Seroprevalence of fasciolosis in Hokkaido sika deer (Cervus nippon yesoensis) from Hokkaido Prefecture,Japan revealed by ELISA using recombinant cathepsin L1

Fasciolosis, a zoonotic disease caused by liver flukes of the genus Fasciola, has been reported in Hokkaido (Yezo) sika deer (Cervus nippon yesoensis) in Hokkaido Prefecture, Japan; however, the actual seroprevalence in the animal has not been adequately evaluated. The objective of the present study was to analyze the seroprevalence of the disease among Hokkaido sika deer. Recombinant cathepsin L1 (rCatL1) was used as an antigen for an indirect enzyme-linked immunosorbent assay (ELISA) to detect antibodies against Fasciola flukes. The sensitivity and specificity of the ELISA were 84.6% and 100%, respectively. The average seroprevalence in 1109 Hokkaido sika deer from 20 locations in Hokkaido Prefecture was 43.9%. Mature deer showed higher seroprevalence than younger individuals; however, even younger animals may act as a reservoir for the disease. Monitoring infection levels in the Hokkaido sika deer population is important not only for the livestock industry, but also for preventing human fasciolosis.     

Efficient management for the Hokkaido population of sika deer Cervus nippon in Japan: accounting for migration and management cost

We evaluated the fourth stage of the “Conservation and Management Plan for Sika Deer (Cervus nippon) in Hokkaido, Japan (CMPS4)”, focusing on its cost-effectiveness and sika deer migration between two management areas of eastern and western Hokkaido. To clarify these factors, we constructed a stochastic matrix population model that accounts for deer migration and several uncertainties. We assumed four different budget scenarios and simple rules regarding nuisance control, and simulated four alternative management strategies. In the stochastic simulation, we calculated the probability of successfully satisfying the population target given by the CMPS4, an average total actual management cost, and a cost-effectiveness index given four budget conditions of migration rate and budget allocation ratio. The simulation results suggest the following. First, the current management budget is so small that the probability of successfully satisfying the population targets in both areas is only 26–30 %. If the total budget remains small, it should be almost entirely invested in one area, regardless of migration situation, to maximize the probability of successfully meeting the target density in at least that area. However, these probabilities of success decrease with greater migration rate. Second, when the government invests more of its budget in the early management stage, the expected total actual cost decreases and the probability of management success increases. These findings represent cost-effective management strategies for satisfying the CMPS4 targets.     

Genetic variation and population structure of the Japanese sika deer (Cervus nippon) in Hokkaido Island, based on mitochondrial D-loop sequences

Mitochondrial DNA (mtDNA) D -loop region sequences (602 bp) from 141 samples of the sika deer Cervus nippon collected from Hokkaido Island of Japan were investigated to elucidate population genetic structure. All animals possessed seven repeat units (38 or 39 bp each) in the sequences. Comparison of the 602-bp sequences showed four sites of transitional mutations (A↔G or C↔T). Based on combination of the substitutions, six D -loop haplotypes (a–f types) were identified in the Hokkaido population, suggesting the occurrence of at least six maternal lineages. Distribution maps of the haplotypes constructed using the Geographic Information System showed that the distribution patterns differed from haplotype to haplotype. In particular, distribution of the major three types (a-, b-, and c-types) almost overlapped with three main areas of coniferous forests in Hokkaido. These results suggest that expansion of the sika deer population could have occurred through the habitat of coniferous forests after the historical bottleneck in Hokkaido.     

Alarm signals of the Sichuan sika deer Cervus nippon sichuanicus

Sichuan sika deer (Cervus nippon sichuanicus) is an endangered and endemic subspecies of sika deer to Sichuan Province, China. According to our observations in the wild, the Sichuan sika deer makes alarm signals in the presence of actual or potential predators. In order to test the variation of the rhythmic alarm calls in some sex/age classes and different risk contexts, we recorded alarm calls of Sichuan sika deer from 2 October to 30 November 2008 and from 4 April to 5 September 2009 in the Tiebu Nature Reserve, Zoige County, Sichuan Province, China, and made acoustic analysis of these alarm calls. The results showed that the fundamental frequencies of alarm signals of Sichuan sika deer tended to decrease with age, and were significantly higher for females than for males. Duration tended to increase with age, and was significantly longer for males than for females. The fundamental frequencies and duration of alarm calls in adults were significantly higher and shorter respectively in high-risk than in moderate-risk contexts.     

