'ww-r 7* ———————v— THE INFLUENCE OF SOME NUTRITIONAL FACTORS ON EXPERWENTAL HOG-CHOLBRA INFECTION Thais Ger the Doom bf M. S. MICHIGAN STATE WIVERSITY Glenn L. Waxlar 1959 rH' ' ' L 13 R :1 R Y Mmhigan Sta t6 University "7/ ’/ ’ J 7 {:7 {/9 7 THE INFLUENCE OF SOLE NUTRITIONAL FACTORS ON EXPERIMENTAL HOG-CHOLERA INFECTION BY Glenn L. waxler AN ABSTRACT Submitted to the College of Veterinary Medicine of Michigan.State University of Agriculture and Applied.Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Veterinary Pathology 1959 Approved by Glenn L. Waxler ABSTRACT A series of experiments was conducted in an effort to determine possible relationships between some nutritional factors and eXperimental hog-cholera infection in baby pigs. Ascorbic acid was administered to groups of animals in doses ranging from 25 to 200 mg. per pound body weight per day. This was ad- ministered by the intraperitoneal, subcutaneous, or oral routes. In 1 exp periment the ascorbic acid appeared to lengthen the survival time of the infected pigs somewhat. Other evidences of beneficial effects were not noted. 0f the 19 pigs given ascorbic acid, only 1 survived, and it is probable that its survival was due to immunity Obtained from ingestion of colostrum. The effects of a tryptOphan-deficient diet on the symptoms, course, and lesions of experimental hog-cholera infection in baby pigs were also investigated. The deficiency delayed the onset of symptoms and time of death in 1 group of animals, and in 2 groups the deficiency caused a slight decrease in the severity of the gross lesions. The administration of tryptazan, a tryptOphan analogue, to a pig inoculated with hog-cholera virus was accompanied by a recovery from the infection. The results were inconclusive, however, due to the possi- bility that the animal had obtained some immunity to the hog-cholera virus from having nursed its dam during the first few days of life. ACKNWNTS The author wishes to express his sincere appre— ciation to Dr. G. K. Whitehair, Dr. C. C. Morrill, and Dr. R. F. Langham for their consideration, encouragement, and guidance throughout the study. I. II. III. INTRODUCTION TABLE OF CONTENTS LITERATURE REVIEW . . . . . . . A. The Role of Ascorbic Acid in Infection B. The Effect of Protein and Tryptophan.Deficiency on Infection . . . . . . . . . . . . . . . . . . . . . . C. The Effect of TryptOphan.Analogues on Infection . . . EXPERIMENTAL . . . . . . . . . . . . . . . . . . . . . . A. The Effect of Ascorbic Acid Infection . . . . . . . . . l. 2. 3. Experiment I . . . . . a. b. 0. Procedure . . . . . Results . . . . . . Discussion . . . . Experiment II . . . . . a. b. C. Procedure . . . . . Results . . . . . . Discussion . . . . Experiment III . . . . a. b. 0. Procedure . . . . . Results . . . . . . Discussion . . . . on.Experimental Hog-Cholera la 11 13 13 13 13 17 18 19 19 22 27 28 28 3o 35 I'l’ltlllilli IV. V. VI. B. The Effect of Protein and Tryptophan Deficiency on Hog-Cholera Infection . . . . . . . 1. Experiment IV . a. Procedure . b. Results . . c. Discussion 2. Experiment V . a. Procedure . b. Results . . c. Discussion C. The Effect of a Tryptophan.Analogue Infection . . . . . 1. Experiment VI . a. Procedure . b. Results . . c. Discussion GENERAL DISCUSSION . . SUMMARY AND CONCLUSIONS REFERENCES . . . . . . on.Hog-Cholera 37 37 37 39 h2 143 AB AS 50 51 51 51 52 52 53 SS 56 I. INTRODUCTION Two factors that play important roles in the life of an animal are nutrition and infection. If the body is to be maintained in a state of good health, it is essential that the dietary intake meet all the nutritional requirements for the Species involved, and it is likewise essential that the individual be at least relatively free from infectious diseases. Not only are these two factors important in themselves, but the interactions of one with the other are also of significance. The be- lief is commonly held that an individual on a high plane of nutrition is more resistant to many of the infectious diseases than an individual whose diet is not adequate in all the essential nutrients. There is some evi- dence, however, that this relationship does not always hold true and that some nutritional deficiencies actually increase resistance to infection. In this study an attempt has been made to investigate certain aspects of the relationship between nutrition and experimental hog-cholera infection in the baby pig. A few of the reasons for choosing the pig as an experimental animal follow. The baby pig is readily available at a reasonable cost. Also, the fact that the baby pig is without circulating antibodies at birth and that these antibodies are obtained from the dam.only through the colostrum, makes this species desirable. By Obtaining colostrumw deprived pigs, one is assured of having animals which have not been inp fluenced by the damfls immunity and areihnrefore relatively uniformly sus- ceptible to infection with certain agents. The pig is, of course, large enough that gross clinical and pathological Observations may be made and tissue samples may be readily obtained. -2- The hog-cholera virus was used as the infectious agent in this work because of its known pathogenicity for the non-immunized pig. Al- most without exception, inoculation of non-immunized pigs with this virus causes death. The incubation period, clinical symptoms, and gross and microscOpic lesions have been well described and are fairly uniform. The objectives of this study were as follows: 1. To determine the effects 93 administration 21; large doses _o_f_ ascorbic 9.2.3 to gigs experimentally infected with the hog-cholera 11313. The role of ascorbic acid (vitamin C) in infection has been in- vestigated by a number of workers. Numerous laboratory investigations have demonstrated the ability of this vitamin to neutralize certain toxins and viruses. Although the results have not been highly consistent, some beneficial effects have been noted from the administration of ascorbic acid to animals experimentally infected with pathogenic viruses. There have also been reports of the successful clinical use of ascorbic acid in human infections. Since the hog-cholera virus produces lesions closely associated with the cardiovascular system in a number of tissues, and since ascorbic acid is considered to be beneficial in maintaining the integrity of the capillary wall, it seemed worth-while to investigate this relationship. 2. To determine the effect of protein and tryptflxan deficiency 23 hog-cholera infection. Experimentally, it has been demonstrated that certain deficiencies tend to increase resistance to infection. This ex— perimental work has been done with animals on low levels of food intake or on diets deficient in protein, certain of the B vitamins, or certain of the amino acids. In most of the experiments, the infectious agent was a virus of known pathogenicity for the species involved. In view of the -3- results reported in the literature, it seemed advisable to determine if the resistance of the baby pig to experimental hog-cholera infection could be increased by feeding a diet deficient in tryptOphan. This amino acid deficiency has been used in a number of the experiments alluded to pre- viously. 3. 1‘2 determine 111.3. M of administration 9_f_ g tryptophan analogue tg_an_animal experimentally infected with hog-cholera virus. It has been fairly well established that viruses are high in nucleopro- teins. The multiplication and growth of a virus in a host animal would be dependent on the synthesis of these specific proteins. The use of an Ianalogue of one of the essential amino acids might serve to ”block” or interfere with the synthesis of these nucleoproteins and thereby interfere with the growth and multiplication of the virus. For this purpose it is essential that the analogue be non-toxic to the host animal and that it be relatively soluble. A.sma11 amount of an analogue of tryptaphan that met these requirements was made available for use in this study. -h- II. LITERATURE REVIEW The interest which has been shown in the possible relationship between nutritional factors and resistance to infection is indicated by the large amount of material which has been published on various aspects of the subject. In preparing this literature review, only those refer- ences which seemed pertinent have been cited. Although much has been published on this subject, it was felt that a great deal of the material, for one reason or another, was not in line with the Objectives of this study as listed previously; Also it became apparent in reviewing the literature that some of the results, especially those based on clinical observations in the field of human medicine, were of a rather indefinite nature and were not derived by use of well—controlled experiments. A. The R_olg g Ascorbic 532E in Infection It is known that most mammals require no dietary source of as- corbic acid (vitamin.C). A series of three enzymes or enzyme systems is involved in the biosynthesis of leascorbic acid from d—glucuronic acid in animal tissues. In those mammals requiring ascorbic acid in the diet - man, monkey, and the