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The entomopathogenic fungi Metarhizium anisopliae and Beauveria bassiana are ubiquitously distributed in soils. As insect pathogens they adhere to the insect cuticle and penetrate through to the insect haemocoel using a variety of cuticle-hydrolysing enzymes. Once in the insect haemocoel they are able to survive and replicate within, and/or evade, phagocytic haemocyte cells circulating in the haemolymph. The mechanism by which these soil fungi acquire virulence factors for insect infection and insect immune avoidance is unknown. We hypothesize that insect phagocytic cell avoidance in M. anisopliae and B. bassiana is the consequence of a survival strategy against soil-inhabiting predatory amoebae. Microscopic examination, phagocytosis assays and amoeba mortality assays showed that these insect pathogenic fungi are phagocytosed by the soil amoeba Acanthamoeba castellanii and can survive and grow within the amoeba, resulting in amoeba death. Mammalian fungal and bacterial pathogens, such as Cryptococcus neoformans and Legionella pneumophila, respectively, show a remarkable overlap between survival against soil amoebae and survival against human macrophages. The insect immune system, particularly phagocytic haemocytes, is analogous to the mammalian macrophage. Our data suggest that the ability of the fungal insect pathogens M. anisopliae and B. bassiana to survive insect phagocytic haemocytes may be a consequence of adaptations that have evolved in order to avoid predation by soil amoebae. DOI 10.1099/mic.0.038216-0

Listeria monocytogenes is a ubiquitous bacterium capable of infecting humans, particularly pregnant women and immunocompromised individuals. Although the intracellular invasion and pathogenesis of listeriosis in mammalian tissues has been well studied, little is known about the ecology of L. monocytogenes, and in particular the environmental reservoir for this bacterium has not been identified. This study used short-term co-culture at 15, 22 and 37[degrees]C to examine the interaction of L. monocytogenes strains with Acanthamoeba polyphaga ACO12. Survival of L. monocytogenes cells phagocytosed by monolayers of trophozoites was assessed by culture techniques and microscopy. A. polyphaga trophozoites eliminated bacterial cells within a few hours post-phagocytosis, irrespective of the incubation temperature used. Wild-type L. monocytogenes and a phenotypic listeriolysin O mutant were unable to either multiply or survive within trophozoites. By contrast, Salmonella enterica serovar Typhimurium C5 cells used as controls were able to survive and multiply within A. polyphaga trophozoites. The data presented indicate that A. polyphaga ACO12 is unlikely to harbour L. monocytogenes, or act as an environmental reservoir for this bacterium. DOI 10.1099/mic.0.031146-0

A parasite's ability to be a specialist vs. a generalist may have consequences for its prevalence within one or more if its host species. In this study we investigated the relationship between host specialization and prevalence in the highly species diverse avian blood parasites of the genera Plasmodium and Haemoproteus. Contrary to trade-off hypotheses that may explain host specialization, within both genera the parasites with the ability to complete their life cycles and be transmitted across a wide host range (broad compatibility) were also the most common parasites within their compatible host species. These patterns remained unchanged when the host species with the highest prevalence were excluded, which reduces the possibility that the observed pattern was caused by parasites reaching high prevalence in a single main host, and being 'spilled over' to other host species. We hypothesize that a positive relationship between parasite host range and prevalence might be explained by an overall higher encounter rate for the parasites with broad host range, which compensates for possibly reduced performance of parasites in each host species. Overall, these results show that parasites with the ability to successfully infect a wide variety of host species of broad ancestry also can have the ability to be the most prevalent in single host species. Key words: avian malaria; blood parasites; encounter rate; Haemoproteus; haemosporidians; host-parasite interaction; host range; Plasmodium; prevalence: specialists vs. generalists.

