Footpad Infection Research Articles

Possible neural basis for age-dependent resistance to neurologic disease from herpes simplex virus

Twenty-week-old mice are known to be resistant to HSV induced neurologic disease, while 5-week-old mice are susceptible. Although age-dependent resistance to disease has been attributed to immunologic maturation, most immunologic development is complete by about 3 weeks of age. We, therefore, postulated that differences in neural spread were involved and we compared the pathogenesis of viral spread in 5-week- and 20-week-old mice. Following footpad infection with 10 5.3 PFU HSV-1, virus was detected in homogenates of sciatic nerve and spinal cord 3–4 days sooner in 5-week-old versus 20-week-old mice. Virus titers in footpad homogenates were 10 5.2 to 10 6.0 in both groups, thus differences in virus replication or immunologic restriction at the initial site of infection could not account for the difference in neural spread. The rate of virus spread to the dorsal root ganglia (DRG) was assessed by ganglia explant/co-cultivation to detect virus presence at various times after footpad infection and by measuring sciatic nerve length. In 5- and 20-week-old mice the rate of virus spread to DRG was 28 mm/day and 4–12 mm/day respectively. We conclude that neural uptake and/or transport of virus may contribute to the difference in susceptibility to neurologic disease.

Cells expressing herpes simplex virus glycoprotein gC but not gB, gD, or gE are recognized by murine virus-specific cytotoxic T lymphocytes.

To determine which viral molecule(s) is recognized by herpes simplex virus (HSV)-specific cytotoxic T lymphocytes (CTL), target cells were constructed which express individual HSV glycoproteins. A mouse L cell line, Z4/6, which constitutively expressed high levels of HSV type 2 (HSV-2) gD (gD-2) was isolated and characterized previously (D. C. Johnson and J. R. Smiley, J. Virol. 54:682-689, 1985). Despite the expression of gD on the surface of Z4/6 cells, these cells were not killed by anti-HSV-2 CTL generated following intravaginal infection of syngeneic mice. In contrast, parental Z4 or Z4/6 cells infected with HSV-2 were lysed. Furthermore, unlabeled Z4/6 cells were unable to block the lysis of HSV-2-infected labeled target cells. Cells which express HSV-1 gB (gB-1) were isolated by transfecting L cells with the recombinant plasmid pSV2gBneo, which contains the HSV-1 gB structural sequences and the neomycin resistance gene coupled to the simian virus 40 early promoter and selecting G418-resistant cell lines. One such cell line, Lta/gB15, expressed gB which was detected by immunoprecipitation and at the cell surface by immunofluorescence. Additionally, cells expressing HSV-1 gC (gC-1) or gE (gE-1) were isolated by transfecting Z4 cells, which are L cells expressing ICP4 and ICP47, with either the recombinant plasmid pGE15neo, which contains the gE structural sequences and the neomycin resistance gene, or pDC17, which contains the gC structural gene coupled to the gD-1 promoter. A number of G418-resistant cell lines were isolated which expressed gC-1 or gE-1 at the cell surface. Anti-HSV-1 CTL generated following footpad infection of syngeneic mice were unable to lyse target cells expressing gB-1 or gE-1. In contrast, target cells expressing very low levels of gC-1 were killed as well as HSV-1-infected target cells. Furthermore, infection of gC-1-transformed target cells with wild-type HSV-1 or a strain of HSV-1 that does not express gC did not result in a marked increase in susceptibility to lysis. These results suggest that murine class I major histocompatibility complex-restricted anti-HSV CTL recognize gC-1 but do not recognize gB, gD, or gE as these molecules are expressed in transfected syngeneic target cells. The results are discussed in terms of recent evidence concerning the specificity of antiviral CTL.

Macrophage activation for antileishmanial defense by an apparently novel mechanism.