guinea pig - enzymes catalyzing the first two reactions are present, but the last reaction cannot be carried out (18). Numerous references are made to the possibility of ascorbic acid affecting the resistance of an organism to infection (3). A number of the functions of ascorbic acid have been pointed out in the literature as related to the defense mechanism of the body. One of the functions of ascorbic acid in the body is related to its high concentration in the adrenal cortex. Although the exact -5- relationship has not been determined, it is postulated that the vitamin is important in the activity of this gland. It has been shown that the ascorbic acid concentration in the adrenal falls markedly when adrenal corticotrOphic hormone is injected into rats (1h). At the same time there is an increase in the amount of ascorbic acid in the adrenal vein (h8). For this reason ascorbic acid may play a part in stress conditions such as those brought about by some of the infectious diseases. Madison and Manwaring (32) report that the injection of rabbits with.1OO mg. of ascorbic acid along with 0.5 ml. of horse serum causes the production of a precipitin titer in the blood which is several times as high as that found in rabbits injected only with the horse serum. Ecker 23 31. (12) have shown that a correlation exists between the con- centration of ascorbic acid in the serum of guinea pigs and the comple- menting activity of the sermn. Ames and.Nungester (l) have shown that the number of exudative polymorphonuclear cells showing phagocytic activity is increased as the concentration of ascorbic acid in the cells is increased. Massa (3h) states that the respiration of leucocytes is increased by the presence of ascorbic acid. Ascorbic acid is also important in the body's defense mechanism because it is necessary for the formation of intercellular substances such as collagen, osteoid, and dentine (36). Roskin (h6) reports that the endothelial cells of capillaries are relatively rich in ascorbic acid. Sokoloff (50) states that one of the most important features of viral in. flammation is the capillary syndrome. The endothelial cells are invaded by the viral particles, and this results in damage to the capillaries. Generalization of the viral infection is enhanced and the inflammatory -6- process intensified. Polozhintseva (ho) mentions that the lowered capil- lary resistance can be demonstrated by means of negative pressure applied to the skin of children during the eruption of measles rash. In 1935 Jungeblut and Zwemer (26), at Columbia University, re- ported the in 11232 inactivation of diphtheria toxin by ascorbic acid. They also fbund that the vitamin helped protect guinea pigs against the fatal outcome of diphtheria intoxication to the extent that about 50 per cent of the animals survived when given 2 M.L.D. of toxin and 5 to 200 mg. of ascorbic acid simultaneously. Jungeblut (2h) preposed that the inp activation of diphtheria toxin.by ascorbic acid occurs as the result of direct interaction by the two substances. He found that this inactivation does not follow the laws of multiple preportions and is limited to rela- tively small doses of toxin and ascorbic acid. He also suggested that the toxin is inactivated during the auto-oxidation of ascorbic acid and that the substance responsible is probably a peroxide. In further work Jungeblut (23) also Observed the inactivation of tetanus toxin in 23352 by ascorbic acid. This is not due to an acid effect, since it occurs when the ascorbic acid has been adjusted to a.pR at which the potency of the toxin is undiminished. Here again, the inactivation is limited to a rather definite quantitative range of toxin and.vitamin. Campillo (8) found that ascorbic acid is effective in the neup tralization of influenza.A and influenza B viruses 32:322529 but it seems to be lacking in either prOphylactic or therapeutic values when used;§g ‘zizg in.mice inoculated with such viruses. He attributed this failure to the presence of catalases in the mouse body which neutralize the normal viricidal powers of ascorbic acid. .-7- Holden and Molloy (20) demonstrated the inactivation of herpes virus by ascorbic acid in 33.32 but not _in_ 33.32. They found that an ex- cess of ascorbic acid was toxic when injected intracerebrally in rabbits and that 20 mg. caused death in a few hours. In 1935 Jungeblut (22) reported the inactivation of poliOmye- litis virus by ascorbic acid when the two were mixed before being injected into monkeys by the intracerebral route. Using 0.1 ml. of the supernatant of a centrifuged 10 per cent poliomyelitis suspension, he found the effec- tive range of the vitamin to fall between S and 10 mg., although there was same protection above and'below this point. In 1937 the same author (25) reported a decrease in the incidence of paralysis when doses of as- corbic acid ranging from 5 to 50 mg. per day were given to monkeys inp fected with the poliomyelitis virus. Prophylactic administration of the 'vitamin failed to protect against subsequent intracerebral infection. Numerous reports have appeared in the literature relative to the clinical use of ascorbic acid in human medicine. McCormick (35) states that there is an unusually broad spectrum of antibiotic activity in ascorbic—acid therapy and that this activity includes all bacterial and viral infections. Hanwaring (33) noted that doses of 1 g. of ascor- bic acid appear to be of marked help in the treatment of the common cold. Brown and associates (7) indicate that ascorbic acid is helpful in checking the further development of colds under certain conditions when given in two l-g. doses 2h hours apart. Klenner (30) has reported quite favorable clinical results in treating such diseases as diphtheria, poliomyelitis, herpes zoster, herpes simplex, chickenpox, influenza, viral encephalitis, measles, mumps, and viral pneumonia with massive doses of vitamin C. He used 1 to 2 g. of the vitamin intravenously or -3- intramuscularly every 2 to h hours and gradually increased the time in- terval with the subsiding of the infection as evidenced by return of the body temperature to normal and disappearance of symptoms. In a later re- port (29) he indicated that the usual dose of vitamin C is 65 mg. per kg. of body weight every 2 to h hours but that under certain conditions a satisfactory response was obtained by giving 250 mg. per kg. of body weight as a single intramuscular dose. In some cases it was necessary to repeat with half this amount 8 hours later. The vitamin was given in a concentration of 500 mg. per ml. of solution. Klenner refers to vita- min C as a ”super-antibiotic" and attributes some of the reported failures of ascorbic acid therapy to inadequate amounts of the vitamin. Egan (13) obtained beneficial effects from the use of ascorbic acid in combination with sulfonamides in the treatment of 6.Afghan hounds affected with chorea resulting from distemper. The daily dose was six loo-mg. tablets per animal. Recovery occurred in 2‘to 6 months. A recent report (h) indicates that the citrus fruit flavonoids, commonly found associated with ascorbic acid in natural sources of the ‘vitamin, play an important role in reSistance to such infections as the common cold, acute follicular tonsillitis, and influenza. The effective- ness of the flavonoids is thought to be related to their ability to re- store to normal the increased permeability and fragility of capillaries. Boines (5) observed abnormal capillary fragility in severe, acute polio- myelitis. The capillary integrity was improved by the daily administration of 600 mg. each of hesperidin (a flavonoid) and ascorbic acid in divided doses. In 80 per cent of the cases improvement occurred in an average of 5 weeks. An improvement in the patient's sense of'wellpbeing and an inp creased appetite were noted within 1 week. B. 1133 Effect of Protein and Tqutophan Deficiency 3 Infection The belief has been expressed that any deficiency in nutrition, 'whether it be of a quantitative or qualitative nature, is invariably ac- companied by increased susceptibility to infection (31). The increased incidence of tuberculosis in the central European countries following Ibrld‘War I has also been attributed, at least in part, to the severe food restrictions imposed upon the pepulation (10). This increased sus- ceptibility may be due to the fact that protein depletion interferes with antibody production (2). There has, however, been considerable evidence to support the idea that deficiencies do not always cause increased susceptibility, but, on the other hand, sometimes decrease susceptibility to disease. This has been especially true with diseases ovairal origin. Rous (h7), working with the experimental transmission of an avian tumor by cell-free filtrate, made the observation that the developing tumor nodule showed retrogression when the host became sick. If the health of the experi- mental animal returned, the tumor frequently reappeared and grew rapidly. Olitsky 23 51. (37) made similar observations in work with the foot-and, mouth disease virus. They found that guinea pigs suffering