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Antifungal constituents of the essential oil fraction of Artemisia dracunculus L. Var. dracunculus.

The isolation and structure elucidation of antifungal constituents of the steam-distilled essential oil fraction of Artemisia dracunculus are described. Antifungal activities of 5-phenyl-1,3-pentadiyne and capillarin against Colletrotichum fragariae, Colletrotichum gloeosporioides, and Colletrotichum acutatum are reported for the first time. The relative abundance of 5-phenyl-1,3-pentadiyne is about 11% of the steam-distilled oil, as determined by GC-MS. Methyleugenol was also isolated and identified as an antifungal constituent of the oil.

Artemisia↗

The role of tadpoles and frogs as paratenic hosts in the life cycle of Dracunculus insignis (Nematoda: Dracunculoidea).

The possibility exists that paratenic hosts play a role in the life cycle of various Dracunculus species. In the present study, we established that tadpoles of 2 genera of frogs (Xenopus and Rana) were capable of ingesting copepods infected with third-stage larvae (L3) of Dracunculus insignis. Once ingested, the L3s migrated from the gut to the somatic tissues of the tadpoles. In Xenopus, the dracunculid larvae persisted through the metamorphosis of the tadpoles into adult frogs. These observations confirm the concept that paratenic hosts, such as tadpoles or frogs, may be important means of transporting infective larvae of Dracunculus species up the food chain and facilitate transmission to the definitive hosts.

Animals↗

Identification of benzodiazepines in Artemisia dracunculus and Solanum tuberosum rationalizing their endogenous formation in plant tissue.

Sterile cultivated plant cell tissues and cell regenerates of several species were tested for their binding affinity to the central human benzodiazepine receptor. Binding activity was found in extracts of Artemisia dracunculus cell tissue (IC(50) = 7 microg/ml) and, to a lesser extent, in plant regenerates of potato herb (Solanum tuberosum). Preparative HPLC led to the isolation of fractions with a significant displacing potency in the benzodiazepine receptor binding assay. Using on-line HPLC-electrospray-tandem mass spectrometry (HPLC-ESI-MS/MS) in the "selected reaction monitoring" (SRM) mode, delorazepam and temazepam were found in amounts of about 100 to 200 ng/g cell tissue of Artemisia dracunculus, whereas sterile potato herb contained temazepam and diazepam ranging approximately from 70 to 450 ng/g cell tissue. It is the first report on the endogenous formation of benzodiazepines by plant cells, as any interaction of microorganisms and environmental factors was excluded.

Artemisia↗

Fatty acid composition of Dracunculus vulgaris Schott (Araceae) seed oil from Turkey.

Dracunculus vulgaris Schott is only one taxon of the genus Dracunculus (Araceae) in Turkey. The tubers and the fruits with the seeds of D. vulgaris have long been in use for the treatment of rheumatism and hemorrhoids, respectively. The fatty acid composition of D. vulgaris seeds have been analyzed as their methyl esters by GC and GC-mass spectrometry. C16:0, C16:1n-7, C18:1n-9, C18:1n-7 (cis -vaccenic acid), C18:2n-6 and 13-phenyl tridecanoic acids were found to be the main components in the seed oil.

Araceae↗

The existence of Dracunculus medinensis (Linnaeus, 1758) in Turkana, Kenya.

Dracontiasis has been previously reported in southern Sudan, northern Uganda and north-west Eritrea but this is the first report of autochthonous cases in Turkana, Kenya. Five Turkana tribesmen, three females from the same family, one young boy and an adult male were being treated for guinea-worm at the dispensary in Lokichogio, northern Turkana District. The three women had recently returned from southern Sudan, where the disease is endemic, but the two male tribesmen had only lived in and around the Lokichogio region. The methods used in obtaining water from water-holes dug in dry river beds provide an ideal situation for Dracunculus transmission amongst the tribesmen; this parasite may therefore become a problem in this remote impoverished area.

Adult↗

Human serum albumin and immunoglobulin on Dracunculus medinensis.

Dracunculus medinensis recovered from infected humans were examined for the occurrence and localization of human serum albumin and immunoglobulins. Immunoelectrophoretic examination of homogenates prepared from adult female worms (ADGW) and first stage larvae (LVGW) showed that anti-human albumin antibodies reacted to both stages of the parasite. By direct fluorescence antibody technique, antigens resembling human albumin and human immunoglobulins (isotype IgG) were identified on the surface of adult female worms. The occurrence of host-like compounds on the parasites may be an adaptation for survival of the parasites. A possible interference of these compounds should be considered in attempts to develop methods for immunological diagnosis of D. medinensis infections.

