Plant Biotests for Soil and Water Contaminated with Oil and Oil Products

Reactions of higher plants (mustard, oat, rye, lettuce, dill and barley) and microalgae (Euglena gracilis) to the contamination of soil and water with oil and oil products was studied. The germination of seeds was analyzed. The length of sprouts, dry biomass and length of plant roots, as well as the optical density of microalgal broth culture were determined. Negative effects of soil and water contamination with oil and oil products on plant and microalgal parameters examined was shown. After biological destruction of contaminants by an association of destructor strains (Acinetobactersp., Mycobacterium flavescens andRhodoccocussp.), the toxicity of contaminated mediums decreased. The data suggest that the integral toxicity of soil and water contaminated with oil and oil products and toxicity changes during biodestruction of these pollutants can be analyzed by using plant test organisms.     

An initial assessment of the genotoxic impact of the Sea Empress oil spill by the measurement of DNA adduct levels in the intertidal teleost Lipophrys pholis

The Sea Empress oil spill resulted in the release of vast quantities of potentially genotoxic contaminants into the coastal environment of the county of Pembrokeshire (UK). We are at present attempting to determine the potential genotoxic impact of the incident upon the native marine species of the area. Here we describe the levels of DNA adducts in specimens of the intertidal teleost, Lipophrys pholis, exposed to extensive oil contamination as an indication of exposure to potential genotoxins. We detected elevated levels of adducts in L. pholis specimens from an area that underwent heavy oil contamination as compared to specimens from a clean reference area devoid of oil contamination. These preliminary studies indicated that the oil contamination induced DNA adducts in the L. pholis specimens, which could potentially cause genetic damage in this native marine species. Further studies are now required to assess the full extent of the genotoxic impact of the oil spill upon the Pembrokeshire area's native marine life.     

Review on the effects of pollution on marine fish life and fisheries in the North Sea

Information on possible effects of pollution on fish life is reviewed with special emphasis to the North Sea. Concentrations of heavy metals are high in onshore waters, sediments and organisms. Offshore regions are considered to be not endangered. In estuaries concentrations of certain heavy metals are often surpassing thresholds, which have been experimentally demonstrated to produce sublethal effects on marine organisms. Also mussel culture areas had to be closed due to excess of cadmium contamination. Mercury in fillets of cod in the southern North Sea sometimes reaches 0.3 ppm representing 1/3 of the legal limit. Reductions of inputs are necessary due to the bioaccumulative and toxic potential of heavy metals. Organochlorine contamination of water, sediments and organisms is higher in the southern than in the northern North Sea. In offshore areas clearly elevated levels of PCB's, which are the dominating organochlorine compound in marine organisms, have been analyzed. At present only a minor fraction of total organochlorine residues accumulated in marine biota is analyzed. Margins between experimentally derived lethal levels and those occurring in marine biota are narrow suggesting that sublethal effects might occur. One of the special concerns in the context of organochlorine contamination has to be seen in the potential of these substances affecting the reproductive capacity of marine organisms. Reduction measures are urgently necessary and should include substances with bioaccumulative and toxicological potential, which are not presently included in Marine Monitoring Programmes. Impact of oil pollution is considered to be a regional problem, although especially in estuaries concentrations in the water column might be high. Input through oil platforms has led to local reductions in benthic communities. Measures have to be taken, to further reduce these inputs also in view to avoid contamination of fish in the vicinity of oil fields. Oxygen depletion in German and Danish coastal waters has been shown to be a severe degradation in coastal water quality resulting in significant impact on fish populations. Indications for large-scale avoidance reactions of low DO-areas and mortalities of fish and benthos organisms have been demonstrated. It is suspected that the low DO-situations are related to increased nutrients input and high phytoplankton production. Improvement of the situation can only be expected, if input of nutrients is reduced. Reasons for the occurrence of toxic plankton blooms are not understood. An intensified research is necessary, to avoid damage to fisheries and mariculture. Although activity of radionucleids in North Sea waters is increased due to input of radioactive material from La Hague and Sellafield reprocessing units, the present levels of activity are not considered to be a threat for marine life. The same holds for thermal pollution, which is, depending on the recipient area, expected to have only local effects. Sewage sludge dumping and discharge of untreated sewage introduce significant amounts of accompanying heavy metals, which can be expected to have a negative influence on the total metal budget of recipient waters. Nutrient input through sewage will exhibit only local effects. Dumping of wastes from titanium dioxide industry are suspected to be causally linked to the occurrence of increased prevalences of certain diseases of dab in the German Bight. Present management strategies should take account of the fact that through dumping of waste from titanium dioxide production significant quantities of heavy metal are introduced into the southern North Sea. Also the incineration of chemical wastes at sea should be reduced due to uncertainties concerning quantities and toxicity of incineration products and their potential impact on marine life. It is concluded that clear evidence exists for pollution impact in estuarine regions. Most of the major estuaries at the North Sea coast show benthos reductions, which can be related to water quality and it has to be assumed that areas of a sublethal effect are located in the vicinity of those areas of well demonstrated effects. They are of unknown size and attempts have to be undertaken to quantify areas of sublethal impact. The occurrence of fish diseases is interpreted as an expression of degraded water quality. Some of the diseases detected can produce mortality, thereby having an as yet unquantified impact on fish populations. The presence of fish diseases also reduces the marketability of catches. It cannot be excluded that changes in species composition of fish populations in German Wadden Sea areas are related to pollution. Concentrations of organochlorine substances in fishes of the southern North Sea give rise to the concern that their reproductive capability might be impaired. Future work has to be directed to this problem. Impact of pollution on populations of marine organisms has so far not been demonstrated.     

