Microfilarial Antigens Research Articles

Onchocerca volvulus: Molecular cloning, primary structure, and expression of a microfilarial surface-associated antigen

Although the filarial nematode parasite Onchocerca volvulus is an important human pathogen in large areas of Africa and Latin America, little is known of the molecular interactions that govern the clinical status of patients with this chronic, debilitating disease. As a step toward defining the parasite molecules important to the immunobiology of host-parasite interactions, we have identified and cloned a major surface-associated antigen expressed by O. volvulus microfilariae. Radiolabeling experiments demonstrated that O. volvulus microfilariae have a limited repertoire of peptides at the surface. Prominent among these labeled peptides is an 18-kDa component. Immunological crossreactivity between a surface-associated component of Diroftlaria immitis microfilariae and the 18-kDa surface-associated molecule from O. volvulus was exploited in a strategy to clone this potentially important O. volvulus microfilarial antigen. The cross-reacting antibodies were used to immunoscreen O. volvulus cDNA expression libraries. One clone, M2f.e, contained an open reading frame of 495 bp encoding an 18.1-kDa protein (OVMS18). Antibodies produced against the expression product of M2f.e recognized an 18-kDa component in extracts of O. volvulus microfilariae and bound to the surface of intact O. volvulus and D. immitis microfilariae. Southern blot analyses showed that M2f.e-like sequences are present in the genomic DNA of a number of filarial nematode species, but not in DNAs from nonfilarial nematode species.

Comparison of Dot-Elisa with Sandwich-Elisa for the Detection of Circulating Antigens in Patients with Bancroftian Filariasis

We compared the performance of a newly developed Dot-ELISA with that of a previously described Sandwich-ELISA to detect parasite antigens in sera from patients with bancroftian filariasis. The same monoclonal antibody and the same sera were used in both tests. In the Dot-ELISA, 67 of 70 sera from microfilaremic donors were deemed to contain filarial antigens when screened at a dilution of 1:50. End titers were 1:80-1:1280. With the Sandwich-ELISA, 64 of the same sera were positive at a dilution of 1:10 and 42 were positive at a dilution of 1:50. End titers were 1:10-1:320. The specificity of both assays was greater than 95%, but their sensitivity was remarkably different. The Dot-ELISA could detect as little as 0.055 ng/ml microfilarial antigen added to normal human sera, whereas the lower limit with the Sandwich-ELISA was 10 ng/ml parasite antigen. Additionally, the Dot-ELISA does not require radioactivity or sophisticated equipment and, therefore, can be performed in virtually all filariasis-endemic areas.

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.

19s and 7s antibody response of Mastomys natalensis in experimental filarial (Litomosoides carinii, Acanthocheilonema viteae, Brugia malayi, B. pahangi) infections.

Specific total antibody (ab), 19s and 7s ab levels in the serum of M. natalensis were investigated after infection with L. carinii, A. viteae, B. malayi and B. pahangi for period of about 500 days p.i., using ELISA (homologous adult antigen) and indirect immunofluorescence tests (IIFT: homologous adult and microfilariae antigen). Total ab levels in L. carinii infected animals rose moderately during prepatency Maximum levels occurred during patency. The response during prepatency was stronger in A. viteae and Brugia spp. infected hosts. Lateron ab levels increased continuously in Brugia infections; in A. viteae infection they decreased with decreasing parasitaemia. 19s abs were stimulated during prepatency and at the beginning of patency, or were found at moderate levels throughout to period of investigation (Brugia infections). 7s abs predominated beginning at the period of late prepatency (IIFT) or at the beginning of patency (ELISA). The time courses of 7s abs corresponded to those of total abs. As obvious by IIFT (adult worm antigen) total and 19s titres were higher against cuticle antigens, egg shell antigens and intrauterine amorphous material than against antigens located in the hypodermis and musculature. 7s abs showed best reactivity with cuticle antigens. Using microfilarial antigens 19s abs reacted predominantly with cuticle antigens whereas 7s abs often showed higher titres against antigens which were localized within the larvae than against cuticle antigens.

Persistence of parasite antigenemia following diethylcarbamazine therapy of bancroftian filariasis.

