Studies in helminth enzymology. IV. Immune responses to malic dehydrogenase from Ascaris suum

EXPERIMENTAL

PARASITOLOGY

Studies

16,373-381

in

(1965)

Helminth

Responses

Enzymology. to

from Marvin

B.

Rhodes,

Department

of

MARVIN

Ascaris

suum’ W.

Kelley,

Jr.,

Veterinary Science, College of Agriculture, of Nebraska, Lincoln, Nebraska

(Submitted

for

B.,

DEBI

NAYAK,

Immune

Dehydrogenase

Dehi P. Nayak, George and Connell L. Marsh

University

RHODES,

Malic

IV.

publication, P.,

25 February

KELLEY,

GEORGE

1964) W.,

AND

MARSH,

CORNELL

L.

1965. Studies in helminth enzymology IV. Immune responses to malic dehydrogenase from Ascaris suum. Experimental Parasitology 16, 373-381. Purified malic dehydrogenase from Ascaris SZLWZ when injected into guinea pigs and swine stimulated the production of specific antibodies, which were demonstrated by enzyme inhibition and immunodiffusion analysis. Fractionation of swine serum on a cellulose ion exchange column resulted in the separation of the inhibiting and precipitating antibodies.Malic dehydro-

genases from closelyrelatedparasiteswereinhibited by the antiserumand gave crossreactions did not genase

in immunodiffusion analyses. However, malic dehydrogenases from swine heart react with the antiserum. Guinea pigs receiving injections of malic dehydrofrom A. suum did appearto receivesomeprotectionagainstmigratingA. suum

larvae. The use of various extracts of Ascaris suum as antigens to induce the formation of antibodies in experimental animals has been included in reviews by Fairbairn (1957), Kent (1963), and Soulsby (1962). However, the use of isolated native proteins from A. suum extracts as antigens appears to be limited to the report by Kent (1960) in which five protein fractions were partially characterized and evaluated for their respective antigenicity. One approach to the further understanding of A. sullrn is a study of the various enzymes of this parasite compared with the host (swine) enzymes. Several enzymes are currently being studied in our laboratory and 1 Published

with the approval of the Director as No. 1500, Journal Series, Nebraska ExperiStation. The investigation reported in this paperwas supported by U.S.P.H.S. grant E-1221.

the purification and partial characterization of malic dehydrogenase ( MDH)2 isozymes from A. suum were reported by Rhodes et al. (1964). As an extension of these studies, the use of specific enzymes of A. suum as immunizing antigens was initiated. The present report concerns primarily the use of MDH in these studies. MATERIALS

AND

METHODS

The preparation of extracts of A. mum and fractionation of MDH isozymes and trypsin inhibitor from these extracts was described by Rhodes et aZ. (1963, 1964). Extracts of all other worms used in this study z Abbreviations used: MDH, malic dehydrogenase; DEAE-cellulose, diethylaminoethylcellulose; NADH, nicatinamide adenosine dinucleotide, reduced form Tris, tris (hydroxymethyl) aminomethane.

Paper ment

373

;

374

RHODES,

NAYAK,

KELLEY,

were made by homogenizing the worms in 10 volumes (w/v) of 0.02 M tris (hydroxymethyl) aminomethane (Tris), pH 8.5. The homogenates were centrifuged at approximately 5,000 g for 15 minutes to remove coarse particles. Materials were treated in the following manner before injection into animals. Ascaris suwz body wall extract and perienteric fluid were dialyzed against phosphate buffered saline, pH 7.3, for 24 hours at 4°C. The MDH and trypsin inhibitor preparations were made 0.2 11/1in phosphate and titrated to pH 7.3 with NaOH. Each of the materials was mixed with an equal volume of complete Freund’s adjuvant.” The injected preparations contained the following concentration of protein expressed in mg per milliliter: body wall extract, 3.9; perienteric fluid, 16.5; MDH, 2.5: and trypsin inhibitor, 2.4. The MDH contained all three of the isozymes present in crude extracts of A. suum and had an activity of 43 units” per milligram. The trypsin inhibitor would inhibit approximately 2 mg of trypsin per milligram of inhibitor. Eggs were collected from living A. suunz. The eggs were decoated with sodium hypochlorite and incubated in 0.1 IV sulfuric acid at 30°C. The vessel containing the eggs was shaken during the incubation period of 20 days. Infectivity of the eggs was checked in mice before they were given to the test guinea pigs or swine. Young guinea pigs (200 to 400 gm weight) were randomly placed in treatment lots. Each animal in a treatment received one ml of one of the above preparations intramuscularly or 10,000 infective eggs subcutaneously on days 0, 10, and 20. Guinea pigs received no treatment in one lot in each experiment. The swine were obtained by hysterectomy, deprived of colostrum, and maintained in individual isoa Purchased from Difco Laboratories, Detroit 1, Michigan. 4 One unit is equal to one p mole of NADH oxidized per minute at 25°C in phosphate buffer, pH 7.5.

