Brugia pahangi infections in cats: antibody responses which correlate with the change from the microfilaraemic to the amicrofilaraemic state

The humoral responses of eight cats infected with Brugia pahangi to somatic antigens from all life-cycle stages were examined quantitatively by ELISA and qualitatively by immunoblotting for almost a year post infection. Six cats spontaneously became amicrofilaraemic: their production of IgG antibodies against somatic antigens of microfilariae, adults, and infective larvae was not statistically higher than that of the two cats which remained microfilaraemic. However, immunoblotting revealed that those cats which spontaneously became amicrofilaraemic selectively recognized certain microfilarial, adult and infective larval somatic antigens prior to disappearance of microfilariae from the peripheral circulation. The data suggest that selective recognition of antigens by some cats is responsible for the production of antibodies which may then promote microfilarial death.     

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 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 μl 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 IgGl 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.     

Secretory acetylcholinesterase of Setaria cervi microfilariae and its antigenic cross-reactivity with Wuchereria bancrofti

Setaria cervi , a bovine filarial parasite, secretes acetylcholinesterase during in vitro cultivation. A significant amount of enzyme activity was detected both in culture media and somatic extracts of different developmental stages of the parasite. The microfilarial stage showed a higher level of AChE activity than adult worms, with females being considerably more active than males. The secretory enzyme from microfilariae preferentially utilized acetylthiocholine iodide as substrate and showed two electrophoretically distinct isoforms in native PAGE. Secretory enzyme was purified from the excretory/secretory products of microfilariae using edrophonium chloride linked to epoxy-activated sepharose. Analysis of purified acetylcholinesterase by SDS-PAGE revealed the existence of two proteins of 75kD and 45kD under non-reducing conditions. These secretory enzymes are antigenic and cross-reactive with Wuchereria bancrofti -infected asymptomatic microfilaraemic human sera when tested by enzyme linked immunosorbent assay and immunoblotting. The secretory AChE(s) from S. cervi microfilariae may be utilized for diagnosis of early filarial infections.     

A study of the antigen, antibody and immunecomplex levels in Wuchereria bancrofti filariasis with reference to clinical status

Circulating antigens, antibodies to somatic and sheath components of microfilariae (mf) and immune complexes were determined in parallel in different categories of Wuchereia bancrofti infection using, respectively, reverse indirect haemagglutination (RIHA), indirect haemagglutination (IHA), indirect immunofluorescent (IFA) and polyethylene glycol (PEG) precipitation assays. Rabbit hyperimmune anti-W. bancrofti mf serum and mf homogenates were used as reagents. Appreciable levels of antigens and antibodies were detected in all categories; endemic normals, asymptomatic carriers, acute and chronic filarial cases. For example, even amongst endemic normals, i.e. those with neither clinical nor parasitological evidence of infection, 66%, 71% and 74% had mf antigens, anti-mf antibodies and anti-sheath antibodies, respectively. Notably, only a small proportion (9.4%) of microfilaraemic individuals had detectable level of anti-sheath antibodies. The relationship of these parameters with the spectrum of filariasis is discussed.     

The observation of microfilarial rate and density in cats inoculated with increasing numbers of Brugia pahangi infective larvae

Having close kinship to Brugia malayi, B. pahangi is a member of the family Filariidae, which causes lymphatic filariasis in dogs and cats. Although this nematode is unlikely to cause a zoonotic disease in humans, study of the B. pahangi life cycle may help control human filariasis. The objective of this study was to examine microfilarial rates and densities of B. pahangi in experimentally induced infections in cats as a relative measurement. Cats were infected with 3 different amounts of 3rd-stage larvae (L3); 100, 300 and 500. Cats infected with 100 L3 became patent for microfilariae longer than the other groups (mean100 = 99+/-44 days). In comparison, the pre-patent period of B. pahangi was somewhat shorter in cats with 300 and 500 L3 infections (mean300 = 76+/-13 and mean500 = 63+/-5 days). The microfilarial densities of these cats were also determined; the density of microfilariae (mf/1 ml blood) increased relative to the duration of infection. One-way ANOVA tests were used to compare the microfilarial densities of the cats with varying numbers of L3. We found that the microfilarial density of cats with 500 L3 exhibited significant differences (p < 0.05) from cats with 300 and 100 L3. However, we concluded that the amount of microfilariae produced in the blood circulation of these cats were not increasing relative to the numbers of L3 taken by the host.     

