THE RELATIONSHIP OF SPECIFIC NUTRIENT DEHcsmcEEs To: ANTIsooY iEsnoNsE EN SWINE [figs 14 mm“ ;.a f {E PAmTI-IENIC ACID; nmooxmt oi RIBOFLAVIN V ' Thesis'fbr‘fhobgm g; phi-b; ' MICHEGAN STA?! UNIVERSITY Bud G Harmon I962 114E513 0-169 This is to certify that the thesis entitled THE RELATIONSHIP OF SPECIFIC NUTRIENT DEFICIENCIES TO ANTIBODY RESPONSE IN SWINE I. VITAMIN A II. PANTOTHENIC ACID, PYRIDOXINE OR RIBOFLAVIN presented by BUD G. HARMON has been accepted towards fulfillment of the requirements for £12— degree inwbandm 7w. Nae/M— Major Professor Date ”1‘61 181.1963. LIB R A R Y Michigan State University ABSTRACT THE RELATIONSHIP OF SPECIFIC NUTRIENT DEFICIENCIES TO ANTIBODY RESPONSE IN SWINE ! I l I. VITAMIN A II. PANTOTHENIC ACID, PYRIDOXINE OR RIBOFLAVIN by Bud G. Harmon Four trials were conducted to study and evaluate the relationship of specific deficiencies of vitamin A, pantothenic acid, pyridoxine or ribo- flavin to antibody production in swine. Each of the trials involved pigs which were weaned to semi-synthetic diets at two weeks of age or less. The first two trials in which 30 pigs were employed were designed to study the antibody response of positive control pigs and pigs deficient in vi- tamin A. Additional data obtained were weight gain, feed efficiency, se- rum vitamin A level, serum protein concentration and the electrophoretic components of serum protein. The pigs receiving the vitamin A free diet exhibited significantly lower (P U.Ol) serum vitamin A levels at six weeks of age after having been weaned to the experimental diet at ages of six days and l2 hours re— spectively in Trials I and II. The pigs in a marginal vitamin A condition (less than 20 micrograms per lOO milliliters of serum) produced signifi— cantly lower (P 0.01) antibody titers to experimental intraperitoneal in— troduction of killed cultures of Salmongila REIIEEET than did the control pigs. In Trial I the average daily gain and feed efficiency values of the vitamin A deficient and control pigs were not statistically significantly different. However, in Trial II the daily gain was significantly greater and more efficient (P 0.01) in the control pigs. Analysis of the electro- phoretic components of serum protein at the time the antibody production __4 Bud G. Harmon was measured,disclosed that the deficient pigs had significantly higher (P\0.0l) percentages of alpha and gamma globulin than did the control pigs. This was accompanied by a significant decrease (P 0.01) in the percent al- bumin in the deficient pigs. Following a repletion phase during which all pigs received a com— plete natural diet the immunological response was measured with human erythrocytes. All pigs from both previous treatments responded with sim- ilar hemagglutination titers. In the second trial the control pigs con- tinued to gain significantly faster (P 0.0l) during the repletion phase than did the pigs which were previously vitamin A deficient. Serum vi- tamin A and protein values were similar in all pigs following the reple- tion phase of each trial. Trials III and IV, which involved #8 pigs, were designed to study the antibody response of positive control pigs and pigs deficient in pan- l l l l l l tothenic acid, pyridoxine or riboflavin. Additional data collected were measures of weight gain, feed efficiency, blood cellular components, se- rum protein concentration and electrophoretic components of serum protein. Also, urinary xanthurenic acid values were obtained from the pyridoxine deficient and control pigs. The pigs in both trials were placed on one of the four experimental diets at four weeks of age after having been weaned to a semi-synthetic diet, dcficientin the three B vitamins under consider- ation,at two weeks of age. In Trial III Salmonella pyllorum antigen was injected after the pigs on experimental treatment had been deprived of the particular vitamin for four weeks. In Trial IV the experimental feeding period was extended one week and human erythrocytes were employed as the antigen. The agglutination titers in Trial III and hemagglutination titers in Trial IV were significantly greater (P 0.0l) in control pigs than for Bud G. Harmon any of the deficient groups. A series of equated feeding groups (a group included a pig from each of the four treatments) established that inani- tion was not reSponsible for decreased antibody production in the defi- cient pigs since the control pigs on limited feed produced significantly greater (P 0.0l) antibody titers. At the conclusion of the depletion phase of the trials the weight gain of the control pigs was significantly greater and more efficient (P-O Ol) than that of any deficient group. Pyridoxine deficient pigs had lower hematocrit, hemoglobin, total erythrocytes and total leukocytes than did the other pigs. The pyridoxine deficient pigs also had signifi- cantly higher concentrations (P 0.05) of urinary xanthurenic acid than did the controls, both before as well as after additions of tryptophan to the regular diet. Analyses of the serum proteins at the conclusion of the depletion feeding phase of the trials established that the alpha globulin was sig- nificantly greater (P 0.05) in the pantothenic acid deficient pigs than Following repletion periods of six to seven weeks all pigs respond- ed with similar antibody titers to human erythrocytes in Trial III and Salmonella BEIIEEET in Trial IV. Also at this time, similar serum protein values were measured in all treatments. In Trial III the weight gain of the control pigs was significantly greater (P 0.05) after six weeks on a complete natural diet than all the pigs formerly fed the deficient diets. However, in Trial IV after extending the repletion feeding period one Week, the control pigs remained only significantly heavier (P-0.05) than the pigs l l l l l in the controls. formerly receiving the pantothenic acid deficient diet. THE RELATIONSHIP OF SPECIFIC NUTRIENT DEFICIENCIES TO ANTIBODY RESPONSE IN SWINE n-c . VITAMIN A II. PANTOTHENIC ACID, PYRIDOXINE 0R RIBOFLAVIN By BudiCi'Harmon A THESIS Submitted to the School for Advanced Graduate Studies of Michigan State University of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Animal Husbandry l962 G. ZSIOG 5"!le ACKNOWLEDGMENT The author wishes to express his very sincere appreciation to Dr. J. A. Hoefer and Dr. E. R. Miller for their invaluable guidance and assistance throughout this study, and for their critical reading of this manuscript. The writer also wishes to extend a sincere note of thanks to the members of his guidance committee, Dr. J. A. Hoefer, Dr. E. R. Miller, Dr. R. W. Luecke, Dr. E. P. Reineke and Dr. C. K. Whitehair for their generous attitude during the completion of his studies. Sincere gratitude is eXpressed to Dr. R. H. Nelson and Dr. D. E. Ullrey for the materials and facilities which made this research possi- ble. The author wishes to thank the herdsman, Mr. G. B. Stafford,and his staff for their assistance in the care and management of the pigs. Gratitude is expressed to the Rackham Foundation for SUpport of this study. The author also wishes to express his thanks to the National Institute of Health for providing him a Public Health Service Fellowship. The writer wishes especially to acknowledge his gratitude, appre- ciation and indebtedness to his wife, Mary Lynne, whose sacrifices and encouragement made this study possible. ii Bud G. Harmon candidate for the degree of Doctor_of Philosophy DISSERTATION: The Relationship of Specific Nutrient Deficiencies to Antibody Response in Swine I. Vitamin A II. Pantothenic Acid, Pyridoxine or Riboflavin OUTLINE OF STUDIES: Main area of study: Animal Husbandry (Animal Nutrition) Supporting areas of study: Biochemistry, Physiology BIOGRAPHICAL ITEMS: Born: July 2, l93l. Camden, Indiana Undergraduate studies: Purdue University, l955-l958 Graduate studies: Michigan State University, l958-l962 EXPERIENCE: Member United States Navy, l95l-l955 Assistant Instructor, Michigan State University, l958-l96l National Institute of Health Fellow, Michigan State University, 1961-1962 ' MEMBER: American Society of Animal Science Society of Sigma Xi American Association for the Advancement of Science II. III. IV. VI. VII. VIII. TABLE OF CONTENTS INTRODUCTION . REVIEW OF LITERATURE . A. Vitamin A. B. Pantothenic acid, pyridoxine and riboflavin. EXPERIMENTAL PROCEDURE . A. General. . . . . . . . . . . . . . . . . . . . . . . . . B. Trials I and II. The effect of vitamin A deficiency in swine upon specific serum antibody titer response . C. Trials III and IV. The effect of pantothenic acid, pyridoxine or riboflavin deficiency in swine upon specific serum antibody titer response . RESULTS AND DISCUSSION . A. Vitamin A studies. Trial I. Trial II B. Pantothenic acid, pyridoxine and riboflavin studies. Trial III. Trial IV . SUMMARY. . . . . . . . . . . . . . . . . . . . . . . . . CONCLUSION 0 O O O O O O O O O 0 O O O O O O O O O O O O O 0 BIBLIOGRAPHY . APPENDIX . iv l7 33 33 Al LE2 LE6 46 [+6 51 58 58 61+ 76 79 Bl 9l \C ll. l2. l4. LIST OF TABLES Influence of diet 0; number of ascarids . . . . . . . Ingredient composition of semi—synthetic ration Mineral mixture of semi-synthetic ration. . . . . Vitamin mixture of semi-synthetic rati0n. Ingredient composition of repletior ration. Trial Trial Trial I. II. III. Summary of data in vitamin A study. Summary of data in vitamin A study Summary of growth and serum protein values in B vitamin study. Trial Trial III. III. Blood cellular values in B vitamin study. Reciprocals of net antibody titer in B vitamin study Trial IV. Summary of growth and serum protein values in B vitamin study. Trial Trial IV. IV. Blood cellular values in B vitamin study Reciprocals of net antibody titer in B vitamin study Trial IV. Pair fed - B vitamin study 59 6l 63 67 6? 7l LIST OF FIGURES figure Page l. Trial 1. Growth curves of pigs on vitamin A deficient and control diets. . . . . . . . . . . . . . . . . . . . . A6 2. Trial I. Examples of a control and a vitamin A deficient pig after being allotted to the respective treatment for nine weeks . . . . . . . . . . . . . . . . . . . . . . 48 3. Trial I. Serum vitamin A values of pigs on vitamin A deficient and control diets. . . . . . . . . . . . . . . . 50 A. Trial II. Serum vitamin A values of pigs on vitamin A deficient and control diets. . . . . . . . . . . . . . . . 53 5. Trial II. Growth curves of pigs on vitamin A deficient and control diets. . . . . . . . . . . . . . . . . . . . . SA 6. Trial II. Examples of a control and a vitamin A deficient pig after being allotted to the respective treatment for six weeks. . . . . . . . . . . . . . . . . . . . . . . 55 7. Trial I and II. Individual net antibody titers produced against Salmonella pullorum in the depletion phase and human erythrocytes in the repletion phase by vitamin A deficient and control pigs . . . . . . . . . . ... . . . . 57 8. Trial III. Growth curves of pigs on pantothenic acid, pyridoxine, riboflavin deficient or control diets. . . . . 6O 9. Trial III. Individual net antibody titers produced against Salmonella pullorum in the depletion phase and 3 human erythrocytes in the repletion phase by pigs on pantothenic acid, pyridoxine, riboflavin deficient or control diets. . . . . . . . . . . . . . . . . . . . . . . 64 l0. Trial IV. Growth curves of pigs on pantothenic acid, pyridoxine, riboflavin deficient or control diets. . . . . 