Riemerella Anatipestifer Infection Research Articles

Occludin and collagen IV degradation mediated by the T9SS effector SspA contributes to blood–brain barrier damage in ducks during Riemerella anatipestifer infection

Riemerella anatipestifer infection is characterized by meningitis with neurological symptoms in ducklings and has adversely affected the poultry industry. R. anatipestifer strains can invade the duck brain to cause meningitis and neurological symptoms, but the underlying mechanism remains unknown. In this study, we showed that obvious clinical symptoms, an increase in blood‒brain barrier (BBB) permeability, and the accumulation of inflammatory cytokines occurred after intravenous infection with the Yb2 strain but not the mutant strain Yb2ΔsspA, indicating that Yb2 infection can lead to cerebrovascular dysfunction and that the type IX secretion system (T9SS) effector SspA plays a critical role in this pathological process. In addition, we showed that Yb2 infection led to rapid degradation of occludin (a tight junction protein) and collagen IV (a basement membrane protein), which contributed to endothelial barrier disruption. The interaction between SspA and occludin was confirmed by coimmunoprecipitation. Furthermore, we found that SspA was the main enzyme mediating occludin and collagen IV degradation. These data indicate that R. anatipestifer SspA mediates occludin and collagen IV degradation, which functions in BBB disruption in R. anatipestifer-infected ducks. These findings establish the molecular mechanisms by which R. anatipestifer targets duckling endothelial cell junctions and provide new perspectives for the treatment and prevention of R. anatipestifer infection.

A case study of a Riemerella anatipestifer infection on a commercial turkey farm in Croatia

Abstract In this study, Riemerella anatipestifer was isolated in a commercial turkey farm in Croatia. The flock showed signs of respiratory distress, lethargy and elevated mortality. In total twenty poults were examined and the most frequent pathomorphological findings were polyserositis and fibrinous airsacculitis. R. anatipestifer strains were isolated from the air sacs, lungs and spleen from three different birds from the same poultry house. The isolates were compared based on the 16S rRNA gene sequences, and the results of the phylogenetic analysis indicated the infection was caused by serotype 6. The flock received an antibiotic therapy via the drinking water consisting of enrofloxacin, after which the symptoms ended without recurrent infections. This is the first report of Riemerella infection found on a commercial turkey farm in Croatia.

Pathogenicity of E. coli O78 Riemerella anatipestifer Chicken Isolate either alone or in combination in Ducks

The objective of this study was to find the relation between the pathogenicity of (E. coli) O78 chicken isolate alone or mixed with Riemerella anatipestifer infection by conventional methods and modern techniques. The results of this study revealed that the pathogenicity of all groups of Riemerella anatipestifer (RA) was 100% while it was 90% among the ducks inoculated with E. coli and was 100% in the injected with both strains of Riemerella anatipestifer and E. coli together and 0% in the control negative group. RA and E. coli strains were correctly identified using the VITEK 2 compact system with 99% and 94% probability, respectively. The results of polymerase chain reaction confirmed the identification results using specific primers either for E. coli O78 or Riemerella anatipestifer giving products 720bp and 665bp, respectively. Histopathological examination revealed that liver showed congestion in blood vessels and hepatic cell necrosis in the group inoculated within Riemerella anatipestifer, While liver showed only congested blood vessels in E. coli inoculated group. On the other hand, Liver showed severe vacuolar degeneration with focal amyloid infiltration in E. coli and Riemerella inoculated group.

Riemerella anatipestifer infection affects intestinal barrier structure and immune reactions in the duck caecum

ABSTRACT Riemerella anatipestifer (RA) infection causes high mortality and poor feed conversion, leading to great economic losses to the duck industry. This study investigated the effects of RA on the intestinal morphology and immune response of ducks. Histological examination showed that RA infection caused intestinal injury, including significantly reduced mucosal thickness on days 2, 3 and 5, significantly reduced villus height on days 1, 2, 3 and 5 (P < 0.05) and significantly reduced villus height to crypt depth ratios on days 2, 3, 5 and 9 of RA infection (P < 0.05). The expression of intestinal mucosal layer construction-associated genes and tight junction genes was significantly altered on at least one time point (day 1, 2, 3, 5, 9 or 14) after RA infection. Quantitative real-time polymerase chain reaction revealed that RA infection affected intestinal mucosal immune function. The genes encoding TLR4 (toll like receptor-4), TRAF6 (TNF receptor-associated factor 6), MYD88 (myeloid differentiation factor 88), IFN-γ (interferon-γ), IL (interleukin)-4 and IL-8 were significantly upregulated on day 2 of RA infection. Taken together, these results indicate that RA infection negatively affects intestinal barrier function in ducks due to impaired mucosal and villus-crypt structure and alters the mRNA expression of mucous layer construction-, intestinal tight junction-, and intestinal mucosal immunity-related genes.