18S rDNA Sequences from Microeukaryotes Reveal Oil Indicators in Mangrove Sediment

Background

Microeukaryotes are an effective indicator of the presence of environmental contaminants. However, the characterisation of these organisms by conventional tools is often inefficient, and recent molecular studies have revealed a great diversity of microeukaryotes. The full extent of this diversity is unknown, and therefore, the distribution, ecological role and responses to anthropogenic effects of microeukaryotes are rather obscure. The majority of oil from oceanic oil spills (e.g., the May 2010 accident in the Gulf of Mexico) converges on coastal ecosystems such as mangroves, which are threatened with worldwide disappearance, highlighting the need for efficient tools to indicate the presence of oil in these environments. However, no studies have used molecular methods to assess the effects of oil contamination in mangrove sediment on microeukaryotes as a group.

Methodology/Principal Findings

We evaluated the population dynamics and the prevailing 18S rDNA phylotypes of microeukaryotes in mangrove sediment microcosms with and without oil contamination, using PCR/DGGE and clone libraries. We found that microeukaryotes are useful for monitoring oil contamination in mangroves. Our clone library analysis revealed a decrease in both diversity and species richness after contamination. The phylogenetic group that showed the greatest sensitivity to oil was the Nematoda. After contamination, a large increase in the abundance of the groups Bacillariophyta (diatoms) and Biosoecida was detected. The oil-contaminated samples were almost entirely dominated by organisms related to Bacillariophyta sp. and Cafeteria minima, which indicates that these groups are possible targets for biomonitoring oil in mangroves. The DGGE fingerprints also indicated shifts in microeukaryote profiles; specific band sequencing indicated the appearance of Bacillariophyta sp. only in contaminated samples and Nematoda only in non-contaminated sediment.

Conclusions/Significance

We believe that the microeukaryotic targets indicated by our work will be of great applicability in biomonitoring hydrocarbons in mangroves under oil contamination risk or during recovery strategies.     

Ratiometric fluorimetric and colorimetric probe for sensing and imaging pH changes in living cells

Cell, enzyme, and tissue activity in living organisms are closely related to intracellular pH. Detecting the changes of intracellular pH is important to understanding the physiological and pathological changes in the process of crucial cell metabolism. A pH probe (HTBI) based on hemicyanine was synthesized. The probe solution displayed a marked colour change from yellow to amaranth with the pH increase from neutral to basic; simultaneously, the emission spectra showed a significant red shift. The probe exhibited a ratiometric fluorescence emission (F_586nm/F_542nm) characteristic of pK_a 8.82. As expected, HTBI exhibited high sensitivity and selectivity for pH, fine photostability, reversibility, and low cytotoxicity. Therefore, it would be a very useful tool for measuring the intracellular pH changes.     