66 ll. Trial IV. Individual net antibody titers produced against human erythrocytes in the depletion phase and Salmonella pullorum in the repletion phase by pigs on pantothenic acid, pyridoxine, riboflavin_deficient or control diets. . 7O l2. Examples of a control pig and pigs deficient in pantothenic acid, pyridoxine or riboflavin after being allotted to the respective treatment for five weeks, , , , , , , . . . . . 72 vi 19.th 10. ll. l2. 14. 15. 16. LIST OF APPENDIX TABLES Trial I. Pig weights in vitamin A study(lb.) Trial 1. Pig weights, gain and feed efficiency in vitamin A study(lb.). . . . . . . Trial I. Serum vitamin A levels(mcg./l00 ml.). . . - - a Trial I. Serum protein values in vitamin A study, experimental depletion phase. . . . . . . . . . . . Trial 1. Serun protein values in vitamin A study, experimental repletion phase. Trial I. Reciprocals of antibody titers in vitamin A study . ' Trial II. Serun vitamin A levels(mcg./l00 ml.) Trial II. Pig weights in vitamin A study(lb.). Trial II. Pig weights, gain and feed efficiency in vitamin A study(lb.). Trial II. Serum protein values in vitamin A study, experimental depletion phase. Trial II. Serum protein values in vitamin A study, experimental repletion phase. Trial II. Reciprocals of antibody titers in vitamin A study . . . . . . . . . . . . . . . . . Trial III. Pig weights, gain and feed efficiency in B vitamin study(lb.). Trial III. Blood cellular data in B vitamin study experimental depletion phase. Trial III. Serum protein values in B vitamin study, experimental depletion phase, at time of antigen injection. Trial III. Serum protein values in B vitamin study, experimental depletion phase, at time of antibody determination . . . . . . . . . . . . . . . . . . . . . vii BEES Ql J 92 93 9A 96 97 98 lOO lOl 103 lOA l05 l07 l09 lll 19.913. 17. l8. 20. 2l. 22. 23. 2A. 25. LIST OF APPENDIX TABLES (CONTINUED) Trial III. Serum protein values in B vitamin study, experimental repletion phase . . . . . . . . . . . . . Trial III. Reciprocals of antibody titers in in B vitamin study . . . . . . . . . . . . . . . . . . Trial IV. Pig weights, gain and feed efficiency in B vitamin study(lb.) . . Trial IV. Blood cellular data in B vitamin study, experimental depletion phase . Trial IV. Serum protein values in B vitamin study, experimental depletion phase, at time of antigen injection. Trial IV. Serum protein values in B vitamin study, experimental depletion phase, at time of antibody determination. Trial IV. Serum protein values in B vitamin study, experimental repletion phase . Trial III and IV. Urinary xanthurenic acid values(mcg./mlJ. Trial IV. Reciprocals of antibody titers in B vitamin study. viii Bass 113 ll5 ll7 ll9 l2l 123 l25 l27 128 I. INTRODUCTION The search for uiritional compele'ts which may modify the resistance or susceptibility of a List to infectious diseases has been pursued nxter- sively since the turn of the century. Evaluation of altered resistarce or susceptibility has progressed from simply a percert mortality ir the early studies to a series of more sophisticated ard specific though less dramatic criteria of host modifica- tions in later irvestigations. ACtive antibody response to a specific antigen is but one of several means of defense by the host in resisting infection. However, antibody formatiO' and iiwunity are readily measured and the importance of the de— fense menhanism has 3een reengnized since the ti e of Jenner(Baron, l927). Tne study of tie relationship of dietary factors to antibody produc- thL has progressed toward two main purpOSuS: (l) To dgtermi:e and iden— tify essential compo.e ts of a nutritional GUXIFO!mUPt for e ha:cing ac- tively acquired immu itv, ard (2) To supply possible approaches of investi- gatiOn for elucidating mecha:1sms of antibody formation. Early Worknrs such as Wcr'na (l923a) could fi'd little irfluence of diet upo: a tibody productior. The early literature, howeveg does contain many reports i: whitL particular diets Were predisposing to increased sus- ceptibility to infection. As various essential nutrients were identified and diets became available in purer forms variwus groups began to report some relationship betWeen diet and antibody production. Axelrod and his co-workers (l955a) have conducted and reported many studies in which the relationship of vitamin deficiencies and antibody production was examined ir rats and guinea pigs. However, relatively few investigations have been conducted in swine relative to specific antibody production in various nutrient deficiency states. This study was initiated to determine: (l) The effect that defi— ciencies of vitamin A, pantothenic acid, pyridoxine or riboflavin would have on specific antibody production, and (2) The ability of pigs to pro- duce specific antibodies on a complete diet following a sustained period of consuming diets deficient in the above mentioned vitamins. II. REVIEW OF LITERATURE A. Vitamin A The voluminous amount of research that has been reported concern- ing the relationship between nutrition and resistance or susceptibility to infection has in many areas been quite contradictory. In one of the earliest published reports McCollum (l9l7) reported that rats on a diet low in vitamin A develOped severe spontaneous infec- tion. Drummond (l9l9) also reported an increase in spontaneous infection in vitamin A deficient rats. Mellanby (l919) observed increased suscep- tibility to pneumonia in pups fed a diet deficient in vitamin A. In a study with kittens McKay (l92l) observed that when the fat of milk was replaced by olive oil there was a high incidence of infections of leylidium caninum. Daniels t al. (l923) also neported an increased susceptibility of xerOphthalmic rats to spontaneous infections of the res— piratory tract. On the other hand Cramer and Kingsbury (l924) were able to demonstrate only small differences between the resistance of vitamin A deficient rats and rats fed a complete diet when exposed to Mycobacte— rium tuberculosis. These same workers observed no spontaneous outbreaks of pneumonia in the vitamin A deficient rats. Green and Mellanby (l928) found evidence of infection in a majority of the rats suffering from a vitamin A deficiency. The control animals exhibited no such infections. In 72 percent of the deficient rats, ab- scesses were observed at the base of the tongue. Also, infections in the urinary tract were quite common. Other areas of infection included the ocular apparatus, respiratory and alimentary tracts, and mastoid and na- sal sinuses. Green and Mellanby (l930) fed graded levels of carotene to -3- II. REVIEW OF LITERATURE A. Vitamin A The voluminous amount of research that has been reported concern- ing the relationship between nutrition and resistance or susceptibility to infection has in many areas been quite contradictory. In one of the earliest published reports McCollum (1917) reported that rats on a diet low in vitamin A develOped severe spontaneous infec- tion. Drummond (1919) also reported an increase in Spontaneous infection in vitamin A deficient rats. Mellanby (1919) observed increased suscep- tibility to pneumonia in pups fed a diet deficient in vitamin A. In a study with kittens McKay (1921) observed that when the fat of milk was replaced by olive oil there was a high incidence of infections of leylidium caninum. Daniels 35 al. (1923) also Reported an increased susceptibility of xerOphthalmic rats to Spontaneous infections of the res- piratory tract. On the other hand Cramer and Kingsbury (1924) were able to demonstrate only small differences between the resistance of vitamin A deficient rats and rats fed a complete diet when exposed to Mycobacte- rium tuberculosis. These same workers observed no spontaneous outbreaks of pneumonia in the vitamin A deficient rats. Green and Mellanby (1928) found evidence of infection in a majority of the rats suffering from a vitamin A deficiency. The control animals exhibited no such infections. In 72 percent of the deficient rats, ab- scesses were observed at the base of the tongue. Also, infections in the urinary tract were quite common. Other areas of infection included the ocular apparatus, respiratory and alimentary tracts, and mastoid and na- sal sinuses. Green and Mellanby (1930) fed graded levels of carotene to -3- -b,_ rats on a vitamin A free diet. At levels of .OA milligrams carotene per day and higher, no observable Spontaneous infections were present at au- topsy. Infections were found in all rats receiving .01 milligrams caro- tene per day or less. The lesions most often seen were tongue abscesses and upper respiratory infections. Greene (1933) found spontaneous cultures of Streptococcus pyogenes in nasal passages in rats on vitamin A deficient diets. Cultures of this organism were not isolated from the rats fed the control diets. At au- topsy Streptococcus pyogenes was isolated from the lungs of 90 percent of the animals on the deficient diet and from blood taken from the heart in 60 percent of the deficient animals. Greene also found increased Sponta— neous infections of pneumococcus and Salmonella leptosepticum. Gerriets (1960) investigated the coccidiostatic effect of vitamin A using graded dosages of vitamin A in a study involving 800 pullets. Ap- pendix coccidiosis was observed to occur Spontaneously in many five week old pullets. An experiment was designed with Six treatment groups two of which received the vitamin A deficient diet. The mortality to coccidiosis was 100 percent in these two groups. The other lots received the vitamin A deficient basal diet plus 15, 30, 45, and 60 IU (international units) reSpectively, of vitamin A per day. The mortality in these groups was 67 percent, 38 percent, 2A percent and 6 percent respectively. Gerriet cred- its much of the prOphylactic effect of vitamin A to the maintenance of the epithelium. Erasmus gt g1. (1959) found that coccidiosis was more severe in chickens which were on a diet deficient in vitamin A than birds receiving a diet meeting the National Research Council requirements. -5- Turner t al. (1930) investigated the relationship of vitamin A to the frequency of spontaneous infection of the middle ear and the upper res- piratory tract involving Staphlococcus aureus, Escherichia coli, Micro- coccus catarrhalis A and Chromagen 6. The latter two, the pyogenic gram negative cocci, were present in greater numbers in the rats on the vitamin A deficient diet. Stoerk it al. (1952) have stated that the metaplasia of the corneal epithelium invariably led to keratitis and iritis. Further, that trache- itis, pyelitis, cystitis and endometritis were found in association with squamous cell metaplasia of the respective tissue. To further study the cause of Spontaneous infection, Stoerk gave daily injections of antibi— otics to vitamin A deficient animals. The deficient animals responded to the antibiotic treatment with increased growth and survival which led these workers to conclude that secondary infection is one of the predis- posing factors of death in vitamin A deficient animals. Other workers chose to study the effect of a vitamin A deficiency upon resistance or susceptibility by experimentally infecting the animals. Werkman (1923b) was able to bring about the death of vitamin A deficient rats by intraperitoneal injections of Bacillus anthracis. Positive control rats were not susceptible to anthrax. Howeven following experimental in- fection in rats Verder (1928) could isolate Salmonella enteritidis cultures with equal frequency from various tissues of rats fed diets either