Assessment of immunization regimens of duck Riemerella anatipestifer vaccines

Riemerella anatipestifer infections of goslings and ducklings can result in high mortality. Since there are at least 21 serotypes of R. anatipestifer, cross-protection is an important goal for vaccine development. In this study, we evaluated the immunostimulatory effect of different immunization regimens - the traditional inactivated vaccine vs prime-boost regimens using DNA and protein subunit vaccines (DNA+subunit, subunit+subunit, subunit+inactivated and DNA+DNA). Results showed that, when compared to the inactivated vaccine, prime-boost regimens induced higher and up to 16-week longer lasting levels of antibody responses, significantly elevated the percentage of the cytotoxic CD8+ T cell and higher expression levels of IFN-γ, IL-6 and IL-12 mRNAs. Furthermore, as an indication of cross-protection, sera from prime-boost regimens were able to recognize lysates of R. anatipestifer serotypes 1, 2 and 6. Prime-boost regimens especially DNA-prime and protein-boost, induce strong long-term immune response and may prove protective for breeder ducks requiring long-term protection. It is worth mentioning that the subunit+inactivated regimen group also elicited strong immune response. The cost of this regimen may only be half of the other prime-boost regimens, making this subunit+inactivated combination an attractive option.

The protective efficacy of specific egg yolk immunoglobulin Y(IgY) against Riemerella Anatipestifer infections

Riemerella anatipestifer (RA) is the significant pathogen of septicemia and duck infectious serositis, diseases which can result in high mortality for ducklings. However, these diseases are difficult to treat because of the bacteria’s broad resistance to multiple drugs. The purpose of this study was to produce a specific egg yolk immunoglobulin Y (IgY) targeted to RA, and to evaluate the protective efficacy of this IgY against RA infection. An RA-inactivated vaccine was produced via centrifugation and formalin treatment, using the most predominant serotype 2 wild-type strains in terms of worldwide prevalence. Anti-RA IgY was produced by immunizing Beijing Red No.1 hens with the inactivated vaccine. Enzyme-linked immunosorbent assays showed that the titer levels of anti-RA IgY antibodies increased significantly after exposure. Specific IgY isolated and purified from yolks effectively inhibited the growth of RA in the antibacterial activity assay, which revealed an 80 % reduction of bacteria populations. Animal experiments showed that duckling survival rates were able to reach up to 100 % after the ducklings were treated with 10 mg intramuscular injections of anti-RA IgY from 1 to 12 h after infection. However, the survival rates of ducklings treated with 30 mg of nonspecific IgY at 1 h after infection were 0%. Additionally, ducklings injected once with anti-RA IgY received complete protection in the first week, but the efficacy of this protection almost entirely disappeared after two weeks. The results suggested that specific anti-RA IgY has the potential to improve the degree of protection and responsiveness of ducklings to RA infections and provide them with passive immunity to RA. With further study, this is expected to become a new method for controlling RA infections.

Characterization of the hemolytic activity of Riemerella anatipestifer.

Riemerella anatipestifer infection causes serious economic losses in the duck industry worldwide. Acute septicemia and high blood bacterial loading in R. anatipestifer infected ducks indicate that R. anatipestifer may be able to obtain iron and other nutrients by lysing duck erythrocytes to support its rapid growth and proliferation in the blood. However, so far, little is known about the hemolytic activity of R. anatipestifer to duck erythrocytes. In this study, 29 of 52 R. anatipestifer strains showed hemolytic activity on duck blood agar, whereas all the tested dba+ (with hemolytic activity on duck blood agar) and dba- strains created pores in the duck red blood cells, with 4.35-9.03% hemolytic activity in a liquid hemolysis assay after incubation for 24 h. The concentrated culture supernatants of all the tested R. anatipestifer strains and the extracted outer membrane proteins (OMPs) from dba+R. anatipestifer strains showed hemolytic activity on duck blood agar. These results, together with the median lethal dose (LD50) of some dba+ and dba-R. anatipestifer strains in ducklings, suggested that there was no direct relationship between the hemolytic capacity of R. anatipestifer on duck blood agar and its virulence.

