Angiostrongylus cantonensis (Nematode: Metastrongiloidea): In Vitro Cultivation of Infective Third-Stage Larvae to Fourth-Stage Larvae

The present study to attempt to cultivate Angiostrongylus cantonensis from third-stage larvae (AcL3) to fourth-stage larvae (AcL4) in vitro in defined complete culture medium that contained with Minimum Essential Medium Eagle (MEM), supplemented amino acid (AA), amine (AM), fatty acid (FA), carbohydrate (CA) and 20% fetal calf serum (FCS) was successful. When AcL3 were cultured in the defined complete culture medium at 37°C in a 5% CO₂ atmosphere, the larvae began to develop to AcL4 after 30 days of cultivation, and were enclosed within the sheaths of the third molts of the life cycle. Under these conditions, the larvae developed uniformly and reached to the fourth-stage 36 days. The morphology of AcL3 develop to AcL4 were recording and analyzing. Then comparison of A. cantonensis larval morphology and development between in vitro cultivation in defined complete culture medium and in vivo cultivation in infective BALB/c mice. The larvae that had been cultivated in vitro were smaller than AcL4 of infective BALB/c mice. However the AcL3 that were cultured using defined incomplete culture medium (MEM plus 20% FCS with AA+AM, FA, CA, AA+AM+FA, FA+CA, CA+AA+AM or not) did not adequately survive and develop. Accordingly, the inference is made that only the defined complete medium enable AcL3 develop to AcL4 in vitro. Some nematodes have been successfully cultured into mature worms but only a few researches have been made to cultivate A. cantonensis in vitro. The present study is the first to have succeeded in developing AcL3 to AcL4 by in vitro cultivation. Finally, the results of in vitro cultivation studies herein contribute to improving media for the effective development and growth of A. cantonensis. The gap in the A. cantonensis life cycle when the larvae are cultivated in vitro from third-stage larvae to fourth-stage larvae can thus be solved.     

Trichinella spiralis: site selection by the larva during the enteral phase of infection in mice

Mice, belonging to two strains, were infected by the oral route with muscle larvae of Trichinella spiralis. Host animals were killed at various times up to 48 hr after administration of larvae, and the infected small intestines were fixed immediately in 10% neutral formalin. Sections of infected gut, embedded in paraffin and cut at 5 μm, or in methacrylate and cut at 0.5 μm, revealed that all stages (i.e., 1 to 4) of T. spiralis were embedded between the lamina propria and the columnar epithelium. First-stage muscle larvae occupied this niche as early as 10 min after introducing them into the host by the oral route.     

Brugia malayi: intravenous injection of microfilariae in ferrets as an experimental method for occult filariasis

Microfilaremia, immune responses, and pathology were compared in ferrets infected with 100 third-stage larvae of Brugia malayi (subperiodic strain) or injected intravenously with 10(6) microfilariae. Ferrets (Mustela putorius furo) inoculated with third-stage larvae typically became patent during the third month after infection, with a mean patency of 123 +/- 25 (SE) days. Ferrets injected intravenously with microfilariae exhibited a relatively constant microfilaremia for 3-4 weeks and usually cleared microfilariae before the fourth month. Ferrets that cleared microfilariae after intravenous injection of microfilariae or after infection with third-stage larvae failed to become patent or became amicrofilaremic within 3 weeks after a challenge intravenous injection of 10(6) microfilariae. Clearance of circulating microfilariae was associated with eosinophilia and serum antibody specific for the microfilarial sheath in ferrets injected with microfilariae and in most ferrets infected with third-stage larvae. Ferrets infected with third-stage larvae and necropsied after clearance of microfilariae had tissue inflammatory reactions to microfilariae characteristic of occult filariasis (tropical eosinophilia) in man; these ferrets exhibited immediate cutaneous hypersensitivity and circulating reaginic antibody to antigens of microfilariae. In ferrets necropsied following two intravenous injections of microfilariae, the majority of ferrets examined within 10 days after clearance of microfilariae had visible liver lesions to microfilariae identical to those of the ferrets infected with third-stage larvae; immediate cutaneous hypersensitivity and reaginic antibody were not consistently detected in ferrets injected with microfilariae. Sera from ferrets that had cleared circulating microfilariae were transferred passively into ferrets made microfilaremic by intravenous injection of microfilariae. Sera with microfilarial sheath-reactive IgG antibody titers (greater than or equal to 1:200) and microfilarial agglutination titers (greater than or equal to 1:40) rapidly cleared injected microfilariae (less than 24 hr); this serum also cleared or greatly reduced circulating microfilariae established by an infection with third-stage larvae; only the IgG-containing fraction of the sera was active in immune clearance. Sera that cleared microfilariae of B. malayi did not clear circulating microfilariae of Dirofilaria immitis or prevent recurrence of circulating microfilariae of B. malayi in ferrets infected with adult filariae.(ABSTRACT TRUNCATED AT 400 WORDS)     

