Effect of cyclosporin A and some derivatives in Litomosoides carinii-infected Mastomys natalensis

Litomosoides carinii-infected Mastomys natalensis were treated 85 days post infection with cyclosporin A (CyA) or 8 derivatives with different immunosuppressive capacities. CyA (oral doses of 5 X 25 mg/kg, 5 X 50 mg/kg, 5 X 80 mg/kg on consecutive days) reduced parasitaemia levels in a dose dependent way, beginning 3 weeks after first drug administration. Using 5 X 50 and 5 X 80 mg/kg animals were free from circulating microfilariae on the day of necropsy (day 56). Derivatives were administered in 5 daily oral doses of 50 mg/kg. Compounds B-5-49 and G-7-53 had similar effects as CyA. Compounds A-4-16 and E-6-44 caused mean microfilaraemia reductions of about 80% until day 56. Compounds C-5-34, D-6-45, F-7-62 and H-7-94 were only marginally effective (10-40%). None of the drugs affected the number or the motility of adult worms. However, in the case of efficacious compounds the number of intrauterine microfilariae was considerably reduced and most of the intrauterine stages were pathologically altered. The efficacy of the various derivatives was independent of their immunosuppressive activity in vivo and in vitro, their anti-inflammatory activity and their activity against Plasmodium berghei. Effects on intrauterine stages were first detectable 7 days after treatment with 5 X 80 mg CyA/kg when the number of intrauterine microfilariae had decreased and the proportion of pathologically altered stages had increased. Alterations increased with time after treatment. Additionally, the uteri contained relatively large amounts of highly active microfilariae which were still included in an ovoid sheath.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cultivation of excysted metacercariae of Echinostoma caproni (Trematoda) to ovigerous adults on the chick chorioallantois

Excysted metacercariae of Echinostoma caproni were cultivated on the chick chorioallantoic membrane (CAM) maintained at 38.5 +/- 1 C and a relative humidity of 60-65%. Of 59 6-day-old embryos, each inoculated with 25 metacercariae, 29 (49.2%) were infected 2-12 days postinoculation. The total number of worms recovered from the infected eggs was 163 or 22.5% of the 725 inoculated metacercariae. Eggs contained from 1 to 12 (average 5.6) worms per CAM. Worm length increased rapidly from an average of 0.5 mm at 2 days to about 3.0 mm at 6 days postinoculation. Ovigerous worms first were seen on day 8 PI, but fluke eggs did not develop embryos. Worm development in ovo lagged about 1 day behind that of in vivo worms. One worm maintained for 17 days on 2 successive CAMs reached 6 mm in length, contained about 100 eggs in its uterus, and laid an additional 100 eggs on the CAM surface.     

Suppression of the parasitemia in rodent filariasis (Litomosoides carinii) by immunization with BCG and microfilaria. II. Intravenous BCG application

By intravenous (i.v.) inoculation of living tuberculosis bacteria (BCG) non-specific resistance to microfilariae of Litomosoides carinii (Filarioidea) is induced in cotton rats. This is only possible using the preparation "Immune-BCG Pasteur F" (suspended germs), but not with "Vaccin-BCG pour scarifications" (lyophilized tuberculosis bacteria). After inoculation of Immune-BCG, followed by a challenge infection by 60 infective larvae 6 weeks later, a patent infection develops. However, the level of microfilaraemia is constantly lower than in the control. After challenge infection 12 weeks later, this effect has disappeared. Immune-BCG has no influence on the worm load or the output of microfilariae by the adult worms. If i.v. inoculation of Immune-BCG is combined with a subcutaneous injection of specific antigen--living embryos from the uteri of adult worms--the BCG-activated immune system undergoes specific sensitization. Upon challenge infection 6 weeks later, the microfilaraemia is completely suppressed, but the worm load and production of microfilariae by the adult female worms are normal. If Immune-BCG is injected i.v. 3 days before intraperitoneal injection of freeze-killed microfilariae, there is still constantly reduced microfilaraemia when challenge infection follows 12 weeks later. Obviously, the effect of this relatively weak antigen may be increased by BCG stimulation.     

Effect of host sex and litter on the population dynamics of Echinococcus granulosus in dogs.

