Mirta A. Carlomagno

The influence of dietary protein on the protective effect of BCG in guinea pigs

Specific pathogen-free, Hartley guinea pigs were vaccinated with viable Bacille Calmette-Guerin (BCG) and given isocaloric diets identical in every nutrient except protein (control = 30%; low protein = 10%). A non-vaccinated group was maintained on the control diet. Five weeks later, all animals were infected with an aerosol containing virulent M. tuberculosis H37Rv. On the same day, half of the protein-deficient guinea pigs were transferred to the control diet, while the remainder were maintained on the low protein (10%) diet. Animals from each diet treatment were tuberculin tested and sacrificed 1,2,3 and 4 weeks post-challenge. Protein-deficient guinea pigs exhibited diminished tuberculin reactions and loss of BCG-induced protection against virulent challenge as measured by the number of viable M. tuberculosis recovered from the lung and spleen. Renourished animals expressed normal levels of delayed hypersensitivity within 1 week of initiating the normal diet and were protected as well as vaccinated control guinea pigs against virulent respiratory challenge.

Chronic zinc deficiency in rats: Its influence on some parameters of humoral and cell-mediated immunity

Abstract The effect of dietary zinc deficiency on immune function was studied in specific pathogen-free rats. The spleen cell response to sheep red blood cells (srbc) and serum hemolysins were measured in rats maintained on the depleted or normal diet for periods of time ranging from 3 to 10 weeks and immunized with srbc six days prior to sacrifice. The in vitro response of splenic lymphocytes to the polyclonal T-cell mitogen, phytohemagglutinin (PHA), was also investigated. There was no significant effect of the diet on spleen plaque-forming cells, although values for zinc-deficient rats tended to vary widely as the time on the diet increased. Hemolysin titers were unaffected by diet at all intervals. A significant reduction in lymphocyte blastogenesis was observed in the zinc-deficient rats after 5 weeks on the diet. Control animals responded with four times the stimulation observed in the low zinc group. Thymus weight and serum zinc levels were significantly reduced by the zinc-deficient diet. Chronic, moderate zinc deficiency appears to have a differential effect on humoral and cell-mediated lymphocyte functions in this model. The normal plaque-forming cell and hemolytic antibody response to srbc suggests that helper T-cell and B-cell functions are intact. In contrast, the impaired mitogenesis in response to PHA stimulation indicates that zinc deficiency is affecting other T-cell subset(s).

Role of Immunoglobulin Classes in Experimental Histoplasmosis in Bats

Pooled normal bat serum was separated by gel filtration to give fractions rich in IgG-, Iga- and IgM-like proteins. These fractions were analogous to the corresponding human immunoglobulin classes by immunoelectrophoresis and SDS-polyacrylamide gel electrophoresis. Rabbits were immunized with the fractions and the antisera absorbed. Neotropical bats (Artibeus lituratus) were infected with Histoplasma capsulatum and serum samples were collected weekly and tested for specific serologic response to the fungus. A radial immunodiffusion test was devised to monitor changes in concentrations of IgG, IgA and IgM in the same sera. Bats infected with a low dose of fungus had significantly increased levels of IgM and IgA between 2-6 weeks post-infection. Bats receiving a high dose maintained elevated levels of IgM and IgA through the end of the study. Significantly elevated levels of IgG were not detected until late in the disease (8-9 weeks). In bats with histoplasmosis, IgM and IgA appeared to contribute primarily to the early positive serologies, while precipitating antibodies of the IgG class were detectable later in the disease. These results are similar to the serologic profile seen in human histoplasmosis, and extend our understanding of comparative immune responses in an important wildlife reservoir of human mycotic pathogens.

