On the 12th, 20th and 21st day after infecting goats orally with sporocysts of S. capracanis from the faeces of dogs which had been infected with raw meat containing Sarcocystis of naturally infected goats, mature schizonts were found in the cytoplasma of endothelial cells of the veins in the liver, spleen, kidney and brain. The nucleus of the young schizont increases markedly in size, forming several protrusions which tore away simultaneously giving rise to numerous merozoites. The schizonts were about 30 microns in size and retained their three-layered pellicle till the merozoites matured. After rupture the schizonts set the 6 microns long daughter cells free, which were distributed by means of the blood. Though degenerated schizonts were present in the brain, the cells around the parasitized cells showed no reaction. In muscles as well as in the brain the second phase of the asexual reproduction (cyst formation) took place. The merozoites first became spherical in parasitophorous vacuoles in parasitized cells. The unit membrane of the parasitophorous vacuole which formed the primary cyst wall developed protrusions which were mostly straight, giving the aspect of a thick, striated cyst wall. A secondary cyst wall was never formed according to light microscopical studies. In a single animal thin-walled cysts occurred, which probably belonged to a second Sarcocystis species. The protrusions of both cyst-types contained no filaments. Cyst maturation took about 1 month, so that on the 65th day p.i. numerous infectious, banana-shaped merozoites (15 microns long) were still present as well as some metrocytes (12 microns long) which were constantly reproducing by endodyogeny.

NMRI mice were infected orally with S. typhimurium. Specific antibody titers determined by indirect hemagglutination, and cellular immune response tested by blastogenesis transformation of lymphocytes and enhanced bactericidal action on Listeria monocytogenes were studied in relation to persistence of bacteria up to one year after the infection. About three weeks after the infection cell mediated immune reactions were no longer detected and in the course of a year detection of serum antibodies often failed despite persistence of Salmonella.

Shortly after an open-heart operation a 5-year-old girl died of an infection caused by M. fortuitum. Strains of this species are often isolated from human specimens, but generally they are not correlated with pulmonary tuberculosis. Nevertheless M. fortuitum produces relatively often infections after transplantations of different kind. The diagnosis is difficult to find, especially because nobody thinks of the possibility that a rapid growing mycobacterium is able to cause such infections. -- The therapy is very problematical. That is why these infections are not seldom fatal.

Alkali-treated lipopolysaccharides of a range of enterobacterial R mutants with complete and incomplete core structures were investigated by agar gel and microcapillary precipitation onto their reactivity with three selected commercial lectins, namely Concanavalin A, Ricinus communis agglutinin and Wheat germ agglutinin. R lipopolysaccharides with complete R cores of the six so far established core types were differentiable from each other by their characteristic lectin reactivity pattern. One core type showed no reaction with all the three lectins. R lipopolysaccharides with incomplete core structure showed in general the reactivity pattern predictable from their individual chemical configuration. It was further shown that substitution of complete R cores by haptens (T1, T2 or ECA) or by a single repeating unit (SR mutant) did not change the reactivity pattern, although some interactions were less pronounced. It will be demonstrated that lectin precipitation can be of help for structural studies in recognizing, for example, terminal sugar units and their anomeric linkages.

The aerobic gram-negative faecal flora of 38 bats consisting of 10 species and genera respectively, of Microchiroptera, and of 4 species and genera respectively, of Megachiroptera was studied (Table 1 and 3). There were no specific differences between Insectivora and Frugivora: E. coli 15-24%, Citrobacter 8-10%, Enterobacter-Klebsiella-group 40-43% and Proteus-group 28-30% (Table 2). The overwhelming majority of the isolated bacteria were lactose-positive (Table 3), corresponding to the membership of the bats to the mammals. The vampire bats (Desmodus rotundus), however, nourishing exclusively with mammalian blood, possess a fundamental other faecal flora. Here we always found Aeromonas hydrophila sometimes as a pure culture and sometimes in combination with E. coli, Enterobacter, Providencia, and Arizona. The normal habitat of Aeromonas hydrophila in vampire bats suggests that these bacteria are necessary for digest the drunken blood in a similar manner as in leechs. The observations were discussed regarding their ecological, epidemiological, and phylogenetic significances.

