Prevalence and Intensity of Sarcocystis spp. Infections in Alpine Chamois ( Rupicapra r. rupicapra ) in Germany

: Chamois are mountain ungulates (Artiodactyla: Caprinae) which inhabit several medium-and high-altitude mountain ranges from southern Europe to the Near East. The first findings of Sarcocystis cysts in the musculature of chamois were reported in the 1970s. However, only limited work on the epidemiology of sarcocystosis and the identification of the species of Sarcocystis in chamois has been carried out in the past. The present study aimed to provide, for the first time, data on the prevalence and intensity of Sarcocystis spp. Infection in native Alpine chamois using a histology examination of heart and/or diaphragm tissue samples collected from 216 chamois (40 kids [<1 year] and 176 chamois ranging up to 18 years of age). Sarcocysts were detected in either the heart or diaphragm of 167/216 chamois (77.3%), with 131 of 183 heart samples and 127 of 215 diaphragm samples testing sarcocyst-positive. Of the 181 chamois with both heart and diaphragm samples available (34 kids and 147 older animals), sarcocysts were detected in the heart and/or diaphragm of 142 animals, translating to an overall 78.5% prevalence of Sarcocystis spp. infection (95%CI 72.5–84.4%). Sarcocysts were more frequently recorded in the heart vs. diaphragm (72.4% vs. 56.4%; p = 0.0021), and diaphragm positivity was associated with heart positivity ( p = 0.0001). The sarcocyst prevalence (heart and/or diaphragm) was significantly ( p < 0.001) lower in the kids than in the older chamois (27.1% vs. 88.6%, respectively); however, it did not differ between the sexes, regardless of the chamois’ age ( p > 0.3). The intensity of infection was generally low (<10 sarcocysts per cm 2 muscle cut) in both heart-positive and diaphragm-positive animals (94.7% and 93.7%, respectively). The heart tissue yielded higher sarcocyst counts than the diaphragm tissue ( p < 0.001). Both the heart and diaphragm sarcocyst counts were significantly ( p < 0.001) lower in the kids than in the older chamois. Sarcocystis spp. infection was demonstrated to be prevalent in chamois in Germany, but its intensity is apparently low. Further studies are desired to identify the species of Sarcocystis parasitizing the chamois using both phenotypic and molecular characteristics.


Introduction
Chamois are mountain ungulates (Genus Rupicapra, Subfamily Caprinae, Family Bovidae) which inhabit numerous medium-and high-altitude mountain ranges from southern Europe to the Near East.The taxonomy of the chamois has been revised several times since the end of the 19th century.Currently, there is consensus to recognize two species of chamois: the northern chamois, Rupicapra rupicapra (with seven subspecies, occurring from central-eastern Europe to the Near East) and the southern chamois, Rupicapra pyrenaica (with three subspecies, occurring in south-western Europe).The Alpine chamois, R. r. rupicapra, is the most abundant subspecies of the two species of chamois.It is native to the whole Alpine mountain range and has also been successfully introduced into former Czechoslovakia (parts of both today's Czech Republic and Slovakia) and into New Zealand, where it forms a significant population on the South Island.However, several subspecies of R. rupicapra and R. pyrenaica require conservation measures [1].
With respect to the intracellular-cyst-forming protozoan parasites of the genus Sarcocystis, a review of the literature has shown that findings of sarcocysts (Sarcocystis intramuscular stages) in chamois were apparently for the first time reported at almost the same time in Alpine chamois (R. r. rupicapra) from Switzerland [2,3], former Czechoslovakia [4,5] and Italy [6].Subsequent work, mainly based on conventional histology, reported further findings of sarcocysts in Alpine chamois, Tatra chamois (R. r. tatrica), Carpathian chamois (R. r. carpatica), Cantabrian chamois (R. p. parva) and Pyrenean chamois (R. p. pyrenaica) (Table 1).However, only limited work regarding the adequate identification of the species of Sarcocystis parasitizing chamois using ultrastructural and molecular methods has been conducted in the past.Two species were identified and named: S. cornagliai, described in fine structural studies among Alpine chamois from Austria, Germany and Italy [12,19], and S. tenella, from Tatra chamois from Poland, using both microscopical and molecular characteristics [22].Sarcocysts, which are morphologically similar to S. tenella/S.capracanistype cysts in sheep and goats, were previously described in Alpine chamois from Italy [12] and from Austria and Germany [19].The sarcocysts of S. cornagliai are ultrastructurally very similar to one sarcocyst type reported from the Rocky Mountain goat (Oreamnos americanus) [19,29] and were recorded too in Alpine ibex (Capra ibex) [30].Interestingly, a brief conference abstract from 1980 stated that sheep, goat and chamois are each the hosts of three sarcocyst forms which are microscopically indistinguishable [21].It is further worth mentioning that there are short descriptions of two transmission experiments where sarcocyst-containing muscle tissues of chamois were fed to dogs, and the sporocysts shed by the dogs developed into sarcocysts of the S. tenella/S.capracanis type in both sheep and goats [21,31].
In addition to the scientific interest in identifying and characterizing the species of Sarcocystis parasitizing the (sub)species of chamois, which is also important for the understanding of the phylogenetic relationship of the species of intermediate hosts belonging to the Caprinae subfamily [32], several aspects of the epidemiology of these muscle parasites are still not well known in chamois and other wild ungulates, which constitute, at least regionally, an important source of meat for human consumption [16,33].Therefore, this paper presents investigations on the prevalence, distribution and intensity of Sarcocystis spp.infection in Alpine chamois from Germany, where the chamois occur as native species in the Bavarian Alps, and, after the successful transfer in the 1930s of chamois from the Alps, in the Black Forest mountains [34].