Assessment LOPU-IVF in Japanese sika deer (Cervus nippon nippon) and application to Vietnamese sika deer (Cervus nippon pseudaxis) a related subspecies threatened with extinction

In mammals, recovery of oocytes by laparoscopic ovum pick-up (LOPU) coupled with in vitro production (IVP) of embryos represents a promising strategy for both amplification and genetic management of sparse animals from captive endangered wild species. As integrated technique developed mainly for domestic livestock, LOPU-IVP requires several studies to set up protocols for follicular stimulation or optimization of IVP before envisaging successful transposition to wild species. In deer, many endangered subspecies would be potentially concerned by applying such an approach using common subspecies for protocols optimization. The aim of the present study was to assess efficiency of follicle stimulation using ovine FSH (oFSH) for recovery of oocytes by LOPU in common sika deer (Cervus nippon nippon) before transposition of an optimized methodology for IVP of embryos from endangered Vietnamese sika deer hinds (Cervus nippon pseudaxis). In common sika deer, two doses of oFSH (0.25 and 0.5 U) and two frequencies of administration (12 and 24 h) were compared by monitoring of subsequent ovarian response, quality of oocytes recovered by LOPU, and in vitro developmental competence. In a first experiment, the dose of oFSH administered did not significantly affect the total number of follicles aspirated per hind per session (8.6 ± 1.0 vs. 8.2 ± 1.6 with 0.5 vs. 0.25 U oFSH, respectively; not significant). In a second experiment, frequency of 0.25 U oFSH administration did not affect ovarian response. Efficiency of IVP determined on blastocysts rates after in vitro maturation, fertilization, and development in oviduct epithelial cells coculture was increased when FSH was administered at 12-h intervals. Immune response after several follicular stimulations was detected against exogenous oFSH in plasma from the majority of sika deer hinds but was not associated with decreased ovarian response. When 0.25 U oFSH was administered at 12-h intervals to Vietnamese sika deer (N = 4), good quality cumulus oocyte complexes with complete and compact cumulus investments were recovered allowing a high cleavage rate after in vitro maturation and fertilization. Development to the blastocyst stage occurred in a high proportion (30% of oocytes) after coculture with ovine epithelial cells allowing cryobanking of transferable embryos from Vietnamese sika deer. These results confirm that LOPU-IVF after ovarian stimulation with oFSH may be a successful tool for cryobanking transferable embryos from endangered sika deer subspecies.     

Prevalence and phylogenetic analysis of Cryptosporidium infections in Yezo sika deer (Cervus nippon yesoensis) in the Tokachi sub-prefecture of Hokkaido,Japan

The Yezo sika deer (Cervus nippon yesoensis) on the island of Hokkaido, Japan are currently recognized as overabundant. Hunting is used to control the deer population, and this has increased the supply of game meat, which is associated with a high risk of various food-borne infections. Additionally, the sub-prefecture Tokachi has a dense population of livestock, which are potentially at risk of cross-species infections from the deer. In this study, we undertook the first analysis of the incidence of Cryptosporidium infection in the Yezo sika deer in the Tokachi area using polymerase chain reaction testing and phylogenetic analysis. Polymerase chain reaction analysis showed Cryptosporidium species present in 7.5% of fecal samples (13/173) collected from deer hunted between 2016 and 2017. However, the zoonotic Cryptosporidium paruvm parasite was not detected in the phylogenetic analysis; when sequenced, all species in the positive samples matched the Cryptosporidium deer genotype. However, deer may act as a reservoir of the zoonotic Cryptosporidium parvum parasite, which affects both humans and livestock. Therefore, we recommend the continuation of surveys of the incidence of Cryptosporidium infections in Yezo sika deer.     

