Journal of Helminthology (1978) 52, 99-107
Studies on humoral immunity against Taenia taeniaeformis infection in rats B. H. KWA and F. Y. LIEW Departments of Zoology and Genetics and Cellular Biology, University of Malaya, Kuala Lumpur, Malaysia ABSTRACT
Rats were infected with doses of 100,1000, 5000 and 10 000 eggs of Taenia taeniaeformis. Haemagglutinating antibody to cysticercus antigen was detected at the 4th week of infection. The appearance and levels of antibody titre did not vary greatly with the infective dose. An IgM peak appeared at the 6th week, with IgG appearing slightly later and continuing to rise. Transfer of serum from the 1st week onwards from infections with 1000 eggs however could confer significant protection. Dilutions of hyperimmune serum (1 ml volumes) of up to 1/32 conferred significant protection on normal recipients. Hyperimmune serum transferred up to 4 days before challenge could confer 80% protection whereas serum transferred 4 days after challenge was totally non-protective. The significance of this finding is discussed in the light of current knowledge of metacestode immunity.
Studies on humoral immunity in rats infected with Taenia taeniaeformis have previously been studied by Miller (1933), Miller and Gardiner (1932, 1934), Campbell (1938a), Kraut (1956), Murrell (1971), Rickard and Bell (1971), Heath and Pavloff (1975), Leid and Williams (1974a, b), Musoke and Williams (1975a, b) and Musoke et ah (1975). Kwa and Liew (1975) have established that cell mediated immunity plays a protective role in immunity against T. taeniaeformis. The present work extends previous studies and critically analyses the role of humoral immunity in protection against challenge infection. An attempt is made to find out when established larvae become resistant to the lethal effects of antibody. MATERIALS AND METHODS Experimental Animals Outbred female albino rats (3-4 weeks old) from the Central Animal Facility, Faculty of Medicine, University of Malaya, were used in all experiments. Cats from the same source were used to obtain adult worms. Taenia taeniaeformis An infection of T. taeniaeformis was maintained in the laboratory by oral inoculation of rats using a tube after light anaesthesia with ether. Strobilocerci were liberated after 8 weeks and 2 each were fed to cats to obtain the adult worms and complete the cycle. Mature oncospheres were obtained from the infected cats (at least 60 days after infection) after autopsy. Egg numbers were estimated by means of a haemocytometer. Somatic Antigens Somatic antigens (Som-Ag) were prepared from freshly liberated strobilocerci washed several times in saline and ground up with phosphate buffered saline (PBS) pH 7.2 in an ice cooled mortar and pestle. Extraction was achieved by continuous stirring at 4°C overnight. The homogenate was then centrifuged at 3000 rpm for 1 hour at 4°C and the supernatant obtained. The protein concentration was determined (1.35 mg/ml) using Lowry's method and stored at -10°C until used. 99
B. H. KWA and F. Y. LIEW Antibody Production
Four groups of rats (5 rats/group) were infected orally with 100, 1000, 5000 and 10 000 eggs per rat respectively. The rats were then bled at weekly intervals over a period of 5-8 weeks, and the sera separated and stored at -10°C. Antibodies produced were estimated using a passive haemagglutination technique based on that described by Jandl and Simmons (1957). Briefly, sheep red blood cells (SRBC) stored in Alsever's solution at 4CC were washed 4 times with saline and resuspended to 5 % in saline. To 5 mis of 5 % SRBC in saline was added 200 mg of Som-Ag. The solution was stirred and 2mls of 0.01 % CrCl 3 solution (pH 5.2) added with further mixing. After standing at room temperature for 5 to 10 min the cells were washed 3 times with PBS and resuspended to 2% in PBS. Titrations were done in V-bottom micro-titration wells, using PBS as diluent. Sera from infections with 5000 oncospheres per rat were titrated using PBS and 0.1M 2-mercaptoethanol (2ME) in PBS as diluents in parallel titrations. Passive transfer of immunity using sera from different time intervals after infection
