Modelling variability in lymphatic filariasis: macrofilarial dynamics in the Brugia pahangi--cat model.

A striking feature of lymphatic filariasis is the considerable heterogeneity in infection burden observed between hosts, which greatly complicates the analysis of the population dynamics of the disease. Here, we describe the first application of the moment closure equation approach to model the sources and the impact of this heterogeneity for macrofilarial population dynamics. The analysis is based on the closest laboratory equivalent of the life cycle and immunology of infection in humans--cats chronically infected with the filarial nematode Brugia pahangi. Two sets of long-term experiments are analysed: hosts given either single primary infections or given repeat infections. We begin by quantifying changes in the mean and aggregation of adult parasites (inversely measured by the negative binomial parameter, kappa in cohorts of hosts using generalized linear models. We then apply simple stochastic models to interpret observed patterns. The models and empirical data indicate that parasite aggregation tracks the decline in the mean burden with host age in primary infections. Conversely, in repeat infections, aggregation increases as the worm burden declines with experience of infection. The results show that the primary infection variability is consistent with heterogeneities in parasite survival between hosts. By contrast, the models indicate that the reduction in parasite variability with time in repeat infections is most likely due to the 'filtering' effect of a strong, acquired immune response, which gradually acts to remove the initial variability generated by heterogeneities in larval mortality. We discuss this result in terms of the homogenizing effect of host immunity-driven density-dependence on macrofilarial burden in older hosts.

Brugia pahangi in cats: the passive transfer of anti-microfilarial immunity from immune to non-immune cats.

Serum from cats (Felis catus) that were repeatedly infected with Brugia pahangi and had become amicrofilaraemic (mf-ve) was injected intravenously into microfilaraemic (mf+ve) cats. If more than 1 microliter of immune serum per 1000 mf was injected, microfilarial counts fell dramatically within minutes and, in some cats, mf completely disappeared. In most cases mf reappeared 21-44 days later. However, in two experiments mf never reappeared and circulating antigen (indicative of the presence of living adults) could not be detected. At autopsy no adult worms were found, but in one cat 6 mf/ml were detected by filtration of cardiac blood. Passive transfer of single Ig isotypes showed that IgG is the immunoglobulin responsible for the mf killing effect of immune serum, and that IgG1 is probably the most active isotype. Mf killing and destruction, occurred in the lungs in an antibody dependent cell mediated reaction involving neutrophils, eosinophils and mononuclear cells. Three of the 20 recipient cats died from what appeared to be anaphylactic shock while under anaesthesia probably due to the sudden release of inflammatory mediators in the lung.

The sheath of the microfilaria of Brugia malayi from human infections has IgG on its surface.

Microfilariae (mf) of Brugia malayi from microfilaraemic people had human IgG on their sheath. Fluorescent antibody studies showed that the predominant IgG isotype was IgG3, with IgG4 and IgG1 being present in lower quantities. Human albumin could not be detected. The sera of patients with chronic disease contained high levels of an IgG2 antibody that reacted with the sheath of mf taken from other people.

Isolation and characterization of three subpopulations of IgG in the common cat (Felis catus).

Although the cat is an important model for a number of human diseases little is known of its basic humoral immunity. In this study we have isolated three subpopulations of IgG from cat serum by DEAE anion-exchange chromatography and protein A affinity chromatography. Subpopulation 3 did not bind to DEAE equilibrated in 2 mM phosphate buffer pH 8.0 while subpopulations 1 and 2 were eluted, as a mixed population, with a salt gradient between 3 mM and 25 mM. When this fraction was loaded onto protein A-Sepharose two distinct peaks were always generated in a pH gradient (pH 8.0-2.1). The first of these, subpopulation 2, was eluted on average at pH 4.3 while the second, subpopulation 1, was eluted on average at pH 3.4. Subpopulations 1 and 2 had similar immunoelectrophoretic mobilities and isoelectric points while subpopulation 3 was more cathodic in both tests. Antisera produced in both mice and rabbits were rendered specific, by absorption of cross-reacting antibodies, for determinants on the heavy chain of each subpopulation as shown by Western blotting. The data suggest, but are not conclusive, that each of these subpopulations are distinct subclasses of IgG.

