Analysis of polymorphism of 18S rRNA gene in Wuchereria bancrofti microfilariae

The polymorphism of the 18S rRNA gene in Wuchereria bancrofti microfilariae (mf) collected from three different zones in India was analyzed by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). The RFLPs of the amplified products obtained after digestion with restriction enzymes Ssp I, Msp I and Hha I showed no difference in the banding patterns among the mf isolates from different endemic zones. Further the sequencing of PCR products did not show any difference in the nucleotide sequence either. The phylogenetic analysis of the sequences of W. bancrofti mf isolates from different endemic zones has shown branching with the earlier reported sequences of W. bancrofti and its close relative Brugia malayi.     

Variation in microfilariae and infective stages of two types of Wuchereria bancrofti from the Thai-Myanmar border

Comparative morphometric and morphological studies of microfilariae and infective stages were undertaken in nocturnally periodic and subperiodic Wuchereria bancrofti. For microfilariae, the body dimensions of nocturnally periodic (NP) were significantly smaller than nocturnally subperiodic (NSP), i.e., body length 268.03+/-14.75 microm (NP), 307.61+/-11.52 microm (NSP); cephalic space length 4.21+/-0.62 microm (NP), 5.32+/-0.79 microm (NSP); head to nerve ring 49.39+/-5.43 microm (NP), 57.40+/-4.46 microm (NSP); innenk?rper length 33.05+/-5.89 microm (NP), 44.02+/-8.71 microm (NSP); cephalic space width 4.28+/-0.59 microm (NP), 6.04+/-0.68 microm (NSP); body width at nerve ring 5.01+/-0.57 microm (NP), 7.45+/-0.75 microm (NSP). The number of nuclei between the cephalic space and nerve ring of NP (66.67+/-5.19) was also significantly less than in NSP (94.74+/-6.95). For infective stages, the body dimensions of NP were significantly smaller than NSP, i.e., body length 1632.50+/-131.48 microm (NP), 2002.63+/-222.60 microm (NSP); head to nerve ring 103.09+/-7.47 microm (NP), 122.44+/-9.62 microm (NSP); head to oesophago-intestinal junction 567.69+/-94.84 microm (NP), 666.75+/-110.08 microm (NSP); body width at oesophago-intestinal junction 23.15+/-1.55 microm (NP), 26.78+/-1.62 microm (NSP). It is too early to infer the NP type as an additional sibling species of W. bancrofti but it is reasonable to treat it as a new variety and additional work is needed to clarify its status.     

Cytologic detection of Wuchereria bancrofti microfilariae in urine collected during a routine workup for hematuria

A 23-year-old Guyanese man experienced intermittent, total, painless, gross hematuria for a month for which he sought medical attention at the Kings County Hospital Center in Brooklyn. Hematuria was accompanied by weakness but not by frequency of urination or burning on urination. Catheterized urine at the time of cystoscopy and each of two subsequent voided specimens examined cytologically contained sheathed microfilariae. Distinguishing features of the microfilariae were well demonstrated with the Papanicolaou stain. The well-stained nuclei, which did not extend into the clear zone, and the distinct, pale-stained sheath led to the positive identification of the microfilariae as Wuchereria bancrofti. The Papanicolaou stain may well be the stain of choice for the identification of microfilariae in the blood. The excellent detail obtained with this routine cytologic stain is as good as that with Giemsa, which does not stain the sheath.     

Transmission dynamics of lymphatic filariasis: density-dependence in the uptake of Wuchereria bancrofti microfilariae by vector mosquitoes

Gaining a better understanding of parasite infection dynamics in the vector mosquito (Diptera: Culicidae) population is central to improving knowledge regarding the transmission, persistence and hence control of lymphatic filariasis. Here, we use data on mosquito feeding experiments collated from the published literature to examine the available evidence regarding the functional form of the first component of this parasite-vector relationship for Wuchereria bancrofti (Filarioidea: Onchocercidae) causing Bancroftian filariasis, i.e. the rate of microfilariae (mf) uptake from the blood of infected humans by the feeding mosquito vector. Using a simple logarithmic regression model for describing the observed relationships between the mean numbers of mf ingested per mosquito and parasite load in humans in each study, and a linear mixed-effects meta-analytical framework for synthesizing the observed regressions across studies, we show here for the first time clear evidence for the existence of density-dependence in this process for all the three major filariasis transmitting mosquito vectors. An important finding of this study is that this regulation of mf uptake also varies significantly between the vector genera, being weakest in Culex, comparatively stronger in Aedes and most severe and occurring at significantly lower human mf loads in Anopheles mosquitoes. The analysis of the corresponding mf uptake prevalence data has further highlighted how density-dependence in mf uptake may influence the observed distributions of mf in vector populations. These results show that whereas strong regulation of mf uptake, especially when it leads to saturation in uptake at low human parasite intensities, can lead to static distributions of mf per mosquito with host parasite intensity, a weaker regulation of mf ingestion can give rise to changes in both mean mf loads and in the frequency distribution of parasites/mosquito with increasing human parasite intensity. These findings highlight the importance of considering local vector infection dynamics when attempting to predict the impacts of community-based filariasis control. They also emphasize the value of developing and applying robust meta-analytic methods for estimating functional relationships regarding parasitic infection from population ecological data.     

