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PubMed2005

Mobility of protozoa through narrow channels.

Microbes in the environment are profoundly affected by chemical and physical heterogeneities occurring on a spatial scale of millimeters to micrometers. Physical refuges are critical for maintaining stable bacterial populations in the presence of high predation pressure by protozoa. The effects of microscale heterogeneity, however, are difficult to replicate and observe using conventional experimental techniques. The objective of this research was to investigate the effect of spatial constraints on the mobility of six species of marine protozoa. Microfluidic devices were created with small channels similar in size to pore spaces in soil or sediment systems. Individuals from each species of protozoa tested were able to rapidly discover and move within these channels. The time required for locating the channel entrance from the source well increased with protozoan size and decreased with channel height. Protozoa of every species were able to pass constrictions with dimensions equal to or smaller than the individual's unconstrained cross-sectional area. Channel geometry was also an important factor affecting protozoan mobility. Linear rates of motion for various species of protozoa varied by channel size. In relatively wide channels, typical rates of motion were 300 to 500 microm s(-1) (or about 1 m per hour). As the channel dimensions decreased, however, motilities slowed more than an order of magnitude to 20 microm s(-1). Protozoa were consistently observed to exhibit several strategies for successfully traversing channel reductions. The empirical results and qualitative observations resulting from this research help define the physical limitations on protozoan grazing, a critical process affecting microbes in the environment.

Animals↗
PubMed1998

Identification of macrophage-specific infectivity loci (mil) of Legionella pneumophila that are not required for infectivity of protozoa.

We have recently shown that many mutants of Legionella pneumophila exhibit similar defective phenotypes within both U937 human-derived macrophages and the protozoan host Acanthamoeba (L.-Y. Gao, O. S. Harb, and Y. Abu Kwaik, Infect. Immun. 65:4738-4746, 1997). These observations have suggested that many of the mechanisms utilized by L. pneumophila to parasitize mammalian and protozoan cells are similar, but our data have not excluded the possibility that there are unique mechanisms utilized by L. pneumophila to survive and replicate within macrophages but not protozoa. To examine this possibility, we screened a bank of 5,280 miniTn10::kan transposon insertion mutants of L. pneumophila for potential mutants that exhibited defective phenotypes of cytopathogenicity and intracellular replication within macrophage-like U937 cells but not within Acanthamoeba polyphaga. We identified 32 mutants with various degrees of defects in cytopathogenicity, intracellular survival, and replication within human macrophages, and most of the mutants exhibited wild-type phenotypes within protozoa. Six of the mutants exhibited mild defects in protozoa. The defective loci were designated mil (for macrophage-specific infectivity loci). Based on their intracellular growth defects within macrophages, the mil mutants were grouped into five phenotypic groups. Groups I to III included the mutants that were severely defective in macrophages, while members of the other two groups exhibited a modestly defective phenotype within macrophages. The growth kinetics of many mutants belonging to groups I to III were also examined, and these were shown to have a similar defective phenotype in peripheral blood monocytes and a wild-type phenotype within another protozoan host, Hartmannella vermiformis. Transmission electron microscopy of A. polyphaga infected by three of the mil mutants belonging to groups I and II showed that they were similar to the parent strain in their capacity to recruit the rough endoplasmic reticulum (RER) around the phagosome. In contrast, infection of macrophages showed that the three mutants failed to recruit the RER around the phagosome during early stages of the infection. None of the mil mutants was resistant to NaCl, and the dot or icm NaCl(r) mutants are severely defective within mammalian and protozoan cells. Our data indicated that in addition to differences in mechanisms of uptake of L. pneumophila by macrophages and protozoa, there were also genetic loci required for L. pneumophila to parasitize mammalian but not protozoan cells. We hypothesize that L. pneumophila has evolved as a protozoan parasite in the environment but has acquired loci specific for intracellular replication within macrophages. Alternatively, ecological coevolution with protozoa has allowed L. pneumophila to possess multiple redundant mechanisms to parasitize protozoa and that some of these mechanisms do not function within macrophages.

Acanthamoeba↗
PubMed1993

Kingdom protozoa and its 18 phyla.