AND

MARSH

lation units by the method of Toung et al. (1955). Swine were 3 days and 30 days old, respectively, in the first and second experiments. Each animal in a treatment received a 5 ml injection of MDH intramuscularly or 10,000 infective eggs per OS on days 0, 10, and 20. Swine received no treatment in one lot in each experiment. All animals were challenged on day 35 with 50,000 infective eggs by gavage. Clinical reactions were observed for the next 8 days. All animals were anesthetized with sodium pentobarbital and exsanguinated on day 43. At necropsy, pathological changes in the livers and lungs were noted. The number of larvae in the livers and lungs were estimated by the method of Kelley et al. (1957). A representative number of larvae was measured with the aid of an ocular micrometer. Each animal was bled by cardiac puncture at weekly intervals throughout the experiments. Serum from this blood was used for serological studies. Serum obtained at day 42 was used in fractionation studies. Anti-MDH guinea pig serum and swine serum were fractionated on DEAE-cellulose. The guinea pig serum was equilibrated by dialysis with 0.0175 M sodium phosphate, pH 6.5. The exchange column was equilibrated with the same buffer. The serum was applied to the column and the unadsorbed proteins were eluted with the starting buffer. The remaining serum proteins were eluted from the exchanger by increasing concentrations of sodium phosphate buffer, pH 6.5, applied in a stepwise manner. Buffers were changed to 0.03 M, 0.1 ll4, and 0.5 M concentrations when the absorbancy of the eluate at 280 rn\t became less than 0.15. Swine serum was equilibrated by dialysis with 0.01 M sodium phosphate buffer, pH 7.5. The exchange column was equilibrated with the same buffer. The unadsorbed proteins were eluted from the column with the starting buffer. The remaining serum proteins were eluted from the exchanger by a sodium chloride gradient in 0.01 M phosphate. pH 7.5.

STUDIES

IN

HELMINTH

The gradient was produced through the use of a separatory funnel, and two mixing chambers connected in series to the exchange column. Initially, the separatory funnel contained 0.2 M NaCl. The mixer (250 ml) connected to the separatory funnel contained 0.1 2M NaCl, and the mixer (250 ml) attached to the column only contained 0.01 M phosphate. Stepwise changes were made in the contents of the separatory funnel at fraction 30 to 0.3 M NaCl and to 0.5 J4 NaCl at fraction 49. An automatic fraction collector was used and fractionations were conducted at 4’C. Estimation of protein in fractions were routinely obtained by measuring the absorbancy of the eluate at 280 ml1 with a Beckman model DB spectrophotometer. Swine serum fractions were concentrated approximately 10 times by dialysis against 15 s polyvinylpyrrolidone at 4°C. Paper and starch gel electrophoretic analyses; MDH and MDH inhibitor analyses; and immunodiffusion analyses were run on the concentrated fractions. Immunodiffusion, immunoelectrophoretic, and electrophoretic analyses in Ionagar No. 2j were performed on microscope slides (Smith, 1960). The agar was made 1% concentration in phosphate buffered saline for immunodiffusion analyses or in 0.050 r/2 Verona1 buffer, pH 8.6, for immunoelectrophoretic and electrophoretic analyses. Precipitin arcs were stained with a protein stain or for enzymatic activity (Uriel, 1963) using nitro blue tetrazolium as previously described for localization of MDH activity following electrophoresis (Rhodes et al., 1964). Electrophoresis in starch was done by the method of Marsh et al. (1964). Paper electrophoretic analyses were run on a Spinco Durham type cell in 0.075 r/2 Verona1 buffer, pH 8.6. A constant current of 4.5 ma were used for a period of 17 hours. Enzymatic analyses for MDH were performed essentially according to Mehler et al. (1948). The assays were performed at 25°C 5 Purchased from Consolidated Chicago Heights, Illinois.

Laboratories,

Inc.,

ENZYMOLOGY.