Results
Overall, of the 216 chamois examined, 167 (77.3%) yielded evidence of infection with sarcocysts, as demonstrated by the histological detection of intramuscular sarcocysts in either the heart or diaphragm muscle specimens (Figure 1).There was no difference in sarcocyst positivity between the chamois from the Bavarian Alps and those from the Black Forest mountains (140/179, 78.2% and 27/37, 72.9%; p = 0.5196).
Overall, of the 216 chamois examined, 167 (77.3%) yielded evidence of infect sarcocysts, as demonstrated by the histological detection of intramuscular sarco either the heart or diaphragm muscle specimens (Figure 1).There was no diffe sarcocyst positivity between the chamois from the Bavarian Alps and those from t Forest mountains (140/179, 78.2% and 27/37, 72.9%; p = 0.5196).For the prevalence of infection in the heart and/or diaphragm, the chi-squar vealed a significant (p < 0.001) difference between the kids (27.5%) and the junior-, and senior-class chamois (87.5%, 89.4% and 89.5%, respectively).Sarcocysts were cantly (p < 0.05) more often recorded in the samples from the junior-, middle-and class chamois than in those from kids, but there was no difference in the preva Sarcocystis spp.infection between the three classes of sexually mature chamois (p No significant difference in the prevalence of Sarcocystis spp.infection was found the male and female chamois in the four age classes combined (80.7% vs. 74.7%;p or in the three classes of sexually mature chamois combined (89.2% vs. 94.3%;p = Similar results were obtained when considering sarcocyst positivity in the h diaphragm samples separately: there was a significant (p < 0.001) difference betw kids (17.1% and 25.0%, respectively) and the junior-, middle-and senior-class (82.5% and 70.8%, 86.7% and 63.1%, and 81.3% and 68.4%, respectively), and sa were significantly (p < 0.05) more often recorded in the samples from the junior-, For the prevalence of infection in the heart and/or diaphragm, the chi-square test revealed a significant (p < 0.001) difference between the kids (27.5%) and the junior-, middle-and senior-class chamois (87.5%, 89.4% and 89.5%, respectively).Sarcocysts were significantly (p < 0.05) more often recorded in the samples from the junior-, middle-and senior-class chamois than in those from kids, but there was no difference in the prevalence of Sarcocystis spp.infection between the three classes of sexually mature chamois (p = 0.941).No significant difference in the prevalence of Sarcocystis spp.infection was found between the male and female chamois in the four age classes combined (80.7% vs. 74.7%;p = 0.3585) or in the three classes of sexually mature chamois combined (89.2% vs. 94.3%;p = 0.3762).
Similar results were obtained when considering sarcocyst positivity in the heart and diaphragm samples separately: there was a significant (p < 0.001) difference between the kids (17.1% and 25.0%, respectively) and the junior-, middle-and senior-class chamois (82.5% and 70.8%, 86.7% and 63.1%, and 81.3% and 68.4%, respectively), and sarcocysts were significantly (p < 0.05) more often recorded in the samples from the junior-, middleand senior-class chamois than in those from the kids, but there was no significant difference in the prevalence of Sarcocystis spp.infection between the junior-, middle-and senior-class chamois (p > 0.5).No significant difference in the prevalence of Sarcocystis spp.infection was found between the male and female chamois in the four age classes combined and in the three classes of sexually mature chamois combined (p > 0.5).
The results of the examination of the samples of the 181 chamois with both heart and diaphragm tissues available for the intensity of infection are summarized in Table 2.As there was no statistical difference for the sarcocyst counts between the three classes of sexually mature chamois, overall and for the two sexes (p < 0.05, Kruskal-Wallis test), the data for the three classes were combined.
Of the 131 heart-sarcocyst-positive chamois, 124 and 7 had <10 and ≥10 sarcocysts per cm 2 of muscle cut, respectively.Of the 127 diaphragm-sarcocyst-positive chamois, 119 had <10 and 8 had ≥10 sarcocysts per cm 2 of muscle cut.The maximum intensity recorded was 18.7 sarcocysts per cm 2 for the heart tissue and 14.6 sarcocysts per cm 2 for the diaphragm tissue in a one-and-a-half-year-old male and a two-and-a-half-year-old female, respectively.The diaphragm tissue had significantly lower counts of sarcocysts than the heart tissue in the four age classes combined and in the three classes of sexually mature chamois combined (p < 0.001), while the counts in the two tissues from the kids did not differ (p = 0.662).
There was a significantly positive correlation between the sarcocyst counts in both the heart and diaphragm tissues and the age of the chamois (r s = 0.4038, p < 0.001, and r s = 0.2093, p = 0.005, respectively).The kids had significantly lower counts of sarcocysts in both the heart and diaphragm tissues than the older chamois (p < 0.001).
There was no statistical difference between the male and female animals for the heart sarcocyst counts in the four age classes combined, the three classes of sexually mature chamois combined and the kids, or between the diaphragm sarcocyst counts in the four age classes combined and the kids (p > 0.1); however, among the three classes of sexually mature chamois combined, female animals had significantly higher diaphragm sarcocyst counts than male animals (p < 0.01).
There was a significant medium-size relationship between the sarcocyst counts of the two tissues for the four age classes combined (Spearman's r = 0.4038; p < 0.001).

