Genetic heterogeneity of Wuchereria bancrofti populations at spatially hierarchical levels in Pondicherry and surrounding areas, south India

Lymphatic filariasis (LF), caused by Wuchereria bancrofti is widely distributed in areas of India with variable geoclimatic factors. These factors, coupled with chemotherapeutic pressure exerted for past half a century may have influenced the genetic structure of the parasite populations. A complex genetic structure of parasite populations will have major consequences to the on-going global LF elimination programme. Hence, it is of interest to understand the genetic heterogeneity of W. bancrofti parasite populations. We studied the genetic heterogeneity of populations of W. bancrofti populations from mf carriers residing in an urban area of Pondicherry and surrounding villages through molecular (RAPD) fingerprinting. The analysis showed that W. bancrofti populations of an urban area were mostly highly heterogeneous, while those of rural areas were homogenous. The urban parasite populations appeared to be a pool of parasite population originating from surrounding rural areas. At least two genotypes, exhibiting high genetic differentiation and minimum gene flow between them, existed in Pondicherry urban areas. There was a minimum gene flow between parasite populations of villages. The genetic heterogeneity of parasite population in an adult microfilariae carrier was significantly high, possibly due to accumulation of different genotypes of the parasite with increasing age. The genetic heterogeneity of W. bancrofti populations in an individual mf carrier, in urban and rural areas, within an endemic area at large, and in different geographical regions of India may have far reaching implications to the epidemiology and strategies of chemotherapy control being adopted for LF elimination programme launched on a global scale.     

Factors affecting transmission of Wuchereria bancrofti by anopheline mosquitoes. 4. Facilitation, limitation, proportionality and their epidemiological significance.

Quantitative understanding of the transmission dynamics of lymphatic filarial parasites is essential for the rational planning of control strategies. One of the most important determinants of transmission dynamics is the relationship between parasite yield, the success rate of ingested microfilariae (mf) becoming infective larvae in a mosquito vector, and mf density in the source of the human blood meal. Three types of relationship have been recognized in human filaria/mosquito couples--limitation, facilitation and proportionality; facilitation has hitherto been observed only in the couple Wuchereria bancrofti/Anopheles gambiae in Burkina Faso, in experimental studies on a high density mf carrier. The present paper demonstrates facilitation in W. bancrofti/An. gambiae and W. bancrofti/An. arabiensis in lower mf density carriers in The Gambia and Tanzania, and in W. bancrofti/An. funestus in Tanzania. Facilitation was not found in An. melas in The Gambia nor in An. merus in Tanzania. Analysis of published data shows limitation at low level mf densities in W. bancrofti/Culex quinquefasciatus in Sri Lanka, and in the same couple in India. Limitation also occurs in Brugia malayi/Aedes togoi in experimental cats; proportionality occurs in B. malayi/Mansonia bonneae in Malaysia. The epidemiological significance of these host/parasite relationships is discussed, and supporting evidence for its validity is presented from the published results of large-scale control programmes.     

Progression of lymphatic vessel dilatation in the presence of living adult Wuchereria bancrofti

Bancroftian filariasis, a mosquito-transmitted disease commonly known as elephantiasis, is caused by infection with the parasite Wuchereria bancrofti. Infection with this parasite can induce a broad array of chronic debilitating and socially stigmatizing conditions, but the pathogenesis of this morbidity remains obscure. Recent evidence indicates that in filariasis-endemic areas the primary lesion is not lymphatic vessel obstruction but, rather, dilatation. To determine the extent to which lymphatic dilatation occurs in the presence of living adult W. bancrofti, we performed longitudinal ultrasonographic measurements in 80 men (mean age 24 years) in Brazil who had a total of 107 W. bancrofti nests detectable by ultrasound. Initial mean lymphatic vessel diameter at the site of the worms was 3.4 mm (range, 0.7-11.3), and was greater in men with 2 or more nests (3.9 mm) than in those with only one nest (3.0 mm, P = 0.003). During the study period (2-35 months, mean, 13.7), lymphatic vessel diameter increased at the site of 92 (86.0%) adult worm nests. Mean rate of increase of lymphatic vessel diameter was 1.2 mm per person-year (range, 0-0.93 mm per month). In a general linear model, no factors, including treatment with antifilarial drugs, were significantly associated with rate of vessel diameter increase. Thus, lymphatic vessel dilatation progress in the presence of living adult W. bancrofti; the rate of this progression is heterogeneous. These data suggest that lymphatic dilatation will continue to progress in most infected persons even after mass treatment with currently recommended antifilarial drugs. In addition to interrupting transmission, the global programme for elimination of lymphatic filariasis should address the potential for disease progression in persons who remain infected with adult W. bancrofti.     

