Aquaporin expression dynamics in osmoregulatory tissues of Atlantic salmon during smoltification and seawater acclimation.

Osmotic balance in fish is maintained through the coordinated regulation of water and ion transport performed by epithelia in intestine, kidney and gill. In the current study, six aquaporin (AQP) isoforms found in Atlantic salmon (Salmo salar) were classified and their tissue specificity and mRNA expression in response to a hyperosmotic challenge and during smoltification were examined. While AQP-1a was generic, AQP-1b had highest expression in kidney and AQP-3 was predominantly found in oesophagus, gill and muscle. Two novel teleost isoforms, AQP-8a and -8b, were expressed specifically in liver and intestinal segments, respectively. AQP-10 was predominantly expressed in intestinal segments, albeit at very low levels. Transfer from freshwater (FW) to seawater (SW) induced elevated levels of intestinal AQP-1a, -1b and -8b mRNA, whereas only AQP-8b was stimulated during smoltification. In kidney, AQP-1a, -3 and -10 were elevated in SW whereas AQP-1b was reduced compared with FW levels. Correspondingly, renal AQP-1a and -10 peaked during smoltification in April and March, respectively, as AQP-1b and AQP-3 declined. In the gill, AQP-1a and AQP-3 declined in SW whereas AQP-1b increased. Gill AQP-1a and -b peaked in April, whereas AQP-3 declined through smoltification. These reciprocal isoform shifts in renal and gill tissues may be functionally linked with the changed role of these organs in FW compared with SW. The presence and observed dynamics of the AQP-8b isoform specifically in intestinal sections suggest that this is a key water channel responsible for water uptake in the intestinal tract of seawater salmonids.

Thyroid nodularity and cancer among Chernobyl cleanup workers from Estonia.

Thyroid examinations, including palpation, ultrasound and, selectively, fine-needle aspiration biopsy, were conducted on nearly 2,000 Chernobyl cleanup workers from Estonia to evaluate the occurrence of thyroid cancer and nodular thyroid disease among men with protracted exposure to ionizing radiation. The examinations were conducted in four cities in Estonia during March-April 1995, 9 years after the reactor accident. The study population was selected from a predefined cohort of 4,833 cleanup workers from Estonia under surveillance for cancer incidence. These men had been sent to Chernobyl between 1986 and 1991 to entomb the damaged reactor, remove radioactive debris and perform related cleanup activities. A total of 2,997 men were invited for thyroid screening and 1,984 (66%) were examined. Estimates of radiation dose from external sources were obtained from military or other institutional records, and details about service dates and types of work performed while at Chernobyl were obtained from a self-administered questionnaire. Blood samples were collected for assay of chromosomal translocations in circulating lymphocytes and loss of expression of the glycophorin A (GPA) gene in erythrocytes. The primary outcome measure was the presence or absence of thyroid nodules as determined by the ultrasound examination. Of the screened workers, 1,247 (63%) were sent to Chernobyl in 1986, including 603 (30%) sent in April or May, soon after the accident. Workers served at Chernobyl for an average of 3 months. The average age was 32 years at the time of arrival at Chernobyl and 40 years at the time of thyroid examination. The mean documented radiation dose from external sources was 10.8 cGy. Biological indicators of exposure showed low correlations with documented dose, but did not indicate that the mean dose for the population was higher than the average documented dose. Ultrasound examinations revealed thyroid nodules in 201 individuals (10.2%). The prevalence of nodules increased with age at examination, but no significant associations were observed with recorded dose, date of first duty at Chernobyl, duration of service at Chernobyl, building the sarcophagus or working on the roof of neighboring buildings or close to the damaged reactor. Nodularity showed a nonsignificant (p(1) = 0.10) positive association with the proportion of lymphocytes with chromosome translocations, but associations with the frequency of variant erythrocytes in the GPA assay were weak and unstable (p(1) > or = 0.46). The majority of fine-needle biopsies taken on 77 study participants indicated benign nodular disease. However, two cases of papillary carcinoma and three benign follicular neoplasms were identified and referred for treatment. Both men with thyroid cancer had been sent to Chernobyl in May of 1986, when the potential for exposure to radioactive iodines was greatest. Chernobyl cleanup workers from Estonia did not experience a markedly increased risk of nodular thyroid disease associated with exposure to external radiation. Possible reasons for the apparent absence of effect include low radiation doses, the protracted nature of the exposure, errors in dose measurement, low sensitivity of the adult thyroid gland or the insufficient passage of time for a radiation effect to be expressed.

