Trichinella spiralis: behavior, structure, and biochemistry of larvae following exposure to components of the host enteric environment

Four layers are present on the surface of infective larvae of Trichinella spiralis isolated from host muscle in pepsin-HCl. Trypsin treatment of pepsin-HCl isolated worms caused partial degradation and removal of large patches of the two outer surface layers. Following exposure to bile, only traces of the outer layers remained on the worms surface. These changes in the worm surface were accompanied by a shift from Type I behavior, typical of pepsin-HCl isolated larvae, to Type II behavior, (snakelike) following exposure to either trypsin or bile. Worm behavior was also temperature dependent. Type I behavior was typical of worms maintained at room temperature regardless of treatment, while Type II behavior displayed by worms held at 37 C was treatment dependent. The absorption of in vitro glucose or beta-methyl-D-glucoside was lowest in pepsin-HCl isolated first stage infective larvae, significantly higher in trypsin treated worms and greatest in worms following exposure to bile. Sugar uptake by worms isolated from the host small intestine after 1 hr of enteral infection was similar to that seen in worms isolated from host muscle in pepsin-HCl. Sugar uptake in vitro in worms 2 hr following enteral infection was similar to worms following exposure to bile. The highest levels of sugar absorption in vitro occurred in worms which had resided in the small intestine for 3 hr. The lowest rates of incorporation of label into worm tissues was seen in 1 hr enteral and pepsin-HCl isolated worms. Infective larvae treated with trypsin or bile incorporated significantly greater amounts of label than the two former groups. The highest levels of incorporation of label into worm tissues was seen in 3 hr enteral worms. These findings support the view that trypsin, bile, and temperature serve as environmental cues which lead to alteration of the parasite's behavioral and nutritional status.     

Sequential production of alpha and beta interferons and gamma interferon in the circulation of Listeria monocytogenes-infected mice after stimulation with bacterial lipopolysaccharide

Sequential production of interferon (IFN)-alpha/beta and IFN-gamma in the circulation of mice which had been previously infected with viable Listeria monocytogenes was induced by injection of lipopolysaccharide (LPS) derived from Salmonella typhimurium. IFN-alpha/beta production occurred 2 hr after injection of LPS, thereafter IFN-gamma appeared and the maximum titer was demonstrated at 6 hr. At that time, almost all of the IFN was IFN-gamma. IFN-gamma production in response to LPS was observed from the 5th through the 11th day after infection with Listeria, but it was not demonstrated in either mice infected with lower doses of viable Listeria or mice immunized with heat-killed bacteria. IFN-alpha/beta production was not drastically affected by treatment with hydrocortisone, cyclophosphamide, carrageenan, antithymocyte serum, or anti-asialo GM1 antibody, whereas IFN-gamma production was suppressed by administration of all those agents. Noteworthily, IFN-alpha/beta, but not IFN-gamma, was produced even 6 hr after stimulation with LPS in cyclophosphamide- or antithymocyte serum-treated mice. IFN-gamma induction by LPS was markedly suppressed in mice in which IFN-alpha/beta produced by Listeria infection itself had been depleted by treatment with anti-mouse IFN-alpha/beta antibody, but it was not inhibited in mice when IFN-alpha/beta induced not by Listeria infection but by LPS had been depleted by treatment with anti-mouse IFN-alpha/beta antibody.     

Studies of latent cytomegalovirus infection: the macrophage as a virus-harboring cell

During chronic infection of mice with mouse cytomegalovirus (MCMV), the virus was isolated from various tissues by cocultivation with allogeneic mouse embryonic fibroblasts (MEF). Infectious virus was recovered from over 15% of the pancreases, salivary glands, kidneys, lacrimal glands, and spleens. When activated macrophages were obtained by intraperitoneal injection of peptone into mice infected 3 months earlier, they harbored MCMV. Macrophages or lymphocytes were infected with MCMV in vitro and injected into normal mice intravenously. The peritoneal cavities of these mice were then stimulated by peptone injection 3 months after the transfer, and peritoneal or splenic macrophages and lymphocytes were cocultured with allogeneic MEF. MCMV was recovered from the peritoneal and splenic macrophages and not from the lymphocytes.     