Twenty-one Beagle dogs consisting of 10 males and 11 females and belonging to 3 litters were infected with 60,000 E. granulosus protoscolices each. They were killed on day 40, the parasites from their intestines recovered, and the number of worms, average number of proglottides per worm, average length per worm, percentage of worms with a uterine cavity, and percentage of egg-bearing worms were determined for each dog and analyzed per sex and litter. On average, the dogs had 1,253 +/- 339 worms (means +/- standard error) with 2.42 +/- 0.1 proglottides, were 1.59 +/- 0.07 mm long, and 25.6 +/- 4.8% of the worms presented a uterine cavity and 1.2 +/- 0.6% bore eggs. The number of worms exhibited a bimodal distribution with 19 dogs having less than or equal to 2,565 worms and 2 greater than or equal to 5,520 worms. Average number of proglottides also showed a bimodal distribution with 7 dogs having less than or equal to 2.1 proglottides per worm and 14 dogs having greater than or equal to 2.4 proglottides per worm. The parasites were significantly more numerous in females than in the males (1,964 +/- 573 vs. 681 +/- 202), had more proglottides (2.67 +/- 0.08 vs. 2.15 +/- 0.16), and were longer (1.72 +/- 0.07 vs. 1.44 +/- 0.11 mm). The percentages of parasites with a uterine cavity (27.8 +/- 5.9 vs. 23.2 +/- 8.1) or bearing eggs (1.0 +/- 0.5 vs. 1.5 +/- 1.8) were comparable in females and males.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sexual dimorphism and female reproductive cycle in the scincid lizard Trachylepis vittata (Olivier, 1804) in Lebanon (Reptilia: Scincidae)

Sexual dimorphism and the female reproductive cycle were studied in a population of the viviparous lizard Trachylepis vittata at 2000 m a.s.l. on Mount Sannine, Lebanon. Females have larger body sizes than males and males have relatively larger heads than females. Females reach maturity at 56 mm snout-vent length. They spend at least six months in hibernation, from October to March. Adult females emerge from hibernation in April. Fertilization occurs mid-May and gestation lasts for 8-10 weeks. All females collected on the same date had embryos at the same embryonic developmental stage. Females produced 1-4 embryos. There is a significant positive relationship between female body size and number of embryos. Parturition lasts for two weeks and new-borns achieve adult size within about ten months.     

Studies on acquired resistance of the cotton rat against microfilariae of Litomosoides carinii. 2. Injection of microfilariae during prepatency

Cotton rats infected by infective third-stage larvae of Litomosoides carinii were treated at increasing time intervals by a threefold injection of living homologous microfilariae (mf) during the prepatent period. Starting with the first treatment 3, 4 or 5 weeks p.i. seven animals remained completely and two almost mf-negative (1 or 2 mf/mm3 each only once) until 16 weeks p.i. Starting 6, 7 or 8 weeks p.i. six animals developed a normal level of parasitaemia between 42 and 436 mf/mm3, two animals developed a continuous level of 1-2 mf/mm3. The number of fertile adult worms shedding great numbers of microfilariae in the pleural cavity was equal in all animals. However, in mf-negative animals the lung capillary blood showed, in the geometric mean, only 0.6% of the mf-concentration seen in mf-positive animals. The hypothesis is proposed that microfilariae accumulating primarily in the lung capillaries absorb all aggressive components specifically reacting with microfilarial antigens, i.e. neutralize the immune response against them to enable the development of the parasitaemia in the peripheral blood.     

Pathology and epizootiology of Dirofilaria scapiceps (Leidy, 1886) (Nematoda: Filarioidea) in Sylvilagus floridanus (J.A. Allen) and Lepus americanus erxleben

Dirofilaria scapiceps was found between the synovial sheath and tendons, i.e., within the tendon sheath, in the ankle region of eastern cottontail rabbits (Sylvilagus floridanus) and snowshoe hares (Lepus americanus). In cottontail rabbits, tendons and sheaths appeared normal and all worms were adults. Only one (4%) of 24 infected rabbits contained dead worms. All female worms were gravid in rabbits killed in late winter or early spring. Microfilaremias in rabbits were high (approximately 30-100 microfilariae/60 microliter blood) and of long duration (at least 8-28 mo), and rabbits were considered normal hosts of D. scapiceps. In some snowshoe hares, tendons and sheaths also appeared normal; however, in other hares a chronic proliferative tenosynovitis, characterized by fibrinous exudate, hyperplasia and hypertrophy of the intima and inflammatory cell (predominantly lymphocytes and plasma cells) infiltration of the intimal and fibrous layers of the synovial sheath led to encapsulation of worms. Dead subadult, dead adult, and live adult worms were found in the ankles of hares; 86 (46%) of 186 infected hares contained some or only dead worms. Fibrosis commonly occurred around dead worms. Dead subadults were also found in subcutaneous connective tissues over the trunk of the body. Degenerate embryos and amorphous material were observed in uteri of some female worms in hares killed in late winter or early spring. Few (1-5 microfilariae/60 microliter blood) or no microfilariae were observed in the peripheral blood of hares and microfilaremias were of short duration (less than 8 mo). Microfilariae in hares are probably trapped and destroyed in the chronic inflammatory lesions in the tendon sheaths since normal, degenerate, and calcified microfilariae were observed in the capsules around adult worms. Some microfilariae might also be destroyed in lymph nodes. Although D. scapiceps can be maintained within snowshoe hare populations, hares are considered abnormal hosts of D. scapiceps. Dirofilaria scapiceps may have spread from cottontail rabbits to snowshoe hares relatively recently.     

