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The study reports diversity in nitrifying microbial enrichments from low (0·5–5‰) and high (18–35‰) saline ecosystems. Microbial community profiling of ammonia‐oxidizing bacteria (AOB) and nitrite‐oxidizing bacteria (NOB) enrichments was analysed by sequencing 16S rRNA and was processed using Mothur pipeline. The α‐diversity indices showed the richness of nitrifying bacterial consortia from the high saline environment and were clustering based on the source of the sample. AOB and NOB enrichments from both the environments showed diverse lineages of phyla distributed in both groups with 38 and 34 phyla from low saline and 53 and 40 phyla in high saline sources, respectively. At class level, α‐ and γ‐proteobacteria were found to be more dominant in both the enrichments. AOBs and NOBs in enrichments from low saline environments were dominated by Nitrosomonadaceae, Gallionellaceae (Nitrotoga sp.) and Ectothiorhodospiraceae and Nitrospira, respectively. Though Chromatiaceae were present in both AOB and NOB enrichments, Nitrosoglobus and Nitrosococcus dominated the AOBs while NOBs were dominated by uncultured genera, whereas Rhizobiales were found in both the enrichments. AOBs and NOBs in enrichments from high saline environments were dominated by Nitrospira‐like AOBs, Nitrosomonas and Nitrosococcus genera, whereas ammonia‐oxidizing archaea (AOA) group included Nitrosopumilus and Nitrososphaera genera comprising and Nitrospirae, respectively. The majority of the genera obtained in both the salinities were found to be either uncultured or unclassified groups. Results of the study suggest that the AOB and NOB consortia have unique and diverse microbes in each of the enrichments, capable of functioning in aquaculture systems practised at different salinities (0–60 ppt). [ABSTRACT FROM AUTHOR]

Abstract: Beggiatoa species are filamentous sulfide-oxidizing bacteria belonging to the family Beggiatoaceae that contains several largest bacteria known today. These large sulfur bacteria occur in diverse ecosystems and play an important role in the global sulfur, nitrogen and phosphorus cycle. In this study, sediment samples from brackishwater shrimp culture ponds and other brackishwater ecosystems from Tamil Nadu, southeast coast of India, were enriched for Beggiatoa species. Extracted hay medium supplemented with catalase was used and were incubated for two weeks at 28°C. Out of seven set-ups, four yielded positive growth of filamentous sulfide-oxidizing bacteria. The filaments were several millimeters long, ranged in width between 2 and 15μm and exhibited typical gliding motility. The 16S rRNA gene of four single filaments representing the four positive enrichments was subjected to PCR-DGGE followed by sequencing. All four filaments were affiliated to the Beggiatoaceae, but showed less than 89% identity with the Beggiatoa type strain Beggiatoa alba and less than 93% identity with any other sequence of the family. One of the four filaments revealed a nearly full-length 16S rDNA sequence (1411bp) and it formed a monophyletic cluster with two of the partial DGGE-16S rRNA gene sequences (99–100% identity) within the Beggiatoa species cluster. These organisms could possibly represent a novel genus within the family Beggiatoaceae. The fourth partial sequence affiliated with less than 93% sequence identity to the genera Parabeggiatoa, Thioploca and Thiopilula, and was likewise strongly delineated from any sequence published in the family. [Copyright &y& Elsevier]

Clostridium chauvoei causes fatal black quarter infection in cattle and buffaloes. The quorum sensing (QS) system, a bacterial cell to cell communication process, of the pathogen was characterized in the current study. The results indicated that C . chauvoei lacked luxS (autoinducer-2) based quorum sensing as detected by the sensor strain Vibrio harveyi BB170. This was supported by absence of luxS gene in C . chauvoei genome. However, the genomic analysis indicated the presence of agrBD system in all three genomes of C. chauvoei available at the NCBI database. The AgrD, which synthesizes QS messenger auto-inducing peptide, was a 44 amino acid protein which shared 59% identity and 75% similarity with AgrD of C. perfringens strain 13 and 56% identity (20% coverage) with Staphylococcus aureus N315. The functional cysteine amino acid was conserved in all the strains. The genomic organisation further suggests the presence of diguanylate cyclase, a gene responsible for synthesis of secondary messenger cyclic di-GMP, at 3’ immediate downstream of agrD gene. The real time expression analysis for agrD gene indicated that expression was better at 37 °C (1.9–3.7 fold increase) compared to a higher temperature of 40 °C. However, stable expression was observed at different growth stages (log and early stationary phase) with 0.8–1.4 fold changes in expression pattern. The results indicate the presence of a constitutively expressed agrBD quorum sensing system in C. chauvoei . [ABSTRACT FROM AUTHOR]

