Elementary Body Suspensions Research Articles

In-cell western assay as a high-throughput approach for Chlamydia trachomatis quantification and susceptibility testing to antimicrobials.

Chlamydia trachomatis, the leading cause of bacterial sexually transmitted diseases in developed countries, with around 127 million new cases per year, is mainly responsible for urethritis and cervicitis in women, and urethritis and epididymitis in men. Most C. trachomatis infections remain asymptomatic (>50%) and, hence, untreated, leading to severe reproductive complications in both women and men, like infertility. Therefore, the detection of C. trachomatis as well as the antimicrobial susceptibility testing becomes a priority, and, along the years, several methods have been recommended, like cell culture and direct immunofluorescence (DFA) on cell cultures. Herein, we described the application of In-Cell Western assay (ICW) via Odyssey CLx as a fast, more accessible, and high-throughput platform for the quantification of C. trachomatis and the screening of anti-chlamydial drugs. As a first step, we set up a standard curve by infecting cell monolayers with 2-fold serial dilutions of C. trachomatis Elementary Body (EB) suspension. Then, different unknown C. trachomatis EB suspensions were quantified and the chlamydial susceptibility testing to erythromycin was performed, using the DFA as comparison. Our results showed a very high concordance between these two assays, as evidenced by the enumeration of chlamydial IFUs as well as the determination of erythromycin Minimum Inhibitory Concentration (MIC). In conclusion, the ICW assay may be a promising candidate as an accurate and accessible methodology for C. trachomatis antimicrobial susceptibility testing.

An attenuated Ehrlichia ruminantium (Welgevonden stock) vaccine protects small ruminants against virulent heartwater challenge

Heartwater is a tick-borne disease of ruminants caused by the intracellular rickettsia Ehrlichia ruminantium. The only commercially available immunization procedure involves infecting animals with cryopreserved sheep blood containing virulent E. ruminantium organisms, followed by treatment with tetracyclines when fever develops. The virulent Welgevonden stock of E. ruminantium was attenuated by continuous propagation of the organisms in a canine macrophage-monocyte cell line (DH82), followed by re-adaptation to grow in a bovine endothelial cell line (BA 886). The material used for the present experiments consisted of the attenuated stock between passages 43 and 64 after re-adaptation. When inoculated into sheep or goats the attenuated organisms did not produce disease, and the only symptom observed was a rise in body temperature in most, but not all, animals. All sheep injected with 2 ml of culture suspension were subsequently found to be fully protected against a lethal needle challenge with the virulent homologous stock or with one of four different heterologous stocks (Ball 3, Gardel, Mara 87/7, Blaauwkrans). Titrations of elementary body suspensions showed that 2 ml of a 1:10,000 dilution of culture suspension injected into sheep or goats was still sufficient to trigger an immune response which resisted a lethal needle challenge with the virulent Welgevonden stock. Adult Amblyomma hebraeum ticks, fed as nymphs on sheep immunized with DH82-derived organisms of passage 111, were able to transmit the attenuated stock to a naïve sheep, which was found to be protected against a subsequent lethal homologous needle challenge.

Overview of Chlamydia trachomatis infection and the requirements for a vaccine.

Chlamydia trachomatis is an important human pathogen. The development of vaccines designed to prevent C. trachomatis-associated morbidity would be highly desirable. In the developing world the major goal is the prevention of blindness due to trachoma; in industrialized societies and in some developing countries, the major goal is the prevention of chlamydial salpingitis and its consequences. In early trials of trachoma vaccine composed of a suspension of elementary bodies (the infectious particles), short-lived protective immunity was induced, but deleterious effects, i.e., hyperreactivity to infection, were also noted. Thus it is likely that separation of the protective from the sensitizing antigens and the development of a subunit vaccine will be necessary. Chlamydial virulence factors include the surface structures (presumably antigenic) that are responsible for specific attachment of the organism to a susceptible host cell, induction of phagocytosis by the host cell, or inhibition of phagolysosomal fusion. Serotype-specific neutralizing antigens might also be candidate antigens. Purification of such subunit antigens from chlamydial particles is probably impractical. It is likely that genetic engineering will be required for the production of cloned antigens after such antigens have been identified.

