Regional and seasonal effects on the gastrointestinal parasitism of captive forest musk deer

Acta Tropica 177 (2018) 1–8

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Regional and seasonal effects on the gastrointestinal parasitism of captive forest musk deer

MARK

Xiao-Long Hua,1, Gang Liub,1, Yu-Ting Weia, Yi-Hua Wanga, Tian-Xiang Zhanga, Shuang Yanga, ⁎ ⁎ De-Fu Hua, , Shu-Qiang Liua, a Laboratory of Non-invasive Research Technology for Endangered Species, College of Nature Conservation, Beijing Forestry University, No. 35 Tsinghua East Road, Haidian District, Beijing 100083, China b Institute of Wetland Research, Chinese Academy of Forestry, Haidian District, Beijing 100091, China

A R T I C L E I N F O

A B S T R A C T

Keywords: Moschus berezovskii Gastrointestinal parasites Coinfection Shedding capacity Infracommunity

Parasite infections can cause adverse effects on health, survival and welfare of forest musk deer. However, few studies have quantified the parasite infection status and evaluated the parasite temporal dynamics and differences between breeding centers for captive forest musk deer. The purpose of this study was to assess seasonal and regional effects on the parasite prevalence, shedding capacity, diversity, aggregation and infracommunity to establish baseline data on captive forest musk deer. The McMaster technique was applied to count parasite eggs or oocysts in 990 fecal samples collected at three breeding centers located in Qinling Mountains and Tibetan Plateau during spring, summer, and winter. Five gastrointestinal parasite groups were found in musk deer, and Eimeria spp. were dominant (mean oocysts per gram = 1273.7 ± 256.3). A positive correlation between Eimeria spp. and Strongyloides spp. (r = 0.336, p < 0.001) based on shedding capacity data was found, as well as a negative correlation between Eimeria spp. and Moniezia spp. (r = −0.375, p = 0.003). Both seasonal and regional differences in diversity, prevalence, shedding capacity, aggregation and infracommunity were observed for five parasite groups. The low level of aggregation and high shedding capacity of Eimeria spp. and Strongyloides spp. might reflect the contaminated environment, and indicate that host-parasite relationships are unstable. The high degree of aggregation of Trichuris spp., Ascaris spp., and Moniezia spp. also suggests that some individual hosts had less ability to resist pathogens and greater transmission potential than others. These conclusions suggest that a focus on disease control strategies could improve the health of forest musk deer in captivity.

1. Introduction Parasite infection poses major threats to the productivity, welfare, and survival of hosts. The spread and outbreak of parasites can reduce host survival and cause declines in population size in some cases (Ebert et al., 2000; Strona, 2015). Because of the poor immune response of the abomasum, ruminants are often more vulnerable to gastrointestinal parasites (Gasbarre, 1997). Many studies have explored the host-parasite relationship of ruminants, such as cattle (Li et al., 2015), sheep (Fthenakis et al., 2015), and deer (Davidson et al., 2014; Hernández and González, 2012), but few have investigated forest musk deer (FMD). Musk deer are shy ruminants that are distributed throughout the forests and mountains of Asia, and China is a particularly important distribution area (Green, 1986; Yang et al., 2003). The musk secreted

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by adult males, is a raw material used in the perfume industry and Traditional Chinese Medicine. Steep declines in wild musk deer populations have resulted from over-exploitation and habitat destruction, particularly those of FMD, which has the largest yield and highest quality musk (Sheng and Liu, 2007). The breeding programs for FMD are a national ex situ protection strategy, which began in the 1950’s and aims to provide sustainable musk resources. Though the farming of FMD has lasted for several decades, this species is still difficult to domesticate mainly because of its timidity and preference for being solitary (Zhang, 1983). With the progress in knowledge of FMD’s management, the space for each captive FMD has increased a lot from little houses, to individual cells with an outdoor yard, and finally the semifree-range. Nevertheless, the population size of captive FMD remains still low partly because of parasite infection. Based on the anatomy of dead FMD, Moniezia spp. and Eimeria spp. have a negative effect on

Correspondence authors. E-mail addresses: [email protected] (D.-F. Hu), [email protected] (S.-Q. Liu). Equal contributors.

http://dx.doi.org/10.1016/j.actatropica.2017.09.021 Received 28 May 2016; Received in revised form 7 September 2017; Accepted 25 September 2017 Available online 28 September 2017 0001-706X/ © 2017 Elsevier B.V. All rights reserved.