The demarcation of protist kingdoms is reviewed, a complete revised classification down to the level of subclass is provided for the kingdoms Protozoa, Archezoa, and Chromista, and the phylogenetic basis of the revised classification is outlined. Removal of Archezoa because of their ancestral absence of mitochondria, peroxisomes, and Golgi dictyosomes makes the kingdom Protozoa much more homogeneous: they all either have mitochondria and peroxisomes or have secondarily lost them. Predominantly phagotrophic, Protozoa are distinguished from the mainly photosynthetic kingdom Chromista (Chlorarachniophyta, Cryptista, Heterokonta, and Haptophyta) by the absence of epiciliary retronemes (rigid thrust-reversing tubular ciliary hairs) and by the lack of two additional membranes outside their chloroplast envelopes. The kingdom Protozoa has two subkingdoms: Adictyozoa, without Golgi dictyosomes, containing only the phylum Percolozoa (flagellates and amoeboflagellates); and Dictyozoa, made up of 17 phyla with Golgi dictyosomes. Dictyozoa are divided into two branches: (i) Parabasalia, a single phylum with hydrogenosomes and 70S ribosomes but no mitochondria, Golgi dictyosomes associated with striated roots, and a kinetid of four or five cilia; and (ii) Bikonta (16 unicellular or plasmodial phyla with mitochondria and bikinetids and in which Golgi dictyosomes are not associated with striated ciliary roots), which are divided into two infrakingdoms: Euglenozoa (flagellates with discoid mitochondrial cristae and trans-splicing of miniexons for all nuclear genes) and Neozoa (15 phyla of more advanced protozoa with tubular or flat [usually nondiscoid] mitochondrial cristae and cis-spliced spliceosomal introns). Neozoa are divided into seven parvkingdoms: (i) Ciliomyxa (three predominantly ciliated phyla with tubular mitochondrial cristae but no cortical alveoli, i.e., Opalozoa [flagellates with tubular cristae], Mycetozoa [slime molds], and Choanozoa [choanoflagellates, with flattened cristae]); (ii) Alveolata (three phyla with cortical alveoli and tubular mitochondrial cristae, i.e., Dinozoa [Dinoflagellata and Protalveolata], Apicomplexa, and Ciliophora); (iii) Neosarcodina (phyla Rhizopoda [lobose and filose amoebae] and Reticulosa [foraminifera; reticulopodial amoebae], usually with tubular cristae); (iv) Actinopoda (two phyla with axopodia: Heliozoa and Radiozoa [Radiolaria, Acantharia]); (v) Entamoebia (a single phylum of amoebae with no mitochondria, peroxisomes, hydrogenosomes, or cilia and with transient intranuclear centrosomes); (vi) Myxozoa (three endoparasitic phyla with multicellular spores, mitochondria, and no cilia: Myxosporidia, Haplosporidia, and Paramyxia); and (vii) Mesozoa (multicells with tubular mitochondrial cristae, included in Protozoa because, unlike animals, they lack collagenous connective tissue).

Animals↗
PubMed1993

Changes in the rumen microbial population and its activities during the refaunation period after the reintroduction of ciliate protozoa into the rumen of defaunated sheep.

Changes in the microbial populations, their activities, and the ruminal fermentation were monitored for 50 d following the reintroduction of ciliate protozoa into four defaunated sheep. A protozoal population was reestablished successfully in each recipient, using a washed inoculum containing approximately 10(3) cells, although there were between-animal differences in the rates of recolonization and genus establishment. Entodinium spp. predominated in the initial stages of the refaunation period and had an apparent maximal generation time of 9-10 h. Bacterial and fungal numbers did not decline following the reintroduction of protozoa and a small transient increase in the numbers of amylolytic and xylanolytic bacteria and fungal zoospores occurred in the early stages of refaunation when the protozoal population was < 10(5)/g ruminal contents, but these subsequently declined as the protozoa established. Although the fibrolytic bacterial population was lowest in period 3 (> 10(5) protozoa/g), the in sacco ruminal digestion of Lolium perenne hay and polysaccharolytic enzyme activities in the solids-associated populations were either maintained or increased when protozoa were present confirming the important contribution of protozoa to fibre breakdown in the rumen. Significant changes in ruminal microbial activities occurred after protozoal reinoculation but before the rumen had refaunated completely. Arylamidase activities in the liquor-phase population and ruminal ammonia concentrations increased significantly within 48 h of transfaunation; the magnitude of the effects became more pronounced as the protozoal population developed. However, volatile fatty acid formation and ruminal pH were not affected after the reintroduction of protozoa.

