Sequence analysis of the Bs-Ag1 gene of Baylisascaris schroederi from the giant panda and an evaluation of the efficacy of a recombinant Baylisascaris schroederi Bs-Ag1 antigen in mice

The Baylisascaris schroederi infection rate among wild giant pandas may reach over 50% or even 100%, making it one of the leading causes of death from primary or secondary infection in wild populations. Until now, little was known about how protective immunity to B. schroederi infection could be achieved. The present study was conducted to evaluate the immunogenicity and protective efficacy of recombinant Bs-Ag1 from B. schroederi, by cloning the full-length Bs-Ag1 gene of B. schroederi and expressing it in a heterologous host, Escherichia coli BL21. In mice vaccinated with rBs-Ag1 coupled with Freund's complete adjuvant (FCA), there was a significant reduction (69.2%) in the recovery of challenged B. schroederi L3 compared with either nonvaccinated controls or mice vaccinated with FCA alone. Our study supports the use of Bs-Ag1 as a potential candidate for vaccination against B. schroederi infection and provides basic data for further vaccination trials with mixtures of antigens (with Bs-Ag2 and Bs-Ag3) to B. schroederi.     

3种苯并咪唑类药物对大熊猫西氏贝蛔虫的驱虫效果初步观察

西氏贝蛔虫Baylisascaris schroederi是圈养大熊猫Ailuropoda melanoleuca最常见、危害最严重的一种体内寄生虫,采用药物驱虫是目前控制圈养大熊猫蛔虫病的主要措施。为了筛选有效的驱虫药物,本研究观察了3种苯并咪唑类药物(阿苯达唑片剂、芬苯达唑膏剂和甲苯咪唑片剂)对大熊猫蛔虫的驱虫效果,统计了驱虫前后粪检蛔虫卵转阴率及排虫情况。结果表明,除芬苯达唑按5 mg·kg^-1体质量口服,每天1次,连续服用2 d,效果较差外,3种药物按10 mg·kg^-1体质量口服,每天1次,连续服用2 d,用药安全且有较好的驱虫效果。     

基于线粒体12S对中国两大山系大熊猫西氏贝蛔虫种群遗传多样性的分析

大熊猫是中国特有的珍稀濒危物种,而西氏贝蛔虫是大熊猫体内最为常见的肠道寄生虫,对野生和圈养大熊猫危害极大。考虑到大熊猫因分布区域的差异而形成了不同的亚种以及寄生虫与宿主间广泛的协同进化关系,西氏贝蛔虫是否也存在与大熊猫相适应的亚种分化一直是野生动物学家极其关注和热议的话题。为此,本文选择中国两大山系（岷山和邛崃）大熊猫种群体内共计34株西氏贝蛔虫虫体样本进行种群遗传多态性研究。利用PCR技术扩增出了岷山（14株）和邛崃（20株）西氏贝蛔虫的线粒体12S基因全序列并对其做了遗传多样性分析。结果表明：（1）34个样本包含9个单倍型,呈现出一个高单倍性多样性和低核苷酸多样性的特点;（2）负的Tajima's D和Fu's Fs中性检验值及“多峰型”的种群歧点分布图暗示种群不久前曾经历过突增长的现象;（3）低的种群间的分化系数和高的基因流表明两个地理种群间未形成显著的遗传分化;（4）系统发育树和单倍型网络图表明两山系种群分布无区域特异性。因此,岷山和邛崃山系的大熊猫体内的西氏贝蛔虫种群遗传变异性较低,分化不明显。该发现不仅暗示了西氏贝蛔虫与其宿主（大熊猫）的进化不同步,而且还为不同区域大熊猫西氏贝蛔虫病的监控提供了理论依据。     

A sensitive and specific PCR assay for the detection of Baylisascaris schroederi eggs in giant panda feces

Baylisascaris schroederi is one of the most common intestinal nematodes in giant pandas. It can cause severe baylisascariasis which is highly infectious in its natural hosts. A rapid and reliable diagnosis of parasite infections is crucial to protect giant pandas, as well as for environmental monitoring and disease surveillance. Here, we established a specific PCR assay for B. schroederi detection which was targeting a 331-bp long fragment of the mitochondrial cytochrome c oxidase subunit II (COII) gene. Fifty fresh fecal samples collected from captive giant pandas were tested by the established PCR assay and the traditional flotation technique. DNA extracted from a single B. schroederi egg could be successfully amplified, while no cross-reactivity was found with DNA from Ancylostoma caninum eggs. The detection rate of the PCR assay was 68%, which was higher than that of the traditional egg flotation (46%). Our findings demonstrated that the PCR assay is sensitive and specific for the detection and identification of B. schroederi eggs. Therefore, it could become a useful tool for the investigation of B. schroederi infections in giant pandas.     

