Early intracellular trafficking and subsequent activity of programmed cell death in channel catfish macrophages infected with edwardsiella ictaluri

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. The development of Edwardsiella-containing-vacuoles (ECV) and the ability of Edwardsiella ictaluri to survive and replicate within macrophages suggests a unique process relative to normal phagosomal/lysosomal maturation and programed cell death. Developing ECV showed that endosomal membrane markers Rab5, EEA1, and Rab7 were all detected in both the wild type (WT) and an E. ictaluri type-3 secretion system (T3SS) mutant, 65ST. Co-localization with Lamp1, however, was significantly lower in the WT. The host cell endoplasmic reticulum marker, calnexin, co-localized to 65ST ECV significantly more than WT ECV, while Golgi vesicle marker, giantin, was recruited to WT ECV significantly more than 65ST. The autophagosomal marker LC3 was significantly lower in WT than in 65ST and Western blotting demonstrated significantly greater induction of the membrane localized, lipidated form, LC3-II, in 65ST ECV than in WT ECV. Activity of the apoptosis initiator caspase-8 increased post-infection in 65ST and was significantly lower in WT-infected cells. Executioner caspase-3/7 activity also increased significantly in 65ST-infected cells compared to WT-infected cells. Repression of apoptosis was further demonstrated with flow cytometry using Alexa Fluor 647-labeled Annexin V and propidium iodide. Results indicate that WT ECV fused with early and late endosomes but that phagosomal/lysosomal fusion did not occur. Additionally, WT-infected cells recruited Golgi vesicles for vacuolar size increase and bacterial growth material, and both autophagy and apoptosis were repressed in the WT. This activity was all based on the function of the E. ictaluri T3SS

Edwardsiella ictaluri encodes an acid-activated urease that is required for intracellular replication in channel catfish (Ictalurus punctatus) macrophages

Genomic analysis indicated that Edwardsiella ictaluri encodes a putative urease pathogenicity island containing the products of nine open reading frames, including urea and ammonium transporters. In vitro studies with wild-type E. ictaluri and a ureG::kan urease mutant strain indicated that E. ictaluri is significantly tolerant of acid conditions (pH 3.0) but that urease activity is not required for acid tolerance. Growth studies demonstrated that E. ictaluri is unable to grow at pH 5 in the absence of urea but is able to elevate the environmental pH from pH 5 to pH 7 and grow when exogenous urea is available. Substantial production of ammonia was observed for wild-type E. ictaluri in vitro in the presence of urea at low pH, and optimal activity occurred at pH 2 to 3. No ammonia production was detected for the urease mutant. Proteomic analysis with two-dimensional gel electrophoresis indicated that urease proteins are expressed at both pH 5 and pH 7, although urease activity is detectable only at pH 5. Urease was not required for initial invasion of catfish but was required for subsequent proliferation and virulence. Urease was not required for initial uptake or survival in head kidney-derived macrophages but was required for intracellular replication. Intracellular replication of wild-type E. ictaluri was significantly enhanced when urea was present, indicating that urease plays an important role in intracellular survival and replication, possibly through neutralization of the acidic environment of the phagosome. Copyright © 2009, American Society for Microbiology. All Rights Reserved

Streptococcus iniae infection in cultured Asian sea bass (Lates calcarifer) and red tilapia (Oreochromis sp.) in southern Thailand

Streptococcal infections are becoming an increasing problem in aquaculture and have been reported worldwide in avariety of fish species. Here we describe the isolation and characterization of Streptococcus iniae from Asian sea bass (Latescalcarifer) and red tilapia (Oreochromis sp.) cultured in southern Thailand. Conventional and rapid identification systems,as well as the polymerase chain reaction (PCR), were used to determine that the isolate was S. iniae. The virulence of thisS. iniae was higher in Asian sea bass than in red tilapia, as shown by the 10 day-LD50 in Asian sea bass and red tilapia, being1.08x104 and 1.14x107 CFU, respectively. Histopathological changes in Asian sea bass are more severe than those observedin red tilapia. The changes can be found in several organs including liver, pancreas, heart, eye and brain. Histopathologicalfindings included cellular necrosis, infiltration of lymphocytes and granuloma formation in the infected organs

