The ultrastructure of the tegument and the digestive caeca of in vitro cultured metacercariae of Fasciola hepatica

Davies C. 1978. The ultrastructure of the tegument and digestive caeca of in vitro cultured metacercariae of Fasciola hepatica. International Journal for Parasitology8: 197–206. The ultrastructure of the tegument and digestive caeca of metacercariae of Fasciola hepatica grown in vitro in two different media is described and compared with the development of these two systems during maturation in vivo. Although the tegument of metacercariae grown in Medium RC showed no development, that of flukes cultured in Medium CS began to produce T-1 and T-2 granules typical of the liver phase of development in vivo. The gastrodermal cells showed some degree of conversion to an adult-like morphology in vitro with the production of typical secretory granules, a limited amount of orientation of the GER and the development of junctional complexes with adjacent parenchyma cells—this was particularly evident in flukes from Medium CS. The growth achieved in each of the culture media is correlated to the degree of development of the tegument and the digestive caeca.     

Development in vitro of the metacercaria of Bucephaloides gracilescens (Trematoda: Bucephalidae)

The development of Bucephaloides gracilescens metacercariae was studied using a range of cultivation conditions. The most rapid development occurred at 18°C in a medium containing NCTC 135 supplemented with 25% chicken serum, 25% hen egg yolk and 25% hen egg albumen, with a gas phase of air. Under these conditions, shell-protein synthesis was triggered by day 3 in culture; secondary oocytes were apparent in the ovary by day 10; and egg production began by day 14. Survival of worms in media containing chicken serum was twice as long as that achieved with either whiting or angler fish serum. The ingestion of yolk (feeding) appeared to be a necessary prerequisite to development and egg production. The presence of yolk in the culture medium greatly increased the amount of ³H-thymidine incorporated by the reproductive system of freshly excysted metacercariae but had little effect on the uptake and incorporation of tyrosine. The eggs produced in vitro failed to embryonale and were abnormal in appearance, being non-operculate with irregularly thickened shells.     

Trypanosoma musculi: In vitro cultivation of blood forms in cell culture media

Blood forms of Trypanosoma musculi were grown at 37°C in media containing adherent peritoneal cells from normal or from T. musculi immune mice. Rat peritoneal cells and Hela cells supported growth equally well which was surprising in view of the strict mouse-host specificity of this parasite in vivo. Cultures showed a 12- to 46-fold increase in numbers over the inoculum during periods up to 23 days whereas control (cell-free) media did not promote growth, the inoculated organisms merely surviving for about 7 days. Sub-culture through several passages was successful and cultured trypanosomes remained fully infective to mice. Foetal calf serum was an essential constituent of the medium.     

In vivo and in vitro studies of the effects of immune rat serum on Fasciola hepatica

Significant protection against infection with 10 or 30 metacercariae of Fasciola hepatica was conferred on naive rats by the passive transfer of serum derived from rats which had been exposed to primary and challenge infections with 5 or 10 and 30 or 20 metacercariae respectively. Immune serum did not have a pronounced effect on the mortality of metacercariae in vitro. However, its presence was associated with the formation of a precipitate on the tegument of each metacercaria and in the culture medium. The precipitate contained rat antibody and other components, presumably parasite antigens, which elicited the formation of antibody when the precipitate was injected into rats. Viability of metacercariae cultured in immune and normal sera as well as freshly excysted specimens was tested in rats by intraperitoneal infection. Metacercariae cultured in immune serum did not develop. By comparison with the viability of freshly excysted metacercariae, that of some metacercariae cultured in normal serum was impaired; this was attributed to inadequacies in the culture technique. A relationship between precipitate formation in vitro and impaired viability of metacercariae in vivo has yet to be established.     

