An ultrastructural study of central nervous system disease produced by wild-type and temperature-sensitive mutants of vesicular stomatitis virus.

Outbred Swiss mice 3 to 4 weeks of age were injected intracerebrally with wild-type vesicular stomatitis virus or its temperature-sensitive (ts) mutants ts 11, ts 22, ts 31, and ts 41. Brain and spinal cord were then studied for pathologic changes by electron microscopy. All mice infected with wild-type vesicular stomatitis virus died within 2 days of inoculation. Diffuse ependymal alterations often culminating in necrosis in brain and especially spinal cord and rare foci of necrosis and mononuclear cell infiltration in the injected hemisphere were the main pathologic changes in these animals. In contrast, mice infected intracerebrally with ts 22 and ts 31 showed their first clinical signs, consisting of hind limb paralysis, on day 4 and did not die until day 7 or 8 after inoculation. Ependymal alterations were of less severe degree in these animals, whereas the most striking changes were those of status spongiosus limited to the gray matter of the spinal cord. Such status spongiosus was mainly due to ballooning of dendrites and astrocytic processes, although myelin and neurons were also occasionally involved. Mice infected with ts 11 and ts 41, on the other hand, remained clinically well and failed to show significant pathologic features at 4 and 8 days after intracerebral inoculation. This study would indicate that some ts mutants of vesicular stomatitis virus are capable of altering the fulminating disease produced by the parent virus and of producing strikingly different pathologic changes.     

Pathogenesis of acute myocardial necrosis in inbred mice infected with coxsackievirus B3

The pathogenesis of myocardial necrosis due to CB3W infection was studied in BALB/c and C3H/HeJ mice. BALB/c mice infected with 5 x 10(4) pfu were found to die of massive hepatic coagulative necrosis before myocardial changes occurred. Reducing the inoculum size to 5 x 10(2) pfu resulted in sublethal hepatic involvement and multifocal myocardial coagulative necrosis by day 7 p.i. In contrast, C3H/HeJ mice survived infection and developed multifocal myocardial coagulative necrosis, but not liver disease following inoculation with as much as 5 x 10(6) pfu of CB3W. As with BALB/c mice infected with 5 x 10(2) pfu, myocardial lesions became apparent in C3H/HeJ mice a few days after peak cardiac virus titer was attained. Minimal inflammatory infiltrate was seen following development of cellular necrosis and was restricted to the areas of virus-induced pathologic change. However, no evidence was found for virus-specific cytotoxic T cell activity or for delayed type hypersensitivity responses. Furthermore, myocardial necrosis in CB3W-infected, T cell-depleted C3H/HeJ mice was as severe as in CB3W-infected, immunocompetent mice. These data have led us to conclude that cardiac lesions were due to virus-induced cytopathology rather than immunopathogenic mechanisms.     

Inhibition of acquired resistance in cowpea chlorotic mottle virus-infected hypersensitive soybean by 2-thiouracil

Cowpea chlorotic mottle virus infection of hypersensitive soybean [Glycine max (L.) Merr. cv Bragg] led to the development of acquired resistance in noninfected halves of primary leaves and in newly formed trifoliolate leaves. The level of acquired resistance was dependent on time, inoculum concentration, temperature, and host age. Application of 1 mm 2-thiouracil via roots to plants 0–24 hr after primary inoculation abolished the development of acquired resistance. The time of application of thiouracil relative to that of primary inoculation was critical for thiouracil to inhibit acquired resistance. During the inhibition of acquired resistance, thiouracil appears to act on an event(s) which takes place between 0 and 24 hr after primary inoculation.     

