Association between nutritional indicators and infectivity of dogs seroreactive for Trypanosoma cruzi in a rural area of northwestern Argentina.

The association between the nutritional state of mongrel dogs naturally infected with Trypanosoma cruzi and their infectivity to Triatoma infestans bugs and immune response to Trypanosoma cruzi were studied in the rural village of Amamá, northwestern Argentina. All of the 97 evaluated dogs were classified into one of three categories of external clinical aspect (ECA) based on the degree of muscle development, external evidence of bone structures, state of the hair of the coat, existence of fatty deposits, and facial expression. ECA was significantly associated with two nutritional indicators, hematocrit and skin-fold thickness, but not with total serum proteins. For all dogs, hematocrit was significantly correlated with skin-fold thickness. The 2-year survival probability decreased significantly from 60.7% for dogs with good ECA to 45.9% and 31.2% for those with regular and bad ECA, respectively. The age-adjusted relative odds of infection for Triatoma infestans xeno-diagnosis nymphs that fed once on a dog seroreactive for Trypanosoma cruzi decreased significantly as ECA improved, when tested by multiple logistic regression analysis. A delayed hypersensitivity reaction was observed in all of the seroreactive dogs with good ECA but only in 45-50% of those with regular or bad ECA. Dogs with bad ECA had a 2.6 and 6.3 times greater probability of infecting triatomines after a single full blood meal than dogs with regular or good ECA, respectively. Our study shows that the reservoir competence of dogs for Trypanosoma cruzi was associated with ECA, which is a surrogate and valid index of nutritional state.

[Molecular and immunologic bases for the development of a vaccine against Chagas disease]. / Bases moleculares e inmunológicas para el desarrollo de una vacuna contra la enfermedad de Chagas.

Subcellular fractions of T. cruzi epimastigotes (Epi) were studied for their capability to induce protective or aggressive effects in animals. The flagellar fraction (F) showed the best immunoprotective properties without tissular aggression. Monoclonal antibodies were raised against F. Two of them, FCH-F8-1 and 4, were able to neutralize the infectivity of bloodstream forms, to mediate lysis by complement of cell culture derived[trypomastigotes (Tripo) and to recognize the surface of Tripo and Epi. FCH-F8-1 reacted with a 85 kDa protein from Tripo (assayed by immunoprecipitation) and with peptides of 43 kDa on Epi and Tripo (tested by immunoblotting). FCH-F8-4 recognized several proteins ranging from 50 to 150 kDa on Epi and two molecules of 15 and 48 kDa on Tripo. Mice immunized with antigens purified by affinity chromatography by using FCH-F8-4 were protected against the infection. Several recombinant clones were detected on a cDNA lambda gt11 expression library constructed from T. cruzi Epi (Tulahuén strain): three with FCH-F8-4 and two with FCH-F8-1. One clone recognized by each monoclonal antibody was studied gamma (FCH-F8-1) 1 and gamma (FCH-F8-4) 1. Both inserts were of 150 base pairs each; they detected a 3.5 and 5.0 kilobases Epi mRNA, respectively. Both inserts were sequenced, and the amino acid sequences were inferred. gamma (FCH-F8-4) 1 codified for a 19 aa peptide, PAFLGCSSRFSGSFSGVEP, and gamma (FCH-F8-1) 1 for a 29 aa peptide EFLERGRISCORHSYTSYTSCSDEHNVTPFC. The whole 19 aa peptide was synthesized. This peptide (SP4) inhibited the ELISA reactivity against the parasite of chronically infected and F immunized mouse sera.(ABSTRACT TRUNCATED AT 250 WORDS)

Bases moleculares e inmunológicas para el desarrollo de una vacuna contra la enfermedad de Chagas. / [Molecular and immunologic bases for the development of a vaccine against Chagas disease]

Subcellular fractions of T. cruzi epimastigotes (Epi) were studied for their capability to induce protective or aggressive effects in animals. The flagellar fraction (F) showed the best immunoprotective properties without tissular aggression. Monoclonal antibodies were raised against F. Two of them, FCH-F8-1 and 4, were able to neutralize the infectivity of bloodstream forms, to mediate lysis by complement of cell culture derived[trypomastigotes (Tripo) and to recognize the surface of Tripo and Epi. FCH-F8-1 reacted with a 85 kDa protein from Tripo (assayed by immunoprecipitation) and with peptides of 43 kDa on Epi and Tripo (tested by immunoblotting). FCH-F8-4 recognized several proteins ranging from 50 to 150 kDa on Epi and two molecules of 15 and 48 kDa on Tripo. Mice immunized with antigens purified by affinity chromatography by using FCH-F8-4 were protected against the infection. Several recombinant clones were detected on a cDNA lambda gt11 expression library constructed from T. cruzi Epi (Tulahuén strain): three with FCH-F8-4 and two with FCH-F8-1. One clone recognized by each monoclonal antibody was studied gamma (FCH-F8-1) 1 and gamma (FCH-F8-4) 1. Both inserts were of 150 base pairs each; they detected a 3.5 and 5.0 kilobases Epi mRNA, respectively. Both inserts were sequenced, and the amino acid sequences were inferred. gamma (FCH-F8-4) 1 codified for a 19 aa peptide, PAFLGCSSRFSGSFSGVEP, and gamma (FCH-F8-1) 1 for a 29 aa peptide EFLERGRISCORHSYTSYTSCSDEHNVTPFC. The whole 19 aa peptide was synthesized. This peptide (SP4) inhibited the ELISA reactivity against the parasite of chronically infected and F immunized mouse sera.(ABSTRACT TRUNCATED AT 250 WORDS)