from.ma1nup trition were more resistant to infection when inoculated with the virus than were normal animals. Sprunt (51) found that simple undernourishment decreased the susceptibility of the rabbit's skin to infection with vaccinmavirus and theorized that poorly neurished cells may be lacking in some of the materials necessary fer the formation of new viral parti- cles. He also found (52) that, when well-nourished animals are placed on a poor diet, they become first more susceptible to viral infection, then -10- less, and terminally more susceptible again. Foster gt 5;. (15) report that the feeding of about to per cent of the usual daily consumption definitely extended the time before the onset of paralysis and the time of death in mice infected with the Lansing strain of poliomyelitis virus. However, the difference between the deficient and control mice had disappeared by the end of 28 days. Several workers (16 ,17 , 27 ,h3) report that thiamine-deficient mice are less susceptible to infection with the Lansing strain of polio- nwelitis virus, Theiler's virus, and Western-equine-encephalomyelitis virus. Lowering of the incidence of paralysis, lower mortality rate, and lack of the characteristic signs of infection were noted. Sprunt and Sands (53) found that chickens, kept on an adequate diet for weight gain but moderately reduced in protein for 3 to 6 weeks, deve10ped more tumors when inoculated with the Rous sarcoma virus than did those on a diet adequate in protein. If, however, the chickens were kept on the diet for a longer period of time, they developed statistically fewer tumors than did the controls. Jones at 51. (21) reported. that pro- tein deficiency delayed the onset of symptoms when mice were inoculated with poliomyelitis virus. Kearney 9t gl_. (33), on the other hand, have reported that a low—protein diet has no influence on the infection of mice with Theiler's encephalomyelitis virus. Jones and co—workers at Pennsylvania (21) inoculated tryptophan- deficient mice with poliomyelitis virus and noted that symptoms were de- layed to a greater extent than in the case of protein deficiency. Pond 23': E. (141) reported that a deficiency of any one of the amino acids, tryptophan, isoleucine, methionine, and valine reduced the incidence of paralysis and other signs of infection in mice inoculated with .11- Theiler's-GDAVII-encephalomyelitis virus. They also found that trypto- phan deficiency caused a slower multiplication of the virus in the brain and spinal cord. Davies and his group (11) found that deficiencies of all the amino acids except lysine affected the course of experimental poliomyelitis in mice in similar fashion. There was an increase in the incubation period, a decrease in the number of animals paralyzed.at the end of 28 days, and an increase in the number of animals which died.with~ out characteristic signs of disease. 0. The Effect g£_Tryptophan.Analogues on Infection It has been demonstrated that tryptOphan is required for viral multiplication and that the addition of Semethyl tryptophan inhibits viral growth in the Escherichia coliATZ bacteriOphage system. This analogue apparently interferes with viral utilization of the tryptOphan produced by the bacteria. Cohen and Fowler (9) report that this inhibition is overcome by the addition of tryptophan to the system. Rasmussen 23 El. (h2) found that the inclusion of 0.h per cent 6—methyl tryptOphan in the diet of tryptOphan-deficient mice increased the time of survival in mice infected 'with.Lansing-poliomyelitis virus. The control animals lived 8.3 days while those treated with 6-methyl tryptophan lived 12.5 days. They attri— buted this increase to either an immediate requirement for tryptophan by the virus or to a manifestation of a more profound change in protein metabolism. The same group of workers (hh) found that the addition of the analogue to a lowbtryptophan diet influenced the incidence of paralysis in monkeys infected orally with poliomyelitis virus. In one experiment 7 of 9 monkeys receiving 6emethyl tryptOphan.became paralyzed, while 9 of 9 controls became paralyzed. Using a smaller amount of virus as the -12... infecting dose, it was found that l of 20 in the treated group showed paralysis while 12 of 20 in the control group became paralyzed. It is evident from this literature review that certain nutrients and.metabolites have an effect on the course, symptoms, and lesions of certain infections. It is also evident that the data on this subject are in conflict. This is probably due, at least in part, to the wide variety of factors involved in experiments of this nature, such as susceptibility of the host to infection, virulence of the pathogen, lesions produced, and the specific nutrient under study. III. EXPERIMENTAL The work for this study was conducted in the facilities .of the Department of Veterinary Pathology. The animals were kept in semi- isolation, entrance to the room being made through a small anteroom. A pan containing an antiseptic solution was placed on the floor of this small room for disinfecting footwear. The main room was provided with an exhaust fan, and air entered the room only through or around the doors. The baby pigs, after being delivered by caesarian section or removed from the dam at l to 3 days of age were brought to the room and placed in individual metabolism cages (Figure 1). Each cage was provided with a floor of one-half inch wire mesh, and a sheet-metal pan below this floor drained liquid waste material into a glass jar. Additional heat was provided for the baby pigs during the first few days of life by sus- pending a ZSO-watt heat lamp over 2 adjacent cages. A. _T_h_e_ _E_f_'£_e_g_t of Ascorbic Acid an Experimental Hog-Cholera Infection 1. Experiment I a. Procedure. This experiment, involving only 2 animals, was conducted as a preliminary investigation to observe the effects of high doses of ascorbic acid on baby pigs. The 2 animals were obtained from a registered Yorkshire herd. One pig was from the fourth litter farrowed by a sow which had been immunized against hOg cholera at weaning age. This pig was removed from the dam at 3 days of age and placed in an in- dividual metabolism cage. It was fed a daily ration of 200 ml. of pasteurized, homogenized, whole milk with 1 egg yolk added. The ration was divided into h feedings. After 13 days the diet was changed to . n v 1" r ‘ - .. f . . ’9 f , ' O 't n o - < ‘v n . 1 . ‘4 . |. n , x v .v r-v - 7 I _.. -1 ~- ! \' .p,\ _...- .mqm5.5u* 11.92 3.83 100 mg. 0.82 >s.su* - - Control 1.2h 1.28 0.80 0.h6 Uninoculated 1.15 1.79 1.02 1.15 * 5.5h represents the upper limits of this deter- mination. These samples contained.more than 5.5h mg. per 100 ml. animals receiving ascorbic acid but not in those inoculated only with the virus. The lesions of peritonitis appeared to be more severe in the ani- mals receiving 50 mg. ascorbic acid per pound body weight than in the .25—mg. group. Gross lesions of hog cholera were seen during necropsy 0f the animals receiving only virus and in the groups receiving 25 mg. and 50 mg. of ascorbic acid. The changes observed in the lymph nodes included edema, congestion, and peripheral hemorrhage. These lesions were seen.most fre— quently in the internal iliac and the prefemoral lymph nodes. Petechial hemorrhages were seen on the surface of the kidneys of all pigs. The spleens of 6 of the 9 pigs contained lesions varying from slight enlarge- ment to numerous infarcts. Lesions of enteritis or gastroenteritis were seen in all but 1 pig. Ulcers around the ileo-cecal valve were found -27- quite consistently. Grossly, the lungs of some pigs were normal while others exhibited a moderate degree of pneumonia. There was an indication that the lesions of hog cholera were slightly'more severe in the pigs receiving only virus than in those re- ceiving ascorbic acid, but the differences were not great and were con- fined to the lymph nodes and kidneys. c. Discussion. The slightly decreased food consumption of the animals receiving virus and ascorbic acid as compared to those receiving only the virus was probably due to the developing peritonitis. The lens kocyte counts and body temperatures did not differ greatly in the various inoculated groups. The plasma ascorbic acid values were quite high in the animals receiving injections of the vitamin. According to Grummer 32 El. (19) the level of plasma ascorbic acid for young pigs up to weaning age is about 0.8 mg. per 100 ml. The control pigs and the uninoculated animal had ascorbic acid levels above this figure before the onset of hog-cholera symptoms. These differences were not great when one considers the fact that a variation of one point on the transmittance scale of the colorimeter used in determining ascorbic acid represents a difference of more than 0.1 mg. per 100 ml. and that the needle sometimes fluctuates l or 2 points as readings are being made. The fact that plasma ascorbic acid levels of more than 7 times the normal values were obtained in both the 50— and 100hmg. level pigs with no significant differences being produced in either the symptoms or lesions of hog cholera indicates that the ascorbic acid was of no value in altering the outcome of the infection. However, the complications produced by the peritonitis in all animals receiving -28- ascorbic acid cannot be disregarded. The deaths of the lOO-mg. pigs were apparently due primarily to peritonitis rather than to hog—cholera in- fection. 