Phagocytosis by catfish neutrophils

Channel catfish peripheral blood leucocytes were separated on a Percoll gradient to establish the phagocytic function of the neutrophils. Four fractions of leucocytes were formed on the Percoll gradient, including a fraction that contained 50–80% neutrophils at a density of 1.08–1.09 g ml⁻¹ and a fraction that contained 10% monocytes at a density of 1.071–1.074 g ml⁻¹. Phagocytic assays, using ³H-uridine, showed that the two fractions had similar phagocytic indices, although neutrophils were less phagocytic than monocytes. Neutrophils were confirmed to be phagocytic when examined with transmission electron microscopy. Staining with 3,3-diaminobenzidine-tetrahydrochloride demonstrated peroxidase-positive granules in the cytoplasm of actively phagocytic cells as well as peroxidase reaction products in a number of phagosomes containing bacteria. Phagocytosis of bacteria by channel catfish neutrophils was further confirmed by differential staining of external bacteria and cell surfaces with ruthenium red during the fixation process.     

Evaluation of in vitro endocytosis and antibody synthesis by rainbow trout head kidney cells treated with bovine lactoferrin

Bovine lactoferrin (LF) was evaluated for its capacity to modulate the in vitro endocytosis (phagocytosis and pinocytosis) and antibody synthesis by head kidney cells of rainbow trout Oncorhynchus mykiss . Phagocytic activity and phagocytic index of head kidney macrophages, determined by measurement of ingested yeast, were influenced by bovine LF starting from the LF concentration of 1 and 0·1 μg ml⁻¹, respectively. Endocytosis, determined by the evaluation of droplet uptake of neutral red dye solution, was significantly enhanced by 10 μg ml⁻¹ of LF. In contrast, antibody synthesis by head kidney cells, evaluated by immunoenzymatic assay, from fish immunized against human‐γ‐globulins (HGG) in vivo was not affected by bovine LF. Although these results showed that bovine LF had no effect on specific immunoglobulin production in vitro , an enhancement of the acquired immune response may be assumed in LF‐treated fish in vivo , as observed in higher vertebrates.     

Effect of inhibitors on ammonium assimilation in Chlorella sorokiniana in light and darkness

In N-sufficient cells of Chlorella sorokiniana Shihira and Krauss strain 211/8K (CCAP of Cambridge University), assimilation of ammonium was strictly dependent on light and CO₂, and was severely inhibited by 100 μ M atrazine or 10 μ M 3-(3,4-dichlorophenyl)-1, l-dimethylurea (DCMU). In N-limited cells, assimilation of NH₄⁺ took place at similar rates in both light and darkness, which were 1.6-fold higher than the rate of light-dependent assimilation by N-sufficient cells. Assimilation by N-limited cells was inhibited by l -methionine- dl -sulfoximine (MSX), but not by atrazine or DCMU.
The rate of photosynthetic O₂ evolution was 2.9±0.9 mmol ml⁻¹ packed cell volume (PCV) h⁻¹ in N-sufficient cells, and 0.64±0.12 mmol ml⁻¹ PCV h⁻¹ in N-limited cells. In the latter resupply of ammonium resulted in a rapid activation by 22%;, followed by a time-dependent increase of the photosynthetic O₂ evolution, which after 12 h reached the same rate as in N-sufficient cells.
Respiratory consumption of oxygen in darkness in N-sufficient and N-limited cells was 0.10±0.03 and 0.11±0.02 mmol ml⁻¹ PCV h⁻¹, respectively. Addition of ammonium was without effect on respiration of N-sufficient cells, but resulted in a 4-fold stimulation of respiration of N-limited cells. Such stimulation took place also in cells treated with DCMU, atrazine, or MSX, and it was also promoted by methylammonium. The stimulation of respiration lasted for several hours.     

