Edwardsiella ictaluri as the Causative Agent of Mortality in Cultured Nile Tilapia

Abstract

Edwardsiella ictaluri was consistently isolated from the spleens, livers, and head kidneys of diseased Nile tilapia Oreochromis niloticus from a farm experiencing mortality events in several culture ponds. We describe the first published outbreak of E. ictaluri–induced edwardsiellosis in Nile tilapia. Pure cultures of the isolated bacteria were characterized both biochemically and molecularly. Biochemical analysis was performed using the API-20E and RapID One systems, and antimicrobial susceptibility was determined by the broth microdilution method. Molecular analysis involved sequencing of the 16S rRNA gene, species-specific real-time polymerase chain reaction (PCR), and PCR-mediated genomic fingerprinting (rep-PCR). Pairwise sequence analysis of the 16S rRNA gene identified the case isolates to be a 100% match to E. ictaluri cultured from channel catfish in the southeastern United States. However, rep-PCR analysis identified the case isolates to be genetically different from representative strains isolated from disease outbreaks in cultured channel catfish in Mississippi. Infectivity challenges (intraperitoneal injection and immersion) demonstrated that a representative E. ictaluri strain isolated from tilapia was pathogenic to naïve tilapia, reproducing clinical signs and mortality, thereby establishing Koch's postulates.

Received August 30, 2011; accepted January 30, 2012     

In vitro evaluation of the susceptibility of Edwardsiella ictaluri, etiological agent of enteric septicemia in channel catfish, Ictalurus punctatus (Rafinesque), to florfenicol.

In vitro studies were conducted to assess the sensitivity of Edwardsiella ictaluri, the etiological agent of enteric septicemia of catfish (ESC), to the antibacterial drug florfenicol (FFC). Twelve different E. ictaluri isolates from cases submitted between 1994 and 1997 to the Thad Cochran National Warmwater Aquaculture Center fish diagnostic laboratory (Stoneville, MS) were used for testing. These isolates originated from channel catfish (Ictalurus punctatus) infected with E. ictaluri through natural outbreaks of ESC in the commercial catfish ponds in Mississippi. Seven hundred sixty-seven additional cultures of E. ictaluri were obtained from channel catfish infected experimentally with E. ictaluri. In some of these experimental infections, FFC was used for treatment. These cultures of E. ictaluri were identified by morphological and biochemical tests. Kirby-Bauer zones of inhibition (in mm) for FFC against E. ictaluri were determined using standard methods. The minimum inhibitory concentration (MIC) of FFC was determined for the natural outbreak E. ictaluri isolates and arbitrarily selected experimental cultures. The zones of inhibition for FFC tested with E. ictaluri ranged from 31 to 51 mm. The MIC for FFC tested with E. ictaluri was consistently 0.25 microg/ml. Edwardsiella ictaluri tested in these studies were highly sensitive to FFC in vitro.     

Outer membrane protein profiles of Edwardsiella ictaluri from fish.

Outer membrane proteins (OMP) prepared with sodium N-lauroyl sarcocinate (SLS) from 33 Edwardsiella ictaluri isolates from fish were examined by electrophoresis. Twenty-eight isolates from channel catfish (Ictalurus punctatus) had similar OMP profiles. Ten bands (71 kilodaltons [kD] to 19.5 kD) were identified in all isolates from channel catfish. One major 35-kD protein comprised most of the protein content of the outer membrane of isolates from channel catfish. Differences existed among isolates in the amount of protein within minor OMP bands. Edwardsiella ictaluri ATCC 33202 contained larger quantities of the 38.5- and 37-kD proteins than did the other isolates. Outer membrane protein profiles of E ictaluri derived from Bengal danio (Danio devario) and walking catfish (Clarias batrachus) were identical to OMP profiles of isolates from channel catfish. In contrast, OMP profiles from single isolates from green knife fish (Eigemannia virescens) and white catfish (Ictalurus catus) were different. Variations in incubation time, SLS extraction time, SLS extraction number, and in vivo and in vitro passage had no effect on the OMP profile of E ictaluri ATCC 33202. An increase in duration of sample solubilization did affect the OMP profile of E ictaluri ATCC 33202 by decreasing the amount of protein in 52-, 46-, and 43.5-kD bands. Accompanying the decrease were increased staining intensity in the 31.5- and 28.5-kD bands and the appearance of 4 new bands (34, 33, 25.5, and 22.5 kD). Edwardsiella ictaluri, a gram-negative bacterium in the family Enterobacteriaceae, is the cause of enteric septicemia of catfish.(ABSTRACT TRUNCATED AT 250 WORDS)     