19 weakly saccharolytic Bacteroides strains of different species were tested by thin-layer chromatography (TLC) for their fermentative abilities for fructose, sucrose, lactose, galactose, and raffinose. Conventional fermentation tests were run parallel. In general, a good agreement between both methods was recorded. Two strains, however, showed a degradation in the TLC test without an acidification. With some strains, sucrose as a substrate yielded a fructose spot, lactose a galactose spot, and raffinose a melibiose spot, indicating an incomplete degradation of these carbohydates.

The influence of incubation temperature (37, 30 and 22 degrees C) on antibiotic susceptibility, beta-lactamase activity and growth characteristics was studied on 43 unselected strains of Y. enterocolitica (Serovar O:3 and O:9) freshly isolated from cliical specimens. Antibiotic susceptibility was measured by the disc diffusion technique and by a broth dilution test (MIC). Beta-lactamase activity was detected with chromogenic cephacetrile using standard curves prepared for 37, 30 and 22 degrees C. Continuous increase of beta-lactamase activity was found when incubation temperatures were lowered. All strains were found to be resistant by ampicillin and cephalothin at the three temperatues tested. Some strains showed an intermediate susceptibility to carbenicillin in the disc diffusion test. A temperature reduction of 37 to 30 degrees C significantly decreased the inhibitory zone diameters for the beta-lactam antibiotics ampicillin, carbenicillin and cephalothin, but also for other substances like tetracycline, chloramphenicole and cotrimoxazole. This suggests, that the observed decrease is caused by a better growth of Y. enterocolitica at 30 degrees C rather than increased beta-lactamase production. From 30 to 22 degrees C a further decrease in inhibitory zone diameters was only seen with ampicillin and carbenicillin. This seems to be mainly due to the increased B-lactamase activity observed at 22 degrees C. In contrast the resistance to cephalothin was apparently not influenced by this additional beta-lactamase activity. Resistance to cephalothin therefore depends probably more on other, not beta-lactamase-related, factors such as permeability variations of the outer membrane or modifications of binding proteins involved in the peptidolycan biosynthesis. The correlation between beta-lactamase activity at various incubation temperatures and resistance to beta-lactam antibiotics was less pronounced when the broth dilution test (MIC) was applied. Only carbenicillin showed significantly increasing MIC values from 30 to 22 degrees C. All the Y.e. strains investigated could be divided into two groups with respect to their beta-lactamase production characteristics. The first group showed continuously increasing beta-lactamase values at lower incubation temperatures. In the second group generally lower amounts of beta-lactamase values were found and temperature dependence was not observed. On the other hand variations in cell wall permeability, resulting in a diminished accessability of the cell wall bound enzymes must also be considered.

96 strains of facultatively anaerobic actinomycetes and 2 Propionibacterium acnes strains were studied for their ability to deaminate and/or decarboxylate 13 amino acids, to reduce nitrate and nitrite, and to produce indole, using specially adapted micro-methods. Several of the tests performed were found to provide information which may aid in improving the classification and in facilitating the identification of these organisms.

Two procedures to improve virus cultivation from clinical material were evaluated: an intensified adsorption with 0.2 ml inoculum and 20 h rocking in a Bellco rocker and a low-speed centrifugation of the material onto the cultured cells. Prototype strains from 5 virus families (adenoviruses, herpes simplex virus, vaccinia virus, enteroviruses, parainfluenza 2) as well as original specimens from patients (adenovirus, herpesvirus, enterovirus) were studied by endpoint titration in comparison with the standard procedure. In adenoviruses, a quantitative immunofluorescence was performed too. In endpoint titrations, the centrifugation method did almost never lead to an increased virus titer, as compared with the standard method. However, in the immunofluoresence evaluation of adenoviruses the values attained were 3- to 4-fold higher. On the other hand, the intensified adsorption method led to an increased sensitivity in most adenovirus titrations with 4- to 25-fold titer increment, with prototype and original material. The procedure was ineffective in all other viruses studied.

