Parasite Spores Research Articles

The expression of virulence during double infections by different parasites with conflicting host exploitation and transmission strategies

In many natural populations, hosts are found to be infected by more than one parasite species. When these parasites have different host exploitation strategies and transmission modes, a conflict among them may arise. Such a conflict may reduce the success of both parasites, but could work to the benefit of the host. For example, the less-virulent parasite may protect the host against the more-virulent competitor. We examine this conflict using the waterflea Daphnia magna and two of its sympatric parasites: the blood-infecting bacterium Pasteuria ramosa that transmits horizontally and the intracellular microsporidium Octosporea bayeri that can concurrently transmit horizontally and vertically after infecting ovaries and fat tissues of the host. We quantified host and parasite fitness after exposing Daphnia to one or both parasites, both simultaneously and sequentially. Under conditions of strict horizontal transmission, Pasteuria competitively excluded Octosporea in both simultaneous and sequential double infections, regardless of the order of exposure. Host lifespan, host reproduction and parasite spore production in double infections resembled those of single infection by Pasteuria. When hosts became first vertically (transovarilly) infected with O.bayeri, Octosporea was able to withstand competition with P.ramosa to some degree, but both parasites produced less transmission stages than they did in single infections. At the same time, the host suffered from reduced fecundity and longevity. Our study demonstrates that even when competing parasite species utilize different host tissues to proliferate, double infections lead to the expression of higher virulence and ultimately may select for higher virulence. Furthermore, we found no evidence that the less-virulent and vertically transmitting O.bayeri protects its host against the highly virulent P.ramosa.

PREDATOR‐INDUCED DEFENSE MAKESDAPHNIAMORE VULNERABLE TO PARASITES

Inducible defensive traits against herbivores or predators are widespread in plants and animals. Theory predicts that defended morphs have greater fitness in the presence of predators, but lower fitness than undefended morphs in the absence of predators. If such costs did not exist, then a constitutively defended morph would be favored by natural selection; yet, evidence for such costs has been elusive. Our current work reveals a significant cost to inducible defenses. Using the waterflea (Daphnia) model system, we show that induced defended morphs are significantly more vulnerable to infection by a virulent yeast parasite than undefended morphs. In two independent experiments, the proportion of successful infections and the number of parasite spores were higher among defended versus undefended Daphnia. Thus, by demonstrating a previously unknown and environmentally relevant cost to inducible defenses, this study enhances our understanding of adaptive phenotypic plasticity and its evolution.

Variation in Resource Acquisition and Use among Host Clones Creates Key Epidemiological Trade‐Offs

Parasites can certainly harm host fitness. Given such virulence, hosts should evolve strategies to resist or tolerate infection. But what governs those strategies and the costs that they incur? This study illustrates how a fecundity‐susceptibility trade‐off among clonally reared genotypes of a zooplankton (Daphnia dentifera) infected by a fungal parasite (Metschnikowia) arises due to variation in resource acquisition and use by hosts. To make these connections, we used lab experiments and theoretical models that link feeding with susceptibility, energetics, and fecundity of hosts. These feeding‐based mechanisms also produced a fecundity‐survivorship trade‐off. Meanwhile, a parasite spore yield–fecundity trade‐off arose from variation in juvenile growth rate among host clones (another index of resource use), a result that was readily anticipated and explained by the models. Thus, several key epidemiological trade‐offs stem from variation in resource acquisition and use among clones. This connection should catalyze the creation of new theory that integrates resource‐ and gene‐based responses of hosts to disease.

Crowding and disease: effects of host density on response to infection in a butterfly–parasite interaction

Abstract. 1. Hosts experiencing frequent variation in density are thought to benefit from allocating more resources to parasite defence when density is high (‘density‐dependent prophylaxis’). However, high density conditions can increase intra‐specific competition and induce physiological stress, hence increasing host susceptibility to infection (‘crowding‐stress hypothesis’).2. We studied monarch butterflies (Danaus plexippus) and quantified the effects of larval rearing density on susceptibility to the protozoan parasite Ophryocystis elektroscirrha. Larvae were inoculated with parasite spores and reared at three density treatments: low, moderate, and high. We examined the effects of larval density on parasite loads, host survival, development rates, body size, and wing melanism.3. Results showed an increase in infection probability with greater larval density. Monarchs in the moderate and high density treatments also suffered the greatest negative effects of parasite infection on body size, development rate, and adult longevity.4. We observed greater body sizes and shorter development times for monarchs reared at moderate densities, and this was true for both unparasitised and parasite‐treated monarchs. We hypothesise that this effect could result from greater larval feeding rates at moderate densities, combined with greater physiological stress at the highest densities.5. Although monarch larvae are assumed to occur at very low densities in the wild, an analysis of continent‐wide monarch larval abundance data showed that larval densities can reach high levels in year‐round resident populations and during the late phase of the breeding season. Treatment levels used in our experiment captured ecologically‐relevant variation in larval density observed in the wild.