Small Subunit rRNA Gene Comparisons of Four Actinosporean Species to Establish a Polymerase Chain Reaction Test for the Causative Agent of Proliferative Gill Disease in Channel Catfish

Abstract

Proliferative gill disease (PGD) causes high morbidity and mortality in cultured channel catfish Ictalurus punctatus. The presence of the myxozoan Aurantiactinomyxon ictaluri (class Actinosporea) is strongly associated with PGD. This parasite, shed as an actinospore from the aquatic oligochaete Dero digitata, infects channel catfish by an undetermined route. Several other actinosporeans have been identified that are shed from D. digitata isolated from catfish ponds, including those designated A. mississippiensis, Helioactinomyxon sp., and the actinospore stage of Henneguya exilis. By the use of multiple sequence alignment of polymerase chain reaction (PCR)-amplified small subunit ribosomal RNA (SSU rRNA) genes of A. ictaluri, A. mississippiensis, and H. exilis, we identified two variable regions. The largest variable region was PCR amplified, sequenced from the Helioactinomyxon sp., and used in addition to the other three sequences in multiple-sequence alignment comparison to develop PCR primers specific for A. ictaluri. This PCR specific for A. ictaluri produced 104-base-pair products from a plasmid clone containing the SSU rRNA gene of A. ictaluri, spore DNA of A. ictaluri, and DNA prepared from channel catfish gill and D. digitata infected with A. ictaluri. The PCR assay was able to detect as few as 100 copies of the cloned gene. There was no detectable product from the genomic DNA of H. exilis, A. mississippiensis, or Helioactinomyxon sp., specific pathogen-free channel catfish gill, and noninfected D. digitata. The PCR assay will be useful as a diagnostic tool for PGD in channel catfish and will aid in the elucidation of the life cycle of A. ictaluri.     

Enhanced susceptibility of channel catfish to the bacterium Edwardsiella ictaluri after parasitism by Ichthyophthirius multifiliis

Bacterium Edwardsiella ictaluri and parasite Ichthyophthirius multifiliis (Ich) are two common pathogens of cultured fish. The objective of this study was to evaluate the susceptibility of channel catfish Ictalurus punctatus to E. ictaluri and determine bacterial loads in different fish organs after parasitism by Ich. Fish received the following treatments: (1) infected by I. multifiliis at 5000 theronts/fish and exposed to E. ictaluri; (2) infected by I. multifiliis alone; (3) exposed to E. ictaluri alone; and (4) non-infected control. E. ictaluri in fish organs were quantified by quantitative real-time polymerase chain reaction and reported as genome equivalents per mg of tissue (GEs/mg). The results demonstrated that the Ich-parasitized catfish showed significantly (P<0.05) higher mortality (91.7%) when exposed to E. ictaluri than non-parasitized fish (10%). The bacterial loads in fish infected by 5000 theronts/fish ranged from 6497 to 163,898 GEs/mg which was between 40 and 2000 fold higher than non-parasitized fish (49-141 GEs/mg). Ich infection enhanced the susceptibility of channel catfish to bacterial invasion and increased fish mortality.     

Effects of variable periods of food deprivation on the development of enteric septicemia in channel catfish

Enteric septicemia of catfish (ESC), caused by the bacterium Edwardsiella ictaluri, is the most significant bacterial disease affecting channel catfish Ictalurus punctatus. Withholding feed during outbreaks of ESC is a widely accepted industry practice used to control losses from the disease. Scientific evidence concerning the validity of the practice is contradictory. Two studies were conducted to further evaluate the survival of channel catfish fingerlings following variable periods of feed deprivation before and after exposure to E. ictaluri in controlled aquarium experiments. In the first study, feed was withheld for varying time periods before bacterial challenge. After bacterial challenge, feed was either withheld or fish were fed daily. The second study utilized fish fed daily or fish deprived of feed 7 d before bacterial challenge. Daily feeding was resumed 4, 48, and 96 h after fish were exposed to E. ictaluri. In both experiments, the prechallenge feed treatments did not affect mortality. In contrast, withholding feed after bacterial challenge reduced mortalities by 52% in experiment 1 and by 45% in experiment 2. The highest mortality was observed when fish were fed immediately after immersion exposure and the lowest when fish were completely denied feed or fed daily starting 96 h after challenge. This reduction in mortality occurred when the concentration of E. ictaluri in aquarium water was negligible. These data suggest that when E. ictaluri is present in the water, feeding fish increases mortality by enhancing oral exposure to the pathogen.     