This study was designed to reexamine the efficacy of diethylcarbamazine for bancroftian filariasis with special reference to changes in serum parasite antigen levels and antifilarial antibody titers after treatment. Patients with asymptomatic microfilaremia were treated with 6 mg/kg diethylcarbamazine daily for 12 days. Microfilaria counts fell dramatically after treatment, as expected. IgG antibody titers to adult and microfilarial antigens of B. malayi were increased 1 month after treatment in most patients. Titers fell slowly to or below pretreatment levels, but remained positive during subsequent months. Parasite antigen levels, measured by monoclonal antibody-based enzyme immunoassay, decreased to 72%, 58%, 53%, and 48% of pretreatment values 1, 3, 6, and 12 months after diethylcarbamazine treatment, respectively. Parasite antigen levels decreased similarly in subjects with and without residual microfilaremia after treatment. These results suggest that diethylcarbamazine has only partial macrofilaricidal activity against W. bancrofti with this dosage schedule. The sustained, impressive reductions in microfilaria counts after treatment despite significant persistence of parasite antigenemia may be explained by sublethal effects of the drug on adult worms. We believe that parasite antigen detection represents a valuable new approach for monitoring the efficacy of antifilarial drug therapy which we hope will lead to improved use of existing drugs and aid in the evaluation of new drugs for filariasis.

Analysis of B. malayi microfilarial antigens by immunoblotting.

BALB/C mice were immunized with heavy or low infection of live B. malayi microfilariae or immunised with different fractions of the microfilarial antigens. Antibody levels and antibody dependent macrophage mediated cytotoxicity to B. malayi microfilariae were determined in the immunized sera. Antigens responsible for induction of antibodies were recognised in B. malayi microfilarial extract by immunoblotting. Appearance of cytotoxic antibodies correlates with recognition of certain common antigens in microfilarial extract such as 45, 54, 62, 66 and 76 KDa mol. wt. proteins.

Filariasis due to Loa loa and Manspnella perstans: distribution in the region of Okondja, Haut-Ogooué Province, Gabon, with parasitological and serological follow-up over one year

The prevalence of Loa loa and Mansonella perstans filariasis has been determined in 6 rural villages in eastern Gabon. Between 18·9 and 27·2% of people carry L. loa microfilariae with an overall microfilarial rate of 25·1%. The microfilarial rate for M. perstans was more variable, between 33·3 and 62·2% (average 49·1%). No significant difference was seen in the microfilarial rate with age over 15 years for either parasite, but men were infected more frequently than women. Anti- L. loa antibody titres were measured, using a homologous microfilarial antigen in ELISA. Taking the parasitological and immunological evaluations together, only 10% of the sample population appear to be free of these filarial infections. L. loa and M. perstans microfilaraemia and corresponding serology were also investigated twice in 150 people at a one-year interval. 99·1% of the cases who had no circulating L. loa microfilaria in March 1984 still did not show any 12 months later. Similarly, 97·1% of the untreated, microfilaraemic cases still harboured this parasite a year later. The same was not observed for M. perstans, since microfilariae appeared or disappeared in 26·7% of the cases. This suggests different dynamics for the two filarial infections. Variation in individual anti- L.loa antibody titres was low. The possibility of a genetic influence on the expression of loiasis is discussed.

Biochemical and immunologic characterization of a major surface antigen of Dirofilaria immitis infective larvae.

A 35 kD major surface antigen of Dirofilaria immitis third-stage larvae was characterized biochemically and immunologically. Living larvae were iodinated by using Iodo-gen, iodosulfanilic acid, lactoperoxidase-glucose oxidase, and Bolton-Hunter reagents. Detergent extracts of larvae labeled by the first three methods showed one major 35 kD component and a number of smaller components of about 6 kD, as analyzed by one-dimensional SDS-PAGE. In contrast, extracts from larvae labeled with the Bolton-Hunter reagent showed multiple bands on gels. The 35kD molecule was shown to be exposed on the larval surface, insofar as it was accessible to trypsin-proteolysis on living radiolabeled larvae. Two-dimensional gel electrophoresis resolved the 35 kD band into two components: a major one with a pI of 3.8, and a minor one of pI 7.3. The lower m.w. bands were resolved into about 12 constituents with pI values from 3.5 to 8.0. Of all these surface molecules, the only one that was antigenic was the 35 kD component. It could be immunoprecipitated with sera from dogs carrying an occult experimental D. immitis infection or with sera from dogs immunized with irradiated third-stage larvae of this parasite. Similarly, sera from rabbits immunized repeatedly with normal unirradiated larvae also precipitated the 35 kD antigen. None of these sera, however, contained detectable antibodies to the surface-labeled low m.w. molecules. Sera from rabbits immunized with D. immitis adult worms and microfilariae precipitated the 35 kD antigen, which is therefore not stage specific. In contrast, sera from dogs experimentally infected with Toxocara canis and Ancylostoma caninum or with Uncinaria stenocephala (a canine hookworm) did not contain antibodies to the 35 kD antigen, but did cross-react with many other D. immitis adult and microfilarial antigens. This molecule may therefore be species specific. Evidence for glycosylation of the 35 kD molecule was not found: it did not bind to peanut, wheat germ, lentil, or Ulex europeus lectins, and its electrophoretic mobility was not altered after treatment with endoglycosidase-F or mild alkali solutions.

