The isolation and partial characterization of a protective antigen from developing larvae of Ascaris suum.

An antigen (ACF antigen) obtained by in vitro cultivation of third-stage larvae of Ascaris suum to the fourth stage in defined medium induced significant protection in guinea pigs. The antigen was further characterized by ultrafiltration and gel filtration and was shown to be a single component by gel precipition and immunoelectrophoresis. It induced active cutaneous anaphylaxis in sensitized animals. The ACF antigen is estimated to contain about 79% protein and 22% carbohydrate and to have a molecular weight of approx 67,000.     

Development-Specific Differences in the Proteomics of Angiostrongylus cantonensis

Angiostrongyliasis is an emerging communicable disease. Several different hosts are required to complete the life cycle of Angiostrongylus cantonensis. However, we lack a complete understanding of variability of proteins across different developmental stages and their contribution to parasite survival and progression. In this study, we extracted soluble proteins from various stages of the A. cantonensis life cycle [female adults, male adults, the fifth-stage female larvae (FL5), the fifth-stage male larvae (ML5) and third-stage larvae (L3)], separated those proteins using two-dimensional difference gel electrophoresis (2D-DIGE) at pH 4–7, and analyzed the gel images using DeCyder 7.0 software. This proteomic analysis produced a total of 183 different dominant protein spots. Thirty-seven protein spots were found to have high confidence scores (>95%) by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Comparative proteomic analyses revealed that 29 spots represented cytoskeleton-associated proteins and functional proteins. Eight spots were unnamed proteins. Twelve protein spots that were matched to the EST of different-stage larvae of A. cantonensis were identified. Two genes and the internal control 18s were chosen for quantitative real-time PCR (qPCR) and the qPCR results were consistent with those of the DIGE studies. These findings will provide a new basis for understanding the characteristics of growth and development of A. cantonensis and the host–parasite relationship. They may also assist searches for candidate proteins suitable for use in diagnostic assays and as drug targets for the control of eosinophilic meningitis caused by A. cantonensis.     

Comparative studies on the life history of Angiostrongylus mackerrasae Bhaibulaya, 1968 and Angiostrongylus cantonensis (Chen, 1935)

The life history of A. mackerrasae was found to differ from that of A. cantonensis as follows: (1) the moulting times of A. cantonensis in the definitive host occurred a few days earlier than those of A. mackerrasae; (2) the growth rate of A. cantonensis was more rapid than that of A. mackerrasae. However, there were no differences in the migratory pattern of the third-stage larvae of both species in experimentally-infected definitive hosts. It is concluded that Mackerras & Sandars (1955) described the life history of A. mackerrasae and not A. cantonensis.     

Onchocerca volvulus: biochemical and morphological characteristics of the surface of third- and fourth-stage larvae

The annulated cuticles of third- and fourth-stage larvae of Onchocerca volvulus have the typical structure of other nematodes but the cuticle of fourth-stage larvae was thinner. The surface of the third-stage larva was wrinkled and fuzzy, while that of the fourth-stage was smooth. Intermediate stages in the formation of the new cuticle and epicuticle beneath the old basal layer and of the separation of the cuticles are shown. Monoclonal antibodies specific to the surface of third-stage larvae did not react with the surface of the fourth-stage larvae. Binding of the monoclonal antibodies to the third-stage larvae was abrogated by treatment of the worms with trypsin and proteinase K, but was unaffected by treatment with periodate or the detergents sodium deoxycholate and SDS. The lectins RCA120 and WGA, but not any of the other lectins tested, bound only to the surface of fourth-stage larvae, and not to that of third-stage larvae. The surfaces of third- and fourth-stage larvae were shown to be different and contained stage-specific surface epitopes.     

Changes in antigen and glycoprotein patterns during the development of Oesophagostomum dentatum

During its development from free-living infectious third-stage larvae to the adult worms in the large intestines of pigs, Oesophagostomum dentatum experiences several environmental changes. Differences in protein patterns can reflect such changes. Somatic and ES antigens and glycoproteins of pre-parasitic, histotropic and intestinal stages were compared by single-dimension SDS–PAGE and stage-specific proteins were defined. Furthermore, fourth-stage larvae derived from different sources—in-vitro cultivation and intestinal contents—were compared and also found to be different. It is hypothesised that O. dentatum reacts to environmental stimuli by differential expression of specific proteins as a possible mode of adaptation to the host.     