Isolation, characterization, and molecular cloning of cryptic plasmids isolated from Edwardsiella ictaluri

Fifty-five isolates of Edwardsiella ictaluri were examined for the presence of plasmid DNA by a rapid alkaline extraction procedure. All 49 isolates from channel catfish and a single isolate from Bengal danio carried 2 plasmids with molecular masses of approximately 3.2 and 3.7 megadaltons (Mdal). Five E ictaluri isolates from other fish contained 1 to 3 plasmids, which had molecular masses ranging from 2.5 to 45 Mdal. The 2 plasmids (3.2 and 3.7 Mdal) from the type strain of E ictaluri (ATCC 33202) were ligated into pUC19 cloning vectors, and restriction endonuclease maps of each insert were prepared.     

Spleen index and mannose-binding lectin levels in four channel catfish families exhibiting different susceptibilities to Flavobacterium columnare and Edwardsiella ictaluri

Edwardsiella ictaluri and Flavobacterium columnare are two bacterial pathogens that affect channel catfish Ictalurus punctatus aquaculture. At the Catfish Genetics Research Unit (U.S. Department of Agriculture, Agricultural Research Service), some progress has been made in selectively breeding for resistance to E. ictaluri; however, the susceptibility of these families to F. columnare is not known. Our objectives were to obtain baseline information on the susceptibility of channel catfish families (maintained as part of the selective breeding program) to E. ictaluri and F. columnare and to determine whether the spleen index and plasma levels of mannose-binding lectin (MBL) are predictive indicators of susceptibility to these pathogens. Four channel catfish families were used: family A was randomly chosen from spawns of fish that were not selectively bred for resistance; families B, C, and D were obtained after selection for resistance to E. ictaluri. All four families were immersion challenged with both bacterial pathogens; the spleen index and plasma MBL levels of unchallenged fish from each family were determined. Mean cumulative percent mortality (CPM) after E. ictaluri challenge ranged from 4% to 33% among families. Families A and B were more susceptible to F. columnare (mean CPM of three independent challenges = 95% and 93%) than families C and D (45% and 48%), demonstrating that there is genetic variation in resistance to F. columnare. Spleen index values and MBL levels were not significantly different, indicating that these metrics are not predictive indicators of F. columnare or E. ictaluri susceptibility in the four tested families. Interestingly, the two families that exhibited the highest CPM after F. columnare challenges had the lowest CPM after E. ictaluri challenge. Further research on larger numbers of families is needed to determine whether there is any genetic correlation between resistance to E. ictaluri and resistance to F. columnare.     

禽霍乱病原菌的鉴定与主要特性分析

从河北某养鸡场80日龄左右鸡群所发生的禽霍乱病死鸡中，分离到了相应病原菌多杀巴斯德氏菌（pasteurella multocida）。对分离获得的16株菌（HPs-1至HPs-16）进行了形态特征、培养特性、理化特性等方面的鉴定，同时选取代表菌株（HPs-1）进行了16SrRNA基因的分子鉴定，测定了16SrRNA序列，构建了系统发育树。结果表明分离鉴定的16株细菌均为多杀巴斯德氏菌败血亚种（P．multocida subsp．septica），所测代表菌株的16SrRNA基因长度142bp，在GenBank登录号为AY999017，该菌株与检索出的22株巴斯德氏菌属（Pasteurella Trevisan，1887）细菌16SrRNA基因序列同源性均在98％～100％。另外，药敏试验结果显示分离株对供试的青霉素G等33种抗菌药物高敏、对克林霉素敏感、对苯唑青霉素等3种耐药。     

Isolation and Pathogenicity of Streptococcus iniae in Cultured Red Hybrid Tilapia in Malaysia