3. Experiment III a. Procedure. Twelve pigs were used in this experiment to con- tinue the study of the effects of administration of ascorbic acid on hog- cholera infection. Six of the animals were purchased at about 10 days of age and 6 at 7 days of age. They were from 2 Hampshire gilts bred to a Yorkshire boar. Neither of the dams had previously been immunized against hog cholera. After they were removed from the sows, these pigs were placed in individual cages and were fed pasteurized, homogenized, whole milk with 1 egg added per quart. The amount fed was gradually increased from 50 ml. 1; times a day to 200 ml. 3 times a day. One iron-copper-cobalt tablet (FeCuCo, Ft. Dodge) was given each pig at approximately 2 weeks of age. When the pigs were 18 and 20 days of age, they were divided into 5 lots, with animals of both age groups being represented in each lot. Two of the animals were removed from the group and placed in a large stall in another part of the building where they were cared for by a separate attendant. The remaining 10 animals were inoculated with the titrated virus used in Experiment II (serial no. 313). Each animal was given 0.8 ml. of the virus subcutaneously in the axillary space. Using the original titra- tion values, each animal received 600,000 infective doses. The actual dosage, however, may have been somewhat less than this, considering the fact that the virus had been thawed for use in Experiment II and then re- frozen. The animals were treated as indicated in Table VII. -29- TABLE VII Treatments Applied to Pigs in Experiment III Group No. of Pigs Treatment 50 mg. 2 Virus plus 50 mg. ascorbic acid* 100 mg. 3 Virus plus 100 mg. ascorbic acid* 200 mg. 3 Virus plus 200 mg. ascorbic acid** Control 2 Virus only - Uninoculated 2 Isolated, no treatment *.Aecorbic acid values are mg. per pound body weight per day. The dose was divided into 3 equal parts and was administered subcutaneously in the axillary space every 8 hours. ‘** 200 mg. ascorbic acid per pound body weight per day divided into 3 equal parts and mixed with the ration at 8—hour intervals. The treatments as indicated were started at the time of inocup lation with the virus and were continued on each individual animal until the time of death. .An injectable product (Ascorbic Acid and Sodium Ascorbate, JensenFSalsbery'Laboratories, Inc.) was used for subcutaneous administration. The concentration of this product is such that each 25 ml. of solution represents 2 g. of crystalline ascorbic acid. The pro- duct used for oral administration was prepared by dissolving crystalline ascorbic acid (merck) in distilled water so that the concentration was equal to that of the injectable compound (2 g. per 25 ml.). \_,) u u m... l - . a n n e , v r . v I u A . . ‘ ‘nl e ‘a wt ‘. -, n I -30. Records of body weight, body temperature, food consumption, and white blood cell counts were made at selected intervals. Ascorbic acid determinations were made on the plasma every 2 to h days. Necropsies 'were perfonned on the animals at the time of death. Blocks of tissues were fixed in Zenker's fluid, except for the brain which was fixed in 10 per cent neutral formalin. These materials were imbedded in paraffin, sectioned, and stained with Harris's alum hematoxylin and.eosin. b. Results. The food consumption began to drOp in the younger group of animals on the second day following inoculation, but in the older group the decrease was not enough to be considered significant until the fourth day. The amount consumed was quite variable, but most animals were taking 50 per cent or less of the normal amount by the seventh day. Symptoms suggestive of infection.were first noticed on the third day in the younger group. These included diarrhea, lethargy, weakness, and some trembling. Similar symptoms in the older animals were observed 1 or 2 days later. Convulsions were seen in S of the animals at some time during the course of the infection. This condition was seen in all the groups inoculated with the virus. The animals gradually became weaker but did.not show the extreme weakness observed in those of Experiment II. The leukocyte counts, body temperatures, and plasma ascorbic acid values are presented in Tables VIII, IX, and.X, reSpectively. The values in the tables are averages of the values obtained for the number of animals in that particular group on any given day; As in Experiment II the total leukocyte counts and body temperatures are similar to what might be expected when susceptible pigs are inoculated with hog-cholera virus. 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M. m. w. um” m. m. m. m. .mu coaumasoocH enema when a.aov HHH unmeasmaxm an mmfim mo nonmemnmaame hpom NH mqmda Umpmadoocfimb Homecoo .wa 00m .wEVOOH .ma om empmasoocaee Homecoo .9: 08 .mg 02 .m3 om mmmmm. -32- 00.0 Hm.0 04.0 Hm.0 uu nu ma.0 H0.0 0~.H ~m.m bb.a m~.~ uu uu 0:.“ am.w un uu 0m.a ~0.~ Hm mm. mm. _mm cowpmfisooaH menus when mw.o H0.0 HN.N po.m ~m.~ m. m~.o m~.o m4.~ Ho.m mo.~ \OI AwEmmHm .He OOH pom .wsv a0.0 a0.0 N0.H m~.H H:.H ml 0m.0 04.0 0~.0 00.0 am.0 H' HHH HCGEHHQMNH CH mWHm HO w0§Hm> UHO4 OHQHOOm< dEdem N mamde UmpmamooCch Homvmoo .me oom .wa 00H .ma om a -3 3- sixth days after injection of the virus. One apparent discrepancy was noted in the results obtained on the sixth day following inoculation. At this time the average leukocyte count of the uninoculated animals was well below that of the control group receiving only the virus. It is difficult to account for this discrepancy other than by assuming that the counts were in error. 0f the h animals involved in.these 2 groups, only 1 had a relatively high count (19,000) at this time. It is possible that the accidental shifting of this count from the uninoculated group to the control group may have caused the error. Body temperatures rose between the second and.fourth days and in most cases remained above normal until the death of the animal. Table I reveals that the plasma ascorbic acid values in this experiment were not as high as those obtained in Experiment II. Gross lesions suggestive of hog cholera were seen upon necropsy of all the animals inoculated with the hog-cholera virus. These lesions included edema, congestion, and hemorrhage of the lymph nodes; congestion, petechial hemorrhage, and ecchymotic hemorrhage on the surface of the kidney; infarcts involving the edge of the spleen; enteritis or gastro- enteritis of varying extent and degree; and varying degrees of congestion and pneumonia in the lungs. Severe submeningeal hemorrhage was present in the area of the cerebellum of 1 animal in the 506mg. group. The lesions were quite variable, but there appeared to be no correlation between the extent and severity of the lesions and the level of ascorbic acid administered to the individual animals. There was more variation within groups than.between groups. Microscopic examination of sections of lymph nodes, kidneys, and four regions of the brain (medulla oblongata, brain stem at the level -3h- of the cerebral peduncles, cerebellum, and cerebrum) revealed numerous lesions suggestive of hog cholera. In the lymph nodes the changes were predominantly those of edema, congestion, and hemmorrhage, most of the lesions being confined to the periphery of the nodes. However, in a few 2 cases, the lesions were spread rather uniformly throughout the nodes. The kidney lesions were most prominent in the area of the cortex adjacent to the capsule. Congestion, varying from slight to marked, was seen both in the glomeruli and in the vessels supplying the tubules. Hemorrhagee were also found in this area, some of them being just under the capsule, and in others the blood had escaped into the area between the tubules. Microscopically, congestion was a more prominent feature than hemorrhage, and apparently many of the lesions appearing grossly to be petechial hemorrhages were actually areas of congestion. The microscopic lesions found in the brain included both peri- vascular cuffing and swelling and proliferation of the endothelial cells. These changes were most commonly found in the sections of the medulla and the sections of the anterior brain stem taken at the level of the cerebral peduncles. However, in a few animals, the lesions were also seen in sec- tions of the cerebrum and cerebellum. The perivascular cuffing consisted of the collection of lymphocytes within the VirchowaRobin spaces. This cuffing varied from a condition in which only a few lymphocytes were seen to the presence in 1 animal of great numbers of lymphocytes. The animal in which extensive cuffing was found was from the lOObmg. group. A more consistent lesion was that involving the endothelial cells themselves. These cells exhibited swelling, and in some cases there appeared to be an increase in the numbers