Texas pulmonary tuberculosis to study the effects of dietary deficiencies. on M. bovis BCG vaccine efficacy (17).

Transcription

1 INFECTION AND IMMUNITY, Nov. 1985, p /85/ $2./ Copyright C) 1985, Americn Society for Microbiology Vol. 5, No. 2 Mycobcterium bovis BCG Vccine Fils to Protect Protein-Deficient Guine Pigs ginst Respirtory Chllenge with Virulent Mycobcterium tuberculosis DAVID N. McMURRAY,* MIRTA A. CARLOMAGNO,t CAROLE L. MINTZER, AND CHRISTINE L. TETZLAFF Deprtment of Medicl Microbiology nd Immunology, College of Medicine, Texs A&M University, College Sttion, Texs Received 31 My 1985/Accepted 3 July 1985 Specific-pthogen-free Hrtley guine pigs were mintined on isocloric-purified diets either dequte (3%) or modertely deficient (1%) in protein. Hlf of ech diet group ws vccinted with vible Mycobcterium bovis BCG. Six weeks lter, ll nimls were chllenged by the respirtory route with virulent Mycobcterium tuberculosis H37Rv. At intervls of 1, 2, nd 3 weeks postchllenge, guine pigs from ech diet nd vccintion group were skin tested with tuberculin nd scrificed. Protein deficiency resulted in loss of tuberculin hypersensitivity. Vccintion with M. bovis BCG protected control nimls, s determined by significnt reductions in the number of M. tuberculosis H37Rv orgnisms recovered from lungs, spleen, nd bronchotrchel lymph nodes 2 nd 3 weeks postchllenge. Bsed upon the sme criteri, the degree of protection fforded protein-deficient nimls by M. bovis BCG vccine rnged from prtil (spleen nd lymph nodes) to none t ll (lungs). Approximtely the sme numbers of tubercle bcilli were recovered from nonvccinted guine pigs in both diet groups. Protein deficiency ppers to impir M. bovis BCG-induced immunity while not ffecting primry pulmonry infection with virulent M. tuberculosis. Tuberculosis is mjor cuse of morbidity nd mortlity in the developing world tody despite the vilbility of vccine, Mycobcterium bovis BCG. While there is very good evidence for the efficcy of M. bovis BCG in protecting vccine recipients ginst clinicl tuberculosis, the recent field tril filure in South Indi suggests tht unknown environmentl or host fctors my render M. bovis BCG less efficcious in certin Third World popultions (2, 19). Mlnutrition coexists with tuberculosis in mny prts of the world in which vccintion is the only cost-effective mens of controlling the disese. Dietry deficiencies of specific nutrients hve been ssocited with ltertions in immunologicl competence in humns nd experimentl nimls (1, 6). Cell-medited immunity in generl nd thymus-dependent (T) lymphocyte numbers nd functions in prticulr pper to be predictbly nd profoundly ltered in the mlnourished host (8, 9). The extrpoltion of these observtions to T cell-medited infectious disese resistnce, however, hs not been strightforwrd. Some mcrophge-dependent host defense mechnisms re pprently unffected or even enhnced in nutritionlly deprived individuls (13, 2). Given the centrl role of T lymphocytes nd mcrophge ctivtion by their products in resistnce ginst tuberculosis, it is likely tht nutritionl deficiencies would lter mycobcteril immunity (2, 3, 7). Erly studies in mlnourished, M. bovis BCG-vccinted children demonstrted tht such children did not develop norml tuberculin skin rectivity fter vccintion nd presumbly were not s protected ginst virulent chllenge (6, 11, 16). The ltter hypothesis hs never been tested in humns or experimentl nimls. We hve dpted guine pig model of experimentl * Corresponding uthor. t Present ddress: Consejo Ncionl de Investigciones Cientifics y Technics (CONICET), Rivdvi 1917, Buenos Aires, Argentin. 