Activities of therapeutic agents against Naegleria fowleri in vitro and in a mouse model of primary amebic meningoencephalitis

Inhalation of water contaminated with Naegleria fowleri may lead to a potentially fatal infection of the central nervous system known as primary amebic meningoencephalitis (PAM). Amphotericin B (AMB), an antifungal drug, is the only agent with established clinical efficacy in the treatment of PAM, though therapy with this drug is not always effective and has been associated with adverse effects on the kidneys and other organs. We investigated the activity of various therapeutic agents against N. fowleri in an attempt to identify other useful agents for treating PAM. Several of these agents exhibited in vitro activity against the Lee (M67) strain of N. fowleri. The minimum inhibitory concentrations of these agents were 0.1 microg/ml (ketoconazole), 1 microg/ml (liposomal AMB), and 10 microg/ml (minocycline, quinupristin-dalfopristin, and trifluoperazine). Other agents had a minimum inhibitory concentration > 10 microg/ml (linezolid) or > 100 microg/ml (rifampin). In a mouse model of PAM, none of the untreated control mice survived, whereas the survival of treated animals was 50% (quinupristin-dalfopristin), 30% (ketoconazole and liposomal AMB), 20% (trifluoperazine), and 10% (linezolid and minocycline). Further studies are needed to ascertain whether these agents have synergistic activity with AMB in vitro and in vivo.     

Diagnosis of the primary amoebic meningoencephalitis due to Naegleria fowleri

Trophozoites of the free-living amoeba, Naegleria fowleri, were isolated from the cerebrospinal fluid of meningoencephalitis patient. The infecting agent was identified as N. fowleri based on morphologic, serologic and molecular techniques carried out on the isolated organisms.     

A light microscopy study of the migration of Naegleria fowleri from the nasal submucosa to the central nervous system during the early stage of primary amebic meningoencephalitis in mice

The migratory pathway of Naegleria fowleri from the nasal submucosa to the central nervous system (CNS) during the early stage of primary amebic meningoencephalitis (PAM) was investigated in mice. Twenty-one-day-old CD-1 mice were inoculated by intranasal instillation of 1 x 10(6) amebas. Animals were divided into 3 groups of 5 and, after being anesthetized, were killed at intervals of 24, 32, and 48 hr postinoculation by transcardial perfusion with formaldehyde, acetic acid, and methanol. The heads were decalcified, divided in the midsagittal plane, and the area of the cribriform plate removed and embedded in paraffin. Serial sections were cut at 8 microm and stained with a combination of celestin blue, Harris' hematoxylin, and acid fuchsin for light microscopy. Focal inflammation and amebas were observed in the submucosal nerve plexus, olfactory nerves penetrating the cribriform plate, and the olfactory bulb of the brain as early as 24 hr postinoculation. The time periods selected assured that the disease process would not obliterate soft tissue structures. Earlier studies used moribund mice in which the inflammation and the number of amebas were overwhelming. The present study provides convincing evidence that amebas gain initial access to the CNS through olfactory nerves within the cribriform plate during the early stages of PAM.     

Ultrastructural observations of experimental Naegleria meningoencephalitis in mice: intranuclear inclusions in amebae and host cells.

Primary amebic meningoencephalitis was experimentallly produced in mice through intranasal instillation of pathogenic Naegleria fowleri. Experimental animals had a 64% mortality with average time of onset of symtoms of death occurring on the 7-8th day following inoculation. Ultrastructural studies of the olfactory lobes from brains of dead (or sacrificed) animals revealed major concentrations of amebae in the perivascular regions; amebae were also seen to be under attack by host polymorphonuclear leukocytes, and in the lumina of blood vessels. Amebae in brain tissue contained 30 nm intranuclear particles arranged in clusters. In the brains of some mice, dead presumably as a result of amebic meningoencephalitis, particles and crystalloids were observed in the nuclei of degenerating cells of the central nervous system. Some alternatives are examined to explain a possible relationship between ameba intranuclear particles and mouse brain cell intranuclear inclusions.     

Assessing the risk of primary amoebic meningoencephalitis from swimming in the presence of environmental Naegleria fowleri.