Aujeszky's disease: blocking ELISA for the detection of serum antibodies.

A blocking ELISA for the detection of serum antibodies to Aujeszky's disease virus has been developed. The test was designed in particular with a view to examining large numbers of blood samples. It has been found to be sensitive, specific and precise. In comparison with the serum neutralization test it was superior in detecting low levels of antibodies in pig sera. The blocking ELISA gave higher titre values in serum samples taken early after experimental infection of pigs with Aujeszky's disease virus, than did the serum neutralization test.

Monoclonal blocking ELISA detecting serum antibodies to the glycoprotein gII of Aujeszky's disease virus.

A monoclonal blocking enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to Aujeszky's disease virus (ADV) in porcine serum was developed. This ELISA is based on the reaction between virus antigen immobilized in a microdilution plate and a monoclonal antibody (MAb) reactive with a highly stable epitope on a glycoprotein complex, gII, of ADV. The viral epitope was expressed by 18 European field, laboratory and vaccine strains of ADV. The MAb used in the test was selected among 15 MAbs all reactive with viral epitopes apparently recognized by the porcine immune system as well. Good agreement was found when serum samples from 375 pigs were tested in both a polyclonal and the monoclonal blocking ELISA.

The genomic diversity and stability of field strains of suid herpesvirus 1 (Aujeszky's disease virus).

The genomic diversity among isolates of suid herpesvirus 1 (SHV-1) collected in the same herd and among clones from the same isolate was studied by restriction fragment pattern (RFP) analysis using BamHI. Tentatively defining a field strain as a transmissible entity, it was concluded that strains of SHV-1 commonly comprise distinguishable genomic variants. Contrary to the hypothesis of genomic lability, it is suggested that the pool of variants is sufficiently stable to specifically characterize a strain. The impact of the results on epizootiological methods based on identification of field isolates is discussed in connection with Aujeszky's disease in Denmark.

Experimental in utero infection of pig foetuses with porcine parvovirus (PPV).

Pig foetuses of various gestational ages were exposed to experimental infection with porcine parvovirus (PPV) in utero. Inoculation of 40-, 50- and 60-day-old foetuses with PPV caused foetal death and mummification and spread of the infection to non-inoculated foetuses. Inoculation at 80 and 100 days gestation caused pathological lesions of various degrees whereas spread of infection occurred only sporadically. Serological examinations of foetuses of different ages suggest that immunocompetence for PPV develops before 70 days gestation. The present results strongly indicate that intrauterine spread of PPV is a route of transmission of this virus between pig foetuses.

Foot-and-mouth disease: detection of antibodies in cattle sera by blocking ELISA.

A blocking ELISA was developed for the detection of antibodies to foot-and-mouth disease virus SAT1, SAT2 and SAT3 and for the quantification of antibodies on a single dilution of serum. The avidin-biotin system was used. The test was compared with the liquid-phase ELISA executed at the World Reference Laboratory for foot-and-mouth disease. It was found to have favourable logistics and combined high specificity with high sensitivity. The quantitative test using a single dilution of serum was resource saving and proved to be a reliable and precise method for the assessment of antibody levels.

Evaluation of a blocking Elisa for screening of antibodies against porcine reproductive and respiratory syndrome (PRRS) virus.