Physiological stress effects of continuous- and pulsed-DC electroshock on juvenile bull trout

Juvenile bull trout Salvelinus confluentus exposed to continuous- or pulsed-DC electroshock exhibited rapid elevations in plasma cortisol and glucose, but plasma chloride did not change. In a 1-h experiment using 240 V at 1·4 A of 60-Hz pulsed DC (voltage gradient 0·81 V cm⁻¹), which proved lethal, plasma cortisol and glucose rose significantly within 15 min of a 10-s electroshock. Plasma cortisol reached a peak level of 156 ± 18 ng ml⁻¹ at 45 min and then decreased, whereas plasma glucose reached its highest level of 179 ± 7·5mg dl⁻¹ at 1 h. In a 24-h experiment using lower dosages, plasma cortisol increased from 6·1-16 ng ml⁻¹ to peak levels of 155–161 ng ml⁻¹ in 1 h in response to a 10-s electroshock of continuous (130 V, 0·5 A, 1·45 V cm⁻¹) or pulsed (120 V, 0·5 A, 60 Hz, 0·55 V cm⁻¹) DC. Although plasma concentrations declined thereafter, levels remained above control values at 24 h. Plasma glucose was elevated from 60–65 to 120–134 mg dl⁻¹ after 1h by both electroshock treatments and remained near or above those levels for the 24-h duration. Plasma cortisol and glucose levels were much higher in electroshocked bull trout at 1 h compared with those in fish 1 h after receiving a 30-s handling stressor (cortisol, 90 ± 12 ng ml⁻¹; glucose, 82 ± 6·1 mg dl⁻¹). The results indicate that both continuous and pulsed DC were more stressful to juvenile bull trout than handling and that recovery, at least for pulsed DC, may take longer than 24 h.     

Haematology of swordtail, Xiphophorus helleri. I: blood parameters and light microscopy of blood cells

The main blood parameters of the swordtail, Xiphophorus helleri , were studied. Morphology, granulation staining and cytochemistry of leucocytes in peripheral blood, kidney, spleen and gills were investigated by light microscopy. Blood parameters are similar to other fish species: Red blood cell count (4.5 × 10⁶μl), white blood cell count (15.2 × 10³μl), haema-tocrit (33.8%) haemoglobin (7.8 mg ml⁻¹), MCV (mean corpuscle volume, 75.1 μm³). MCH (mean content of haemoglobin, 17.3 pg), MCHC (mean percentage haemoglobin/erythrocyte, 23.1%/100 ml erythrocytes). Leucocytes can be classified into lymphocytes, thrombocytes, neutrophilic and eosinophilic gra-nulocytes, monocytes macrophages and melanomacrophages.
Morphological and cytochemical features of the cells are described and compared with results from other fish species.     

Symbiotic effectiveness, rate of respiration and glutamine synthetase activity of sodium azide-resistant strains of Rhizobium leguminosarum biovar trifolii

Nitrogen fixing efficiency of sodium azide-resistant strains of Rhizobium leguminosarum bv. trifolii was studied in symbiosis with berseem clover plants in chillum jars. Rate of respiration and glutamine synthetase activity were tested in cultured cells and nodules, respectively. It was observed that shoot dry weight and percentage shoot nitrogen were maximum in plants inoculated with strains resistant to 15 μg ml⁻¹ sodium azide. Rate of respiration in cultured cells was lowest in strains resistant to 15 μg ml⁻¹ sodium azide and highest in strains resistant to 5 μg ml⁻¹ sodium azide. A negative correlation was observed between rate of respiration (in cultured cells) and shoot dry weight of host plants. Glutamine synthetase activity was maximum in nodule extracts of host plants inoculated with strains resistant to 5 and 10 μg ml⁻¹ sodium azide, whereas it was minimum for strains resistant to 15 μg ml⁻¹ sodium azide. Hence, resistance to low doses (15 μg ml⁻¹) of sodium azide, together with lower respiratory and glutamine synthetase activities, could be used as a potential method for isolating the symbiotically effective strains of Rh. leguminosarum bv. trifolii.     