Activation of macrophages by lymphokines (including interferon-gamma; IFN-gamma) is presently considered to be a major host defense mechanism against a number of intracellular microorganisms. In a series of earlier studies that made use of mice undergoing spontaneous resolution of footpad infections with Leishmania major, we obtained evidence suggesting that a subpopulation of Leishmania-sensitized lymph node T lymphocytes could activate antimicrobial effects in Leishmania-infected macrophages by an apparently lymphokine-independent mechanism. These effector lymphocytes are not cytotoxic to host cells, and their effects are antigen specific and genetically restricted. To more rigorously investigate this apparently novel mechanism of macrophage activation, we examined the effect of blocking lymphokine production with cyclosporin A (CSA) on the capacity of these effector lymphocytes to exert macrophage activating function. Although CSA blocked lymphokines that activate antileishmanial effects, it did not inhibit the antimicrobial capacity of the effector lymphocytes. We also confirmed that IFN-gamma is the major macrophage-activating lymphokine that induces antileishmanial effects; treatment of lymphokine-containing supernatants with anti-IFN-gamma antibody markedly reduced their antimicrobial effects. In contrast, treatment of effector lymphocytes with this antibody failed to reduce their macrophage-activating capacity. We conclude that there exists an apparently novel macrophage-activating mechanism for antimicrobial defense that is independent of soluble lymphokine mediators.

IgG-mediated viral clearance in experimental infection with herpes simplex virus type 1: role for neutralization and Fc-dependent functions but not C' cytolysis and C5 chemotaxis.

For determination of whether the Fc moiety is required for antibody effectiveness in models of herpes simplex virus type 1 (HSV-1) infection, the effects of immune IgG and F(ab')2 fragments were compared by using a passive transfer model of footpad infection. In the IgG- and the F(ab')2-treated groups illness developed in 2 (10%) of 20 and 6 (25%) of 24 mice, respectively, compared with 10 (63%) of 16 controls. IgG treatment markedly, and F(ab')2 treatment moderately, reduced footpad viral titer and viral spread to sciatic nerve and spinal cord. The marked viral clearance by IgG was not attributable to C'-dependent lysis because rapid viral clearance was observed in C5-deficient B10.D2/oSn mice. Viral latency as a consequence of acute infection occurred in 38 (63%) of 60 lumbosacral dorsal root ganglia in the control group, 5 (8%; P less than .001) of 60 in the IgG-treated group, and 26 (33%; P less than .01) of 78 in the F(ab')2-treated group.

Antileprotic action of macromolecular immunomodifiers in the mouse foot-pad model

Infectious drug resistance denotes the transmission of drug resistance from one bacterium (so far only Gram-negatives) to another of the same or different strain, species, or genus. This is accomplished by conjugation that leads directly to a new recipient strain with a comparable level of resistance. The surprising facet of this phenomenon is the simultaneous transfer of resistance to an entire series of chemically and biochemically unrelated antimicrobials; most commonly in sulfanilamides, tetracyclines, streptomycin, and chloramphenicol, but also more recently penicillins, kanamycin, neomycin, nalidixic acid, and nitrofurans. Resistant bacteria can be stripped of infectious drug resistance with various acridines in vitro, albeit with low frequency; usually the method applies to all the resistance factors, but some segregation has been achieved. Several sulfanilamides and other synthetic antibacterials as well as the antibiotics are involved with infectious drug resistance. No new antibacterial screening methods are reported. Recent success in experimental animals in vitro with growing the leprosy bacillus has led to significant and promising additions in this field. Over 100 Myco leprae strains from all over the world and from the several clinical types of leprosy have now produced mouse foot-pad infections. The relationship of bacterial morphology, administration of antileprotic agents, BCG vaccination, and immune-response suppression to production of increased infectivity in mice has been studied.

Reovirus‐induced tenosynovitis in chickens: The effect of breed

The effect of breed of chicken on infection with an arthrotropic avian reovirus strain R2 was studied by oral or footpad inoculation of 1-day-old chicks of the following breeds: (1) specific pathogen-free (SPF) light-hybrid, (2) commercial White Leghorn egg-layer, and (3) commercial Ross I broiler, and observed to 12 weeks of age. Although most inoculated birds of all three breeds developed swelling of one or both legs below the hock joint at 3 to 4 weeks of age, gross lesions of tenosynovitis became progressively more severe and extended above the joints only in broilers, whereas in most orally-infected SPF and commercial light chickens gross lesions were intermittently severe and regressed with time. Cloacal virus shedding continued up to 2 weeks in the lighter breeds and 3 weeks after infection in broilers. From a small proportion of infected chickens, reovirus was also reisolated from heart, pancreas and caecal tonsils. In all breeds, the tissue in which virus persisted longest was the hock joint/tendon. There was a poor correlation between isolation of virus and the presence of gross lesions in chickens of 12 weeks of age, especially in broilers. Virus-neutralisation tests demonstrated that seroconversion in the lighter breeds occurred predominantly at 3 weeks and in broilers at 4 weeks after infection. In all three breeds the footpad infection gave significantly lower growth rates than were found in the control and oral-infection groups. Oral infection had no apparent effect on growth rates. The greater susceptibility of broilers to reovirus infection is discussed.

