Isolation Of Amoebae Research Articles

First Report of Isolation and Characterization of Acanthamoeba spp. from the Milk Used for Calf Feeding.

Acanthamoeba spp. can be found in natural and artificial environments, which reflects their high adaptability to different conditions. Based on the available data, there is scarce information about the isolation of amoeba from milk. This study aimed to investigate the probable presence of Acanthamoeba in milk used for calf feeding. 200 milk samples from 50 industrial and traditional farms were collected. The samples were filtered and cultured on the 1.5% Non-nutrient agar medium. The amoebic growth was examined with an inverted microscope daily. DNA was extracted from the positive plates, and a PCR reaction was undertaken using the primers amplifying the Acanthamoeba 18S rRNA gene. Five samples were purified and sequenced using specific primers. Maximum likelihood reconstructions were performed using the phylogenetic program MEGA software. The osmo and thermotolerance of isolated trophozoites were examined as well. Out of 200 milk samples, Acanthamoeba was isolated from 27 (13.5%). The phylogenetic tree represents that all the isolates belonged to the genotype T4. Results of thermo and osmotolerance tests showed that isolates could develop at 37 and 43 ◦C. Besides, trophozoites survived at 0.5M mannitol and 1M. For the first time, Acanthamoeba spp. were isolated from milk used to feed dairy calves. Due to Acanthamoeba's neglected role in pathogen persistence and survival, hygiene instructions should be reconsidered.

Growth characteristics and morphology of Paramoeba perurans from Atlantic salmon Salmo salar L. and ballan wrasse Labrus bergylta in Norway

BackgroundParamoeba perurans is the causative agent of amoebic gill disease (AGD) in Atlantic salmon Salmo salar L. and many other farmed marine fish species worldwide. The first cases of AGD in Norway were reported in 2006, and it has subsequently become established as a significant gill disease that affects the country’s salmonid aquaculture industry. Despite several decades of research on AGD, there is still a lack of knowledge of the biology of P. perurans and its interactions with its hosts and the environment.MethodsThe growth and morphology of 10 clonal isolates of P. perurans were studied. The isolates were from farmed Atlantic salmon and ballan wrasse that had been obtained from different sites along the Norwegian coast between 2013 and 2015. The morphology and population growth patterns of these clonal amoeba isolates were examined in vitro using light microscopy and real-time reverse transcription polymerase chain reaction under a range of temperatures (4, 12, 15 and 21 °C) and salinities (20, 25, 30 and 34 ‰).ResultsWe found distinct morphological differences between both locomotive and floating forms of the amoeba isolates. The locomotive amoebae of the clonal isolates varied in size (area) from 453 µm2 to 802 µm2. There were differences in the growth patterns of the clonal amoeba isolates under similar conditions, and in their responses to variations in temperature and salinity. While most of the isolates grew well at salinities of 25–34 ‰, a significant reduction in growth was seen at 20 ‰. Most of the amoeba isolates grew well at 12 °C and 15 °C. At 4 °C, amoebae grew slower and, in contrast to the other temperatures, no extended pseudopodia could be seen in their floating form. The isolates seemed to reach a plateau phase faster at 21 °C, with a higher number of smaller, rounded amoebae.ConclusionsThe differences observed here between clonal isolates of P. perurans should be further examined in experimental in vivo challenge studies, as they may be of relevance to the virulence and proliferation potential of this amoeba on gills. Potential differences in virulence within P. perurans could have implications for management strategies for AGD.Graphical

Differential Growth Rates and In Vitro Drug Susceptibility to Currently Used Drugs for Multiple Isolates of Naegleria fowleri.