of the endothelial cells. Other microsc0pic lesions such as varying degrees of vacuolation, apparent degeneration of -35.. Purkinje fibers in the cerebellum, and extensive hemprrhage in the care- bellum of one animal in the SO—mg. group were also found. These changes 'were not, however, consistent enough to be considered important. Gross lesions in the uninoculated animals were negligible. Microscopically, however, there were areas of congestion in the kidneys, and slight congestion and hemorrhage in the internal iliac lymph nodes of both pigs. The level of ascorbic acid administered appeared to have some influence on the length of survival after inoculation with the virus. As the amount of the vitamin was increased, the survival time in both litters was increased, with the exception of one animal in the younger litter (Table XI). TABLE XI Survival Time of Pigs in Experiment III after Inoculation Survival Time (Days) ‘ggggp Younger'Litter Older Litter Virus 8 1h 50 mg. 10 l? 100 mg. 8, 12 19 200 mg. 12 21, 3h c. Discussion. The differences in time of onset of symptoms and survival time between the two litters may have been due to the small age difference present or possibly to a slight immunity obtained from -36. the dam of the older litter, although neither had been vaccinated against hog cholera. The plasma ascorbic acid levels were not as high using the in- jectable product subcutaneously as those obtained when a solution made from crystalline ascorbic acid was administered intraperitoneally in Experiment II. It is not known whether this was due to less complete ah-‘ sorption of the subcutaneously administered product, a more rapid elimina- tion, or some other factor. The apparent correlation between level of ascorbic acid ad- ministered and time of survival after inoculation with the virus suggests that a partial or transient protection was offered by the vitamin. If one attributes the survival of the pig in Experiment I to a carry-over of immunity acquired from the dam, this constitutes the only evidence of ascorbic acid exerting an effect on the hog-cholera virus in these experi- ments. The lack of significant beneficial effects from ascorbic acid in this experiment is in contrast to some of the results cited in the re- view of literature. .0ne possible explanation is that the vitamin used in at least part of the reported work may have been from natural sources and may have contained some of the citrus fruit flavonoids. It is quite possible that the beneficial results were due to a combination of the vitamin and the flavonoids. It would be desirable at some future time to investigate the influence of a combination of ascorbic acid and citrus fruit flavonoids on hog-cholera infection. B. The Effect of Protein and Tryptophan Deficiency 22_Hog-Cholera Infection 1. Experiment I! a. Procedure. This experiment was conducted in an attempt to determine if the feeding of a ration deficient in tryptOphan to baby pigs ‘would have any influence on resistance of the animals to hog-cholera in- fection. Pigs for this experiment were removed from the dam by caesarian section at the time of the appearance of milk in the udder. Twelve pigs were procured from 1 Yorkshire gilt and 9 from another. The second litter was delivered 15 days after the first. The pigs were kept either in ine dividual cages or 2 pigs to each cage with heat lamps being placed above the cages. These lamps were used for the first several days. The ration for the first week consisted of pasteurized, homogenized, whole milk with 1 egg yolk added to each 200 ml. of milk. A SO—ml. portion of this mix, ture was offered to each pig 5 times a day'for the first few days and then h times a day. After the first week the diet was gradually changed to the con- trol ration listed in Table XII. This diet was prepared in liquid form so that it contained 20 per cent solids, and it was then homOgenized. The mineral mixture was that formulated by Phillips and Hart (38). To each 100 ml. of the control diet, 2 ml. of vitamin solution B (Table II) ‘were added. The vitamins were dissolved in 30 per cent ethyl alcohol. Additional amounts of vitamins A and.D in cod-liver oil were given in the milk every third day. The tryptOphan-deficient diet was prepared from the ingredients listed in Table XII. Since the gelatin caused solidification when the ration was cooled, it was found necessary to dissolve the ingredients in -38- TABLE XII Composition of Rations Used in Experiments IV and.V Control Ration Deficient Ration Ingredient % % Casein 35.0 - Zein -— 17.5 Gelatin - 17.5 Lard no.0 no.0 Lactose 20.0 19.3 Mineral mixture 5.0 5.0 Lysine - 0.6 Methionine - 0.1 enough hot water to make half of the required volume. The vitamin solus tion was added in the amount of h ml. per 100 ml. of the concentrate. This was then homogenized and divided into the desired volume for indi— vidual feedings. At the time of feeding an equal volume of hot water was added and the ration was mixed in a‘Waring Blendor. Twenty mg. of ferric citrate were given in the ration of each pig approximately every week. Nine animals from the first litter and 3 from the second litter were used in this experiment. Eight of the older pigs were placed on the . tryptOphanedeficient diet when they were 17 days of age. The remaining animal from the first litter and.3 from the second litter were left on the control ration. One of the animals from the control group died 5 days after it was placed on the control ration. The amount of ration 'was gradually increased until each animal received 85 ml. h times a day. -39- After they had been on the deficient ration for h weeks, these pigs along with the h control animals were injected with 2 ml. of commercial hog- cholera virus (Pitmore Moore, serial no. 265k). The treatments are listed in Table XIII. TABLE XIII Treatments Applied to Pigs in Experiment IV Group No. of Pigs Treatment Deficient 8 Deficient ration plus hog-cholera virus Control h Control ration plus hog-cholera virus Records were kept of food consumption, body weight, and body temperatures. Blood counts were also made, and post-mortem examinations ‘were conducted soon after death of the pigs. b. Results. The pigs on the tryptophan-deficient ration began to consume less food after about 5 days. They began to eat rather slowly and deve10ped diarrhea with some vomiting. After 19 days on the ration, the pigs were consuming only about 70 per cent of the amount offered to them and were becoming quite weak. Additional food in the form of 10 to 20 ml. of homogenized, whole milk was given at each feeding to those ani- mals severely weakened. This was done only when it was thought necessary to prevent the pigs from dying of the deficiency. The food consumption of the pigs on the control ration remained quite good, and they consumed all the ration offered them. —lno— After consuming the deficient ration for h weeks, the pigs weighed an average of 32 g. each less than they did when the deficient ration was started. The control pigs, on the other hand, gained an average of more than 1 kg. each during the same interval. Body temperatures of the deficient pigs became quite low, and at the time of inoculation with the virus, the deficient animals had average temperatures of 98.80 F. as compared to 102.00 F. for the control animals. Average hematologic values for the 2 groups of animals at the time of inoculation with the virus are shown in Table XIV. TABLEuXIV Hematologic Values of Pigs in Experiment IV at Time of Inoculation with Virus Control Animals Deficient Animals Erythrocyte counts 7,530,000 per cmm. 7,38h,000 per cmm. HemoglObin 11.8 g. per 100 ml. 9.28 g. per 100 ml. Leukocyte counts 17,812 per cmm. 23,106 per cmm. Symptoms of hag cholera began deve10ping in the control pigs in h days and included profuse, fetid diarrhea, respiratory difficulty, mild conjunctivitis, and nervous manifestations in 1 animal. Symptoms in the deficient animals were much less marked and consisted primarily of a gradually increasing weakness. Food consumption in the control animals remained nearly normal until about 1 day'before death. There was a gradual decline in food consumption in the deficient group. Three days after the -h1- virus was injected, it was thought advisable to change the deficient pigs to a.mixture of the control ration and homogenized, whole milk. This was done because 1 pig died the day following inoculation and another on the third day. There was a temporary increase in the amount consumed. The average body temperatures of the control pigs increased to 105.50 F. on the third day after inoculation, while it rose to 105.3° F. on the fourth day in the deficient pigs. Leukopenia developed in both groups, but the values were not consistent, due in part to the fact that 1 pig in the con- trol group developed a transient leukocytosis. Leukocyte counts are shown in Table IV. TABLE IV Total Leukocyte Counts of Pigs in Experiment IV (Leukocytes per cmm. blood) Days after Inoculation .EESEB .9 . l. .3 .2 .E 2. .9 Control 20,250 12,h88* 6,638* 8,262* 12,725* t,150 -- Deficient 21,606 22,686* 13,879* 8,371* 10,058* 