Stage and isotype specific immune responses in a rat model of filariasis.

Inbred PVG rats infected with 100 Brugia pahangi infective larvae (L3) divide into rats that develop microfilaraemic infection (Mf+), and those that remain microfilaria negative (Mf-). All rats had high levels of specific IgG to adult worm and L3 extracts, however, after the onset of microfilaraemia, Mf+ rats had significantly higher levels of IgG to Mf extract. Mf+ rats recognized several antigenic components of each developmental extract that were not responded to by Mf- rats. In particular Mf+ rats recognized a triplet of proteins of 61-67 kD in microfilarial extract from day 74 post infection onwards. This indicated that these proteins were stage specific to Mf and Mf- rats had not been exposed to Mf rather than Mf absence being due to a protective antibody response. Analysis of the immunoglobulin isotype usage during infection revealed that each isotype was independent both in its period of induction and the developmental stage to which it responded. The predominant isotypes responding throughout infection were IgG1, IgG2a and IgM. Specific IgG2b and IgG2c were elevated early in infection but after the onset of microfilaraemia antibody of these subclasses was suppressed. The antigenic profiles recognized on immunoblots by IgG1, IgG2a and IgM were very similar.

IgE responses in cats infected with Brugia pahangi.

Passive cutaneous anaphylaxis tests were used to examine the IgE responses of cats repeatedly infected with the filarial nematode Brugia pahangi. Specific IgE was usually detected only in those cats that killed their adult worms and rarely in those cats in which adult worms survived for long periods. We suggest that this specific IgE is actively involved in killing adult worms in the lymphatics.

The sheath of the microfilaria of Wuchereria bancrofti has albumin and immunoglobulin on its surface.

Using direct fluorescent antibody analysis it was shown that the sheath of live microfilariae of Wuchereria bancrofti has human albumin and the immunoglobulin G subclasses IgG1 and IgG4 on its surface.

Brugia pahangi infections in immune-compromised rats demonstrate that separate mechanisms control adult worm and microfilarial numbers.

The immunological basis of resistance to Brugia pahangi infection in rats was studied. Infections were investigated in athymic rnu/rnu rats and in rats treated with the immuno-suppressive agents cyclosporin A (CsA) or cyclophosphamide (Cy). The recovery of adult worms in normal rats was 1-2% in comparison to 12.2% recovery in athymic rats. CsA and Cy treated rats did not have increased adult worm burdens. Microfilarial (Mf) levels (expressed as Mf per ml per adult worm) were highly elevated in both athymic and Cy treated rats but not in CsA treated rats. IgG and IgM levels to B. pahangi antigens were severely depressed in both athymic and Cy treated rats. IgG levels but not IgM levels were abrogated in CsA treated rats. These results implied that control of larval establishment or adult killing, and regulation of Mf levels are separate T-cell dependent mechanisms and act independently of IgG antibody. Control of Mf levels is associated with a specific IgM response which is Cy sensitive but CsA resistant.

Repeated infection of cats with Brugia pahangi: parasitological observations.

