Cellular and humoral immune responses in calves experimentally infected with salmonella dublin

Zbl. Bakt. Hyg., 1. Abt. Or ig. A 249, 494-503 (1981) Departm ent of Veterinary Micro biology, College of Veterinary Sciences, H aryana Agricultu ral University, Hissar, Indi a

Cellular and Humoral Immune Responses in Calves Experimentally Infected with Salmonella dublin 1 Zellulare und humorale Immunantwort in experimentell mit Salmonella dublin infizierten Kalbern G. C. CHATURVEDI and V. K. SHARMA With 4 Figures' Received Janu ary 16, 1981

Abstract Both hu mor al and cellular immune responses ha ve been found to be opera tive in calves surviving smooth Salmo nella dublin infection. Such calves showed rises in agglutinin tit re and seru m immunoglobul in as compare d to uninfected contro ls. Th ese calves at 3 weeks post infect ion showed a 3- 4 fold increase of initial skin thickness at 6 h post antigen inoculation - an ind ication of immediate type of skin hypersensitivity reaction. Th e maximum increase in skin thickness of 2-3 fold was observed at 48 h post antigen inoculation - a criterion of delayed type of hypersensitivity reaction. There was a moderat e increase, 28.1 per cent in the migrat ion inhibition of peripheral leucocytes during the first week and a shar p increase, 45.7 and 66.6 per cent dur ing the second and th ird week post infection, respectively. Passively immunized calves on challenge exhibited delayed clinical symptoms and mo rtality as compared to control animals, thou gh the calves could not be protected agains t challenge. The serum im munoglobulin levels in passively immunized calves decreased moderately du ring the first week and markedly duri ng the second week post challenge while in the control calves the levels dropped abruptly in the first week followed by death in both groups.

Zusammenfassung Sowohl das Wirken hum oraler als auch zellularcr Immunreakt ionen ist in Kalbern, die eine unk ompl iziert e Salmonella dublill-Infek tion uberlebt barren, nachgewiesen wo rden. Irn Gegensatz zu nichtinfizierten Kontrollt ieren zeigten diese Kalber einen Anstieg im Agglutinin-Ti ter und im Seru m-Immunglobulin. Die infizierten Kalber zeigten dre i Wochen nach der Infektion eine drei- bis vicrfache Verstarkung der anfa nglichen H aut schwellung und zwar 6 Std. nach der Antigenino kulation - ein Kriterium fur eine un mitt elbare H aut hypersensitivirarsreaktion. Eine maximale, zwei- bis fiinffache Verstarkung der Haut1 Th is paper was presented in a symposium on " Recent Advances in Enterob acteriaceae Research" held in November 1978 at Hau, Hissar.

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schwellung wur de 48 Std. nac h der Antigengabe beobachtet, was ein Kriterium fiir eine Spa t-Hypersensitivi tatsreaktion ist, Ein leichter Anstieg von 28, 1% in der Migrationshemmun g der periphe ren Leuk ozyten wurde in der ersten Woche und ein sta rker Anstieg von 45,7 bzw, 66,6 % wurde in der zweiten und dritten Woche nach der Infekti on verzeichnet. Passiv-immunisierte Kalb er zeigten im Gegensa tz zu den Kontrolltieren nach der Infektion klini sche Spatsymptorne und Mortalitat , wenn auch die Kalb er gegen eine Infekt ion nicht geschurzt werden konnten. Die Serumimmung lobulinspiegel in passiv-imrnunisierten Kiilbern fielen in der ersten Woc he leicht und in der zweiten Woc he nach der Infektion stark ab, wah rend bei den Kont rollen diese Spiegel in der ersten Woche sta rk ab fielen und bei beid en Gru ppen zum Tod fiihrte.

