Renate Reimschuessel

Identification of mycobacteria infecting fish to the species level using polymerase chain reaction and restriction enzyme analysis

An assay is described utilizing PCR technology for a rapid diagnostic test to identify fish infection with Mycobacterium marinum, M. fortuitum and M. chelonae. A 924 bp DNA fragment from a highly conserved area of the mycobacterial 16S rRNA gene was amplified using mycobacteria genus-specific primers and digested with restriction enzymes (BanI and ApaI). This examination yielded unique restriction patterns for each mycobacterial specie enabling identification of mycobacteria infecting fish to the species level. The protocol can be applied to purified DNA, a simple colony preparation or infected fish tissue. This protocol can be completed in 1-2 days.

Transgenic fish and its application in basic and applied research.

Since 1985, transgenic fish have been successfully produced by microinjecting or electroporating desired foreign DNA into unfertilized or newly fertilized eggs using many different fish species. More recently, transgenic fish have also been produced by infecting newly fertilized eggs with pantropic, defective retroviral vectors carrying desired foreign DNA. These transgenic fish can serve as excellent experimental models for basic scientific investigations as well as in biotechnological applications. In this paper, we will review the current status of the transgenic fish research and its potential application in basic and applied research.

New nephron development in fish from polluted waters: a possible biomarker

Recent evidence has shown that fish have the ability to develop new nephrons following renal injury. This study evaluated the usefulness of quantifying developing nephrons in mature fish as an ecotoxicological assessment tool. Histological sections of kidney were prepared from Atlantic tomcod and brown bullhead specimens collected from reference and contaminated streams. The numbers of developing nephrons and basophilic cell clusters from which the nephrons arise per section area were determined by image analysis. The numbers of basophilic clusters and developing nephrons in tomcod kidney were found to be one to two orders of magnitude higher than for the bullhead. In tomcod from the Hudson River, the number of both basophilic clusters and developing nephrons were elevated relative to samples from the less contaminated Saco and Royal Rivers. In bullheads, when analysis was conducted over several seasons, the number of basophilic clusters and developing nephrons from Cuyahoga River samples were elevated relative to samples from the less-contaminated Old Women Creek and Toussaint River. Developing nephrons and basophilic clusters seem to have potential as general indicators of ecological condition, but may be better suited for detection of nephrotoxicity at specific sites.

Molecular and Physiological Effects of Mycobacterial oxyR Inactivation

The majority of slow-growing mycobacteria have a functional oxyR, the central regulator of the bacterial oxidative stress response. In contrast, this gene has been inactivated during the evolution of Mycobacterium tuberculosis. Here we inactivated the oxyR gene in Mycobacterium marinum, an organism used to model M. tuberculosis pathogenesis. Inactivation of oxyR abrogated induction of ahpC, a gene encoding alkylhydroperoxide reductase, normally activated upon peroxide challenge. The absence of oxyR also resulted in increased sensitivity to the front-line antituberculosis drug isoniazid. Inactivation of oxyR in M. marinum did not affect either virulence in a fish infection model or survival in human macrophages. Our findings demonstrate, at the genetic and molecular levels, a direct role for OxyR in ahpC regulation in response to oxidative stress. Our study also indicates that oxyR is not critical for virulence in M. marinum. However, oxyR inactivation confers increased sensitivity to isonicotinic acid hydrazide, suggesting that the natural loss of oxyR in the tubercle bacillus contributes to the unusually high sensitivity of M. tuberculosis to isoniazid.

Development of New Nephrons in Adult Kidneys Following Gentamicin-Induced Nephrotoxicity

Gentamicin-induced nephrotoxicity results in necrosis of proximal tubular epithelial cells. In mammals, the injured nephron can be repaired by migration and division of surviving cells. We examined this repair process in the fresh-water goldfish, Carassius auratus. Fish were given a single intraperitoneal injection of 50 mg/kg gentamicin and sacrificed at 1, 4, 14, and 21 days. Injured nephrons showed regeneration along the basement membrane several days following gentamicin administration. In addition, 2-3 weeks following the injection, entirely new nephrons formed in the renal interstitium. Development of new nephrons in adult fish kidneys provides an excellent model for studying renal development and toxicity, and could provide insights into new therapies for chronic renal disease.

