Epizootics of epizootic ulcerative syndrome among estuarine fishes of Kerala, India, under post-flood conditions.

Epizootic ulcerative syndrome (EUS), primarily caused by the water mold Aphanomyces invadans, is an OIE-notifiable disease, having potential impacts on fisheries. We report EUS epizootics among estuarine fishes of Kerala, India, during 2018, under post-flood conditions 3 decades after its primary outbreak. Six fish species (Mugil cephalus, Platycephalus sp., Scatophagus argus, Arius sp., Planiliza macrolepis and Epinephelus malabaricus) were infected, including the first confirmed natural case in E. malabaricus and P. macrolepis. Salinity, surface temperature, dissolved oxygen and pH of resident water during the epizootic were <2 ppt, 25°C, 4.1 ppm and 7.0. The presence of zoonotic bacterial pathogens (Aeromonas veronii, Shewanella putrefaciens, Vibrio vulnificus and V. parahaemolyticus) in tissues of affected fish indicates that EUS-infected fish may pose a public health hazard if not handled properly. Lack of clinical evidence in the region during the last 3 decades, a high number of affected fishes, including 2 new fish species, the severity of skin lesions and very low water salinity (<2 ppt) during the outbreak in contrast to historical water salinity records suggest relatively recent invasion by A. invadans. Phylogenetic analysis based on the internal transcribed spacer region of the rRNA gene showed that the same clone of pathogen has spread across different continents regardless of fish species and ecotypes (fresh/estuarine environments). Altogether, the present study provides baseline data which can be applied in EUS management strategies within brackish-water ecosystems. We recommend strict surveillance and development of sound biosecurity measures against the disease.

Effect of immunization of rohu Labeo rohita with inactivated germinated zoospores in providing protection against Aphanomyces invadans.

Infection with Aphanomyces invadans is one of the most destructive diseases of freshwater fishes. Indian major carps, the dominant cultured species in the Indian sub-continent are highly susceptible to this disease. Till date, there is no effective treatment for control of this disease and immunization can be one of the strategies to reduce disease-related losses. In the present study, inactivated germinated zoospores of A. invadans were evaluated as antigen in conjunction with and without adjuvant Montanide™ ISA 763 A VG, for assessing their efficacy in rendering protection against A. invadans infection. For the experiment, rohu Labeo rohita, (n = 160, 74 ±â€¯12 g) were divided into 4 groups (C, A, G and GA) with 40 fish in each group. The fish in groups i.e., C, A, G and GA were injected intraperitoneally with PBS, adjuvant emulsified with PBS, inactivated germinated zoospores, and inactivated germinated zoospores emulsified with adjuvant, respectively. After 21 days of immunization, the fish were given a booster dose as above. After 7 days of the booster dose, the fish were challenged with zoospores of A. invadans to determine the relative percent survival (RPS). The results revealed that all the fish in C, A and G group succumbed to infection (0% RPS), although there was delayed mortality in fish from A and G groups in comparison to the C group. However, the fish in GA group showed significantly higher (P < 0.05) protection (66.7% RPS). In addition, following booster immunization (before challenge), the antibody level in the GA group was significantly higher (P < 0.05) than the control group. In western blotting, sera from G and GA groups showed reactivity with peptides of about 54 KDa. Following challenge (on 14th day), the antibody level as well as total antiprotease activity in fish of all the groups was significantly decreased in comparison to pre-challenge, except fish in GA group not exhibiting any gross lesions. In addition, sera of surviving fish of GA group showed significant inhibition of germination of zoospores and germlings growth in comparison to other groups (P < 0.05). Further, histopathological examination of the muscle tissue revealed that, in fish of GA group without any gross lesions, there were well developed granulomas and extensive mononuclear cell infiltration restricted to the site of injection, whereas in other groups, there was extensive myonecrosis with proliferating hyphae. These preliminary findings indicate that inactivated germinated zoospores of A. invadans in combination with adjuvant could stimulate good immune response and confer remarkable protection in rohu.

Candidatus Actinochlamydia pangasiae sp. nov. (Chlamydiales, Actinochlamydiaceae), a bacterium associated with epitheliocystis in Pangasianodon hypophthalmus.

Chlamydial infections are recognised as causative agent of epitheliocystis, reported from over 90 fish species. In the present study, the farmed striped catfish Pangasianodon hypophthalmus (14-15 cm, 70-90 g) with a history of cumulative mortality of about 23% during June and July 2015, were brought to the laboratory. The histopathological examination of gills from the affected fish revealed presence of granular basophilic intracellular inclusions, mostly at the base of the interlamellar region and in gill filaments. A concurrent infection with Trichodina spp., Ichthyobodo spp. and Dactylogyrus spp. was observed in the gills. The presence of chlamydial DNA in the gills of affected fish was confirmed by amplification and sequencing of 16S rRNA gene. BLAST-n analysis of these amplicons revealed maximum similarity (96%) with Candidatus Actinochlamydia clariae. On the basis of phylogenetic analysis, it was inferred that the epitheliocystis agents from striped catfish were novel and belonged to the taxon Ca. Actinochlamydia. It is proposed that epitheliocystis agents from striped catfish will be named as Ca. Actinochlamydia pangasiae. The 16S rRNA gene amplicons from novel chlamydiae were labelled and linked to inclusions by in situ hybridisation. This is the first report of epitheliocystis from India in a new fish host P. hypophthalmus.

