Histopathological characterization and in situ hybridization of a novel spiroplasma pathogen in the freshwater crayfish Procambarus clarkii

Ding, Zhengfeng; Yao, Wenjing; Du, Jie; Ren, Qian; Li, Wenjie; Wu, Ting; Xiu, Yunji; Meng, Qingguo; Gu, Wei; Xue, Hui; Tang, Jianguo; Wen, Weidong

doi:10.1016/j.aquaculture.2012.12.007

Cited by 26 publications

(9 citation statements)

References 23 publications

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“…It coincided with the published histological descriptions reporting that hemocytes and tissues rich in hemocytes were the main infection targets of S. eriocheiris (Ding et al, 2013). This result is also significant, as hemolymph sampling is a non-lethal procedure, which is important to monitoring breeders and the selection of resistant strains.…”

Section: Quantitative Detection Of S Eriocheiris In Asymptomatic Crasupporting

confidence: 92%

Quantitative detection and proliferation dynamics of a novel Spiroplasma eriocheiris pathogen in the freshwater crayfish, Procambarus clarkii

Ding

Tang

Xue

et al. 2014

Journal of Invertebrate Pathology

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Section: Quantitative Detection Of S Eriocheiris In Asymptomatic Crasupporting

confidence: 92%

Quantitative detection and proliferation dynamics of a novel Spiroplasma eriocheiris pathogen in the freshwater crayfish, Procambarus clarkii

Ding

Tang

Xue

et al. 2014

Journal of Invertebrate Pathology

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“…Traditionally, a Spiroplasma genus‐specific in situ hybridization (ISH) was set up according to the spiroplasma 16S rDNA (Ding et al . ). However, the extremely slow rate of evolution of 16S rDNA makes it unsuitable for design of probes for interspecies discrimination, whereas the 16S‐23S rRNA ISRs are universally present in all the species in the genus Spiroplasma and have more variations in their sequences enabling the design of species‐specific probes (Bi, Gu & Wang ; Ding et al .…”

Section: Discussionmentioning

confidence: 97%

Direct visualization of the novel pathogen, Spiroplasma eriocheiris, in the freshwater crayfish Procambarus clarkii (Girard) using fluorescence in situ hybridization

Ding

Xia

Xue

et al. 2014

Journal of Fish Diseases

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Spiroplasma eriocheiris is the first spiroplasma strain known to be pathogenic to freshwater crustaceans. It has caused considerable economic losses both in the freshwater crayfish Procambarus clarkii (Girard) and in some other crustaceans. The monitoring of the pathogen in crustacean populations and study of its behaviour in the laboratory require the development of reliable diagnostic tools. In this article, we improved microscopic identification of S. eriocheiris by combining in situ hybridization with specific fluorescently labelled oligonucleotide probes. The established fluorescence in situ hybridization (FISH) allowed simultaneous visualization, identification and localization of S. eriocheiris in the tissues of diseased crayfish P. clarkii and exhibited low background autofluorescence and ideal signal-to-noise ratio. With the advantages of better tissue penetration, potentially more specific and stable, we designed three species-specific oligonucleotide probes utilizing the sequences of 16S-23S rRNA intergenic spacer regions (ISRs) of S. eriocheiris. Positive hybridization signals were visualized in haemocytes and connective tissues of hepatopancreas, cardiac muscle and gill from diseased crayfish. This unique distribution pattern matched the pathological changes when diagnosed by H&E staining and indicated that S. eriocheiris probably spread throughout the tissues in P. clarkii by hemokinesis. This assay will facilitate our understanding of the pathogenesis of S. eriocheiris and enhance the early diagnosis of the novel pathogen.

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“…Chromogenic in situ hybridization (ISH) allows reliable visualization of pathogens directly in tissues and in association with lesions (Ding et al . ). However, a technical disadvantage of ISH or other histopathology methods is their qualitative nature.…”

Section: Introductionmentioning

confidence: 97%

“…For practical reasons, and perhaps greater precision, it is necessary to develop a method to measure parasites in tissues for information on the correlation of infection and lesions. Chromogenic in situ hybridization (ISH) allows reliable visualization of pathogens directly in tissues and in association with lesions (Ding et al 2013). However, a technical disadvantage of ISH or other histopathology methods is their qualitative nature.…”

Section: Introductionmentioning

confidence: 99%

Development of In situ hybridization and real‐time PCR assays for the detection of Hepatospora eriocheir, a microsporidian pathogen in the Chinese mitten crab Eriocheir sinensis

Ding

Chen

Lin

et al. 2016

Journal of Fish Diseases

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A microsporidian parasite, Hepatospora eriocheir, is an emerging pathogen for the Chinese mitten crab Eriocheir sinensis. Currently, there is scant information about the way it transmits infection in the crustacean of commercial importance, including its pathogenesis, propagation and infection route in vivo. In this study, chromogenic in situ hybridization (ISH) and quantitative real-time PCR (qPCR) assays were developed to address this pressing need, and we provided an advance in the detection methods available. Pathogens can be seen in situ with associated lesions using ISH. Positive hybridization signals were noted inside the epithelial cells of the hepatopancreas, and putative free parasite spores were observed within the tubule lumen, which were associated with lesions detected by electron microscopy and haematoxylin and eosin (H&E) analysis. qPCR allows the determination of parasite loads in infected tissues, which is important for understanding disease progression and transmission. The hepatopancreas displayed the biggest statistical copy numbers among different tissues of infected crabs, confirming a tissue-specific pathogen infection characteristic. The qPCR assay also proved to be suitable for the diagnosis of asymptomatic carrier crabs. Combination of the two methods could facilitate the study of H. eriocheir infection mechanism in E. sinensis, enhance the early diagnosis of the pathogen and improve the management of microsporidian diseases in commercial crustaceans.

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Histopathological characterization and in situ hybridization of a novel spiroplasma pathogen in the freshwater crayfish Procambarus clarkii

Cited by 26 publications

References 23 publications

Quantitative detection and proliferation dynamics of a novel Spiroplasma eriocheiris pathogen in the freshwater crayfish, Procambarus clarkii

Quantitative detection and proliferation dynamics of a novel Spiroplasma eriocheiris pathogen in the freshwater crayfish, Procambarus clarkii

Direct visualization of the novel pathogen, Spiroplasma eriocheiris, in the freshwater crayfish Procambarus clarkii (Girard) using fluorescence in situ hybridization

Development of In situ hybridization and real‐time PCR assays for the detection of Hepatospora eriocheir, a microsporidian pathogen in the Chinese mitten crab Eriocheir sinensis

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