A Note on the Diagnosis of Ratoon Stunting Disease of Sugarcane by Negative-Stain Electron Microscopy of the Associated Bacterium

After negatively staining with 1% (w/v) sodium phosphotungstate (pH 6·5) or 1% (w/v) ammonium molybdate (pH 6·5), the cell wall, cytoplasmic membrane and mesosomes of the RSD-associated bacterium obtained from the fibrovascular fluid of infected sugarcane were usually clearly displayed. The cells measured 0·19–0·39 μm (av. 0·27 μm) in width and 0·6–3·4 μm in length. Few mesosomes were visible and the cells were approx. 40% wider (0·27–0·52 μm, av. 0·38 μm) when stained with 1% (w/v) uranyl acetate (pH 3·0–4·2). Freezing and thawing the suspension before negative staining with sodium phosphotungstate did not greatly affect the size of the cells or resolution of the mesosomes. Glycine (0·25 M) as the suspending medium, fixation in 2% (w/w) glutaraldehyde, or placing wet instead of dry specimen grids in the electron microscope resulted in wider cells usually lacking mesosomes.     

Life Cycle of Eimeria ferrisi Levine & Ivens, 1965 in the Mouse, Mus musculus

SYNOPSIS. The life cycle of Eimeria ferrisi is described from experimentally infected Mus musculus. The prepatent period was 3 days and the patent period was 3–4 days. The endogenous stages were found only in the cecum and colon. Three generations of schizonts were found. Mature 1st-generation schizonts first seen 24 hr postinoculation (PI) measured 10.9 (7–14) × 10.2 (6–13) μm and had 9.6 (7–14) merozoites. Some 2nd-generation schizonts had uninucleate merozoites and others had multinucleate merozoites. The former were first seen in small numbers 36 hr PI and were most abundant 48 hr PI. They measured 9.6 (5–13) × 7.9 (6–12) μm and had 18 (6–25) merozoites. Schizonts with multinucleate merozoites were seen 72 hr PI. Mature 3rd-generation schizonts were seen 72 hr PI. They measured 14.0 (12–18) × 11-0 (9–13) μm and had 12.5 (5–16) merozoites. Macrogamonts were first seen in 72 hr sections. Each young macrogamont had a large nucleus with a prominent nucleolus. Only one type of cytoplasmic granule appeared to be involved in the formation of the oocyst wall. Mature macrogamonts were 11.0 (5–14) × 10.0 (6–13) μm. Crescent-shaped bodies were observed in the parasitophorous vacuole of trophozoites and young macrogamonts. Early microgamonts were first recognized at 96 hr by the presence of darkly stained and irregularly shaped nuclei. Usually, mature microgametes were arranged in long, narrow whorls at the periphery of the microgamont or in whorls at the surface of 2–5 compartments.     

A new technique to gather 3‐D spatial information using a single camera

A new technique using a single camera and shadows to determine 3‐D spatial positions of fishes in the laboratory is described. The apparatus consisted of a large aquarium (2·0 × 1·5 × 0·4 m), a wide‐angle camera mounted above and two light sources to cast shadows to either side of the fish. Using image analysis and vector mathematics, aquarium objects were plotted within 1·5 cm of their actual location along the x ‐, y ‐ and z ‐axis. The technique was also successful in quantifying changes in 3‐D spatial pattern of juvenile fish, Atlantic cod Gadus morhua (7·4–8·6 cm standard length, L _S) and cohabitant piscivorous shorthorned sculpin Myoxocephalus scorpinus (12·0–25·8 cm L _S), at these same viewing fields. The new technique should have a wide application, largely because it is potentially less expensive, laborious and invasive than alternative methods for determining 3‐D positions of fishes.     

