Pathogenicity of white spot syndrome virus (WSSV) after multiple passages in mud crab, Scylla olivacea.

White spot syndrome virus (WSSV) is a highly virulent shrimp pathogen with a broad host range. Among the hosts, though mud crab, Scylla olivacea is reported to be more susceptible to WSSV than S. serrata and S. paramamosain, a detailed study on the pathogenicity and genome stability of the virus after multiple passages has yet to be reported. Firstly, to test the pathogenicity of the virus, WSSV was intramuscularly injected into healthy shrimp, Penaeus vannamei. Experimentally infected P. vannamei showed the first mortality at 36 h post-injection (hpi), followed by 100 % cumulative mortality in 7 days post-injection (dpi). However, S. olivacea injected with the WSSV inoculum derived from infected shrimp showed the first mortality at 48 hpi and a cumulative mortality of 70 % at the end of the ten days experiment. Subsequently, WSSV was sequentially passaged five times in Scylla olivacea to find out any change in the virulence of the virus in each passage. S. olivacea groups injected with 1st, second, third and fourth passages derived from the crab recorded the first mortality between 48 and 56 hpi and the cumulative mortality of 60 to 70 % at the end of the ten days experiment. Injection of WSSV inoculum in P. vannamei derived from multiple passages in S. olivaceae revealed the retention of the pathogenicity of the virus. Shrimp groups injected with WSSV derived from different passages showed first mortality between 24 and 36 hpi and cumulative mortality of 100 % between 6 and 7 dpi. The average viral load in the shrimp groups injected with WSSV inoculum derived from shrimp was 3.6 × 108, whereas in shrimp injected with the inoculum derived from 1st, third and fifth passages from crab showed 4.0 × 108, 4.7 × 108 and 4.3 × 108 copies per 100 ng DNA. Histological examination of the gill and stomach tissue of shrimp injected with inoculum prepared from shrimp as well as the inoculum derived from 1st, third and fifth passages in S. olivacea revealed characteristic pathological manifestations of the WSSV infection in gill and stomach tissues such as hypertrophied nuclei, Cowdry A-type inclusions as well as massive basophilic intranuclear inclusions. Further, to study the genome stability, the primers targeting highly variable regions of the WSSV genome (ORF94, ORF125, ORF75, variable region (VR) 14/15 and VR 23/24) were used to amplify WSSV derived from different passages and the amplified PCR products were sequenced. The sequence analysis revealed the WSSV genome stability after multiple passages in mud crab, S. olivacea.

Gain of function studies on predicted host receptors for white spot virus.

Three decades after its first outbreak, the shrimp white spot virus (WSV) is still a global cause of concern due to considerable losses and lack of effective control measures. Several candidate host receptor proteins have been identified, but the pathogenesis is not clearly understood, although the key role of the WSV envelope protein VP28 in virus internalization is established. Here, protein-protein docking is applied to evaluate the interaction of VP28 trimeric extracellular region with four host (Penaeus monodon) receptors reported earlier, Rab7 GTPase (PmRab7), glucose transporter 1 (PmGLUT1), C-type lectin (PmCTL) and calreticulin (PmCRT). The stability of predicted complexes evaluated in terms of binding energy per unit buried surface area ranged from -8.46 to -11.82 cal mol-1/Å2, which is not sufficient for functional interaction. Nevertheless, each of these host proteins was tested by a gain-of-function approach by observing their ability to make a fish cell line permissive to the shrimp WSV. Full-length expression constructs of the four receptors were transfected into SSN1 snakehead fish cells that are non-permissive to WSV. Transfected SSN1 cells and WSV permissive insect Sf9 cells were challenged with purified WSV. After 24 h, the presence of receptor transcripts was confirmed in the treated SSN1 cells, and not in the non-transfected SSN1 cells. Further, vp28 transcript was detected in Sf9 cells, but not in any of the treated SSN1 cells, indicating that none of the receptors were singly sufficient to make SSN1 cells permissive to WSV, even though PmRab7 was a strong candidate that alone showed >85% protection in virus neutralization experiments. For the other 3 candidates, previous reports predicted the involvement of co-receptors, which is confirmed here by their inability to act singly.

Monodon baculovirus (MBV) infects wild mud crab, Scylla serrata.

