Vertical transmission and prevalence of white spot syndrome virus (WSSV) in the wild spawning population of the Indian white shrimp, Penaeus indicus.

White spot disease, caused by white spot syndrome virus (WSSV), has historically been the most devastating disease in shrimp aquaculture industry across the world. The mode of virus transmission is the most crucial stage in the dynamics and management of virus infection. This study explored the mechanism of vertical transmission of WSSV in Indian white shrimp, Penaeus indicus, potential native species for domestication and genetic improvement, using quantitative real time PCR (q RT PCR), light and electron microscopy, and in situ hybridization. Wild brooders of P. indicus (n = 2576) were sampled along the South east coast of India, during 2016 to 2021. Of these âˆ¼ 58 % of the brooders were positive for WSSV, and almost 50 % of infected wild brooders were at the various stages of reproductive maturation. WSSV-PCR positive brooders (n = 200) were analysed for vertical WSSV transmission. The q RT PCR studies of reproductive tissues revealed that 61 % (n = 13) of spermatophore, 54 % (n = 28) of immature ovaries and 48 % (n = 27) of ripe ovaries were infected with WSSV. The lowest level of infection was recorded in females with ripe ovaries (6.84 × 101 ± 9.79 × 100 ng genomic DNA) followed by fertilized eggs (1.59 × 102 ± 3.69 × 101 ng genomic DNA), and larvae (nauplius and zoea). The histology of gravid females with high WSSV copies showed pyknotic and karyorrhectic germinal vesicle with degenerated cortical rods. Conversely, the gravid females with low WSSV copies showed fully developed ovary without characteristic signs of WSSV infection. Transmission electron microscopic studies clearly established the presence of WSSV particles in both ovaries and spermatophores. When subjected to in situ hybridization, WSSV-specific signals were observed in connective tissues of spermatophore, although gravid ovary and fertilized eggs were failed to produce WSSV specific signals. The present study provides the first molecular and histological evidence for trans-ovarian vertical transmission of WSSV. Development of disease-free base population being the cornerstone and first step in establishing the breeding program, the present findings could be a basis for development of such programs.

Bioaugmentation of biofloc system with enzymatic bacterial strains for high health and production performance of Penaeus indicus.

The beneficial effects of two probiotic bacterial strains Marinilactibacillus piezotolerans and Novosphingobium sp. during the culture of Indian white shrimp, Penaeus indicus, under biofloc and clear water system were evaluated. The experimental variation were CW1 (M. piezotolerans in clear water), BFT1 (biofloc + M. piezotolerans), CW2 (Novosphingobium sp. in clear water), BFT2 (biofloc + Novosphingobium sp.) and control (without bacterial strains and biofloc). Growth and survival considerably increased in probiotic bio-augmented treatments. Probiotic incorporation significantly improved water quality, especially ammonia reduction. Microbiota analysis from gut samples taken from different treatments revealed varied microbial population structure among clear water culture, biofloc culture and control. Proteobacteria and Firmicutes were the top phyla observed in the treatments which were significantly higher in bio-augmented systems than the control. Vibrio genera were predominantly observed in control and clear water system compared to that of biofloc systems. Immune genes were significantly altered in response to probiotic gut microbial supplementation than the control. Higher gene expression profile of important immune genes was observed in the biofloc reared shrimps. Expression of digestive enzyme related genes such as trypsin, chymotrypsin, cathepsin L, cathepsin B and alpha amylase were also upregulated significantly in probiotic supplementation especially in the biofloc treatments. Proteomic analysis of hepatopancreas of shrimps from different treatments was carried out by using 2D gel electrophoresis and MALDI-TOF analysis. The proteins were mostly related to growth and stress tolerance. Eukaryotic initiation factor 4E binding protein was expressed in all the groups and it was high in biofloc treated animals followed by animals treated solely with probiotics compared to those of control groups. The results concludes that biofloc already proved as an effective culture method for healthy shrimp production and supplementation of probiotic bacterial strains registered additional benefit for growth, survival, microbial, immunological status of P, indicus culture.

Carbon: Nitrogen (C:N) ratio level variation influences microbial community of the system and growth as well as immunity of shrimp (Litopenaeus vannamei) in biofloc based culture system.