Dauer Larvae of Caenorhabditis briggsae in Axenic Culture

The free-living hermaphroditic nematode, Caenorhabditis briggsae, enters a dauer stage under certain conditions in axenic culture. Dauer larvae differ from directly-developing third-stage larvae in internal structure, size at time of second molt, morphology of second and third cuticles, separation zone of cuticular caps, and survival at 4 C and 37 C, temperatures fatal to other stages. Males, which occur rarely in liquid medium, may mature under conditions which cause most of the hermaphrodites to go into the dauer stage, resulting in a culture with increased male-to-hermaphrodite ratio.     

Effects of plumbagin on development of the parasitic nematodes Haemonchus contortus and Ascaris suum

1. Plumbagin (5-hydroxy,2-methyl-l,4-napthoquinone) inhibited the motility and survival of Haemonchus contonus first-stage larvae (L1) with an ed₅₀ of 1 μg/ml, but was less effective in preventing the development of H. contortus to infective third-stage larvae in a faecal slurry assay.2. Of the structural analogs tested, plumbagin was the most potent in preventing development of L1 followed in decreasing order of potency by 1,4-napthoquinone, 5-hydroxy-1,4-napthoquinone (juglone) and 1,2-napthoquinone.3. Plumbagin had a biphasic effect on development of the fourth-stage Ascaris suum larvae that caused an increase in growth at low concentrations but was lethal at higher doses.4. Plumbagin and 1,2-napthoquinone partially inhibited embryonation of A. suum eggs.     

Effect of Monochamus carolinensis on the Life History of the Pinewood Nematode,Bursaphelenchus xylophilus

The development of Bursaphelenchus xylophilus in pine wood infested with and free of Monochamus carolinensis was investigated. Formation of third-stage dispersal juveniles occurred in the presence and absence of pine sawyer beetles. The proportion of third-stage dispersal juveniles in the total nematode population was negatively correlated with moisture content of the wood. Formation of nematode dauer juveniles was dependent on the presence of the pine sawyer beetle. Dauer juveniles were present in 3 of 315 wood samples taken from non-beetle-infested Scots pine bolts and 81 of 311 samples taken from beetle-infested bolts. Nematode densities were greater in wood samples taken adjacent to insect larvae, pupae, and teneral adults compared with samples taken from areas void of insect activity. Nematodes recovered from beetle larvae, pupae, and teneral adults were mostly fourth-stage dauer juveniles, although some third-stage dispersal juveniles were also recovered. Dauer juvenile density was highest on teneral adult beetles.     

Transplantation of young adult Angiostrongylus cantonensis into the rat pulmonary vessels and its application to the assessment of acquired resistance

Yoshimura K., Aiba H. and Oya H. 1979. Transplantation of young adult Angiostrongylus cantonensis into the rat pulmonary vessels and its application to the assessment of acquired resistance. International Journal for Parasitology9: 97–103. A simple procedure for the transplantation of young adult Angiostrongylus cantonensis into the rat pulmonary vessels was described and its potential usefulness was discussed. Acquired resistance to A. cantonensis was conferred on rats by infection with 30 third-stage larvae or by a similar infection followed by treatment with thiabendazole during 14–19 days postinfection. Nearly comparable levels of resistance to reinfection were also observed in rats that were transplanted with 30 young adult worms, collected from the brain surface of donor animals at 21 and 22 days postinfection. The time-course development of reaginic, hemagglutinating and precipitating antibodies differed in these groups of rats although there appeared to be no positive correlation between the appearance of these antibodies and acquired resistance.     

Angiostrongylus cantonensis: Scanning Electron Microscopic Observations on the Cuticle of Moulting Larvae

Angiostrongylus cantonensis is a parasitic nematode that needs to develop in different hosts in different larval stages. Freshwater snails, such as Pomacea canaliculata, are the intermediate host, and rats are the definitive host. Periodic shedding of the cuticle (moulting) is an important biological process for the survival and development of the parasite in the intermediate and definitive hosts. However, there are few studies on the cuticle alterations between different stages of this parasite. In this study, we observed the ultrastructural appearance and changes of the cuticle of the 2nd/3rd stage larvae (L2/L3) and the 3rd/4th stage larvae (L3/L4) using a scanning electron microscope. We also first divided L2/L3 into late L2 and early L3. The late L2 lacked alae, but possessed a pull-chain-like fissure. Irregular alignment of spherical particles on the cuticle were noted compared to the L3. Alae appeared in the early L3. The old cuticle turned into a thin film-like structure which adhered to the new cuticle, and spherical particles were seen regularly arranged on the surface of this structure. Regular rectangular cavities were found on the surface of L3/L4. The caudal structure of L3/L4 was much larger than that of L3, but caudal inflation, such as seen in L4, was not observed. These results are the first to reveal the ultrastructural changes of the cuticle of A. cantonensis before and after moulting of L2/L3 and L3/L4.     