Cell fusion induced by a plant virus

Cytopathic effects of a plant rhabdovirus on established cultures of leafhopper cell monolayers were studied. When potato yellow dwarf virus inoculum with multiplicities of about 800 infectious units per cell was used to inoculate Aceratagallia sanguinolenta vector-cell monolayers, damage to the cells could be observed under a phase contrast microscope as early as 30 min after the start of inoculations. Subsequent development of damage caused gradual detachment of cells from the surface of culture flasks. However, surviving infected cells began to exhibit some morphological changes 8 to 10 hr after inoculation. The distinctness of the perimeter of the cells began to disappear. A reduction in cytoplasm and a congregation of nearby nuclei occurred 20 to 24 hr after inoculation. Extensive cell fusion was observed by phase contrast microscopy 40 hr after inoculation and was also evident by fluorescence microscopy when infected cells were stained with fluorescein-conjugated antibodies to the virus. Similar cytopathic effects and cell fusion were also observed in Dalbulus elimatus non-vector-cell monolayers inoculated with concentrated virus inoculum. Virus inoculum rendered noninfectious by exposing it to ultraviolet light still caused cell fusion, and the longer the uv irradiation, the greater the capacity of the preparation to cause cell fusion.     

Gemeinsame Verwertung von Glucose und n-Alkanen bei der Citronensäuresynthese durch Saccharomycopsis lipolytica

Fermentations for the overproduction of citrate and isocitrate with S. lipolytica in media containing both glucose and n-alkanes as mixed C-source have been performed. Biomass and product yields strongly depend on the C-source of the inoculation culture. If the inoculation culture had been taken from media containing glucose as sole C-source both glucose and n-alkanes were utilized for cell growth in the main culture whereas only glucose was utilized if the inoculation medium contained only n-alkanes. For idiophasic citrate and isocitrate production both glucose and n-alkanes were consumed independently of the C-source of the inoculum but that C-source was preferentially utilized which has been the C-source of the inoculation culture. These findings are reflected by the activities of the isocitrate lyase and the pyruvate carboxylase, respectively. In S. lipolytica both anaplerotic pathways are coexisting but the C-source of the inoculation culture determines the level of the specific activities even if the ratio of the cell-mass of the inoculum to the cell-mass of the main culture at the end of the growth phase is about 1:35.     

Infectivity of respiratory syncytial virus by various routes of inoculation.

To understand the transmission of respiratory syncytial virus, we examined the frequency of infection in volunteers after inoculation by different routes with varying doses of virus. Thirty-two adult volunteers received serial dilutions of a safety-tested live strain of respiratory syncytial virus instilled into nose, eye, or mouth. The highest inoculum, 5.2 log10 50% tissue culture infective dose (TCID50), was administered to four groups of four subjects each, by nose to one group, by eye to one group, and by mouth to two groups. Subsequently, 1:100 and 1:1,000 dilutions of this inoculum were administered by nose and eye. At the highest inoculum, infection occurred in three of four subjects inoculated by nose and in three of four subjects inoculated by eye. Infection occurred in one of eight subjects inoculated by mouth, but this subject most likely was infected by secondary spread. With an inoculum of 3.2 log10 TCID50, the proportion of subjects infected by either route diminished to 25%. When the inoculum was further reduced to 2.2 log10 TCID50, no infections occurred by either route. Infections after the highest inoculum were characterized by earlier and greater shedding. These findings suggest that respiratory syncytial virus may infect by eye or nose and that both of these routes appear equally sensitive. In comparison, the mouth appears to be an insensitive route of inoculation. This is of potential import in infection control procedures and in the development of vaccines or other prophylactic measures.     

Successful colonization and immunization of adult rabbits by oral inoculation with Vibrio cholerae O1.