Microfilaraemia, filarial antibody, antigen and immune complex levels in human filariasis before, during and after DEC therapy. A two-year follow-up

A study on the effect of DEC therapy on microfilaraemia and the immune status in 27 patients with W. bancrofti infection was carried out for two years. Persistence of microfilaraemia was observed in 4 out of 27 cases after one course of DEC therapy and were treated again for one week. On further follow-up, none was microfilaraemic upto Day 60. The mean filarial antibody titres of IgM and IgG showed a gradual decrease as assessed by enzyme linked immunosorbent assay (ELISA). The mean titres of circulating microfilarial excretory-secretory (ES) antigens and immune complexes (ICs) showed an initial increase during therapy, followed by a gradual fall upto Day 60. Filarial antigen was detected in urine of all the carriers during therapy. Excretion pattern of antigen in urine showed correlation with DEC dose. Reappearance of microfilariae (mf) in circulation in 12 patients after a year showed that DEC had temporary attenuating effect on adult worms or no effect on developing larvae, suggesting further treatment and follow-up of patients. Parasitological and immunoscreening at the end of 2 years showed that the presence of mf ES antigen in blood correlated with the appearance of microfilariae in blood.     

Localization and immunogenicity of tubulin in the filarial nematodes Brugia malayi and B. pahangi

Tubulin was identified in the filarial nematodes Brugia malayi and B. pahangi by several approaches. Initially, a monoclonal antibody (6D8) was selected for its unusual binding to B. malayi microfilariae in indirect immunofluorescence assays: 6D8 showed granular, heterogeneously dispersed fluorescence on fixed parasites but did not bind to unfixed microfilariae. The microfilarial sheath did not bind 6D8, although it did bind fluoresceinated wheatgerm agglutinin. By Western blotting against microfilarial sonicate, 6D8 reacted with a 50,000-55,000 mol. wt protein, and also bound to purified chicken brain beta-tubulin. Additionally, this monoclonal antibody reacted with a recombinant fusion protein expressed by a clone (Bpa-7) originally isolated from an adult B. pahangi cDNA expression library by its reaction with chronic human filariasis serum. This clone encodes a small 40 amino acid C-terminal segment corresponding to residues 409-449 of beta-tubulin, and shows complete amino acid sequence homology with vertebrate beta-tubulin from 409 to 430 but 55% divergence (six amino acid substitutions, four insertions and one deletion) from human and chicken beta-tubulin over positions 431-449 at the C terminus. Antibody to both parasite and vertebrate (chicken) tubulin was found in filarial infection sera, with higher levels of autoreactive antibody apparent in amicrofilaraemic individuals. Immunogold electron microscopy was then used to localize beta-tubulin in B. malayi microfilariae and adult worms. Tubulin was shown not to be exposed on the microfilarial sheath or in the cuticle of either stage, but was found to be abundant in the somatic tissues. In microfilariae, 6D8 bound myofibril structures under the hypodermal layer, and also bound within cell nuclei. In the adult stage, tubulin was associated with muscle blocks, as well as the intestinal brush border and the embryonic uterine microfilariae.     

Identification of different radiolabelled antigens of the developmental stages of Onchocerca volvulus

By using radioiodination methods which are thought to label preferentially the surface followed by SDS-PAGE and autoradiography, components of different developmental stages of O. volvulus have been identified. Between 2 and 10 polypeptide antigens were revealed on infective larvae (L3), females, males, eggs, nodular and skin microfilariae by using immunoblotting assays with human onchocerciasis sera. Antigen recognition did not vary with the density of skin microfilariae in the patients from whom the sera were obtained. Some of the antigens seemed to be stage specific; for example, antigens of 31 kDa which were detected only on skin microfilariae, or the 67.5 and 25 kDa components that occurred on the adult females, but were absent from adult males. Some of these antigens were also identified as glycoproteins. A 68 kDa glycoprotein was found in adult females, males and nodular microfilariae. Two glycoproteins of 74 and 45 kDa were found on egg shells, and a 18.5 kDa glycoprotein was recovered from L3. Type VI collagen was found with a specific antiserum on skin microfilariae, but not on eggs and females. Laminin was found on nodular mf. It is concluded that the changing antigenic profiles of the worm stages and the coating of these worms with connective tissue epitopes contribute to the evasion of host immunity.     

