6株鱼源嗜水气单胞菌的分子鉴定与毒力基因分析检测

为了分子鉴定6株鱼源嗜水气单胞菌,并从分子层面验证通过检测毒力基因以推测嗜水气单胞菌潜在致病性的可行性。实验采用PCR扩增16 S rDNA和gyrB基因并结合系统发育树的构建和分析进行菌种的分子鉴定,检测气溶素（ aerolysin, aer）、溶血素（ haemoly-sin, hly）、丝氨酸蛋白酶（ serine protease, ahp）、热稳定细胞肠毒素（ heat-stable cytotonic enterotoxin, ast）和热敏感细胞肠毒素（ heat-labile cytotonic enterotoxin, alt）5种毒力基因,且使用Mega 5．2对核苷酸和氨基酸序列进行分析。结果显示6株菌均为嗜水气单胞菌Aero-monas hydrophila,检测出5种毒力基因中的至少4种,其中均检测出溶血素和2种肠毒素,序列分析表明气溶素、溶血素和丝氨酸蛋白酶的氨基酸序列高度保守。本研究基于16 S rD-NA和gyrB基因可以准确地对嗜水气单胞菌进行分子鉴定,6株菌的毒力基因丰富预示着一定的致病性, aer、 hly和ahp基因相对保守,编码的毒力因子高度同源,在临床分子诊断中建议使用aer、 ahp和hly基因对嗜水气单胞菌的潜在致病性进行检测。     

流水式养殖动物中弧菌和嗜水气单胞菌的PCR法监测

为了解流水式养殖场养殖动物中弧菌和气单胞菌的感染状况,分别针对霍乱孤菌肠毒素A亚单位(ctxA)基因、0139和01群群特异基因、毒力协同调节菌毛A亚单位基因(tcpA)、中心调节蛋白基因(toxR)、副溶血弧菌不耐热溶血素基因(tlh)、创伤弧菌Vibrio vulnificus溶细胞素基因(vvhA)和嗜水气单胞菌的气溶素基因(aerA)设计引物,建立了聚合酶链式反应(PCR)检测技术,PCR产物经电泳,根据扩增条带的大小和数目,检测和区分霍乱孤菌、嗜水气单胞菌和副溶血孤菌.对南昌沿岸霍乱流行水域及附近养殖场的养殖动物进行了18个月的连续监测,对1 440份水产品的增菌液进行PCR检测,17份检出副溶血弧菌,25份检出霍乱弧菌,创伤弧菌和嗜水气单胞菌均未检出.扩增结果与传统培养检测法的结果一致.监测结果表明,南昌附近地区霍乱弧菌和副溶血弧菌检出率分别为1.74%和1.18%,总体感染状况处于较低水平,但个别养殖品种弧菌检出率较高.     

PCR扩增特异性16SrDNA和溶血素基因检测致病性嗜水气单胞菌

根据已发表的气单胞菌16S rDNA基因序列及气单胞菌气溶素(aerolysin)基因序列，设计了2对引物，建立了检测致病性嗜水气单胞菌的PCR方法。通过对12株气单胞菌的检测，发现16S rDNA引物具有高度的特异性，仅对嗜水气单胞菌扩增阳性。而Aero基因引物检测结果与采用生物学方法(鲜血平板法)检测的结果符合率为97．2％，且具有高度的敏感性，可检测最低1fg的模板。将16S rDNA与Aero基因结合PCR方法检测致病性嗜水气单胞菌与用致病性嗜水气单胞菌检测试剂盒的符合率为94．4％。该方法的建立为致病性嗜水气单胞菌的检测提供了一种简便、快速的途径，是一种比较实用的致病性嗜水气单胞菌的检测方法。     

鳗源嗜水气单胞菌β—溶血素基因的克隆及表达

应用PCR技术,从1株鳗源嗜水气单胞菌ML316中扩增得到β 溶血素基因AHL316HEM,将AHL316HEM克隆到pGEM TEasyVector,经分析验证后重组到pcDNA3.0中,构建了重组质粒PDLH。转化重组质粒PDLH的大肠杆菌(Escherichiacoli)DH5α能在血琼脂培养皿中形成明显的β 溶血斑,重组菌纯化的胞外产物溶血价为1.28×104HU·mg-1,同时重组菌的胞外产物能被原始菌株的高免血清特异性地识别。结果证实,克隆到鳗源嗜水气单胞菌β 溶血素基因,重组质粒PDLH能够表达具有天然生物学活性的β 溶血素,为构建嗜水气单胞菌核酸疫苗奠定了基础。     

