海水鱼源美人鱼发光杆菌美人鱼亚种对小鼠的致病性研究

美人鱼发光杆菌美人鱼亚种(Photobacterium damselae subsp.damselae)能感染多种海洋动物,并且对人也具有致病性。为验证我国近海环境中的美人鱼发光杆菌美人鱼亚种对哺乳动物的致病能力,以从患病许氏平鲉(Sebastes schlegeli)分离的1株美人鱼发光杆菌美人鱼亚种菌株为对象,用昆明系小鼠为实验动物,研究该菌对小鼠的致病力。结果发现,灌胃和注射该菌均能造成实验小鼠发病和死亡。感染小鼠出现了精神萎靡、被毛杂乱、呼吸急促和对外界刺激迟钝等临床症状,个别小鼠体表还出现了溃疡性伤口。该菌对昆明系小鼠的半数致死剂量(LD_(50))为5.52×10~5CFU/g。组织病理学研究发现,死亡小鼠的多个器官均出现了病理变化,如心脏心肌组织肌纤维束相互解离、断裂;肝脏细胞出现了炎性病变,细胞核轮廓变得模糊;肺实质组织崩解,组织结构变得稀松、零落;肾脏和脾脏都出现局灶性的溃疡;胃和肠道的黏膜层和黏膜下层发生了大面积的溃疡性坏死;心脏、肝脏和肾脏均有大量的血细胞浸润。病理学的研究结果证实该菌对小鼠具有较强的致病力。     

大菱鲆源美人鱼发光杆菌杀鱼亚种的分离鉴定

从秦皇岛昌黎县某海水养殖场患病大菱鲆体内分离到1株细菌,经过生理生化特性和16SrDNA序列分析,鉴定为美人鱼发光杆菌杀鱼亚种,并命名Phdp QHD-1,在国内尚属首次。该菌在绵羊血平板上呈α溶血,利用PCR方法检测到Phdp QHD-1携带1种重要的溶血素hlyAch基因,与传统对杀鱼亚种无溶血素基因的特点不同。通过耐药性分析,确定了Phdp QHD-1对左氧氟沙星、头孢曲松钠、链霉素和氟苯尼考高度敏感,选取左氧氟沙星和头孢曲松钠进行联合用药,取得了良好的治疗效果。最后,攻毒试验显示大菱鲆死亡率高达85%,并从心血中分离到该菌,证明其为此次发病的病原菌。     

大菱鲆源美人鱼发光杆菌胞外产物生物学特性及蛋白成分分析

为了研究美人鱼发光杆菌胞外产物对大菱鲆的致病作用以及美人鱼发光杆菌的致病机理,本试验以大菱鲆源美人鱼发光杆菌为研究对象,提取其胞外产物,采用打孔法测定其胞外产物的酶活性,并对溶血活性进行溶血谱分析,同时分析其致病性。应用LC-MSMS方法对其胞外产物蛋白成分进行鉴定,利用Gene Ontology(GO)对已鉴定蛋白进行生物学过程、分子功能和细胞组分的分类分析。结果表明:美人鱼发光杆菌胞外产物具有淀粉酶活性、脂肪酶活性、蛋白酶活性、卵磷脂酶活性和溶血活性,不具有明胶酶活性和脂肪酶活性;其可溶解多种动物红细胞,尤以对鱼类红细胞溶血性更强,但对鸡、鸭红细胞无溶血活性。通过对菌株胞外产物蛋白成分分析显示,有45种蛋白共参与34种生物学过程,主要涉及碳水化合物代谢过程、运输等;共有41种分子功能,主要涉及脱氢酶、磷酸酶、氧化还原酶和金属离子结合等;包括15种细胞组分,主要有细胞质和细胞外膜等。     

