AN EXPERIMENTAL APPROACH TO THE PROBLEM OF ACQUIRED IMMUNITY IN HUMAN HOOKWORM (NECATOR AMERICANUS) INFECTIONS1

BY A. J. SHELDON2 AND M. E. GROOVER, JR.3 (Received for publication January 19th, 1942) Downloaded from https://academic.oup.com/aje/article/36/2/183/127626 by guest on 28 September 2021 Epidemiological studies of human periments of these workers definitely hookworm disease have long suggested suggested that the infected dogs became that host reactions play an important generally sensitized and developed a role in the regulation of infection. It generalized immunity. The mechanism has been observed repeatedly that per- of this immunity seemed to depend on sons exposed to large numbers of infec- the presence of a humoral antibody. tive Necator americanus larvae over long That such was the case was shown by periods of time do not all build up heavy Otto, 1940, who found that the serum of worm burdens. In fact, in such popula- dogs actively immunized by repeated in- tions the majority of the infections are fections with larvae of A. caninum con- moderate or subclinical. The intrinsic tained an antibody of considerable po- factor that prevented overwhelming in- tency against the larvae. He obtained fections was assumed to be the develop- a typical precipitin reaction around and ment of acquired immunity as the result in the oral opening and around the ex- of previous infection with hookworms. cretory and anal openings of some of the There is no clear-cut experimental evi- larvae in vitro. This report of a serum dence of such a specific immunity in man. antibody against the larvae of A. cani- However, in recent years (Otto and num suggested an experimental approach Kerr, 1939; Otto, 1941) it has been to the problem of acquired immunity in clearly demonstrated that dogs acquire human hookworm infections. It is the a marked specific immunity following object of this paper to report an attempt repeated infections with the dog hook- to demonstrate, microscopically, evi- worm, Ancylostoma caninum. The ex- dence of the presence of a specific pre- cipitin in serum samples collected from 'This work was made possible by a summer a small group of white and colored school training grant from the International Health children, and to correlate its presence or Division of The Rockefeller Foundation. The authors wish to express their appreciation to absence with the severity of hookworm Dr. J. Andrews, Director of the Division of infection in these children. Malaria and Hookworm Service, Georgia De- partment of Public Health, for aid in obtaining the hookworm egg counts. The authors are also MATERIALS AND METHODS indebted to Dr. D. L. Augustine, Harvard Med- ical School, and Drs. W. W. Cort and G. F. Otto, The cases were chosen from an un- Johns Hopkins University, School of Hygiene treated group of white and colored school and Public Health, for their suggestions con- children infected with hookworms, living cerning presentation of data. 1 in Brooks County, Georgia. School Department of Preventive Medicine and Epi- children were used as subjects because of demiology, Harvard Medical School and School of Public Health, Boston, Mass. their availability and the fact that they •Health Officer, Brooks County Board of represent the age group (6-19 years) of Health, Quitman, Ga. highest incidence of hookworm infection. 183 184 A. J. SHELDON AND M. E. GROOVER, JR.

These children had previously been ex- modification of the Baermann technique amined for hookworms by the Division (Cort, 1922) and washed 3 times in sterile of Malaria and Hookworm Service of the normal saline. They were then placed Georgia Department of Public Health. in 0.1 per cent mercuric chloride for 30 The number of hookworms harbored was minutes, after which they were again estimated by the Stoll egg-counting washed 3 times in sterile normal sa- method (Stoll, 1923a). The children line. Approximately 100 larvae (dilu- were divided into arbitrary groups ac- tion count) were added to 5 ml of serum Downloaded from https://academic.oup.com/aje/article/36/2/183/127626 by guest on 28 September 2021 cording to the intensity of their infec- in a sterile Petri dish, which was placed tions, as suggested by Smillie and Augus- in a moist chamber at 37 C. At the tine, 1926. These authors studied 2,000 times when observations were made on Alabama school children having varying the larvae, a few drops of the serum con- intensities of hookworm infection and taining the larvae were placed on a sterile divided them as follows: glass slide and covered with a sterile coverslip. The coverslip was ringed with Group I, no hookworms. vaseline to prevent evaporation. These Group II, very light infections, 1-25 preparations were then studied with the worms. microscope. In addition to the sera Group III, light infections, 26-100 from infected children, larvae were incu- worms. bated in sera collected from uninfected Group IV, moderate infections, 101-500 children from a white and a colored fam- worms. ily, the remaining members of which Group V, heavy infections, 501-1,000 harbored various intensities of hookworm worms, infections. Larvae were also placed in Group VI, very heavy infections, 1,001- sera collected from 5 uninfected white 3,000 worms. children from Boston, Mass., aged 10 to The figures for the approximate number 13 years, who had never been exposed to of worms was arrived at by dividing the human hookworm larvae, so far as is number of eggs per gram of feces by the known, and in sterile normal saline to factor 22 (Stoll, 1923b). serve as controls. The children were visited at their homes and serum for study was obtained EXPERIMENTAL RESULTS from blood collected by venapuncture. The blood was immediately placed on ice Microscopic observations were made where it was kept from 1 to 4 hours until at hourly intervals, using a 10X ocular it was possible to return to the laboratory and a 4 millimeter objective (400X mag- and place it in the refrigerator at 4 C. nification) . No change in the appearance The length of stay in the refrigerator of the larvae was noted until 6 hours after varied from 48 to 72 hours before use. incubation. At this time some of the Active infective (filariform) larvae of larvae had lost their sheaths. A fine Necator americanus were obtained from granular precipitate was seen around and charcoal cultures of feces collected from in the oral opening and around the geni- an untreated white child with a heavy tal and anal pores of these larvae. The hookworm infection. The cultures were precipitate was not present in and incubated at room temperature (30-32 around all the exsheathed larvae in the C) for 8 days. The larvae were isolated same serum, and it did not appear to from the cultures by the use of the Cort interfere with the activity of the larvae. IMMUNITY IN HOOKWORM INFECTIONS 185