Neutrophils are short-lived leukocytes that die by apoptosis, necrosis, and NETosis. Upon death by NETosis, neutrophils release fibrous traps of DNA, histones, and granule proteins named neutrophil extracellular traps (NETs), which can kill bacteria and fungi. Inoculation of the protozoan Leishmania into the mammalian skin causes local inflammation with neutrophil recruitment. Here, we investigated the release of NETs by human neutrophils upon their interaction with Leishmania parasites and NETs' ability to kill this protozoan. The NET constituents DNA, elastase, and histones were detected in traps associated to promastigotes by immunofluorescence. Electron microscopy revealed that Leishmania was ensnared by NETs released by neutrophils. Moreover, Leishmania and its surface lipophosphoglycan induced NET release by neutrophils in a parasite number- and dose-dependent manner. Disruption of NETs by DNase treatment during Leishmania--neutrophil interaction increased parasite survival, evidencing NETs' leishmanicidal effect. Leishmania killing was also elicited by NET-rich supernatants from phorbol 12-myristate 13-acetate-activated neutrophils. Immunoneutralization of histone during Leishmania--neutrophil interaction partially reverted Leishmania killing, and purified histone killed the parasites. Meshes composed of DNA and elastase were evidenced in biopsies of human cutaneous leishmaniasis. NET is an innate response that might contribute to diminish parasite burden in the Leishmania inoculation site. histone | protozoa | trypanosomatid

Leishmania is unable to synthesize heme and must acquire it from exogenous source, the mechanism of which is not known. We have shown that Leishmania endocytoses hemoglobin (Hb) and subsequently degrade it probably to generate heme. To understand how internalized Hb is degraded, we have cloned and expressed Rab7 homolog from Leishmania donovani. Interestingly, Rab7 in Leishmania is found to be localized both on early and late endocytic compartment and regulates both uptake and degradation of endocytosed Hb demonstrating that Rab7 in Leishmania play a very unique role connecting both early and late events of Hb endocytosis. Our data also indicate that overexpression of Rab7:VVT in Leishmania induces transport of Hb to lysosomes and rapidly degrade internalized Hb. Whereas Hb transport to lysosomes and its degradation is significantly inhibited in cells overexpressing Rab7:T21N, a GDP locked mutant of Rab7. Moreover, cells overexpressing Rab7:T21N grow at a slower rate (

Bacillus spores are highly resistant to many environmental stresses, owing in part to the presence of multiple 'extracellular' layers. Although the role of some of these extracellular layers in resistance to particular stresses is known, the function of one of the outermost layers, the spore coat, is not completely understood. This study sought to determine whether the spore coat plays a role in resistance to predation by the ciliated protozoan Tetrahymena, which uses phagocytosis to ingest and degrade other microorganisms. Wild-type dormant spores of Bacillus subtilis were efficiently ingested by the protozoan Tetrahymena thermophila but were neither digested nor killed. However, spores with various coat defects were killed and digested, leaving only an outer shell termed a rind, and supporting the growth of Tetrahymena. A similar rind was generated when coat-defective spores were treated with lysozyme alone. The sensitivity of spores with different coat defects to predation by T. thermophila paralleled the spores' sensitivities to lysozyme. Spore killing by T. thermophila was by means of lytic enzymes within the protozoal phagosome, not by initial spore germination followed by killing. These findings suggest that a major function of the coat of spores of Bacillus species is to protect spores against predation. We also found that indigestible rinds were generated even from spores in which cross-linking of coat proteins was greatly reduced, implying the existence of a coat structure that is highly resistant to degradative enzymes. Bacillus spore | phagocytosis | spore resistance

A protozoa-specific primer (P-SSU-342f) was designed and paired with a eukarya-specific primer to amplify a 1,360-bp fragment of DNA encoding protozoal small subunit (SSU) ribosomal RNA from ruminal fluid of cows fed a mixed forage:grain diet or alfalfa hay. Sequencing of clones showed that P-SSU-342f is specific to ruminal protozoa and, with slight modifications, the primer will be useful for ecological studies of ruminal protozoa. Key Words: Microbial Ecology, Ribosomal Ribonucleic Acid, Rumen Protozoa

Research demonstrates that Francisella tularensis grows faster in cocultures containing Acanthamoeba castellanii, which results in the decrease in the number of latter organism. Data indicate that F. tularensis cells are located intracellularly in A. castellanii cells. Results also suggest that the protozoa might be an essential environmental reservoir for F. tularensis growth and survival.