IV

375

with the use of a Beckman model DB spectrophotometer connected to a Sargent model SR recorder. The following solutions were pipetted into a cuvette in the order listed: 0.00-0.10 ml of diluted antiserum; 0.10 ml MDH (0.15 units); 0.10 ml NADH (1.5 mJf) ; and O.lC-0.00 ml 0.1 M phosphate buffer, pH 7.5. The cuvette was placed in the spectrophotometer and 3 ml of oxalacetate (2.5 m&f) in 0.1 2M phosphate buffer, pH 7.5, was added. Adequate mixing of solutions was accomplished by blowing the substrate-buffer solution into the cuvette using a fine-tipped pipette. Recordings at 340 rnp were made for 1 to 2 minutes. Determination of MDH inhibitor activity consisted of enzymatic assays of MDH activity of worm extracts; of serum; and of the mixture of worm extract and serum. In the latter case, worm extract, serum, and NADH were mixed and allowed to stand at 25°C for at least 2 minutes prior to adding the substrate-buffer solution to the cuvette and recording the results. Calculations were made according to the following equation: Units of worm MDH inhibited = (units of worm MDH + units of serum MDH) - units of mixture. RESULTS

Serological Reactions The results of preliminary assays indicated that inhibiting antibodies to MDH were present in the sera of guinea pigs receiving injections of MDH. As expected, the amount of inhibition measured was dependent on the length of time of pre-incubation of mixture of MDH, antiserum, and NADH before adding the substrate. The amount of inhibition increased rapidly during the first minute, but at 2 minutes was 85 to 90% of that measured at 20 minutes. Consequently a pre-incubation period of at least 2 minutes was used for measuring enzyme inhibition in this study. Other results indicated that the amount of enzyme inhibition was not entirely proportional to the amount of antiserum. A plot of the results of assays on mixtures, containing

376

RHODES,

NAYAK,

KELLEY,

a given amount of MDH (0.15 units) and varying amounts of antiserum, gave a curve that was linear to approximately SOY0 inhibition, Beyond this point the inhibition per unit of antiserum became progressively smaller. Only 8575 inhibition occurred when the amount of antiserum was increased to 20 times that required to cause half-inhibition. Thus for quantitative purposes, measurement of inhibition was restricted to using that amount of anti-serum that caused less than 50% inhibition of the MDH. Similar results were obtained with antiserum from swine with regard to rate of reaction of enzyme and antibody, and the type of inhibition curve. Figure 1 shows the increase in MDH inhibitor activity in guinea pig serum with time. Maximum inhibitor levels occurred between 30 and 35 days from the time of the first injection of MDH. The level of inhibitor activity was somewhat reduced at 42 days, which was 7 days following challenge with 50,000 infective eggs. The sera of guinea pigs that had received injections of infective eggs, trypsin inhibitor, body wall extract, perienteric fluid, or no injections were assayed for the presence of MDH inhibitor activity. No significant inhibition was found. Likewise with swine, only sera of animals receiving MDH contained inhibitor to MDH.

0

5

IO DAYS

FIG.

inhibition.

1.

Development of antibodies 0 Expt. 1. X Expt. 2.

15

AND

Multiple arcs were obtained in agar when purified MDH from A. suum was diffused against antiserum from guinea pigs (Fig. 2 (A) ) . Immunoelectrophoretic analyses of

FIG. 2. (A) Immunodiffusion of MDH in agar against guinea pig serum. MDH, center well; control sera, left; immune serum, 4 wells on the right. (B) Electrophoresis of MDH in agar. Stained with Ponceau S for protein. (C) Electrophoresis of MDH in agar. Stained for enzymatic activity with nitro-blue tetrazolium. (D) Immunoelectrophoresis of MDH and diffused against anti-MDH guinea pig serum. (E) Immunoelectrophoresis of MDH and diffused against anti-MDH swine serum, Electrophoretic separations on microscope slides with a current of 50 ma per 8 slides for 130 minutes.