Cats were repeatedly inoculated with infective larvae of Brugia pahangi. On parasitological grounds they could be divided into 5 groups. Group I--most cats (some 70%) became microfilaraemic (mf+) and retained high levels of microfilariae (mf) in their blood for over 2 years. In some Group I cats mf counts stabilized at high levels whilst in others mf counts continued to increase. Large numbers of fecund adult worms were recovered from their lymphatics. Adult counts were not made on the cats in the current experiments but over 100 adults have been recovered from 'super-susceptible' cats. Large amounts of B. pahangi adult antigen were consistently present in the serum of all Group I cats. About 30% of cats became amicrofilaraemic (mf-). In these cats the peak mf levels were seldom above 10,000 mf/ml. Group II--these cats had less than 10,000 mf/ml and low antigen levels. After more than 1 year of being repeatedly infected B. pahangi adult antigen slowly declined and eventually could no longer be detected in their serum and the number of mf declined very slowly after the fall in antigen levels. This shows that in Group II cats the adult worms die and as the cats are resistant to the development of the continuing weekly inoculation of L3 no new adults can develop. Group III--these cats became mf--during the first year of infection but remained B. pahangi antigen-positive for many weeks after this and, at autopsy, had living adults in their lymphatics.(ABSTRACT TRUNCATED AT 250 WORDS)

Cats with single Brugia pahangi infections: relationship between parasitological status and humoral responses to somatic and surface parasite antigens.

Cats given a single inoculation of Brugia pahangi infective larvae (L3) were retrospectively allocated into three groups according to parasitological outcome of infection. Recognition of somatic and surface antigens of B. pahangi by sera from each group was compared by ELISA, immunoelectroblotting, and immunoprecipitation techniques. In cats that never became microfilaraemic mean serum IgG antibody levels against somatic extracts from adult male worms, L3, and microfilariae (mf) were higher than levels in cats that initially became microfilaraemic (mf + ve) then spontaneously became nonmicrofilaraemic (mf - ve). The lowest levels of antibody against each stage were found in cats that remained persistently mf + ve. Antigenic components of 18 kD and 22 kD in somatic extracts of adult worms and L3 were recognized by sera from cats that never became mf + ve and by spontaneously mf - ve cats, but not by sera of persistently mf + ve cats. When radioiodinated surface antigens of mixed adult worms and microfilariae were immunoprecipitated by sera from cats in the three groups, no correlation was observed between recognition of individual antigen components and parasitological outcome of infection.

Diethylcarbamazine (DEC): immunopharmacological interactions of an anti-filarial drug.

Anti-parasitic drugs may achieve their therapeutic effect either by direct activity against the pathogenic organism, or by altering host factors which lead to parasite killing. In this review, we discuss the evidence for an indirect mode of action for one major anti-filarial drug, diethylcarbamazine (DEC). The interpretation most consistent with existing data is that DEC alters arachidonic acid metabolism in microfilariae and in host endothelial cells. These changes may result in vasoconstriction and amplified endothelial adhesion leading to immobilization of microfilarial parasites, enhanced adherence and cytotoxic activity by host platelets and granulocytes. These events would represent activation of the innate, non-specific immune system, independent of the adaptive, antigen-specific, immune response. This model explains the paradox between rapid clearance in vivo and the lack of an in vitro effect, as well as the efficacy of DEC in non-immune animals. It may also account for the inconsistencies in the effects of DEC against different filariae in different host species. In addition, we discuss the significant side-effects often associated with treatment of heavily infected patients, and the longer-term changes in T-cell reactivity and the host-parasite relationship which follow successful treatment with DEC.

A model for the dynamics of human lymphatic filariasis.

In this paper, Bryan Gren fell, Edwin Michael and David Denham review the appropriateness of feline filariasis as a model of the population dynamics of human lymphatic filarial infection and disease. Because of the longevity of infection and our inability to measure the adult parasite population in humans, research in filariasis is particularly dependent on the use of laboratory animal models. We demonstrate that Brugia pahangi infection patterns in the cat closely parallel those of Brugia and Wuchereria in humans. Although primary infections in 'susceptible' cats are long-lived, repeatedly infected animals show evidence of concomitant immunity which prevents the establishment of later cohorts of infective larvae. Furthermore, there is some evidence from macro filarial length distributions of 'stunting' of adult worms during long-term repeat infections. Cats can also show an 'acute' response that spontaneously eliminates infections, and this appears to be due to a combination of intrinsic and dynamic mechanisms. As in humans, pathology in cat filariasis develops as a sequel to the asymptomatic microfilaremic state, largely as a result of re-expression of immunity. The relationship between macro filarial burdens and microfilariae in blood is positive but portrays a high degree of variability. The cat model provides an important tool for elucidating the relationships between infection, immunity and disease dynamics in lymphatic filariasis, and we conclude by suggesting directions for further work in this area.