Photosynthesis in dynamic light: systems biology of unconventional chlorophyll fluorescence transients in Synechocystis sp. PCC 6803

Photosynthetic organisms live in a dynamic environment where light typically fluctuates around a mean level that is slowly drifting during the solar day. We show that the far-from-equilibrium photosynthesis occurring in a rapidly fluctuating light differs vastly from the stationary-flux photosynthesis attained in a constant or slowly drifting light. Photosynthetic organisms in a static or slowly drifting light can be characterized by a steady-state quantum yield of chlorophyll fluorescence emission F′ that is changing linearly with small and slow variations of the incident irradiance I+ΔI(t): F′(I+ΔI(t))≈ F_mean^′(dF)/(dI)·ΔI(t). In Synechocystis sp. PCC 6803, the linear approximation holds for an extended interval covering largely the static irradiance range experienced by the cyanobacteria in nature. The photosynthetic dynamism and, consequently, the dynamism of the chlorophyll fluorescence emission change dramatically when exposing the organism to a fluctuating irradiance. Harmonically-modulated irradiance I+ΔI · sin(2πt/T), T ≈ 1–25 s induces perpetual, far-from-equilibrium forced oscillations that are strongly non-linear, exhibiting significant hysteresis with multiple fluorescence levels corresponding to a single instantaneous level of the incident irradiance. We propose that, in nature, the far-from-equilibrium dynamic phenomena represent a significant correction to the steady-state photosynthetic activity that is typically investigated in laboratory. Analysis of the forced oscillations by the tools of systems biology suggests that the dynamism of photosynthesis observed in fluctuating light can be explained by a delayed action of regulatory agents.     

Preferential distribution of excitation energy into photosystem I of desiccated samples of the lichen Cladonia impexa and the isolated lichen-alga Trebouxia pyriformis

Abstract The effect of desiccation on distribution of excitation energy between the two photosystems has been studied in the lichen Cladonia impexa Harm., in the green alga Trebouxia pyriformis Archibald, isolated from Cladonia squamosa; and in the non-lichen green alga Scenedesmus obliquus, strain D₃. The method used was to compare the low temperature fluorescence emission of samples equilibrated with air with different humidity prior to freezing in liquid nitrogen. Desiccation of Cladonia and Trebouxia caused a pronounced increase of the height of the far red fluorescence emission band, F 715, over the short wave bands, F685 and F697; the ratio between the two short wave bands remained essentially constant. Upon rewetting, these species regained normal fluorescence emission properties, indicating that they are desiccation-tolerant. Scenedesmus, which was used as a desiccation intolerant species, also showed an increase of the far red fluorescence band over the two short wave bands upon desiccation, but the original fluorescence spectrum was not restored upon rewetting. These results are interpreted as showing that desiccation of tolerant species such as Cladonia and Trebouxia causes a preferential energy distribution into photosystem I. We tentatively believe that desiccation induces conformational changes within the chloroplast thylakoids, thereby controlling distribution of energy between the two photosystems. Furthermore, this change in energy distribution may be of ecological significance as the mechanism by which desiccated lichens or algae avoid photo-dynamic destruction of the photosynthetic apparatus when photosynthesis is inhibited under dry conditions. By a preferential distribution of absorbed energy into photosystem I, the organisms avoid the formation of strong, harmful oxidants in photosystem II when photosynthesis is inhibited. It is suggested that β-carotene associated with the far red-absorbing chlorophyll a fraction of the reaction center antenna of photosystem I is the final sink for excess excitation energy in dry, desiccation-tolerant lichens and algae.     

Atoxoplasma Associated with an Isosporan Oocyst in Canaries*

SYNOPSIS Coccidia-free and mite-free domestic canaries Serinus canarius given sporulated Isospora oocysts of canary origin and autopsied at intervals thereafter had numerous organisms in cells of the macrophage system structurally similar to organisms called Atoxoplasma. Judging from the sequence of intensity of infection, the mucosa of the upper intestine was parasitized first and the organisms spread from there down the intestine and to the internal organs. The prepatent period of 10 days for the Isospora infection could be started in one bird by oocyst administration and completed by transfer of infected liver cells from this bird to a 2nd bird. Isospara oocysts from sparrows were not infective for a canary.     