555 pulmonry tuberculosis to study the effects of dietry deficiencies on M. bovis BCG vccine efficcy (17). Our previous results reveled specific lesions in the immune responses of protein-deficient guine pigs vccinted with M. bovis BCG. In generl, protein-mlnourished guine pigs filed to mount significnt tuberculin rection, even to incresed doses of tuberculin, lthough these nimls eventully controlled infection with M. bovis BCG nd other ttenuted mycobcteri (12, 1). The experiments we describe here were designed to determine whether protein-mlnourished guine pigs could be protected by M. bovis BCG vccine ginst respirtory chllenge with virulent Mycobcterium tuberculosis. (This work ws presented in prt t the 85th Annul Meeting of the Americn Society for Microbiology, Ls Vegs, Nev., 3 to 7 Mrch, 1985.) MATERIALS AND METHODS Experimentl nimls. Outbred, lbino, specific-pthogenfree femle guine pigs, weighing 15 to 2 g ech, were obtined from commercil source (Hrtley-COBS, Crl:(HA)BR; Chrles River Breeding Lbortories, Inc., Wilmington, Mss.). They were housed individully in polycrbonte cges on stinless-steel grid floors nd provided food in stinless-steel feeders nd tp wter d libitum. Ech niml ws rndomly ssigned to n experimentl diet nd vccintion tretment group. Body weights were recorded weekly during the experiment. Experimentl diets. The experimentl diets, bsed upon ovlbumin s the protein source, were designed to meet current recommended nutritionl requirements for guine pigs (14). The biotin content of the diet ws incresed to counter the effects of vidin, biotin-binding protein found in ovlbumin. Two diets representing n dequte level of protein (3%) nd protein-deficient level (1%) were obtined from commercil source (Dyets, Inc., Bethlehem, P.). The diets were isocloric, with the proportion of corn Downloded from on April 3, 218 by guest

2 556 McMURRAY ET AL. INFECT. IMMUN. TABLE 1. Influence of diet on growth nd protein sttus Dietry Vccintion Body weight (g) t wk: Serum lbumin (g/dl) t wk: tretment sttusb Control VACC 387 ± 32c 391 ± 57" 413 ± 17C 3.62 ±.17c 3.35 ±.18c 2.93 ±.16c NV 368 ± 22" 365 ± 2" 342 ± 24d 3.57 ±.3c 3.24 ±.16" 2.88 ±.1c Low protein VACC 287 ± 23d 297 ± 14d 257 ± 25e 2.77 ±.9d 2.9 ±.13d 2.74 ±.14c"d NV 262 ± 21d 248 ± 18e 23 ± 21e 3.5 ±.7d 2.87 ±.22d 2.61 ±.9d Vlues re men + stndrd error of the men of four to six observtions t indicted week post-respirtory chllenge with virulent M. tuberculosis. b VACC, Vccinted guine pigs; NV, nonvccinted guine pigs. cad,e Mens within ech intervl lbeled by the sme superscript letter re not sttisticlly different (P >.5); mens lbeled with different superscript letters re sttisticlly different. strch nd ovlbumin vrying inversely to provide the desired protein content. The formultion of the diets hs been published previously (12). All nimls were given mixture of the fully supplemented diet (3% protein) nd decresing proportions of powdered commercil guine pig stock diet (Rlston Purin Co., St. Louis, Mo.) over 2-week dpttion period before ssignment to one of the experimentl diets. The food ws given s powder, nd fresh diet ws provided every other dy. Food intke ws monitored periodiclly throughout the study. M. bovis BCG vccintion. On the sme dy the experimentl diets were strted, hlf of the guine pigs in ech diet tretment were vccinted with vible M. bovis BCG vccine (Copenhgen 1331; Sttens Seruminstitut, Copenhgen, Denmrk). Ech niml received.1 ml of sline solution contining pproximtely 13 vible bcilli subcutneously over the left inguinl region. The vibility of the vccine ws determined by plting pproprite dilutions on M7H1 gr (Difco Lbortories, Detroit, Mich.). Respirtory infection. M. tuberculosis H37Rv (ATCC 27294) ws obtined from the Americn Type Culture Collection, Rockville, Md. The chllenge inoculum ws prepred nd stored t -7 C by published procedure (5). After 6 weeks on the experimentl diets (i.e., 6 weeks postvccintion with M. bovis BCG), ll guine pigs were infected vi the respirtory