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survival (Liu et al., 2002; Yang et al., 2001b), especially for the young animals. The principal transmission routes for gastrointestinal parasites are soil (Bethony et al., 2006), diet (Vitone et al., 2004), social contact (MacIntosh et al., 2012), and intermediate hosts (Bush et al., 2001; Mbora and McPeek, 2009), whereas the spread of parasites can be affected by season (Viljoen et al., 2011), temperature (Tinsley et al., 2011), and humidity (Altizer et al., 2006; Setchell et al., 2007). A limited number of earlier studies on gastrointestinal parasites in FMD focused on diagnosis of single parasite species (Yang et al., 2001b), anthelmintic treatment (Yang et al., 2001a; Wang et al., 2011), and epidemiological characteristics of parasites in separate host populations (Liu et al., 2002; Wang et al., 2015b), yet comprehensive baseline data remains insufficient. Longitudinal studies of several populations in various geographical regions and details of temporal and spatial variations in parasitic infections would provide a baseline from which potential pathogenic changes could be identified (Bjork et al., 2000). This study set out to determine the baseline parasite community and identify how this parasitism impacts on the health of captive FMD. Three FMD breeding centers located in southern Qinling Mountains and eastern Tibetan Plateau were chosen as study sites. The McMaster technique was applied to count parasite eggs or oocysts in 990 fecal samples collected at these three breeding centers during spring, summer, and winter of 2014, and the parasite prevalence, diversity, shedding capacity, infracommunity and parasite aggregation were measured to assess how seasonal and regional factors affect five measures of parasitism. The shedding capacity of different parasite groups was investigated to reveal patterns of coinfection.

Table 1 The number of forest musk deer used for collecting fecal samples from Huangniupu breeding center during summer, Huoshaodian breeding center during summer, Miyaluo breeding center during summer, Miyaluo breeding center during winter, Miyaluo breeding center during spring. Sampling sites

Huang Huo Mi Total (M:F)

Sampling time

Total (M:F)

Summer (M:F)

Winter (M:F)

Spring (All males)

A 45(25:20) B 83(45:38) C 88(48:40) 216(118:98)

–a –a D 70(40:30) 70(40:30)

–a –a E 44 44

45(25:20) 83(45:38) 202(132:77) 330(202:128)

M, males; F, females. a No sample collected.

temperature. All FMD included in the trial were aged between 3 and 5 years. 2.3. Sample analysis and data analysis The mean eggs per gram (EPG) or oocysts per gram (OPG) of parasites in fecal samples were counted using the McMaster technique (Cringoli et al., 2004). The copromicroscopic analysis was conducted within 2 weeks according to our previous study (Hu et al., 2016, Supplementary file 1). The five measures of parasitism were defined for this study as: i) prevalence, the percentage of infected FMD within a host population; ii) shedding capacity, the number of eggs or oocysts that are shed from parasites per host (whilst the number of eggs or oocysts released cannot directly inform on the number of parasites present, this can give us an indication of the transmission potential for a given host); iii) parasite diversity, which is expressed as parasite groups richness (S), reflecting the number of parasite groups per deer; iv) parasite aggregation, is used to indicate the distribution pattern of parasites within FMD populations. Here we used the corrected moment estimate of k as per the following equation (Sherrard-Smith et al., 2015): k = (x2 − s2/N)/ (σ2 − x), where x, σ2 and N represent mean shedding capacity, variance, and sample size, which is an inverse measure of aggregation. Several measures including the corrected moment estimate of k (Gregory and Woolhouse, 1993), Poulin’s Index of Discrepancy (Poulin, 1993), Boulinier’s J (Boulinier et al., 1996) and Taylor’s Power Law (Taylor and Taylor, 1977), had been developed to explore parasite aggregation. The k is the most commonly used measure because it was found to vary least with parasite abundance and sample size. v) infracommunity composition, is a comprehensive measure which is based on the number and shedding capacity of parasite groups in an individual FMD. The one-sample Kolmogorov-Smirnov test was used to test the normality of the data. The effects of sex, season, and region on the prevalence were investigated using binomial generalized linear models (GLMs), and the negative binomial GLMs were used to model the effects of sex, season and region on shedding capacity. The shedding capacities of other parasites were included as explanatory variables in above GLMs. As for parasite diversity, we used multinomial GLMs to test the effects of factors. The two-sample Kolmogorov-Smirnov (K-S) test was applied to test the variation of distributions (aggregation) among sample groups. The paired-sample Friedman test was used to indicate differences in prevalence, shedding capacity, and aggregation among the parasite groups found in the fecal samples. Meanwhile, the correlation among shedding capacity of parasite groups was conducted using Spearman correlation analysis. All above statistical analyses were performed in SPSS ver. 20.0 software (IBM Corp., Armonk, NY, USA). Furthermore, non-metric multidimensional scaling (NMDS) based on the Bray-Curtis similarities of the log (x + 1) transformed shedding capacity was applied to rank the infracommunity, and one-way analysis