Abstract

This study describes the isolation and pathogenicity of Streptococcus iniae in cultured red hybrid tilapia (Nile Tilapia Oreochromis niloticus × Mozambique Tilapia O. mossambicus) in Malaysia. The isolated gram-positive S. iniae appeared punctiform, transparently white, catalase and oxidase negative and produced complete β-hemolysis on blood agar, while a PCR assay resulted in the amplification of the 16 S rRNA gene and lactate oxidase encoded genes. The isolate was sensitive to tetracycline, vancomycin, and bacitracin but was resistant to streptomycin, ampicillin, penicillin, and erythromycin. Pathogenicity trials conducted in local red hybrid tilapia (mean ± SE = 20.00 ± 0.45 g) showed 90.0, 96.7, and 100.0% mortality within 14 d postinfection following intraperitoneal exposure to 10⁴, 10⁶, and 10⁸ CFU/mL of the pathogen, respectively. The clinical signs included erratic swimming, lethargy, and inappetance at 6 h postinfection, while mortality was recorded at less than 24 h postinfection in all infected groups. The LD_{50-336 h} of S. iniae against the red hybrid tilapia was 10² CFU/mL. The post mortem examinations revealed congested livers, kidneys, and spleens of the infected fish. This is the first report of S. iniae experimental infection in cultured red hybrid tilapia in Malaysia.

Received January 20, 2017; accepted July 16, 2017 Published online October 11, 2017     

Sphaerospora infection of American catfishes (Ictalurus punctatus and I. nebulosus) in Europe.

The occurrence in Europe of a Sphaerospora species described in North America is reported. Based upon its morphological characteristics, the parasite could be identified with the species S. hankai described from brown bullhead in Canada. This parasite was found to infect channel catfish (Ictalurus punctatus) cultured in farm ponds in Italy and brown bullhead (Ictalurus nebulosus) living in the supply channels of fish ponds in Hungary. The spores and sporogonic developmental stages were situated in the lumen of the renal tubules. In the authors' opinion, S. ictaluri described from channel catfish can be considered synonymous with S. hankai.     

A real-time polymerase chain reaction assay for quantification of Edwardsiella ictaluri in catfish pond water and genetic homogeneity of diagnostic case isolates from Mississippi

A quantitative polymerase chain reaction (qPCR) assay was developed for the detection and quantification of Edwardsiella ictaluri in channel catfish Ictalurus punctatus pond water using modifications to a published E. ictaluri-specific qPCR assay and previously established protocols for the molecular detection of myxozoan parasites in catfish ponds. Genomic DNA equivalents indicative of the number of bacteria in a sample were determined and standard curves correlating to bacterial numbers were established. The assay was found to be highly repeatable and reproducible, with a linear dynamic range of five orders of magnitude. There was no interference of the assay from the presence of large quantities of nontarget DNA. Known quantities of bacteria were added to sample volumes of 40 or 500 mL of pond water collected from several different ponds. The minimum level of detection was approximately 100 cell equivalents (CE) in 40 (2.5 CE/mL) or 500 mL of pond water (0.2 CE/mL). Sample volumes of 40 mL yielded the most consistent results, which were not significantly different from those obtained from broth culture alone. Cell equivalents determined by qPCR in 40-mL pond water samples spiked with known quantities of bacteria were within one order of magnitude of the actual number of cells added. Repetitive element-based polymerase chain reaction analysis of archived isolates demonstrated the genetic homogeneity of E. ictaluri, and consistent amplification of these isolates by qPCR analysis demonstrated the stability of the PCR target. The assay described here provides a reliable method for the detection and quantification of E. ictaluri in pond water and will be an invaluable tool in epidemiological studies. Additionally, the assay provides a way to evaluate the effects that vaccination, antibiotic treatments, and restricted feeding practices have on E. ictaluri populations during an outbreak. Information obtained with these tools will aid in optimizing disease management practices designed to maximize productivity while minimizing losses.     

