Bacteriostatic effects of horse sera and serum fractions on Clostridium welchii type A, and the abolition of bacteriostasis by iron salts

Under a variety of conditions of concentration, Eh, and pH, horse anti-Clostridium welchii serum and normal horse serum exerted similar bacteriostatic effects against Cl. welchii Type A. Ferric iron abolished the bacteriostatic effect when added during the first 2 hours of incubation at Eh+60 mV. Ferrous iron abolished the bacteriostatic effect when added after 3 hours. Ferric iron abolished the bacteriostatic effect at—140 mV. A mixture consisting of horse β₂- and γ-globulins together with human transferrin exerted a bacteriostatic effect similar to that of whole serum. This system responded in the same way as whole serum to the addition of iron. A mixture of horse β₂- and γ-globulins exerted an immediate bactericidal effect which could not be prevented by ferric iron.     

Iron compounds and resistance to infection: Further experiments with Clostridium welchii type A in vivo and in vitro

Transferrin has a bacteriostatic effect on Clostridium welchii type A provided it is stabilized by the presence of serum albumin. However, the bacteriostatic properties of whole serum cannot be attributed entirely to transferrin since it has been found that a mixture of transferrin and β₂- or γ-globulin is required to imitate exactly the behaviour of whole serum.

Fe³⁺, haematin, and haemoglobin abolish bacteriostasis in vitro, but large amounts of Cl. welchii α-antitoxin or Cl. tetani antitoxin tend to inhibit bacterial growth in the presence of excess Fe³⁺ when the environment is maintained at a high Eh (+110 mV, pH 7.5).

A number of factors have been shown to influence the outcome of Cl. welchii infections in guinea-pigs given intravenous Fe³⁺ (5 mg/kg). In the case of limb infections, 1300 units of α-antitoxin protected animals receiving 10⁵ bacteria but failed to protect animals receiving 10⁷ bacteria; 1300 units of α-antitoxin failed to protect animals injected with 10⁵ bacteria in the lumbar region.

The limb lesion has been found to contain an Eh gradient rising from –400 mV at its centre to +300 mV in normal tissue. Changes in the size of the area of low Eh can be correlated with the outcome of the infection. The results of these experiments provide some indication of the factors that are likely to influence the outcome of Cl. welchii infections.

     

The abolition of the protective effect of Pasteurella septica antiserum by iron compounds

Ferric ammonium citrate, haematin hydrochloride, soluble haematin, lysed mouse red cells and a variety of purified haemoglobins abolished the protective effect of Pasteurella septica antiserum in mice when the iron compounds were injected intraperitoneally with P. septica. Ferric ammonium citrate was less effective than haematin or lysed red cells when the dose of P. septica was reduced to less than 10⁵. The ability of lysed red cells to abolish protection was greatly reduced if given 4 hours or more after infection. P. septica grew rapidly in unimmunized normal mice. In passively immunized mice the number of viable bacteria declined rapidly after infection. In passively immunized mice given haematin or lysed red cells the growth of bacteria was identical to that seen in unprotected normal mice. Large numbers of dead P. septica or carbon particles did not interfere with passive protection.     

The effect of iron compounds on the virulence of Escherichia coli for guinea-pigs

Ferric ammonium citrate, haematin hydrochloride, lysed guinea-pig red cells and crystalline human haemoglobin greatly enhanced the virulence of Escherichia coli 0111/B4/H2 when injected intraperitoneally into normal guinea-pigs. The viable counts of E. coli in the peritoneal fluid of normal guinea-pigs given a sub-lethal infection were very variable and the bacteria eventually disappeared. In lethal infections in animals treated with iron compounds the bacteria grew extremely rapidly and death occurred when the counts reached 10⁹–10¹⁰/ml of peritoneal fluid. It is suggested that the mechanisms underlying non-specific immunity of guinea-pigs to E. coli may not be dissimilar to those involved in passive immunity to Clostridium welchii Type A in guinea-pigs, and in passive immunity to Pasteurella septica in mice.     