route in n exposure chmber described previously (1, 2). The infection ws crried out in biohzrd fcility for use with clss 3 humn microbil pthogens. The concentrtion of vible M. tuberculosis H37Rv orgnisms in the nebulizer fluid ws djusted empiriclly to result in the inhltion nd retention of bout 12 vible mycobcteri per guine pig. The vible count ws determined by plting pproprite dilutions of the chllenge culture on M7H1 gr pltes (Difco). The ctul chllenge level ws estimted from the number of primry tubercles observed grossly in the lungs of nonvccinted nimls t 3 weeks postinfection. Diet did not influence significntly the number of tubercle bcilli inhled nd retined by this criterion. Groups of guine pigs, selected rndomly from ech of the vccintion nd dietry tretments, were exposed repetedly to result in uniform, reproducible infection of ll nimls with mycobcteri (18, 2). PPD skin tests. One dy before scrifice, guine pigs received two intrderml injections of.1 ml of solution contining either 5 or 1 tuberculin units (TU) of purified protein derivtive (PPD) (PPD-RT23; Sttens Seruminstitut, Copenhgen, Denmrk) on shved re of the side. The rections were mesured with trnsprent plstic ruler 24 h lter, nd the men dimeter of indurtion ws recorded in millimeters. Necropsy procedure. One, two, nd three weeks fter respirtory infection with virulent M. tuberculosis H37Rv, groups of four to six guine pigs from ech tretment were killed by cervicl disloction. A blood smple (5 to 7 ml) ws drwn immeditely from ech niml by crdic puncture into 1-ml syringe. The serum ws seprted from ech smple nd stored t -2 C. The right lower lobe of the lung, the spleen, nd the bronchotrchel lymph nodes were removed septiclly, plced in sterile petri dishes, nd weighed. All infected tissues were hndled in biohzrd hood with pproprite precutions for protection of personnel. The tissues were then homogenized seprtely in 4.5 ml of sterile physiologic sline in Teflon-glss homogenizers. Approprite dilutions were inoculted onto duplicte M7H1O gr pltes which were incubted t 37 C for 3 to 4 weeks. The number of colonies ws counted, nd the results were expressed s men log1 vible orgnisms per grm (wet weight) of tissue to ccount for differences in orgn size due to dietry deficiency nd smpling procedures. Sttisticl nlysis. The nlysis of vrince ws used to test for the effects of the tretment vribles (diet nd vccintion) on the dependent vribles (extent of disese nd delyed hypersensitivity). Men differences between tretment groups were tested for sttisticl significnce by Student's t test or Duncn's new multiple rnge test. The ccepted level of probbility ws set t 95% for ll tests. RESULTS Dietry influence on nutritionl sttus. The low-protein diet hd the expected effects on growth nd metbolism of guine pigs (Tble 1). Body weights of protein-deficient nimls were significntly lower t ll scrifice intervls. Nonvccinted nimls ppered to lose more weight thn vccinted nimls in both diet groups. The significnt reduction in serum lbumin concentrtions in the low-protein group is evidence of the protein-deficient sttus of those guine pigs. No differences in food intke were noted between the diet groups t ny time during the experiment. Effect of diet nd vccintion on tuberculin hypersensitivity. Derml rections in both doses of tuberculin were profoundly impired in protein-deficient guine pigs. The sme tretment effects were seen in response to both the 5- nd 1-TU doses, lthough the bsolute rection size nd the mgnitude of the differences between tretments were greter with the lrger dose of tuberculin, nd therefore only those dt re presented (Fig. 1). Vccinted guine pigs consuming the control diet exhibited strong positive rections t ll scrifice intervls, while their nonvccinted counterprts developed significnt derml rections to PPD s their disese developed. In contrst, protein-deficient nimls were hyporesponsive t ll intervls. Neither previous vccintion with M. bovis BCG nor extensive repliction of virulent M. tuberculosis ws ccompnied by significnt Downloded from on April 3, 218 by guest