Aminopeptidases↗
PubMed2005

Cell death and human intestinal protozoa: a brief overview.

Protozoan programmed cell death or apoptosis is an important factor in the survival of the parasite and its pathogenicity. The most amazing aspect of protozoan cell death is in its molecular architecture. To date, protozoa lack most of the components of the highly complex cell death machinery studied in multicellular organisms. Hence the unique apoptotic machinery in protozoa can be exploited for the development of therapeutic drugs and diagnostic markers. This review focuses on human intestinal protozoa undergoing cell death and inducing or inhibiting host cell apoptosis. The first part of this review focuses on intestinal protozoa that undergo PCD under various stress conditions. The second part focuses on protozoa that induce or inhibit PCD in their host cell. Although these intestinal parasites differ in their mechanism of infection and intracellular localization, they may activate conserved cell death pathways within themselves and in the host cell. Understanding conserved cell death pathways in the intestinal protozoa and their host-parasite PCD relationship may lead to drug targets which can be used for a broad range of parasitic diseases.

Animals↗
PubMed2006

Rumen ciliate protozoa contain high concentrations of conjugated linoleic acids and vaccenic acid, yet do not hydrogenate linoleic acid or desaturate stearic acid.

Conjugated linoleic acids (CLA) have been shown to improve human health. They are derived from the microbial conversion of dietary linoleic acid (cis-9,cis-12-18 : 2 (LA)) in the rumen. An investigation was undertaken to determine the role of ruminal ciliate protozoa v. bacteria in the formation of CLA and its precursor in animal tissues, vaccenic acid (trans-11-18 : 1 (VA)). Mixed protozoa from the sheep rumen contained at least two to three times more unsaturated fatty acids, including CLA and VA, than bacteria. Different species had different composition, with larger fibrolytic species such as Epidinium ecaudatum caudatum containing more than ten times more CLA and VA than some small species, including Entodinium nanellum. In incubations with ruminal microbial fractions (bacterial fraction (BAC), protozoal fraction (PRO)), LA metabolism was very similar in strained ruminal fluid (SRF) and in the BAC, while the PRO had LA-metabolising activity an order of magnitude lower. Using PCR-based methods, no genes homologous to fatty acid desaturase genes were found in cDNA libraries from ruminal protozoa. The absence of an alternative route of VA/CLA formation via desaturation of stearate was confirmed by incubations of SRF, BAC or PRO with [14C]stearate. Thus, although protozoa are rich in CLA and VA, they appear to lack the ability to form these two fatty acids from LA or stearate. The most likely explanation is that protozoa preferentially incorporate CLA and VA formed by bacteria. The implication of the present findings is that the flow of unsaturated fatty acids, including CLA and VA, from the rumen could depend on the flow of protozoa rather than bacteria.

Animals↗
PubMed1996

In vitro metabolism of the stereoisomers of 2,6-diaminopimelic acid by mixed rumen protozoa and bacteria.

Formation of lysine from stereoisomers (SI) of 2,6-diaminopimelic acid (DAP) and the epimerization between the three SI of DAP (DAP-SI) by rumen protozoa and bacteria were examined. Mixed rumen protozoa (P) and bacteria (B) were isolated from the rumen of goats given a concentrate and hay cubes and incubated separately with and without a mixture and a single one of the three DAP-SI. In P suspensions, mixed DAP-SI decreased by 10.59% as a whole and converted mainly to lysine by 8.41% during 12 h incubation. When meso-, L- and D-DAP were added singly to the media, the results showed that each DAP-SI interconverted and produced lysine. This means that mixed rumen protozoa have an ability to synthesize lysine from not only meso-DAP but also from D- and L-DAP, though probably via meso-DAP, and hence have DAP epimerase activities for the reversal conversion of each DAP-SI. This is the first discovery to show the interconversion of DAP-SI and synthesis of lysine from them by protozoa. In B suspensions, mixed DAP-SI decreased by 10.92% as a whole and converted to lysine by 4.20% during 12 h incubation. When a single DAP-SI was added to the media, meso-, L- and D-DAP were interconverted and then converted to lysine by the rumen bacteria as well as the protozoa. This also means that mixed rumen bacteria have DAP epimerase activities to interconvert DAP-SI and have an ability to synthesize lysine from not only meso-DAP but also from L- and D-DAP, and this is also the first finding in rumen bacteria.