TRACHOMA VIRUSES ISOLATED IN THE UNITED STATES. 8. SEPARATION OF TRIC VIRUSES FROM RELATED AGENTS BY IMMUNOFLUORESCENCE.

SummaryYolk sacs infected with trachoma, inclusion conjunctivitis, lymphogranuloma venereum, psittacosis 6BC or meningopneumonitis were extracted repeatedly with fluorocarbon. The resulting suspension of elementary bodies contained little egg material. The particles were distinct, and reacted with strain-specific rabbit antisera as measured by indirect fluorescence. The homologous antiserum usually yielded the highest titer; heterologous antisera showed equal, intermediate or no reaction; antisera to normal yolk sac were negative. Psittacosis 6BC and meningopneumonitis could be separated easily from TRIG agents. LGV, an IC isolate, and BOUR, a trachoma isolate, showed much cross-reaction, but BOUR could be differentiated regularly from another trachoma isolate (ASGH).

Demonstration of complement-fixation titres against the speciesspecific trachoma antigen in sera of trachoma patients.

Semi-purified elementary body suspensions, prepared or preserved in three different ways, have been compared as to their species-specific CF-activity as revealed both against Bedsonia-negative sera of trachoma patients (of known stage), and against psittacosis-positive sera of men and animals.Two of these antigens (the SPG-formol-antigen and the SPG-glycerol-antigen) revealed a positive species-specific CF-reaction in 61–100% of the Bedsonia-negative serum samples obtained from trachoma patients, with titres ranging from 1/2 to 1/128.Various parameters (as percentage proportion of positive reactors, CF-titre, duration of the disease, clinical stage of trachoma, etc.) have been analysed for correlation, and the results obtained with the different antigens are compared with one another.The most useful of the three antigens, showing a good species-specific CF-reactivity and no anticomplementary activity, was found to be the ‘SPG-formolantigen’. However because of its group-specific reactivity, for detection of species-specific trachoma antibodies in routine, it could be used only with Bedsonia-negative samples of sera. Because of the thermolability of its species-specific component, the SPG-formol-antigen proved to be liable to deterioration.We have pleasure in thanking to Dr F. B. Gordon for help in procuring bibliography and the viable strain of trachoma virus. We wish to express our thanks also to Mrs R. Šepetavic and to Miss M. Krečo for their technical assistance.

Loss of immunogenic properties of vaccinia virus inactivated by formaldehyde.

In the experiments here reported, vaccinia virus in the form of partly purified elementary body suspensions was exposed to 0.006 M formaldehyde in the presence of 0.02 M glycine at 20–24 °C. Samples were taken at intervals to determine the rate of inactivation. Assays were carried out by intracutaneous inoculation of rabbits and by the plaque count method in monkey kidney cell monolayers. In addition, the virus suspensions so treated were injected into rabbits and the resulting immune response was studied by serum–virus neutralization tests performed on the rabbit skin and in tissue cultures.It was found that the rate of inactivation by HCHO decreased with time in accordance with the usual dynamics of disinfection. Similarly, the immunogenic properties decreased in proportion to the fall in infectivity. Minimal quantities of virus could still be detected after 150–250 hours; and samples removed at these times were capable of producing a feeble immune response. Later samples were neither infective nor immunogenic. These experiments lend no support to the belief that HCHO-inactivated vaccinia virus could be employed as a substitute for living virus in immunization against smallpox.