The circulating immunoglobulins involved in protective immunity to the intestinal stage of Nippostrongylus brasiliensis in the rat

Antisera taken from rats after one, two or three larval infections with Nippostrongylus brasiliensis or after one infection of adult worms were separated by chromatography and the fractions were tested for passive protective activity.

Irrespective of the number or type of infections given, protection against the parasite was most frequently obtained with fractions of antisera containing predominantly 7Sγ₁-globulin. γM- and γA-globulins sometimes appeared to contribute to protective activity, but γM-globulin was not the major source of protective antibodies in antisera taken after a primary infection either of adult worms or of larvae. 7Sγ₂-protective antibodies were found only in a pool of antiserum taken after three infections.

Rats were passively protected with antiserum or fractions of antiserum, which were free of reagins and of other identifiable anaphylactic antibodies. It appears that rats can be passively immunized with antisera which do not have anaphylactic activity.

     

A Vaccine and Monoclonal Antibodies That Enhance Mouse Resistance to Candida albicans Vaginal Infection

We previously reported that a vaccine composed of liposome-mannan complexes of Candida albicans (L-mann) stimulates mice to produce protective antibodies against disseminated candidiasis. An immunoglobulin M (IgM) monoclonal antibody (MAb), B6.1, specific for a β-1,2-mannotriose in the complexes protects against the disease, whereas MAb B6 does not. In the present study, the vaccine and MAbs B6.1 and B6 were tested for the ability to protect against Candida vaginal infection, established by intravaginal (i.vg.) inoculation of yeast cells in mice maintained in pseudoestrus. Fungal CFU in each vagina was determined to assess the severity of infection. Mice vaccinated before infection developed about 62% fewer vaginal CFU than nonimmunized controls. Naive mice that received polyclonal antiserum (from vaccinated mice) i.vg. before infection had 60% fewer CFU than controls. The serum protective factor was stable at 56°C, but C. albicans cells absorbed this factor. Mice given MAb B6.1 i.vg. after infection was established had fewer Candida CFU in vaginal tissue than control mice given buffer instead of antibody. MAbs B6.1 and B6 given intraperitoneally before infection protected mice, but MAbs preabsorbed with yeast cells did not. MAb B6.1 also protected against C. tropicalis vaginal infection, but MAb B6 did not. The protective activities of MAbs B6.1 and B6 appeared to be specific because an irrelevant IgM carbohydrate-specific MAb and an irrelevant IgG protein-specific MAb were not protective; also, MAb B6.1 did not affect development of vaginal chlamydial infection. These studies show that an appropriate antibody response, or administration of protective antibodies, can help the host to resist Candida vaginal infection.     

The bacteriostatic effect of serum on Pasteurella septica and is abolition by iron compounds

Methods are described for controlling the oxygen tension and pH of bacterial cultures in vitro. In fresh normal rabbit serum or plasma at a Po₂ of 80–90 mmHg and a pH of 7.5, the presence of specific antibody has a powerful bacteriostatic effect against Pasteurella septica. It is suggested that the serum components involved in this reaction consist of transferrin, specific antibody, and complement. Bacteriostasis can be abolished by a variety of iron compounds. This evidence together with that obtained in vivo (Bullen, Wilson, Cushnie and Rogers, 1968a) strongly suggests that an interference with bacterial iron metabolism plays an important role in resistance to infection against P. septica.     

The effect of digestion with papain and pepsin upon the antitoxic activity of rabbit antibody

Rabbit antibodies to the α and ε toxins of Cl. welchii have been hydrolysed with papain. It has been found that in some sera there is a loss of molar antitoxic activity which may be as high as 50 per cent. Partial digestion with pepsin does not affect the molar antitoxic activity. Reaction with anti-globulin sera increases the antitoxic activity of the digested globulins. It is suggested that the changes in activity are due to changes in the effective size of the antitoxic molecules and a consequent variation in the extent of steric inhibition of the toxins.     