Antler growth and morphology in a feral sika deer (Cervus nippon) population in Killarney, Ireland

Antler growth and form was examined for a sample of 303 sika stags (Cervus nippon) culled over an eight-year period from a feral population in Killarney National Park, County Kerry, Ireland. Most antler measurements taken were highly correlated with each other and growth was complete after the sixth year. Few abnormalities were recorded and the degree of asymmetry in structure was less than previously reported for other sika populations. Variability in structure decreased with age and there was no effect of year of birth or cull on antler size for either adult or yearling stags. The growth pattern was similar to that of other populations except that the proportion of 'eight pointers' was low. An examination of antler damage was used to estimate fighting frequency across age classes. The results are discussed in relation to the genetic history of the herd and the use of antlers as weapons for intra-specific combat.     

Artificial insemination, hybridization and pregnancy detection in sika deer (Cervus nippon )

Artificial insemination (AI) was performed on sika hinds (Cervus nippon ) receiving various dosages of pregnant mare serum gonadotropin (PMSG; Year 1: 0, 50 and 100 IU; Year 2: 100 and 150 IU) and using semen collected from elk and 1 2 elk x 1 2 sika stags. The time from synchronization device removal (CIDR vs norgestomet) to estrus was determined through observations of mounting activity. Methods for pregnancy detection, serum progesterone (P4), estrone sulfate (E1S), pregnancy-specific protein B (PSPB) and ultrasonography, following AI (Year 1: AI, Days 28 and 48 after AI; Year 2: AI, Days 42, 53 and 100 after AI) and a 90-d natural breeding season were investigated. From available production data, body weights were compared among sika and 1 4 elk x 3 4 sika hybrids relative to age. Pregnancy rates tended (P < 0.10) to differ relative to PMSG treatment and sire; administration of 0 IU PMSG resulted in fewer hinds becoming pregnant to AI than 50 or 100 IU of PMSG. Hinds receiving 100 IU of PMSG had higher (P < 0.05) pregnancy rates than hinds receiving 150 IU PMSG. Time to standing estrus did not differ (P > 0.10) between the CIDR and norgestomet groups. Pregnancy rates 50 d after a 90-d breeding season were similar (P > 0.10) between ultrasound (70.9%) and PSPB (61.6%). Serum P4 after 90 d in breeding groups and 50 d after stag removal were higher (P < 0.05) for pregnant than open hinds. Pregnancy rates (Year 1) 48 d after AI were similar (P > 0.10) between ultrasound (49.0%) and PSPB (37.3%). Serum P4 28 and 48 d after AI were higher (P < 0.05) for pregnant than open hinds. Serum E1S was higher (P < 0.01) for pregnant than open hinds 48 d after AI. Pregnancy rates (Year 2) 100 d after AI did not differ (P > 0.10) between ultrasound and PSPB (66.7%). Serum P4 was higher (P < 0.03) in pregnant than open hinds at 42, 53 and 100 d after AI. At 100 d after AI, pregnant hinds had higher (P < 0.002) serum E1S than open hinds. At 6 to 8 and 11 to 13 mo of age, 1 4 elk x 3 4 sika males tended (P < 0.08) to be heavier than sika males, while 1 4 elk x 3 4 sika females were heavier (P < 0.05) than sika females at all ages. In summary, this study documents the use of AI and methods for pregnancy detection in sika hinds as well as preliminary information regarding the production of elk-x-sika hybrids.     

Microsatellite analysis of genetic diversity of the Vietnamese sika deer (Cervus nippon pseudaxis)

The Vietnamese sika deer (Cervus nippon pseudaxis) is an endangered subspecies of economic and traditional value in Vietnam. Most living individuals are held in traditional farms in central Vietnam, others being found in zoos around the world. Here we study the neutral genetic diversity and population structure of this subspecies using nine microsatellite loci in order to evaluate the consequences of the limited number of individuals from which this population was initiated and of the breeding practices (i.e., possible inbreeding). Two hundred individuals were sampled from several villages. Our data show both evidence for limited local inbreeding and isolation by distance with a mean F(ST) value of 0.02 between villages. This suggests that exchange of animals occurs at a local scale, at a rate such that highly inbred mating is avoided. However, the genetic diversity, with an expected heterozygosity (H(e)) of 0.60 and mean number of alleles (k) of 5.7, was not significantly larger than that estimated from zoo populations of much smaller census size (17 animals sampled; H(e) = 0.65, k = 4.11). Our results also suggest that the Vietnamese population might have experienced a slight bottleneck. However, this population is sufficiently variable to constitute a source of individuals for reintroduction in the wild in Vietnam.     

