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In vitro Development of Strongylus edentatus to the Fourth Larval Stage with Notes on Strongylus vulgaris and Strongylus equinus
Abstract
Strongylus edentatus was successfully cultured in vitro to the fourth larval stage (L4). Some growth continued for periods of 40-50 days at which time reductions in viability were observed in some of the culture systems tested. Various combinations of media, sera, buffers and organ explant cultures were tested. All cultures were incubated at 37 C in an atmosphere of 95% air and 5% CO2. Larvae underwent growth and differentiation to the L4 in all medium-serum combinations with and without organ explant cultures. Development and growth did occur but viability was reduced to insignificant levels in media without serum or cells. Optimal growth, differentiation, and longevity were observed in bicarbonate buffered RPMI-1640 containing 10% fetal calf serum and gerbil (Meriones unguiculatus) cecum explant cultures. Observations indicated that Strongylus vulgaris and Strongylus equinus also developed to the L4 stage using similar techniques. However, viability of S. vulgaris L4 was markedly limited. Specific morphological changes marked phases of development of S. edentatus, categorized as early, middle and late third stage, third molt and early fourth stage. Strongylus equinus appeared to follow the same developmental pattern in vitro as S. edentatus. Distinct differences in morphological features during differentiation were observed between S. edentatus and S. vulgaris.
... vulgaris L4" in accordance with previous definitions. 9,19,20 Both exsheated L3 and L4 larvae were washed three times in RPMI 1640 medium (BioWhittaker, Cambrex Bioscience, Verviers, Belgium) containing 400 IU/mL penicillin, 200 µg/mL streptomycin and 1 µg/mL amphotericin B before UV irradiation. ...
... This is in accordance with previous observations 20, 22 although it is not possible to distinguish between the late L3 and the newly moulted L4 based on morphology. 20 These exsheated S. vulgaris L3 and L4 preparations only resisted a lower dose of UV irradiation, 3 minutes at 30 MJ, to remain intact. ...
Aims:
To generate different larval stages of Strongylus vulgaris and to study cytokine responses in cultures of eqPBMC exposed to defined larval stages of S. vulgaris and cyathostomins with the aim to understand the early immune reaction to these parasites.
Methods and results:
EqPBMC were exposed to S. vulgaris larvae (L3, exsheated L3 and L4) as well as cyathostomin L3 and analysed for cytokine gene expression. Procedures for decontamination, culturing and attenuation of larvae were established. Transcription of IL-4, IL-5 and IL-13 were induced by both S. vulgaris and cyathostomin L3. Moulting of S. vulgaris from L3 to L4 stage was accompanied by a shift to high expression of IL-5 and IL-9 (exsheated L3 and L4) and IFN-γ (L4 only). In parallel, the adjuvant G3 modified the cytokine profile induced by both parasites by reducing the expression of IL-4, IL-5 and IL-10 while concomitantly enhancing the expression of IFN-γ.
Conclusion:
The L4 stage of S. vulgaris generated a cytokine profile different from that induced by the earlier L3 stage of S. vulgaris and cyathostomins. This diversity depending on the life cycle stage will have implications for the choice of antigen and adjuvant in future vaccine design.
... L3 recovery from fecal collection, culture, purification and further exsheathing were essentially carried out as described by Farrar & Klei., (1985) and Klei et al, (1982). Larvae were cultured using media and methods as described by Chapman et al, (1994) to obtain L4 stages. ...
Transglutaminases (E.C. 2.3.3.13) are a family of Ca(2+)-dependent enzymes that stabilize protein structure by catalyzing the formation of isopeptide bonds. A novel form of transglutaminase has been identified and characterized that seem to play an important role in growth, development, and molting in adult and larval stages of filarial nematodes. The aim of this study was to identify the ubiquitous nature of this enzyme in other nematodes and to measure its significance to larval growth, molting, and development. For this purpose, equine Strongylus spp. were used. Activity of this enzyme was identified in extracts of larvae and adults of Strongylus vulgaris, S. edentatus, Parascaris equorum and Cylicocyclus insigne. The significance of transglutaminase in the early growth and development of Strongylus vulgaris, S. edentatus and S. equinus was tested by adding specific inhibitors, monodansylcadaverine (MDC) or cystamine (CS), to in vitro cultures of third (L3) and fourth stage larvae (L4). The viability, molting and growth of these nematode species were affected by both inhibitors. Cystamine promoted abnormal development of Strongylus edentatus L3, resulting in an aberrant expansion of the anterior end. Addition of these inhibitors to cultures of L4 also reduced growth of the three species. The results indicated that transglutaminase is present in a wide array of nematode parasites and may be important in growth and development of their larval stages.
... 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 Lustigman et al., , 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.
