Crayfish (Cherax quadricarinatus) susceptibility to acute hepatopancreatic necrosis disease (AHPND).

Acute hepatopancreatic necrosis disease (AHPND) is an OIE-listed enteric disease that has continued to plague the shrimp aquaculture industry since its first discovery in 2009. AHPND is one of the biggest disease threats to the shrimp aquaculture industry along with white spot disease (WSD) which has severely impacted both crayfish and shrimp aquaculture. AHPND is caused by specific marine Vibrio spp. which carry plasmid-borne binary toxins PirAVp and PirBVp. This research investigated if crayfish are susceptible to AHPND-causing Vibrio parahaemolyticus (VpAHPND) to discern the potential risk that AHPND may pose to the crayfish aquaculture industry. Susceptibility was investigated by challenging Cherax quadricarinatus (Australian red claw crayfish) and Penaeus vannamei (Pacific white shrimp) with VpAHPND in a cohabitation immersion bioassay. Upon termination of the bioassay, crayfish survival was significantly higher than shrimp survival (87% vs. 33%). Hepatopancreas dissected from experimentally challenged animals were screened for the binary toxin genes pirAVp and pirBVp by real-time and duplex conventional PCR assays, and also were examined by H&E histology for the detection of characteristic AHPND pathology. Although AHPND toxin genes pirAVp and pirBVp were detected in a subset of crayfish samples, histopathology did not reveal any pathognomonic lesions that are characteristic of AHPND in any crayfish samples examined. These findings suggest that crayfish are likely resistant to AHPND.

Detection and quantification of Hepatobacter penaei bacteria (NHPB) by new PCR and quantitative PCR assays.

Necrotizing hepatopancreatitis (NHP) is a bacterial disease caused by a Gram-negative bacterium classified as Hepatobacter penaei. H. penaei affects cultured penaeid shrimp in several countries from the western hemisphere, including the USA, and most Central and South American countries that farm shrimp. The current PCR and quantitative PCR (qPCR) assays based on the amplification of the 16S rRNA gene developed at the University of Arizona Aquaculture Pathology Laboratory (UAZ-APL) are the only techniques recommended in the World Organisation for Animal Health (OIE) manual for H. penaei detection. Although these techniques are quite sensitive and specific to H. penaei detection in shrimp, in recent years, rare non-specific amplifications have been observed in the end-point PCR when screening for H. penaei in Artemia cyst samples submitted to the UAZ-APL. To avoid these non-specific amplifications, new end-point PCR and qPCR assays were developed based on the H. penaei flagella gene, flgE. Unlike the current OIE methods, the new H. penaei PCR assay did not provide any non-specific amplification, and the qPCR assay had a detection limit of 100 copies and a log-linear range up to 108 copies. Because the previous PCR-based assay using the 16S rRNA was showing non-specific amplification, the new non-specific product of around 400 bp was sequenced to determine its identity. A phylogenetic analysis revealed 2 clusters of H. penaei: Ecuador and Central-North America. This information will enable us to determine the genetic diversity and possible origin of H. penaei and emphasizes the need to evaluate H. penaei PCR detection methods to avoid inaccurate detection of H. penaei.

Phage Application for the Protection from Acute Hepatopancreatic Necrosis Disease (AHPND) in Penaeus vannamei.

Acute hepatopancreatic necrosis disease (AHPND) caused by Vibrio parahaemolyticus has been one of the most problematic diseases in marine shrimp aquaculture throughout Southeast Asia and Latin America. To evaluate the effectiveness of a bacteriophage (phage) treatment for AHPND, a series of bioassays were carried out in a marine shrimp (Penaeus vannamei) model using an AHPND-V. parahaemolyticus strain that is highly pathogenic to shrimp. We monitored the mortality and histopathological changes during phage treatment. Shrimps treated with phage prophylaxis and phage therapy displayed significant protection from AHPND and survived a lethal bacterial challenge.