Discussion
The present work was part of a comprehensive study on the parasites of chamois in Germany [35,36], aiming to provide, for the first time, epidemiological data on the prevalence and intensity of Sarcocystis spp.infection in chamois from Germany using a histology examination of heart and/or diaphragm muscle samples collected from 216 chamois.
For the study of the prevalence and intensity of infection with Sarcocystis species in intermediate hosts, standard histology was used, which has been shown to be suitable for epidemiological studies of Sarcocystis spp.infections, although enzymatic digestion was more sensitive for the detection of the infection, as discussed previously [17,37].Therefore, the histologic examination used to determine the prevalence of infection represents a minimal estimate.Apart from the technique used for the detection of infection, the type of muscle tissue examined for sarcocysts may influence the outcome of the testing, so that the results of various studies are not easily comparable [37].
While the majority of publications do not necessarily allow for conclusions as to the prevalence of Sarcocystis spp.infection in chamois, mainly because of the small sample size examined, a lack of quantification of information or accidental findings [2][3][4][5][6][7][9][10][11][14][15][16][17][18][19][20][21][22][23]27,28], the overall prevalence of Sarcocystis infection established in this study with almost 80% of the animals testing sarcocyst-positive is in line with the highest prevalences reported previously in the literature [12,25].Cornaglia et al. [12] examined the heart tissue of 198 Alpine chamois from the western Alps in Italy by conventional histology and recorded sarcocysts in 79.8% of the animals, and Díez-Baños et al. [25], using tissue compression, recorded sarcocysts in the muscle tissues of 78.1% of 32 Cantabrian chamois originating from the Cantabrian range in northern Spain.A slightly lower prevalence of Sarcocystis spp.infection, 65%, was reported based on a histological examination of heart and diaphragm samples from 49 Alpine chamois from the central-eastern Alps in Italy [16].Overall, however, it seems reasonable to assume that Sarcocystis spp.infections are common in chamois of both species and have been demonstrated in animals of several subspecies of chamois.In agreement with a study by Cornaglia et al. [12] on chamois and several studies on other wild ungulates in Europe, including mouflon and several species of cervids [18,[37][38][39][40][41][42][43][44][45], the prevalence of sarcocyst positivity was positively correlated with the age of the animals, which reflects an age-and nutrition-associated risk of greater exposure to the infectious Sarcocystis stages for the older animals related to a lack of development of a robust protective immunity.Interestingly, the sarcocyst positivity of chamois kids found by Cornaglia et al. [12] was more than twice the frequency recorded in the present study-~57% vs. ~28%, respectively-while the sarcocyst positivity in the older animals was comparable.
Similarly, consistent with previous studies evaluating sex-related differences in the prevalence of Sarcocystis spp.infection in wild ungulates (see 37), no difference between male and female chamois was found.
To the best of the authors' knowledge, only one previous study in chamois provided an estimation of the intensity of Sarcocystis spp.infection, based on counts of sarcocysts in pieces of muscle tissue examined by tissue compression [25].The present investigation generated sarcocyst counts following standard histology, and it is not known to what extent histology sarcocyst counts correlate with the counts established by Díez-Baños et al. [25].According to a score proposed for the classification of sarcocyst counts in histology muscle samples of red deer as 'low intensity infection' (<10 sarcocysts per cm 2 ; [46]), the intensity of infection in the heart and diaphragm samples of almost all the Sarcocystis spp.-positivetested chamois was low.This may also be concluded for the Cantabrian chamois examined by Díez-Baños et al. [25], given that the mean sarcocyst counts in 15 small pieces of muscle in chamois and roe deer were 1.9, with a range of 1 to 9, vs. 15.1, with a range of 1 to 155, respectively.In agreement with studies conducted on mouflon and cervids [38,45,47], the heart tissue of the Alpine chamois from Germany yielded higher sarcocyst counts than the diaphragm samples.
There are many studies which have found that the prevalence and intensity of parasite infections, especially infections with helminths, are higher among male than female hosts of different taxa of animals; however, male-biased parasitism is not necessarily the general rule, as there are several factors, ecological and physiological, which may influence the susceptibility and exposure to infection [47].Regarding sarcocystosis, there are a couple of studies which indicate, at least partly, that male-biased parasitism may play a role in this infection.However, this did not apply to the prevalence of infection but only to the intensity of infection measured in terms of sarcocyst counts (but not for all tissues examined in a study), while no difference in the prevalence of infection between the male and female animals was found [37,44,45].This study did not find male-biased sarcocyst counts.In contrast, the diaphragm sarcocyst counts of the sexually mature female chamois were significantly higher than the counts in the male animals.
The results of the present study demonstrated that sarcocystosis is highly prevalent in Alpine chamois in Germany, but the intensity of infections is apparently low.Further studies are desired to characterize the species of Sarcocystis parasitizing chamois using the current adequate methodology in Germany, but also in chamois in general, including experimental transmission studies to understand the role of carnivores or other animals in the life cycle