During a survey of farmed and wild crustaceans from India for viruses, spherical baculovirosis otherwise known as Penaeus monodon-type baculovirus (MBV) was detected in field-collected juvenile/sub-adult mud crab, Scylla serrata using a nested polymerase chain reaction (PCR)-based amplification of the hepatopancreatic DNA. Eight out of 115 mud crab (7.0%) examined during the study were found to be positive in the nested PCR resulting in a 361 nt amplicon. Mud crab, S. olivacea and other crustaceans such as marine crab, Portunus sanguinolentus and farmed penaeid shrimp, Penaeus vannamei and P. monodon were tested negative for the virus. Further, degenerate primers reported to amplify polyhedrin protein gene of MBV also showed PCR amplification in one of the MBV-positive crab samples resulting in a 250 nt amplicon. Sequencing of the two target amplicons (MBV- 361 nt and MBV polyhedrin - 216 nt) revealed more than 97.5 % and 92.8% sequence identity, respectively with the Penaeus monodon nudivirus and Penaeus monodon nucleopolyhedrovirus (MBV) reported from shrimp. Further, histological analysis of mud crab revealed nuclear hypertrophy, chromatin margination and intranuclear eosinophilic/basophilic inclusions in tubule epithelium of hepatopancreas. The hepatopancreatic tissue also showed unusually large, eosinophilic/basophilic inclusion-like structures. These inclusions resembled the viral inclusions reported from S. serrata from Australia. This is the first record of monodon-type baculovirus from a crab host and the second from a non-penaeid crustacean. Interestingly, some of the crab samples also showed deeply basophilic intranuclear inclusion-like bodies resembling hepatopancreatic parvovirus group of viruses (HPV). However, none of the crab samples subjected to PCR amplification using HPV-specific primers showed any amplification. The histological observations made in the present study indicate the possibility of the presence of two hepatopancreas-infecting viruses in S. serrata from India.

Dose-dependent co-infection of Argulus sp. and Aeromonas hydrophila in goldfish (Carassius auratus) modulates innate immune response and antioxidative stress enzymes.

Co-infection with parasites and bacteria is of frequent occurrence in aquaculture, leads to growth impedance otherwise mortality in fish depending on the varying degree of a load of primary pathogen either parasite or bacteria. The mechanistic regulation of immune response during co-infection in fish has merely documented. The aim of this study was to determine the impact of co-infection with Aeromonas hydrophila at three exposure doses of Argulus sp. on the innate immune responses and antioxidative stress enzymes of goldfish (Carassius auratus). The experimental fish were randomly distributed into eight treatment groups viz. T1 (control group without Argulus and A. hydrophila infection), T2 (fish exposed to a sub-lethal dose of A. hydrophila), T3 (low Argulus-infested fish), T4 (T3 + sub-lethal dose of A. hydrophila), T5 (moderate Argulus-infested fish), T6 (T5 + sub-lethal dose of A. hydrophila), T7 (high Argulus-infested fish) and T8 (T7+ sub-lethal dose of A. hydrophila) in duplicates. After distributing experimental fish into their respective treatment group, A. hydrophila was injected to T2, T4, T6 and T8. After the bacterial challenge, four fish from each experimental group were randomly sampled on 24, 72, and 168 h and subjected to the hematological, innate immune parameters and enzymatic analysis. In the co-infection group T8, a high degree of enhanced pathogenicity of A. hydrophila was noticed with increased mortalities (84.2%) in comparison to other groups. The current study shows a declining pattern in RBC, PCV and Hb values with the degree of parasite infestation without co-infection groups. Moreover, in the T8 group, exposure of a sub-lethal dose of bacteria resulted in a drastic reduction of the recorded parameters. Furthermore, a decreased value for WBC, monocyte and neutrophil was found in higher parasite group co-infected with a sub-lethal dose of bacteria relative to other co-infected groups during the experimental period. Also, a decrease in innate immune parameters and antioxidative stress enzymes were observed in the T8 group compared to T7 and T2 groups throughout the trial period. These findings indicate that a rise in the dose of Argulus infection improves A. hydrophila colonization in goldfish and contributes to suppression of the innate immune system and increased mortality.

Transcriptome analysis of liver elucidates key immune-related pathways in Nile tilapia Oreochromis niloticus following infection with tilapia lake virus.

