Evaluation of the immunogenicity of attenuated feline calicivirus vaccines by ELISA.

An ELISA test was developed to measure the levels of IgG antibody in specific-pathogen-free (SPF) cats immunised with two doses of an attenuated feline calicivirus (FCV) vaccine. All eight vaccinates were protected from virus challenge, but four out of five non-vaccinates were not. There was a significant difference in respect of protection from virus challenge between SPF cats with and without three-fold or greater increase in antibody units (P = 0.01). Each serum absorbance was standardised against the reference positive which has an arbitrary value of 100 antibody units. In SPF cats, the 99% confidence level for seropositivity to FCV was determined as greater than or equal to 2.5 antibody units. The results suggest that the sensitive ELISA test can be used to monitor the antibody status of SPF cat colonies prior to FCV vaccine trials, and to measure the immunogenicity of attenuated FCV vaccines. Thus, the ELISA test may replace the need for virus challenge, with consequent reduction in animals used in future FCV vaccine trials.     

Detection of foot-and-mouth disease virus by nucleic acid sequence-based amplification (NASBA)

A study was conducted to evaluate the performance of a nucleic acid sequence-based amplification (NASBA) assay for the detection of foot-and-mouth disease virus (FMDV). Two detection methods: NASBA-electrochemiluminescence (NASBA-ECL) and a newly developed NASBA-enzyme-linked oligonucleotide capture (NASBA-EOC) were evaluated. The diagnostic sensitivity of these assays was compared with other laboratory-based methods using 200 clinical samples collected from different regions of the world. Assay specificity was also assessed using samples (n=43) of other viruses that cause vesicular disease in livestock and genetic relatives of FMDV. Concordant results were generated for 174/200 (87.0%) of suspect FMD samples between NASBA-ECL and real-time RT-PCR. In comparison with the virus isolation (VI) data, the sensitivity of the NASBA-ECL assay was 92.9%, which was almost identical to that of the real-time RT-PCR (92.4%) for the same set of samples. There was broad agreement between the results of the NASBA-ECL and the simpler NASBA-EOC detection method for 97.1% of samples. In conclusion, this study provides further data to support the use of NASBA as a rapid and sensitive diagnostic method for the detection and surveillance of FMDV.     

Three-colour flow cytometric detection of PrP in ovine leukocytes

PrP(c) (cellular prion protein, CD230) expression by subpopulations of lymphoid cells has been widely investigated in a variety of species, possibly because of the possible link between transmissible spongiform encephalopathies (TSE) transmission and blood transfusion. However, the role of the immune cells in the transmission of the disease is still unclear. Here we describe the optimisation and standardisation of a three-colour staining procedure to detect PrP in association with phenotypic and activation markers in ovine immune cells. We demonstrate a reproducible, flexible and sensitive method and that the combination of isotype-specific antibodies and Fab fragments is feasible. To our knowledge, this is the first report of such labelling of ovine cells. Using this method, we were able to detect differences in levels of PrP expression between blood and lymph node cells of the same animal, and considerable variability between animals. Moreover, we were able to explore possible associations between PrP expression and cellular activation and to identify cell subsets with different labelling patterns. We are currently employing this approach to evaluate variations in immunological parameters during experimental infection in sheep.     

Characterization of vitellogenin induced by estradiol-17β in protandrous black porgy,Acanthopagrus schlegeli

The objectives of this study were to isolate and characterize vitellogenin from plasma of estradiol-treated protandrous black porgy,Acanthopagrus schlegeli. Vitellogenin concentrations in plasma measured by a validated enzyme-linked immunosorbent assay (ELISA) were also compared. Two-year-old black porgy (n=20) were fed with estradiol-17 (4 mg kg^–1 of feed). Plasma was collected for purification of vitellogenin. Two forms of vitellogenin were found in plasma after chromatography on Sepharose CL-2B column and hydroxylapatite, and polyacrylamide gel electrophoresis. Black porgy vitellogenins are phospho-lipo-glycoproteins based on their chemical staining properties. The apparent molecular weights of the two forms of vitellogenin were 636 kDa and 321 kDa, respectively. The amino acid composition of purified vitellogenin was also analyzed after acid hydrolysis. The presence of immunoreactive vitellogenin was identified in the plasma and mucus extract from control and estradiol-induced females on the basis of Western blotting. Serial dilution of the plasma and mucus extract taken from estradiol-induced black porgy showed reactivity to an antiserum against lipovitellin in the ELISA, whereas mucus extract and plasma from male fish did not. Significantly higher concentrations of plasma vitellogenin were detected in estradiol-stimulated black porgy than in the control males.     