11,050 h,t10 ‘* Blood samples drawn from ear vein. Other samples drawn from anterior vena cava. One of the control pigs died on the fifth day following inocue lation with the virus, 2 on the sixth day, and 1 on the seventh day. In the deficient group, 1 pig died on the day following inoculation, 1 on the third day, and 1 on the fifth day (Table XVI). The other 5 animals ‘were killed on the seventh day when they were in a moribund condition, and post-mortem examinations were made. -hz- TABLE XVI Survival Time of Pigs in Experiment IV after Inoculation Groupv Survival Time (Days) Deficient 1, 3, 5* Control 5, 6, 6, 7 *‘Remaining 5 animals killed on seventh day. Necropsy of the pigs revealed lesions suggestive of hog cholera in all except the deficient animal which died on the day following inocu- lation. Lesions included edema, congestion, and hemorrhage of the lymph nodes; petechial hemorrhages on the surface of the kidney; infarcts in the Spleen; pneumonia; and enteritis. These lesions, especially those in the kidneys, were slightly more severe in the control than in the deficient animals. Edema was present in all but 1 animal. In the deficient pigs this edema was quite extensive in the subcutaneous tissues of the ventral part of the body, often extending from the mandible posteriorly to the flank region. Edema was also found in the mesentery of the coiled part of the colon. The control pigs were not so severely affected but 3 of the h exhibited some edema. c. Discussion. Although the ration fed to the deficient pigs in Experiments IV and V was theoretically lacking only in trypt0phan, it is highly probable that the deficiency state produced was actually one of lack of protein as well as of tryptophan. This ration seemed to be ab- sorbed rather poorly and was evidently not digested as thoroughly as the control ration in which protein was supplied in the form of casein. The -h3- extensive edema observed in the deficient group of pigs is also evidence of a protein deficienoy. The fact that some edema was also Observed in the control group of pigs suggests that the control ration may have been lacking in some way or perhaps was not fed in sufficient quantities. The time of death was delayed in the deficient animals in this experiment, if one assumes that the deficient animals dying on the first and third days died of the deficiency rather than from effects of the hog- cholera virus. This is in agreement with the findings of Kearney st 21. (28), Davies gt El‘ (11), and Pond gt 2}. (bl) in that the deficiency was effective in prolonging the incubation period and lengthening the survival time. 2. Eicperiment V a. Procedure. This experiment was a continuation of the study of the tryptOphan-deficient diet and its effects on hog-cholera infection. Ten pigs were purchased at 3 days of age from a Yorkshire herd. These pigs were litter mates of those used in Experiment I and were from a gilt and third-litter sow, both of which had been immunized against hog cholera at weaning age. These animals were fed egg yolk and.whole milk as were those in.Experiment IV for the first few days and then changed to the con- trol ration. Vitamin solution A (Table II) in the amount of 1 ml. per liter was added to the ration. Two of these animals were lost before the deficient diet was started. Six pigs were placed on the tryptOphan- deficient ration when they were h weeks of age. This ration was formulated as in Experiment IV, except that 2 ml. of vitamin solution A per liter were substituted for the vitamin solution B. The amount of ration fed was gradually increased until the animals on the control ration were consuming -hh- 300 ml. h times a day while those on the deficient ration were consuming about 125 ml. h times a day. When these pigs were h6 days of age, a litter mate which had been left with its dam was removed from the sow and added to the experi- mental group. It was given the control ration. At the age of 59 days all the pigs were inoculated with a ti- trated hog-cholera virus furnished'by the United States Department of Agriculture Hog Cholera Research Station,.Ames, Iowa. This virus (serial no. 312) had been found to kill pigs at dilutions of 1/10,000 and 1/100,000. Two ml. of this agent were diluted with sterile physiological saline to a total volume of 100 m1. Each pig was then given 5 m1. of this solution subcutaneously in the axillary space. Using the 1/10,000 dilution as a practical end point, each animal received 1000 infective doses. Two pigs of the deficient group were also given 20 ml. each of commercial anti-hog- cholera serum ( Fort Dodge, serial no. h59). The treatments are listed in Table XVII. TABLE XVII Treatments Applied to Pigs in Experiment V Group No. of Pigs Treatment Deficient h Deficient ration plus hog-cholera virus Deficient 2 Deficient ration plus plus serum hog-cholera virus and anti-hog—cholera serum Control 2 Control ration plus hog- cholera virus Control 1* Control ration plus hog- cholera virus * Removed from sow at h6 days of age. -hg- Records were kept of food consumption, body weight, and body temperatures. Blood counts were made, and post-mortem examinations were carried out at the time of death of the animals. The animals surviving were given 1 m1. of the original undiluted virus (10,000 infective doses) 22 days after being inoculated in an effort to determine if they had acquired an immunity to the hog-cholera virus. b. Results. The food consumption began to decrease in the de- ficient pigs about 11 days after they were changed to the deficient ration. The animals became lethargic and ate quite slowly, but they did not become as weak as did those in Experiment IV. At the time they were inoculated with the virus, they were consuming approximately 75 per cent of the 125 ml. offered at each feeding. The control animals were at this time consuming 300 m1. of the control ration at each feeding. After consuming the deficient ration for 32 days, the pigs weighed an average of h22 g. each less than they did when the deficient ration was started. The control pigs, on the other hand, gained an average of 6h22 g. each during the same interval. The body temperatures of the deficient pigs did not become as low as did those in.Experiment IV, and at the time of inoculation with the virus, the temperature of both groups of animals averaged 102.7o F. Average hematologic values for the 2 groups of animals at the time of inoculation with the virus are shown ianable XVIII. Symptoms of hog cholera began developing in the control pigs on the third day following inoculation with the virus. At this time 1 pig developed anorexia, had a black, pasty stool, exhibited respiratory diffi- culty, and became lethargic. Some convulsions were also noted in this ani— mal. The deficient pigs became rather lethargic at this time and developed 4.6. diarrhea, although the stool was not as dark nor as fetid as that of the control animals. The remaining control pig began to exhibit diarrhea and became lethargic on the fourth day. It developed convulsions on the eighth day, and conjunctivitis was noted at this time. TABLE XVIII Hematologic Values at Time of Inoculation Control Animals Deficient Animals .Erythrocyte counts 7,865,000 per cmm. 7,555,000 per cmm. Hemoglobin 11.2 g. per 100 ml. 9.7 g. per 100 ml. Leukocyte counts 1h,838 per cmm. 13,933 per cmm. Average food consumption of the deficient pigs receiving only the virus followed about the same pattern as that of the 2 animals re- ceiving both the virus and serum. There was a drop of approximately 20 ml. per pig per feeding the day following inoculation. Following this there was a fluctuation in the amount consumed for several days until, at the end of 8 days, the consumption was slightly above the original value. Food consumption of the control group gradually declined after the second day. The pig left with the sow until it was h6 days of age exhibited a drop in consumption on the fourth day but returned to normal by the eleventh day. Average body temperatures and leukocyte counts for the pigs are shown in Tables XIX and.XX, respectively. These results indicate that the temperature reaction was not so severe in the deficient animals as in those receiving the control diet. The body temperatures of the pigs .mwm no name on am too sonm eo>osom.* H.moa m.:oa m.:oa m.:oa m.©OH m.40a m.moa ~.moa 0.:oa 0.:oa ~.moa w.m0H w.moa *Homeeoo III III III III o.m0H ©.moa ©.moa o.~oa m.ooa m.m0H m.moa w.:oa ~.NOH Houpooo a.m0H m.m0H ~.N0H a.N0H 0.m0a m.N0H H.m0H ~.