Genetic diversity analysis by microsatellite markers in four captive populations of the sika deer (Cervus nippon)

The Chinese sika deer (Cervus nippon) is a rare and vulnerable animal in China for medical use. In this study, the genetic diversity and population genetic structure of 113 Chinese sika deer from 4 populations (Linyi Farm, LF; Linyi Park, LP, Yangzhou Farm, YF; Yangzhou Zoo, YZ) were investigated with 14 microsatellite loci. Eighty-three alleles were detected at the 14 loci in all populations. The expected heterozygosity ranged from 0.257 to 0.863 and the observed heterozygosity from 0.226 to 0.821. The polymorphism information content at different loci ranged from 0.217 to 0.825. The results of the HWE (Hardy–Weinberg equilibrium) tests indicated that only four loci (CEH-5, BL42, Mber70, and CEH-2) were in HWE (P > 0.01). The mean number of alleles per population ranged from 3.21 to 5.64, observed and expected heterozygosities ranged from 0.568 to 0.685, respectively. Positive inbreeding coefficient (F_IS) values were found in every population. F_ST values ranged from 0.101 in the LF to 0.155 in the YZ. The genetic identity ranged from 0.1236 to 0.1645. The genetic distance ranged from 0.4746 to 0.6025. The results of this study indicate moderate genetic variation and polymorphism across the loci. Appropriate breeding strategies should be designed for deer in captivity.     

东北梅花鹿初级卵母细胞的超微结构

研究东北梅花鹿初级卵母细胞发育的超微结构变化,目的是为探索东北梅花鹿初级卵母细胞的发育规律提供组织学和形态学依据。本研究于2003年和2004年的6月初到8月末取3只、9月中旬到10月初取4只,共计7只健康经产2~3胎的成年东北梅花鹿卵巢;卵巢经2·5%戊二醛固定液固定后,切取约1mm3的卵巢皮质和直径0·5~1·5mm及1·5~3mm的卵泡作为电镜观察用材料;该材料经0·1MpH7·2的PBS漂洗、1%锇酸固定、不同浓度乙醇脱水后,再经Epon812和丙酮等量混合液浸透,最后用Epon812包埋制块,并用半薄切片机切成0·5~2μm半薄切片;再经亚甲基兰-天青Ⅱ染色后,在光镜下进行卵泡分类和卵母细胞定位;将经定位的材料用超薄切片机切成厚度为700~800的切片,经醋酸铀和柠檬酸铅双重染色后,用透射电镜观察、记录并照相。观察时将卵泡依其直径大小、透明带的形成、卵泡腔的出现等分为原始卵泡、初级卵泡、次级卵泡和三级卵泡4类。研究结果表明,在原始卵泡阶段,卵母细胞为较规则的圆形,质膜与卵泡细胞膜紧密相贴,有时形成桥粒,细胞器多分布于近核区,高尔基体不典型,线粒体多为圆形,嵴较少;在次级卵泡阶段,2~4层的卵泡细胞局部开始形成透明带,4层以上时形成薄的透明带,微绒毛斜伸入透明带内,方向不规律;在直径为0·5~1·5mm的三级卵泡阶段,卵母细胞的透明带增厚,各种细胞器在皮质区内数量较多,皮质区内高尔基体的数目增多,粗面内质网明显减少;在直径为1·5~3mm的三级卵泡阶段,卵母细胞的透明带继续加厚,微绒毛缩短变弯,开始从透明带退出,许多皮质颗粒开始排列在卵母细胞膜下。     

Costs of parturition and rearing in female sika deer (Cervus nippon)