ade- quate or deficient in vitamin A. In an extremely interesting Study Boynton and Bradford (1931) were able to Show that vitamin A deficient rats experienced a higher mortality rate than did the control animals when injected intraperitoneally with a -6— bacillus of the Mucosu§_cap§ulatus group. In this study 46 rats were di- vided into two groups and fed either a diet deficient in vitamin A or the same diet SUpplemerted with cod liver oil as a vitamin A carrier. Start- ing after four weeks on test and at biweekly intervals from five to eight rats per treatment were selected and injected with the bacillus. Survival time following the injection was measured and this time interval served as a criterion for evaluating the effect of vitamin A deficiency upon resis- tance of the animal. The induced infection was fatal to all but one defi- cient rat and to 75 per cent of the control animals. Of the rats that died the average survival time was 31 hours for the control animals and 10 hours for the deficient. The growth rate of the vitamin A deficient rats was equal to that of the positive control rats until the experiment had progressed eight weeks. At that time weight gain became suppressed in the deficient animals. In contrast,the deficient animals had depressed resistance to the bacillus injections at least by four weeks after the ex- periment had started. McClung and Winters (1932) developed vitamin A deficient rats by feeding a semi-syrthetic diet for seven weeks. At that time intraperi- toneal injections of Salmonella enteritidis were begun with 1 cc. of a 2h hour broth of which 1.2 cc. had been found to be an MLD at two to four days. At the end of 216 hours one control rat of 20 had died and 13 of 20 of the vitamin A deficient rats had died. The most characteristic 1e- sion was a hemorrhage within the intestinal tract. Tisdall (1950) injected Salmonella typhimurium into rats fed a vi- tamin A deficient or control diet for four weeks. Forty percent of the rats on the deficient diet survived while 50 percent of the controls sur- vived. In similar studies with mice using Salmonella typhosa the mortality was 90 percent for the vitamin A deficient mice and 15 percert for the con- trol mice. Orskox and Moltke (1928) have studied the manner in which an oral in- fection of Salmonella paratyphi transpired in mice. The bacilli rapidly disappear from the alimentary tract reappearing in the lymph nodes of the mesentery. From the mesentery the bacilli pass via the thoracic duct and blood stream to the lower spleen and peripheral lymph nodes. In normal animals these three areas carry out the destruction of the bacilli. In vitamin A deficient animals the bacilli are not destroyed and a severe sec- ondary infection of the blood stream may follow. Diehl (1960) investigated the severity of experimental hepatic cocci- diosis infection in rabbits receiving a complete diet or one deficient in either vitamin A or vitamin E. The infection was more severe in the vitamin A deficient grOUp than ir either of the other treatments. Niilo and Beyeau (1961) fed chickens a diet deficient in vitamin A i: which carotene was added at either 1340 or 5340 IU per kilogram of diet. After four weeks on trial the drinking water was contaminated on three suc- cessive days with cultures of Pseudomonas aeruginosa. On the low carotene diet 56 percent of the chickens died while the mortality of the chickens on the high carotene diet was only 14 percent. The vitamin A levels from liv- ers of birds exhihiting positive cultures were 40.4 and 0.9 micrograms per 100 grams respectively in the control and deficient birds. Sayedain and Kinsy (1960) observed that vitamin A deficient chickens were more susceptible to experimental Candida albicans infections than were the control birds. Sixty percent of the vitamin A deficient birds showed -8- lesions while only 7 percent of the control birds had moniliasis lesions. In a more quantitative study Guggenheim and Buechler (1946) made peri- odic bacteria counts at various intervals following intra-abdomiral injections of Salmonella typhimurium. They found significantly larger numbers of ex— tracellular bacteria in the cell-free peritoneal fluid of the vitamin A de— ficient rats than in the controls. These researchers concluded that the bactericidal action by the vitamin A deficient rats was significantly di- minished. Phagocytosis was measured and determined to be significantly greater in the rats receiving vitamin A than in the deficient rats. Orskov t 1. (1928) have concluded that the principal defenses of mice against Salmonella typhimurium and other salmonellas are phagocytosis by leukocytes and by macrophages of the reticuloendothelial system. Mellanby and Green (1929) reported that the administration of vitamin A in quantities greater than normally included in a diet provided increased resistance to puerperal sepsis and septicemia. These workers concluded from this and other studies that vitamin A is an ”anti-infective“ vitamin. Hess gt al. (1933) and Clausen (1934) studied the “anti-infective“ influence of high levels of vitamin A superimposed upon a diet assumed to provide adequate vitamin A. Neither group could Show any increased resist— alice from the high levels of vitamin A. Schneider (1946) was quite criti— cal of the feeding of additional vitamin A above that normally added to a complete diet as an ”anti-infective” measure. He suggests that vitamin A is no more "anti—infective'l than many of the B vitamins, and analogizes that to call pyridoxine the llgrowth vitamin” would be just as proper. Wohlbach (1942) casts further doubt on the term llanti-infective“ that Mellanby and Green (1929) applied to vitamin A. The sublingual abscesses _ g - which the latter workers described in vitamin A deficient animals were shown by Wohlbach (1942) to be simple cysts containing desquamated epithelial cells and detritus. Other investigators have studied the effects of vitamin A deficiency upon resistance or susceptibility to viruses. Rous (1911) observed that well nourished chicks were more susceptible to fowl sarcoma virus than were chicks which were undernourished. Squibb and Veros (1961) have studied the effect of varying levels of vitamin A upon the resistance of young chickens to Newcastle disease virus (NOV). All birds were fed a diet free of vitamin A for three to four weeks. The birds were then lotted but continued to receive the same diet. The re- sultant groups included four which were dosed with from 6250 to 50,000 IU of vitamin A into the crOp and two which received no supplemental vitamin A. The mortality following experimental infection was quite high in all treat- ment groups receiving NDV. In another trial all birds received a complete starter diet for four weeks. At this time seven treatment groups received vitamin A into the crop at levels of from 780 to 50,000 IU of vitamin A. Again two treatment groups received no additional vitamin A other than that contained in the complete starter diet. NOV was again given orally into all birds except the noninfected control group. The mortality again was high in all infected birds. Squibb and Veros (1961) concluded that sup- plemental vitamin A did not alter the mortality of birds either on a vitamin deficient diet or on a diet that meets National Research Council standards. Underdahl and Young (1956) studied the influence of dietary intake of fat-soluble vitamins on the intensity of experimental swine influenza virus infection in mice. Mice received diets which were either adequate or -10- low in vitamin A. After four weeks all rats were intranasally inoculated with swine influenza virus. The mice receiving supplemental vitamin A in the diet showed increased resistance to the swine influenza virus. This was indicated by a lower mortality rate and less severe lesions in the mice showing infection. These workers stated that the increased morbid- ity of rats on inadequate vitamin A was not due to a lowering of the a- bility of the mice to produce antibodies. Several authors have reported the effect of vitamin A deficiency up- on resistance of the host animal to parasitic infection. Zinsser _t al. (1931) found that rickettsia infections were difficult to initiate in rats receiving vitamin A, whereas, rats exhibiting xerophthalmia developed an exudate rich in the organisms following intraperitoneal injections of the rickettsia. Ackert 23 al. (l93l)have shown that vitamin A included in a diet in- creased the resistance of thi:kens to parasitism by Ascaridia lineatia. In a study involving 200 chickc.s, Ackert SE al. (1931) separated the birds into two treatment groups one of which received a semi—synthetic diet free of «itamin A, and the other the same diet supplemented with vi- tamir A. After only two weeks on test each bird received 500 embryonated eggs of Ascaridia lineatia. Three weeks later all birds were killed and the intestines removed and flushed clean of the worms. The following ta— ble indicates the increased incidence and size of the ascarids in the birds on the vitamin A free diet. TABLE 1. INFLUENCE OF DIET ON NUMBER OF ASCARIDS No. gf_worms Length of worms (cm) Vitamin A deficient diet 58.4 49 Positive control 23.6 12 Commercial diet 11.3 - 11 _ Hirashi (1928) of Japan investigated the resistance to human ascaris of swine in an avita"inosis A condition and when fed adequate dietary vi- tamin A. The pigs on the vitamin A deficient diet became parasitized with the human ascaris while the positive control pigs failed to become in— fested. Payne 33 al. (l925) had previously reported that human ascarids would ot establish a normal reproductive cycle in swine. Wright (l935) subjected dogs infected with ascar'ids to a diet ei- ther adequate or deficient in vitamin A for periods up to l06 days. The dogs on the deficient diet harbored about five times as many worms as did the control dogs on at adequate diet. Pande and Krishnamurty (l959) became interested in the inter-rela- tionship between hypovitaminosis and Ascaridia galli infestation in poul— try. The birds deficient in vitamin A developed the characteristic clin— ico-pathological changes of the epithelium. The altered epithelium favor— ed infestation by the Ascaridis galli which in turn further altered the mucosa of the epithelial tissue. Mori (l922), Wolhach and Howe (l925), Goldblatt and Benischek (l927), SicFried (l930), Castellanos and Beato (l9hl) and Follis (l955) have re- ported the histological changes observed in various tissues of the vitamin A deficient animal. In these pathological studies the vitamin A defi- cient animals exhibit a metaplasia of the normal columnar type of epithe- lium to the squamous kerati ized type in areas of the respiratory, ali- mentary, and genito-urinary tracts, the para-ocular glands and the eyes. Richards (l935) stated that changes in the intestinal epithelium are visible to the naked eye following three weeks feeding of a vitamin A free diet. Cramer and Kingsbury (l924) reported that the intestinal mu- - 12 - cosal glands undergo atrophy in severe vitamin A deficiency. Bacterial infections were reported to follow the alteration of the epithelium. Results of intraperitoneal and intravenous injections of bacterial cultures as reported by Boynton and Bradford (l93l), Sayedain and Kinsy (l960), Tisdall (l950) and McClung and Winters (l932) indicated that the deleterious effect of a vitamin A deficiency can not be explained entirely on the basis of altered epithelium or