Leishmania donovani: action of excreted factor on hydrolytic enzyme activity of macrophages from mice with genetically different resistance to infection

The effect of purified excreted factor from promastigotes of Leishmania donovani upon the activity of four enzymes from lysed peritoneal exudate cells of mice (C3H and C57BL) was determined. There was no demonstrable effect on acid phosphatase (EC3.1.3.2), β-glucuronidase (EC3.2.1.21), and N-acetyl-β-glucosaminidase (EC3.2.1.29), but β-galactosidase (EC3.2.1.23) was inhibited up to 72% after 3 hr of incubation at 37 C. Inhibition of C57BL mouse enzymes was not significantly different from that of C3H mice. Protamine sulfate combined with the highly negatively charged excreted factor of L. donovani to migrate as a single positively charged band on immunoelectrophoresis. Protamine sulfate also reversed the β-galactosidase inhibition, though this was without direct effect on the enzyme. The excreted factor did not change or lose its charge or antigenicity with regard to precipitating antibody, when incubated with extracts of mouse peritoneal exudate cells, splenocytes, or liver homogenate—irregardless of whether the mice had been infected with leishmaniasis for 1 or 2 weeks or were uninfected.     

Effects of Carbon Dioxide on Coccidian Oocysts from 6 Host Species*

SYNOPSIS Activation of sporozoites in oocysts of Eimeria acervulina (chicken), E. intricata (sheep), and E. scabra (swine) occurred after pretreatment in aqueous 0.02 M cysteine hydrochloride under an atmosphere of CO₂, followed by incubation in a trypsin-bile mixture. Sporozoites of E. stiedae (rabbit), E. bilamellata (squirrel), and Isospora canis (dog) became activated when incubated in trypsin and bile with or without prior CO₂-pretreatment of oocysts; however, when CO₂-pretreatment was used, activation of these species in trypsin and bile was greatly enhanced. For E. acervulina, 12% of the oocysts were activated after 4 hr CO₂-pretreatment and 10 hr incubation in trypsin and bile at 43 C; higher temperatures or longer pretreatment times did not cause greater activation. Eimeria intricata oocysts became activated after 1 hr pretreatment and 10 hr incubation in trypsin and bile at 37, 39 or 41 C, respectively. The highest activation (31%) occurred after 20 hr pretreatment and 10 hr incubation in trypsin and bile at 41 C. Ninety percent of E. scabra oocysts contained active sporozoites after 1 hr CO₂-pretreatment and 10 hr incubation in trypsin and bile at 37 C. At 39 or 41 C, 100% activation occurred with this species after similar pretreatment and treatment periods. With E. bilamellata, 64% activation occurred in nonpretreated oocysts incubated 10 hr in trypsin and bile at 41 C, whereas 100% activation occurred if oocysts were pretreated with CO₂ for 1 hr before treatment with trypsin and bile. Thirty-one, 35, and 36% of CO₂-pretreated E. stiedae oocysts were activated after 1 hr incubation in trypsin and bile at 37, 39 or 41 C, respectively, whereas 1, 2, and 20% activation occurred in nonpretreated oocysts incubated at the same temperatures. Sporozoites in 99-100% of I. canis oocysts were activated after 10 hr treatment in trypsin and bile with or without 1 hr CO₂-pretreatment at 23, 37, 39 or 41 C.     

Evaluation of bluetongue virus (BTV) decontamination techniques for caprine embryos produced in vivo

The objective of this study was to investigate methods of decontaminating early goat embryos that had been infected in vitro with bluetongue virus (BTV). Embryos were isolated from in vivo-fertilized BTV-free goats. Zona pellucida (ZP)-intact 8 to 16 cell embryos were cocultured for 36 h in an insert over a Vero cell monolayer infected with BTV serotype 8. The embryos were then treated with one of five different washing procedures. The treatment standard (TS) comprised phosphate-buffered saline (PBS) + 0.4% BSA (five times over for 10 s), Hank's +0.25% trypsin (twice for 45 s), and then PBS + 0.4% BSA again (five times for 10 s). The four other washing procedures all included the same first and last washing steps with PBS but without BSA (five times for 10 s) and with PBS + 0.4% BSA (five times for 10 s), respectively. The intermediate step varied for each washing procedure. Treatment 1 (T1): 0.25% trypsin (twice for 45 s). Treatment 2 (T2): 0.25% trypsin (twice for 60 s). Treatment 3 (T3): 0.5% trypsin (twice for 45 s). Treatment 4 (T4): 1% hyaluronidase (once for 5 min). After washing, the embryos were transferred and cocultured with BTV indicator Vero cell monolayers for 6 h, to detect any cytopathic effects (CPE). The effectiveness of the different washing techniques in removing the virus was evaluated by RT-qPCR analysis. The TS, T1, T3, and T4 trypsin or hyaluronidase treatments did not eliminate BTV; Treatment 2 eliminated the virus from in vitro infected goat embryos.     