Interaction of monoclonal antibodies with cuticular antigens of filarial parasites, Brugia malayi and Wuchereria bancrofti

Monoclonal antibodies (mAbs) have been prepared against excretory-secretory-metabolic (ESM) antigens of microfilariae (mf) of Wuchereria bancrofti (WbmfESM) and against third stage larvae (L3) of Brugia malayi (BmL3), and purified from ascites fluids with ammonium sulphate. Both antibodies were of the IgM type and did not react with phosphorycholine. The mAb against BmL3 (F46) reacted in ELISA with antigens of L3 of B. malayi, B. pahangi and W. bancrofti and of adults of B. malayi. The mAb raised against wbmfESM (F32) resembled F46 in this respect, though with a lower titer towards the antigens, and in addition reacted with the ESM-antigens of mf and of L3 of W. bancrofti. F46 was able to detect L3 antigens of filarial parasites in spiked serum samples with a detection limit of 8-16 ng in absolute amount. The antibody was found to label the cuticular portion of L3 and adults of the lymphatic parasites, and not the epicuticular surface, in immunoelectron microscopic studies. The antibody recognized a 36 kDa component of the beta-mercaptoethanol extracts of B. pahangi-adults in Western blot analysis.     

IgG subclass recognition of Loa loa antigens and their correlation with clinical status in individuals from Gabon

In endemic areas for Loa loa, a significant percentage of actively infected individuals have no circulating microfilariae, an observation which implies the existence of a stage-specific immune response. In an attempt to define the immunological basis of the amicrofilaraemic state, the reactivity of antigens from adult, microfilariae and infective larvae of L. loa was examined by Western blotting with individual serum samples from four clinically defined groups (high microfilaraemic, low microfilaraemic, amicrofilaraemic and endemic controls) using IgG subclass-specific reagents and IgE. In the adult parasite, a complex of antigens at 28-31 kDa was exclusively recognized by IgG1 from amicrofilaraemic individuals and, to a lesser extent, by IgG1 from endemic controls. However, this complex of antigens was recognized by IgG4 antibodies in serum samples from all individuals, including microfilaraemics. A microfilarial antigen of 21 kDa was recognized by IgG1 antibodies present in serum from amicrofilaraemic, endemic control and low microfilaraemic individuals. Persons with high levels of microfilariae did not recognise this antigen. In both the L3 and the microfilariae, a ladder antigen with increments of 15 kDa was the main target of IgG4 antibodies in amicrofilaraemic and microfilaraemic individuals. IgE antibodies recognized more antigens in the microfilarial stage than in the adult of L3. These results suggest that immunological differences between clinically defined groups are associated with the recognition of different antigens or epitopes .     

Differential recognition of microfilarial antigens by sera from immigrants into an area endemic for brugian filariasis

Studies in animal models indicate that antibodies to surface antigens of microfilariae participate in the control of parasitaemia resulting from infections with lymphatic filarial nematodes. In an attempt to identify parasite antigens that elicit such 'protective' host responses, we compared the antigen recognition patterns of persons who remained amicrofilaraemic after 3-6 years of exposure to Brugia malayi with those of individuals who developed patent filariasis during the same period. IgG antibodies in sera from immigrants identified between 0 and 25 microfilarial antigens on Western blots. The highest degree of reactivity was observed with antigens in the 65-75 kD and 20-30 kD ranges, and with a group of high mol. wt antigens (greater than 180 kD). Sera from amicrofilaraemic donors preferentially reacted with 70/75 kD microfilarial antigens. A proportion of such sera inhibited the binding of monoclonal antibody MF1 to its target epitope; eight of nine inhibitory sera were from patients with active infections, evidenced by the presence of microfilariae or filarial antigens in the donors' blood, but who were amicrofilaraemic. These results indicate that some amicrofilaraemic residents of areas where brugian filariasis is endemic develop immune reactions to a microfilarial stage-specific antigen that was previously identified as a potentially 'protective' parasite antigen in animal models of lymphatic filariasis.     

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.     

