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Home , Yersinia, Yersinia enterocolitica, Yersinia ruckeri

enterocolitica as a cause of septicemia in crucian carp (Carassius carassius)

Item Type article

Authors Wang, L.

Download date 02/10/2021 21:02:35

Link to Item http://hdl.handle.net/1834/37623 Iranian Journal of Fisheries Sciences 15(1) 402-414 2016

Yersinia enterocolitica as a cause of septicemia in crucian carp (Carassius carassius)

Wang L.*

Received: April 2014 Accepted: May 2015

Abstract is an important to animals and humans. Thirteen strains of Y. enterocolitica were isolated from diseased fish. The partial 16SrDNA gene, five virulence genes, pathogenicity, and drug resistance of Y. enterocolitica strain G6029 were studied using molecular biological technology and toxicological method. Results showed that the length of amplified 16SrDNA sequence was 1448bp, and revealed 99% homology with Y. enterocolitica. Its GenBank accession number was JX855135. Five virulence genes (ail, ystB, yadA, virF and HPIINT) were detected, and only yadA gene was not seen. Twenty-eight crucian carps that were injected with strain G6029 died within a week, and the mortality was 93.3%, indicating highly pathogenic attribute of this strain. In addition, the strain G6029 was resistant to four antibiotics (sulfafurazole, furazolidone, enrofloxacin and norfloxacin), but it was susceptible to six antibiotics (florfenicol, vibramycin, cefaloridine, ciprofloxacin, streptomycin, and ). Further study of pathogenicity of Y. enterocolitica in teleost fish is suggested.

Keywords: Yersinia enterocolitica; Virulence gene; Pathogenicity; Carassius auratus

Key Laboratory of Animal Genetics and Breeding of State Ethnic Affairs Commission and Ministry of Education, College of Life Science and Technology, Southwest University for Nationalities, Chengdu 610041, China. * Corresponding author's Email: [email protected]. 403 Wang, Yersina enterocolitica as a cause of septicemia in crucian carp (Carassius carassius)

Introduction pathologic effects and clinical Y. enterocolitica is a widespread member manifestations occur. Temperature and of Yersinia, which belongs to the calcium concentration regulate phylum , class expression of virulence factors that guide Gammaproteo , order the invading yersinia and allow them to Enterobacteriales, family survive and disseminate (Fàbrega and (Bottone, 1997). Y. Vila, 2012; Asadishad et al., 2013). The enterocolitica is Gram-negative, genome sequencing of a representative of rod-shaped, non-sporulating, the most epidemiologically successful Y. facultatively anaerobic c-proteobacteria. enterocolitica subsp. palearctica strain Y. enterocolitica is most often transmitted Y11, serotype O:3, biotype 4 has been by consumption of contaminated food, finished and annotated (Batzilla et al., unpasteurized or inadequately 2011). Some virulence genes (i.e., yadA, pasteurized milk, untreated water, or by virF, ysa, inv, ail, and yst) located in direct or indirect contact with animals chromosome or plasmid of pathogenic Y. (Fredriksson et al., 2003; Sabina et al., enterocolitica has been widely used to 2011;). It is classified using biochemical identify pathogenic strains in characteristics along with serotyping. epidemiological studies (Fredriksson et There are six major biotypes designated al., 2006). However, the pathogenesis as 1A (the non-pathogenic type), and and epidemiology of Y. enterocolitica are there are pathogenic biotypes 1B, 2, 3, 4, complex and remain incompletely and 5 (Kot et al., 2010). understood as most cases are sporadic Y. enterocolitica, a classical enteric and reported without an apparent source. pathogen, causes human and animals Y. enterocolitica had been isolated from infections whose symptoms include humans, alpine ibex, piglets, sheep, birds, , terminal ileitis, intestinal , African green monkeys, and intussusception,mesenteric lymphadenitis, other animals, as well as from the , and septicemia (Imoto et al., environment, such as water and soil (; 2012). Moreover, Y. enterocolitica can Neubauer and Sprague, 2003; Fàbrega occasionally be present as a primary skin and Vila, 2012; Joutsen et al., 2012; and soft tissue abscess from direct Söderqvist et al., 2012; Virtanen et al., inoculation and extend to cause regional 2012; Soto et al., 2013; Virtanen et al., suppurative adenitis (Menzies, 2010). 2014). However, few reports existed in The primary pathogenic event of Y. the literature regarding infections caused enterocolitica is colonization of the by this organism in fish (Andrew et al., intestinal tract where most of the 2001). Crucian carp (C. auratus L., Iranian Journal of Fisheries Sciences (15) 2016 404