The effect of Fosfomycin on K. pneumoniae ATCC 10031 was studied. The morphology on the electron microscopical level and the viability were markedly altered after application of 6 micrograms/ml and 60 micrograms/ml of Fosfomycin, respectively. These were chosen because they can be attained in man by oral or parenteral administration. Until 30 min after the administration of 6 micrograms/ml, and 10 min after administration of 60 micrograms/ml the turbidity increased in the same range as in the control. Thereafter the turbidity decreased but did not fall below its minimal values; after application of 6 micrograms/ml of Fosfomycin the OD remained at higher levels than after applying 60 micrograms/ml of Fosfomycin, at all corresponding times. The number of viable cells, after application of 6 micrograms/ml of Fosfomycin, was maximally reduced for 70% of the value at the time of administration. 60 micrograms/ml quickly impaired the ability of reproduction. Consequently, the CFU were reduced continuously, e.g. by 80% after 30 min and by more than 99% after 180 min. The finestructural alterations were characterized by loss of contrast and regular shape. The occurrence of protruded protoplasts and defects in the cell wall indicate the action of Fosfomycin on the bacterial envelope, preferably on the peptidoglycan layer.

With regard to that of other facultatively pathogenic microorganisms, the rate of isolation of Pseudomonas aeruginosa was 12.5%. Most frequently the pathogen was isolated from wound swabs, tracheal secretion, throat swabs and urine. In 2.8% of the cases it was identified as the causative pathogen of sepsis. The serotype most commonly identified was the O antigen type 6, followed by types 11 and 1. The distribution of the serotypes observed in specimens and hospitals varied altogether. Epidemiologically striking was the incidence of serotype 1 in the urological division, of type 11 in the gynecological division, of type 6 in the pediatric division, and of type 4 in the dermatological division. Between 4 to 5% of the 618 strains examined exhibited resistance to gentamicin, tobramycin and sisomicin. Insensitivity to amikacin has not been observed. There were no statistically significant differences in incidence of resistance or the pattern of resistance as far as individual serotypes or isolates from the above mentioned divisions are concerned.

A loss of nerve cells in the cortex after encephalitis was reported already in the classical work by Nissl, Spielmeyer, and Spatz. A loss of nerve cells will become only noticeable if it amounts to at least 50%. But as such clear pictures are rarely found, estimations were always considered as doubtful and incorrect. Not only the number of cells is important in consideration of the morphological change in the cortex but also the size of cells. The development of a new apparatus made is possible to consider two structural parameters: the surface and the perimeter of cells. In 497 histological serial preparations obtained from 43 mouse brains we determined the number, the surface, and the perimeter of nerve cells. 39 animals were infected intracerebraly with yellow fever 17 D; 4 normal animals served as controls. Among the infected animals, 8 were treated with a mucopolysaccharide. The cells were counted within a determined area (standard unity); this area was taken from the angle between the curbura exterior and the sulcus anterior-posterior of the brain. There was a significant difference between the number of nerve cells in normal (278) and in infected (202) animals. The animals treated with mucopolysaccharide showed a normal quantity of nerve cells but surface and perimeter corresponded to the data of the infected ones. The surface of normal animals was at 23.39, that of infected at 14.29. There was also a significant difference with regard to the cell perimeter: normal 14.97, infected 12.02. This means a shrinking of cells. The cell shrinkage revealed that the nerve cells were affected. The measurement of these three parameters presents new and exact statistical findings which enable a reconsideration of neurovirulence.

Macrophages are highly differentiated mononuclear phagocytes which originate from stem cells of the bone marrow. The secretory potential of these cells has been recognized in recent years. Major secretory products comprise lysosomal enzymes, complement proteins, prostaglandins and interferon. Secretion of lysosomal hydrolases and proteinases is most prominent in macrophages stimulated in vivo or in vitro (Fig. 4). Lysosomal enzyme secretion may be an important factor in the induction and maintenance of inflammatory reactions. Complement (C) proteins secreted by macrophages belong to the classical activation unit (C1, C4 and C2), alternative activation unit (C3, B, D, P) and to the group of delayed-acting C proteins (Fig. 7). Therefore macrophages produce at local sites the C component C3 from which biologically active C3 fragments (C3a, C3b, C3e) can be generated. These C3 fragments mediate inflammatory and cytotoxic reactions and also promote phagocytic processes (Fig. 6). Cleavage of secreted C3 into the active fragments may occur by enzymes derived from both C activation units or by secreted lysosomal proteinases (Fig. 8). Stimulated macrophages also synthesize and release prostaglandins. These compounds which have inflammatory as well as antiinflammatory effects (Fig. 12) may play an important regulatory role in inflammatory processes. Interferon has been also recognized as a secretory product of macrophages. This substance supports antimicrobial resistance by its phagocytosis-increasing effect and its antiviral activity. The secretory function of macrophages as well as the biological effects of secreted mediators are highly susceptible to modulation. Thus, C3 fragments stimulate the secretion of lysosomal enzymes (Fig. 6) whereas prostaglandins inhibit their release (Fig. 12). The inflammatory reactions induced by lysosomal enzymes may be further increased by the generation of C3b which stimulates additional lysosomal enzyme release (Fig. 4). These and other examples suggest that endogenous control mechanisms may have a strong influence on the secretory function of macrophages as well as on the biological activity of secreted mediators.