Reduced dietary protein content suppresses infection with Babesia microti.

The influence of acute dietary protein restriction on the development of Babesia 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 with B. 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 antibody-mediated 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 x 10(3) 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.

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 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 with Listeria monocytogenes five days prior to sacrifice. Zinc-deficient rats demonstrated 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 viable L. monocytogenes 10 days prior to respiratory challenge. Vaccination resulted in successful control of bacteria in both dietary groups.

Mycobacterium bovis BCG vaccine fails to protect protein-deficient guinea pigs against respiratory challenge with virulent Mycobacterium tuberculosis.

Specific-pathogen-free Hartley guinea pigs were maintained on isocaloric-purified diets either adequate (30%) or moderately deficient (10%) in protein. Half of each diet group was vaccinated with viable Mycobacterium bovis BCG. Six weeks later, all animals were challenged by the respiratory route with virulent Mycobacterium tuberculosis H37Rv. At intervals of 1, 2, and 3 weeks postchallenge, guinea pigs from each diet and vaccination group were skin tested with tuberculin and sacrificed. Protein deficiency resulted in loss of tuberculin hypersensitivity. Vaccination with M. bovis BCG protected control animals, as determined by significant reductions in the number of M. tuberculosis H37Rv organisms recovered from lungs, spleen, and bronchotracheal lymph nodes 2 and 3 weeks postchallenge. Based upon the same criteria, the degree of protection afforded protein-deficient animals by M. bovis BCG vaccine ranged from partial (spleen and lymph nodes) to none at all (lungs). Approximately the same numbers of tubercle bacilli were recovered from nonvaccinated guinea pigs in both diet groups. Protein deficiency appears to impair M. bovis BCG-induced immunity while not affecting primary pulmonary infection with virulent M. tuberculosis.

Influence of early weaning and dietary fat on immune responses in adult rats.

Equal numbers of female rats, either prematurely weaned at 14 days of age or allowed to nurse for 21 days, were pair-fed a diet containing either vegetable oil or cholesterol-enriched animal fat for 95 days. Thereafter all animals received the animal fat diet until 11 months of age. Rats were then immunized with sheep erythrocytes and the antibody response quantified. There was no significant effect of early weaning or diet on the number of plaque-forming splenocytes or on serum hemolysins. A significant positive correlation between HDL cholesterol and both plaque-forming cells and hemolysin titres was detected in the groups fed animal fat. Significant impairment in splenocyte blastogenic response to phytohemagglutinin was observed in rats receiving animal fat prior to 95 days. Separate groups of rats were infected 5 days before death with Listeria monocytogenes. Splenocyte blastogenesis was impaired in the group fed animal fat to a degree similar to that observed in uninfected rats fed the same diet, and there were increased numbers of bacteria recovered from the spleen and kidney of animals whose early diet contained animal fat. We conclude that the fat content of the early postweaning diet has an impact on immune responses which persists into adulthood.

Respiratory infection with attenuated Mycobacterium tuberculosis H37Ra in malnourished guinea pigs.

Specific pathogen-free guinea pigs were infected via the respiratory route with viable, attenuated Mycobacterium tuberculosis H37Ra and maintained on purified isocaloric diets. The control diet contained 30% protein (ovalbumin) and 50 ppm of added zinc (50 micrograms/g), the low protein diet contained 10% protein and 50 ppm of added zinc, and the low zinc diet contained 30% protein and no added zinc. Guinea pigs from each diet treatment were skin tested with purified protein derivative 48 h before sacrifice at 3, 4, and 5 weeks postinfection. Protein-deficient animals exhibited significantly reduced body weight, spleen weight, serum total proteins, and serum albumin. Zinc deficiency was characterized by loss of weight and progressive reductions in plasma zinc concentrations. The number of viable M. tuberculosis H37Ra cells was significantly higher in the lungs of both malnourished groups at 3 weeks, but fell below control viable counts by 5 weeks postinfection. A similar pattern was seen in the spleens and bronchotracheal lymph nodes. Both the proportion and intensity of delayed hypersensitivity reactions increased steadily between 3 and 5 weeks in control animals, whereas the two malnourished groups were essentially anergic at all intervals, despite systemic infection. These results demonstrate that both protein and zinc deficiencies exert a significant influence on the development of pulmonary tuberculosis but that the nature of the influence depends upon the interval studied. In both malnourished groups, the pulmonary infection tended to peak early and decline, whereas the disease developed more slowly in control animals. Apparent control of mycobacterial populations in the tissues was accomplished by malnourished animals in the absence of demonstrable delayed hypersensitivity.