Stability of ectromelia virus strain NIH-79 under various laboratory conditions

Ectromelia virus strain NIH-79 was suspended in fetal bovine serum (FBS), minimum essential medium, Hanks' base plus 10% FBS (MEMH + FBS), phosphate-buffered saline (PBS) or PBS plus 50% glycerol (PBS + G). Suspensions were held as liquids or as dry spots at various temperatures. Virus was most stable in FBS and least stable in PBS + G at 4 degrees C, room temperature (23-25 degrees C) or 37 degrees C. Virus held at 4 degrees C was more stable than virus held at higher temperatures, irrespective of supporting medium. Dried spots of blood or serum from ectromelia virus-infected mice remained infectious at room temperature for 11 days and 4 days, respectively. Dried spots of FBS that contained virus were infectious for 5 days, whereas virus retained infectivity for 1 day after drying in other media. Virus was inactivated completely in 10% serum in PBS exposed to 60 degrees C for 30 minutes. Virus was inactivated completely in slices of infected liver and spleen immersed in 10% neutral buffered formalin for 20 hours. These results show that the stability of ectromelia virus strain NIH-79 is medium and temperature dependent and that rapid inactivation occurs after treatments routinely used in diagnostic and research procedures.     

Candida glabrata and Candida albicans; dissimilar tissue tropism and infectivity in a gnotobiotic model of mucosal candidiasis

Germ-free transgenic epsilon 26 (Tgepsilon26) mice, deficient in both natural killer (NK)- and T-cells, were inoculated (orally) with each of two Candida glabrata (BG2 or BG1003) or Candida albicans (CAF2-1 or SC5314) strains. Candida glabrata- or C. albicans-colonized mice exhibited similar numbers of viable Candida in the alimentary tract. Neither C. glabrata nor C. albicans caused systemic candidiasis of endogenous (alimentary tract) origin. Candida albicans invaded oroesophageal (tongue, palate, esophagus) and keratinized gastric tissues, evoked hyperkeratosis and a prominent, chronic, granulocyte-dominated, inflammatory response in all infected tissues, stimulated the production of splenic granulocytes and was lethal for the mice within 3-5 weeks after oral colonization. The two C. glabrata strains colonized the alimentary tract and penetrated into the keratinized (cardia-antrum) gastric tissues, but in contrast to C. albicans, were unable to infect oroesophageal tissues. Furthermore, C. glabrata strains were not lethal for the Tgepsilon26 mice, and did not evoke an inflammatory response in colonized gastric tissues or stimulate the production of splenic granulocytes. This 'stealth-like' behavior could explain the ability of C. glabrata to persist in infected tissues and survive as a commensal in the alimentary tract.     

Human beta-interferon incubated with muscle homogenate is protected by albumin but not by proteinase inhibitors.

The scarce bioavailability of beta-interferon (IFN-beta) after intramuscular administration is probably due either to the binding of IFN-beta to interstitial matrix, or to lymphatic absorption and/or to local breakdown by lysosomal proteinases from muscle. In this work, we first showed that after intramuscular injection, the apparent bioavailability of natural human IFN-beta is about 10% of that of recombinant IFN-alpha 2 and then we evaluated the effects of proteinase inhibitors and albumin on IFN-beta incubated at 37 degrees C with muscle homogenate. IFN biological activity decreased spontaneously by about 20% after incubation for 6 hr at 37 degrees C in Hanks' solution, but it was almost completely lost after incubation with muscle homogenate. Proteinase inhibitors (alpha 1-antitrypsin, alpha 2-macroglobulin, aprotinin, soybean trypsin inhibitor, leupeptin, EP-459, and EP-475) failed to block the inactivation of IFN-beta by muscle proteinases, whereas albumin exerted a partial but consistent protection.     

Endotoxin increases hepatic susceptibility to lipid peroxidation: a possible role of iron