A 7-month-old female mixed breed foal with a 2-day history of recumbency and inability to open its mouth convulsed acutely and died and was submitted for necropsy examination. The foal was thin and large patches of haemorrhage were present throughout the peritoneal wall, the diaphragmatic surfaces and the retroperitoneum. Numerous nematode larvae were visible on the serosal surfaces and penetrated and embedded into the subserosa associated with the haemorrhages. The dorsal portion of the abdominal diaphragm had a partial tear and large numbers of nematodes were within the muscle fibres. Histologically, the larvae had a smooth cuticle, polymyarian/coelomyarian musculature and multinucleated intestinal cells, and were typically surrounded by haemorrhage, neutrophils, dense fibrovascular connective tissue and rare multinucleated giant cells. Parasitological examination identified the larvae as Strongylus edentatus based on the morphology of the buccal capsule. Additionally, there was severe muscle necrosis of the tongue and liver tissue analysis detected selenium deficiency. S. edentatus infections are uncommon in California, USA, and are typically non-lethal. In this case, the selenium deficiency may have led to immunosuppression, resulting in the hyperinfection with S. edentatus, and to the muscle damage and tear of the diaphragm. Although ivermectin treatment was indicated in the history, inadequate deworming or anthelmintic resistance may have played a role in the severity of infection.
Third-stage larvae of equine cyathostomes were cultured in various culture systems for 57 days. Culture systems containing the medium NCTC-135, chick embryo extract, foetal calf serum and casein hydrolysate and incubated in an atmosphere of 10% CO2, 5% O2 and 85% N2 were most successful in supporting growth from early third stage to early fourth stage larvae. Moulting began 22 days post inoculation in 40% of larvae and approximately 20% of infective larvae exsheathed.
This study was designed to determine how the behaviors of horses on a fenced-in pasture in the northeastern United States may contribute to infection by Strongylus vulgaris. The infective stages of strongylid parasites of horses develop in the feces, and they must be ingested for infection to occur. Domestic horses are thought to be coprophobic, and to avoid grazing in areas contaminated with their feces. 1,2 In this study, nine Standardbred colts were observed on two pastures three times a week during two observation periods (January, 1995 and February through mid-March, 1995). Nine adult Standardbred mares and geldings were also observed on the same pastures during one observation period from April through mid-May, 1995. On each pasture, the roughs (areas of group defecation associated with long grass), lawns (short-cropped grass) and bare areas (where hay was fed in racks), were mapped. Grazing occurred equally on roughs, lawns, and bare patches of the pasture in two of the three observation periods (January and April/May). In the February/mid-March observation period, most of the grazing activity occurred on the bare areas, where hay was provided, but there was still grazing in the roughs. Defecation and urination activities occurred at similar frequencies in all areas of the pastures during all observation periods. These data suggest that horses kept at high grazing intensities will graze near feces in the roughs, and will defecate in the “grazing” areas. It was also determined that the infective larvae of S. vulgaris are attracted to horse feces, but show no responses to grass, in laboratory chemical migration assays. These data suggest that the parasites may not migrate from the feces to the grass as part of their transmission strategy as previously reported. Thus, parasite infection may be occurring when horses graze in the roughs or near feces in the lawns and bare areas, and pasture management strategies for controlling these parasites may be most effective if focused on reducing the infective larvae present on the pasture.
Tese (doutorado) - Instituto de Ciências biomédicas da Universidade de São Paulo. Inclui referências.
A mixed population of equine cyathostomin (Nematoda, Strongyloidea) infective third stage larvae (L3) was cultured in vitro using a cell-free medium. Some L3 were cultured immediately after Baermann collection from fecal cultures, while others were kept in water at 4 degrees C for 7 days before initiating the in vitro cultures. Cultures were examined daily for viability. At days 2, 7, 14 and 21 larvae were collected for identification of developmental stage and morphological changes, using both light and scanning electron microscopy. Larvae were classified as early L3 (EL3), developing L3 (DL3), late L3 (LL3) and fourth stage larvae (L4) on the basis of morphological features. Viability remained high throughout the entire study period in cultures of both non-refrigerated (84.7%) and refrigerated (77.4%) larvae. However, viability of the non-refrigerated was significantly greater from 7 through 21 days of culture. Significant differences were also observed in the percentage of DL3 between the non-refrigerated and refrigerated larval cultures by day 7. The highest percentage of DL3 larvae (22.5%) was reached at the end of study in those larvae that were not previously refrigerated. The data suggests that prior refrigeration decreases viability and slows L3 development. At day 21 LL3 larvae were only a small percentage of the DL3: 6.9 and 5% in non-refrigerated and refrigerated cultures, respectively. Few of these larvae freed themselves from the L3 cuticle and moulted to L4 stage. Characteristics of individual species in vitro developmental patterns were determined by the molecular identification of individual larvae in pools of larvae randomly collected at days 0 and 21. Seven species (Coronocyclus coronatus, Cylicostephanus goldi, Cylicostephanus longibursatus, Cyathostomum catinatum, Cylicocyclus nassatus, Cylicocyclus ashworthi, Petrovinema poculatum) were identified in the day 0 pool. The greatest tendency to develop in vitro was shown by the genus Cylicostephanus with the species C. goldi and C. longibursatus that developed to the LL3-L4 stages. C. nassatus, C. ashworthi and C. coronatus did not progress in their development beyond the EL3 stage, while no apparent signs of development were registered for C. catinatum.
