Effects of temperature on Renibacterium salmoninarum infection and transmission potential in Chinook salmon, Oncorhynchus tshawytscha (Walbaum).

Renibacterium salmoninarum is a significant pathogen of salmonids and the causative agent of bacterial kidney disease (BKD). Water temperature affects the replication rate of pathogens and the function of the fish immune system to influence the progression of disease. In addition, rapid shifts in temperature may serve as stressors that reduce host resistance. This study evaluated the effect of shifts in water temperature on established R. salmoninarum infections. We challenged Chinook salmon with R. salmoninarum at 12 °C for 2 weeks and then divided the fish into three temperature groups (8, 12 and 15 °C). Fish in the 8 °C group had significantly higher R. salmoninarum-specific mortality, kidney R. salmoninarum loads and bacterial shedding rates relative to the fish held at 12 or 15 °C. There was a trend towards suppressed bacterial load and shedding in the 15 °C group, but the results were not significant. Bacterial load was a significant predictor of shedding for the 8 and 12 °C groups but not for the 15 °C group. Overall, our results showed little effect of temperature stress on the progress of infection, but do support the conclusion that cooler water temperatures contribute to infection progression and increased transmission potential in Chinook salmon infected with R. salmoninarum.

Persistence of external signs in Pacific herring Clupea pallasii Valenciennes with ichthyophoniasis.

The progression of external signs of Ichthyophonus infection in Pacific herring Clupea pallasii Valenciennes was highly variable and asynchronous after intraperitoneal injection with pure parasite preparations; however, external signs generally persisted through the end of the study (429 days post-exposure). Observed signs included papules, erosions and ulcers. The prevalence of external signs plateaued 35 days post-exposure and persisted in 73-79% of exposed individuals through the end of the first experiment (147 days post-exposure). Among a second group of infected herring, external signs completely resolved in only 10% of the fish after 429 days. The onset of mortality preceded the appearance of external signs. Histological examination of infected skin and skeletal muscle tissues indicated an apparent affinity of the parasite for host red muscle. Host responses consisted primarily of granulomatous inflammation, fibrosis and necrosis in the skeletal muscle and other tissues. The persistence and asynchrony of external signs and host response indicated that they were neither a precursor to host mortality nor did they provide reliable metrics for hindcasting on the date of exposure. However, the long-term persistence of clinical signs in Pacific herring may be useful in ascertaining the population-level impacts of ichthyophoniasis in regularly observed populations.

Infecting Pacific Herring with Ichthyophonus sp. in the Laboratory.

The protistan parasite Ichthyophonus sp. occurs in coastal populations of Pacific Herring Clupea pallasii throughout the northeast Pacific region, but the route(s) by which these planktivorous fish become infected is unknown. Several methods for establishing Ichthyophonus infections in laboratory challenges were examined. Infections were most effectively established after intraperitoneal (IP) injections with suspended parasite isolates from culture or after repeated feedings with infected fish tissues. Among groups that were offered the infected tissues, infection prevalence was greater after multiple feedings (65%) than after a single feeding (5%). Additionally, among groups that were exposed to parasite suspensions prepared from culture isolates, infection prevalence was greater after exposure by IP injection (74%) than after exposure via gastric intubation (12%); the flushing of parasite suspensions over the gills did not lead to infections in any of the experimental fish. Although the consumption of infected fish tissues is unlikely to be the primary route of Ichthyophonus sp. transmission in wild populations of Pacific Herring, this route may contribute to abnormally high infection prevalence in areas where juveniles have access to infected offal.

Bench-top validation testing of selected immunological and molecular Renibacterium salmoninarum diagnostic assays by comparison with quantitative bacteriological culture.