Induction of protection against Brugia malayi infection in jirds by microfilarial antigens.

The development of immunologic methods to reduce transmission of human lymphatic filariasis depends on measures that will enhance the host's ability to eliminate infective larvae, adult worms, or blood-borne microfilariae (mf). The present study was designed to assess the capacity of a crude extract of Brugia malayi mf to decrease the level of microfilaremia and adult worm burden in jirds inoculated with infective larvae, and to identify the filarial antigens that elicit antibody responses in these animals. Thirty weeks after subcutaneous inoculation with 75 infective larvae, 100% of control jirds were patent (i.e., had microfilaremia) compared with 60% of the group immunized with 10 micrograms of crude microfilarial extract (p less than 0.05). In addition, microfilaremia was lower in patent immunized animals compared with controls (p less than 0.05). The mean total number of adult female B. malayi per jird recovered at necropsy in control animals was 16.0 vs 7.0 in immunized jirds (p less than 0.05). Serum of immunized jirds contained anti-mf antibodies with an end titer of 1:8000, a value similar to that of animals with chronic B. malayi infection. Microfilarial antigens of Mr approximately 150,000, 75,000, 42,000, and 25,000 were identified in immunoblotting studies by reactivity with antibodies in sera of immunized jirds. Antibodies induced by immunization with microfilarial extract were not specific for this stage of the parasite life cycle, as jird anti-mf antibodies reacted with a Mr approximately 150,000 and several Mr 50,000 to 110,000 antigens derived from immature and mature adult parasites of both sexes. These data indicate that immunization of jirds with a water soluble microfilarial extract enhances the host's ability to eliminate adult worms and blood-borne mf. The filarial antigens that induce antibodies in immunized jirds have been identified.

Expression of cross-reactive surface antigens by microfilariae and adult worms of Brugia pahangi during infections in cats

Microfilariae of Brugia pahangi were labelled with 125-Iodine using the reagent IODOGEN. Electron microscope autoradiographs of sections of iodinated microfilariae showed that the label was strictly confined to their sheath. Adult worms were also iodinated by the same procedure. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of detergent extracts of radio-labelled parasites revealed components of molecular weights 113, 81-71, 46 and 33 kDa in microfilariae, and of molecular weights 29, 20 and 16 kDa in adult worms. All but the 33 kDa component of microfilariae were immunoprecipitable with sera of infected cats and therefore antigenic. Antibodies to the 81-71 kDa and the 46 kDa microfilarial antigens were detected by immunoprecipitation before patency. Similarly, the 29 kDa antigen of adult worms was immunoprecipitable before the fourth moult. Therefore, during infection in cats, these antigens cross-react with epitopes present on earlier developmental stages.

Sharing of antigens among filarial species revealed by antibody dependent cell-mediated reactions

Antisera raised in albino rats against microfilariae ofLitomosoides carinii, Brugia pahangi, Brugia malayi and sera fromBancroftian elephantiasis patients promoted rat neutrophil-mediated adherence and cytotoxicity to the microfilariae. Pre-treatment of the immune sera, with microfilarial antigen at a final concentration of 5 and 25μg per ml blocked cellular adherence and cytotoxicity to the microfilariae indicating the presence of cross-reactive antibodies. The heterologous immune sera were effective in eliminating the circulatingLitomosoides carinii microfilariae inMastomys natalensis.