The effect of cryptolepine on the morphology and survival of Escherichia coli, Candida albicans and Saccharomyces cerevisiae

The antimicrobial activity of the indoloquinoline alkaloid, cryptolepine, isolated from Cryptolepis sanguinolenta (Fam. Periplocaceae) was determined against selected micro-organisms. The minimum inhibitory concentration (MIC) ranges obtained, expressed as μg ml⁻¹, were: 5–10 for Saccharomyces cerevisiae NCPF 3139; 10–20 for S. cerevisiae NCPF 3178; 20–40 for Escherichia coli NCTC 10418; 40–80 for E. coli NCTC 11560, Candida albicans ATCC 10231 and C. tropicalis NCPF; and 80–160 for C. albicans NCPF 3242 and NCPF 3262.
Biocidal effects were noted at concentrations 2–4 times those of the MIC of the alkaloid following challenge with 10⁶ cfu ml⁻¹ of micro-organisms. Time-kill studies showed a reduction in viable count from 10⁶ to < 10 cfu ml⁻¹ in 4 h in C. albicans ATCC 10231 exposed to 320 μg ml⁻¹ of the agent; 3 log cycle reductions were recorded for the 6 h counts of E. coli NCTC 10418 and S. cerevisiae NCPF 3139 exposed to 40μg ml⁻¹ and 160 μg ml⁻¹ of the alkaloid respectively.
These results were consistent with findings using scanning electron microscopy. Exposure of cells to biocidal concentrations of cryptolepine produced filamentation prior to lysis in E. coli NCTC 10418 and extreme disturbance of surface structure, including partial and total collapse, followed by lysis in C. albicans ATCC 10231 and S. cerevisiae NCPF 3139.     

Cortisol and glucose responses in juvenile brown trout subjected to a fluctuating flow regime in an artificial stream

In an artificial stream channel, wild 1 year old brown trout Salmo trutta were exposed to fluctuations in flow and water level to simulate hydro-peaking conditions downstream of a hydropower installation. Blood plasma cortisol concentrations reached a maximum of 59·4 ± 35·3 ng ml⁻¹ (mean ± 95% CL) 2h after the end of down-ramping. Return to the pre-exposure cortisol level was achieved within 6 h. When subjected to daily cyclical fluctuations over 7 days, plasma cortisol levels were significantly elevated (61·3 ± 26·8 ng ml⁻¹) on the first day compared to undisturbed fish (4·9 ± 3·7 ng ml⁻¹). On the fourth and seventh day, no elevation in plasma cortisol above control levels was observed. No changes in blood glucose that could be attributed to the stressor were found. There was no correlation between plasma cortisol and blood glucose levels. The short-lived cortisol response to daily fluctuations indicates a rapid habituation to this Stressor.     

Rapid, extensive and reversible vacuolation of Schizosaccharomyces pombe induced by amphotericin B

Abstract The fission yeast Schizosaccharomyces pombe has no large vacuoles under normal growth conditions, although budding yeasts usually have large central vacuoles. The minimum inhibitory concentration of amphotericin B to S. pombe was 0.5 μg ml⁻¹; treatment with 0.2 μg ml⁻¹ for 20 min induced rapid and extensive vacuolation in S. pombe exponential phase cells. Growth rate of the cells with 0.2 μg ml⁻¹ amphotericin B was much reduced for 6 h, showing extensive vacuolation. Vacuolation in itself was not fatal: on removal of the drug, most cells recovered gradually and eventually multiplied.     

In vitro suppression of the phagocytic response of fish macrophages by tetracyclines

Tetracycline (TC) and oxytetracycline (OTC) caused a dose-dependent suppression of the chemiluminescence (CL) emitted by phagocytes from the kidney of rainbow trout, Salmo gairdneri , using zymosan or latex beads as the stimulus. Compared to the control response without antibiotics, partial but significant suppression was found after exposing the cells to OTC concentrations of 0.1–50.0 μg ml⁻¹. Cells exposed to 100 or 500 μg ml⁻¹ OTC showed CL responses below the base levels elicited by control cells. Comparable results were obtained with cells exposed to TC and stimulated by the same stimuli. The kinetics of the CL response and the suppressive effects of the antibiotics were similar in cells from individual fish but the magnitude of responses varied. No acclimation occurred following extended exposure to the drugs.     