2. Materials and methods 2.1. Study sites and animals Qinling Mountains and the Tibetan Plateau are renowned for their rich biodiversity and also provide natural food and suitable climate for FMD, as such, these regions are ideal for breeding centers. The Huangniupu center (Huang) (34°11′N, 106°50′E) is located in Baoji, Shaanxi Province, a region of southern Qinling Mountains with an altitude of 1500 m. The Huoshaodian center (Huo) (33°35′N, 106°49′E) located in Hanzhong, Shaanxi Province, is also on the south slope of the Qinling Mountains with an altitude of 1400 m. The Miyaluo center (Mi) (34°11′N, 106°50′E) is located in Aba, Sichuan Province, a region of eastern Tibetan Plateau with an altitude of 2800 m. All three breeding centers have multiple adjacent enclosures and adopted the following reproductive system. One male and three females were kept in the same enclosure consisting of several individual brick cells (2 × 2 × 1.5 m3) and an outdoor yard (15 × 15 m2). The FMD were kept together during the daytime but separated at night, so feces could be collected from individuals. Musk deer were fed with leaves collected from their natural habitat, twice daily at 7:00 and 18:00. The plants mainly included Ulmus pumila, Picrasma chinensis, Anacardiaceae rhus, Morus alba and Usnea diffracta. Supplementary artificial food consisted of soybean flour, wheat bran, corn flour, milk, seasonal vegetables, and fruits. Fresh water was provided ad libitum. All musk deer are dewormed twice annually using albendazole tablets, and the treatment is ceased three months prior to the study through to the end of sampling. 2.2. Feces collection A total of 990 fresh fecal specimens were collected from 330 FMD during spring, summer, and winter 2014, and each individual was sampled once daily and continuously for 3 days (Table 1). The feces from all houses was cleaned out every evening from 18:00 to 20:00 h, which could allow collection of fresh feces from each musk deer the next day at 7:00. Ear tags were used to distinguish each musk deer. All fresh fecal samples were preserved in 10% formalin solution at room 2