美人鱼发光杆菌MCCC 1K03226株的全基因组测序及生物信息学分析

为了探究美人鱼发光杆菌MCCC 1K03226株的功能。本研究利用Megalign构建美人鱼发光杆菌16S r DNA遗传进化树,利用电镜观察菌株,并进行生化鉴定;采用PacBio RSII高通量测序平台对美人鱼发光杆菌进行全基因组测序;利用SMRT portal软件对测序所得基因进行组装拼接;利用NR、KEGG、COG等数据库和SignalP、EffectiveT3、antiSMASH等软件对MCCC 1K03226株进行基因功能注释;利用Circos软件绘制基因组环形图谱。通过遗传进化树、序列比对及生化鉴定结果显示,该菌为美人鱼发光杆菌美人鱼亚种;测序结果显示该菌基因组由2条染色体和1个质粒组成,共编码4 298个基因,GC含量为41.59%;基因功能分析显示,2 392个基因对应美人鱼发光杆菌,1 980个基因参与34类代谢通路;3 452个基因编码蛋白质;3 570个基因与分子功能相关;2 884个基因与细胞成分相关;6 526个基因与生物学过程相关;501个基因编码转运蛋白;5个基因与耐药性相关;97个基因编码的蛋白与致病性相关;317个基因编码分泌蛋白,其中192个蛋白为III型分泌系统(T3SS)效应蛋白。本研究对美人鱼发光杆菌分离株MCCC 1K03226进行全基因组测序和生物信息学分析,为该菌的致病机制研究和疾病防控奠定基础。     

死亡仔猪脏器中致病性大肠杆菌的分离鉴定及药敏试验

从送检的死亡猪组织病料中分离到1株病原菌,经病原菌的分离、生化试验、动物致死试验、药物敏感性试验和PCR扩增及测序,对该菌株进行了鉴定。结果表明:该病原菌为致病性大肠杆菌;药敏试验表明在检测的17种抗生素中该菌株仅对阿奇霉素和克林霉素药物敏感;对强力霉素、多粘菌素B药物中敏;对杆菌肽、氯霉素和多粘附素E等耐药。说明该猪死亡前感染了耐药性较强的致病性大肠杆菌。     

肉雏鹅源多杀性巴氏杆菌分离鉴定与致病性试验

为了鉴定引起肉雏鹅死亡的病原,无菌采集病死肉雏鹅心血、肺脏、肝脏等病料组织中分离到1株病原菌,通过细菌培养特性、形态学观察、生化试验、Kmt2基因序列测序等方法进行鉴定,分离菌株被鉴定为多杀性巴氏杆菌。采用PCR方法、人工感染致病性试验、K-B药敏纸片法分别对分离菌株进行血清型鉴定、致病性检测及耐药性分析。结果显示,多杀性巴氏杆菌血清型为A型,对14日龄肉雏鹅具有很强的致病性,半数致死量(LD_(50))为5.86×10~6 CFU/mL;分离菌株对对头孢曲松、头孢噻呋、恩诺沙星等6种药物高度敏感;对其他药物有不同的程度耐药性。     

西藏日喀则地区绵羊源醋酸钙不动杆菌的分离鉴定及药敏试验

为了解致西藏日喀则市某村死亡绵羊感染的主要病原,试验对采集的绵羊肝脏、肺脏、脾脏等病料组织进行病原菌的分离培养,对分离纯化后的细菌进行革兰氏染色;根据染色结果选择细菌鉴定卡和药敏鉴定卡进行鉴定,并对分离菌进行致病性分析;通过16S rRNA引物对分离菌进行PCR鉴定和遗传进化分析。结果表明:分离菌为革兰氏阴性;对分离的1株优势菌进行全自动微生物鉴定系统VITEK2 Compact System的GN卡鉴定,结果为鲍曼不动杆菌复合物;分离菌对氨苄西林/舒巴坦、亚胺培南、庆大霉素、复方新诺明等8种药物敏感,对氨苄西林、头孢呋辛酯、安曲南、呋喃妥因等7种药物不敏感,对头孢曲松中度敏感;动物致病性试验结果表明,本菌具有一定的毒力;PCR产物测序比对后鉴定该菌为醋酸钙不动杆菌;遗传进化分析结果显示,该菌与序列号为MG011594.1的醋酸钙不动杆菌相似性达100%,确定为醋酸钙不动杆菌。说明西藏日喀则市某村死亡绵羊感染了醋酸钙不动杆菌,应加强防控。     

西藏牛源多杀性巴氏杆菌分离鉴定及耐药性分析研究

多杀性巴氏杆菌是一种能感染多种动物甚至人的重要的病原菌。研究对西藏某地临床症状疑似出败病死亡的牛进行了巴氏杆菌的分离鉴定、致病性、荚膜分型及耐药性分析,掌握西藏牛源多杀性巴氏杆菌的生物学特性为临床合理用药提供科学依据。采集死亡牛肺脏、肝脏、脾脏、肾脏等脏器及淋巴结,分离细菌并进行培养,研究其形态学特征及其生理生化分析。随后对分离菌株进行荚膜分型、动物实验和药敏实验。结果表明,从肺脏组织中成功分离鉴定出1株牛源巴氏杆菌且分离菌株为A型巴氏杆菌,且动物致病性试验证明该细菌具有致病作用且病例变化较明显,说明分离菌株为致病菌;该菌株对苯唑西林、万古霉素和克林霉素不敏感;庆大霉素、多粘菌素B和氯霉素中敏;对哌拉西林、头孢呋辛、氧氟沙星等其他大多数抗菌药物均敏感。     