Excystation and development in the chick and on the chick chorioallantois of the metacercaria of Sphaeridiotrema globulus (Trematoda)

Fried B. and Huffman J. E. 1982. Excystation and development in the chick and on the chick chorioallantois of the metacercaria of Sphaeridiotrema globulus (Trematoda). International Journal for Parasitology12: 427–431. Encysted metacercariae of Sphaeridiotrema globulus were obtained from naturally infected Goniobasis virginica snails in Morris County, New Jersey, U.S.A. These metacercariae were successfully excysted within l h in an alkaline bile salts trypsin medium maintained at 41°C in the absence of acid pepsin pretreatment. Encysted metacercariae treated in 3% sodium bicarbonate produced successful infections in day-old domestic chicks and worms became ovigerous in the lower ileum by day 4. Excysted metacercariae transferred to the chorioallantois of 6–10-day-old chick embryos maintained at 41°C developed into ovigerous adults by day 4.     

Observations on the in vitro culture of Cotylurus erraticus (Trematoda: Strigeidae)

Mtitchell J. S., Halton D. W. and Smyth J. D. 1978. Observations on the in vitro culture of Cotylurus erraticus (Trematoda: Strigeidae). International Journal for Parasitology8: 389–397. Cotylurus erraticus metacercariae obtained from around the heart of rainbow trout were excysted and grown in vitro and in vivo to egg-producing adults. For in vitro development, tissue culture media M199 or NCTC 135 was used, together with varying amounts of chicken serum. Worms grown in media containing the highest concentration of serum (80% per volume) showed the fastest rate of development, measured by the time taken for the first eggs to appear in the uterus. The testes, ovaries and vitellaria of these worms were comparable in structure and histochemistry with those of worms reared in gulls. Eggs were produced by worms in all media containing chicken serum, but the eggs had abnormal shells and failed to embryonate.     

The immunological response of CBA mice to Trypanosoma musculi: mechanisms of protective immunity

CBA mice which had recovered from infection with Trypanosoma musculi were immune to challenge with all strains of the homologous species that were tested but were still full susceptible to challenge with T. cruzi, T. brucei or T. evansi. A heavy challenge inoculum of T. musculi was cleared rapidly from the blood of mice which had recently recovered from infection but, in mice which had recovered 11 months earlier, the parasitaemia changed very little for 3–4 days but then fell abruptly within a few hours. Immunization with a parasite extract in multiple emulsion conferred a strong though not complete protection against homologous challenge.Serum from mice which had recovered from infection had a marked neutralizing effect in vitro on the infectivity of the homologous parasites although the numbers of live organisms were not reduced during the period of in vitro incubation. The test did not reveal antigenic differences among three isolates of the parasite.A summary is given of the sequence of events that is thought to make up the immune response of mice to T. musculi.     

Fasciola hepatica: Simple,large-scale, in vitro excystment of metacercariae and subsequent isolation of juvenile liver flukes

A simple method has been developed for the in vitro excystment of metacercariae of Fasciola hepatica, and for the isolation of large numbers of juvenile liver flukes free from intact metacercariae and from cyst-wall material. In this method, the outer cyst wall was removed by gently grinding the metacercariae between small glass plates. The metacercariae were activated by incubation for 1 hr under $\text{60\%}\text{CO}{}_2\text{40\%}\text{N}{}_2$ and excysted by the addition of 10% sterilized sheep bile or an equivalent amount of taurocholic acid. Excystment was accomplished in an experimental apparatus allowing the newly excysted juveniles to escape from the bile-containing excystment medium into a medium with low bile content. The yield of isolated liver flukes was 60–80%; their protein content was about 125 ng. Both bile and taurocholic acid, though necessary for excystment, were detrimental to the survival of the juvenile liver flukes. The presence of bile in the host intestine may be a stimulus for juveniles to leave the gut and enter the abdominal cavity.     

Vasoprotective activity of standardized Achillea millefolium extract

We investigated the effects of Achillea millefolium extract in vitro on the growth of primary rat vascular smooth muscle cells (VSMCs) as well as the potential involvement of estrogen receptors (ERs) in this process. In addition, the ability of A. millefolium extract to modulate the NF-κB pathway was tested in human umbilical vein endothelial cells (HUVECs). The fingerprinting of the extract was carried out by HPLC-DAD and LC-MSⁿ and main constituents were flavonoids (10%) and dicaffeolylquinic acid derivatives (12%). The extract enhanced VSMC growth at least in part by acting through ERs and impaired NF-κB signaling in HUVECs. The various compounds may act with different mode of actions thus contributing to the final effect of the extract. Our findings support some of the traditional uses of A. millefolium, and suggest potential modes of action as related to its effects on vascular inflammation. Therefore, A. millefolium may induce novel potential actions in the cardiovascular system.     