3 VOL. 5, 1985 M. BOVIS BCG VACCINE IN PROTEIN-DEFICIENT GUINEA PIGS 557 P 18-2 z 16_ 14T 12 O 1- lo FIG. 1. Influence of dietry protein nd M. bovis BCG vccintion on delyed hypersensitivity rections to 1-TU PPD in vccinted (open symbols) nd nonvccinted (solid symbols) guine pigs mintined on either norml (squres) or protein-deficient (circles) diets nd chllenged t zero time with virulent M. tuberculosis H37Rv; men ± stndrd error of the men of four to six nimls per dt point is shown. delyed hypersensitivity rections to 1-TU dose of tuberculin. Impct of diet on protective efficcy of M. bovis BCG vccine. Vccine-induced protection ws defined s significnt reduction in the number of virulent M. tuberculosis orgnisms recovered quntittively from the tissues. In the lung (Fig. 2), vccintion with M. bovis BCG conferred protection on normlly nourished guine pigs, s evidenced z o 7 JI _ T by the 1- to 1-fold reduction in the number of virulent tubercle bcilli, compred with their nonvccinted counterprts t 2- nd 3-week intervls. Protein deficiency, on the other hnd, completely eliminted ny benefit from prior vccintion. There ws no difference between the bcillry lods in the lungs of M. bovis BCG-vccinted, low-protein nimls nd either nonvccinted group t ny scrifice intervl. In the spleen (Fig. 3), similr results were obtined. Compred with nimls consuming the control diet, proteindeprived guine pigs were significntly less protected by M. bovis BCG vccintion, lthough the loss of protection in the spleen ws prtil rther thn complete. Figure 4 illustrtes the effects of dietry protein nd vccintion on the number of virulent M. tuberculosis orgnisms in the lymph nodes drining the lungs. Both vccinted groups hd significntly fewer orgnisms in their lymph nodes 2 weeks postchllenge, while this protection hd begun to wne by 3 weeks in the protein-mlnourished group. There were no significnt differences in the bcillry lods of protein-deprived vccinted nd nonvccinted guine pigs t the lst intervl. In ll orgns, no dietry influence ws observed on the bility of nonvccinted nimls to control primry, pulmonry infection with virulent tubercle bcilli. DISCUSSION We hve demonstrted tht chronic, moderte dietry protein deficiency impirs the bility of M. bovis BCG vccine to protect guine pigs ginst respirtory chllenge with virulent M. tuberculosis H37Rv. Protein deprivtion ws chrcterized by weight loss or reduced growth velocity nd modest but sttisticlly significnt reductions in the concentrtion of serum lbumin (Tble 1). Protein-deprived nimls were incpble of mounting significnt delyed hypersensitivity rections even to lrge dose of tuberculin (1 TU) nd even in the presence of extensive, extrpulmonry tuberculosis (Fig. 1). Although we did not skin z w w -j. en 5- E I: Downloded from on April 3, 218 by guest. 35 w 2- _1 j FIG. 2. Effect of dietry protein nd M. bovis BCG vccintion on the number of vible M. tuberculosis H37Rv orgnisms recovered from the lungs of vccinted (open symbols) nd nonvccinted (solid symbols) guine pigs mintined on either norml (squres) or protein-deficient (circles) diets; men + stndrd error of the men of four to six nimls per dt point is shown. 1 3 FIG. 3. Effect of dietry protein nd M. bovis BCG vccintion on the number of vible M. tuberculosis H37Rv orgnisms recovered from the spleens of vccinted (open symbols) nd nonvccinted (solid symbols) guine pigs mintined on either norml (squres) or protein-deficient (circles) diets; men + stndrd error of the men of four to six nimls per dt point is shown.

