A simple method for maintaining large, aging populations of Caenorhabditis elegans.

This paper describes a technique capable of establishing and maintaining large, age-synchronous populations of the nematode Caenorhabditis elegans. The technique has three essential components: a rich chemical medium; a method for producing and harvesting mass quantities of eggs; and 5-fluorodeoxyuridine (FUdR), an inhibitor of DNA synthesis. A culture of worms is filtered through glass wool or a wire screen to isolate young larvae. Eggs laid by these worms after they mature are collected over a period of 4-6 hours and allowed to hatch. A low level of FUdR (25 microM) is added just before the larvae reach maturity. This timing is important to avoid developmental abnormalities. The adults lay eggs in the presence of FUdR but the eggs do not hatch, which maintains the synchrony of the culture. Many aging characteristics appear to be similar in treated and untreated worms, such as the time of cessation of egg production, the appearance of visible and behavioral age-related changes, and the mean lifespan. This system thus seems suitable for large-scale biochemical analysis of certain aspects of aging in C. elegans.

Purification, characterization, and immunochemical studies of beta-N-acetyl-D-hexosaminidase from the parasitic nematode Trichinella spiralis.

The exoglycosidase, beta-N-acetyl-D-hexosaminidase was purified 600-fold from the muscle-stage larvae (L1) of Trichinella spiralis. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the purified enzyme-active fraction contained 4 polypeptides with apparent molecular weights of 100,000, 68,000, 58,000 and 54,000. The beta-N-acetyl-D-hexosaminidase corresponds to the Mr 100,000 polypeptide as demonstrated by SDS-PAGE analysis of the enzyme-stained region isolated from a non-denaturing polyacrylamide gel. In addition, rabbit antiserum to a homogeneous preparation of the Mr 100,000 polypeptide (isolated by electroelution from an SDS-PAGE gel) specifically immunoprecipitated beta-N-acetyl-D-hexosaminidase activity from an extract of L1. Isoelectrofocusing (pH 3-10) resolved 4 isoenzymes of T. spiralis beta-N-acetyl-D-hexosaminidase with isoelectric points (pI) of 5.35, 5.49, 5.63 and 5.79. The T. spiralis beta-N-acetyl-D-hexosaminidase is a glycoprotein based on its binding to lentil-lectin Sepharose affinity column and its specific binding of concanavalin A on Western blots. The IgG fraction of T. spiralis-infected mouse serum specifically immunoprecipitated T. spiralis beta-N-acetyl-D-hexosaminidase. The removal of carbohydrate from T. spiralis beta-N-acetyl-D-hexosaminidase significantly reduced its antigenicity. Immunocytochemical analysis of L1 tissue sections with polyclonal rabbit antisera to the homogeneous beta-N-acetyl-D-hexosaminidase enzyme indicated localization on cell membranes and the epicuticle.

Dioctophyme larva in the subcutaneous tissues of a woman in Ohio.

A nematode larva undistinguishable from Dioctophyme renale was found in the subcutaneous tissues of the abdomen of a 23-year-old woman from Ohio. This is the third case of Dioctophyme larva reported in humans. Although it is not known how the infection was acquired, we suggest that ingestion of raw fish was the probable source. We further hypothesize that such a larva could eventually migrate to the kidney and grow to the adult state.

Population dynamics of microfilarial production and eosinophilic levels in slow lorises infected with Breinlia sergenti. Petter (Filarioidea: Dipetalonematidae).

Population dynamics of microfilarial production and eosinophilic levels in slow lorises infected with Breinlia sergenti, Petter (Filarioidea: Dipetalonematidae). International Journal for Parsitology 4: 383388. Observations have been made on microfilarial and eosinophilic levels in slow lorises infected with Breinlia sergenti. Animals given a single inoculation of 100-150 infective larvae exhibited three different patterns of microfilaraemia while superinfected animals showed enhanced microfilarial levels. It appeared that the number of inoculations as well as the interval between inocula are important factors in enhancing microfilarial levels. Two different types of incubation periods were seen, one at 100-120 days and the other at 200 days. The eosinophilic levels were investigated in some of the animals and an attempt was made to correlate these levels with the microfilaraemia. Cortisone injection appeared to promote a vigorous eosinophilia in some of the infected animals tested.

Two cases of enteric myiasis in man. Pseudomyiasis and true intestinal myiasis.