Detection of Wuchereria bancrofti in mosquitoes by the polymerase chain reaction: a potentially useful tool for large-scale control programmes

Focally endemic bancroftian filariasis is targeted for elimination in the Nile delta of Egypt. Improved methods are needed for identifying endemic villages to be included in the control programme and for monitoring its success. We have evaluated the performance of a polymerase chain reaction (PCR) assay in estimating Wuchereria bancrofti infection in pools of Culex pipiens (1-25 females) from 2 adjacent villages with high (El Qolzom, 10.8%) and low (Kafr Shorafa, 2.1%) prevalence rates of human filariasis. This assay detects a repeated sequence in W. bancrofti deoxyribonucleic acid (DNA). Mosquitoes resting within houses were captured by aspiration and pooled by house. Houses were classified as positive or negative for human filarial infection based on night blood examinations of residents. The assay detected parasite DNA in mosquitoes from 60% of 25 infected houses and 24% of 25 uninfected houses. PCR processing of mosquitoes caught within houses of unknown filariasis infection status (44 in El Qolzom, 37 in Kafr Shorafa) identified 31.8% and 8.1% of houses, respectively, as containing infected mosquitoes. These results support the validity of the PCR assay for evaluating filarial prevalence in different villages. C. pipiens collected outdoors in dry ice-baited traps and tested by PCR (266 in Qolzom, 82 in Kafr Shorafa) did not contain parasite DNA. Pools of female mosquitoes (296 in Qolzom, 240 in Kafr Shorafa) captured in oviposition traps were also negative. We concluded that the PCR based assay is a powerful epidemiological tool that can be used for evaluating W. bancrofti infection in villages in the Nile delta and for monitoring the application of control programmes in filariasis endemic areas.     

Immune reactions to Wuchereria bancrofti infections in Tanzania. II. Characterization of the serum-mediated adherence reaction of leucocytes to microfilariae in vitro

Investigations to characterize the in vitro reaction of serum-mediated leucocyte-adherence to microfilariae of Wuchereria bancrofti were carried out in Tanzania. The adherence reaction took place within one hour and at least two serum factors were involved: a heat-labile factor, present also in normal serum (probably complement), and a more heat-stable factor, present in positive serum only (probably an antibody). Neutrophils and eosinophils were involved in the reaction proportionately to Their numbers in the cell solutions used; the reaction killed significantly more microfilariae, than did adherence-negative tests. Microfilariae could be used for at least one week after their isolation from the donor, but a significant decrease in the percentage of reactive microfilariae occurred within 16 hours of isolation. A high degree of species specificity was observed when positive sera were tested against other nematodes. The involvement of complement and the speed with which the reaction took place is in contrast to the results of similar studies from India, and may indicate a difference in the parasite in the two continents.     