abbreviated CC) belongs to the genus of under aseptic processing. Afterwards, the Carassius within the family of samples were subcultured onto general Cyprinidae. Due to its good survival rate, nutrition agar plate and incubated at 28ºC high reproduction rate and good disease for 17h. Single colonies of bacterial resistance, it is widely bred in Eurasia strains were picked and further grown and America. In China, crucian carp is and sub-cultured several times to obtain a one of the most important freshwater pure culture. Biochemical species for Chinese aquaculture and has characterizations of the strains was been found in most provinces (Xiao et al., performed using API 20E system 2011). In this study, the case of fatal Y. (Simmons et al., 2014). The isolate was enterocolitica infections was reported in biotyped by special biochemical crucian carp, and the partial 16SrDNA characterizations, and serotyped by slide gene, virulence genes, pathogenicity and agglutination with antisera (O:3, O:9 and antibiotic resistance were studied by O:8). molecular biological technology and toxicological method. DNA isolation of bacteria After 18h cultivation in lysogeny broth Materials and methods (LB) medium at 28ºC, bacterial DNA Bacterial isolates was extracted using genomic extraction In May, 2012, thirteen crucian carps kit for bacterium (Axygen, China) (body weight 46-68g) were obtained according to the manufacturer’s from a local fish farm in Chengdu city, instruction. The integrity of DNA was Sichuan Province. The symptoms of checked using 1.0% (w/v) agarose gel in these carps included: sluggishness, TBE buffer. DNA was quantified and extensive dermatorrhagia mainly around stored at -20°C until required. lower jaw and abdomen, abdominal swelling, and red swelling of the anus. PCR amplification of 16SrDNA gene The morbidity and mortality were about The 16SrDNA gene was partially 15% and 10%, respectively. Necropsy amplified in PCR reactions using the was performed on these crucian carps universal primers 27F and 1492R. The within 30 min of death. Typically, the 27F primer was 5'-AGA GTT TGA TCC hearts and showed hyperaemia TGG CTC AG-3' and the 1429R primer and swelling, the intestines were enlarged was 5'-ACG GCT ACC TTG TTA CGA with diffuse hemorrhagy. The heart CTT-3'. The PCR reaction mixture samples of thirteen crucian carps were included 21μL of ddH2O, 2μL of immediately inoculated in blood plates bacterial DNA, 2μL of each primer (10 405 Wang, Yersina enterocolitica as a cause of septicemia in crucian carp (Carassius carassius) umol), and 23μL of rtaq Mix (Takara, reference (Thoerner et al., 2003). These Japan) in a 50 μL final volume. primers are shown on Table 1 and Amplifications were performed at 94°C synthetized by Shanghai Sangon Biotech for 4min, 94°C for 30s, 60°C for 30s, Companies. The PCR reaction mixture 72°C for 4min (5 cycles); 94°C for 30s, included 12μL of rtaq Mix (Takara, 55°C for 30s, 72°C for 4 min (5 cycles); Japan), 1 μL of bacterial DNA, 11μL of