ABSTRACTThe free-living amoeba Naegleria fowleri, which typically dwells within warm, freshwater environments, can opportunistically cause primary amoebic meningoencephalitis (PAM), a disease with a mortality rate of >97%. The lack of positive treatment outcomes for PAM has prompted the discovery and development of more effective therapeutics, yet most studies utilize only one or two clinical isolates. The inability to assess possible heterogenic responses to drugs among isolates from various geographical regions hinders progress in the discovery of more effective drugs. Here, we conducted drug efficacy and growth rate determinations for 11 different clinical isolates by applying a previously developed CellTiter-Glo 2.0 screening technique and flow cytometry. We found significant differences in the susceptibilities of these isolates to 7 of 8 drugs tested, all of which make up the cocktail that is recommended to physicians by the U.S. Centers for Disease Control and Prevention. We also discovered significant variances in growth rates among isolates, which draws attention to the differences among the amoeba isolates collected from different patients. Our results demonstrate the need for additional clinical isolates of various genotypes in drug assays and highlight the necessity for more targeted therapeutics with universal efficacy across N. fowleri isolates. Our data establish a needed baseline for drug susceptibility among clinical isolates and provide a segue for future combination therapy studies as well as research related to phenotypic or genetic differences that could shed light on mechanisms of action or predispositions to specific drugs.IMPORTANCE Naegleria fowleri, also known as the brain-eating amoeba, is ubiquitous in warm freshwater and is an opportunistic pathogen that causes primary amoebic meningoencephalitis. Although few cases are described each year, the disease has a case fatality rate of >97%. In most laboratory studies of this organism, only one or two well-adapted lab strains are used; therefore, there is a lack of data to discern if there are major differences in potency of currently used drugs for multiple strains and genotypes of the amoeba. In this study, we found significant differences in the susceptibilities of 11 N. fowleri isolates to 7 of the 8 drugs currently used to treat the disease. The data from this study provide a baseline of drug susceptibility among clinical isolates and suggest that new drugs should be tested on a larger number of isolates in the future.

Isolation and identification of free-living amoeba from contact lenses: Thermal and osmotic tolerance in relation to their pathogenicity.

Isolation and identification of free-living amoeba from contact lenses: Thermal and osmotic tolerance in relation to their pathogenicity.

Isolation, Morphotyping, Molecular Characterization and Prevalence of Free-Living Amoebae from Different Water sources in Makkah city, Saudi Arabia

Free-living amoebae (FLA) are protozoa that can be found in a wide range of habitats around the world. Some species have been found to be pathogenic to humans in addition to their normal distribution. There is no research on the prevalence of FLA in various water sources in Makkah city that we are aware of. The aim of this study was to investigate the prevalence of FLA from various water sources in a different part of the city. A total of 86 water samples were processed and cultured with Escherichia coli overlay on non-nutrient agar medium (NNA). Microscopic examination was used to examine the samples. Fifty (58.1%) of the 86 samples tested were positive for FLA. Amoebae identified by morphology belonging to the genus Acanthamoeba were 30 (34.9%) and that belonging to the genus Naegleria, were 20 (23.3%). Among the positive cases, 100% of ponds, 80% of air conditioner exhaust, 50% of water fountains and tape water filters, 40% of swimming pools and 33.3% of stored tanks. The absence of FLA in the examined wells was a striking feature. Polymerase chain reaction (PCR) on DNA showed a 229-bps fragment using Nelson primers and a 500-bps fragment using Acanthamoeba genus-specific primer pair JDP1/JDP2. In conclusion, Acanthamoeba spp. was the most predominant type of free-living amoeba and more prevalent in ponds water absent in wells as shown by culture, microscopy and PCR.

Transfer of the thecate amoebae Lecythium spinosum and Pamphagus armatus to Rhizaspis (Thecofilosea, Cercozoa, Rhizaria)

Thecofilosea is a class in Cercozoa (Rhizaria) comprising mainly freshwater-inhabiting algivores. Recently, numerous isolates of thecofilosean amoebae have been cultured and were characterized by an integrated morphological and molecular approach. The captivating spine-bearing taxa in Thecofilosea were not yet molecularly characterized due to being very rare. There are only two known spine-bearing species, Pamphagus armatus and Lecythium spinosum, which were synonymized by Penard in 1902. Due to a morphological difference of those taxa, we discuss here that we disagree with this taxonomical act. We further isolated single cells of Pamphagus armatus directly from their habitat and successfully sequenced their SSU rDNA, which we subjected to phylogenetic analyses. We show that Pamphagus armatus branches within the Rhizaspididae (Tectofilosida, Thecofilosea). Accordingly, we transfer Pamphagus armatus and the assumingly closely related species Lecythium spinosum to Rhizaspis.

Isolation and identification of free-living amoebae isolated from well water in Istanbul.

