No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
In vitro Cultivation of Dipetalonema viteae Third-Stage Larvae: Effect of the Gas Phase
Abstract
The effect of the gas phase on the in vitro growth and development of Dipetalonema viteae (Nematoda: Filarioidea) third-stage larvae obtained from the tick vector and 3 day infections of jirds was examined. Measurements of the oxygen (pO2) and carbon dioxide (pCO2) tensions and the pH in the medium were made for each gas phase. In cultures gassed with 5% carbon dioxide in nitrogen the pO2 was between 32 and 50 mm Hg, the pCO2 ranged from 25 to 40 mm Hg and the pH was between 7.2 and 7.4. This gas phase resulted in the best growth and development of third-stage larvae to the fourth-stage. Survival and development of larvae were decreased in cultures with oxygen tensions less than 20 mm Hg and greater than 50 mm Hg.
... One-tenth milliliter of the sample was inoculated into a tube of thioglycollate broth to test for sterility. The remaining 0.2 ml was injected into the injection port of the blood-gas analyzer (Franke and Weinstein, 1984). Readings of pH, PO2, and pCO2 were taken with water in the circulating water bath of the analyzer maintained at 25 C. ...
The effect of oxygen and carbon dioxide concentration on the development of the free-living stages of Strongyloides ratti in axenic culture was examined. Hatching of S. ratti eggs was inhibited at very low oxygen levels. Development of all organisms was inhibited in medium gassed with less than 4% O2 in 0.03% CO2 with the remainder N2. Between 8 and 21% O2 there was no significant difference in the percentage developing directly to filariform larvae, free-living females, or free-living males. Cultures gassed with CO2 concentrations greater than 1% in 21% O2 with the remainder N2 manifested inhibited development to filariform larvae, whereas concentrations between 5 and 7.5% enhanced development to free-living females. There was a positive effect on increasing the CO2 concentration from 0.03% to 5% on development to filariform larvae and male worms at levels of 1% and 2% oxygen in nitrogen, respectively. Direction of development, either directly to the filariform larva or to the free-living female, was influenced by CO2 and O2 concentrations encountered by the egg or the newly hatched larva of the parasitic female in axenic culture. The axenic culture system permits an important experimental approach to the study of factors modulating the differentiation of the free-living stages of Strongyloides.
... Basic among these is their programmed developmental arrest followed by reactivation upon encountering the host or host-like conditions. In vitro culture methods have allowed some organism-scale studies of extrinsic factors governing the reactivation of L3i (Franke and Weinstein, 1984a, b; Farrar and Klei, 1985; Abraham et al., 1987; Wisnewski and Weinstein, 1993) and of the early events following reactivation (Abraham et al., 1987; Lustigman et al., 1995, 1996). Pioneering microsurgical techniques have been used recently to study the neuronal control of arrest and reactivation of Strongyloides stercoralis L3i (Ashton et al., 1998). ...
A forkhead transcription factor gene, fktf-1, which we propose to be orthologous to the Caenorhabditis elegans dauer-regulatory gene daf-16 has been discovered in the parasitic nematode Strongyloides stercoralis. Genomic and cDNA sequences from both species predict alternately spliced a and b message isoforms. In contrast to C. elegans, where two a isoforms, daf-16a1 and daf-16a2, are found, a single fktf-1a isoform is found in S. stercoralis. Five of the 10 introns found in the C. elegans gene are found in the proposed S. stercoralis ortholog. Functional motifs common to DAF-16 and several mammalian forkhead transcription factors are conserved in FKTF-1. These include the forkhead DNA binding domain, four Akt/protein kinase B phosphorylation sites and a C-terminal domain that may associate with factors such as the steroid receptor coactivator and other factors necessary for transcriptional regulation. An N-terminal serine-rich domain found in DAF-16A is greatly expanded in FKTF-1A. This domain is missing in DAF-16B, FKTF-1B and all mammalian orthologs. FKTF-1 shows the closest phylogenetic relationship to DAF-16 among all known mammalian and nematode forkhead transcription factors. Like its proposed Caenorhabditis ortholog, the fktf-1 message is expressed at all stages of the life cycle examined thus far. Discovery of fktf-1 indicates the presence of an insulin-like signalling pathway in S. stercoralis similar to that known to regulate dauer development in C. elegans. This pathway is a likely candidate to control infective larval arrest and reactivation as well as regulation of the switch between parasitic and free-living development in the parasite.