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summer (3.34 ± 0.10), but the lowest were at Mi during winter (1.86 ± 0.11). The study indicated significantly higher parasite richness during summer than spring (GLMs, χ2 = 47.51, df = 2, p < 0.001) and winter (GLMs, χ2 = 47.51, df = 2, p < 10−3), and significantly higher parasite richness were investigated at the Huo center than those at the Huang (GLMs, χ2 = 17.31, df = 2, p = 0.017) or Mi center (GLMs, χ2 = 17.31, df = 2, p < 10−3). 2) The interactions between season and region on prevalence were significant for all five parasite groups (GLMs, χ2 = 95.38–2356.30, df = 4, p < 10−3). The highest prevalence occured at the Huo center during summer (Eimeria spp., 100%; Strongyloides spp., 98.8%; Trichuris spp., 35.0%; Ascaris spp., 65.1%; Moniezia spp., 34.9%), whereas the lowest at the Mi center during winter (Eimeria spp., 90.0%; Strongyloides spp., 64.3%; Trichuris spp., 5.7%; Ascaris spp., 21.4%; Moniezia spp., 4.3%). Significantly greater prevalence was observed for all five parasite groups (GLMs, χ2 = 50.47–605.48, df = 2, p < 10−3) during summer than those during spring or winter, and Eimeria spp. (GLMs, χ2 = 605.48, df = 2, p < 0.001), Strongyloides spp. (GLMs, χ2 = 413.93, df = 2, p < 0.001), and Moniezia spp. (GLMs, χ2 = 111.92, df = 2, p = 0.032) was found a significantly higher prevalence during spring than those during winter (Table 2). Significantly greater prevalence was detected for five parasite groups (GLMs, χ2 = 5.47–636.41, df = 2, p = 10−3–0.023) at the Huo center than those at the Mi center, whereas only Eimeria spp. (GLMs, χ2 = 636.41, df = 2, p < 0.001) and Trichuris spp. (GLMs, χ2 = 7.31, df = 2, p = 0.027) showed a significantly higher prevalence at the Huang center than those at the Mi center (Table 2). 3) The interactions between season and region on shedding capacity were significant for five parasite groups (GLMs, χ2 = 10.75–55.56, df = 4, −3 p = 10 –0.030). The Huo center during summer yielded the highest mean shedding capacity (Eimeria spp., 1990.4 ± 423.0; Strongyloides spp., 360.8 ± 77.9; Trichuris spp., 32.3 ± 7.6; Ascaris spp., 108.8 ± 22.0; and Moniezia spp., 84.4 ± 17.1), while the lowest for the Mi center during winter (Eimeria spp., 511.0 ± 92.5; Strongyloides spp., 205.7 ± 33.5; Trichuris spp., 3.1 ± 1.6; Ascaris spp., 14.2 ± 5.8; and Moniezia spp., 9.0 ± 6.6). Significantly greater shedding capacity was investigated for all five parasite species during summer than spring (GLMs, χ2 = 11.10–165.55, df = 2, p = 10−3–0.039) or winter (GLMs, χ2 = 11.10–165.55, df = 2, p = 10−3–0.002). The study suggested a significantly lower shedding capacity for all five parasite groups at the Mi center than those at the Huang (GLMs, χ2 = 10.88–40.43, df = 2, p = 10−3–0.048) and Huo centers (GLMs, χ2 = 10.88–40.43, df = 2, p = 10−3–0.003; Table 3). 4) Significantly higher aggregation levels were detected for Eimeria spp. (K-S, t = 1.600, p spring = 0.012; t = 1.589, p winter = 0.013), and Strongyloides spp. (K-S, t = 2.523, p spring < 0.001; t = 2.232, p winter < 0.001) during summer than spring and winter, whereas Ascaris spp. (K-S, t = 1.416, p summer = 0.036; t = 1.855, p winter = 0.002) showed significantly higher aggregation levels during spring than summer and winter. Meanwhile, significantly greater aggregation levels were found at the Mi center than those at the Huang (K-S, Eimeria, t = 2.115, p < 0.001; Strongyloides, t = 1.740, p = 0.005) and Huo (K-S, Eimeria, t = 2.141, p < 0.001; Ascaris, t = 2.704, p < 0.001; Moniezia, t = 1.764, p = 0.004) centers, but the regional differences among other groups were not significant (Table 4). 5) The ANOSIM was statistically significant between summer and winter (R = 0.121, p = 10−3) and the NMDS ranking showed a clear similarity in the distribution of parasite infracommunities between spring and summer (R = 0.029, p = 0.215), and spring and winter (R = 0.008, p = 0.340; Fig. 3). Significant differences in the distribution of parasite infracommunities among three breeding center (Huang vs Huo, R = 0.075, p = 0.009; Huang vs Mi, R = 0.122, p = 0.005; Huo vs Mi, R = 0.050, p = 10−3) were found, and the NMDS ranking showed clear dissimilarities (Fig. 4).

Fig. 1. The relationships between aggregation (the corrected moment estimate of k) and shedding capacity and how these change with coinfection (the hosts infected with 1, 2, 3, 4 or 5 parasite groups). The mean shedding capacity data was calculated using the total data of all parasite groups infected the hosts. The corrected moment estimate of k is an inverse measure of aggregation.