No gold standard assay exhibiting error-free classification of results has been identified for detection of Renibacterium salmoninarum, the causative agent of salmonid bacterial kidney disease. Validation of diagnostic assays for R. salmoninarum has been hindered by its unique characteristics and biology, and difficulties in locating suitable populations of reference test animals. Infection status of fish in test populations is often unknown, and it is commonly assumed that the assay yielding the most positive results has the highest diagnostic accuracy, without consideration of misclassification of results. In this research, quantification of R. salmoninarum in samples by bacteriological culture provided a standardized measure of viable bacteria to evaluate analytical performance characteristics (sensitivity, specificity and repeatability) of non-culture assays in three matrices (phosphate-buffered saline, ovarian fluid and kidney tissue). Non-culture assays included polyclonal enzyme-linked immunosorbent assay (ELISA), direct smear fluorescent antibody technique (FAT), membrane-filtration FAT, nested polymerase chain reaction (nested PCR) and three real-time quantitative PCR assays. Injection challenge of specific pathogen-free Chinook salmon, Oncorhynchus tshawytscha (Walbaum), with R. salmoninarum was used to estimate diagnostic sensitivity and specificity. Results did not identify a single assay demonstrating the highest analytical and diagnostic performance characteristics, but revealed strengths and weaknesses of each test.

Integrated circuit-based instrumentation for microchip capillary electrophoresis.

Although electrophoresis with laser-induced fluorescence (LIF) detection has tremendous potential in lab on chip-based point-of-care disease diagnostics, the wider use of microchip electrophoresis has been limited by the size and cost of the instrumentation. To address this challenge, the authors designed an integrated circuit (IC, i.e. a microelectronic chip, with total silicon area of <0.25 cm2, less than 5 mmx5 mm, and power consumption of 28 mW), which, with a minimal additional infrastructure, can perform microchip electrophoresis with LIF detection. The present work enables extremely compact and inexpensive portable systems consisting of one or more complementary metal-oxide-semiconductor (CMOS) chips and several other low-cost components. There are, to the authors' knowledge, no other reports of a CMOS-based LIF capillary electrophoresis instrument (i.e. high voltage generation, switching, control and interface circuit combined with LIF detection). This instrument is powered and controlled using a universal serial bus (USB) interface to a laptop computer. The authors demonstrate this IC in various configurations and can readily analyse the DNA produced by a standard medical diagnostic protocol (end-labelled polymerase chain reaction (PCR) product) with a limit of detection of approximately 1 ng/microl (approximately 1 ng of total DNA). The authors believe that this approach may ultimately enable lab-on-a-chip-based electrophoretic instruments that cost on the order of several dollars.

A scalable and modular lab-on-a-chip genetic analysis instrument.

We demonstrate a new and extremely inexpensive, multipurpose desktop system for operating lab-on-a-chip (LOC) devices. The system provides all of the infrastructure necessary for genetic amplification and analysis, with orders of magnitude improvement in performance over our previous work. A modular design enables high levels of integration while allowing scalability to lower cost and smaller size. The component cost of this system is ca. $600, yet it could support many diagnostic applications. We demonstrate an implementation of genetic amplification via polymerase chain reaction (PCR), and analysis using capillary electrophoresis (CE). The PCR is able to amplify from single or several copies of target DNA and the CE performance (e.g. sensitivity) is comparable to that of commercial photomultiplier-based confocal lab-on-chip instrumentation. We believe this demonstrates that the cost of infrastructure need no longer be a barrier to the wide-spread application of LOC technologies in healthcare and beyond.

Infectious haematopoietic necrosis virus genogroup-specific virulence mechanisms in sockeye salmon, Oncorhynchus nerka (Walbaum), from Redfish Lake, Idaho.

Characterization of infectious haematopoietic necrosis virus (IHNV) field isolates from North America has established three main genogroups (U, M and L) that differ in host-specific virulence. In sockeye salmon, Oncorhynchus nerka, the U genogroup is highly virulent, whereas the M genogroup is nearly non-pathogenic. In this study, we sought to characterize the virus-host dynamics that contribute to genogroup-specific virulence in a captive stock of sockeye salmon from Redfish Lake in Idaho. Juvenile sockeye salmon were challenged by immersion and injection with either a representative U or M viral strain and sampled periodically until 14 days post-infection (p.i.). Fish challenged with each strain had positive viral titre by day 3, regardless of challenge route, but the fish exposed to the M genogroup virus had significantly lower virus titres than fish exposed to the U genogroup virus. Gene expression analysis by quantitative reverse transcriptase PCR was used to simultaneously assess viral load and host interferon (IFN) response in the anterior kidney. Viral load was significantly higher in the U-challenged fish relative to M-challenged fish. Both viruses induced expression of the IFN-stimulated genes (ISGs), but expression was usually significantly lower in the M-challenged group, particularly at later time points (7 and 14 days p.i.). However, ISG expression was comparable with 3 days post-immersion challenge despite a significant difference in viral load. Our data indicated that the M genogroup virus entered the host, replicated and spread in the sockeye salmon tissues, but to a lesser extent than the U genogroup. Both virus types induced a host IFN response, but the high virulence strain (U) continued to replicate in the presence of this response, whereas the low virulence strain (M) was cleared below detectable levels. We hypothesize that high virulence is associated with early in vivo replication allowing the virus to achieve a threshold level, which the host innate immune system cannot control.