Serotypes,Virulence Factors,and Antimicrobial Susceptibilities of Vaginal and Fecal Isolates of Escherichia coli from Giant Pandas

Although Escherichia coli typically colonizes the intestinal tract and vagina of giant pandas, it has caused enteric and systemic disease in giant pandas and greatly impacts the health and survival of this endangered species. In order to understand the distribution and characteristics of E. coli from giant pandas, 67 fecal and 30 vaginal E. coli isolates from 21 giant pandas were characterized for O serogroups, phylogenetic groups, antimicrobial susceptibilities, and pulsed-field gel electrophoresis (PFGE) profiles. In addition, these isolates were tested for the presence of extraintestinal pathogenic E. coli (ExPEC) and diarrheagenic E. coli (DEC) by multiplex PCR detection of specific virulence genes. The most prevalent serogroups for all E. coli isolates were O88, O18, O167, O4, and O158. ExPEC isolates were detected mostly in vaginal samples, and DEC isolates were detected only in fecal samples. Phylogenetic group B1 predominated in fecal isolates, while groups B2 and D were frequently detected in vaginal isolates. Resistance to trimethoprim-sulfamethoxazole was most frequently observed, followed by resistance to nalidixic acid and tetracycline. All except five isolates were typeable by using XbaI and were categorized into 74 PFGE patterns. Our findings indicate that panda E. coli isolates exhibited antimicrobial resistance, and potentially pathogenic E. coli isolates were present in giant pandas. In addition, these E. coli isolates were genetically diverse. This study may provide helpful information for developing strategies in the future to control E. coli infections of giant pandas.     

Extraction,PCR amplification,and sequencing of mitochondrial DNA from scent mark and feces in the giant panda

To expand the feasibility of applying simple, efficient, non-invasive DNA preparation methods using samples that can be obtained from giant pandas living in the wild, we investigated the use of scent markings and fecal samples. Giant panda–specific oligonucleotide primers were used to amplify a portion of the mitochondrial DNA control region as well as a portion of the mitochondrial DNA cytochrome b gene and tRNA^Thr gene region. A 196 base pair (bp) fragment in the control region and a 449 bp fragment in the cytochrome b gene and tRNA^Thr gene were successfully amplified. Sequencing of polymerase chain reaction (PCR) products demonstrated that the two fragments are giant panda sequences. Furthermore, under simulated field conditions we found that DNA can be extracted from fecal samples aged as long as 3 months. Our results suggest that the scent mark and fecal samples are simple, efficient, and easily prepared DNA sources. Zoo Biol 17:499–504, 1998. © 1998 Wiley-Liss, Inc.     

Phylogenetic study of Baylisascaris schroederi isolated from Qinling subspecies of giant panda in China based on combined nuclear 5.8S and the second internal transcribed spacer (ITS-2) ribosomal DNA sequences

The nuclear ribosomal DNA (rDNA) region spanning 5.8S rDNA and the second internal transcribed spacer (ITS-2) of Baylisascaris schroederi isolated from the Qinling subspecies of giant panda in Shaanxi Province, China were amplified and sequenced. Sequence variations in the two rDNA regions within B. schroederi and among species in the family Ascarididae were examined. The lengths of B. schroederi 5.8S and ITS-2 rDNA sequences were 156 bp and 327 bp, respectively, and no nucleotide variation was found in these two rDNA regions among the 20 B. schroederi samples examined, and these ITS-2 sequences were identical to that of B. schroederi isolated from giant panda in Sichuan province, China. The inter-species differences in 5.8S and ITS-2 rDNA sequences among members of the family Ascarididae were 0-1.3% and 0-17.7%, respectively. Phylogenetic relationships among species in the Ascarididae were re-constructed by Bayesian inference (Bayes), maximum parsimony (MP), and maximum likelihood (ML) analyses, based on combined sequences of 5.8S and ITS-2 rDNA. All B. schroederi samples clustered together and sistered to B. transfuga with high posterior probabilities/bootstrap values, which further confirmed that nematodes isolated from the Qinling subspecies of giant panda in Shaanxi Province, China represent B. schroederi. Because of the large number of ambiguously aligned sequence positions (difficulty of inferring homology by positions), ITS-2 sequence alone is likely unsuitable for phylogenetic analyses at the family level, but the combined 5.8S and ITS-2 rDNA sequences provide alternative genetic markers for the identification of B. schroederi and for phylogenetic analysis of parasites in the family Ascarididae.     