致病性嗜水气单胞菌多重PCR检测方法的建立

致病性嗜水气单胞菌(Aeromonas hydrophila)是近年中国各地大规模流行的淡水养殖鱼类暴发性疾病的主要病原,本研究针对GenBank中登录的致病性嗜水气单胞菌的气溶素基因(hlyA)、溶血素基因(aerA)以及为气单胞菌属所特有的内参照基因16S rRNA保守区设计了3对特异性引物,通过进行多重PCR反应体系优化,多重PCR产物的测序鉴定与特异性和敏感性实验,试图建立一种检测致病性嗜水气单胞菌的多重PCR检测方法。对8株嗜水气单胞菌、16株相关菌株进行多重PCR检测,结果显示,非致病性分离株均未扩增出毒力基因hlyA和aerA,而致病性分离株则至少含有hlyA基因;对40份送检的水产动物样品进行多重PCR检测,结果与常规微生物学检测符合率为97.5%。多重PCR检测方法具有较高的敏感性与特异性,最低可检测模板量为10 ng的样品。该方法的建立对水产动物嗜水气单胞菌病的快速诊断和分子流行病学的调查有重要意义。     

白斑狗鱼(Esox lucius)致病性嗜水气单胞菌的分离与鉴定

近些年来,新疆许多养殖场发生白斑狗鱼暴发性疾病而死亡。为鉴定病因,本研究对乌鲁木齐周边地区的自然患病白斑狗鱼(Esox lucius)的肝脏、肾脏等组织进行分离,经形态观察、生理生化试验、16S r DNA序列分析进行细菌鉴定。并通过人工回归感染白斑狗鱼和鲫,来验证分离菌株的致病性。结果表明,分离出的9株优势菌为致病性嗜水气单胞菌,命名为PK001-PK009。经PCR特异性检测发现,所得的9个菌株分别含有丝氨酸蛋白酶基因(ahp A)、溶血素基因(hly A)和气溶素基因(aer A)中的0-3个,从而导致了菌株间致病性的差异。通过人工回归感染实验发现,白斑狗鱼患病症状与自然病例相似,并重新分离得到原感染菌,经细菌学鉴定,其性状与原菌株一致,为嗜水气单胞菌。     

嗜水气单胞菌J-1株丝氨酸蛋白酶基因克隆与序列分析

根据已发表的气单胞菌胞外蛋白酶基因核苷酸序列，设计和合成了一对引物，以嗜水气单胞菌AhJ—1的基因组DNA为模板，通过PCR技术，扩增到约900bp的丝氨酸蛋白酶基因片段，并克隆到质粒载体pGEM—T中进行测序和分析，结果表明扩增的丝氨酸蛋白酶基因片段与已发表的嗜水气单胞菌丝氨酸蛋白酶Ahe2的同源性有87％，扩增片段编码343个氨基酸，推测的分子量为35700，计算机软件分析表明编码的氨基酸有较高的抗原性，可作为核酸疫苗的侯选基因片段。     

白斑狗鱼(Esox lucius)致病性嗜水气单胞菌的分离与鉴定

近些年来,新疆许多养殖场发生白斑狗鱼暴发性疾病而死亡。为鉴定病因,本研究对乌鲁木齐周边地区的自然患病白斑狗鱼(Esox lucius)的肝脏、肾脏等组织进行分离,经形态观察、生理生化试验、16S r DNA序列分析进行细菌鉴定。并通过人工回归感染白斑狗鱼和鲫,来验证分离菌株的致病性。结果表明,分离出的9株优势菌为致病性嗜水气单胞菌,命名为PK001-PK009。经PCR特异性检测发现,所得的9个菌株分别含有丝氨酸蛋白酶基因(ahp A)、溶血素基因(hly A)和气溶素基因(aer A)中的0-3个,从而导致了菌株间致病性的差异。通过人工回归感染实验发现,白斑狗鱼患病症状与自然病例相似,并重新分离得到原感染菌,经细菌学鉴定,其性状与原菌株一致,为嗜水气单胞菌。     