Introduction Several studies have been made on experimental S. dublin infection in calves, they are, however, mostly limited to bacteriological aspects (29, 12, 13). Little information is available on the immune response of calves to S. dublin infection (14, 3, 26). In the present investigation an attempt has been made to demonstrate immun e responses to experimental S. dublin 188 infection in calves employing an agglutination test, quantitation of immunoglobulin levels, skin hypersensitivity, leucocyte migration inhibition and passive protection tests. Materials and Methods Calves. Thi rty thre e, cross bred male cow calves one day to 12 weeks old were procured from H ar yana Agricult ura l University, Animal Farm, Hissar ; they were free fro m salmonella infection as determined by fecal exam ination and seru m H and 0 agglutinin titre exa minat ion . Bacterial strain. A fre eze-dr ied smooth vir ulent culture of S. dublin 188 was obtained, through th e cou rtesy of Dr. H. W .Smith, Hou ght on Poultry Research Station, Hou ghton , Huntington , U. K. The cult ure was revived in broth, nutrient aga r an d modified MacConkey's medium (MMM) of Kaura et al. (17). The colonies were tested by slide agglutinat ion test (SAT) wi th 0 9, 12 and H , g, P anti sera. Later, a single colony was streaked on Dorset egg medium and also stabbed in semi-solid agar fo r storage. One p assage was given in two, 2 weeks old, calves by ora l infection with 101 0 via ble cells in acco rda nce with the procedure described by Smith (26) and th e organism, re-isolated fro m th e liver were used for pr eparin g antigens, for rais ing hyperimmune serum and for challenge p urposes. Experimental design. Ten healthy 8-12 weeks old calves pretested for agglutinin titres and serum immunoglobulin levels, were infected with 10' viabl e cells of S. dublin 188 ora lly as per procedure described earlier. A subsequent dose comprising 109 viable cells of S. dublin 188 was admi r.istered subcuta neously a week later into th ese calves. The studies on humoral and cellular imm une response were conducted on th ese infected calves at weekly interva ls up to 3 weeks, th e maximum period of observation. Th e hyperimmune serum fro m these calves was collected, Seitz-filtered and stored at - 20 °C fo r use in the passive prot ection test. Th e infectio n due to S. du blin 188 was recor ded based on clinic al symptoms, re-isolation and biochemical (Urease, Triple sugar iro n aga r) and sero lcgical (SAT) ch ara cterisati on of the culture isolated from th e diarrheal feces. Agglutination test. The prep arat ion of " H" and "0" antigens from smoo th S.dublin 188 an d agglutination test was performed as describ ed by Kauffmann (16). Th e seru m samples were obtai ned at 0 da y and at weekly intervals up to 3 weeks fro m calves surviv ing infect ion as well as calves passively immun ized with hyperimmune serum, and controls after challenge.

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Immuno globulin levels. Th e immunoglobulins from the serum of a 6 months old health y calf were precipitated and dialysed following the method of Cam pbell et a!' (7). Later , immunoglobulin supernatant was dried in vacuum chamber. Fetal calf serum (Flow Laboratori es Ltd., Scotland) was used as diluent for dried immunoglobulin reconstitution. A sta ndard curve based on turb idit y was determined through Spectronic-20 at 660 nm and levels of immunoglobulin, in the sera obtained at 0 day and at weekly intervals up to 3 weeks from calves surviving infect ion as well as passively immunized with hyperimmune serum and control , after challenge were also estimated following the meth od of Pfeiffer et a!' (24). A solut ion of zinc sulphate (208 rng of ZnSo. , 7 H 2 0 /L) was prepared in distilled water that had been boiled to remove carbon di-oxide (20). Leucocyte migration inhib ition (LM I) test. Th e veronal extract (VE) ant igen was prepared fro m smooth culture of S. dubl in 188 accordi ng to th e method of Barber et a!' (2) and Barber and Eylan (1) with slight modifications. The culture was grown over the surface of trypt ic soy agar (Difco). Th e VE antigen was distributed in 1-2 ml aliquores in screw capp ed vials and kept at - 20 "C. The protein of VE antigen was estimated by the method of Lowry et a!' (19). The nontoxic dose of VE antigen was 5 Ilg/ml of suspending fluid (Minimum essential medium, Eagle; Bios, Bombay), which was standardi zed according to the technique of Cameron and Van Rensburg (6) which was used for studying the LMI test. Th e leucocytes from peripheral blood of controls and surviving calves were separated at weekly int ervals up to 3 weeks post infection by the method of Naylor and Little (22) with the following modifications. The lysis of the erythrocytes by tripl e glass distilled water was carried out for 1 min and ph osphate buffer (pH 6.8) was used. Th e final pack of leucocytes was reconstituted in 0.5 ml of suspending fluid. The viability of the cells was 95 per cent when tested by the trypan blue dye exclusion technique. After filling with cell suspension by means of capillarity, the capillary tubes (100 x 1 mm) were sealed with plasticine and centrifu ged at 250 g for 3- 4 min at 4 "C. The duplicate capillarie s containing the pack of sensitized or unsensitized Ieucocytcs were fixed in the Mackaness chambers with the help of silicon grease. Mack aness chamber s with the pack of sensitized and unsensitized leucocytes with and without VE antigen in suspending fluid, respectively, were incubated at 37 °C for 18 h. The area of migrati on was dr awn with the help of a Camera lucida at x 20 magnification and was measured with a planimeter. The for mula of Cameron and Van Rensburg (6) was emplo yed to calculate the actual per cent leucocyte migration inhib ition . Skin hypersensitivity reactions. At the third week post infection 0.1 ml VE anti gen was intradermall y inoculated into the surviving calves, at both sides of neck and hypersensitivit y reactions were recorded. On each side of neck 0.1 ml distilled water was inoculated as cont rol. The skin thickness was measured at 3,4,5,6, 12,24, 30,48, 54 and 72 h post inoculation of antigen. An average of two readings of swelling on both sides of neck due to VE antigen, was taken after deductin g the swelling due to distilled water. Biopsy material from the test area of the skin was studied histopathologically after staining the paraffin block sections with hematoxylin and eosin (H and E) stain. Passive protection test. Another batch of a 6 weeks old health y calves comprising 5 control and 5 experimental animal s in two batches, was passively immuni zed with hyper. immun e serum and challenged as per procedure of Smith (26). Percenta ge protection, agglutinin titre s, immunoglobulin levels as well as bacteri ological findings (viable count from diarrho eic feces and vital organ s) were assayed in the passively immun ized calves and in the controls.