Epinephrine, norepinephrine, and cortisol concentrations in cannulated seawater-acclimated rainbow trout (Oncorhynchus mykiss) following black-box confinement and epinephrine injection

The present study investigates the effect of cannulation and chronic'black-box' confinement, as well as epinephrine administration (4–0 μg kg⁻¹), on the degree and time-course of alterations in trout ( Oncorhynchus mykiss ) catecholamine and cortisol concentrations. Plasma cortisol concentrations in seawater trout acclimated to 3–6° C reached 104 ng ml⁻¹ 1 day after cannulation/confinement and remained elevated above resting levels (8 ng ml⁻¹) until 6 days post-confinement. Although plasma epinephrine and norepinephrine generally declined over the period of confinement (day 1 approx. 12 nM; day 7 approx. 6 nM), norepinephrine titres were usually higher and more variable. Epinephrine injection caused elevations in plasma epinephrine levels but not in norepinephrine levels; epinephrine titres reaching 107 ± 26 nM (range 65–238 nM) at 2 min post-injection and returning to pre-injection levels by 30 min post-injection. Plasma cortisol increased by 20 ng ml⁻¹ following epinephrine administration. Based on the time-course for post-confinement alterations in plasma cortisol, it appears that up to a week may be required before cannulated fish are completely acclimated to 'black-box' confinement. The findings suggest that meaningful results from experiments utilizing epinephrine injection and 'black-box confinement are contingent upon: (1) knowledge of circulating epinephrine levels shortly after injection (i.e. within 2 min post-injection); and (2) an experimental design that takes into account the elevated cortisol titres that are inherent with cannulation/confinement and epinephrine injection.     

Immunodot detection of nisin Z in milk and whey using enhanced chemiluminescence

A highly specific antisera was produced in New Zealand white rabbits against nisin Z, a 3400 Da bacteriocin produced by Lactococcus lactis ssp. lactis biovar. diacetylactis UL 719. A dot immunoblot assay was then developed to detect nisin Z in milk and whey. As few as 1·5 10⁻¹ international units per ml (IU ml⁻¹), corresponding to 0·003 μg ml⁻¹ of pure nisin Z, were detected in carbonate-bicarbonate buffer within 6 h using chemiluminescence. When milk and whey samples were tested, approximately 0·155 μg ml⁻¹ (7·9 IU ml⁻¹) of nisin Z was detected. The detection limit obtained was lower than that of traditional methods including microtitration and agar diffusion.     

Effects of triploidy on rainbow trout myogenesis in vitro

To examine the effects of triploidy on rainbow trout myogenesis in vitro , mononuclear cells were liberated enzymatically from the lateralis muscles of diploid and triploid trout. The muscle of diploids yielded 1 × 10⁶± 1 × 10⁵ (± s.e.m. ) mononuclear cells g⁻¹ muscle compared to 0.7 × 10⁶± 8 × 10⁴ cells g⁻¹ from triploids ( P <0.01). The plating efficiencies of diploid and triploid mononuclear cells on Matrigel™ following 18 h of culture in Leibovitz's L-15 + 10% foetal bovine serum were not significantly different, 35.0 ± 3.5% and 33.0 ± 2.9%, respectively. For most time points examined, the proportion of nuclei in multinucleated cells and the proportion of nuclei in myosin positive cells were not significantly different for diploid and triploid trout. Taken together, these data suggest that diploid and triploid myogenic cells will differentiate similarly when compared under identical, in vitro conditions.     

