Studies on American Paragonimiasis. II. Serological Observations of Infected Cats

Abstract

Extracts prepared from adult worms of Paragonimus kellicotti contain a minimum of five soluble precipitating antigens. The possibility of two or more metabolic antigens is also discussed. Sera from cats infected for as long as 210 days contained precipitating and complement fixing antibody, but failed to immobilize miracidia or show extensive Cercarienhiillen reaction, cercarial immobilization, or agglutination reactions. There have been few or no attempts to characterize the antigens present in various stages of the life cycle of Paragonimus kellicotti Ward, 1908. Indeed, little or no work has been done to study the immunological aspects of animals infected with P. kellicotti. Serological tests have been developed for the clinical diagnosis of human infections (Sadun, Buck, and Walton, 1959; Yokogawa, Cort, and Yokogawa, 1960; Kent, 1963). The complement fixation and the intradermal skin test have been most extensively studied using an antigen prepared from adult worms. Sadun et al. (1959) also studied the complement fixation test with sera from infected cats. However, in none of this work has the number or type of antigens present in Paragonimus extracts been determined. Nor has there been any report on the use of other stages in the life cycle of P. kellicotti as the antigenic material. Reasonable quantities of all stages of the life cycle of P. kellicotti can be maintained in the laboratory (Sogandares-Bemal, 1965). The availability of such laboratory-raised material and the lack of prior experimentation in this area suggested that an immunological study of Paragonimus kellicotti infection in the cat might be fruitfully explored. In addition, it was of interest to determine whether the serology of P. kellicotti infections are similar to schistosome and other Paragonimus species infections. This work describes our serological observations on sera obtained from infected cats, and includes a preliminary characterization of antigens found in the adult worms. Received for publication 30 August 1965. * This work was supported by a research grant from the NIH (AI 03386-TMP). METHODS Domestic cats (7 to 10 lbs) were infected by feeding them varying numbers of metacercariae of P. kellicotti. The cats were killed after a predetermined period of time, and the serum and adult worms were collected. All sera were stored at -30 C until needed. In addition, the cyst wall and the fluid from the inside of the cyst were collected from several cats. Various pieces of tissue surrounding the cyst were also obtained for use as antigens. Cats were necropsied for helminth infections of the intestine. Other stages in the life cycle of P. kellicotti were mai tained as previously described (SogandaresBernal, 1965). Paragonimus extracts were prepared by homogenizing 15 to 25 adult worms in 6 to 8 ml distilled water with the aid of a hand glass homogenizer. The adult worms used in extracts were relatively free of eggs due to the removal of their uteri. The homogenate was then centrifuged at 12,000 g for 10 min in a refrigerated centrifuge. The supernatant was used as the antigen. In one experiment, the adult worms were homogenized in 0.1 M borate buffer (pH 8.3). The homogenate was centrifuged and the supernatant collected. Then 0.2 N HC1 was added to the supernatant which was chilled in a crushed ice bath and stirred with a magnetic stirrer to pH 4.0. The precipitate which formed was removed by centrifugation at 12,000 g for 10 min. The acid soluble supernatant (acid soluble fraction) was saved and the sediment resuspended in the original volume of borate buffer, pH 8.3. This suspension was centrifuged at 12,000 g for 10 min, the sediment discarded, and the supernatant retained (acid insoluble-alkali soluble fraction). All antigens were stored at -30 C until needed. In addition, Paragonimus extract (0.8 mg protein/ml) was treated with trypsin (0.1 mg/ml, 2X crystallized, Sigma Chemical Company, St. Louis, Mo., 30 C) or chymotrypsin (0.1 mg/ml, crystalline, Type IV, Sigma Chemical Company, 30 C) for 1 hr. The treated extract was chilled and then tested for the presence of Paragonimus antigens. Protein was assayed by the method of Lowry et al. (1951). All antigen suspensions were made to contain from 0.4 to 0.45 mg protein/ml.