[Occurrence of struma in cattle in the Czech Republic]. / Strumavorkommen bei Kälbern in der Tschechischen Republik.

In the years 1988 to 1995 among 1355 cattle examined in the frame of iodine deficiency studies in the Czech Republic 404 animals (30%) showed congenital struma. Clinical and postmortal findings are described. A monitoring of iodine content in milk showed lower values in herds with struma prevalence. The insufficient supply of iodine in these animals results from low iodine content of feed or from the goitrogenic influence of nitrate in drinking water or high content of crucifera in feed or other stress factors. An increased iodination of mineral mix in feed is highly recommended.

The secondary antibody response induced in tissue cultures.

The conditions for induction of memory cells (B-MC) and evocation of the secondary antibody (Ab) response in tissue cultures (TC) were estimated. (1) In vivo primed B-MC cells were isolated 6-150 d after priming and stimulated in TC with different doses of sheep red blood cell (SRBC) antigen. The Ab response has a strict time and dose dependence: only small doses (10(5)) evoke a secondary response, high doses (10(8), 10(9)) a state of immediate tolerance. (2) Antigen added to TC directly with B-MC rescued their Ab production for a long period. Addition of the antigen 1 or 2 d after setting the TC, follows the Ab-response decay, comparable with virgin cells (B-ICC). (3) Primed B-MC stimulated in TC responded preferentially with an IgM secondary response; the same cell suspension adoptively transferred into isologous recipients switched into IgG cells. (4) Virgin, immunocompetent, B-ICC were primarily stimulated in TC with a small dose of antigen (10(5) SRBC); after 7 d of cultivation the cells were transferred into isologous recipients, SCID mice and into TC. In all cases, the secondary response of IgM was determined, 10 times higher than in the primary controls.

The severe combined immunodeficient mouse as a model for Encephalitozoon cuniculi microsporidiosis.

Microsporidia have been recently recognized as opportunistic pathogens in AIDS patients. In attempt to develop an animal model with features similar to the infections observed in the immunodeficient patients, the adult severe combined immunodeficient mice (SCID) were administered both intraperitoneally and perorally by 2 x 10(7) spores of the murine isolate of E. cuniculi. The experimental inoculation caused a severe, fatal disease characterized by the dissemination of microsporidia into the host tissues. The dominant route of E. cuniculi dissemination in the SCID mice was continual direct extension from the site of inoculation to adjacent tissues and organs, terminating in hematogenous spread of infection in the host. The different courses of microsporidiosis in SCID mice relative to the mode of inoculation (i.p. vs. p.o.) was observed. The survival time of i.p. infected SCID mice was 3 weeks--vs. 5 weeks in p.o. infected SCID mice. Experimental microsporidiosis in SCID mice should provide a useful model for studies in microsporidial pathogenesis, mechanisms of resistance, immunotherapy, and in evaluating potential antimicrosporidial agents.

Prophylactic and therapeutic immune reconstitution of SCID mice infected with Encephalitozoon cuniculi.

Severe combined immunodeficient (SCID) mice develop lethal infections, resembling opportunistic microsporidiosis of immunocompromised patients, after intraperitoneal (i.p.) inoculations of spores of Encephalitozoon cuniculi. In the present study, SCID mice reconstituted i.p. with 5 x 10(7) spleen cells from naive adult BALB/c mice 14 days prior to the i.p. injection of 10(7) spores were completely resistant to the infection, whereas control infected SCID mice developed clinical disease and died within 17 days post infection (DPI). In another experiment, SCID mice infected i.p. with 10(7) spores of E. cuniculi and after that (on DPI 7) injected i.p. with 5 x 10(7) spleen lymphocytes isolated from immune adult BALB/c mice were partially protected against the parasite (40% of the reconstituted mice survived). In both experiments, high levels of parasite-specific serum antibodies (mostly of the IgG-isotype) were detected in the infected immunocompetent BALB/c mice, whereas virtually no antibodies were found in the infected SCID mice. However, SCID mice reconstituted with either naive spleen cells or immune lymphocytes revealed humoral immune responses comparable with those of immunocompetent mice.

