Differences between the microfilariae of Wuchereria malayi and Wuchereria bancrofti in giemsa-stained thick blood films.
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[Development of Wuchereria bancrofti (Cobbold) and Wuchereria malayi (Brug). II].
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On the adult morphology of Wuchereria sp. (malayi?) from a monkey (Macaca irus) and from cats in Malaya, and on Wuchereria pahangi n.sp. from a dog and a cat.
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Development of Wuchereria bancrofti (Cobbold) and Wuchereria malayi (Brug).
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Animals↗
The vectors of human infections by Wuchereria species in endemic areas and their biology.
In this paper the author has compiled an up-to-date list of the principal natural vectors of human Wuchereria infections, arranged by zoogeographical regions and countries, and gives data on natural and experimental filarial infection rates in vectors in different parts of the world. As an aid to workers in areas where Wuchereria and Plasmodium infections co-exist, he also provides a list of anophelines as filarial vectors, including in this list the results of natural and experimental studies of Wuchereria infection both alone and in combination with malaria.Finally, the author suggests a number of aspects of the biology of infected and uninfected vectors on which further studies might to advantage be conducted.
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The preferential site of adult Wuchereria bancrofti: an ultrasound study of male asymptomatic microfilaria carriers in Pondicherry, India.
BACKGROUND: The traditional method of detection of microfilaria in night blood specimens for the diagnosis of Wuchereria bancrofti infection is being replaced with circulating filarial antigen in day blood specimens, which has a high sensitivity. However, both methods are indirect tests to detect the presence of adult worms in vivo. Localization of adult worms in vivo in their natural habitat may help in understanding better the end-point of drug treatment, the adulticidal action of antifilarial drugs, and in locating the site of lymphatic pathology. We used ultrasound examination to assess the preferential location of adult worms in an area endemic for lymphatic filariasis. METHODS: Ultrasound examination was done in 36 asymptomatic male carriers of Wuchereria bancrofti microfilaria to detect the location of adult worms. Both sides of the scrotum (root of the scrotum, epididymis, spermatic cord, testis and the adjoining area), lymphatic vessels and inguinal, popliteal, axillary and epitrochlear lymph nodes were examined using a 7.5 MHz probe in real-time B mode. RESULTS: The 'filaria dance sign (FDS)' suggesting the presence of adult worms was observed in 22 carriers (61%). The preferential site of location of the adult worms was the intrascrotal juxtatesticular lymphatic vessels in 'nests' along the lymphatic vessels of the epididymis, spermatic cord and paratesticular region. The number of nests varied between 1 and 4 with a mean size of 0.3 cm2. In 95% of cases, localization of the worms was unilateral. The mean microfilaria (SD) count-positive cases for those with the filarial dance sign (264 [199]) was significantly higher (p<0.05) than for the negative cases (171 [196]). CONCLUSION: Ultrasound visualization of adult worms of Wuchereria bancrofti in vivo is possible and confirms the concept that the worms have their own territory and reside in 'nests'. The preferential site of localization of the adult worms in men is the intrascrotal juxtatesticular lymphatic vessels.
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Filarid parasites of South Kalimantan (Borneo) Indonesia. Wuchereria kalimantani sp. n. (Nematoda: Filarioidea) from the silvered leaf monkey, Presbytis cristatus Eschscholtz 1921.
On the basis of morphological characteristics of adult and microfilarial stages, Wuchereria kalimantani sp. n. (Nematoda: Filarioidea) is described. The parasites were recovered from the inguinal lymph nodes and testes of silvered leaf monkeys (Presbytis cristatus) from a rubber estate, Banjar Regency, South Kalimantan (Borneo), Indonesia. Adult males are characterized by having more than 24 caudal papillae, a left spicule with a simple lamina, four pairs of sessile caudal papillae, a blunt tail, a mean overall length of 41 mm, and sheathed microfilariae with a nude tail tip; accordingly, the parasite is placed in the genus Wuchereria. This is the second species of Wuchereria described on the basis of both adult and embryonic forms, and is the first such species that has been found to infect primates naturally.
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[Further study on in vitro culture of third-stage larvae of Wuchereria bancrofti and Brugia malayi].