20

AFTER

START

to MDH

from

MARSH

OF

25

30

35

40

EXPERIMENT

A. suum

in guinea

pig

serum

as measured

by enzyme

STUDIES

IN

HELMINTH

MDH in agar showed that the precipitin arcs formed by diffusing against antiserum from guinea pigs or swine fall in the same area as enzymatic activity and protein (Fig, 2 (B, C, D, and E)). No antibodies to MDH could be demonstrated by immunodiffusion analyses in sera from guinea pigs that had received injections of trypsin inhibitor, body wall extract, perienteric fluid, or infective eggs. However, precipitin arcs were obtained when antiserum to purified MDH was diffused against body wall extract or perienteric fluid. These extracts contain relatively high levels of MDH activity. When antiserum from guinea pigs was submitted to electrophoresis in agar and then diffused against MDH, precipitin arcs were observed in the gamma and beta-2 regions. Similar observations regarding the distribution of antibodies in sera of guinea pigs has been previously reported (Yagi et al., 1962). Extracts of several other parasites and a common earthworm (Eisenia foetida) were assayed for MDH activity and for inhibition by the antiserum against MDH from A. suum (Table I). Relatively high MDH activity was found in extracts of all the worms with the exception of Metastrongylus spp., Moniezia expansa, and E. foetida. As shown in Table I, approximately an equal number of units of MDH from A. sum, Toxocara cati, Toxocara canis, Ascaridia galli, and Ascaridia dissimilis were inhibited per milliliter of antisera. Only a relatively few units of MDH of the other worms were inhibited per milliliter of antisera indicating very little cross reaction outside of the ascarid group. Immunodiffusion analyses in agar yielded similar results. Only extracts of T. cati, T. canis, A. galli, and A. dissimilis gave cross reactions with the anti-MDH sera. MDH isozymes prepared from swine heart extract (Rhodes et al., 1964) were not inhibited by antiserum of guinea pig or swine origin, nor did they give cross reactions in immunodiffusion analyses in agar. Sera from guinea pigs injected with body

ENZYMOLOGY.

Inhibition

377

IV

TABLE I of MDH from Worms by Antisera to MDH from Ascaris suum MDH activity inhibited/ml of serum

MDH activity/

(units) b

ml extracta (units) b

Species Ascaris suum Toxocara cati Toxocara canis Ascaridia galli Ascaridia dissimilis Trichuris ovis Haemonchus contortus Metastrongylus spp. Moniezia expansa Eisenia foetida a Worms M Tris, pH b Bmole phosphate the assay. C Values

Guinea Pig

Swine

34 29 30 26

12c

12 12

9c 6 8 6

35 27

12 1.6

1.7

12

1.3 0.5 0.1 0.2

10

1 .o 2.3 1.6

1.5 0.3

0.4 0.2

extracted with 10 volumes (w/v) of 0.02 8.5. NADH oxidized per minute at 25°C in buffer, pH 7.5. See text for details of obtained

using

purified

MDH.

wall extracts or perienteric fluid gave precipitin arcs when diffused in agar against these extracts. The antigenic components in these extracts have not been identified. However, perienteric fluid does contain protein(s) that appears to be strongly antigenic. Partial purification of this protein has been achieved and further studies are in progress. The trypsin inhibitor showed only faint precipitin arcs when diffused against its antiserum from guinea pig. Fractionation

of Anti-MDH

Serum

At this point in the investigation, it was assumed that the precipitin antibodies were identical with the inhibiting antibodies. Preliminary fractionations of antiserum from guinea pigs on DEAE-cellulose showed that the initial unadsorbed peak contained inhibitor activity, but precipitin arcs in agar could not be demonstrated. Fractions eluted with 0.035 M and 0.1 M sodium phosphate, pH

378

RHODES,

NAYAK,

KELLEY,

AND

MARSH

5-84

2.5

1.5 5 z a

f a a5

E

0.1.03 a

‘i FIG. 3. Fractionation 1 ml per minute with at 280 mu of original

2'30

I

VOLUME 2’0

’ FRACTION

of MDH antiserum a 2.2 X 18 cm column fractions. l inhibitor

4’0

400 MLS ’ NUMBER

600 6’0

’

from swine on DEAE-cellulose. .4 flow rate of approximately used. Gradient described in text. Key to curves: 0 absorbance activity. Shaded area = Serum MDH activity.

6.5, contained both precipitating antibodies and inhibitory activity. A somewhat more extensive fractionation of MDH antiserum from swine was performed. Results are presented in Fig. 3. The initial peak contained primarily gamma globulin as shown by electrophoresis in starch and on paper. However, only a small portion of the inhibitor activity was found in this peak. The main peak of inhibitory activity was eluted with the beta globulins and somewhat ahead of the serum albumin which emerged from the column starting at fraction 37. The serum MDH was eluted under the sameconditions as the bulk of the MDH inhibitor. Some attempts have been made to separate the MDH inhibitor activity from the serum MDH but without success. Precipitin arcs were obtained in immunodiffusion analyseswith fractions o-15, but no arcs could be demonstrated with frac-

tions 28-36, which contained the maximum inhibitory activity. The precipitin arcs were stained for enzymatic activity after the agar had been properly dried and washed to remove the soluble, unprecipitated proteins. All arcs present were stained. Inhibition of ilscaris MDH by material from fractions 28-36 was linear to nearly 100% inhibition. Reactions of Animals to Challenge The results of the challenge dose of A. suz~n infective eggs in guinea pigs are presented in Table II. Respiratory distress occurred in all guinea pigs regardlessof treatment; however, it was more intense in some lots than in others. Thumping” occurred in 6 Thumping consists of rhythmic spasms of the diaphragm resulting from effects of migration of dscaris SUUWL, baby pig anemia, or some respiratory infection.