肺炎克雷伯氏菌强毒株的分离鉴定及16-23SrRNAITS序列分析

为确诊疑似仔猪肺炎克雷伯氏菌(K.pneumonia)感染,并研究其病原的致病性、耐药性、16-23SrRNA ITS系统进化特征,本研究从云南因肺炎、腹泻而大量死亡的仔猪中分离到1株革兰氏阴性短粗杆菌,命名为KP14013,对其进行生化鉴定、16SrRNA鉴定,研究其对小白鼠和仔猪的致病性,并对其16-23SrRNA ITS基因进行测序和遗传进化分析。结果显示,KP14013分离株生化特征与肺炎克雷伯氏菌相符,其16SrRNA与GenBank中23株肺炎克雷伯氏菌代表株之间的同源性均为99%,将KP14013鉴定为肺炎克雷伯氏菌。KP14013对小白鼠半数致死量(LD50)为3×101.8 CFU,腹腔注射3×108 CFU可使仔猪100%致死。16-23SrRNA ITS系统进化关系结果表明,KP14013与GenBank中收录的15株肺炎克雷伯氏菌形成进化树的一个分支,属于同一个亚群,它们之间的核苷酸同源性为98.4%~99.2%。本研究证实了肺炎克雷伯氏菌是该起仔猪腹泻大量死亡的病原;KP14013分离株为毒力极强菌株,具有多重耐药性,其16-23SrRNA ITS与GenBank中收录的肺炎克雷伯氏菌代表株之间核苷酸存在差异,可用于肺炎克雷伯氏菌菌株间的鉴别。     

Evaluation of zebrafish Danio rerio as a model for enteric septicemia of catfish (ESC)

Zebrafish (also known as zebra danio) Danio rerio were injected intramuscularly with Edwardsiella ictaluri at doses of 6 x 10(3), 6 x 10(4), or 6 x 10(5) colony-forming units per gram (CFU/g) or sterile phosphate-buffered saline (sham) or were not injected. Mortality occurred from 2 to 5 d postinjection (dpi) at rates of 0, 76.6, and 81.3% for the low, medium, and high doses, respectively, and E. ictaluri was isolated from dead fish. Survivors were sampled at 10 dpi and E. ictaluri was not isolated. Sham-injected and noninjected controls did not suffer mortality. Histopathology trials were performed in which zebrafish were injected with 1 x 10(4) CFU/g or sham-injected and sampled at 12, 24, 48, 72, and 96 h postinjection for histological interpretation. Collectively, these zebrafish demonstrated increasing severity of splenic, hepatic, cardiac, and renal interstitial necrosis over time. To evaluate the progression of chronic infection, zebrafish were injected with 1 x 10(2) CFU/g and held for 1 month postinjection. Beginning at 12 dpi and continuing for an additional 2 weeks, zebrafish demonstrated abnormal spiraling and circling swimming behaviors. Histopathology demonstrated necrotizing encephalitis. In immersion trials, zebrafish were exposed to low, medium, and high doses (averaging 1.16 x 10(5), 1.16 x 10(6), and 1.16 x 10(7) CFU/mL of tank water) of E. ictaluri for 2 h. Mortality occurred from 5 to 9 d postexposure at rates of 0, 3.3, and 13.3% for the low, medium, and high doses, respectively; E. ictaluri was isolated from dead fish. Channel catfish Ictalurus punctatus exposed to the medium doses suffered 100% mortality, and E. ictaluri was isolated from these fish. This study demonstrates the potential use of zebrafish as a model for E. ictaluri pathogenesis.     

豫陕两省14株鸡杆菌的gyrB、16S rRNA和rpoB基因序列比较分析

分析河南、陕西分离的14株鸡杆菌(Gallibacterium)之间的进化关系,及gyrB基因序列比较在该菌进化分析中的作用。PCR扩增鸡杆菌分离株的gyrB、16SrRNA和rpoB3个看家基因,PCR产物纯化后直接测序。将鸡杆菌分离株、国外参考株的3个看家基因序列进行比较分析,用Phylip 3.67软件构建进化树。结果表明,14株鸡杆菌与鸭源鸡杆菌(Gallibacterium anatis)模式株间的相似性为96.3%～98.0%(gyrB)、97.7%～99.6%(16SrRNA)和97.7%～99.0%(rpoB);14株鸡杆菌与鸡杆菌复合群1(Gallibacterium genomosp.1)参考株间的相似性为88.8%～89.9%(gyrB)、96.2%～97.5%(16SrRNA)和92.6%～93.6%(rpoB)。基于3个看家基因序列的进化分析,均显示14株鸡杆菌和鸭源鸡杆菌模式株形成单独的一个群。14株鸡杆菌分离株均属于鸭源鸡杆菌种;在3个看家基因位点,鸡杆菌河南株与陕西株之间、鸡杆菌输卵管炎病鸡分离株与健康鸡分离株之间均无明显遗传上的差异;gyrB基因序列分析可用于鸡杆菌分离株的种类鉴定,且对14株鸡杆菌与复合群1参考株的区别能力优于另外2个看家基因。     