This paper reported on improved in vitro culture system of human lymphatic filarial larvae. Four culture systems were used. Third-stage larvae of Brugia malayi were best maintained, developing and molted twice in the medium containing modified RPMI-1640 medium, supplemented with 20% newborn calf serum and human embryonic kidney cell line as feeder layer. This culture system kept larvae alive up to 54 days. Brugia malayi third-stage larvae began to moult on the 8-10th day and again on the 32-36th day. Wuchereria bancrofti third stage larvae grew and developed to the fourth-stage and juvenile and survived to 57 days. They began to moult on the 12-18th days and again on the 32-44th day. This culture system was thought to be useful for studies on morphology and sensitivity to drugs. We also studied several cell-free culture systems. Among them, Best survival, growth and development were obtained in 1:1 mixture of modified RPMI-1640 and TC199 medium supplemented with 20% newborn calf serum. Both Wuchereria bancrofti and Brugia malayi third-stage larvae grew and developed to the fourth stage larvae and juveniles and survived to 36 days and 42 days respectively. The availability of such culture systems for human filariasis would facilitate studies of biochemistry, immunology, production of monoclonal antibodies and vaccine.
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Development of Brugia malayi in Mongolian gerbils previously exposed to Wuchereria bancrofti.
Twelve Mongolian gerbils, Meriones unguiculatus, were infected with 100 third-stage larvae of Wuchereria bancrofti. One month later these animals, along with 4 control animals, were given 100 third-stage larvae of Brugia malayi. Eleven of the 12 experimental animals and the 4 controls survived, and 8 of the experimental animals and all of the controls demonstrated microfilaremia after 3 months. The animals were killed at 6-months post-infection and examined for parasites. One W. bancrofti larva was found in one of the experimental animals, and 15% of the B. malayi given were recovered as adults from the testes, viscera, and carcass. Thirty-eight percent of the worms given to the controls were recovered from the testes, viscera, and pelt. The worms from the experimental animals also appeared to be smaller. This study suggests that gerbils are able to develop partial resistance to Brugia malayi following a previous infection with Wuchereria bancrofti.
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Rapid detection of Wuchereria bancrofti in mosquitoes by LightCycler polymerase chain reaction and melting curve analysis.
A LightCycler real-time polymerase chain reaction (PCR) assay was developed to detect Wuchereria bancrofti DNA in blood-fed mosquitoes. The assay is based on fluorescence melting curve analysis of the PCR product generated from a family of repeated DNA elements: the 182 bp SspI repeat, specific to the genus Wuchereria. According to the melting temperature, W. bancrofti infected-mosquitoes were differentiated from Brugia malayi-infected and non-infected mosquitoes as well as from genomic DNA of Dirofilaria immitis and human DNA. The method proved to be 100% sensitive in all W. bancrofti-infected mosquitoes. Melting curve analysis offers a rapid alternative for the specific detection of W. bancrofti in mosquitoes. It is very accurate and sensitive, allows a high throughput and can be performed on very small samples. The method therefore has great potential for application in epidemiological studies.
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Development of a quantitative, competitive polymerase chain reaction--enzyme-linked immunosorbent assay for the detection of Wuchereria bancrofti DNA.
A quantitative, competitive polymerase chain reaction (QC-PCR) assay for the sensitive detection of Wuchereria bancrofti DNA was developed. A competitor sequence was constructed by an exchange of nucleotides in the Wuchereria-specific Ssp I repeat. The PCR products were hybridized to specific DNA probes and their amounts, determined by an enzyme-linked immunosorbent assay (ELISA). In laboratory-prepared samples the QC-PCR-ELISA assay was capable of detecting the amount of DNA equivalent to 0.1 microfilaria (mf) added to 200 microl of blood lysate. The assay was also tested on 78 blood samples collected in endemic areas in Egypt. All 28 samples that were positive both for mf and for circulating antigen were also QC-PCR-ELISA-positive. In addition, one mf-negative but antigen-positive sample was also positive as determined by QC-PCR-ELISA. A positive correlation of mf density with the QC-PCR-ELISA was observed. Samples containing 10 or fewer mf/ml had a mean relative amount of Ssp I PCR product of 19.7 units, whereas samples with 11-100 mf/ml had a mean of 36.3 units and those with more than 100 mf/ml had a mean of 84.6 units. Because of the high standard deviation within each group, estimates of worm burdens in infected individuals using the QC-PCR-ELISA are not recommended. However, we present data indicating that the W. bancrofti QC-PCR-ELISA is a powerful new tool for evaluation of parasitic loads for community-based diagnosis of bancroftian filariasis.
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Characterization of a muscle-associated antigen from Wuchereria bancrofti.
A recombinant clone, WbN1, isolated from a genomic expression library of Wuchereria bancrofti and showing restricted specificity at the DNA level (Southern and PCR analyses) for Wuchereria bancrofti and Brugia malayi has been previously described. Sequence analysis of WbN1 indicated that it had notable similarity to myosin. Further characterization using in situ hybridization has localized the mRNA in the muscle of the adult parasite and in the microfilariae. Rabbit polyclonal antiserum, raised against the recombinant WbN1 fused to the maltose-binding protein, recognized a 200-kDa polypeptide in immunoblots containing B. malayi antigen extracts. The same antibody also recognized myosin extracted from Brugia pahangi, Onchocerca volvulus, and Caenorhabditis elegans. Localization using the rabbit antiserum revealed the presence of the antigen in the adult muscle tissue and in the microfilariae; the same antibody inhibited the binding of a monoclonal antibody 28.2 (directed toward MHC B of C. elegans myosin) to the recombinant WbN1 antigen and also to purified C. elegans myosin. Based on homology data, structural location, competitive ELISA, and immunoblot we conclude that WbN1 is related to myosin or a similar myofibrillar protein.
