Shotgun Sequencing of Bacteria from AHPNS A New Shrimp Disease Threat for Thailand SUMMARY The most threatening new problem in Asian shrimp aquaculture at this time is serious mortality from unknown causes that began in China in 2009, then Vietnam since 2010 and more recently in 2011 to the eastern coast of Malaysia and the eastern coast of the gulf of Thailand. So far no potential causative pathogen has been found and possible etiologies include toxins (biotic or abiotic), bacteria and viruses. A case definition for the syndrome affecting shrimp in the outbreak ponds has been described by D.V. Lightner from the University of Arizona (unpublished) and this is consistent with the specimens that have been received and analyzed by Centex Shrimp in Thailand (T.W. Flegel, unpublished). Dr. Lightner has referred to this syndrome as acute hepatopancreatic necrosis syndrome (AHPNS) that can be described as acute progressive degeneration of the hepatopancreas (HP) with the following histological features: lack of mitotic activity in generative E cells of the HP; central dysfuncton of hepatopancreatic B, F and R cells; prominent karyomegaly and massive sloughing of central HP tubule epithelial cells; terminal stages including massive untertubular hemocytic aggregation followed by secondary bacterial infections. To test for the possible involvement of bacteria in causing AHPNS, we carried out shotgun sequencing of bacterial ssrDNA fragments amplified from HP of shrimp from AHPNS ponds. From analysis of approximately 100,000 fragments from 6 disease ponds and one control pond, a total of 8327 unique sequences (taxonomic units or TU) of approximately 400 bp were obtained. Eliminating singleton TU left 1205 TU with reads from 2 or more ponds. A comparison of the read frequency for these yielded 5 TU with the highest difference between test ponds and the control pond. These sequences will be used to design specific probes for in situ hybridization assays with HP tissues from AHPNS shrimp. AHPNS histopathology Sloughing of HP cells Lack of E-cell mitosis Lack of B, F & R cells INTRODUCTION Since the serious mortality from unknown causes that began in China in 2009, there have been rumors that infectious myonecrosis (IMN) caused by the virus IMNV may have been the cause. IMNV was first described from Indonesia (Senapin et al. 2007. Aquaculture 266:32-38) as a probable example for disease translocation with aquaculture stocks from Brazil. However, continual testing at Centex Shrimp has revealed that these have been false rumors (Senapin et al. 2011. BMC J Negative Results in Biomedicine. 10:10). Most of these rumors were probably caused by muscle cramp syndrome that can commonly cause whitened muscles in stressed Penaeus (Litopenaeus) vannamei. Additonal rumors may have resulted from light, false-positive RT-PCR results using the IQ2000 test kit with DNA extracts from shrimp pleopods rather than excised, infected muscle tissue or hemolymph (i.e., internal material). Since samples from the outbreaks in Vetnam and China have tested negative for IMNV by RT-PCR, histopathology and immunohistoghemistry, IMNV can be ruled out as the cause of the problems in Vietnam and China. In addition, other pathogens newly discovered in P. vannamei from Thailand and Vietnam [including Macrobrachium rosenbergii nodavirus (MrNV) and the microsporidian Enterocytozoon hepatopenaei] have also proven not to be the cause of the massive disease outbreaks based on low prevalence in tested specimens from the outbreak ponds as revealed by histological analysis, PCR testing and immunohistochemistry. In addition, tests for nectotizing hepatopancreatic necrosis (NHP) have proven negative by PCR testing, histological analysis and transmission electron microscopy (TEM). karyomegaly Enlarged HP nuclei Hemocytic infiltration 2o bacterial infection Semi-thin HP tissue sections Thai samples of P. vannamei collected from Chantaburi and Rayong provinces in late 2011 and early 2012 showed histological signs of AHPNS and HP tissues were embedded in epoxy resin to prepare for transmission electron microscopy. In prelmnary semi-thin sections stained with toluidine blue, abnormal blebbing of the HP tubule margins was seen. Since it occurred in many tubules in the absence of visible bacteria, supportng Dr. Lightner’s earlier proposal for a possible toxic origin. This phenomenon was also seen in similar HP tissue sections of AHPNS shrimp specimens from Vietnam and Malaysia, indicating that the phenomenon could serve as an additional characteristic of the AHPNS case definition. We are cooperating with Dr. Niti Chuchird and Dr. Chalor Limsuwan at Kasetsart Universty to investigate the possibility of novel bacteria that might be present in shrimp from AHPNS ponds using “shotgun” samples originating from normal ponds and AHPNS ponds. Since this is a new technological approach, we have already begun with the simplest method of screening for bacterial pathogens. Briefly, the process involves use of universal bacterial primers to amplify a 500 bp small subunit ribosomal RNA (ssu rRNA) gene fragment from all bacteria present in shrimp HP tissue samples. These are then sequenced in mass followed by computer analysis to collate the sequences. The objective is to be able to compare the sequences from normal and disease ponds to search for any unusual bacteria characteristic of the disease ponds only. RESULTS Sequencing and bioinformatics Using sets of shrimp pooled from each of 7 ponds (3 diseased and one control pond from Vietnam, and 5 diseased ponds from Thailand), DNA was extracted and used as the template for PCR amplification of an rRNA gene fragment of an expected size of approximately 500 bp. The amplified bands from each pond were cut from the agarose gel, eluted from the gel and subjected to DNA barcode addition for each pond sample. Next, the pooled samples were subjected to Roche 454 sequencing. A total number of 41 million bases were read with an average read of 450 bp yielding approximately 100,000 sequences. At 97% identity for sequences of approximately 400-500 bp with primers at both ends, a total of 8327 unique sequences (taxonomic units or TU) were obtained. Of these, 7123 TU were singletons (i.e., arising from only one pond as single or as multiple reads). Eliminating the singleton TU left 1205 TU with reads from 2 or more ponds. A comparison of the read frequency for each of the 1205 TU for each of the ponds yielded the following 5 TU with the highest difference between test ponds and the control pond. The genus names are numbered and shown in yellow with example species names for Blast hits given in brackets. The latter may or may not be the actual species in the target samples. CONCLUSIONS The bacterial sequences obtained from AHPNS shrimp by shotgun sequencing were not related to sequences normally reported from diseased shrimp, particularly with respect to Vbrio species. Of the genera showing significant homology, Delftia was the most prevalent in our samples, but aside from one report for a new species (Delftia litopenaei) described from normal shrimp in Taiwan (Chen et al. 2011. IJSEM, in press doi:10.1099/ijs.0.037507-0), no species of Delftia has previously been reported from shrimp. Similarly, none of the other 4 genera from the homology search have previously been reported from shrimp and all 5 are most frequently reported from water or wastewater samples. Their possible role in AHPNS will be assessed by preparation of labeled probes for in situ hybridization tests using AHPNS shrimp samples. Acknowledgements Order Burkholderiales: Family Burkholderiaceae Genus Ralstonia (1) Family Comamonadaceae (all found were former Pseudomonas species) Genus Delftia (acidovorans & tsuruhatensis) (2) Genus Pelomonas (aquatica) (3) This work was supported by Mahidol Universty, Charoen Pokphand Co. Ltd., the Higher Education Research Promotion and National University Development, Office of the Thailand Higher Education Commission, the Surathani Shrimp Farmer’s Club, the Thai Frozen Foods Association and the National Research Council of Thailand.