Chronic zinc deficiency and listeriosis in rats: acquired cellular resistance and response to vaccination

The functional significance of zinc deficiency on primary and secondary host responses to infection with a facultative intracellular pathogen was studied in specific pathogen free rats. Groups of female rats fed either a low zinc or normal diet for 8 or 10 weeks were infected withListeria monocytogenes five days prior to sacrifice. Zinc-deficient rats demostrated thymic atrophy, reduced delayed hypersensitivity responses to listeria antigen, and impaired lymphocyte response of spleen cells to phytohemagglutinin, but not to Concanavalin A. Separate groups of zinc-deficient or control rats were vaccinated with viableL. monocytogenes 10 days prior to respiratory challenge. Vaccination resulted in successful control of bacteria in both dietary groups.

Differential effect of protein and zinc deficiencies on lymphokine activity in BCG-vaccinated guinea pigs

Abstract Pathogen-free guinea pigs maintained on isocaloric, purified low protein (LP), low zinc (LZ) and control (C) diets and commercial chow (Ch) were vaccinated with viable Mycobacterium bovis BCG and sacrificed 4, 5, and 6 weeks later. Both groups of malnourished animals showed weight loss and zinc-deficient guinea pigs had reduced plasma zinc levels. Serum proteins and albumin levels were reduced in LP guinea pigs. Animals on LP and LZ diets showed significantly impaired delayed hypersensitivity to dinitrochlorobenzene and tuberculin when compared to animals consuming the C and the CH regimen. The ability of peritoneal exudate cells to produce macrophage migration inhibition factor (MIF) when stimulated with PPD in vitro was diminished in animals maintained on the LZ diet for more than 5 weeks, but was unaffected by protein deficiency. Reduced capacity to produce functional lymphokines may contribute to skin test anergy in chronic zinc deficiency, but does not explain loss of delayed hypersensitivity in protein-deprived guinea pigs.

Reduced dietary protein content suppresses infection withBabesia microti

The influence of acute dietary protein restriction on the development ofBabesia microti infection in the mouse model was investigated. Female mice consuming a diet either devoid of protein or adequate with respect to protein were infected withB. microti-parasitized erythrocytes and sacrificed 7 days later. Absence of dietary protein resulted in a delay in the onset of infection and a significantly reduced peak parasitemia. Non-specific antibody responses to heterologous erythrocytes and specific anti-babesial antibody titers were impaired in mice consuming the protein-free diets, suggesting that the enhanced resistance to experimental babesiosis observed in protein-malnourished mice is not an antibodymediated phenomenon. In addition, protein-malnourished mice did not demonstrate significantly lower concentrations of the serum complement component, C3, which has been implicated as a participant in the invasion process of host erythrocytes by parasites. Serum C3 levels were significantly reduced in infected mice consuming both diets. The mechanism by which acute protein deprivation protects mice against lethal babesiosis remains to be determined.

Effect of protein and zinc deficiencies on vaccine efficacy in guinea pigs following pulmonary infection with Listeria

Specific pathogen-free guinea pigs were maintained for 3 weeks on purified diets containing 30% protein (ovalbumin) and 50 ppm added zinc (Control-C), 10% protein and 50 ppm added zinc (low protein-LP), or 30% protein and no added zinc (low zinc-LZ). Half of the animals in each diet group were vaccinated intraperitoneally with 2.5 × 103 viable Listeria monocytogenes organisms after 8 days of diet treatment. Ten days later, all animals received an aerosol challenge of 250 L. monocytogenes organisms and were killed 4 days later. Both zinc and protein deficiency resulted in animals that were growth retarded as compared to controls. Specific nutrient effects were observed as significant reductions in total serum proteins (LP group) and plasma zinc concentrations (LZ group). In vaccinated guinea pigs, both protein and zinc deprivation resulted in significant impairment of delayed-type hypersensitivity (DTH) responses following the intradermal injection of listeria antigen. Diet did not exert a measurable impact on the response of nonvaccinated guinea pigs to pulmonary listeriosis. Prior vaccination allowed both malnourished groups to control the challenge infection successfully as measured by significant reductions in viable bacilli recovered from the lung, spleen and hilar lymph nodes. The diet and vaccine effect varied depending on the tissue examined. Thus, although both protein and zinc deficiencies resulted in loss of peripheral antigen-specific T lymphocyte function (DTH), vaccine efficacy was not impaired.