of similarity (ANOSIM) was used to determine the differences of grouping patterns among seasons and regions in the NMDS ranking. The NMDS and ANOSIM were carried out using the PRIMER ver.6 software (Clarke and Gorley, 2006). 3. Results 3.1. Associations between parasite groups The gastrointestinal parasite groups detected in FMD were Eimeria spp., Strongyloides spp., Trichuris spp., Ascaris spp., and Moniezia spp. The percentage of uninfected (S = 0) FMD was 2.12%, and the infected with one parasite group (S = 1), two (S = 2), three (S = 3), four (S = 4) and five (S = 5) were 6.67%, 35.76%, 34.55%, 17.88% and 3.33%, respectively. The highest mean shedding capacity and lowest aggregation were both observed when four parasite groups infected the FMD (Fig. 1). Eimeria spp. was the dominant parasite group, with higher prevalence (Friedman, χ2 = 22.325, df = 4, p < 10−3) and shedding capacity (Friedman, χ2 = 495.666, df = 4, p < 10−3) than other parasite groups, but showed the lowest degree of aggregation (0.1124–0.4553). However, the parasite group Moniezia spp. was highly aggregated (0.0276 − 0.2810). The shedding capacity of Trichuris spp. (GLMs, χ2 = 2.384, df = 1, p = 0.123), Strongyloides spp. (GLMs, χ2 = 0.973, df = 1, p = 0.324), Ascaris spp. (GLMs, χ2 = 0.853, df = 1, p = 0.356) and Moniezia spp. (GLMs, χ2 = 0.445, df = 1, p = 0.505) were found no significant effects on prevalence of Eimeria spp., the same for the prevalence of other four parasite groups. The relationships of shedding capacity between Eimeria spp. and Trichuris spp. (GLMs, χ2 = 8.401, df = 1, p = 0.004), Eimeria spp. and Strongyloides spp. (GLMs, χ2 = 62.221, df = 1, p < 10−3) were significant. The shedding capacity of Eimeria spp. was positively correlated with that of Strongyloides spp. (Spearman, r = 0.336, t = 6.461, df = 328, p < 10−3), while a markedly negative correlation was found between Eimeria spp. and Moniezia spp. (Spearman, r = − 0.375, t = 7.326, df = 328, p = 0.003; Fig. 2). 3.2. Seasonal and regional variation of parasitism The effects of sex on five measures of parasitism and the interactions between sex and region, sex and season were not significant. 1) The significant interactions between season and region on parasite richness (GLMs, χ2 = 134.68, df = 6, p < 0.001) were found in FMD. The highest parasite richeness were observed at the Huo center during 3

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Fig. 2. The correlations between Eimeria spp. and Moniezia spp. (solid cycles), and Eimeria spp. and Strongyloides spp. (open cycles) based on the EPG (eggs per gram) or OPG (oocysts per gram) data. The values of r and p were determined using Spearman correlation analysis.

4. Discussion

are particularly susceptive to stressors after encountering in artificial breeding, which included high population density, human disturbances, and changing food types. Moreover, the cross-contamination are also significant factor that increases the chance of parasite infections. Helminths and protozoa are integral components of the ecological communities and most hosts are likely to be infected with more than one parasite species (Cox, 2001; Pedersen and Fenton, 2007). The patterns of coinfection become an important question because infection by one parasite species may affect susceptibility to others by directly competing for resources or indirectly via the host immune system (Telfer et al., 2010), and studies on wildlife populations have confirmed that positive and negative associations can occur between parasites (Behnke, 2008). The diversity data indicate that 91.21% of FMD were

There are clear differences in parasitism of FMD between management centers in Qinling mountains and the Tibetan Plateau in summer. Whilst the sex of deer was not associated with differences in parasitism, region and season were key. Across parasite species, highest prevalence estimates were observed in summer when shedding capacity was also high for the majority of parasite species. The prevalence and shedding capacity values of the parasites investigated in FMD were higher than those reported in wild alpine musk deer and Tianshan red deer (Lu et al., 2010; Amila et al., 2014). The chronic stress suffered by captive FMD could weaken their immunity, and then increase parasitic infection rates (Oppliger et al., 1998). FMD

Table 2 Summary statistics of prevalence (%) for host-parasite interaction between the musk deer and Eimeria spp., Strongyloides spp., Trichuris spp., Ascaris spp., Moniezia spp. The sub-groups are differentiated by season and region. Bold type-face refers to the sub-groups with higher prevalence among factors, and superscript letters represent which sub-groups were significantly lower. Factor

Sample group

Sub-population (number of hosts)

Eimeria spp. C

Strongyloides spp. C

Trichuris spp.

Ascaris spp.

Moniezia spp.

Season

A B C

Spring (44) Summer (216) Winter (70)

96.6 97.7AC 90.0

84.1 96.3AC 64.3

13.6 26.9AC 5.7

25.0 57.4AC 21.4

6.8C 27.8AC 4.3

Region

D E F

Huang (45) Huo (83) Mi (202)

100F 100F 94.1

95.6 98.8F 81.7

31.1F 35.0F 12.4

55.6 65.1F 35.1

26.7 34.9F 12.4

4

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Table 3 Summary statistics of mean shedding capacity ( ± Standard Error) for host-parasite interaction between the musk deer and Eimeria spp., Strongyloides spp., Trichuris spp., Ascaris spp., Moniezia spp. The sub-population are differentiated by season and region. Bold type-face refers to the sub-groups with higher mean shedding capacity among factors, and superscript letters represent which sub-groups were significantly lower. Factor

Sample group

Sub-population (number of hosts)

Eimeria spp.