Coccidia from the Tiger Salamander, Ambystoma tigrinum, in Northeastern Colorado and in Northern New Mexico

SYNOPSIS. Oocysts of Eimeria ambystomae Saxe, 1955, Eimeria microcapi sp. n., and Eimeria urodela sp. n. are described from the tiger salamander, Ambystoma tigrinum , collected in Colorado and New Mexico. The oocysts of E. ambystomae are ellipsoid, 29.8 × 17.3 (24–38 × 15–25) μm, and the sporocysts lanceolate, 22.6 × 5.4 (16–27 × 5–7) μm. Oocyst and sporocyst residua are present, but not a polar granule and a micropyle. The oocysts and sporocysts of E. microcapi are ellipsoid, measuring respectively 38.1 × 25.3 (35-41 × 23-26) μm and 18.1 × 7.4 (16-19 × 6–8) μm. Oocyst and sporocyst residua, a micropyle (mean 3 μm), and a distinct micropyle cap (2 μm high) are present, but not a polar granule. The oocysts of E. urodela are spheroid, 22.2 (14-26) μm, and the sporocysts lanceolate, 16.3 × 5.8 (12-19 × 4-7) μm. Oocyst and sporocyst residua are present, but a polar granule and a micropyle are absent.     

Scanning Isoelectric Focusing and Isotachophoresis of Clostridium perfringens Type A Enterotoxin

Fractionation of highly purified Cl. perfringens type A enterotoxin by scanning isoelectric focusing (SIF) and isotachophoresis (IT) in polyacrylamide gels is described for the first time. The use of 2% ampholytes pH 3–6 allowed the separation of enterotoxin into 2 species. The major component had an isoelectric point of 4·5 and possessed antigenic as well as functional activity. The minor component of enterotoxin, at equivalent concentrations, was devoid of any demonstrable biological activity had an isoelectric point of 4·6 and appeared to represent approximately 15% of the purified enterotoxin. With ampholytes pH 3·5–10 the minor and major components were focused at different times than when ampholine pH 3–6 was employed. Electrofocusing of enterotoxin in the presence of 6 M-urea did not alter the SIF pattern. During IT the major component of enterotoxin migrated ahead of the minor component. The 2 proteins were completely separated. Isotachophoretic separations required 0·023 M-phosphate pH 6·0 as the leading ion, 0·079 M-Tris as the counter-ion, 0·2 M-glycine (in Tris pH 8·1) as the terminating ion, 30 γ carrier ampholytes pH 3·5–10, 263 μg enterotoxin, 4% acrylamide and a current of 5 mA per gel column.     

Coccidian Parasites (Apicomplexa: Eimeriidae) of Nerodia spp. (Serpentes: Colubridae), with a Description of a New Species of Eimeria

Eimeria conanli n. sp. (Apicomplexa: Eimeriidae) is described from intestinal contents and feces of Nerodia erythrogaster transversa and N harteri harteri from northcentral Texas. Oocysts of the new species are ellipsoid in shape. 17.9 × 13.0(15–21 × 12–15) μm, with a smooth, thin, single-layered wall; shape index 1.4 (1.2–1.5). One to several (usually 2) polar granule(s) and an oocyst residuum are present, but a micropyie is absent. Sporocysts are elongate, 12.9 × 5.2 (13–15 × 5–6) -m, apparently without a true Stieda body structure. Each sporoeyst contains an ellipsoid residuum, 3.9 × 3.2 (3–6 × 2–4) μm, and elongate sporozoites, 11.4 × 2.5 (10–14 × 2–3) μm in situ, each with a spherical or subspherical anterior refractile body and spherical to ellipsoid posterior refractile body. In addition to the new species, oocysts of 4 previously described eimerians from colubrid snakes were found in these hosts.     

The Structure of the Oocyst and the Excystation Process of Isospora marquardti sp. n. from the Colorado pika, Ochotona princeps

SYNOPSIS. Oocysts of Isospora marquardti sp. n. from the Colorado pika, Ochotona princeps , have spheroid oocysts, 30.5 (23–36) μm in diameter, and ovoid sporocysts, measuring 19.3 × 12.0 (17–22 × 10–14) μm. A polar body, 2 × 4 μm, a spheroid sporocyst residuum (8.3 μm in diameter), a Stieda body, and a distinct substiedal body (3 × 3 μm) are present. A micropyle and an oocyst residuum are absent. Excysted sporozoites, averaging 3.0 × 18.5 (2–4 × 15–20) μm, contain 2 refractile globules, 1 on each side of the nucleus with a prominent nucleolus.
The sporozoite excystation process using a trypsin-sodium taurocholate fluid is described.     