Biofloc technology (BFT) is a novel modern aquaculture farming technique used to reduce toxic nitrogen concentration, act as in situ food source and eradicate pollutants using carbon and therefore to control C:N ratio in an aquaculture system. In this study, effect of different C:N ratios of a biofloc based system on water quality such as the level of Total ammonia nitrogen (TAN) nitrite-nitrogen (NO2--N) and nitrate nitrogen (NO3--N) were explored. Further, the growth and immunity status of shrimp L. vannamei under the influence of different C:N ratios were evaluated. Two of the C:N ratios (15 and 20) could significantly (P < 0.05) reduce TAN, NO2-N and NO3-N levels (0.456 ±â€¯0.01, 0.145 ±â€¯0.09, and 0.102 ±â€¯0.02 ppm) compared to control (1.45 ±â€¯0.1, 0.749 ±â€¯0.14 and 0.675 ±â€¯0.16 ppm). Large variations in the frequency distribution of operational taxonomic units (OTUs) for the bacterial community in water with different C:N ration (BFT) and control were observed. Vibrios often considered as opportunistic pathogens, where the most dominant bacterial flora of water in control (79%) and C:N5 (37%) group. In C:N10, Thauera (62%) was most represented genus. Similarly, Attheyaceae (56%), followed by Peridiniaceae (30%) were the most dominant groups in C:N15 treatment. The diversity of bacterial flora was more spread in C:N20 treatments with Psychrobacter (26%), Proteobacteria (25%) and Peridiniaceae (20%) as the major groups. The trend of Vibrio dominance decreased with the increase in C:N ratios and thus confirming the dominance of heterotrophic bacteria in high C:N ratio groups. Upon challenge with pathogens, shrimps from C:N10, C:N15 and C:N20 groups showed significantly higher survival (P < 0.05) compared to the C:N5 and control group. Similarly, better growth rate was also observed in BFT tanks compared to control both during the culture and at harvest. Comparatively higher expression of four immune-related genes (ras-related nuclear gene (RAN), serine proteinase gene (SP), prophenoloxidase activating enzyme (PPAE), and crustin were observed in different C:N ratio ponds than control and these were in increasing trend with the C:N ratio. Gene expression analysis showed that the transcripts of those immune genes were significantly increased among all C:N treatments than that of control. Overall, these findings demonstrated that with optimum C:N ratio, BFT can be used to optimize the bacterial community composition for both optimal water quality and optimal shrimp health. This study thus indicates the possibility of obtaining better performance of L. vannamei culture with proper adjustment of C:N ratio in a biofloc based system.

First Report of a Complete Genome Sequence of White spot syndrome virus from India.

White spot syndrome virus is a major pathogen of shrimp, causing economic loss to the aquaculture industry. For the first time, a complete de novo genome of an Indian isolate of this virus has been deciphered using Illumina and Nanopore sequencing technologies. The genome has 280,591 bp with 442 predicted coding genes.

Immune responses of whiteleg shrimp, Litopenaeus vannamei (Boone, 1931), to bacterially expressed dsRNA specific to VP28 gene of white spot syndrome virus.

In this study, dsRNA specific to VP28 gene of white spot syndrome virus (WSSV) of shrimp was synthesized in Escherichia coli in large scale and studied the immune response of shrimp to dsRNA-VP28. The haematological parameters such as clotting time and total haemocytes counts, and immunological parameters such as prophenoloxidase (proPO), superoxide dismutase (SOD), superoxide anion (SOA) and malondialdehyde content, as well as the mRNA expression of ten immune-related genes were examined to estimate the effect of dsRNA-VP28 on the innate immunity of Litopenaeus vannamei. The activities of proPO, SOA and SOD significantly increased in haemocyte after dsRNA-VP28 treatment, whereas MDA content did not change significantly. Among the ten immune-related genes examined, only the mRNA expression of proPO, cMnSOD, haemocyanin, crustin, BGBP, lipopolysaccharides (LPs), lectin and lysozyme in haemocytes, gill and hepatopancreas of L. vannamei, was significantly upregulated at 12 h after dsRNA-VP28 treatment, while no significant expression changes were observed in Toll receptor and tumour receptor genes. The increase of proPO and SOD activities, and SOA level and mRNA expression level of proPO, cMnSOD, haemocyanin, crustin, BGBP, LPs, lectin and lysozyme after dsRNA-VP28 stimulation indicate that these immune-related genes were involved in dsRNA-VP28-induced innate immunity in shrimp.

White spot syndrome virus (WSSV) genome stability maintained over six passages through three different penaeid shrimp species.