Amino Acid Isomerases↗
PubMed2006

Klebsiella to Salmonella gene transfer within rumen protozoa: implications for antibiotic resistance and rumen defaunation.

The rumen has long been thought to be a site of gene transfer for microorganisms. Rumen protozoa (RPz) are active predators of bacteria that can harbor antibiotic resistance genes. In this study, RPz were assessed as sites of gene transfer between two bacterial species, Salmonella and Klebsiella. One Klebsiella isolate carried a plasmid bearing bla(CMY-2), encoding an extended-spectrum beta-lactamase conferring ceftriaxone resistance, while the Salmonella was susceptible to ceftriaxone yet capable of thriving within protozoa. In vitro studies revealed that ceftriaxone-resistant Salmonella could be isolated following co-incubation of Salmonella and Klebsiella with RPz obtained from adult cattle and goats. Ceftriaxone-resistant Salmonella were not recovered in the presence of an inhibitor of protozoa engulfment or when a protozoa-sensitive Salmonella was part of the co-incubation. This transfer event was additionally observed in vitro for protozoa-independent stressors although at a significantly lower frequency. The gene transfer event was related to bacterial conjugation since a conjugation inhibitor, nalidixic acid, perturbed the phenomenon. Ceftriaxone-resistant Salmonella were recovered from calves, sheep, and goats co-challenged with ceftriaxone-resistant Klebsiella and ceftriaxone-sensitive Salmonella. However, the transfer event was not observed in calves and sheep that were defaunated prior to the co-challenge. Moreover, Salmonella transconjugants were isolated from separate bovine in vivo studies involving a Klebsiella donor carrying a plasmid conferring colicin activity while no such transconjugants were obtained from defaunated calves. These results provide an important basis for evaluating and preventing the spread of antibiotic resistance and other selective advantages for pathogens present in ruminants.

Animals↗
PubMed1997

Colonization of transplant unit water supplies with Legionella and protozoa: precautions required to reduce the risk of legionellosis.

Organ transplant recipients and other immunosuppressed patients are known to be at increased risk of nosocomial Legionnaires' disease. Although the ecology of Legionella in hospital water storage and distribution systems (including a protozoonotic relationship with free-living protozoa) has been well documented, little is known regarding the quality of water supplied to high-risk units. Hot- and cold-water samples (two first draw and one run to waste for 5 min) were taken from 69 (85%) of the 81 United Kingdom organ transplant units (31 renal, 24 bone marrow, nine cardiopulmonary and five liver transplant units) and cultured for Legionella and protozoa. Legionella spp. were isolated from the water supplies of 38 (55%) units and Legionella pneumophila from 31 (45%). The blue-white fluorescent group of Legionella (Legionella gormanii, Legionella bozemanii and others) was isolated from 18 (26%) units. Free-living protozoa were isolated from 47 units (68%) and genera of the protozoa known to permit the intracellular growth of Legionella (PGIGL), from 40 units (58%). Possible associations between Legionella and the variables Protozoa; PGIGL; water pH; and circulating water temperature (recorded after running to waste for 5 min) were examined by logistic regression analysis. In cold-water supplies, a significant association was found between the isolation of Legionella and PGIGL (P = 0.032; OR = 1.81; 95% CI 1.1-3.1). In hot-water supplies, an inverse association was found between the isolation of Legionella and circulating water temperature (P = 0.034; OR = 1.0719 per degree C; 95% CI 1.0052-1.1432). (We failed to isolate Legionella when the circulating hot water was > 58 degrees C. No other associations were significant. We recommend the active surveillance of water quality in high-risk patient areas, and that transplant units, either with a history of nosocomial Legionnaires' disease, or where active surveillance indicates a persistently high Legionella colony count, take remedial action. The quality of cold water may be improved by provision of a dedicated supply taken directly from the incoming mains; and of hot water by the use of a dedicated calorifier, able to maintain a minimum circulating hot water return temperature of 60 degrees C.

Cross Infection↗
PubMed2006

Contribution of rumen protozoa to duodenal flow of nitrogen, conjugated linoleic acid and vaccenic acid in steers fed silages differing in their water-soluble carbohydrate content.