Free-living amoebae (FLA) are ubiquitous protozoa commonly found in water and soil environments. FLA belonging to various genera, including Acanthamoeba, Balamuthia, Naegleria, and Vermamoeba, can cause opportunistic and non-opportunistic infections in humans and animals such as keratitis or meningoencephalitis. In addition, some of them serve as hosts for a large number of pathogenic bacteria, yeasts, and viruses. The purpose of the present study was to assess the prevalence and molecular characterization of FLA in well water samples in İstanbul. Ten well water samples were collected from the taps and the presence of FLA was monitored both by the culture and polymerase chain reaction methods. FLA were isolated in 8 out of the 10 samples (80%) included in this study. Morphological analysis and partial sequencing of the 18S rDNA revealed the presence of Acanthamoeba genotypes T3 and T4, and Vermamoeba vermiformis in the investigated well water samples. This study reports for the first time the detection of Acanthamoeba genotype T3 in well water samples in İstanbul. The presence of potentially pathogenic amoebae in habitats related to human activities supports the relevance of FLA as a potential public health concern.

Morphology and Ecology of Two New Amoebae, Isolated From a Thalassohaline Lake, Dziani Dzaha

Dziani Dzaha is a hypersaline lake (Mayotte island), whose microbial community is dominated by photosynthetic microorganisms. Here, we describe two new free-living heteroloboseans. One belonging to the Pharyngomonas genus and the other, whose 18S rRNA gene sequence shares only 85% homology to its closest relatives Euplaesiobystra hypersalinica, was proposed as a new species of this genus being called Euplaesiobystra dzianiensis. Both strains were salt tolerant to 75‰ and grew between 25 and 37°C. Their distribution patterns varied seasonally and depended also on depth. Noticeably, both free-living amoebae isolates were able to graze on Arthrospira filaments, which are found within the same water layer. In conclusion, we document for the first time the presence and ecology of free-living amoebae in the thalassohaline lake Dziani Dzaha, and describe a new species of the Euplaesiobystra genus.

Occurrence of parasites in salads in restaurants in Aparecida de Goiânia, Goiás, Brazil

Parasitoses are the most widespread diseases in the world. They are transmited via contaminated water or food. Considering that the daily consumption of vegetables is estimated at 142g per person, the purpose of this study was to evaluate the occurrence of parasites in salads available for consumption in restaurants in Aparecida de Goiânia, Goiás State. Salad samples were collected from the restaurants and parasitological analysis was performed using the Willis, Hoffman, Faust and Ziehl Neelsen techniques as well as cultures for the isolation of free-living amoebae. 51 samples were analyzed, 16 (31.4%) were positive. The parasites detected were: Acanthamoeba spp. in 12 (23.5%); free-living larvae, Schistosoma mansoni and Entamoeba coli in 1 (2.0%); Endolimax nana in 2 (3.9%). The PCR technique determined that 17.6% of the samples presented Toxoplasma gondii DNA. These techniques evidenced that the salad samples presented parasite contamination not only in the restaurants with the lowest price per Kg, but also in the most expensive ones. Therefore, in addition to effective sanitary surveillance, prophylactic measures are necessary regarding suppliers, handlers and restaurant owners to prevent the spread of these and other parasites.KEY WORDS: Food contamination; food parasitology; polymerase chain reaction.

Various dictyostelids from the environment can produce multilamellar bodies.

Multilamellar bodies (MLBs), structures composed of concentric membrane layers, are known to be produced by different protozoa, including species of ciliates, free-living amoebae, and Dictyostelium discoideum social amoebae. Initially believed to be metabolic waste, potential roles like cell communication and food storage have been suggested for D. discoideum MLBs, which could be useful for the multicellular development of social amoebae and as a food source. However, among dictyostelids, this phenomenon has only been observed with D. discoideum, and mainly with laboratory strains grown in axenic conditions. It was thought that other social amoebae may also produce MLBs. Four environmental social amoeba isolates were characterized. All strains belong to the Dictyostelium genus, including some likely to be Dictyostelium giganteum. They have distinctive phenotypes comprising their growth rate on Klebsiella aerogenes lawns and the morphology of their fruiting bodies. They all produce MLBs like those produced by a D. discoideum laboratory strain when grown on K. aerogenes lawns, as revealed by analysis using the H36 antibody in epifluorescence microscopy as well as by transmission electron microscopy. Consequently, this study shows that MLBs are produced by various dictyostelid species, which further supports a role for MLBs in the lifestyle of amoebae.

