By Timothy William Flegel*
Cultivation of domesticated and genetically selected stocks of the American whiteleg shrimp Penaeus (Litopenaeus) vannamei remains the first choice in Asia with the black tiger shrimp P. monodon a far second. Importance of pathogens and levels of threat depend on the species of shrimp cultivated and on the geographical location of farms. For viral pathogens in Asia, white spot syndrome virus (WSSV) and yellow head virus type-1 (YHV-1) are still the most lethal for both species, although the latter has so far been confined to Thailand. However, a new, lethal variant (YHV-8) has been found in China, and it is recommended that a disease card for this, together with a specific detection method be posted at the NACA website. Also from China, another new virus called covert mortality nodavirus (CMNV) was recently reported [Zhang et al. 2014. A new nodavirus is associated with covert mortality disease of shrimp. J Gen Virol. in press]. We have found that it also occurs in at high prevalence (approximately 40%) in Thai shrimp farms and we have recently also received RT-PCR positive material from India. Its species range and impact on culture in the region have not yet been determined, but it is of urgent concern to do so. Again, it is recommended that a disease card, including the specific RT-PCR detection method be posted at the NACA website and that member countries work together to study the prevalence and impact of this virus.
For P. vannamei only, the next most important viral threat is infectious myonecrosis virus (IMNV) (fortunately still confined to Indonesia) while Taura syndrome virus (TSV) and infectious hypodermal and hematopoietic necrosis virus (IHHNV) are not serious threats to the tolerant shrimp stocks being cultivated. P. vannamei sometimes exhibits abdominal segment deformity disease (ASDD), associated with a retrovirus-like agent [Sakaew et al. 2013. Discovery and partial characterization of a non-LTR retrotransposon that may be associated with abdominal segment deformity disease (ASDD) in the whiteleg shrimp Penaeus (Litopenaeus) vannamei. BMC Veterinary Research. 9, 189].
For P. monodon only, the next most important viral pathogen is Laem Singh virus (LSNV) and an integrase-containing element (ICE) that are together associated with monodon slow growth syndrome (MSGS), but so far, only in Thailand [Panphut et al. 2011. A novel integrase-containing element may interact with Laem-Singh virus (LSNV) to cause slow growth in giant tiger shrimp. BMC Vet Res. 7, 18]. Less important are hepatopancreatic parvovirus (HPV) and monodon baculovirus (MBV), but only when captured P. monodon are used for postlarval production without implementation of proper preventative measures.
The most important non-viral disease threat for both species since 2009 has been called (unadvisedly) early mortality syndrome (EMS). It is characterized by massive sloughing of hepatopancreatic epithelial cells followed by death, and it is called acute hepatopancreatic necrosis disease (AHPND). The causative agent comprises unique isolates of Vibrio parahaemolyticus that carry an approximately 69 kbp plasmid that contains two toxin genes capable of acting together to kill shrimp. They pose no threat to human health. AHPND began in China around 2009 and spread to Vietnam in 2010, Malaysia in 2011, Thailand in 2012 and Mexico in 2013. Two interim PCR detection methods (AP1 and AP2) were introduced at the NACA website in December 2012 based on detection of the 69 kbp plasmid, and AP2 turned out to be the best with about 3% false positive results. Despite this weakness, the method was used successfully to reveal a high prevalence of AHPND bacteria in living broodstock feeds (e.g., polychaetes and bivalves), in broodstock and in post larvae used to stock rearing ponds. One of the 2 toxins (approximately 13 kDa) resembling the Pir binary insect toxins, located on the 69 kbp plasmid and found to act together to cause AHPND was used to develop a new PCR method (AP3). This was released at the NACA website in June 2014. It gave no false positive or false negative results with 104 bacterial isolates tested. It is recommended that the AP3 method be used to identify sources of AHPND bacteria and that positive shrimp or other materials be excluded from shrimp production facilities. It is also recommended that the practice of feeding living marine animals to broodstock shrimp be strongly discouraged unless they have been proven free of AHPND bacteria and other pathogens.