Strongyloides spp.

Trichuris spp.

Ascaris spp.

Moniezia spp.

Season

A B C

Spring (44) Summer (216) Winter (70)

574.6 ± 121.3 1663.3 ± 250.9A C 511.0 ± 92.5

230.3 ± 54.5 322.0 ± 50.3A C 205.7 ± 33.5

6.8 ± 3.2 24.5 ± 4.2A C 3.1 ± 1.6

24.2 ± 14.4 90.5 ± 15.8A C 14.2 ± 5.8

12.9 ± 7.1 62.4 ± 10.7A C 9.0 ± 6.6

Region

D E F

Huang (45) Huo (83) Mi (202)

1941.5 ± 568.9F 1990.4 ± 423.0F 830.5 ± 166.6

322.2 ± 94.9F 360.8 ± 77.9F 245.7 ± 41.7

25.6 ± 7.4F 32.3 ± 7.6F 9.8 ± 2.9

91.8 ± 28.3F 108.8 ± 22.0F 41.8 ± 13.5

66.3 ± 20.2F 84.4 ± 17.1F 23.3 ± 8.1

and transmission rates decrease in a cold, dry environment. The Huang and Huo centers are located on the south slope of the Qinling Mountains with similar climates and altitudes, whereas the Mi center is located on the Western Sichuan Plateauat an altitude of 2800 m, and the lowest temperature during winter is always below −20 °C. Liu et al. (2002) reported a high incidence of coccidiosis during August and September in the Dujiangyan area, which is located in the Sichuan Plain, but outbreaks of coccidiosis are rare in the Maerkang area, which is located on the Western Sichuan Plateau. Moreover, the management may be another reason for the higher egg or oocyst counting excluding the same stocking density. For instance, some of the food plants for FMD paly a positive role as anthelmintic (Wang et al., 2015a), so the food types can affect the parasitism. In addition, whether the feces can be cleaned timely is a key factor for cross-contamination, whose effects on parasitism is also considerable. There is an increasing recognition that parasites can affect the dynamics of vertebrate populations, and parasites are typically aggregated within their host populations (Tompkins et al., 2002). Aggregation of Graphidium strigosum and Trichostrongylus retortaeformis in mountain hares is strongly affected by season and region, but not sex (Newey et al., 2005). Meanwhile, region and season significantly affect aggregation of Pseudamphistomum truncatum in otters, but not host age, sex and body condition (Sherrard-Smith et al., 2015). Eimeria spp.and Strongyloides spp. aggregated in fewer FMD in summer and were more dispersed in spring and winter, and higher levels of aggregation were obserevd for Trichuris spp., Ascaris spp., and Moniezia spp. during spring and winter. The parasites displayed higher aggregation levels at the Mi center than those at the Huang and Huo centers. Aggregation of parasites within individual hosts is caused by several factors, such as differences in exposure history, differences in pathogen susceptibility, health condition of the host, or simply chance events of infection (Holt and Boulinier, 2005; Sherrard-Smith et al., 2015). Highly aggregated parasite populations might be beneficial to hosts because the majority of animals would then, by definition, have parasite loads that are lower than the mean (Holt and Boulinier, 2005). Parasite abundance and aggregation were always considered together to identify critical factors related with the host-parasite relationship and identify individual hosts with the greatest transmission potential. Parasite abundance is defined as the number of parasites per host, which is distinct from shedding capacity used in this study because it is