A new fish cell line derived from the caudal fin of freshwater angelfish Pterophyllum scalare: development and characterization.

In this study, a new cell line derived from the caudal fin of the freshwater angelfish Pterophyllum scalare was developed and characterized. The cell line was designated angelfish fin (AFF) and subcultured 44 times since its development. These cells grew well in Leibovitz's -15 medium supplemented with 10% foetal bovine saline (FBS) at 28° C and the modal chromosome number (2n) was 48. The AFF cell-line is mainly comprised of epithelial cells as confirmed by immunocytological technique using anti-cytokeratin antibodies, an epithelial cell marker. This cell line was tested for growth in a temperatures range from 20 to 37° C and at various FBS concentrations of 5-20% at 28° C. The cell line was cryopreserved at different passage levels and revived successfully with 80% survival rate. Polymerase chain reaction amplification and sequencing of partial mitochondrial 16s rRNA and coI genes confirmed that the AFF cell-line originated from angelfish. Mycoplasma sp. contamination was not detected in AFF cells and checked by Hoechst 33258 fluorescence staining. At the 42nd passage the cells were transfected with 2 µg of pAcGFP1-N1 expression vector. The AFF cells exhibited cytotoxic effects when exposed to the bacterial extra cellular products from Serratia marcescens and Proteus hauseri. The AFF cells and cells from kidney and brain did not show cytopathic effect when exposed to cyprinid herpes virus2 and viral nervous necrosis virus. The newly developed AFF cell line will be useful for the isolation of viruses affecting angelfishes, such as iridoviruses, in the future.

Reassortment and evolutionary dynamics of tilapia lake virus genomic segments.

The tilapia lake virus (TiLV), a highly infectious negative-sense single-stranded segmented RNA virus, has caused several outbreaks worldwide since its first report from Israel in 2014, and continues to pose a major threat to the global tilapia industry. Despite its economic importance, little is known about the underlying mechanisms in the genomic evolution of this highly infectious viral pathogen. Using phylogenomic approaches to the genome sequences of TiLV isolates from various geographic regions, we report on the pervasive role of reassortment, selection, and mutation in TiLV evolution. Our findings provided the evidence of genome-wide reassortment in this newly discovered RNA virus. The rate of non-synonymous (dN) to synonymous (dS) substitutions was less than one (dN/dS = 0.076 to 0.692), indicating that each genomic segment has been subjected to purifying selection. Concurrently, the rate of nucleotide substitution for each genomic segment was in the order of 1-3 × 10-3 nucleotide substitutions per site per year, which is comparable to the rate of other RNA viruses. Collectively, in line with the results of the previous studies, our results demonstrated that reassortment is the dominant force in the evolution and emergence of this highly infectious segmented RNA virus.

Development, characterization and application of a new epithelial cell line from caudal fin of Pangasianodon hypophthalmus (Sauvage 1878).

A cell line, designated as PHF, has been established from caudal fin of Pangasianodon hypophthalmus. The cell line was developed using explant method and PHF cells have been subcultured for more than 72 passages over a period of 14 months. The cells were able to grow at temperatures between 24 and 32°â€¯C, with an optimum temperature of 28°â€¯C. The growth rate of PHF cells was directly proportional to FBS concentration, with optimum growth observed at 20% FBS concentration. On the basis of immunophenotyping assay, PHF cells were confirmed to be of epithelial type. Karyotyping of PHF cells revealed diploid number of chromosomes (2n = 60) at 39th and 65th passage, which indicated that the developed cell line is chromosomally stable. The origin of the cell line was confirmed by amplification and sequencing of cytochrome oxidase c subunit I and 16S rRNA genes. The cell line was tested for Mycoplasma contamination and found to be negative. The cells were successfully transfected with GFP reporter gene suggesting that the developed cell line could be utilized for gene expression studies in future. The cell line could be successfully employed for evaluating the cytotoxicity of heavy metals, namely mercuric chloride and sodium arsenite suggesting that PHF cell line can be potential surrogate for whole fish for studying the cytotoxicity of water soluble compounds. The result of virus susceptibility to tilapia lake virus (TiLV) revealed that PHF cells were refractory to TiLV virus. The newly established cell line would be a useful tool for investigating disease outbreaks particularly of viral etiology, transgenic as well as cytotoxicity studies.