Several parameters concerning the reproduction ofLitomosoides carinii were assessed using quantitatively infected cotton rats (Sigmodon hispidus). The course of embryogenesis from the fertilization of eggs to the delivery of the first microfilariae was observed by daily autopsies during prepatency. The duration of embryogenesis in vivo could thus be determined as 182 days. The contents of embryos in the uteri of female worms had been examined at various intervals. At the onset of patency 7–8 weeks p.i. the females were 716 mm long and on average contained 308103 embryos/female, of which 19% were pathologically altered. In the middle of patency 16–20 weeks p.i. the females had grown up to 10011 mm in length and now contained 509103 embryos/female, 25% of them were pathologically altered, the others were normally developed. A positive correlation between the body length of a female worm and its number of embryos in utero was evident. Additionally the percentage of pathologically altered embryos was increased with respect to the age of the worms. The calculated fecundity of a femaleL. carinii in vivo of around 20103 microfilariae/female per day had been confirmed with worms maintained in vitro. Three combinations of media and serum supplements were used and their influence on embryogenesis evaluated.
Living third- and fourth-stage larvae (L3 and L4) of Acanthocheilonema viteae were recovered quantitatively from adult Meriones unguiculatus within the first 10 days after subcutaneous inoculation of 60 arthropod-derived larvae (mL3). The average recovery of the inoculated larvae was about one third (28.5%), and the majority (87.7%) were found in muscular tissues. Seventy-two hours after inoculation, larvae could be isolated from all body locations, although the majority still was found near the site of inoculation. Morphological and biometrical data indicated that, at least until molting, the development of the larval population was not synchronous, with molting occurring over a period of 48 hr on days 7 and 8 postinoculation. The stomatal rings of postinvasive L3's and L4's were distinguishable structurally and could be used as stage-specific determinants. Immediately after infection, L3's showed a linear growth in diameter; rapid longitudinal growth started after the molt, leading to a doubling in the length of L4's within 4 days. The time course of shedding was reconstructed in detail using isolated L3/L4 intermediates.
A primary constraint in the culture of human filariae is in obtaining starting material--either microfilariae (mfs), which infect invertebrates, or third stage larvae (L3s), which are infective to humans. Cryopreservation methods which partially overcome this difficulty have been developed for both mfs and L3s. Complete development of mfs to L3s outside an intact host was obtained recently when mosquito thoraces infected by Brugia malayi (24 h after the bloodmeal) were maintained in vitro. In another recent study in which no host tissues were present, a semidefined culture medium was used to investigate the properties of reduced glutathione (GSH) that stimulate early development of Onchocerca lienalis mfs. An extended cysteinyl backbone and a free sulfhydryl were identified as the key structural elements provided by GSH. Stimulation also required the presence of low and high molecular weight components of serum as well as oxygen. Molting of Onchocerca spp. L3s to the fourth stage at the rate of 50 to 70% has been reported by several researchers. Key factors identified in those successes have been temperature and serum lot. Improved long-term viability occurred with cellular co-culture. Beneficial effects of co-culture were shown to be due both to cellular conditioning of the medium as well as to lowered dissolved oxygen levels as a result of cellular metabolism. With the use of cell-conditioned medium and decreased incubator oxygen levels, long-term viability of Onchocerca larvae in culture exceeded that previously reported. Recently, Brugia malayi adults of both sexes were cultured from L3s using a semidefined medium supplemented with human serum. Many of these sexually matured adults mated and produced viable microfilariae.
A series of experiments examined the effects of various media, serum supplements, gas phases and the incorporation of mammalian cell feeder layers on the survival of Onchocerca gutturosa adult worms in vitro. The survival of male worms was poor in all media tested that were not supplemented with inactivated foetal calf serum (IFCS), with improved but variable survival in media supplemented with 10-30% IFCS. Using a cell-free system in an atmosphere of 5% CO2 in air, good results were obtained in medium NCTC 135 + 10% IFCS (median survival time 39 days, range 25-41). Marginally better survival was obtained with the same medium in an atmosphere of 95% N2/5% CO2 (median 45 days, range 25-56) and with a 1:1 mixture of media NCTC 135 and IMDM + 10% IFCS (median 38 days, range 38-51). Survival was enhanced in culture systems which incorporated bovine kidney (MDBK) cells, bovine trachea (EBTR) cells and monkey kidney (LLCMK2) cells. Exceptionally long survival was obtained using medium MEM + 10% IFCS + LLCMK2 cells under a gas phase of 5% CO2 in air, in which male worms survived from approximately 6 to over 7 months. Under similar conditions, female worms were also maintained for periods of up to 6 months and 5 out of 18 specimens released microfilariae into the culture system. The long-term culture described in this study will be useful for basic biochemical, chemotherapeutic and immunological studies in vitro.