Strength in numbers: high parasite burdens increase transmission of a protozoan parasite of monarch butterflies (Danaus plexippus)

Parasites often produce large numbers of offspring within their hosts. High parasite burdens are thought to be important for parasite transmission, but can also lower host fitness. We studied the protozoan Ophryocystis elektroscirrha, a common parasite of monarch butterflies (Danaus plexippus), to quantify the benefits of high parasite burdens for parasite transmission. This parasite is transmitted vertically when females scatter spores onto eggs and host plant leaves during oviposition; spores can also be transmitted between mating adults. Monarch larvae were experimentally infected and emerging adult females were mated and monitored in individual outdoor field cages. We provided females with fresh host plant material daily and quantified their lifespan and lifetime fecundity. Parasite transmission was measured by counting the numbers of parasite spores transferred to eggs and host plant leaves. We also quantified spores transferred from infected females to their mating partners. Infected monarchs had shorter lifespans and lower lifetime fecundity than uninfected monarchs. Among infected females, those with higher parasite loads transmitted more parasite spores to their eggs and to host plant leaves. There was also a trend for females with greater parasite loads to transmit more spores to their mating partners. These results demonstrate that high parasite loads on infected butterflies confer a strong fitness advantage to the parasite by increasing between-host transmission.

Staying alive: The post‐consumption fate of parasite spores and its implications for disease dynamics

Studies on the effects of selective predation by fish on disease dynamics in Daphnia generally assume that consumption by fish means the death of the parasite. I use a combination of feeding trials and infection assays to test this assumption using the host Daphnia dentifera and its common, virulent yeast parasite, Metschnikowia bicuspidata. Approximately 50% of Metschnikowia spores consumed by bluegill sunfish were released in fecal pellets. These spores remained infective to D. dentifera. Therefore, consumption of infected hosts by fish is not necessarily a dead end for the parasite, and in some cases, fish predation may actually help fuel epidemics in natural populations.

Haplosporidian parasite in Diporeia spp. amphipods from the Great Lakes region, USA

Light microscope observations of a haplosporidian in Diporeia spp. amphipods from Lakes Michigan and Huron, USA, found that the parasite spore is operculate and measures 8.1 microm in length and 6.1 microm in width. Round to amorphous sporocysts averaging 23.6 microm were present throughout hemal sinuses of infected amphipods. The number of developing spores within a sporocyst was highly variable and spores were observed in various stages of development. Electron microscopy revealed a lid over the operculum of developing spores and documented spores with fairly large and extensive ornamentation. This is the first haplosporidian reported in Diporeia spp. amphipods and only the second haplosporidian from freshwater species.

The Unique Adaptation of the Life Cycle of the Coelomic Gregarine Diplauxis hatti to its Host Perinereis cultrifera (Annelida, Polychaeta): an Experimental and Ultrastructural Study

The coelomic gregarine Diplauxis hatti exhibits a unique adaptation of its life cycle to its polychaete host Perinereis cultrifera. Experimental and ultrastructural observations on natural populations from the English Channel showed that release of parasite spores is concomitant with the polychaete spawning. As the development of P. cultrifera is direct, the notochete larva ingest parts of the jelly coat covered with numerous sporocysts of D. hatti during hatching. Transepithelial migration of the sporozoites takes place in the gut of three- or four-segment notochete larvae and syzygies of about 20 microm are observed in the coelom. Growth of these young syzygies is slow: after 18-24 mo they reach only 60-70 microm. They exhibit active pendular movements. In the English Channel, female and male gametogenesis of P. cultrifera begins at 19 mo and 2 yr, respectively; the somatic transformations (epitoky) in the last 4 mo of their 3-year life. During epitoky, the syzygies undergo an impressive growth and reach 700-800 microm within a few weeks. A shift from pendular to active peristaltic motility is observed when the syzygies reach 200-250 microm. When gamogony occurs, syncytial nuclear divisions are initiated and cellularization produces hundred to thousands of male and female gametes of similar size. The male gametes exhibit a flagellum with 3+0 axoneme. The mixing of the gametes ("danse des gametes") and fertilization are observed during 4-5 h. Zygotes differentiate sporoblasts with eight sporozoites. The sporozoites exhibit the canonical structure of Apicomplexa, a polarized cell with micronemes and rhoptries.