Surgical experience of total anomalous pulmonary venous connection with mid-term follow-up in a developing country.

OBJECTIVE: To illustrate the incidence, the different age group, varied clinical presentation, incremental risk factors for surgery and follow-up results from this part of the world. EXPERIMENTAL DESIGN: Retrospective study with follow-up from 3 months to 5 years. SETTING: Institutional practice with hospitalised care. SELECTION PROCEDURE: Hospitalised patients with a diagnosis of total anomalous pulmonary venous connection. INTERVENTIONS: All patients who were diagnosed to have total anomalous pulmonary venous connection underwent corrective surgery. RESULTS: There were 20 hospital deaths postoperatively. The mean follow-up was 1.9 year. Logistic regression analysis identified age group, timing of surgery, associated anomalies preoperative symptoms, anatomical type of lesion, pulmonary artery hypertension, duration of ventilation, low cardiac output as risk factors for surgery. The mean survival time was 4-8 years. CONCLUSION: Mortality continues to be higher from this part of the world due to severe malnourishment, late presentation. Sociodemographic factors also play a major role in affecting surgical outcome apart from the identified risk factors for surgery.

Acquisition and analysis of brainstem auditory evoked responses of normal and diseased subjects by spectral estimation.

Human brainstem auditory evoked responses (BAERs) are sensory evoked potentials that can be recorded within a few milliseconds following a transient acoustic stimulus (click signal). This paper suggests a novel technique to clearly demarcate normals and patients with complaints of vertigo and deafness by computing hitherto unused power spectral parameters from the BAER signals recorded on them. The BAER spectrum of normal subjects contains three main frequency components, i.e. low-, mid- and high-frequency components around 100, 500 and 1000 Hz, respectively, which is not so in the case of diseased subjects. The spectral parameters, i.e. the mean power frequency, median frequency, the ratios of the integrated power at dominant frequencies to that of the total power in spectrum and change in spectral power (CP) between these dominant frequency components are used to classify the recorded BAER signals into those of normals and the patients, and aid the clinician in quick and better diagnosis. The ranges of CP are estimated for the different groups and appear to be the most dominant parameter in the classification of the BAER signals.

Survival of cardiac function after brain death in patients in Kuwait.

BACKGROUND: Persistent cessation of all cerebral and brainstem function (brain death) is accepted in most countries as legal evidence of death. It is presumed that cardiac function will cease within a short time after brain death has occurred. In some countries, such as Kuwait, tradition and practice discourage application of the brain death criteria despite legal acceptance. OBJECTIVE: The study was designed to assess the duration of persistence of cardiac function in patients after the diagnosis of brain death had been made on the basis of generally accepted criteria. METHODS: We evaluated how long cardiac function persisted after brain function had ceased in 40 patients in Kuwait who were admitted to hospital and died during the 10-year period 1992-2001. RESULTS: It was found that the mean persistence of cardiac function after brain death was 8.20 days and the median survival time was 6 days. Two thirds of the patients survived longer than a week, but none had cardiac function for longer than 30 days. CONCLUSION: The study confirms that brain death is not automatically followed immediately by cessation of all other body functions. It may be speculated therefore that whole-body homeostasis is not as intimately associated with brain function as has hitherto been thought.

Risk factors, subtypes and outcome of ischaemic stroke in Kuwait--a hospital-based study.

OBJECTIVE: To report on stroke subtypes, associated risk factors and outcome in Kuwait. METHODS: The records of 62 patients (30 male, 32 female) admitted with diagnosis of stroke to Kuwait Oil Company Hospital, Kuwait, a tertiary care hospital, during a 5-year period (1995-1999), were retrospectively reviewed. RESULTS: Small artery infarction was the most common subtype and occurred in 37 subjects (59.7%); less common were atherosclerotic large artery strokes (19 patients, 30.6%) and strokes of cardio-embolic origin (6 patients, 9.7%). Identifiable risk factors or associated morbidities were hypertension (72.5%), diabetes mellitus (69.4%), ischaemic heart disease (14.5%), history of migraine (8.1%), lone atrial fibrillation (5.0%), and valvular heart disease (1.6%). The most important determinants of a deleterious 30-day outcome, as indicated by severe disability or death, were female gender, lack of use of anti-platelet drugs, presence of a large artery infarction stroke subtype, and cardio-embolic stroke. CONCLUSION: Prevalence of hypertension and diabetes is high among patients with stroke in Kuwait, with rates higher than those found in any previous reports from the Gulf region. Two unusual observations were that women had a rather high frequency of stroke, and infarction of the small artery was more common than that of the large artery. Outcome, as indicated by severe disability or death, was worse among women, elderly patients, and those with large artery atherosclerotic and cardio-embolic strokes. There is some evidence that such a deleterious outcome might be ameliorated with use of anti-platelet drugs.