Development of a microtitre ELISA to quantify development of Cryptosporidium parvum in vitro

Abstract An in situ enzyme-linked immunosorbent assay (ELISA) was developed to evaluate growth of Cryptosporidium parvum in vitro. Ninety-six-well tissue culture microtitre plates were each seeded with 4.0 X 104 human ileocecal adenocarcinoma (HCT-8) cells, then infected with CsCl-purified oocysts 24 h later. The growth medium consisted of RPMI 1640 supplemented with 10% fetal bovine serum, 15 mM HEPES (JV-2-hydroxyethylpiperazine N −2-ethanesulfonic acid), 50 mM glucose, 1 μg ml⁻¹ folic acid, 4 μg ml⁻¹ 4-aminobenzoic acid, 2 μg ml⁻¹ pantothenic acid and 35 μg ml⁻¹ ascorbic acid. Incubation conditions were at 37 ° C in a 5% CO₂/95% humidified air incubator. Oocysts were allowed to excyst in situ so that sporozoites could infect cells directly. Monolayers were then washed, new medium added, and infected cells re-incubated. Levels of infection were assessed 48 h later using a rat anti-C. parvum polyvalent antiserum directed against purified parasite membranes, followed by a goat anti-rat IgG conjugated to horseradish peroxidase and 3,3',5,5'-tetramethyl-benzidine as substrate. Using various parasite inoculating doses and incubation times, optimal results were obtained using a 90-min exposure of host cells to 2.5−3.0 × 10⁴ oocysts/well. Evaluation of various concentrations of four anti-microbials (monensin, lasalocid, paromomycin and sulfadimethoxine) in the system resulted in the acquisition of precise dose-response curves for each compound.     

The effect of sublethal endrin exposure on rainbow trout, Salmo gairdneri Richardson. I. Evaluation of serum cortisol concentrations and immune responsiveness

Two-hundred-and-forty rainbow trout, Salmo gairdneri , were exposed for 30 days to sublethal concentrations of the pesticide endrin. On day 30, ten fish from each treatment group were sacrificed and examined for the ability of peritoneal macrophages to phagocytize latex beads. The remaining fish were immunized with 10 μg of Yersinia ruckeri O-antigen, and exposure to endrin continued. The migration inhibition factor assay (MIF), plaque forming cell assay (PFC), and serum agglutination titres (SAG) were performed 2, 14, and 30 days post-antigen inoculation. Serum was collected from all fish for serum cortisol concentrations. Endrin exposure had no effect on the phagocytic ability of peritoneal macrophages. However, the MIF, PFC and SAG responses were significantly reduced from control values. Serum cortisol concentrations were found to be significantly elevated in endrin-exposed fish. Serum cortisol concentrations were found to be significantly higher on days 44 and 60 (192 and 194 ng ml⁻¹, respectively) when compared to days 30 and 32 (159 and 141 ng ml⁻¹, respectively). Cortisol values for days 30 and 32 did not differ significantly, nor did those of days 44 and 60. The relationship between elevated serum cortisol concentrations and endrin exposure on the immune response is discussed.     

Evaluation of biochemical methods for the non‐destructive identification of sex in upstream migrating salmon and sea trout

Female‐specific markers of reproductive activity [plasma 17β‐oestradiol (E2), vitellogenin (VTG) and alkali‐labile phosphoprotein phosphorous (ALP)] were measured over 12 months in a captive population of brown trout Salmo trutta . During the early months of the reproductive season (February to May) and using the concentration of plasma E2 or plasma ALP as a marker for females the proportion of fish in which sex was misidentified was high (15–50%). The misidentification rate was considerably lower (1–8%) using plasma VTG. Preliminary evaluation of a commercial immunochromatographic VTG test system as a screen for the presence or absence of VTG in plasma from brown trout provided results that were consistent with those obtained from direct measurement of plasma VTG levels by enzyme‐linked immunosorbent assay (ELISA). These preliminary conclusions were verified by sampling upstream‐migrating anadromous brown trout, sea trout, and Atlantic salmon Salmo salar trapped over a 6 month period. Plasma E2 levels did not satisfactorily discriminate between male and female sea trout and Atlantic salmon. Plasma VTG levels in both species, however, were bimodally distributed and it was assumed that this divergence corresponded to male (plasma VTG levels <10 μg ml⁻¹) and female (plasma VTG levels >800 μg ml⁻¹) fishes. Plasma ALP provided a more accurate indication of sex in the wild Atlantic salmon and sea trout than was suggested by the pilot study on captive brown trout. The commercial immunochromatographic VTG test system provided results that were wholly consistent with the data obtained from the trapped fishes by direct measurement of plasma VTG.     