Dipterous larvae were found in the appendices from two postmortem examinations. The single maggot in the first case was not immediately identified in the sections. However, when the posterior end of the larva was recovered from the unsectioned portion of the appendix, it was identified as a maggot of the genus Sarcophaga. The first case was believed to be a case of "pseudomyiasis,+ i.e., the accidental entrapment of a swallowed larva passing through the digestive tract. In the second case, the appendix contained numerous larvae with great variation in sizes and stages of development of the mouth hooks, pharyngeal sclerites, and spiracular breathing plates. Such development was considered to have occurred within the intestinal tract, which indicated that this was a case of true intestinal myiasis.

An evaluation of the hypothesis of transovarial transmission of eastern equine encephalomyelitis virus by Culiseta melanura.

Evidence for and against the hypothesis of transovarial transmission by Culiseta melanura was obtained during an ongoing eastern equine encephalomyelitis (EEE) surveillance and control program. Evidence inconsistent with transovarial transmission included failure to isolated virus from 1,047 larvae, from 2,140 first-brood adults, or from 8,919 males collected at the same time as 3,977 nonblooded females which yielded 12 EEE isolates. Evidence supporting the hypothesis was the isolation of virus from both blooded and nonblooded adults simultaneously and also from a population with a parity rate so low that the infection rate for parous specimens would have been 1:8. Two alternative hypotheses which assume transovarial transmission are advanced to explain these results, but they seem so unlikely that the data are interpreted as opposing the concept.

Concentration from continuous culture of erythrocytes infected with trophozoites and schizonts of Plasmodium falciparum.

Gelatin, from readily available sources, at specified concentrations in RPMI 1640 medium enhances sedimentation rates of suspensions of Plasmodium falciparum-infected erythrocytes from continuous cultures, resulting in an enrichment of the trophozoite- and schizont-infected cells to parasitemias of 75% or more with no deleterious effects to the parasites.

Larvae of Anisakidae in marine fish of coastal waters near Jakarta, Indonesia.

Three species of marine fish were collected from the waters around Seribu Islands, near Jakarta, Indonesia, and examined for nematode larvae of the family Anisakidae. Larvae were found in 719 (49%) of 1.459 Rastrelliger kanagurta, 445 (50%) of 884 Decapterus russelli, and 217 (41%) of 531 Sardinella sirm. Larvae from a subsample of 150 infected fish, 44 R. kanagurta, 86 D. russelli, and 20 S. sirm, were examined microscopically and only Anisakis type I and Terranova type B larvae were found. In all three species of fish the Anisakis larvae predominated. The Anisakis larvae found in these fish are a potential source of infection for humans in Indonesia; however, human anisakiasis has not yet been reported from this country.

Transmission of Onchocerca volvulus by secondary vectors in Guatemala.

A comprehensive study of the transmission of Onchocerca volvulus at 4 locations in Guatemala with different prevalence rates of onchocerciasis included observations on potential secondary vectors, the most prevalent of which were Simulium metallicum, S. callidum, and S. gonzalezi. Filariae encountered in S. metallicum were primarily of a Dipetalonema-like species, but third-stage larvae indistinguishable from O. volvulus were found in 4 flies of this species. Our findings suggest that O. volvulus may occasionally be transmitted by S. metallicum, but such transmission is likely limited to areas having both a high parasite prevalence maintained by S. ochraceum and a relatively high host-seeking density of S. metallicum. Two third-stage larvae that could not be differentiated from O. volvulus were found once in S. gonzalezi; however, transmission by this species appears to be inconsequential.

Anisakid larva from the throat of an Alaskan Eskimo.

An anisakid nematode, identified as Phocanema sp. fourth-stage larva, was "pulled" from the throat of a 46-year-old Alaskan Eskimo. The nematode is described and illustrated.

The effects of repetitive community-wide ivermectin treatment on transmission of Onchocerca volvulus in Guatemala.

The effects of biannual ivermectin treatment at the community level on transmission of Onchocerca volvulus during the dry season were measured over a 30-month period in Guatemala. In the Los Tarrales Transmission Zone, an area encompassing three villages, significant changes occurred in both the prevalence and quantity of infection in the Simulium ochraceum vector population. These included a 76% reduction in females with infective stage larvae (L3S) and an 80% reduction in number of L3S per 1,000 parous flies. Significant reductions in both the mean infective biting density (IBD) and mean transmission potential (TP) also occurred. In Santa Emilia, the prevalence of infection with L3S in S. ochraceum was significantly reduced by 77% from the baseline value. The number of O. volvulus L3S per 1,000 parous flies was also reduced by 92%. Changes in both the IBD and TP were substantial but not significant due to the high degree of variance in the occurrence of O. volvulus L3S in the vector population. This was due, in part, to the movement of infected migrant workers into the finca (coffee farm). In Los Andes, four recurrent treatments successfully blocked transmission of infective stage larvae. Prevalence (flies with all stages of developing larvae) in the vector population was reduced by 89% over the two-year period; yearly reductions in both the IBD and TP were also highly significant, ultimately ending in zero values. This finding is particularly striking since prior to treatment, Los Andes exhibited the highest IBD of the three study locations and the second highest TP.