~0H m.m0a 0.N0H m.m0e n.40H 0.00H assoc + scoaoaocn «.moa ~.NOH «.moa ~.moa o.m0H ~.moa w.m0H ©.moa :.moa 4.moa H.30H m.mOH ©.NOH psoaoflmon Mm um fl w m .M m w. m m m m m muons sowpmfisoosH poems when A.m ov > peosahmaxm cw emam mo momspeaooeoa hpom NHN mqmda la. .0me no name 0: pm_aom Seem reboEom * 0m0.0H 00m.ma 0ma.0 00a.m 0ma.o 0mH.0H 0mm.a 0m:.aa sacnscco uu uu uu 0ma.ma 0mm.a 00m.sa mma.ma mun.sa Honscco mm0.0a mam.am maa.ma m-.ma mmm.HH mao.sa 0mm.aa 00m.oa spoon + sumacaacn 00m.ma mNN.sH maa.aa 00m.NH mma.r 004.:H 0aH.ma 0m0.NH scoaoaaca m. We. w. m .a. m. M. m malomm soapmfisoocH poems when “ocean .eeo nod mophooxsoqv > poosaamaxm cw emam mo moosoo ophooxsog Hence NM mnmda -h9- receiving the deficient diet and anti-hog-cholera serum remained normal. All pigs except those receiving the anti-hog-cholera serum deve10ped leukopenia. The transient nature of this leukopenia in the group of 3 control pigs which died between the eighth and eleventh day may possibly have resulted from hemoconcentration before death of the animals. One of the control pigs died on the fourth day after inoculation and the other on the ninth day. One deficient pig died on the fourth day, 1 died on the fifth day, and 2 survived. Both of the deficient pigs re- ceiving antiserum survived as did the animal left with the sow until it was 146 days of age (Table XXI). TABLE XXI Survival Time of Pigs in Experiment V after Inoculation §£o_up Survival Time (Days) Deficient h, 5* Deficient + serum Survived Control A, 9 ControlML Survived * Remaining 2 survived. “Removed from sow at 116 days of age. NecrOpsy of the 2 control animals revealed hemorrhage involving the lymph nodes of both animals, petechial hemorrhage on‘the surface of the kidney of 1 animal, pneumonia in 1 animal, enlargement of the Spleen with several infarcts, and enteritis. The hemorrhage of the lymph nodes found upon necrOpsy of the 2 deficient pigs was not as severe as in the -50. control animals. Enteritis was seen in both deficient animals, but other gross lesions suggestive of hog cholera were not present. Inoculation of the 5 surviving pigs with 10,000 infective doses of hog-cholera virus 22 days after the original inoculation produced no significant temperature elevation or other suggestions of hog cholera in any of the animsls. c. Discussion. The survival of the 2 deficient pigs which were given anti-hog-cholera serum at the time of inoculation with the virus in- dicates that the 2 deficient pigs which failed to survive died either from the effects of the hog-cholera virus or from a combination of the effects of the virus and the deficiency rather than from the, deficiency alone. The fact that 2 deficient pigs in this experiment, along with the animal left with the sow until it was ’46 days of age, the animal given ascorbic acid in Experiment I, and the animal given a tryptOphan analogue in Experiment VI, were all littermates and all survived suggests that a high degree of immunity against hog cholera, was probably carried over from the dam. The results of these 2 experiments indicate that the tryptOphan deficiency had a slight effect on the outcome of hog-cholera infection. The longer incubation period and survival time in Experiment IV was not demonstrated in Experiment V. 0n the other hand, the slightly less severe gross lesions in the deficient pigs were seen in both experiments. -51.. C. The Effect o_f_‘ _a_ TryptOphan Analogue 23 Pkg-Cholera Infection 1. Experiment VI a. Procedure. This experiment was a preliminary study of the possible inhibiting effects of a tryptOphan analogue on hogbcholera virus. Only a small amount of the analogue was available. The 1 animal used in this experiment was a litter mate of part of those used in Experiments I and V. It was removed from the sow at 3 days of age, started on homoge- nized milk and egg yolk, and later changed to the control diet in Table XII. 'When the animal was 59 days of age, it was inoculated subcutaneously with 1000 infective doses of the titrated hog—cholera virus (serial no. 312) described in.Experiment V. .At the same time it was given 100 mg. of the tryptOphan analogue tryptazan (dl-Ut -amino-!-(3-1ndazole) -- prepionic acid. This product has the following chemical formula: 0 CHZ-CH-C-OH I I NH N’N 2 l H One g. of the analogue was dissolved in 50 ml. of sterile physiological saline. Five ml. of the solution were given intravenously once a day to this pig for a total of 10 days. Food consumption, body weight, body tem- perature, and blood counts were recorded at selected intervals. Twenty- two days following inoculation the animal was given 1 ml. of the original undiluted virus in an effort to determine if it had acquired an immunity to hog-cholera virus. -52- b. Results. The food consumption of the pig receiving the tryptaphan analogue (tryptozan) began to decline approximately 72 hours after inoculation with the virus. 0n the fifth day it reached a low of approximately one—sixth of the amount normally consumed. During the next 10 days there was a gradual return to normal. The body temperature of this pig reached a high of 105.10 F. on the fourth day following inoculation. This was followed by a gradual de- cline and a return to normal by the twelfth day. The leukocyte count was l3,h50 per cmm. at the time of inocula- tion, and it remained normal until the sixth day when it dropped to 7,300 per cmm. The counts on the eighth.and eleventh days were 9,950 and 9,900 per cmm., reSpectively. The subsequent counts were normal or above normal. A dark, fetid diarrhea developed in the animal receiving trypta- zan on the fourth day after inoculation. The pig also became somewhat lethargic for a number of days, but it gradually became more active as the food consumption increased and the body temperature became normal. c. Discussion. ‘While it appears that administration of trypta— zan in this experiment modified the course of the hog-cholera infection and.the final outcome, it must again be pointed out that this animal was from a litter in which several other animals under various treatments survived inoculation with the hog-cholera virus. .Any further conclusions will have to await the results of future experiments. h. old n'er -53- IV. GENERAL DISCUSSION The techniques used in.maintaining baby pigs on artificial rations for this series of experiments were fairly successful. Some pigs, eSpecially those receiving no colostrum, were found to be quite susceptible to bacterial infections. Some difficulty was encountered with these in- fections in Experiment IV, and several pigs from the original litters were lost. A number of different micro-organisms were isolated from these ani- mals. Less difficulty was encountered in.Experiment V, probably because of the fact that the animals had received colostrum. These results point out the desirability of working with animals in a pathogen-free environment or using animals which have acquired at least sane degree of immunity by receiving colostrum for the first few days of life. 'Wbrk is being con- tinued to determine the effect on colostrumedeprived pigs of some species of bacteria not commonly thought to be pathogenic. The growth rates obtained in these studies were less than de- sirable. This is partially explained by the fact that the consumption was limited in some of the experiments in an attempt to reduce the incidence of diarrhea. This diarrhea apparently was caused in some cases by bac- terial infection and in other cases by improper homogenization of the prepared ration. For the most part, the ration prepared from homogenized, whole milk and egg caused less difficulty than the one prepared from casein, lard, lactose, minerals, and vitamins. The whole milk ration was also much more convenient to prepare. 'Wbrk is being continued to improve the methods of raising baby pigs in artificial surroundings, and at the present time it appears that -51,.. the incorporation of some of the techniques used by the Nebraska workers (55,56,57,58) would.be desirable. Statistical analyses were not carried out in this series of ex- periments due to the small numbers of animals involved and due to the fact that the various groups changed in size as the experiments progressed and animals died. -55- V. SUMMARY AND CONCLUSIONS A series of experiments was conducted in an effort to determine possible relationships between some nutritional factors and experimental hog—cholera infection in baby pigs. Ascorbic acid was administered to groups of animals in doses ranging from 25 to 200 mg. per pound body weight per day. This was ad- ministered by the intraperitoneal, subcutaneous, or oral routes. In 1 ex- periment the ascorbic acid appeared to lengthen the survival time of the infected pigs somewhat. Other evidences of beneficial effects were not noted. Of the 19 pigs given ascorbic acid, only 1 survived, and it is probable that its survival was due to immunity obtained from ingestion of colostrum. The effects of a trypt0phan-deficient diet on the symptoms, course, and lesions of experimental hog-cholera infection in baby pigs were also investigated. The deficiency delayed the onset of symptoms and time of death in 1 group of animals, and in 2 groups the deficiency caused a slight decrease in the severity of the gross lesions. The administration of tryptazan, a tryptophan analogue, to a pig inoculated with hog-cholera viruS'was accompanied by a recovery from the infection. The results were inconclusive, however, due to the possi- bility that the animal had Obtained some immunity to the hog-cholera virus from having nursed its dam during the first few days of life. 3. h. 5. 9. 10. ll. 12. 