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Ecological factors determining establishment of cellulolytic bacteria and protozoa in the rumens of meroxenic lambs.

Ecological factors that control the establishment of cellulolytic bacteria and ciliate protozoa in the lamb rumen were studied in meroxenic animals. Axenic lambs received dilutions of rumen liquor from either conventional lambs and sheep (pool A) or meroxenic lambs (pool B). The total number of bacteria established in the rumen was between 10(9) and 5 x 10(10) g-1. In lambs inoculated with dilutions (10(-6), 10(-7), 10(-8)) of pool A, cellulolytic bacteria did not become established. However, subsequent inoculation with Bacteroides succinogenes, resulted in colonization in lambs that had received 10(-6) and 10(-7) dilutions of pool A. However, B. succinogenes became established in only one of three lambs that received the 10(-8) dilution. Similar results were obtained for the protozoan Entodinium sp. With pool B, lambs were inoculated earlier and cellulolytic bacteria were established directly from the 10(-6) and 10(-7) inocula. Polyplastron multivesiculatum establishment occurred readily when inoculated into the lambs which had received the 10(-6) dilution of pool B. Results obtained in this study suggest that establishment of cellulolytic bacteria and protozoa requires an abundant and complex flora and is favoured by early animal inoculation.

Animals↗

Poly(ethylene oxide), a new slowing agent for protozoa.

The water-soluble, viscoelastic resin Polyox WSR 301), a poly(ethylene oxide) of high molecular weight (approximately 4 million) is introduces as a new slowing agent for protozoa. Generally, as the kinetic viscosity of the resin increased from 0.25% to 1% (w/v), the swimming velocity of Euglena gracilis, Didnium nasutum, Paramecium aurelia, Blepharisma undulans, and Prorodon platyodon decreased. The 1.0% solution had the highest viscosity and decreased velocity more effectively than 1.0% methyl cellulose and Protoslo solutions. The Polyox solutions differed from those of methyl cellulose and Protoslo by having, in addition to viscous drag, an elastic recoil that pulled the protozoa backwards when their swimming efforts stopped. The toxicity of these slowing agents was determined using 10 P. aurelia/test slide preparation. Paramecium numbers decreased in 1.0% methyl cellulose and Protoslo to nearly zero by 24 hr; in Polyox, not only were most these ciliates alive after 24 hr, but many survived for 96 hr and divisions occurred in 0.25% and 0.50% solutions.

Animals↗

Ciliate protozoa in the rumen of Brazilian water buffalo, Bubalus bubalis Linnaeus.

Total numbers and distribution of genera, subgenera and species were determined for the ciliate protozoa in rumen contents of 4 Brazilian water buffalo Bubalus bubalis Linnaeus. The fauna of one animal, housed in close proximity to European and zebu-type cattle, differed considerably from that of the remaining animals, which were somewhat isolated on a large ranch. Several of the protozoan species observed in the semi-isolated animals were first described in rumen contents from humped Indian cattle, and their subsequent occurrence in other hosts and geographic locations has been limited or absent. In all, 49 different species of protozoa were found, 8 of which have not been previously described. Three of the new species belong to the genus Entodinium: E. ciculum sp. n., E. spinonucleatum sp. n. and E. triangulum sp. n.; 4 to Diplodinium (Ostracodinium): D. (O.) brazili sp. n., D. (O.) esalqum sp. n., D. (O.) nucleolobum sp. n., and D. (O.) tiete sp. n.; and one to Diplodinium (Eudiplodinium): D. (E.) bubalus sp. n.

Animals↗

A newly revised classification of the protozoa.

The subkingdom Protozoa now inclues over 65,000 named species, of which over half are fossil and approximately 10,000 are parasitic. Among living species, this includes approximately 250 parasitic and 11,300 free-living sarcodines (of which approximately 4,600 are foraminiferids); approximately 1,8000 parasitic and 5,100 free-living flagellates; approximately 5,600 parasitic "Sporozoa" (including Apicomplexa, Microspora, Myxospora, and Ascetospora); and approximately 2,5000 parasitic and 4,700 free-living ciliates. There are undoubtedly thousands more still unnamed. Seven phyla of PROTOZOA are accepted in this classification--SARCOMASTIGOPHORA, LABYRINTHOMORPHA, APICOMPLEXA, MICROSPORA, ASCETOSPORA, MYXOSPORA, and CILIOPHORA. Diagnoses are given for these and for all higher taxa through suborders, and reporesentative genera of each are named. The present scheme is a considerable revision of the Society's 1964 classification, which was prepared at a time when perhaps 48,000 species had been named. It has been necessitated by the acquisition of a great deal of nex taxonomic information, much of it through electron microscopy. It is hoped that the present classification incorporatesmost of the major changes that will be made for some time, and that it will be used for many years by both protozoologist and non-protozoologists.