Experimental life cycle of Lagochilascaris major leiper, 1910 (Nematoda: Ascarididae) in cats (Felis domesticus)

The life cycle of Lagochilascaris major was studied using eggs collected from a natural clinical case in a domestic cat. Twenty-seven white mice (Mus musculaus), 5 hamsters (Mesocricetus auratus), and 1 vesper mouse (Calomys callosus) were orally inoculated with 800-1,300 embryonated eggs. When examined from 73 to 246 days postinoculation (PI), encysted third-stage larvae were seen in skeletal muscles and less frequently in connective tissue, liver, and lungs. Twenty-two of the 23 cats orally inoculated with 40-430 encysted larvae from these rodents, and necropsied from 1 hr to 185 days PI, became infected. Third-stage larvae were located in the stomach, esophagus, and oropharynx from 1 to 24 hr PI. At 48 hr, larvae, from mainly the fourth stage, were only found, unilaterally or bilaterally, inside a "sac" in the region of the semilunar fold of the palatine tonsil at the base of the tongue. Adult worms were found in this location from 10 to 175 days PI. No fistulated abscess to the outside medium was found. Adult worms were also found in the middle ears of 2 cats showing purulent otitis. Eggs in the ear secretion were under different stages of development. Eggs in feces were first observed on days 14 and 15 PI, and 1 cat shed them until 178 days PI. Six infected cats were treated with fenbendazole at 50 mg/kg of body weight for 3 consecutive days, eliminating all the parasites present in the tonsils. The drug was not effective against the parasites present in the middle ear. No stage of the parasite was found in the tissues of 5 cats given 4,000-5,200 eggs orally and examined after 19 and 50 days PI. This indicates that the life cycle of L. major requires an obligate paratenic host and is characterized by heteroxenic cycle.     

Changes in the structure of Nippostrongylus brasiliensis intestinal cells during development from the free-living to the parasitic stages

The ultrastructure of Nippostrongylus brasiliensis intestinal cells was examined in free-living, feeding second-stage larvae, infective, nonfeeding third-stage larvae, and parasitic, feeding third-stage larvae. The intestinal cells of second-stage larvae were characterized by a well-developed microvillar border, large numbers of ribosomes, Golgi complexes, rough endoplasmic reticulum, and nuclei with prominent nucleoli. The intestinal cells of infective, third-stage larvae had very few microvilli and the cells were extremely narrow. Few ribosomes, Golgi complexes, and little rough endoplasmic reticulum were present. Nuclei did not contain nucleoli. When worms were introduced into an in vitro culture system, development of intestinal cells began. By 36 hr, microvilli were well differentiated and the cysoplasm contained numerous ribosomes and Golgi complexes, and rough endoplasmic reticulum, mitochondria, and nucleoli were prominent. These morphological changes were related to changes in the physiology of Nippostrongylus brasiliensis which occur during development from a free-living to parasitic form.     

The tissue reactions of mice to infection with Heligmosmoides polygyrus

The intestines of normal and resistant LAF1 mice were subjected to histologic study to determine the timing and mechanisms of resistance to reinfection by Heligmosmoides polygyrus. During reinfection third-stage larvae are less able to penetrate the intestinal wall. Larvae which are able to encyst develop at a slower rate and provoke an increase in nonspecific inflammation around their cysts. After emergence from intestinal cysts, preadults are rapidly lost, but at no time were injured or destroyed larvae or adults noted. Exsheathed larvae were injected via tail vein into control, sensitized and resistant BALB/c mice. The inflammatory response around entrapped larvae in the lung was measured at 1, 2, 4, and 8 days. A heightened inflammatory response, consisting primarily of polymorphonuclear cells with some round cells which peaked in size on day 2, was observed in both sensitized and resistant mice. A similar heightened inflammatory response was also observed in both AKR (non-resistant) BALB/c (resistant) mice vaccinated subcutaneously with exsheathed larvae.     

Life cycle of Gnathostoma nipponicum Yamaguti, 1941.