Increased susceptibility to Mycobacterium avium in hemochromatosis protein HFE-deficient mice

Mycobacterium avium is an opportunistic infectious agent in immunocompromised patients, living inside macrophage phagosomes. As for other mycobacterial species, iron availability is a critical factor for M. avium survival and multiplication. Indeed, an association between host secondary iron overload and increased susceptibility to these mycobacteria is generally acknowledged. However, studies on the impact of primary iron overload on M. avium infection have not been performed. In this work, we used animal models of primary iron overload that mimic the human disease hereditary hemochromatosis. This pathology is characterized by increased serum transferrin saturation with iron deposition in parenchymal cells, mainly in the liver, and is most often associated with mutations in the gene encoding the molecule HFE. In this paper, we demonstrate that mice of two genetically determined primary iron overload phenotypes, Hfe^−/− and β2m^−/−, show an increased susceptibility to experimental infection with M. avium and that during infection these animals accumulate iron inside granuloma macrophages. β2m^−/− mice were found to be more susceptible than Hfe^−/− mice, but depleting Hfe^−/− mice of CD8⁺ cells had no effect on resistance to infection. Overall, our results suggest that serum iron, rather than total liver iron, levels have a considerable impact on susceptibility to M. avium infection.     

Oral administration of antibodies as prophylaxis and therapy in Campylobacter jejuni-infected chickens

Passive immunity against gastrointestinal infections has recently been successfully applied as prophylaxis and therapy in patients in a variety of virally and bacterially induced infections. Campylobacter jejuni is frequently associated with acute diarrhoea in humans, and several species of animals have been shown to transmit the disease, although birds have been implicated as the main source of infection. We used bovine and chicken immunoglobulin preparations from the milk and eggs, respectively, of immunized animals for prophylactic and therapeutic treatment of chickens infected with C. jejuni. A marked prophylactic effect (a >99% decrease in the number of bacteria) was noted using either antibody preparation, whereas the therapeutic efficacy, i.e. when antibodies were given after the infection was established, was distinctly lower (80–95%) as judged by faecal bacterial counts. These observations may serve as a starting point for experiments aimed at elimination of the infection in an industrial or farm setting. It may also encourage future attempts to treat, prophylactically or therapeutically, patients with Campylobacter-induced diarrhoea.     

Haemolytic activity of human blood monocytes. Lysis of human erythrocytes treated with anti-A serum

Purified monocytes and lymphocytes from peripheral blood of healthy human donors were tested in vitro for cytotoxicity against blood group A erythrocytes (RBC) treated with a human hyperimmune anti-A serum. Haemolysis was quantitated by the release of radioactivity from RBC pre-labelled with ⁵¹Cr-chromate.

The antiserum contained high titre antibodies which agglutinated A-RBC. After separation of serum on a Sephadex G-200 column the IgG containing fraction agglutinated A-RBC and precipitated blood group A substance. No or only weak antibody activity was detected in the IgA- and IgM-containing fractions.

Purified monocytes added to a 100-fold excess of RBC in the presence of 0·1% antiserum induced some haemolysis. It was calculated that one monocyte was able to lyse 2–3 RBC within 18 hr incubation. In contrast, lymphocyte suspensions containing more than 97% small lymphocytes had no or only weak haemolytic activity at a lymphocyte-RBC ratio of 25: 1. The effector cells of the monocyte fraction adhered to glass and were eliminated by incubation with carbonyl iron. Phagocytosis by monocytes of antibody-treated RBC was frequently observed. Loading of monocytes by treatment with heat-killed Candida albicans or carbonyl iron particles suppressed their haemolytic action. Cytotoxicity was impaired after treatment of monocytes with 10⁻⁴ M sodium iodo acetate. After separation of serum on Sephadex G-200 column all monocyte induced haemolytic activity was found in the IgG containing fraction. It is assumed that haemolysis is induced by the interaction of monocytes with an IgG anti-A antibody of the antiserum.