Circulating filarial antigen in cats infected with Brugia pahangi is indicative of the presence of adult worms.

Using counterimmunoelectrophoresis with rabbit antisera raised against soluble extracts of adult females of Brugia pahangi parasite antigen was detected in the serum of all cats repeatedly infected with B. pahangi. Antigen was never detected in uninfected cats. The antigen was associated with the presence of adult worms. Antigen was detected consistently in a cat that was amicrofilaraemic but at autopsy harboured only two or three adult worms. Conversely, some cats showed slowly declining numbers of microfilariae and, in these, circulating antigen declined before the number of microfilariae. Eventually no antigen was detectable in circulation whereas microfilariae, although in diminishing numbers, were still present. At autopsy no adult worms were found in these cats. Antigen also appeared in several cats before they became microfilaraemic.

Brugia pahangi in rats: increasing the number of infective larvae inoculated increases the percentage of microfilaremic rats.

One hundred Brugia pahangi infective larvae (L3) caused microfilaremic (mf + ve) infection in 56% of inbred PVG rats. Adult worms were recovered consistently from infected rats but worm recovery was very low, only 1-3% of L3 inoculated survived to adulthood and the worms were dispersed in a wide range of anatomical sites. This suggested that lack of microfilaremia may be due to the low probability of male and female worms meeting in the same site and thus may be numerically and topographically based. When the number of infective larvae inoculated was increased to 500, the percentage of mf + ve infections in rats also increased to 94%, corroborating the hypothesis that lack of mf was not due to an immune response. In a further experiment all infected rats had lost both mf and adult worms by day 420. It has yet to be established whether final rejection of the parasite is due to immunity.

Comparative susceptibility to anthelmintics of Brugia pahangi in jirds infected by different methods.

It is possible to infect jirds with Brugia pahangi by three methods. Infective larvae (L3) can be injected either intraperitoneally (ip), when adults develop in the peritoneal cavity, or sub-cutaneously (sc), when they develop in the lymphatics or the heart and blood vessels associated with the lungs. Alternatively adult worms which have been grown in the peritoneal cavities of jirds can be implanted into the peritoneal cavities of other jirds. This latter system has been widely used for screening for new filaricides. We have compared the activity of 9 macrofilaricidal compounds against these 3 types of infection. Mebendazole and albendazole were more active against implanted adults than against L3 induced adults in the peritoneal cavity. Oxibendazole, flubendazole, CGP24588A and oxfendazole were equally active against both types of worm. CGP20376, Mel Ga and Mel Ni were more active against adult worms derived from inoculated L3 than implanted worms. When comparing intra-lymphatic and ip adults (both derived from L3 infections and in the same jirds) albendazole and CGP20376 were active at the same levels against both types of infection. Mebendazole, flubendazole, oxfendazole, CGP24588A, Mel Ga and Mel Ni were more active against ip adults than intra-lymphatic adults. No drug was more active against intra-lymphatic adults than against adults.

The anthelmintic activity of a novel organic arsenical, R7/45, upon Brugia pahangi in Meriones unguiculatus.

The new organic arsenical R7/45 is a rapidly acting and very potent anthelmintic against adult Brugia pahangi in jirds. Against adult worms implanted into the peritoneal cavity 5 subcutaneous (SC) injections at 2.5 mg/kg of R7/45 killed 100% of adult worms. A single dose SC of 20 mg/kg was 100% effective and 10 mg/kg 76% effective against adult worms. When jirds were autopsied at different times after treatment at 20 mg/kg SC 89% of worms were dead within three days. R7/45 was not active when given by stomach intubation. Pretreatment of jirds with R7/45 had no effect on adult worms subsequently implanted into jirds. R7/45 was highly active against third and fourth stage larvae of B. pahangi in jirds.