The purpose of this study was to investigate the possible mechanism by which endotoxin enhances peroxidative damage to membrane lipids. Male B6C3 mice were treated with endotoxin intraperitoneally 0 or 20 mg/kg body weight for 24 h. Freshly prepared liver homogenate was incubated with either 1-5 mM of reduced glutathione (GSH), glucose, H(2)O(2), ascorbic acid (AA), FeSO(4), FeCl(3), EDTA, FeCl(3) plus AA, AA plus EDTA or EDTA plus FeCl(3) in phosphate-buffered saline (PBS), pH 7.0, or PBS, at 37 degrees C for 60 min. The levels of lipid peroxidation products, thiobarbituric acid reactants (TBAR), were significantly higher in the liver of endotoxin-treated mice, and the values were markedly increased following incubation. Compared to PBS, incubation with H(2)O(2), FeCl(3), FeSO(4), and AA, but not glucose, significantly enhanced TBAR formation. The greatest increase of TBAR was found when AA and FeCl(3) were added together. On the other hand, EDTA and GSH inhibited the formation of TBAR during incubation. When added before AA, EDTA completely inhibited the peroxidative effect of AA or FeSO4, and when added subsequent to AA, EDTA partially prevented the adverse effect of AA. The results obtained suggest that ionic iron plays an important role in initiating endotoxin-induced peroxidative damage to membrane lipids, and that AA may be involved in releasing iron from its protein complex and/or maintaining ionic iron in a reduced or catalytic state.     

Cross-reacting antigens of human thymus myoid cells and stable L-forms of group A streptococci

Indirect immunofluorescence has shown a similarity between the antigen components of group A streptococcus L-forms and human thymus myoid cells. An analogous antigen (or antigens) is present in the cytoplasmic membrane of human myocardial cell fibers. The depletion of antiserum to the streptococcal L-forms both by the culture of L-forms grown in meat or casein media and by the homogenate of the cardiac muscle leads to the inhibition of immunofluorescence. The depletion of serum by the homogenate of other tissues (liver) or by L-form culture does not virtually affect the immunofluorescence intensity. According to the authors' opinion, the similarity of antigens of group A streptococcus L-forms to the antigenic components of organ tissues is likely to be responsible for long-term persistence of the microorganisms under consideration and to favour, in some cases, the occurrence of autoantibodies. The latter circumstance might lead to pathological changes in organs containing cross-reacting antigens.     

Reaginic antibody formation in the mouse. IX. Enhancement of suppressor and helper cell activities of primed spleen cells.

Attempts were made to increase the activity of suppressor cells in vitro. Antigen-specific suppressor cells were induced by i.v. injections of urea-denatured ovalbumin (UD-OA) into OA-primed mice. Nonadherent splenic lymphocytes from the UD-OA-treated mice were incubated at 37 degrees C for 24 hr with either OA or OA-bearing macrophages and lymphocytes harvested from the culture were examined for the ability to suppress primary anti-hapten antibody response on nonirradiated mice to DNP-OA. The results showed that the suppressive activity of the lymphocytes increased after culture of the cells with OA or OA-bearing macrophages. Similar results were obtained when nylon column-purified T cell-rich fraction of the lymphocytes were similarly cultured. The suppressive activity was associated with theta-bearing lymphocytes and was specific for OA. Suppressor cells were not induced by the culture of OA-primed lymphocytes with OA. The helper function of splenic lymphocytes from both UD-OA-treated mice and OA-primed mice was enhanced by the culture of the cells with OA-bearing macrophages but not by culture with OA in the absence of macrophages.     

ENHANCEMENT OF SALMONELLOSIS AND EMERGENCE OF SECONDARY INFECTION IN MICE EXPOSED TO COLD.

Miraglia, Gennaro J. (Bryn Mawr College, Bryn Mawr, Pa.) and L. Joe Berry. Enhancement of salmonellosis and emergence of secondary infection in mice exposed to cold. J. Bacteriol. 84:1173-1180. 1962.-The ld(50) dose for mice of Salmonella typhimurium, strain RIA, is 4.1 x 10(5) for animals individually housed without bedding and maintained at 25 C. It is 3.8 x 10(3) for animals similarly housed but kept at 5 C. An intravenous injection of 0.1 ml of Proferrin 2 hr prior to infection with RIA lowers the ld(50) to 4.9 x 10(3) and to 4.0 x 10(1) for mice kept, respectively, at 25 and at 5 C. Low environmental temperature and "blockade" of the reticuloendothelial system (RES) lower the resistance of mice to about the same degree, but low temperature and RES impairment together lower resistance as if each were acting independently. No effect of cold could be detected in mice infected with the highly virulent SR-11 strain of S. typhimurium, since all animals died after infection with only a few cells. Mice that were natural carriers of salmonellae, as judged by fecal discharge, were highly resistant to challenge and responded to cold in a manner similar to normal mice infected with RIA. Strain RIA could be isolated from the tissues of infected animals with greater frequency and persisted longer in mice maintained at 5 C than those at 25 C. Staphylococci were isolated from livers of animals that survived salmonella infection for 14 days at 5 C, and the incidence of staphylococci was proportional to the number of salmonellae injected. At 25 C, only a small percentage of mice had staphylococci in tissues and these occurred independent of the infectious dose of salmonellae. These observations were made on normal mice infected with RIA and on carrier mice infected with SR-11.     