Spore ornamentation of Minchinia occulta n. sp. (Haplosporidia) in rock oysters Saccostrea cuccullata (Born, 1778)

A Minchinia sp. (Haplosporidia: Haplosporidiidae) parasite was identified infecting rock oysters and morphologically described by Hine and Thorne (2002) using light microscopy and transmission electron microscopy (TEM). The parasite was associated with up to 80% mortality in the host species and it is suspected that the parasite would be a major impediment to the development of a tropical rock oyster aquaculture industry in northern Western Australia. However, attempts to identify the parasite following the development of a specific probe for Haplosporidium nelsoni were unsuccessful. The SSU region of the parasite's rRNA gene was later characterized in our laboratory and an in situ hybridization assay for the parasite was developed. This study names the parasite as Minchinia occulta n sp. and morphologically describes the parasite using histology, scanning electron microscopy and transmission electron microscopy. The non-spore stages were unusual in that they consisted primarily of uninucleate stages reminiscent of Bonamia spp. The parasite's spores were ovoid to circular shaped and measured 4.5 microm-5.0 microm x 3.5-4.1 microm in size. The nucleus of the sporoplasm measured 1.5-2.3 microm and was centrally located. The spores were covered in a branching network of microtubule-like structures that may degrade as the spore matures.

THE EFFECTS OF MULTIPLE INFECTIONS ON THE EXPRESSION AND EVOLUTION OF VIRULENCE IN ADAPHNIA‐ENDOPARASITE SYSTEM

Multiple infections of a host by different strains of the same microparasite are common in nature. Although numerous models have been developed in an attempt to predict the evolutionary effects of intrahost competition, tests of the assumptions of these models are rare and the outcome is diverse. In the present study we examined the outcome of mixed-isolate infections in individual hosts, using a single clone of the waterflea Daphnia magna and three isolates of its semelparous endoparasite Pasteuria ramosa. We exposed individual Daphnia to single- and mixed-isolate infection treatments, both simultaneously and sequentially. Virulence was assessed by monitoring host mortality and fecundity, and parasite spore production was used as a measure of parasite fitness. Consistent with most assumptions, in multiply infected hosts we found that the virulence of mixed infections resembled that of the more virulent competitor, both in simultaneous multiple infections and in sequential multiple infections in which the virulent isolate was first to infect. The more virulent competitor also produced the vast majority of transmission stages. Only when the less virulent isolate was first to infect, the intrahost contest resembled scramble competition, whereby both isolates suffered by producing fewer transmission stages. Surprisingly, mixed-isolate infections resulted in lower fecundity-costs for the hosts, suggesting that parasite competition comes with an advantage for the host relative to single infections. Finally, spore production correlated positively with time-to-host-death. Thus, early-killing of more competitive isolates produces less transmission stages than less virulent, inferior isolates. Our results are consistent with the idea that less virulent parasite lines may be replaced by more virulent strains under conditions with high rates of multiple infections.

NONADDITIVE GENETIC COMPONENTS IN RESISTANCE OF THE RED FLOUR BEETLETRIBOLIUM CASTANAEUMAGAINST PARASITE INFECTION

Genetically coupled antagonistic coevolution between host and parasites can select for the maintenance of recombination in the host. Mechanistically, maintenance of recombination relies on epistatic interactions between resistance genes creating linkage disequilibria (LD). The role of epistasis in host resistance traits is however only partly understood. Therefore, we applied the joint scaling principle to assess epistasis and other nonadditive genetic components of two resistance traits, survival, and parasite spore load, in population crosses of the red flour beetle Tribolium castanaeum under infections with the microsporidian Nosema whitei. We found nonadditive components only in infected populations but not in control populations. The genetic architecture underlying survival under parasite infection was more complex than that of spore load. Accordingly, the observed negative correlation between survival and spore load was mainly based on a correlation between shared additive components. Breakdown of resistance was especially strong in F2 crosses between resistant lines indicating that multiple epistatic routes can lead to the same adaptation. In general, the wide range of nonoverlapping genetic components between crosses indicated that parasite resistance in T. castanaeum can be understood as a multi peaked fitness landscape with epistasis contributing substantially to phenotypic differentiation in resistance.

