Vibrio parahaemolyticus becomes lethal to post-larvae shrimp via acquiring novel virulence factors.

IMPORTANCE: As a severe emerging shrimp disease, TPD has heavily impacted the shrimp aquaculture industry and resulted in serious economic losses in China since spring 2020. This study aimed to identify the key virulent factors and related genes of the Vp TPD, for a better understanding of its pathogenicity of the novel highly lethal infectious pathogen, as well as its molecular epidemiological characteristics in China. The present study revealed that a novel protein, Vibrio high virulent protein-2 (MW >100 kDa), is responsible to the lethal virulence of V. parahaemolyticus to shrimp post-larvae. The results are essential for effectively diagnosing and monitoring novel pathogenic bacteria, like Vp TPD, in aquaculture shrimps and would be beneficial to the fisheries department in early warning of Vp TPD emergence and developing prevention strategies to reduce economic losses due to severe outbreaks of TPD. Elucidation of the key virulence genes and genomics of Vp TPD could also provide valuable information on the evolution and ecology of this emerging pathogen in aquaculture environments.

Clinical protective effects of polyhexamethylene biguanide hydrochloride (PHMB) against Vibrio parahaemolyticus causing translucent post-larvae disease (VpTPD) in Penaeus vannamei.

A new emerging disease called "translucent post-larvae disease" (TPD) in Penaeus vannamei, caused by a novel type of highly lethal Vibro parahaemolyticus (VpTPD), has become an urgent threat to the shrimp farming industry in China. In order to develop an effective disinfectant for the prevention and control of the VpTPD, the clinical protective effects of polyhexamethylene biguanide hydrochloride (PHMB) against VpTPD in Penaeus vannamei were investigated by carrying out an acute toxicity test of PHMB on post-larvae of P. vannamei and its effect of treatment test on VpTPD infection. The results showed that the median lethal concentration of disinfectant (LC50) values of PHMB to post-larvae of P. vannamei after treatment for 24 h, 48 h, 72 h, 96 h were 16.13 mg/L (14.18-18.57), 10.77 mg/L (9.93-11.72), 9.68 mg/L (8.53-11.64), 9.14 mg/L (7.70-10.99), respectively. In addition, a clinical trial showed that 1 mg/L PHMB showed a strong protective effect on the post-larvae of shrimp challenged with 101-104 CFU/ml of VpTPD. The relative percentage survival (RPS) of 1 mg/L PHMB on post-larvae of P. vannamei challenged with VpTPD at 101, 102, 103 and 104 CFU/ml were 63.65 %±6.81, 62.96 %±5.56, 60.00 %±3.75 and 66.67 %±3.75 at 96 hours post infection. The results highlight the clinical protective effects of the PHMB and therefor PHMB can be used as a preventive measure to control early TPD infection in shrimp culture. This study also provides valuable information for the prevention of other bacterial diseases in shrimp culture.

Investigation on Natural Infection of Covert Mortality Nodavirus in Farmed Giant Freshwater Prawn (Macrobrachium rosenbergii).

Covert mortality nodavirus (CMNV), from the Nodaviridae family, is characterized by its unique cross-species transmission and wide epidemic distribution features. In this study, Macrobrachium rosenbergii was proved to be infected naturally by CMNV, which further expand the known host range of CMNV. Here, 61.9% (70/113) of the M. rosenbergii samples collected from Jiangsu Province were CMNV positive in the TaqMan RT-qPCR assay, which indicated the high prevalence of CMNV in M. rosenbergii. Meanwhile, the sequences of CMNV RdRp gene cloned from M. rosenbergii were highly identical to that of the original CMNV isolate from Penaeus vannamei. In situ hybridization (ISH) and histology analysis indicated that the intestine, gill, hepatopancreas and ovary were the targeted organs of CMNV infection in M. rosenbergii, and obvious histopathological damage including vacuolation and karyopyknosis were occurred in the above organs. Notably, the presence of CMNV in gonad alerted its potential risk of vertical transmission in M. rosenbergii. Additionally, numerous CMNV-like particles could be observed in tissues of hepatopancreas and gill under transmission electron microscopy. Collectively, our results call for concern of the potential negative impact of the spread and prevalence of CMNV in M. rosenbergii on its aquaculture, as well as providing a renewed orientation for further investigation and exploration of the diverse pathogenic factors causing M. rosenbergii diseases.

A Novel Picornavirus Discovered in White Leg Shrimp Penaeus vannamei.

Global shrimp farming is increasingly threatened by various emerging viruses. In the present study, a novel picornavirus, Penaeus vannamei picornavirus (PvPV), was discovered in moribund White leg shrimp (Penaeus vannamei) collected from farm ponds in China in 2015. Similar to most picornaviruses, PvPV is non-enveloped RNA virus, with a particle diameter of approximately 30 nm. The sequence of the positive single-stranded RNA genome with a length of 10,550 nts was characterized by using genome sequencing and reverse transcription PCR. The existence of PvPV related proteins was further proved by confirmation of viral amino acid sequences, using mass spectrometry analysis. Phylogenetic analysis based on the full-length genomic sequence revealed that PvPV was more closely related to the Wenzhou shrimp virus 8 than to any other dicistroviruses in the order Picornavirales. Genomic sequence conservative domain prediction analysis showed that the PvPV genome encoded a large tegument protein UL36, which was unique among the known dicistroviruses and different from other dicistroviruses. According to these molecular features, we proposed that PvPV is a new species in the family Dicistroviridae. This study reported the first whole-genome sequence of a novel and distinct picornavirus in crustaceans, PvPV, and suggests that further studies of PvPV would be helpful in understanding its evolution and potential pathogenicity, as well as in developing diagnostic techniques.

Determination of the Infectious Agent of Translucent Post-Larva Disease (TPD) in Penaeus vannamei.

A new emerging disease called "translucent post-larvae disease" (TPD) or "glass post-larvae disease" (GPD) of Penaeus vannamei, characterized by pale or colorless hepatopancreas and digestive tract, has become an urgent threat to the shrimp farming industry. Following this clue that treatment of an antibacterial agent could alleviate the disease, systematic investigation of the potential infectious agent of TPD was conducted using bacterial identification and artificial challenge tests to fulfill Koch's postulates. A dominant bacterial isolate, Vp-JS20200428004-2, from the moribund individuals was isolated and identified as Vibrio parahaemolyticus based on multi-locus sequence analysis. However, Vp-JS20200428004-2 differed from the V. parahaemolyticus that caused typical acute hepatopancreatic necrosis disease. Immersion challenge tests revealed that Vp-JS20200428004-2 could cause 100% mortality within 40 h at a dose of 1.83 × 106 CFU/mL, and experimental infected shrimp showed similar clinical signs of TPD. The Vp-JS20200428004-2 could be re-isolated and identified from the experimental infected individuals. Moreover, histopathological analysis of diseased samples indicated that Vp-JS20200428004-2 caused severe necrosis and sloughing of epithelial cells of the hepatopancreas and midgut in shrimp individuals both naturally and experimentally infected. Our present results indicated that Vp-JS20200428004-2 is a highly virulent infectious agent associated with the TPD and deserves further attention.