A Capillary Tube Agglutination Test for Detecting Antibodies against Ornithosis in Turkey Serum

Summary and Conclusions A capillary tube agglutination test using a Giemsa-stained, buffered suspension of elementary bodies prepared from a toxigenic turkey strain of ornithosis virus propagated in the allantoic cavity of chicken embryos was compared with the indirect complement fixation test, and the results were promising. The CTA test seemed to be more sensitive in the first week or two after infection, and thereafter the two tests seemed equally sensitive. Both were a little more sensitive than the direct complement fixation test when the test material was turkey serum. In the CTA test false positive reactions were encountered, usually in low serum dilutions, but occasionally in dilutions as high as 1:32, when the serum was hemolyzed, contaminated or contained residual fibrin. Reactions at dilutions of 1:16 or higher constituted good presumptive evidence of infection or of some previous encounter with ornithosis agents; reactions at dilutions of less than 1:16 were less specific and were interpreted cautiously. Preliminary testing of serum from infected parrots and pigeons indicated that the CTA test can detect ornithosis antibodies in these species. The CTA test is economical and simple to perform. According to the results of these tests it could be a useful flock test, of value in epizootiologic surveys, and a rapid screening test of flocks before marketing in enzootic or epizootic areas. Flocks undergoing acute or subacute epizootics of ornithosis, the most dangerous from the standpoint of public health, could be detected easily.

The antigenic structure of psittacosis and feline pneumonitis viruses: I. Isolation of complement-fixing antigens with group and species specificity

Alkali-soluble and ether-soluble complement-fixing antigens were isolated by chemical fractionation of purified psittacosis and feline pneumonitis elementary body suspensions disrupted by sonic vibration. Both group- and species-specific serological activity were demonstrated in the alkali-soluble fractions by means of cross absorption and cross complement fixation tests. The destruction of the group activity with either potassium periodate or lecithinase produced psittacosis and feline pneumonitis species-specific antigens. In comparable experiments, the ether-soluble antigens exhibited only group serological activity. The alkali-soluble and ether-soluble group-specific complement-fixing antigens are serologically related. However, they show differences in heat stability, hemagglutinating properties, and prozone manifestation.

Studies on the soluble antigen of influenza virus: I. The release of S antigen from elementary bodies by treatment with ether

Soluble (S) antigen is readily released from influenza virus particles by exposure to ether. In order to obtain reproducible results with maximal yields of S and minimal damage to or loss of hemagglutinating and virus (V) antigen activities the technique described by Hoyle was modified and standardized. Elementary body suspensions which contained at least 5120 HA units per milliliter, and which failed to react per se with anti-S, were exposed to 1 2 volume of anesthetic ether at room temperature for periods up to 2 hours under constant agitation by a magnetic stirrer. Under these conditions, the hemagglutinating activity, as measured with guinea pig red cells, increased up to sixteenfold with all four influenza A strains tested; agglutination of chicken red cells was slightly enhanced with one of the strains and reduced twofold or more with the other three. The V antigen levels remained essentially unaltered. On the average, about 40 HA units of standard elementary bodies were required to yield one unit of S. Release of S became apparent within a few minutes of treatment. This was accompanied by nearly complete loss of infectivity and the sedimentation of the HA activity by high-speed centrifugation was markedly reduced. These results do not necessarily imply that the virus particles are disintegrated, as has been discussed.

Studies on the soluble antigen of influenza virus: II. A comparison of the effects of sonic vibration and ether treatment of elementary bodies

A comparison was made of the effects of sonic vibration and of exposure to ether on the release of S antigen from influenza virus. Elementary body suspensions (EB) obtained by high-speed centrifugation or by one cycle of adsorption onto and elution from red cells frequently reacted directly with anti-S sera. The presence of such “external” S depended upon the length of in ovo incubation and upon the strain of virus employed. Following sonic vibration the S titers increased in the EB preparations which initially revealed some S activity, and antigen became detectable in those originally free of measurable quantities. After sonic vibration or even only after second adsorption-elution cycles, the EB suspensions, as a rule, failed to react with anti-S. Although the data indicate that sonic vibration liberated some internal S, its release was not immediately accompanied by detectable losses in infectivity nor was a change noted in the degree of sedimentation of the virus particles by high-speed centrifugation. In any event, exposure to ether proved to be a more potent means of releasing internal S than sonic vibration since no additional S was liberated from ether-treated virus by the latter technique, whereas, in contrast, sonically vibrated EB suspensions on treatment with ether yielded apparently as much S as untreated virus particles. The implications of these results have been discussed.

