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.

Development of a Novel RT-qPCR Detecting Method of Covert Mortality Nodavirus (CMNV) for the National Proficiency Test in Molecular Detection.

Covert mortality nodavirus (CMNV), the pathogen of viral covert mortality disease (VCMD), has caused serious economic losses of shrimp aquaculture in Southeast Asian countries and China in the past decade. In view of that the rapid and accurate laboratory detection of CMNV plays a major role in the effective control of the spread of VCMD. The national proficiency test (NPT) for the detection of covert mortality nodavirus (CMNV) started in China from 2021. In this study, a novel TaqMan real-time reverse transcription quantitative PCR (RT-qPCR) detection method for CMNV with higher sensitivity than previous reports was established based on specific primers and probe designing from the conserved regions of the CMNV coat protein gene for using molecular detection of CMNV in NPT. The optimized RT-qPCR reaction program was determined as reverse transcription at 54.9 °C for 15 min and denaturation at 95 °C for 1 min, followed by 40 cycles including denaturation at 95 °C for 10 s, and annealing and extension at 54.9 °C for 25 s. The detection limit of the newly developed RT-qPCR method was determined to be as low as 2.15 copies of CMNV plasmids template per reaction, with the correlation coefficient (R2) at above 0.99. The new method showed no cross reaction with the six common aquatic animal pathogens and could be finished in one hour, which represents a rapid detection method that can save 50% detection time versus the previously reported assay. The CMNV TaqMan probe based RT-qPCR method developed in present study supplies a novel sensitive and specific tool for both the rapid diagnosing and quantitating of CMNV in NPT activities and in the farmed crustaceans, and will help practitioners in the aquaculture industry to prevent and control VCMD effectively.

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.

Investigation of Pathogenic Mechanism of Covert Mortality Nodavirus Infection in Penaeus vannamei.

Viral covert mortality disease (VCMD), also known as running mortality syndrome (RMS), is caused by covert mortality nodavirus (CMNV) and has impacted the shrimp farming industry in Asia and Latin America in recent years. The pathogenic mechanism of CMNV infecting Penaeus vannamei was investigated in this study. In the naturally infected shrimp, histopathological and in situ hybridization (ISH) analysis verified that CMNV infection and severe cellar structural damage occurred in almost all cells of the ommatidium. Under transmission electron microscopic (TEM), vacuolation and necrosis, together with numerous CMNV-like particles, could be observed in the cytoplasm of most cell types of the ommatidium. The challenge test showed that a low CMNV infectious dose caused cumulative mortality of 66.7 ± 6.7% and 33.3 ± 3.6% of shrimp in the 31-day outdoor and indoor farming trials, respectively. The shrimp in the infection group grew slower than those in the control group; the percentage of soft-shell individuals in the infection group (42.9%) was much higher than that of the control group (17.1%). The histopathological and ISH examinations of individuals artificially infected with CMNV revealed that severe cellar damage, including vacuolation, karyopyknosis, and structural failure, occurred not only in the cells of the refraction part of the ommatidium, but also in the cells of the nerve enrichment and hormone secretion zones. And the pathological damages were severe in the nerve cells of both the ventral nerve cord and segmental nerve of the pleopods. TEM examination revealed the ultrastructural pathological changes and vast amounts of CMNV-like particles in the above-mentioned tissues. The differential transcriptome analysis showed that the CMNV infection resulted in the significant down-regulated expression of genes of photo-transduction, digestion, absorption, and growth hormones, which might be the reason for the slow growth of shrimp infected by CMNV. This study uncovered unique characteristics of neurotropism of CMNV for the first time and explored the pathogenesis of slow growth and shell softening of P. vannamei caused by CMNV infection.