Journal of Helminthology (1976) 50, 53—57

The detection of antibodies in human and animal filariases by counterimmunoelectrophoresis with Dirofilaria immitis antigens ROBERT S. DESOWITZ and STEVEN R. UNA Department of Tropical Medicine and Medical Microbiology, School of Medicine, University of Hawaii, Honolulu, Hawaii 96816, USA

ABSTRACT Counterimmunoelectrophoresis revealed the presence of precipitin antibody in all of 6 dogs and the 1 cat infected with Dirofilaria immitis and in the serum of 17 of 24 individuals living in a setting of hyperendemic subperiodic bancroftian filariasis. Antigens used in the test were prepared from microfilariae and adult male D. immitis. Some humans and animals had antibodies to both antigens while others had antibodies against microfilariae or adult worms only. The presence of soluble circulating antigen was detected in the sera of two dogs with high microfilaraemias.

The occurrence of precipitation antibody in the serum of humans and animals infected with filarial worms has been reported by several investigators. Ellsworth and Johnson (1973) ,. observed precipate formation when sera of Dirofilaria /m/m'/w-infected dogs were reacted, by diffusion in capillary tubes, with a crude antigen extracted from adult heartworms. Wheeling and Hutchinson (1971) reported the immunoelectrophoresis (IEP) of serum from a patient with a "tentative diagnosis of filariasis" produced one and two precipitin arcs against antigens prepared from D. immitis microfilariae and adults respectively. Kaeuffer et al. (1974) however, did not detect any positive reactions when the sera of 14 Tahitians infected with Wuchereria bancrofti were tested by IEP with adult D. immitis antigen. IEP has also been successfully employed for the diagnosis of onchocerciasis with antigen extracted from adult worms of that infection (Biguet et al, 1962,1964). By this technique Gentilini et al. (1973) obtained positive reactions with the serum of 8 out of 9 patients with onchocerciasis but no precipitin bands developed when the sera of 3 patients with bancroftian filariasis were reacted against the Onchocerca volvulus antigen. The average number of arcs produced are considered by D'Haussy et al. (1972) to be related to the clinical severity of the onchocercal infection. The specificity of these precipitin reactions is not known to any degree of certainty. Whereas Wheeling and Hutchinson obtained a positive reaction to D. immitis antigen in a case of human filariasis, the results were entirely negative to the same antigen in the series of Tahitians with patent \V. bancrofti microfilaraemia investigated by Kauffer's group. That there may be a broad spectrum of antigenic specificity is indicated by the investigation of Niel et al. (1972) who found that the antigens prepared from Setaria labiato-papillosa and Dipetalonema viteae produced precipitin arcs, by double diffusion and IEP, when reacted against the serum of patients with loiasis, onchocerciasis, bancroftian filariasis, and dracunculiasis. It should also be noted that these authors reported positive precipitin reactions with an antigen of Ascaris suum and the sera from these filariasis patients. However, since intestinal hclminthiases are usually highly prevalent in areas of endemicfilariasesit is difficult to determine whether the positive heterologous precipitin reaction represents a common antigen relationship or a reaction with A. suum antibody. 53

R. S. DESOWITZ and S. R. UNA

Of the various techniques employed for the visualization of precipitin antibody, counterimmunolectrophoresis (CIEP) seems to be the most sensitive. For example, Krupp (1974) found that CIEP was superior to immunodiffusion for. the diagnosis of amoebiasis. Despomier et al. (1974) reported CIEP to be a sensitive, specific immunodiagnostic test for trichinosis. CIEP has the further advantage of simplicity of performance, rapidity and the capability of processing relatively large numbers of serum samples at the same time. The test has not before been applied to the serology of filarial infections and this present paper reports the results gained with CIEP using sera from humans infected with W. bancrofti and from D. immitis-infected animals and soluble antigens prepared from Dirofilaria adults and microfilariae.

MATERIALS AND METHODS Antigens

The antigens used in this study were prepared from adult D. immitis collected at autopsy of naturally infected dogs. The worms were separated by sex and then washed in two changes of phosphate-buffered saline (PBS) pH 7*2, at room temperature. It had previously been observed that when female Dirofilaria were immersed in cold PBS they rapidly expressed copious numbers of microfilariae and ova and this phenomenon was exploited to obtain microfilarial antigen (MA). After being washed, the female worms were immersed in 200 ml cold (4°C) PBS and incubated at that temperature for 1 hour. The adult worms were then removed and the PBS containing the microfilariae was centrifuged at 2500 rpm for 10 minutes at 4°C. The supernatant was discarded and the deposit washed again by centrifugation. The deposit was resuspended in 10 volumes of PBS and then extracted by passing through a Hughes-Colab press maintained at —20°C. The extract was centrifuged at 10 000 rpm for 20 minutes. The supernatant antigen was collected and adjusted by means of Biuret analysis, to contain an estimated 15 mg protein/ml. When not used immediately the antigens were stored in 100 mm3 capillary tubes at —70°C. Adult male antigen (AMA) was prepared by finely mincing the washed male worms, passing them through the Hughes-Colab press, and adjusting the centrifuged soluble extract to the same protein concentration as that of the MA. Antisera Canine and feline sera were obtained from animals born and bred in Oahu, Hawaii, an area of enzootic D. immitisfilariasis.The microfilarial density in these animals was determined by examination of a 20 mm3 stained thick-bloodfilmand by membranefilterconcentration (Desowitz and Southgate, 1973). Sera were collected from humans living in Te'ekiu village, Kingdom of Tonga, a setting of hyperendemic sub-periodic bancroftian filariasis (Desowitz and Berman, 1973; Desowitz and Hitchcock, 1974). The microfilarial density was determined for each donor by membrane filter concentration of 1 ml of citrated venous blood. Each individual was given a skin test with Sawada's antigen. Clinical histories were taken and physical examinations performed. Antisera to MA and AMA were obtained by immunizing rabbits with a mixture of 1 ml antigen and 0«5 ml Freund's complete adjuvant. Four such doses, spaced one week apart, were inoculated and the rabbit bled for serum one week after the last injection. 54