Effects of aluminium and cortisol on in vitro calcium deposition on otoliths in rainbow trout

Aluminium (1–100 μM) reduced in vitro calcium deposition on otoliths concentration-dependently in rainbow trout, but cortisol (1 and 10 μg ml⁻¹) had no effect.     

In vitro susceptibility of Clostridium perfringens isolated from farm animals to growth-enhancing antibiotics

Minimal inhibitory concentration (MIC) determinations were carried out with seven growth-enhancing antibiotics against 95 Clostridium perfringens field isolates obtained during 1991 and 1992 from poultry, pigs and calves. All were resistant to 64 μg ml⁻¹ of the bambermycin antibiotic, flavomycin (flavophospholipol) and susceptible to avoparcin (MIC₉₀ 0.25 μg ml⁻¹), avilamycin (MIC₉₀ 0.5 μg ml⁻¹) and salinomycin (MIC₉₀≤ 0.12 μg ml⁻¹). Acquired resistance against bacitracin was detected in some isolates from poultry and bovines and resistance to tylosin and virginiamycin in some strains from all species investigated. Overall, the prevalence of resistance was comparable to the low levels recorded in 1979 in Cl. perfringens isolates from the same animal host species.     

Sequence of gonadal events and oestradiol levels in Sparus aurata (L.) under two photoperiod regimes

Individually tagged Sparus aurata were kept in tanks with running sea water (21°± 2°C) during their second and third years of life. Gonads were biopsied and blood was sampled at monthly intervals. Thirteen of 50 fish in the second year and 24 of 35 fish in the third year were phenotypically females. Fish kept under 16L/8D photoperiod from July 1981 to August 1982 did not reach maturation; waves of initial ovarian growth alternated with waves of atresia. When photoperiod was shortened as from August 1982, gonadal development commenced within a month and proceeded at a rate higher than that of the control fish reared under natural photoperiod. Under natural photoperiod oestradiol serum levels (E₂) were relatively low during the resting phase, May-October (108 ± 11 pg ml⁻¹), and during the late vitellogenic phase (502 ± 76 pg ml⁻¹). High levels (1669 ± 312 and 1240 ± 172pg ml⁻¹) occurred during the early vitellogenic and the maturational phases. The high level of E₂ during the spawning season of S. aurata is explained by earlier reports indicating a prolonged breeding season with daily release of eggs, and alternating daily surges of E₂ and 17α, 20β, dihydroxy-4-pregnen-3-one, possibly a 'maturational progestin' in this fish. In phenotypic males, E₂ was highest during early spermatogenic phases (745 ± 142pg ml⁻¹) but was low (155 ± 11 pg ml⁻¹) in males with running sperm.     

Antibacterial activity of bovine lactoferrin and its peptides against enterohaemorrhagic Escherichia coli O157:H7

The antimicrobial activities of bovine lactoferrin (bLF), its pepsin hydrolysate (bLFH) and the active peptide lactoferricin^® B (LFcinB) against four clinical isolates of enterohaemorrhagic Escherichia coli O157:H7 were studied. The MICs against these isolates were 3 mg ml⁻¹ for bLF, 0·1–0·2 mg ml⁻¹ for bLFH and 8–10 μg ml⁻¹ for LFcinB in 1% Bactopeptone broth. LFcinB killed these bacteria within 3 h at concentrations above 10 μg ml⁻¹. Transmission electron microscopy findings suggested that LFcinB acts on the bacterial surface and affects cytoplasmic contents. LFcinB was shown to influence the levels of verotoxins in the culture supernatant fluid of an E. coli 0157:H7 strain. These results demonstrate that E. coli O157:H7 strains are susceptible to the antimicrobial effects of bLF and its peptides.