Temperature-sensitive photoreactivation of cyclobutane thymine dimer in soybean

UV radiation induces the formation of two classes of photoproducts in DNA, the cyclobutane pyrimidine dimer (CPD) and the pyrimidine 6-4 pyrimidone photoproduct. CPDs in plants are repaired by class II CPD photolyase via a UV-A/blue light-dependent mechanism. The genes for the class II CPD photolyase have been cloned from higher plants such as Arabidopsis, Cucumis sativus (cucumber), Oryza sativa (rice) and Spinacia oleracea (spinach). Flavin adenine dinucleotide (FAD) has been identified as a cofactor. Here we report the isolation and characterization of the CPD photolyase cDNA from soybean (Glycin max). The sequence of amino acids predicted from the cDNA sequence was highly homologous to sequences of higher plant class II CPD photolyases. When the cDNA was expressed in a photolyase-deficient Escherichia coli, photoreactivation activity was partially restored by illumination with a fluorescent light. The purified enzyme showed CPD binding and light-dependent photoreactivation activities in vitro. When soybean CPD photolyase was heat-treated in vitro from 25 degrees C to 45 degrees C for 3 min, thymine dimer-binding activity and photoreactivation activity were decreased, and FAD was released from the enzyme. On the other hand, when the enzyme-CPD complex was heat-treated, photoreactivation activity was stable. We argue that FAD in the soybean CPD photolyase is labile for temperature, but once the enzyme-CPD complex has formed, FAD becomes tightly bound to the enzyme or complex.     

A qPCR-based multiplex assay for the detection of Wuchereria bancrofti, Plasmodium falciparum and Plasmodium vivax DNA

The purpose of this study was to develop real-time multiplex quantitative PCR (qPCR) assays for the simultaneous detection of Wuchereria bancrofti (Wb), Plasmodium falciparum (Pf) and P. vivax (Pv) in mosquitoes. We optimized the assays with purified DNA samples and then used these assays to test DNA samples isolated from Anopheles punctulatus mosquitoes collected in villages in Papua New Guinea where these infections are co-endemic. Singleplex assays detected Wb, Pf and Pv DNA in 32%, 19% and 15% of the mosquito pools, respectively, either alone or together with other parasites. Multiplex assay results agreed with singleplex results in most cases. Overall parasite DNA rates in mosquitoes, estimated by PoolScreen 2 software, for Wb, Pf and Pv were 4.9%, 2.7% and 2.1%, respectively. Parasite DNA rates were consistently higher in blood-fed mosquitoes than in host-seeking mosquitoes. Our results show that multiplex qPCR can be used to detect and estimate prevalence rates for multiple parasite species in arthropod vectors. We believe that multiplex molecular xenodiagnosis has great potential as a tool for non-invasively assessing the distribution and prevalence of vector-borne pathogens such as W. bancrofti and Plasmodium spp. in human populations and for assessing the impact of interventions aimed at controlling or eliminating these diseases.     

Vector competence of autogenous and anautogenous Culex pipiens mosquitoes for Wuchereria bancrofti

Vector competence of autogenous and anautogenous Culex pipiens; derived from North Sinai Governorate, Egypt, for the human filaria parasite Wuchereria bancrofti was studied. After feeding on the same microfilaremic volunteers both biotypes were readily infected with the parasite (infection rates > 80%) and supported its development to the infective stage within 11 - 12 days. Infective rates of both autogenous and anautogenous mosquitoes were similar (> 95%). However, autogenous Cx. pipiens developed significantly less number of infective stage larvae (4.7 +/- 1.4 L3 / female) than did anautogenous siblings (6.7 +/- 3.6 L3 / female) (P < 0.05). Moreover, autogenous females were observed to contain twice the number of L3 larvae in the thoracic muscles and less larvae in the head region compared to autogenous counterparts. Vector competence characteristics of Cx. pipiens derived from a filariasis endemic area in Qalubiya Governorate were similar to those of anautogenous mosquitoes of North Sinai. These findings indicate that autogenous Cx. pipiens may be less efficient vector of W. bancrofti in endemic areas of Egypt.     

Polymorphism of gp15/400 allergen gene of W. bancrofti from different regions of India endemic for lymphatic filariasis.