Nile tilapia (Oreochromis niloticus) is one of the most important aquaculture species farmed worldwide. However, the recent emergence of tilapia lake virus (TiLV) disease, also known as syncytial hepatitis of tilapia, has threatened the global tilapia industry. To gain more insight regarding the host response against the disease, the transcriptional profiles of liver in experimentally-infected and control tilapia were compared. Analysis of RNA-Seq data identified 4640 differentially expressed genes (DEGs), which were involved among others in antigen processing and presentation, MAPK, apoptosis, necroptosis, chemokine signaling, interferon, NF-kB, acute phase response and JAK-STAT pathways. Enhanced expression of most of the DEGs in the above pathways suggests an attempt by tilapia to resist TiLV infection. However, upregulation of some of the key genes such as BCL2L1 in apoptosis pathway; NFKBIA in NF-kB pathway; TRFC in acute phase response; and SOCS, EPOR, PI3K and AKT in JAK-STAT pathway and downregulation of the genes, namely MAP3K7 in MAPK pathway; IFIT1 in interferon; and TRIM25 in NF-kB pathway suggested that TiLV was able to subvert the host immune response to successfully establish the infection. The study offers novel insights into the cellular functions that are affected following TiLV infection and will serve as a valuable genomic resource towards our understanding of susceptibility of tilapia to TiLV infection.

A novel myxozoan parasite, Ellipsomyxa boleophthalmi sp. nov. (Myxozoa: Ceratomyxidae) in the brackishwater fish, Boleophthalmus dussumieri Valenciennes, 1837 (Perciformes: Gobiidae) from India.

A novel myxozoan parasite is identified and described from mudskipper, Boleophthalmus dussumieri, collected from a brackishwater ecosystem in Maharashtra, India. Ellipsomyxa boleophthalmi sp. nov. was found in the gallbladder of 58 of 60 fish examined (96.7%). The parasite formed disporous plasmodia that varied in size and shape, and the thin-walled, ellipsoidal and elongated myxospores measured 9.0-10.7 × 6.0-7.8 µm. The two, spherical polar capsules measured 2.7 µm in diameter and enclosed 3-4 coils of polar tubules. Histological observations of infected gallbladder revealed the attachment of disporous plasmodial stages of the parasite to the gallbladder wall with fine pseudopodia. Under the scanning electron microscope (SEM), the myxospores showed a distinct central sutural line and two distinct depressions on the opposite sides at the openings of polar capsules. SEM also revealed the engulfment of microvilli of gallbladder wall by pseudopodia of the plasmodial stages. Analysis of the partial fragment of the SSU rDNA region (1386 bp) showed less than 98% sequence similarity with the other reported Ellipsomyxa spp. In the phylogenetic tree, the present species formed as a distinct subclade within the major clade of Ellipsomyxa spp. The unique morphological and morphometric features of the myxospore, together with the molecular analysis, allowed us to conclude that the present myxozoan is a new species and is named Ellipsomyxa boleophthalmi sp. nov., after the generic name of the host. This is the first report on the occurrence of the genus Ellipsomyxa in B. dussumieri.

TB diagnostic cascade among patients registered under the Revised National TB Control Programme in Chennai, South India.

OBJECTIVE: To discern and quantify the TB diagnostic cascade among patients registered under the Revised National TB Control Programme, Chennai city, Tamil Nadu, South India. METHODS: This cross-sectional study was conducted in metropolitan Chennai from February 2017 to March 2018. We interviewed TB patients retrospectively on their diagnostic attempt in different health facilities. RESULTS: Of 455 TB patients, only 4.4% received their diagnosis at their first health facility. Of 1250 visits to health facilities, the vast majority (79.4 vs. 20.6%) was in the public rather than the private sector. 56% of patients went to a public facility as the first point of care, of whom 1.6% shifted to private facilities subsequently. The remaining 54.4% shifted between up to five government health facilities. Male patients and those with a higher family income were more likely to shift from private to public. CONCLUSION: Most shifts between diagnostic facilities occurred in the public sector. This necessitates interventions at public health facilities for strengthening and extending services to TB patients at their first point of care.

OBJECTIF: Discerner et quantifier la cascade de diagnostic de la TB chez les patients enregistrés dans le Programme National Révisé de lutte contre la TB, dans la ville de Chennai, dans le Tamil Nadu, dans le sud de l'Inde. MÉTHODES: Cette étude transversale a été menée dans la région métropolitaine de Chennai de février 2017 à mars 2018. Nous avons interviewé rétrospectivement des patients TB sur leur tentative de diagnostic dans différents établissements de santé. RÉSULTATS: Sur 455 patients TB, seuls 4,4% ont reçu leur diagnostic dans le premier établissement de santé visité. Parmi 1250 visites dans les établissements de santé, la grande majorité (79,4 vs 20,6%) était dans le secteur public plutôt que le privé. Parmi les 56% des patients qui sont allés dans un établissement public comme premier point de soins, dont 1,6% sont ensuite passés dans des établissements privés. Les 54.4 restants se sont déplacés entre cinq établissements différents de santé publics. Les patients de sexe masculin et ceux dont le revenu familial était plus élevé étaient plus susceptibles de passer du privé au public. CONCLUSION: La plupart des changements entre les établissements de diagnostic se sont produits dans le secteur public. Cela nécessite des interventions dans les établissements de santé publique pour renforcer et étendre les services aux patients TB à leur premier point de soins.