Egg production of Oesophagostomum bifurcum, a locally common parasite of humans in Togo.

In northern Togo and northeastern Ghana, Oesophagostomum bifurcum is a common parasite in humans. Diagnosis is based on coproculture because the eggs of hookworm and Oesophagostomum are indistinguishable. To determine the level of egg production, 12 subjects were treated with 2 x 10 mg/kg of pyrantel pamoate and the worms they evacuated were then counted. Pretreatment and post-treatment species-specific egg counts were calculated on the basis of larval and total egg counts. The median worm burden was 81 (range 12-300) per person. The calculated median egg production was 33.7 egg/gram of feces per female worm. Assuming a total daily stool production of 150 g/day, this amounts to 5,055 eggs/day, which is comparable with the production of other nematodes of the same superfamily.

Arrested development in Ancylostoma duodenale: course of a self-induced infection in man.

The course of self-induced infection with about 100 Ancylostoma duodenale larvae was followed by twice-weekly blood and stool examinations. After a small initial increase, no further change in eosinophil counts occurred until the 33rd week of infection when they began to increase sharply. Seven weeks later, eggs were first observed in stools. Thus, in this infection, the prepatent period was five times the expected. It is suggested that a strain of A.duodenale exists which either 1) has an inherent, abnormally long prepatent period or 2) has a prepatent period which is susceptible to change depending upon the environmental conditions experienced by the free-living larvae. In either case, the third stage larvae probably enter a hypobiotic state within the host which lasts for about 8 months.

The finding of Angiostrongylus cantonensis in rats in New Orleans.

Twenty of 94 (21.4%) Rattus norvegicus trapped in New Orleans, Louisiana, between April 1986 and February 1987 were infected with Angiostrongylus cantonensis (3-62 worms per rat). This is the first report of the parasite from North America. A carnivorous snail, Euglandina rosea, was found experimentally to be able to serve as both an intermediate and a paratenic host. Other locally occurring gastropods that were successfully infected experimentally included Mesodon thyroidus, Anguispira alternata, Bradybaena similaris, Subulina octona, Polygyra triodontoides, Vaginulus ameghini, Philomycus carolinianus, Deroceras laeve, Limax flavus, and Lehmannia poirieri. Laboratory reared, 4- to 5-week-old M. thyroidus and D. laeve were able to support the development of small numbers of larvae to the third stage. First stage larvae of A. cantonensis in the feces of experimentally infected rats were found not to migrate out of the fecal pellet; this behavior favors the infection of feces-consuming gastropods. Twenty heavily infected L. flavus were observed over a period of 2 months, and shedding of third stage larvae of A. cantonensis was never seen. While factors support the spread of A. cantonensis in rats in the southern United States, the probability of human infection is uncertain since the parasite is transmitted primarily by ingestion of raw intermediate and paratenic hosts.

A case of human strongyloidosis apparently contracted from asymptomatic colony dogs.

A 63-year-old, white, animal caretaker suffered intermittent abdominal pain and diarrhea of 3 weeks duration. Hematological findings included leukocytosis (18,600) with eosinophilia (73%). Fecal examination revealed Stongyloides rhabditiform larvae which on culture yielded a preponderance of filariform larvae and a few free-living adults. Thiabendazole therapy resulted in rapid recovery. Epidemiological investigation yielded no history of previous Strongyloides infection or exposure; his wife and pet dog were not infected, but about one-third of the dogs in the colony under his care were found to be discharging Stongyloides rhaditiform larvae in their feces. Strongyloides infection was successfully trasmitted to specific pathogen-free pups using filariform larvae derived both from the human case and from the dog colony. Specimens recovered from one pup infected with filariform larvae of human origin were identified as Strongyloides stercoralis.

Transmission indices of Loa loa in the Chaillu Mountains, Congo.