卧龙圈养大熊猫遗传多样性现状及预测,

以中国最大的大熊猫圈养种群—四川卧龙中国大熊猫保护中心的圈养种群为对象,以８个大熊猫微卫星位点为分子标记, 探讨了大熊猫圈养种群的遗传多样性, 并与邛崃野生种群及其他７个濒危物种进行比较。微卫星数据表明, 圈养种群的遗传多样性水平(A＝5.5, He ＝0.620, Ho＝0.574) 低于邛崃野生种群(A＝9.8,He＝0.779,Ho＝0.581),但高于其他7 个濒危物种的种群(He＝0.13~0.46)。在此数据的基础上对未来100个世代内圈养种群遗传多样性的变化情况做出了预测。结果表明假设种群数量比现在扩大一倍, 经历100个世代后也只会使平均等位基因数少减少0.4。因此继续增加野生个体对保持遗传多样性的意义已经不大, 建议该圈养种群的保护策略应将重点放到制定更有效的繁殖计划以避免近交上。     

粪样在大熊猫研究上的应用

大熊猫(Ailuropoda melanoleuca)数量少,传统取样法难以实施,样品的缺乏给大熊猫研究带来很大困难。而采集大熊猫粪样对大熊猫自身无损伤,粪样易获得和保存。本文总结了应用粪样进行的大熊猫食性与消化吸收、种群与个体识别、类固醇激素、寄生虫与病原微生物等方面的研究进展,对大熊猫粪样的应用前景进行了展望。     

Microbial Diversity and Evidence of Novel Homoacetogens in the Gut of Both Geriatric and Adult Giant Pandas (Ailuropoda melanoleuca)

Recent studies have described the bacterial community residing in the guts of giant pandas, together with the presence of lignocellulolytic enzymes. However, a more comprehensive understanding of the intestinal microbial composition and its functional capacity in giant pandas remains a major goal. Here, we conducted a comparison of bacterial, fungal and homoacetogenic microbial communities from fecal samples taken from two geriatric and two adult captive giant pandas. 16S rDNA amplicon pyrosequencing revealed that Firmicutes and Proteobacteria are the most abundant microbiota in both geriatric and adult giant pandas. However, members of phylum Actinobacteria found in adult giant pandas were absent in their geriatric counterparts. Similarly, ITS1 amplicon pyrosequencing identified developmental changes in the most abundant fungal classes from Sordariomycetes in adult pandas to Saccharomycetes in geriatric pandas. Geriatric pandas exhibited significantly higher abundance of a potential probiotic fungus (Candida tropicalis) as compared to adult pandas, indicating their importance in the normal digestive physiology of aged pandas. Our study also reported the presence of a lignocellulolytic white-rot fungus, Perenniporia medulla-panis, and the evidence of novel homoacetogens residing in the guts of giant pandas.     

贮存温度和时间对大熊猫粪便样品皮质醇浓度的影响

通过非损伤性取样比如粪便样品监测野生动物受胁迫的水平已成为一个重要的手段,然而针对从野外采集的样品,其运输和贮存温度条件是否干扰分析结果,尚未有过报道。为评估不同贮存温度和天数对野生动物粪便样品中皮质醇浓度的影响,我们将大熊猫的新鲜粪便置于3 种模拟条件(0℃ 、10℃ 和22℃ ),并进行连续6 d的检测分析。结果表明,贮存天数对粪样中皮质醇浓度有显著影响(F _5,107 = 7.501,P = 0.001),但温度则无显著影响(F _2,107 = 1.094,P = 0.366)。粪样在0℃ 、10℃ 和22℃ 条件下贮存6 d,皮质醇浓度与贮存前的基准值之间均不存在显著差异(0℃ :F _6,41 = 1.274,P = 0.290;10℃ :F _6,41 = 2.027,P = 0.084;22℃ :F _6,41 =1.009, P = 0.434)。结果提示在室温条件下短期运输和贮存(不超过6 d),不会引起大熊猫粪便样品中皮质醇浓度的显著变化。该结果对于野外所采集的粪便样品运输和贮存具有一定的指导意义。     