Experimental evolution of field populations of Daphnia magna in response to parasite treatment

Although there is little doubt that hosts evolve to reduce parasite damage, little is known about the evolutionary time scale on which host populations may adapt under natural conditions. Here we study the effects of selection by the microsporidian parasite Octosporea bayeri on populations of Daphnia magna. In a field study, we infected replicated populations of D. magna with the parasite, leaving control populations uninfected. After two summer seasons of experimental evolution (about 15 generations), the genetic composition of infected host populations differed significantly from the control populations. Experiments revealed that hosts from the populations that had evolved with the parasite had lower mortality on exposure to parasite spores and a higher competitive ability than hosts that had evolved without the parasite. In contrast, the susceptibility of the two treatment groups to another parasite, the bacterium Pasteuria ramosa, which was not present during experimental evolution of the populations, did not differ. Fitness assays in the absence of parasites revealed a higher fitness for the control populations, but only under low population density with high resource availability. Overall, our results show that, under natural conditions, Daphnia populations are able to adapt rapidly to the prevailing conditions and that this evolutionary change is specific to the environment.

Male-biased sex-ratio distortion caused by Octosporea bayeri, a vertically and horizontally-transmitted parasite of Daphnia magna

Female-biased sex-ratio distortion is often observed in hosts infected with vertically-transmitted microsporidian parasites. This bias is assumed to benefit the spread of the parasite, because male offspring usually do not transmit the parasite further. The present study reports on sex-ratio distortion in a host–parasite system with both horizontal and vertical parasite transmission: the microsporidium Octosporea bayeri and its host, the planktonic cladoceran Daphnia magna. In laboratory and field experiments, we found an overall higher proportion of male offspring in infected than in uninfected hosts. In young males, there was no parasite effect on sperm production, but, later in life, infected males produced significantly less sperm than uninfected controls. This shows that infected males are fertile. As males are unlikely to transmit the parasite vertically, an increase in male production could be advantageous to the host during phases of sexual reproduction, because infected mothers may obtain uninfected grandchildren through their sons. Life-table experiments showed that, overall, sons harboured more parasite spores than their sisters, although they reached a smaller body size and died earlier. Male production may thus be beneficial for the parasite when horizontal transmission has a large pay-off as males may contribute more effectively to parasite spread than females.

Relationship between Marteilioides chungmuensis infection and reproduction in the Pacific oyster, Crassostrea gigas

Marteilioides chungmuensis, a protozoan paramyxean parasite, infects the oocytes of the Pacific oyster, Crassostrea gigas. The effects of infection on the reproductive cycle of C. gigas were investigated over two consecutive years at Okayama Prefecture, Japan. In male oysters, gonadal development began during February/March, maturity was achieved in June and spawning activity extended from July to September. In November and December, male oysters were not seen, probably because their gonads regressed to connective tissue and they transformed into undifferentiated oysters. By contrast, female oysters, in which parasite spore formation occurred, were still carrying oocytes until the following March and the spawning process of female oysters took 5 months longer than that of males in epizootic areas. The prevalence of M. chungmuensis infection increased from July to September, when most female oysters had their spawning period, and declined from October to the following April when oysters were at the spent stage. The prevalence of infection increased again in May of the following year and high prevalence was observed in the following July. When prevalence was compared between oysters of different age classes, higher prevalence was detected in older than in younger oysters. Histological examination showed that infected oysters produced oocytes continuously and spawned repeatedly from October to March, during which period healthy oysters were reproductively inactive. Parasites can infect the oocytes of infected oysters throughout the longer spawning period. These observations suggest that M. chungmuensis extends the reproductive period of infected oysters for its own reproductive benefit.