The present experiment was designed to estimate the quantitative contribution of rumen protozoa to the total N, conjugated linoleic acid (CLA) and vaccenic acid (VA; trans-11-18 : 1) flow to the duodenum of steers fed two silage diets: control silage (CS) and silage high in water-soluble carbohydrates (HS). Protozoal duodenal flows were estimated using a real-time PCR assay to quantify the genes encoding protozoal 18S ribosomal RNA. Denaturing gradient gel electrophoresis was used to confirm that the rumen protozoa populations were similar to the protozoal population flowing to the duodenum. Estimated duodenal flow of protozoal N was 14.2 and 18.2 g/d (P>0.05) for animals fed the CS and HS diets respectively. Protozoal flow thus represented between 12 and 15 % of the total N duodenal flow. In terms of fatty acid flow, protozoa accounted for between 30 and 43 % of the CLA and 40 % of the VA reaching the duodenum. The contribution of protozoa to 16 : 0 and 18 : 0 flows to the duodenum was less than 20 and 10 %, respectively. These results show that the fatty acids within protozoa make up a significant proportion of the CLA and VA reaching the duodenum of ruminants.

Animals↗
PubMed1998

Risk assessment of waterborne protozoa: current status and future trends.

Throughout the past decade much research has been directed towards identifying the occurrence, epidemiology, and risks associated with waterborne protozoa. While outbreaks are continually documented, sporadic cases of disease associated with exposure to low levels of waterborne protozoa are of increasing concern. Current methodologies may not be sensitive enough to define these low levels of disease. However, risk assessment methods may be utilised to address these low level contamination events. The purpose of this article is to provide an introduction to microbial risk assessment for waterborne protozoa. Risk assessment is a useful tool for evaluating relative risks and can be used for development of policies to decrease risks. Numerous studies have been published on risk assessment methods for pathogenic protozoa including Cryptosporidium and Giardia. One common notion prevails: microbial risk assessment presents interesting complications to the traditional chemical risk assessment paradigm. Single microbial exposures (non-threshold) are capable of causing symptomatic illness unlike traditional chemical exposures, which require a threshold to be reached. Due to the lack of efficient recovery and detection methods for protozoa, we may be underestimating the occurrence, concentration and distribution of these pathogenic micro-organisms. To better utilize the tool of microbial risk assessment for risk management practices, future research should focus in the area of exposure assessment.

Animals↗
PubMed1985

Effect of protozoa on rumen protein degradation in sheep.

We have studied the contribution of ciliate protozoa to the degradation of dietary protein, utilizing 5 defaunated and 6 faunated sheep. Rumen samples from these animals were used as inocular for in vitro determination of the degradability of different protein sources: lupine grain, peanut and soybean cake, and fish meal. We also applied the in sacco method to measure the digestibility of soybean proteins. Two pore sizes (50 and 100 microns) were used in the in sacco study. The nitrogen degradation curve was constructed according to the mathematical model: P(t) = a + b (1-e-ct), and the parameters a, b, and c were determined for both faunated and defaunated animals. The proteolytic activity of the rumen contents was also determined using azocasein. The in vitro degradability of proteins was significantly less in defaunated than in faunated animals: 17, 19, 28 and 64% with lupine grain, peanut cake, soybean cake and fish meal proteins, respectively. The protozoal effect was greater when protein solubility was low. The in sacco study showed that neither the protozoa nor bag pore size had any effect on the protein fraction (a) immediately soluble in the rumen juice. However, both factors increased the insoluble, potentially degradable fraction "b" (P less than or equal to .01). Protozoa had a positive effect on the rate of degradation (c) of the fraction b. This effect was most clear with the 100 micron bags. The significant interaction between protozoa and pore size on factor "c" showed that large ciliate protozoa (100 microns) were directly involved in determining this parameter.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1978

Effects of diet on the protozoa population in permeable continuous cultures of rumen contents.

