Identification of a gene set to evaluate the potential effects of loud sounds from seismic surveys on the ears of fishes: a study with Salmo salar.

Functional genomic studies were carried out on the inner ear of Atlantic salmon Salmo salar following exposure to a seismic airgun. Microarray analyses revealed 79 unique transcripts (passing background threshold), with 42 reproducibly up-regulated and 37 reproducibly down-regulated in exposed v. control fish. Regarding the potential effects on cellular energetics and cellular respiration, altered transcripts included those with roles in oxygen transport, the glycolytic pathway, the Krebs cycle and the electron transport chain. Of these, a number of transcripts encoding haemoglobins that are important in oxygen transport were up-regulated and among the most highly expressed. Up-regulation of transcripts encoding nicotinamide riboside kinase 2, which is also important in energy production and linked to nerve cell damage, points to evidence of neuronal damage in the ear following noise exposure. Transcripts related to protein modification or degradation also indicated potential damaging effects of sound on ear tissues. Notable in this regard were transcripts associated with the proteasome-ubiquitin pathway, which is involved in protein degradation, with the transcript encoding ubiquitin family domain-containing protein 1 displaying the highest response to exposure. The differential expression of transcripts observed for some immune responses could potentially be linked to the rupture of cell membranes. Meanwhile, the altered expression of transcripts for cytoskeletal proteins that contribute to the structural integrity of the inner ear could point to repair or regeneration of ear tissues including auditory hair cells. Regarding potential effects on hormones and vitamins, the protein carrier for thyroxine and retinol (vitamin A), namely transthyretin, was altered at the transcript expression level and it has been suggested from studies in mammalian systems that retinoic acid may play a role in the regeneration of damaged hair cells. The microarray experiment identified the transcript encoding growth hormone I as up-regulated by loud sound, supporting previous evidence linking growth hormone to hair cell regeneration in fishes. Quantitative (q) reverse transcription (RT) polymerase chain reaction (qRT-PCR) analyses confirmed dysregulation of some microarray-identified transcripts and in some cases revealed a high level of biological variability in the exposed group. These results support the potential utility of molecular biomarkers to evaluate the effect of seismic surveys on fishes with studies on the ears being placed in a priority category for development of exposure-response relationships. Knowledge of such relationships is necessary for addressing the question of potential size of injury zones.

Use of recombinant antigens for detection of Toxoplasma gondii infection in swine.

Five recombinant Toxoplasma gondii antigens, designated B427, C51, C55, V22, and MBP30 were assessed for their potential use in an enzyme-linked immunoassay (EIA) for detection of T. gondii infection in swine. The antigens were evaluated with sera from young pigs that had been fed 1-10,000 T. gondii oocysts of the VEG or GT-1 strains. Results were compared with an EIA using a native T. gondii antigen extract. All 5 recombinant antigens, as well as native antigen, detected antibody responses as soon as 3 wk after infection in pigs inoculated with 1 or 10 oocysts of the VEG strain. This antibody response persisted, at varying levels, for 14 wk when the experiment was terminated. All antigens also detected antibody responses in pigs 4 wk after inoculation with 10,000 oocysts of the GT-1 strain. The antibody response recognized by native antigen remained high through 51 wk after inoculation. However, there was considerable animal-to-animal variation in responses to the individual recombinant antigens. Only antigens C51 and MBP30 consistently detected a positive antibody response over the entire 51-wk course of the experiment. These results suggest that these antigens might be useful for the serological detection of T. gondii infection in pigs.

Toxoplasma gondii recombinant antigens H4 and H11: use in ELISAs for detection of toxoplasmosis in swine.

Toxoplasma gondii recombinant antigens H4 and H11 were assessed for their potential for use in ELISA for diagnosis of toxoplasmosis in swine. The antigens were evaluated with sera from young pigs experimentally infected with T. gondii. Results were compared with ELISAs based on a native T. gondii antigen extract. Although recombinant antigen ELISAs showed a sharp rise in response with some sera very early after infection, they were relatively non-reactive with late (chronic) infection sera.