Adult rabbits were inoculated orally (or duodenally) with virulent Vibrio cholerae O1. Jejunal colonization occurred only when hypoperistalsis was induced at the time of inoculation by tincture of opium given intraperitoneally (or by temporary ileal obstruction). For oral inoculation, prior neutralization of gastric acid was also required. Inoculation with 10(9) V. cholerae caused jejunal colonization for 1 to 2 days and ileal colonization for 5 to 6 days. The extent of small bowel colonization 18 h after oral inoculation was related to inoculum size but also reflected limited multiplication of small inoculum sizes and net death, clearance of large inoculum sizes, or both. Serious diarrhea occurred only in rabbits fed large inoculum sizes, i.e., 10(10) V. cholerae, and then rarely. Rabbits colonized once with 10(10) V. cholerae became highly resistant to recolonization with either the same or opposite serotype. After 18 weeks, these rabbits were still partially protected, whereas twice-colonized rabbits were highly protected. Protection against recolonization appeared to be due, at least partly, to interference with the adherence of V. cholerae to the bowel mucosa, thus allowing rapid removal of V. cholerae when peristalsis resumed. Prior colonization also protected against cholera-like diarrhea in rabbits challenged by the removable intestinal tie-adult rabbit diarrhea technique, the 50% effective dose for severe or lethal diarrhea being increased more than 100-fold, and probably more than 10,000-fold, for challenge with either the homologous or heterologous serotype of V. cholerae. The described rabbit model appears well suited for the study of immunity evoked by enteric colonization with V. cholerae O1.     

Establishment of rat pneumococcal meningitis models: a histopathological analysis

The aim of this study was to perform a preliminary investigation of the pathogenesis of bacterial meningitis-induced brain injury by establishing rat pneumococcal meningitis models. Infant Wistar rats were intracranially inoculated with different concentrations of Streptococcus pneumoniae. Rats were sacrificed at different time points to observe clinical symptoms and pathological changes in brain tissues. Twenty-four hours after intracranial inoculation with Streptococcus pneumoniae, regardless of high or low concentrations of bacterial inoculation, all rats developed bacterial meningitis with manifestations such as lethargy and seizures. Pathological changes in brain tissues included subarachnoid and intraventricular inflammation, vasodilation and vascular congestion, and cortical neuronal necrosis. The number of rats with seizures, the degree of cerebral vascular disease, and the extent of neuronal damage were associated with the concentration of bacterial inoculum. Thirty days after infection, brain tissue weight significantly reduced. The pathological changes induced by inoculation with pneumococcal meningitis in Wistar rats were similar to those seen in the human brain. The possible mechanisms of brain damage caused by meningitis are cerebrovascular inflammation and disruption of regional cerebral blood flow.     

The pathogenesis of pseudorabies in mice: virus replication at the inoculation site and axonal uptake.

Three-week-old mice were inoculated in the right ear pinna with pseuforabies virus. Ears were surgically removed at various times after inoculation and changes from the normal pathogenesis were observed. Virus replication in the ear tissue and cervical dorsal root ganglia was also monitored. Followed inoculation with a small dose of virus, local multiplication of the virus was necessary before the infection spread to the nerves. With larger infecting doses there was probably direct uptake of virus from the inoculum into the nerve endings. After these larger doses virus was first detected in the dorsal root ganglia 17 h agter infection, suggesting a retrograde axonal flow rate of at lease 1-7 mm/h.     

Factors affecting griseofulvin susceptibility testing of Trichophyton rubrum in microcultures.

A microculture broth assay system for griseofulvin susceptibility testing of Trichophyton rubrum was further characterized. The effects of mass and number of colony-forming units of a fragmented mycelial inoculum, 5- or 8-day incubation periods, 25 or 32 degrees C incubation temperatures, and the solvents used to dissolve griseofulvin on the minimal inhibitory concentration (MIC) of griseofulvin were determined. An inoculum density with an absorbance of 0.600 at 450 nm ensured successful inoculation of all microcultures. Reduction of the inoculum mass to an absorbance of 0.200 lowered the number of colony-forming units in the inoculum by 60 to 80%. This decreased the efficiency of inoculation but did not alter the resulting MIC. There was no correlation between MIC and the number of colony-forming units used to initiate growth. Neither incubation temperature nor the length of incubation affected the MIC. The use of either acetone or ethanol to solubilize griseofulvin likewise had no effect on the MIC. The mean reproducibility of the MICs determined with the microculture method was 96%.     