STUDIES

Larvae Experiment 1

2

3

in Lungs

of “Immunized”

IN

HELMINTH

ENZYMOLOGY.

TABLE II Pigs That Received

Guinea

50,000

Infective Mean

Treatment Control Eggs MDH Control Eggs MDH Trypsin inhibitor Control Em MDH Body wall extract Perienteric &id

Animal@ 2 2 2 4 3 3 2 3 1 3 4 4

Mean

number

20,500 7,200 9,800 23,600 5,900 10,600 17,300 8,200 2,500 10,600 7,000 7,100

of larvae

in lungs

(13,100-27,800)b (4,000-10,300) (8,000-11,600) (13,6OC-39,800) (3,40(x9,700) (9,700-12,200) (7,90&26,600) (6,00&12,300) (9,500-12,100) (4,8&10,600) (5.000-10.000)

379

IV

Ascaris

suum

length of larvaeb (mm)

.72 .55 58 .92 .62 .70 .44 .71 .62 .65 .73 .72

(.25-l.Ol)c (.24-.72) (.31X.76) (.48-1.34) (.25-.92) (.24-1.11) (.16-.80) (.34-1.20) (.30-.72) (.30-.90) (.34-1.02) (.26-1.20)

Eggs Mortality guinea

of pigs

100% 0 0 50% 0 0 100%

50% 0 67% 50% 75%

a Number of guinea pigs at time of challenge. b 25 larvae were measured in each treatment. C Range.

all controls but not in animals injected with eggs; it was slight in MDH-injected guinea pigs, but occurred with the same intensity as in the controls in all other treatments. Deaths occurred in each of the control lots but not in lots injected with eggs or MDH (except in Experiment 3 where the injections produced abscesses in each hind leg). Mortality in the trypsin inhibitor, body wall, and perienteric fluid-injected guinea pigs was similar to the controls. Severe pneumonia and hemorrhage occurred in the lungs of the controls. Egg-inoculated guinea pigs had no pneumonia and only a few petechiae in the lungs. Severity of the lung lesions of MDH-injected guinea pigs was between that of controls and egg-inoculated. Lesions in the lungs of animals in other lots were of the same severity as those observed in the lungs of the controls. Figure 4 shows the relative lung damage in guinea pigs injected with MDH and eggs. No larvae were found in the livers of any guinea pigs. Except for Experiment 3, more larvae were found in the lungs of the control animals than in any other lots. Animals that received eggs had the fewest larvae in their lungs, In Experiments 1 and 2 the guinea pigs that received MDH had a reduced number of larvae in their lungs. The guinea pigs

that had been injected with trypsin inhibitor, body wall extract, and perienteric fluid had a slightly lower number of larvae migrating than the controls (Table II). The length of larvae from the MDH-injected guinea pigs was consistently shorter than larvae from the controls. In the first experiment with swine, the one animal receiving MDH appeared to have some immunity against Ascaris larvae on challenge. The control animal and two animals receiving previous doses of infective eggs had more severe clinical symptoms and more larvae in the lungs. In the second experiment, the control swine (2 animals) had the least severe clinical symptoms and fewest number of larvae in the lungs. The 2 swine receiving MDH and one animal receiving previous doses of eggs showed no evidence of immunity. DISCUSSION

The results presented in this paper clearly demonstrate that purified MDH from adult A. suum when injected into guinea pigs or swine stimulates the production of specific antibodies. The antibodies in swine serum were of two types, namely, inhibiting and precipitating. Complete inhibition was not obtainable when whole antiserum was used, but it

380

RHODES,

NAYAK,

KELLEY,

AND

MARSH

FIG. 4. Lungs of guinea pigs in Experiment 1. Left, control: middle, immunized with eggs; right, immunized with MDH. Note the hemorrhagic pneumonia in control lungs (not immunized), while those immunized with MDH and embryonated eggs had relatively few localized hemorrhagic spots on lungs.

