Parasitism by protozoan Ichthyophthirius multifiliis enhanced invasion of Aeromonas hydrophila in tissues of channel catfish

Protozoan Ichthyophthirius multifiliis Fouquet (Ich) and bacterium Aeromonas hydrophila are two common pathogens of cultured fish, which cause high fish mortality. Currently there is no information available for the effect of parasitism by Ich on survival of channel catfish and invasion of A. hydrophila in fish tissues following exposure to A. hydrophila. A trial was conducted in this study to: (1) determine whether A. hydrophila increased fish mortality in Ich-parasitized channel catfish; and (2) compare the bacterial quantity in different tissues between non-parasitized and Ich-parasitized catfish by real-time polymerase chain reaction (qPCR). The results demonstrated that the Ich-parasitized catfish showed significantly (P<0.05) higher mortality (80%) when exposed to A. hydrophila by immersion than non-parasitized fish (22%). Low mortality was observed in catfish exposed to Ich alone (35%) or A. hydrophila alone (22%). A. hydrophila in fish tissues were quantified by qPCR using a pair of gene-specific primers and reported as genome equivalents per mg of tissue (GEs/mg). Skin, gill, kidney, liver and spleen in Ich-parasitized fish showed significantly higher load of A. hydrophila (9400-188,300 GEs/mg) than non-parasitized fish (4700-42,100 GEs/mg) after exposure to A. hydrophila. This study provides evidence that parasite infections enhance bacterial invasion and cause high fish mortality.     

The prevalence of enteric pathogens in diarrhoeic thoroughbred foals in Britain and Ireland.

A survey of 77 normal and 326 diarrhoeic foals in Britain and Ireland from 1987 to 1989 revealed a significantly higher prevalence of Group A rotaviruses and Aeromonas hydrophila in diarrhoeic foals. The prevalence of cryptosporidia, potentially pathogenic Escherichia coli, Yersinia enterocolitica and Clostridium perfringens was similar in normal or diarrhoeic foals. Rotaviruses had a similar prevalence in all age groups of scouring foals up to three months of age, with an overall prevalence of 37 per cent among diarrhoeic foals. The number of cases of diarrhoea varied considerably from year to year, but in all three years of the survey rotavirus was a significant pathogen. A comparison of diagnostic tests for rotavirus in the faeces showed electron microscopy (EM) and polyacrylamide gel electrophoresis (PAGE) to have similar sensitivity. The Rotazyme ELISA test kit was found to have the same sensitivity as a combination of EM and PAGE. A. hydrophila had an overall prevalence of 9 per cent among diarrhoeic foals, although its prevalence was higher in some age groups. A. hydrophila has not been established previously as a significant enteric pathogen in foals. Other putative pathogens found at very low prevalence were coronavirus, the putative picobirnavirus, Campylobacter spp. and Salmonella spp. No evidence was found of synergistic effects between rotavirus, cryptosporidia and potentially pathogenic E. coli. Neither coccidia nor non-Group A rotaviruses were found in any of the samples examined.     

Antibodies to spotted fever-group rickettsiae in dogs in North Carolina

A seroepidemiologic survey was conducted to determine the prevalence of antibodies reactive with 4 spotted fever-group (SFG) rickettsiae in sera of dogs from various geographic regions in North Carolina. Serum specimens were obtained from 600 dogs, and antibody titers were determined, using microimmunofluorescence. Data analysis (setting as the criterion for a positive result, a Rickettsia rickettsii titer greater than or equal to 1:64) overestimated the actual prevalence of canine exposure to this rickettsia. When data were analyzed by considering each dog's serologic response to all 4 rickettsial antigens simultaneously, the prevalence rate for exposure to R montana was 15%, to R rhipicephali was 11%, and to R rickettsii was 5%. A definitive exposure to R bellii was not observed, and the identification of the specific inciting rickettsia could not be established for 13% of the dogs, because of identical highest titers to 2 or more antigens. Our data indicate that canine exposure to R rhipicephali is prevalent in the eastern coastal region, whereas exposure to R montana takes place uniformly throughout the state. Rickettsia rickettsii exposure appears to be more prevalent in the central Piedmont region, but rarely is encountered in the western mountains. Regional seroprevalence for canine R rickettsii exposure approximates that for human exposure. Our findings support earlier suggestions that dogs may serve as environmental sentinels for establishing the geographic prevalence of foci of spotted fever.     