Diagnosis offilariasisby counterimmunoelectrophoresis Counterimmunoelcctrophoresis (CIEP) CIEPwas performed with an Austigen apparatus (Hyland Corp., Costa Mesa, California). The agar plates were prepared from 0-75% Noble agar in veronal buffer of pH 8-4 to which merthiolate was added to a concentration of 1 : 10 000. The wells were spaced 5 mm apart and 12 samples were run on each plate. The serum sample was placed in the anodic well and the antigen in the cathodic well. For determination of soluble antigen the serum sample was placed in the cathodic well and the rabbit anti-filaria serum in the anodic well. Electrophoresis was carried out for 1 hour at a setting of 30 mA. Following completion of electrophoresis, the plates were allowed to remain at room temperature for 1 hour and the precipitin bands visualized and recorded. The plates were then immersed in physiological saline for 24 hours and reexamined. In a number of instances precipitin bands developed only after this procedure. Permanent preparations were made by washing the plates in several changes of distilled water over a period of 24 hours, staining with acid fuchsin followed by decolorization with an acetic acid-methanol solution, and finally drying in a 37°C incubator. RESULTS D. immitis-infectcd animals

Table 1 summarizes the results obtained with the sera from dogs and cats infected with D. immitis reacted against MA and AMA antigens and against rabbit anti-filaria antibody for the detection of soluble circulating antigen. The serum of all infected animals produced precipitin lines to one or both antigens whereas the uninfected controls, although harbouring helminth parasites other than D. immitis, were serologically negative. The amicrofilaraemic dog from the endemic area was positive to the MA only. The serum of the cat without demonstrable circulating microfilariae but with 2 adult worms found in the heart was positive to both MA and AMA antigens. The sera of dogs with a low to moderate microfilaraemia (25—92 mf/20 mm3) were positive to AMA only while the two dogs with high microfilaraemia (480 and 696 mf/20 mm3) had precipitating antibody to both MA and AMA. The serum of these two dogs also showed the presence of soluble circulating filarial antigen. Tongans residing in an area of hyperendemic bancroftian filariasis Table 2 summarizes the CIEP reactions with MA and AMA and the serum of 24 Tongans of various ages and with various degrees of microfilaraemia. There is an indication that children and adults who are exposed to filarial infection but harbour either no or low densities of microfilariae have antibodies against MA or MA and AMA together, but not against AMA alone. In contrast, the individuals with moderate to high microfilaraemias produced precipitin lines only against the adult antigen. There was no correlation between skin test reactivity and the absence or presence of precipitin antibody. Some individuals with a positive skin test were also CIEP positive to MA or AMA while others were precipitin negative. Conversely, some skin test-negative individuals were CIEP positive and others negative. DISCUSSION Precipitin antibody to D. immitis antigens was regularly present, as detected by CIEP, in both animals infected with or exposed to D. immitis and humans infected with subperiodic W. bancrofti. However, stage-specific antibody appears to be elicited by both human and animal hosts. Some serum samples reacted only to microfilarial antigen, others to antigen derived from adult male D. immitis, and others still to both antigens. The relationship between the degree of microfilaraemia and the stage specificity of the antibody elicited is not 55

Rl S. DESOWITZ and S. R. UNA TABLE 1 The CIEP reactions with various D. immitis antigens of sera from dogs and cats infected with D. immitis

Animal

microfilaraemia mf/20 mm3

Description

CIEP reaction AMA

MA

soluble antigen (Host serum as antigen)

Control. No D. immitis found at autopsy. Infected with hookworm and Trichuris.

0

0

0

0

Dog 2 , Control. No D. immitis found at autopsy

0

0

0

0

Control. No D. immitis found at autopsy. Infected with Fasclola gigantica.

0

0

0

0

Control. No D. immitis found at autopsy. Infected with Platynosum conicum.

0

0

0

0

Control. No D. immitis found at autopsy. Infected with Aelurostrongylus abstrusus.

0

0

0

0

Cat 4

Two adult D. immitis recovered at autopsy.

0

+

+

not done

Dog 3

Adult dog from endemic area of Oahu.

0

+

0

0

Dog 4

Not killed

25

0

0

Dog 5

Not killed

28

0

Dog 6

Not killed

92

0

Dog 7

Not killed

482

+

Dog 8

Not killed

696

+ + + + +

.. Dog 1

Cat 1 Cat 2 Cat 3

0 0

+ +

TABLE 2 CIEP reactions in sera from Te'ekiu villagers to D. immitis antigens. CIEP reactions (percentages in brackets) Microfilaraemia

Age group

The detection of antibodies in human and animal filariases by counterimmunoelectrophoresis with Dirofilaria immitis antigens.

Counterimmunoelectrophoresis revealed the presence of precipitin antibody in all of 6 dogs and the 1 cat infected with Dirofilaria immitis and in the ...
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