Fifty donor rats were infected with 5000 eggs each and bled weekly from week 1week 6. The sera from each weekly collection were polled and stored at -10°C. Seven groups of rats (5 rats/group) were used as the recipients of the serum transfer. Each rat was injected intravenously with 1 ml of antiserum with 6 groups receiving sera from week 1-week 6 of infection respectively. The 7th group acted as controls. All rats were then immediately challenged with 1000 eggs each. Autopsy was performed 3 weeks after challenge. Counts of cyst numbers in the liver were expressed as percentage of control. Passive transfer using dilutions of hyperimmune serum
The donor rats described in the previous experiment were killed after 8 weeks infection and the sera were collected, pooled and stored at -10°C. This serum was designated as hyperimmune serum. A series of two fold dilution of the hyperimmune serum was made in saline containing 5 % normal rat serum. Recipient rats were divided into 9 groups (5 rats/group) and each rat received intravenously 1 ml of the respective dilution of hyperimmune serum, with a control group injected with 1 ml of the diluent. All rats were immediately challenged with 1000 eggs each. Autopsy was performed 3 weeks after challenge. Counts of cyst number in the liver were expressed as percentage of control. Time course of protection conferred by passive serum transfer before and after challenge
Ten groups of rats (5 rats/group) were used as recipients of the serum transfer. Four groups were injected intravenously with 1 ml hyperimmune serum each 4, 3, 2 and 1 days respectively before challenge infection. One group received the same dose of hyperimmune serum on the same day as the challenge. Four other groups of rats received the serum 1, 2, 3 and 4 days respectively after challenge. The control group received no serum. In all cases the challenge dose was 1000 eggs per rat. Autopsy was performed 3 weeks after challenge. Statistical Methods Standard errors of the means and P values were calculated according to Student's "t" Test.
100
Immunity to T. taeniaeformis in rats
RESULTS The results from the experiment on antibody production are shown in Fig. 1. Detectable titres of antibody appeared around the 4th and 5th weeks of infection, rising sharply thereafter. The levels of antibody in the circulation and the time course of antibody production did not appear to vary greatly with the infective dose of eggs. The rise of antibody production from the 4th to the 6th weeks was remarkably consistent in all 4 doses of egg used. 8r-
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Studies on humoral immunity against Taenia taeniaeformis infection in rats B. H. KWA and F. Y. LIEW Departments of Zoology and Genetics and Cellular Biology, University of Malaya, Kuala Lumpur, Malaysia ABSTRACT
Rats were infected with doses of 100,1000, 5000 and 10 000 eggs of Taenia taeniaeformis. Haemagglutinating antibody to cysticercus antigen was detected at the 4th week of infection. The appearance and levels of antibody titre did not vary greatly with the infective dose. An IgM peak appeared at the 6th week, with IgG appearing slightly later and continuing to rise. Transfer of serum from the 1st week onwards from infections with 1000 eggs however could confer significant protection. Dilutions of hyperimmune serum (1 ml volumes) of up to 1/32 conferred significant protection on normal recipients. Hyperimmune serum transferred up to 4 days before challenge could confer 80% protection whereas serum transferred 4 days after challenge was totally non-protective. The significance of this finding is discussed in the light of current knowledge of metacestode immunity.