Effect of Dietary Zinc Oxide on Morphological Characteristics, Mucin Composition and Gene Expression in the Colon of Weaned Piglets

The trace element zinc is often used in the diet of weaned piglets, as high doses have resulted in positive effects on intestinal health. However, the majority of previous studies evaluated zinc supplementations for a short period only and focused on the small intestine. The hypothesis of the present study was that low, medium and high levels of dietary zinc (57, 164 and 2,425 mg Zn/kg from zinc oxide) would affect colonic morphology and innate host defense mechanisms across 4 weeks post-weaning. Histological examinations were conducted regarding the colonic morphology and neutral, acidic, sialylated and sulphated mucins. The mRNA expression levels of mucin (MUC) 1, 2, 13, 20, toll-like receptor (TLR) 2, 4, interleukin (IL)-1β, 8, 10, interferon-γ (IFN-γ) and transforming growth factor-β (TGF-β) were also measured. The colonic crypt area increased in an age-depending manner, and the greatest area was found with medium concentration of dietary zinc. With the high concentration of dietary zinc, the number of goblet cells containing mixed neutral-acidic mucins and total mucins increased. Sialomucin containing goblet cells increased age-dependently. The expression of MUC2 increased with age and reached the highest level at 47 days of age. The expression levels of TLR2 and 4 decreased with age. The mRNA expression of TLR4 and the pro-inflammatory cytokine IL-8 were down-regulated with high dietary zinc treatment, while piglets fed with medium dietary zinc had the highest expression. It is concluded that dietary zinc level had a clear impact on colonic morphology, mucin profiles and immunological traits in piglets after weaning. Those changes might support local defense mechanisms and affect colonic physiology and contribute to the reported reduction of post-weaning diarrhea

Invasion and Replication of Photobacterium damselae subsp. piscicida in Fish Cell Lines

The objective of this study was to evaluate the ability of Photobacterium damselae subsp. piscicida to invade and replicate within different fish cell lines. Channel catfish ovary (CCO), fathead minnow (FHM), and epithelioma papillosum cyprini (EPC) cell lines were all susceptible to invasion and supported replication of P. damselae in an in vitro invasion assay in which extracellular growth was controlled with gentamicin. The number of bacteria recovered from EPC and CCO cells increased significantly after 6, 12, and 18 h, indicating that P. damselae replicated within those cells. There was also a significant increase in EPC cells at 3 h. Although the number of bacteria recovered from FHM cells increased slightly after 3 and 6 h, it declined at 12 and 18 h. The decline, however, was due to the early release of bacteria from FHM cells associated with a greater susceptibility to initial infection, which resulted in early cell lysis and subsequent exposure to gentamicin in the media. Using light and electron microscopy, we observed the invasion of bacteria as early as 30 min after infection. Intracellular bacteria were initially contained in small, close-fitting vacuoles that developed into large, clear, spacious vacuoles over time. There was no evidence of bacteria free in the cytoplasm. The intracellular location of P. damselae was confirmed by means of transmission electron microscopy with ruthenium red staining to discriminate between the extra- and intracellular spaces. This is the first report that provides evidence of intracellular replication of P. damselae in cell lines

Cloning and characterisation of a channel catfish (Ictalurus punctatus) Mx gene

A 2.5 kb full-length cDNA clone of a channel catfish (Ictalurus punctatus) Mx gene was obtained using RACE (rapid amplification of cDNA ends) polymerase chain reaction (PCR) from RNA extracted from the liver of poly I:C stimulated channel catfish. The gene consists of an open reading frame of 1905 nucleotides encoding a 635 amino acid protein. The predicted protein is 72.5 kDa and contains the dynamin family signature, a tripartite GTP binding motif and a leucine zipper, characteristic of all known Mx proteins. The catfish Mx protein exhibited 79% identity with perch Mx and between 71% and 74% identity with the three Atlantic salmon and the three rainbow trout Mx proteins. Mx mRNA was constitutively expressed in channel catfish ovary (CCO) cells, but in higher quantities in response to poly I:C treatment. Mx was induced in channel catfish following injection with channel catfish virus (CCV) and poly I:C. © 2003 Elsevier Ltd. All rights reserved