The costs of parturition and lactation of female sika deer on Kinkazan Island (9.6 km(2) in size), northern Japan, which live at a high density (about 50 deer/km(2)), were evaluated by comparison of body weights of 481 females measured during a 15 year study (1993-2008). Weight data were chosen from only females that did not give birth in the preceding year. The mean body weight of females that did not give birth ("yelds") was significantly lower (P < 0.001) than that of females who gave birth ("milks"); yelds' body weight was 93.1% and 83.5% that of milks in the preceding and parturition years, respectively. The yelds increased in body weight by the following March by 8.2% (P < 0.001), whereas milks did not. Among the milks, those whose fawns survived until the following May ("rearing milks") lost body weight by 14.9% (P < 0.001). Milks who lost fawns within a week after birth ("early fawn-less milks") did not lose body weight (P = 0.583), while those whose fawns died after the first autumn but died before May ("late fawn-less milks") lost body weight by 19.9% (P < 0.001). These results indicate that sika deer females do not enter estrus unless they are heavy enough, and that both parturition and rearing are costly for sika deer mothers living in high-density conditions.     

Successful in vitro production of embryos in the red deer (Cervus elaphus) and the sika deer (Cervus nippon)

The aim of our study was to define the conditions for IVM and IVF of oocytes in 2 common deer species as models for endangered related subspecies. Immature oocytes were recovered during the breeding season from postmortem ovaries (red deer) or by repeated laparoscopic follicular aspiration (sika deer). Oocytes were cultured for 24 h in IVM medium supplemented with EGF or FSH and follicular fluid. Stag semen was collected by electroejaculation (both species) or by epididymal flushing (red deer) and cryopreserved. For IVF, oocytes were exposed to different concentrations of thawed spermatozoa in a modified Tyrode albumin lactate pyruvate medium supplemented with 20% (v/v) estrus sheep serum for 18 h. After IVF, presumptive zygotes were allowed to develop in vitro for 7 days in synthetic oviduct fluid (SOF) supplemented with fetal calf serum (10%, v/v). In both species, the presence of ovine FSH and follicular fluid improved the in vitro maturation rate. In the sika deer, the optimal sperm concentration for IVF was 10(6)/mL and some fertilized oocytes reached the early morula stage (20 to 25 cells). In the red deer, after IVF with ejaculated or epididymal spermatozoa (2.0 x 10(6)/mL), 20% of zygotes developed to the blastocyst stage (50 to 80 cells).     

Tracing the genetic roots of the sika deer Cervus nippon naturalized in Germany and Austria

To determine the geographical origin of the sika deer (Cervus nippon) naturalized in Germany and Austria, we sequenced the mitochondrial control region for 214 individuals. Adding these sequences to previously published data from native sika deer across its natural geographic range, the total comes to 245, extending what is already known about the geographical variation in this sequence in Cervus nippon. From these sequences, a neighbour-joining tree was constructed. This tree showed that the 49 different mitochondrial (mt)DNA types are grouped into three distinct phylogenetic clusters, which correspond to different geographic areas. Similarities between sequences of the naturalized sika deer and those described from native sika deer from both southern Honshu, Kyushu with associated islands, and northern Honshu suggest that the ancestors of the sika deer populations in Germany and Austria originated from the Japanese archipelago. In contrast, there is no evidence that female sika deer of Chinese, Taiwanese or north Vietnamese origin were involved in the ancestry of the present sika population in Germany and Austria.     

Helminthes detected from wild sika deer (Cervus nippon centralis) in Kanto-Chubu region,Japan

The helminth fauna of 105 sika deer (Cervus nippon centralis) captured in Yamanashi, Kanagawa and Nagano Prefectures, Japan was investigated during 2014–2019. As a result, 12 helminthes, i.e. 3 digeneans (Ogmocotyle sikae, Dicrocoelium chinensis and D. dendriticum), 8 nematodes (Gongylonema pulchrum, Dictyocaulus sp., Pygarginema sp., Spiculopteragia houdemeri, Chabaudstrongylus ninhae, Trichuris discolor, Oesophagostomum sikae and Oes. asperum), and 1 cestode (Moniezia sp.) were detected. To our knowledge, this is the first report of Pygarginema sp., Cha. ninhae, and Oes. asperum from sika deer in Japan. Some helminthes detected in the present study can infect livestock. Considering the possibility of the spread of the helminthes to livestock through deer excrement, it is important to promote understanding the parasite fauna in wild deer.     

