Veterinary Immunology and Immunopathology

Not AvailableThe accuracy of quantitative real time PCR (RTqPCR) can be attained only when a suitable reference gene is used. The gene expression for a particular gene may vary within different cells at different conditions. Hence, the suitability and stability of various potential reference genes have to be determined for expression studies. In this study, we have examined the potential of four different reference genes including ?-Actin (ACTB), 18S ribosomal RNA (18S), glyceraldehyde-3P-dehydrogenase (GAPDH), and elongation factor 1 alpha (EF1AA) in seven different tissues including gill, liver, kidney, spleen, heart, muscle and intestine of goldfish (Carassius auratus). The housekeeping genes were analyzed from healthy fish and in CyHV-2 challenged fish. Based upon the real time PCR results the gene expression varied among the genes and in tissues. The expression levels of the housekeeping genes were then compared and evaluated with the RefFinder web tool which analyses results using four different algorithms ? BestKeeper, delta Ct, geNorm and NormFinder. EF1AA was ranked to be the best gene in healthy fish by BestKeeper and geNorm analysis. The delta Ct and NormFinder algorithm have found 18S to be a stable gene in healthy fish but 18S was given to be least expressed in challenged fish. ACTB was also given as a stable gene by geNorm analysis in both healthy and challenged fish. Also, in CyHV-2 challenged fish, EF1AA was identified as the best gene by all the three analysis except by BestKeeper analysis, where it has ranked GADPH as the best housekeeping gene. Expression of the four candidate reference genes differed across all tissue types tested, inferring that a thorough study of the reference genes is necessary for cross tissue comparison. These results can be further used in the immune gene response study of goldfish infected with any viral pathogen to develop better health strategies in the disease management of goldfish aquaculture

Squeeze me, but don't tease me: Human and mechanical touch enhance visual attention and emotion discrimination

10.1080/17470919.2010.507958Social Neuroscience63219-23

Natural outbreak of infectious spleen and kidney necrosis virus threatens wild pearlspot, Etroplus suratensis in Peechi Dam in the Western Ghats biodiversity hotspot, India

A large-scale mortality of pearlspot, Etroplus suratensis was reported from Peechi Dam, an artificial tropical lake made for irrigation and drinking water supply in Kerala, India during 2018. This dam is located in the premises of Western Ghats, recognized as one of the biodiversity hotspots of the world. The objective of this study was to identify the aetiological agent of this large-scale mortality of E. suratensis by systematic diagnostic investigation and identification of the pathogen. Virus isolation was carried out on a species-specific pearlspot fin (PSF) cell line. Infected PSF cells showed cytopathic effects (CPEs) like cell shrinkage, rounding, enlargement, clustering, and subsequent detachment of cells with a high viral titre of 106⋅95 TCID50 ml−1 at 8 days post-inoculation (dpi). Histopathological examination of the fish showed the presence of numerous abnormal enlarged basophilic cells and intracytoplasmic eosinophilic inclusions in the liver. Moreover, transmission electron microscopy (TEM) analysis revealed the presence of large numbers of 125–132 nm viral particles in the spleen tissues. PCR amplification and phylogenetic analysis of the major capsid protein (MCP) gene sequence confirmed that the causative agent was infectious spleen and kidney necrosis virus (ISKNV) of the genus Megalocytivirus. The experimental infection recorded 86.7 ± 2.7% mortality in the E. suratensis (body weight 11.01 ± 2.7 g; body length 8.01 ± 2.23 cm) injected with 1 × 104⋅25 TCID50 ml−1 ISKNV per fish. Our detailed investigation provided definitive diagnosis of ISKNV in the severe mass mortality event in wild E. suratensis in Peechi Dam, India, adding one more species to expanding host range of ISKNV infection. The high mortality rate of ISKNV infection in pearlspot suggests the perilous nature of this disease, particularly among the wild fish population

Co-infection of Lactococcus garvieae and Tilapia lake virus (TiLV) in Nile tilapia O. niloticus cultured in India