鱼源气单胞菌的毒力基因检测、分型及致病力

为调查鱼源气单胞菌毒力基因与其致病力的相关性,以2009—2018年从不同患病鱼分离的173株气单胞菌为研究对象,通过检测毒力相关基因、测定溶血活性、腹腔注射感染异育银鲫等方法开展评价。通过管家基因gyrB分子鉴定结果显示,173株气单胞菌中维氏气单胞菌(119/173,68.9%)和嗜水气单胞菌(50/173,28.9%)是主要流行的菌株。10个毒力基因aer(162/173,93.64%)、act(131/173,75.72%)、ast(55/173,31.79%)、alt(58/173,33.53%)、lip(152/173,87.86%)、exu(154/173,89.02%)、fla(143/173,82.66%)、gcaT(148/173, 85.55%)、 eprCAI(41/173, 23.70%)和ahyB(51/173, 29.48%)普遍存在于173株气单胞菌中。依据检测到的毒力基因数量从多到少分布情况,这些菌株可分为7大类(Ⅰ～Ⅶ)53个毒力基因型。大部分嗜水气单胞菌检测到8～10个毒力基因,主要分布于Ⅰ、Ⅱ和Ⅲ类基因型;维氏气单胞菌的eprCAI、ahyB、 ast和alt等4个毒力基因检测率低,主要分布于Ⅳ、Ⅴ和Ⅵ类基因型。大部分气单胞菌(94.22%,163/173)具有溶血活性。代表性毒力基因型的38株维氏气单胞菌和20株嗜水气单胞菌腹腔注射异育银鲫攻毒结果显示,3.0×106 CFU/尾的剂量下,3株维氏气单胞菌使鲫死亡率达80%～100%,16株嗜水气单胞菌使鲫死亡率达90%～100%。研究表明,维氏气单胞菌是目前最主要流行的气单胞菌,但其检测到的毒力基因普遍少于嗜水气单胞菌,且对异育银鲫的致病力普遍弱于嗜水气单胞菌。本研究能够为气单胞菌败血症的流行病学调查和疫苗研究提供理论依据。     

循环水养殖系统中高毒力嗜水气单胞菌的鉴定与药敏试验

为探究中华鳖（Pelodiscus sinensis）循环水养殖过程中出现体表穿孔和溃疡的病因，对患病中华鳖的肝、脾、肺和肾等部位取样并进行病原菌分离及革兰氏染色，利用16S rRNA测序和生化鉴定相结合进行细菌鉴定，通过毒力基因PCR检测和人工回感试验评估细菌的致病性，以纸片扩散法评估几种抗生素的抑菌效果，利用琼脂打孔法评估中药提取物的抑菌效果。结果显示，从病鳖的脏器中分离到一株优势菌AH0421，该菌为革兰氏阴性菌，菌体呈短杆状；菌株AH0421 与嗜水气单胞菌（Aeromonas hydrophila）模式菌株ATCC 7966 的16S rRNA 序列相似性高达99.66%。菌株AH0421 能利用葡萄糖、蔗糖和麦芽糖，具有O/129 耐受性，其携带act、aerA、aha、ahh、ahp、ahpA、alt、ast、hlyA、lip、ompA毒力基因。回感试验出现与自然发病相似病症，菌株AH0421半致死浓度(LC50)为1.7×106CFU/mL。中药五味子提取物、诺氟沙星、多西环素、恩诺沙星、新霉素以及头孢克肟、头孢曲松等头孢类抗生素对该菌有较强的抑制作用。研究表明，五味子和多数头孢类抗生素可作为治疗菌株AH0421感染的参考药物。     

Comparative genomic analysis of five high drug‐resistance Aeromonas hydrophila strains induced by doxycycline in laboratory and nine reference strains in Genbank