infected with two or more parasite groups, which is much higher than that reported for alpine musk deer at 23.95% (Lu et al., 2010). Furthermore, the relationships of shedding capacity were found between Eimeria spp. and Strongyloides spp., Eimeria spp. and Trichuris spp., and Eimeria spp. and Moniezia spp., respectively. Eimeria spp. and Strongyloides spp. are the most prevalent parasite groups in FMD, while Moniezia spp. is a less prevalent but harmful group, the outbreaks of which have resulted in a large number of deaths (Yang et al., 2001a,b). Eimeria spp. and Strongyloides spp. have always attracted the attentions of veterinarians and breeders because of their high prevalence and intensity for captive or domestic ruminant, and the death of FMD caused by them have been reported (Liu et al., 2002). Cestode infections of ruminants were previously regarded to be relatively harmless. However, some studies have found potential pathogenicity of Moniezia in ruminants associated with gastrointestinal disorders or even deaths (Tsotetsi and Mbati, 2003; Yan et al., 2013). The present results indicate that the infection of Strongyloides spp., Moniezia spp., Trichuris spp. and Eimeria spp. could be influenced by each other. Furthermore, the relationships between shedding capacity, aggregation and coinfection indicate that the most stable coinfection pattern was that when the hosts were infected with four parasite groups. The present study did not identify any host sexual differences in parasite prevalence, shedding capacity, or diversity in captive FMD, which is consistent with red deer (Davidson et al., 2014). Changes in the social position of males caused by human activities may be one reason. Furthermore, males and females have equal risks of exposure to pathogens under the captive breeding. Season has a prominent effect on parasite composition and load (Popiołek et al., 2007; Titi et al., 2014). The parasite composition displays greater prevalence, shedding capacity, and diversity during summer than during spring and winter, and similar results have been reported for pampas deer (Hernández and González, 2012) and mountain hare (Newey et al., 2005). Stromberg (1997) reported that temperature, moisture, and rainfall are the main environmental factors influencing survival and transmission of gastrointestinal nematode eggs. The warm, moist environment and abundant rainfall during summer facilitate the parasite development and transmission, whereas cold and dry weather during winter and spring inhibits development and transmission. Regional factors appear to have an effect on the epidemiology of parasitosis, which could be attributed to climatic conditions, as prolonged low temperature reduces survival,

Table 4 Summary statistics of the corrected moment estimate of k for host-parasite interaction between the musk deer and Eimeria spp., Strongyloides spp., Trichuris spp., Ascaris spp., Moniezia spp. The sub-groups are differentiated by season and region. Bold type-face refers to the sub-groups with higher aggregation levels among factors, and superscript letters represent which subgroups were significantly lower. Factor

Sample group

Sub-group

Eimeria spp.

Strongyloides spp.

Trichuris spp.

Ascaris spp. BC

Season

A B C

Spring Summer Winter

0.4553 0.1124AC 0.4343

0.3575 0.1102AC 0.4633

0.0718 0.0717 0.0422

0.0375 0.0576 0.0728

Region

D E B

Huang Huo Mi

0.2366 0.2547 0.1124DE

0.2339 0.2466 0.1102D

0.2462 0.2092 0.0717

0.2112 0.2821 0.0576E

5

Moniezia spp. 0.0485 0.0346 0.0276 0.2178 0.2810 0.0346E

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Fig. 3. Non-metric multidimensional scaling (NMDS) plot of a two-dimensional solution of 202 parasite infracommunities (spring, 44; summer, 88; winter, 70) based on the Bray-Curtis similarities of the log (x + 1) transformed parasite shedding capacity data (stress = 0.14). Symbols and cycles denote sample membership as follows: ▲ and dash dot line cycle, samples from Miyaluo center during spring; ● and solid line cycle, samples from Miyaluo center during summer; ■ and dash line cycle, samples from Miyaluo center during winter.

spp. in FMD might be explained as a reflection of a contaminated environment, and indicate that the host-parasite relationship might be unstable, which could lead to an outbreak of parasitosis. The low shedding capacity and high aggregation of Trichuris spp., Ascaris spp., and Moniezia spp. indicate stable host-parasite dynamics. Breeders should pay more attention to individual hosts with intense parasite infections because their ability to resist pathogens maybe lower than others in this captive population. The high prevalence or shedding capacity most probably indicates the unhealthy of FMD. However, a diverse parasite community may be a good thing in consideration of the density dependent effects on each parasite groups as a consequence of the presence of another. In the present study, the lowest parasite prevalence and shedding capacity at Miyaluo breeding center were found, whereas the parasite community was the most diverse. Thus, we can conclude that Miyaluo center

not always true that a heavily infected host will be the one to shed the most infectious eggs or oocysts. However, as important and useful measures of parasitism, the number of parasites per host has direct impact on the health of the individual host, whilst the number of infectious stages that are shed per host can significantly affect parasite transmission through the host population. The transmission of parasite is always dependent on the amount of transmissible stages that are shed by an infectious host, indicating the shedding capacity data is more suitable for evaluating the parasite transmission potential. A low level of aggregation, along with the parasite-induced breeding failure and time delays in life cycle of parasites may indicate a relatively stable host-parasite relationship (Newey et al., 2004). Some studies have suggested that parasites can reduce fecundity of female hosts but the effect on host survival is limited (Newey and Thirgood, 2004). The high shedding capacity but low aggregation of Eimeria spp. and Strongyloides