THE PROLIFERATION OF LYMPHOID CELLS IN GUINEA-PIG BONE MARROW

A double isotope DNA labelling method has been used to determine the duration of DNA synthesis (S) in bone marrow lymphoid cells classified by their nuclear diameters in smears. Incorporation of ³H-thymidine was confined almost entirely to marrow lymphoid cells of 8·0-15·0 μm nuclear diameter (large lymphoid cells). After exposure to ³H-thymidine in vivo and ¹⁴C-thymidine 40-104 min later in vitro , the proportion of cells labelled with ³H alone to those labelled with ¹⁴C(±³H) in radioautographic smears, plotted against time indicated the efflux from S per hour. Collectively, 28·3 ± 1·1% of all large lymphoid cells were in S and the efflux from S was 15·1% per hour. With decreasing cell size (nuclear diameter) the efflux fell progressively from 28·3% per hour (11·0 μm) to 9·2% per hour (8·0-8·9 μm) and the proportion of cells in S declined from 54·9 ± 2·3% to 14·8 ± 1·6%. Influx into S, measured in vitro by reversing the sequence of isotopes, closely resembled the corresponding efflux values in vivo relative to cell size. Most DNA synthesizing marrow large lymphoid cells belonged to a subgroup with deeply basophilic cytoplasm. The results demonstrate that basophilic large lymphoid cells in the marrow are actively proliferating and have a mean S phase duration of 6·6 hr. The largest marrow lymphoid cells (11·0 μm) proliferate most rapidly (S phase, 3·5 hr; maximum cell cycle time, 6·4 hr) while S duration is prolonged progressively to 10·9 hr for the smaller cells (8·0-8·9 μm).     

Morphology and fine structure of the heart of the burbot, a cold stenothermal fish

The ventricle of the burbot Lota lota heart comprised 0·148 ± 0·006% of the body mass which is nearly two-fold heavier than the relative ventricular mass ( M _V) of other similarly sized teleosts. The shape of the ventricle is pyramidal and the wall is exclusively composed of spongious muscle without a distinct compact layer. The atrium forms 0·017 ± 0·002% of the body mass. Length, width, sarcolemmal surface area and volume of enzymatically isolated myocytes from burbot ventricle were 147·2 ± 10·2 μm, 6·3 ± 0·4 μm, 2440·8 · 251·5 μm² and 2356·8 ± 316·6 μm³, respectively. The myofibrils were peripherally located and their volume density was remarkably high: 65 ± 2 and 68 ± 3% in ventricle and atrium, respectively ( P >0·05). Although not particularly conspicuous, some nonjunctional and junctional sarcoplasmic reticulum (SR) was present in both atrial and ventricular myocytes. The SR formed peripheral couplings with the sarcolemma and the junctional clefts were frequently occupied by foot processes. These findings suggest that cold-adaptation is achieved by cardiac enlargement, high volume density of myofibrils and well-developed peripheral couplings in the SR in the heart of stenothermal burbot.     

Eimeria galateai sp. n. from the Paradise Kingfisher, and Eimeria duncani sp. n. from the Sacred Kingfisher in Papua New Guinea

SYNOPSIS. Eimeria galateai sp. n. from the paradise kingfisher ( Tanysiptera galatea Gray) and Eimeria duncani sp. n. from the sacred kingfisher ( Halcyon sancta Vigors & Horsfield) have been described from Papua New Guinea. Four of 11 paradise kingfishers were infected with E. galateai oocysts, measuring 13 (11–16) × 9 (8–11) μm. The oocysts were ovoid with nipple-like protrusion at one pole. Micropyle and polar granule were absent, while oocyst residuum (5 × 4 μm) was present. Sporocysts, measuring 5 (4–6) × 2 μm, were elongate-ovoid, and had a distinct convex Stieda body; the sporocyst residuum was absent. Two of 9 sacred kingfishers were infected with ovoid-truncated, 22 (19–25) × 16 (12–18) μm oocysts of E. duncani . Polar granule (5 × 2) was present in the oocysts, but there was no micropyle or oocyst residuum. Sporocysts were ovoid, measuring 9 (8–10) × 5 (4–6) μm, with a prominent Stieda body, and granular sporocyst residuum. Eimeria galateai and E. duncani are the first species of this genus to be described from birds of the order Coraciiformes.     

