Analysis of the bacterial diversity in the fecal material of the endangered Yangtze finless porpoise, <Emphasis Type="Italic">Neophocaena phocaenoides asiaeorientalis</Emphasis>

The aim of this study was to determine the bacteria present in the fecal material of the endangered Yangtze finless porpoise, Neophocaena phocaenoides asiaeorientalis. Fecal samples were collected from 12 Yangtze finless porpoises living in the wild at Poyang Lake, located in Jiangxi Province, China. To determine the bacterial diversity, a 16S rRNA gene clone library using the bacterial PCR primers fD1 and rP2, was prepared. A total of 138 near-full-length sequences were analyzed and 39 operational taxonomic units (OTUs) were identified. Sequences showing ≥97% similarity were grouped together as an OTU. Six different phyla were identified in which 38 OTUs were classified. Most of the OTUs contained sequences belonged to the phylum Firmicutes (51.3%), followed by Tenericutes (17.9%), Proteobacteria (15.4%), Actinobacteria (7.7%), Deinococcus-Thermus (2.6%) and Cyanobacteria (2.6%). A phylum could not be assigned for one clone within one OTU (2.6%). It appears that the Yangtze finless porpoise has a more diverse range of bacteria compared to other aquatic mammals, such as seals.     

Seasonal changes in the local distribution of Yangtze finless porpoises related to fish presence

The Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis) is an endangered freshwater porpoise subspecies unique to the Yangtze River basin. Seasonal variations in local distribution of the animal, as well as fish presence, sand dredging, ship navigation, and bridges were examined as potential factors affecting the occurrence of the animals. Passive acoustic surveys were performed regularly from May 2007 to August 2010, near the conjunction of the Yangtze River and Poyang Lake. The distribution of the porpoises was seasonally site‐specific. In May and August, the animals were detected more often at river junctions than in the lake, but vice versa from November to February. The rate of the porpoise detection was significantly higher in areas of fish presence than in areas of absence. The number of porpoises detected did not differ significantly between the sand dredging operation and the prohibition period (in 2008), although the number of vessels obviously declined in 2008. Ship traffic and bridges also did not appear to affect the presence of porpoises. These results showed the relative importance of the various environmental factors, which is important for conservation of not only Yangtze finless porpoise but also endangered isolated cetaceans.     

SNP discovery based on CATS and genotyping in the finless porpoise (Neophocaena phocaenoides)

Single nucleotide polymorphisms (SNPs) discovery and genotyping were performed for the finless porpoises (Neophocaena phocaenoides). About 202 comparative anchor tagged sequence primers derived from genomes of human, mouse and some other mammals were used to screen the finless porpoise population. Of the 51 SNPs discovered, 25 were further characterized with ideal genotyping primers and using fragment length discrepant allele specific PCR assay. This is the first report of SNP loci for the finless porpoise, which is helpful to provide some novel molecular markers and new genetic information relevant to the conservation and management of this endangered species.     

长江江豚微卫星DNA分离的初步研究

为了开发物种特异性微卫星标记,本文采用一种改良的快速微卫星分离法(FIASCO)从长江江豚(Neophocaena phocaenoides asiaeorientalis)的基因组中筛选得到72条微卫星DNA序列。根据重复单元的排列特点,完美型、非完美型及复合型序列所占的比例分别为58.3%、22.2%和19.5%。选择其中30条序列设计PCR扩增引物,并用12个随机选择的长江江豚样品进行多态性筛选。初步结果表明其中14对引物的扩增产物稳定并且具有多态性;在每个座位上获得2-13个等位基因,平均等位基因数为5.87个;14个微卫星座位的平均观察杂合度(Ho)和期望杂合度(He)分别为0.560和0.709。本研究获得了长江江豚的第一批物种特异性微卫星座位及其扩增引物,这些微卫星标记将在后续的保护遗传学研究中发挥重要作用。       