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The pathology of lymphoreticular and genital tissues of silvered leaf monkeys (Presbytis cristata) experimentally infected with Wuchereria bancrofti.
The discovery of the silvered leaf monkey, Presbytis cristata, as a suitable experimental host for the human filarial parasite, Wuchereria bancrofti, opened the door for major advances in our understanding of the disease caused by this parasite. To study the pathogenesis of bancroftian filariasis in this model, 15 adult P. cristata which had been experimentally infected with 250 infective third-stage larvae of the parasite were examined. After inoculation with larvae, the monkeys were maintained under study for periods of 2 to 3 years, at which time all had achieved patent infections. At necropsy, Wuchereria-induced macroscopic lesions were not detected in experimentally infected monkeys. Microscopic findings included nematodiasis, microfilariasis, lymphadenitis, lymphangiectasis, perilymphangitis, splenitis, orchitis, periorchitis, epididymitis and funiculitis. Sections of normal adult worms were most often found in lymphatic vessels near lymph nodes, or in the lymphatics of the male genital system. These worms caused microfiliariasis in some regional lymph nodes. Inflammatory responses to filariae were most prominent in proximity to degenerated and dead worms, whereas intact, normal appearing adult worms elicited only minimal cellular response.
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Wuchereria bancrofti infection in human and mosquito populations of a Polynesian village ten years after interruption of mass chemoprophylaxis with diethylcarbamazine.
In 1991, a study on Wuchereria bancrofti microfilariae (mf) and infection rates was carried out in the human and mosquito populations of a Polynesian village where, 10 years before, the mf prevalence rate was 6.4% and twice-yearly mass treatment with 3 mg/kg of diethylcarbamazine (DEC) was interrupted. Venous blood samples were collected from 575 (97%) individuals aged 15 years or more, of whom 122 (21.4%) were mf positive. The mf carrier prevalence rate was 27.4% in males, significantly higher than that of 14% in females; it increased from 7-12% in the youngest age group (15-19 years) to 40-50% in the oldest (> or = 60 years) for both males and females. 387 mosquito collections were performed and 1748 female Aedes polynesiensis were dissected, of which 1176 were parous. Among the latter, 114 (9.7%) were infected with Wuchereria bancrofti larvae at L1, L2 or L3 stages. The mean number of larvae per mosquito was 2.46 (range 1-15). Of the 114 infected mosquitoes, 30 harboured L3 larvae, giving a 2.55% infective rate; the mean number of L3 larvae per mosquito was 1.15 (range 1-2). Such findings indicate that the interruption of systematic twice-yearly mass treatment with DEC (3 mg/kg) has resulted, after 10 years, in a substantial increase of microfilarial prevalence in humans, and in high infection rates in mosquitoes.
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Isolation of Wuchereria bancrofti microfilariae from archived stained blood slides for use in genetic studies and amplification of parasite and endosymbiont genes.
Information on change in genetic diversity of Wuchereria bancrofti is important in view of the launching of the Global Lymphatic Filariasis Elimination Programme, as it may have important consequences on the control operations and on the potential resurgence after their withdrawal. Since attention was not paid to generate such information when the programme was launched, use of archived parasite material will provide an opportunity to derive this information in a prospective manner. In this paper a simple and effective technique is reported for isolation of microfilariae of W. bancrofti from dried and stained slides archived for several years and their utility in analysis of genetic structure and amplification of certain genes of the parasite is tested. The method was found to be efficient in purifying mf from the dried smears and the DNA of the parasite found to be useful in studying the genetic structure of Wuchereria bancrofti populations using random amplified polymorphic DNA (RAPD)-PCR and for amplifying genes of the parasite and its endosymbiont, Wolbachia sp.
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Wuchereria bancrofti: effect of single and multiple larval inoculations on infection dynamics and development of clinical manifestations in non-human primate Presbytis entellus.
