In vitro immunization and culture: persistence and specificity of antibody producing cells from rainbow trout
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The thymus is believed to be a central component of haematopoiesis and immune function in teleosts. Hence, chemically-elicited adverse effects to the thymus may result in immunomodulation and organ dysfunction. The objective of this research was to assess the levels of active (apoptotic) and passive (necrotic) cell death in untreated and organochlorine treated fish thymocytes. Lake trout ( Salvelinus namaycush ) thymocytes were challenged with Aroclor 1254 (concentration range 1·5–10·5 μg ml −1 ) and alpha, beta, gamma, delta isomers of hexachlorocyclohexane (concentration range 10–100 μ M ). The resulting maintenance or loss of viability was assessed by cytofluorometry (expression of phosphatidylserine and exclusion of propidium iodide) and confirmed with fluorescence microscopy. The results indicate that 20–60% of thymocytes in healthy fish undergo apoptosis, whereas thymocytes treated for 6–24 h with organochlorines exhibit increased levels of apoptotic cell death. This study demonstrates that given sufficient concentration, contact time and cellular receptors, organochlorines such as Aroclor 1254 and hexachloro-cyclohexanes may induce direct or indirect toxicity, altered functionality, or cell death to an organ important for fish immunocompetence.
The immunotoxicity of chemical combinations commonly encountered by the lake trout ( Salvelinus namaycush ) immune system was the focus of this study. It was hypothesised that combinations of an environmental contaminant (mercuric chloride or Aroclor 1254) and an immunomodulatory agent (bacterial endotoxin or cortisol) might interact to produce a greater toxicity than that of the environmental contaminant alone at concentrations typically encountered in piscine blood and other tissues. Thus lake trout thymocytes were isolated and treated with mercuric chloride or Aroclor 1254 in the presence and absence of cortisol or lipopolysaccharide. Incubations were performed for 6 or 20 h at 4° C or 10° C. Lipopolysaccharide did not affect the toxicity of either contaminant. In contrast, cortisol enhanced the toxicity of both environmental contaminants. Hence, stressors that lead to increased cortisol production, but not lipopolysaccharide directly, may increase the toxicity of mercury and Aroclor 1254 to lake trout thymocytes.
We obtained two channel catfish estrogen receptor (ccER) cDNA from liver of female fish using RT–PCR. The two fragments were identical in sequence except that the smaller one had an out-of-frame deletion in the E domain, suggesting the existence of ccER splice variants. The larger fragment was used to screen a cDNA library from liver of a prepubescent female. A cDNA was obtained that encoded a 581-amino-acid ER with a deduced molecular weight of 63.8 kDa. Extracts of COS-7 cells transfected with ccER cDNA bound estrogen with high affinity ( K d = 4.7 nM) and specificity. Maximum parsimony and Neighbor Joining analyses were used to generate a phylogenetic classification of ccER on the basis of 18 full-length ER sequences. The tree suggested the existence of two major ER branches. One branch contained two clearly divergent clades which included all piscine ER (except Japanese eel ER) and all tetrapod ERα, respectively. The second major branch contained the eel ER and the mammalian ERβ. The high degree of divergence between the eel ER and mammalian ERβ suggested that they also represent distinct piscine and tetrapod ER. These data suggest that ERα and ERβ are present throughout vertebrates and that these two major ER types evolved by duplication of an ancestral ER gene. Sequence alignments with other members of the nuclear hormone receptor superfamily indicated the presence of 8 amino acids in the E domain that align exclusively among ER. Four of these amino acids have not received prior research attention and their function is unknown. The novel finding of putative ER splice variants in a nonmammalian vertebrate and the novel phylogenetic classification of ER offer new perspectives in understanding the diversification and function of ER.
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Volcanic activity releases sulphur dioxide (SO₂) gas and fine particulate matter that may degrade air quality in populated areas, yet the effects of combined exposure remain insufficiently characterised. We examined the biological effects of acute combined exposure to volcanic ash and SO₂ in human alveolar epithelial cells (A549) maintained at the air-liquid interface. Cells were exposed to respirable ash from the 2021 Tajogaite eruption (La Palma; 100 µg/cm²) and SO₂ gas (nominal concentration 50 ppm; 1, 2, or 3 h), separately or sequentially. Cellular outcomes evaluated after exposure included cytotoxicity, pro-inflammatory and redox responses. Tajogaite ash alone had limited effects on cells, while exposure to SO 2 altered inflammatory responses (increasing gene transcription while reducing extracellular cytokine levels). Co-exposure responses were largely comparable to SO₂ alone, with no statistically significant evidence of synergistic effects under the conditions tested. These findings highlight the importance of considering gas-phase exposures in volcanic environments.