Experiments in vitro with Litomosoides carinii (Nematoda: Filarioidea). I. Maintenance of adult females and microfilariae as well as release of microfilariae in different culture media (author's transl)

Embryos of L. carinii continue intrauterine development to microfilariae and are totally released into the medium within 5--6 days when the latter (Tc 199) is changed daily and air is used as the gas phase. Oogenesis or further fertilization of eggs, however, does not occur in vitro in any of the media examined by us. One female releases 140 X 10(3) microfilaria/day on an average in vitro within 5--6 days. Mean initial numbers of 300 X 10(3) Mf/female/day are observed. Addition of equine serum inhibits microfilarial release in vitro; normal cotton rat serum prolongs survival of females while total numbers of released microfilariae or retained embryonic stages are not increased. The serum of post-patent animals does not influence the numbers of released microfilariae or their viability or survival of females. Microfilariae released in vitro in Tc 199 + 33% normal cotton rat serum survive for more than 8 days, when air is used as the gas phase and the medium is changed daily. Microfilariae isolated from the blood of patent animals survive for at most 6 days, at a 48-hourly change of medium survival does not even exceed 4 days.     

Dipetalonema vitae: survival of adult females and microfilarial release in both a chemically defined and serum-supplemented medium

Studies were conducted on survival and microfilarial release of afult Dipetalonema viteae in culture, using worms of various ages derived from jirds. In chemically defined NI medium (a 1:1 mixture of NCTC 135 and Iscove's modified Dulbecco's medium) under a gas phase of 5% CO2 in nitrogen (pO2 of medium approximately 40 mm Hg), the peak of microfilarial release of several thougsand microfilariae per female per 24 hr occurred at approximately day 10. Thereafter, microfilarial release declined and generally ended about 1 mo after the start of culture. The adult females moved actively for about 50 days or more and survived up to 82 days in NI medium alone. The females in NI medium supplemented with fetal bovine serum showed serpentine movement for approximately 2 mo. Some of the worms survived more than 83 days. The total number of microfilariae deposited in culture by D. viteae increased as adult females grew in size (volume) over time. Microfilarial deposition continued to increase after worms reached maximum size, deposition reaching a plateau between approximately 300 and 400 days of age. Thereafter, microfilarial deposition decreased as females continued to age. Addition of fetal bovine serum to the NI medium increased the number of microfilariae released and extended the period of release.     

The Effects of Ivermectin on Brugia malayi Females In Vitro: A Transcriptomic Approach

BackgroundLymphatic filariasis and onchocerciasis are disabling and disfiguring neglected tropical diseases of major importance in developing countries. Ivermectin is the drug of choice for mass drug administration programs for the control of onchocerciasis and lymphatic filariasis in areas where the diseases are co-endemic. Although ivermectin paralyzes somatic and pharyngeal muscles in many nematodes, these actions are poorly characterized in adult filariae. We hypothesize that paralysis of pharyngeal pumping by ivermectin in filariae could result in deprivation of essential nutrients, especially iron, inducing a wide range of responses evidenced by altered gene expression, changes in metabolic pathways, and altered developmental states in embryos. Previous studies have shown that ivermectin treatment significantly reduces microfilariae release from females within four days of exposure in vivo, while not markedly affecting adult worms. However, the mechanisms responsible for reduced production of microfilariae are poorly understood.Conclusion/SignificanceThese changes provide insight into the mechanisms involved in ivermectin-induced reduction in microfilaria output and impaired fertility, embryogenesis, and larval development.     

