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Liang T.,Nanjing Normal University | Ji H.,Nanjing Normal University | Du J.,Nanjing Normal University | Ou J.,Nanjing Normal University | And 6 more authors.
Molecular Biology Reports

To investigate the interaction between Chinese mitten crab Eriocheir sinensis hemocytes and the pathogen Spiroplasma eriocheiris, a system for in vitro culture of E. sinensis hemocytes with high viability was developed. Following optimization of conditions, hemocytes survived for >35 days. After challenge with the novel crustacean pathogen S. eriocheiris, E. sinensis hemocytes began to develop vacuoles, and then they began to die (within 60 h). Real-time RT-PCR analysis of S. eriocheiris infected hemocytes identified increased expression levels of antilipopolysaccharide factor (ALF), peroxinectin (Pox) and clip domain serine protease (cSP) genes. The expression levels of ALF, Pox, and cSP genes in hemocytes of E. sinensis demonstrated that all three immune genes were significantly induced by challenge with S. eriocheiris. Increases in Pox mRNA levels were highest (up to 36-fold) and peaked at 24-48 h post-challenge (pc) (P < 0.05) and lesser increases were evident with ALF and cSP, peaking at 24 h and at 12-48 h pc, respectively. The hemocytes culture method described herein provides a feasible in vitro research model of E. sinensis that can be used to study its immune reactions against various crab pathogens. © Springer Science+Business Media B.V. 2012. Source

Wu T.,Nanjing Normal University | Wu T.,Baoying Center for Control and Prevention of Aquatic Animal Infectious Disease | Ding Z.,Nanjing Normal University | Ren M.,Nanjing Normal University | And 6 more authors.

Instead of the usual host, the goldfish, cyprinid herpesvirus 2 (CyHV-2), has been detected in the Prussian carp (Carassius gibelio) from Hungary, the Czech Republic and mainland China in recent years, which has caused the massive mortality of C. gibelio and appears to be spreading worldwide. From May to July of 2011 and 2012, the massive mortality of C. gibelio was observed in many aquatic breeding ponds in northern Jiangsu province, eastern China. Moribund fish showed petechial and eccymotic hemorrhages in gills and around the base of the fins and white color of fins. Histological examination revealed that tissues from gill, kidney and spleen presented symptoms of infiltration of hemocytes and the lesions. Transmission electron microscopy of these cells demonstrated naked herpes-like virus nucleocapsids (95-110. nm) in their nuclei, while the cytoplasm contained multiple aggregates of enveloped viral particles (170-200. nm). CyHV-2 infected gill, kidney and spleen and multiplied or enveloped in both nucleus and cytoplasm of leukocytes or hematopoietic cells. Maturation of CyHV-2 occurred in the cytoplasm, particularly associated with Golgi apparatus, indicating that the virus from C. gibelio probably contains viral glycoproteins. PCR method was used for detecting the virus by using CyHV-2 primers and produced positive results in the tissues of gill, kidney and spleen but negative results in the fins, liver and intestine, which coincided with histological and morphological results. The present work presents the ultrastructural pathology of this serious disease in C. gibelio caused by CyHV-2. It will be helpful to further understand the mechanism of CyHV-2 infection of this new fish host. © 2013. Source

Liang T.,Nanjing Normal University | Wu T.,Nanjing Normal University | Wu T.,Baoying Center for Control and Prevention of Aquatic Animal Infectious Disease | du J.,Nanjing Normal University | And 4 more authors.
African Journal of Biotechnology

In this study, a virus similar to the causative agent of white spot syndrome virus (WSSV) but without tail-like extension was identified and characterized from diseased Penaeus vannamei and moribund Procambarus clarkia. Contrary to previous reports, white spots were not observed on the carapace of the diseased P. vannamei but with ulceration on the carapace and red tail. All samples were analyzed for WSSV and Sporoplasma eriocheiris using polymerase chain reaction (PCR) methods. Samples were negative for S. eriocheiris but positive for WSSV. Following the World Organization for Animal Health (OIE) standard protocol, the result of nest-PCR showed a characteristic band of 1447 bp, suggesting that the pathogen of P. vannamei and P. clarkii was similar to the causative agent of WSSV. Interestingly, transmission electron microscopy of sectioned tissues and negatively stained samples revealed an elliptical shaped virus-like particle but without tail-like extension, which was different from previous reported cases of WSSV. The major envelope proteins, VP19, VP26 and VP28 were cloned and sequenced. Results show that the present isolates had over 97% DNA and 100% amino acid sequence similarity to the known WSSV. These results suggest that this tail less virus may be a different strain of the WSSV virus exhibiting a different disease sign but equally virulent. Results of this study broaden our understanding of WSSV symptoms and diversity. ©2011 Academic Journals. Source

Xiu Y.,Nanjing Normal University | Xiu Y.,Baoying Center for Control and Prevention of Aquatic Animal Infectious Disease | Wu T.,Nanjing Normal University | Wu T.,Baoying Center for Control and Prevention of Aquatic Animal Infectious Disease | And 3 more authors.

Spiroplasmas are small, wall-less, helical, and motile bacteria classified within the class Mollicutes. Spiroplasma has been identified as a lethal pathogen of four freshwater crustaceans in previous studies. Here, the fifth crustacean host of spiroplasma was found. A novel disease of oriental river prawn Macrobrachium nipponense appeared in the summer of 2012 in Jiangsu province of China. Morphological observation, molecular biological methods and infection experiments identified the pathogen as a spiroplasma. The agent isolated from diseased prawns was able to pass through membrane filters with pores 220. nm in diameter and cultivated by R2 medium. A 16S rRNA complete sequence was cloned from the isolation and alignment results revealed that the spiroplasmas from freshwater crustaceans were highly related. Phylogenetic analysis of these sequences showed that the five freshwater crustacean spiroplasma strains had a close relationship with Spiroplasma mirum. The pathogenicity of the agent, evaluated by the mortalities, was determined to be 65% through a challenge experiment. The above results indicated that M. nipponense is another new spiroplasma host in aquatic crustaceans, thus further indicating that more attention should be paid to this pathogen. © 2014 Elsevier B.V. Source

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