Effects of osmolality and ions on the motility of stripped and testicular sperm of freshwater-and seawater-acclimated tilapia, Oreochromis mossambicus

A significantly higher concentration of testicular spermatozoa was obtained from freshwater Oreochromis mossambicus (9·9×10⁹ spermatozoa ml⁻¹) than seawater O. mossambicus (4·6×10⁹ spermatozoa ml⁻¹). The mean osmolality of the urine of freshwater fish (78·5 mOsmol kg⁻¹) was significantly different from that of seawater fish (304·8 mOsmol kg⁻¹). The mean length of the mid-piece of the spermatozoa together with the tail was more variable in freshwater O. mossambicus (8·80±0·23μm) than in seawater specimens (8·27±0·18 μm). Stripped sperm of freshwater O. mossambicus was highly contaminated by urine which was a good activator of sperm motility in O. mossambicus held in both fresh and sea water. The osmolality for initiation of motility in freshwater O. mossambicus spermatozoa was from 0 to 333 mOsmol kg⁻¹ while for seawater O. mossambicus spermatozoa it was from 0 to 1022 mOsmol kg⁻¹. The optimum osmolality for motility was from 70 to 333 mOsmol kg⁻¹ for freshwater O. mossambicus spermatozoa and from 333 to 645 mOsmol kg⁻¹ for seawater fish. In freshwater O. mossambicus spermatozoa, the presence of 20 mM CaCl₂ increased the permissive osmolality of NaCl from 184 to 645 mOsmol kg⁻¹. For seawater O. mossambicus spermatozoa, solutions of NaCl devoid of CaCl₂ were unable initiate motility, but the addition of 1·5 to 30 mM CaCl₂ to the NaCl solution (0–934 mOsmol kg₁) had a full motility initiating effect.     

Eimeria procyonis sp. n., an Isospora sp., and a Redescription of E. nuttalli Yakimoff and Matikaschwili, 1932 (Protozoa: Eimeriidae) from the American Raccoon (Procyon lotor)

SYNOPSIS. Thirty-two of 48 raccoons examined were infected with a previously undescribed species of Eimeria which is herein named E. procyonis. Of the 32 infected animals, 10 also harbored E. nuttalli and 1 had Isospora sp. oocysts.
The ellipsoid to ovoid oocysts of E. procyonis measured 23.4 × 18.0 (16–29 × 13–24) μm; its sporocysts measured 12.1 × 9.3 (11.5–15 × 7–10) μm, each containing a slightly flattened substiedal body. The sporocyst residuum consisted of numerous scattered granules each ∼1 μm in diameter. The oocyst wall was double-layered. The outer layer appeared rough and pitted, measuring 1.5 μm, except at the micropyle where it was 1 μm thick.
The oocysts of the Isospora sp. measured 16.8 × 13.7 (16–18.5 × 12.5–15.5) μm. The wall consisted of a single layer ∼0.5 μm thick. The sporocysts measured 11.2 × 9.1 (9.5–11.5 × 8–10) μm, and each contained 4 elongate sporozoites. The oocysts of E. nuttalli measured 17.5 × 13.6 (12-21 × 11-15) μm, with a smooth single-layered wall approximately 0.7 μm thick. The sporocysts measured 12.2 × 7.1 (9-13 × 5.5–11) μm. Each sporocyst had a thin, dark, Stieda body and the sporocyst residuum consisted of many fine granules.     

Ultrastructure of Tachyzoites, Bradyzoites and Tissue Cysts of Neospora caninum

. Dividing tachyzoites of Neospora caninum were 4x3 μm and had ultrastructural characteristics typical for the cyst-forming coccidia. Unusual ultrastructural characteristics of fully-formed tachyzoites included no micropores, 8–12 anterior and 4–6 posterior rhoptries, and a few posterior micronemes. Most tachyzoites were located free in the host cell cytoplasm; only a few occurred within a parasitophorous vacuole. Parasite multiplication appeared to be rapid because most organisms were in various stages of endodyogeny. Neural tissue cysts of N. caninum were 24.3 × 19.2 μm and contained 50–200 bradyzoites (7.3 × 1.5 μm), which lacked micropores. The cyst wall was 0.74–1.12 μm thick and consisted of the primary cyst wall (the parasitophorous vacuole membrane) and a thick granular layer with electron-dense vesicles.     