Nematode polyprotein allergens (NPA) are lipid binding/transport molecules that elicit elevated levels of IgE response in the infected host, leading to Th2 type of immune response. They also transport arachidonic acid and its metabolites that are known to be involved in the action of antifilarial drug, Diethylcarbamazine and hence are of great significance for the control of lymphatic filariasis. We investigated the polymorphism of gp15/400 polyprotein of 35 isolates of lymphatic filarial parasite Wuchereria bancrofti collected from different geographic locations of India. The repeat sub-unit of the gene was found to be highly conserved in all the isolates with only two nucleotide synonymous changes at positions 286 (A-G) and 337 (C-T). Since this molecule is highly conserved and has multifarious roles in the survival and pathogenesis of the parasite it has good potential as a target for drug, immunodulation tool and immunotherapy development.     

Polymorphism of gp15/400 allergen gene of Wuchereria bancrofti from different regions of India endemic for lymphatic filariasis.

Nematode polyprotein allergens (NPA) are lipid-binding/transport molecules that elicit elevated levels of IgE response in the infected host, leading to Th2 type of immune response. They also transport arachidonic acid and metabolites that is known to be the action of antifilarial drug, diethylcarbamazine, and hence are of great significance to the control of lymphatic filariasis. We investigated the polymorphism of gp15/400 polyprotein of 35 isolates of lymphatic filarial parasite Wuchereria bancrofti collected from different geographic locations of India. The repeat subunit of the gene was found to be highly conserved in all the isolates with only two nucleotide synonymous changes at positions 369 (A-G) and 375 (C-T). As this molecule is highly conserved and has multifarious roles in the survival and pathogenesis of the parasite, it has good potential as a target for drug, immunodulation tools and immunotherapy development.     

The transmission dynamics of bancroftian filariasis: the distribution of the infective larvae of Wuchereria bancrofti in Culex quinquefasciatus and Anopheles gambiae and its effect on parasite escape from the vector

The anatomical distribution of the infective larvae (L3) of Wuchereria bancrofti in Culex quinquefasciatus and Anopheles gambiae, and its influence on L3 escape, was evaluated by exposing the vectors to human individuals infected with W. bancrofti. After the extrinsic incubation period of W. bancrofti, a random sample of the infected mosquitoes was dissected to determine the distribution of infective larvae in the body of the mosquitoes and the proportion of mosquitoes that were infected. The remaining mosquitoes were exposed to an experimental definitive host (mouse skin). The infective larvae on and in the host tissues were counted. The engorged mosquitoes were dissected to determine the proportion infected and the distribution of L3 in mosquitoes after exposure. The results show that 53.4% of L3 escaped from Cx. quinquefasciatus to enter the experimental host whereas only 4% of L3 escaped from An. gambiae. Analysis of the results indicates that the number of L3 in the vector and the proportion of L3 in the head and mouthparts influence the number of L3 escaping from the vector to enter the definitive host and consequently the number that gain access to host tissues. The implication of these findings on the transference of the parasite from the vector to the definitive host is discussed.     

Does longevity of adult Wuchereria bancrofti increase with decreasing intensity of parasite transmission? Insights from clinical observations

To interrupt transmission of Wuchereria bancrofti, a parasite that causes lymphatic filariasis, mass treatment of at-risk populations with antifilarial drugs is recommended for 4-6 years, the minimum estimated adult worm lifespan. Factors associated with adult worm longevity are unknown. In Recife, Brazil, we conducted a retrospective cohort study of 57 men whose adult W. bancrofti were not sensitive to diethylcarbamazine and who were followed with semi-annual physical examinations (to detect intrascrotal nodules, indicative of adult worm death) and ultrasound examinations (to detect the 'filaria dance sign' (FDS), indicative of living adult worms). After 5 years, the FDS remained detectable in 10 (24.4%) of 41 adult worm nests in 25 men from areas of high filariasis transmission intensity and in 30 (90.9%) of 33 nests in 32 men from areas of low transmission (P<0.001). New nodules and adult worm nests were detected only in men from high-transmission areas. Of 30 men who were microfilaria-positive initially and whose FDS remained detectable after 5 years of follow-up, 19 (63.3%) remained microfilaria-positive in 5 ml blood (mean density, 0.4 per ml). In conclusion, survival of adult W. bancrofti is inversely associated with transmission intensity. These findings have implications for filariasis elimination and research.     