Animals↗

Intestinal ciliated protozoa of African rhinoceros: two new genera and five new species from the white rhino (Ceratotherium simum Burchell, 1817).

This report represents the first published information on intestinal ciliated protozoa in the African white rhinoceros (Ceratotherium simum Burchell, 1817). Two new genera which do not relate to any known ciliated protozoa from the intestines of mammals and five new species are described. The ciliates were found in the colon of three of these free-living hindgut-fermenting grazers that were shot in widely spaced districts in southern Africa. Phalodinium digitalis n. gen., n. sp., Arachnodinium noveni n. gen., n. sp., Monoposthium vulgaris n. sp., M. bracchium n. sp., and M. latus n. sp. constituted between 1% and 10% of the total ciliate population (ca. 1 X 10(5)/ml digesta) in the ascending colon. Exceedingly small numbers were observed in the descending colon, indicating temporary accommodation only.

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Comparison of sequence differences in a variable 23S rRNA domain among sets of cryptic species of ciliated protozoa.

Studies were undertaken to discover the relative molecular distances separating some familiar forms of ciliated protozoa, and the genetic species they include. Sequences of 190 bases of the D2 domain of the large ribosomal nucleic acid molecule were obtained by polymerase chain reaction from protists of three distinctive groups of ciliated protozoa-Colpoda, Paramecium and Tetrahymena. Evolutionary trees were constructed for each set of sequences using the PHYLOGEN 1.0 string programs. All three groups of ciliates manifested large molecular diversity among strains difficult or impossible to distinguish morphologically. The largest single evolutionary distance within a group was the 75 differences separating Tetrahymena paravorax from the other tetrahymenids. The largest mean distance for a group was the 21.2 for the colpodids. In all the protist groups the large molecular diversity is obscured by morphological conservatism associated with constraints of ancient designs. The molecular diversity within morphotypes argues for long evolutionary coexistence of species differentiated from each other in significant physiological, ecological, or nutritional ways.

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Ciliated protozoa in the rumen of Turkish domestic cattle (Bos taurus L.).

Rumen contents obtained from 28 domesticated cattle (Bos taurus L.) slaughtered at abatoirs near Izmir, Turkey were surveyed for ciliate protozoa. Protozoa are known to make an appreciable contribution to ruminal fiber digestion in many different geographical areas; however, little if any information is available on their occurrence in Turkish cattle. As a result of our survey, 13 genera including 52 species were identified. Nine of the species were further divided into 36 forma. The average ciliate density in our cattle (52.44 X 10(4)/ml) was higher than that of Turkish domestic sheep and other domestic cattle reported previously from different geographical areas. Entodinium basoglui, Entodinium williamsi f. williamsi, E. williamsi f. turcicum, E. dalli f. rudidorsospinatum, Entodinium imai, Entodinium oektemae, Eudiplodinium dehorityi, Epidinium graini, Ophryoscolex purkynjei f. bifidobicinctus, and Ophryoscolex purkynjei f. bifidoquadricinctus have previously been reported from Turkey and appear to be endemic. All ofthe remaining species represent a new host record for domestic Turkish cattle. This study also reports for the second time the presence of Entodinium constrictum in herbivorous mammals, and is the first record of its occurrence in domesticated cattle.

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Ancient weapons for attack and defense: the pore-forming polypeptides of pathogenic enteric and free-living amoeboid protozoa.

Pore-forming polypeptides have been purified from several amoeboid protozoans that are well-known human pathogens. Obligate enteric parasites, such as Entamoeba histolytica, and free-living but potentially highly pathogenic species, such as Naegleria fowleri, contain these cytolytic molecules inside cytoplasmic granules. Comprehensive functional and structural studies have been conducted that include isolation of the proteins from their natural sources, monitoring of their biological activity towards different targets, and molecular cloning of the genes of their precursors. In the case of the most prominent member of the protein family, with respect to protozoans, the three-dimensional structure of amoebapore A was solved recently. The amoebic pore-forming polypeptides can rapidly perforate human cells. The antibacterial activity of amoebapores and of related polypetides from free-living protozoa points to a more vital function of these molecules: inside the digestive vacuoles they combat growth of phagocytosed bacteria which are killed when their cytoplasmic membranes are permeabilized. The concommitant activity of these proteins towards host cells may be due to a coincidental selection for an efficient effector molecule. Nonetheless, several lines of evidence indicate that these factors are involved in pathogenesis of fatal diseases induced by amoeboid protozoa.