In the present study, a suitable carrier system was developed for the delivery of dsRNA into Penaeus monodon ( P. monodon ) post larvae to silence the Monodon baculovirus (MBV) structural gene of p74. The carrier system was developed by layer by layer adsorption of oppositely charged chitosan-dextran sulfate, on charged silica nanoparticles. The silica template was removedto produce multilayered hollow nanocapsules (CS-DS) that were utilized for dsRNA loading at an alkaline pH. The capsule's surface was modified by conjugating with shrimp feed for enhanced cellular uptake. In vivo cellular uptake of CS-DS/FITC loaded nanocapsules conjugated with feed was studied after oral administration into post-larvae. The results revealed that the encapsulated FITC was effectively delivered and exhibited a sustained release into the cytoplasm of shrimp post-larvae. The MBV challenge study for structural gene p74was conducted after 3–25 days of post infection (dpi) with respective CS-DS/dsRNA coated with feed. The results showed a significant survival rate of 86.63% and effective gene silencing in P. monodon . Our findings indicated that the delivery of dsRNA using shrimp feed coatedCS-DSnanocapsules could be a novel approach to prevent viral infections in shrimp. [ABSTRACT FROM AUTHOR]

Enterocytozoon hepatopenaei (EHP), a microsporidian parasite, has emerged as a serious pathogen reported to be associated with retarded growth in cultured shrimp in many of the shrimp growing countries in Asia. As a part of ongoing disease surveillance among the farmed shrimp, we have investigated Penaeus (Litopenaeus) vannamei cultured in the south-east coast of India for EHP infection using light and scanning electron microscopy, histopathology, polymerase chain reaction (PCR) and in situ hybridisation. Squash preparation of hepatopancreas and white faecal strings showed large number of microsporidian spores. Spores under scanning electron microscope appeared oval and measured 1.7 × 1.0 μm. Histology of infected animals showed severe degeneration of hepatopancreatic tubules. Basophilic inclusions resembling the developmental stages of EHP were found in the epithelial cells and large number of spore aggregations was observed in the tubular lumen. Enlargement of haemal sinuses and encapsulation of hepatopancreatic tubules were also observed in some cases. DNA extracted from hepatopancreas was subjected to PCR amplification using primers targeting microsporidian ssu rRNA gene. The PCR yielded an expected product of ~ 951 bp and the sequences showed 100% identity with the EHP reported from Vietnam, Thailand and China. Further screening of field samples was carried out using nested PCR employing EHP-specific primers. Of the 137 juvenile P. vannamei samples tested, 10 were found to be positive in the first step and 77 in the nested PCR. Overall prevalence of EHP was estimated to be 63.5%. However, only first step PCR-positive samples showed discernible number of spores in the hepatopancreas under a light microscope. Post-larvae of P. vannamei collected from a hatchery were found to be PCR negative for EHP. In situ hybridisation using EHP-specific DIG-labelled probe showed positive signals in infected hepatopancreatic tissue. Animals collected from white faeces syndrome (WFS)-affected pond showed higher prevalence of EHP (96.4%) compared to those from the unaffected pond (39.7%). On the contrary, slow growing animals showed low prevalence (58.5%) compared to normally growing animals (80.8%). Although EHP could be detected from slow-growing as well as WFS-affected animals, the present study could not conclusively elucidate the association of EHP with these clinical signs through experimental infection trials. This report forms the first record of the emergence of EHP infection in cultured SPF P. vannamei in India. Statement of relevance E. hepatopenaei (EHP), a microsporidian parasite, is an emerging pathogen having serious economic consequences to cultured shrimp in Asia. This report forms the first record of the emergence of the parasite in farmed P. vannamei from India, and presents a comprehensive account of the pathogen and its impact on the host. [ABSTRACT FROM AUTHOR]

Abstract: White spot syndrome virus (WSSV), is a highly virulent rapidly replicating large, enveloped, double-stranded DNA (dsDNA) virus, causing an estimated losses of over US$6billion since its emergence in 1992. As part of the better management practices (BMPs), ploughing, tilling and sun-drying of shrimp culture ponds are advocated for prevention and control of this disease. Despite this, instances of outbreaks of white spot disease (WSD) recur, causing loss to shrimp farmers. Some studies have indicated that WSSV DNA is reported to persist for over 20months in sediments as detected by PCR. Since mere detection of virus using PCR methods does not indicate its viable nature and ability to infect, information on its viability outside the host in water and pond sediment is necessary. Hence, in this study, the viability of WSSV in seawater and shrimp pond sediments under experimentally simulated drainable and non-drainable pond conditions was examined by shrimp infectivity experiments. WSSV with an initial viral load of 1000virionsmL−1 was found to be viable for a period of 12days in seawater of 27ppt salinity, pH of 7.5 at 29–33°C as revealed by its ability to infect juvenile shrimp, whereas, in shrimp pond sediment (with initial viral load of 211,500copiesg−1), the virus was viable and infective up to 19days despite sun-drying. In the case of non-drainable conditions, WSSV (753,600copiesg−1) remained infective for a period of 35days. Although the sediment samples tested nested PCR positive after 19days of sun-drying and 40days under water-logged conditions, shrimps did not develop WSD, suggesting that WSSV was not viable. Over a period of time after 21days under sun-drying and 40days under non-drainable experimental conditions, due to reduction in viral load, sediments were positive only by nested PCR, and by this time, viability of WSSV was almost lost as revealed by shrimp infectivity. Hence, PCR testing of shrimp farm sediment before starting culture as one of the BMPs may help in ensuring biosecurity from WSSV. The information generated here would help in the improvement of better management practices (BMPs) with regard to pond preparation protocols for shrimp aquaculture. [Copyright &y& Elsevier]