IL-17A treatment influences murine susceptibility to experimental Riemerella anatipestifer infection

Riemerella anatipestifer causes infectious disease and considerable economic loss in the duck industry worldwide. Our previous studies demonstrated an association between proinflammatory cytokine interleukin (IL)-17A and R. anatipestifer infection. Here, we provide evidence for IL-17A involvement in R. anatipestifer infection using a mouse model. Mice showed higher resistance to R. anatipestifer infection than ducks, with median lethal doses (LD50) of 3.5 × 1010 and 5 × 107 colony-forming units (CFU), respectively. Twenty-four hours after infection, mice with a sub-lethal dose (3.5 × 109 CFU) exhibited levels of IL-17A and IL-23 expression similar to uninfected mice. Thus, we hypothesized that exogenous IL-17A or IL-23 administration affects susceptibility of mice to R. anatipestifer. Mice pretreated with IL-17A or IL-23 prior to sub-lethal dose infection of R. anatipestifer exhibited increased bacterial burden and spleen weights compared to untreated infected mice, confirming the involvement of IL-17A in susceptibility to R. anatipestifer infection in vivo.

Riemerella anatipestifer infection in ducks induces IL-17A production, but not IL-23p19

R. anatipestifer (RA) is one of the most harmful bacterial pathogens affecting the duck industry, and infection is associated with the production of proinflammatory cytokines, including IL-17A. Another proinflammatory cytokine, IL-23, is critical for the development of Th17 cells, which produce IL-17. However, IL-23 roles have not been studied in this infection. Here, we describe the identification and mRNA expression analysis of duck IL-23p19 (duIL-23p19) in splenic lymphocytes and macrophages stimulated with killed RA and in spleens of RA-infected ducks. Expression of duIL-23p19 transcript identified in this study was relatively high in livers of healthy ducks and was upregulated in mitogen-activated splenic lymphocytes as well as in splenic lymphocytes and macrophages stimulated with killed RA. In spleens of RA-infected ducks, expression levels of duIL-23p19 transcript were unchanged at all time points except on days 4 and 7 post-infection; however, duIL-17A and IL-17F expression levels were upregulated in both spleens of RA-infected ducks and splenic lymphocytes and macrophages stimulated with killed RA. In sera collected at 24 h after this infection, duIL-23p19 expression levels were unchanged, whereas IL-17A significantly upregulated. These results suggest that IL-23p19 does not play a critical role in the IL-17A response in early stages of RA-infected ducks.

Cas9 regulated gene expression and pathogenicity in Riemerella anatipestifer

Riemerellosis, a Riemerella anatipestifer infection, can cause meningitis, pericarditis, parahepatitis, and airsacculitis in ducks, leading to serious economic losses in the duck meat industry. However, the molecular mechanism of the pathogenesis and virulence factors of this infection are poorly understood. In the present study, we created a mutant strain RA-YMΔCas9 using trans-conjugation. Bacterial virulence tests indicated that the median lethal dose (LD50) of RA-YMΔCas9 was 5.01 × 107 CFU, significantly lower than that of the RA-YM strain, which was 1.58 × 105 CFU. The distribution and blood bacterial load from the infection groups showed no significant difference in the brain between the RA-YMΔCas9 mutant and the wild-type RA-YM strains, however, the number of mutant strains were significantly reduced in the liver, heart, and blood. Animal immunization experiments demonstrated that the intranasal administration of RA-YMΔCas9 in ducklings provided 80% protection after challenge with the wild-type strain, showing potential use as a live mucosal vaccine. RNAseq analysis indicated that Cas9 protein played a regulatory role in gene expression. This study is the first to report on the involvement of Cas9 in the regulation and pathogenesis of R. anatipestifer, and provides a theoretical basis for the development of relevant genetic engineering vaccines.

Immunogenicity and safety of a live Riemerella anatipestifer vaccine and the contribution of IgA to protective efficacy in Pekin ducks

Riemerella anatipestifer (RA) infections cause major economic losses in the duck industry.In this study, we developed an RA vaccine to control virulent serotype 1 and 2 RA, which predominate in worldwide prevalence. We established a strategy for vaccine candidate screening, and selected strains D15-RDA-92 (serotype 1) and D14-RDA-8 (serotype 2). These strains were characterized by ≤50% embryo mortality and <3.0 serum resistance assay values in in vitro screening. We evaluated the protective efficacy of live bivalent RA vaccines against virulent homologous serotype RA. Ducklings received two oral immunizations with the bivalent vaccine and showed significant protection against two virulent strains (serotypes 1 and 2) at 21 days post-immunization. No death or clinical signs of diarrhea, tremors, or limb swelling were observed in the immunized ducks. In a safety evaluation, ducks immunized with 100 times higher doses showed no clinical signs, mortality, gross lesions, or histological lesions, and body weight of the ducks showed no significant difference compared to that of negative controls. In addition, IgA analysis showed a significant increase in secretory IgA antibodies generated in the trachea and duodenum of orally immunized ducks at 28 days of age. The IgA might be involved in one of the major immune responses to RA and contributes to protecting against virulent RA. In this study, we developed monovalent and bivalent RA vaccines that were safe in ducks and provided significant protective efficacy against virulent homologous RA strains.