Aeromonas hydrophila is an opportunistic pathogen and the leading cause of fatal haemorrhagic septicaemia in fish and shellfish. Doxycycline, one of the second generation tetracyclines, has been used in fish farming to fight against infectious diseases caused by A. hydrophila due to its broad‐spectrum antimicrobial activity and lower cost. However, progressive increase in resistance of Aeromonas strains to doxycycline aroused serious concern. In this report, drug‐resistant A. hydrophilaAH10 strains were induced and selected by using a consecutive batch culture system in Mueller‐Hinton Broth (MHB) supplemented with varying concentrations of doxycycline. Five isolates (AH101‐105) were obtained from the bacterial culture induced by 25 μg/ml doxycycline for drug‐resistance analysis. Minimal inhibitory concentrations (MIC) values of all five isolates were 50 times higher than that of the parental strain AH10. All of them also displayed high‐level resistance to sulphonamides and amides. We sequenced five isolates and performed comparative genomic analysis of these draft genomes with nine A. hydrophila complete genomes from GenBank. Results showed that the pan‐genome of 14 strains contains 4,730 genes, 3,056 genes of which present in all strains. The drug‐resistance genes also showed significant difference in these genomes, which indicated dangers of indiscriminate use of antibiotics in aquaculture and the necessity of understanding the variation of antibiotic resistance of A. hydrophila. Pan‐genome analysis further revealed that no specific SNP (single nucleotide polymorphism) or InDel (insertion and deletion variation) was identified in any functional gene locus among the genomes of AH10 mutated strains, in contrast, significant CNVs (copy number variations) and SV (structure variations) for gene groups were identified in all the mutant genomes.     

Immunocytochemical identification of adenohypophyseal cells in the pirarucu (<Emphasis Type="Italic">Arapaima gigas</Emphasis>), an Amazonian basal teleost

The adenohypophysis (AH) of juvenile pirarucu (Arapaima gigas), a representative species of the Osteoglossomorpha (bonytongue fishes, one of the oldest living groups of the teleosts), was studied using histochemical and immunocytochemical methods. The AH is comprised of the pars distalis (PD), without a clear distinction between rostral pars distalis (RPD) and proximal pars distalis (PPD), and the pars intermedia (PI). The neurohypophysis (NH) is positioned on top of the PD and penetrates and branches into the PI. In the most rostral dorsal portion of the PD, adrenocorticotropic cells and fusiform gonadotropic cells were found. In the central PD, scarce prolactin-producing cells and growth-hormone-producing cells were located mainly in the dorsal part, whereas round gonadotropic cells were abundant in the ventral portion of this region. Human thyrotropin immunoreactive cells were not found in the entire AH. In the PI, melanotropic, some adrenocorticotropic, and somatolactin-producing cells were located intermingled surrounding the neurohypophyseal branches. Our results showed that the A. gigas pituitary has some basal characteristics between the ancient Actinopterygii and the more derived teleosts.     

高度耐药嗜水气单胞菌的定向诱导及其交叉耐药性分析

为了分析比较不同抗生素对耐药嗜水气单胞菌(Aeromonas hydrophila)的诱导效率,从五大类抗生素中各挑选出一种代表性的抗生素,在针对嗜水气单胞菌菌株AH10的防耐药突变浓度(mutant prevention concentration,MPC)的琼脂平板上,分别筛选出五株耐药菌株,并对筛选的耐药菌株进行交叉耐药性分析,制定出这些耐药菌株对常用抗生素的最小抑菌浓度(minimal inhibitory concentrations,MIC)图谱。结果显示:四环素、氟苯尼考、磺胺嘧啶、硫酸新霉素、诺氟沙星对AH10的MIC分别为:1.0、0.5、8.0、8.0、0.25μg/m L;MPC分别为4.0、3.0、128、88、2.0μg/m L,分离的耐药菌株对所使用抗生素的MIC均提高100倍以上。4℃划线平板冰箱保存条件下,一个月内个别耐药菌株耐药性下降。在药物筛选压力下,菌株耐药性随着传代次数增多而表现出递增的趋势。同属一类抗生素耐药相关性较高;不同大类抗生素压力下筛选出来的耐药菌株表现出不同的交叉耐药性。     

Effects of delayed first feeding on the survival and growth of tiger grouper,Epinephelus fuscoguttatus (Forsskål, 1775), larvae