Isolation and identification of free-living amoeba from the hot springs and beaches of the Caspian Sea

Free-living amoeba (FLA) such as Acanthamoeba, Naegleria, Balamuthia, and Vermamoeba have been identified from both natural and human-made environments such as Hot springs and spa. Naegleria fowleri causes Primary Amoebic Meningoencephalitis (PAM), while Acanthamoeba and Balamuthia cause chronic granulomatous encephalitis. Acanthamoeba also can cause cutaneous lesions and Amoebic Keratitis (AK) that is associated with contact lens use or corneal trauma. FLA are known to serve as host of and vehicles for diverse intracellular organisms. This study aimed was to identify the presence of FLA in the hot springs and beaches of the Caspian Sea in Ramsar tourist town located in the northern part of Iran. Water samples were collected in sterile bottles and were transferred to the laboratory. One litre of each sample passed through the nitrocellulose membrane filter. Each filter insert was then placed in non-nutrient agar plates already seeded with lawn culture of Escherichia coli. Positive samples were analyzed by morphological keys and Polymerase chain reaction (PCR) using 18S rDNA gene and ITS region to identify amoeba isolates. A total of 81 water sampled were tasted. After identified using the morphological key and PCR assay, 54 (66.6%) of the samples were positive for FLA. Ten of the samples were identified as Acanthamoeba (belong to T3, T4, and T5 genotypes), three as Vermamoeba vermiformis, four as Naegleria (3 N.australiensis and 1 N.grubery). Only one sample was positive Vahlkampfia. The presence of thermotolerant FLA in the Hot springs and beaches of the Caspian Sea as places for recreational purposes or wellness may be a potential health risk.

Isolation and molecular identification of free-living amoebae (Naegleria spp., Acanthamoeba spp. and Vermamoeba spp.) from mineral springs in Guilan Province, northern Iran.

Free-living amoebae (FLA) include many genera which cause serious diseases such as sight-threatening keratitis, cutaneous ulcers and fatal encephalitis. This study was conducted due to the lack of research regarding genotypes Acanthamoeba, Naegleria and Vermamoeba in mineral springs of Guilan Province in northern Iran. Twenty-five water samples were collected from mineral springs in Guilan Province. After filtration through nitrocellulose membrane, samples were cultured on non-nutrient agar plates. The morphological key of Page was used to identify free-living amoebae (FLA) using an inverted microscope. Positive cultures were analyzed by polymerase chain reaction (PCR) and genotypes based on the NCBI database. Eleven (44%) samples were positive for Acanthamoeba, Naegleria and Vermamoeba. By sequencing the positive isolates, the strains were shown to belong to Acanthamoeba castellanii (three case isolates belonged to T4 genotype), three cases of Vermamoeba vermiformis, and two cases of N. australiensis, two cases of N. pagei and one case of N. gruberi. According to our research the occurrence of Acanthamoeba, Naegleria spp. and Vermamoeba spp. in mineral springs could be hazardous for high risk persons. Regular monitoring and posting warning signs of these waters by health planners could prevent free-living amoebae mediated diseases.

Transfer of the Thecate Amoeba Lecythium mutabilis to a Novel Genus Omnivora (Fiscullidae, Thecofilosea, Cercozoa).

Thecofilosea is a class in Cercozoa (Rhizaria) comprising mainly freshwater-inhabiting algivores. Recently, numerous isolates of thecofilosean amoebae have been cultured and were characterized by an integrated morphological and molecular approach. As attempts to establish a culture of Lecythium mutabilis repeatedly failed, it was not yet investigated by molecular means. We isolated single cells of L.mutabilis directly from their habitat and successfully sequenced the V4 region of their SSU rDNA. Phylogenetic analyses showed that L.mutabilis is not directly related to the genus Lecythium and instead branches within the Fiscullidae (Tectofilosida, Thecofilosea). Accordingly, we transfer the species L.mutabilis to a novel genus Omnivora gen. nov.

Growth-promoting effects of the hydrogen-sulfide compounds produced by Desulfovibrio desulfuricans subsp. desulfuricans co-cultured with Escherichia coli (DH5α) on the growth of Entamoeba and Endolimax species isolates from swine.