Brugia pahangi adults implanted into mice: a possible screen for filaricidal activity.

Brugia pahangi adults grown in the peritoneal cavities of jirds (Meriones unguiculatus) were implanted into the peritoneal cavities of six inbred mouse strains to investigate this system as a screen for detecting filaricidal activity. The mice were given 15 adult B. pahangi and autopsied 35 days later. The recoveries of adult worms were 25%, 35%, 49%, 33%, 26% and 27% of the number implanted respectively for the MF1, TO, NIH, CBA, BALB/c and C3H/HE strains. There was great variation in the number of worms recovered from each strain of mouse. It is concluded that the variation in recoveries was so high that this system is not useful in detecting low level filaricidal activity.

Heat shock cognate 70 is a prominent immunogen in Brugian filariasis.

A cDNA expression library constructed from RNA derived from adult stage Brugia pahangi (mixed sexes) was screened with pooled sera from chronic, amicrofilaremic cases of human lymphatic filariasis from the Indonesian island of Tanjungpinang, where Brugia malayi is endemic. Polyclonal antisera raised to purified beta-galactosidase fusion proteins from two of the most highly reactive clones identified a protein of Mr 70,000 in all stages examined (microfilariae, L3 and adults) of both B. malayi and Brugia pahangi. Derivation of the amino acid sequence from these two overlapping cDNAs identified the encoded protein as a member of the heat shock protein 70 family, and showed the closest similarity to the constitutively expressed "heat shock cognate 70" (hsc70) protein. Hybridization of hsc70 cDNAs to RNA and DNA from B. pahangi under stringent conditions identified a major transcript of 2.4 kb and revealed the existence of a family of related genes. In vitro culture of larval stages of B. pahangi at elevated temperatures (43 degrees C) resulted in increased expression of hsc70, and a classic heat shock response in which five proteins (mr 18,500, 22,000, 62,000, 70,000, and 85,000) were exclusively synthesized in microfilariae. Analysis of cross-reactivities by Western blotting implied that antibody generated by infection with B. malayi was directed at filarial-specific determinants of Brugia hsc70. However, ELISA with recombinant fusion proteins for both Plasmodium falciparum and Schistosoma mansoni hsc70 indicated that some individuals with Brugian or Bancroftian filariasis did produce antibodies which cross-reacted with plasmodial and schistosomal homologs. Thus filarial-specific antibody responses were not generated in all individuals, indicating that this molecule would not be suitable for diagnostic purposes. ELISA with a purified beta-galactosidase fusion protein from B. pahangi showed antibody responses to hsc70 across the clinical spectrum of filariasis. Alignment of the derived amino acid sequences from B. pahangi, P. falciparum, S. mansoni and rat hsc70 homologs, and comparison of the immunologic reactivity of the products of the two cDNA clones by Western blotting and ELISA suggested that these determinants were located primarily at the C terminus of the protein.

Experimental Brugia pahangi and B. malayi infections of callitrichid primates.

The callitrichid primates, Callithrix jacchus jacchus (the marmoset) and Saguinus labiatus (the tamarin) were inoculated with infective larvae of Brugia malayi and B. pahangi. Microfilaraemia at low levels developed in 3 out of 4 C.j. jacchus infected with B. malayi and living or dead adult worms found in all 4. Only one of 4 C.j. jacchus became microfilaraemic (mf + ve) when given B. pahangi and adults were found in two. Of 4 S. labiatus given B. pahangi one became very lightly mf + ve and adults were found in 3. It is concluded that these animals are not suitable hosts for chemotherapeutic experiments.