The life cycle of Gnathostoma nipponicum was examined by field survey and by experimental infection of animals with the larvae. Naturally infected larval G. nipponicum were found in loaches, catfish, and snakes. Experimentally, loaches, killifishes, frogs, salamanders, mice, and rats were successfully infected with the early third-stage larvae of G. nipponicum obtained from copepods (the first intermediate host), whereas snakes, quails, and weasels were not. Frogs, snakes, quails, and rats were experimentally infected with the advanced third-stage larvae (AdL3) from loaches. These results reveal that some species of fishes, amphibians and mammals can act as the second intermediate host and that some species of reptiles, birds and mammals can act as a paratenic host. The life cycle was completed in weasels, the definitive host, which were infected with AdL3 from loaches and started to evacuate eggs of G. nipponicun in faeces on days 65-90 postinfection.     

Histopathology of a nuclear polyhedrosis infection in Aedes epactius with observations in four additional mosquito species

Aedes epactius larvae were utilized to study the infection sequence of the nuclear polyhedrosis virus (NPV) from Aedes sollicitans. From 30 min to 6 hr postinoculation, polyhedra and many free virions were observed in the larval midgut lumen. Penetration of the midgut cells by virions was not observed. The first infected nuclei were observed 12 hr postinoculation. Nucleocapsids initially exhibited electron translucent cores which became electron dense before the nucleocapsids acquired an envelope. Envelope acquisition occurred through a process of de novo membrane morphogenesis. Occlusion of the singly embedded virions began by 18 hr postinoculation with the mature rough-surfaced polyhedra averaging approximately 1 by 2 μm. Unusually long nucleocapsids (approximately two or three times the length of other nucleocapsids) were only observed in late infection period nuclei. There was no evidence that long nucleocapsids represented an early developmental stage for nucleocapsids of standard length. Infection was restricted to midgut nuclei and gastric caecae cells. Infected early instar A. epactius larvae became moribund 36 to 40 hr postinoculation and infected midgut nuclei were observed to undergo lysis. The late stages of NPV infection were observed in larvae of A. annandalei, Wyeomyia smithii, Toxorhynchites brevipalpus, and Eretmapodites quinquevittatus. Virion development and occlusion in these species was basically identical to the sequence observed in A. epactius larvae.     

Arrested development and the histotropic phase of Ascaridia galli in the chicken

Studies on 300 worm-free chickens infected with Ascaridia galli indicated that the histotropic phase is a normal part of the life cycle and that it involves both second- and third-stage larvae. The duration of the histotropic phase was dose-dependent. At low dose rates (50 eggs) it occurred from days 3 to 16 and was terminated abruptly by the third ecdysis. At high dose rates (2000 eggs) it was prolonged until at least day 54, because third-stage larvae became arrested in their development and the third ecdysis was delayed. Arrested development of Ascaridia galli was significantly suppressed following treatment of birds with cyclophosphamide, but expulsion of worms was not prevented. This result suggests the involvement of host antibodies in the induction of the arrested state.     

Tissue eosinophil numbers and phospholipase B activity in mice infected with Trichinella spiralis

Wilkes S. D. and Goven A. J. 1984. Tissue eosinophil numbers and phospholipase B activity in mice infected with Trichinella spiralis. International Journal for Parasitology14. 479–482. Tissue eosinophils were counted and phospholipase B activity was assayed in the intestines of mice infected with 200 Trichinella spiralis larvae. The numbers of intestinal eosinophils and phospholipase B activity increased, peaked and returned to normal levels during the same time period. The findings support the hypothesis that a parasite-induced tissue eosinophilia is the source of elevated phospholipase B activity present in parasitized tissues.     

Inhibition of the accumulation of viral polypeptides in the densonucleosis virus-infected midgut of the silkworm,Bombyx mori,reared at a supraoptimal temperature

Effect of a supraoptimal temperature on the accumulation of viral polypeptides in the midgut was examined by immunoblot analysis in the larvae of the silkworm, Bombyx mori, infected with Bombyx densonucleosis virus type 2. In the larvae reared continuously at 25°C, viral polypeptides were first detected in the midgut at 2 days postinfection (pi) and in the feces at 4 days pi. When the larvae inoculated per os with the virus for 24 hr at 25°C were immediately shifted to 35°C, there were no detectable viral polypeptides in both the midgut and feces throughout the experiment. In the infected larvae shifted from 25° to 35°C at 48 hr pi, viral polypeptides preexisting in the midgut decreased to an undetectable level within 48 hr after the temperature shift, and no viral polypeptides were detected thereafter. Viral polypeptides in the feces of these larvae became detectable at 48 hr (4 days pi) after the temperature shift, as in the larvae at 25°C, and disappeared by 96 hr (6 days pi). These results indicate that a supraoptimal temperature inhibits accumulation of viral polypeptides in the midgut. It is likely that inhibited production of viral polypeptides rather than enhanced discharge of the infected midgut cells is responsible for the inhibited accumulation of viral polypeptides in the midgut at 35°C.     