A longitudinal entomological survey of the vectors of loiasis was conducted in the Missama area (Lekoumou region) in the Congo from September 1987 to August 1989. The principal catching site was a palm grove surrounded by forest 3 km from the village. Landing/biting densities of Chrysops were measured by standardized fly catches lasting 11 hr carried out twice a month. Vector landing densities were also assessed in the Bantu and Pygmy villages and in the fields. Populations of Chrysops from the palm grove were examined 6 times a month for infection with the infective stage of Loa loa. Chrysops silacea was the predominate vector except at the beginning of the rainy season, when C. dimidiata was the prevailing species. Chrysops were caught throughout rainy season, from October to June. The host-seeking activity of C. silacea was greatest in the middle of this season (February), but occurred sooner (October) for C. dimidiata. The following variables associated with transmission were calculated from our observations in the palm grove (the first figure corresponds to the first year of the study and the figure in parentheses corresponds to the second year). It was calculated that 2.658 (2.185) C. silacea and 1.412 (1.182) C. dimidiata could bite a person in the palm grove per year, including an average of 14.4 (12.7) infective C. silacea and 9.8 (7.2) infective C. dimidiata. The percentage of all dissected flies with third stage larvae in the head and the mean number of larvae in the head/infective fly were 0.57% and 10.1 +/- 6.8 for C. silacea and 0.66% and 11.2 +/- 6.5 for C. dimidiata, respectively. The estimated annual transmission potentials were 171.1 (102.9) for C. silacea and 116.1 (73.8) for C. dimidiata. In the palm grove, transmission was ensured by 2 effective vectors during the rainy season (October to May). Although the annual biting rate for both species was twice as low in the village as in the forest, our data suggest that effective transmission occurs there also.

Cutaneous manifestations of strongyloidiasis.

Strongyloides stercoralis is a small intestinal nematode that has the ability to multiply within the human host. Because of the potential opportunistic behavior of this parasite, immunocompromised patients may develop fatal disseminated infections. Chronic strongyloidiasis may last decades and give rise to various dermatologic lesions, the most characteristic of which is larva currens, a serpiginous creeping urticarial eruption caused by the intradermal migration of the infective filariform larvae. Rarely recognized is the presence of widespread petechiae and purpura that may develop in patients with disseminated infections. A 64-year-old immunosuppressed man developed fatal extraintestinal S stercoralis infection with extensive purpura associated with massive invasion of the skin by migrating larvae.

Conjunctival ophthalmomyiasis caused by the sheep nasal botfly (Oestrus ovis)

Three patients had conjunctival ophthalmomyiasis caused by the ovine nasal botfly. All patients had a sudden onset of redness, tearing, and foreign-body sensation of the affected eye. One to nine Oestrus ovis first-instar larvae were removed from the bulbar or palpebral conjunctiva of each patient. Symptoms and clinical signs resolved after mechanical removal of the larvae. Specific taxonomic diagnosis of O. ovis larvae was determined on the basis of characteristic conformation of the terminal end of the larval caudal segment as seen by use of light microscopy.

Identification of nematode larvae by coal cultures in closed boxes.

A method is described for the identification of concentrated larvae from coal cultures in closed boxes. The condensation drops formed beneath the lid of the culture box are taken up with an L-shaped pipette. The sample taken up is added to a drop of Lugol's solution on a slide and examined under the microscope. The details of the nematode structure are clearly visible. Great care should be taken to avoid a possible cutaneous contamination.

Population biology of hookworms in west Bengal: analysis of numbers of infective larvae recovered from damp pads applied to the soil surface at defaecation sites.

The aims of this study were to analyze the seasonal distribution of infective larvae on the soil surface, to determine whether numbers of infective larvae near faeces were related to the faecal egg count of individuals, and to relate the distribution of larvae to environmental characteristics. Larvae were recovered from damp pads, applied to the soil surface in an annulus around fresh, identified stools of individuals who were participating in a larger epidemiological study. This provided an estimate of exposure to infection at the time of defaecation. Transmission was restricted to the rainy season and large aggregations of larvae were encountered earlier rather than later in the rainy season. Frequency distributions for the number of larvae extracted from each pad showed a high degree of aggregation, with most monthly counts showing good fits to the negative binomial probability distribution. Despite variations in monthly sampling means, the degree of aggregation in the population of larvae was remarkably stable over the 18 month sampling period (k of negative binomial = 0.01 to 0.08). Estimates of the degree of aggregation of the parasites in the human population were also available, and comparisons suggest that the infective larvae were much more aggregated than the parasitic stages. There was no relationship between the mean daily egg output of individuals and the number of larvae which developed and were recovered from the soil surface near the faeces. Thus, people who contribute large numbers of eggs to the environment are not necessarily those who are the greatest source of infection for others.(ABSTRACT TRUNCATED AT 250 WORDS)