Studies on humoral immunity in rats infected with Taenia taeniaeformis have previously been studied by Miller (1933), Miller and Gardiner (1932, 1934), Campbell (1938a), Kraut (1956), Murrell (1971), Rickard and Bell (1971), Heath and Pavloff (1975), Leid and Williams (1974a, b), Musoke and Williams (1975a, b) and Musoke et ah (1975). Kwa and Liew (1975) have established that cell mediated immunity plays a protective role in immunity against T. taeniaeformis. The present work extends previous studies and critically analyses the role of humoral immunity in protection against challenge infection. An attempt is made to find out when established larvae become resistant to the lethal effects of antibody. MATERIALS AND METHODS Experimental Animals Outbred female albino rats (3-4 weeks old) from the Central Animal Facility, Faculty of Medicine, University of Malaya, were used in all experiments. Cats from the same source were used to obtain adult worms. Taenia taeniaeformis An infection of T. taeniaeformis was maintained in the laboratory by oral inoculation of rats using a tube after light anaesthesia with ether. Strobilocerci were liberated after 8 weeks and 2 each were fed to cats to obtain the adult worms and complete the cycle. Mature oncospheres were obtained from the infected cats (at least 60 days after infection) after autopsy. Egg numbers were estimated by means of a haemocytometer. Somatic Antigens Somatic antigens (Som-Ag) were prepared from freshly liberated strobilocerci washed several times in saline and ground up with phosphate buffered saline (PBS) pH 7.2 in an ice cooled mortar and pestle. Extraction was achieved by continuous stirring at 4°C overnight. The homogenate was then centrifuged at 3000 rpm for 1 hour at 4°C and the supernatant obtained. The protein concentration was determined (1.35 mg/ml) using Lowry's method and stored at -10°C until used. 99
B. H. KWA and F. Y. LIEW Antibody Production
Four groups of rats (5 rats/group) were infected orally with 100, 1000, 5000 and 10 000 eggs per rat respectively. The rats were then bled at weekly intervals over a period of 5-8 weeks, and the sera separated and stored at -10°C. Antibodies produced were estimated using a passive haemagglutination technique based on that described by Jandl and Simmons (1957). Briefly, sheep red blood cells (SRBC) stored in Alsever's solution at 4CC were washed 4 times with saline and resuspended to 5 % in saline. To 5 mis of 5 % SRBC in saline was added 200 mg of Som-Ag. The solution was stirred and 2mls of 0.01 % CrCl 3 solution (pH 5.2) added with further mixing. After standing at room temperature for 5 to 10 min the cells were washed 3 times with PBS and resuspended to 2% in PBS. Titrations were done in V-bottom micro-titration wells, using PBS as diluent. Sera from infections with 5000 oncospheres per rat were titrated using PBS and 0.1M 2-mercaptoethanol (2ME) in PBS as diluents in parallel titrations. Passive transfer of immunity using sera from different time intervals after infection
Fifty donor rats were infected with 5000 eggs each and bled weekly from week 1week 6. The sera from each weekly collection were polled and stored at -10°C. Seven groups of rats (5 rats/group) were used as the recipients of the serum transfer. Each rat was injected intravenously with 1 ml of antiserum with 6 groups receiving sera from week 1-week 6 of infection respectively. The 7th group acted as controls. All rats were then immediately challenged with 1000 eggs each. Autopsy was performed 3 weeks after challenge. Counts of cyst numbers in the liver were expressed as percentage of control. Passive transfer using dilutions of hyperimmune serum
The donor rats described in the previous experiment were killed after 8 weeks infection and the sera were collected, pooled and stored at -10°C. This serum was designated as hyperimmune serum. A series of two fold dilution of the hyperimmune serum was made in saline containing 5 % normal rat serum. Recipient rats were divided into 9 groups (5 rats/group) and each rat received intravenously 1 ml of the respective dilution of hyperimmune serum, with a control group injected with 1 ml of the diluent. All rats were immediately challenged with 1000 eggs each. Autopsy was performed 3 weeks after challenge. Counts of cyst number in the liver were expressed as percentage of control. Time course of protection conferred by passive serum transfer before and after challenge
Ten groups of rats (5 rats/group) were used as recipients of the serum transfer. Four groups were injected intravenously with 1 ml hyperimmune serum each 4, 3, 2 and 1 days respectively before challenge infection. One group received the same dose of hyperimmune serum on the same day as the challenge. Four other groups of rats received the serum 1, 2, 3 and 4 days respectively after challenge. The control group received no serum. In all cases the challenge dose was 1000 eggs per rat. Autopsy was performed 3 weeks after challenge. Statistical Methods Standard errors of the means and P values were calculated according to Student's "t" Test.
100
Immunity to T. taeniaeformis in rats
RESULTS The results from the experiment on antibody production are shown in Fig. 1. Detectable titres of antibody appeared around the 4th and 5th weeks of infection, rising sharply thereafter. The levels of antibody in the circulation and the time course of antibody production did not appear to vary greatly with the infective dose of eggs. The rise of antibody production from the 4th to the 6th weeks was remarkably consistent in all 4 doses of egg used. 8r-
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