Total anaerobiosis during in vitro culture in conventional cell culture media is required for retaining infectivity of Trichinella spiralis L1 larvae

The infectivity of Trichinella spiralis L1 larvae was examined in Swiss CD-1 mice after their maintenance in conventional cell culture media under different atmospheric conditions. Larvae isolated from the infected mouse carcasses were cultured for 24 hr in Roswell Park Memorial Institute (RPMI) medium, minimum essential medium (MEM), 199 medium, and Hank's balanced salt solution (HBSS) medium under anaerobic, microaerobic, and 5% CO2 conditions. Only those larvae maintained under anaerobiosis in all media retained their infectivity in mice. The larvae maintained microaerobically and under 5% CO2 lost more of their infectivity when cultured in RPMI medium and MEM (> 96%) than in 199 and HBSS (> 78%).     

Trans-ovum transmission of a cytoplasmic polyhedrosis virus of Heliothis virescens (Lepidoptera: Noctuidae)

Adults of Heliothis virescens infected with a cytoplasmic polyhedrosis virus (CPV) produced healthy offspring when their eggs were surface sterilized with either 15% formaldehyde or 0.2% sodium hypochlorite solution. Larvae from infected parents (1) cultured on a vitamin-deficient medium, (2) exposed to cold treatment (5°C, 24 hr), or (3) as progeny of adults from diapaused infected pupae, produced the same number of infected individuals as larvae reared in the customary way. Field studies indicated that the percent of CPV infection in larvae originating from virus-infected parents was density dependent.     

Heligmosomoides polygyrus: excretory/secretory antigens released in vitro by exsheathed third-stage larvae

The excretory/secretory antigens released during in vitro culture of infective third-stage Heligmosomoides polygyrus larvae were analyzed by enzyme-linked immunosorbent assay and immunoblotting using sera from repeatedly infected mice. During the first 8-10 hr of culture at 37 C, freshly exsheathed larvae released only one antigen that cosedimented with trypsin (24 kDa) upon ultracentrifugation and was composed of a single 23-kDa polypeptide chain. After 10 hr of culture, the larvae released additional antigens identified by bands equivalent to polypeptides of approximately 18, 25, 26, 32, 58, and 76 kDa on nonreduced Western blots. The release of these molecules was maintained for up to 60 hr. Their staining intensity on blots was in the order 23 much greater than 25 greater than 76 greater than 18 greater than or equal to 58 greater than or equal to 32 greater than or equal to 26 kDa. Velocity sedimentation analysis showed that the 76-kDa component exists as a monomeric 76-kDa "native" antigen. The 32-, 58-, and 76-kDa antigens were specifically adsorbed by concanavalin A (Con A)-Sepharose and the 76-kDa molecule was detected on blots incubated with alkaline phosphatase-conjugated Con A, indicating the presence of mannose-like residues on these molecules. The 18-, 23-, 25-, and 26-kDa antigens did not bind to Con A-Sepharose. Hyperimmune antisera raised against lyophilized larvae had negligible antibody activity against the larval ES antigens, suggesting that the ES antigens are released soon after synthesis rather than being stored in significant quantities within the larvae.     

Nematospiroides dubius: susceptibility to infection and the development of resistance in hypothymic (nude) BALB/c mice.

BALB/c-nu/nu mice and their intact nu/+ littermates are equally susceptible to infection with third-stage larvae of Nematospiroides dubius. Unlike their heterozygous littermates, however, the nu/nu mice are unable to form ganulomata in the intestinal wall and become only partially resistant to rechallenge. Following two or more infections, nu/nu mice maintain a high burden of adult intestinal worms, whereas worms are lost from immune nu/+ mice. Studies in T cell-injected nu/nu mice suggest that a full complement of T cells is needed to develop maximum resistance against the infective third-stage larvae and to expel adult worms. Measurement of serum immunoglobulin levels indicate that infected nu/+ mice have very high levels of IgG1 whereas the levels of IgG2a are reduced. In infected T cell-injected nu/nu mice, IgG1 levels increase with the number of T cells injected, whereas IgG2a levels are variable but always higher than in infected nu/+ mice.     