Downregulation of common cytokine receptor γ chain inhibits inflammatory responses in macrophages stimulated with Riemerella anatipestifer

Th17-cell-mediated inflammation is affected by the soluble form of common cytokine receptor γ chain (γc). We previously suggested that inflammatory cytokines including interleukin (IL)-17A are associated with Riemerella anatipestifer infection, which a harmful bacterial pathogen in ducks. Here, the expression profiles of membrane-associated γc (duγc-a) and soluble γc (duγc-b) in R. anatipestifer-stimulated splenic lymphocytes and macrophages, and in the spleens and livers of R. anatipestifer-infected ducks, were investigated. In vitro and in vivo results indicated that the expression levels of both forms of γc were increased, showing that marked increases were detected in the expression of the duγc-b form rather than the duγc-a form. Treatment with γc-specific siRNA downregulated mRNA expression of Th17-related cytokines, including IL-17A and IL-17F, in duck splenic macrophages stimulated with R. anatipestifer, whereas the expressions of interferon (IFN)-γ and IL-2 were enhanced. The results showed that the upregulation of γc, especially the duγc-b form, was associated with expression of Th17-related cytokines during R. anatipestifer infection.

The Role of the Regulator Fur in Gene Regulation and Virulence of Riemerella anatipestifer Assessed Using an Unmarked Gene Deletion System

Riemerella anatipestifer, an avian pathogen, has resulted in enormous economic losses to the duck industry globally. Notwithstanding, little is known regarding the physiological, pathogenic and virulence mechanisms of Riemerella anatipestifer (RA) infection. However, the role of Ferric uptake regulator (Fur) in the virulence of R. anatipestifer has not, to date, been demonstrated. Using a genetic approach, unmarked gene deletion system, we evaluated the function of fur gene in the virulence of R. anatipestifer. For this purpose, we constructed a suicide vector containing pheS as a counter selectable marker for unmarked deletion of fur gene to investigate its role in the virulence. After successful transformation of the newly constructed vector, a mutant strain was characterized for genes regulated by iron and Fur using RNA-sequencing and a comparison was made between wild type and mutant strains in both iron restricted and enriched conditions. RNA-seq analysis of the mutant strain in a restricted iron environment showed the downregulation and upregulation of genes which were involved in either important metabolic pathways, transport processes, growth or cell membrane synthesis. Electrophoretic mobility shift assay was performed to identify the putative sequences recognized by Fur. The putative Fur-box sequence was 5′-GATAATGATAATCATTATC-3′. Lastly, the median lethal dose and histopathological investigations of animal tissues also illustrated mild pathological lesions produced by the mutant strain as compared to the wild type RA strain, hence showing declined virulence. Conclusively, an unmarked gene deletion system was successfully developed for RA and the role of the fur gene in virulence was explored comprehensively.

Downregulation of inflammatory cytokines by berberine attenuates Riemerella anatipestifer infection in ducks

Riemerella anatipestifer, an important infectious bacterium affecting the duck industry, has 5–75% mortality, depending on strain virulence. We previously demonstrated that proinflammatory cytokines are involved in inflammation during, and regulating susceptibility to, R. anatipestifer infection. We investigated the effects of the anti-inflammatory compound berberine in duck splenic lymphocytes stimulated with killed R. anatipestifer, and in R. anatipestifer-infected ducks. IL-17A, IL-17F, and IL-1β transcripts were downregulated, and IFN-γ and IL-10 transcripts enhanced, in berberine-treated stimulated splenic lymphocytes, compared to stimulated untreated splenic lymphocytes. Similarly, IL-17A, IL-17F, IL-6, and IL-1β expressions were significantly reduced, and IFN-γ and IL-10 expressions significantly upregulated, in spleens and livers of R. anatipestifer-infected berberine-treated ducks, compared to infected untreated birds. Moreover, infected and treated birds showed increased survival rates and significantly decreased bacterial burdens compared to infected untreated birds, confirming that inflammatory cytokines are strongly associated with R. anatipestifer infection in ducks.