94°C for 30s, 50°C for 30s and 72°C for ddH2O, 1μL of each primer (10 μmol) in 4 min (30 cycles). PCR products were a 25 μL final volume. Amplifications examined for size and yield using 1.2% were performed with the following PCR (w/v) agarose gel in TBE buffer. The parameters: 94°C for 5 min; 94℃for 60s， obtained product was sequenced on both 48-56℃ for 60s，72℃for 60s (35 cycles); strands using an ABI PRISM 3730 72°C for 10 min. The optimal annealing automated sequencer (ABI, America). temperatures for yadA genes were proposed by gradient PCR, which varied Phylogenetic analysis from 40°C to 60°C. PCR products were DNA sequences were edited and examined for size and yield using 1.2% assembled using the programs DNA Star. (w/v) agarose gel in TBE buffer. A comparison of the 16SrDNA gene sequence of the test strain with the Artificial infection experiment non-redundant collection of sequences The pathogenicity of the Y. enterocolitica was performed using BLAST program was performed in sixty crucian carps (http://www.ncbi.nlm.nih.gov/BLAST) weighing 49-72g. These crucian carps (Raja et al., 2015). Multiple sequence were artificially cultivated in Chengdu alignments were generated using city, Sichuan province. Each treatment ClustalW. A phylogenetic tree was group had 10 apparently healthy crucian constructed using neighbor joining carps randomly allocated, with 2 method with MEGA5 package. The replicate tanks. A volume of 0.1 mL of reliability of the neighbor joining tree bacterial concentration (3×108 cfu/mL) was estimated using bootstrap analysis was given to each fish by intraperitoneal with 1000 replicates. injection. While, the control groups (30 fishes) were treated in the same way but Amplification of virulence gene were inoculated with 0.1 mL of PBS Five virulence genes (ail, yadA, ystB, (pH7.4). This challenge lasted for a week virF and HPIint) of Y. enterocolitica and during this time the fishes were were designed according to relevant normally maintained and not fed. The sequences in Genbank database and mortality was given as number of dead Iranian Journal of Fisheries Sciences (15) 2016 406 animals /total number of animals tested. Results Bacteriological analyses of dead crucian Bacterial isolates carp were carried out in all the cases. Samples were taken from the hearts of Samples were taken from the thirteen crucian carps and inoculated in experimental group and cultured using blood nutrient agar plate and nutrition cefsulodin-irgasan-novobiocin (CIN) agar plate. After incubation at 28°C, the agar. Then, the pure culture was thirteen pure cultures were identified. On identified by biochemical characteristics nutrient agar, single isolates produced and PCR. Death was considered caused smooth transparent colonies with an by inoculated bacteria only if the strain entire edge. These isolates displayed used for inoculation was isolated in pure morphologic characteristics typical of the culture. Yersinia appearing as small rod-shaped, Gram-negative coccobacilli. Biochemical Susceptibility testing characterization identified these isolates Susceptibility of the isolated strain to as Y. enterocolitica (probability 99.9%). twelve antibiotics was determined by For further study, one strain of these Kirby-Bauer disk diffusion method isolates was named G6029, and identified according to the clinical laboratory to be biotype 1B and serogroup 0:8. standards institute guidelines. The test organism was picked up with sterile loop PCR amplification and analysis of 16S and suspended in peptone water and rDNA gene incubated at 37°C for 2 hours. Standard In order to further characterize the isolate, 0.5 McFarland saline suspensions of 16SrDNA of strain G6029 was partially bacteria were used to inoculate Mueller sequenced. This 16SrDNA sequence was Hinton agar media confluently with a 1448bp, which showed 99% homology cotton swab. Antibiotic disks were placed with Y. enterocolitica. It was submitted to on the agar, and incubated at 37°C NCBI, and gained a GenBank accession overnight. The zone of inhibition was number (JX855135). A phylogenetic measured and interpreted as per the CLSI analysis to assess the relationship of guidelines. strian G6029 with other bacterium was (ATCC25922) was used as quality performed (Fig.1). The result revealed control strains for the susceptibility the relationships between strain G6029 testing method. and Yersinia. Strain G6029 belonged to the group of Y. enterocolitica, and grouped together with Y. enterocolitica (FJ717343), as the closest neighbour. 407 Wang, Yersina enterocolitica as a cause of septicemia in crucian carp (Carassius carassius)