Kinetics and genetics of herpes simplex virus-induced antibody formation in mice.

The kinetics of antibody synthesis was investigated after intraperitoneal, subcutaneous, and footpad infection of various strains of mice with herpes simplex virus. Immunoglobulin M antibodies appeared 5 days after and immunoglobulin G antibodies appeared 10 to 12 days after intraperitoneal infection with herpes simplex virus type 1. The major histocompatibility complex and the background genome of inbred mice were not found to have a systematical influence on antibody synthesis. Female mice, however, consistently produced more antibodies than did male if the infection was done intraperitoneally, but not if it was done subcutaneously or into footpads. Castration considerably increased the amount of antibodies produced by male mice. The difference in antibody formation between females and males could be abolished by injection of silica; moreover, antibody titers were enhanced by this treatment. This has also been found by immunization with a Formalin-inactivated herpes simplex virus vaccine. The effect of silica in enhancing antibody formation could be observed up to 12 days after infection. Infectious virus could be detected up to 2 days after infection, and herpes simplex virus type 1 antibody-stimulating antigens could be detected up to 4 days in ultrasonicates of macrophages. The assumption is made that androgen-sensitive cell populations, including macrophages and their soluble products, are involved in antibody-depressing mechanisms.

Viability of Mycobacterium leprae after multiplication in mice.

To measure the rate at which Mycobacterium leprae are killed in the course of the mouse footpad infection after the maximum of multiplication has been achieved, M. leprae were harvested shortly before and at intervals after multiplication had reached the level of 10(6) organisms per footpad, serially diluted, and inoculated into the footpads of passage mice. Beginning 1 year later, foot-by-foot harvests of M. leprae were performed from passage mice, and the proportion of viable organisms in the passage inocula was calculated by means of a most-probable-number calculation. In addition, the proportion of solidly staining M. leprae was measured in the passage inocula. The proportion of viable M. leprae in the passage inocula was found to decrease with the time after multiplication to 10(6) organisms per footpad of donor mice; the half-time of loss of viable M. leprae was 25 days. The proportion of solidly staining organisms appeared to be directly related to the proportion of viable organisms, as measured by mouse passage, and inversely proportional to the time after multiplication to 10(6) organisms per footpad.

Response in distant lymph nodes of mice to infection in the hind footpad with Mycobacterium marinum.

In an attempt to demonstrate the importance of the popliteal lymph node in limiting the progress of infection with Mycobacterium marinum in the hind footpads of C57BL mice, such infections were studied in mice subjected to popliteal or popliteal and inguinal adenectomies. In the absence of the popliteal node, the footpad infection was only slightly enhanced compared with infections of sham-operated control mice; the inguinal node was found to be greatly enlarged and appeared to have substituted for the absent popliteal node. In the absence of both popliteal and inguinal nodes, the disease process in the footpads was again only slightly enhanced, and the axillary node appeared to have enlarged greatly and to have functionally replaced the missing, more proximate nodes. In additional experiments, mice subjected to adenectomy only on one side and injected in that hind footpad with phytohemagglutinin or India ink demonstrated hypertrophy or deposition of carbon particles in the more distant node only on the side of the injection. Thus, there appear to be rather direct functional connections among popliteal, inguinal, and axillary nodes that do not depend on blood circulation.

Listeria pneumonitis: influence of route of immunization on resistance to airborne infection.

