and adult animals.

The prevalence of AMDV antibody was defined as the percentage of individuals with a positive CIEP test. For animals recaptured more than once per year, the latest result was used. Animals recaptured during the 45 d following a previous capture were not subjected to anesthesia or blood extraction. The true incidence of AMDV infection was calculated by dividing the number of animals that seroconverted by the period at risk (days between two negative tests or days between a negative and a positive test divided by two). For this purpose, one animal that showed a negative result between two positive tests was considered a false negative result.

Necropsies of the American mink included the routine extraction of one canine tooth from each specimen for age determination. Annuli in the dental cementum were counted and the age was estimated using the standardized, species-specific age analysis models of Matson’s Laboratory (Milltown, Montana, USA) following their own internal protocol (Matson 1981). Individuals were divided into age classes at 1-yr intervals. Animals without cementum annuli and with open apical foramen were classified as a subadult or in the 0+ group (animals <1 yr old) and samples with one or more cementum annuli were classified as adult. We assumed a fixed date of birth of 1 May, based on bibliography (Sidorovich 1993; Bravo and Bueno 1999; García-Díaz and Lizana 2013).

American mink mating takes place in the first 3 wk of March and kits are born by late April or early May, with an average litter size of six kits (Espregueira-Themudo et al. 2011). Considering these dates, the uteri from 27 American mink (4+ yr or less age class) trapped in April were examined for embryos by eye and microscopically.

EpiCalc 2000 (http://www.brixtonhealth.com/epicalc.html) was used to estimate the 95% confidence intervals (CI). A chi-square analysis was used to test for significance of differences in AMDV antibody prevalence between sexes. A one-sample Z-test was used to estimate differences in proportions, and analysis of variance was used to compare weight differences between positive and negative European mink using SPSS 19 (IBM Corporation, Armonk, New York, USA).

From 1997 to 2012, 410 individual European mink were captured (245 males and 165 females, P<0.01) in the Spanish distribution area, and 492 serum samples were obtained. For animals recaptured more than once during the same year, the prevalence was calculated using the latest sample obtained (475 serum samples, 278 males and 197 females). One hundred fifty-two samples were antibody positive (32%; 95% CI, 27.8–36.4%). There were no statistically significant differences between females and males (33.5% and 30.93%, respectively) or between prereproductive and postreproductive periods (34.8%, n=181 and 30.9%, n=288) (P> 0.05 in all cases). Figure 2 shows the locations where positive and negative European mink were captured, and the two active American mink farms present in the area (MAGRAMA 2014).

The prevalence of AMDV antibody by year is summarized in Figure 3. Samples obtained between years 1997–2000, 2004–05 and 2006–07 were combined to avoid collection intervals with <10 samples. The total number of samples processed in each period ranged from 16 to 60. The prevalence estimates suggested an increase during the study period, but the simple linear regression was not significant (R²=0.551; P=0.79).

Body weights of antibody-positive and -negative European mink during the prereproductive period were similar. The mean weights for female mink were 513 g for positive (n=17, SD=72) and 508 g for negative animals (n=33, SD=62). The mean weights for male mink were 863 g for positive (n=42, SD=103) and 856 g for negative animals (n=83, SD=108). These differences were not statistically significant (F=0.063, P=0.8 for females and F=0.122, P=0.7 for males).

Fifty-nine mink (14.4% of the sampled animals) were recaptured during the study period: 42 were captured twice, 12 captured three times, four captured four times, and one was captured five times. The AMDV antibody results from the first sample were negative in 41 animals (70%) and positive in 18 (30%). The antibody prevalence in recaptured animals was similar to the prevalence in the overall population (z-score=0.23; P=0.8).

The mean number of days between consecutive captures was 326 (range, 45–1,076). The mean distance between consecutive captures was 3,780 m (n=82; range, 0–39,727 m). Six recaptures (7%) were more than 10,000 m from the previous capture site and only three (4%) of the animals were recaptured more than 30,000 m from the previous capture site. Of the 82 recaptures, 55 animals maintained the previous serology result (34 negative and 21 positive in the two controls), whereas 20 animals seroconverted from negative to positive and seven positive animals became negative by CIEP. Thus, recaptured mink during the study period showed an incidence of AMDV infection of 0.46 per animal-year at risk, while the incidence of loss of detectable antibody was 0.18 per animal-year at risk.

We analyzed 1,735 American mink serum samples. These mink were captured in the five Spanish distribution areas of the species and the range expansion area between two of these populations (La Rioja population; Fig. 1). The majority of sampled animals were males (61.5%). The prevalence of AMDV antibody in this species was almost identical to that obtained for European mink: 32.4% (n=1,735; 95% CI, 30.2–34.6%). The prevalence in females was 33.2% (n=668; 95% CI, 29.6–37.0%), and in males it was 31.9% (n=1,067; 95% CI, 29.0–34.7%).

However, prevalence varied geographically. Significant differences were detected between Catalonia and all of the other regions. The prevalences from Basque Country and La Rioja populations were also different from the prevalences in Central Spain and Galicia (Table 2).

Subadult animals (0+ age class) represented 52.5% of all animals (551 samples, subadult:adult ratio = 1.1). The prevalence in subadults was lower than in adults (22.3%; CI 95%, 19–26 vs. 35.5%; CI 95%, 31.3–40, z-score=−4.73, P<0.001). The prevalence increased with age (R²=0.88; P<0.05; Table 3).

Of 27 females captured in April, 15 were AMDV antibody negative and 12 were positive. Age determination suggested that 50% of females were in their first reproductive period in both cases. No pregnant females were detected in three (20%) of the antibody-negative and in six (50%) of the antibody-positive animals (P=0.05). There was no significant difference in the mean number of embryos per pregnant female between the positive (mean=6.0) and negative (mean=5.6) animals.

