Comparative cytotoxicity of secondary hydatid cysts, protoscoleces, and in vitro developed microcysts of Echinococcus granulosus.

Infection with the metacestode of Echinococcus granulosus is characterized by a concomitant immunity. Survival of established and developing hydatid cysts in the intermediate host implies a mechanism to modulate its immunological reactions. In order to investigate this mechanism, secondary hydatid cysts were isolated from intraperitoneally infected laboratory white mice (strain NMRI) 12 months p.i. A number of hydatid cysts were freed from the surrounding host adventitial tissue. Monolayer cultures of non-stimulated peritoneal macrophages of NMRI mice were prepared and incubated in the presence of the hydatid cysts. By means of a trypan blue exclusion test and by measuring the incorporation of tritium labelled uridine, it was found that the presence of hydatid cysts reduced the viability of the macrophages in vitro. Toxic substances are probably secreted since the medium of cultured hydatid cysts also displayed cytotoxic activity. Hydatid cysts with adventitia, as well as culture medium of those cysts, were less toxic. When toxins, partially purified from hydatid cyst fluid, were previously incubated on a collagen coated surface, a reduced level of toxicity was found, suggesting that collagen of the host adventitia may play a role in controlling the liberation of toxins by the hydatid cyst. Virtually no toxicity was exerted by protoscoleces or by the medium of cultured protoscoleces, in contrast to in vitro vesiculated protoscoleces (so called microcysts). The results reveal a novel feature of hydatid cysts that may play a role in the survival of the parasite in the immunized host.     

An improved method of isolating Pneumocystis carinii from infected rat lungs

A new method of isolating Pneumocystis carinii from infected lungs of cortisonized rats is described. Clumping of parasites and host lung material was diminished by suspension of macerated Pneumocystis-laden rat lung in a modified calcium, magnesium-free Hanks' balanced salts solution at physiologic pH and osmolality, containing the wetting agent G-acid. After washing, this material was suspended in a second buffer system for digestion. The digestion step was done in the same buffer but with the addition of calcium, magnesium, collagenase, hyaluronidase and deoxyribonuclease. These innovations allowed enumeration of trophozoites as well as cysts. Following digestion, the parasites were separated from particulate host lung debris by Percoll density gradients designed to pellet the debris, leaving parasites in the gradient. Density studies done prior to this step revealed that trophozoites and non-nucleated cysts had similar densities, 1.028 g/ml, whereas nucleated cysts were heavier at 1.030 g/ml. Particulate host lung debris could be removed due to its heavier density, 1.040 g/ml. The significance of this study includes: relatively clump-free suspensions of infected rat lung, enumeration of trophozoites as well as cysts, and characterization of nucleated cysts.     

Substratum preference of Philophthalmus sp. cercariae for cyst formation under natural and experimental conditions

Selection on parasites should favor adaptations that maximize the probability of transmission to the definitive host, such as the preference for and use of intermediate hosts or encystment substrata that are likely to be consumed by the definitive host. Eye flukes in the genus Philophthalmus are passed to their definitive avian host through the ingestion of metacercariae encysted on hard substrata. The life cycle of these parasites is generally well understood; however, there is almost no information on substratum use or preference of the cercariae of these parasites. In this study, we combine a survey of naturally occurring substrata with experimental, laboratory-based choice tests to determine the preferred substratum of Philophthalmus sp. and whether this preference is affected by the presence and density of pre-existing cysts. A concordance between natural and experimental data show a preference for the shells of multiple species of snail over other hard substrata that are common at the field site, including seaweed, other molluscs, and crustaceans. In addition, we found that cercariae preferred substrata with pre-existing cysts and that this preference seemed to increase with increasing cyst density. Such a preference should lead to an aggregated distribution of cysts among snail shells that may benefit the parasite by increasing the number of potential mates that become established in the definitive host. The identification of a preferred substratum also may help to identify potential definitive hosts that were previously unknown.     

Genetic differences between cysts of Echinococcus granulosus from the same host

Isozyme differences were found between protoscoleces derived from different cysts in three sheep and three macropod marsupials. Isozymes were interpreted as the products of different alleles at corresponding enzyme loci, indicating that the same host may contain cysts derived from genetically different embryos. Genetic variation on this scale may cause confusion in epidemiological studies, if protoscoleces from several cysts are pooled prior to strain-typing.     