Animals↗

Dracunculus insignis: experimental infection in the ferret, Mustela putorius furo.

The laboratory study of dracunculiasis has suffered from the lack of a suitable, readily available animal model. We have been able to experimentally infect ferrets. Mustela putorius furo, with the North American dracunculid, Dracunculus insignis. Ferrets were inoculated with 75 to 100 infective larvae and were necropsied 90 to 240 days later. Guinea worms were recovered from 10 (56%) of 18 ferrets. A total of 44 worms were recovered, for an average of 4.4 worms per infected ferret. Gravid female worms were recovered as early as 128 days postinoculation. Thirteen (87%) of 15 gravid female worms were recovered from the extremities. Living male worms were recovered at 200 days of age, indicating that not all male worms die shortly after mating. First-stage larvae recovered from gravid females as early as 200 days of age were found to be infective to the copepod, Acanthocyclops vernalis. These observations suggest that the ferret is an excellent laboratory animal for dracunculiasis research.

Animals↗

Chemoprophylactic drug trials for treatment of dracunculiasis using the Dracunculus insignis-ferret model.

Groups of ferrets inoculated with Dracunculus insignis were treated with various anthelminthic compounds to evaluate the potential use of drugs in controlling the human parasite D. medinensis. The three primary compounds tested were diethylcarbamazine (DEC), albendazole (ALBZ), and invermectin (IVER); they were administered in dosages of 60 mg/kg, 10 mg/kg, and 1 mg/kg, respectively. Groups of animals received treatment either once at 60 days after inoculation, once at 60 and 90 days, or for 3 days at either 60 or 90 days postinoculation. The most marked decrease occurred in the animals that received treatment once at 60 and 90 days after inoculation. This was observed for all three drugs tested. Increased dosages, i.e., 3 days of treatment at 60 or 90 days did not result in decreased worm burdens. In no group was there a statistically significant reduction in worm burden when compared with controls. Two other compounds, metrifonate and CGP 6140, were tested in a more limited manner, but again the worm recovery rates were comparable with those in control groups. It would appear that existing drugs commonly used to treat helminthic infections are poor candidates for use in the campaign to eradicate guinea worm disease.

Albendazole↗

Susceptibility of four species of copepods, from areas of endemic Dracunculus medinensis, to the North American D. insignis.

Studies of the relative importance of different copepod species in areas of endemic dracunculiasis have been restricted, in part, by limited access to Dracunculus medinensis. However, development of the D. insignis-ferret model provides a source of larvae of a species closely related to D. medinensis. Using larvae derived from this model, the susceptibility of four species of copepods from D. medinensis-endemic areas was compared to that of Canadian Acanthocyclops vernalis, which is fully susceptible to D. insignis. Cryptocyclops linjanticus, from Cameroon, was susceptible but highly sensitive to infection, resulting in high mortality. Mesocyclops aequatorialis similis, also from Cameroon, was fully susceptible, but M. leuckarti leuckarti from Pakistan was partially refractory to D. insignis, as was Thermocyclops emini from Cameroon. Comparisons of infection rates and mean and maximum larval recoveries at three to four and 16-20 days after infection indicated that exposure to 15 first-stage larvae exceeded the threshold of tolerance of A. vernalis, C. linjanticus and M. a. similis to infection with D. insignis. This may account for a decreased survival rate in these copepods which is not evident in the partially refractory T. emini and M. l. leuckarti. The patterns of susceptibility of these copepods to D. insignis are the same as those of copepods exposed to D. medinensis, and indicate that D. insignis can be used to examine factors influencing the susceptibility of copepods to dracunculid infection.

Animals↗

The intermediate hosts of Dracunculus medinensis in northern region, Ghana.