Characterization of cofactor-independent phosphoglycerate mutase isoform-1 (Wb-iPGM) gene: a drug and diagnostic target from human lymphatic filarial parasite, Wuchereria bancrofti

The inter-conversion of 3-phosphoglycerate and 2-phosphoglycerate during glycolysis and gluconeogenesis in filarial nematodes, is catalyzed by a co-factor-independent phosphoglycerate mutase (iPGM). The gene encoding iPGM isoform-1 was amplified from Wuchereria bancrofti, the major causative agent of human lymphatic filariasis. Partial genomic DNA (gDNA) fragment of the gene was also amplified from periodic and sub-periodic forms of W. bancrofti and Brugia malayi and sequenced. The Wb-iPGM isoform-1 gene encodes an ORF of 515 amino acids and is found to share 99.4%, 96.0%, and 64.0% amino acid sequence identity with iPGM of B. malayi, Onchocerca volvulus, and Caenorhabditis elegans, respectively. Serine and all the other 13 amino acid residues involved in the catalytic function of iPGM are highly conserved. Further comparison of iPGM nucleotide and amino acid sequences of Wolbachia of B. malayi with Wb-iPGM showed 41% and 54.4% similarity, respectively. The analysis of partial genomic and amino acid sequences and phylogenetic tree of Wb-iPGM indicated that this gene, apart from being a potential drug target, could provide diagnostic, taxonomical, and evolutionary markers. This is the first report of the characterization of iPGM gene from W. bancrofti.     

Expression and properties of human liver beta-ureidopropionase

A cDNA encoding beta-ureidopropionase (BUP) was isolated from a human liver cDNA library, expressed in E. coli, and purified from the culture extract. The 2,006 bp cDNA contained a 1,152 bp open reading frame encoding a protein of 384 amino acids with a molecular weight of 43,165 Da. The subunit molecular weight of the enzyme expressed was about 43,000 Da. The enzyme was inhibited by 1 mM propionate, but not by 10 mM beta-alanine. Chemical analysis of the purified human BUP showed 0.54 zinc atoms per subunit, and the sequence of BUP cDNA contained one putative zinc-binding site motif. The purified enzyme had a pI of 5.65, and exhibited positive cooperativity with N-carbamoyl-beta-alanine as the substrate with a Hill coefficient 2.0. These properties of human BUP, except the inhibition by beta-alanine, were similar to the rat liver purified enzyme. Beta-alanine inhibits rats BUP activity. The complex regulatory function and the negative cooperative mechanism of BUP by beta-alanine have been observed in rats. This kind of mechanism may not exist in humans, because beta-alanine did not inhibit human BUP.     

EPIFIL: the development of an age-structured model for describing the transmission dynamics and control of lymphatic filariasis

Mathematical models of transmission dynamics of infectious diseases provide a useful tool for investigating the impact of community based control measures. Previously, we used a dynamic (constant force-of-infection) model for lymphatic filariasis to describe observed patterns of infection and disease in endemic communities. In this paper, we expand the model to examine the effects of control options against filariasis by incorporating the impact of age structure of the human community and by addressing explicitly the dynamics of parasite transmission from and to the vector population. This model is tested using data for Wuchereria bancrofti transmitted by Culex quinquefasciatus in Pondicherry, South India. The results show that chemotherapy has a larger short-term impact than vector control but that the effects of vector control can last beyond the treatment period. In addition we compare rates of recrudescence for drugs with different macrofilaricidal effects.     

Antigenic activity in adult Dipetalonema viteae in the indirect immunofluorescent test against sera from filariasis patients--the immunofluorescent histological search for "pure" antigen.

Using IFAT, it has been shown that isolated egg-shells and uterine fluid of Dipetalonema viteae are the most potent antigens in heterologous systems using human sera from patients infected with Wuchereria bancrofti, Onchocerca volvulus and Loa loa, as well as in homologous systems using sera from animals infected with D. viteae. It is suggested that these antigens are unlikely to be highly species-specific, and that anatomical isolation of antigens is a necessary prerequisite to immunochemical analysis aimed at the preparation of a "pure" antigen.     