Haemocyte population and ultrastructural changes during the immune response of the mosquito Culex quinquefasciatus to microfilariae of Wuchereria bancrofti

Abstract Haemocytes circulating in the haemolymph protect insects against pathogens that enter the haemocoel. Changes in haemocyte morphology and differences in haemocyte counts during the immune response of Culex quinquefasciatus Say (Diptera: Culicidae) to microfilariae of Wuchereria bancrofti (Cobbold) (Spirurida: Onchocercidae) were investigated in the present study. The mean number of total haemocytes was significantly elevated in infected mosquitoes (P < 0.001), reaching a peak on the third day post‐infection. Differential counts show that mean numbers of prohaemocytes, plasmatocytes, granular cells and oenocytoids increased significantly after infection with microfilariae granulocytes compared to the control and näive groups of Cx. quinquefasciatus (P < 0.05). Changes in proportional counts of haemocytes were also analysed in haemolymph perfusates of Cx. quinquefasciatus infected with W. bancrofti. On the first day post‐infection, infected mosquitoes showed an increase in the proportion of prohaemocytes (18.8% compared to 9.6% for the control) and of oenocytoids (7.1% compared to 4.7% control); however, they exhibited lower levels of plasmatocytes (36.6% compared to 42.1% control) and granular cells (36.1% compared to 41.4% control). On day 14 post‐infection, similar changes were observed for these haemocyte types, except that the proportion of granular cells was significantly greater than the control (41.2% compared to 31.3% control). Although an enhancement of prohaemocyte numbers was observed, this cellular type did not show any ultrastructural alteration. On the other hand, granular cells, plasmatocytes and oenocytoids presented morphological alterations indicative of innate immunological activation in mosquitoes infected with W. bancrofti.     

Bloodmeal microfilariae density and the uptake and establishment of Wuchereria bancrofti infections in Culex quinquefasciatus and Aedes aegypti.

The relationship between ingestion of microfilariae (mf), production of infective larvae (L3) and mf density in human blood has been suggested as an important determinant in the transmission dynamics of lymphatic filariasis. Here we assess the role of these factors in determining the competence of a natural vector Culex quinquefasciatus and a non vector Aedes aegypti to transmit Wuchereria bancrofti. Mosquitoes were infected via a membrane feeding procedure. Both mosquito species ingested more than the expected number of microfilariae (concentrating factor was 1.28 and 1.81 for Cx. quinquefasciatus and Ae. aegypti, respectively) but Cx. quinquefasciatus ingested around twice as many mf as Ae. aegypti because its larger blood meal size. Ae. aegypti showed a faster mf migration capacity compared to Cx. quinquefasciatus but did not allow parasite maturation under our experimental conditions. Similar proportions of melanized parasites were observed in Ae. aegypti (2. 4%) and Cx. quinquefasciatus (2.1%). However, no relationship between rate of infection and melanization was observed. We conclude that in these conditions physiological factors governing parasite development in the thorax may be more important in limiting vectorial competence than the density of mf ingested.     

Assessing density dependence in the transmission of lymphatic filariasis: uptake and development of Wuchereria bancrofti microfilariae in the vector mosquitoes

Understanding density dependence in the transmission of lymphatic filariasis is essential for assessing the prospects of elimination. This study seeks to quantify the relationship between microfilaria (Mf) density in human blood and the number of third stage (L3) larvae developing in the mosquito vectors Aedes polynesiensis Marks and Culex quinquefasciatus Say (Diptera: Culicidae) after blood-feeding. Two types of curves are fitted to previously published data. Fitting a linearized power curve through the data allows for correction for measurement error in human Mf counts. Ignoring measurement error leads to overestimation of the strength of density dependence; the degree of overestimation depends on the accuracy of measurement of Mf density. For use in mathematical models of transmission of lymphatic filariasis, a hyperbolic saturating function is preferable. This curve explicitly estimates the Mf uptake and development at lowest Mf densities and the average maximum number of L3 that can develop in mosquitoes. This maximum was estimated at 23 and 4 for Ae. polynesiensis and Cx. quinquefasciatus, respectively.     

Microfilariae of Wuchereria bancrofti in cytologic smears

Microfilariae of Wuchereria bancrofti were observed in cytologic material in 35 cases. The material included cervicovaginal smears (17 cases), effusions (14), urine (2), bronchial washings (1) and ovarian cyst fluid (1). The initial diagnosis was made from the cytologic smear in all cases; none had clinical filariasis. Symptomatic vaginal bleeding in 9 of the 17 cases with microfilaria-positive cervicovaginal smears was reflected in the large numbers of red blood cells found in the smear. Blood eosinophilia was present in 11 of 19 cases investigated. Eosinophils were seen in the smears in 20 cases. In the majority of the cases of effusions with microfilariae the effusions were malignant. Significant adherence of inflammatory cells and macrophages to microfilariae was present in 7 of the 35 cases. The significance of these findings is discussed.     