An efficient and reliable method is described for the culture of equine strongyles from the third (L3) to the fourth (L4) larval stage. Medium consists of 50% fetal calf serum and 50% NCTC with additions of L-glutamine, NaHCO3, yeast extract, bactopeptone, and dextrose. The gas phase used is of prime importance; it is a mixture of 10% CO2, 5% O2, and 85% N2. Strongylus vulgaris, Strongylus edentatus, Strongylus equinus, Triodontophorus brevicauda, Triodontophorus serratus, Triodontophorus tenuicollis, Oesophagodontus robustus, Cylicocyclus insigne, and mixed species of cyathostomes were cultured to the L4 stage. Oesophagodontus robustus was cultured to the fifth larval stage. Depending on species, 44-95% of Strongylinae L3 inoculated into this system molted to L4. Although some development of the Cyathostominae L3 occurred, only a small portion (1%) completed ecdysis to L4. Viability in cultures of all species remained high (> 60-70% larvae surviving) for at least 4 wk (cyathostomes) and as long as 6 mo (S. edentatus). The addition of equine hemin to cultures of S. vulgaris and O. robustus L4 enhanced development and prolonged viability of these larvae. Hemin had no effect on cultures of S. edentatus or S. equinus, and it was not tested in cultures of other species.
Infective larvae (L3) of Strongylus vulgaris have limited energy stores for host finding and for infection. For transmission to occur, the larvae must have sufficient energy to (a) migrate onto grass, where they are ingested by their equine host (host finding), and (b) penetrate into the host gut. This study is designed to test the hypothesis that L3 larvae of S. vulgaris partition their energy stores between locomotory activity (used in host finding) and infection activity (penetration). Chronic locomotory activity was stimulated by incubating S. vulgaris L3 larvae at a constant temperature (38 C). After 8 days of treatment, locomotory activity ceased (exhaustion). Exhausted L3 larvae had significantly decreased total lipid when compared to controls (P < 0.05), but there was no decrease in levels of protein of carbohydrate. Lipids of S. vulgaris L3 larvae are comprised of 9 fatty acids, some of which are depleted in exhausted worms (14:0, 14:1, 16:0, 16:1, 18:1, 18:2), whereas others (18:0, 20:4, 24:0) remain unchanged. These data suggest that specific fatty acids provide the energy source for locomotory activity in S. vulgaris. Exhausted L3 larvae were also less able to penetrate host cecal tissue in in vitro penetration assays when compared to controls (P < 0.05), suggesting that the depletion of individual fatty acids during locomotory activity also reduced infectivity. These data do not support the hypothesis that S. vulgaris L3 larvae partition their energy stores between host-finding and infection activities. A comparison of lipid storage profiles in the L3 larvae of 4 nematode species with similar transmission strategies (S. vulgaris, Strongylus edentatus, Strongylus equinus, and Haemonchus contortus) revealed similarities in the fatty acid composition of these species. These data suggest a relationship between transmission patterns and energy storage strategies in the L3 larvae of nematode parasites of vertebrates.
Stephanurus dentatus was grown to advanced parasitic stages in a new static in vitro system, free of bacteria and fungi. Criteria for evaluation were based on anatomical characters of the head, stoma, excretory system, and intestine. Descriptions of these characters and observations on morphogenesis not previously reported from in vivo specimens are given in detail. Development to late fourth stage was obtained in cultures consisting of primary monolayers of swine kidney cells overlaid with the following medium (KW-1S): yeast extract (BBL), 112.5 mg; Bacto-peptone (Difco), 140.65 mg; dextrose (BBL), 140.65 mg; swine serum, 50 ml; NCTC 109, 50 ml; and antibiotics to give a final concentration of 1,000 units penicillin G potassium, 1 mg dihydrostreptomycin, and 10 μg Amphotericin B per ml of medium. When bovine serum was substituted (KW-1) development halted at mid fourth stage. In addition, with both fluid media, the following were observed: only larvae in fourth stage fed on the kidney cell cultures; a definite feeding pattern occurred; larvae fed on rolled cell layers (presumably dead) as well as living cells; digestion and egestion took place, and there was a correlation between feeding behavior and anatomical development of the stoma, excretory system, and intestine. In either medium, without the cell culture, only early fourth stage was attained and yields of this phase of development were reduced to one-fortieth. Media KW-1 and KW-1S evolved from attempts to culture S. dentatus in modifications of Pitts' and Ball's (1953) medium, or in NCTC 109 (McQuilkin et al., 1957) used alone or supplemented with calf serum. The prototype cell-free media, one of which was also supplemented with nonliving tissue, had the following effects on development: (1) the most advanced stage attained was early fourth stage; (2) larvae advanced to late parasitic third stage in media supplemented with either NCTC 109 or serum; (3) development proceeded to early fourth stage when both NCTC 109 and serum were present; (4) a more rapid rate of development to third molt and early fourth stage ensued when NCTC 109 and serum were present at concentrations of 50%, each; and (5) although fourth-stage larvae ingested nonliving tissues when available, development was not enhanced.