Brugia malayi: Clearance of microfilaremia induced by diethylcarbamazine independently of antibody

The microfilaricidal effect of diethylcarbamazine was studied in the murine model of Brugia malayi microfilaremia. CFI mice with circulating microfilariae were treated with either diethylcarbamazine (6 mg kg −1 day −1) for 10 days or with normal saline. Although antibodies against crude microfilarial antigen were detected in both groups at the time of completion of therapy, the chemically induced microfilarial clearance occurred prior to the development of these antibodies. In addition, the passive transfer of antibody just prior to chemical treatment did not enhance diethylcarbamizine-induced microfilarial clearance. In vitro studies confirmed previous observations that diethylcarbamazine enhanced antibody-dependent neutrophil adherence. Microfilarial clearance was found to be due to trapping and lysis in the liver on pathologic examination. These studies demonstrate that diethylcarbamazine leads to microfilarial clearance in the liver by mechanisms that are not dependent on host antibody.

Pathologic changes of human onchocerciasis: implications for future research.

Onchocerciasis--infection by Onchocerca volvulus--has four cardinal manifestations: dermatitis, subcutaneous nodules, sclerosing lymphadenitis, and eye disease. The first three are discussed here. The dermatitis begins when microfilariae degenerate in the dermis. This process is accompanied by inflammation, with degranulation of eosinophils and deposition of the major basic protein of the eosinophil granules on the cuticle of the microfilariae. So far as is known, the chronic effects of onchocerciasis are all a consequence of the degeneration of microfilariae. Subcutaneous nodules contain coiled adult worms and have an outer layer of fibrous scar and a central inflammatory cell exudate, which may cavitate. Perfusion of India ink reveals arborization of capillaries around adult worms, which derive nutrition from these networks. Onchocercal lymphadenitis is characterized initially by histiocytic hyperplasia and follicular atrophy and later by fibrosis and obstruction of lymph flow, a condition causing adenolymphocele ("hanging groin") and elephantiasis of the genitalia. Some patients appear to have immune tolerance to degenerating microfilariae, perhaps as a result of exposure in utero to microfilarial antigens in the maternal circulation. In contrast, other patients (Yemenites, for example) have a localized but intense response to a few microfilariae; these patients are hypersensitive--perhaps because they were not exposed to microfilarial antigens in utero. Autopsy data on infection of deep organs are limited.

Brugia malayi: Intravenous injection of microfilariae in ferrets as an experimental method for occult filariasis

Microfilaremia, immune responses, and pathology were compared in ferrets infected with 100 third-stage larvae of Brugia malayi (subperiodic strain) or injected intravenously with 10 6 microfilariae. Ferrets ( Mustela putorius furo) inoculated with third-stage larvae typically became patent during the third month after infection, with a mean patency of 123 ± 25 (SE) days. Ferrets injected intravenously with microfilariae exhibited a relatively constant microfilaremia for 3–4 weeks and usually cleared microfilariae before the fourth month. Ferrets that cleared microfilariae after intravenous injection of microfilariae or after infection with third-stage larvae failed to become patent or became amicrofilaremic within 3 weeks after a challenge intravenous injection of 10 6 microfilariae. Clearance of circulating microfilariae was associated with eosinophilia and serum antibody specific for the microfilarial sheath in ferrets injected with microfilariae and in most ferrets infected with third-stage larvae. Ferrets infected with third-stage larvae and necropsied after clearance of microfilariae had tissue inflammatory reactions to microfilariae characteristic of occult filariasis (tropical eosinophilia) in man; these ferrets exhibited immediate cutaneous hypersensitivity and circulating reaginic antibody to antigens of microfilariae. In ferrets necropsied following two intravenous injections of microfilariae, the majority of ferrets examined within 10 days after clearance of microfilariae had visible liver lesions to microfilariae identical to those of the ferrets infected with third-stage larvae; immediate cutaneous hypersensitivity and reaginic antibody were not consistently detected in ferrets injected with microfilariae. Sera from ferrets that had cleared circulating microfilariae were transferred passively into ferrets made microfilaremic by intravenous injection of microfilariae. Sera with microfilarial sheath-reactive IgG antibody titers (⩾1:200) and microfilarial agglutination titers (⩾1:40) rapidly cleared injected microfilariae (<24 hr); this serum also cleared or greatly reduced circulating microfilariae established by an infection with third-stage larvae; only the IgG-containing fraction of the sera was active in immune clearance. Sera that cleared microfilariae of B. malayi did not clear circulating microfilariae of Dirofilaria immitis or prevent recurrence of circulating microfilariae of B. malayi in ferrets infected with adult filariae. Ferrets were examined at 1 and 3 weeks after transfer of sera and injection of microfilariae for lesions on the surface of the liver typical of occult infection. Ferrets that received sera inducing a rapid clearance of microfilariae did not develop liver lesions or eosinophilia. Only certain sera containing reaginic antibody to microfilarial antigens were associated with an accelerated development of liver lesions and eosinophilia. By the third week after injection, ferrets receiving normal sera had liver pathology and eosinophilia comparable to those receiving sera that induced the early development of lesions. There was a significant linear correlation between the numbers of surface liver lesions and the absolute numbers of blood eosinophils in all the ferrets injected with microfilariae.