Free-living Naegleria fowleri amoebae cause primary amoebic meningoencephalitis (PAM). Because of the apparent conflict between their ubiquity and the rarity of cases observed, we sought to develop a model characterizing the risk of PAM after swimming as a function of the concentration of N. fowleri. The probability of death from PAM as a function of the number of amoebae inhaled is modeled according to results obtained from animals infected with amoeba strains. The calculation of the probability of inhaling one or more amoebae while swimming is based on a double hypothesis: that the distribution of amoebae in the water follows a Poisson distribution and that the mean quantity of water inhaled while swimming is 10 ml. The risk of PAM for a given concentration of amoebae is then obtained by summing the following products: the probability of inhaling n amoebae x the probability of PAM associated with inhaling these n amoebae. We chose the lognormal model to assess the risk of PAM because it yielded the best analysis of the studentized residuals. Nonetheless, the levels of risk thereby obtained cannot be applied to humans without correction, because they are substantially greater than those indicated by available epidemiologic data. The curve was thus adjusted by a factor calculated with the least-squares method. This provides the PAM risk in humans as a function of the N. fowleri concentration in the river. For example, the risk is 8.5 x 10(-8) at a concentration of 10 N. fowleri amoebae per liter.     

Cell-mediated immunity in experimental amoebic meningoencephalitis

Cell-mediated and humoral immune reactions in mice infected with pathogenic Acanthamoeba culbertsoni were observed according to the period of time after amoebic infection by intranasal inoculation. The degrees of blastogenesis of spleen cells induced by mitogens, which were measured using radioactive [3H]-thymidine, were compared between infected and non-infected control groups. The mitogens used in this blastogenesis experiment were concanavalin A (Con A) and lipopolysaccharide(LPS). On the other hand, enzyme-linked immunosorbent assay(ELISA) was employed for the detection of humoral antibodies against A. culbertsoni. The levels of blastogenesis of splenocytes and serum titres in the experimental group showed increasing tendency a week after inoculation of A. culbertsoni, although there was no difference between the experimental and control groups in other periods of the experimental time.     

An experimental model for chronic lymphedema

Although a multitude of operations exist for the treatment of lymphedema, none is highly successful. An experimental model that reliably and easily produces chronic lymphedema in an extremity would be useful to study treatments in a controlled and comparative manner and would enhance our understanding of the physiology and treatment of lymphedema. Many models that simulate clinical lymphedema have been described, but they suffer from cumbersome protocols, high laboratory costs, and an inconsistent yield of permanent lymphedema. We describe an experimental model for chronic lymphedema in the lower extremity of the rat that creates a lymphatic block in the groin induced by radiation treatment and one operation--surgical division of the superficial and deep lymphatics. All animals develop stable chronic lymphedema of the lower extremity within days of operation, with swelling that persists for at least 9 months. A mortality rate of 8 percent was associated with this technique. Methods for quantification of limb swelling are described, as is analysis of the lymphatic block by lymphoscintigraphic imaging of lymph channels and nodes. This model has the advantages of simplicity of technique, cost-effective use of rodent subjects, reproducibility of lymphedema, and quantification of results.     

Ischemia in experimental acute amebic liver abscess in hamsters.

In experimental acute amebic liver abscess, produced in hamsters by the intraportal inoculation of 1 x 10(6) axenic trophozoites of Entamoeba histolytica strain HM-1, we examined the blood perfusion of the lesions 5, 10, 24 and 72 h after injection of the parasites. India ink introduced into the portal circulation filled all liver vessels but was systematically excluded from even the earlier amebic lesions. The absence of serum proteinase inhibitors from the lesions may allow the participation of amebic proteinases in the causation of tissue necrosis.     

Growth of calvarial width. An experimental investigation in rabbits

This investigation was conducted to analyze growth in width of the rabbit calvarium. 15 male New Zealand white rabbits were subjected to regular stereometric examinations from 31 to 141 days of age after implantation of tantalum bone markers. The sagittal suture complex, i.e. the interfrontal and interparietal sutures, and the bilateral temporal sutures demonstrated similar growth rates in magnitude which moderately decelerated throughout the observation period. Transverse growth exhibited local growth fluctuations and short-term negative growth values in a well-balanced manner.     

Naegleria australiensis ssp. italica: experimental study in mice

A subspecies of Naegleria australiensis, N. australiensis italica, pathogenic for mice, was recently isolated and identified from an Italian thermal spa. We describe the histopathological changes of the central nervous system with experimental infection of albino mice. The histopathological patterns are intermediate to those seen with infection caused by N. fowleri and N. australiensis or Acanthamoeba spp. An acute inflammatory reaction was present within the choroid plexus, ependyma, midbrain, cerebellum, and basal ganglia. Occasional single amebic trophozoites were found within some microabscesses. Cysts were not identified. Involvement of the olfactory neuroepithelium and of the nasal mucosa was not detected.     