Four ruminally cannulated dairy cows were used to examine the effect of diet on the AA composition of rumen bacteria and protozoa, and the flow of microbial and nonmicrobial AA entering the omasal canal. Cows were offered grass-red clover silage alone, or that supplemented with 5.1 kg DM of barley, 1.9 kg DM of rapeseed meal, or 5.1 kg DM of barley and 1.9 kg DM of rapeseed meal according to a 4 x 4 Latin square design with a 2 x 2 factorial arrangement of treatments. During the first 10 d of each period, cows had free access to silage and, thereafter intake was restricted to 95% of ad libitum intake. Postruminal digesta flow was assessed using the omasal canal sampling technique in combination with a triple marker method. Liquid- (LAB) and particle- (PAB) associated bacteria were isolated from digesta in the reticulorumen and protozoa from digesta entering the omasal canal. Microbial protein flow was determined using [sup.15]N as a microbial marker. Flows of AA entering the omasal canal were similar in cows fed silage diets supplemented with barley or rapeseed meal. However, rapeseed meal increased nonmicrobial AA flow while barley increased the flow of AA associated with LAB and protozoa. Diet had negligible effects on the AA profile of microbial fractions. Comparison of AA profiles across diets indicated differences between LAB and PAB for 10 out of 17 AA measured. Rumen bacteria and protozoa were found to be different for 14 out of 15 AA measured. For grass silage-based diets, energy and protein supplementations appear to alter postruminal AA supply through modifications in the proportionate contribution of microbial and nonmicrobial pools to total protein flow rather than as a direct result of changes in the AA profile of microbial protein. Key Words: Amino Acids, Dairy Cows, Grass Silage, Rumen Bacteria, Rumen Protozoa

Research has been conducted on the dynamics of the active and encysted protozoa in the decomposing organic material soil. The use of the ciliates as the model organisms and the effect of the fresh organic material on the ciliate excystment and growth are discussed.

Research has been conducted on the apicidins, cyclic tetrapeptides which inhibit deacetylase and antiprotozoal agents. The isolation and elucidation of these apicidins from the Fusarium species, temperature-dependent NMR studies of the apicidins, molecular and NMR based conformation of their macrocyclic ring and apicidin's selected chemical modification are discussed.

Research has been conducted on the intracellular protozoan parasite Cryptosporidium oocysts which is responsible for the gastrointestinal infections in humans. The use of the nested PCR-restriction fragment length polymorphism assay developed for the oocyst detection and human pathogen differentiation is described.

Research has been conducted on the Cryptosporidium oocysts. The method establishing the state of the river contamination by these oocysts in western Japan has been discussed, and the relationship between the contamination intensity and the numbers of the domestic animal kept in the river valleys has been investigated.

The amino acid sequences, specificities for substrate, and enzyme kinetics of aminotransferases in the three parasitic protozoans identify them as aspartate aminotransferases, involved in methionine formation.

Trichomonas vaginalis, a urogenital parasite, and its dependence on polyamine backconversion are discussed as is the potential of bis(alkyl) polyamine analogs as antitrichomonad agents. A study has shown that growth of T. vaginalis with DENSpm also brings on polyamine depletion and significant inhibition of growth by a mechanism that involves competition with the transport of spermine and inhibition of SSAT effectively blocking spermidine synthesis. The parasite differs significantly from other eukaryotes in several aspects of its polyamine metabolism. The spermine taken up is backconverted to spermidine through a spermidine:spermine N(super.1)-acetyltransferase (SSAT)/polyamine oxidase-coupled pathway.

Steroisomeric substrate analogues and their parasite-specific inhibition of the glycosylphosphatidylinositol biosynthetic pathway are discussed. Study has shown that a diastereoisomer, D-GlcN(alpha)1-6-L-myo-inositol-1-P-sn-1,2-diacyl-glycerol, is an inhibitor of Trypanosoma brucei enzyme in a trypanosomal cell-tree system.