White spot syndrome virus (WSSV) replicates rapidly, can be extremely pathogenic and is a common cause of mass mortality in cultured shrimp. Variable number tandem repeat (VNTR) sequences present in the open reading frame (ORF)94, ORF125 and ORF75 regions of the WSSV genome have been used widely as genetic markers in epidemiological studies. However, reports that VNTRs might evolve rapidly following even a single transmission through penaeid shrimp or other crustacean hosts have created confusion as to how VNTR data is interpreted. To examine VNTR stability again, 2 WSSV strains (PmTN4RU and LvAP11RU) with differing ORF94 tandem repeat numbers and slight differences in apparent virulence were passaged sequentially 6 times through black tiger shrimp Penaeus monodon, Indian white shrimp Feneropenaeus indicus or Pacific white leg shrimp Litopenaeus vannamei. PCR analyses to genotype the ORF94, ORF125 and ORF75 VNTRs did not identify any differences from either of the 2 parental WSSV strains after multiple passages through any of the shrimp species. These data were confirmed by sequence analysis and indicate that the stability of the genome regions containing these VNTRs is quite high at least for the WSSV strains, hosts and number of passages examined and that the VNTR sequences thus represent useful genetic markers for studying WSSV epidemiology.

Association of dual viral infection with mortality of Pacific white shrimp (Litopenaeus vannamei) in culture ponds in India.

Pacific white shrimp, Litopenaeus vannamei has been introduced recently for culture practice in India. Though SPF stocks are imported for larval production and thereafter culture practice, these are prone to infection with the existing viruses in the environment. Here we report mortality of L.vannamei in several farms in India with minimum biosecurity. The shrimp were harvested early within 50-72 days of culture due to the onset of disease and consequent mortality. As per the analysis carried out, the shrimp were infected with two virus, white spot syndrome virus (WSSV) and infectious hypodermal and hematopoietic necrosis virus (IHHNV). About 80 % of the samples collected had either or both of the viruses. A majority of these samples (60 %) had dual infection with WSSV and IHHNV. Infection of shrimp with WSSV and IHHNV could be detected both by PCR and histopathology. Some of the samples had either exclusively WSSV infection or IHHNV infection and were also harvested before the completion of the required culture period. All the samples analyzed were negative for taura syndrome virus, yellow head virus and infectious myonecrosis virus. While it is difficult to point out the exact etiological agent as the cause of mortality, strict biosecurity measures are advisable for the continuity of L. vannamei culture in India.

Successful umbilical cord blood stem cell transplantation in a patient with Rothmund-Thomson syndrome and combined immunodeficiency.

The ATP-dependent DNA helicase Q4 (RECQL4) belongs to a family of conserved RECQ helicases that are felt to be important in maintaining chromosomal integrity (Kitao et al., 1998, Genomics: 54 (3): 443-452). Deletions in the RECQL4 gene located on chromosome 8 region q24.3 have been associated with Rothmund-Thomson syndrome (RTS, OMIM 268400), a condition characterized by poikiloderma, sparse hair, small stature, skeletal abnormalities, cataracts and an increased risk of malignancy. We present a patient with a molecularly confirmed diagnosis of RTS with two unique genetic alterations in RECQL4 (IVS16-2A>T and IVS2+27_51del25), who at the age of 7 months nearly succumbed to Pneumocystis carinii pneumonia. Evaluation of his immune system demonstrated a T- B+ NK- phenotype with agammaglobulinemia consistent with combined immunodeficiency (CID). Studies to evaluate for known genetic causes of CID were not revealing. The patient received an umbilical cord blood (UCB) transplant with complete immune reconstitution. This report represents the first description of a CID phenotype and UCB transplantation in a patient with RTS.

Typing of Listeria monocytogenes isolates by random amplification of polymorphic DNA.

BACKGROUND & OBJECTIVES: Listeria monocytogenes is an important food-borne pathogen causing meningitis and septicaemia in newborns and immunocompromised persons, abortion and preterm labour in pregnant women. Though various methods are available for typing L. monocytogenes, RAPD analysis has been used for epidemiological purposes in developed countries due to its greater discriminating ability. However, as there are no published reports from India on the typing of L. monocytogenes by RAPD technique the present study was undertaken to type isolates of L. monocytogenes from clinical, food and veterinary samples. METHODS: Isolates of L. monocytogenes were subjected to RAPD using four decamer random primers R1, R2, R3 and R4. Amplified products were analysed by agarose gel electrophoresis. RESULTS: Eight strains of L. monocytogenes on RAPD analysis generated 4 distinct profiles each with R1 and R4 primers and 3 different profiles with R2 and R3 primers. The isolates from fish, clinical and veterinary samples showed different profiles with respect to each other. Isolate from flat fish (serovar 4) showed a different profile from that of clams (serovar 1). Two isolates from placenta (serovar 1) showed similar profiles and all the isolates from veterinary samples generated similar profiles. INTERPRETATION & CONCLUSION: RAPD analysis in the present study allowed discrimination of isolates among the same serotype but from different sources. Since RAPD is a rapid technique and offers greater discrimination of strains, this method may be used for typing L. monocytogenes in India.