A blocking Elisa was developed for the detection of antibodies against PRRS virus with a view to satisfying the need for examination of blood samples on a large scale. The test was evaluated in comparison with an indirect Elisa and the immunoperoxidase monolayer assay. The blocking Elisa was sensitive and specific. It had a higher capacity and was cheaper to perform than the immunoperoxidase monolayer assay and the indirect Elisa. It was comparable to the immunoperoxidase monolayer assay and better than the indirect Elisa in detecting antibodies formed early after infection, and it was superior to both the immunoperoxidase monolayer assay and the indirect Elisa in detecting antibodies at a late stage of infection.

Blocking ELISA's for the distinction between antibodies against European and American strains of porcine reproductive and respiratory syndrome virus.

A double blocking ELISA was developed in order to satisfy the need for large scale serological screening for PRRS and simultaneous distinction between infection with European and American strains of PRRSV in pig herds. The Immunoperoxidase monolayer assay (IPMA) and the double blocking ELISA enabled distinction on serological basis between infection with European and American strains of PRRSV. The distinction was possible from about day 7 after infection of pigs with PRRSV. The double blocking ELISA enabled the distinction at later stages of infection compared to the IPMA, irrespective of the strain involved.

The in vivo dose rate effect of chronic gamma radiation in mice: translocation and micronucleus analyses.

The in vivo effects of chronic, ultra low dose rates of gamma radiation in mice were evaluated using fluorescence in situ hybridization and the in vivo micronucleus test. SWRxC57BL/6 mice were divided into nine exposure groups and continuously exposed to 0.5, 2.0 or 4.0cGy 137Cs per day for 30, 60 or 90 days; unexposed control mice were also included. Following exposure, blood samples were taken from each animal and the frequencies of micronucleated polychromatic erythrocytes (MPCE) and micronucleated normochromatic erythrocytes (MNCE) were determined using flow cytometry. Peripheral blood lymphocytes were cultured and analyzed by chromosome painting to determine translocation frequencies. A significant dose rate response was seen in translocations and both MPCE and MNCE. Comparisons were made between the three chronic dose rates and it was determined that there was no significant difference among translocation frequencies for each rate. However, a significant difference was found between the chronic exposures reported here and the fractionated daily exposures reported previously. Dose rate reduction effects, ranging from 3 at low doses to 14 at high doses, were found for chronic versus acute exposures. The possibility of gender effects was investigated in both micronucleus and translocation data. No gender effect was found in translocation induction, but a slight effect was suggested in micronucleus induction.

Differential effect of acetyltransferase expression on the genotoxicity of heterocyclic amines in CHO cells.

We earlier developed the Chinese hamster ovary UV5P3 cell line that expresses cytochrome P4501A2 and lacks nucleotide excision repair for studying metabolism and mutagenicity of heterocyclic amines. The Chinese hamster ovary UV5P3 cells are approximately 50-fold more sensitive to the cooked food mutagen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) than 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), another genotoxic compound found in cooked food, with respect to cytotoxicity and mutation induction at the adenine phosphoribosyltransferase (aprt) locus. To test the hypothesis that the important missing activity in our CHO system for IQ genotoxicity was acetyltransferase, we transfected the UV5P3 cells with cDNA plasmids of either the human NAT2 N-acetyltransferase gene or a bacterial O-acetyltransferase gene. Functionally transformed clones were determined by the differential cytotoxicity assay using IQ, and confirmed by measuring the enzyme activity with isoniazid as substrate. Two clones designated 5P3NAT2 and 5P3YG (expressing human and bacterial transferases, respectively) were characterized. Both cell lines were sensitive to killing by IQ at concentrations as low as 4 ng/ml. Based on the D37 value, the dose that reduced the survival to 37% relative to untreated controls, the acetyltransferase expressing lines showed approximately 1000-fold increase in sensitivity to the killing effect of IQ over the parental UV5P3 cell line. The same dramatic change in sensitivity was also seen in mutation response at the aprt locus and with chromosomal aberrations and sister chromatid exchanges. In contrast, these cell lines showed cytotoxicity to PhIP similar to that of the parental line UV5P3. These results suggest that PhIP does not require acetyltransferase for metabolic activation leading to genotoxicity in these cells. These new cell lines constitute a sensitive cell system for assessing genotoxicity of compounds requiring metabolic activation by both P450IA2 and acetyltransferase, as well as for studying the molecular processes by which DNA damage can lead to mutation and cancer.