1. Responses of the protozoa population to the composition and the components of the diet were studied in permeable continuous cultures of rumen contents. 2. In Expt 1 a study was made of responses to diets containing different combinations of rice straw, lucerne meal and mixed concentrates given to the cow supplying inocula for cultures. It was found that (1) when a diet devoid of concentrates was fed, entodiniomorphs decreased in numbers; (2) their numbers increased with the increase in the amount of concentrates; (3) holtrich numbers were hardly affected by the type of diet; (4) rice straw and lucerne meal were not essential for protozoa to survive in culture medium. 3. In Expt 2 responses were determined to diets containing different combinations of maize, maize starch, sugars (glucose-sucrose (1:1, w/w)), soya-bean meal and isolated soya-bean protein. The results suggested the following relationship between dietary component and protozoa population. (1) The diet rich in both starch and sugars sustains the increased numbers of protozoa on the whole. (2) The diet rich in starch and deficient in sugars decreases selectively Dasytricha and small species of Entodinium such as Entodinium minimum and E. nanellum. The other species of protozoa, especially large entodiniomorphs such as Ophyroscolex and Polyplastron, are maintained at relatively high levels. (3) The diet deficient in starch and abundant in sugars decreases general entodiniomorphs except small species, while the numbers of Holotrichs are kept at relatively high levels.

Animal Feed↗
PubMed2001

Emerging and reemerging intestinal protozoa.

The intestinal protozoa have gained importance to physicians practicing medicine in the United States, Canada, and Europe during recent years as a result of increasing world travel, the globalization of the world's economy, and the growing number of chronically immunosuppressed people. During the spring of 1996, Cyclospora cayetanensis caused diarrhea in approximately 1500 people exposed to Guatemalan raspberries. This epidemic recurred in 1997, emphasizing the risks of the global economy and food supply on which we depend. In addition to importation of intestinal protozoa from the tropics, AIDS and the increasing use of organ transplants have created a new population of people at risk for chronic infection by ubiquitous protozoa previously not known to cause serious human disease. These infections include cryptosporidiosis, isosporiasis, and microsporidiosis. Finally, Entamoeba histolytica, the etiologic agent of invasive amebiasis, has only recently been recognized to be a distinct species from a nonpathogenic but indistinguishable (by light microscopy) intestinal commensal, Entamoeba dispar. The rapidly changing epidemiology of these intestinal protozoa, as well as new approaches to diagnosis and treatment of these protozoa, are discussed.

Journal Article↗
PubMed2006

Portable continuous flow centrifugation and method 1623 for monitoring of waterborne protozoa from large volumes of various water matrices.

AIMS: The aims of this study were to validate a portable continuous flow centrifuge (PCFC) as an alternative concentration step of US-EPA Method 1623 and to demonstrate it's efficacy for recovery of low numbers of protozoa from large volumes of various water matrices. METHODS AND RESULTS: Recoveries of Cryptosporidium parvum oocysts, Giardia intestinalis cysts and Encephalitozoon intestinalis spores spiked into 10-1000 l volumes of various water matrices were evaluated during in-house and collaborative trials. Spiked protozoa were either approved standards or diluted stock samples enumerated according to USEPA Method 1623. Cryptosporidium recoveries exceeded method 1623 criteria and substantially high recoveries were observed for Giardia and E. intestinalis. CONCLUSIONS: Portable continuous flow centrifuge methodology exceeded method 1623 acceptance criteria for Cryptosporidium and could be easily adopted for other protozoa. SIGNIFICANCE AND IMPACT OF THE STUDY: The PCFC could be adopted as an alternative user-friendly concentration method for Cryptosporidium and for monitoring of large volumes of source and tap water for accidental or deliberate contamination with protozoa and potentially with other enteric pathogens. It is anticipated that PCFC would also be equal or superior to filtration for protozoa monitoring in wastewater and effluents.

Animals↗
PubMedDate not supplied

In vitro production of lysine from 2,2'-diaminopimelic acid by rumen protozoa.

Rumen protozoa can produce lysine from free 2,2'-diaminopimelic acid (DAP). However, the quantitative importance of this transformation has been disputed; lysine contents of protozoal incubation supernatants reported by Onodera & Kandatsu and Masson & Ling show a 26-fold difference. The in vitro experimental methods of both groups were compared to determine the causes of this difference. Lysine production was proportional to DAP concentration. Results with rumen protozoa from sheep or goats were similar. The incubation medium and deproteinizing procedure of the Welsh group gave a two-fold increase in lysine production compared with Japanese protocols. Omissions of rice starch from protozoal incubations slightly increased lysine production, whereas omissions of antibacterial agents resulted in varying, yet relatively small changes. The greatest cause of the difference was the number of rumen protozoa incubated. When this factor was taken into account, the difference in the maximum rates of lysine production between the Welsh and Japanese groups was only three-fold, namely 4.5 versus 15.0 nmol lysine/10(5) protozoa/h. Adding other amino acids to the incubations suggested that DAP uptake by rumen protozoa may occur via transport system ASC. The importance of DAP metabolism by protozoa as a source of lysine for ruminant host animals is discussed.