绿色荧光蛋白基因标记嗜水气单胞菌的研究

嗜水气单胞菌广泛存在于水体环境中,可引起人和动物发病,尤其是引起鱼类的败血症。本文构建了pWSK129-gfp重组载体,并分别导入到嗜水气单胞菌强毒株J-1和无毒株4332中。在荧光显微镜下观察,菌体呈现绿色荧光。稳定性试验表明,Ah4332GFP传至70代,质粒稳定率可达100%;而AhJ-1GFP在同样条件下培养10代后,质粒的稳定率仅为15.1%。绿色荧光蛋白基因的导入为研究嗜水气单胞菌与宿主的相互关系提供了一种简单而直观的方法。     

Dot-ELISA法检测致病性嗜水气单胞菌

在鱼类致病性气单胞菌诊断试剂盒的基础上,用斑点酶联免疫吸附试验(Dot-ELISA)检测致病性嗜水气单胞菌(Aeromonas hydrophila,Ah)胞外蛋白酶ECPase54,同时用脱脂奶平板、PCR特异性扩增气溶素基因aer和16S rRNA基因检测72株气单胞菌分离株.结果显示致病性嗜水气单胞菌检测阳性率分别如下:Dot-ELISA法90.3％(65/72)、脱脂奶平板法75％(54/72)、aer基因PCR法94.4％(68/72)、16S rRNA PCR法81.9％(59/72),Dot-ELISA与其他3种方法的符合率分别为79.2％(57/72)、91.6％(66/72)、81.9％(59/72).在ECPase54兔抗血清制备后的2、4、6、12、18个月,用Dot-ELISA检测72株分离株,检测结果重复性好.结果表明Dot-ELISA法敏感、特异、实用,可用于鱼类致病性Ah的临床诊断.     

嗜水气单胞菌Ⅲ型分泌系统ascV全长基因的克隆及其在不同菌株中的分布

根据已发表的嗜水气单胞菌Ⅲ型分泌系统（TTSS）的ascV基因保守区核苷酸序列设计合成1对引物,以国内疫苗菌株J-1的基因组为模板,通过PCR扩增得到331bp保守基因片段,将目的片段进行测序。在此基础上,分4段克隆J-1株的Ⅱscv基因并进一步拼接,全长为2166bp,同时PCR检测ascV基因在66株嗜水气单胞菌中的分布情况。测序分析发现,扩增出的J-1株ascV全长基因与嗜水气单胞菌AH-1、SSU、AH-3的同源性分别为97％、86％、86％,与杀鲑气单胞菌杀鲑亚种A449的同源性为86％,与温和气单胞菌的同源性为87％。PCR检测表明,64株能扩增出n5fV基因的目的片段,包括2株无毒菌株,而在2株有毒菌株中却未能检测到,说明TTSS在嗜水气单胞菌致病机制上的作用值得进一步探讨。     

嗜水气单胞菌分离株ERIC-PCR及RAPD分型比较

采用ERIC-PCR和RAPD 2种方法对嗜水气单胞菌2009年分离菌株及实验室保存菌株共83株进行分子分型。参考相关文献设计并合成引物,分别进行PCR扩增及琼脂糖凝胶电泳,电泳结果经Quantity One软件数值化后利用SPSS软件进行聚类分析。结果显示,除少数分离株外,使用2种方法对大多数菌株分型,均能得到多态性较好的指纹图谱。RAPD的AP12H引物能扩增出1～12个200～4 500bp之间的条带,可将嗜水气单胞菌分离株分为4群、12类。ERIC-PCR能扩增出4～16个100～5 800bp之间的条带,将嗜水气单胞菌分离株分为4群、17类,同一年代、同一地域分离株分别有聚成一类的趋势。2种分型方法均体现很好的分型能力。但ERIC-PCR分型与RAPD分型相比,重复性和稳定性更好,分辨力更高,且具备RAPD不要求模板序列已知而直接进行扩增的优点,更适合嗜水气单胞菌的分型研究。     

Class 1 integrons in Aeromonas hydrophila isolates from farmed Nile tilapia (Oreochromis nilotica)