     

Development and passive transfer of immunity to gonococcal infection in Guinea pigs

Gonococcal infections of relatively long duration were produced in guinea pigs with minimal infective doses ranging from 10¹ to 10² colony-forming units. After spontaneous eradication and upon rechallenge with ≥10⁸ gonococcal colony-forming units, guinea pigs were refractory to infection. Serum from these guinea pigs was bactericidal in vitro and protected virgin guinea pigs from in vivo challenge. The 7S peak, but not the 19S peak, from gel filtered immune serum demonstrated both bactericidal and passive protective properties. In vitro bactericidal activity of whole immune serum and its 7S peak was abolished after heating at 56 C for 30 min. The in vivo protective ability of the 7S peak was not abolished after heating.     

Host Defenses in Murine Malaria: Immunological Characteristics of a Protracted State of Immunity to Plasmodium yoelii

Random-bred ICR mice recovered from infection with avirulent Plasmodium yoelii were challenged at various later times with virulent P. yoelii or with another species of Plasmodium, P. berghei, to characterize the immunological nature of the long-term state of immunity generated in response to the avirulent infection. It was found that recovered mice resisted lethal challenge with virulent P. yoelii through at least 416 days after primary infection. However, the quality of this immunity changed as the time after avirulent infection increased. Mice challenged early after recovery were able to prevent the development of patent parasitemia. Later, these immune animals lost this capacity and after challenge infections progressed to patency at the same rate as did nonimmune controls. However, after the establishment of parasitemia, those animals which had encountered the homologous parasite a long time before controlled, and then eliminated, blood infection and survived. The “early” state of immunity was expressed by animals which may have harbored small numbers of viable avirulent parasites and possessed a protective humoral factor which could passively transfer anti-P. yoelii activity to naive recipients. In contrast, animals with “late” immunity showed evidence of neither persisting avirulent parasites nor serum anti-P. yoelii activity. The results support the proposition that immunity to this parasite exists as two distinct but interrelated states of immunological reactivity: an early “active” immunity and a later state which has characteristics suggestive of a state of immunological memory wherewith the animals were capable of anamnestically responding to P. yoelii challenge. Little evidence of heterologous immunity to P. berghei was observed for animals recovered from P. yoelii.     

The Decisive Period in the Primary Infection of Muscle by Escherichia Coli

Escherichia coli superinfected with a non-replicating phage was used to study the course of infection in the adductor muscle of mice in terms of the content of viable bacilli and the in vivo multiplication rate. The infection was characterized by a steady decrease of bacterial numbers to 5% of the inoculum within 4 hours without any compensating multiplication. Within the next 3 hours there was slight multiplication (2 generation or less) followed by a slow decrease of numbers to nil in 72 hours. In terms of the viable count, the infection was temporarily enhanced between three- and eight-fold when the following modifiers were given at the time of inoculation: local adrenaline, liquoid and ferric iron, systemic malonate and ferric iron, and hypovolaemic shock. Within 1-2 hours the inoculum was preserved from the bactericidal action of the muscle and multiplied to a limited extent (up to 3 generations). Given 2 hours after the inoculation, all the modifiers enhanced infection, but not when given 4 hours afterwards.The results confirm the hypothesis, based on studies of local intracutaneous infections in the guinea-pig, that during the first few hours of infection, there is an extensive kill of the primary lodgement of bacteria by local defences that cease to operate after this period; and that the subsequent course of the local infection is determined by the number of bacteria surviving after this early decisive period.     

β Toxin of Clostridium welchii : Cytotoxic Effects Produced by the • on Guinea-Pig Monocytes

The cytopathic effects of Cl. welchii β toxin on guinea-pig monocytes in vitro have been studied using the uptake of eosin-Y^* as evidence of cell death.

The experiments show that these effects are due to antigen—antibody reaction probably on the cell surface, and that these reactions are not dependent on the presence of complement. Monocytes can be actively sensitized in vivo, and passively sensitized in vitro. The serum used to passively sensitize the monocytes need not possess a precipitating antitoxin titre.

Comparable experiments using an ovalbumin antigen—antibody system produced the same cytopathic effects on the monocytes as those which occurred in the β toxin—cellular system.