a defense at the outer surface of the body. Guggenheim and Buechler (l9h6) reported that in vitamin A deficient rats phagocytosis was not altered when measured two hours after bacterial infection, but after four hours the rats receiving vitamin A exhibited a significantly greater level of phagocytosis. Werkman (l932a) concluded that phagocytosis was not altered by a lack of vitamin A in the diet. Cottingham and Mills (l9h3) considerably later found that a lack of the combination of vitamin A and vitamin D resulted in a decreased rate of phagocytosis. Osborn (l932) found a lowering of complement activity in vitamin A deficient rats. However Axelrod and Pruzansky (l955a) have shown comple- ment level to be inhibited by inanition which could possibly account for the results by Osborn and others. Greene (1933) carried out complement fixation upon blood from vi- tamin A deficient rabbits and from rabbits on adequate levels of vitamin A. No differences were recorded between the control rabbits and those on a vitamin A deficient diet. Feller _t _l. (l942) found no reduction of complement in humans with hypovitaminosis A. Experiments to measure the effect of vitamin A deficiency upon anti- - 13 _ body production have been carried out by many investigators. Werkman (l923a) was one of the first investigators to turn to measurements of antibody production as a criterion of nutritional adequacy. In a study with ll rabbits, six of which were fed a diet low in vitamin A, Werkman measured antibody titer to Salmonella typhosa. The diet in this study, consisted of white corn, linseed oil meal, ground oats, casein (alcohol extracted), tankage, calcium carbonate and sodium chloride. Cod liver oil was used as a source of vitamin A. The rabbits remained on this diet for a period of seven weeks before initiating antigen injections. Five injec- tio s of a tige: (.2, .3, .4, .6 and l.0 ml.) were administered to the rabbits at seven day intervals. No differences were found between the antibody titers ofthe control group and the grOUp on a vitamin A defi- cient diet. Werkman (l923a) then repeated the experiment with rats using a diet corsisting of casein (alcohol extracted), dextrin, salt mixture and yeast. Again the antibody titer was just as high in the rats receiv- ing no supplemental vitamin A. With the same diets as above, Werkman (l923a) next measured hemoly- sins using rat erythrocytes in rabbits and rabbit erythrocytes in rats. The vitamin A deficient rabbits immunized with the rat erythrocytes show- ed the same hemolysis response as the controls. However, the vitamin A deficient rats showed slightly lower hemolysin titers than did the con- trols. Cramer and Kingsbury (l924) fed diets to rats deficient in either vitamin A or B complex. They reported no decreased agglutirin formation against Escherichia coli or Salmonella typhosa. However, neither the diet nor the length of time the animals were maintained on the diet was discussed. Blackberg (l928) has studied the effect of avitaminosis A and B on the immunity of rats. In the first study, Blackberg injected killed cul- tures of Salmonella typhosa into rats which were on diets either defi- cient or adequate in vitamin A. The vitamin A deficient rats consistent— ly developed a lower antibody titer than that measured in the controls. In a second study with treatment groups as before Blackberg injected a broth media culture of Salmonella typhosa. The antibody titer showed a higher value for the rats receiving adequate vitamin A when measured one week after the final injection. With further live Salmonella typhosa culture injections the difference in the antibody titer of the two treat— ment groups diminished. The deficient animak with sufficient stimulation eventually produced antibody titers equivalent to the control rats. In a third study Blackberg used tetanus toxin as an antigen. The vitamin A deficient rats responded with a very meager antibody production compared to a very high titer in the control animals. Greene (l933) conducted an extensive study of changes of actively ac— quired immunity under conditions of a vitamin A deficiency. Greene meas— ured hemolysins to ox and sheep erythrocytes and agglutinins to Salmonella typhosa in rabbits receiving either a vitamin A deficient diet or one supplemented with vitamin A. The rabbits were fed the dietary regime un- til xerophthalmia had been observed in the vitamin A deficient group for several weeks. Two injections of washed sheep erythrocytes were intra— venously injected into the rabbits. Eight days after the second injection the hemolysin titer was measured. In the first trial the deficient ani— mals had from 250- 2000 hemolytic units and the controls from 5000—200,000 - 15 _ units. In a study with ox erythrocytes the deficient rabbits had from 7.5 - 3O hemolytic units and the controls had from l25 - 1250 units. The same author (1933) injected Salmonella typhosa into xerophthalmic and con- trol rabbits and found a somewhat lower antibody titer in the vitamin A deficient rabbits when compared to the controls. Lassen (l930,l93l) working with Salmonella paratyphoid in vitamin A deficient and control rats reported some reduction in agglutination titers in the vitamin A deficient rats. Natvig (l942) conducted a three year study on the influence of vitamin A and B complex upon resistance of rats to Salmonella danycz and Leptospira icterohemorrhagia. He could find no evidence of reduced antibody production in vitamin A deficient rats. Simola and Brunius (l933) reported that the hemolysin titer to sheep erythrocytes was just as high in vitamin A deficient guinea pigs as in controls. Feller _t _l. (l942) examined the relationship between low vitamin A levels in humans and antibody production. This study involved only three human patients studied over a period of one year. The antibody ti- ter for these individuals was not different from control patients; how- ever, the plasma vitamin A level of the three patients never fell below lOO IU per lOO milliliters of blood plasma. Ludovici and Axelrod (l95la) have compared the level of circulating antibodies in rats receiving a complete semi-synthetic diet or diets lacking in pteroylglutamic acid, niacin-tryptophan, vitamins 812, A or D. After the rats had been maintained on the various diets for four weeks immunization was initiated. A l0 percent suspension of washed type 0, Rh positive,human erythrocytes was injected intraperitoneally as the antigen. _ 16 _ Five days after the second of two injections the rats were exsanguinated and the antibody titers were determined. The average hemagglutination ti- ter for the control rats was 1984 as compared to 533 for the vitamin A de— ficient animals. Axelrod (1953) and Axelrod and Pruzansky (1955a) again measured he- magglutination response by rats on vitamin deficient diets. Axelrod in- cluded ad libitum,pair fed and pair weighed controls. The antigen as in the previous study was type 0, Rh positive,human erythrocytes injected in- traperitoneally. Once again the vitamin A deficient rats were able to pro- duce antibodies but the hemagglutination response was significantly less than in the control animals. The ad libitum,pair fed and pair weighed con- trols all produced similarly high levels of hemagglutinins. A later study was carried out by Pruzansky and Axelrod (1955) in which antibody production was measured to diptheria toxoid in vitamin de- ficient rats. Inanition controls and ad libitum controls were maintained in this study as well as vitamin deficient rats. The rats received a semi-synthetic diet based on vitamin free casein and sucrose. After the rats had been on feed for 12 days, each was given a single intraperitoneal injection of .15 milligrams of alum precipitated diptheria toxoid. Seven- teen days later the rats were bled by cardiac puncture. The antibody ti- ters were determined by hemagglutination of sheep red cells treated with tannic acid and coated with diphtheria toxoid. Just as Axelrod (1953) re- ported in the previous paper, the vitamin A deficient rats produced a con— siderable lower antibody titer than did the control rats, 1300 and 3900, respectively. The literature contains much conflicting data as to the importance _ 17 _ of vitamin A in antibody production. One contributing factor may be that an intended vitamin A deficient diet may not always have resulted in such a deficiency. The early work was subject to error in that the true com- position of the natural diet as well as quantitative and qualitative re- quirements were not well established. Some authors have been prone to I use only gross criteria in determining a deficiency status. Levels of serum or liver vitamin A were reported only in rare instances. B. Pantothenic Acid,gPyridoxine and Riboflavin In an early study Verder (1928) reported that Salmonella enteritidis did not cross the intestinal wall unless the rats were deprived of vitamin B complex of yeast. Rose (1928) reported that many cases of bacteremia caused by Clostridium perfringes were cured by injections of vitamin B complex. Zinsser 35 al. (1931) using a diet lacking in all the then known B vitamins found that rats and guinea pigs were much more susceptible to murine typhus infection. Ross and Robertson (1932) placed rats either on diets deficient in the B complex or diets in which supplemental B complex had been added. Salmonella murotitis organisms were introduced orally into the rats after the rats had been on trial one week. The mortality was much greater in the rats receiving a diet deficient in the B vitamin com- plex. Rose and Rose (1936) observed that dogs receiving from 13 — 33 per- cent of the required amounts of the B vitamins known at that time became much less resistant to infections of Staphlococcus aureus than were dogs receiving the recommended amounts of B vitamins. Pinkerton and Bessey (1939) reported that rats maintained on a ribo- flavin free diet for seven weeks were more susceptible than controls to -18- murine typhus and were more severely affected by the infection. Seeler and Ott (1944) have reported a decreased susceptibility to Plasmodium lOphurae infection in chicks suffering from riboflavin defi- ciency. In this study two levels of riboflavin were fed, 20 and 2000 mi- crograms per 100 grams of diet respectively. After 11 days on the respec- tive diets one-half of the chicks in each treatment was infected with Plas- modium lophurae. The progress of the infection was measured by determining the frequency of parasitized erythrocytes in circulation and mortality of the birds. Three percent of the erythrocytes from the riboflavin deficient birds were parasitized while 17 percent of the cells from the chicks fed high riboflavin levels were parasitized. In a second trial the feed of the chicks on the high riboflavin diet was limited to the quantity of that con- sumed by the chicks on the low riboflavin diet. The results with the pair fed controlsiwwwzthe same as that reported for the ad libitum feeding tri- al. Although the parasitism of the riboflavin deficient birds was low, the mortality of these birds was higher (61 percent) than it was among the birds on the high riboflavin diet (29 percent). Uninfected controls on either dietary treatment had a low mortality. The increased mortality of the in- fected birds was manifested in a manner other than increased parasitism of erythrocytes. < Wooley and Sebrell (1942) found that mice maintained on a diet defi- cient either in