The growth of vaccinia virus in the chorioallantois of the developing chick embryo and the production of complement-fixing antigen and haemagglutinin.

1. Elementary body suspensions of vaccinia virus placed on the chorio-allantoic membrane of the developing chick embryo showed immediately an enhanced infectivity which may amount to a several-fold increase in titre.2. In studying the growth curve of vaccinia virus in the chorio-allantois the distribution of virus between the liquid and the membrane had to be taken into account.3. Some virus disappeared after inoculation, but this did not necessarily indicate a non-infective phase in a growth cycle.4. Virus began to increase in 4–5 hr. and proceeded without stepwise increments, reaching a maximum in about 36 hr.5. Haemagglutinin and complement-fixing ‘soluble’ antigen were formed during the growth of virus, and the titre of each was directly related to the amount of virus. They were detectable when the titre of virus reached respectively 106.5–10.70 and 105.5–106.0 pocks per ml.6. Haemagglutinin may have been acting as a complement-fixing antigen.7. Neither haemagglutination nor complement-fixation provided evidence for the occurrence of a non-infective phase of virus as part of a growth cycle.

Electron Microscopic Counting of Elementary Bodies of the Virus of Meningo-Pneumonitis

Summary and Conclusions The counting method of Backus and Williams is described as applied to enumeration of the elementary bodies of the agent of meningo-pneumonitis. The necessity of counting particles of biologic origin but of “indeterminate” nature, present in elementary body suspensions, is described. Homogeneity of the distribution of latex spheres, elementary bodies and other particles of biologic origin appears to be present in the results of this counting technic, at several concentrations of the elementary body suspensions. The estimates of concentration of biologic particles generally, or of elementary bodies alone, have a minimum standard deviation of about 3% resulting from instrumental limitations in determining diameter of latex spheres. Values for concentrations of elementary bodies, and of biologic particles generally, are reported, in which one standard deviation represents approximately 5% of the figure for concentration.

The serological relationship between vaccinia and ectromelia viruses

1. Elementary body suspensions of vaccinia and ectromelia were obtained from rabbit skin and chick embryo or mouse liver and chick embryo respectively and used to immunize rabbits.2. Judged by the complement fixation and haemagglutination tests, the viruses from different hósts were very similar antigenically. Ectromelia and vaccinia appeared closely related, the heterologous virus titres being usually lower than the homologous.3. Elementary body suspension agglutination tests did not reveal a similar relationship between the two viruses, but were not satisfactory on account of the low antibody content of the antiectromelia sera.4. Highly active ectromelia antisera were not obtained and there was no evidence of multiplication of this virus in rabbits.5. Only slight neutralizing activity was demonstrated in the antisera.6. Elementary body suspensions of ectromelia virus prepared from mouse livers, as well as the crude liver extracts, were soon inactivated. Egg membrane preparations were relatively stable.

Transformation of Virus of Rabbit Fibroma (Shope) into that of Infectious Myxomatosis (Sanarelli)

Berry,1 Hoffstadt,2 and Hurst3 have reported the transformation of fibroma virus into myxoma virus employing crude tissue suspensions of myxoma virus. Meck4 and Hyde5 were unable to confirm these observations. Recently Gardner and Hyde6 have accomplished the transformation with 3 of 7 elementary body suspensions of myxoma virus. Hurst3 has obtained positive results in 5 of 7 rabbits and Hoffstadt2 in 7 of 20. The purpose of the experiments here reported was to amplify these observations and to study factors which might account for the inconsistent results.A 10% suspension of a dermal tumor of virulent myxoma virus was heated for 35 min at 60°C in a water-bath, then kept in sealed ampoules at 4°C until used. This tissue suspension of heat-inactivated myxoma virus was submitted to the following procedures:1. Serial testicular passage.2. Mixture with (a) active fibroma virus, (b) attenuated fibroma virus, (c) agents producing reduction, inflammation, increase in virulence; and subsequent testicular injection...