Tilapia lake virus (TiLV) and Lactococcus garvieae are 2 major pathogens of cultured Nile tilapia Oreochromis niloticus. In June-July 2018, a disease outbreak was reported in Nile tilapia cultured in brackish water floating cages in Kerala, India. Affected fish died gradually, and cumulative mortality reached ~75% within 1 mo. In the present study, TiLV and L. garvieae were isolated from the infected fish and confirmed. Nucleotide analysis of the partial sequence of segment 3 revealed that the present TiLV isolate showed 100% similarity with TiLV MF574205 and 97.65% similarity with TiLV KU552135 isolated in Israel. The partial 16S rDNA nucleotide sequence of L. garvieae shared 99% similarity with the 16S rDNA nucleotide sequence of L. garvieae isolated from Nile tilapia in Brazil. Eight virulence genes (hly1, hly2, hly3, NADH oxidase, adhPav, LPxTG-1, LPxTG-4, adhC1) were amplified in the present isolate. In the experimental challenge study, the onset of mortality started earlier in fish co-infected with TiLV and L. garvieae (3 d post-infection [dpi]) compared to other groups. Cumulative mortality (90% at 12 dpi) was significantly higher in the co-infected group than in fish infected with TiLV (60% at 12 dpi) and L. garvieae (40% at 12 dpi) alone. This study reveals that synergistic co-infection with TiLV and other bacteria may increase mortality in disease outbreaks. To the best of our knowledge, this is the first reported co-infection of L. garvieae with TiLV associated with mass mortality in Nile tilapia in India

Journal of Fish Diseases

Not AvailableMegalocytivirus cause diseases that have serious economic impacts on aquaculture, mainly in East and South-East Asia. Five primary genotypes are known: infectious spleen and kidney necrosis virus (ISKNV), red sea bream iridovirus (RSIV), turbot reddish body iridovirus (TRBIV), threespine stickleback iridovirus (TSIV) and scale drop disease virus (SDDV). ISKNV-mediated infectious spleen and kidney necrosis disease (ISKND) is a major viral disease in both freshwater and marine fish species. In this study, we report the isolation of ISKNV from diseased giant gourami, Osphronemus goramy, in India. Transmission electron microscopy of ultrathin sections of kidney and spleen revealed the presence of numerous polygonal naked viral particles having an outer nucleocapsid layer within the cytoplasm of enlarged cells (115-125 nm). Molecular and phylogenetic analyses confirmed the presence of ISKNV and the major capsid protein (MCP) (1,362 bp) gene in the infected fish had a high similarity to the other ISKNV-I isolates. Moreover, ISKNV was propagated in the Astronotus ocellatus fin (AOF) cell line and further confirmed genotypically. A high mortality rate (60%) was observed in gourami fish injected with ISKNV-positive tissue homogenate through challenge studies. Considering the lethal nature of ISKNV, the present study spotlights the implementation of stringent biosecurity practices for the proper control of the disease in the country

FAD-I, a Fusobacterium nucleatum Cell Wall-Associated Diacylated Lipoprotein That Mediates Human Beta Defensin 2 Induction through Toll-Like Receptor-1/2 (TLR-1/2) and TLR-2/6

We previously identified a cell wall-associated protein from Fusobacterium nucleatum, a Gram-negative bacterium of the oral cavity, that induces human beta defensin 2 (hBD-2) in primary human oral epithelial cells (HOECs) and designated it FAD-I (Fusobacterium-associated defensin inducer). Here, we report differential induction of hBD-2 by different strains of F. nucleatum; ATCC 25586 and ATCC 23726 induce significantly more hBD-2 mRNA than ATCC 10953. Heterologous expression of plasmid-borne fadI from the highly hBD-2-inducing strains in a ΔfadI mutant of ATCC 10953 resulted in hBD-2 induction to levels comparable to those of the highly inducing strains, indicating that FAD-I is the principal F. nucleatum agent for hBD-2 induction in HOECs. Moreover, anti-FAD-I antibodies blocked F. nucleatum induction of hBD-2 by more than 80%. Recombinant FAD-I (rFAD-I) expressed in Escherichia coli triggered levels of hBD-2 transcription and peptide release in HOECs similar to those of native FAD-I (nFAD-I) isolated from F. nucleatum ATCC 25586. Tandem mass spectrometry revealed a diacylglycerol modification at the cysteine residue in position 16 for both nFAD-I and rFAD-I. Cysteine-to-alanine substitution abrogated FAD-I's ability to induce hBD-2. Finally, FAD-I activation of hBD-2 expression was mediated via both Toll-like receptor-1/2 (TLR-1/2) and TLR-2/6 heterodimerization. Microbial molecules like FAD-I may be utilized in novel therapeutic ways to bolster the host innate immune response at mucosal surfaces