The relative abundance of brown trout in acidic softwater lakes in relation to water quality in tributary streams

The influence of the water quality of tributary streams on the relative abundance in benthic gillnet catches (catch per unit effort, cpue) of allopatric brown Salmo trutta was assessed in associated acidic, softwater lakes. The study was carried out over 6 years (1989–1994) in 15 lakes located at altitudes between 230–715 m a.s.l. in two Norwegian catchments. The water quality of the main inlets and outlets varied little, as indicated by their of pH range (4·93–5·51) and calcium concentrations (0·19–0·44 mg 1⁻¹), but varied more with respect to concentrations of inorganic, monomeric aluminium (7·0–41·0 μg l⁻¹). Most of the lakes were also fed by secondary streams with better water quality: a maximum pH of 6·56, calcium levels of up to 0·74mg 1⁻¹, and inorganic aluminium levels as low as l·0 μg 1⁻¹. The cpue was inversely correlated with lake altitude ( r ²=0·50), and thus was adjusted to a mean altitude. The calcium concentration in the richest secondary stream to each lake, its richness judged on the basis of its acid-neutralizing capacity, had the highest predictive power of the variability in cpue ( r ² = 0·49).The calcium content in the other secondary streams or in the main inlets and outlets did not correlate with cpue. Alkalinity in the main outlets correlated to some extent with cpue ( r ² = 0·27). It is suggested that secondary streams with good water quality provide important refuges for the recruitment of brown trout in acidic softwater lakes.     

Modelling the growth of Weissella cibaria as a function of fermentation conditions

Aims:  To investigate the effect of pH, water activity ( a _w) and temperature on the growth of Weissella cibaria DBPZ1006, a lactic acid bacterium isolated from sourdoughs.
Methods and Results:  The kinetics of growth of W. cibaria DBPZ1006 was investigated during batch fermentations as a function of pH (4·0–8·0), a _w (0·935–0·994) and temperature (10–45°C) in a rich medium. The growth curve parameters (lag time, growth rate and asymptote) were estimated using the dynamic model of Baranyi and Roberts (1994. A dynamic approach to predicting bacterial growth in food. Int J Food Microbiol 23, 277–294). The effect of pH, a _w and temperature on maximum specific growth rate (μ_max) were estimated by fitting a cardinal model. μ_max under optimal conditions (pH = 6·6, a _w = 0·994, T  = 36·3°C) was estimated to be 0·93 h⁻¹. Minimum and maximum estimated pH and temperature for growth were 3·6 and 8·15, and 9·0°C and 47·8°C, respectively, while minimum a _w was 0·918 (equivalent to 12·2% w/v NaCl).
Conclusions:  Weissella cibaria DBPZ1006 is a fast-growing heterofermentative strain, which could be used in a mixed starter culture for making bread.
Significance and Impact of the Study:  This is the first study reporting the modelling of the growth of W. cibaria , a species that is increasingly being used as a starter in sourdough and vegetable fermentations.     

Two New Species of Isospora from the Desert Skink (Chalchides ocellatus) from the Egyptian Desert

Isospora chalchidis n. sp. and Isospora eimanae n. sp. were isolated from the desert skink, Chalchides ocellatus , order Squamata, from Egypt. Sporulated oocysts of I. chalchidis n. sp. were spheroidal 19 × 19 (18–20.5 × 18–20.5) μm with a 2–layered walled. Oocysts lacked a micropyle and residuum and few polar granules were present. Sporocysts were lemon-shaped 12.2 × 6.5 (9.5–13 × 5–8) μm with a Stieda body and sporocyst residuum but without a substiedal body. Isospora eimanae n. sp. were spheroidal 18.5 × 18.5 (17–19.5 × 17–19.5) μm with a 2–layered wall. There were oocysts without residuum or polar granule, but with small micropyle at 1 end. Sporocysts were ovoid 12 × 8.5(11–13 × 7.5–9) μm with a small Stieda body and sporocyst residuum but without a substiedal body.     