Results A gglutinin titres. The cal ves surviving sm ooth S. dublin 188 infection show ed peak H titre 1: 16,000 and 0 titre 1: 40 at 3 weeks post infection. The sera of calves before infection were negative for both Hand 0 agglutinins.

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Immunoglobulin levels. Except for a transient decline in the levels of immunoglobulin in the 1st week, all the calves exhibited increases in the levels of total immunoglobulin at 2 weeks, 3 weeks, post infection which ranged from 53.57 to 61.28 mg/ml of serum, while control calves showed almost constant levels of immunoglobulin which ranged from 59.0-59.33 mg/ml of serum. Leucocyte migration inhibition. The results of migration of peripheral leucocytes of calves at 1, 2 and 3 weeks post infection with S. dublin 188 are shown in Fig. 1. The average leucocytic migration inhibition percentage 1 week post infection was 28.13, which increased to 45.69 after 2 weeks and to 66.61 after 3 weeks.

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Skin hypersensitivity reactions. The results of skin hypersensitivity reactions (immediate and delayed type) exhibited by calves surviving the infection were recorded at different time intervals. The maximum increase in skin thickness as a criterion of immediate-type hypersensitivity reaction, recorded at 6 h post inoculation of VE antigen at both sides of neck, was characterized by erythema and edema. The thickness at the site of inoculation of antigen at 0 h was 4.2-4.6 mm (without deducting the swelling due to distilled water) which increased to 12.0 to 16.6 mm at 6 h. Whatever of little reaction was present at the control site, that too subsided subsequently. Histopathologically the immediate-type hypersensitivity reaction revealed infiltration of neutrophils and edema in the dermis and subcutaneous layer of the skin. At the control site, the cellular reaction was absent. The maximum skin thickness as a criterion of delayed type hypersensitivity reaction was observed at 48 h as 11.0-17.0 mm in calves surviving active infection. At 72 h, the induration was more prominent and the thickness of the skin reaction was slightly decreased as compared to 48 h reaction when it ranged between 7.5 and 13.0 mm. The swelling was indurated while induration was not perceptible

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Fig. 2. Skin section showing infiltration of macrophages, lymphocytes and few neutrophils in dermis at 72 h post inoculation of VE antigen in a surviving calf at three weeks post infection with S. dublin 188. Hand E x 400. at the control site. Histopathologically the skin reaction revealed massive infiltration of mononuclear cells in the subcutaneous tissue (Fig. 2). There was no cellular reaction in the skin section taken from the site inoculated with distilled water. Passive protection experiments. Before passive immunization and challenge all the calves were in good physical condition. The rectal temperature of calves before and after receiving 450 ml hyperimmune serum ranged from 100.2 to 102.4 OF. These calves, when challenged exhibited a body temperature after 24 h and onward ranging from 106.0 to 107.0 OF which later came down to 100.6-99.0 OF with severe diarrhoea in terminal stages of death. All the five calves died within

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Fig. 3. Effectof passive transfer of homologous hyperimmune serum in calves on protection against challenge with smooth S. dublin 188. ------- control calves, - - - immunized calves.