The water content of the particle of vaccinia virus proteid

When vaccinia virus proteid particles agglutinated in a weakly acid solution were centrifuged by an ordinary centrifuge for a long period of time, and the water contents of the precipitate thus sedimented were measured successively after every period of centrifuging, then it was found that the water content of the sediment diminished rapidly at the beginning of the centrifuging, but that at later periods of sedimentation the diminution rate became low and constant, decreasing rectilinearly. If we assume that the decrease in the water content with a constant rate is due to the squeezing out the water contained in the particle itself and that the rapid decrease at the beginning of the centrifugation with an inconstant rate is due to the pressing out of the water interposing among proteid particles, the water content of the particle can be estimated by measuring the content at the time when the decreasing rate becomes constant. From this point of view, the proteid particles are estimated as containing about 90% water. In the same way coli-phage proteid particles are found to have a similar water content. Sugar-insoluble space of the proteid particle was calculated by measuring the quantity of sugar added to the suspension of elementary bodies which were centrifuged off after the addition of the sugar; the water content was estimated from this sugar-insoluble space to be likewise about 90%, although the space varies over a wide range with the concentration of sugar added. Also from the viewpoint of the filtrability of the particles it is reasonable to consider that they strongly swells on absorbing water of about 10 times as great as its mass. These conclusions can be applied also generally to minute-bodyforming proteid (protoplasm-lipoproteid particles) having no virus activity.

The Immunity Following Intracutaneous and Subcutaneous Vaccination with Elementary Body Suspensions of Vaccinia

The knowledge of the nature of vaccinia virus gained in recent years and the methods of preparing relatively pure suspensions of the virus which have been developed justify the hope that the technique of vaccination against smallpox may be improved. The potency of vaccine lymph and its relative freedom from bacterial contaminants have been regulated under the Therapeutic Substances Act for several years past; nevertheless, for prophylactic purposes, it is desirable to introduce the use of pure bacteria-free virus in a measured volume of known potency. The usual method of vaccination which involves scratching the skin and applying to the scratch a variable amount of virus-containing lymph cannot be described as an accurate procedure and, unless proper precautions are taken, may invite local secondary bacterial infection. If it could be shown that a satisfactory immunity follows the intracutaneous injection of a measured amount of a bacteria-free suspension of virus, this method would possess obvious advantages; the usual dressing could be dispensed with, the amount of scarring, and the local secondary bacterial infection which is associated with vaccination would be reduced or abolished. In our opinion, the value of vaccinial scars has been exaggerated; they may be useful as visible evidence that the patient has been successfully vaccinated sometime in the past, but they are not an indication of existing immunity to smallpox. It is not usually suggested that any other measure of prophylactic inoculation should be accompanied by a permanent scar as a record of immunization.

Further observations on the value of heated elementary body suspensions in immunization against experimental influenza

Further evidence has been obtained that E. B. S. heated at 57° C. for 30 minutes constitute a satisfactory immunizing agent. They produce a high degree of immunity in mice; they are sterile, non-infective and relatively stable. They are consequently very suitable for field experiments in man.