Aleutian mink disease virus is currently endemic, highly prevalent, and widely distributed in the Spanish population of European and American mink. The observed AMDV antibody prevalence in European mink (32%) was higher than that reported in France (18%, n=99; Fournier-Chambrillon et al. 2004) or in a previous study from Navarra, Spain (0 of 84 tested animals; Sanchez-Migallon et al. 2008). Our study included 30 serum samples from Navarra that were obtained before the study by Sanchez-Migallon et al. (2008); in these samples, we identified six antibody-positive animals (20% prevalence; 95% CI, 8.4–39.1%). Our results and the virus characteristics (highly stable and persistent in the environment) suggest that the presence of AMDV in Navarra should be re-evaluated.

The year 2010 was a notable exception to the trend of increasing prevalence with time in the European mink population (Fig. 3). Samples from 2010 were primarily from Zaragoza and were collected shortly after the colonization of this area by European mink (Gómez et al. 2011). The variation in antibody prevalence in American mink populations among regions might be related to time since introduction of AMDV. The highest prevalence was in Galicia (Table 2), which has 80% of the active mink farms in Spain (MAGRAMA 2014). The two areas with the lowest prevalence were Catalonia, where the virus was recently introduced (the first antibody-positive animal was detected in 2005, after >70 mink had been investigated) and La Rioja, an area recently colonized by American mink invading from the Basque Country and Central Spain populations.

Our finding of a bias toward males in the sampling, supports results of Buskirk and Lindstedt (1989) who attributed the male bias in sampled mustelid species to differences in capturability resulting from sex-specific home-range sizes and higher mobility in males. The home-range sizes of European and American male mink are larger than those of females (Palazón and Ruiz-Olmo 1993; Garin et al. 2002; Zabala et al. 2007). An absence of differences in the prevalence of AMDV antibody between sexes was also reported in European and American mink by other investigators (Yamaguchi and Macdonald 2001; Fournier-Chambrillon et al. 2004; Hossain-Farid 2013). Mink are solitary species, especially European mink. This behavior might increase the relative importance of vertical or perinatal transmission and infection during mating contacts, which equally affects both sexes, minimizing other transmission modes related to other social behaviors.

As expected for a chronic infection, the prevalence in American mink increased with the age of the animal. Similar results have been previously described (Yamaguchi and Macdonalds 2001; Persson et al. 2015).

Low reproductive parameters were detected in infected American mink females reared in captivity (Jahns et al. 2010). This supports our finding of a higher proportion of nonpregnant females in the antibody-positive group (50%, n=12) versus the antibody-negative animals (20%, n=15), although the mean number of embryos per pregnant female was similar.

Our finding of no differences in weight between positive and negative animals in the prereproductive period suggests that infection has no detectable effect on mink body weight. Animals with progressive AMD might have low trapability or, alternatively, this effect cannot be appreciated until progressive AMD ensues.

Since our study began, AMDV-positive European mink have been found throughout the distribution area and not only in areas where there are fur farms. Fournier-Chambrillon et al. (2004) also found antibody-positive animals outside of the areas where American mink are found. These findings also suggest that infection from the invasive American mink is not currently affecting the transmission of AMDV to native mink. Nevertheless, mink farms can act as sources of AMDV transmission to the wild (Nituch et al. 2011).

European mink have linear territories along watercourses with maximum ranges of 17 km for males and 6 km for females (Palazón and Ruiz-Olmo 1993; Garin et al. 2002; Palazón and Ceña 2007). In the 82 recaptures that occurred for 59 mink, the mean distance between captures was 3.7 km, but some animals were recaptured nearly 40 km from the previous capture site (linear distance, much longer following watercourses).

Due to the critical status of the European mink, any factor that negatively affects the population can have severe repercussions on the conservation of the species. Despite that we have not observed any obvious negative effects of AMDV among the antibody-positive animals in this study, AMD can potentially affect the survival of the species and serious efforts should be undertaken to reduce the transmission of AMDV in the European mink population. Thus, it would be advisable to use biosecurity protocols in American mink farms and in captured European mink to avoid the spread of infection. Only antibody-negative European mink should be incorporated into captive breeding programs. Determination of the specific viral strains present in populations and pathology studies need further attention in the endangered European mink.

Special thanks to D. Camps, J. M. López-Martín, C. Fernández, M. Ferrer, M. Pérez-Haro, S. Oreca, C. Aguilar, J. López de Luzuriaga, J. Pinedo, J. González-Esteban, B. Minobis, P. Lizarraga, Y. Melero, L. Lopo, J. Carreras, C. Temiño, S. Brizuela, I. Molina, J. M. Meneses, M. Alcántara, J. M. González, L. Lobo, G. Belamentia, E. Castién, L. M. González and all of the rehabilitation centers in Spain, Kopenhagen Fur, and Matson’s Laboratory. Funding for the field research, the control of American mink, and this study was provided by Generalitat de Catalunya, the Spanish Environment Department, Gobierno de La Rioja, Diputación Foral de Álava, Junta de Castilla y León, Gobierno de Aragón, Generalitat Valenciana, Ayuntamiento de Vitoria-Gasteiz and European LIFE programs; LIFE 00/NAT/E 7335, LIFE 00/NAT/E 7299, LIFE 00/NAT/E 7331, LIFE 02/NAT/E 8604, LIFE CO-OP 2003/NAT/CP/E/000002. We are grateful to M. Bloom for revising the manuscript and for making useful comments.