Antibody responses to experimental Brugia malayi infections in patas and rhesus monkeys

Humoral antibody responses in experimental infections with Brugia malayi (subperiodic strain) were compared in two primate species. Erythrocebus patas and Macaca mulatta. Antibody responses were related to the infection protocol and the duration and magnitude of microfilaremia. Patas monkeys were uniformly susceptible to infection and characteristically exhibited prolonged microfilaremia; infections in Rhesus monkeys produced low and usually microfilaremia. Antibody, measured by enzyme linked immunosorbent assay with extracts of adult Brugia and microfilariae as antigens, declined at patency in Patas monkeys and there was an inverse relationship between serum antibody concentration and the number of circulating microfilariae. Rhesus monkeys generally had high, sustained antibody levels relative to Patas monkeys, but antibody levels were comparable in the two species when the numbers of circulating microfilariae were similar. By fluorescent antibody technique, antibodies reactive with somatic antigens of microfilariae were detected in all infected monkeys; antibodies reactive with the cuticle of infective larvae were also present in both primates and were consistently detected in monkeys receiving multiple infections. Antibodies (IgG, IgM) reactive with the sheath of microfilariae were detected only in certain Rhesus monkeys which were essentially amicrofilaremic and sera with antibodies specific for microfilarial sheath promoted in vitro microfilarial agglutination and leukocyte adherence.     

Infection of inbred and nude (athymic) rats with Brugia spp.

Infective larvae of Brugia pahangi were injected subcutaneously into inbred PVG (-RTIc) rats, and 'nude' (PVG-rnu/rnu) (athymic) rats. Adult worms or circulating microfilariae were recovered from 20/34 (59%) of PVG-RTIc rats and from 30/30 (100%) of 'nude' rats. Fertile worms were regularly found in the lumbar lymphatics and hearts of both strains of rat. Blood eosinophilia first developed in PVG-RTIc rats about 17 days, and in all such animals by 6 weeks. High circulating eosinophil counts persisted only in patent animals, proving a useful hallmark for the presence of microfilariae. Nude rats despite patency, developed eosinophilia only latterly and then to a lesser extent. Specific anti-B. pahangi IgG antibody was first detected at 7 days in all infected PVG-RTIc rats, with levels rising until 8 weeks and remaining high only in microfilaraemic animals; total IgE showed a similar response. Specific IgE rose in all the eight patent rats inconsistently and only to low levels in eight non-patent infected rats. IgG and IgE were undetectable in nude rats. Other strains of inbred rats of different RTI haplotype were also successfully infected with B. pahangi and the human parasite B. malayi, a total of 10/23 (43%) and 5/15 (33%) becoming patent respectively. In the small numbers tested no major influence of RTI haplotype was detected. Infection by the intraperitoneal route did not result in the development of microfilariae. The difference in patency rates between 'nude' and normal PVG rats supports the contention that the development of filarial infections is T lymphocyte dependent. Inbred and 'nude' rats provide a valuable model of human filariasis, in which many features of filarial immunopathology can be studied.     

Phosphorylcholine-bearing antigens in filarial nematode parasites: analysis of somatic extracts, in-vitro secretions and infection sera from Brugia malayi and B. pahangi

A set of cross-reactive antigens is described which are present in somatic extracts and in-vitro secretions of the filarial nematodes Brugia pahangi and B. malayi. A monoclonal antibody reactive with a repeating epitope on these molecules readily detects circulating antigen in the serum of animals infected with lymphatic filariae, using either an immunoradiometric assay or enzyme-linked immunosorbent assay (ELISA). This epitope has the immunological reactivity and chemical characteristics of the phosphorylcholine (PC) hapten. The anti-PC monoclonal has been used to define the antigens bearing this epitope, and in chromatographic studies on material from extracts of Brugia adult worms, a heterogeneous profile of PC-positive molecules are found. In sera from Brugia-infected jirds, an antigen with a native molecular weight of approximately 500,000 is observed, which displays limited sensitivity to protease degradation. However, denatured samples on Western blots show a major parasite circulating antigen of Mr 90,000. The detection of this antigen in the presence of excess host antibody is also demonstrated, taking advantage of the stability of the target epitope to a range of treatments designed to dissociate and eliminate immune complexes.     