Figure 1 .
Figure 1.Thin-walled sarcocyst in hematoxylin-eosin-stained diaphragm muscle section.The examination for sarcocysts in the 181 chamois with both heart and diaphragm tissues available revealed a collective prevalence of Sarcocystis infection of 78.5% (95%CI 72.5-84.4%).Sarcocysts were more frequently recorded in the heart vs. diaphragm samples (131/181, 72.4% vs. 102/181, 56.4%; p = 0.0021).Diaphragm sarcocyst positivity was associated with heart sarcocyst positivity (diaphragm positive [heart positive]/heart positive: 91/131, 69.5% vs. diaphragm positive [heart negative]/heart negative: 11/50, 22.0%; p = 0.0001).For the prevalence of infection in the heart and/or diaphragm, the chi-square test revealed a significant (p < 0.001) difference between the kids (27.5%) and the junior-, middle-and senior-class chamois (87.5%, 89.4% and 89.5%, respectively).Sarcocysts were significantly (p < 0.05) more often recorded in the samples from the junior-, middle-and senior-class chamois than in those from kids, but there was no difference in the prevalence of Sarcocystis spp.infection between the three classes of sexually mature chamois (p = 0.941).No significant difference in the prevalence of Sarcocystis spp.infection was found between the male and female chamois in the four age classes combined (80.7% vs. 74.7%;p = 0.3585) or in the three classes of sexually mature chamois combined (89.2% vs. 94.3%;p = 0.3762).Similar results were obtained when considering sarcocyst positivity in the heart and diaphragm samples separately: there was a significant (p < 0.001) difference between the kids (17.1% and 25.0%, respectively) and the junior-, middle-and senior-class chamois (82.5% and 70.8%, 86.7% and 63.1%, and 81.3% and 68.4%, respectively), and sarcocysts were significantly (p < 0.05) more often recorded in the samples from the junior-, middleand senior-class chamois than in those from the kids, but there was no significant difference in the prevalence of Sarcocystis spp.infection between the junior-, middle-and senior-class chamois (p > 0.5).No significant difference in the prevalence of Sarcocystis spp.infection was found between the male and female chamois in the four age classes combined and in the three classes of sexually mature chamois combined (p > 0.5).The results of the examination of the samples of the 181 chamois with both heart and diaphragm tissues available for the intensity of infection are summarized in Table2.As there was no statistical difference for the sarcocyst counts between the three classes of sexually mature chamois, overall and for the two sexes (p < 0.05, Kruskal-Wallis test), the data for the three classes were combined.Of the 131 heart-sarcocyst-positive chamois, 124 and 7 had <10 and ≥10 sarcocysts per cm 2 of muscle cut, respectively.Of the 127 diaphragm-sarcocyst-positive chamois, 119 had <10 and 8 had ≥10 sarcocysts per cm 2 of muscle cut.The maximum intensity recorded was

Table 1 .
Summary of publications reporting on the finding of sarcocysts or occurrence of sarcocystosis in chamois.

Table 2 .
Intensity of infection by histology (number of sarcocysts per cm 2 of muscle cut) in the heart and diaphragm muscle tissues of 181 chamois from the Bavarian Alps and Black Forest mountains in Germany (animals with both heart and diaphragm muscle samples examined).