A new high performance liquid chromatographic method for quantitative analysis of doxycycline has been compared with the microbiological assay. Both analytical methods yielded the same results of nearly 100% recovery when doxycycline was incubated with serum, lung-, or liver-homogenate (Table 1). The advantages of the chromatographic method over the microbiological method are i. short and simple way of analysis (Fig. 1), ii. need of only small amounts of biological material. In a pilot study doxycycline - after i.v. application to mice - could be analyzed in good agreement by both methods (Fig. 2). The microbiological analysis yielded in slightly lower serum- or organ-levels what might be due to an inactivation of the antibiotic attached to proteins to some extend.

369 staphylococcal strains isolated from clinical material were examined for tubeagglutination with sensitized sheep red cells in a standardized assay to study its reliability for routine identification of S. aureus. Colonies isolated from blood agar plates correlated in 99.5% with the (optimized) coagulase reaction. The test is easily to perform and results can be read after 2 hours, whereas the reference methods coagulase, hyaluronidase and deoxyribonuclease took as much as 24 h. The reliability of these tests is discussed.

Phenol-water-extracted lipopolysaccharide (LPS) from Yersinia enterocolitica (strain 75 in smooth form; O-group I), after growth at 10 degrees C, contains approximately 40% (w/w) 6-deoxy-L-altrose. By increasing the cultivation temperature a significant decrease in the O-specific sugar is observed. LPS from cells grown at 40 degrees C contains about 12% 6-deoxy-L-altrose. Thin-section micrographs of cells grown at lower temperatures reveal a distinct layer corresponding to the O-specific side chains of LPS in contrast to cells grown at higher temperatures where a decrease in thickness is observed. It is assumed that this observation is due to the decreasing amount of the O-specific sugar at a higher cultivation temperature. The results obtained by the indirect immunoferritin test indicate that the O-specific polysaccharide layer covers the cell surface completely up to 37 degrees C. However, this layer no longer covers the entire surface of all cells as soon as an incubation temperature of 40 degrees C is applied.

Concentrations of alpha 2-macroglobulin could be determined in the sera of 215 blood donors and 94 patients with various internal diseases by quantitative inhibition of an acid protease from Staphylococcus aureus, M 135 (fig. 1, 2). The determinations agreed closely with those obtained by immunodiffusion (tab. 1, fig. 3). However, the alpha 2-macroglobulin-measurements by the protease method required only 4 h and 40 microliter serum. This procedure would also be suitable for the determination of alpha 2-macroglobulin in sera from experimental and domestic animals.

Neuraminidase activity was discovered in 32 of 38 strains of Propionibacterium acnes. Enzyme production was studied in yeast extract bouillon of different pH containing various amounts of human milk as neuraminidase inductor. Enzyme activity was found in the bacterial sediments as well as in the culture filtrates. Since neither ultrasonic treatment nor lysozyme incubation of bacterial sediments did release reasonable amounts of enzyme, culture filtrates were used for enzyme preparation. Neuraminidase was isolated by 40% ammonium sulfate precipitation, dialysis, concentration and repeated gel chromatography on Sephadex G-100. The enzyme posesses a molecular weight of about 33 000 and a pH-optimum around 5.0. The Michaelis constants are 1.8 x 10(-3) M for alpha 2 leads to 3 linked N-acetylneuraminic acid (NeuAc) in II3NeuAc-Lac, 3.7 x 10(-3) M for the alpha 2 leads to 6 linkage in II6NeuAc-Lac, and 2.1 x 10(-3) M for the alpha 2 leads to 8 linkage of II3 (comes from 2 alpha NeuAc8)2-Lac, respectively. Among the different groups of naturally occurring NeuAc-containing substrates, i.e. oligosaccharides, glycolipids and glycoproteins, the enzyme exhibits its highest activity towards low molecular weight oligosaccharides. Activity is considerably lower on glycoproteins. Glycolipids (gangliosides) are only little attacked under conditions used in the test. However, there is no remarkable specificity towards one of the different linkage types of N-acetylneuraminic acid. In general, the enzyme reveals a specificity pattern similar to that found in other bacteria of low pathogenicity towards man.