Of mudsnails,terrapins, and flukes: Use of trematodes as a field‐based project in parasitology research

We present an exercise for counting trematode cysts on mudsnails that can be implemented as a field‐based project in course work by students or by volunteers. The exercise involves the digenetic trematode Pleurogonius malaclemys, which infects diamondback terrapins (Malaclemys terrapin) as its definitive host, and eastern mudsnails (Tritia obsoleta) as its intermediate host. The trematode forms macroscopic metacercarial cysts on the shells and opercula of the mudsnails, and the life cycle is completed when terrapins ingest the intermediate hosts with cysts. Previous research has suggested that quantifying cysts on mudsnails can be a relatively simple method to estimate terrapin population sizes where terrapins co‐occur with mudsnails. Three non‐professional scientist groups (including students and volunteers) were tested in their estimates of prevalence and intensity data compared to verified cyst counts. Prevalence data were relatively accurate (~76–78%) and reliable (68–89%) across the groups. The intensity of cysts (on shells and opercula of mudsnails) were mostly within 12% of the verified counts (total mean intensity of 1.81–2.97 cysts/parasitized mudsnail) but varied significantly in two cases due to false positives (likely due to sand grains or debris scored as cysts) or negatives (likely due to missed cysts behind the operculum). The exercise can be completed as a lab or series of labs in courses, and data from such projects could be used in terrapin conservation efforts, particularly in helping scientists determine where terrapins occur. We discuss modification of the exercise for other trematode species in regions around the world and hope this expands recognition by the public of the important role of parasites in ecosystems.     

Proteomic characterisation of Echinococcus granulosus hydatid cyst fluid from sheep, cattle and humans

The hydatid cyst fluid (HCF) of Echinococcus granulosus is a complex biological mixture containing a wide range of proteins of both parasite and host origin. Using a combination of in- and off-gel protein fractionation techniques and tandem mass spectrometry 130 HCF proteins were identified from fertile cysts of sheep and human origin and infertile cysts from cattle. Forty-eight proteins were of parasite origin including Antigen 5 and Antigen B--the most abundant parasite proteins, thioredoxin, low-density lipoprotein receptors, cyclophilin and ferritin. Across the three host species the identified HCF proteins were broadly similar although, based on spectral counts, three proteins, including an antigen B isoform, were more abundant in sheep HCF compared with the fluids of cattle and human origin. Eighty-two host proteins were identified in HCF from the three species. Host plasma proteins were the most abundant, although approximately thirty of the host proteins that were identified are not considered constituents of plasma. The identification of parasite heat shock proteins and annexin A13 exclusively in infertile cysts, along with an increased spectral count for cathepsin B, supports the hypothesis of increased cellular stress and apoptosis as the cause of their infertility.     

鳙鳃上台湾棘带吸虫囊蚴的感染

从1993年8月到1994年11月,对感染台湾棘带吸虫囊蚴的鳙鳃丝、鳃耙进行了为期16个月的调查研究表明;鳃丝、鳃耙的感染率范围为0-100%(83.33±25.47%)、(61.13±44.45%),感染强度范围分别为0-109.15(54.89±34.09)、0-125.57(41.18±49.34),囊蚴的平均密度范围分别为0-97.76(46.94±26.25),0-125.57(40.61±49.71);当鳙体长小于90mm时,鳃丝、鳃耙的感染率、感染强度、囊蚴的平均密度随着其体长的增大而增长;当体长大于90mm时,三项指标随着其体长的增大具有减小的趋势,其中鳃耙的变化规律明显;当体长大于141.7mm时,鳃耙的感染率、感染强度、囊蚴的平均密度都为0;随着鳙的生长,鳃丝囊蚴所占的比例逐渐增大,从0增加到100%,而鳃耙囊蚴所占的比例逐渐减小,从100%下降到0.       

Some histochemical observations on the effects of chorioallantoic grafts on the spleen, bursa, and peripheral blood of chicken embryos