The aim of this study was to determine antimicrobial resistance of Aeromonas hydrophila isolated from farmed Nile Tilapia. A total of 50 A. hydrophila isolates from clinical cases were screened for the presence of class 1, 2 and 3 integrons and all the strains resistant to enrofloxacin and/or ciprofloxacin (n=19) examined for mutation in the quinolone resistance-determining regions (QRDRs) of gyrA and parC. The intI1 gene was detected in 23 A. hydrophila strains (46%) but no intl2 and intl3 were detected. Among these, 14 isolates (60.8%) carried gene cassettes inserted in variable regions i.e., partial aadA2, aadA2, dfrA1-orfC and dfrA12-aadA2, of which the most common gene cassette array was dfrA12-aadA2 (26.09%). Conjugal transfer of class 1 integrons with resistance gene array was detected. All the A. hydrophila strains resistant to enrofloxacin and/or ciprofloxacin possessed mutations in the QRDRs of gyrA and parC. Only a Ser-83-Ile substitution was identified in GyrA and only a Ser-80-Ile amino change was found in ParC. The data confirms that A. hydrophila from farm-raised Nile Telapia serve as a reservoir for antimicrobial resistance determinants.     

Adjuvant and immunostimulatory effects of beta-glucan administration in combination with lipopolysaccharide enhances survival and some immune parameters in carp challenged with Aeromonas hydrophila

Combined effects of beta-glucan and lipopolysaccharide (LPS) on survival and immune response were studied in Cyprinus carpio that were challenged with the pathogen Aeromonas hydrophila. beta-Glucan from Saccharomyces cervisiae and LPS from a virulent strain of A. hydrophila were used in this study. Different concentrations of beta-glucan+LPS mixture were administered on days 1, 7, and 14 through different routes (intraperitoneal injection, bathing, and oral administration). Control and test fish were challenged by intraperitoneal injection of LD50 concentration of A. hydrophila on day 16 and subsequently, mortality and relative percent survival (RPS) were recorded. Intraperitoneal injection elicited 100% RPS even at the lowest concentration (100 microg beta-glucan+10 microg LPS); whereas, oral administration improved RPS rate of carps at higher concentration (1% beta-glucan+0.25% LPS). Bathing did not improve the RPS. Test animals injected with even the minimum dose of the immunomodulators (100 microg beta-glucan+10 microg LPS/fish) had a significant increase in total blood leukocyte counts and an increase in the proportion of neutrophils and monocytes. Superoxide anion production by macrophages was also elevated, which presumably aided the efficient killing of bacterial pathogen. Lower concentration of beta-glucan+LPS had an adjuvant effect on antibody production as pretreatment by injection of 100 microg beta-glucan+10 microg LPS/fish resulted in higher antibody titer against A. hydrophila following vaccination. RT-PCR analyses showed that the expression of interleukin-1beta mRNA did not increase in test fish when compared with the control. Classical and alternative complement pathways were not affected by either the dose or the route of administration of the compounds. It may be concluded that intraperitoneal injection and oral administration, and not the bathing, of beta-glucan+LPS mixture in carp could enhance resistance to challenge by A. hydrophila through changes in several non-specific and specific immune responses.     

Isolation and haemolytic activity of Aeromonas species from domestic dogs and cats.

Rectal swabs from 120 domestic dogs and 15 domestic cats were examined for Aeromonas species using alkaline peptone water (pH 8.6) as the enrichment medium and blood agar containing 15 mg/l ampicillin as the plating medium. Aeromonads were isolated from 13 (10.8%) dogs and from 1 (6.7%) cat. Of the 14 aeromonads isolated in the present study only 9 were available for speciation and testing in the haemolysin assay. Of these 5 were A. sobria (including one from a cat), 2 were A. hydrophila and 2 were A. caviae. Six were positive in the haemolysin assay; 4 A. sobria (one from a cat) and 2 A. hydrophila. The presence of haemolysin producing-Aeromonas species in the faeces of domestic dogs and cats may pose a public health problem for humans who come into contact with such animals.     

罗非鱼出血病病原鉴定及疫苗

从惠出血病罗非鱼血液、肝脏、脾脏中分离到一株细菌,经理化鉴定,该菌为嗜水气单胞菌,动物回归试验阳性,认为该罗非鱼出血病是嗜水气单胞菌引起。用该分离菌制成的饵料及附型疫苗田间免疫罗非鱼成活率达87．1％,未免疫组成活率仅59．9％。     

Effect of Dietary Supplementation with Achyranthes aspera Seed on Larval Rohu Labeo rohita Challenged with Aeromonas hydrophila