Cyclopoid copepods were collected over a period of 17 months from six man-made water reservoirs in an area of northern Ghana where dracunculiasis is endemic. The numbers and species of cyclopoid copepods as well as the species naturally infected with Dracunculus medinensis larvae were recorded at fortinightly intervals. The occurrence of copepods was compared with concurrent rainfall and with monthly incidences of dracunculiasis in the year after the reservoir survey. Seven species and two subspecies of copepods were found in the study area: Thermocyclops neglectus decipiens, T. crassu' consimilis, T. incisus, T. inopinus, T. emini, Mesocyclops major, M. ogunnus, M. kieferi and Cryptocyclops linjanticus. More than 60% of all the cyclopoids found were T. n. decipiens and only four species (T. incisus, T. inopinus, T. n. decipiens and M. kieferi) were found infected. Infection rates were very low (< or = 0.3%). The seasonal fluctuations of cyclopoid copepods showed different patterns in the six reservoirs, perhaps related to local circumstances not investigated in the present study. As the people in the study area are very mobile during the dracunculiasis transmission season, it seems likely that some of them become infected at water sources other than the ones investigated. It was therefore difficult to correlate occurrence of cyclopoid copepods in the local water sources with the prevalence of the human infection in the catchment area. No conclusion as to the most important intermediate host(s) in the area could be made.

Animals↗

Dracunculus medinensis and Schistosoma mansoni contain opiate alkaloids.

The results of analysis, by high-performance liquid chromatography coupled with electrochemical detection and by nano-electrospray-ionization, double quadrupole/orthogonal-acceleration, time-of-flight mass spectrometry, indicate that adult Dracunculus medinensis and Schistosoma mansoni both contain the opiate alkaloid morphine and that D. medinesis also contains the active metabolite of morphine, morphine 6-glucuronide. From these and previous observations, it would appear that many helminths are probably using opiate alkaloids as potent immunosuppressive and antinociceptive signal molecules, to down-regulate immunosurveillance responsiveness and pain signalling in their hosts.

Animals↗

Attempted experimental cross infections with mammalian guinea-worms, Dracunculus spp. (Nematoda: Dracunculoidea).

Dracunculus medinensis and D. insignis are morphologically indistinguishable. Experiments to test the susceptibility of various mammalian hosts to these two guinea worm species are described. Infective 3rd-stage larvae of D. medinensis were administered to each of four raccons (Procyon lotor): infective 3rd-stage larvae of D. insignis were administered to a rhesus monkey (Macaca mulatta), two dogs, two ferrets (Mustela putorius furo), and a marten (Martes americana). D. medinensis was not found in the raccoons when necropsies were performed on days 247, 283, 354, and 390 post-infection, respectively. Nine female D. insignis containing eggs, embryos, and motile 1st-stage larvae were found in the rhesus monkey 180 days post-infection. Lesions had not formed and the larvae were presumed to be immature and not yet infective as they were comparatively inactive and attempts to infect suitable copepods failed. D. insignis was not found in the dogs or the marten, although both ferrets were successfully infected. Variations in susceptibility of various mammalian species to the guinea worm are discussed together with comments on variations in migration routes and sites of emergence in hosts which may be partially refractory. D. insignis and D. medinensis may represent physiological strains of a single species, or they may in fact be two distinct species which have evolved in different geographical locations.

Animals↗

Red Dracunculus medinensis.

Two instances of the emergence of a bright-red worm from humans in Pakistan are described. A 25-year-old man and a 26-year-old woman, unrelated and living in separate households, were observed in 1987 and 1988, respectively, to have emergent worms typical in size and location for Guinea worm disease, except for the color of the worms. The posterior end of 1 worm was recovered and appeared to be a female Dracunculus medinensis. Histologic sections provided insufficient information to determine the cause of the red color, although we could exclude blood as the cause.

Adult↗

Specific and cross-reacting antibodies in human responses to Onchocerca volvulus and Dracunculus medinensis infections.

Immunoelectroblotting and enzyme-linked immunosorbent assay were used to identify non-cross-reacting antigenic components of Dracunculus medinensis and the filarial worms Onchocerca volvulus, Loa loa, Wuchereria bancrofti, Brugia malayi, and Mansonella ozzardi. Parasite specific serodiagnostic ELISA systems for onchocerciasis and dracunculiasis were devised based on these findings. Phosphate buffered saline extracts of adult worms were passed through a column of monoclonal antibodies to phosphorylcholine (PC). Crude and PC-depleted extracts were reacted on ELISA plates with individual sera from subjects infected with a range of nematodes. Binding of total antibody (Ig) or IgG class antibody and IgG4 subclass antibody was revealed using goat antihuman-Ig-phosphatase conjugate, or appropriate mouse monoclonal antihuman-Ig-type-specific reagents, followed by goat antimouse-Ig-phosphatase conjugate. Specificity of ELISA was improved by restricting reaction to the host's IgG4 antibody subclass, and/or by removing PC determinants from crude antigens. In parallel immunoelectroblots, crude and PC-depleted extracts probed with pooled sera showed potentially useful diagnostic antigens, including a 12 kDa protein from D. medinensis and 14, 18, and 27 kDa proteins from O. volvulus. Two Onchocerca specific ELISA systems non-reactive with antibodies to D. medinensis were devised.