T and B lymphocyte function in response to a protein-free diet.

Groups of female adult rats were fed either isocaloric protein-free or 18% protein diets for various intervals. Four days before sacrifice, the animals were immunized either with sheep erythrocytes or with a trinitrophenyl-lipopolysaccharide (TNP-LPS) conjugate. Spleen lymphoid cell populations, spleen plaque-forming cells, and serum hemolysins were measured. A persistent diminution, proportional to the duration of protein deprivation, was observed in all parameters studied after immunization with the T-dependent antigen, sheep erythrocytes. The immune dysfunction was more pronounced for hemolysin titers, which became undetectable after 15 days of protein-free diet. The response of the protein-free group to the T-independent antigen (TNP-LPS) after 15 days of diet was only 34% of the control. When a T-cell lymphokine, macrophage migration inhibitory factor, was measured, a normal response was observed in the protein-free group. Feeding a normal diet rapidly restored the spleen plaque-forming cell populations to 60% of normal after 4 days and to 100% after 6 days. Protein starvation influenced the production of antibodies more than it did the number of antibody-forming cells. The nutritional impairment of immunoglobulin synthesis appears to be reversible.

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.

B-cell immune response during total protein deprivation.

Female adult rats fed with a protein-free normocaloric diet were antigenically stimulated with 1.3 x 10(-9) sheep red blood cells. Lymphoid spleen population, spleen plaque forming cells and serum haemolysins were measured in experimental as well as in control animals fed with a normal (18% protein) diet. A persistent diminution, proportionally related to the period of protein deprivation, of all parameters studied was observed; the fall was more prominent for haemolysin titre which became undetectable after 15 days of protein-free diet. Also, animals exposed to the aproteic diet during 15 days were afterwards returned to a normal diet and maintained on it during variable periods of time. A rapid restoration of the humoral response capacity, up to 60% of normal after 4 days of protein intake, and completely normal, following 6 days of refeeding, was observed. These findings suggest that protein starvation influences the production of circulating antibodies more than the antibody-forming cells. The aproteic induced impairment involved in the immunoglobulin synthesis does not appear to be irreversible, at least during the dieting period explored in the present study.

B-cell immune response during total protein deprivation

Female adult rats fed with a protein-free normocaloric diet were antigenically stimulated with 1.3 x 10(-9) sheep red blood cells. Lymphoid spleen population, spleen plaque forming cells and serum haemolysins were measured in experimental as well as in control animals fed with a normal (18

protein) diet. A persistent diminution, proportionally related to the period of protein deprivation, of all parameters studied was observed; the fall was more prominent for haemolysin titre which became undetectable after 15 days of protein-free diet. Also, animals exposed to the aproteic diet during 15 days were afterwards returned to a normal diet and maintained on it during variable periods of time. A rapid restoration of the humoral response capacity, up to 60

of normal after 4 days of protein intake, and completely normal, following 6 days of refeeding, was observed. These findings suggest that protein starvation influences the production of circulating antibodies more than the antibody-forming cells. The aproteic induced impairment involved in the immunoglobulin synthesis does not appear to be irreversible, at least during the dieting period explored in the present study.

B-cell immune response during total protein deprivation.

Female adult rats fed with a protein-free normocaloric diet were antigenically stimulated with 1.3 x 10(-9) sheep red blood cells. Lymphoid spleen population, spleen plaque forming cells and serum haemolysins were measured in experimental as well as in control animals fed with a normal (18

protein) diet. A persistent diminution, proportionally related to the period of protein deprivation, of all parameters studied was observed; the fall was more prominent for haemolysin titre which became undetectable after 15 days of protein-free diet. Also, animals exposed to the aproteic diet during 15 days were afterwards returned to a normal diet and maintained on it during variable periods of time. A rapid restoration of the humoral response capacity, up to 60

of normal after 4 days of protein intake, and completely normal, following 6 days of refeeding, was observed. These findings suggest that protein starvation influences the production of circulating antibodies more than the antibody-forming cells. The aproteic induced impairment involved in the immunoglobulin synthesis does not appear to be irreversible, at least during the dieting period explored in the present study.