Dense populations of the microsporidian Enterocytozoon hepatopenaei (EHP) in feces of Penaeus vannamei exhibiting white feces syndrome and pathways of their transmission to healthy shrimp.

White feces syndrome (WFS) is an emerging problem for penaeid shrimp farming industries in SE Asia countries, Thailand, Malaysia, Vietnam, Indonesia, China, and in India. This occurrence of this syndrome is usually first evidenced by the appearance of white fecal strings floating on surface of the shrimp ponds. The gross signs of affected shrimp include the appearance of a whitish hindgut and loose carapace, and it is associated with reduced feeding and growth retardation. To investigate the nature of the white feces syndrome, samples of white feces and shrimp hepatopancreas tissue were collected from Penaeus vannamei in affected farms in Indonesia, and these were examined histologically. Within the white feces, we found densely packed spores of the microsporidian Enterocytozoon hepatopenaei (abbreviated as EHP) and relatively fewer numbers of rod-shaped bacteria. From WFS ponds, hepatopancreas samples form 30 individual shrimp were analyzed by histology and in situ hybridization. The results showed that all of the shrimp examined were infected with EHP accompanied by septic hepatopancreatic necrosis (SHPN). Midgut epithelial cells were also infected and this increased the number of tissue types being affected by EHP. By PCR, EHP was detected in all the samples analyzed from WFS-affected ponds, but not in those sampled from healthy shrimp ponds. To determine the modes of transmission for this parasite, we performed feeding and cohabitation bioassays, the results showed that EHP can be transmitted through per os feeding of EHP-infected hepatopancreas tissue to healthy shrimp and through cohabitation ofinfected and healthy shrimp. In addition, we found the use of Fumagillin-B, an antimicrobial agent, was ineffective in either reducing or eliminating EHP in infected shrimp.

Characterization of a new strain of Taura syndrome virus (TSV) from Colombian shrimp farms and the implication in the selection of TSV resistant lines.

Prior to 2004, Colombian shrimp farming benefited from a selection program in which Penaeus vannamei stocks were developed with resistance to Taura syndrome disease (TS). However since 2004, TS reappeared as a significant disease. In 2010, an apparently new strain of TSV (designated as CO 10) was collected in Colombia. Its genome was sequenced and compared with six other fully sequenced isolates. This analysis revealed that the TSV CO 10 is closely related to the isolates from Hawaii and Venezuela. Phylogenetic analysis based on capsid protein 2 (CP2) region from 59 TSV isolates shows that the recent Colombian isolates (2006-2010) form a new cluster and differ from the previous Colombia isolates (1994-1998) by 4% in nucleotide sequence. The virulence of this CO 10 isolate was similar to a Belize TSV determined through experimental infection in P. vannamei showing 100% mortalities and similar survival curves. By RT-qPCR for TSV, the viral loads were also close in the infected shrimp from both CO 10 and Belize at the order of 1×10(10) copies per µl RNA. To develop TSV-resistant lines, the candidate shrimp should be challenged with virus strains that have been isolated most recently from the regions where they will be cultured. This study suggests that the TSV present in Colombian shrimp farms during the last 5 years is a new TSV strain with high virulence.

A novel agent (Endozoicomonas elysicola) responsible for epitheliocystis in cobia Rachycentrum canadum larvae.

Aquaculture of cobia has gained popularity in the last decade, and this species is now farmed in several countries in Latin America and Asia. Despite recent improvement in production techniques that allowed the expansion of the industry, little is known about the diseases that affect cobia during the larviculture stage. In this article we investigated the cause of mass mortalities occurring 13-20 d post-hatching in 3 cycles of cobia larviculture. Wet mounts from diseased larvae gills revealed the presence of cyst-like basophilic inclusions. DNA from the cysts was extracted and PCR amplified using the 16S rRNA gene universal primers for prokaryotes. The amplified products were sequenced and analyzed using BLAST, finding a similarity of 99% with Endozoicomonas elysicola, a Gram-negative bacterium. Confirmation of E. elysicola was conducted by designing a specific probe for in situ hybridization. Specific primers were also designed for diagnostic purposes. This is the first report of epitheliocystis in cobia larvae and also the first report of E. elysicola as an epitheliocystis-causing agent.