4 558 McMURRAY ET AL. w z I E cm._. - : -i co CD -i I 1 2 FIG. 4. Effect of dietry protein nd M. bovis BCG vccintion on the number of vible M. tuberculosis H37Rv orgnisms recovered from the bronchotrchel lymph nodes of vccinted (open symbols) nd nonvccinted (solid symbols) guine pigs mintined on either norml (squres) or protein-deficient (circles) diets; men ± stndrd error of the men of four to six nimls per dt point is shown. test nimls prior to respirtory chllenge, previous experience indictes tht the protein-mlnourished guine pigs would hve been responsive to PPD with rections similr to those seen t 1 week (12). These observtions confirm our previous reports of tuberculin nergy in proteinmlnourished guine pigs infected with M. bovis BCG nd ttenuted M. tuberculosis H37R (1, 12) nd support the contention tht mlnutrition interferes with the dignostic usefulness of PPD skin tests in individuls with clinicl tuberculosis (D. N. McMurry, Editoril, Chest 77:4-5, 198). Among the most consistent nd predictble reports of impired cell-medited immune function in mlnourished humns hve been the observtions of filure to develop norml tuberculin skin rectivity fter M. bovis BCG vccintion (6, 11, 16). While it is tempting to suggest tht lck of conversion to PPD postvccintion implies tht the vccine hs been unsuccessful in eliciting protective response in the mlnourished vccine recipient, tht hypothesis hs never been formlly tested either in humn field trils or in experimentl nimls. Using guine pig model similr to the one described here, Smith nd his collegues (4, 18) hve demonstrted tht successful vccintion with M. bovis BCG results in control of mycobcteril ccumultion in the primry lung lesions s well s limittion of bcillemic spred of virulent orgnisms to the spleen nd the lymph nodes. A sttisticlly significnt inverse correltion hs been estblished between the number of vible M. tuberculosis H37Rv orgnisms recovered from the lungs between 3 nd 5 weeks postchllenge nd the ultimte survivl of vccinted nd nonvccinted guine pigs (2). Bsed upon tht rtionle, we observed tht M. bovis BCG did not protect protein-deficient guine pigs ginst virulent M. tuberculosis. The loss of vccine-induced protection ws complete in the lung nd prtil but significnt in the spleen nd the lymph nodes drining the lung. It ppers tht protein deprivtion not only prevents control of mycobcteril ccumultion in the pulmonry lesions but interferes with the bility of vccinted nimls to limit extrpulmonry dissemintion. Although we believe tht these dt re predictive of the ultimte outcome of infection, studies of longer chllengescrifice intervls my be desirble to confirm the deleterious effect of protein deficiency on vccine efficcy. Given the filure of protein-deficient guine pigs to mount protective immune response fter vccintion with M. bovis BCG, it ws surprising to observe no effect of diet on the course of primry tuberculosis in nonvccinted nimls. Identicl numbers of virulent mycobcteri were recovered from lung, spleen, nd lymph nodes of low-protein nd control, nonvccinted groups. We hd observed previously tht protein-deficient guine pigs ultimtely controlled primry infections with M. bovis BCG or ttenuted M. tuberculosis H37R s successfully s normlly nourished nimls (1, 12). In those experiments, s in the ones reported here, control of mycobcteril popultions in nonvccinted nimls ws seen in the bsence of significnt tuberculin hypersensitivity. The development of pprently effective resistnce to progressive tuberculosis hs been observed occsionlly in nergic ptients (McMurry, Editoril, Chest, 198). These observtions suggest tht derml rectivity nd ntimicrobil resistnce in tuberculosis my be dissocible events, possibly medited by different T lymphocyte subpopultions, s reported in mice (15). The extrpoltion of