Strain G6029 was confirmed again to be bacterial suspension. The time of death Y. enterocolitica in light of the sequence ranged from two to seven days after the analysis of 16S rDNA. challenge. A number of 28 crucian carps that had been injected with strain G6029 Amplification of virulence gene died within a week, while all normal PCR-based assays were developed for controls (30 healthy crucian carps) were the detection of five virulence genes in alive, and had no obvious symptom. The strain G6029. The yadA and virF were mortality of the crucian carps was 93.3%. plasmid-borne genes, while the ail, ystB Y. enterocolitica strains were reisolated and HPIint were chromosomal virulence using CNI-1 culture medium from all genes. In the results the ail, ystB, virF moribund and dead crucian carp after the and HPIint were detected, however, yadA challenge. Y. enterocolitica colonies on gene was not seen after optimizing the CIN formed the characteristic deep red experimental conditions of PCR (Fig. 2). center (bull’s eye) with a transparent Additionally, the lengths of ail, ystB, virF margin and diameter 2-4 mm. and HPIint gene were about 351bp, 146 bp, 561bp and 714bp, respectively, which Susceptibility testing were in line with the anticipant results. The antibiotic resistance of Y. enterocolitica strain G6029 was screened Artificial infection experiment by Kirby-Bauer disk diffusion method. In order to investigate the pathogenicity This stain was resistant to four antibiotics of Y. enterocolitica strain G6029, it was (sulfafurazole, furazolidone, enrofloxacin, injected into abdominal cavity of thirty and norfloxacin). However, it was crucian carps. The symptoms observed in insensitive to two antibiotics intraperitoneally inoculated fish consisted (erythromycin, and penbritin). In addition, of inactivity, anorexia, and red swelling it was susceptible to six antibiotics of the anus, which accorded with the (florfenicol, vibramycin, cefaloridine, natural cases observed. The mortality ciprofloxacin, streptomycin, and was observed 48 h after inoculation of ampicillin). Table 1: Primers of virulence genes for Y. enterocolitica Gene Forward primer 5’-3’ Reverse primer 5’-3’ Tm(℃) Size(bp) ail taa tgt gta cgc tgc gag gac gtc tta ctt gca ctg 50 351 ystB gta cat tag gcc aag aga cg gca aca tac ctc aca aca cc 56 146 yadA ctt cag ata ctg gtg tcg ctg t atg cct gac tag agc gat atc c 56 849 virF ggc aga aca gca gtc aga cat a ggt gag cat aga gaa tac gtc g 48 561 HPIINT tgc gcc atg cgg tcc atc ggt gca taa gat tct cgg 50 714

Iranian Journal of Fisheries Sciences (15) 2016 408

Figure 1: Phylogenetic tree of strain G6029 and other Yersinia.

Figure 2: The virulence genes of Y. enterocolitica strain G6029 1.ail, 2. ystB, 3. virF, 4. yadA, 5.HPI, M.DNA Marker

409 Wang, Yersina enterocolitica as a cause of septicemia in crucian carp (Carassius carassius)

Discussion confirmed the CIN agar was useful for Y. enterocolitica is a Gram-negative, the isolation and identification of Y. predominantly extracellularly located enterocolitica. pathogen that causes food-borne acute or Y. enterocolitica is an important cause chronic gastrointestinal diseases in of acute gastrointestinal disease and animals and humans. Rapid and reliable post-infectious complications in humans isolation and identification of strains of and animals. Y. enterocolitica biotype 1B, the species Y. enterocolitica within the serotype O:8 (1B/O:8), is the most genus Yersinia and the differentiation of pathogenic of the Yersinia species the pathogenic from the non-pathogenic because of the presence of biotypes are increasingly important. high-pathogenicity island and the Isolation of pathogenic Y. enterocolitica Yersinia virulence plasmid (pYV). There can be done using CIN agar which is was a pediatric case of Y. enterocolitica useful to expedite the recovery of Y. 1B/O:8 bacteremia and . A enterocolitica and mVYE agar to 20-month-old girl was admitted to differentiate virulent from non-virulent hospital with fever, pharyngitis, and isolates. The characteristic deep red abdominal pain on day 2. Blood culture center (“ bull’s eye”) with a transparent on admission was positive for Y. margin and appearance of Yersinia enterocolitica 1B/O:8 (Ito et al., 2012). colonies with a diameter 2-4 mm on CIN In this study, Y. enterocolitica strain 6209 incubated at 30°C for 24h is important was also identified to be biotype 1B and for identification and is due to the serogroup 0:8, and it was highly presence of mannitol (Fukushima et al., pathogenic to crucian carps. Fresh fish 2011). from France, Great Britain and Portugal Salmonella-Shigella-desoxycholate-calci were examined for the Y. um chloride (SSDC) agar, and Y. enterocolitica. It showed that Y. enterocolitica chromogenic medium enterocolitica was detected from both (YeCM) were also used as selective agar salmon and trout in Great Britain media. For enrichment, (Andrew et al., 2001). Many materials irgasan-ticarcillin-potassium chlorate about infection in fish (ITC) broth and peptone-sorbitol-bile has been widely reported (Tobback et al., (PSB) broth were incubated at 25°C for 2007; Bastardo et al., 2012; Jaafar et al., 48h (Premaratne et al., 2012). In this 2012). However, there is still little study, Y. enterocolitica strains were information about the Y. enterocolitica reisolated from artificial infection infection in fish by now. experiment using CIN agar that There are some researches Iranian Journal of Fisheries Sciences (15) 2016 410