Certain Desulfovibrio sp. (anaerobic sulfate-reducing bacteria) are indigenous to swine cecum and colon, which are also common habitats for parasitic amoebae such as Entamoeba polecki and Entamoeba suis. In this study, we evaluated the growth-promoting effects of D. desulfuricans co-cultured with Escherichia coli (DH5α) and its products [e.g., hydrogen sulfide (H2S) and certain iron-sulfide (FeS) compounds] using Robinson's medium, on the 4 amoeba isolates from swine-Entamoeba polecki subtype (ST)-1, E. polecki ST-3, Entamoeba suis, and Endolimax sp., and, consequently, a continuous culture system for these amoebae was established. However, this novel culture system was required to regulate the excess H2S dissolved in the medium by increasing air space as amoeba isolates thrive only in large air spaces (30-40%). The effects of air space and H2S and FeS compounds on the growth of E. polecki ST-1 (TDP-5) were determined. E. polecki ST-1 (TDP-5) thrived well in culture bottles with an air space of 30-40% (aerobic) (H2S: ~250-400 μmoles/L), but did not grow at all in an air space < 5% (microaerobic) ( H2S:~800 μmoles/L) and in anaerobic vessels (H2S: 20-30 μmoles/L). In both H2S-depleted and FeS compound-depleted conditions, the amoebae sp. could not thrive either. It was hypothesized that an appropriate concentration of H2S and FeS compounds might function as important physiologically active components of electron carriers such as FeS and ferredoxin.

Isolation and Phylogenetic Analysis of Free-Living Amoebae (Acanthamoeba, Naegleria, and Vermamoeba) in the Farmland Soils and Recreational Places in Iran.

Free-living amoeba (FLA) including Acanthamoeba spp., Balamuthia mandrillaris, and Naegleria are among the soil-born parasites. There are reports of FLA-related keratitis with a history of contact with soil and dust sources, particularly among the farmers. Due to lack of the previous studies on the farmland soils and a limited number of researches conducted on recreational soils in Iran, the present study was conducted. A total of 93 soil samples including farming lands and recreational places were tested for the presence of Acanthamoeba spp. Balamuthia mandrillaris, Naegleria, and Vermamoeba using morphological key and sequencing-based tools. Pathogenicity of Acanthamoeba positive strains was also evaluated. To verify genetic associations and taxonomic status of isolated amoeba, a phylogenetic tree was built by MEGA 5.05 software inferred by the 18S rRNA gene based on maximum likelihood algorithm. Overall, 28 samples (30%) were contaminated with potentially pathogenic FLA, and according to the sequencing data, 17 strains were successfully sequenced. The isolated Acanthamoeba belonged to T2, T4, T5, mixed T4 and T5 contaminations, and T11. ITS sequencing revealed the occurrence of one strain of Naegleria canariensis. Four strains of Vermamoeba vermiformis were also confirmed. Morphological survey and PCR assay failed to show any positive results for Balamuthia mandrillaris. Pathogenic potential of the Acanthamoeba strains showed that T2, T4, and T11 genotypes were highly pathogenic, whereas T5 genotypes demonstrated lower pathogenic potential. The results indicate that soil could be a serious hazard to human health, and therefore, further studies are expected to investigate the source of infection in patients developing FLA-related diseases. The present study is the first to investigate FLA in the farmland soils in Iran and the first to report the presence of N. canariensis in the country.

Isolation and molecular identification of free-living amoebae from dishcloths in Tenerife, Canary Islands, Spain.

In this work, the presence of free-living amoebae (FLA) in dishcloths collected from human activity related places was evaluated. Once in the laboratory, 6cm2 pieces of each dishcloth were cut and washed with Page's Amoeba Solution (PAS) in sterile tubes. After washing, the dishcloth pieces were removed, and the tubes were centrifuged (1500rpm for 10min). The obtained pellets were seeded onto 2% non-nutrient agar (NNA) plates, incubated at room temperature and were monitored daily an inverted microscope. Once clonal cultures were obtained (only one type of FLA observed), molecular analyses were carried out in order to characterize the isolated FLA strains at the genus/genotype level. From the 31 dishcloths which were processed, FLA strains were isolated in NNA plates in 13 the samples (13/31, 42%). However, and due to bacterial overgrowth, only six strains were characterized at the molecular level (PCR and sequencing). Among the PCR positive strains, 83.33% (5/6) of the PCR positive samples belonged to Acanthamoeba genus (80% (4/5) to genotype T4 and 20% (1/5) to genotype T11). Furthermore, one strain was identified as a member of Allovahlkampfia genus using both morphological and molecular approaches. To the best of our knowledge, this is the first report on the isolation of Allovahlkampfia genus from dishcloths and in the Spanish territory. The presence of FLA in dishcloths should raise awareness to improve hygienic strategies in food- and domestic-related environments, in order to prevent contamination with these protozoa, which are able to be pathogenic and even to act as vehicles of other pathogenic agents.