Smallmouth bass ( Micropterus dolomieu ) are used as an indicator species in environmental monitoring and assessment studies. However, laboratory-based studies for methods development and effector assessment are limited for this species. Nutrition, a known modulator of teleost physiological responses including immune function, is a critical knowledge-gap sometimes overlooked in the design of laboratory studies. We report the results from a study evaluating a commercially available artificial pelleted diet for bass and live feed (fathead minnows). Following a six-month diet-acclimation period, age-0 smallmouth bass were assessed using morphometric measures, histologic and immune-function end points using conventional methods, miniaturized cell isolation and assay methods as well as imaging flow cytometry. Fish on the two diets did not significantly differ in length, weight, or condition factor, indicating that growth was similar in the two groups. Histologic examination revealed relatively higher levels of macrophage aggregates and accumulation of ceroid/lipofuscin in the spleen as well as hepatocyte changes in the pellet-fed group. Leukocytes from the pellet-fed group exhibited significantly elevated bactericidal activity and significantly depressed mitogen response compared to fish fed live feed. Following exposure to a known inducer of inflammatory responses, bacterial lipopolysaccharide, responses including the transition of leukocytes to an apoptotic/necrotic condition differed significantly based on diet. Histologic findings were consistent with the occurrence of diet-related oxidative stress in the pellet-fed fish. Oxidative stress can be induced by multiple factors including environmental pollutants. For a diet to be useful in laboratory-based studies, it cannot elicit response that could also be induced by experimental treatments. To do so greatly complicates the detection of experimental effects. Until an artificial diet is developed for smallmouth bass that does not produce potentially confounding conditions for laboratory-based studies, use of a live feed appears to be the best option.
Exposure to fine particulate matter (PM) is acknowledged as a risk factor for human morbidity and mortality. Epidemiology and toxicology studies have focused on anthropogenic sources of PM and few consider contributions produced by natural processes (geogenic), or PM produced from natural sources as a result of human activities (geoanthropogenic PM). The focus of this study was to elucidate relationships between human/ecosystem health and dusts produced by a system transitioning from a dominantly natural to a geoanthropogenic PM source. As part of a larger study investigating the relationship between atmospheric transportation of African dust, human health, and coral reef declines, we examined dust samples sourced in Mali, Africa, collected using high-volume samplers from three sites (Mali, Tobago and U.S. Virgin Islands). Inhalation and ingestion exposure pathways were explored by filter extractions using simulated lung and gastric fluids. Bioaccessibility varied by metal and extraction fluid. Although too few samples were analyzed for robust statistics, concentrations for several metals decreased slightly while bioaccessibility increased at downwind sites.
Rapid detection and delineation of contaminants in urban settings is critically important in protecting human health. Cores from trees growing above a plume of contaminated groundwater in Verl, Germany, were collected in 1 day, with subsequent analysis and plume mapping completed over several days. Solid-phase microextraction (SPME) analysis was applied to detect tetrachloroethene (PCE) and trichloroethene (TCE) to below nanogram/liter levels in the transpiration stream of the trees. The tree core concentrations showed a clear areal correlation to the distribution of PCE and TCE in the groundwater. Concentrations in tree cores were lower than the underlying groundwater, as anticipated; however, the tree core water retained the PCE:TCE signature of the underlying groundwater in the urban, populated area. The PCE:TCE ratio can indicate areas of differing degradation activity. Therefore, the phytoscreening analysis was capable not only of mapping the spatial distribution of groundwater contamination but also of delineating zones of potentially differing contaminant sources and degradation. The simplicity of tree coring and the ability to collect a large number of samples in a day with minimal disruption or property damage in the urban setting demonstrates that phytoscreening can be a powerful tool for gaining reconnaissance-level information on groundwater contaminated by chlorinated solvents. The use of SPME decreases the detection level considerably and increases the sensitivity of phytoscreening as an assessment, monitoring, and phytoforensic tool. With rapid, inexpensive, and noninvasive methods of detecting and delineating contaminants underlying homes, as in this case, human health can be better protected through screening of broader areas and with far faster response times.
Ten isolates of infectious haematopoietic necrosis from salmonid fishes of different locations on the West Coast of North America from California to Alaska were compared by plaque size, single-step growth curves at 15 and 18 °C, rate of appearance of cytopathic effects in cell cultures, and growth over a range of temperatures. All isolates were distinguishable on the basis of each growth characteristic examined. The CO isolate from the Sacramento River drainage of California was the most singular of the 10 because of its diminutive plaque size and sensitivity to slightly elevated temperatures. The mean plaque diameter of the l0 isolates increased as the latitude of the geographic source of the isolate increased. Although the maximum titre obtained by all isolates was depressed at temperatures above approximately 18 °C, half of the isolates were not inhibited by temperatures as low as 0.5 °C. t.