Inexpensive, universal serial bus-powered and fully portable lab-on-a-chip-based capillary electrophoresis instrument.

Capillary electrophoresis is a cornerstone of lab-on-a-chip (LOC) implementations for medical diagnostics. However, the infrastructure needed to operate electrophoretic LOC implementations tends to be large and expensive, hindering the development of portable or low-cost systems. A custom-designed and highly integrated microelectronic chip for high-voltage generation switching and interfacing is recently developed. Here, the authors integrate the microelectronic chip with a microfluidic chip, a solid-state laser, filter, lens and several dollars worth of electronic components to form an inexpensive and portable platform, which is the size of a mobile telephone. This compact system has such reduced power requirements that the complete platform can be operated using a universal serial bus link to a computer. It is believed that this system represents a significant advancement in practical LOC implementations for point-of-care medical diagnostics.

High-Voltage CMOS Controller for Microfluidics.

A high-voltage microfluidic controller designed using DALSA semiconductor's 0.8-mum low-voltage/high-voltage complementary metal-oxide semiconductor/double diffused metal-oxide semiconductor process is presented. The chip's four high-voltage output drivers can switch 300 V, and the dc-dc boost converter can generate up to 68 V using external passive components. This integrated circuit represents an advancement in microfluidic technology when used in conjunction with a charge coupling device (CCD)-based optical system and a glass microfluidic channel, enabling a portable and cost-efficient platform for genetic analysis.

An integrated CMOS high voltage supply for lab-on-a-chip systems.

Electrophoresis is a mainstay of lab-on-a-chip (LOC) implementations of molecular biology procedures and is the basis of many medical diagnostics. High voltage (HV) power supplies are necessary in electrophoresis instruments and are a significant part of the overall system cost. This cost of instrumentation is a significant impediment to making LOC technologies more widely available. We believe one approach to overcoming this problem is to use microelectronic technology (complementary metal-oxide semiconductor, CMOS) to generate and control the HV. We present a CMOS-based chip (3 mm x 2.9 mm) that generates high voltages (hundreds of volts), switches HV outputs, and is powered by a 5 V input supply (total power of 28 mW) while being controlled using a standard computer serial interface. Microchip electrophoresis with laser induced fluorescence (LIF) detection is implemented using this HV CMOS chip. With the other advancements made in the LOC community (e.g. micro-fluidic and optical devices), these CMOS chips may ultimately enable 'true' LOC solutions where essentially all the microfluidics, photonics and electronics are on a single chip.

Guide to the identification of fish protozoan and metazoan parasites in stained tissue sections.

The identification of protozoan and metazoan parasites is traditionally carried out using a series of classical keys based upon the morphology of the whole organism. However, in stained tissue sections prepared for light microscopy, taxonomic features will be missing, thus making parasite identification difficult. This work highlights the characteristic features of representative parasites in tissue sections to aid identification. The parasite examples discussed are derived from species affecting finfish, and predominantly include parasites associated with disease or those commonly observed as incidental findings in disease diagnostic cases. Emphasis is on protozoan and small metazoan parasites (such as Myxosporidia) because these are the organisms most likely to be missed or mis-diagnosed during gross examination. Figures are presented in colour to assist biologists and veterinarians who are required to assess host/parasite interactions by light microscopy.

Mortality and kidney histopathology of chinook salmon Oncorhynchus tshawytscha exposed to virulent and attenuated Renibacterium salmoninarum strains.