E. coli rough strains (as determined by spontaneous agglutination of in boiled saline suspension) isolated in significant amounts (>10(5)/ml) from from patients with urinary tract infections were investigated for some factors possibly related to virulence. The frequencies of capsular antigen, colonization factor antigen (CFA I), dulcitol fermentation, and hemolysin production are summarized in table 1. 270 (66.2%) out of 408 strains were found to be encapsulated as determined by the inagglutinability of the living strains in saline. 180 (44.1%) strains were able to ferment dulcitol, and evidence for hemolysin production could be demonstrated in 146 (35.8%) strains. The CFA I (demonstrated by a mannose resistant hemagglutination of human red blood cells) was detected in 62 (15.2%) of the E. coli rough strains tested. The frequency of single properties in the presence or absence of capsular antigen is shown in table 2 demonstrating no significant differences in both groups. The most frequent single factor was found to be the capsular antigen with 22.1%, followed by the double combination capsular antigen/dulcitol fermentation (16.7%), dulcitol fermentation alone (10%), and capsular antigen/hemolysin production (9.8%). There were only 5 strains possessing all four factors tested. On the other hand we found 35 (8.6%) rough strains with none of the properties. Some aspects of virulence of E. coli rough strains are discussed.

In a clinical routine study 304 yeasts were examined with the aid of API 20 C Auxanogram (Analytab Products Inc.) Mycotube (Roche) and Bacto-Candida-albicans-Antiserum (Difco) (=CAA). API (in parenthesis the result of the Mycotube test) identified 99% (92.4%) correctly; without considering C. albicans, the result was correct in 96.5% (75.8%). The CAA indicated a high specificity against C. albicans. For the Routine Laboratory we recommend screening of all yeasts with the CAA; an additional morphological examination is also recommended as cross-reactions with C.tropicalis and Torulopsis species are found. The non-agglutinating yeasts can then be further examined with the API 20 C.

NMRI mice were vaccinated by the aerosol technique, using the ethylethylenimine inactivated and polyethylenglycol concentrated virus strain A/PR/8/34 (HO/N1) with or without addition of Bordetella pertussis extract (BPE) as an adjuvant. The immune response of the vaccinated animals was controlled by challenge infection via aerosol technique and by examination of HAI antibodies in the serum and in the washings of lungs. After a single aerosol vaccination a weak protection was observed only, if the vaccines contained BPE. But a second immunization with a vaccine containing BPE induced a high degree of immunity, even if a reduced amount of antigen was used for booster vaccination. After three aerosol vaccine doses in two-weeks intervals, however, we were unable to infect the immunized animals even with largest amounts of challenge virus (approximately 50 000 LD50). The addition of BPE as an adjuvant induced a significantly better protection and resulted in much higher titres of HAI antibodies in the serum and in the respiratory tract compared to mice vaccinated by the same procedure but without BPE.