Molecular characterization and expression analysis of secretory immunoglobulin M (IgM) heavy chain gene in rohu, Labeo rohita.

Immunoglobulin M (IgM) is the major isotype among teleost immunoglobulins. The present study was aimed to explore IgM heavy chain gene and its expression profile in rohu. Full-length IgM heavy chain cDNA of rohu consisted of 1994 bp encoding a polypeptide of 576 amino acid residues including a leader peptide, variable (VH) and constant (CH1-CH2-CH3-CH4) domains confirming the secretory form of IgM. The sequence carries conserved residues such as cysteine, tryptophan and amino acid motifs like 'YYCAR' and 'FDYWGKGT-VTV-S'. The predicted 3 D model confirmed various domains of rohu IgM heavy chain. Phylogenetic tree analysis revealed that IgM heavy chain gene of rohu shared the same cluster with that of other cyprinid fishes. Tissue distribution analysis showed the predominant level of IgM heavy chain gene expression in kidney, spleen and intestine. IgM heavy chain gene expression in rohu kidney was found to be up-regulated and reached a maximum at 7 days post-challenge with Aeromonas hydrophila. These findings demonstrate the first report of full-length secretory IgM heavy chain gene in rohu. Besides, IgM heavy chain gene was highly expressed in major lymphoid tissues and bacterial challenge influenced its expression which further confirmed its role in the adaptive humoral immune response.

Analysis of phthalate esters in two different baby care products available in United Arab Emirates.

Phthalates are used as plasticizers in a wide range of products and are known to affect the human health adversely. Hence, the present study was carried out to identify and quantify the presence of four phthalates namely dimethyl phthalate (DMP), dibutyl phthalate (DBP), and diethyl phthalate (DEP), di (2-ethylhexyl) phthalate (DEHP) in the two baby products i.e. baby oils and baby lotions. The daily exposure levels and hazard index of each phthalate were also calculated. It was an analytical study where two different brands of samples of baby oil and baby lotion each, from the date of manufacturing of 3, 10, and 20 months were collected. The extraction of phthalates from different samples was done and analyzed using HPTLC. Results showed the presence of all four phthalates, although some phthalates were not present in 3 M samples. The maximum concentration of all the phthalates was found in 20 M samples. Their concentration increased with the storage time indicating the possibility of leaching and migration of phthalates from the container into the product. The hazard indices for phthalates estimated for baby oil and baby lotion were found below 1, which denotes that the daily phthalate exposures are within the regulatory limits. It is important to consider that the exposure to phthalates can occur not only by dermal contact of these baby products but also through other routes. Hence, the study signifies the importance of phthalates concentration in such regularly used products.

Nucleotide-binding oligomerization domain-containing protein 1 (NOD1) in Asian seabass, Lates calcarifer: Cloning, ontogeny and expression analysis following bacterial infection or ligand stimulation.

NOD1 (Nucleotide-binding oligomerization domain-containing protein 1) is one of the most prominent intracellular Nod-like receptors (NLRs), responsible for detecting different microbial components and products arising from tissue injury. Here, we have identified and cloned NOD1 transcript in the Asian seabass, Lates calcarifer (AsNOD1), which consists of 3749 nucleotides and encodes for a predicted putative protein of 900 AA. The AsNOD1 possesses the typical structure of NLR family, consisting of N-terminal CARD domain, centrally located NACHT domain and C-terminal LRRs. The AsNOD1 showed ubiquitous tissue expression in 11 different tissues of healthy animals tested with high levels of expression in hindgut and gill. From the ontogenetic expression profile of AsNOD1, it is quite evident that this gene might follow a maternally-transferred trend in euryhaline teleosts, as it is highly abundant in embryonic developmental stages. The constitutive immunomodulation of AsNOD1 in terms of expression level was clearly evident in the different tissues of Asian seabass-injected either with Vibrio alginolyticus or poly I:C. However, injection with Staphylococcus aureus did not elicit similar immunomodulation except for the up-regulation noticed at few time-points in some tissues. SISK-cell line induced with different ligands such as poly I:C, LPS and PGN also showed up-regulation of AsNOD1 in certain time-points in vitro. Based on the results obtained in the present study, it can be inferred that the AsNOD1 might play an immunoregulatory role upon exposure to different bacterial as well as viral PAMPs and also might be an important component of innate immune element during embryonic and larval development in the euryhaline teleost Asian seabass.