Gut microbiota in reintroduction of giant panda

Reintroduction is a key approach in the conservation of endangered species. In recent decades, many reintroduction projects have been conducted for conservation purposes, but the rate of success has been low. Given the important role of gut microbiota in health and diseases, we questioned whether gut microbiota would play a crucial role in giant panda's wild‐training process. The wild procedure is when captive‐born babies live with their mothers in a wilderness enclosure and learn wilderness survival skills from their mothers. During the wild‐training process, the baby pandas undergo wilderness survival tests and regular physical examinations. Based on their performance through these tests, the top subjects (age 2–3 years old) are released into the wild while the others are translocated to captivity. After release, we tracked one released panda (Zhangxiang) and collected its fecal samples for 5 months (January 16, 2013 to March 29 2014). Here, we analyzed the Illumina HiSeq sequencing data (V4 region of 16S rRNA gene) from captive pandas (n = 24), wild‐training baby pandas (n = 8) of which 6 were released and 2 were unreleased, wild‐training mother pandas (n = 8), one released panda (Zhangxiang), and wild giant pandas (n = 18). Our results showed that the gut microbiota of wild‐training pandas is significantly different from that of wild pandas but similar to that of captive ones. The gut microbiota of the released panda Zhangxiang gradually changed to become similar to those of wild pandas after release. In addition, we identified several bacteria that were enriched in the released baby pandas before release, compared with the unreleased baby pandas. These bacteria include several known gut‐health related beneficial taxa such as Roseburia, Coprococcus, Sutterella, Dorea, and Ruminococcus. Therefore, our results suggest that certain members of the gut microbiota may be important in panda reintroduction.     

Major histocompatibility complex alleles associated with parasite susceptibility in wild giant pandas

Major histocompatibility complex (MHC) polymorphism is thought to be driven by antagonistic coevolution between pathogens and hosts, mediated through either overdominance or frequency-dependent selection. However, investigations under natural conditions are still rare for endangered mammals which often exhibit depleted variation, and the mechanism of selection underlying the maintenance of characteristics remains a considerable debate. In this study, 87 wild giant pandas were used to investigate MHC variation associated with parasite load. With the knowledge of the MHC profile provided by the genomic data of the giant panda, seven DRB1, seven DQA1 and eight DQA2 alleles were identified at each single locus. Positive selection evidenced by a significantly higher number of non-synonymous substitutions per non-synonymous codon site relative to synonymous substitutions per synonymous codon site could only be detected at the DRB1 locus, which leads to the speculation that DRB1 may have a more important role in dealing with parasite infection for pandas. Coprological analyses revealed that 55.17% of individuals exhibited infection with 1–2 helminthes and 95.3% of infected pandas carried Baylisascaris shroederi. Using a generalized linear model, we found that Aime-DRB1*10 was significantly associated with parasite infection, but no resistant alleles could be detected. MHC heterozygosity of the pandas was found to be uncorrelated with the infection status or the infection intensity. These results suggested that the possible selection mechanisms in extant wild pandas may be frequency dependent rather than being determined by overdominance selection. Our findings could guide the candidate selection for the ongoing reintroduction or translocation of pandas.     

大熊猫粪便宽径与咬节平均长度的关系

粪便咬节是大熊猫数量调查中最常用的指标。然而,大熊猫在主食竹叶的季节有时在粪便中未留下咬节而使数量调查难以进行。为此,本文对大熊猫粪便宽径与咬节平均长度的关系进行了初步探讨。结果表明,大熊猫粪便宽径呈正态分布,同一个体的含咬节粪便与全由竹叶组成的粪便在宽径上无显著差异。大熊猫咬节平均长度与粪便宽径存在显著的回归关系。在不考虑年龄的情况下,粪便咬节平均长度与粪便宽径的回归方程为:Y=0.38X十14.40。成体和亚成体组的粪便咬节平均长度与粪便宽径的回归方程为:Y=0.22 X+22.79。年龄对大熊猫粪便咬节平均长度与粪便宽径的回归关系有显著影响。     

The parasites of giant pandas: individual-based measurement in wild animals

There is a growing recognition of parasites as a significant factor in the successful conservation of endangered species. Determining parasite infection and load in free-ranging populations traditionally is done via necropsy or coproscopy. For studies of wild animals, fecal sample collection can result in bias because the individual identity of animals is unknown and multiple samples may be collected from the same individual, yet treated as unrelated samples. We studied parasite load in wild giant pandas (Ailuropoda melanoleuca) across six mountain ranges in China. Genetic identification was used to determine the exact number of individuals sampled. The parasite fauna consisted of five species, dominated by Baylisascaris shroederi. The pattern of statistical difference between mountains was artificially inflated when animal identity was not included in the model. Our results suggest that caution should be exercised in inferring patterns from comparative parasitologic studies when samples cannot be attributed to specific individuals. Using noninvasive genetic sampling to avoid such bias should form a standard tool in the management of endangered species and their parasites.     