Expression of messenger RNAs for insulin-like growth factors and their receptors in bovine fetuses at early gestation from embryos produced in vivo or in vitro

The objective of this study was to determine the effects of in vitro embryo production on physical development and levels of expression of mRNAs for insulin-like growth factor (IGF) ligands (IGF1, IGF2), their receptors (IGF1R, IGF2R), and IGF binding protein-2 (IGFBP2) in bovine fetuses during early gestation. In vivo embryos were recovered from superovulated Holstein cows. For production of embryos in vitro, Holstein oocytes were matured, fertilized, and subsequently cultured in M199 with 10% serum to 168 hpi. On Day 70 of gestation, fetuses (in vivo, n = 14; in vitro, n = 13) were recovered, serum samples collected, and physical measurements recorded. Semi-quantitative RT-PCR assays were used to determine the levels of expression of mRNAs for IGF1, IGF2, IGF1R, and IGF2R in fetal liver and skeletal muscle. Western blots were used to assess levels of IGFBP2 in fetal serum. Fetal body weight did not differ with treatment; however, production of embryos in vitro was associated with decreased crown-nose length and a tendency for increased paired kidney weight, which became significant when expressed on a per bodyweight basis. There was no effect of treatment on levels of IGFBP2 in fetal serum. Levels of IGF1 mRNA in fetal liver were decreased (P < 0.001) in the in vitro group. Levels of IGF2R mRNA in both liver and skeletal muscle were also decreased (P < 0.01) in fetuses from the in vitro group. In summary, fetuses at Day 70 of gestation from embryos produced in vitro had shortened crown-nose length and increased kidney weight on a per bodyweight basis, as well as decreased expression of mRNAs for IGF1 in liver and IGF2R in both liver and skeletal muscle, compared with fetuses from embryos produced in vivo. In conclusion, in vitro embryo culture was associated with subtle changes in fetal development as well as altered expression of both imprinted and non-imprinted genes.     

Toxocara canis: Potential activity of natural products against second-stage larvae in vitro and in vivo

The anthelmintic activity of extracts from Chenopodiumambrosioides, Pycnanthusangolensis and Nutridesintox® was in vitro and in vivo investigated, against Toxocaracanis larvae. The in vitro assays results showed that the aqueous extract of Nutridesintox® was the most effective, followed by C. ambrosioides extracts, hexane, dichloromethane and the infusion. P. angolensis extracts showed a lower anthelmintic activity compared to the other natural products. For the in vivo assays, Nutridesintox®, the hexane extract and the infusion of C. ambrosioides were administered orally to T. canis-infected mice, in single doses, during three consecutive days. The efficacy was evaluated on the 17th day post-infection, not only by counting T. canis larvae in the tissues but also by ELISA detection of IgM and IgG antibodies and histological analysis of liver and lungs. The different treatments did not reduce the larvae burden and had no influence on the antibodies dynamic. Interestingly, a reduction on the inflammatory infiltrates was observed in the liver and lung sections of the group treated with the hexane extract of C. ambrosioides. In conclusion, the hexane extract of C. ambrosioides is of further research interest, as it showed an anthelmintic activity in vitro and a reduction on the inflammatory reaction produced by the infection of T. canis larvae in vivo.     

Cultivation in vitro of Schistosomatium douthitti (trematoda: Schistosomatidae)

Cultivation in vitro of Schistosomatium douthitti (Trematoda : Schistosomatidae). International Journal for Parasitology12: 541–545. We grew S. douthitti in vitro from the cercaria to pairing ovigerous adults, using methods and media previously reported for cultivation of Schistosoma mansoni. Growth of S. douthitti in vitro was much more rapid than that of S. mansoni, but the cultures achieved a similar stage of maximal development. Cultured S. douthitti worms were smaller than those from animals and eggs were inviable. We determined that females of this species, known to produce eggs in unisexual animal infections, will also do so in culture in the absence of males.     