Abstract: A Pseudomonas sp PS-102 recovered from Muttukkadu brackish water lagoon, situated south of Chennai, showed significant activity against a number of shrimp pathogenic vibrios. Out of the 112 isolates of bacterial pathogens comprising Vibrio harveyi, V. vulnificus, V. parahaemolyticus, V. alginolyticus, V. fluvialis, and Aeromonas spp, 73% were inhibited in vitro by the cell-free culture supernatant of Pseudomonas sp PS-102 isolate. The organism produced yellowish fluorescent pigment on King''s B medium, hydrolysed starch and protein, and produced 36.4% siderophore units by CAS assay and 32 μM of catechol siderophores as estimated by Arnow''s assay. The PS-102 isolate showed wide ranging environmental tolerance with, temperatures from 25 to 40 °C, pH from 6 to 8, salinity from 0 to 36 ppt, while the antagonistic activity peaked in cultures grown at 30 °C, pH 8.0 and at 5 ppt saline conditions. The antagonistic activity of the culture supernatant was evident even at 30% v / v dilution against V. harveyi. The preliminary studies on the nature of the antibacterial action indicated that the antagonistic principle as heat stable and resistant to proteolytic, lipolytic and amylolytic enzymes. Pseudomonas sp PS 102 was found to be safe to shrimp when PL-9 stage were challenged at 107 CFU ml−1 and by intramuscular injection into of ∼5 g sub-adults shrimp at 105 to 108 CFU. Further, its safety in a mammalian system, tested by its pathogenicity to mice, was also determined and its LD50 to BALB/c mice was found to be 109 CFU. The results of this study indicated that the organism Pseudomonas sp PS 102 could be employed as a potential probiont in shrimp and prawn aquaculture systems for management and control of bacterial infections. [Copyright &y& Elsevier]

Histopathology of Y-organ (moulting gland) in Indian white shrimp Fenneropenaeus indicus was studied after challenging the shrimp with virulent white spot syndrome virus (WSSV). Histopathological investigation on the Y-organ collected from moribund shrimps revealed the presence of intranuclear basophilic inclusions, characteristic of WSSV. More than 70% of the Y-organ cells were infected, suggesting the degenerated state of the organ. The cellular integrity of the Y-organ was completely destroyed by the WSSV. Further, Y-organ tissue samples collected from all the 16 experimental shrimp were positive by one-step polymerase chain reaction (PCR), confirming severe WSSV infection. The infected Y-organ in F. indicus with majority of the organ cells observed in the lysed condition suggests a physiological dysfunction of the organ. In uninfected and healthy controls, the lobulated Y-organ showed closely packed normal cells with prominent nuclei and sparse cytoplasm. Physiological implication of a degenerated Y-organ in the moulting and reproduction of the penaeid shrimp is discussed. [Copyright &y& Elsevier]

[Display omitted] • This LAMP is a closed tube and comparatively simple and specific EHP diagnostic assay. • This LAMP assay is developed based on EHP spore wall protein (SWP) gene sequence. • This LAMP can detect EHP in all clinical samples such as Hepatopancreas, faeces, pond water and soil. • In combination with FTA cards this assay can detect EHP within 1 h (LAMP-40 min, DNA elution – 17 min). • This EHP LAMP can be possibly performed by the farmer himself with a mini dry bath. Hepatopancreatic microsporidiosis (HPM) is an infectious shrimp disease caused by the microsporidian Enterocytozoon hepatopenaei (EHP). In recent years, the widespread occurrence of EHP poses a significant challenge to the shrimp aquaculture industry. Early, rapid and accurate diagnosis of EHP infection is very much essential for the control of HPM crop-related losses. Loop-mediated isothermal amplification (LAMP) is a robust, sensitive, cost-effective disease diagnostic technique. Here, we demonstrate an improved, simple, closed-tube, colorimetric EHP LAMP diagnostic assay. LAMP assay was illustrated with the specific EHP spore wall protein (SWP) gene primers. Naked eye visual detection of LAMP amplicons was achieved using Hydroxy naphthol blue (HNB) or Phenol red dye without opening the tubes. This LAMP assay is efficient in detecting the EHP pathogen in all clinical samples include shrimp hepatopancreas, FTA card samples, feces, pond water, and soil. Also, the elution of EHP DNA from FTA cards was demonstrated within 17 min using a simple dry bath. In clinical evaluation, the visual LAMP assay established 100% diagnostic sensitivity and 100% diagnostic specificity. The visual LAMP assay is rapid, can detect the EHP pathogen within 40 min using a simple dry bath, and does not require any expensive instruments and technical proficiency. In conclusion, this visual LAMP protocol is a user-friendly, specific assay that can be conceivably operated at the farm-site/ resource-limited settings by the farmer himself with simple equipment. [ABSTRACT FROM AUTHOR]