Animals↗

Immunoepidemiology of Dracunculus medinensis infections I. Antibody responses in relation to infection status.

The specific serum IgG1, IgG4, and IgE responses to Dracunculus medinensis and the level of total IgE of individuals living in a highly endemic area of northern Ghana were measured by ELISA. Sera were obtained in the high transmission season from individuals with prepatent, patent, or postpatent infection as well as from individuals from the same endemic area who claimed to have never had a patent infection (i.e., endemic normal individuals). Individuals with prepatent or postpatent infections responded with a significantly lower mean level of specific IgG1 and IgG4 compared with individuals with a patent infection, and with a significantly higher mean level of specific IgG1 and IgG4 compared with endemic normal individuals. For specific IgE, no differences were found in the mean antibody level between the infection status categories. Individuals with a patent infection had a significantly lower mean serum level of total IgE compared with prepatent, postpatent, and endemic normal individuals. Endemic normal individuals had the highest mean level of total IgE. Furthermore, in all clinical categories, high responders for specific IgG1 and IgG4 generally had low levels of total IgE, whereas low responders for specific IgG1 and IgG4 generally had high levels of total IgE. A similar dichotomy, although less distinct, was observed between specific IgG1 and IgG4 on the one hand and specific IgE on the other. Thus, similar to what has been suggested for schistosomiasis and lymphatic filariasis, the relationship between the IgG subclasses and IgE appears to play a role in, or at least to reflect, a mechanism for protective immunity in dracunculiasis.

Adolescent↗

Immunoepidemiology of Dracunculus medinensis infections II. Variation in antibody responses in relation to transmission season and patency.

The serum antibody responses (specific IgG1, IgG4, and IgE, and total IgE) to Dracunculus medinensis infection in humans from a highly endemic area of northern Ghana were examined regularly by ELISA over a period of one year in cohorts of individuals who developed a patent D. medinensis infection during the study period (actively infected category), or who claimed to have never had a patent infection (endemic normal category). The results were analyzed in relation to seasonality and time of patency of infection. For individuals in the actively infected category, a clear seasonal variation in the mean levels of specific IgG1 and IgG4 was found, with the highest levels late in the dry season and early in the rainy season, when transmission is high, and the lowest levels late in the rainy season and early in the dry season. Endemic normal individuals responded with low and fluctuating levels of specific IgG1 and with low and nonfluctuating levels of specific IgG4. For specific and total IgE, no seasonal variation was observed in any of the two infection status categories. In relation to time of patency of infection (only involving the category of actively infected individuals), the mean levels of specific IgG1 and IgG4 increased from two months before patency of infection, peaked during patency, and then gradually decreased for four months until a constant level was reached. No significant fluctuations in the levels of specific and total IgE were observed in relation to time of patency. The present study thus showed extensive variation in levels of D. medinensis-specific IgG1 and IgG4 (but not IgE) over time. Seasonal variations in antibody responses may also occur in other helminth infections, especially those with seasonal transmission, and these should be taken into consideration when interpreting the results of immunologic studies.

Adult↗

Massive Dracunculus insignis infection in a dog.

An 11-year-old 13-kg (28.6-lb) spayed female Cocker Spaniel was examined because of subcutaneous nodules on the hind limbs and ventral aspects of the thorax and abdomen. Focal areas of erythema and pyoderma were associated with the nodules, and purulent exudate could be expressed from a fistula in the nodules. A nematode approximately 20.5 cm in length was isolated from a draining fistula in 1 nodule and identified as Dracunculus insignis. The dog was treated with ivermectin, fenbendazole, and metronidazole, but the owner was still able to recover worms from multiple nodules for the next year.

Animals↗