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1-2 weeks post challenge. The control calves exhibited a body temperature of 100.0-101.4 of before and 103.0-106.0 of after challenge, coming down to 100.0 to 98.0 of with severe diarrhoea in the terminal stages of death. All the control calves died wit hin 1 week po st challenge (Fig. 3). The passively immunized calves after challenge ex hibited a delayed development of clinical symptoms such as rise of temperatur e and on set of dia rrhoea as compared to con tro l calves. The mortality was also dela yed in the se passively immunized calves (Table 1). Table 1. Viable count of S. dublin 188 in diarrhoeic faeces in passivelyimmunized and nonimmunized calves after challenge Calf No.

Immunized Batch No .1 1 2 3 Batch No.2 1 2

Average Non-immunized Batch No .1 1 2

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Day of severe diarrhoea and collection of faeces after challenge-

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.• Viable count was done on day shown in column 2. • Since, calculated Mann-Whitney U statistic was zero, the null hypothesis was rejected at C( = 0.05. Hence, there was significant increase in all the three characters under study in immunized batch with respect to non-immunized batch. The Hand 0 agglutinin titres of passively immunized calves were 1,000 and 10, during first and second weeks post challenge, respectively. One our of 5 passively immunized calves and all the control calves died within 1 week post challenge, which is why, th eir serum agglutinin titres could not be estimated. The levels of immunoglobulin in immunized calves ra nged from 40.0-54.0 mgt ml of serum before passive immunization. These levels decreased moderately in the first week (36.0-44.0 mg) and markedly in second week (21.0-38.0 mg) post challenge followed by death. In control calves, the levels of immunoglob ulin before

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G. C. Chaturvedi and V. K. Sharma

challenge were 32.0-60.0 mg/ml of serum, which dropped abruptly to 15.0-35.0mg within 1 week post challenge and all the calves died. The viable count of the challenge organism in the diarrheal feces of passively immunized as well as control calves during the time of severe diarrhoea was on an average 5.93 X 106 and 3.25 X 106 viable organism/g diarrheic feces respectively, as shown in Table 1. The viable count of the challenge organism from internal organs of the immunized and control calves that died was more or less the same. The viable count/g of liver and spleen of experimental calves was on an average 4.58 X 106 and 9.66 X 106 , respectively. The viable count/ml of heart blood was 2.13 X 105 in case of 3 immunized calves, whereas no organism could be isolated in 2 calves which died on 12th and 16th day post challenge, respectively. In the control calves viable count/g of liver, spleen and per ml of heart blood was on an average 5.42 X 106 , 6.02 X 106 and 5.05 X 106 , respectively (Fig. 4). The challenge organism was also isolated from the bile of experimental and control calves. The identity of the challenge organism on post mortem was confirmed by isolation, biochemical and serological characterization. a

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Discussion The basis of immunity against salmonellosis has been a long-standing controversy with respect to the relative importance of cellular versus humoral factors.