Dyakia bioluminescence—1. Bioluminescence and fluorescence spectra of the land snail,D. striata

The luminescent land snail Dyakia striata displayed a bioluminescence spectrum with a maximum wavelength of 515 nm. A green fluorescent substance extracted from the photogenic organ of an adult snail had a similar wavelength maximum but its fluorescence spectrum differed from that of flavin chromophore substances involved in light emission in some other luminescent organisms.     

A CCD-OMA device for the measurement of complete chlorophyll fluorescence emission spectra of leaves during the fluorescence induction kinetics

Summary A new device for the measurement of complete laser induced fluorescence emission spectra (maxima near 690 and 735 nm) of leaves during the induction of the chlorophyll fluorescence is described. In this the excitation light (cw He/Ne laser, 632.8 nm) is switched on by a fast electro-mechanical shutter which provides an opening time of 1 ms. The emitted fluorescence is imaged onto the entrance slit of a multichannel spectrograph through a red cut-off filter (> 645 nm). A charge coupled device (CCD) sensor with 2048 elements simultaneously detects the complete chlorophyll fluorescence emission spectrum in the 650–800 nm wavelength range. Scanning is accomplished electronically and the integration time for a complete fluorescence emission spectrum can be selected from 10 ms up to 260 ms. Shutter, detector system and data acquisition are controlled by an IBM-PC/AT compatible computer. A maximum of 32 spectra can be measured at selected times during the fluorescence induction kinetics with the shortest time resolution of 10 ms. The instrument permits the determination of various fluorescence parameters:a) the rise-time of the fluorescence to the maximum level fm,b) the changes in the shape of the fluorescence emission spectra during the induction kinetics,c) the induction kinetics in the fluorescence ratio F690/F735 as well asd) the fluorescence decrease ratio Rfd at any wavelength between 650 to 800 nm. These fluorescence parameters provide information about the functioning of photosynthesis. The ratio F690/F735 allows the non-destructive determination of the chlorophyll content of leaves. The application of this instrument in ecophysiological research and stress physiology of plants is outlined.     

Adhesive Property of Toxin-Coregulated Pilus of Vibrio cholerae O1

The adhesive property of toxin-coregulated pilus (TCP) to the human intestine (jejunum), and whether or not TCP mediates the adhesion of Vibrio cholerae 395 organisms to the intestinal epithelium were investigated using visually proving methods. The purified TCP did not agglutinate human erythrocytes nor adhere to the surface of human intestinal epithelium. V. cholerae 395 adhered to the epithelium, but the adhesion was not inhibited by blocking the pili with the Fab fraction of anti-TCP IgG. The organisms adhered to the intestine treated with purified TCP in advance, as well as to the intact intestine. These findings suggest that TCP is not involved in the adhesion of these organisms to the intestinal epithelium.     

Temperature dependence and polarization of fluorescence from Photosystem I in the cyanobacterium Synechocystis sp. PCC 6803