Hatching eggs from inbred lines of chickens (inbreeding coefficient exceeds 95%) which show various degrees of resistance and susceptibility to Rous sarcoma, were used for experimentation. Adult tissues were grafted onto the chorioallantois on the tenth day of incubation and tissues of host and control embryos were harvested on the twentieth day of incubation. Enzymes were localized in tissues by histochemical procedures. Small pieces of tissue (thymus or bursa), when grafted onto the chorioallantois, increased the size of the spleen in host embryos although splenomegaly did not invariably occur. Two types of reactions were observed in the spleen, i.e., enlarged spleens with cysts or enlarged spleens which from a morphological point of view were normal. Grafts of either thymus or bursa decreased the size of the host embryo's bursa or were without effect. When weight of the bursa of host embryos was significantly less than that of control embryos on the twentieth day of incubation, this size relationship persisted in chicks four weeks post hatching. Intensity of dehydrogenase and acid phosphatase reactions in cysts of enlarged spleens and in the multinucleated giant cells investing them suggests that they consist of groups of degenerating cells. Intensity of enzyme reaction indicates that enlarged spleens of host embryos in which cysts were absent were normal. Enzyme reactions in the bursae of experimental embryos were more intense than those identified in the same tissues of control embryos. Catabolic reactions were the predominant type in grafts ten days subsequent to implantation. Grafts increased the number of erythrocytes in the peripheral blood of host embryos.     

Sexual, seasonal and tissue variation in the encystment of Cotylurus variegatus metacercariae in perch, Perca fluviatilis

All of the 267 perch sampled from Lough Neagh between 1981 and 1983 were infected with the metacercarial cysts of Cotylurus variegatus. Sites of infection were the swim-bladder, pericardium, septum transversum and, to a lesser extent, the visceral cavity. The swim-bladder, particularly the anterior portion, was the site of heaviest infection. Visceral cysts were found chiefly in female fish and this may be related to reduced immunological defence and/or thinner body wall during the breeding season. The number of cysts recorded was not related to host length, suggesting that further parasite invasion is offset by cyst mortality possibly as a result of the host immunological response to the parasite. Highest mean worm burden was recorded between May and June. This corresponded to increased water temperature necessary for development of eggs and the breeding season (April-June) of the perch.     

Spanning the gap: experimental determination of paratenic host specificity of horsehair worms (Nematomorpha: Gordiida)

Abstract. Details of the life cycle of freshwater nematomorphs (gordiids) remain unclear. Free-living aquatic larval gordiids must make a critical transition from an epibenthic aquatic environment to terrestrial hosts. In order to identify potential hosts capable of bridging this ecological gap, the specificity of paratenic hosts of three common species of North American gordiids was investigated. All three species were characterized by an identical infection pattern: low host specificity. Gordiids were able to encyst within annelids, mollusks, crustaceans, insects and a vertebrate. Three species of putative host (a turbellarian, a water mite, and a mosquito larva) were not infected with any of the gordiid species. Internal defense reactions (IDR) and feeding behaviors are implicated as preventing infection in these species. Several of the other host species produced either an IDR or an immune reaction to the cysts, although reactions to the cysts were highly variable between species. In most species, IDR did not cause noticeable harm to the encysted larvae. It is proposed that although many species are easily infected with gordiid cysts, most do not act as natural paratenic hosts. For some of these host groups, especially snails, a role as reservoir host is suggested. Of all hosts included in this study, aquatic insects were identified as the hosts likely responsible for spanning the ecological gap and acting as true hosts for gordiids.     

Chronic Toxoplasma infection modifies the structure and the risk of host behavior

The intracellular parasite Toxoplasma has an indirect life cycle, in which felids are the definitive host. It has been suggested that this parasite developed mechanisms for enhancing its transmission rate to felids by inducing behavioral modifications in the intermediate rodent host. For example, Toxoplasma-infected rodents display a reduction in the innate fear of predator odor. However, animals with Toxoplasma infection acquired in the wild are more often caught in traps, suggesting that there are manipulations of intermediate host behavior beyond those that increase predation by felids. We investigated the behavioral modifications of Toxoplasma-infected mice in environments with exposed versus non-exposed areas, and found that chronically infected mice with brain cysts display a plethora of behavioral alterations. Using principal component analysis, we discovered that most of the behavioral differences observed in cyst-containing animals reflected changes in the microstructure of exploratory behavior and risk/unconditioned fear. We next examined whether these behavioral changes were related to the presence and distribution of parasitic cysts in the brain of chronically infected mice. We found no strong cyst tropism for any particular brain area but found that the distribution of Toxoplasma cysts in the brain of infected animals was not random, and that particular combinations of cyst localizations changed risk/unconditioned fear in the host. These results suggest that brain cysts in animals chronically infected with Toxoplasma alter the fine structure of exploratory behavior and risk/unconditioned fear, which may result in greater capture probability of infected rodents. These data also raise the possibility that selective pressures acted on Toxoplasma to broaden its transmission between intermediate predator hosts, in addition to felid definitive hosts.     