Riemerella anatipestifer M949_0459 gene is responsible for the bacterial resistance to tigecycline.

Based on its important role in last-line therapeutics against multidrug-resistant bacteria, tigecycline has been increasingly important in treating infections. However, mounting reports on tigecycline-resistant bacterial strains isolated from different sources are of concern, and molecular mechanisms regarding tigecycline resistance are poorly understood. Riemerella anatipestifer is a Gram-negative, non-motile, non-spore-forming, rod-shaped bacterium, which causes fibrinous pericarditis, perihepatitis, and meningitis in infected ducks. We previously constructed a random transposon mutant library using Riemerella anatipestifer strain CH3, in present study, we described that Riemerella anatipestifer M949_0459 gene is responsible for the bacterial resistance to tigecycline. Using the minimum inhibitory concentration assay, a mutant strain showed significantly increased (about six-fold) tigecycline susceptibility. Subsequently, the knocked-down gene was identified as M949_0459, a putative flavin adenine dinucleotide-dependent oxidoreductase. To confirm the resistance function, M949_0459 gene was overexpressed in Escherichia coli strain BL21, and the minimum inhibitory concentration analysis showed that the gene product conferred resistance to tigecycline. Additionally, expression of the M949_0459 gene under treatment with tigecycline was measured with quantitative real-time PCR. Results showed that the mRNA expression of M949_0459 gene was elevated under tigecycline treatment with dose range of 1-10 mg/L, and peaked at 4 mg/L. Moreover, two kinds of efflux pump inhibitors, carbonyl cyanide m-chlorophenyl hydrazone and phenylalanine arginyl β-naphthylamide were tested, which showed no function on tigecycline resistance in the strain CH3. Our results may provide insights into molecular mechanisms for chemotherapy in combating Riemerella anatipestifer infections.

Identification of a wza-like gene involved in capsule biosynthesis, pathogenicity and biofilm formation in Riemerella anatipestifer.

Duck infectious serositis is the most serious bacterial disease of ducks. It is caused by Riemerella anatipestifer (RA) infection. The capsule plays an important role in virulence of many pathogenic bacteria. In addition, the capsule has some key biological features. However, few studies have explored the characteristics of the RA capsule. In this study, we mainly constructed a capsular mutants of RA by inactivating the wza gene using homologous recombination. We found that the mutant was failed to produce a capsule layer. The mutant was less resistant to killing by the host complement or by desiccation and oxidative stress. Furthermore, the mutant strain was more hydrophobic, more able to auto-aggregate and underwent increased biofilm formation. Moreover, the mutant was less virulent than the wild-type invivo studies. In summary, we found that the RA capsule was involved in the desiccation and oxidative stress, surface hydrophobicity, complement-mediated killing, biofilm formation, and virulence.

Evaluation of a quantitative real-time PCR for rapid detection of Riemerella Anatipestifer infection in birds.

To establish an accurate, rapid, and a quantifiable method for the detection of Riemerella anatipestifer infection, a widespread infectious disease in birds, we developed a TaqMan-based real-time PCR assay by using DtxR gene-specific primers and a TaqMan probe. The standard curve established with a linear correlation (R2) of 0.998 and efficiency of 99% between the Ct value and the logarithm of the plasmid copy number. The reproducibility and specificity of the real-time PCR assay were confirmed by using plasmids containing DtxR genes or DNAs extracted from well-known bacteria or viruses causing duck diseases. The real-time PCR assay was 100 times more sensitive than the conventional PCR. The results reveal that the established real-time PCR assay might be a useful method for diagnosis and quantitative detection of Riemerella anatipestifer in birds.

Identification of Ribonuclease Z Gene from an Outbreak of Riemerella anatipestifer Infection in Ducks of Bangladesh

Duck septicemia is a deadly disease of duck causing huge economic losses in duck rearing areas. Despite its importance, no study was devoted to unravel the Riemerella (Pasteurella) anatepestifer in Bangladesh. Therefore, a systemic investigation was carried out to identify the isolate targeting ribonuclease Z gene from an outbreak in 2013-2014. Samples were collected from sixty randomly selected dead ducks from three outbreaks areas of North East Bangladesh (Netrokona district). Histopathological, cultural and sugar fermentation test of all samples revealed the infectivity with R. anatipestifer. Polymerase chain reaction specific to R. anatipestifer identified the 421 bp fragment of ribonuclease Z gene in thirty seven cases. Identified gene of the isolate was sequenced (Accession No. KU992381) and phylogenetic lineage was analyzed for the first time in Bangladesh. The phylogenetic analysis showed that Bangladeshi isolate R. anatipestifer BD 2014 resembles highest similarity with the R. anatipesrifer isolated from ducks of china. This study provide evidence that duck septicemia could be the main culprit of mortality in growing ducks in the duck rearing areas. Further investigation is needed to identify secondary or co-infection state as well as the prevalence of this infection including the economic burdens in duck industry in Bangladesh.