To determine the fluorescence properties of cyanobacterial Photosystem I (PS I) in relatively intact systems, fluorescence emission from 20 to 295 K and polarization at 77 K have been measured from phycobilisomes-less thylakoids of Synechocystis sp. PCC 6803 and a mutant strain lacking Photosystem II (PS II). At 295 K, the fluorescence maxima are 686 nm in the wild type from PS I and PS II and at 688 nm from PS I in the mutant. This emission is characteristic of bulk antenna chlorophylls (Chls). The 690-nm fluorescence component of PS I is temperature independent. For wild-type and mutant, 725-nm fluorescence increases by a factor of at least 40 from 295 to 20 K. We model this temperature dependence assuming a small number of Chls within PS I, emitting at 725 nm, with an energy level below that of the reaction center, P700. Their excitation transfer rate to P700 decreases with decreasing temperature increasing the yield of 725-nm fluorescence.Fluorescence excitation spectra of polarized emission from low-energy Chls were measured at 77 and 295 K on the mutant lacking PS II. At excitation wavelengths longer than 715 nm, 760-nm emission is highly polarized indicating either direct excitation of the emitting Chls with no participation in excitation transfer or total alignment of the chromophores. Fluorescence at 760 nm is unpolarized for excitation wavelengths shorter than 690 nm, inferring excitation transfer between Chls before 760-nm fluorescence occurs.Our measurements illustrate that: 1) a single group of low-energy Chls (F725) of the core-like PS I complex in cyanobacteria shows a strongly temperature-dependent fluorescence and, when directly excited, nearly complete fluorescence polarization, 2) these properties are not the result of detergent-induced artifacts as we are examining intact PS I within the thylakoid membrane of S. 6803, and 3) the activation energy for excitation transfer from F725 Chls to P700 is less than that of F735 Chls in green plants; F725 Chls may act as a sink to locate excitations near P700 in PS I.Abbreviations Chl chlorophyll - BChl bacteriochlorophyll - PS Photosystem - S. 6803 Synechocystis sp. PCC 6803 - PGP potassium glycerol phosphate     

Highly fluorescent carbon dots from wheat bran as a novel drug delivery system for bacterial inhibition

In this study, we prepared carbon dots (CDs) from wheat bran via hydrothermal treatment at 180°C for 3 h. The prepared CDs showed blue‐green fluorescence under UV light. The fluorescence emission study of the CDs revealed that they showed maximum fluorescence emission at 500 nm. The prepared CDs showed a high quantum yield of 33.23%. Solvent‐dependent fluorescence emission analysis of the CDs was performed to study the variation in fluorescence emission characteristics with solvent polarity. The prepared CDs were conjugated with amoxicillin (AMX) to explore its potential for use as a drug delivery agent for AMX. The drug release profile of the CD–AMX conjugates was analyzed at different pH (5.0, 6.8 and 7.2) to study drug release kinetics. CD–AMX conjugates showed notable bacterial inhibition against Gram‐positive (S. aureus) and Gram‐negative (E. coli) strains with minimal cytotoxic effects, indicating its potential as a promising antibacterial drug delivery system.     

The arrangement of chloroplasts in cells influences the reabsorption of chlorophyll fluorescence emission. The effect of desiccation on the chlorophyll fluorescence spectra of Rhizomnium punctatum leaves

Chlorophyll fluorescence spectra measured with leaves are distorted by the effect of fluorescence reabsorption. A heterogeneous theoretical model simulating the effect of chloroplast arrangement in a cell on the distortion of chlorophyll fluorescence spectra due to reabsorption was formulated. Desiccation of leaves of the moss Rhizomnium punctatum was carried out as a simple model experiment. The parameters entering the model (maximal number of chloroplasts forming columns in a cell, chloroplast size and chlorophyll concentration in a chloroplast) were estimated by means of light microscopy and spectrophotometry. During the desiccation, a grouping of chloroplasts was observed by light microscopy and the chlorophyll fluorescence emission and excitation spectra of the leaves were measured at room temperature and at 77 K. The leaves were infiltrated with DCMU. The ratio F685/F735 of the main emission bands decreased by about 50% at room temperature and by about 30% at 77 K upon decreasing the leaf water content. No significant changes were found in the ratio E475/E436 of the bands of the leaf fluorescence excitation spectra at 77 K for both 685- and 735-nm emission wavelengths. The excitation spectra and mechanical dilution experiments indicated that no functional changes appeared upon desiccation at the level of energy transfer. Theoretical simulations were in a good agreement with the experimental dependencies. We were able to conclude that the grouping of chloroplasts in cells may enhance the effect of chlorophyll reabsorption and thereby cause a significant decrease of the F685/F735 ratio in the chlorophyll fluorescence spectrum.     