Abstract

Larval rohu Labeo rohita were fed four different diets: three of the diets contained Achyranthes aspera (prickly chaff-flower) seeds at 0.10% (D1), 0.25% (D2), or 0.50% (D3); the fourth diet was a control diet (D4; no A. aspera supplementation). After 70 d, the rohu were injected intraperitoneally with live Aeromonas hydrophila. Mortality of fish was recorded for 7 d. In the D4 group, the first mortality was observed within 12 h of exposure, whereas in the D1–D3 treatment groups, mortality was first observed at 24 h postexposure. In the D4 group, 50% of fish died within 72 h of exposure, whereas in the D3 group, 10–15% mortality occurred between 72 and 84 h. The cumulative mortality rate was 50% for D4, 40% for D1, 35% for D2, and 15% for D3. Total tissue protein level in the larvae was higher for the D2 and D3 groups than for the other groups. Glutamic oxaloacetic transaminase, glutamate pyruvate transaminase, and thiobarbituric acid reactive substance levels were significantly lower in D3 larvae than in the other groups, whereas lysozyme and nitric oxide synthase levels were significantly higher in D3 larvae compared with the other groups. Dietary supplementation with A. aspera seeds at the 0.50% level provided protection against oxidative stress, prevented tissue damage, and enhanced disease resistance in rohu larvae.

Received December 26, 2011; accepted May 7, 2012     

南京地区池塘水中嗜水气单胞菌的分离鉴定与相关毒力基因检测

嗜水气单胞菌在自然界广泛存在,是一种典型的人-兽-鱼共患病病原菌。本研究收集南京市多处湖泊池塘水样,采用增菌、平板分离培养、生化试验和种特异性16S rRNA基因扩增等进行嗜水气单胞菌的分离鉴定;采用PCR技术检测分离株毒力基因的分布,进一步通过动物试验确定是否为致病性菌株。结果,从28份水样中共检出嗜水气单胞菌7株,检出率达25%;其中致病性菌株4株,占被检嗜水气单胞菌总数的57.14%(4/7)。本研究有助于了解南京市水体环境中嗜水气单胞菌的污染情况,为进一步预防该菌所引起的相关疾病的发生和传播提供理论依据。     

肽聚糖对鲫鱼嗜水气单胞菌灭活疫苗免疫增强效果的研究

为了探讨肽聚糖在鱼类抗嗜水气单胞菌（Aeromonas hydrophila，AH）感染中的免疫佐剂作用，用添加A3a肽聚糖的饲料投喂接种F-AH疫苗的彭泽鲫（Carassius auratus var．pengze）；通过测定鲫白细胞吞噬活性、血清和体表黏液溶菌酶活性、抗体效价以及活菌攻毒后的免疫保护率，证明肽聚糖与F-AH疫苗联合使用，鲫体表黏液和血清溶菌酶活性、白细胞吞噬活性、抗体效价以及活菌攻毒后的免疫保护率均显著提高，表明A3a肽聚糖是F-AH疫苗的良好佐剂，能增强鱼类F-AH疫苗免疫应答。     

鱼源嗜水气单胞菌对氟苯尼考的耐药性分析

为阐明淡水鱼的主要致病菌嗜水气单胞菌对氟苯尼考的耐药现状,本试验采用纸片扩散法和浓度稀释法分别定性、定量检测了2010年9月至2013年5月期间收集的26株鱼源嗜水气单胞菌对氟苯尼考的耐药性,利用PCR法检测菌株携带氟苯尼考耐药基因的情况。结果显示,北京地区的鱼源嗜水气单胞菌对氟苯尼考的耐药率为15.38%,氟苯尼考对其最小抑菌浓度（MIC）主要集中在2～4 μg/mL,5株菌对氟苯尼考的MIC超过8 μg/mL,1株分离菌具有氟苯尼考耐药基因floR。结合以往数据及其他省市的相关数据,结果表明鱼源嗜水气单胞菌对氟苯尼考已呈较高的耐药率和耐药浓度,且位于质粒上的floR基因阳性菌株有传递该基因给其他种属鱼类致病菌的风险。因此,有必要科学规范氟苯尼考在水产养殖中的应用。     

Aeromonas hydrophila-associated skin lesions and septicaemia in a Nile crocodile (Crocodylus niloticus)