Enzyme variation in Plasmodium falciparum in the Gambia

A study has been made of electrophoretic forms of enzymes of P. falcaparum in samples of blood from Gambian women and children. Variation was found in each of the 3 enzymes examined. 2 forms of parasite GPI, labelled GPI-1 and GPI-2, 2 forms of parasite LDH, labelled LDH-1 and LDH-2 and 3 forms of parasite 6PGD, labelled 6PGD-1, 6PGD-2 and 6PGD-3 were identified. Different combinations of enzyme forms occurred among different samples demonstrating genetic diversity of the parasite within and between blood samples. The distribution of enzyme forms among the samples suggested that the parasites belong to a single mendelian population. From statistical considerations of the distribution of enzyme forms among the samples it was estimated that the samples contained an average of about 2 parasite clones each. It was also possible to estimate the frequencies of variant forms of GPI among parasite clones as 0·62 and 0·38 for GPI-1 and GPI-2 respectively and LDH as 0·76 and 0·24 for LDH-1 and LDH-2 respectively. 6PGD-1 occurred in about 95% of blood samples showing activity of this enzyme, the remaining variants of 6PGD occurred in only 2 samples each. In many instances it was possible to identify the enzyme forms characterizing clones of parasite in individual blood samples, but rarely to estimate the actual number of clones present in a given sample. In a few samples, however, in which a single form of each enzyme occurred it could be estimated that probably only 1 parasite clone was present.     

Prophylactic evaluation of recombinant extracellular superoxide dismutase of Brugia malayi in jird model

The immunoscreening of Brugia malayi adult cDNA library with pooled endemic normal sera identified several seroreactive clones including, EC-SOD which contained a 612 bp insert and showed significant nucleotide and deduced amino acid sequence homologies with superoxide dismutase (SOD) of other nematode parasites. The SODs are known to play an important role in the protection of parasite against reactive oxygen species of the host. The coding region of the B. malayi EC-SOD (BmEC-SOD) was cloned and expressed in Escherichia coli followed by affinity purification on nickel agarose resin. Staining of native polyacrylamide gel for SOD activity of the expressed recombinant protein revealed that SOD activity inactivated by potassium cyanide and hydrogen peroxide but not by sodium azide, indicating presence of Cu/Zn-SOD. The rBm EC-SOD protein showed its activity over a broad range of pH.7.0-11.0. Further the immune protective activity of recombinant EC-SOD antigen was evaluated in susceptible host, jirds (gerbils) (Meriones unguiculatus) against B. malayi filarial infection. The immunized jirds showed 33.5% and 36% cytotoxicity against microfilariae and 42.8% and 45.5% cytotoxicity against infective larvae in in vitro antibody dependent cellular cytotoxicity (ADCC) assay and in in situ micropore chamber methods respectively. This study suggests that the rBm EC-SOD antigen could stimulate a partial protective immune response against microfilariae and infective larvae in experimental animals against filarial infection.     

Oral infection of Aedes polynesiensis by Wuchereria bancrofti by using parafilm membrane feeding.

In order to construct a cDNA library from third-stage larvae (L3) of Wuchereria bancrofti var. pacifica, the Parafilm membrane feeding method is proposed for the oral infection of Aedes polynesiensis. Heparinized blood supplemented with 5.10(-3) M ATP was put in the feeder with carbon dioxide provided as additional phagostimulant. The results of this artificial infection feeding method were compared with those obtained when mosquitoes fed directly on the forearm of a microfilaremic patient. The number of females feeding through the artificial membrane was smaller than on the patient's forearm (32.1 vs. 84.8%). The mean number of L3s obtained per female was not statistically different between the 2 feeding methods; however, the total number of L3s obtained from 100 females allowed to feed in each group was twice as high in the natural feeding method.