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An agarose slide method to follow the fate of bacteria within digestive vacuoles of protozoa.

A method to follow the fate of ingested bacteria within digestive vacuoles of protozoa is presented. Tetrahymena pyriformis, previously fed with bacteria, is deposited onto glass microscope slides covered with a film of nutritive agarose. The protozoa lyse and the digestive vacuoles containing the bacteria stay undamaged and can be observed microscopically. After incubation, microcolonies reveal those vacuoles which contained living bacteria. The method can be used to study the survival ability of the ingested bacteria. It is a potentially valuable technique for studies on digestion efficacy, virulence ability, or escape mechanisms of bacteria from digestion.

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Intestinal parasitism--protozoa and helminths--in primates at the Barcelona Zoo.

The faunistic results regarding intestinal parasitism by protozoa and helminths in 21 primate species (three Cebidae, thirteen Cercopithecidae, one Hylobatidae, one Lemuridae, three Pongidae) are reported. The primate species were housed in four separate galleries. Six faecal samples of each host species were subjected to coprological analysis. Fifteen parasite species were detected: 11 protozoa (Entamoeba coli, E. chattoni, E. hartmanni, Iodamoeba bütschlii, Endolimax nana, Giardia intestinalis, Chilomastix mesnilii, Enteromonas hominis, Trichomonas intestinalis, Balantidium coli, and Blastocystis hominis) and 4 helminths (Ancylostoma sp., Strongyloides fuelleborni, Strongyloides sp., and Trichuris trichiura). The results reveal certain parasitic similarities between host species housed in the same gallery; however, these primate species do not always carry identical parasite species.

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Metabolism of aflatoxin, ochratoxin, zearalenone, and three trichothecenes by intact rumen fluid, rumen protozoa, and rumen bacteria.

The effect of rumen microbes on six mycotoxins (aflatoxin B1, ochratoxin A, zearalenone, T-2 toxin, diacetoxyscirpenol, and deoxynivalenol ) considered to be health risks for domestic animals was investigated. The mycotoxins were incubated with intact rumen fluid or fractions of rumen protozoa and bacteria from sheep and cattle in the presence or absence of milled feed. Rumen fluid had no effect on aflatoxin B1 and deoxynivalenol . The remaining four mycotoxins were all metabolized, and protozoa were more active than bacteria. Metabolism of ochratoxin A, zearalenone, and diacetoxyscirpenol was moderately or slightly inhibited by addition of milled feed in vitro. The capacity of rumen fluid to degrade ochratoxin A decreased after feeding, but this activity was gradually restored by the next feeding time. Ochratoxin A was cleaved to ochratoxin alpha and phenylalanine; zearalenone was reduced to alpha-zearalenol and to a lesser degree to beta-zearalenol; diacetoxyscirpenol and T-2 toxin were deacetylated to monoacetoxyscirpenol and HT-2 toxin, respectively. Feeding of 5 ppm (5 mg/kg) of ochratoxin A to sheep revealed 14 ppb (14 ng/ml) of ochratoxin A and ochratoxin alpha in rumen fluid after 1 h, but neither was detected in the blood. Whether such conversions in the rumen fluid may be considered as a first line of defense against toxic compounds present in the diet is briefly discussed.

Aflatoxin B1↗

Contribution of protozoa to lysine synthesis in the in vitro rumen microbial ecosystem.

Isotopic tracer experiments were conducted in vitro to determine contribution of protozoa toward the biosynthesis of lysine in the rumen microbial ecosystem. The presence of protozoa in a rumen microbial suspension always increased lysine synthesis from aspartate. Rumen contents from a faunated goat produced a higher amount of lysine than did those from a defaunated one.

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Ruminal cellulolytic bacteria and protozoa from bison, cattle-bison hybrids, and cattle fed three alfalfa-corn diets.