Doxycycline in the treatment of human onchocerciasis: Kinetics of Wolbachia endobacteria reduction and of inhibition of embryogenesis in female Onchocerca worms

Recently, experts have warned that mass treatment with ivermectin alone may not interrupt the transmission of Onchocerca. Hence, additional drugs are needed, such as antibiotics acting on symbiotic endobacteria of the filariae, the causative agents of onchocerciasis. Based on animal experiments, human onchocerciasis was treated with doxycycline, and preliminary observations published in 2001 in The Lancet showed sterility in female worms by depletion and marked reduction in symbiotic Wolbachia endobacteria from the filariae. Here, a detailed kinetic analysis of the features of the worms, following administration or not of doxycycline to the patients is reported. Sixty-three onchocerciasis patients in Ghana were treated with 100 mg doxycycline daily for 6 weeks and 2 or 6 months later with ivermectin. Onchocercomas were extirpated 2, 6, 11 and 18 months after the onset of treatment and the filariae were examined by immunohistology and PCR. The analysis showed: (i) progressive depletion of Wolbachia from adult worms and microfilariae by doxycycline over a period of 6 months; (ii) inhibition of embryogenesis by doxycycline after 6 months with respect to all embryo stages followed by decline in microfilariae after 11 months; (iii) reduction in spermatozoa in the female genital tract by doxycycline, whereas spermiogenesis was only partly reduced after 11 and 18 months; (iv) no relevant macro- or microfilaricidal activity; (v) depletion/marked reduction in endobacteria and inhibition of embryogenesis were sustained until 18 months after doxycycline and 12 months after co-administration of ivermectin; (vi) no severe adverse side effects were seen. Due to its long-lasting inhibition of embryogenesis, doxycycline presents an additional strategy for the treatment of onchocerciasis and control of Onchocerca microfilariae transmission. Extension of the existing registration will not require much time or high cost. Treatment of individual patients can be considered immediately.     

Colony-forming hematopoietic cells in the mouse embryonic lung

The colony-forming activity of embryo lung cells CBA mice was determined according to the Till and McCulloch technique (1961). After intravenous injection to jung cells (1 x 10(6)) from 14-day embryos the total number of colonies on the area of 1 mm2 of spleen sections from irradiated recipient mice averaged 2.31 +/- 0.39 whereas after transplantation of lung cells from 15-day embryos it averaged 2.34 +/- 0.53. The percent of macrocolonies equalled 52.5% in the former case and only 2.5% in the latter case. Macrocolonies contained cells of the myeloid, erythroid and megakaryocyte lines. Microcolonies predominantly consisted of granulocytes at various stages of differentiation. Thus, polypotent stem hematopoietic cells migrate into the fetal lung in vivo. The colony-forming ability of the lung polypotent stem cells decreases as the period of embryogenesis increases.     

Recovery of adult stages and microfilaraemia after low dose inoculation of third stage larvae of Litomosoides carinii in Sigmodon hispidus

Because of the negative binomial distribution of filarial third stage larvae (L3) in their vectors, under natural conditions only a few are usually transferred per bite. After an inoculation of 5 L3 per animal into eight host animals at least one developed a long lasting patency based on one reproductive female only. After an inoculation of 15 L3, three of eight animals developed long lasting patency, harbouring between two and five fertile females. The rates of adult stages recovered were 0.43 and 0.30 respectively. The parasitaemias of the six patent animals in both experimental groups increased with the number of reproductive females present (r = 0.89, p = less than 0.005). All non-patent animals which were mf-negative in the pleural fluid and lung blood as well had a single sex or no worm load. In only one animal was there an apparently normal but non-reproductive pair of worms without any pathological alterations of the host animal. Encapsulated adult worms were found rarely, but independent of the final worm load or inoculation dose and always beside normal adults. In three of the 16 animals inoculated with 5 or 15 L3 patency passed after 10-12 weeks p.i., in two others it seemed to pass soon. After inoculation of 30, 40 or 60 L3 per animal patency passed early in about one half of 105 animals, when they were observed up to 24-36 weeks p.i. In conclusion all types of host defensive reactions are already visible after inoculation with such small doses.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro antifilarial activity of extracts of the medicinal plant Cardiospermum halicacabum against Brugia pahangi