The accumulation of chromosome aberrations and Dlb-1 mutations in mice with highly fractionated exposure to gamma radiation.

The dichotomy between the doses at which experimental measurements of genetic effects can be made and the doses to which people are exposed is often different by two or more orders of magnitude. This presents a significant problem when determining the effects of low doses of radiation or chemicals. The solution has usually involved extrapolating the data by curve-fitting or by applying theoretical considerations. Both approaches are unsatisfactory due to uncertainties of the assumptions used in each process. The alternative solution has been to increase the sample size enormously at the lower doses. This is impractical beyond a certain point due to the variation in the spontaneous frequency and the need to quadruple the sample size for a doubling of precision. The development of new methods for measuring stable genetic effects, however, permits a simple and effective approach to this problem: if the genetic events being detected have no effect on survival, i.e., are selectively neutral, then the effects of multiple independent treatments will be additive. If the independent treatments are identical, then the effect of each is easily calculated by dividing the total effect by the number of treatments. Here we report a limited test of this approach using mice. Chromosome aberrations induced in lymphocytes and Dlb-1 mutations induced in the small intestine were measured after daily doses of 0.64, 1.85 or 5.5 cGy 137Cs gamma rays administered for 21, 42 or 63 days. The dose response curve for chromosome translocations obtained in this way, combined with the data from single larger acute doses, shows no evidence for a threshold over a 500-fold dose range. Dlb-1 mutations were increased at each dose and time but the results do not permit reliable extrapolations. The results suggest that translocations might be useful for quantifying the effect of doses below 0.05 cGy and that the effect of dose rate and dose fractionation at much lower doses than reported here could be investigated.

Natural transmission of foot-and-mouth disease virus from African buffalo (Syncerus caffer) to cattle in a wildlife area of Zimbabwe.

An outbreak of foot-and-mouth disease (FMD) occurred during April 1991 in a trypanosomiasis sentinel cattle herd by the Rifa River to the east of Lake Kariba, Zimbabwe. Despite the cattle having been vaccinated biannually for the previous five years the disease was severe. The viruses isolated from the affected animals were typed as FMD virus type SAT 1. Free-living African buffalo (Syncerus caffer) which had been using the same watering place as the affected cattle were sampled and FMD type SAT 1 virus was isolated. Partial nucleotide sequencing of the gene coding for the capsid protein 1D (VP1) of one of the viruses isolated from cattle and two of the viruses isolated from buffalo demonstrated a close relationship between the three viruses. Since no other cattle were present in the area and no outbreaks of SAT 1 had occurred in Zimbabwe since 1989, it was concluded that the disease had been transmitted from buffalo to cattle.

Serotype specificity of antibodies against foot-and-mouth disease virus in cattle in selected districts in Uganda.

Uganda had an unusually large number of foot-and-mouth disease (FMD) outbreaks in 2006, and all clinical reports were in cattle. A serological investigation was carried out to confirm circulating antibodies against foot-and-mouth disease virus (FMDV) by ELISA for antibodies against non-structural proteins and structural proteins. Three hundred and forty-nine cattle sera were collected from seven districts in Uganda, and 65% of these were found positive for antibodies against the non-structural proteins of FMDV. A subset of these samples were analysed for serotype specificity of the identified antibodies. High prevalences of antibodies against non-structural proteins and structural proteins of FMDV serotype O were demonstrated in herds with typical visible clinical signs of FMD, while prevalences were low in herds without clinical signs of FMD. Antibody titres were higher against serotype O than against serotypes SAT 1, SAT 2 and SAT 3 in the sera investigated for serotype-specific antibodies. Only FMDV serotype O virus was isolated from one probang sample. This study shows that the majority of the FMD outbreaks in 2006 in the region studied were caused by FMDV serotype O; however, there was also evidence of antibodies to both SAT 1 and SAT 3 in one outbreak in a herd inside Queen Elizabeth national park area.