investigating the distribution of virulence the virulence of Yersinia. However, there genes in clinical isolates of pathogenic Y. was a report about two Y. enterocolitica enterocolitica (Kumar and Virdi, 2012). biotype 1A strains that are usually It also highlights the importance of nonpathogenic and carry the ail gene. addressing genetic and phenotypic The ail gene sequences of biotype 1A variations among closely related bacterial strains displayed similarity to the strains, even those belonging to the same bioserotype 1B/O:8 strain 8081 bioserotype (Garzetti et al., 2012). suggesting that ail-based detection Virulence genes can be applied as methods for Y. enterocolitica alone are distinguishing markers and indicators of insufficient to detect real pathogenic the potential virulence of Y. strains (Sihvonen et al., 2011). A number enterocolitica strains. Plasmid-encoded of 160 isolates of pathogenic Y. targets, like virF and yadA genes, and enterocolitica cultured from stool chromosomally encoded targets, like ail , samples were examined for virulence inv, ystB, and HPIINT genes, have also genes (inv, ail, ystA, ystB, ystC, yadA, been used in PCR to identify Y. virF) by PCR. The positive rate of enterocolitica strains in clinical, food, virulence genes tested in 160 isolates was and environmental samples (Bhaduri et inv (100%), ail (94%), ystA (93%), ystB al., 2005; Thisted and Danielsson, 2005). (7.5%), ystC (5%), yadA (89%) and virF The virulence of Y. enterocolitica can be (82%), respectively (Zheng et al., 2008). increased, by acquiring new genes and/or In this study, five virulence genes (ail, improving the function of essential ystB, HPIINT, yadA, and virF) of Y. virulence proteins, resulting in enterocolitica were analyzed. These permanently hyper-virulent strains. genes except yadA were all detected, The attachment invasion locus (ail) which was basically consistant with the gene, located in the chromosome of above reports. These data showed that pathogenic Y. enterocolitica strains was not all pathogenic Y. enterocolitica the most frequently used targets. The ail necessarily carry all traditional virulence sequence was highly conserved among genes in both plasmids and chromosomes the same serotype strains from different to cause illness. Perhaps, some of them, sources (Huang et al., 2010). The ail and lacking some traditional virulence genes, ystA genes were detected in all the contain other unknown virulence markers strains isolated from humans and animals, that interact with each other and play an but only in two strains isolated from food important role in the diverse (Falcão et al., 2006). A high correlation pathogenecity of pathogenic Y. has been found between the ail gene and enterocolitica. The yadA gene was 411 Wang, Yersina enterocolitica as a cause of septicemia in crucian carp (Carassius carassius) located on the Yersinia virulence plasmid Acknowledgements and its expression is mainly This work was supported by research temperature-regulated occurring at 37ºC. grants from science and technology However, strain G6029 was cultured in department of Sichuan Province 28ºC. So the reason why yadA gene had (2013FZ0014) and construction project not been seen might be due to the low of postgraduate academic degree in temperature or possible plasmid loss on southwest university for nationalities subculture and storage. The HPIint (2016XWD-S071007). (High-Pathogenicity Island) gene is a partial genomic island indispensable for References pathogenicity of Yersinia and was Andrew, R., Davies, Ch., Capell, D., detected in stain G6029, which indicated Jehanno, George, J.E., Nychas, Roy strain G6029 might have high and Kirby, M., 2001. Incidence of pathogenicity. Moreover, the foodborne pathogens on European fish. pathogenicity of the Y. enterocolitica Food Control, 12(2), 67-71 strain G6029 was proved in crucian carps Asadishad, B., Ghoshal, S. and by artificial infection test. This study Tufenkji, N., 2013. Role of cold showedthat this strain had high climate and freeze-thaw on the pathogenicity to crucian carp. survival, transport, and virulence of Furthermore, it also indicated that this Yersinia enterocolitica. strain might be virulent to other aquatic Environmental Science and animals. Technology, 47 (24), 14169-14177. In conclusion, thirteen Y. Bastardo, A., Ravelo, C. and Romalde, enterocolitica strains were isolated from J.L., 2012. Highly sensitive detection C. auratus, and the 16SrDNA, and quantification of the pathogen pathogenicity and antibiotic resistance of Yersinia ruckeri in fish tissues by strain G6029 were studied by molecular using real-time PCR. 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