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Since salmonella organisms are ubiquitous pathogens of human and animal species, an understanding of events during the immune response is of much importance in developing an effective prophylactic agent. Collins (9, 10), Smith and Bigley (27, 28) suggested that protection against salmonellosis in mice can be correlated with the appearance of cell-mediated responses to salmonella antigens and concluded that T lymphocytes played a dominant role in preventing the development of overt disease. Many research workers have shown that passively transferred hyperimmune serum provided little or no protection to mice challenged with salmonella (8, 15). The present studies in calves showed that the elevation in agglutinin and serum immunoglobulin levels might perhaps be correlated with survival and, therefore, passive protection experiments were undertaken to test the role of antibodies in protection if any. Serum from calves surviving S. dublin 188 infection and showing H titre of 16,000 was used to passively immunize another batch of calves in order to study the role of humoral immune response in affording protection. Upon virulent challenge, appearance of clinical symptoms such as rise in temperature, onset of diarrhoea and mortality were delayed, indicating thereby that the humoral immunity might be playing a marginal role in protection by delaying the course of infection. Such observations, about the role of hyperimmune serum in delaying the development of clinical symptoms and a little prolongation of the life of sick calves, are probably not available in the literature. Hochadel and Keller (15), however, could protect 65 per cent of the recipient mice against S. typhimurium infection by adoptive transfer of B lymphocytes from sensitized donor, but on passive transfer of hyperimmune serum none could be protected. Morris et al. (21) while studying immunological responses in mice against salmonellosis maintained that humoral immunity was more important than cellular in affording protection. Logan and Penhale (18) upon inoculation of IgM fraction in calves also found that it delayed the onset of E. coli septicemia and prolonged survival but failed to prevent death. Smith (26), Smith and Bigley (27), Cameron and Fuls (5) also observed marginal protection in passively immunized mice against virulent salmonella challenge. Ushiba (30) and Collins (8, 9) also reported that in the early stages of infection humoral immunity was responsible for the clearance of the challenge salmonella organisms in mice and this may explain the delay in mortality in passively immunized calves in the present study. On the other hand, the incapability of the hyperimmune serum to protect the recipient mice on challenge has also been well documented by other scientists (8,31). Serum immunoglobulin levels in passively immunized calves decreased moderately in the first week and markedly in the second week post challenge while in the control calves the levels dropped abruptly in the first week followed by death in both group of calves. Such a sudden fall of immunoglobulin levels in the control and a gradual fall in the passively immunized calves could be explained on the basis of work of Penhale et al. (23), Boyd (4), Sapre and Ramadwar (25) who reported correlation between the presence of low levels of immunoglobulin in young calves and their susceptibility to infection by gram-negative bacteria. They found that those calves which had low levels of serum immunoglobulin were more susceptible to infection by gram-negative organisms, particularly E. coli, as compared to those which had high levels of immunoglobulin. It may be assumed that the same type of mechanisms, might also be working in salmonellosis. This indicates that in response to a virulent S. dublin infection both humoral and cell33 Zbl. Bakt. Hyg., I. Abr, Orig. A 249

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mediated immunological responses are initiated by the host and that the humoral response probably plays a marginal role in affording protection especially in prolonging the course of disease.

Acknowledgements The financial assistance provided to one of us (GCe) by the Indian Council of Agricultural Research, New Delhi through a project entitled, "Research on Salmonellosis" is duly acknowledged. The senior author is grateful to Dr. M.U.Kharole and Dr. S.N.Dixit for histopathological interpretation of slides, microphotography and useful suggestions during the preparation of the manuscript.

References 1. Barber, C. and KEylan: Immunochimie de Salmonella typhimurium. Variations de la fraction protei que de l'antige'ne somatique. Rev. Immunol. (Paris) 36 (1972) 1-13 (Cited by Cameron, C.M. and I.J. Van Rensburg, 1975) 2. Barber, c., KEylan, and K.Keydar: Contributions a'l'etude des Salmonelles. Proteienes communes des Salmonelles appartenant aux groups. B, C, D et E bur relation avec Citrobacter ballerup. Path. et Microbiol. 31 (1968) 165-174 (Cited by Cameron, C.M. and J.I. Van Rensburg, 1975) 3. Botes, H.I. W.: Calf paratyphoid immunity: Evaluation of formol killed and live attenuated vaccines. Bull. Off. Int. Epiz. 62 (1964) 581-605 4. Boyd, I. W.: The relationship between serum immune globulin deficiency and disease in calves: a farm survey. Vet. Rec. 90 (1972) 645-649 5. Cameron, C.M. and W.I.P.Fuls: Immunization of mice and calves against Salmonella dublin with attenuated live and inactivated vaccines. Onderstepoort J. vet. Res. 43 (1976) 31-38

6. Cameron, C.M. and I.], Van Rensburg: Inhibition of macrophage migration in Salmonella immunity. Onderstepoort J. vet. Res. 42 (1975) 15-24 7. Campbell, D. H., I. s. Garvey, N. E. Cremer, and D. H. Sussdori : Methods in immunology, 2nd edn. W.A.Benjamin, Inc., New York (1970) 8. Collins, F.M.: Effect of specific immune mouse serum on the growth of Salmonella enteritidis in non-vaccinated mice challenged by various routes. J. Bact. 97 (1969) 667 -675 9. Collins, F.M.: Mechanisms in antibacterial immunity. J. Reticuloendothel. Soc. 10 (1971) 58-90 (Cited by Davies, R. and I. Kotlarski, 1976) 10. Collins, F.M.: Vaccines and cell mediated immunity. Bact. Rev. 38 (1974) 371-402 11. Davies,R. and I. Kotlarski: A role fer antibody in the expression of cellular immunity to Salmonella typhimurium. Aust. J. expo BioI. med. Sci. 54 (1976) 207-219 12. Gronstol, H., A. D. Osborne, and S. Pethiyagoda: Experimental Salmonella infection in calves. 1. The effect of stress factors on the carrier state. J. Hyg. (Camb.) 72 (1974a) 155-162 13. Gronstol, H., A. D. Osborne, and S. Pethiyagoda: Experimental Salmonella infection in calves. 2. Virulence and the spread of infection. J. Hyg. (Camb.) 72 (1974b) 163-168 14. Henning, M. W.: Calf paratyphoid. II. Artificial immunization. Onderstepoort. J. vet. Res. 26 (1953) 25-44 15. Hochadel, ]. F. and K. F.Keller: Protective effects of passively transferred immune T or B lymphocytes in mice infected with Salmonella typhimurium. J. infect. Dis. 135 (1977) 813-823