Possible preventative measures against pathogen entry with such feed materials would require treatment that would result in their death and it would include (in declining order of desirability) gamma irradiation (sterilization) of frozen material, pasteurization or freezing. The last of these methods (freezing) was the standard practice for polychaetes fed to shrimp broodstock, and it is still the practice in North and South America. However, the widespread habit of feeding live polychaetes has apparently arisen based on associated increases in nauplii production, at the complete sacrifice of all biosecurity concerns. In my opinion, it would be better to accept decreased nauplius yields in order to insure the integrity of SPF broodstock. This is especially important for the risk of exposure to previously unknown pathogens. Another approach to solve the problem of disease transmission from living polychaetes has been to produce SPF animals in closed culture facilities.
Three other phenomena in the HP have become prominent together with AHPND since 2009. These include high prevalence of the microsporidian Enterocytozoon hepatopenaei in both broodstock and cultivated shrimp [Tangprasittipap et al. 2013. The microsporidian Enterocytozoon hepatopenaei is not the cause of white feces syndrome in whiteleg shrimp P. vannamei. BMC Veterinary Research. 9], of vermiform, aggregated transformed microvilli (ATM) (sometimes mistaken for gregarines) (Sriurairatana et al. 2014. White feces syndrome of shrimp arises from transformation, sloughing and aggregation of hepatopancreatic microvilli into vermiform bodies superficially resembling gregarines. PLos ONE. 9, e99170] and of distorted hepatopancreatic tubules. It is possible that the latter two phenomena may result either from low levels of the toxins that cause AHPND or from separate causes. However, the rapid regional spread of AHPND and the simultaneous increase in prevalence of infections by the distinctly different, endemic pathogen E. hepatopenaei, suggests that the current situation in Asia may have resulted from an industry-wide decrease in rigor of biosecurity measures in shrimp hatcheries and rearing ponds. This could have arisen due to the dramatic reduction in disease outbreaks in cultivated shrimp since the widespread adoption of specific pathogen free (SPF) P. vannamei in Asia since 2001. Even with production based on use of SPF stocks, any decline in biosecurity measures would have left the industry vulnerable to the emergence of any new pathogen.
Although the cause of ATM is unknown and its impact on shrimp production has not been assessed, retarded growth in P. vannamei caused by endemic EHP is rapidly increasing in prevalence in China, Vietnam, Thailand and Malaysia. PCR methods are available for EHP detection [(Tangprasittipap et al. 2013 above) and a LAMP method (Suebsing et al. 2013. Loop-mediated isothermal amplification combined with colorimetric nanogold for detection of the microsporidian Enterocytozoon hepatopenaei in penaeid shrimp. J Appl Microbiol)], and EHP should be added to the list of required pathogens for exclusion from SPF stocks of both P. monodon and P. vannamei. An advisory on the threat from EHP and measures for control has been posted at the NACA website and an accompanying disease card is being prepared.
From 150 ponds in an ongoing Thai study of 200 ponds randomly selected before stocking, the prevalence of ponds affected by AHPND was in the range of 24% while prevalence for the microsporidian Enterocytozoon hepatopenaei (EHP) was 49% and that for vermiform, aggregated transformed microvilli (ATM) (sometimes mistaken for gregarines) was over 80%. The cause of the latter and its impact on production is still unknown, while EHP is associated with severe growth retardation rather than mortality. EHP is an endemic pathogen, generally not present in imported SPF stocks, so contamination occurs in Thailand. Its prevalence in other countries is not yet known.
For all the pathogens described above, the most effective control measures for reducing the risk of disease are to use post larvae derived from domesticated SPF shrimp stocks (with a pathogen exclusion list that includes all major viruses and parasites including E. hepatopenaei), cultivated in biosecure settings under management practices aimed at optimum (not maximum) production.
Dr. Timothy Flegel works in the Department of Biotechnology at Mahidol University in Bangkok Thailand, and heads the Center of Excellence for Shrimp Molecular Biology and Biotechnology. We are pleased to have him contributing as a guest columnist, recommended by Dr. Hui Gong.