Protection from yellow head virus (YHV) infection in Penaeus vannamei pre-infected with Taura syndrome virus (TSV).

Pacific white shrimp Penaeus vannamei that were pre-exposed to Taura syndrome virus (TSV) and then challenged with yellow head virus (YHV) acquired partial protection from yellow head disease (YHD). Experimental infections were carried out using specific-pathogen-free (SPF) shrimp which were first exposed per os to TSV; at 27, 37 and 47 d post infection they were then challenged by injection with 1 × 104 copies of YHV per shrimp (designated the TSV-YHV group). Shrimp not infected with TSV were injected with YHV as a positive control. Survival analyses comparing the TSV-YHV and YHV (positive control) groups were conducted, and significant survival rates were found for all the time groups (p < 0.001). A higher final survival was found in the TSV-YHV group (mean 55%) than in the positive control (0%) (p < 0.05). Duplex reverse transcription quantitative PCR was used to quantify both TSV and YHV. Lower YHV copy numbers were found in the TSV-YHV group than in the positive control in pleopods (3.52 × 109 vs. 1.88 × 1010 copies µg RNA-1) (p < 0.001) and lymphoid organ (LO) samples (3.52 × 109 vs. 1.88 × 1010 copies µg RNA-1) (p < 0.01). In situ hybridization assays were conducted, and differences in the distribution of the 2 viruses in the target tissues were found. The foci of LO were infected with TSV but were not infected with YHV. This study suggests that a viral interference effect exists between TSV and YHV, which could, in part, explain the absence of YHD in the Americas, where P. vannamei are often raised in farms where TSV is present.

Does hyperthermia increase apoptosis in white spot syndrome virus (WSSV)-infected Litopenaeus vannamei?

Apoptosis plays a critical role in development and maintenance of multicellular organisms. It has also been described as an anti-viral mechanism in both insects and vertebrates. In fact, to escape the immune system and to increase their spread, some viruses such as baculovirus produce anti-apoptotic molecules. Conversely, a recent report showing a positive correlation between the number of apoptotic cells and the severity of white spot syndrome virus (WSSV) infection in Penaeus monodon suggested that apoptosis might be the cause of death in viral-infected shrimp. Searching for the mechanisms involved in the beneficial effect of hyperthermia for WSSV-infected Litopenaeus vannamei (also called Penaeus vannamei) and considering that hyperthermia increases apoptosis in other experimental models, we investigated the presence of apoptosis by Tdt-mediated dUTP nick-end label (TUNEL), from 4 of 168 h in 3 groups of 50 L. vannamei juveniles. Group 1 consisted of experimentally infected shrimp (intramuscular injection of 3 x 10(7) viral copies) kept at 25 degrees C, Group 2 of similarly infected shrimp kept at 32 degrees C and Group 3 of uninjected shrimp kept at 32 degrees C. Apoptosis was found only in WSSV-infected individuals. Shrimp at 25 degrees C were positive for apoptotic cells in 48 (16%) of their examined tissues or organs, compared to 62 (21%) for those at 32 degrees C. Moreover, shrimp at 32 degrees C also had a significantly higher overall mean apoptotic index (AI) than shrimp at 25 degrees C (p < 0.05). Comparison of mean AI at 72, 96 and 120 h post-infection showed that individuals at 32 degrees C presented a significantly higher values than those at 25 degrees C. These results suggested that hyperthermia might facilitate apoptosis in WSSV-infected L. vannamei and might be one of the mechanisms responsible for increased survival of infected shrimp maintained at 32 degrees C.