Protozoans and their role in enhancing copepod growth are discussed with consideration of the role of essential lipids. Trophic upgrading of food quality on the part of the protozoans is of interest. Very unsaturated fatty acids and/or sterols appear to be essential compounds and can be produced by protozoans. The protozoans may bridge the gap in essential nutrients between the microbial and higher trophic levels.

We have identified telomerase activity in extracts of three evolutionarily diverse kinetoplastid species: Trypanosoma brucei, Leishmania major, and Leishmania tarentolae. Telomerase activity was initially detected in extracts from insect form cells of all three kinetoplastid species by using a modification of the one-tube telomere repeat amplification protocol [Kim, N, et al. (1994) Science 266, 2011-2015], although better results were subsequently achieved with the two-tube telomere repeat amplification protocol [Autexier, C., Pruzan, R., Funk, W. & Greider, C. (1996) EMBO J. 15, 5928-5935]. The activity in T. brucei extracts was sufficiently robust to enable its detection in a direct assay of telomerase; enzyme processivity was found to be relatively low. The in vitro properties of telomerase suggest a possible templating domain sequence for the telomerase RNA of T. brucei. Telomerase activity is likely to contribute to telomere maintenance in these parasitic organisms and provides a new target for chemotherapeutic intervention.

Spores of Bacillus thuringiensis subsp. israelensis and their toxic crystals germinated, grew and sporulated in excreted food vacuoles of Tetrahymena pyriformis forming new active insecticidal crystal proteins in the process. The spores and crystals are concentrated in the food vacuoles of each T. pyriformis cell and delivered to mosquito larvae which soon die. The cells later excrete vacuoles with undigested material. Excreted food vacuoles gradually aggregated and vegetative growth of B. thuringiensis subsp. israelensis was noted with the outgrowth of filaments, which sporulated between 27 and 42 h.

Marteilia refringens and Bonamia ostreae, parasitic protozoans, have caused the decline in commercial production of the European flat oysters Ostrea edulis in France. In the 1980s a selection program raised two O.edulis strains displaying resistance to B. ostreae. The growth and mortality performances of two crosses involving S85 and S89, the two O. edulis strains, with the aim of estimating genetic variances and heritability for growth.

Results obtained during the past decade indicate clearly that protozoa are actively involved in the degradation of dietary and microbial proteins in the rumen. Because of the great ability of protozoa to ingest the particulate matter suspended in the rumen, protozoa are more active in degrading insoluble than soluble proteins. This indicates that studies carried out using lysed and sonicated protozoa are not appropriate for quantifying the actual contribution of protozoa to protein degradation in the rumen. In vivo trials have confirmed that duodenal flow of both undegraded dietary protein plus bacterial protein generally is increased by defaunation. The decrease in ruminal ammonia concentration consistently observed after defaunation accounts for the lower urinary nitrogen (N) excretion found in defaunated animals, whereas the increase in fecal N excretion in the same animals probably results from a shift of plant cell wall digestion from the rumen to the large intestine. Total N excretion is not altered significantly by defaunation. A summary of literature data indicates there are contradictory effects of defaunation on ruminant performance. This implies that animal response to defaunation may depend on the specific nutrient-limiting performance on the one hand and on the modifications of digestion and metabolism resulting from defaunation on the other. Different methods are proposed to either eliminate or decrease the numbers of ruminal protozoa or to alter their makeup. However, none of these approaches has been tested under practical feeding conditions. J. Nutr. 126: 1335S-1346S, 1996. INDEXING KEY WORDS: rumen, nitrogen, digestion, metabolism, protozoa

High-performance liquid chromatographic analysis of pigment degradation by six species of herbivorous protozoa is presented. Results show that microzooplankton consistently produce both pheophorbides and pheophytins duringherbivorous grazing. Further results show that the loss of pheopigment and decrease in pigment conversion ratios are due to reingestion of fecal matter byprotozoan grazers.