Stimulating effect of Streptococcus faecalis on phagocytosis in gnotobiotic piglets.

The concentration of active phagocytes in peripheral blood remained in germfree pigs up to the age of one year approximately at the same level as found at the age of 7 d and did not exceed 0.3 X 10(9)/L of blood, whereas a steady increase was established in conventional pigs. Monoassociation of gnotobiotic piglets with Streptococcus faecalis increased during 24 h the concentration of circulating granulocytes and the concentration of active phagocytes. An even more pronounced effect was obtained when formolized S. faecalis cells were applied intraperitoneally to germfree piglets. This treatment elevated the phagocytosis index also in conventional piglets, as well as in germfree piglets previously given cyclophosphamide. Escherichia coli O 83 or a mixture of anaerobic bacteria did not cause any serious changes in the activity of phagocytosis in gnotobiotic piglets. S. faecalis seems to be a natural factor stimulating both the release of granulocytes from their depots as well as their phagocytic activity.

Immunological state of adult germfree miniature Minnesota pigs.

Fundamental hematological and immunological data were obtained on sexually mature germfree miniature pigs fed, after the milk diet period, with cereal-type diet sterilized by gamma-radiation, and were compared with data of control conventional animals. Germfree adult pigs had a lower count of peripheral blood leukocytes with a lower percentage of neutrophil granulocytes and without any younger forms, a lower total serum protein level with a negligible amount of gamma and alpha 2 globulin fractions and a higher serum albumin and beta globulin level. In the mesenteric lymph nodes and in spleen, surface IgA-bearing cells predominated over surface IgG-bearing cells. Also a large amount of IgA-containing cells was found in the intestinal lamina propria, where the IgG cells were present in a negligible amount. IgM cells were the most frequent surface isotype in peripheral blood. The count of blood T lymphocytes was more than doubled.

Toxicity of staphylococcal toxic shock syndrome toxin 1 for germ-free and conventional piglets.

The susceptibilities of conventional and germ-free miniature pigs to staphylococcal toxic shock syndrome toxin 1 (TSST-1) were studied and compared. TSST-1 at doses of 100 micrograms/kg evoked reactions similar to those observed in rabbits, i.e., hyperthermia, diarrhea, apathy, conjunctival hyperemia, and various changes indicating toxic damage of different organs, but it was never lethal at this dose. Germ-free piglets were less susceptible than were corresponding conventional animals, a finding that may support the concept that endogenous endotoxin is involved in the pathogenesis of toxic shock syndrome.

Germ-free newborn baby.

Long term experience with germ-free animal experiments and technology encouraged us to apply the know-how in the field of the care of human neonates with presumed immunodeficiency. The program was carried out at the Faculty of Pediatrics of Charles University in Prague. The sterile caesarean section was performed within the plastic surgical isolator overlying the pregnant mother. A new type of this isolator adapted for the use in human medicine was designed and constructed. For the care of the neonate another special isolator was used. The whole isolator system included two rigid supply isolators, too. The newborn was sterile up to the age of 1 month. During this time, the newborns immunological capacity was investigated and found to be almost normal. Afterwards, the infant was gradually colonized with selected strains of bacteria and thus prepared for conventionalization.

The minipig as a model in gnotobiology.

Due to its close physiological and morphological resemblance to man, the pig occupies an important position in biomedical research. The minipig lends itself particularly well to germfree research. It is easy to deliver, maintain, and utilize in isolators. The colostrum-free, newborn germfree piglet lacking passively obtained antibodies is of an exclusive use if distinguishing the factors of natural and adaptive immunity is needed. It fits also well in studies investigating the earliest mechanisms of antibody synthesis. Under germfree conditions, the pig has been proved an excellent model for testing the action of ionizing radiation as well as of selected bacterial strains when monoassociated. Recently, conventional pigs have been used also for studies in total or selective decontamination of the intestinal tract by antibiotics and strict isolation.