Mice that are immunized with an airborne inoculum of BCG are more highly resistant to airborne challenge with Mycobacterium tuberculosis than are mice that are immunized by the subcutaneous or intravenous route. To discover whether this phenomenon is peculiar to tuberculosis, we studied the influence of the route of immunization upon pulmonary resistance in Listeria monocytogenes infection. Mice were immunized by the airborne, intravenous, or footpad route and were subsequently challenged by the same route at 1 to 4 weeks after immunization. Mice were highly and uniformly resistant to intravenous challenge, regardless of the route of immunization. The route of immunization bore no influence upon resistance to footpad infection, but resistance was appreciably better in mice challenged within 2 weeks of immunization than it was at later time points. In mice immunized by the footpad and intravenous routes, the pattern of resistance to airborne and footpad challenges was similar, in that there was substantially less immunity at 4 weeks than at 2 weeks after immunization. However, mice immunized by the airborne route were highly resistant to airborne challenge, regardless of the interval between immunization and reinfection. In this last respect, resistance of the lungs to reinfection was similar after Listeria and tuberculosis pneumonitis. It is suggested that a similar pattern of resistance may prevail in pneumonitis caused by other facultative intracellular parasites.

Antigen specific lymphocyte transformation, delayed hypersensitivity and protective immunity: I. Kinetics of the response

The association between protective immunity, delayed hypersensitivity (DTH) and in vitro antigen specific lymphocyte transformation (ASLT) has been studied during the course of BCG infection in mice. The peak ASLT response was obtained 2 weeks after a subcutaneous footpad infection using lymph node cells draining the immunization site. This response decreased to a low but statistically significant level by week 4 and persisted indefinitely thereafter. A similar course of events was observed in splenic cell cultures, but the size of the response was smaller. The reactive cells were shown to be T-lymphocytes. The ability to express DTH to tuberculin purified protein derivative coincided with the emergence of cells capable of responding in ASLT assays. In contrast, there was a poor association between ASLT and protective immunity: lymphocytes from lymph nodes that did not drain the immunization site conferred substantial protective immunity but nonetheless failed to respond significantly in ASLT assays. The latter failure could not be attributed to insufficiency of sensitized lymphocytes and was independent of the antigen concentration and the culture incubation time. It was concluded that the ASLT reactive cells were either immunoblasts or recent progeny of these cells.

Suppression of immunity to Mycobacterium lepraemurium infection

After injection of 10(8) live Mycobacterium lepraemurium (MLM) into the left hind footpad of mice, there is development of local swelling attributable to a granuloma of the cell-mediated immunity type. Concomitant intravenous inoculation of live MLM delays and may even suppress footpad swelling, the effects being proportional to the intravenous dose of organisms. Concomitant footpad infection and intravenous inoculation of 10(9) dead MLM also delays footpad swelling, but over a period of months the feet become excessively swollen. The excessive swelling is due to local enhancement of infection as evidenced by an increase in the number of MLM per footpad. Attempts were made to prevent such immunosuppression by splenectomy or treatment with BCG. Splenectomy was entirely without effect, but 10(7) live BCG administered intravenously 2 to 4 weeks before dead MLM prevented enhancement of infection. The mediator of the immunosuppressive mechanism that results in enhanced infection remains to be elucidated, but it is unlikely to be antibody or immune complexes.

A simplification of the mouse foot-pad infection using Mycobacterium leprae from skin scrapes

A simplification of the mouse foot-pad infection using Mycobacterium leprae from skin scrapes

Growth of Mycobacterium leprae and M. marinum in congenitally athymic (nude) mice

THE failure to grow the leprosy bacillus, Mycobacterium leprae, in vitro means that there is a requirement for a highly susceptible experimental animal model. The only animal confirmed so far as naturally highly susceptible is the nine-banded armadillo, of which at least some specimens develop, after inoculation, a disseminated disease which resembles human lepromatous leprosy1. The inoculation of M. leprae into the footpads of mice results in a localised, self-limiting infection, with a maximum bacillary yield of about 106.5 organisms per footpad2. Suppression of the immune response of the mouse, by thymectomy and irradiation, results in enhancement of the footpad infection, and dissemination of M. leprae bacilli to internal organs and peripheral tissue such as ears, nose and tail3. This ability to enhance infection by nonspecific depression of T-cell function suggests that the congenitally athymic, or nude, mouse might provide a suitable, naturally susceptible host for M. leprae. In spite of the severe impairment of the cellular immune response of nude mice4–7, the only report of the growth of M. leprae in such mice concluded that there was no significant increase in bacillary proliferation, and no tendency to promote a generalised infection8. This conclusion was based, however, on observations made only 6 months after inoculation—an interval that is almost certainly too short for detection of significant enhancement or dissemination.