The effects of delayed first feeding on the survival and growth of tiger grouper, Epinephelus fuscoguttatus (Forsskål 1775), larvae were examined under controlled conditions. The total length, yolk sac volume, oil globule volume, yolk sac absorption time and nutritional transition period (NTP) of the larvae fed at different first times (0, 6, 12, 18 and 24 h after the mouth opening stage; h AMO) were compared. Larval first feed intake was observed at 54 h after hatching (h AH) at 27.5 ± 0.5 °C. The yolk sac was consumed more rapidly with an increase in delayed first feeding and was significantly different among treatments (P<0.05). Larvae first fed at 0 h AMO had the longest yolk sac absorption (72 h AH) and NTP (20 h) times and had the highest survival and growth rates at the end of the experimental period (360 h AH), being significantly higher (P<0.05) than the other treatments. First mortality was observed at 69 h AH, approximately 2 h after point of no return (PNR) occurred. This study suggests that first feeding of tiger grouper larvae should commence at 0 h AMO for enhancement of larval survival and growth.     

Simple and direct detection of Aeromonas hydrophila infection in the goldfish, Carassius auratus (L.), by dot blotting using specific monoclonal antibodies

A combination of eight isolates of Aeromonas hydrophila was used to produce monoclonal antibodies (MAbs). Ten different groups of MAbs specific to Aeromonas were selected. The first five groups of MAbs demonstrated high specificity and bound to only one or two isolates of A. hydrophila. The sixth and the seventh groups of MAbs were A. hydrophila specific. They recognized seven of eight A. hydrophila isolates (AH1, 2, 3, 4, 5, 6, 8); however, the MAb in the seventh group also showed cross‐reactivity to one isolate of Aeromonas caviae (AC3). The eighth MAb group recognized two isolates of A. hydrophila (AH2 and AH5) and demonstrated cross‐reactivity to one isolate of Aeromonas sobria (AS1) and one isolate of A. caviae (AC3). The tenth group of MAbs bound to all isolates of Aeromonas spp. tested (AH1‐8, AS1‐6, AC1‐5, Aeromonas veronii and Aeromonas jandaei) without cross‐reactivity to any of the other bacteria tested. MAbs in the ninth group showed similar specificity to those in the tenth group but did not recognize two isolates of A. sobria (AS4 and AS6) or A. jandaei. All the MAbs could be used to identify Aeromonas by dot blotting with a sensitivity ranging from 10⁵ to 10⁷ CFU mL^?1. However, the sensitivity of detection was increased to 10²–10³ CFU mL^?1 after inoculation of the sample in tryptic soy broth for 3–6 h before performing the dot blotting. The dot blot method can be used for the direct detection of A. hydrophila infection in symptomatic and asymptomatic goldfish. This study demonstrated a convenient immunological tool that can be used for the direct detection of A. hydrophila and Aeromonas infections in a complex sample without the requirement for separation of the bacteria or isolation and biochemical tests.     

我国泥鳅微卫星遗传多样性初步分析

用6个微卫星标记分析了安徽(AH)、甘肃(GS)、广东(GD)、广西(GX)、黑龙江(HLJ)、江苏(JS)和重庆(CQ)7个泥鳅(Misgurnus anguillicaudatus)群体的遗传多样性。这6个微卫星摘自泥鳅微卫星连锁图谱,不存在连锁不平衡,在HLJ群体中处于哈代-温伯格平衡,适用遗传多样性分析。六个微卫星共检测到69个等位基因,每位点7到16个不等,有效等位基因数(Ne)3.0-5.5个,观察杂合度在0.202和0.408之间,而期望杂合度在0.673和0.820之间,揭示的多样性信息含量(PIC)在0.643和0.796之间。这些数据显示7个泥鳅群体遗传多样性丰富。群体间遗传分化系数(Fst)达到0.499,49.9%的变异可归于群体间差异,而Fis在0.167和0.421之间,表明泥鳅群体存在较明显的近交或斑块化分布。聚类分析显示AH、GD、HLJ和JS聚成一个分支,而CQ、GS 和GX聚为另一个分支。这表明我国泥鳅呈现东西向梯度分布并在南北向可能存在扰动。这些发现将有助于我国泥鳅资源的有效管理和合理利用。     

Genetic differentiation and historical demography of wood stork populations in Brazilian wetlands: Implications for the conservation of the species and associated ecosystems