Selective decontamination, induced colonization resistance and connected immunological changes in piglets.

14-d-old conventional piglets were picked from normal piggery, washed with disinfectants, placed into isolators suitable for germfree work, fed a sterile diet and treated with peroral antibiotics (nalidixic acid, kanamycin, and nystatin). Beginning with day 5 or 7, Enterobacteriaceae were not found in feces. The absence of these bacteria was proved by inoculation of germfree newborn piglets with caecal content. In selectively decontaminated piglets, the white blood cell count in blood had fallen to 6 X 10(9)/L; this decrease was due to an extremely low number of granulocytes (to 0.8 X 10(9)/L). On day 35, IgG-positive cells almost disappeared from the spleen, whereas IgA cells were found in an unusually great amount. Corresponding changes in serum levels were established. The colonization resistance effect in Enterobacteriaceae-deprived piglets was confirmed; settling of introduced various E. coli strains did not occur or was delayed.

Interaction of xenogeneic antiglomerular antibodies with foetal pig tissues.

Rabbit anti-swine-glomerular-immunoglobulin (AG Ig) was administered in utero to 14 pig foetuses 40-103 d post-conception. In nine foetuses AG Ig was injected into the umbilical vein and 60 min later kidneys and other organs were collected. In five others, AG Ig was injected via the intact uterine wall, and the tissues were examined after further 11 or 35 d of uninterrupted pregnancy. No signs of glomerular inflammation were found by light microscopy. In electron microscopy, especially in younger foetuses, focal endothelial defects and subendothelial granular "deposits" were seen in deep glomeruli, but they appeared also in control foetuses of corresponding age. Immunofluorescence microscopy showed a strong diffuse linear positivity of the swine-anti-rabbit-Ig conjugate in the capillary loops of deep juxtamedullary glomeruli irrespective of foetal age and dose of antigen injected. Immature glomeruli and S-bodies yielded a poor to negative staining. Proof of swine Ig was negative in all kidneys as well as the test for rabbit and swine Ig in various other organs. Traces of rabbit Ig were only detected in disseminated platelet aggregates of myocardium in the early post-injection period. Ultrastructural enzyme-antibody assay visualized AG Ig throughout all three layers of glomerular capillary basement membrane, and in the early period signs of transendothelial passage and escape of unbound rabbit Ig molecules were evident. The mesangial matrix, cell membranes, and extraglomerular basement membranes remained all negative. As follows, xenogeneic antiglomerular antibodies specifically react with target structures even at the end of the first third of intrauterine life, afflicted being only the deep mature polyanion-coated glomeruli. The foetal immune system is incapable of an adequate autologous antibody response, and we have failed to find even an early glomerular lesion comparable with the postnatal anti-basement-membrane glomerulonephritis.

The appearance of an IgD-like molecule on pig lymphocytes during ontogeny.

An IgD-like molecule was found on the surface of pig fetal lymphocytes using guinea pig anti-human IgD antisera and indirect immunofluorescence. The binding of antiIgD antisera could be specifically inhibited with human IgD. Surface IgD-like molecules were found later in ontogeny than surface IgM (after about 70 d of gestation in the spleen). There is evidence for their endogenous origin. The number of lymphocytes bearing IgD-like molecules rapidly increased during ontogeny.

Changes in the intestinal epithelial cells in abdominally irradiated germfree piglets.

The renewal time of intestinal epithelial cells was established using tritiated thymidine in 14-day-old conventional piglets as 96 hours, but 192 hours in germfree piglets. In germfree piglets, irradiated by single abdominal exposure to 60Co gamma-rays with 3.87 x 10(-1) C kg (= 1500 R), the first degenerative signs in the ileal epithelial cells were found on the 13th postirradiation day. Since that day, the columnar epithelial cells exhibited progressive degeneration changes in their ultrastructure. Quickly growing, substituting flat epithelial cells occurred beginning with the 15th day after irradiation and covered parts of the leveled denudated intestinal mucosa. These flat epithelial cells also exhibited degenerative changes.