[No abstract available]
Trophozoites of Hexamita salmonis, asserted pathogen of juvenile salmonid fishes, were isolated from two species of Pacific salmon hosts and cultured repeatedly in an organic medium saturated with nitrogen. Primary isolates and serial subcultures usually exhibited five- to tenfold population increases per passage.
Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy (TSE) of cervids now detected in 19 states of the United States, three Canadian provinces, and South Korea. Whether noncervid species can be infected by CWD and thereby serve as reservoirs for the infection is not known. To investigate this issue, we previously used serial protein misfolding cyclic amplification (sPMCA) to demonstrate that CWD prions can amplify in brain homogenates from several species sympatric with cervids, including prairie voles ( Microtus ochrogaster ) and field mice ( Peromyscus spp. ). Here, we show that prairie voles are susceptible to mule deer CWD prions in vivo and that sPMCA amplification of CWD prions in vole brain enhances the infectivity of CWD for this species. Prairie voles inoculated with sPMCA products developed clinical signs of TSE disease approximately 300 days prior to, and more consistently than, those inoculated with CWD prions from deer brain. Moreover, the deposition patterns and biochemical properties of protease-resistant form of PrP (PrP RES ) in the brains of affected voles differed from those in cervidized transgenic (CerPrP) mice infected with CWD. In addition, voles inoculated orally with sPMCA products developed clinical signs of TSE and were positive for PrP RES deposition, whereas those inoculated orally with deer-origin CWD prions did not. These results demonstrate that transspecies sPMCA of CWD prions can enhance the infectivity and adapt the host range of CWD prions and thereby may be useful to assess determinants of prion species barriers.
An electron microscope study was performed on three virus isolates that caused hematopoietic necrosis in salmonid fishes: infectious hematopoietic necrosis (IHN), Oregon Sockeye Disease (OSD), and Sacramento River Chinook Salmon Disease (SRCD). All three isolates were examined by negative staining of fathead minnow (FHM) monolayer tissue culture concentrates and IHN virus was also examined in thin sections of FHM cells. Viruslike particles were observed in infected tissues, but similar structures were not found in uninfected cultures. All three isolates were bullet-shaped, but oval and truncated forms were also observed. Mean measurements of particles from IHN-virus-infected tissue were 158 × 90 mμ. They consisted of an outer coat 15 mμ thick, a core 60 mμ in diameter, subunits about 5 mμ, and an axial pore about 20 mμ in diameter. These particles also were seen budding from the cytoplasmic membrane. Similar particles from SRCD were 159 × 90 mμ and isolates from OSD were 181 × 91 mμ. The three isolates were morphologically indistinguishable from one another and the greater length of OSD was considered insignificant. IHN, SRCD, and OSD viruses were tentatively placed in the rhabdovirus group, but serological studies are needed to determine if they are antigenically identical or should be included as separate members. Biochemical and physical characteristics of these viruses and a comparison with other salmonid viruses is also discussed.
Rapid antemortem tests to detect individuals with transmissible spongiform encephalopathies (TSE) would contribute to public health. We investigated a technique known as protein misfolding cyclic amplification (PMCA) to amplify abnormal prion protein (PrP TSE ) from highly diluted variant Creutzfeldt-Jakob disease (vCJD)-infected human and macaque brain homogenates, seeking to improve the rapid detection of PrP TSE in tissues and blood. Macaque vCJD PrP TSE did not amplify using normal macaque brain homogenate as substrate (intraspecies PMCA). Next, we tested interspecies PMCA with normal brain homogenate of the southern red-backed vole (RBV), a close relative of the bank vole, seeded with macaque vCJD PrP TSE . The RBV has a natural polymorphism at residue 170 of the PrP-encoding gene (N/N, S/S, and S/N). We investigated the effect of this polymorphism on amplification of human and macaque vCJD PrP TSE . Meadow vole brain (170N/N PrP genotype) was also included in the panel of substrates tested. Both humans and macaques have the same 170S/S PrP genotype. Macaque PrP TSE was best amplified with RBV 170S/S brain, although 170N/N and 170S/N were also competent substrates, while meadow vole brain was a poor substrate. In contrast, human PrP TSE demonstrated a striking narrow selectivity for PMCA substrate and was successfully amplified only with RBV 170S/S brain. These observations suggest that macaque PrP TSE was more permissive than human PrP TSE in selecting the competent RBV substrate. RBV 170S/S brain was used to assess the sensitivity of PMCA with PrP TSE from brains of humans and macaques with vCJD. PrP TSE signals were reproducibly detected by Western blot in dilutions through 10 -12 of vCJD-infected 10% brain homogenates. This is the first report showing PrP TSE from vCJD-infected human and macaque brains efficiently amplified with RBV brain as the substrate. Based on our estimates, PMCA showed a sensitivity that might be sufficient to detect PrP TSE in vCJD-infected human and macaque blood.