Viability and induction of tyrosine aminotransferase in rainbow trout hepatocytes cultured on laminin and polylysine in a serum-free medium

The objective of this study was to find an economical substrate for long-term primary culture of rainbow trout hepatocytes in which viability and differentiated liver function could be maintained in a serum-free media. Attachment efficiency of primary cultures of rainbow trout (Oncorhynchus mykiss) hepatocytes was determined for cells grown on plastic and Falcon Primaria dishes, and dishes coated with ECM (extracellular matrix), Matrigel, fibronectin, collagen type 1, laminin and polylysine. On ECM, Matrigel, laminin and polylysine, attachment efficiency exceeded 90%. On plastic, Falcon Primaria, fibronectin and collagen-coated dishes attachment efficiency was 15–20% and the cells were easily dislodged by pipetting. Cells grown on ECM, laminin and polylysine were maintained up to 22 day with 76%, 60% and 80% viability, respectively. On ECM cells flattened and formed a confluent monolayer on day 2 in culture. Cells grown on matrigel, laminin and polylysine had a more rounded appearance with establishment of cell-to-cell contacts on the second day in culture. Induction of TAT (tyrosine aminotransferase), a marker enzyme for differentiated liver cell functions, was studied with DEX (dexamethasone) in cells grown on laminin and polylysine in serum-free media. Cells grown on laminin and polylysine showed TAT inducibility up to 15 and 19 days, respectively.

UDP-Glucuronyltransferase Kinetics for 3-Trifluoromethyl-4-nitrophenol (TFM) in Fish

Abstract Studies were conducted to address glucuronidation of 3-trifluoromethyl-4-nitrophenol (TFM) in sea lampreys Petromyzon marinus, channel catfish Ictalurus punctatus, rainbow trout Oncorhynchus mykiss, and bluegills Lepomis macrochirus. The ability of these species to biotransform TFM was investigated by determining the kinetics of UDP-glucuronyltransferase (UDPGT; also known as glucuronosyltransferase) in vitro from hepatic microsomal preparations. Maximal velocity (V max, nmol/min·mg) for UDPGT activity toward TFM was significantly greater (P rainbow trout (97 μM) > channel catfish (172 μM) > sea lamprey (261 μM). Analysis of V max/K m ratios, a measure of enzyme efficiency (nmol/min-mguM TFM), indicated that the efficiency of UDPGT activities in all species examined was in...

Communications: A Classification System for Histological Lesions

Abstract This paper presents a system for classifying and coding histological data from fish and selected invertebrate species. These codes can be readily entered into a computerized spreadsheet and subsequently imported into statistical software programs. Use of this system can reduce time spent taking notes at the microscope and facilitate data analysis.

Renal histopathological changes in the goldfish (Carassius auratus) after sublethal exposure to hexachlorobutadiene

Abstract Hexachlorobutadiene (HCBD), a chlorinated hydrocarbon, is an acute renal toxicant in mammals. Goldfish (Carassius auratus) were given a single i.p. injection of a sublethal dose (500 mg/kg) of HCBD and sampled daily for one week. No damage was observed by light microscopy 6 h post injection. At 24 h, however, cytoplasmic vacuolation and necrosis occurred in the renal tubules. This damage was localized to the epithelium of the second (P2) and third (P3) segments of the proximal tubule. The damage persisted for seven days. By the sixth day the first segment (P1) of the proximal tubule had small cytoplasmic vacuoles. The ratio of kidney to body weight was significantly greater in the treated fish on the fourth day. Gamma glutamyl transpeptidase (GGT), a histochemical marker of proximal tubule brush border in mammals, was demonstrated in the goldfish kidney. Intense staining was noted only in P2 and P3. GGT staining was also present in the lumen of the damaged, vacuolated tubules of HCBD-treated fish.

Goldfish as an animal model system for mycobacterial infection

Publisher Summary Humans are the primary natural host of M. tuberculosis infection; however, several animal models have been developed to study infection with this pathogen. The primary animal models used are the guinea pig, mouse, and rabbit models. Each of the commonly used mycobacterium models has its advantages and disadvantages. In addition to these small animal models, a primate model has been developed which most closely parallels human disease. It is uncommonly used because of its expense and the limited availability of primates. Tuberculosis is spread from an infected person to a new host through infected droplets made airborne by coughing and talking.In addition to a mycobactefial pathogenesis model, our animal model can be used as a vaccine challenge model. A challenge model is used to demonstrate whether a vaccine strain can elicit protective acquired immunity and therefore protect against a challenge with a fully virulent wild-type strain. This application was demonstrated in an experiment with two groups of fish inoculated intraperitoneally with M. marinurn ATCC 927 organisms at a dose of 10 3 or 105 cfu. However, animals immunized with 105 cfu of M. marinum were completely protected, with 100% of the fish surviving until the end of the experiment. This established that protective immunity develops following an M. marinum infection. This model can thus be used to evaluate the protective efficacy of candidate vaccine strains.