Oesophagostomum radiatum: Glucose metabolism of larvae grown in vitro and adults grown in vivo

Larval stages of Oesophagostomum radiatum grown in vitro and adults grown in vivo were incubated in complex media or in a simple salt solution containing radioactive glucose. Glucose disappearance and end product accumulation of third-stage larvae in a simple salt solution indicated that they excreted CO₂ and acetic, propionic, and lactic acids. Larvae in third molt, fourth stage, and adults all excreted CO₂, acetic, propionic, and lactic acids at twice the rate of third-stage larvae plus an additional product, methylbutyric acid. Carbon dioxide arose primarily from the 3 or 4 carbons of glucose. An anaerobic atmosphere (95% N₂:5% CO₂) had no apparent effect on metabolism. When incubation was done in complex media, isobutyric and 3-methylbutyric acids were seen as major excretion products (10 and 24%, respectively). However, these acids were quantitatively minor when incubations took place in simple salts-glucose medium (1 and 0–3%, respectively).     

Development of Thelastoma bulhoesi (Oxyurata: Thelastomatida) and the Effect of Thiabendazole on the Unembryonated Egg

The embryological and postembryological development of Thelastoma bulhoesi was determined. Initial cleavage was into unequal cells and occurred within 1-2 hours at 25 C. Cell division was holoblastic but no true morula is formed. Gastrulation occurred at approximately 48 hours by epibolic synectic mechanisms. First-stage larvae were fully developed at 96 hours. The molt to second-stage larvae was initiated in the egg and was completed at hatching. Second-stage larvae were first observed in the host at 11 hours postinfection, third-stage larvae at 18 hours, and fourth-stage larvae at 192 hours. Adult female worms were observed at 32 days. Thiabendazole, in even the lowest concentrations, inhibited the developmem of unembryonated ova.     

PCR-Based Detection of Angiostrongylus cantonensis in Tissue and Mucus Secretions from Molluscan Hosts

Angiostrongylus cantonensis is a common cause of human eosinophilic meningitis. Recent outbreaks of this infection have shown that there is a need to determine the distribution of this nematode in the environment in order to control transmission. A. cantonensis is generally identified morphologically in the molluscan intermediate host by microscopic examination, which can be labor-intensive. The aim of this study was to develop a PCR-based method to detect A. cantonensis directly from molluscan tissue. A total of 34 Parmarion cf. martensi (Simroth) semislugs, 25 of which were naturally infected with A. cantonensis, were used to develop this assay. Tissue pieces (approximately 25 mg) were digested with pepsin-HCl to recover third-stage larvae for morphological identification or were used for DNA extraction. PCR primers were designed to amplify 1,134 bp from the Angiostrongylus 18S rRNA gene, and the amplicons produced were sequenced for identification at the species level. Both microscopy and the PCR-DNA sequencing analysis indicated that the same 25 semislugs were positive for A. cantonensis, showing that the two methods were equally sensitive and specific for this application. However, morphological detection requires access to living mollusks, whereas molecular analysis can also be performed with frozen tissue. The PCR-DNA sequencing method was further evaluated using tissue from Veronicella cubensis (Pfeiffer) slugs and mucus secretions from infected P. martensi. To our knowledge, this is the first use of a PCR-based method to confirm the presence of A. cantonensis in mollusks collected in the environment.     

Dirofilaria immitis: molting process of third-stage larvae

The objective of this study was to determine the molting process of Dirofilaria immitis third-stage larvae (L-3) to fourth-stage larvae (L-4), as it occurred in vitro. After 48 hr in vitro, the L-4 epicuticle was completely formed, and by 72 hr there was a clear separation between the L-3 and L-4 cuticles. The thickness of the newly formed L-4 cuticle was significantly less than that which has been described for larvae recovered from dogs after a similar incubation time period. If culture conditions were lacking in bovine albumin or proper temperature, larvae successfully developed the L-4 epicuticle but did not complete ecdysis. The molting process of D. immitis L-3 was thus shown to be multistepped with different factors required to induce the various developmental phases.     

Development of Dipetalonema viteae third-stage larvae (Nematoda: Filarioidea) in micropore chambers implanted into jirds, hamsters, normal and immunized mice

Development of third-stage larvae of Dipetalonema viteae within subcutaneously implanted micropore chambers proceeded in all hosts tested up to the fourth-stage larvae and occasionally to adolescent worms. In the jird the timing of development was comparable to a natural infection. Although the mouse is an insusceptible host, larval development could take place, but was very slow. Two intraperitoneal inoculations of living third-stage larvae into mice induced the production of antibodies against the larval cuticle and against common antigens. In such immune mice the development of third- and fourth-stage larvae within micropore chambers was significantly inhibited, larval mortality was increased, and the larval motility was impaired.     