雏鸡源致病性奇异变形杆菌的鉴定与药敏试验

为了鉴定致雏鸡死亡病原菌,从无菌采集病死雏鸡肝脏组织中分离到1株病原菌。采用常规鉴定方法、16S rRNA PCR和致病性试验等方法,分离菌株被鉴定为致病性奇异变形杆菌。采用K-B药敏纸片法对分离菌株进行药敏试验,结果显示,分离菌株对头孢克肟、头孢曲松、环丙沙星、丁胺卡那霉素、林可霉素5种药物高度敏感;对其他药物有不同程度耐药性。     

赛鸽源鸡杆菌复合群-3的分离鉴定与药敏试验

为分离鉴定引起辽宁省某赛鸽养殖场赛鸽患病及死亡的病原菌及其特性,本实验从采集的病死赛鸽肝脏、肺脏、脾脏、心脏等病料样品中分离到1株优势菌株,并对其进行常规形态学观察、生化鉴定、16S r DNA基因序列测定、动物致病性试验及药敏试验。结果显示,该菌属于鸡杆菌复合群-3,命名为HLD-1;动物致病性试验结果显示HLD-1对种鸽有一定的致病性。药敏试验结果显示,HLD-1对氨苄西林、头孢曲松敏感。本研究首次从辽宁省赛鸽分离得到鸡杆菌复合群-3,为进一步研究赛鸽源鸡杆菌复合群-3的致病机制奠定基础。     

Infection with Photobacterium damselae subspecies damselae and Vibrio harveyi in snapper, Pagrus auratus with bloat

OBJECTIVE: To diagnose the cause of chronic, low mortality associated with bloat in tanks of snapper at an aquaculture facility. DESIGN: A clinical, pathological and microbiological investigation into the cause of a low number of ongoing mortalities associated with bloat in snapper at an aquaculture facility is outlined. Necropsy, histology, microbiology and a comparison of haematology and water analysis from affected and unaffected fish and holding tanks, respectively were conducted. RESULTS: Affected moribund fish were found in lateral or dorsal recumbency floating on the water surface within 24 hours of death. Photobacterium damselae subspecies damselae was isolated from intestinal contents and Vibrio harveyi from the blood of affected fish and both were isolated from culture water. Both V harveyi and P damselae subspecies damselae isolates were sensitive to tetracycline, ciprofloxacin and sulphamethoxazole plus trimethoprim. Environmental parameters such as pH and dissolved oxygen were similar in tanks of affected and unaffected fish. Affected fish had gas distended swimbladders, anaemia, and the intestines were diffusely distended with a clear, pale yellowish fluid. Livers were mottled tan and green in a zonal pattern. Histologically the intestines of fish from tanks suffering mortality had a moderate granulocytic enteritis with oedema and infiltrations with eosinophilic granule cells that were also present as an infiltrate in the gills. There were elevated numbers of melanomacrophage centres and haemosiderin deposits in the spleen, kidney and liver of affected fish. CONCLUSION: Vibrio harveyi and Photobacterium damselae subspecies damselae infection should be recognised as potential pathogens of snapper held in water of less than optimal quality.     

Isolation and Identification of Photobacterium damselae subsp.damselae(PDD) from Tongue Sole

In May 2016,an epizootic occured among cultured tongue soles caused mass deaths in a fish farm in Qinhuangdao,China.In order to find out the etiological agent,a bacterial strain was isolated from ascites and other tissues of sick tongue sole aseptically collected.The isolate was identified as Photobacterium damselae subsp.damsela(PDD) by isolation culture,Gram staining,physiological identification,morohological observation,biochemical identification and 16S rDNA sequence analysis.The results showed that the isolate shared 99.6% homology with the reference strain in GenBank.The animal regression test displayed that the isolate had very strong pathogenicity to tongue sole.The LD_(50) was 3.1 × 10~4 CFU/mL,and it showed pathogenicity to mammals.The antimicrobial susceptibility test showed the isolate was highly sensitive to nrofloxacin,Norfloxacin,Ciprofloxacin,Mequindox;moderately sensitive to Cefradine,Doxycycline;and insensitive to Gentamicin,Ceftriaxone,Tilmicosin,etc..     