Ruminal cellulolytic bacteria and protozoa and in vitro digestibility of alfalfa fiber fractions were compared among bison, bison hybrids, and crossbed cattle (five each) when they were fed alfalfa and corn in a ratio of 100:0, 75:25, and 50:50, respectively. The total number of viable bacteria (2.16 x 10(9) to 5.44 x 10(9)/ml of ruminal fluid) and the number of cellulolytic bacteria (3.74 x 10(7) to 10.9 x 10(7)/ml) were not different among groups of animals fed each diet. The genera of protozoa in all of the animal groups were similar; however, when either the 100:0 or 50:50 diet was used the percentage of Entodinium sp. was lower and the percentage of Diplodiniinae was higher (P less than 0.05) in bison than in bison hybrids or cattle. Bacteroides succinogenes made up the largest number of cellulolytic isolates from bison (58 and 36%, respectively, on the 100:0 and 75:25 diets), which were more numerous (P less than 0.05) than those from bison hybrids (36 and 12%) and cattle (33 and 18%). This was offset by a lower number of cellulolytic Butyrivibrio isolates. The numbers of Ruminococcus albus and R. flavefaciens isolates, in general, were similar among the bovid species, although R. flavefaciens generally made up less than 10% of the cellulolytic isolates. In vitro digestibility coefficients were greater (P less than 0.05) for the bison when the 75:25 diet was used and similar for the other two diets. The concentration of ruminal volatile fatty acids was larger (P less than 0.05) in bison than in bison hybrids and cattle when the 50:50 diet was used.(ABSTRACT TRUNCATED AT 250 WORDS)

Ammonia↗

Two-step freezing procedure for cryopreservation of rumen ciliates, an effective tool for creation of a frozen rumen protozoa bank.

The present study aimed at the long-term storage of rumen protozoa as living cells in liquid nitrogen. The two-step or interrupted slow freezing procedure was used to cryopreserve six of the dominant species of rumen ciliates isolated from monofaunated animals, Dasytricha ruminantium, Entodinium caudatum, Epidinium ecaudatum caudatum, Eudiplodinium maggii, Isotricha prostoma, and Polyplastron multivesiculatum. We optimized the first step in the interrupted slow freezing procedure, from the extracellular ice nucleation temperature to the holding temperature, and studied the effects of the cooling rates on survival. In addition to the nature of the cryoprotectant (dimethyl sulfoxide), the equilibration temperature and equilibration time (25 degrees C and 5 min, respectively), and the holding time at subzero temperature (45 min) recommended previously (S. Kisidayová, J. Microbiol. Methods 22:185-192, 1995), we found that a holding temperature of -30 degrees C, a cooling rate from extracellular ice nucleation temperature to holding temperature of between 1.2 degrees C/min and 2.5 degrees C/min, depending on the ciliate, and rumen juice as the freezing and thawing medium markedly improved the survival rate. Survival rates determined after 2 weeks in liquid nitrogen were 100% for Isotricha, 98% for Dasytricha, 85% for Epidinium, 79% for Polyplastron, 63% for Eudiplodinium, and 60% for Entodinium. They were not significantly modified after a period of 1 year in liquid nitrogen. Four of the five ciliate species cryopreserved for 8 months in liquid nitrogen successfully colonized the rumen when inoculated into defaunated animals. These results have made it possible to set up a bank of cryopreserved rumen protozoa.

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Effects of l-(2-nitro-l-imidazolyl)-3-methoxy-2-propanol and 2-methyl-5-nitroimidazole-l-ethanol against anaerobic and aerobic bacteria and protozoa.

1-(2-Nitro-1-imidazolyl)-3-methoxy-2-propanol (RO 7-0582) and 2-methyl-5-nitroimidazole-1-ethanol (Metronidazole), substances known to be potent trichomonacides, were shown to inhibit obligate anaerobic bacteria in vitro but were essentially without effect at the doses tested against bacteria capable of growing aerobically. A similar effect was noted in vivo in that both substances exhibited good chemotherapeutic activity against infections produced by three species of anaerobic protozoa but were essentially inactive at the doses tested against three species of aerobic protozoa.

1-Propanol↗

Protozoa as agents responsible for the decline of Xanthomonas campestris in soil.

A streptomycin-resistant mutant of Xanthomonas campestris was used to assess the persistence of the plant pathogen in soil and the changes in populations that might be important for its survival. In soil into which large numbers of the organism were introduced, a marked decline in its abundance occurred, but after about 1 week its population density reached a level of about 105 and did not continue to fall during the test period. No such marked decline was evident in sterile soil inoculated with X. campestris. The bacterium did not lose viability if starved for carbon or inorganic nitrogen. Although abundant in soil, the numbers of propagules capable of producing antibiotics or lytic enzymes active against X. campestris did not increase coincident with the pathogen's decline, and no increase in tartrate-extractable toxins was observed. Neither bdellovibrios nor bacteriophages active against the xanthomonad were found in the soil, but a marked increase in the frequency of protozoa paralleled the phase of rapid diminution in the X. campestris population. In actidione-treated soil, in which protozoan activity was severly limited, the high cell density of the pathogen was maintained. On the basis of these data, it is concluded that predation by protozoa is responsible for the abrupt fall in frequency of the bacterium in natural soil.

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