Molecular characterisation of a haplosporidian parasite infecting rock oysters Saccostrea cuccullata in north Western Australia

A haplosporidian parasite was identified in rock oysters ( Saccostrea cuccullata Born, 1778) from the Montebello Islands (latitude −20.4′S longitude 115.53′E) off the northern coast of Western Australia by histopathological examination, PCR amplification and DNA sequencing of a segment of the SSU region of the parasite’s rRNA gene. An oligonucleotide probe was constructed from the parasite’s SSU rRNA gene in order to confirm its presence by in situ hybridisation. The parasite was disseminated throughout the gonad follicles of the host and to a lesser extent in the gills. The only parasite life stages thus far observed in this study were a uninucleate naked cell assumed to be a precursor to multinucleate plasmodial stages and a binucleate plasmodial stage. Whilst no parasite spores were detected in affected rock oysters, a phylogenetic analysis of the SSU region of the parasite’s rRNA gene indicates the parasite belongs to the genus Minchinia. A PCR and in situ hybridisation assay for the Minchinia sp. was used to identify haplosporidians described by Hine and Thorne [Hine, P.M.., Thorne, T., 2002. Haplosporidium sp. (Haplosporidia: Haplosporidiidae) associated with mortalities among rock oysters Saccostrea cuccullata in north Western Australia. Dis. Aquat. Organ. 51, 123–13], in archived rock oyster tissues from the same coastline.

Application of a real-time PCR assay to detect and quantify the myxozoan parasite Ceratomyxa shasta in river water samples

Ceratomyxa shasta is a virulent pathogen of salmonid fishes that is enzootic in the Pacific Northwest of North America. Current parasite detection methods involve sentinel fish exposures that are laborious and time-consuming. As a substitute, a filtering protocol and a quantitative real-time TaqMan polymerase chain reaction (QPCR) assay were developed to detect and enumerate parasite spores in river water. Fluorescence was detected from both the myxospore and actinospore stages of the parasite but not from the fish or polychaete hosts or from 9 other myxozoans tested. Less than 1/1000th of a spore was detected, indicating each had >1000 copies of the target 18S rRNA gene. The assay detected 1 spore in 1 l river water. Inhibition of the assay by some river samples was overcome by reducing the template volume and including bovine serum albumin in the reaction; occasionally a second purification step was required. The QPCR methodology was utilised to investigate the temporal and spatial distribution of C. shasta in the Klamath River, Oregon/ California. The parasite was detected throughout the river, and 2 of 5 tributaries tested contributed parasites to the mainstem. Correlation of QPCR cycle threshold values with a standard curve for known starting numbers of whole spores revealed several sites where parasite abundance was in excess of 20 spores l(-1). Although QPCR data corroborated results of sentinel fish exposures, spore numbers did not correlate consistently with mortality in the exposure groups. The water sampling and filtering protocol combined with the QPCR assay is a simple and relatively rapid method for detection and quantification of parasite levels in environmental water samples.

Empirical Support for Optimal Virulence in a Castrating Parasite

The trade-off hypothesis for the evolution of virulence predicts that parasite transmission stage production and host exploitation are balanced such that lifetime transmission success (LTS) is maximised. However, the experimental evidence for this prediction is weak, mainly because LTS, which indicates parasite fitness, has been difficult to measure. For castrating parasites, this simple model has been modified to take into account that parasites convert host reproductive resources into transmission stages. Parasites that kill the host too early will hardly benefit from these resources, while postponing the killing of the host results in diminished returns. As predicted from optimality models, a parasite inducing castration should therefore castrate early, but show intermediate levels of virulence, where virulence is measured as time to host killing. We studied virulence in an experimental system where a bacterial parasite castrates its host and produces spores that are not released until after host death. This permits estimating the LTS of the parasite, which can then be related to its virulence. We exposed replicate individual Daphnia magna (Crustacea) of one host clone to the same amount of bacterial spores and followed individuals until their death. We found that the parasite shows strong variation in the time to kill its host and that transmission stage production peaks at an intermediate level of virulence. A further experiment tested for the genetic basis of variation in virulence by comparing survival curves of daphniids infected with parasite spores obtained from early killing versus late killing infections. Hosts infected with early killer spores had a significantly higher death rate as compared to those infected with late killers, indicating that variation in time to death was at least in part caused by genetic differences among parasites. We speculate that the clear peak in lifetime reproductive success at intermediate killing times may be caused by the exceptionally strong physiological trade-off between host and parasite reproduction. This is the first experimental study to demonstrate that the production of propagules is highest at intermediate levels of virulence and that parasite genetic variability is available to drive the evolution of virulence in this system.