固化河岸对长江江豚栖息活动的影响

长江江豚主要栖居在近岸浅水水域,喜好泥沙质类型的河岸。但在长江干流,有很多自然河岸被固化,河岸固化给长江江豚分布和栖息活动造的影响仍不甚清楚。2016年3月—2017年1月,对长江干流2个自然河段江豚的数量和分布做了12次考察,并收集了这2个河段岸型的相关数据,来分析固化河岸对江豚栖息活动的影响。12次考察累计发现江豚215头次,平均每次考察观察到江豚(17.92±7.09)头次。研究区域的固化河岸约占岸线总长的59%,分析发现,仅约13.9%的江豚分布在固化河岸水域,86.1%的江豚均分布在自然河岸水域。江豚在单位河岸长度的分布数量与该段固化河岸长度所占的比例呈显著负相关(r=-0.639,P0.01)。在自然河岸,分布在近岸50 m水域的江豚占31.8%,而在固化河岸,仅观察到2头江豚活动在近岸50 m水域内。由此可见,固化河岸对江豚的近岸分布有明显影响。调查还发现,安庆城区建设带约10 km江段12次考察均未发现江豚分布,城区建设带可能对江豚栖息已造成严重影响。长江干流的固化河岸所占比例非常高,研究结果提示这可能导致长江干流江豚栖息地的丧失和破碎化加剧,在制定长江江豚保护措施必需慎重考虑此因素的影响,并据此提出相应的栖息地保护和恢复方案。     

Non-pylori Helicobacteraceae in the upper digestive tract of asymptomatic Venezuelan subjects: detection of Helicobacter cetorum-like and Candidatus Wolinella africanus-like DNA

BACKGROUND: The spectrum of human non-pylori Helicobacter infections is expanding, with species such as H. heilmannii and H. felis occasionally being associated with gastritis. However, the existence of non-pylori Helicobacter colonization in asymptomatic subjects has not been evaluated. The aim of this study was to investigate whether Helicobacter species other than pylori are present in the upper digestive tract of asymptomatic human subjects. MATERIALS AND METHODS: A Helicobacteraceae-specific semi-nested polymerase chain reaction (PCR) assay was used to detect Helicobacter-like organisms in the upper digestive tract of 91 Venezuelan volunteers (aged 18-68 years, 41 females, 50 males). Species were identified by denaturing gradient gel electrophoresis analysis and sequencing of the PCR products. RESULTS: We detected DNA sharing 99-100% sequence identity in over 300-400 bp with the 16S rRNA genes of H. pylori, H. cetorum, and Candidatus Wolinella africanus in 76%, 16%, and 15% of the subjects, respectively. Multiple colonization was documented in 10% of the subjects: H. cetorum and Candidatus W. africanus (4%), H. pylori and Candidatus W. africanus (4%), and H. pylori and H. cetorum (2%). CONCLUSIONS: Our results suggest that non-pylori Helicobacteraceae colonization is relatively common in the Venezuelan asymptomatic population. This is the first report documenting the presence of H. cetorum DNA in the human upper digestive tract, and the second report of the recently discovered Candidatus W. africanus.     

长江安庆段航道整治对长江江豚数量和分布的时空影响

栖息地丧失和破碎化是威胁长江江豚(Neophocaena asiaeorientalis)种群长期发展的主要因素之一。航道整治工程是导致江豚栖息地丧失和破碎化的一个主要原因,研究工程施工和运行对江豚数量和分布的影响可为江豚栖息地管护提供有益信息。通过对湖口-吉阳矶水域江豚数量和分布的持续调查,分析了安庆段航道整治工程施工和运行对江豚的影响。12次目视调查共在研究水域发现江豚258群次613头次,工程施工前、施工期和初步运行期单次考察分别平均观察到江豚(67.0±24.3)头次、(35.6±22.2)头次和(50.0±2.8)头次。在主要施工区共发现江豚79群次185头次,施工前、施工期和初步运行期单次考察分别平均观察到江豚(18.60±12.03)头次、(10.4±7.3)头次和(20.0±4.2)头次。工程不同时期主要施工区江豚的数量及占比在统计学上没有显著差异。主要施工区S2和S3工程段施工期江豚平均分布密度((0.18±0.17)头/km、(0.04±0.08)头/km)较施工前((0.62±0.16)头/km、(0.40±0.29)头/km)显著下降,S5和S6工程段施工期平均...     