Long-term humoral antibody responses by various serologic tests in pigs orally inoculated with oocysts of four strains of Toxoplasma gondii.

Antibody titers to Toxoplasma gondii were determined in 16 pigs orally inoculated with 1000 or 10,000 oocysts of one of the four strains (GT-1, ME-49, TS-2, TC-2) of T. gondii. Pigs were euthanized on postinoculation days 103-875 and their tissues were bioassayed for T. gondii. Antibody titers were measured in the modified agglutination test (MAT) using formalin-preserved (test A) or acetone-preserved (test B) tachyzoites, latex agglutination test (LAT), indirect hemagglutination test (IHA), enzyme-linked immunobsorbant assay (ELISA), and the Sabin-Feldman dye test (DT). Toxoplasma gondii was isolated from all but two (one with GT-1 strain and one with TC-2 strain) of the inoculated pigs. Results of the serologic tests varied by test used, by strain of T. gondii and from pig to pig within groups. One pig inoculated with the TC-2 strain was considered not to be infected because it remained seronegative in all tests and T. gondii was not isolated from its tissues by bioassay. The IHA and LAT did not produce consistently positive results with infected pigs and two pigs remained seronegative (< 1:64) in both tests. At the time of necropsy, IHA titers had declined to < 1:64 in five pigs and LAT titers had declined to < 1:64 in four pigs. The MAT (test A) and ELISA detected antibodies in all infected pigs but ELISA did not detect antibody at the time of necropsy in one pig. Antibody titers peaked earliest in the DT. Antibody titers in the MAT (test B) peaked at 2560 in all seropositive pigs by 6 weeks postinoculation (PI) but declined to < 1:160 by 15 weeks PI; this test may be useful in determining recency of T. gondii infection in pigs. Overall, the MAT (test A) gave most consistent results.

Antibody responses measured by various serologic tests in pigs orally inoculated with low numbers of Toxoplasma gondii oocysts.

OBJECTIVE: To follow antibody responses measured by various serologic tests in pigs orally inoculated with low (< or = 10 oocysts) numbers of Toxoplasma gondii oocysts. ANIMALS: 24, 2- to 3-month-old pigs. PROCEDURE: Pigs (n = 42) were inoculated orally with 10 (14 pigs) or 1 (28 pigs) infective oocysts, and 6 pigs served as uninoculated controls. Blood (serum) samples were obtained at 1- to 3-week intervals until euthanasia. At necropsy, the brain, heart, and tongue of pigs were bioassayed in mice and cats for isolation of T gondii. Modified agglutination test (MAT), using whole, fixed tachyzoites and mercaptoethanol; latex agglutination test (LAT); indirect hemagglutination test (IHAT); Sabin-Feldman dye test (DT); and ELISA were used to evaluate serologic responses to T gondii. RESULTS: T gondii was isolated from tissues of 13 of 14 pigs each fed 10 oocysts, 17 of 28 pigs each fed 1 oocyst, and 0 of 6 control pigs. 29 of 30 T gondii-infected pigs developed antibodies when measured by MAT, DT, and ELISA; the 1 seronegative-infected pig had been fed 10 oocysts and was euthanatized 69 days after inoculation. LAT detected antibodies in 26 of 30 T gondii-infected pigs. IHAT detected antibodies in 11 T gondii-infected pigs. CONCLUSION: MAT, DT, and ELISA were more sensitive serologic assays than LAT and IHAT for detecting antibodies induced by low numbers of T gondii in pigs.

Sensitivity and specificity of various serologic tests for detection of Toxoplasma gondii infection in naturally infected sows.