Several parameters concerning the reproduction of Litomosoides carinii were assessed using quantitatively infected cotton rats (Sigmodon hispidus). The course of embryogenesis from the fertilization of eggs to the delivery of the first microfilariae was observed by daily autopsies during prepatency. The duration of embryogenesis in vivo could thus be determined as 18 +/- 2 days. The contents of embryos in the uteri of female worms had been examined at various intervals. At the onset of patency 7-8 weeks p.i. the females were 71 +/- 6 mm long and on average contained 308 X 10(3) embryos/female, of which 19% were pathologically altered. In the middle of patency 16-20 weeks p.i. the females had grown up to 100 +/- 11 mm in length and now contained 509 X 10(3) embryos/female, 25% of them were pathologically altered, the others were normally developed. A positive correlation between the body length of a female worm and its number of embryos in utero was evident. Additionally the percentage of pathologically altered embryos was increased with respect to the age of the worms. The calculated fecundity of a female L. carinii in vivo of around 20 X 10(3) microfilariae/female per day had been confirmed with worms maintained in vitro. Three combinations of media and serum supplements were used and their influence on embryogenesis evaluated.
At concentrations of 0.1-100 ng/ml ivermectin inhibited L3-L4 molting by Onchocerca lienalis in vitro. The degree of inhibition was dose-dependent with a significant effect apparent at 0.1 ng/ml and complete inhibition occurring at 100 ng/ml. The ED50 for molt inhibition was 0.19 ng/ml. Molt-inhibiting levels of the drug were not acutely toxic to the worms. In the presence of 10 ng/ml, a concentration giving 95% molt inhibition, motility at day 7 postinoculation was 71% of that seen in nontreated controls. A more pronounced effect on motility was apparent in larvae under long-term cultivation in the presence of ivermectin. Kinetic studies indicated that the majority of the larvae respond irreversibly to the drug within the first 2 hr of exposure. Twenty-four hours of exposure were required for a maximal response. The inhibitory effects of ivermectin were less pronounced if larvae were allowed to develop under normal culture conditions for 24 or more hours prior to the initiation of drug treatment.
The objective of the present study was to define culture conditions under which larval Dirofilaria immitis would molt, grow, and survive. Third-stage larvae (L3) survived for over 3 wk with a molt rate of up to 95% in a variety of media supplemented with fetal calf serum. Bovine albumin, added to several media at concentrations of 10-30 mg/ml, also proved to be an effective culture supplement for the induction of molting and for supporting larval survival. Two gas phases were tested, 5% CO2/95% N2 and 5% CO2/air; no differences were noted in larval development based on gas phase. Larvae, maintained in media with FCS or albumin for 48 hr, were capable of completing the molting process and growing in length in unsupplemented media. If the temperature at which cultures were maintained was changed from 37 C to 27 C, L3 did not molt but did survive for several weeks. Two factors required for larval D. immitis molting and growth have been identified, temperature of approximately 37 C and the presence of albumin in the culture medium. The defined culture system developed for D. immitis L3 may provide a source for collection of excretory-secretory antigens, which could prove useful in immunodiagnosis or immunoprophylaxis as well as provide a means of studying the process and requirements of filarial larval molting.
Cultivation of fourth stage Brugia pahangi and B. malayi larvae from infective larvae (stage 3) were obtained in culture medium RPMI 1640 supplemented with 10% human AB serum and an LCC-MK2 rhesus monkey kidney continuous cell line feeder layer. This culture system kept larvae alive in excess of 7 weeks, and served as a source for collection of the worms' secretory, excretory, and moulting antigens.
Third stage larvae of Ascaris suum, recovered from the lungs of rabbits 7 days after oral infection with eggs, were cultured to the fourth larval stage in chemically defined, low molecular weight medium. The medium consisted of tissue culture medium 199, supplemented with glucose and glycyl-hystidyl-lysine and gassed with a mixture of N2-CO2-O2 (90:5:5). Growth and development in this medium were similar to that in media supplemented with whole serum or with a serum dialysate.