Tryptophan fluorescence studies of plasma fibronectin: effects of environmental factors

Steady state fluorescence measurements have been used to study tryptophan fluorescence of plasma fibronectin. The native protein has an emission maximum at 337 nm with a quantum yield of 0.03. A red shift of emission maximum was observed in 3–5M urea and a further red shift in 7–8M urea. The emission maximum shifted from 337 to 345 nm when the temperature was changed from 30 to 80°C, with a midpoint of thermal denaturation at 58°C. Similarly, the emission maximum shifted from 337 to 345 nm when the solution pH was increased from 9 to 12, with a midpoint of pH transition at 10.6. The results obtained from difference absorption spectroscopy studies suggest that the unfolding of fibronectin at alkaline pH is related at least in part to ionization of tyrosine residues. Since most of the tryptophan residues are in invariant positions in homology sequences, it is suggested here that tryptophan residues are useful intrinsic probes for elucidating fibronectin structure in solution.     

Chlorophyll <Emphasis Type="Italic">a</Emphasis> fluorescence induction kinetics in leaves predicted from a model describing each discrete step of excitation energy and electron transfer associated with Photosystem II

Chlorophyll a fluorescence induction (FI) is widely used as a probe for studying photosynthesis. On illumination, fluorescence emission rises from an initial level O to a maximum P through transient steps, termed J and I. FI kinetics reflect the overall performance of photosystem II (PSII). Although FI kinetics are commonly and easily measured, there is a lack of consensus as to what controls the characteristic series of transients, partially because most of the current models of FI focus on subsets of reactions of PSII, but not the whole. Here we present a model of fluorescence induction, which includes all discrete energy and electron transfer steps in and around PSII, avoiding any assumptions about what is critical to obtaining O J I P kinetics. This model successfully simulates the observed kinetics of fluorescence induction including O J I P transients. The fluorescence emission in this model was calculated directly from the amount of excited singlet-state chlorophyll in the core and peripheral antennae of PSII. Electron and energy transfer were simulated by a series of linked differential equations. A variable step numerical integration procedure (ode15s) from MATLAB provided a computationally efficient method of solving these linked equations. This in silico representation of the complete molecular system provides an experimental workbench for testing hypotheses as to the underlying mechanism controlling the O J I P kinetics and fluorescence emission at these points. Simulations based on this model showed that J corresponds to the peak concentrations of Q _A ⁻ Q_B (Q_A and Q_B are the first and second quinone electron acceptor of PSII respectively) and Q _A ⁻ Q _B ⁻ and I to the first shoulder in the increase in concentration of Q _A ⁻ Q _B ²⁻ . The P peak coincides with maximum concentrations of both Q _A ⁻ Q _B ²⁻ and PQH₂. In addition, simulations using this model suggest that different ratios of the peripheral antenna and core antenna lead to differences in fluorescence emission at O without affecting fluorescence emission at J, I and P. An increase in the concentration of Q_B-nonreducing PSII centers leads to higher fluorescence emission at O and correspondingly decreases the variable to maximum fluorescence ratio (F _v/F _m).     

Biophysical analysis of phaseolin denaturation induced by urea, guanidinium chloride, pH, and temperature.

The structural stability of phaseolin was determined by using absorbance, circular dichroism (CD), fluorescence emission, and fluorescence polarization anisotropy to monitor denaturation induced by urea, guanidinium chloride (GdmCl),pH changes, increasing temperature, or a combination thereof. Initial results indicated that phaseolin remained folded to a similar extent in the presence or absence of 6.0 M urea or GdmCl at room temperature. In 6.0 M GdmCl, phaseolin denatures at approximately 65°C when probed with absorbance, CD, and fluorescence polarization anisotropy. The transition occurs at lower temperatures by decreasingpH. Kinetic measurements of denaturation using CD indicated that the denaturation is slow below 55°C and is associated with an activation energy of 52 kcal/mol in 6.0 M GdmCl. In addition, kinetic measurement using fluorescence emission indicated that the single tryptophan residue was sensitive to at least two steps of the denaturation process. The fluorescence emission appeared to reflect some other structural perturbation than protein denaturation, as fluorescence inflection occurred approximately 5°C prior to the changes observed in absorbance, CD, and fluorescence polarization anisotropy.     