Studies on the cell-mediated immunity in experimental Naegleria spp. infections

Observations were made on the differences in cell-mediated immune responses in the mice infected with strongly pathogenic Naegleria fowleri ITMAP 359, weakly pathogenic Naegleria jadini 0400, or non-pathogenic Naegleria gruberi EGB, respectively. Variations in cell-mediated responses and changes in antibody titers according to the duration after infection were noted. Infections were done by dropping 5 microliters saline suspension containing 10 x 10(4) trophozoites cultured axenically in the CGVS medium into the right nasal cavity of ICR mice aging about 6-7 weeks, under the anesthesia by intraperitoneal injection of secobarbital. Following infection, delayed type hypersensitivity(DTH) responses in the footpad and blastogenic responses of the mouse spleen cells using [3H]-thymidine were observed on the day 1, 4, 7, 10 and 14 after infection. For the preparation of amoeba lysates, each of cultured trophozoites were homogenized with an ultrasonicator, and centrifugated at 20,000 g. The supernatants of amoeba lysates were used as the mitogen and antigen for ELISA. Concanavalin A(Con. A) and lipopolysaccharide(LPS) were also used as mitogens in the blastogenic response. 1. The mice infected with N. fowleri showed the mortality rate of 75.7%. The rate was 6.2% for the N. jadini infected group, while no dead mouse was observed for N. gruberi infections. 2. In regard to DTH responses in the N. fowleri infected mice, the level increased in comparison to the control group but declined after 7 days. An increase was also noted for the N. jadini group after 1 day, but gradual decreases were observed through the infection period. In addition, no difference was noted between the N. gruberi infected and control groups. 3. Concerning the blastogenic response of the splenocytes, it increased after 10 days in the experimental group of N. fowleri infection, but the differences were not statistically significant compared with control group. It was evident that N. jadini group was not different from control group either, while there was a tendency of decrease in N. gruberi infected group. In regard to the blastogenic response of the splenocytes by LPS, it was found that the N. fowleri, N. jadini and N. gruberi infected groups had no differences from the control group. 4. The serum antibody titer of N. fowleri and N. jadini infected mice increased from the day 7 and 14 after infection respectively, while the N. gruberi infected mice showed no increase.(ABSTRACT TRUNCATED AT 400 WORDS)     

Intravenous miconazole therapy for experimental keratomycosis in rabbits

Intravenous miconazole (90 mg daily for 3 weeks) reduced the intensity of experimental fungal keratitis due to Candida albicans in a group of 10 rabbits. Clinical scores of affected eyes were statistically significantly lower in the treated group than in a control group of 10 untreated rabbits. All cultures of corneal scrapings were negative on 18th day after inoculation in the treated group, but four cultures were still positive on the 21st day in the control animals. Histopathological examination of eyes from treated and untreated rabbits showed great differences in the severity of inflammatory changes in the two groups.     

An experimental study of translocation success and habitat improvement in wild rabbits

The translocation of wild animals is a strategy frequently used in the conservation and management of natural populations. The aim of this study was to evaluate the impact of translocation (population supplementation) and habitat improvement on the abundance of European wild rabbit Oryctolagus cuniculus . We used eight open plots with different habitat treatments: two with increased shelter, two with increased food, two with increased shelter and food and two without habitat treatment (control plots). We translocated wild rabbits during 3 consecutive years, each year in four of the eight plots, with the remaining plots serving as control for the translocation treatment. Rabbit abundance (translocated plus native rabbits) was calculated by means of pellet counts, and the results were evaluated mainly by generalized linear mixed models. We found that rabbit abundance was determined primarily by habitat improvement. Rabbits were more abundant in treated than in control plots, and most abundant in the plots where food availability was increased. This effect persisted throughout the year. Translocation also increased abundance, but this effect was the strongest where shelter and food had also been improved and declined and disappeared after breeding. These findings suggest that the habitat is an important factor for rabbit abundance, with food availability being the prime factor regulating densities. Moreover, translocations on their own are only effective in the short term in situations in which factors limiting population growth (e.g. disease and predation) have not been corrected. Translocations should be carried out in conjunction with improvements in release habitat as rabbit densities will depend on the maximum carrying capacity of the habitat.