riboflavin or thiamin were more susceptible than ad libitum controls to intranasal infections of Diplococcus pneumoniée. In a similar experiment using intraperitoneal introduction of Diplococcus pneumoniae, Day and McClung (1945) fed a diet deficient in pantothenic acid for from 19 to 38 days. The pantothenic acid deficient rats were no more suscep- _ 1o _ a tible than were §§.LLELEWH control rats. Seventy-four percent of the pan- tothenic acid deficient and 69 percent of the controls died. Mortality was the only reported criteria. Robinson and Siegel (1944) placed rats on a complete control diet and diets deficient either in pantothenic acid or riboflavin. All rats were Sebrell (1942) had done. No differences were observed in the resistance by the rats on any of the diets. West et al. (1944) found that pantothenic acid deficient rats exhibited an increased resistance to experimental in- fection of Qiplogogggs pnegmggiae when introduced by nasal insufflation. They concluded that the pneumococcus required pantothenic acid for growth, and that in the deficient animal the pathogen was unable to obtain suffi— cient pantothenic acid for maximum growth. Kligler et al. (1944) demonstrated that when mice were fed a diet de- ficient in riboflavin the susceptibility to Salmonella typhjmgiigm was greatly increased. Guggenheim and Buechler (1946) supported these findings in a study in which rats were fed a diet low in riboflavin, thiamin or vi- tamin A. All rats were intra—abdominally injected with Salmonella typhi— TEELHE following four to five weeks feeding of the respective diet. A significantly larger number of extracellular bacteria was present in the peritoneal fluid of the riboflavin deficient rats. Guggenheim and Buechler (1946) concluded, without using pair fed controls, that the reduced bac- tericidal activity of the riboflavin, thiamin and vitamin A deficient rats was due to the reduced feed intake. Fitzpatrick (1948) found an increased susceptibility of rats to mur- ine typhus infection when fed diets deficient in pantothenic acid, pyri- - 20 - doxine or riboflavin. A deficiency of thiamin did not appear to increase the severity of the infection from that of the controls. Smith ard Reynolds (1961) studied the influence of dietary levels of riboflavin from O - 150 milligrams per kilogram of diet upon resistance to Leptospjra pgmona in hamsters. The diets were maintained six to eight weeks prior to experimental intraperitoneal infection. The pathogenicity of: the virus culture varied in the three different trials from causing no mortality in the first trial to complete mortality at six days post injec- 't ion in the third trial. No differences in resistance were recorded be- ‘t\~een any of the levels of dietary riboflavin. Watt (1944) introduced infections of Nippostroggylus muris into rats ‘F e:d ad libitum either a control diet or a diet deficient in riboflavin. 'Tfie control rats exhibited more resistance to an infection of the parasites tlwan did the riboflavin deficient rats. A second experimental infection of Nippostrongylus muris was introduced and the control rats presented almost a complete immunity to the organism while the riboflavin deficient rats were severely affected by the second infection. Watt (1944) suggested that the decreased resistance to the second infection may be due to a low anti- body titer against Nippostroggylus muris. Several workers have shown that deficiencies of the B vitamins do h(>1: lower the resistance to experimental viral infections. Rasmussen t l. ( 1 944) studied the influence of riboflavin deficiency in mice upon experi- mental poliomyelitis infection. After the mice had received the experi- n'Iehtal diet for 14 days they were injected with either Lansing poliomyeli- ti 5 strain or encephalomyelitis GD VII. No difference in resistance was ObServed between the control and the riboflavin deficient animals follow- - 21 ing encephalomyelitis GD VII i je.tion; however, the riboflavin deficient mice were less se.ere1y affected by the Lansing strain poliomyelitis injec- tions that were the tontrols. Lichstei _t al. (1944) working on the same research team reported tiie influence of pantothenic acid deficiency on resistance of mice to ex- p>erimenta1 virus irfection. Cultures of either Lansing strain poliomyeli- ‘t is, or Theilero encephalomyelitis were injected intracerebrally follow- i ng two weeks on the pantothenic acid deficient diet. In this study the F>antothenic acid deficient mice were less severely affected by the Theilero eercephalomyelitis than were the controls. No difference in resistance was c>bserved when Lansing strain poliomyelitis was injected. Mirick et al. (1945» injected Lansing strain poliomyelitis into rats rnaaintained on a pyridoxine deficient diet. A slight increase in resistance vveas reported for the pyridoxine deficient rats. Lichstein _t al. (1945) c;c>uld demonstrate no difference between the resistance of pyridoxine defi- czi e:t mice and the controls following an intracerebral injection of Lansing srtrair poliomyelitis. Seronde _t al. (1956) studied the pathogenicity of Corynebacterium £;-l97 in rats maintained on diets deficient in partothenic acid, pyridoxine c>r- thiamin. The incidence of Spontaneous as well as experimental infec- t'i<>n was measured involving this normally non-pathogenic organism. Inani- tii c>n controls, ad libituw controls and infected and non-infected deficient tr"eatments were included in this study. Spontaneous infection of Eoryn - EifiELierium C-197 was observed only in the rats on the pantothenic acid defi- C leant diets. After the rats had received the experimental diet for 30 Cha)’3 the Corynebacterium C-197 was intraperitoneally injected into the \ _ 22 _ rats. The experimental infection was fatal to many pantothenic acid de- Ficient rats. The pyridoxine deficient and thiamin deficient rats were less severely affected and no apparent infection was established in the control rats. In an attempt to determine the humoral factors which may become al- tered in B vitamin deficiencies many authors have turned to studies of antibody production primarily in rodents. Werkman (1923a) as was pointed ()LJt in the vitamin A section, measured antibody production to Salmonella £thosa in rats and rabbits deficient in vitamins A and B. The deficiency OF either vitamin did not result in a decreased antibody titer, when the diets were made up of natural foodstuffs. Morey and Spies (1942) measured antibody response in humans showing clinical manifestation of pellagra, beri beri and riboflavin deficiency. Avirulent forms of Pasteurella tularensis were given in three daily injec- t ions to patients testing negative to this organism prior to the initiation 0F the study. The titers were measured periodically following the antigen i nj ections. The more severe the symptoms of the three B vitamin defici- encies the lower were the measured antibody titers. Also the titers were ma intained a shorter period of time in these patients showing the more seve re symptoms . Ruchman (1946) determined the effect of particular vitamin defici- enci es on the development of neutralizing antibodies against the virus of Wes tern equine encephalomyelitis in mice. Mice received the control diet 0‘” experimental diets deficient in riboflavin, thiamin or total B vitamins For- 17 days prior to vaccination with a 4 percent formalin solution of We313ern equine encephalomyelitis virus. Two weeks later all mice were - 23 _ bled out and the serum antibody titers measured. The antibody titers were similar for all treatments. The results obtained by Ruchman are consistent with the failure of a deficiency of B vitamins to lower resistance of mice to viral infections. Stoerk and Eisen (1946) evaluated the influence of a pyridoxine de— ficiency upon antibody production in rats. In addition to the deficient diet treatment, pair weighed and ad libitum controls were included. All rats reCeived a semi—synthetic diet. During the fifth week of the study 6311 animals were injected with the first of three intraperitoneal injec- tions of sheep erythrocytes. Five days later all rats were exsanguinated éand hemagglutination and hemolysin determinations were made. The average fienmgglutination titers were 0.4 and 64.0 for the pyridoxine deficient and tine oair weighed controls respectively. The hemolysin titers were 13.0 airid 412.0 in the same order as above . Six of the nine pyridoxine defi- c:ilent rats had no measurable antibody titer. Stoerk t 1. (1947) in a similar study measured serum antibody titer i r. rats or diets deficient in pyridoxine, thiamin, riboflavir, partothenic .a-:'id or protein. A semi—synthetic diet was fed to all rats with appropri- .a't<: omissiors for the deficient diets. The first of three intraperitoneal irij ections of sheep erythocytes was begun during the fifth week of the t:r'i.a 1 followed by two additional injections on alternate days. The pyridoxine deficient rats exhibited a significantly lower hem- agglutination titer than did the pair fed or ad libitum controls. The reSponse to the sheep erythrocytes was low in all treatments. Axelrod e l. (1947) measured the titer of circulating antibodies 1r) "éits deficient in pantothenic acid, pyridoxine or riboflavin. Pair g -21.- fed and ad libitum controls were included along with the deficient diets. All diets were semi-synthetic comprised of vitamin free casein and sucrose fortified. After seven weeks on the experimental diets the rats were im- n1unized with intraperitoneal injections of type 0 human erythrocytes. Five ciays later all rats were exsanguinated and hemagglutination titers were (determined. The pantothenic acid and the pyridoxine deficient groupsfailed 1:0 produce a measurable hemagglutination titer. Forty-two percent of the r‘iboflavin deficient rats failed to produce a measurable titer and the r‘emainder produced low titers. In contrast, to the work by Stoerk et al. (1947) with sheep erythrocytes the titers of the pantothenic acid defi— <:ient grOUp was also inhibited. Agnew and Cook (1949) measured antibody production in pynidoxine de- f’icient rats. The study in addition included ad libitum, pair fed and pair vveighed cortrols. The rats remained on the respective experimental treat— nwents for six weeks prior to the antigen injection with sheep erythrocytes i n one study and formalinized Salmonella typhosa in the second study. The Piemagglutination response to the sheep erythrocytes and the agglutin r'esponse to the Salmonella typhosa were significantly lower in the rats freclthe diet free of pyridoxine than for any other treatment. The circulating antibodies were compared by Stoerk (1950) in rats r‘eaceiving a pyridoxine deficient diet and the same diet with deoxypyridoxine added. All rats remained on the experimental treatment three weeks prior 11C) immunization with sheep erythrocytes. Five days following immunization tlfie hemagglutination titers were determined. The rats on the deoxypyri- <3C>xine treatment produced a significantly lower titer than did the un- COmplicated pyridoxine deficient rats. In a second experiment Stoerk(l950) _ 25 _ immunized both rats and mice 20 weeks before the start of the feeding trial. All rats received deoxypyridoxine and one—half the rats received pyridoxine in the diet. The experimental feeding period was continued three weeks before a second injection of the original antigen was admin— i stered. The anamnestic response one week following the injection of the auntigen was high in the rats and mice on the adequate diet and not detect— gable in the deficient animals. Axelrod t l. (1961) determined the circulating antibodies present i n guinea pigs fed diets deficient in pyridoxine,containing deoxypyridoxine c>r control diets. Injections of .15 milliliters of alum—precipitated ciiphtheria toxoid preparation were given parenterally after four weeks Feeeding of the respective diets. Three weeks later the guinea pigs were I) led and a second dosage of diphtheria toxoid was administered for measur— i rig secondary response. The antibody titer in both the deficient group and tine group receiving the antagonist were severly inhibited when the primary r'ezsponse was measured and somewhat less inhibited when the secondary re— sponse was measured. Ludovici et al. (1949) measured the circulating antibodies in rats ciezficient in pantothenic acid and pair weighed controls. The study was C17 \Iided into three groups with each containing both the deficient rats and <3<>r1trol rats. The three groups were maintained on the respective diets for tint—nly in the amount of methionine added to the particular diet. Each tri- eal consisted of a positive control diet, positive control plus DL—methi- c>nine, pantothenic acid deficient diet and the deficient plus methionine. 