The sensitivity and specificity of various serologic tests for antibodies to Toxoplasma gondii were compared in 1,000 naturally exposed sows, using isolation of viable T gondii as the definitive test. Serum samples obtained from heart blood of 1,000 sows from Iowa were examined for T gondii antibodies by use of the modified agglutination test (MAT), latex agglutination test (LAT), indirect hemagglutination test (IHAT), and ELISA. Toxoplasma gondii was isolated from 170 hearts of 1,000 sows by bioassays in mice and cats. The percentage of samples diagnosed as positive for each of the serologic tests was: MAT = 22.2% (titer > or = 1:20), IHAT = 6.4% (titer > or = 1:64), LAT = 10.4% (titer > or = 1:64), and ELISA = 24.1% (OD > 0.360). The sensitivity and specificity of these tests were calculated respectively to be: 82.9 and 90.29% for MAT, 29.4 and 98.3% for IHAT, 45.9 and 96.9% for LAT, and 72.9 and 85.9% for ELISA. The dye test was run at 1:20 dilution on only 893 sera because of bacterial contamination and presence of anticomplement substances. Dye test antibodies were found in 17.8% of the sera, and sensitivity and specificity were 54.4 and 90.8%, respectively. Thus, the MAT had the highest sensitivity among all serologic tests used.

Quality of life in hypertensives treated with atenolol or captopril: a double-blind crossover trial.

AIM: To compare the effects of captopril and atenolol on quality of life of hypertensive patients. METHODS: In a randomly allocated double-blind crossover trial with two 6-week treatment periods captopril at 25 mg twice a day or atenolol at 50 mg once a day were administered to 265 hypertensive patients (mean age 56 years; 55% men). Of these, 65% were newly treated hypertensives and 35% were previously uncontrolled on a diuretic alone. A seated diastolic blood pressure of 95-115 mmHg was required after a 3-week placebo run-in period. Any previous diuretic therapy was changed to hydrochlorothiazide (25 mg once a day) and the dose was kept constant throughout the trial. Newly diagnosed patients did not take a diuretic at any time. Quality of life was assessed from self-completed questionnaires measuring psychological well-being, symptomatic side effects of treatment, and activity and perceived well-being (a health index). A relative's perception of the patients' mood was also obtained where possible. RESULTS: Twelve patients withdrew on atenolol and 10 on captopril. No differences between the drugs were observed in quality of life measures, and 95% confidence intervals suggested that important differences were excluded. CONCLUSION: We conclude that at the doses used in this trial there were no important differences between captopril and atenolol in their effects on quality of life.

National seroprevalence of Toxoplasma gondii in pigs.

Serum samples from 11,842 commercial pigs killed in 1983-1984 throughout the United States were tested for anti-Toxoplasma gondii antibodies by the agglutination test in dilutions of 1:25, 1:50, and 1:500. Anti-T. gondii antibodies were found in 23.9% of pigs. At dilutions of 1:25, 1:50, and 1:500, 13.5%, 6.9%, and 3.5% were serologically positive, respectively. The prevalence of anti-T. gondii antibodies was higher in breeder pigs (42%) than in market pigs (23%). These results indicate that anti-T. gondii antibodies are widespread in the national swine herd.

Effect of Freezing on Infectivity of Toxoplasma Gondii Tissue Cysts in Pork.

To study the effect of low temperatures on infectivity of Toxoplasma gondii tissue cysts, pork from infected pigs was mixed with infected mouse brains and homogenized thoroughly. Twenty-gram samples of infected homogenized meat which were sealed in plastic pouches, pressed to a uniform thickness resulting in samples having the dimensions of ~.2 × 16 × 18 cm, were subjected to temperatures of -1 to -171.1 °C for 1 s to 67.2 d. Treated samples were digested in HCl-pepsin solution and infectivity assayed in mice. A regression model from these data is described by the least squares linear regression: Square root of time for the inactivation of T. gondii (h) = 26.72 + 2.16 temperature (°C) with r = 0.77. T. gondii tissue cysts remained viable usually up to 22.4 d at -1 and -3.9°C and 11.2 d at -6.7°C but were usually rendered nonviable by freezing at -12.37°C. These data demonstrate that T. gondii tissue cysts are inactivated by freezing more readily than encysted Trichinella spiralis larvae.