At 7 and 24 hours after incubation more uninfected children. The latter were of the larvae had shed their sheaths, and from families with other infected mem- a precipitate was observed in and around bers, and it seems likely that they, too, some of them. No further changes were had been exposed to infective hookworm noted until 48 hours after incubation larvae. A serum precipitin was not when most of the larvae were seen to found to be present in the blood of any have lost their sheaths, and all appeared of the 6 children with heavy and very to be dead or disintegrating. At this heavy infections, or in the blood of the Downloaded from https://academic.oup.com/aje/article/36/2/183/127626 by guest on 28 September 2021 time, despite the attempt to keep the 5 uninfected children who had never been incubator moist, the sera had become exposed to infection. At least 3 inter- very viscid. The death of the larvae was pretations of these data are possible: attributed to this fact. The larvae first, that the precipitin reaction is not which had been placed in normal sera specific and is a purely coincidental find- showed no precipitate, although most of ing; second, that the precipitin reaction them had shed their sheaths after 24 is specific, but does not play a role in hours. The larvae which had been acquired immunity against hookworm placed in normal saline showed no pre- infection; last, that the precipitin is spe- cipitate and remained alive and active cific and is part of the mechanism of ac- for many days; only a few of them lost quired immunity developed in some of their sheaths. The results are given in the children against hookworm infection table 1. as the result of previous exposure or TABLE 1 infection. In these children the precipi- Summary of precipitin reactions obtained in the tins formed against the excretions and sera of children harboring varying intensities of secretions of the larvae would account hookworm (Necator americanus) infections and of for the precipitates seen in and around uninfected children. them. Although the precipitates ap- peared to have no effect on the activity Precipitin reaction of the larvae in vitro, it is likely that in Severity of Infection Total Negative Positive vivo they may be instrumental in affect- ing the immobilization, delay in develop- None; never exposed 5 0 5 ment, stunting, prevention of food as- None; presumably similation and inhibition of the enzy- exposed 0 3 3 Very light 4 3 7 matic action of the worms, as suggested Light 1 2 3 by Taliaferro, 1940. The inconstant Moderate 1 3 4 presence of a serum precipitin in the sera Heavy 5 0 5 of these children, if specific, would be Very heavy 1 0 1 difficult to explain. Such factors as Total 17 11 28 technique employed for its demonstra- tion, certain intrinsic host factors, in- cluding heredity, variations in titer, DISCUSSION individual variation and diet, and the In table 1 it will be seen that micro- numerous environmental conditions in- scopic evidence of the action of a serum fluencing dissemination of hookworms, precipitin against hookworm larvae was must all be considered. jbtained in the blood of 8 of the 14 It is not possible at present to deter- jhildren with very light, light and mod- mine which of these interpretations, if erate infections, and in the blood of 3 any, is the correct one. However, since 186 A. J. SHELDON AND M. E. GROOVER, JR. the findings suggest that the precipitin 3 uninfected, but presumably exposed, reaction may be specific, the methods children and of 8 of 14 children harboring employed in this study offer an experi- very light, light and moderate infections, mental approach to the problem of ac- is presented. A precipitin reaction was quired immunity in human hookworm obtained around and in the oral opening infections. an(^ around the genital and anal pores of , some of the larvae placed in the sera of

SUMMARY these children. A precipitin reaction Downloaded from https://academic.oup.com/aje/article/36/2/183/127626 by guest on 28 September 2021 was not obtained in the sera of 6 children Microscopic evidence of the action of with heavy and very heavy infections, or a serum precipitin against infective lar- 5 children who had never been exposed vae of Necator americanus in the blood of to hookworm infection.

REFERENCES Cort, W. W., J. E. Ackert, D. L. Augustine and F. K. Payne 1922 Investigations on the control of hookworm disease. II. The description of an apparatus for isolating infective larvae from soil. Amer. Jour. Hyg., 2: 1-16. Otto, G. F. 1940 A serum antibody in dogs actively immunized against the hookworm, Ancylostoma caninum. Amer. Jour. Hyg., 31: Sec. D, 23-27. 1941 Further observations on the immunity induced in dogs by repeated infections with the hookworm, Ancylostoma caninum. Amer. Jour. Hyg., 33: Sec. D, 39-57. Otto, G. F. and K. B. Kerr 1939 The immunization of dogs against hookworm, Ancylostoma caninum, by subcutaneous injection of graded doses of living larvae. Amer. Jour. Hyg., 29: Sec. D, 25-45. Smillie, W. G. and D. L. Augustine 1926 Hookworm infestations. The effect of varying intensities on the physical condition of school children. Amer. Jour. Dis. Child., 31: 151-168. Stoll, N. R. 1923a Investigations on the control of hookworm disease. XV. An effective method of counting hookworm eggs in feces. Amer. Jour. Hyg., 3: 59-70. 1923b Investigations on the control of hookworm disease. XVIII. On the relation between the number of eggs found in human feces and the number of hookworms in the host. Amer. Jour. Hyg., 3:156-179. Taliaferro, W. H. 1940 The mechanism of acquired immunity in infections with parasitic worms. Physiol. Rev., 20: 469-492.