大熊猫轮状病毒PCR检测方法的建立及应用

轮状病毒是威胁大熊猫健康的主要病原微生物之一。为了对大熊猫轮状病毒进行快速、方便且准确地检测,研发适合于基层饲养单位和保护区的检测方法是非常有必要的。本研究通过合成大熊猫轮状病毒Vp7基因序列,构建了PUC-VP7的重组质粒,并将其作为阳性对照对大熊猫轮状病毒样本进行PCR检测和分析。结果表明,在进行PCR扩增分析时,该质粒和病毒cDNA二者均在340 bp处出现了特异性条带。此外,对收集到的45份大熊猫粪便样本进行轮状病毒抗原检测时,其中2份样品在340 bp处出现条带,该基因片段与大熊猫轮状病毒CH-1株的相似性为99.89%。本研究构建的PUC-VP7质粒不但可以作为大熊猫轮状病毒PCR检测中的阳性质控品,而且还能有效地促进该PCR病毒检测技术在基层饲养单位和保护区的推广和应用。     

大熊猫胃内纤毛虫检测初报

Twenty three captive giant pandas, fecal specimens and stomach juice from five captive giant pandas(Ailuropoda melanoleuca)were tested for ciliate in 2004. Of them, ciliates of Epdinium spp.and Entodinium spp.were found in the stomach of five giant panda. Density of ciliates in each stomach of giant pandas were 4.21×10 4 ind./ml, 6.98×10 4 ind./ml, 3.05×10 4 ind./ml, 4.46×10 4 ind./ml and 4.38×10 4 ind./ml respectively. But all the fecal specimens were negative.     

Yersinia pseudotuberculosis infection in breeding monkeys: detection and analysis of strain diversity by PCR

In the last three decades, several monkeys reared in outdoor/indoor-outdoor breeding colonies and cages of the Primate Research Institute, Kyoto University, died of yersiniosis caused by Yersinia pseudotuberculosis, necessitating introduction of a method to detect the bacteria rapidly and thus allow preventive measures to be undertaken. A rapid nested polymerase chain reaction (PCR) method for identification of Y. pseudotuberculosis in fecal samples and a random amplified polymorphic DNA (RAPD)-PCR approach for distinguishing between bacterial strains were therefore developed. Yersinia pseudotuberculosis isolates from monkey specimens were found to be classifiable into several types. To determine the source of infection, hundreds of fecal samples of wild rats, pigeons, and sparrows were collected from around the breeding colonies and cages, and subjected to PCR analyses. Yersinia pseudotuberculosis was detected in 1.7% of the fecal samples of wild rats. The DNA fingerprints of the bacteria revealed by RAPD-PCR were the same as that of one strain isolated from macaques, suggesting the wild rat to be a possible source of infection.     

大熊猫气味标记DNA的制备和序列分析

大熊猫气味标记在其个体间的通讯中具有重要意义。用不同方法收集了７只大熊猫个体的９个气味标记样品,运用Ｉｎｓｔａｇｅｎｅ Ｋｉｔ制备出了ＤＮＡ。采用ＰＣＲ扩增线粒体Ｄ－环区和细胞色素ｂ基因、Ｔｈｒ－ｔＲＮＡ基因片段并作序列分析。结果提示,不同收集方式所得气味标记样品均有ＤＮＡ,但用干棉花收集样品的方法最佳。该方法为大熊猫的遗传多样性研究提供了新的简捷有效的ＤＮＡ来源。     

Dogs and Disease Threats to Giant Pandas in China

The potential threat of domestic dogs to wildlife habitat in China is not widely recognized, despite their large population, lack of regulations regarding their control, and threat they pose to native species. In a case study in 2017, we surveyed villages surrounding Liziping Nature Reserve, the primary site for the release of captive-born giant pandas (Ailuropoda melanoleuca) into the wild. We conducted surveys of dog owners to assess the population size, demographics, free-roaming status, and vaccination and sterilization history of their dogs. We collected blood and fecal samples to assess the prevalence of viral and parasite disease threats. At least 370 owned dogs lived near the core giant panda habitat; 64% were free-roaming, 21% had positive antibody titers for ≥1 of the 4 viruses we tested (canine distemper, parvovirus, rotavirus, rabies), and 67% were positive for gastrointestinal parasites. The high proportion of free-roaming dogs, uninhibited access to the reserve, and high prevalence of infectious diseases indicate that dogs pose a serious threat to wildlife within Liziping. The extent of this threat throughout the giant panda nature reserve network is unknown and should be assessed. © 2019 The Authors. Journal of Wildlife Management published by Wiley Periodicals, Inc. on behalf of The Wildlife Society.