13. -55- VI . REFERENCES Ames, A. M., and Nungester, W. J .: The Relationship between Ascorbic Acid and Phagocytic Activity. J. Bact., _5_l_u_ (19147): 53. Anonymous: Protein and Antibody Production. Nutr. Rev., 6 (19148): 23h. '- Anonymous: Role of Ascorbic Acid in Infections. Nutr. Rev., 1).; (1956): 215. Biskind, M. S., and Martin, W. C.: The Use of Citrus Fruit Flavonoids in Infections. II. Am. J. Digest. Dis., _2_§ (1955): 111. Boines, G. J .: A Rationale for the Use of Hesperidin and Ascorbic Acid in the Management of Poliomyelitis. Bioflavonoids and the Capillary. Ann. N. Y. Acad. of Sci., 61. (1955): 721. Brambell, F. W. R., Hanmings, W. A., and Henderson, M.: Antibodies and Embryos. The Athlone Press, London, England, 1951. Brown, W. B. , Mahoney, F., Niedringhaus, A., and Locke, 15.: Weather and Susceptibility in Relation to the Spread of Common Cold; Effect of Ascorbic Acid, in Massive Dosage, on Duration. J. Immunol., Virus Res., and Exptl. Chemother., 52 (19145): 161. Campillo, 0.: E1 Acido Ascorbico Asociado a la Sulfanilamida en el Tratamiento de la Influenza Experimental del Raton. Rev. Inst. Salubridad y Enferm. Trop., 2 (19118): 101. Cohen, S. S., and Fowler, 0. B.: Chemical Studies on Host-Virus Interactions. III. Tryptophan Requirements in the Stages of Virus Multiplication in the Escherichia Coli-T2 Bacteriophage System. Jo EXP. Med., £5 (19187): 771. Cowell, S. J .: Discussion on Nutrition and Its Effects on Infectious Disease. Proc. Roy. Soc. Med. (London), 39 (1937): 10116. Davies, W. L., Pond, W. L., Smith, S. C., Rasmussen, A. F., Jr., Elvehjem, C. A., and Clark, P. F.: The Effect of Certain Amino Acid Deficiencies on Lansing Poliomyelitis in Mice. J. Bact., 6].; (1952): 5715 Ecker, E. E. , Pillemer, L., Wertheimer, D., and Gradis, B.: Ascorbic Acid and Complement Function. J. Immunol., 33 (1938): 19. Egan, F. D.: Ascorbic Acid in a Small Animal Practice. Mich. State Coll. Vet., E (191114): 117. Follis, R. E., Jr.: The Pathology of Nutritional Disease. Charles C. Thomas, Springfield, Illinois, l9h8. 15. 16. 17. 18. 19. 20. 21. 22. 23. 25. 26. 27. .57- Foster, C., Jones, F. H., Henle, W., and Dorfman, F.: 'Relation of Food Intake to Response of Mice Inoculated with Lansing Strain of Poliomyelitis Virus. Science, 9_7_ (19113): 207.‘ Foster, C., Jones, J. H., Henle, W., and Dorfman, F.: The Comparative Effects of Vitamin B1 Deficiency and Restriction of Food Intake on the Response of Mice to the Lansing Strain of Poliomyelitis Virus, as Determined by the Paired Feeding Technique. J. Exp. Med., 89 (19111;): 257. Foster, C., Jones, J. H., Henle, W., and Dorfman, F.: The Effect of Vitamin B1 Deficiency and of Restricted Food Intake on the Response of Mice to the Lansing Strain of Poliomyelitis Virus. J. Exp. Med., 1_9_ (19th): 221. Grollman, A. P., and Lehniger, A. L.: Enzymic Synthesis of l—Ascorbic Acid in Different Animal Species. Arch. Biochem. and Biophys., 69 (1957): h58. GruImner, R. H., Whitehair, C. K., Bohstedt, G., and Phillips, P. H.: Vitamin A, Vitamin C, and Niacin Levels in the Blood of Swine. J. Ani. Sci., 1 (19h8): 222. Holden, M., and Molloy, E. A.: Further Experiments on the Inactivation of Herpes Virus by Vitamin C (l-Ascorbic Acid). J. Immunol., _3_3_ (1937): 251. Jones, J. H., Foster, C., Henle, W., and Alexander, B.: Dietary Deficiencies and Poliomyelitis. Effects of Low Protein and of Low Tryptophan Diets on the Response of Mice to the Lansing Strain of Poliomyelitis Virus. Arch. Biochem., 11 (191.6): 1181. Jungeblut, C. W.: Inactivation of Poliomyelitis Virus in vitro by Crystalline Vitamin c (Ascorbic Acid). J. Exp. Med., 52‘ 93 : 517. Jungeblut, C. W.: Inactivation of Tetanus Toxin by Crystalline Vitamin C (l-Ascorbic Acid). J. Immunol., 32 (1937): 203. Jungeblut, C. W.: Studies on the Inactivation of Diphtheria Toxin by Vitamin C (l-Ascorbic Acid). J. Inf. Dis., 39 (19141): 70. Jungeblut, C. W.: Vitamin C Therapy and Preplylaxis in Experimental Poliomyelitis. J. Exp. Med., Q): (1937): 127. Jungeblut, C. W., and Zwemer, R. L.: Inactivation of Diphtheria Toxin in ‘vivo and in vitro by Crystalline Vitamin C (Ascorbic Acid). Proc. sec—m. p. Bi31._d—an Med., _3_g (1935): 1229. Kearney, E. B., Pond, W. L., Plass, B. A., Maddy, K. H., Elvehjem, C. A., and Clark, P. F.: Effect of Thiamine Deficiency on Western Equine Encephalomyelitis in Mice. J. Inf. Dis. , 82 (19h8): 177. 28. 29. 30. ‘ 31. 32. 33. 3h. 35.. 36. 37. 38. 39. W. -58. Kearney, E. B., Pond, W. L., Plass, B. A., Madchr, K. H., Elvehjem, C. A., and Clark, P. F.: The Influence of Varied Protein Intake and of Trypt0phan Deficiency on Theiler's Encephalomyelitis of Mice. J. Bact., _5_5_ (19h8): 89. Klenner, F. R.: Massive Doses of Vitamin C and the Virus Diseases. Southern Med. and Surgery, 193 (1951): 101. Klenner, F. R.: The Treatment of Poliomyelitis and Other Virus Diseases with Vitamin C. Southern Med. and Surgery, _11_l_. (19119): 209. Lassen, H. C. A.: Experimental Studies on the Course of Paratyphoid Infections in Avitaminotic Rats with Special Reference to Vitamin A Deficiency. Levin and Munksgaard, Copenhagen, 1931. Cited by Clark, et al.: Influence of Nutrition in Experimental Infection. Bact. R—V” 1_3_ (19119): 111. Madison, R. R., and Manwaring, W. H.: Ascorbic Acid Stimulation of Specific Antibody Production. Proc. Soc. Exp. Biol. and Med. , 37 (1937): 1102. Manwaring, W. H.: Ascorbic Acid vs. the Common Cold. Calif. and West. Med., E?- (19115): 3090 Massa, V.: Ascorbic Acid and Reapiratory Metabolism of Leukocytes. Bull. Soc. Chim. Biol., 29 (19117): 732. Cited in Biol. Abstr., 22 (l9h8): 1567. McCormick, W. J .: Ascorbic Acid as a Chemotherapeutic Agent. Arch. Pediat., 62 (1952): 151. McLester, J. S., and Darby, W. J .: Nutrition and Diet in Health and Disease. W. B. Saunders, Philadelphia, 1952. Olitsky, P. K., Traum, J ., and Schoening, H. W.: Report of Foot-and- . Mouth Disease Commission. U. S. D. A. Tech. Bu11., No. 76, 1928. Phillips, P. H., and Hart, E. B.: The Effect of Organic Dietary Constituents upon Chronic Fluorine Toxicosis in the Rat. J. Biol. Chem., 192 (1935): 657. Pickens, E. M., Welsh, M. F. , and Poelma, L. F.: The Susceptibility of Young Pigs to Hog Cholera. J.A.V.M.A., 5_8_ (1921): 1103. Polozhintseva, S. P.: Change of Capillary Resistance in Measles and Its Relation to Ascorbic Acid Content of the Blood. Pediatriia, 19M (19M): 21. Cited in Biol. Abstr., $1 (19113): 1209. Pond, W. L., Davies, W. L., Smith, S. C., Elvehjem, C. A., Rasmussen, A. F., Jr., and Clark, P. F.: The Influence of Amino Acid Deficiencies on Theiler's GD VII Encephalomyelitis of Mice. J. Bact., _6_1_1_ (1952): 583. 1:2. h3. 1:5. ’46. 1:7. 1:8. h9. 50. 51. 52. 53. Sh. SS. -59- Rasmussen, A. F., Jr., Clark, P. F., Smith, S. C., and Elvehjem, C. A.: Effect of 6-Methy1 TryptOphan on Lansing Poliomyelitis in Mice. Bact. Proc., (1953): 93. Rasmussen, A. F., Jr., Waisman, H. A., Elvehjem, C. A., and Clark, P. F.: Influence of the Level of Thiamine Intake on the Susceptibility of Mice to Poliomyelitis Virus. J. Inf. Dis., 14 (19th): hi. Rasmussen, A. F., Jr., Weaver, R. W., Elvehjem, C. A., and Clark, P. F.: Influence of Low TryptOphan Diets Containing 6—Methy1— trypt0phan on Oral Infection with Poliomyelitis Virus. Proc. Soc. Expt. Biol. and Med., 81$ (1953): 306. Reber, E. F., Whitehair, C. K., and MacVicar, R.: The Effect of Level of Protein Fed Baby Pigs. J. Nutrition, 59 (1953): 1451. Roskin, G. I.: The Thin Structure of the Wall of Capillaries and the Problem of Carrier Function. Bull. Biol. et Med. Exptl., U.R.S.S., 1_8_ (191114): 11. Cited in Biol. Abstr., _2_]_._ (191:7): 86. Rouse, P.: A Sarcoma of the Fowl Transmissable by an Agent Separable from the Tumor Cells. J. Exp. Med., _12 (1911): 39?. Slusher, M. A., and Roberts, 8.: Fate of Adrenal Ascorbic Acid: Relationship to Corticosteroid Secretion. Endocrin., 6...]; (1957): 98. Smith, H. R., and King, N. B.: Passive Immunity to Hog Cholera in Nursing Pigs. J.A.V.M.A., l_3_2_ (1958): 107. Sokoloff, B.: The Capillary Syndrome in Viral Infections. Treatment with Citrus Flavonoids. Am. J. Digest. Dis., 23 (1955): 7. Sprunt, D. H.: The Effect of Undernourishment on the Susceptibility of the Rabbit to Infection with Vaccinia. J. Exp. Med., 15 (191:2): 297. Sprunt, D. H., and Flanigan, C. 0.: The Effect of Malnutrition on the Susceptibility of the Host to Viral Infection. J. Exp. Med., E (1956): 687. Sprunt, D. H., and Sands, 8.: Effect of Low Protein Diet on Rous Sarcoma in Chickens. Fed. Proc. of Fed. Am. Soc. Exptl. Biol., 13 (1953): h61. Stotz, E.: A Clinical Method for the Determination of Ascorbic Acid in Blood Plasma and Urine. J. Lab. and Clin. Med., 26 (19110-111): 15h2. Underdahl, N. R., and Young, G. A.: An Improved Hood for Swine Hysterectomies. J.A.V.M.A., l_3_l_ (1957): 222. 56. 57. 58. -60- Underdahl, N. R., and Young, G. A.: An Isolation Brooder for Raising Disease-Free Pigs. J.A.V.M.A.,‘lgl (1957): 279. Young, G. A., and Underdahl, N. R.: Isolation Units for Growing Baby APigs‘Without Colostrum. Am. J. Vet. Res., 13 (1953): 571. Young, G. A., Underdahl, N. R., and Hinz, R. W.: -Procurement of Baby Pigs by Hysterectomy. ,Am. J. Vet. Res., 16 (1955): 123. if? Wv fl”- ' "' r1! 3% 7.. M E'nw' - ' “\v? I: Q ‘?3