'The levels of DL-methionine added were 2.7 percent in the first trial and l .4 percent in the second trial. The rats were immunized during the Sxeventh week of the trial with human erythrocytes. None of the unsupple- rntented pantothenic acid deficient animals possessed serum antibody titers Pfi'igher than 320. In contrast 60 percent of the pantothenic acid deficient rfiats SUpplemented with DL-methionine at either level exhibited titers of 6540 or higher. The controls had titers that measured at least 2560. Sup- F>lemental DL-methionine did not alter the antibody titers of the normal Ctbntrols. The addition of methionine to the pantothenic acid deficient ¥ -28- diet did not alleviate the growth depression. In a similar study B-alanine was added to a pantothenic acid deficient diet. The addition had no ob- servable effect upon the antibody titer or increase in weight. Ludovici and Axelrod (1951b) have also substituted pantothenol for gaantothenic acid and found the antibody titer to human erythrocytes was j ust as high as the normal pantothenic acid controls while the deficient ggroup failed to produie a measurable titer. Wertman and Sarandria (1951a) using the same semi-purified diets as LJsed by Stoerk (1946) ard Ludovici (1949) reported no inhibition of anti- t>ody productior in rats receiving 10 percent of normally recommended a- rnannts of all the B vitamins. The diet was fed for six weeks prior to the i:.itiatio: of a series of weeLly injections of formalinized Rickettsiae ‘t>/phi. The rats on the B vitamin limited diet did not grow normally but ttie serum antibody titer was not inhibited. In a second study the treatments included diets totally lacking in B vitamins, deficient in pantothenic acid, deficient in thiamin and a nor— nna 1 control. The procedure was just as before except that the titer was nneéasured one week after the first injection and one week after the third ilfi;iection. Only the pantothenic acid deficient group exhibited a lower t i1:er after the first injection; after the third injection the rats on the deef“icient diets had similar titers which were only slightly lower than the nc>rrnal cortrols. Wertman and Sarandria (1951b) and Wertman _t _l. (1952) compared the ‘afaFfiicts of deficiencies of pyridoxine, nicotinic acid, riboflavin and folic a‘:l Cl upo; levels of complement-fixing murine typhus antibodies. After three v - . . . . . . . “€3€3b~s on the respective diets a series of five weekly injettions of forma- - 29 - linized suspension of Rickettsja§_typhi was commenced. The serum was ex— amined for antibodies one week after the first and fifth injection. The pyridoxine and folic acid deficient rats produced an extremely low comple- ment-fixing titer. The riboflavin deficient rats produced a titer mid-way between that of the pyridoxine deficient rats and the control rats. The niacin deficient, pair fed control and ad libitum controls all produced a LJniformly high complement—fixation titer. Zucker et al. 0956) have measured serum antibody levels in rats fed (diets deficient in pantothenic acid, pyridoxine and thiamin. Following 30 rought about an increase in erythrocytes and hemoglobin of the rats. Beck 35 al. (1950) also reported decreasing erythrocyte counts and tiemoglobin values in pyridoxine deficiencies. They also found a decrease in 'total leukocytes. This is in agreement with Axelrod and Pruzansky (1954) vvho also found a decrease in total leukocytes. Miller a5 al. (l957a)found ea significantly decreased hemoglobin and a slightly decreased red blood <:ell count in pyridoxine deficient pigs. The presence of xanthurenic acid was first identified in the urine c>F pyridoxine deficient pigs by Cartwright a: al. (1944). Wintrobe a: al. ( 1943) a year earlier had reported a green pigment producing substance in tfie urine of pyridoxine deficient animals. Wintrobe _£ al. (1944) reported a moderate anemia in swine fed a I‘ilooflavin free diet. In a study of riboflavin deficient pigs Mitchell SiE.‘_l. (1950) concluded that the most sensitive index of riboflavin defi- <31€ancy is the increase in concentration of neutrOphilic granulocytes. ”411 ler a3 al. (1956) confirmed these findings and also found an increased .-.“. -—_—._ .-._Ir. _ 31 _ total leukocytes in riboflavin deficient pigs. Wertman 35 al. (1957) reported only a slight increase in polymorpho- nuclear neutrophilsand a slight leukopenia in riboflavin deficient rats. No change in the total erythrocyte count was observed. Complement activity was decreased in the riboflavin deficient as well as inanition control rats. Shukers and Day (1943) supported the observation that a riboflavin defi- ciency was followed by a leukopenia. Axelrod and Pruzansky (1954) found a decreased total leukocyte count. Wintrobe (1943) reported that a deficiency of pantothenic acid was associated with the development of only a moderate normocytic anemia. Axelrod and Pruzansky (1954) could find no change in total leukocyte count in pantothenic acid deficient rats. Stothers _£ _1. (1955) observed an in- crease in leukocytes in pantothenic acid deficiency. Recently Furness and Axelrod (1959) have reported a leukopenia and lymphopenia in pantothenic acid deficient animals. The effect of vitamin deficiencies upon disease resistance has been the subject of a multitude of papers. Just as was the case with vitamin l\, research groups have not all agreed as to the importance of particular B vitamins in maintaining resistance to infection. Agglutination provides just one of the many lines of defense within the body. However, due to the importance and also to the ease of deter- niination, antibody production in relationship to nutrient deficiencies liaas been one of the defenses more thoroughly studied. Axelrod and Pruzan- slléasma drawn off. The remaining cellular material was rinsed twice with _ 38 _ physiological saline and centrifuged. Alsevers solution was finally added to the cells in a sufficient quantity to give a 20 percent erythrocyte so- lution. Serum antibody titers were determined according to the tube serial dilution technique described by Stafseth SE 31. (I959). A 0.2 milliliter quantity of serum was serially diluted from l:5 - 1:5120 and tested with an equal volume of diluted antiger suspension. All agglutination titers reported represent net values determined by deducting the pre-injection ti- ter from the final titer. The prepared tubes for the Salmonella pullorum antibody tests were shaken and then allowed to incubate at 37° C. for ZR hours. They were then placed in a cold room (40 C.) for one hour and then the agglutination titer was determined. The tests for the type 0 erythro— cytes were prepared as the above and allowed to incubate for one hour. The tubes were then cooled and the hemagglutination titer determined. Serum Protein and Electrophoresis The determination of serum protein and the electrOphoretic com- ponents was carried out at the time of initiation of antigen injection, at the close of the depletion, and at the initiation of antigen injection in the repletion phase of the trial. The serum protein was determined according to the method first de- scribed by Waddell (l956). Five lambda of serum was diluted to five milli- liters (lleOO) with 0.9 percent NaCl. The reading was made at wavelengths of 2l5 mu and at 225 my on a Beckman Model DU Spectrophotometer. The ab- sorbance at 225 mp was subtracted from that at 2l5 mu. This difference multiplied by lhh gave the protein concentration in the serum expressed in nficrograms per milliliter. This value was then converted to grams per- Cent. - 39 - The serum protein fractions were separated on a Spinco, Model R, paper electrOphoresis system (Spinco Technical Bulletin 6027A) at room temperatdre. A constant current of three milliamperes per cell was main- tained for 16 hours on Spinco number 300-846 paper strips using a veronal buffer of pH 8.6 and an ionic strength of 0.075. This buffer was made up of 2.26 grams of di-ethyl barbituric acid and 15.4 grams of sodium di- ethyl barbiturate per liter of distilled water. Approximately 0.006 milliliters of serum was applied to each pa- per strip. Following the electrophoretic separation, the strips were dried for 30 minutes at llOO C., dyed with brom phenol blue, rinsed with acetic acid and dried once again. The basic color was develOped with am- moniUm hydroxide and the relative intensities of the separated proteins were determined by scanning with a Spinco Model RB Analytrol with a number five cam. Serum Vitamin A Determination Serum samples collected in Trials I and II were analyzed for vitamin A according to the method by Sobel and Snow (l9h7). Slight modifications were made to adjust the reagents since two milliliters of serum were used. The samples were kept in amber glassware throughout the extraction and the evaporation periods. The serum samples were all analyzed colorimetrically using a Beckman Model DU Spectrophotometer. The color producing reagent in all analyses was l,3-dichloro-2 propanol(glycerol dichlorhydrin). The samples were analyzed as rapidly as possible, usually within two weeks. None of the samples remained frozen (-230 C.) longer than six weeks be- fore analysis. Heaney (l960) reported only slight loss in vitamin A con— tent after six months of freezing. E l Hemoglobin Determination Hemoglobin was determined by the cyanmethemoglobin method described by Crosby et al. (l95R). A 0.02 milliliter sample of blood was collected with a Sahli pipette and diluted in five milliliters of Drabkins solution. Drabkins contains NaHCC, l.0 om. KCN 50 cm. K3Fe(CN)6 200 am. diluted to one liter with double distilled water. The light absorbence of the sample was read at 540 mu on a Bausch and Lomb Spectronic 20. Hematocrit Determinations The hematocrit values were determined by the procedure outlined by McGovern et al. (l955). The blood was collected in capillary tubes, sealed with flame and centrifuged at a speed of l2,000 RPM's for five minutes in an International hematocrit centrifuge. The hematocrit readings were taken on an International micro—capillary reader. Erythrocyte and Leukogyte Counts The blood for these counts was