Larval abundance, phytoplankton community structure and chlorophyll a concentration was studied among the larval species of Penaeus merguiensis in Albatross Bay, Gulf of Carpentaria. It was shown that the main diet of the larvae consist of diatoms, green algae and seagrass detritus. Furthermore, it was shown that the diatom flora of the bay can sustain the prawn larvae even during seasonally low levels and therefore is not responsible for the mortality of the species during those periods.

The protozoon Paraphysomonas imperforata has psychrophilic ecotypes that exhibit different responses to different degrees of low temperature. These responses are demonstrated in their growth rates, ingestion rates, and cell sizes as shown in this laboratory study of flagellates isolated from Newfoundland coastal waters and the Arctic Ocean. The adaptability of these protozoa to different environmental conditions seems to have produced ecological types thatare different physiologically and genetically. Adaption of Paraphysomonas imperforata to cold temperature suggests its tolerance to specific temperature it was exposed to over a long period of time. This study shows the similar characteristics of the two flagellate ecotypes that vary in their response rates due to the altitude and different temperatures of their geographic sources.

Protozoan hosts are identified for Legionella pneumophila by using a biofilm batch test (BBT). In vitro studies are required to confirm their role as hosts for L. pneumophila and unidentified protozoa are implicated as hosts for uncultured Legiomella spp. grown in BBT flasks.

An rRNA-derived eukaryote clone library was constructed to identify the active protozoan phylotypes present in activated sludge and RNA-SIP was used to track carbon from bicarbonate and acetate into protozoa under ammonia-oxidizing and denitrifying conditions. The results from RNA-SIP analysis indicated that the peritrich ciliate Epistylis galea dominated the acquisition of carbon from bacteria with access to C[O.sub.2] under ammonia-oxidizing conditions, while no evidence was found for the specific grazing on acetate consumers under denitrifying conditions.

The dynamics and mechanisms involved in the regulation of the cyclic lipopeptide (CLP) biosynthesis by the application of protozoan are discussed. The analysis proves that such interactions play an extremely significant role in the activation of the predation defense mechanism in several different prey populations.

Free-living protozoa are identified in two unchlorinated groundwater supplies by using cultivation-independent molecular approaches and eukaryotic communities are analyzed by using terminal restriction fragment length polymorphism and clone library analyses of partial 18S rRNA gene fragments and a Hartmannella vermiformis-specific quantitative PCR (qPCR). The studies have shown that the application of qPCR is promising for analyzing the relationship between drinking water quality and the presence of specific protozoa.

The authors review the publications on parasitic protozoa's genomes. The topics of interest include the repetitive DNA content of genomes, subtelomeric satellites and gene expression, and retroelements and RNA interference.

The authors have investigated protozoan parazite's amoebapores and porcin cytotoxic lymphocytes' NK-lysin. The study of the protein/lipid interactions for the amoebapore's and NK-lysin's isoforms is described.

The authors have investigated Cyclospora cayetanensis in environmental water samples. They describe the development of the alternative netsted-PCR-restriction fragment length polymorphism protocol for detection of the bacterium which differentiates C. cayetanensis amplified target sequence from those that may cross-react.

The authors describe the study of Tritrichomonas foetus. The investigation of T. foetus intracellular amino acids has indicated that T. foetus exhibits a regulatory volume increase in response to hyperosmotic challenge but does not exhibit a regulatory volume decrease after hypoosmotic cell swelling.

Results show that Mycobacterium bovis survives injestion by Acanthamoeba castellanii but two strains of M. bovis BCG are unable to survive within A. castellanii.

Research has been conducted on H (sub)2 O (sub)2 cysticidal activity. The authors demonstrate that it is possible to enhance this activity via the use of plant peroxidase-hydrogen peroxide-halide antimicrobial system.

Research has been conducted on Entamoeba histolytica protein Ehp53 binding to the human p53 DNA consensus sequence. The authors report that this protein is the p53-like protein found in protozoa.