Effect of high temperature on infectivity of Toxoplasma gondii tissue cysts in pork.

To study the effect of high temperature on infectivity of Toxoplasma gondii tissue cysts, pork from infected pigs was mixed with infected mouse brains and homogenized thoroughly. Twenty-gram samples of infected homogenized meat were sealed in plastic pouches, pressed to a uniform thickness of 2 mm, and subjected to water-bath temperatures of 49, 52, 55, 58, 61, 64, and 67 C for 0.01, 3, 6, 9, 12, 24, 48, and 96 min. Treated samples were digested in HCl-pepsin solution and bioassayed in mice. Toxoplasma gondii tissue cysts remained viable at 52 C for 9.5 min but not for 9.5 min at 58 C; tissue cysts were generally rendered nonviable by heating to 61 C or higher temperature for 3.6 min. Tissue cysts survived once at 64 C for 3 min. These data demonstrate that T. gondii tissue cysts are less heat resistant than encysted Trichinella spiralis larvae.

Continuous in vitro cultivation of Sarcocystis cruzi.

Sporozoites of Sarcocystis cruzi were inoculated onto monolayer cultures of bovine pulmonary artery endothelial (CPA) cells. Sporozoites entered the cells, formed large and small multinucleate schizonts, and produced large numbers of merozoites. Continuous cultivation from the original sporozoite inoculum has been maintained for more than 1,320 days by subinoculating merozoites onto new cultures of CPA cells. During this time, the capacity to produce both types of schizonts was preserved, and schizogony was the only form of reproduction that was observed.

In vivo voltammetric evidence of production of uric acid by rat caudate.

Linear sweep in vivo voltammetry with carbon paste electrodes records a prominent peak at about 340 mV in the anterior caudate of rat brain. This peak is increased by microinfusion of uric acid or xanthine oxidase (which enhances conversion of hypoxanthine and xanthine to uric acid) and is decreased or eliminated by microinfusion of uricase. Allopurinol (a specific xanthine oxidase inhibitor) also decreases this peak when given either intracranially or intraperitoneally. Co-administration of uricase and allopurinol reliably eliminate the peak in question. These data suggest that uric acid, a purine metabolite that has been thought to be absent in brain, is formed locally in rat caudate and that uric acid is the sole component of the peak at 340 mV. In vivo voltammetry may be a useful new tool for studying brain purine metabolism.

The effects of lesions produced by 5,7-dihydroxytryptamine on 5-hydroxytryptamine-mediated behaviour induced by amphetamine in large doses in the rat.

The role of 5-hydroxytryptamine (5-HT)-containing terminals in the spinal cord and basal ganglia in behavioural responses induced by amphetamine in large doses have been investigated using the neurotoxin for 5-HT, 5,7-dihydroxytryptamine (5,7-DHT). The effects of pretreatment with 5,7-DHT were also examined using the 5-HT agonist, 5-methoxy-N,N-dimethyltryptamine (5-MeODMT). d-Amphetamine (25 mg/kg) induced several classical 5-HT-dependent behavioural responses (head weaving , forepaw treading, hind limb abduction, "wet dog" shakes, Straub tail), together with some classical dopamine (DA)-dependent behaviour and backward locomotion which requires both transmitters. Pretreatment with 5,7-DHT, given into the striatum significantly decreased "wet dog" shakes and virtually abolished backward walking. Pretreatment with 5,7-DHT in the nucleus accumbens or substantia nigra did not significantly alter behaviour. Pretreatment with 5,7-DHT intraspinally did not significantly alter behaviour induced by amphetamine, although a decrease of Straub tail just failed to reach significance (P = 0.056). Similar pretreatment in rats given 5-MeODMT (8 mg/kg) significantly enhanced both Straub tail and tremor but did not alter the other behavioural responses induced by this drug (limb abduction, forepaw treading, head weaving ). The results in general suggest that behavioural responses induced by 5-HT can be classified into 3 groups (a) those requiring striatal 5-HT ("wet dog" shakes and backward locomotion), (b) those requiring spinal 5-HT (Straub tail, tremor) and (c) those requiring neither spinal nor striatal 5-HT (hind limb abduction, head weaving and forepaw treading).