Observations on the suitability and importance of the domestic intermediate hosts of Echinococcus granulosus in Uttah Pradesh, India

The present study investigated the suitability and importance of buffaloes, camels, sheep, goats and pigs in maintaining the life-cycle of Echinococcus granulosus in Aligarh, India. A total of 565 (36%) of 1556 buffaloes, 20 (2%) of 1208 goats, 5 (1%) of 559 pigs, 6 (6%) of 109 sheep and two of three camels were found to harbour hydatid cysts. The frequency distribution of the hydatid cysts in each intermediate host species was over-dispersed and in buffaloes cyst fertility increased with increasing cyst size. Of 2171, 95 and four buffalo, goat, and camel cysts examined 327 (15%), two (2%) and three cysts respectively were fertile. No pig or sheep cysts were found to contain protoscoleces. The unfenced buffalo abattoir and the large number of dogs allowed access to the abattoir coupled to the number of buffalo slaughtered in comparison to the other potential hosts, indicates that the buffalo is the most significant host for maintaining the life-cycle of the parasite in this area of India. Applicable control measures for the region are suggested.     

Sarcocystis hemionilatrantis (Sp. N.) life cycle in mule deer and coyotes.

Fifteen coyotes (Canis latrans) shed sporulated sporocysts in their feces after eating freshly ground skeletal muscles from a mule deer (Odocoileus hemionus hemionus) infected with microscopic-sized cysts of Sarcocystis. Sporocysts were shed intermittently from 12 to 36 days after ingestion of the infected meat. Sporocyst size averaged 14.4 X 9.3 mum. Eleven mule deer fawns orally inoculated with these sporocysts became infected and 9 of 11 died between post-inoculation days (PID) 27 and 63. Clinical signs of anorexia, weight loss, pyrexia and weakness were evident prior to death. A calf (Bos taurus) and two lambs (Ovis aries) orally inoculated with these sporocysts did not become infected and remained healthy throughout the experiments. Similarly, uninoculated control animals consisting of three mule deer fawns, two lambs and one calf remained healthy during the experiment. Preliminary histologic examinations conducted on selected tissues from all animals revealed microscopic-sized schizogonous stages in macrophages, between muscle fibers and near blood vessels in the esophagus, heart, biceps femoris, semi-membranosus, diaphragm and tongue from seven of eight fawns which died between PID 27 and 39. Developing or mature muscle cysts were not found in fawn tissue until PID 60. Sarcocysts were found in the three infected fawns examined after this time. Muscle cysts or earlier schizont stages were not found in tissues from the inoculated or uninoculated calves and lambs. A single muscle cyst was found in one control fawn; the other two control fawns were negative for both muscle cysts and other schizogonous stages. These results established that the life cycle of this species of Sarcocystis can be completed with coyotes as the definitive host and mule deer as the intermediate host. Based on the demonstrated host specificity and earlier findings, the name Sarcocystis hemionilatrantis is proposed for this parasite of mule deer and coyotes.     

A Dermocystidium infection in Trichomycterus sp. (Osteichthyes, Trichomycteridae)

Two whitish elongate cysts in the left pectoral fin of Trichomycterus sp. (Osteichthyes, Trichomycteridae) were packed with Dermocystidium sp. spores. The spores were spherical and had a large PAS positive central refractile body, the cytoplasm being restricted to a narrow peripheral layer containing the nucleus. The cysts were surrounded by a thin homogeneous wall of parasite origin, and there was no encapsulation of the cysts by host tissue.     

Prevalence,geographic distribution,and fitness effects of Microphallus turgidus (trematoda: Microphallidae) in grass shrimp (Palaemonetes spp.) from coastal Georgia

Grass shrimp, Palaemonetes pugio Holthuis and P. vilgaris (Say), were collected at 11 localities along the coast of Georgia and surveyed for digenetic trematode metacercariae. The effect of trematode infection on grass shrimp fitness was also examined. Microphallus turgidas (Leigh) was the only trematode observed. The prevalence of metacercarial cysts of this parasite in P. pugio (75%) was higher than in P. vulgaris (24%), as were the mean intensity, abundance, and population density (no. cysts/cm host body length) of the parasite. Infected shrimp were found at every collection locality and parasite prevalence and density were greatest in P. pugio from higher salinity localities (> or = 20 parts per thousand [ppt]). There was no relation between host body size and parasite density in P. vulgaris, and parasite density increased with host body size in P. pugio, suggesting that the parasite does not affect host survival. There was no relation between parasite density and shrimp egg mass, but nonovigerous female P. pugio were more heavily infected than ovigerous ones. In addition, 1 metacercaria of M. turgidus in each of 2 specimens of P. vulgaris was parasitized by the haplosporidian Urosporidium crescens De Turk. This represents a new shrimp host record for this hyperparasite.     