Investigation of TbfA in Riemerella anatipestifer using plasmid-based methods for gene over-expression and knockdown.

Riemerella anatipestifer is a duck pathogen that has caused serious economic losses to the duck industry worldwide. Despite this, there are few reported studies of the physiological and pathogenic mechanisms of Riemerella anatipestifer infection. In previous study, we have shown that TonB1 and TonB2 were involved in hemin uptake. TonB family protein (TbfA) was not investigated, since knockout of this gene was not successful at that time. Here, we used a plasmid based gene over-expression and knockdown to investigate its function. First, we constructed three Escherichia-Riemerella anatipestifer shuttle vectors containing three different native Riemerella anatipestifer promoters. The shuttle plasmids were introduced into Riemerella anatipestifer ATCC11845 by conjugation at an efficiency of 5 × 10−5 antibiotic-resistant transconjugants per recipient cell. Based on the high-expression shuttle vector pLMF03, a method for gene knockdown was established. Knockdown of TbfA in Riemerella anatipestifer ATCC11845 decreased the organism’s growth ability in TSB medium but did not affect its hemin utilization. In contrast, over-expression of TbfA in Riemerella anatipestifer ATCC11845ΔtonB1ΔtonB2. Significantly promoted the organism’s growth in TSB medium but significantly inhibited its hemin utilization. Collectively, these findings suggest that TbfA is not involved in hemin utilization by Riemerella anatipestifer.

Comparative genomic analysis identifies structural features of CRISPR-Cas systems in Riemerella anatipestifer.

BackgroundRiemerella anatipestifer infection is a contagious disease that has resulted in major economic losses in the duck industry worldwide. This study attempted to characterize CRISPR-Cas systems in the disease-causing agent, Riemerella anatipestifer (R. anatipestifer). The CRISPR-Cas system provides adaptive immunity against foreign genetic elements in prokaryotes and CRISPR-cas loci extensively exist in the genomes of archaea and bacteria. However, the structure characteristics of R. anatipestifer CRISPR-Cas systems remains to be elucidated due to the limited availability of genomic data.ResultsTo identify the structure and components associated with CRISPR-Cas systems in R. anatipestifer, we performed comparative genomic analysis of CRISPR-Cas systems in 25 R. anatipestifer strains using high-throughput sequencing. The results showed that most of the R. anatipestifer strains (20/25) that were analyzed have two CRISPR loci (CRISPR1 and CRISPR2). CRISPR1 was shown to be flanked on one side by cas genes, while CRISPR2 was designated as an orphan. The other analyzed strains harbored only one locus, either CRISPR1 or CRISPR2. The length and content of consensus direct repeat sequences, as well as the length of spacer sequences associated with the two loci, differed from each other. Only three cas genes (cas1, cas2 and cas9) were located upstream of CRISPR1. CRISPR1 was also shown to be flanked by a 107 bp-long putative leader sequence and a 16 nt-long anti-repeat sequence. Combined with analysis of spacer organization similarity and phylogenetic tree of the R. anatipestifer strains, CRISPR arrays can be divided into different subgroups. The diversity of spacer organization was observed in the same subgroup. In general, spacer organization in CRISPR1 was more divergent than that in CRISPR2. Additionally, only 8 % of spacers (13/153) were homologous with phage or plasmid sequences. The cas operon flanking CRISPR1 was observed to be relatively conserved based on multiple sequence alignments of Cas amino acid sequences. The phylogenetic analysis associated with Cas9 showed Cas9 sequence from R. anatipestifer was closely related to that of Bacteroides fragilis and formed part of the subtype II-C subcluster.ConclusionsOur data revealed for the first time the structural features of R. anatipestifer CRISPR-Cas systems. The illumination of structural features of CRISPR-Cas system may assist in studying the specific mechanism associated with CRISPR-mediated adaptive immunity and other biological functions in R. anatipestifer.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3040-4) contains supplementary material, which is available to authorized users.