Amino Acids↗
PubMed1984

Protease activities of rumen protozoa.

Intact, metabolically active rumen protozoa prepared by gravity sedimentation and washing in a mineral solution at 10 to 15 degrees C had comparatively low proteolytic activity on azocasein and low endogenous proteolytic activity. Protozoa washed in 0.1 M potassium phosphate buffer (pH 6.8) at 4 degrees C and stored on ice autolysed when they were warmed to 39 degrees C. They also exhibited low proteolytic activity on azocasein, but they had a high endogenous proteolytic activity with a pH optimum of 5.8. The endogenous proteolytic activity was inhibited by cysteine proteinase inhibitors, for example, iodoacetate (63.1%) and the aspartic proteinase inhibitor, pepstatin (43.9%). Inhibitors specific for serine proteinases and metalloproteinases were without effect. The serine and cysteine proteinase inhibitors of microbial origin, including antipain, chymostatin, and leupeptin, caused up to 67% inhibition of endogenous proteolysis. Hydrolysis of casein by protozoa autolysates was also inhibited by cysteine proteinase inhibitors. Some of the inhibitors decreased endogenous deamination, in particular, phosphoramidon, which had little inhibitory effect on proteolysis. Protozoal and bacterial preparations exhibited low hydrolytic activities on synthetic proteinase and carboxypeptidase substrates, although the protozoa had 10 to 78 times greater hydrolytic activity (per milligram of protein) than bacteria on the synthetic aminopeptidase substrates L-leucine-p-nitroanilide, L-leucine-beta-naphthylamide, and L-leucinamide. The aminopeptidase activity was partially inhibited by bestatin. It was concluded that cysteine proteinases and, to a lesser extent, aspartic proteinases are primarily responsible for proteolysis in autolysates of rumen protozoa. The protozoal autolysates had high aminopeptidase activity; low deaminase activity was observed on endogenous amino acids.

Ammonia↗
PubMed1988

Survival of coliforms and bacterial pathogens within protozoa during chlorination.

The susceptibility of coliform bacteria and bacterial pathogens to free chlorine residuals was determined before and after incubation with amoebae and ciliate protozoa. Viability of bacteria was quantified to determine their resistance to free chlorine residuals when ingested by laboratory strains of Acanthamoeba castellanii and Tetrahymena pyriformis. Cocultures of bacteria and protozoa were incubated to facilitate ingestion of the bacteria and then were chlorinated, neutralized, and sonicated to release intracellular bacteria. Qualitative susceptibility of protozoan strains to free chlorine was also assessed. Protozoa were shown to survive and grow after exposure to levels of free chlorine residuals that killed free-living bacteria. Ingested coliforms Escherichia coli, Citrobacter freundii, Enterobacter agglomerans, Enterobacter cloacae, Klebsiella pneumoniae, and Klebsiella oxytoca and bacterial pathogens Salmonella typhimurium, Yersinia enterocolitica, Shigella sonnei, Legionella gormanii, and Campylobacter jejuni had increased resistance to free chlorine residuals. Bacteria could be cultured from within treated protozoans well after the time required for 99% inactivation of free-living cells. All bacterial pathogens were greater than 50-fold more resistant to free chlorine when ingested by T. pyriformis. Escherichia coli ingested by a Cyclidium sp., a ciliate isolated from a drinking water reservoir, were also shown to be more resistant to free chlorine. The mechanism that increased resistance appeared to be survival within protozoan cells. This study indicates that bacteria can survive ingestion by protozoa. This bacterium-protozoan association provides bacteria with increased resistance to free chlorine residuals which can lead to persistence of bacteria in chlorine-treated water. We propose that resistance to digestion by predatory protozoa was an evolutionary precursor of pathogenicity in bacteria and that today it is a mechanism for survival of fastidious bacteria in dilute and inhospitable aquatic environments.

Animals↗