Migration, site selection, and development of Ornithodiplostomum sp. metacercariae (Digenea: Strigeoidea) in fathead minnows (Pimephales promelas)

The metacercarial stage of trematodes is typically considered an encysted, developmentally quiescent, resting stage. Yet the metacercariae of some species of strigeoid trematode undergo extravagant development within specific tissues of their second intermediate host. Our understanding of patterns of migration, site selection and development of these types of metacercariae is known for only a few species. In this study, we characterize the invasion and development of Ornithodiplostomum sp. metacercariae in their second intermediate host, the fathead minnow, Pimephales promelas. Diplostomules completed their migration into the abdominal cavity between 15 min and 48 h p.i. Most diplostomules migrated along muscular and connective tissue then penetrated the peritoneal lining of the abdominal cavity en route to the liver or pancreas. Alternatively, some diplostomules migrated within the host’s circulatory system, including the heart and arteries of the hepatic portal system. Metacercarial development in the liver and pancreas involved distinct growth, encystment and consolidation phases. Metacercarial volume increased 15-fold between 48 h and 4 weeks p.i., presumably due to absorptive and/or ingestive feeding activities within host tissues. By 2 weeks p.i., metacercariae were enveloped within a cyst wall and they were found loosely attached to the surfaces of internal tissues or unattached within the body cavity. These results emphasize the complex nature of metacercarial migration and growth and demonstrate that their growth and encystment phases occur within different habitats within their intermediate hosts.     

Fungal interaction between Trichoderma spp. and Pleurotus ostreatus on the enriched solid media with licorice Glycyrrhiza glabra root extract

The aim of this study is to investigate the antifungal activity of mycelia of Pleurotus ostreatus (white oyster mushroom) and licorice (Glycyrrhiza glabra) root extract against three undesirable fungi. They are Trichoderma spp., Trichoderma harzianum I and Trichoderma harzianum II which was tested on PSA (potato sucrose agar) medium enriched with licorice (Glycyrrhiza glabra) root extract (PSA-G media) using three concentrations (0.05, 0.10 and 0.20 g/L) in alone and dual cultures. Trichoderma spp. showed less mycelial growth of 8.75, 9.17 and 9.50 mm/day on PSA-G0.05, PSA-G0.1 and PSA-G0.2 respectively compared with 10.25 mm/day on fresh PSA (control) in dual culture. The best mycelial growth inhibition was recorded on PSA-G0.2 (14.97%) by T. harzianum II in alone culture opposite 63.72% in dual ones. The lower mycelial growth rate of T. harzianum I was 17.75 mm/day on PSA-G0.1 (0.10 g/L). In dual culture, overgrowth time of T. harzianum I had 5 days compared as approx. 6 days in alone culture. Generally, when the concentration of licorice extract increased, the mycelial growth rate of the undesirable fungi decreased. Also, all PSA-G media, especially PSA-G0.2, indicated low growth averages compared with the control (fresh PSA) against the pathogen while this concentration encourages growth of oyster mushroom. Also, this concentration reduced the density of sporulation of green molds; therefore, this concentration can be applied to reduce influence this pathogen in cultivation farm.     

Temperature effects on hyphal growth of wood-decay basidiomycetes isolated from Pinus densiflora deadwood

Hyphal growth rates were tested on malt extract agar plates at eight different temperatures (5–40?°C) using 36 isolates of 17 basidiomycete species obtained from Pinus densiflora deadwood in Japan. All isolates of four brown rot species showed optimum growth at 30?°C, whereas the optimum growth temperature of white rot species varied from 20?°C to 30?°C. Analysis using a dataset from four cooler sites showed that brown rot fungi grew more rapidly than white rot fungi at higher temperatures (25?°C, 30?°C, and 35?°C). These results suggest that the hyphal growth of brown rot fungi might be physiologically adapted to higher temperatures than those of white rot fungi among the fungal species inhabiting deadwood of P. densiflora in Japan.     