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16. Kauffmann, F.: The bacteriology of Enterobacteriaceae, 2nd edn. Munksgaard, Copenhagen (1969) 17. Kaura, Y.K., V.K.Sharma, I. P. Singh, R.Sakazaki, and R.Rhode: Snakes as reservoirs of Arizona and Salmonella. Zbl. Bakt. Hyg., LAbt. A 219 (1972) 506-513 18. Logan, E. F. and W.]. Penhale: Studies on the immunity of the calf to colibacillosis. IV. The prevention of experimental colisepticaemia by the intravenous administration of a bovine serum IgM rich fraction. Vet. Rec. 89 (1971) 663-667 19. Lowry, O. H., N.]. Rosebrough, A. 1. Farr, and R.]. Randall: Protein measurement with folin phenol reagent. J. BioI. Chern. 193 (1951) 265-275 20. McEwan, A.D., E. W.Fisher, I.E.Selman, and W.].Penhale: A turbidity test for the estimation of immunoglobulin levels in neonatal calf serum. Clin. Chim. Acta 27 (1970) 155-163 21. Morris, ].A., C. Wray, and W.].Sojka: The effect of lymphocytes depletion on the protection of mice vaccinated with a Gal E mutant of Salmonella typhimurium. Brit. J. expo Path. 57 (1976) 354-360 22. Naylor, P. F. and T. W. A. Little: A simple method for isolating lymphocytes from bovine blood and their separation into lymphocyte and granulocyte fractions. Res. vet. Sci. 18 (1975) 336-337 23. Penhale, W.]., G.Christie, A.D.McEwan, E. W.Fisher, and I.E.Selman: Quantitative studies on bovine immunoglobulins. II. Plasma immunoglobulin levels in market calves and their relationship to neonatal infection. Brit. vet. J. 126 (1970) 30-37 24. Pfeiffer, N. E., T. C. McGuire, R. B. Bendel, and ]. M. Weikel: Quantitation of bovine immunoglobulins: Comparison of single radial immunodiffusion, zinc sulfate turbidity, serum electrophoresis, and refractometer methods. Amer. J. vet. Res. 38 (1977) 693-698 25. Sapre, V. A. and D. K. Ramadwar: Serum gammaglobulin in relation to neonatal calf mortality amongst Indian cow calves and buffalo calves. Indian J. Anim. Sci. 47 (1977) 617-624 26. Smith, H. W.: The immunization of mice, calves and pigs against Salmonella dublin and Salmonella cholerae-suis infections. J. Hyg. (Camb.) 63 (1965) 117-135 27. Smith, R. A. and N.]. Bigley: Ribonucleic acid-protein fractions of virulent Salmonella typhimurium as protective immunogens. Infect. Immun. 6 (1972a) 377-383 28. Smith, R.A. and N.].Bigley: Detection of delayed hypersensitivity in mice injected with ribonucleic acid-protein fractions of Salmonella typhimurium. Infect. Immun, 6 (1972b) 384-389 29. Smith, H. W. and ].E. T.]ones: Observations on experimental oral infection with Salmonella dublin in calves and Salmonella cholerae-suis in pigs. J. path. Bact. 93 (1967) 141-156 30. Ushiba, D.: Two types of immunity in experimental typhoid, "Cellular immunity" and "humoral immunity". Keio J. Med. 14 (1965) 45-61 31. Venneman, M.R. and 1.]. Berry: Cell mediated resistance induced with immunogenic preparations of Salmonella typhimurium. Infect. Immun. 4 (1971) 381-387 G. C. Chaturvedi, Ph. D., Assistant Professor of Immunology, Department of Veterinary Microbiology, Hau, Hissar, India V.K.Sharma, Ph. D., College of Veterinary Medicine, University of Baghdad, Baghdad, Iraq