Fluorescent Antibody Reactions against Six Species of Simian Malaria in Monkeys from India and Malaysia

Abstract

Sera from 45 Malaysian and 51 Indian monkeys were tested for fluorescent antibody response to antigens of Plasmodium fieldi, P. gonderi, P. inui, P. coatneyi, P. knowlesi, and P. cynomolgi bastianellii. A total of 17 Malaysian and 33 Indian monkey sera had positive antibody responses with the larger number reacting with P. fieldi and P. gonderi. The results suggest the possibility of prior malaria infection or of nonspecific antibody to other parasites. The intensity of this response to heterologous or common antigens may be sufficient to mask a species-specific response. The use of monkeys having no initial fluorescent antibody titer for Plasmoclium is suggested for studies involving antibody in monkey malaria. The staining of monkey malaria parasites using the fluorescent antibody (FA) technique was first reported by Ingram et al. (1961) using Plasmodium cynomolgi bastianellii. Additional studies by Tobie and Coatney (1961) and Tobie et al. (1962) showed cross-reactions between P. vivax and P. cynomolgi bastianellii. Reported here are the fluorescent antibody reactions of monkey sera from Malaysia and India against six different species of monkey malaria. MATERIALS AND METHODS Thin blood films, containing the parasites of P. fieldi, P. gonderi, P. inzi, P. coatneyi, P. knowlesi, and P. cynomolgi bastianellii, were prepared and stored without fixation at -70 C in a mechanical freezer. Prior to use, they were raised to room temperature in a desiccator and then fixed for 30 min in acetone at -20 C. The slides were dried and then dehemoglobinized by immersion for 5 min in 0.1% HC1, followed by 1-min rinses each in distilled water and phosphate-buffered saline (PBS), pH 7.0. The antigen slides were then ready for immediate use. Rabbit antiserum (globulin fraction) to monkey globulin conjugated with fluorescein isothiocyanate was obtained commercially (Sylvana Chemical Company) and prepared by absorption once with rabbit liver powder and once with rabbit bone marrow powder, and stored at 1: 40 in PBS at -20 C. The 1: 40 dilution was used throughout the studies. Received for publication 28 July 1964. 81 A Leitz SM fluorescence microscope with an Osram HBO-200 light source was used with BG-12 and UG-2 exciter filters and a Wratten 2A barrier filter. For each test, several drops of the serum dilution to be tested were applied to the antigen slide and allowed to react for 20 min at room temperature in a moist chamber. The slide was then given 3 5-min rinses in PBS. Several drops of the labeled rabbit antimonkey globulin were applied and allowed to react for 20 min. The slide was again given three 5-min rinses in PBS and then mounted under a cover slip in buffered glycerine. Fluorescence was graded 1+ to 4+. A reading of 2+ was considered positive and reproducible. Each serum sample was titrated simultaneously against all six antigens. The test sera were from several species of Macaca and Presbytis monkeys collected in the Selangor, Pahang, and Perak states of Malaysia in 1961, and from Macaca mulatta monkeys arriving in our laboratory from New Delhi, India, in 1963. Of the Malaysian monkeys, only one (C-22) was infected with Plasmodium (species undetermined) at the time of collection. Since these were field-collected sera, slides were made only the 1 day. It is therefore impossible to determine if other of these monkeys were suffering from chronic infections. None of the Indian monkeys had malarial infections as determined by the microscopic examination of blood films taken on two separate days. Previous laboratory studies had shown that monkeys having chronic infection with Plasmodium sp. had FA end points of 1: 40 or greater. Even though all the sera were tested starting at the 1: 10 dilution, it was decided to report only those FA reactions of 1 : 40 or greater as being positive. This content downloaded from 157.55.39.249 on Wed, 03 Aug 2016 05:47:52 UTC All use subject to http://about.jstor.org/terms 82 THE JOURNAL OF PARASITOLOGY, VOL. 51, NO. 1, FEBRUARY 1965 TABLE I. Fluorescent antibody test reactions (expressed as reciprocal of titer) of 17 positive sera from Presbytis sp. and Macaca sp. monkeys collected in Malaysia in 1961. Monkey Monkey Species of Plasmodium species no. species no. P. fieldi P. gonderi P. inui P. coatneyi P. obscura 0-61 40 40 P. cristatus C-18 40 C-22 40 40 -25 40 160 C-27 40 C-28 40 40 -29 40 40 C-31 40 C-32 160 C-38 40 M. irus M-312 40 -313 80 M-314 40 M-315 40 M-316 40 M. nemestrina N-93 80 N-94 40 Positive responses 13 4 1 4 Control sera from monkeys known to be free of previous malarial infection gave negative responses. In addition, the labeled antimonkey globulin alone failed to give any response. Monkeys having previous malaria reacted strongly both to homologous and heterologous antigens.