Cultivation of excysted metacercariae of Echinostoma revolutum (Trematoda) in chick embryos

Three techniques were used to study post-metacercarial growth and development of chemically excysted metacercariae of Echinostoma revolutum on the chorioallantoic membrane of domestic chick embryos. The in ovo technique of Zwilling (1959) and the in vitro technique of Auerbach et al. (1974) provided for better worm recovery in chick embryos than the in ovo technique of Woodruff &; Goodpasture (1931). Regardless of the technique used, postmetacercarial development was obtained for this species, and 6- to 8-day-old chorioallantoic-wornis achieved sexual development to the coiled uterus stage comparable to worms grown in domestic chicks for 7 days. However, somatic growth of worms from the chorioallantois was stunted when compared to worms grown in chicks. Worms grown on the chorioallantois voided their excretory concretions and showed histochemical lipid staining identical to that seen in worms grown in chicks.     

The development of the metacercariae of Microphallus similis in vitro and in the mouse

The development of the progenetic metacercariae of Microphallus similis in the mouse and under a range of in vitro cultivation systems was studied and compared. The flukes began egg production after 24 h in the mouse and by Day 4 post infection large numbers of eggs were present. These eggs began to embryonate in utero and contained a ball of cells; however, subsequent incubation in sea water failed to produce recognisable miracidia. In the majority of culture systems, gametogenesis and vitellogenesis occurred. Large numbers of abnormal eggs of three basic types were produced in vitro; these defects were probably related to the premature tanning of the eggshell precursors within the vitellaria and ducts. Apparently normal eggs were also produced, but they were probably unfertilized since sperm were rarely observed in vitro and they failed to embryonate.     

Characterization of an in vitro proliferation response to solubilized Trichinella spiralis antigens: Role of Ia antigens and Ly-1+ T cells

An antigen extract from Trichinella spiralis muscle larvae was prepared and used to immunize strains of mice which were either relatively resistant (B10.S) or susceptible (B10.BR) to oral infection with T. spiralis larvae. Proliferation of cells from the draining lymph nodes was then measured in vitro after stimulation with the T. spiralis extract as well as appropriate control antigens. Primed cells from resistant B10.S mice responded better to challenge than did cells from the susceptible B10.BR strain. Cell-depletion experiments involving B10.S cells indicated that the in vitro cell proliferation response is dependent upon Ly-1⁺ T cells. The data were also consistent with a requirement for Ly-1⁺, -2⁺, and -3⁺ amplifier cells. Administration of anti-I^s serum to the cultures specifically inhibited (nearly 75%) the cell proliferation response. The potential applications of this system as a tool in immunogenetic analyses of immunity to T. spiralis are discussed.     

In vitro excystation of Echinostoma paraensei (Digenea: Echinostomatidae) metacercariae assessed by light microscopy,morphometry and confocal laser scanning microscopy

Trypsin and bile salts have been identified as important triggers for excystation of Echinostoma metacercariae. Although excystation in trematodes is a well-known phenomenon, some morphological developmental changes remain to be elucidated. In order to gain further insight into the in vitro development of metacercariae, we assayed different cultivating conditions: 0.5% trypsin and 0.5% bile salts; 1% trypsin and 1% bile salts; 1% trypsin and 0.5% bile salts; 0.5% bile salts; or 0.5% trypsin. By means of light microscopy and confocal microscopy, we characterized each encysted, activated, breached and excysted stage based on the morphological features. However, breached and excysted stages were not revealed in both bile salts and trypsin-free medium. Excretory concretions (25 ± 3.9) were visualized within excretory tubules, close to the ventral sucker and genital anlage. The oral sucker armed with spines and digestive system was similar to those of adult worms. The reproductive system is composed of a genital anlage and the cirrus sac primordium. In short, trypsin and bile salts associated were fundamental for the in vitro metacercariae excystation of Echinostoma paraensei. This article presents the first detailed information of all stages of metacercariae excystation obtained through light and confocal microscopy.