     

Immunity in cutaneous leishmaniasis of the guinea-pig

This paper describes the course of infection and development of immunity in guinea-pigs after intradermal inoculation of Leishmania enriettii, and the use of in vivo and in vitro techniques to characterize the immunological response to infection and artificial immunization.

Inoculation of 10⁶ amastigotes into the ear produced a nodule which ulcerated in 2–3 weeks and healed in 8–16 weeks. 8% of animals developed cutaneous metastases which healed with the original lesions. Histology of the primary lesions showed epidermal necrosis overlying a mass of parasitized macrophages which, after 4–6 weeks, became surrounded and infiltrated by lymphocytes. Histological changes in the draining lymph node began after 3 days and proceeded for 6 weeks; both germinal centres and paracortical areas were hyperplastic and the medulla contained many plasma cells. Superinfection produced an `isophasic' lesion, but reinfection after healing elicited only a delayed hypersensitivity response. Artificial immunization with soluble and insoluble antigenic extracts of L. enriettii in Freund's complete adjuvant partially protected against infection; extracts of other leishmanial species failed to protect. Immunological paralysis, attempted with intravenous injections of soluble antigen, increased the severity of subsequent infection.

Both infection and immunization were accompanied by delayed hypersensitivity which could be transferred passively by lymphoid cells. Cell-mediated immunity was studied in vitro by the ability of soluble leishmanial antigens to transform lymphocytes, to inhibit macrophage migration, and to induce the production of lymphokine factors from lymphocytes of sensitized animals. A target cell system was devised in which sensitized lymphocytes destroyed monolayers of parasitized macrophages. Cross reactivity of leishmanial with mycobacterial antigens was shown in skin tests and in target cell destruction, but not in cell transfer or in the other cell culture systems. The phagocytic activity of peritoneal macrophages from recovered animals was increased for homologous but not for heterologous species of Leishmania; the growth of ingested organisms was not however reduced.

Circulating antibodies were not demonstrated by passive cutaneous anaphylaxis, or by agglutination of antigen coated sheep erythrocytes, in the sera of infected or convalescent animals, although some convalescent animals showed active cutaneous anaphylaxis. However, antibodies were demonstrated by both these techniques in immunized animals, which also showed anaphylactic and Arthus hypersensitivity when skin tested with the soluble antigens.

The results are taken to indicate that cellular mechanisms are prominent in the development of immunity of the guinea-pig against L. enriettii, and ways in which the host may eliminate the parasite are discussed. It is concluded that this model provides an experimental counterpart of human cutaneous leishmaniasis and that it is suitable for the analysis of the role of cell-mediated specific immunity in resistance to intracellular infection.

     

Immunization against Natural Helicobacter pylori Infection in Nonhuman Primates

Helicobacter pylori infection is widespread in some breeding groups of a rhesus monkey colony (71% H. pylori positive by 1 year), and the rate of seroconversion is also high. As a result, these groups can be used to test the safety and efficacy of an anti-H. pylori vaccine. Nine-month-old female animals were randomized to receive either 8 mg of recombinant urease (rUre) plus 25 μg of Escherichia coli heat-labile enterotoxin (LT) (n = 26) or placebo plus LT (n = 29), given four times at 1-week intervals followed by a booster 1 month later. Ten months after the start of the immunization, the animals were subjected to endoscopy and biopsy samples were obtained. H. pylori negativity was defined as no H. pylori growth by culture and no H. pylori observed at histology. By this criterion, 2 (7%) of 29 animals receiving placebo and 8 (31%) of 26 immunized animals were H. pylori negative (P < 0.035). In addition, antral gastritis score was significantly less in H. pylori-negative immunized monkeys than in H. pylori-positive animals, whether they were given rUre plus LT or placebo plus LT (P < 0.02 or P < 0.01, respectively). Interestingly, antral gastritis was also significantly less in H. pylori-positive animals given rUre plus LT than in H. pylori-positive animals given placebo plus LT (P < 0.02). However, quantitative cultures did not demonstrate significant differences between the two latter groups. It is concluded that oral administration of rUre vaccine plus LT significantly protects nonhuman primates against H. pylori infection while not causing undesirable side effects.     