Isolation and characterization of novel Helicobacter spp. from the gastric mucosa of harp seals Phoca groenlandica

Since the recent discovery of Helicobacter cetorum in cetaceans and its role in the development of gastritis, speculation has existed as to whether pinnipeds have Helicobacter spp. associated gastritis and peptic ulcer disease. The gastric mucosa of 4 stranded harp seals Phoca groenlandica from the Massachusetts coastline were assessed for Helicobacter spp. by culture and PCR. We cultured 2 novel Helicobacter spp. from the pyloric antrum of 1 of the 4 harp seals studied, and identified these by PCR in 2 of the 4 seals. Both gram-negative bacterial isolates were catalase- and oxidase-positive. However, a fusiform helicobacter with flexispira morphology was urease-positive, and a spiral-shaped helicobacter was urease-negative. Slender, spiral and fusiform-shaped bacteria were detected in the gastric mucosa by the Warthin-Starry stain. Histopathologic analysis revealed mild diffuse lymphoplasmacytic gastritis within the superficial mucosa of the pyloric antrum of both infected seals. The 2 bacterial isolates were classified by 16S rRNA analysis; they clustered with other enteric helicobacters and represent 2 novel Helicobacter spp. The urease-negative bacterial isolate clustered with H. canis and the urease-positive isolate clustered with an isolate from a sea lion and isolates from sea otters. This cluster of pinniped isolates has 97 % similarity to a number of Helicobacter species, but appears to be most closely related to other helicobacters with flexispira morphology. These findings suggest that the novel Helicobacter spp. may play a role in the etiopathogenesis of gastrointestinal diseases in pinnipeds. To our knowledge, this represents the first isolation and characterization of a novel Helicobacter spp. from pinnipeds.     

The Diel Rhythms of Biosonar Behavior in the Yangtze Finless Porpoise (Neophocaena asiaeorientalis asiaeorientalis) in the Port of the Yangtze River: The Correlation between Prey Availability and Boat Traffic

Information on the habitat use of the critically endangered Yangtze finless porpoise (Neophocaena asiaeorientalis asiaeorientalis) is critical for its conservation. The diel biosonar behavior of the porpoise in the port areas of the Yangtze River was examined along with simultaneous observations of fish density and boat traffic. Biosonar pulses from the porpoises were detected for 1233 min (5.77%) over a 21,380 min duration of effective observations. In total, 190 (5.63%) buzzes (an indication of prey capture attempts) were recorded among the 3372 identified click trains. Of the 168 echolocation encounters (bouts of click trains less than eight min apart), 150 (89.3%) involved single animals, indicating that solitary porpoises were frequently present and feeding in the port areas. Significant diel patterns were evident involving the biosonar behavior of the porpoises (including click trains and buzzes), fish density and boat traffic. The frequencies of the click trains and buzzes were significantly lower during the day than in the evening and at night, which suggests that porpoises in this region are primarily engaged in crepuscular and nocturnal foraging. The lack of a significant diel pattern in the echolocation encounters indicates the importance of the port in porpoise conservation. A forced feeding schedule may be associated with the lack of a significant correlation between porpoise acoustics and boat traffic. Overall, prey availability appears to be the primary factor that attracts porpoises. Additionally, porpoises tend to migrate or remain downstream in the morning and migrate or remain upstream in the evening, most likely to follow their prey. The findings of this study can be used to improve the conservation of the Yangtze finless porpoise.     

天鹅洲迁地保护江豚群体的遗传评估与发展预测

文章以2008年春季遭受严重雪灾的天鹅洲白鱀豚国家级自然保护区迁地保护江豚群体为研究对象,利用物种特异性微卫星遗传标记对该群体的遗传多样性进行了评估,并通过软件对其发展趋势进行了模拟和预测。研究结果表明,虽然2008年春季雪灾并未导致该群体遗传多样性的严重损失,灾后群体依然表现出中等水平的微卫星遗传多样性(18个座位的平均等位基因数为4.780,平均观察杂合度和期望杂合度分别为0.748和0.674);但模拟结果显示,该迁地保护江豚群体的遗传多样性将快速下降,100年后仅能保留约79%的等位基因和约84%的遗传杂合度。此外,模拟结果表明,通过增加群体数量和调整群体性比可有效减缓该群体遗传多样性的下降速度。根据研究结果,并结合长江江豚自然种群快速下降的现状,作者提出天鹅洲迁地保护江豚群体当前最重要的保护策略应该是尽早补充一定数量的、合适的野生个体,在加强遗传多样性保护的同时使其群体数量快速增长并形成一个更大规模的有效繁殖群体。     