The pathology of experimentally induced cecal amebiasis in gerbils (Meriones unguiculatus). Liver changes and amebic liver abscess formation.

The pathogenesis of experimentally induced cecal amebiasis in gerbils (Meriones unguiculatus) was studied from 5 to 60 days after inoculation. Ulcerative lesions were noted 10 to 60 days after inoculation. The sequential development of lesions was asynchronous and progressed from destruction of the interglandular epithelium and of glandular crypt elements to loss of mucosa and formation of granulomatous lesions in the submucosa involving the muscularis mucosae. Pathologic changes in the liver correlated with the formation of ulcerative cecal lesions. Subacute hepatic changes showed lymphocytic portal infiltrate, Kupffer cell hyperplasia, multinucleated giant cells, granuloma formation, and sinusoidal mononuclear and granulocytic infiltrates. Metastatic amebic liver abscesses occurred as early as 10 days after inoculation, and small abscesses were found in the portal areas of the right liver lobe. The sequential development and pathologic manifestation of the infection and the usefulness of the gerbil for the study of human intestinal amebiasis are discussed.     

A bench-scale, cost effective and simple method to elicit Lycopersicon esculentum cv. PKM1 (tomato) plants against Cucumber mosaic virus attack using ozone-mediated inactivated Cucumber mosaic virus inoculum

Studies were undertaken to evaluate ozone for inactivation of Cucumber mosaic virus present in the inoculum and to stimulate Lycopersicon esculentum cv. PKM1 (tomato) plants against Cucumber mosaic virus infection by using the inactivated Cucumber mosaic virus inoculum. Application of a T(4) (0.4mg/l) concentration of ozone to the inoculum containing Cucumber mosaic virus resulted in complete inactivation of the virus. The inactivated viral inoculum was mixed with a penetrator (delivery agent), referred to as T(4) preparation, and it was evaluated for the development of systemic acquired resistance in the tomato plants. Application of a T(4) preparation 5 days before inoculation with the Cucumber mosaic virus protected tomato plants from the effects of Cucumber mosaic virus. Among the components of the inactivated virus tested, coat protein subunits and aggregates were responsible for the acquired resistance in tomato plants. In field trials, the results of enzyme-linked immunosorbent assay revealed that, Cucumber mosaic virus accumulation was significantly less for all the test plants (16%) sprayed with the T(4) preparation than untreated control plants (89.5%) at 28 days postinoculation (dpi). A remarkable increase in the activities of the total soluble phenolics (10-fold) and salicylic acid (16-fold) was detected 5 days after the treatment in foliar extracts of test plants relative to untreated control plants. The results showed that treatment of tomato plants with inactivated viral inoculum led to a significant enhancement of protection against Cucumber mosaic virus attack in a manner that mimics a real pathogen and induces systemic acquired resistance.     

Clinical, bacteriologic and histopathologic studies on induced leptospirosis in stray dog pups

Eighteen 8-12 days old stray dog (canis familiaris mongrel) pups of either sex; 6 pups each in test groups and control group were infected with lepotspiral serovars autumnalis and canicola. The experimental animals, clinical, bacteriologic and histopathologic kinetics were observed. Both the serovars had evoked typical clinical manifestations. Leptospiraemia could be demonstratedin between the post inoculation (PI) days 1 & 5. Leptospiruria commensed in between the PI days 5 & 7 and lasted throughout the study period. Histopathologic study did not reveal any marked pathologic changes except hydropic changes in the liver of both the test groups.     