Kinetics of primary and secondary infections with Strongyloides ratti in mice

Dawkins H. J. S. and Grove D. I. 1981 Kinetics of primary and secondary infections with Strongyloides ratti in mice. International journal for Parasitology11: 89–96. The kinetics of infection with S. ratti were quantitated in normal and previously exposed C57B1 /6 mice. In primary infections, larvae penetrated the skin rapidly and were seen in peak numbers 12 h after infection. By 24 h after infection, larval numbers had declined appreciably and there was a slow decrease in numbers thereafter. Larvae were first observed in the lungs at 24 h and maximal recovery occurred at 48 h. It is thought that larval migration through the lungs is rapid. Worms were first seen in the intestines two days after infection. Maximum numbers were seen on the fifth day and worm expulsion was complete by day 10. Two moults took place in the small intestine during days 3 and 4 after infection. Rhabditiform larvae were first noted on the fourth day after infection. Mice exposed to S. ratti four weeks previously had significantly less larvae in the skin 4 and 12 h after infection but by 24 h there was no difference when compared with mice with primary infections. Peak recovery of larvae from the lungs occurred 24 h after infection; significantly less larvae were recovered on days 2 and 3 when compared with normal mice. There was a marked reduction in the adult worm burden in the gut; the number of worms recovered was less than one fifth of that seen in primary infections. Those worms which did mature were less fecund and were expelled from the intestines within 7 days of infection. It is suggested that in previously exposed animals, the migration of larvae from the skin is hastened, many of these larvae are destroyed in the lungs and that expulsion of worms which do mature in the intestines is accelerated.     

Embryology and Life Cycle of Tylenchorhynchus claytoni Steiner, 1937 (Nematoda: Tylenchoidea)

Development of Tylenchorhynchus claytoni from unsegmented egg to hatching takes 135 hr at 22-25 C. The fourth molt lasts 5 to 6 days. During exsheathment the cast cuticle of the larva separated into two unequal parts, breaking near either the anterior or posterior end. The life cycle from egg to egg required from 31 to 38 days at 28 C on alfalfa seedlings and included four molts and four larval stages. Sexual differentiation was apparent in third-stage larvae.     

Thelazia lacrymalis in horses in Kentucky and observations on the face fly (Musca autumnalis) as a probable intermediate host.

Eyes from 114 (30.3%) of 376 dead horses, examined from 3 April 1975 to 3 April 1976, were naturally infected with adult Thelazia lacrymalis; 1 horse was also infected with 1 male Thelazia skrijabini. Adult T. lacrymalis from dead horses were successfully transferred mechanically to the eyes of 3 of 4 Shetland ponies raised helminth-free. Larvae from gravid female T. lacrymalis underwent development in experimentally infected, laboratory-raised face flies (Musca autumnalis) and third-stage larvae ranging from 1.82 to 2.94 mm in total length were recovered at 12 to 15 days postexposure. A total of 866 naturally occurring face flies were collected from the head region of horses. Twelve of the face flies harbored larval stages of Thelazia spp. One of the larvae resembled third-stage T. lacrymalis that were recovered from the experimentally infected, laboratory-raised face flies. Introduction of 3 third-stage larvae from 1 face fly onto the cornea of a pony raised helminth-free resulted in the recovery of 1 male T. skrjabini 242 days later. In addition to the eyeworm larvae, other parasites recovered from the face flies included Heterotylenchus autumnalis, hypopi of astigmatid mites and a first instar beetle (Coleoptera: Rhipiphoridae). Data from these investigations indicate the likelihood that face flies are an intermediate host for T. lacrymalis and probably other species of Thelazia in this part of the country.     

Commercial blast-freezing of third-stage Anisakis simplex larvae encapsulated in salmon and rockfish

Sixty-four fish were blast-frozen to -35 C for 15 hr to determine the effects of commercial blast-freezing on the viability of third-stage larvae of Anisakis simplex encapsulated in the muscle and viscera of sockeye salmon (Oncorhynchus nerka) and canary rockfish (Sebastes pinniger). Parallel tests were conducted on larval nematodes in 16 whole (round) salmon, 16 dressed salmon (heads and viscera removed), and 32 whole (round) rockfish. After blast-freezing, 4 in-the-round salmon, 4 dressed salmon, and 8 in-the-round rockfish were examined at 1, 24, 48, and 72 hr. A total of 3,539 dead and 6 live larvae were collected from the fish tissues after standard enzymatic digestion. Salmon were infected with 1,245 of these larvae, and rockfish with 2,300. The 6 live worms, 2 from salmon and 4 from rockfish rounds, were recovered from muscle 1 hr after freezing; they were slightly motile and showed severe internal damage. No viable worms were found at or after 24 hr. The commercial blast-freezing process effectively killed larval nematodes in whole or dressed fish. Market-ready samples of previously blast-frozen silver salmon (O. kisutch) and chum salmon (O. keta) fillets and chum salmon steaks yielded no live worms, thereby confirming the efficacy of this process.