Characterization of thymidine kinase encoded by channel catfish virus\u3csup\u3e1\u3c/sup\u3e

The thymidine kinase (TK) encoded by channel catfish virus (CCV) was demonstrated to be biochemically distinguishable from the TK of the host cell line, channel catfish ovary (CCO), as well as from TKs encoded by other herpesviruses. Extracts of CCO cells and of TK-negative CCO cells infected with CCV were assayed for differences in inhibition by deoxynucleosides, in feedback inhibition mediated by thymidine triphosphate (dTTP), and in phosphate donor specificity. Results indicated that, compared with CCO-encoded TK, CCV-encoded TK was more inhibited by deoxypurines and showed less dTTP-mediated feedback inhibition. The CCV-TK was unique among herpesvirus TKs in its inability to utilize CTP as a phosphate donor. © by the American Fisheries Society 1993

Ectocommensal protozoan infestations of gills of red swamp crawfish, Procambarus clarkii (Girard), from commercial ponds

Crawfish, Procambarus clarkii (Girard), collected from commercial ponds were examined microscopically to determine the presence of ectocommensal protozoans on the gills. The relation between the incidence of gill ectocommensal protozoans and pond water quality was examined. Ninety-four percent of crawfish sampled were infested, with sixty-five percent of the infested crawfish carrying more than 100 ectocommensals per gill filament. Protozoans observed were of the genera Epistylis, Acineta, Lagenophrys, and Cothurnia, the latter being the most prevalent. Significant correlations existed between gill ectocommensal incidence and water quality variables indicative of primary productivity. © 1986

Genomic organisation of the channel catfish Mx1 gene and characterisation of multiple channel catfish Mx gene promoters

In order to further characterise channel catfish (Ictalurus punctatus) Mx1, studies were initiated to amplify and clone the Mx1 promoter into a reporter vector, pGL3basic. Initially the Mx1 gene was amplified from genomic DNA and was found to have 12 exons and 11 introns, spanning a region over 6 kilobases (kb) in length. The Mx1 promoter was amplified using genome walking and during this process four additional Mx promoters were identified, suggesting the presence of five Mx genes in the channel catfish. All five promoters possess an interferon stimulated response element (ISRE) and the Mx1 promoter possessed two potential NF-κβ transcription sites. Following cloning each construct was transiently transfected into COS-7 and EPC cells for 24 h and treated with 5 μg/ml poly I:C for 24 h. An increase in expression of the reporter gene in response to poly I:C was noted in both cell lines in the pGL3Mx1 construct only. However, the reporter gene was also constitutively expressed in these cells. Constitutive expression was also observed in channel catfish ovary cells transiently transfected with pGL3Mx1 only. Treatment with 5 μg/ml poly I:C did not increase this expression, which may be due to high levels of cell death in this difficult to transfect cell line. The constitutive expression observed implies that a repressor element is missing in the 390 base pair sequence of the Mx1 promoter used in this study. These results suggest that only channel catfish Mx1 is involved in the type I interferon pathway and that the presence of an ISRE in a regulatory region is not necessarily indicative of a role in the type I interferon response. © 2008 Elsevier Ltd. All rights reserved

Ciprofloxacin treatment eliminates mycoplasma in contaminated channel catfish ovary cells

A temperature-sensitive mycoplasma that was present in the channel catfish ovary (CCO) cell line and in a thymidine kinase-negative mutant of this cell line (CCOBr) was effectively eliminated from the cultures by adding ciprofloxacin, a 4-fluoroquinolone, to the culture medium. Both cell lines have remained mycoplasma free after 3 years of continuous culture. A commercially available genetic probe to mycoplasma ribosomal RNA was used to monitor mycoplasma contamination and evaluate the effectiveness of the treatment. © by the American Fisheries Society 1994