Effect of dietary protein and zinc on anti-mycobacterial antibody responses in guinea pigs

Abstract An enzyme immunoassay was developed to quantify the levels of antibodies reactive with purified protein derivative (PPD) at 3, 4, 5 and 6 wks following vaccination with Mycobacterium bovis BCG and initiation of diets deficient in either protein (LP) or zinc (LZ). Significant antibody levels were detected in all groups as early as 3 wks post-vaccination compared to non-vaccinated animals. Both LP and LZ guinea pigs had higher levels of antibody than animals consuming an adequate diet at 3 and 4 wks, respectively. By 6 wks post-vaccination, animals on the low protein diet had the highest antibody titers, followed by control, chow and LZ guinea pigs in order of decreasing titers. A highly significant positive correlation was detected between the levels of anti-mycobacterial antibodies and the number of viable BCG recovered from the lymph nodes draining the vaccination site. A significant inverse correlation was seen between antibody levels and PPD skin reactions. High-titer sera from protein-deficient, but not zinc-deficient, BCG-vaccinated pigs significantly suppressed the tuberculin reactions when passively transferred into normal, PPD-positive recipients.

Effect of diet on non-specific antimicrobial resistance in Mycobacterium bovis BCG-vaccinated guinea pigs

Abstract Specific pathogen-free guinea pigs were maintained on purifieddiets low in protein (LP-10% ovalbumin; 50 ppm Zn); low in zinc (LZ-30% ovalbumin; Mycobacterium bovis BCG. Four and five weeks post-vaccination and diet initiation, guinea pigs from each of the 6 diet × vaccination groups were challenged with 10 3 viable Listeria monocytogenes per animal by the respiratory route and sacrificed four days later. Low protein guinea pigs demonstrated weight loss and reduced serum albumin levels. Low zinc animals were growth-retarded and had reduced plasma Zn levels. Neither LZ nor LP guinea pigs developed significant tuberculin reactions post-vaccination at either sacrifice interval. Non-specific acquired resistance against L. monocytogenes was enhanced in LP and LZ animals at 4 wks post- M. bovis BCG vaccination but not at the 5 wk interval. The anti-listeria resistance was most pronounced in the liver and spleen. While M. bovis BCG-vaccinated LP and LZ guinea pigs were incapable of expressing tuberculin hypersensitivity, they did develop a significant non-specific anti-listeria resistance.

Influence of the timing of nutritional insult on alterations of cell-mediated immunity in guinea pigs infected with Mycobacterium bovis BCG by the respiratory route

Abstract Alterations of cell-mediated immunity in nutrient-deficient humans and animals are influenced by the severity and chronicity of the nutritional insult. Previously we have shown that initiation of deficient diets at the time of vaccination with Mycobacterium bovis BCG alters the immunologic status of guinea pigs several weeks later. In the present study, guinea pigs were infected by the respiratory route with M. bovis BCG simultaneously with, or two weeks or four weeks prior to placing groups on either low protein (LP), low zinc (LZ) or control (C) diets. Six weeks post-infection, all guinea pigs were skin tested with tuberculin and sacrificed. Splenocyte suspensions were stimulated in vitro with three doses each of two polyclonal T cell mitogens, phytohemagglutinin (PHA) and concanavalin A (Con A). Tuberculin reactions were completely suppressed in both LP and LZ groups maintained for the entire vaccination interval (6 weeks) and partially impaired in the protein-deficient animals after 4 weeks or 2 weeks on the LP diet. Reduced lymphoproliferative responses to both PHA and Con A were seen in acutely malnourished LP and LZ guinea pigs after 2 weeks on the diet. After 6 weeks on the deficient diets, lymphocytes from LZ animals were hyporesponsive to low concentrations of both mitogens.