Quantitative and qualitative changes in the humoral response of dogs through the course of infection with Dirofilaria immitis.

The present study compared the humoral response of dogs which developed microfilaremic or occult forms of dirofilariasis following experimental infection with Dirofilaria immitis L3 larvae. Quantitative analysis by ELISA revealed that antibody levels to adult somatic (AS), excretory-secretory (ES), and microfilarial (MF) antigens were highest during the patent phase of infection in dogs with either form of dirofilariasis. Patent sera from dogs destined for occult infections contained anti-AS and anti-MF antibody concentrations of 1,572 and 1,004 micrograms/ml, respectively, while microfilaremic-bound dogs contained 1,044 and 906 micrograms/ml, respectively. Chronic sera (430 days post infection) from occult dogs contained anti-AS and anti-MF antibody levels of 982 and 600 micrograms/ml, respectively, which were higher than in microfilaremic dogs. The antibody response to ES antigen was generally 10-fold less in absolute antibody concentrations at all time points tested. Immunoperoxidase staining of antigens transferred to nitrocellulose revealed the presence of several antigenic proteins which were recognized by occult, and to a lesser extent or not at all, by microfilaremic dogs. Sera drawn from occult-bound dogs 280 days post-infection, a time corresponding to microfilarial clearance (transition phase), contained higher antibody activity to microfilarial proteins weighing 47.5, 42.0, 34.2, and 22.4 kilodaltons compared to the microfilaremic dogs. This difference in antigen recognition became more apparent during the chronic phase of infection.

Monoclonal antibodies againstWuchereria bancrofti microfilarial excretory-secretory antigens

A battery of monoclonal antibodies were produced againstWuchereria bancrofti microfilarial excretory-secretory antigens and their specificity was studied using different filarial antigens. Among the 1116 wells plated out, 42 % of the wells developed hybrids and 5 % of the hybrids showed antiWuchereria bancrofti microfilarial excretory-secretory antigens. Specificity studies on the antibodies produced from 63 cloned and expanded hybrids showed 10 clones which were specifically positive only toWuchereria bancrofti microfilarial excretory-secretory antigens.

Immunodiagnosis of bancroftian filariasis—Problems and progress

The immunodiagnosis of bancroftian filariasis is a major challenge to the immunoparasitologist. Significant progress is yet to be made in developing convenient laboratory animal model and inin vitro cultivation of filarial parasites making it very difficult to obtain required amount of parasite material for research. Parasitological examination techniques are not useful in low microfilaraemia, occult or chronic.filarial infections. A precise and accurate immunodiagnostic technique is very much needed for successful filaria control programmes. Such a test will also avoid the need for laborious night blood examination in bancroftian filariasis. Due to comparatively easy availability, a good amount of work has been done to explore immunodiagnostic potential of heterologous filarial antigens isolated fromLitomosoide carinii, Dirofilaria immitis, Brugia malayi, Setaria digitata, Setaria cervi and number of other filarial species. However, there has been limited or no significant success due to number of false negative and false positive reactions. Extensive study has also been made with antigens isolated fromWuchereria bancrofti microfilariae. Soluble antigens of microfilariae have been used in different immunological techniques such as skin test, counter immuno electrophoresis, indirect haemagglutination test, indirect fluorescent antibody test and enzyme linked immunosorbent assay. Fractionation of Wuchereria bancrofti microfilarial soluble antigens yielded mfS3e antigen fraction which was found to be highly reactive in microfilaraemia by enzyme linked immunosorbent assay, but the yield of the purified antigen was not sufficient enough to make it a practical proposition for large scale isolation of antigen. Wuchereria bancrofti microfilarial excretory-secretory antigens were found to be specific and highly sensitive requiring as little as 0.35 ng antigen protein per well in penicillinase enzyme linked immunosorbent assay for detection of filarial antibody. One ml of culture fluid was found to be sufficient for 400,000 tests. Field evaluation of this test showed that it can replace laborious night blood examination. Assay systems have been developed for detection of filarial antigen in serum, urine, hydrocele fluid and immune complexes using immunoglobulins from chronic filarial sera and antisera to excretory filarial antigens. Further purification of excretory-secretory antigens by affinity chromatography and production of monoclonal antibodies should hopefully give suitable reagents for use in sensitive assays such as enzyme immuno assay and immuno radiometric assay, providing an ideal assay system for detection of active filarial infection in the not too distant future.