1. Wetlands are increasingly threatened by human activities worldwide. Genetic monitoring of associated wildlife provides valuable data to support their conservation. Waterbirds such as the wood stork (Mycteria americana) are good bioindicators of wetland disturbance and destruction.
2. This study investigated past and contemporary levels of genetic diversity, differentiation and demographic processes in 236 wood storks from two major wetlands in Brazil in which breeding colonies are concentrated, using nine microsatellite loci and a 237‐bp untranslated fragment of the mitochondrial Control Region.
3. Amapá populations (northern region) showed slightly higher levels of genetic diversity than Pantanal populations (central western region) and both populations had a low number of effective breeders.
4. Results from assignment tests, F‐statistics, AMOVA, spatial and non‐spatial Bayesian clustering analyses support the hypothesis of gene flow among colonies within regions, but significant differentiation between regions.
5. The better supported Bayesian coalescent models based on both markers indicated that the northern population exchanged migrants with unsampled populations, and that the central western population was founded by individuals from the north. Mitochondrial estimates revealed that the timing of population divergence broadly overlapped the Last Glacial Maximum (LGM), and that the central western population expanded more recently than the northern population.
6. The results support the hypothesis that the coastal wetlands in northern Brazil remained stable enough to shelter large wood stork populations during the LGM and storks colonized freshwater wetlands in the central western region following deglacial warming.
7. Conservation policies and protective measures should consider Amapá and Pantanal wood stork populations as genetically differentiated units and priority should be given to Amapá populations that represent the source gene pool. Continuous genetic monitoring of wood storks would help detect genetic signs of changes in demographic trends that may reflect alterations or degradation in wetlands.

     

The effects of wood on stream habitat and native fish assemblages in New Zealand

Historic deforestation has deprived many river systems of their natural wood loadings. To study the effects of the loss of wood from waterways, a field trial was conducted in three small forested streams in New Zealand. The objectives were to (i) examine differences in fish assemblages among wooded pools (where wood provided cover), open pools and riffles and (ii) measure the effects of wood removal on channel morphology and fish assemblages. In the first part of the study, no significant differences were found in total fish density among the three habitats. However, total fish biomass was significantly higher in wooded pools (64% of total fish biomass) compared with open pools and riffles. Mean density and biomass of banded kokopu (Galaxias fasciatus) and mean biomass of longfin eel (Anguilla dieffenbachii) were highest in wooded pools, whereas the density and biomass of bluegill bully (Gobiomorphus hubbsi) and torrentfish (Cheimarrichthys fosteri) were highest in riffles. In the second part of the study, wood was removed from a 200‐m section (treatment) in each stream, significantly reducing pool area and increasing the proportion of channel area and length in riffles. At the habitat scale, banded kokopu and large longfin eel were the two species mostly affected by wood removal. At the reach scale, banded kokopu biomass was significantly lower in the treatment sections. Although wooded pools were a small portion of total habitat, they provided important habitat for two of New Zealand's larger native fish taxa.     

Freshwater catfish jundiá (Rhamdia quelen) larvae are prepared to digest inert feed at the exogenous feeding onset: physiological and histological assessments

This study assessed the morphological development of jundiá larvae’s digestive system and digestive proteolytic activity. Specific serine proteinases activities varied over time, with the highest peak at 12 h after hatching (AH), which corresponded to 296.38 ± 84.20 mU mg^?1 for trypsin and 315.45 ± 42.16 mU mg^?1 for chymotrypsin. Specific aspartic proteinases activities increased up to the start of weaning, oscillated during that phase, but showed a consistent increase after that, resulting in the highest specific activity at 252 h AH (7.88 ± 0.68 mU mg^?1). Gel assays showed different molecular forms, especially of serine proteinases. Histology showed the gastrointestinal tract development onset at 0 h AH and open mouth at 4 h AH. At 16 h AH, the following differentiation of the digestive tract was evident: oropharyngeal cavity, esophagus, liver, pancreas, stomach, and intestine. At 40 h AH, zymogen granules in the pancreas were observed, and at 48 h AH, mucus in the digestive tract and gastric glands in the stomach. Findings indicate that jundiá has a functional stomach before the end of vitelline reserves. Therefore, jundiá larvae are probably capable to digest inert feed at the exogenous feeding onset.