湖北天鹅洲故道试养江豚生活习性的初步观察

为抢救濒危水生兽类白暨豚,建立保护区无疑是最有效的途径,１９９０年３月到１９９３年１０月,我们对引进白暨豚保护区湖北天鹅洲故道中试养的１１头江豚的生活状况和对环境的适应性,进行了较为详细的研究,力求在引入白暨豚前,对此地江豚的生活情况及白暨豚保护区的建区工作做进一步的生态评价。结果表明,江豚非常适应故道环境,它们能在其中正常生活,完成妊娠过程,顺利分娩,抚幼,幼体也能正常发育和成长。此外,故道还存     

渤海江豚种群现状初探

渤海是黄渤海江豚种群的重要分布区,但是有关该区域江豚的种群数量的资料极其匮乏。2015年至2016年,采用截线抽样法在渤海水域开展了6个航次的目视调查,收集了渤海江豚的分布数据,首次评估了区域内的江豚种群数量,为制定有效的渤海江豚种群生态保护策略提供了基础资料。调查结果显示,在2015年8月、2016年5~7月、9月和11月进行的6个调查航次共计8972 km的有效航程中,发现江豚87次、136头次。江豚在渤海各海湾和中部水域都可观察到;14:00-18:00时段、离岸15~35km范围内较易观察到江豚;黄河口外、秦皇岛东北侧老龙口和莱州外沿岸河口水域,江豚出现频次较多。各季节中,以5月在黄河口外20~40km范围内发现江豚的频次和数量最多。采用Distance 7.0软件估算渤海江豚种群数量,其中探测概率分别取值0.399、0.85和1时,渤海江豚种群数量估算值分别为7883头（CV=0.21）、3701头（CV=0.21）和3124头（CV=0.21）,对应的种群密度分别为1.441头/km2、0.066头/km²和0.056头/km²,均低于已报道的南黄海和日本海江豚种群相应值。受海上天气、调查条件和经验所限,本文估值可能存在偏差,但限于渤海江豚历史资料缺乏的现状,本文的结果对于认识渤海江豚的种群状况仍具有重要参考意义。     

Post-mortem investigations on stranded dolphins and porpoises from Hong Kong waters

Stranded cetaceans reported from the territorial waters of Hong Kong during the period May 1993 to March 1998 were examined to establish factors that may have contributed to their death. During the current study, 28 Indo-Pacific hump-backed dolphins (Sousa chinensis), 32 finless porpoises (Neophocaena phocaenoides), and four bottlenose dolphins (Tursiops truncatus) were necropsied. Bacteria (15 species) were isolated from nine animals. Of these bacteria, 47% were of possible fecal origin reflecting the high level of sewage contamination in Hong Kong's waters. One finless porpoise displayed wounds caused by a shark attack, and two female finless porpoises presented prolapsed uteri. At least 10 finless porpoises showed evidence of moderate to heavy lungworm infections (Halocercus pingi), and this appears to have been a factor contributing to death in at least six animals. Evidence suggesting blunt traumatic injury (probably caused by boat collisions) was found in six cetaceans (three finless porpoises and three hump-backed dolphins). Signs of fishery-related mortality were detected in at least nine animals (six hump-backed dolphins, two finless porpoises, and one bottlenose dolphin). Of these two human-caused mortality types, pre-existing disease or bacterial infection were detected in 29% of cases. Results indicate that human factors may have played a significant role in the death of at least 15 animals (32% of hump-backed dolphins, 15% of finless porpoises, and 25% of bottlenose dolphins).     