In vivo morphological and antigenic characteristics of Photobacterium damselae subsp. piscicida

The present study was conducted to examine the morphology and antigenicity of Photobacterium damselae subsp. piscicida by culturing the bacterium in vivo in the peritoneal cavity of sea bass (Dicentrarchus labrax) within dialysis bags with either a low molecular weight (LMW) cut-off of 25 kDa or a high molecular weight (HMW) cut-off of 300 kDa. Differences were observed in the growth rate between the bacteria cultured in vivo or in vitro. Bacteria cultured in vivo were smaller and produced a capsular layer, which was more prominent in bacteria cultured in the HMW bag. Antigenicity was examined by Western blot analysis using sera from sea bass injected with live Ph. d. subsp. piscicida. The sera recognised bands at 45 and 20 kDa in bacteria cultured in vivo in the LMW bag. Bacteria cultured in vivo in the HMW bag did not express the 45 kDa band when whole cell extracts were examined, although the antigen was present in their extracellular products. In addition, these bacteria had a band at 18 kDa rather than 20 kDa. Differences in glycoprotein were also evident between bacteria cultured in vitro and in vivo. Bacteria cultured in vitro in LMW and HMW bags displayed a single 26 kDa band. Bacteria cultured in the LMW bag in vivo displayed bands at 26 and 27 kDa, while bacteria cultured in vivo in the HMW bag possessed only the 27 kDa band. These bands may represent sialic acid. The significance of the changes observed in the bacterium''s structure and antigenicity when cultured in vivo is discussed.     

Invasion and Replication of Photobacterium damselae subsp. piscicida in Fish Cell Lines

Abstract

The objective of this study was to evaluate the ability of Photobacterium damselae subsp. piscicida to invade and replicate within different fish cell lines. Channel catfish ovary (CCO), fathead minnow (FHM), and epithelioma papillosum cyprini (EPC) cell lines were all susceptible to invasion and supported replication of P. damselae in an in vitro invasion assay in which extracellular growth was controlled with gentamicin. The number of bacteria recovered from EPC and CCO cells increased significantly after 6, 12, and 18 h, indicating that P. damselae replicated within those cells. There was also a significant increase in EPC cells at 3 h. Although the number of bacteria recovered from FHM cells increased slightly after 3 and 6 h, it declined at 12 and 18 h. The decline, however, was due to the early release of bacteria from FHM cells associated with a greater susceptibility to initial infection, which resulted in early cell lysis and subsequent exposure to gentamicin in the media. Using light and electron microscopy, we observed the invasion of bacteria as early as 30 min after infection. Intracellular bacteria were initially contained in small, close-fitting vacuoles that developed into large, clear, spacious vacuoles over time. There was no evidence of bacteria free in the cytoplasm. The intracellular location of P. damselae was confirmed by means of transmission electron microscopy with ruthenium red staining to discriminate between the extra- and intracellular spaces. This is the first report that provides evidence of intracellular replication of P. damselae in cell lines.     

Cloning and Nucleotide Sequence Analysis of the Ampicillin Resistance Gene on a Conjugative R Plasmid from the Fish Pathogen Photobacterium damselae subsp. piscicida

Abstract

Transferable resistance to various drugs was investigated in strains of Photobacterium damselae subsp. piscicida from Japan. Drug resistance was transferred via two plasmids of 100 and 50 kilobases (kb) or three plasmids of 100, 50, and 40 kb. Resistance to ampicillin was transferred on the 50-kb plasmid. An ampicillin resistance gene in the 50-kb plasmid pPDP8517 from strain PP8517 was cloned from a 1.8-kb Hinc II fragment of the plasmid. The nucleotide sequence of the coding and flanking region of the ampicillin resistance gene was determined to be 1,736 base pairs. The nucleotide sequence identified an open reading frame (ORF) encoding 282 amino acid residues with a calculated molecular mass of 31,292 daltons. The ampicillin resistance gene's ORF was found to be a β-lactamase bearing low levels of amino acid identities (<63%) relative to other β-lactamases. This ORF was very high in amino acid identities with class A β-lactamases compared with those of class B, C, and D β-lactamases. Four important structural features were conserved in all class A β-lactamases present in the deduced amino acid sequence of the ampicillin resistance gene. These results suggest that the P. damselae subsp. piscicida ampicillin resistance gene is a class A β-lactamase encoded on an R plasmid that is distinctly different from other known β-lactamases. Moreover, Southern blot analysis suggested that P. damselae strains with the ampicillin resistance gene are widely distributed in marine fish farms in the Kyushu area in Japan.     