could be achieved with relatively small amounts of the isolated inhibiting antibodies. Such evidence indicates that the precipitating antibodies compete with the inhibiting antibodies for the MDH. However, further work needs to be done to characterize each type of antibody found in this study. The fact that these antibodies did not inhibit or precipitate the host (swine) heart MDH isozymes indicates that the MDH of the parasite and host animal are distinctly different. However, the antigenic portion must be similar in MDH from phylogentically related parasites. This was evident from the amount of inhibition of their MDH by the MDH-antiserum and the cross-reactions in immunodiff usion analyses. Staining of precipitin arcs in agar for enzymatic activity was reported by Uriel (1963) for a number of enzyme-antibody

systems, and it worked for enzyme-antibody system reported here. The multiple arcs obtained are probably due to antibodies to the different isozymes of MDH. Multiple precipitin arcs having enzymatic activity could also result from the presence of more than one kind of precipitating antibody. Evidence for the multiplicity of antibodies, including inhibiting and precipitating antibodies, was presented in a number of papers of the results of a conference edited by Cinader (1963). The lack of detectable antibodies to MDH in the serum of guinea pigs injected with crude extracts of A. suum or perienteric fluid emphasizes the need for using purified proteins or enzymes if specific antibodies are to be produced. Guinea pigs did appear to receive some

STUDIES

IN

HELMINTH

protection against A. suum larvae from injected MDH compared to the untreated guinea pigs or animals receiving injections of trypsin inhibitor or extracts. The protection was not as good as in animals receiving infective eggs as judged by respiratory distress, number of larvae in lungs, and condition of the lungs. Further experiments with swine will be necessary to ascertain the value of MDH from A. suum in protecting the animals against migrating larvae. Several enzymes of A. suum may be useful in immunizing swine against A. suum infections. Purification of other enzymes of A. suum is currently in progress in our laboratory. REFERENCES

B. enzymes-A the New 1154.

(Consulting Ed.) 1963. Antibody to three-component system. Annuls of York Academy of Sciences 103, 493-

CINADER,

D. Experimental

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A. of of 24,

G.

W.

1957. The biochemistry of Ascaris. Parasitology 6, 491-554. JR.,

OLSEN,

L.

S.,

AND

HUERLEIN,

B. 1957. Rate of migration and growth larval Ascaris suullz in baby pigs. Proceedings the Helminthological Society of Washington 133-136. KENT, H. N. 1960. Isolation of specific antigens from Ascaris lumbricoides (var. suum). Experimental Parasitology 10, 313-323. KENT, H. N. 1963. Seminar on immunity to para-

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IV

sitic helminths. V. Antigens. Experimental Parasitology 13, 45-56. MARSH, C. L., JOLLIFF, C. R., AND PAYNE, L. C. 1964. A rapid micromethod for starch-gel electrophoresis. American Journal Clinical Pathology 41, 217-223. MEHLER, A. H., KORNBERG, OCHOA, S. 1948. The

oxidation-reductions and pyruvate. The istry 174, 961-977.

A.,

GRISOLIA,

M. B., MARSH, C. L., AND KELLEY, Trypsin and chymotrypsin JR. 1963. itors from Ascaris suum. Experimental sitology 13, 266-272.

RHODES,

S.,

AND

enzymatic mechanism of between malate or isocitrate Journal of Biological ChemG. W. inhibPara-

B., MARSH, C. L., AND KELLEY, G. W. 1964. Studies on helminth enzymology III. Malic dehydrogenases from Ascaris suum. Experimental Parasitology 15, 403-409. SMITH, I. 1960. Chromatographic and electrophoretie techniques. Vol. 2. Wiley (Interscience), New York. SOULSBY, E. J. L. 1962. Antigen-antibody reactions in helminth infections. In “Advances in Immunology” (W. H. Taliaferro and J. H. Hymphrey, eds.), Vol. 2, pp. 265-308. Academic Press, New York. URIEL, J. 1963. Characterization of enzymes in specific immune-precipitates. Annals of the Xew York Academy of Sciences 103, 956-979. YAGI, Y., MAIER, P., AND PRESSMAN, D. 1962. Two different anti-insulin antibodies in guinea pig antisera. Journal of Immunology 89, 442451. YOUNG, G. A., UNDERDAHL, N. R., AND HINZ, R. W. 1955. Procurement of baby pigs by hysterectomy. Journal American Veterinary Medical Association 58, 123-131. RHODES,

JR.

M.