Lymphatic filariasis in man: demonstration of circulating antigens in Wuchereria bancrofti infection

Summary Five hundred and twenty-nine sera obtained from people dwelling in an area endemic for bancroftian filariasis were analysed for the presence of soluble circulating antigens (SCA) of filarial origin by counter Immunoelectrophoresis and 303 were found positive. It, therefore, appears that 57.3% subjects could be diagnosed by the detection of SCA irrespective of their clinical status. Of the three groups investigated, microfilaraemic, amicrofilaraemic and clinical, SCA could be demonstrated in maximum number of sera obtained from clinical cases. The parasite specificity of the SCA was determined by enzyme linked immunosorbent assay (ELISA), immunofluorescent antibody test (IFAT), counter Immunoelectrophoresis (CIEP) and gel diffusion (GD) using antisera raised against Litomo-soides carinii in rabbits and antigens derived from Wuchereria bancrofti microfilariae or antigens present in sera of patients with bancroftian filariasis. This anti-carinii hyperimmune serum under study did not show positive reactions with parasites other than filaria, or with the sera obtained from pre-immunized rabbits and non-endemic controls. A demonstration of a distinct precipitin band in CIEP and GD indicates that either monospecific hyperimmune sera or monoclonal antibodies could be raised for the development of a simple method for immunodiagnosis of bancroftian filariasis.     

Infection of inbred and nude (athymic) rats with Brugia spp.

Summary Infective larvae of Brugia pahangi were injected subcutaneously into inbred PVG (-RIT) rats, and ‘nude’ (PVG -rnu/rnu) (athymic) rats. Adult worms or circulating microfilariae were recovered from 20/34 (59%) of PVG -RTI^C rats and from 30/30 (100%) of ‘nude’ rats. Fertile worms were regularly found in the lumbar lymphatics and hearts of both strains of rat. Blood eosinophilia first developed in PVG -RTI^c rats about 17 days, and in all such animals by 6 weeks. High circulating eosinophil counts persisted only in patent animals, proving a useful hallmark for the presence of microfilariae. Nude rats despite patency, developed eosinophilia only latterly and then to a lesser extent. Specific anti-2?. pahangi IgG antibody was first detected at 7 days in all infected PVG -RTT rats, with levels rising until 8 weeks and remaining high only in microfilaraemic animals; total IgE showed a similar response. Specific IgE rose in all the eight patent rats inconsistently and only to low levels in eight non-patent infected rats. IgG and IgE were undetectable in nude rats. Other strains of inbred rats of different RTI haplotype were also successfully infected with B. pahangi and the human parasite B. malayi, a total of 10/23 (43%) and 5/15 (33%) becoming patent respectively. In the small numbers tested no major influence of RTI haplotype was detected. Infection by the intraperitoneal route did not result in the development of microfilariae. The difference in patency rates between ‘nude’ and normal PVG rats supports the contention that the development of filarial infections is T lymphocyte dependent. Inbred and ‘nude’ rats provide a valuable model of human filariasis, in which many features of filarial immunopathology can be studied.     

Comparative efficacy of some benzimidazoles and amoscanate (Go.9333) against experimental filarial infections

The comparative efficacy of mebendazole, fenbendazole, oxibendazole, oxfendazole, albendazole, flubendazole and micronized amoscanate (particle size 5-8 micron) against Litomosoides carinii and Brugia pahangi infections in Mastomys natalensis was studied on administration of the compounds per os (150 mg/kg/day for 5 days) and subcutaneous (100 mg/kg/day for 5 days) routes. It was found that benzimidazoles when given by the oral route had no effect on adults of L. carinii and B. pahangi. With most of these compounds there was a rise in microfilariae before registering a fall to varying degrees in the peripheral circulation. There was a gradual but effective reduction of microfilariae of L. carinii in animals treated orally with mebendazole (99%), flubendazole (95%) and oxfendazole (85%). No such effect was seen against B. pahangi microfilariae. On subcutaneous administration, all the benzimidazoles with the exception of fenbendazole exhibited marked macrofilaricidal activity against L. carinii. Such activity was not seen with oxibendazole, oxfendazole and fenbendazole against adults of B. pahangi. Amoscanate exhibited superiority over the benzimidazoles in that the compound eliminated microfilariae and adult worms of both L. carinii and B. pahangi species when given by oral and subcutaneous routes.     

Successful vaccination of cats against Brugia pahangi with larvae attenuated by irradiation with 10 krad cobalt 60

Cats were vaccinated by the inoculation on 10 occasions of approximately 300 larvae of Brugia pahangi which had been irradiated with 10 krad cobalt 60. They were challenged on 3 occasions with normal larvae of either B. pahangior B. patei. The vaccinated cats were resistant to challenge as demonstrated by either longer pre-patent periods or failure to become microfilaraemic and by having fewer third, fourth or adult worms than normal controls. Although the vaccination procedure was unpractically heavy these results lend encouragement to the possibility of developing vaccines against filarial infections.