these results to the effects of mlnutrition on M. bovis BCG vccine efficcy in humn popultions must be qulified. Vribles such s the severity nd chronicity of protein deprivtion, the virulence of the prevlent strins of M. tuberculosis, nd the endpoint used to define protection (quntittive reduction of bcillry lods versus bsence of clinicl tuberculosis) might ffect the conclusions reched if similr study were crried out in humns. However, the fct tht our observtions were mde in n niml model designed specificlly to pproximte the relevnt humn condition, with respect to both diet nd infection, suggests tht nutritionl vribles my hve contributed to previous filures of M. bovis BCG vccine in humn trils. ACKNOWLEDGMENTS This work ws supported by Public Helth Service grnt AI from the Ntionl Institutes of Helth. We thnk Dinne Hkri for secretril support. LITERATURE CITED INFECT. IMMUN. 1. Beisel, W. R Single nutrients nd immunity. Am. J. Clin. Nutr. 35: Chprs, S. D Immunity in tuberculosis. Bull. W.H.O. 6: Collins, F. M The immunology of tuberculosis. Am. Rev. Respir. Dis. 125: Fok, J. S., R. S. Ho, P. K. Aror, G. E. Hrding, nd D. W. Smith Host-prsite reltionships in experimentl irborne tuberculosis. V. Lck of hemtogenous dissemintion of Mycobcterium tuberculosis to the lungs in nimls vccinted with Bcille Clmette-Guerin. J. Infect. Dis. 133: Grover, A. A., H. K. Kim, E. H. Wiegeshus, nd D. W. Smith Host-prsite reltionships in experimentl irborne tuberculosis. II. Reproducible infection by mens of n inoculum preserved t -7 C [sic]. J. Bcteriol. 94: Hrlnd, P. S. E. G Tuberculin rections in mlnourished children. Lncet ii: Downloded from on April 3, 218 by guest

5 VOL. 5, 1985 M. BOVIS BCG VACCINE IN PROTEIN-DEFICIENT GUINEA PIGS Lefford, M. J Mcrophge ctivtion nd resistnce to pulmonry tuberculosis. Infect. Immun. 28: Mrtin, T. R., L. C. Altmn, nd. Alvrez Proteinclorie mlnutrition impirs cell-medited immunity in the rt lung. Chest 83: McMurry, D. N Cell-medited immunity in nutritionl deficiency. Prog. Food & Nutr. Sti. 8: McMurry, D. N., M. A. Crlomgno, nd P. A. Cumberlnd Respirtory infection with ttenuted Mycobcterium tuberculosis H37R in mlnourished guine pigs. Infect. Immun. 39: McMurry, D. N., S. A. Loomis, L. J. Cszz, H. Rey, nd R. Mirnd, Development of impired cell-medited immunity in mild nd moderte mlnutrition. Am. J. Clin. Nutr. 34: McMurry, D. N., nd E. A. Yetley Response to Mycobcterium bovis BCG vccintion in protein- nd zinc-deficient guine pigs. Infect. Immun. 39: McMurry, D. N., E. A. Yetley, nd T. Burch Effect of mlnutrition nd BCG vccintion on mcrophge ctivtion in guine pigs. Nutr. Res. 1: Nvi, J. M., nd C. E. Hunt Nutrition, nutritionl diseses nd nutrition reserch pplictions, p In J. E. Wgner nd P. J. Mnning (ed.), The biology of the guine pig. Acdemic Press, Inc., New York. 15. Orme, J. M., nd F. M. Collins Adoptive protection of the Mycobcterium tuberculosis-infected lung. Cell. Immunol. 84: Sinh, D. P., nd F. B. Bng Protein nd clorie mlnutrition, cell-medited immunity, nd BCG vccintion in children from rurl West Bengl. Lncet ii: Smith, D. W., nd G. E. Hrding Animl model of humn disese. Pulmonry tuberculosis. Am. J. Pthol. 84: Smith, D. W., D. N. McMurry, E. H. Wiegeshus, A. A. Grover, nd G. E. Hrding Host-prsite reltionships in experimentl irborne tuberculosis. IV. Erly events in the course of infection in vccinted nd nonvccinted guine pigs. Am. Rev. Respir. Dis. 12: Tuberculosis Prevention Tril, Mdrs Tril of BCG vccines in South Indi for tuberculosis prevention. Indin J. Med. Res. 7: Wiegeshus, E. H., D. N. McMurry, A. A. Grover, G. E. Hrding, nd D. W. Smith Host-prsite reltionships in experimentl irborne tuberculosis. III. Relevnce of microbil enumertion to cquired resistnce in guine pigs. Am. Rev. Respir. Dis. 12: Downloded from on April 3, 218 by guest