Delayed Influenza Treatment in Children With False-Negative Rapid Antigen Test: A Retrospective Single-Center Study in Korea 2016–2019

BackgroundWe aimed to examine the delay in antiviral initiation in rapid antigen test (RAT) false-negative children with influenza virus infection and to explore the clinical outcomes. We additionally conducted a medical cost-benefit analysis.MethodsThis single-center, retrospective study included children (aged < 10 years) with influenza-like illness (ILI), hospitalized after presenting to the emergency department during three influenza seasons (2016–2019). RAT-false-negativity was defined as RAT-negative and polymerase chain reaction-positive cases. The turnaround time to antiviral treatment (TAT) was from the time when RAT was prescribed to the time when the antiviral was administered. The medical cost analysis by scenarios was also performed.ResultsA total of 1,430 patients were included, 7.5% were RAT-positive (n = 107) and 2.4% were RAT-false-negative (n = 20). The median TAT of RAT-false-negative patients was 52.8 hours, significantly longer than that of 4 hours in RAT-positive patients (19.2–100.1, P < 0.001). In the multivariable analysis, TAT of ≥ 24 hours was associated with a risk of severe influenza infection and the need for mechanical ventilation (odds ratio [OR], 6.8, P = 0.009 and OR, 16.2, P = 0.033, respectively). The medical cost varied from $11.7–187.3/ILI patient.ConclusionAntiviral initiation was delayed in RAT-false-negative patients. Our findings support the guideline that children with influenza, suspected of having severe or progressive infection, should be treated immediately.     

Antigenic Homology of the Inducible Ferric Citrate Receptor (FecA) of Coliform Bacteria Isolated from Herds with Naturally Occurring Bovine Intramammary Infections

Expression of ferric citrate receptor FecA by Escherichia coli and Klebsiella pneumoniae isolated from bovine mastitis was investigated. Transformant E. coli UT5600/pSV66, which produces large quantities of FecA in the presence of citrate, was constructed. The FecA of E. coli UT5600/pSV66 was purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and used to prepare polyclonal antiserum in rabbits. All coliform isolates of E. coli (n = 18) and K. pneumoniae (n = 17) from naturally occurring bovine intramammary infections in five herds induced iron-regulated outer membrane proteins when grown in Trypticase soy broth containing 200 μM α-α′-dipyridyl and 1 mM citrate. Polyclonal antiserum against FecA was used in conjunction with an immunoblot technique to determine the degree of antigenic homology of FecA among isolates. In the presence of citrate, each isolate expressed FecA that reacted with the anti-FecA polyclonal antiserum. The molecular mass of FecA (~80.5 kDa) was also highly conserved among isolates. Therefore, the ferric citrate iron transport may be induced in coliform bacteria and utilized to acquire iron in milk for survival and growth. The FecA is an attractive vaccine component for controlling coliform mastitis during the lactation period.     

Cellular reactions in guinea-pigs following primary and challenge infection with Trichostrongylus colubriformis with special reference to the roles played by eosinophils and basophils in rejection of the parasite

The cellular changes which occur in blood, bone marrow, and small intestines of guinea-pigs during a primary infection and after a challenge infection with Trichostrongylus colubriformis are described. These studies show that (1) the dominant haematological and histological changes which follow infection with the parasites are proliferation of eosinophil and basophil leucocytes in the bone marrow, a circulating eosinophilia and basophilia, and an accumulation and degranulation of these cells at the site of infection. (2) The eosinophil and basophil changes occur at an accelerated rate in animals reinfected with T. colubriformis. These reach their peak 5–7 days after reinfection compared with 21–28 days in previously uninfected animals, and these peaks are correlated with the time of rejection of the parasite.

In a previous study (Rothwell, Dineen and Love, 1971) it was shown that histamine and/or 5-hydroxytryptamine participate in the rejection of T. colubriformis in the guinea-pig. As the results of the present study show that the accumulation and degranulation of eosinophils and basophils at the site of infection is correlated with rejection of the parasite, it is concluded that these cells are the source of the pharmacologically active amines.