北部湾印太江豚密度估算及适宜栖息地预测

目前我国印太江豚 (Neophocaena phocaenoides) 的基础信息十分有限,2018—2020年对北部湾离岸较近的广西壮族自治区合浦儒艮国家级自然保护区及离岸较远的涠洲岛水域进行了截线抽样法船只考察。应用DISTANCE估算出儒艮国家级自然保护区印太江豚密度约为每平方千米0.273 (0.133 ~ 0.561) 头,数量约88头 (95%CI：43 ~ 181);涠洲岛水域印太江豚密度约为每平方千米0.100 (0.048 ~ 0.210) 头,数量约137头 (95%CI：65 ~ 286)。利用最大熵模型 (MaxEnt) 分析北部湾水域印太江豚的适宜栖息地分布及面积。MaxEnt模型训练集和测试集AUC值分别为0.980和0.927,模型拟合度较好,结果表明离岸距离和海洋深度是影响印太江豚栖息地分布的主要因子,印太江豚的适宜栖息地在北部湾东北部近岸区和海南西侧,适宜度超过0.5的栖息地面积达14 630.62 km²。2018—2020年印太江豚适宜栖息地面积较2013年大幅度下降,0.5适宜度的栖息地面积减少了40%。参考印太江豚2个群体的平均密度和北部湾适宜栖息地面积,粗略估计北部湾水域印太江豚约1 463 ~ 3 994头。由此可见,北部湾水域内印太江豚密度比较高,应引起足够的重视。建议首先进行区域性海洋环境的整体保护,重点关注鱼类资源的恢复及对人类活动尤其渔船的管控,并尽快建立新的自然保护区。     

Mitochondrial control region variability of baiji and the Yangtze finless porpoises,two sympatric small cetaceans in the Yangtze river

BaijiLipotes vexillifer (Miller, 1918) and the Yangtze finless porpoiseNeophocaena phocaenoides asiaeorientalis (Pilleri and Gihr, 1972) are two sympatric small cetaceans inhabiting the middle and lower reaches of the Yangtze River. In this study, a fragment (420–428 bp) of the mitochondrial control region was sequenced to provide the first comparative survey of genetic variability and population structure in these two endangered species, with samples of finless porpoises from the Yellow/Bohai Sea, East China Sea, and South China Sea also included. Low values of haplotype diversity and nucleotide diversity were found for both species, especially for the baiji and the Yangtze River and South China Sea populations of finless porpoises. The analysis of molecular variance (AMOVA) supported a high level of overall genetic structure among three porpoise populations in Chinese waters, with greatest differences found between either the Yangtze River population or the Yellow Sea population and the South China Sea population. The differentiation between the Yangtze and Yellow Sea populations was not significant, and the males have higher genetic differentiation than the females, suggesting a significant female-biased dispersal between these two populations. This study showed that the Yangtze finless porpoise, unlike the sympatric baiji, was not a genetically isolated population. The Yangtze and Yellow Sea porpoises should be included in the same management unit, but further studies using more samples and especially based on more molecular markers are urgently needed to confirm this.     

长江江豚脐带永生化成纤维细胞系建立及细胞生长特性研究

取长江江豚(Neophocaena phocaenoids asiaeorientalis)产后胎盘脐静脉, 经组织块培养, 差异贴壁法纯化, 构建长江江豚的原代细胞系; 经外源癌基因SV40 T antigens（猿猴病毒T抗原）转染构建稳定脐带细胞系, 并对长江江豚的永生化后的成纤维细胞的细胞形态、转染效率、生长曲线和活率等进行探究。结果表明: (1)原代脐静脉细胞大约14d从组织边缘分离, 20d形成单层, 细胞呈现典型的成纤维细胞状, 梭形、不规则星行或多边形。原代细胞传代14代后出现老化和凋亡。(2)通过SV40 T antigens转染构建的细胞系可传代40—50次, 证实转染SV40 T antigens后可增加细胞的增殖能力。(3)不同世代永生化的成纤维细胞复苏前后细胞活性并无明显差异, 复苏细胞活性在90%以上, 表明永生化后的细胞可以稳定保存。利用CCK-8法测得细胞的生长曲线呈现“S”型。(4)对永生化的细胞系进行后续基因表达尝试, 通过转染外源基因绿色和红色荧光蛋白, 转染外来质粒效率约为15%, 说明外源基因可以表达, 并能被成功检测。研究构建长江江豚的脐静脉来源的永生化后细胞系, 为江豚的其他组织细胞系建立以及江豚细胞库的建立提供基础, 成功对永生化后的细胞系进行外源基因的转染, 为以后深入长江江豚相关基因和分子机制研究打下基础。     

Yangtze finless porpoises along the main channel of Poyang Lake,China: Implications for conservation