Antigenic structure of influenza viruses; the preparation of elementary body suspensions and the nature of the complement-fixing antigen

There is much evidence to indicate that viruses, in certain cases at least, have an antigenic structure of comparable complexity to that of the bacteria. Hughes (1933) found that the serum of animals immunized with the yellow-fever virus contained two independent antibodies—precipitins and protective antibodies; the precipitinogen was distinct from the virus. Craigie & Wishart (1936) in investigations of the vaccinia virus have shown that, in addition to the elementary bodies, virus suspensions contain two soluble precipitable substances, the “L” antigen which is labile at 56° C. and the “S” antigen which is stable at 95° C. These antigens were readily demonstrated by precipitation, agglutination and complement fixation. Their nature and origin have not, however, been precisely determined. Bedson (1936), working with the psittacosis virus, prepared a soluble antigen, which was independent of the elementary bodies. It was most satisfactorily demonstrated by complement fixation.

ELEMENTARY BODIES OF VACCINIA FROM INFECTED CHORIO-ALLANTOIC MEMBRANES OF DEVELOPING CHICK EMBRYOS

By a method of differential centrifugation and tryptic digestion suspensions of elementary bodies have been prepared from chorioallantoic membranes of chick embryos infected with vaccine virus. The infective titer of the final suspension of elementary bodies was usually the same as that of the original tissue emulsion. Elementary bodies from infected chick membranes were agglutinated as well by antivaccinal serum obtained from different mammalian species as were bodies prepared from inoculated rabbit skin. Seitz filtrates of infected chick material contained soluble precipitable substances of vaccinia; these filtrates and filtrates from infected rabbit skin, respectively, reacted equally well with rabbit serum which contained either L or S antibodies.

IMMUNOLOGICAL AND CHEMICAL INVESTIGATIONS OF VACCINE VIRUS

Previous investigations of the metabolism of viruses have been hindered by the difficulty or impossibility of securing adequate amounts of the active agents in a pure state. However, by the application of recently developed technics, it is possible to prepare large quantities of vaccine virus free from living host cells, and to concentrate the suspensions to any desired degree. Advantage has been taken of this in the present investigation. Large quantities of washed elementary bodies of vaccinia were prepared, and suspended in small volumes of liquid. The amounts of oxygen consumed aerobically and of acid produced anaerobically were measured, the latter as carbon dioxide released from a buffer solution containing sodium bicarbonate and carbon dioxide. Even when large amounts of virus were used (as much as 26 mg., dry weight, of elementary bodies) the quantities of oxygen consumed and of acid liberated were very small. Furthermore, the greater part of the gaseous exchange which occurred took place in the first hour of observation; during the succeeding periods no absorption of oxygen or liberation of carbon dioxide was demonstrated. No increased absorption followed the addition of glucose, glucose monophosphate, or methylene blue. At the conclusion of the experiments the virus was shown to be fully active. Such findings are in sharp contrast to the results to be expected if true respiration were taking place, as for example in resting bacteria, in which case the quantities of oxygen consumed are much greater and are relatively constant during the period of observation. It was considered that the failure of elementary bodies to consume oxygen might be due to lack of a proper substrate, or of respiratory supplements. In an effort to supply these essentials, a tissue extract was prepared which was shown to contain respiratory supplements, and this was added to the suspension of elementary bodies. It had, however, no effect on the rate of utilization of oxygen by the elementary bodies. Since elementary bodies alone, and in the presence of simple and complex substrates, showed no evidence of continued respiration, it was decided to ascertain whether they contained substances capable of stimulating the metabolism of other cells. Rabbit erythrocytes were used for this purpose; and the amounts of oxygen consumed under aerobic conditions and of acid produced under anaerobic conditions, respectively, by the red blood cells were determined. In neither case was any consistent stimulation of metabolism demonstrated. In the interpretation of the results of our experiments it must be borne in mind that the conditions under which they were performed are highly artificial, and while they are compatible with the survival of virus, there is no reason to suppose that they would permit its growth (3, 4). It may be said, however, that under the conditions which have been described above, no evidence was secured that vaccine virus, in considerable amounts, freed from viable host cells and bacteria, is capable of continued utilization of measurable quantities of oxygen, or of continued release of appreciable amounts of acid.