Survival of Listeria monocytogenes in Experimentally Infected Mice

Physiological saline was found to be very detrimental to the viability of Listeria monocytogenes. The LD(50) value was substantially reduced when peptone was used as the suspending fluid rather than saline. Normal splenic tissue was not inhibitory to the survival of Listeria. In experimentally infected mice, L. monocytogenes survived for 8 days in the peritoneal cavity and for at least 11 days in the spleen.     

Seasonal rhythmicity in lymphocyte blastogenic responses of mice persists in a constant environment

Young C57BL/6 mice were housed in an environment in which the ambient temperature (22.5 degrees C +/- 1 degree) and photo-period were constant for the duration of the observations. At weekly intervals, animals were sacrificed, and splenic single cell suspensions were tested for percentage of viable cells. The functional capacity of T and B lymphocytes was assessed in vitro by the mitogen-induced incorporation of tritiated thymidine by dividing cells. The lymphocytes were incubated in RPMI 1640 containing 10% fetal calf serum for 68 hr at 37 degrees C. T and B lymphocytes were stimulated with phytohemagglutinin-P and concanavalin A and with lipopolysaccharide, respectively. Seasonal rhythmicity in the incorporation of tritiated thymidine was exhibited by both lymphocyte subpopulations, with peak responses two to five times higher in March to April 1978 and February to March 1979 than during the previous two Decembers. Factors such as birth date of the mice and components of the culture medium had no influence on the results. Because the annual cycles persisted in the absence of known environmental signals or Zeitgebers, they bear similarities with circannual rhythms. It is suggested that a relationship may exist in mice and in other species between seasonally depressed immune functions and increased incidence of infectious agents.     

Isolation of Neospora caninum from the brain of a pregnant sheep.

Neospora caninum was isolated from the brain of a naturally infected pregnant sheep by inoculation of immunodeficient mice with a homogenate of the brain tissue. The ewe showed no clinical signs. Tachyzoites were observed in the tissues of the nu/nu mice injected with the brain tissue homogenate and the diagnosis was confirmed by immunohistochemical staining with anti-N. caninum antibodies and by detecting N. caninum-specific DNA by polymerase chain reaction.     

Mitogenic Activity of Cytoplasmic Membranes Isolated from L-Forms of Staphylococcus aureus

Cytoplasmic membranes of L-forms of Staphylococcus aureus exerted a strong mitogenic effect on splenocytes of athymic nude mice as well as normal mice, while a cytoplasmic fraction of the same bacteria did not show definite mitogenicity. The mitogenic principle(s) of the membrane fraction was resistant to treatment with trypsin and was heat stable (at 100 C for 10 min). The active principle(s) in the insoluble residue of the membrane fraction digested with trypsin was not extracted with cold acetone, but could be solubilized by extraction with a cold chloroform-methanol mixture (2:1, v/v). The mitogenic principle(s) in the extract was fractionated by silicic acid column chromatography. Among five fractions separated by chromatography, fractions eluted with chloroform-methanol mixtures (1:1 and 1:20, v/v) were found to be strongly mitogenic. The cytoplasmic membranes of the L-forms also exerted a definite mitogenic effect on guinea pig splenocytes, but not on the thymocytes.     

Role of lymphocyte surface determinants in lymph node homing

Thoracic duct lymphocytes briefly incubated in vitro with trypsin and then infused into syngeneic rats are unable to migrate into lymph nodes. Trypsin-treated lymphocytes incubated at 37 °C in the absence of enzyme for 12 hr recovered their lymph node homing properties. In vitro recovery did not occur if the cells were cultured at 17 °C. Evidence was obtained that trypsin cleaved sialyglycoproteins from the surface of lymphocytes and that these determinants reappeared after the cells were maintained at 37 °C for 24 hr.Puromycin added to cultures of normal lymphocytes for 3 hr before infusion markedly reduced the recovery of donor cells in lymph nodes. The results suggest that surface determinants of recirculating lymphocytes essential for homing into lymph nodes may be rapidly turned over.     

金葡菌及其L型感染荷瘤小鼠的实验研究

本文应用金黄色葡萄球菌及Ｌ型分别感染肿瘤和非肿瘤５７ＢＬ／６Ｎ小鼠,病理学和细菌学检测发现细菌及Ｌ型感 染种植肿瘤的小鼠组,肿瘤转移率和诱癌能力均显著提高。