We earlier reported the successful experimental transmission of Wuchereria bancrofti from humans to the Indian leaf monkey (Presbytis entellus) [Misra, S., Tyagi, K., Chatterjee, R.K., 1997. Experimental transmission of nocturnally periodic Wuchereria bancrofti to Indian leaf monkey (Presbytis entellus). Experimental Parasitology 86,155-157.; Dube, A., Murthy, P.K., Puri, S.K., Misra-Bhattacharya, S., 2004. Presbytis entellus: a primate model for parasitic disease research. Trends in Parasitology 20(8), 358-360.] using a small number of animals. The present study, involving 27 langur monkeys, found the development of pathological manifestations ranging from filarial fever, lymphangitis, lymphadenitis, hydrocoele, and limb edema to minor histopathological changes in tissues after single, double, triple, or multiple inoculations of infective larvae of W. bancrofti recovered from Culex quinquefasciatus fed on human microfilaraemic blood. Thirty-eight percent of the infected langurs developed detectable microfilaraemia in their blood. Single or double larval exposure resulted in better worm establishment than multiple exposures with small numbers of larvae. All of the langurs receiving a single large inoculum and 85.71% of those receiving two inoculations harbored adult parasites. Worm establishment decreased with increasing number of larval inoculations. In all, 60% of infected langurs developed classical gross-pathological symptoms of lymphatic filariasis. Of these, 29.16% developed thickening of the lymphatics, 25% suffered from periodic rise in rectal temperature, and 16.66% developed scrotal swelling with presence of microfilariae in the hydrocoele fluid. Only one out of 25 langurs (4%) developed acute limb edema. It appeared that a single inoculum of a large number of infective larvae was able to induce maximum pathology. Fifty-six percent of the infected langurs acquired a peculiar sitting posture, retracting both the hind limbs, usually after 4-5 months of larval inoculation and eventually resulting in reduced mobility. Oral administration of diethylcarbamazine (citrate) at 12mg/kg for 12 consecutive days to one langur caused 80% suppression in microfilaraemia on day 8, killing all the adult parasites. Of the two immunosuppressed (cortisone- or prednisolone-treated) male rhesus monkeys included in the study, neither developed any of the above lymphatic symptoms or parasites after receiving larval inoculations on two or three occasions; however, a rise in rectal temperature in one of the animals was noticed. The present study thus reveals that the Indian leaf monkey, P. entellus, may serve as an ideal non-human primate model of human bancroftian filariasis for carrying out longitudinal studies on pathology, host-parasite interactions, and preclinical evaluation of candidate anti-filarial drugs or vaccines.
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Antibody response to a filarial antigen fraction in individuals exposed to Wuchereria bancrofti infection in India.
An antigen fraction (DssdI) was isolated from the aqueous-insoluble components of adult Setaria digitata. Rabbit antiserum to DssdI labeled the sheaths of Wuchereria and Setaria microfilariae suggesting DssdI to be surface associated. Antibody responses to DssdI were determined in Wuchereria bancrofti exposed individuals. IgM titre was highest in endemic normals (EN; asymptomatic amicrofilaraemic) followed by chronic filarial patients (CP) and asymptomatic microfilaraemics (AS). IgG titer was high in both chronic filariasis and endemic normals. The microfilaraemic group had the lowest titer. The seropositivity rate in AS was 28% for IgG and 10% for IgM, in CP 100% for IgG and 33% for IgM, and in EN 86% for both antibodies. An age-dependency of IgM but not of IgG antibodies was observed in endemic normals, with IgM prevalence reaching a plateau by 12 years of age. The subclass composition of the antibody response to DssdI appeared to be primarily IgG3 in endemic normals, IgG1 in chronic filariasis and IgG4 in asymptomatic microfilaraemics. Diethylcarbamazine (DEC) treatment led to a significant increase in IgG and IgM levels in microfilaraemic individuals. Of interest, the expression of IgG subclasses altered. There was an increased IgG3 and IgG1 response associated with a reciprocal decrease in IgG4 following DEC therapy.
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Microfilarial periodicity of Wuchereria bancrofti in Vanuatu.
A study on the relationship between the microfilarial periodicity of Wuchereria bancrofti and vector biting activity was carried out in Penama province, Vanuatu from February to April 1999, to enable the design of a more efficient strategy to control filariasis transmission. The microfilarial periodicities of 22 W. bancrofti antigen-positive volunteers were studied. Microfilariae (mf) were counted every hour for 24 h for 6 volunteers and every hour for 12 h (from 18:00 to 06:00) for 16 volunteers. At the same time as the preparation of mf test slides, indoor human landing catches of the vector mosquito, Anopheles farauti, were conducted to assess the vector biting activity. The time of peak microfilaraemia was 01:32 and the microfilarial periodicity index was 112.3, confirming the nocturnal periodicity of Wuchereria bancrofti in Vanuatu. Nearly all (98.5%) of the mf appeared during the time periods when A. farauti were collected. The timing of vector biting activity corresponded to the time of mf circulation.
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