Detection of Shiga-toxigenic Escherichia coli (STEC) in fresh seafood and meat marketed in Mangalore, India by PCR.

AIMS: To study the incidence of Shiga-toxigenic Escherichia coli (STEC) in seafoods from India. METHODS AND RESULTS: Escherichia coli isolated from various seafoods such as fresh fish, clams and water were screened for the presence of stx, hlyA and rfbO157 genes by PCR; 5% of clams and 3% of fresh fish samples were positive for non-O157 STEC. CONCLUSIONS: STEC is prevalent in seafoods in India, and non-O157 serotype is more common. SIGNIFICANCE AND IMPACT OF THE STUDY: Seafood could be a vehicle for transmission of STEC even in tropical countries.

Biofilm formation by salmonella spp. on food contact surfaces and their sensitivity to sanitizers.

Biofilm formation by two poultry isolates of Salmonella on three commonly used food contact surfaces viz plastic, cement and stainless steel were studied. Biofilm formation of both the isolates showed a similar trend with the highest density being on plastic followed by cement and steel. Salmonella weltevreden formed biofilm with a cell density of 3.4 x 10(7), 1.57 x 10(6) and 3 x 10(5) cfu/cm2 on plastic, cement and steel respectively while Salmonella FCM 40 biofilm on plastic, cement and steel were of the order of 1.2 x 10(7), 4.96 x 10(6) and 2.23 x 10(5) cfu/cm2 respectively. The sensitivity of the biofilm cells grown on these surfaces to different levels of two sanitizers namely hypochlorite and iodophor for varying exposure times was studied. Biofilm cells offered greater resistance when compared to their planktonic counterparts. Such biofilm cells in a food processing unit are not usually removed by the normal cleaning procedure and therefore could be a source of contamination of foods coming in contact with such surfaces.

Polymerase chain reaction in detection of Gymnodinium mikimotoi and Alexandrium minutum in field samples from southwest India.

Polymerase chain reaction (PCR) primers were constructed for the detection of two toxic dinoflagellate species, Gymnodinium mikimotoi and Alexandrium minutum. The primers amplified a product of expected size from cultured cells of G. mikimotoi and A. minutum. The species-specific primers targeting G. mikimotoi did not yield any product with a wide range of other cultured algae used as negative controls. Primers designed for A. minutum were species-group-specific since it PCR yielded a product from the closely related species A. ostenfeldii and A. andersonii, but not from other species of this genus tested. The confirmation of PCR products was performed by digestion of the products with restriction enzymes. Sensitivity analyses of the primers on DNA template from cultured cells was positive by PCR at a DNA template concentration of 1.5 x 10(-4) ng/microl (0.3 cells/L) for A. minutum, and at a DNA concentration of 2.5 x 10(-2) ng/microl (697 cells/L) for G. mikimotoi. The PCR method for detection of G. mikimotoi and A. minutum was applied on field samples collected with a plankton net. Gymnodinium mikimotoi could be detected in 11 field samples by microscopy, and all these field samples were positive by PCR. The cell counts of G. mikimotoi in simultaneously collected water samples ranged from 306 to 2077/L. Alexandrium minutum could be detected by microscopy in 3 different field samples. The cell counts in water samples collected at the same time as the net samples ranged from 115 to 1115 cells/L. Alexandrium minutum was detected by PCR in these field samples, with the exception of the sample displaying the lowest cell count (115 cells/L). Plankton samples that were negative by microscopy for any of the two target species were also negative by PCR. All the PCR products from field samples were confirmed by restriction enzyme digestion. The application of PCR-based detection of harmful algal bloom species for aquaculture and monitoring purposes in natural field samples is discussed.

Immunization with bacterial antigens: infections with motile aeromonads.

Motile aeromonads are a heterogeneous group of organisms which are involved in a number of disease syndromes of warm water fish. They are commonly associated with bacterial haemorrhagic septicaemia, infectious dropsy, red mouth disease, ulcerative conditions etc. A variety of factors has been considered to be associated with virulence including haemolysins, proteases, surface array protein, acetylcholinesterase etc. We have studied the immune response of Indian major carps to antigens of motile aeromonads. The Indian major carp, Labeo rohita, showed immunological memory and secondary response on booster administration. The extent of protection showed good correlation with titres of agglutinating antibody. When polyvalent vaccine was used, the fish showed antibody titres against all the component strains. However, the level of antibody was less compared to immunization by monovalent vaccines. Cross-reacting antibodies induced by monovalent vaccines showed varying degrees of protection.