QUANTITATIVE HISTOCHEMISTRY OF γ-AMINOBUTYRIC ACID IN CAT SPINAL CORD WITH SPECIAL REFERENCE TO PRESYNAPTIC INHIBITION

Abstract— A method is described for quantifying the GABA distribution in cat spinal cord at 200–500 μn resolution. Isolated spinal cord (L5–S1) was frozen and sectioned at about 150 μm thickness. The frozen tissue section was cut into 200 or 500 μm square blocks. The GABA content of each square tissue block was determined by enzymic micromethods and GABA distribution was mapped quantitatively. Average GABA concentrations were: 0·4 mmol/l. in white matter, 1·2 mmol/l. in ventral horn and 1·7 mmol/l. in dorsal horn. The highest concentrations of GABA (2–3 mmol/l.) were found in the dorsolateral part of dorsal horn. In order to destroy the interneurons of dorsal horn, the blood vessels supplying the dorsal horn of the lumbar enlargement were unilaterally cauterized. Seven to 30 days after operation, both the size of dorsal root potential and the GABA level in the dorsal horn were markedly reduced on the cauterized side. These results suggest that GABA is highly concentrated in the interneurons of dorsal horn and functions as a transmitter of presynaptic inhibition.     

Growth characteristics of skeletal muscle tissue in Oreochromis niloticus larvae fed on a lysine supplemented diet

The effect of lysine amino acid supplementation on the growth characteristics and morphological pattern of skeletal muscle tissue in Nile tilapia Oreochromis niloticus larvae was evaluated. There were four treatments (T) with increasing levels of lysine supplement (T1 = 0·0%; T2 = 1·1%; T3 = 1·7%; T4 = 4·0%) and one treatment with a commercial diet (T5). In all treatments, morphological and histochemical muscle tissue analyses were similar. Two distinct layers were identified: a superficial red layer, more developed in the lateral line region, formed by fibres with intense to moderate NADH‐TR reaction and strong acid‐stable mATPase activity, and a deep white one, most of the muscle mass, formed by fibres with weak NADH‐TR reaction and strong alkali‐stable mATPase activity. There was an intermediate layer between these two layers with fibres exhibiting either weak acid‐stable or acid‐labile mATPase activity. Body mass increase was significantly higher in T5 than in the lysine treatments (T1–T4). There was no difference in number and diameters of muscle fibres between lysine treatments. In T5, muscle fibre diameter and number were higher. The frequency of red fibres with diameters ≤8 μm was higher in the lysine treatments, and with diameters between 16 and 24 μm, was higher in T5. Most white fibre diameters in T5 were significantly larger than 24 μm and in T1–T4 were between 8 and 16 μm. Cell proliferation was higher in the lysine treatments and muscle growth in T5 was mainly by fibre hypertrophy.     

An efficient transformation system for Bifidobacterium spp.

This study describes a broad host transformation protocol that enables the uptake of plasmid DNA into 10 different species of Bifidobacterium , some of which have never been transformed before. The vector pNC7 (4·9 kb) was used to optimize the electroporation protocol. Transformation efficiencies ranged from 3·6×10⁻¹ to 1·2×10⁵ transformations per μg DNA. The impact of growth medium composition and electric field strength on transformation efficiency were independently optimized. Electrocompetent cells were grown in Iwata medium broth enriched with Actilight^RP 16%, harvested during the early exponential growth phase, and pulsed at 12·5 kV cm⁻¹, 100 Ω and 25 μF.     

Haematology and leucocyte morphology of wild caught Thunnus maccoyii

The haematology of wild southern bluefin tuna Thunnus maccoyii was described using blood samples collected from fish immediately after they were caught. Cytology and cytochemistry revealed that the blood in peripheral circulation is comprised of erythrocytes, reticulocytes, ghost cells, lymphocytes, thrombocytes, eosinophilic granulocytes, neutrophilic granulocytes and monocytes. Reference ranges established were 41·09–55·50% for haematocrit, 0·62–3·00% for leucocrit, 13·25–17·92 g dl⁻¹ for haemoglobin and 2·1–2·9 million erythrocytes μl⁻¹ for erythrocyte count. Differential cell counts showed 94·58 ± 2·15% erythrocytes, 3·99 ± 1·44% leucocytes and 1·43 ± 1·03% thrombocytes (mean ±  s . d .). Normal ranges for differential leucocyte counts were 0·00–5·45% for neutrophils, 0·69–12·06% for eosinophils, 0·00–5·03% for monocytes, 46·97–74·32% for lymphocytes and 14·47–43·92% for thrombocytes. Erythrocyte indices, leucocyte types and cytochemistry were comparable to other species of scombrids. Packed cell volume was sensitive to the physiological state of the fish and to sample handling technique.