An isolate of Renibacterium salmoninarum (strain MT 239) exhibiting reduced virulence in rainbow trout Oncorhynchus mykiss was tested for its ability to cause bacterial kidney disease (BKD) in chinook salmon Oncorhynchus tshawytscha, a salmonid species more susceptible to BKD. Juvenile chinook salmon were exposed to either 33209, the American Type Culture Collection type strain of R. salmoninarum, or to MT 239, by an intraperitoneal injection of 1 x 10(3) or 1 x 10(6) bacteria fish(-1), or by a 24 h immersion in 1 x 10(5) or 1 x 10(7) bacteria ml(-1). For 22 wk fish were held in 12 degrees C water and monitored for mortality. Fish were sampled periodically for histological examination of kidney tissues. In contrast to fish exposed to the high dose of strain 33209 by either injection or immersion, none of the fish exposed to strain MT 239 by either route exhibited gross clinical signs or histopathological changes indicative of BKD. However, the MT 239 strain was detected by the direct fluorescent antibody technique in 4 fish that died up to 11 wk after the injection challenge and in 5 fish that died up to 20 wk after the immersion challenge. Viable MT 239 was isolated in culture from 3 fish that died up to 13 wk after the immersion challenge. Total mortality in groups injected with the high dose of strain MT 239 (12%) was also significantly lower (p < 0.05) than mortality in groups injected with strain 33209 (73 %). These data indicate that the attenuated virulence observed with MT 239 in rainbow trout also occurs in a salmonid species highly susceptible to BKD. The reasons for the attenuated virulence of MT 239 were not determined but may be related to the reduced levels of the putative virulence protein p57 associated with this strain.

Small cell carcinoma of the uterine cervix.

From 1962 to 1985, 2201 patients with invasive cervical cancer were staged, evaluated, and treated at the University of Kentucky Medical Center. After a thorough evaluation, 25 cases (1.1%) fulfilled the histologic criteria for small cell cancer defined by Reagan and coworkers. These patients were computer-matched for age, disease stage, and lesion size to 25 patients with large cell nonkeratinizing cancer and 25 patients with keratinizing squamous cell cancer. Morphometric analyses of nuclear size and maximum nuclear diameter were performed on all cases without knowledge of cell type. Small cell cancers were characterized by a nuclear area of 160 mu 2 or less and a maximum nuclear diameter of 16.2 mu, which was significantly lower than that for large cell tumors. Thirty-three percent of the small cell carcinomas stained positively for the neuroendocrine markers (neuron-specific enolase [NSE] and chromogranin [CGR]), whereas the remainder contained only epithelial markers such as cytokeratin (CYK) and epithelial membrane antigen (EMA). Small cell cancers were associated with a high frequency of lymph-vascular space invasion and a diminished lymphoplasmacytic response. Patients with small cell cancer had a significantly higher recurrence rate, particularly to extrapelvic sites, than the matched patients with large cell cancers, and their survival was lower. Clinical trials to determine the efficacy of adjuvant chemotherapy in the treatment of small cell cervical cancer are needed.

Thermo-magnetic surgery for experimental renal cancer.

Thermo-magnetic surgery is an innovative modality in the treatment of malignancies. This unique combination can produce selectively controlled destruction of deep tumors. Heating of the magnetic metallic compounds of ferrosilicone by hysteresis produces temperatures which are incompatible with cancer cell survival. The intense focal heating causes a coagulation necrosis. Damage to surrounding normal tissue can be avoided by careful temperature monitoring and power control of the magnetic field. Cell destruction in the target tissue has been demonstrated by light and electron microscopy. Reproducible and predictable VX2 renal carcinomas in rabbits have been produced and used as the tumor model in the initial experiments of this research project. A clinical trial with selected cancer patients, performed with this technique of thermo-magnetic surgery, is now a reasonable and feasible procedure.

Thermomagnetic surgery for cancer.

Thermomagnetic Surgery is a unique technique that takes advantage of the phenomenon of hysteresis heating of a ferromagnetic material to produce intense but controlled temperatures within solid organs or tumors to cause coagulation necrosis. By controlling the power of the electromagnetic coil system, the degree of heating of the tumor can be controlled through temperature monitoring that allows limitation of the area of destruction to the disease process and avoids damage to surrounding structures. If the ferromagnetic material is delivered by the arterial route to the tumor or organ, there is an additional beneficial effect of ischemic necrosis of the tissue and in time more concentration of the ferromagnetic particles. This new technique is applicable to selected cases of human cancer because no ill effect has been shown to exposure of the electromagnetic field or the ferromagnetic material in experimental animals.