drawn into the respective ”Zero error Hellige tru count“ pipettes and diluted accordingly with physiological sa- lire for the erythrocytes and three percent acetic acid for the leucocytes. The counts for erythrocytes and leucocytes Were made on a Neubauer count- ng chamber by the method of Ham (l956). Slides for differential counts Were prepared according to the procedure of Wintrobe (l956) using Wright’s Stain. Values are presented for the percent lymphocytes and polymorpho— nLJclear neutrophils. Two counts of l00 cells each were made on the blood Sniears by the method described by Ham (l956). -41- B. Trials I and II. The Effect of Vitamin A Deficiengy In Swine Upon Specific Serum Antibody Titer Response This experiment consisted of two studies involving pigs from three litters in each trial. In Trial I, 2l Hampshire and Hampshire-Duroc pigs were weaned to semi-synthetic milk diet free of vitamin A at five days of age and placed in individual metal cages. The diet was bottle fed five times daily until the pigs were two weeks old. At that time the l6 thrif- tiest pigs were allotted according to weight, sex and litter to either a complete semi-synthetic milk diet containing 3960 IU vitamin A per kilo- gram of dry diet or to a vitamin A free diet. The number of feedings was reduced to four per day at this time. Weights were taken weekly throughout the depletion study. During the fifth week the diet was gradually switched to a dry form. Serum vitamin A levels were determined at weekly intervals starting when the pigs were six weeks old. The original plan had been to initiate antigen injections when the average serum vitamin A for the deficient pigs of a litter dropped to lo micrograms percent. However, it became obvious that the mortality rate of such pigs would be quite high. In subsequent litters the initiation of antigen injection began when the serum vitamin A level was in the range of l5 — 20 micrograms per milliliter. Prior to the first antigen injection,blood was drawn for initial or background antibody response, total serum protein and electrophoretic protein fractionation. Serum protein and antibody titers for Salmonella pullorum were determined as described, and the pigs were then moved to the Doane-type house and put on the starter ration based primarily on corn and soybean oil meal. The repletion period continued from 45 — 60 days. Once again the serum vitamin A level was determined and the human erythrocyte injections were initiated. _ 02 _ Measurement of the serum hemagglutination titer concluded the repletion phase of the trial. In Trial II the pigs were again selected from three different litters. However, in an attempt to produce a low serum vitamin A at an earlier age the pigs were removed from the sows at 12 hours of age. It was hoped that by limiting the intake of colostrum which Braude (l95l) and Davis St al. (l950) have shown to be extremely high in vitamin A, the pig would acqui re less vitamin A for liver storage. As in Trial I all pigs were bottle fed the vitamin A free synthetic milk until two weeks of age at which time they were allotted to either the vitamin A free diet or to the complete diet. Serum vitamin A level determinations were commenced at six weeks of age. In contrast to the findings in Trial I the serum vitamin A values of all the deficient pigs dropped below 15 micrograms per lOO milliliters at six weeks of age. The intraperitoneal injections of Salmonella pullorum were commenced immediately. As in the previous trial pre-injection serum anti- body titers and protein values were obtained. Following the serum agglutina- tion determinations the pigs began the repletion phase which continued for 30 - 50 days. Once again antibody response to human erythrocytes was deter- mined. C. Trials III and IV. The Effect of Deficiencies of Pantothenic Acid, Pyridoxine or Riboflavin in Swine Upon Specific Serum Antibody Titer Response The studies with the particular B vitamin deficiencies were carried out in two trials. The pigs were weaned to a synthetic milk diet at two Weeks of age. The only change in the synthetic diet in trials III and IV Other than omission of the particular B vitamin was to provide vitamins ancd D in cod liver oil rather than specific sources of each. A -43- Miller e: 31. (l954,l957a) and Stothers SE 31' (l955) have shown that deficiencies of the three B vitamins under question can be brought about much more rapidly than can a vitamin A deficiency. Also Harmon 5: al. (l959), Brown et al. (l96l) and Miller et al. (l962) have shown that the young pig is inefficient in producing measurable quantities of antibodies before six weeks of age. In an earlier study Miller _t al. (l957b) were unable to show an effect of pantothenic acid, pyridoxine or riboflavin de- ficiency upon hemagglutinin levels of three to four week old pigs. How- ever, extremely low antibody titer values were measured in all the pigs. Therefore, to avoid developing a deficiency before the pigs were capable of producing measurable antibody titers, weaning to a deficient synthetic diet was delayed until the pigs were two weeks old. Twenty—four Yorkshire—Hampshire pigs from three litters were started on a synthetic milk diet deficient in the three B vitamins under considera- tion. The milk was pan fed four times a day until the pigs were four weeks old. At this time 23 of the pigs were allotted to four different dietary treatments: positive control, pantothenic acid deficient, pyridoxine defi— cient, and riboflavin deficient. The pigs were then gradually switched to the dry form of the diet. After two weeks on the respective diets or a total period for each vitamin deficiency of four weeks, a series of six daily injections of Salmonella pullorum was begun. Immediately prior to the antigen injections the pigs were bled for determinations of initial or background antibody response, erythrocytes, leukocytes, differentials, total protein and electrophoretic protein frac- tions. Also at this time urinary xanthurenic acid levels were determined 7 n the pyridoxine deficient group and the controls, according to the meth- P -44- 0d of Wachstein and Gudaitis (l952), to give an indication of the severity of the deficiency. Twenty—four hour urine collections were made before and after the feeding of lOO milligrams of DL-tryptophan per kilogram of body l l weright. The pigs were housed in individual metabolism units during the per‘iod of urine collection. Seven days after the last antigen injection, the pigs were bled and the serum agglutination titer, total serum protein and electrophoretic pro- teiri fractions were determined once again. The pigs were then moved to the Doarie-type house and put on the starter ration. After six weeks the pigs were bled to determine background antibody response and protein values and chdl lenged with six daily injections of a 20 percent solution of type 0, Rh positive, human erythrocytes. The hemagglutination titers were measured one week after the last injection to conclude the repletion phase of trial three. In the final trial 25 Yorkshire-Hampshire pigs from three litters were weaned at two weeks to a synthetic milk diet deficient in pantothenic acid, pyridoxine or riboflavin. At four weeks of age the pigs were placed on dry diets and allotted to the four treatments as in Trial III. In addition, three groups of four pigs (one from each of the three deficient rations and a control constituting a group) were established as pair fed groups with the intake by all pigs within one group being limited to that amount eaten by the pig consuming the least. All pigs were bled at seven weeks of age in this trial for antibody background titer, protein values and cellular com- POnents, At that time a series of six daily injections of type 0, human erYthl'ocytes was commenced. The order of the use of the antigens was re— verSeCi in this study to determine if the choice of antigens had any influ- , -55- erlce or. the antibody response. The hemagglutination titers were determined seven days post—injection and then a repletion period of between seven and eight weeks was begun. At the close of this period serum antibody levels to Salmonella £19332 were determined one week after the last of Six daily injections of the antigen. r'r-‘--'I'_r—'-: T I'D-F'- ir fi—v- ... .. 1- IV. RESULTS AND DISCUSSION A. Vitami! A Studies Trial I A summary of the results obtained in this trial is contained in Ta- t>le 6. The differences between means throughout this trial as well as all the: vitamin A and specific 8 vitamin trials were tested for significance by the: studentized range method of Duncan(l955). In particular instances, lit- ter' effect differences were removed by using the method of Snedecor (l956) fCN‘ analyzing multiple classification variance. As evidenced in Figure l 35 l- —— Control Diet --——— Vitamin A Deficient Diet Weight in Pourds l l 1 l I I L, L 9 IO Age in Weeks FIGURE l GROWTH CURVES OF PIGS ON VITAMIN A DEFICIENT AND CONTROL DIET -46- A_o.ovav pcoEummcp cospo Lem o:_m> mcwpcoomoccoo cmgu coumocm >_p;mo.-w cmmme o3_m> cmmE cope: mwumzucocmo cw cmoE ecu co cecco ocmpcmpm: onmxe cowpo_;oc mc_c3p pomp _mc3um: opo_ano bo>woooc xmwo __mleom l :owpo_ooo "muowom A:.m_nv Am.Nva Am many Am.mnv _.mk m.mm moo.nmm _._m cmo_o seaswpeu om: .>< A_o._wv ANO.NMV Aom._wv A_a._wv Amm.wv Ao:._wv _.MN o.mm m.m em.e_ m.o_ N.N_ cc_sso_m m5;mm x .>< Aeo.wv Aka.“ “om._wv “we._nv Ank.wv ANN.MV «.s_ :.e_ _.m_ o.m_ o.N_ m.k_ :w_sso_m moss \ .>< Akm._nc Ask.“ A_N._nv A_m._nv Amm._nc A_m._nc . J.mm _.mN m.wm U©.:m w.:N b.~m tw_3po_o mca_m \ .>< Ame.mnv ANO._HV Ama._wv Akm.muv Aom.an AHm._nV o.km e.km ss.ms e._m :.ms M.m: .cgss_m \ ..e AON.HV 2mm.“ . Amm.nc Ams.nc Amm.nc A_N.nc - om.m km.m o_.m Nu.: .m.: mo.m A\ .Eov ecoBOLs Escwm ..< Am._nv Am._nv Am.mnv Aa._nv Am.mnv AN.NHV . m.mm m.mm em.mm :.m_ e_.em w.N_ A\ .ouqu :cgmocs Escmm .»< Aoo.nv Am_.wv es._ u:._ .ma_ .kcet.accco ewwc Ak_.n Am_.n Aeo.n, Aeo.nv - __._ o_._ em. 0:. .m;_ .ccm. s_cme .,< AN.O_MV Am.:_wv Aa.enc A_.enc Am.snv .Am.snv m.mm N.Nm m.mm w.:m _.NN , m.NN .mn_ .uemcmz .:< m w m m m m mmwo .oz oowm_uc0u “cowomcmo ommcwuc0m “cowowcop po_cmccoc “cowomeop < ccsmoc> < CWEmoc> < CWCmbc> < cwsmo_> < cmEmom> < enamom> >_m:ow>mcm >_mpo%>ocm cowumcwecoaop co_uoohcw comwucm o:_m> _mcmu cowwmcwu3_mmm mo oEwo u< Lo o.mw u< >osnm < 2H2«HHmlzc m < cz< Cum: ..omkzoo < no mmgngfi .H .__ucmowmw;:wmw “cospmocu cosuo cow o:_m> memocoomMLLOU :mzu Amo.o my LoumoLm >_ucmowmwcmwmo acmsumocu coLwo cow o:_m> mzwozoamoccoo :mzu “—0.0 av coumocm >_u:movmw;mmmt o3_m> cmoE cope: mwmospcocma 5? come esp we cocco tcmbccumo ommca compo—awe mcwcsp wove _oc:um: mum—aeoo po>mwooc mama __< ANn.w Amm.w Amc._wv Ack.wv Awm.mv Acm.wv a.» m.e_ :.o_ um.m_ ck.w m.a_ :c_sno_m magma x .»< Akm._wv Ak_._nv Aoe._nv Aoo._nv Ace._nv Ame.nv N.m_ ;.k_ m.o_ m.k_ N. _ o.k_ c__sno_a mbme \ .>< A; .nc “mo._nc A k.nc z_o.Nnc A- .nc Asm.nc 3.0N s.om m.km ee.mm _.:N as.km cc_sso_m aea_m \ .>< Amx._w Amm._wv Aeo._wv A_N.NHV Adm._wv A_o._wv *~._m m.n: p©.mq 0.3m pm.m: a.m_c cmE33_m \\ f.< A__.n Ao_.n Amc.nv A__.Hv Aso.wv Asa.wv on.m mn.m MN.J :N.m mo.¢ :m.; A\ .Emv conOLQ Eocom .>< Amm._nc Amm._nc AN_._HV A__._nc Asm.nc Aos._flc . J.Nm a.0m Us.em ..o_ Tm.eN e.N_ A\ ...swt ccgmsc. as... .>< Akmo.n Aoem.n 3N._ m 0.N .mp_ .mocowowmco boom Amdo.w Ame.“ Amoo.w AmN9.H. vmm._ _an. ewe. _N. .mg. . com s_cme .>< Aom.snv Amo.env Ask.mv fl_o._nw Amm.flv UAoe.wv JnN.m~ o.mm 1N._N :.m_ m.:_ m.mz .me_ .oemcmz .