Development of a method to extract protozoan DNA from black soil

ObjectivesMicroorganisms in environmental samples are identified by sequential screening, isolation, and culture steps, followed by the verification of physiological characteristics and morphological classification. Isolation and purification of Amoebae from soil samples is extremely complex, laborious, and time-consuming and require considerable expertise for morphological evaluation. PCR testing of soil DNA seems to be an effective means for protozoa habitat screening. In this study, we added Acanthamoeba sp. (MK strain) to soil and developed a method of extracting protozoan DNA from the soil. MethodsSoil allophane is a known DNA adsorbing substance that inhibits the PCR reaction. After comparing the soil properties and allophane contents of 7 soil samples, we attempted to combine multiple cell disruption and DNA purification methods to design an optimal soil DNA extraction method that can be used for downstream PCR analysis. ResultsWe compared five different crushing/refining methods. Amplification of the gene was confirmed by Acanthamoeba specific PCR in protocol V where the concentration of Acanthamoeba in soil (1.0 × 102/g) was the detection limit of PCR. ConclusionThe soil DNA extraction method following protocol V allows DNA amplification of protozoa, including Amoeba, which is difficult to cultivate, thus simplifying the investigation of protozoa habitats and genetic analyses.

Isolation of Free-living amoeba and Molecular Characterization of Acanthamoeba From Stagnant Water, Kashan, Iran

Isolation of Free-living amoeba and Molecular Characterization of Acanthamoeba From Stagnant Water, Kashan, Iran

Amoebic gill disease outbreak in marine fish cultured in Korea.

In 2015, 6.7-60% mortality was observed in black seabream ( Acanthopagrus schlegelii), rock bream ( Oplegnathus fasciatus), and gray mullet ( Mugil cephalus) farmed in the southern coast of Korea. On examination, numerous amoebae were found on the gills of these 3 fish species with detection rate of 100%. Some rock bream and gray mullet were coinfected with bacteria ( Pseudomonas anguilliseptica, Vibrio tapetis, or Vibrio anguillarum). Histologic examination revealed extensive hyperplastic epithelium and lamellar fusion in the gills. Numerous amoebae were seen between gill filaments. The amoebae collected from the 3 fish species had specific 630 bp of a partial 18S rRNA gene fragment for Neoparamoeba perurans. Phylogenetic analysis based on partial 18S rRNA gene nucleotide sequences revealed that these Korean amoeba isolates belonged to the N. perurans group. Based on our results, black seabream, rock bream, and gray mullet were added as new hosts for N. perurans.

Response of the protistan community of a rice field soil to different oxygen tensions.

Heterotrophic protists in soil are grazers that control the biomass and community structure of bacteria, thereby enhancing nutrient recycling. Oxygen regulates the microeukaryotic community, but little is known about its response to microoxic conditions. Here we studied the impact of oxygen tension on culturable heterotrophic protists in a rice field soil. The number of protists, dominated by amoeba and flagellates, under oxygen tensions ranging from atmospheric level (21%) to below the Pasteur point (0.08%) were similar (10(4) cellsg(-1) dry soil); no protists were detected under anoxic conditions. DGGE fingerprinting of microeukaryotes demonstrated a shift in the community structure depending on the oxygen tension during growth. Both common and specific amoeba and flagellates were identified at different oxygen tensions. Amoeba isolates (Acanthamoeba sp. and Hartmannella sp.) grew to the same extent under the oxygen tensions tested; the Acanthamoeba sp. isolate migrated more slowly under the lowest tension (0.08%). Our results demonstrated that amoeba and flagellates in soil adapt to a wide range of oxygen tensions with a shift in community structure. This suggests an ability to search for food in soil environments such as the oxic-anoxic interface of flooded soil or inside soil aggregates that are inaccessible to ciliates.