Evaluation of fractionatedWuchereria bancrofti microfilarial excretory-secretory antigens for diagnosis of bancroftian filariasis by enzyme linked immunosorbent assay

TheWuchereria bancrofti microfilarial excretory-secretory antigens were fractionated into ES1, ES2, ES3 and ES4 by ultra-membrane filtration and evaluated for their diagnostic utility by enzyme linked immunosorbent assay. Three of the four fractions showed antigenic activity (ES2, ES3 and ES4). The antigen fractions ES2 and ES4 were highly active in the detection of filarial IgM antibody in clinical filariasis and microfilaraemia respectively. The chemical characterization of the ES2 and ES4 antigen fractions showed that they were glycoproteins

Brugia pahangi: Antibodies and microfilaremias in Lewis rats

Male and female Lewis rats were inoculated subcutaneously in the left groin with 75 infective larvae of Brugia pahangi and microfilaremias were followed for as long as 420 days postinoculation. Patent infections developed in 64% of the female rats and 95% of the male rats. Mean prepatent periods were similar (65.9 and 63.9 days, respectively), but mean microfilaremias in males rose much higher, to a mean of 218 mf/0.25 ml blood at 270 days postinoculation. IgG titers, as measured by enzyme-linked immunosorbent assay (ELISA), to adult worm somatic antigen were higher than those to microfilariae in almost all rats. For both sexes, the most consistently microfilaremic rats had highest titers to these antigens. Granulomas with degenerating microfilaria were present in the spleen of male rats with high microfilaremias (>100–300 mf/0.25 ml blood). Ouchterlony precipitin reactions suggested that most rats with spleen granulomas responded to microfilarial antigen components to which most rats without granulomas did not. Neither spleen granulomas nor antibody responses measured in this study appeared to have protective (microfilaremia-lowering) value. As measured by microfilaremias, the male Lewis rat is not as susceptible as some conventional hosts of B. pahangi, but it does consistently become infected and remains microfilaremic for more than a year. Preferential male susceptibility indicates that this model may be useful for studying this aspect of human lymphatic filariasis.

The ferret (Mustela putorius furo) as an experimental host for Brugia malayi and Brugia pahangi.

Ferrets inoculated subcutaneously with 150--200 infective larvae of Brugia malayi (subperiodic strain) usually developed patent infection during the 3rd month post inoculation. Microfilaremia was transient, and most animals became amicrofilaremic after the 6th month of infection. Ferrets developed a persistent eosinophilia at the time of patency. At necropsy, 5--8 months post infection, adult worms were recovered principally from lymphatic vessels and recovery ranged from 0.5--13% of the inoculated larvae. The inflammatory response of ferrets to microfilariae was characterized by nodules 1--5 mm in diameter in the liver, lungs, spleen, and submucosa of the gastrointestinal tract. The center of these lesions contained a degenerated microfilaria or the cast of a microfilaria embedded in Splendore-Hoeppli substance. The Splendore-Hoeppli substance was surrounded by eosinophils and/or foreign body giant cells. Identical lesions were observed in ferrets experimentally infected with Brugia pahangi. Sera from ferrets infected with B. malayi demonstrated a 3- to 5-fold increase in IgG by the 4th month of infection and these sera produced 2--3 precipitin bands in double gel diffusion assays with an extract of B. malayi microfilarial antigen. Skin tests with B. malayi microfilarial antigen showed that the majority of the infected ferrets had immediate hypersensitivity responses, but none had Arthus or delayed hypersensitivity responses.