斑点叉尾鮰中一种病原菌的分离与鉴定

目的未知病菌对江苏地区斑点叉尾鮰的养殖业造成了巨大的危害,本文对该地区某养殖场病鱼中分离的细菌进行了鉴定及微生物学特性研究。方法采用VIA培养基分离优势菌株;采用回归感染实验确认病原菌;采用形态学观察、生理生化特征测定、细菌特异性引物PCR扩增检测及细菌基因测序分析鉴定细菌;采用药敏实验测定它对部分抗生素的敏感性。结果从病鱼症状及菌株的培养特性、形态特征与嗜麦芽寡养单胞菌感染斑点叉尾鮰症状十分相似,病鱼有典型的肠套叠和肝脏变性现象。分离菌株为革兰氏阴性杆菌,氧化酶阴性、硝酸还原阴性,不发酵糖类,无动力。最初判为嗜麦芽寡养单胞菌,而经过VITEK2生化及16S rRNA基因系统发育进化显示,与不动杆菌16S rRNA核苷酸同源性最高,为98.7%～99.8%,该菌株为约翰逊不动杆菌。结论分离出的约翰逊不动杆菌grp2580083能导致斑点叉尾鮰患病,症状与嗜麦芽寡养单胞菌感染具有类似性,对渔业养殖造成影响。     

貂源肺炎克雷伯菌的分离与鉴定

本研究从疑似感染肺炎克雷伯菌(Kpn)的病死水貂病料中分离得到5株革兰氏阴性杆菌,通过对其进行细菌形态学、培养特征、生化特性和分子生物学鉴定,确认分离得到的菌株为Kpn。16S rRNA基因序列分析结果显示:该分离菌株与已知的Kpn相应序列的同源性为100%;药敏试验显示分离株对头孢曲松和舒巴坦高度敏感。动物致病性试验显示该分离株对小鼠具有较强的致病性。     

16S ribosomal RNA sequence-based identification of veterinary clinical bacteria.

This study evaluated 16S rRNA gene sequence analysis methods as tools for identification of 22 phenotypically difficult to identify veterinary clinical bacterial isolates in a veterinary diagnostic laboratory. The study compared 16S rRNA gene sequencing and conventional phenotypic identification methods. Using 16S rRNA full-gene sequencing, 95% (21/22) of the isolates were identified to the genus level and 86% (19/22) to the species level. The conventional or commercially available manual identification phenotypic characterization methods presumptively identified 91% (20/22) of the isolates to the genus level and 1 isolate to the species level. However, only 55% (12/22) or 4.5% (1/22) of the phenotypic identifications were correct at the genus or species level when they were compared with the 16S rRNA full-gene sequencing. This study also compared 16S rRNA full-gene and partial-gene sequencing. The results demonstrated that the best 16S rRNA gene-sequencing approach is full-gene sequencing because it gives the most precise species identification. Sequencing of the variable regions 1, 2, and 3 of the 16S rRNA gene could be used for tentative identification because the ability of this sequencing to identify bacteria to the genus level is similar to that of the 16S rRNA full-gene sequencing. This method identified only 14% (3/22) isolates differently to the species level compared with the 16S rRNA full gene sequence. Sequencing of the variable regions 7, 8, and 9 is not recommended because it gives more ambiguous identifications. The cost of a 16S RNA full-gene-sequencing analysis was Can 160 dollars and Can 60 dollars for a partial 16S rRNA gene sequence, i.e., sequencing of variable regions 1, 2, and 3 or variable regions 7, 8 and 9.     

红腹锦鸡大肠杆菌病的细菌学诊断

无菌采集病死红腹锦鸡肝脏、脾脏和心血,接种多种培养基分离细菌。以生化试验和小鼠攻毒试验,鉴定分离细菌及其致病性。采用PCR方法扩增分离细菌16S rRNA,测定核苷酸序列,与GenBank中大肠杆菌相应基因序列进行分析比较,绘制系统进化树。结果表明,分离细菌为大肠杆菌,可以致小鼠死亡。其16S rRNA基因序列与GenBank登录的大肠杆菌相应基因同源性超过99%。由此证明,该红腹锦鸡死于大肠杆菌病。