Lois Norman

The maintenance of Echinococcus multilocularis in gerbils (Meriones unguiculatus) by intraperitoneal inoculation.

(Em) have been produced by feeding eggs to several strains and species of laboratory rodents (Rausch, 1954; Mankau, 1956, 1957; Vogel and Schumacher, 1957; Sadun et al, 1957; Yamashita et al, 1958 Lubinsky, 1960a, b). Sadun et al (1957), in our laboratory, reported that the CFW strain of albino mouse fed eggs of Em was resistant to infection, but that the cotton rat, Sigmodon hespidus, was very susceptible. Infection in cotton rats develops rapidly (in 1 to 2 months) and eventually kills the host. Cysts grow to 1 cm in diameter and emanate first from liver and lung tissue, and finally invade other viscera until the organs are compressed or displaced. Host and parasite tissue are so intimately mingled that complete separation is impossible. In continuing our search for a practical mIethod of obtaining Em cyst material as free from host tissue as possible and also for a practical technique of maintaining the parasite in the laboratory without the use of eggs of Em, experiments were initiated to produce infections by means of intraperitoneal inoculation of viable cyst cells frorll animal to animal. The transplantation of Echinococcus grandnlosis (Eg) and Em cyst tissue via intraperitoneal inoculation has recently been reported by Yamashita, Ohbayashi and Konno (1957); Schwabe, Schinozi and Kilejian (1959), and Lubinsky (1960a, b). Continued attempts to render our CFW mice susceptible to infection by treatment with cortisone either before or simultaneously with oral feeding of eggs rXesulted in production of only very limited parasitic material; however, feeding of eggs to gerbils resulted in fairly good infections, suggesting that these rodents mlight be suitable animals for further investigation. Using infected gerbils and cotton rats as the sources of viable cyst tissue, intraperitoneal infections in gerbils

ANALYSIS OF HELMINTH ANTIGENS (ECHINOCOCCUS GRANULOSUS AND SCHISTOSOMA MANSONI) BY AGAR GEL METHODS.

Although parasitologists have worked with unknown and crude extracts of parasitic worms as antigens in their immunologic and serologic studies, the desirability for more specific and sensitive reagents has become increasingly apparent. There have been excellent studies reported on the isolation and characterization of helminth antigens employed in immunodiagnostic tests which were recently reviewed by J. F. Kent (1963). In this paper, selected studies on the characterization of helminth antigens by agar-gel methods will be reviewed and the work in progress in our laboratory on hydatid and schistosome antigens will be outlined.

IMMUNOLOGIC STUDIES ON HAMSTERS INFECTED WITH ENTAMOEBA HISTOLYTICA

The serologic and cell-mediated immune responses of hamsters exposed to 2 strains of Entamoeba histolytica (HM-1 and HM-19) were evaluated by a series of in vitro tests. The pathogenicity of the 2 strains was evaluated in terms of their ability to produce liver abscesses and spleen enlargement. Antibody response was evaluated by the indirect hemagglutination test. The cellular immune response was assayed by increased DNA synthesis by lymphocytes and migration inhibition of macrophages.

Bentonite, latex, and cholesterol flocculation tests for the diagnosis of trichinosis.

AT THE Communicable Disease Center of the Public Health Service the bentonite flocculation test (BFT) is used routinely for the diagnosis of trichinosis. This test has been under continual evaluation for sensitivity and specificity since 1952. As part of this evaluation and to keep abreast of new developments, we have attempted, whenever possible, to compare the BFT with new tests and reagents as they have become commercially available. The trichinalatex antigen sold by Hyland Laboratories has been tested intensively. The antigen for the Suessenguth and Kline flocculation test marketed by the LaMotte Company and the reagents for a bentonite flocculation test and a latex test prepared by the Difco Laboratories have received limited evaluations. The accumulated results of our studies may be of help to local laboratories in interpreting their tests. They do not constitute an endorsement or rejection of the proiducts examined. Several particle agglutination tests have been used for the diagnosis of trichinosis. Suessenguth and Kline (1) adapted the Kline test for syphilis to a test for trichinosis and used cholesterol particles Icoated with various trichina antigens. They reported high sensitivity for both human and animal trichinosis (2-4). Campbell (5) and Coudert and Coly (6) used coated collodion particles, and Leikina and Poliakova (7), carmin particles in their tests. Vogel and co-workers (8) employed cholesterollecithin particles. Bozicevich and associates (9) introduced the use of bentonite particles. A series of papers from the CDC. laboratory has reported evaluations of the bentonite test for detection of infection in pigs (10) and in humans (11). The efficacy of Melchers antigen (13) and of metabolic antigen (12) has been reported as well as that of lyophilized reagents (14). Innella and Redner (15) were the first to describe the use of latex particles coated with trichina antigen. Their test was essentially a modification of the latex test for rheumatoid arthritis developed by Singer and Plotz (16). Muraschi and co-workers have been using the latex test since 1957 and recently published their evaluation of a slide latex-particle agglutination test for trichinosis (17).

Biologic identification of the cestode Echinococcus multilocularis isolated from foxes in North Dakota.

The eggs from Echinococcus multilocularis recovered from red foxes in North Dakota were fed to cotton rats (Sigmodon hispidus) and infection in this host was obtained. This is biological verification of the occurrence of this species in the continental United States. Adult cestodes isolated from red foxes (Vulpes vulpes) in North Dakota were identified morphologically by Leiby and Olsen (1964) as Echinococcus multilocularis, Leuckart, 1863. This was the first time this species had been found in the continental United States and marked an extension of its geographic range. Biologic and morphologic studies of the cystic stages which developed in an experimentally infected rodent host have confirmed the identification of the organisms as E. multilocularis, indistinguishable from the species isolated in Alaska (Rausch, 1956). Gravid worms collected in North Dakota were suspended in water and given by stomach cannula to groups of cotton rats (Sigmodon hispidus). Individual rats received 10 to 15 worms each. The first three rats were inoculated 2 days after receipt of the worm material; the second group of three was fed 7 days later, and the final group was fed after the adult worms and some of the expelled eggs had been stored in water in the cold for 3 weeks. All but one of the cotton rats became infected. Figure 1 is a picture of an animal autopsied 8 weeks after experimental infection with small cystic clumps of E. multilocularis which are typical for infections in cotton rats (Sadun et al., 1957). In this animal there were abundant scolices in the cysts, many of which were mature although the infection was only 8 weeks old. A second animal autopsied at 6 weeks also contained some mature cysts. The scolices in cysts from two 4-month-old infections were examined with low-power magnification and could not be differentiated from scolices collected from rats infected for the Received for publication 29 March 1965. * U. S. Department of Health, Education, and Welfare, Public Health Service, Communicable Disease Center, Atlanta, Georgia. t Minot State College, Minot, North Dakota. same length of time with the Alaskan isolates which are being maintained at the Communicable Disease Center. The number of scolices per cyst of the North Dakota isolate was more numerous than that of the Alaskan strain. Two animals died with heavy experimental infections. The number of rostellar hooks on the scolices of E. multilocularis recovered from two animals with primary infections autopsied 8 months after feeding varied from 24 to 33 with a mean of 26.9 hooklets. This is comparable to the observations made by Cameron (1960). When the scolices were washed and subinoculated by intraperitoneal injection into four young cotton rats and three young gerbils FIGURE 1. Experimental infection of a cotton rat (Sigmodon hispidus) with eggs of Echinococcus multilocularis collected from a red fox (Vulpes vulpes) in North Dakota, USA.