Detection and identification of Theileria infection in sika deer ( Cervus nippon ) in China

The sika deer ( Cervus nippon ) is a first-grade state-protected animal in China and designated a threatened species by the World Conservation Union. To detect hemoparasite infection of sika deer, blood samples were collected from 24 animals in the Hubei Province Deer Center. Genomic DNA was extracted, and the V4 hypervariable region encoding 18S rRNA was analyzed by reverse line blot hybridization assay. PCR products hybridized with Babesia / Theileria genus-specific probes but failed to hybridize with any of the Babesia or Theileria species-specific probes, suggesting the presence of a novel, or variant, species. Here 18S rRNA and internal transcribed spacer (ITS) genes were amplified, cloned, and sequenced from 7 isolates. Alignment and BlastN of the cloned sequences revealed high similarities to the homologous 18S rRNA genes and ITS genes of Theileria cervi (AY735122), Theileria sp. CNY1A (AB012194), and Theileria sp. ex Yamaguchi (AF529272). Phylogenetic analysis based on the 18S rRNA gene and ITS sequences showed that all cloned sequences were grouped within the Theileria clade. Phylogeny based on the 18S rRNA gene divided the organisms into 2 groups. Group 1 was closest to Theileria sp. ex Yamaguchi (AF529272), and group 2 was distinct from all other identified Theileria and Babesia species. These results suggest the existence of Theileria sp. infection in sika deer in China. To our knowledge, this is the first report of cervine Theileria sp. in China.     

Bottlenecks, drift and differentiation: the population structure and demographic history of sika deer (Cervus nippon) in the Japanese archipelago

We assessed genetic differentiation and diversity in 14 populations of sika deer (Cervus nippon) from Japan and four populations of sika deer introduced to the UK, using nine microsatellite loci. We observed extreme levels of differentiation and significant differences in diversity between populations. Our results do not support morphological subspecies designations, but are consistent with previous mitochondrial DNA analyses which suggest the existence of two genetically distinct lineages of sika deer in Japan. The source of sika introduced to the UK was identified as Kyushu. The underlying structure of Japanese populations probably derives from drift in separate glacial refugia and male dispersal limited by distance. This structure has been perturbed by bottlenecks and habitat fragmentation, resulting from human activity from the mid-nineteenth century. Most current genetic differentiation and differences in diversity among populations probably result from recent drift. Coalescent model analysis suggests sika on each of the main Japanese islands have experienced different recent population histories. Hokkaido, which has large areas of continuous habitat, has maintained high levels of gene flow. In Honshu the population is highly fragmented and is likely to have been evolving by drift alone. In Kyushu there has been a balance between gene flow and drift but all the populations have experienced high levels of drift. Habitat fragment size was not significantly associated with genetic diversity in populations but there was a significant correlation between habitat fragment size and effective population size.     

日本日光地区森林中梅花鹿的夏季巢区面积和内部利用

为了估算梅花鹿(Cervusnippon)巢区面积和内部利用以及雌雄个体间的比较,于1993-1997年在日本国日光国立公园使用无线电遥测定位法测定了8头成年雌鹿和4头雄鹿(包括1头亚成年)在森林生境中的活动定位点。按照当地梅花鹿离开和返回越夏地的日期,使用每年6月1日至10月25日的无线电遥测定位数据,采用定值核心法(Fixed kernel)计算了梅花鹿的夏季巢区面积和内部利用密度分布。梅花鹿雄鹿夏季巢区面积(192·5±50·5 hm2)显著大于雌鹿(115·7±56·7 hm2,P=0·017) ,其半家区面积(HRS0·50)和核心区面积(HRS0·25)也显著大于雌鹿(依次为P=0·027和P=0·042) ,但巢区内部利用集中度指标Ah(HRS0·50/HRS0·95)和Ac(HRS0·25/HRS0·50)在雌雄鹿之间没有显著性差异(依次为P=0·999和P=0·234)。在无线电遥测的12头梅花鹿中, 11头个体的巢区为单核心型,只有1头成年雄鹿的巢区为双核心型。11头梅花鹿个体的混合数据显示巢区面积和体重之间存在显著性相关(P=0·049) [动物学报52 (2) : 235 -241 , 2006]。