A rapid and efficient inoculation method for Tomato spotted wilt tospovirus

A rapid and efficient method of inoculation for Tomato spotted wilt tospovirus (TSWV) was achieved by applying the inoculum with a device consisting of a spray gun, an atomizer and a CO2-powered sprayer. The inoculum contained infected leaf sap prepared in 0.1M phosphate buffer, pH 7.0, 0.2% sodium sulfite and 0.01 M 2-mercaptoethanol (1g: 10 ml) and 1% each of Celite 545 and Carborundum 320 grit. The spray application of chilled inoculum at the rate of 1.1 ml/plant and at an air pressure of 4.1 bar resulted in systemic infection nearly to a 100% of the tobacco (Nicotiana tabacum) plants inoculated. The inoculation procedure was successfully applied to two other important host species of TSWV, peanut (Arachis hypogaea) and tomato (Lycopersicon esculentum), where 75.0-100% and 72.2-91.6% plants developed systemic infection, respectively. The approach facilitated a much faster inoculation of test plants with TSWV as it was estimated to be about 50 times quicker (depending on the plant species) than the hand inoculation. The procedure is suitable for rapid and simultaneous inoculation of a large number of test plants with TSWV and should facilitate screening of germplasm and breeding lines for virus resistance.     

Secondary degeneration of oligodendrocytes in canine distemper virus infection in vitro

To study the pathogenesis of demyelination in canine distemper virus (CDV) infection, primary canine brain cell cultures were infected with CDV to examine specific virus-induced glial cell changes. Cultures were harvested at regular intervals after inoculation and were immunostained for the specific demonstration of astrocytes, oligodendrocytes, and CDV antigen. The infection spread slowly with moderate cytolysis and cell fusion. Soon after inoculation, infection of astrocytes was found by means of double immunofluorescent labeling. One week after inoculation, CDV-induced astrocytic fusion and rearrangement of astroglial fibrils became apparent. The astrocytic changes progressed during the observation period. Double immunofluorescent labeling failed to show oligodendroglial infection. Despite clear absence of viral replication within oligodendrocytes at all stages of the experiment, these cells exhibited marked pathologic changes starting at about 20 days after inoculation and progressed to complete cytolysis within 10 days. The degeneration of the oligodendrocytes was thought to be secondary to CDV-induced changes in other cell types of the culture probably through the release of toxic factors in the tissue culture medium. Since little evidence has been found for oligodendroglial infection in demyelinating lesions in canine distemper in vivo, the present tissue culture findings suggest that demyelination in vivo could be the result of indirect oligodendroglial damage caused by CDV-induced changes in other cell types such as astrocytes.     

Accumulation dynamics of the agent of amyotrophic leukospongiosis and the development of degenerative changes in the central nervous system of guinea pigs

After retrobulbar inoculation into guinea pigs, the agent of amyotrophic leukospongiosis was detected in the central nervous system (CNS) as early as 7 days post-infection (p.i.), whereas specific morphological changes appeared since days 14 to 21 p.i. Virological and morphological findings suggested that motoneurons of the spinal cord were first damaged in the course of the pathologic process which then proceeded upwards.     

Experimental infection of Tamarins with human non-A, non-B hepatitis virus

Non-A, non-B (NANB) viral hepatitis was successfully transmitted to two colony-born Tamarins following inoculation with antihaemophilic factor VIII concentrate or the "H" inoculum. Both animals showed histological and ultrastructural evidence of viral hepatitis, with raised alanine aminotransferase (ALT) levels from the second week after inoculation through to the end of follow-up, 5 months later.     

Experiments in human adults with a recently isolated virus associated with respiratory disease

Summary A controlled infection experiment on the effect of inoculation in adult females of a virus recently isolated from non-diphtheritic croup cases and from Day-Nursery children is reported. Mild respiratory disease developed between 1 and 3 days after inoculation in 4 of 7 patients receiving virus inoculum. The virus was recovered in all infected subjects from throat for 3 – 9 days and from faeces for 0 – 21 days post-inoculation. All infected showed a fourfold or higher increase in neutralizing and haemagglutination-inhibiting antibodies within 1 – 2 weeks postinfection and a decrease in both types of antibodies within 7 weeks after infection. Among the 5 controls, 2 patients developed mild respiratory disease on the 11th day after receiving the control inoculum. One of these infections was shown by virologic and serologic examination to be associated with the agent. The isolation period of the infected cases was 8 days, and there was virologic evidence that the virus persisted in the infected patients after that time and thus it may have spread to the controls. Consequently, the infections among the controls were assumed to be secondary to the infected cases.     