The in vitro effects of ethanol and aqueous extracts of the medicinal plant Cardiospermum halicacabum on adult worms and microfilariae of Brugia pahangi were investigated. With or without the plant extracts in culture medium, the motility of adult worms, microfilariae and microfilarial release from female worms were monitored daily. After 7 days of culture, viability or tissue damage of adult worms was assessed using the MTT assay. At > 500 microg ml-1, the aqueous extract significantly reduced motility of adult females after 24 h of exposure and adult males after 3 days. The aqueous extract, at > 500 microg ml-1, also significantly reduced microfilarial release from female worms, starting on day 2. The reduction in the motility of adult worms and the pattern of microfilarial release from female worms were concentration and time dependent. The MTT assay results revealed that adult worms cultured in the presence of aqueous extracts at > 500 microg ml-1 were damaged. However, the aqueous extract did not affect the motility of microfilariae with the exception of those in higher concentration extracts. Higher concentrations of ethanol extracts (2 mg ml-1) inhibited both the motility of adult worms and the release of microfilariae from females. Little effect of ethanol extracts was detected by the MTT assay, as only slight damage was caused to worms exposed only to the highest concentration (2 mg ml-1). However, ethanol extract at 500 microg ml-1 rapidly reduced the motility of microfilariae on day 2. The present study revealed that an aqueous extract of C. halicacabum has mild but definite direct macrofilaricidal action on B. pahangi.     

In vitro effects of antibiotics on Brugia malayi worm survival and reproduction

Recent studies have suggested that intracellular Wolbachia spp. endobacteria are necessary for the reproduction and survival of filarial nematodes. The effects of antibiotics that are active against related bacteria on adult worms and microfilariae (Mf) of Brugia malayi in vitro were investigated. Antibiotics tested were doxycycline (Doxy), tetracycline (Tet), rifampicin (Rif), azithromycin (Azith), and chloramphenicol (Chlor). Doxy, Tet, Rif, and Azith reduced release of Mf by adult female worms. The minimum effective concentrations that reduced Mf release by 50% were 5 microg/ml for Doxy, 20 microg/ml for Tet, 40 microg/ml for Rif, and 100 microg/ml for Azith. The same drugs (at higher concentrations) killed adult worms and Mf. Embryograms showed that Tets blocked embryogenesis in female worms. Electron microscopy (EM) showed that the Tets, Rif, and Azith cleared Wolbachia spp. from adult worms and damaged developing embryos. These studies show that antibiotics active against Rickettsiaceae affect adult B. malayi worms and Mf in vitro. Additional studies will be needed to elucidate the mechanisms of action of these antibiotics on Wolbachia and filarial worms.     

Cytomorphology of filariasis revisited. Expansion of the morphologic spectrum and coexistence with other lesions

OBJECTIVE: To review the cytomorphologic spectrum of the filarial worm and associated tissue response in 33 cases. STUDY DESIGN: Retrospective analysis was carried out in clinically unsuspected cases of filariasis diagnosed on cytology over a period of 10 years. Twenty-nine aspirate smears from 28 patients were air dried and stained with May-Grünwald-Giemsa stain. Four routine cervical smears and one centrifuged smear of urine were stained with Papanicolaou stain. RESULTS: Microfilariae alone and along with adult gravid females were present in 25 and 4 cases, respectively. In one case both adult male and female worms with microfilariae and eggs were seen. The diagnosis was based on the presence of eggs alone in one case and fragments of female worms in two. Four of these cases were neoplastic lesions, and microfilariae were found incidentally. In one case of splenomegaly microfilariae were seen along with Leishman-Donovan bodies. CONCLUSION: Filariasis can be diagnosed on cytology by demonstrating microfilariae, a male or female worm, or eggs alone. It can be seen in association with neoplastic lesions and rarely with other parasitic infections.     

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)     

Dipetalonema viteae: primary, secondary and tertiary infections in hamsters.

Hamsters were given primary infections of 100, 200, and 300 D. viteae larvae and groups killed at various intervals after infection. In addition, hamsters were sequentially infected with 100, 200, and 300 larvae and groups killed at 100 or 75 days after the secondary and tertiary infection, respectively. Blood microfilariae were detected on Day 60 following a primary infection, reached a maximum on Day 75, declined to low levels by Day 105, and were negative on Day 120. No microfilariae reappeared in the blood of hamsters given secondary or tertiary infections.Between 20–30% of the infecting larval dose had reached the adult stage by Days 75 or 100 postinfection in hamsters given primary, secondary, or tertiary infections. There was no evidence of arrested larval development in hamsters receiving a second or third challenge infection. Almost half of the tertiary infection hamsters developed subcutaneous nodules and their numbers varied greatly among individual animals. The nodules variously contained living worms, pus, and fragmented worms, or pus only. Hamsters given primary infections of 100, 200, or 300 larvae and killed 375 days after infection had no subcutaneous nodules; however, hamsters given the 200 and 300 larval infections were seen to have dead worms in the subcutaneous tissues. No stunting of adult worms was noted and all female worms had uteri packed with microfilariae.