Turning behaviour following nigral injections of dopamine agonists and glycine.

Glycine, ADTN and ergometrine, but not LSD, induced strong dose-related contraversive turning following unilateral microinjections into the substantia nigra (zona reticulata) of conscious rats. The responses to glycine and ADTN were blocked by low doses of systemically injected cis-Z-flupenthixol but were unaffected by pretreatment with a variety of dopamine antagonists injected intranigrally. Turning behaviour following glycine was antagonised by strychnine but that to ADTN was not. Possible ways by which the rotational responses may be induced in view of the postulated neuronal circuitry of the substantia nigra and its connections with other brain areas are discussed.

A reliable multichannel commutator for making electrical contact with conscious, freely moving rats.

A counterbalanced mercury swivel arrangement is often used to make connections between electrodes implanted in the brain of conscious laboratory animals and electronic equipment so that movement is not restricted. There are certain disadvantages associated with this system and an alternative is described which is based on a rhodium plated slip ring. The device is reliable, robust, causes minimal hindrance to the movement of the animal and causes virtually no interference noise to low level electrical signals. The slip ring based commutator has been incorporated into an automated system for making in vivo voltammetric recordings from conscious rats where currents of less than 100 nA are measured. Some of the results are shown.

Differential involvement of dopamine-containing tracts in 5-hydroxytryptamine-dependent behaviours caused by amphetamine in large doses.

Dopamine (DA) concentrations in the rat striatum and/or nucleus accumbens were decreased by lesions in the nigra, ventral tegmentum, striatum and accumbens induced by 6-hydroxydopamine. These affected a range of DA- and 5-hydroxytryptamine-(5HT) dependent behaviours induced by 25 mg/kg of D-amphetamine as follows. Changes in DA-dependent behaviour (forward locomotion, head bobbing) occurred which were consistent with DA terminals in the accumbens and striatum being required for locomotor and stereotypical behaviour, respectively. A repetitive 5HT-dependent behaviour (head weaving) was decreased by lesions which depleted striatal DA. A 5HT-dependent behaviour with repetitive and locomotor elements (reciprocal forepaw treading) was only decreased by the nigral and tegmental lesions which depleted DA in both the striatum and the accumbens (and presumably also in other regions). These results suggest that 5HT-dependent behaviours of stereotypical character also require DA. Conversely, two 5HT-dependent behaviours, without obvious stereotypical or locomotor elements (body shakes, hind limb abduction), were not decreased by any of the lesions (and increased by some). Backward walking was markedly inhibited by lesions which decreased striatal DA. The above evidence for different relationships between each of the 5HT-dependent behaviours studied and DA-containing systems implies that these behaviours are separately mediated. It may clarify previous disagreement on the role of DA in these behaviours.

Dopamine receptors in rat striatum and nucleus accumbens; conformational studies using rigid analogues of dopamine.

A study was made of the actions of dopamine and of some 2-amino-1,2,3,4-tetrahydronaphthalenes on dopamine-sensitive adenylate cyclase in homogenates of rat striatum and nucleus accumbens. The compounds were also tested for their ability to stimulate motor activity following bilateral injection into the nucleus accumbens of conscious rats. The most active compounds on adenylate cyclase from both striatum and nucleus accumbens were dopamine and 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene(6,7-diOHATN). The 5,6-dihydroxy analogue (5,6-diOHATN) was 50 times less active than 6,7-diOHATN in striatal homogenates and 350 times less active in homogenates of nucleus accumbens. All dihydroxy compounds tested were active in causing stimulation of motor activity, the most active compounds being 6,7-and5,6diOHATN. Both dimethoxy derivatives tested were inactive on the adenylate cyclase and as locomotor stimulants.