In vitro and in vivo studies of the action of rifampicin, clofazimine and B1912 on Mycobacterium marinum.

Rifampicin (RMP) and two riminophenazine compounds (B663 and B1912) suppressed the growth of Mycobacterium marinum in vitro and in a mouse footpad infection. At appropriate concentrations and dietary dosage, all three drugs showed bactericidal activity in vitro and in vivo. On the basis of minimum inhibitory concentration, RMP was considerably more active than the other two substances in vitro. However, the rate of bactericidal effect was similar for all three drugs. In vivo, the minimum bactericidal dietary dosages of RMP, B663 and B1912 were 0.03, 0.03 and less than or equal to 0.01%, respectively. Results suggest that these drugs may be of value for the treatment of clinical M. marinum infections.

Herpes simplex virus infection in guinea pigs: an animal model for studying latent and recurrent herpes simplex virus infection

Footpad infection of guinea pigs with herpes simplex virus led to an acute local inflammatory reaction, followed by a persistent latent infection of lumbosacral dorsal root ganglia. Spontaneous reactivation of the latent virus occurred, leading to recurrent lesions at the site of the initial infection. Clinical observations and virological studies during the acute latent and recurrent infection are reported.

Growth and immunogenicity of photochromogenic strains of mycobacteria in the footpads of normal mice.

Specific pathogen-free CD-1 mice were infected subcutaneously in the footpad with mycobacterium kansasii, three strains of M. marinum, and two strains of M. simiae-habana, and the growth of the organisms in the footpad, the draining popliteal lymph node, and the lung and spleen was followed quantitatively for up to 60 days. The ability of a footpad inoculum of M. marinum to spread to the lungs and spleen correlated with the ability of the organism to survive and multiple at 37 C in vitro cultures. The amount of footpad swelling which developed in the M. kansasii- and M. marinum-infected mice varied depending upon the strain of organism and the size of the original footpad inoculum. Injection of dead M. marinum into the footpad also induced an extensive amount of swelling which varied with the strain used, as well as being dose dependent. M. marinum- and BCG-vaccinated mice were protected against a later footpad challenge with M. marinum or the highly mouse virulent M. tuberculosis strain ERDMAN. The significance of this finding is discussed in relation to cross-protection studies using a variety of mycobacteria in the footpad infection model.

The activity of chaulmoogra acids against Mycobacterium leprae.

The activity of the crude sodium salts of the fatty acids of chaulmoogra oil and of hydnocarpic and chaulmoogric acids against Mycobacterium leprae was studied in mouse footpad infection. Multiplication of the organisms was inhibited when the salts were administered intraperitoneally and subcutaneously 3 times per week, and when chaulmoogric acid was administered intraperitoneally 5 times per week in half the equivalent dose. Dihydrochaulmoogric acid was also active, whereas palmitic acid was not. Hydnocarpic acid administered intraperitoneally once per week in a dose equivalent to half that of the sodium salts of the chaulmoogra fatty acids was not effective. The demonstration that chaulmoogra fatty acids possess activity against M. leprae lends weight to our earlier suggestion that a study of compounds analogous to these acids may yield effective antimicrobial agents with a unique mechanism of action.

Staphylococcal Footpad Infection in Mice

Local footpad infection in mouse was investigated with 55 clinically isolated strains of Staphylococcus aureus. When 10(7) viable cells were inoculated into the footpad, local swelling and bacterial growth resulted after 24 hr. With a dose of 10(6) cells, moderate swelling was observed after a few hours but the reaction had almost disappeared after 24 hr. About 75% of the staphylococcal strains tested caused footpad edema in mice at doses of 10(7) cells. A statistical comparison of the virulence of the organisms on intravenous and intraperitoneal injection with that in inducing footpad swelling is also reported.

Latent herpes simplex virus in the central nervous system of rabbits and mice.

Herpes simplex virus (HSV) type 1 induces a long-standing latent infection in the central nervous system of mice and rabbits. The infection was extablished in the brain stems of rabbits after corneal inoculation of the virus, and in the spinal cords of mice after rear footpad infection. In these animals, infectious virus could not be recovered by direct isolation from tissues; it was detected only after the tissues were maintained as organ cultures in vitro.