A NEW MYXOZOAN FROM FERAL GOLDFISH (CARASSIUS AURATUS)

In February 2004, a mass die-off of common goldfish Carassius auratus L., presumptively caused by bacterial coldwater disease (Flavobacterium psychrophilum), occurred at Fern Ridge Reservoir, Oregon. A range of size classes was affected, but all mature fish were female and all fish were infected with a single myxozoan, Chloromyxum auratum n. sp. No histological changes were observed associated with the parasite. Infection was represented by mictosporic plasmodia and free-floating spores in the gall bladder. Parasite spores were nearly spherical, 13.6 microm long x 12.6 microm wide x 13.1 microm thick, and possessed 4 equal-sized polar capsules. Spores had a coglike appearance in apical view because of distinct ridges 2.1 microm high protruding from the valve cells. There were 6-9 extrasutural ridges per valve (15-20 ridges per spore), aligned along the longitudinal axis, with some branching, and convergence at both poles. Morphologically, spores identified most closely with Chloromyxum cristatum Léger, 1906; however, 18S rDNA sequence data indicated only 97.5% similarity over 2,076 bp with Chloromyxum cyprini, the only synonym of C. cristatum for which DNA data are available; additional sequence data may reveal the other synonyms to be distinct species. This is the first record of a species of Chloromyxum from goldfish.

Assessing the accuracy of a polymerase chain reaction test for Ichthyophonus hoferi in Yukon River Chinook salmon Oncorhynchus tshawytscha

Ichthyophonus hoferi Plehn & Mulsow, 1911, is a cosmopolitan, protistan pathogen of marine fishes. It is prevalent in mature returning Chinook salmon Oncorhynchus tshawytscha in the Yukon River watershed, and may be associated with prespawning mortality. We developed and evaluated a polymerase chain reaction (PCR) test for I. hoferi using primers specific to the parasite's small subunit rDNA. The test has a minimum detection limit of approximately 10(-5) parasite spores per reaction and does not cross-react with the closely related salmon parasites Dermocystidium salmonis or Sphaerothecum destruens. Sensitivity and specificity of the PCR test used on somatic muscle and heart tissue for detecting infected fish were determined using 334 Chinook salmon collected from the Yukon River at 2 locations (Tanana and Emmonak) in 2003 and 2004. The true infection status of the fish was determined by testing somatic muscle, heart and kidney tissue using histological evaluation, culture, and PCR. The severity of infection was grouped into 2 categories, light and heavy infection. The probability of detecting a heavily infected fish (sensitivity of the test) was generally much higher than the probability of detecting light infection, suggesting that more than one tissue and/or method should be used to accurately detect light or early infection by I. hoferi. The probability of correctly identifying a negative fish (specificity of the test) was always greater than 94% regardless of the tissue used, infection severity, sampling site or year of collection.

Apical Spore Phagocytosis Is Not a Significant Route of Infection of Differentiated Enterocytes by Encephalitozoon intestinalis

Encephalitozoon intestinalis is a microsporidian species that infects the intestinal mucosal epithelium, primarily in immunodeficient individuals. The present study employed undifferentiated and differentiated human colonic carcinoma cell lines to determine if this parasite species infected polarized epithelial cells by spore phagocytosis or by impalement with the deployed spore polar tube. Apical surface spore attachment differed between cell lines such that SW480>HT-29>Caco-2>HCT-8, with attachment being greater to undifferentiated Caco-2 cells than differentiated cells and greater to partially differentiated HCT-8 cells than differentiated HCT-8 cells. Attachment was inhibited by chondroitin sulfate A, suggesting that it was mediated by host cell sulfated glycoaminoglycans. Infection rates 3 days postinfection paralleled spore attachment in the various cell lines. The undifferentiated cell line SW480 and undifferentiated Caco-2 and HCT-8 cells exhibited modest spore phagocytosis while the more differentiated cell line HT29 and differentiated Caco-2 and HCT-8 cells did not. All cell lines were impaled by the polar tubes of germinating spores. When normalized to the number of spores attached to the apical membrane, such impalement was greatest in the more differentiated Caco-2 and HCT-8 cells. The host cell apical surface influenced parasite spore germination, as in populations of large undifferentiated Caco-2 cells to which >3 spores had attached, the frequency distribution of the percentages of spores germinated per cell was bimodal, indicating that the surface of some cells favored germination, while others did not. This study suggests that phagocytosis is not a biologically significant mode of infection in differentiated intestinal epithelial cells.