In vitro cultivation of Dipetalonema viteae third-stage larvae: effect of the gas phase

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.     

Microtopography of Nippostrongylus brasiliensis (Nematoda: Heligmosomatidae): Parasitic larval stages and adults

Specimens of the rat hookworm, Nippostrongylus brasiliensis (Nematoda) were recovered from lungs (third- and fourth-stage larvae) and intestine (fourth-stage larvae and adults). The following features were studied in the different stages by scanning electron microscopy: cephalic structures, especially sense organs, synlophe, cervical region, and caudal part. The main differences between the third and fourth stages concerned the lip-like structures around the oral aperture, the appearance of the cephalic space with the presence of a cephalic cap in fourth-stage larvae, the pattern of longitudinal ridges, and sexual differentiation. Pore-like papillae, not seen in third-stage larvae, developed in later stages. Deirids were observed only in adults, and phasmids were poorly discerned. Some of these morphological features, such as the cephalic sense organ apertures and cuticle pores and micropores, can be observed only by scanning electron microscop y. The possible functions of these different structures and their relationship with the behavior of the worms during their life cycle are discussed. © 1993 Wiley-Liss, Inc.     

Morphological studies of Ancyracanthopsis winegardi Wong & Anderson, 1990 (Nematoda: Acuarioidea) and larval stages of acuariid nematodes parasitic in Larus dominicanus Lichtenstein (Aves: Laridae) from Argentina

Ancyracanthopsis winegardi Wong & Anderson, 1990 (Nematoda: Acuarioidea) is described from Larus dominicanus Lichtenstein (Aves: Laridae) on the Southwest Atlantic coast (38° 42S, 59° 47W). The main character used to distinguish species of Ancyracanthopsis is the morphology of the ptilina. Thus, although the specimens described here have some differences in the morphology and size of the spicules and in the female genitalia, they were referred to A. winegardi because they have a very similar ptilina. This is the first record of a member of Ancyracanthopsis from larid birds and for A. winegardi in the Southwest Atlantic coast. We have also studied acuariid larvae found inhabiting the gizzard alongside adult specimens of A. winegardi. Among those larvae, two morphological groups were clearly distinguished. The first group was characterised by the absence of ptilina and the presence of spicular primordia and rectal cells (third-stage larvae). The second group could be distinguished by the presence of ptilina and partly-developed genitalia (fourth-stage larvae). In order to identify the larvae, a Principal Component Analysis was applied to morphometric data taken from the third-stage larvae. These results and the morphology of the partly-developed ptilina of the fourth-stage larvae indicated that the larval stages found in L. dominicanus appear to belong to Sciadiocara haematopodi, Cremonte, Navone & Etchegoin, 1999.     

Kinetic analysis of oil biosynthesis by an arachidonic acid-producing fungus, Mortierella alpina 1S-4

 Kinetic analysis of arachidonic acid (AA)-oil biosynthesis by Mortierella alpina 1S-4 growing under lipid-accumulating (LN medium) and non-lipid-accumulating (HN medium) conditions was investigated and compared with industrial AA fermentation. Various kinetic parameters of these cultivation processes demonstrate a characteristic pattern of the lipogenesis in this fungus, where growth phase, phase of oil accumulation and phase of AA synthesis are distinct from each other. The fungus utilizing LN medium synthesized 32.3 g fatty acid 100 g⁻¹ glucose on the 4th day of cultivation and reached the maximum daily fatty acid accumulation (expressed as differential specific rate q _D(FA/B)) of 9.5%. Our results also indicate that a q _D(FA/B) value of about 2.5% might be critical for lipid overproduction in M. alpina. AA was rapidly incorporated into triacylglycerols (90% of total AA) at the later cultivation phase and overall AA yield was directly related to the total yield of fatty acid. Received: 10 December 1999 / Accepted: 13 February 2000     

Development of Ascaris suum larvae from the third to fourth stage, in vitro

Larvae of Ascaris suum recovered from the lungs of experimentally infected guinea pigs or rabbits, seven to ten days after infection, underwent the third moult in vitro to become fourth-stage larvae. Ecdysis was first observed on the first and second day of culture and was completed by the eighth day. The culture medium consisted of tissue culture Medium 199 supplemented with either guinea pig or porcine serum and glucose in a gaseous atmosphere of Nitrogen:Oxygen:Carbon dioxide (90:5:5). By the twelfth day in culture, early development of both the male and female reproductive systems was observed.