A comparison of the isolation rates of Salmonella and thermophilic Campylobacter species after direct inoculation of media with a dilute faecal suspension and undiluted faecal material

Regardless of media used, dilution of faecal samples before direct plating may improve isolation rates and reduce subcultures by freeing organisms from the faecal mass and diminishing competing flora. Despite the routine use of dilution in many laboratories, it has never been established properly whether direct or dilute inocula should be used in primary plating of faeces. A total of 3764 faecal samples was examined in four laboratories with a standardised methodology. The isolation rates, competing flora and confirmatory work performed for Salmonella spp. and Campylobacter spp. from primary plating media with a dilute faecal inoculum were compared with those after direct inoculation of faecal material. Inoculum effects on the isolation of Shigella spp. could not be assessed as only one isolate occurred during the study period. The overall isolation rates of both major enteric pathogens were unaffected by the inoculum. However, significantly fewer wasted subcultures were recorded with a dilute inoculum for Campylobacter spp., and competing florawas reduced in all cases without diluting out small numbers of the pathogen.     

Influence of macrophage resistance gene Lsh/Ity/Bcg (candidate Nramp) on Toxoplasma gondii infection in mice.

Functional studies have shown that the murine macrophage resistance gene Lsh/Ity/Bcg (candidate Nramp) regulates macrophage priming/activation for antimicrobial activity via the tumour necrosis factor-alpha (TNF-alpha)-dependent production of reactive nitrogen intermediates. Since Toxoplasma gondii also parasitizes macrophages, is a stimulator of endogenous TNF-alpha release, and is sensitive to nitric oxide-mediated killing in activated macrophages, studies were carried out using chromosome 1 congenic mouse strains to determine whether Lsh influences T. gondii infection. Two interesting observations were made: (i) contrary to expectation, mice carrying the Lsh-resistant allele died earlier over the acute phase of infection than Lsh-susceptible mice; and (ii) Lsh-resistant mice which survived this acute phase of infection showed lower brain cyst numbers than the Lsh-susceptible mice. Whilst the latter occurred independently of route of inoculation (oral, intraperitoneal, or subcutaneous), the former was influenced both by the route of inoculation and the genetic background on which the Lsh-resistant allele had been isolated. Hence, following oral administration of 20 brain cysts of the RRA strain of T. gondii, mice carrying the Lsh-resistant allele on a B10 genetic background showed a significantly enhanced rate of mortality over the acute (first 8-12 days) phase of infection than B10 Lsh-susceptible mice. Although this acute phase of infection in B10 background mice was accompanied by an increase in serum TNF-alpha levels in both Lsh-resistant and -susceptible mouse strains, early mortality preceded the TNF-alpha peak, and administration of neutralizing rabbit anti-TNF-alpha did not significantly enhance survival. Hence, inflammatory mediators other than TNF-alpha appear to be responsible for the increased rate of acute mortality observed in resistant mice. Infection intraperitoneally led to delayed mortality in B10 mice, with the mean time to 50% mortality now being significantly longer in Lsh-resistant than in Lsh-susceptible mice. On a BALB genetic background, it was the i.p. route of infection which led to acute mortality and more rapid death in the Lsh-resistant strain. When a less virulent inoculum was used and mortality delayed, Lsh-susceptible mice died more rapidly, and i.p. administration of rabbit anti-TNF-alpha led to 100% mortality between days 8 and 10 of infection in both susceptible and resistant mouse strains, consistent with a crucial protective role for TNF-alpha during this phase of infection.(ABSTRACT TRUNCATED AT 400 WORDS)