Hyperparasitism by Paragordius varius (Nematomorpha: Gordiida) larva of Monostome redia (Trematoda: Digenea)

Gordiid larvae enter and encyst within a large variety of aquatic vertebrates and invertebrates. Cysts of a single species of gordiid have been found in such diverse hosts as insect larvae, snails, crustaceans, fish, and many others. One particularly surprising host report was made nearly 100 yr ago, i.e., cysts within adult trematodes. The origin of these cysts remained unclear, but it was suggested that the gordiid larvae gained entry into the adult flukes via their definitive, vertebrate host. In the present study, the finding is reported of gordiid cysts within monostome redia within a Physa gyrina snail. This is the first time gordiid cysts have been reported within redia, and this provides an alternative explanation for the presence of cysts within adult trematodes.     

Primary infection with mouse-derived cysticercoids of Hymenolepis nana prepared from baby or adult mice and secondary infections with eggs or cysticercoids

Oral infection with mouse-derived cysticercoids (cysts) of Hymenolepis nana on day 0 did not make the 5 ± 1 week old mouse host immune to egg challenge by day 7 of the prepatent period, although the number of cyst-derived tapeworms was 1000 times greater than that of egg-derived tapeworms sufficient to make the host immune by day 7. Neither cysts recovered from immunologically competent 5 ± 1 week old donor mice, which should have become immune within 2 days of egg inoculation, nor those from incompetent 5–7 day-old baby mice given eggs when 0–2 days old made the host immune. The time course of differentiation of cysts in baby mice was not different from that in 5 ± 1 week-old mice. Mice infected twice with cysts on days 0 and 4 did not become immune either. Rapid protective immunity against egg challenge was acquired by inoculation exclusively with eggs but not with cysts. Apparently cysts differ from oncospheres in their immunogenicity. The importance of cysts for analysing the mouse—H. nana system from the immunological point of view is discussed.     

The development of brood capsules and protoscolices in secondary hydatid cysts of Echinococcus granulosus. A histological study.

Using the Mongolian jird (Meriones unguiculatus) as a laboratory host for the cystic stage of the British horse strain of Echinococcus granulosus, the mode of development of brood capsules and protoscolices was studied histologically. The successive stages in development in secondary hydatid cysts are described and shown to correspond closely with previous observations on primary hydatid cysts. The usefulness of this experimental model for future ultrastructural studies is emphasized.     

食蚊鱼鳃上台湾棘带吸虫囊蚴内种群的月变化

1993年7月-1994年6月,作者对食蚊鱼鳃上台湾棘带吸虫囊蚴内种群的变化进行了为期12个月的研究,结果表明:食蚊鱼种群数量和种群重量的月变化相似,但两者与水温没有相关性;食蚊鱼种群的体长结构呈季节性动态变化规律;食蚊鱼的感染与其自身的生长紧密相关,感染率、感染强度表现出了与体长相似的变化趋势;中间的两组鳃片寄生的囊蚴较多,不同鳃片组囊蚴平均密度的月变化规律一致,食蚊鱼任何两个鳃片组分布的台湾棘带吸虫囊蚴的平均数差异都显著(P0.001);囊蚴在食蚊鱼鳃上的分布为聚集分布。       

Culture and filtration methods for obtaining Pneumocystis carinii trophozoites and cysts.

Two methods for acquisition of Pneumocystis carinii (Pc) trophozoites and cysts are reported. One method, the isolation of Pc from infected rat lung, provides large numbers of trophozoites and cysts but retains rat proteins. Ground lung is filtered through a series of Nucleopore filters from 10 to 3 microns; 1 g of rat lung yields an average of 1.1 x 10(9) Pc trophozoites and 1 x 10(7) cysts. The second method, propagation of Pc in culture with human embryonic lung cells on microcarrier beads, provides Pc trophozoites which are relatively free of host lung material. Cultured organisms may be filtered to remove rare culture monolayer cells. Organisms harvested from filtered lung are free from intact host cells and cell nuclei, however, host cell proteins and host DNA remain. Organisms from culture have minimal host contamination.