Poyang Lake is the largest freshwater lake in China and flows into the Yangtze River. It is a traditional habitat for the endangered Yangtze finless porpoise, which has not been well investigated. To reveal the distribution of the porpoise in Poyang Lake, 12 passive acoustic surveys were conducted along 123 km of the main channel of the lake during different seasons (spring transition season, wet season, autumn transition season, and dry season) from 2008 to 2012. We counted the number of phonating porpoises encountered and calculated the detection rate (encountered individuals detected per kilometer). The median porpoise detection rates ranged from 0 to 0.65 individuals per kilometer during the different surveys. The highest median detection rate of 0.50 was detected in the autumn transition season. The seasonal shrinking of the lake during the dry season may cause a concentration of porpoises in the narrow channels and a high incidence of collisions with cargo ships and fishing boats. Conservation actions should be focused on the main channel of the lake during the dry and transition seasons. In addition, the expansion of the existing reserve to include areas with high porpoise detection rates is necessary.     

Helicobacter anseris sp. nov. and Helicobacter brantae sp. nov., isolated from feces of resident Canada geese in the greater Boston area

Numbers of nonmigratory Canada geese have increased substantially in the past decade, and they have become a nuisance in some urban areas. Because of their close contact with humans in parks and areas adjacent to surface waterways, contact with their feces poses a zoonotic risk. A total of 97 geese from 10 separate geographic locales in the greater Boston area had their feces sampled for detection of Helicobacter spp. Identification of Helicobacter spp. based on 16S rRNA genus-specific helicobacter primers was noted in 39 of 97 (40.2%) DNA fecal extracts. Twenty-seven (27.8%) of these geese had helicobacters isolated from their feces. A urease-positive novel species, Helicobacter anseris, based on phenotypic, biochemical, and 16S rRNA analyses, was isolated from 20 geese from seven different flocks. A second, novel, urease-negative Helicobacter sp., H. brantae, was identified in seven geese. Four geese had both novel Helicobacter spp. cultured from their feces. Whether these two novel helicobacters pose a zoonotic risk, similar to other enteric helicobacters (e.g., H. canadensis, previously isolated from diarrheic and bacteremic humans and from geese in Europe), will require further studies.     

长江江豚细菌性疾病的诊治

人工饲养或易地保护长江江豚(Neophocaena asiaeorientalis asiaeorientalis), 其水体中常含有病原菌且易引起江豚发病, 探讨长江江豚的细菌性疾病诊疗方法十分必要。文章利用细菌的分离培养与鉴定, 结合血液检测结果, 对安庆西江围网内一头患病江豚进行了病原确诊, 结果发现患病江豚的致病原为金黄色葡萄球菌(Staphylococcus aureus)和魔氏摩根菌(Morganella morganii)。依据致病菌的药敏试验结果, 综合患病江豚的整体状况制定了治疗方案并对其进行系统治疗, 结果预后良好。研究的成功开展为江豚、海豚等鲸类动物细菌性疾病的诊断与防治提供了良好借鉴。     

Identification of enterohepatic Helicobacter species by restriction fragment-length polymorphism analysis of the 16S rRNA gene

Background. Restriction fragment-length polymorphism (RFLP) analysis of a 1,200-bp polymerase chain reaction–amplified DNA fragment of gene coding for 16S rRNA was used to generate restriction profiles of 11 enterohepatic Helicobacter spp. isolated from various animals and humans.
Methods. The amplicon from each Helicobacter sp. was digested with four restriction endonucleases: Alu I, Hinf I, Hha I, and Dde I. Alu I digestion produced five patterns that were useful for initial differentiation.
Results. Most Helicobacter spp. isolated from rodents had the same RFLP profiles by Alu I digestion (except H. rodentium and H. cholecystus ), but they had different RFLP profiles by Hha I digestion. Only H. bilis and " H. rappini" mouse isolates could not be readily distinguished by the polymerase chain reaction-RFLP method. However, these two species can be distinguished using H. bilis specific primers. Some of the Helicobacter spp. have an intervening sequence in their 16S rRNA gene, which changes the RFLP patterns; in these cases, sequencing is the preferred method to make an appropriate diagnosis.
Conclusions. The RFLP method used in this study was straightforward and rapid and should prove useful as an adjunct for identification and classification of multiple enterohepatic Helicobacter spp.