>< h J m m N m mmmq .oz vowumeOm “zomommmp pommwpcow ucwmowmoc bowmwbccc “nowowmwc < cmEmum> < ;wemu_> < nwEme> < cwEmuw> < :wEmum> < :wEmuw> >_m30m>mc¢ >_m:om>mcm :owumszcwump cowBUWch somwutm m:_m> .mcwc cowcmewus_mmm c9 as?“ u< co mews b< nfimwmmxa cowum_omm-._uawm moves; :owuo_awo ._fime >o3em < sz 2H aVVEer (P<0.0l) levels by the time vitamin A determinations were begun at - 57 _ six weeks of age. Apparently the drop in serum vitamin A was most rapid during early deprivation. Foot gt al. (l939) found that the liver vitamin A stores of baby pigs became exhausted at eight weeks of age when the dam re- ceived a vitamin A deficient diet for two weeks prior to parturition. In the second trial weaning to a synthetic vitamin A free diet at l2 hours of age (thereby limiting the total intake of vitamin A rich colostrum) result— ed in a considerably lower serum vitamin A level at six weeks than was meas- 640 0000 6&0 320 00 320 160 00000 160 0 XX _Ireatments Reciprocals 00 0 0 - Control8 80 0 XX 80 000 X of 000 X X — Vitamin A 000 Deficienta Antibod 40 0 X y LIo XX XX 0 Titers 20 XXX 20 XX XXX 10 XXXX 10 5 XX 5 Exptl. Depletion Exptl. Repletiona Phase Phase FTITGURE 7 TRIAL I AND II. INDIVIDUAL NET ANTIBODY TITERS PRODUCED AGAINST SALMONELLA PULLORUM IN THE DEPLETION PHASE AND HUMAN ERYTHROCYTES IN THE REPLETION PHASE a All pigs received complete natural diet during repletion I r_____e_wfi - 58 - ured in Trial I. Moore and Berry (l9hh) with dairy calves and Heaney (1960) with baby pigs have reported as much as a five fold increase in se- rum vitamin A the first ZA hours of life. The deficient pigs upon receiving the complete natural diet contain- ing 2300 IU per pound responded rapidly in appearance and in serum vitamin A levels. The correlation between antibody titer and serum vitamin A lev- els in Trials I and II were similar, .60 and .67 respectively. However, the correlation between weight gain and serum vitamin A was low (.AA) in the Trial I and quite high (.86) in Trial II. The results of Trial II strongly suggest that serum vitamin A and antibody titers respond more rapidly to vitamin A additions than does weight gain. A scatter diagram in Figure 7 clearly shows the differences in antibody production of con— trol and deficient pigs in the depletion phase and the increased similarity following repletion. The inhibiting effect of vitamin A deficiency Upon antibody produc- tion in pigs was even more pronounced in this study than was reported by lXxelrod _£ 31. (1947) with rats. However, Ludovici and Axelrod (l951) 'Fed a deficient diet for only four weeks and no attempt to measure either seyrum or liver vitamin A values was reported. 8.. Pantothenic Acid, Pyridoxine, and Riboflavin Studies Trial III A summary of the results obtained in this trial is presented in Ta- t>leas 8, 9 and l0. The riboflavin and pyridoxine deficient pigs were sig- rllfricantly (P<0.05) lighter than the control pigs after having received tile“ deficient diet for only three weeks. One week later, at six weeks of Amo.QV¢vmasoLm ucwmuwmou acmXOUmcxa Lo _oLucou cmgu swam mo tczoa Lon boom mo mncsoa uLoe >_ucmowmmcmvwo -59- masoLm acmsumoeu stuo __m Low o:_m> mcwvcoammLLou cmzp Amo.mev LopmoLm >_ucmowmmcmwm ommsa compo—dwu mchsp pomp —mL:umc mum—deco m m_nmh cm topmw_ pomp —mczymz - o3_m> cmoE Loos: mwmosucmcma cw cows 0:» comuo_amm p mo LoLLo vcmtcmpmo po>moomu mmwa __< Amn.w Ame.“ Amn.wv Amm.wv Am_._wv “mm.“ “mm.“ Am:.Hv :.N_ . ..N_ m.__ _.m_ :.m_ N.m_ _.:_ u_.m_ cu_gno_m mums x .>< AN_._MV Aoa.wv A__._hv A_m.nv “km.awv Ame.“ Ak_.NHV “om.“ w.m_ m.m_ m.w_ m.m_ _.mN 0.:N m.NN o.mm cu_:no_m m£a_m x .>< Amm._wv A_m.NHV Amo.muv Am_._uv Amm.duv Amm.mnv AmN.NHV Amm._wv m.w: :._m m._m m.m¢ N._: m.w: :.m: N._: cw533_m x .>< Am_.w A_N.MV ANN.“ AJN.H A_m.w AN_.HV AmN.H Ame.“ ox.m Nm.w Nw.m co.“ _o.m No.w mm.o am.m x .2m .cwmuota Estmm .>< AON.HV “km._mv Amm.wv A__.MV am.m mmN.m _w.N mm._ .xucmwuwccm name “mo.“ Ame.“ AN_.H Aqo.w “do.w Amo.wv Ao_.w “mo.“ :N._ m_._ om._ uo:._ m_. k_. mm. ems. .ma_ .c_mm s__me .>< Amm.~wv Am_.swv Ao:.ewv Amm.NHV Amw._uv AmN.NHV Amo.mwv ufikm._Hv N.Ok m.mw m.on em.km m.m_ m.m_ :.om nm.m~ .u;m_m3 .mc_c : a m m m m m m mama .02 .< .m 02mm mm opo_anU .< .m onmm mm mum—deco Nacwmomwoo xocwmuwmmo pcoEumoLH xumuwmo “cosummck xcmuowo L.momm;m compo—mwa ._ume mmmmcm cowuo_aoc ._pmxm >ascm zuz 11.1 4 1:: m 2H mmDu<> szHomm zsmmw oz< rpzoxu co >¢v:L_—“” /’ ’ / // fl/ ’/ ’/ ’f‘./ :-—“=—-—" l2 h 5 6 7 8 Age in Weeks FIGURE 8 GROWTH CURVES OF PIGS ON PANTOTHENIC ACID, PYRIDOXINE, RIBOFLAVIN DEFICIENT OR CONTROL DIETS TRIAL III. - 6l TABLE 9 I BLOOD CELLULAR VALUES IN B VITAMIN 3Tuny_, Experimental Depletion Phase Dietary Treatmenti Deficiency Complete 86 Ribo P. A. Hematocrit, % 44.1 34.0C 44.8 37.7 (t1.20) (t3.14) (t1.72) (t3.67) Hemoglobin, gm. % l2.93 lO.lO l3.l6 13.04 (tl.00) (tl.05) (t.5l) (tl.3h) Erythrocytes, 106 7.13 6.39 8.69 8.61 (t.h2) (t.70) (t.5h) (fl 05) Leukocytes, 103 25.86 18.57C 26.58 24.09 (fl.86) (tl.9l) (t2.hh) (t5.50) Differential Lymphocytes, % 49.0 6l.2 37.8d 52.2 (t2.6o) (t6.60) (t5.44) (£2.06) Neutrophils, % 48.2 37.5 61.0e 44.4 (t2.05) (t5.55) (t5.60) (t2.82) aDiets listed in Table 2 bStandard error of the mean in parenthesis under mean value CSignificantly lower (R<0.05) than corresponding value for ribo- flavin deficient or control pigs dSignificantly lower (P>mplete diet,the pigs formerly designated controls were still signifi- carthy heavier (P<0.05) than were the pigs that had been on the three -62 - deficient diets. Two pigs which were on the pantothenic acid deficiency treatment died during the course of this study. The data obtained from blood cellular studies as presented in Ap- pendix Table lh and summarized in Table 9 show the hematocrit of the pyridoxine deficient pigs to be significantly lower than for the control or riboflavin deficient pigs. The pyridoxine deficient pigs had a lower average value for hemoglobin and total erythrocytes though the differ- ences were not significant. The average leukocyte count in the pyridoxine deficient pigs was significantly lower (P<0.0E) than in the riboflavin deficient or control pigs. A further analysis of the leukocytes by dif- ferential count showed the riboflavin deficient pigs to have signifi- cantly lower (P<0.0S) lymphocytes than pyridoxine deficient pigs and higher (P<0.0S) polymorphonuclear neutrophils than the pantothenic acid or pyridoxine deficient pigs. The latter observation agrees with the findings of Mitchell et al. (l950) of an increased percent of neutrophils in a riboflavin deficient condition. Miller (l956) also reported an in- creased percentage of neutrophils in baby pigs fed no riboflavin. The feeding of diets deficient in pantothenic acid, pyridoxine or r-iboflavin for either four or six weeks did not result in a change of t<3tal serum protein, albumin, alpha globulin or gamma globulin. However, tfie percentage of serum beta globulin was significantly higher (P<0.05) iii the control pigs than in any other treatment. The statistical dif- f=earences between mean values of the serum beta globulin fraction were no 1<3nger evident after four weeks feeding of a complete natural diet. The ‘itfidividual protein determinations made periodically in this trial are <2Ontained in Appendix Tables 15, 16 and 17. _ 53 _ TABLE 10 TRIAL III. RECIPROCALS OF NET ANTIBODY TITERS IN B VITAMIN STUDY __Dietary_Treatmen£:________d_r . Deficiengy____m_ _____ Complete 86 Ribo P. A. Experimental depletion I76C b 33 23 l5 phase (216.0) (t9 5) (67.2) (t6.3) Experimental repletion 6O 65 S6 33 phase (I12.6) (IZA.5) (flh.h) (t4.8) dDiets: Depleti'n — Semi—synthetic diets listed in Table 2 Rujlttior - Natural diet listed in Table 5 o . . “Standard error of the mean in parenthe51s under mean value C... . Sign1f1cantly greater (P 0.0l) than corresponding value for all other treatment groups Data obtained from uri e studies indicate that urinary xanthurenic acid concentration is an excellert indication of pyridoxine insufficiency. ‘The pigs receiving a diet free of pyridoxine for six weeks had average xanthurenic acid levels of 278 micrograms per milliliter while the control Fed pigs had only 20 micrograms per milliliter aafter feeding of an oral dose of tryptophan However, even without addi- t ional tryptophan the pyridoxine deficient pigs excreted significantly Egr'eater amounts (P=0.05) of xanthurenic acid than did the controls. The antibody production to the §almgnella puLloium antigen in pigs C>r1 the three vitamin deficiency treatments was extremely low as is shown 1' r71 Table 10 and Appendix Table 18 and the means were highly significantly 1 C>VJer (P 0.01) than for the control pigs. However, as had been observed 1 r1 the vitamin A study all pigs were able to form hemagglutinins after r'63Ceiving complete natural diets for si x Weeks. Figure 9 presents the These values were obtained L -64- different net agglutinin and hemagglutinin titers in a graphic form. Trial [1 The data compiled in this study are summarized in Tables ll, l2 and 13. In contrast to the-previous study the average weight of the control pigs did not'become significantly heavier (R<0.0S)_until the deficient pigs had been on test seven weeks. The pigs were nine weeks of age at this time. Appendix Table l9 contains the individual feed and growth data for this trial and Figure lO presents the growth data graphically. The centrol pigs 640- 640- 3201» 320f O 160‘ CCOO 160+ Treatments . Y a Rec1procals 0 - Control 80- 80' ZZ 000 of Y Y Z X - Pantothenic Acid Deficienta Artibody [+0. X Z2 40- XX Y O Y - P ridoxine x Y zz Y Titers Y Y Deficienta 20 ,- 20- YY X 0 Z - Riboflavin X Z Deficienta lOP' Y' Z 10* XXX Y Z 5' 5’ Exptl. Depletion ’ Exptl. Repletiona Phase Phase FIGURE 9 TRIAL III. INDIVIDUAL NET ANTIBODY TITERS PRODUCED AGAINST §A£z MQEEEIE.EHLEPBPN.IN THE DEPLETION PHASE AND HUMAN ERYTHROCYTES IN THE REPLETION PHASE a All pigs received complete natural diet during repletion - 65 _ mmwa _oLucoo Lou o:_m> mcmvcoamoLLou cmcp Amo.o7¢v Lopmoem >_ucmowmwcmmmm masoLm acoEummLu Lmzuo __m cmgu cmmm mo vczoa Loo team we mvcsoa Amo.Qva mmO. >_ucmommwcmwme mama ucowummop ovum omcosu0ucma Low o:_m> mcmvcoammccoo cmgu “mo.Qva Loumoem >_ucmommwcmmmm masocm ucoEumoLu Losuo __m Lou w:_m> mcmpcoammeeoo cmsu Amo.o,mv Emummcm >_ucmowmwcmwmp m:_m> come Lone: memospcoLma cm cmme osu mo LoLLo oLOUCMHmo ommca compo—awe mchsv Home _mL:uwc mum—asoo oo>wouoe mmwa __< Aom._Mv “we.“ “mm.“ Aka.“ A_N.Fhv Ame.“ Amo._nv Akm.h _.:_ m.:_ e.m_ m.m_ _.:_ m.m_ N.N_ o.:_ ce_aeo_a meme x .>< Amm._ev ANA._hV Amk._nv 2mm.“ Aao.mnv 2mm.“ Amm.Nnv Aea.w m.m_ o._N e.m_ _.m_ m:.m~ _.NN A.UN m.x_ ce_:eom m£e_e x .>< flak.ehv A_o.mev Ame.“ A_e._hv Ame.knv Aek._hv Ree.ehv Amm.Nev 0.x: ..m: m.@: q.mq m.m: m.mm :.m: m.mm cease—m x .>< Aa_.n 2mm.“ Am_.h A_m.H Adm.“ Ame.“ “mm.“ Ace.“ om.e ow.e 56.0 Ne.e om.: om.: NN.m No.m x .5m .ceeeoLe Ezeem .>< ANN.“ “mo.“ “am.“ “we.“ Ne.N mk._ mm._ LNm._ .mg. .seceee_eee eeee Ama.uv Ae_.nv Ame.nv Aka.“ Ae_o.u Aona.wv Amea.nv Aema.wv N_._ mm._ ::._ emm._ om. mu. mN. e_:. .mg. .ceem x_eee .>< 20.7.; “8.2“; 873.; €0.me Amen: $_.N..L Amman; 6:97.; o.mm m.mm o.mo_ eo.k__ m.m_ m.om m._m em.em .me_ .egmeez _ecee N e m e e e e 6 meme .62 .< .m onwm mm mum—deco .< .m 03mm mm oum_a50u mocomowmoo xucmmommoo “coeummeh xempmwo l. “coepmoep >Lmumwo .mOmmxm compo—mom ._uaxm mmmcm comuo_moo ..uaxm m >03Hm sz m 2H mm34<> szHomm 23mmm oz< IFZOmo no >mH 4HL mow mkszkmE. m>HHommmmm Ht 8. omtbj< wszm mmt< zH><._n_omHm mo mszoon>m «Boa. quthohz