The metabolisms and effects of several anthelmintics on 3 adult filarial nematodes, L. carinii, D. viteae, and B. pahangi, have been compared. The latter 2 differ from the obligate erobe, L. carinii, in that they survive for extended periods of time anerobically, indicating that they obtain their metabolic energy by utilizing erobic pathways. In accord with their apparent anerobic nature, fermentation balance studies indicate that D. viteae and B. pahangi are homolactate fermenters. L. carinii, on the other hand, forms appreciable quantities of acetate in addition to lactate and CO2. Isotope studies indicate that the tricarboxylic acid cycle does not constitute a quantitatively significant energy yielding pathway in L. carinii. Instead, findings suggest that the erobic requirement of L. carinii may reside completely in one system, the oxidative decarboxylation of pyruvate to acetate and CO2. Cytochrome c oxidase activity could not be demonstrated in any of the parasites as assayed by the oxidation of reduced cytochrome c. l Tetramisole at low concentrations (4.2 x 10-7 M) completely stops motility of all 3 species within 1.5 min. d Tetramisole also inhibits motility effectively, but it is considerably more readily reversible than the corresponding levo isomer.
1.1. Protocollagen proline hydroxylase (PPH) activity in developing eggs of Ascaris lumbricoides first appeared in the late blastula-early gastrula stages, was most active at or near the molting stage and disappeared in infective eggs. PPH activity was closely correlated with the amount of soluble collagen.2.2. Little hydroxyproline was present in gelatin extracted from the insoluble proteins of one-celled eggs. It increased forty-hold during development, whereas the gelatin increased only three-fold.3.3. The apparent Michaelis constant () of egg PPH was strongly affected by temperature, being minimal at 30°C. That of PPH from adult A. lumbricoides muscle was less strongly affected, with a minimum at 38°C.4.4. Oxygen concentrations exceeding 5 vol. per cent inhibited muscle but not egg PPH. Catalase did not relieve the inhibition.5.5. Egg and muscle PPH gave electrophoretically distinct peaks on acrylamide gel columns.6.6. The protocollagen proline hydroxylases of eggs and muscle seem to be isoenzymes which are adapted to the developmental physiology of A. lumbricoides.
Infective third-stage larvae of Dipetalonema viteae (Nematoda: Filarioidea) were cultured to young adults in a cell-free culture
system. Third-stage larvae from the tick vector grew, developed, and molted twice in a medium containing NCTC 135 and Iscove's
modified Dulbecco's medium supplemented with fetal bovine serum under a gas phase of 95 percent nitrogen and 5 percent carbon
dioxide. The availability of such a culture system for filariids should facilitate studies of their immunology, biochemistry,
and sensitivity to drugs.
The in vitro cultivation of Ascaris larvae was carried out by supplementing either Eagle's medium or medium 199 with 20% calf or bovine serum. Growth was observed only when an initial concentration of 20,000 or more larvae/10 ml of medium were cultured at 37.5 C under partially anaerobic conditions. By this procedure a small percentage of the larvae reached sizes which made their isolation feasible. Continued development of the isolated larvae occurred only after the normal atmosphere was replaced by one of a nitrogen-5% CO2 mixture. Larvae were maintained for as long as 110 days and reached sizes up to 2,300 by 83 μ.
Trichinella spiralis larvae from rat muscles were cultured axenically in roller tubes. In chick embryo extract and rabbit serum medium, male and female external genitalia appeared after the second incomplete molt. Molting was preceded by cuticular swelling around the neck, followed by constriction and ultimate rupture. The worms did not increase in size. Digestive enzymes and bile salts did not promote exsheathment and were often toxic. With added vitamins, the second molt was reached after 24 hours in vitro (as against 48 hours in the basic medium), but amino acids and glucose had no effect on survival and development. Under nitrogen or 5% CO2 in nitrogen the worms survived longer than in the presence of 5% oxygen or under air. Development was better in the presence of 5% CO2 than under 5% oxygen, and still better under higher CO2 tensions. Because CO2 tension in the mammalian intestine is often high, this gas and (or) carbonic acid probably play an important part in the biology of intestinal parasites. The two incomplete molts represent advancement through two stages and the worms with external genitalia are believed to be immature adults (that is, fifth stage). Because direct microscopic evidence of molting prior to encapsulation in muscles of infected animals has been obtained, the infective larvae are believed to be in the third stage.