Presence of Wolbachia endosymbionts in microfilariae of Wuchereria bancrofti (Spirurida: Onchocercidae) from different geographical regions in India

In view of the recent discovery of rickettsial endosymbionts, Wolbachia in lymphatic filarial parasites, Wuchereria bancrofti and Brugia malayi and subsequently of their vital role in the survival and development of the latter, antibiotics such as tetracycline are being suggested for the treatment of lymphatic filariasis, by way of eliminating the endosymbiont. But, it is essential to assess their presence in parasites from areas endemic for lymphatic filariasis before such a new control tool is employed. In the present communication, we report the detection of Wolbachia endosymbionts in microfilariae of W. bancrofti parasites collected from geographically distant locations of India, such as Pondicherry (Union Territory), Calicut (Kerala), Jagadalpur (Madhya Pradesh), Thirukoilur (TamilNadu), Chinnanergunam (TamilNadu), Rajahmundry (Andhra Pradesh), and Varanasi (Uttar Pradesh), using Wolbachia specific 16S rDNA polymerase chain reaction.     

Susceptibility of aposymbiotic Culex quinquefasciatus to Wuchereria bancrofti

Larvae of the mosquito Culex quinquefasciatus originating from Kenya were reared in 0.025 mg/ml tetracycline hydrochloride. Some of the resulting progeny were shown, by electron microscopy and crossing experiments, to have been rendered free of the rickettsia like symbiont Wolbachia pipientis and from these progeny, symbiont-free lines were established. In experimental feedings on infected human volunteers and on cryopreserved microfilariae, the aposymbiotic stocks were found to be fully susceptible to the filaria Wuchereria bancrofti. This contrasts with some recently published data on Aedes polynesiensis, from which it has been suggested that rickettsia like symbionts have an important role in the development of filaria in the mosquito.     

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.     

Effectiveness of annual ivermectin treatment for Wuchereria bancrofti infection

Using estimates for the anthelmintic efficacy of a single dose of ivermectin in the treatment of lymphatic filariasis patients, Anton Plaisier, Wilma Stolk, Gerrit van Oortmarssen and Dik Habbema here present and discuss model predictions of the impact of a five-year programme of annual community treatment on the intensity of infection. They show that the effectiveness of such programmes in terms of reductions in the microfilarial density depends critically on the treatment coverage and the pattern of attendance at repeated mass administrations. Improving these factors will possibly be more important than improving the efficacy of ivermectin by increasing its dosage or by adding other drugs.     

Molecular characterisation of the actin gene of the filarial parasite Wuchereria bancrofti

Wuchereria bancrofti is the major cause of lymphatic filariasis in humans. Although it is responsible for this immensely morbid and debilitating disease, very little is known of the basic molecular biology of this parasite, and there is a vast lack of knowledge on its gene organisation. In this study, the actin gene of W. bancrofti has been characterised by sequencing a clone isolated from a genomic DNA library of this parasite. The 5' flanking region had a potential TATA box and a putative mRNA initiation site. The gene had five exons encoding 376 amino acids, and four introns ranging in size from 109 to 190bp. The 3' flanking region had a potential polyadenylation signal with the sequence ATTAAA which is a common natural variant of the conventional sequence AATAAA. The gene was AT-rich, with a GC content of 37.2%. Southern blot analysis of W. bancrofti genomic DNA indicated that the gene is possibly found as a single copy. The actin amino acid sequence of W. bancrofti showed a high degree of homology to the actin of many organisms of different taxonomic groups, but the highest homology was observed with the free-living nematode Plectus acuminatus. This suggests that P. acuminatus may bear a close evolutionary relationship to W. bancrofti.     

Population genomics of the filarial nematode parasite Wuchereria bancrofti from mosquitoes

Wuchereria bancrofti is a parasitic nematode and the primary cause of lymphatic filariasis – a disease specific to humans. W. bancrofti currently infects over 90 million people throughout the tropics and has been acknowledged by the world health organization as a vulnerable parasite. Current research has focused primarily on the clinical manifestations of disease and little is known about the evolutionary history of W. bancrofti. To improve upon knowledge of the evolutionary history of W. bancrofti, we whole genome sequenced 13 W. bancrofti larvae. We circumvent many of the difficulties of multiple infections by sampling larvae directly from mosquitoes that were experimentally inoculated with infected blood. To begin, we used whole genome data to reconstruct the historical population size. Our results support a history of fluctuating population sizes that can be correlated with human migration and fluctuating mosquito abundances. Next, we reconstructed the putative pedigree of W. bancrofti worms within an infection using the kinship coefficient. We deduced that there are full‐sib and half‐sib relationships residing within the same larval cohort. Through combined analysis of the mitochondrial and nuclear genomes we concluded that this is likely a results of polyandrous mating, the first time reported for W. bancrofti. Lastly, we scanned the genomes for signatures of natural selection. Annotation of putative selected regions identified proteins that may have aided in a parasitic life style or may have evolved to protect against current drug treatments. We discuss our results in the greater context of understanding the biology of an animal with a unique life history and ecology.