Chlorophyll fluorescence emission spectroscopy of oxygenic organisms at 77 K

Photosynthetic fluorescence emission spectra measurement at the temperature of 77 K (–196°C) is an often-used technique in photosynthesis research. At low temperature, biochemical and physiological processes that modulate fluorescence are mostly abolished, and the fluorescence emission of both PSI and PSII become easily distinguishable. Here we briefly review the history of low-temperature chlorophyll fluorescence methods and the characteristics of the acquired emission spectra in oxygen-producing organisms. We discuss the contribution of different photosynthetic complexes and physiological processes to fluorescence emission at 77 K in cyanobacteria, green algae, heterokont algae, and plants. Furthermore, we describe practical aspects for obtaining and presenting 77 K fluorescence spectra.     

Cyst-Induced Toxoplasmosis in Cats*

SYNOPSIS. The life cycle of Toxoplasma gondii is described from cats orally inoculated with Toxoplasma cysts. Five new structural stages of Toxoplasma designated as “types” A-E were found in the epithelial cells of the small and large intestine. Type A is the smallest of all 5 intestinal Toxoplasma types. It occurs as collections of 2-3 organisms in the jejunum 12–18 hr after infection. Type B organisms are characterized by a centrally located nucleus, a prominent nucleolus and dark blue cytoplasm giving rise to the appearance of bipolar staining with Giemsa. Type B occurs 12–54 hr after infection and appears to divide by simple endodyogeny and by multiple endodyogeny (endopolygeny). Type C organisms are elongate with subterminal nuclei and strongly PAS-positive cytoplasm. They occur at 24–54 hr and divide by schizogony. Type D organisms are smaller than type C and contain only a few PAS-positive granules. They occur from 32 hr to 15 days after inoculation and account for over 90% of all parasites in the small intestine during this time. Three subtypes divide by endodyogeny, schizogony and by splitting of their merozoites from the main nucleated mass without leaving a residual body. Type E organisms resemble one of the subtype D which divide by schizogony, but they leave a residual body. They occur from 3–15 days after inoculation. Gametocytes occur thruout the small intestine but more commonly in the ileum 3-15 days after infection. Male gametocytes contain on an average of 12 microgametes and comprise 2–4% of the gametocyte population. The prepatent period after cystinduced infection is 3–5 days with the peak oocyst production between 5–8 days and a patent period varying from 7–20 days. Variable numbers of trophozoites are present in the lamina propria of the small intestine and in the extra-intestinal tissues within a few hr after inoculation. After 9–10 days cysts were seen in the heart and later in the brain. The lesions of toxoplasmosis are compared in newborn and weanling kittens and in adult cats after oral and subcutaneous inoculation with cysts. After the ingestion of cysts, newborn kittens developed enteritis, hepatitis, myocarditis, myositis, pneumonitis and encephalitis and were moribund by the 9th day. Kittens aged 2 weeks and older developed enteritis, myocarditis, encephalitis and myositis but often survived; adult cats usually remained asymptomatic. After subcutaneous inoculation of cysts, newborn and weanling kittens died of acute toxoplasmosis with severe pneumonia, myocarditis, encephalitis and hepatitis.     

Microbial Succession During “Kunun Zaki” Production with Sorghum (Sorghum bicolor) Grains

Microorganisms associated with the production of kunun zaki using sorghum grains were isolated and characterized. They consisted mainly of lactic acid bacteria, Lactobacillus plantarum, Leuconostoc mesenteroides, Corynebacterium, Lactococcus lactis, Pediococcus cerevisiaeand yeast Saccharomyces cerevisiae. The organisms occurred in various numbers at different stages of production but persisted to the end of the production process. The growth of lactic acid bacteria brought about a gradual decrease of pH of the steeping medium from 6.87 to 4.78 in the final product. Although it is clear that specific microorganisms are involved in the production process, the chances of contamination seem to be unavoidable, as the process is not standardized and the processing environment is unhygienic. There is lack of precautionary measures against contamination and crude household materials are used in processing. Since the use of local household materials cannot be avoided, it is suggested that good manufacturing practices at all stages of production and application of the hazard analysis critical control point (HACCP) concept be applied to reduce the level of contamination.