白术、微米白术和白术多糖对断奶仔猪生长性能和肠道形态及微生态区系的影响

试验选用96头平均体重14.82 kg左右的杜×长×大断奶仔猪,随机分成4组,每组3栏,每栏8头(公母各半)。对照组饲喂基础日粮,试验1、2、3组分别添加1%80目白术、0.2%白术多糖和1%微米白术。试验期30 d。结果表明:在生长性能方面,与对照组相比,1%微米白术添加组可显著提高日增重(P0.05)、降低饲料增重比和腹泻率,而且效果优于1%80目白术组和0.2%白术多糖组,在肠道形态和肠道微生态区系方面,与对照组相比,日粮添加1%80目白术、0.2%白术多糖、1%微米白术均可不同程度的提高十二指肠和空肠的绒毛高度,加深十二指肠和空肠的隐窝深度,并且增加肠道微生态区系的多样性,其中以1%微米白术添加组的效果最佳。     

Prevalence and seasonal incidence of nematode parasites and fluke infections of sheep and goats in eastern Ethiopia

Sissay, M.M., Uggla, A. and Waller, P.J., XXXX. Prevalence and seasonal incidence of nematode parasites and fluke infections of sheep and goats in eastern Ethiopia. Tropical Animal Health and Production, XXXX. A 2-year abattoir survey was carried out to determine the prevalence, abundance and seasonal incidence of gastro-intestinal (GI) nematodes and trematodes (flukes) of sheep and goats in the semi-arid zone of eastern Ethiopia. During May 2003 to April 2005, viscera including liver, lungs and GI tracts were collected from 655 sheep and 632 goats slaughtered at 4 abattoirs located in the towns of Haramaya, Harar, Dire Dawa and Jijiga in eastern Ethiopia. All animals were raised in the farming areas located within the community boundaries for each town. Collected materials were transported within 24 h to the parasitology laboratory of Haramaya University for immediate processing. Thirteen species belonging to 9 genera of GI nematodes (Haemonchus contortus, Trichostrongylus axei, T. colubriformis, T. vitrinus, Nematodirus filicollis, N. spathiger, Oesophagostomum columbianum, O. venulosum, Strongyloides papillosus, Bunostomum trigonocephalum, Trichuris ovis, Cooperia curticei and Chabertia ovina), and 4 species belonging to 3 genera of trematodes (Fasciola hepatica, F. gigantica, Paramphistomum {Calicohoron} microbothrium and Dicrocoelium dendriticum) were recorded in both sheep and goats. All animals in this investigation were infected with multiple species to varying degrees. The mean burdens of adult nematodes were generally moderate in both sheep and goats and showed patterns of seasonal abundance that corresponded with the bi-modal annual rainfall pattern, with highest burdens around the middle of the rainy season. In both sheep and goats there were significant differences in the mean worm burdens and abundance of the different nematode species between the four geographic locations, with worm burdens in the Haramaya and Harar areas greater than those observed in the Dire Dawa and Jijiga locations. Similar seasonal variations were also observed in the prevalence of flukes. But there were no significant differences in the prevalence of each fluke species between the four locations. Overall, the results showed that Haemonchus, Trichostrongylus, Nematodirus, Oesophagostomum, Fasciola and Paramphistomum species were the most abundant helminth parasites of sheep and goats in eastern Ethiopia.     

Y‐chromosomal variation of local goat breeds of Turkey close to the domestication centre

Genetic variations in chromosome Y are enabling researchers to identify paternal lineages, which are informative for introgressions and migrations. In this study, the male‐specific region markers, sex‐determining region‐Y (SRY), amelogenin (AMELY) and zinc finger (ZFY) were analysed in seven Turkish native goat breeds, Angora, Kilis, Hair, Honaml?, Norduz, Gürcü and Abaza. A SNP in the ZFY gene defined a new haplotype Y2C. All domestic haplogroups originate from Capra aegagrus, while the finding of Y1A, Y1B, Y2A and Y2C in 32, 4, 126 and 2 Turkish domestic goats, respectively, appears to indicate a predomestic origin of the major haplotypes. The occurrence of four haplotypes in the Hair goat and, in contrast, a frequency of 96% of Y1A in the Kilis breed illustrate that Y‐chromosomal variants have a more breed‐dependent distribution than mitochondrial or autosomal DNA. This probably reflects male founder effects, but a role in adaptation cannot be excluded.