The effects of protozoa on ruminal [NH.sub.3] N kinetics and bacterial N recycling were measured in five sheep (57.6 [+ or -] 7.1 kg BW, [bar]x [+ or -] SD) with ruminal and duodenal cannulas in naturally faunated, defaunated, and refaunated periods. The sheep were fed a diet of 239 g of alfalfa haylage and 814 g of barley concentrate per day (DM basis) divided into 12 equal portions and allocated at 2-h intervals. A pulse dose of 300 mg of [.sup.15]N as [[.sup.15]N][NH.sub.4]Cl was administered into the rumen (on d 1 and 15) and 300 mg of [.sup.15]N as [[.sup.15]N]urea was administered intravenously to the blood (d 8). Enrichment of [.sup.15]N was measured in ruminal [NH.sub.3]-N, bacterial N, and plasma urea N over a period of 35 h. Total collection of urine was made for 5 d and analyzed for purine derivatives to calculate the flow of microbial N. Ruminal parameters and nutrient digestibilities were also measured. Sheep were defaunated using a rumen washing procedure 50 d prior to measurements in the defaunated period. Sheep were refaunated with ruminal contents from a faunated sheep receiving the same diet. Measurements began 26 d following refaunation, at which time protozoal numbers had returned to those in the originally faunated sheep. Data reported in parentheses are for faunated, defaunated, and refaunated sheep, respectively. Total culturable and cellulolytic bacterial numbers were unaffected by defaunation, but there was an increase in flow of microbial N from the rumen (10.8, 17.3, and 11.1 g N/d; P [is less than] .05) in the defaunated period. Flux, irreversible loss, and intraruminal recycling of [NH.sub.3]-N and recycling of [NH.sub.3]-N from plasma urea N were not affected by defaunation. Defaunation had no effect on reducing the absolute amount (13.8, 10.0, and 11.3 g N/d; P [is greater than] .20) of bacterial N recycling and the percentage of N flux through the bacterial N pool. Total-tract digestion was reduced in defaunated compared with faunated sheep by 8, 17, 15, and 32% for OM, N, NDF, and ADF, respectively. In conclusion, defaunation improved ruminal N metabolism through the enhancement of bacterial protein synthesis, and improvement in the flow of microbial protein to the host animal. Key Words: Bacterial Protein, Nitrogen Balance, Protozoa, Ruminant Flora, Sheep

Three Holstein heifers and one nonlactating cow, fitted with ruminal and duodenal cannulas, were arranged in a 4 x 4 Latin square design to determine the effects of degree of fat saturation on ruminal neutral detergent fiber digestion and microbial protein synthesis and to determine whether changes in the efficiency of microbial protein synthesis were related to protozoal populations in the rumen. Corn silage-based diets contained no added fat or 4.85% of diet dry matter as partially hydrogenated tallow, tallow, or animal-vegetable fat. Iodine values of fat sources were 12.8, 50.6, and 109.7 for partially hydrogenated tallow, tallow, and animal-vegetable fat, respectively. Cattle were fed every 2 h and consumed 1.5% of body weight as dry matter daily. Ruminal neutral detergent fiber digestibility was decreased by added fat but was not affected by increasing iodine value. Flows of microbial N and non-[NH.sub.3]-nonmicrobial N to the duodenum were not affected by treatment. Ruminal protozoa concentration decreased linearly as the iodine value of fats increased. The efficiency of microbial protein synthesis was increased and protozoa concentrations tended to decrease when fat was fed. Decreased ruminal protozoa concentration may have decreased intraruminal N recycling. Biohydrogenation of added fat may result in a low ruminal concentration of unsaturated fatty acids when cows are fed frequently, reducing the negative effects of unsaturated fat sources on ruminal neutral detergent fiber digestibility. Protozoa were inhibited by unsaturated fat, but it is not clear if biohydrogenation and frequent feeding lessened inhibition. Key Words: Cattle, Feeding Frequency, Protein Synthesis, Protozoa, Unsaturated Fatty Acids