Aeromonas hydrophila is one of the most common bacteria associated with the aquatic environment. There are, however, limited data on A. hydrophila infection in crocodilians. The aim of this report is to describe a case of skin lesions and septicaemia associated with A. hydrophila in a Nile crocodile (Crocodylus niloticus). A captive male crocodile in the Zoological Park of Antalya (Turkey) was found dead without showing signs of any disease. Gross examination showed brown or red-spotted skin lesions of varying size. These lesions were mostly scattered over the abdomen and occasionally on the tail and feet. At necropsy, numerous white, multifocal and randomly distributed areas were seen on the liver. Gram-stained smears from skin and liver lesions showed Gram-negative bacilli arranged in clusters. Pure cultures of A. hydrophila were recovered from skin, internal organs and blood. Isolates were found to be susceptible to ceftiofur, amoxicillin + clavulanic acid, oxytetracycline, enrofloxacin, danofloxacin, neomycin, gentamicin, and lincomycin + neomycin. A pathogenicity test was performed using this isolate on 4 male 2-year-old New Zealand white rabbits. Local abscesses formed in 2 rabbits injected subcutaneously and the 2 that were injected intraperitoneally died as a result of septicaemia. In conclusion, this report has shown that A. hydrophila may cause skin lesions and even death due to septicaemia in crocodiles.     

Gut microbial status induced by antibiotic growth promoter alters the prebiotic effects of dietary DVAQUA® on Aeromonas hydrophila-infected tilapia: production, intestinal bacterial community and non-specific immunity

The purpose of the present study was to investigate whether dietary antibiotic-induced changes in the fish intestinal microbiota altered host physiological responses to the infection with Aeromonas hydrophila in hybrid tilapia (Oreochromis niloticus ♀ × O. aureus ♂). After an 8-week induction period with an antibiotic-supplemented or antibiotic-non-supplemented diet, 160 hybrid tilapias in 16 tanks were each injected with phosphate buffered saline (PBS) or A. hydrophila at a dose of one-half of the LD(50). Then, all of the diets were changed to a prebiotic-supplemented one for the sequential 8-week response period. Parameters including production, gut microbial diversity and count, and non-specific immunity were determined at the end of the response period. Our results showed that A. hydrophila infection had no effects on the growth and diet conversion of tilapia, but it caused the decrease of the gut bacterial count, the number of visual bands, and the Shannon diversity and equitability indexes of gut bacteria in antibiotic-non-supplemented fish based on PCR-DGGE fingerprints. Infection with A. hydrophila reduced the gut bacterial evenness (lower Shannon equitability index), and slightly improved the gut bacterial richness (more visual bands) in antibiotic-supplemented tilapia. In addition, A. hydrophila infection affected non-specific immunity such as serum lysozyme activity and serum alternative complement pathway (C3 and C4) activities regardless of hybrid tilapia fed antibiotic-supplemented diets. These changes varied based on the intestinal microbial status of the fish before infection with A. hydrophila.     

嗜水气单胞菌选择培养基鉴别效果的比较

本试验比较了嗜水气单胞菌（Ａｅｒｏｍｏｎａｓｈｙｄｒｏｐｈｉｌａ,Ａｈ）三种选择培养基ＡＨＭ、ＡＰＭ和ＲＳ培养基的鉴别效果,并用于１２１株细菌检测,Ａｈ的阳性检出率分别是５４．７６％、８１．３３％和８３．５４％。经过交叉比较分析,确定ＡＨＭ和ＲＳ可作为适用于生产实践的Ａｈ的选择培养基。     

Identification and expression profiles of multiple genes in Nile tilapia in response to bacterial infections

To understand the molecular mechanisms involved in response of Nile tilapia (Oreochromis niloticus) to bacterial infection, suppression subtractive cDNA hybridization technique was used to identify upregulated genes in the posterior kidney of Nile tilapia at 6h post infection with Aeromonas hydrophila. A total of 31 unique expressed sequence tags (ESTs) were identified from 192 clones of the subtractive cDNA library. Quantitative PCR revealed that nine of the 31 ESTs were significantly (p<0.05) upregulated in Nile tilapia at 6h post infection with A. hydrophila at an injection dose of 10(5)CFU per fish (≈ 20% mortality). Of the nine upregulated genes, four were also significantly (p<0.05) induced in Nile tilapia at 6h post infection with A. hydrophila at an injection dose of 10(6)CFU per fish (≈ 60% mortality). Of the four genes induced by A. hydrophila at both injection doses, three were also significantly (p<0.05) upregulated in Nile tilapia at 6h post infection with Streptococcus iniae at doses of 10(6) and at 10(5)CFU per fish (≈ 70% and ≈ 30% mortality, respectively). The three genes induced by both bacteria included EST 2A05 (similar to adenylate kinase domain containing protein 1), EST 2G11 (unknown protein, shared similarity with Salmo salar IgH locus B genomic sequence with e value of 0.02), and EST 2H04 (unknown protein). Significant upregulation of these genes in Nile tilapia following bacterial infections suggested that they might play important roles in host response to infections of A. hydrophila and S. iniae.