Fig. 4. Non-metric multidimensional scaling (NMDS) plot of a two-dimensional solution of 216 parasite infracommunities (Huang, 45; Huo, 83; Mi, 88) based on the Bray-Curtis similarities of the log (x + 1) transformed parasite shedding capacity data (stress = 0.16). Symbols and cycles denote sample membership as follows: ▲ and dash dot line cycle, samples from Huangniupu center during summer; ● and solid line cycle, samples from Huoshaodian center during summer; ■ and dash line cycle, samples from Miyaluo center during summer.

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represents a better option for FMD. 5. Conclusion Captive breeding has helped recover several endangered species from near extinction, but there is increased contact between infectious parasite eggs or oocysts and hosts in a restricted space. In summary, this study provides comprehensive baseline data on the prevalence, shedding capacity, diversity, infracommunity and aggregation of gastrointestinal parasites within captive FMD populations, and confirmed that they are suffering from serious parasite infections, and these captive populations might benefit by treating infected individuals to alleviate any parasite-induced morbidity or mortality. Moreover, the temporal dynamics and differences between breeding centers of parasites within the host population was investigated in captive FMD for the first time in this study. The present study indicates that differences in five measures of parasitism are better explained by region and season than gender. Our findings highlight the importance of identifying the regional and temporal factors that are associated with heavily infected hosts and/or highly fecund parasites, which could help to find and optimize treatment strategies. This study has implications for the future conservation management of FMD, and some suggestions for the breeders and managers were given. Firstly, the selected anthelmintic should not be only the albendazole, which is more useful for roundworm. Secondly, only the infected animals should be dewormed rather than all the animals, and summer maybe the best anthelmintic season. Thirdly, the feces must be cleaned up timely to avoid cross-contamination. Finally, the better location for the breeding centers might be the area with a high attitude just like the Miyaluo breeding center. Fundings This work was supported by the State Forestry Administration of China (grant number musk deer 2014), and China’s Ministry of Science and Technology (grant number 201004054). Acknowledgements We thank Xuhua Pan, Cunxuan Li, Youbin Li, and Baoqing Liu for their valuable suggestions on samples collection. Special thanks to all the breeders of the Breeding Center of Forest musk deer in Shaanxi and Sichuan Province. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.actatropica.2017.09. 021. References Altizer, S., Dobson, A., Hosseini, P., Hudson, P., Pascual, M., Rohani, P., 2006. Seasonality and the dynamics of infectious diseases. Ecol. Lett. 9, 467–484. Amila, A., Risalat, T., Aysajan, T., Rizihan, A., Zhou, C., Mahmut, H., 2014. Intestinal parasites of Tianshan red deer in nanshan mountains areas in Xinjiang. Acta Theriol. Sin. 34, 87–92 (in Chinese). Behnke, J.M., 2008. Structure in parasite component communities in wild rodents: predictability, stability, associations and interactions… or pure randomness? Parasitology 135 (7), 751–766. Bethony, J., Brooker, S., Albonico, M., Geiger, S.M., Loukas, A., Diemert, D., Hotez, P.J., 2006. Soil-transmitted helminth infections: ascariasis, trichuriasis, and hookworm. Lancet 367, 1521–1532. Bjork, K.E., Averbeck, G.A., Stromberg, B.E., 2000. Parasites and parasite stages of freeranging wild lions (Panthera leo) of northern Tanzania. J. Zoo Wildl. Med. 31, 56–61. Boulinier, T., Ivers, A.R., Danchinn, E., 1996. Measuring aggregation of parasites at different host population levels. Parasitology 112, 581–587. Bush, A.O., Fernandez, J.C., Esch, G.W., Seed, J.R., 2001. Paras Itism: The Diversity and Ecology of Animal Parasites, first ed. Cambridge University Press, Cambridge. Clarke, K.R., Gorley, R.N., 2006. PRIMER v6: User Manual/Tutorial. PRIMER-E, Plymouth.

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