Two S-form-revertant strains were isolated from a S. typhimurium Rd1 culture on account of their phage resistance. In microbiological and serological (O-agglutination) characterization - as well as in stability tests (agglutination in auramin and saline and heating at 100 degrees C) - the behaviour of the two strains was the same as that of the wild type. The two strains were found to be indistinguishable from the wild type strain also with respect to the chemical composition of their lipopolysaccharides. Thus the amount and proportion of fatty acids and sugar residues as well as the number of repeating units in the O-chain were all identical. In contrast, the isolated revertants were similar to the Rd1 mutant with respect to their auxotrophic markers methionine and tryptophane, to the absence of flagella as well as to the reduced content of cyclopropane fatty acids (C17, C19). Protein analysis revealed no significant qualitative or quantitative differences between the wild type strain and the two revertants with respect to the major proteins of their outer membranes. The sensitivity of the revertants to crystal violet, erythromycin and rifamycin SV was intermediate between the wild type and the Rd1 mutant. Their temperature maximum in nutrient broth was 43 degrees C, the retardation in growth at this temperature corresponding to that of the Rd1 mutant. At 37 degrees C, however, the growth rate of the revertants was identical to that of the wild-type, while that of the Rd1 mutant was slower. Addition of sodium chloride to the growth medium rendered the temperature dependent behaviour of the mutants and revertants similar to that of the wild type. Studies in NMRI mice revealed that the revertants, also with regard to their virulence, occupy an intermediate position between the mutant and the wild type. Nevertheless their ability to afford protection to Salmonella typhimurium infection following active immunization with acetone killed cells was as high as that of the wild type. The results show that the biologic behaviour of S. typhimurium is determined by the type of lipopolysaccharide it contains but also to a large extent by other cell-wall constituents.

Antibodies of Leptospirae were demonstrated in the sera of immunized rabbits by the indirect immunofluorescence test, only after a suitable dilution of the "anti-rabbit-gamma-globulin fluorescin conjugated" was carried out in a cross reference titration ("chessboard method"). A distinct crossreaction was observed when anti-icterohaemorrhagiae - and anti-bratislava-immunsera reacted with several serotypes out of the "interrogans complex". But only a weak crossreaction was found when both sera reacted with the serotyp patoc from the "biflexa complex". The evaluation of the indirect immunofluorescence test was complicated by a streaky background of fluorescence. The agargeldiffusion tests (Ouchterolony) exhibited four, three and two precipitation bands according to the conbination of antigens and immunsera.

IN the State of Bahia (Brazil) the leptospirin produced in Germany for experimental use by the Institute for Veterinary Medicine, Federal Health Office, Berlin, was administered to humans and animals in order to diagnose leptospirosis in collaboration with this Institute. The results were compared with the microscopic agglutination reaction. The total number of test persons or animals was 268; this group included 81 human patients. 60 heads of cattle, 50 goats, 40 pigs, 25 horses, and 12 dogs. All were tested serologically and simultaneously the intracutaneous test was carried out. This test was positive when the erythema formed had a diameter of more than 9 mm. The evaluation took place 8 to 10 h and 24 h after the injection of leptospirin. 1. 52 humans reacted serologically; out of this group 44 were positive in the leptospirin test. The allergy test was also negative in the 29 serologically negative patients. 2. Out of the 21 heads of cattle with a positive agglutination test 7 reacted to leptospirin whereas 39 animals which did not react serologically were also negative in the skin test. 3. Although 10 goats out of 50 reacted serologically, all were negative in the intracutaneous test. 4. 9 out of 40 pigs reacted serologically; however, 8 reacted only to apathogenic leptospires (L.patoc, L.rufino, L.andamana). Out of these 9 animals 4 were positive in the intracutaneous test; among them the pig which reacted serologically to L.autumnalis. Out of 31 serologically negative pigs 2 were allergologically positive. 5. Out of 21 serologically positive horses 15 exhibited an erythema which was considered positive in the skin test. 4 serologically negative animals also were negative in the intracutaneous test. 6. Out of 6 serologically positive dogs, 4 reacted to leptospirin. 6 that had reacted serologically were all negative in the intracutaneous test. A comparison of the serological and allergological findings judged by the serological standard showed that out of all cases tested, 0.7% had at the same time a serologically negative and allergologically positive reaction and 16.8% had a serologically positive and allergologically negative reaction. In some cases, the administration of leptospirin caused the formation of antibodies which could only be detected in low dilutions and usually disappeared after 6-8 weeks. In humans, pigs, and horses the leptospirin also showed positive reactions which serologically could be attributed only to biflexa leptospires. Serotypes not contained in the leptospirin were accounted for to a varying degree in the individual animal species. These reactions and the results of other studies carried out in pig stocks have been the basis for studies performed at the Institute for Veterinary Medicine. These studies are expected to contribute to an improvement of the sensitivity of the leptospirin.