How the brain translates money into force: a neuroimaging study of subliminal motivation

Unconscious motivation in humans is often inferred but rarely demonstrated empirically. We imaged motivational processes, implemented in a paradigm that varied the amount and reportability of monetary rewards for which subjects exerted physical effort. We show that, even when subjects cannot report how much money is at stake, they nevertheless deploy more force for higher amounts. Such a motivational effect is underpinned by engagement of a specific basal forebrain region. Our findings thus reveal this region as a key node in brain circuitry that enables expected rewards to energize behavior, without the need for the subjects;awareness.     

A complete inventory of long-chain polyunsaturated fatty acid biosynthesis pathway enzymes in the miniaturized cyprinid Paedocypris micromegethes

Fish Physiology and Biochemistry - The capacity for long-chain polyunsaturated fatty acid (LC-PUFA) biosynthesis activity in a species depends on the enzymatic activities of fatty acyl desaturase...     

Activin stimulates goldfish FSH biosynthesis by enhancing FSHβ promoter activity

Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) play critical roles in controlling vertebrate gonadal development and function. Activin, a dimeric growth factor initially identified in the gonads, is important in the differential regulation of the two gonadotropins in mammals. Using goldfish as a model, we have demonstrated that activin stimulates FSHβ but suppresses LHβ expression. The present study demonstrated that the 5′-flanking region of goldfish FSHβ gene is functional in the mouse gonadotrope cell line, LβT2 cells. Similar to its effect on the cultured pituitary cells, activin stimulated FSHβ promoter activity in the LβT2 cells and the effect could be blocked by its binding protein follistatin. Follistatin also significantly suppressed the basal FSHβ promoter activity, suggesting secretion of endogenous activin by the LβT2 cells. Further characterization of the cis-regulatory elements responsible for activin stimulation is now under way in our laboratory.     

Recovery,survival and dissemination of human enteric viruses in ocean waters receiving sewage in Hawaii

A commercial model of a portable virus concentrator operating on the principle of the membrane virus adsorption-elution technique was used to examine the recovery and distribution of human enteropathogenic viruses in Hawaiian waters receiving sewage wastes. Although operating at an efficiency of 20%, a variety of human enteric viruses was isolated, including polioviruses, Coxsackievirus and Echoviruses. Positive virus isolations were consistently made and at high concentrations at sampling stations closest to the sewage outfall. The frequency of virus isolations decreased with increasing distance from the discharge site. viruses were recovered in all directions from the outfall and even as far as 3.2 km east but not 6.4 km away from the discharge site. Tidal conditions to some degree were found to influence the dissemination of viruses. However, since sampling was generally performed at the surface of the water (0.3 to 0.6 m depth), the influence of winds and surface currents was more difficult to assess. Although a positive correlation was found between the presence of high concentrations of indicator coliforms and viruses in waters closest to the discharge site, this relationship was not seen at sampling stations farther away. At these latter stations, positive virus isolations with corresponding low to negligible coliform counts were made which reaffirmed previous published observations that human enteric viruses are relatively more stable in ocean water than coliform bacteria. Furthermore, the instability of the coliform bacteria consequently affected the fecal coliform/fecal streptococci ratios which thus invalidated the use of such ratios to indicate contamination by human wastes.     

Transmission dynamics of the etiological agent of SARS in Hong Kong: impact of public health interventions

We present an analysis of the first 10 weeks of the severe acute respiratory syndrome (SARS) epidemic in Hong Kong. The epidemic to date has been characterized by two large clusters-initiated by two separate "super-spread" events (SSEs)-and by ongoing community transmission. By fitting a stochastic model to data on 1512 cases, including these clusters, we show that the etiological agent of SARS is moderately transmissible. Excluding SSEs, we estimate that 2.7 secondary infections were generated per case on average at the start of the epidemic, with a substantial contribution from hospital transmission. Transmission rates fell during the epidemic, primarily as a result of reductions in population contact rates and improved hospital infection control, but also because of more rapid hospital attendance by symptomatic individuals. As a result, the epidemic is now in decline, although continued vigilance is necessary for this to be maintained. Restrictions on longer range population movement are shown to be a potentially useful additional control measure in some contexts. We estimate that most currently infected persons are now hospitalized, which highlights the importance of control of nosocomial transmission.