Defense Date

1979

Document Type

Thesis

Degree Name

Master of Science

Department

Microbiology & Immunology

First Advisor

David T. John

Abstract

Primary amebic meningoencephalitis is a fatal disease of man caused by the free-living ameboflagelate Naegleria fowleri. In general, the victims have been active, healthy, young adults with a recent history of swimming or other fresh water-related activity. Infection with N. fowleri is apparently by way of nasal introduction of amebae-containing water. After nasal installation, electron microscopic and histopathologic studies with experimental animals reveal that amebae reside in the olfactory mucosa and then invade and migrate through the submucosal structures into the nerve plexuses. Amebae pass through pores of the cribiform plate and into the subarachnoid space. Subsequently, amebae invade the olfactory bulbs and lobes and spread to more distant areas of the brain causing massive hemorrhage, necrosis and edema. Frequently, the amebae aggregate in the perivascular spaces where they provoke a predominantly neutrophilic cellular response. Within seventy-two hours after the onset of symptoms, a rapid deterioration of the patient ensues resulting in coma and death (Carter, l972; Martinez et al., l977).

Standard amebocides, such as diodohydroxyquin, chloroquin and metronidazole are ineffective in treating N. fowleri infections. Amphotericin B, an antibiotic used to treat systemic fungal infections, has been shown to be an effective antinaeglerial agent in vitro. Its ability to provide protection in vivo is unclear (Carter, l969; Padilla and Padilla, 1974; Schuster and Rechthand, l975). However, in Australia, Anderson and Jamieson (1972) used amphotericin B to successfully treat a l4 year old boy with confirmed primary amebic memingoencephalitis. Duma et al. (l97l), using the same drug and similar regimen, were unsuccessful with two patients at the Medical College of Virginia, Richmond. Other drugs, such as penicillin, suphadiazine, chloramphenicol, oxytetracycline HCl, streptomycin, methotrexate and emetine have had no effect on N. fowleri in vitro at levels in excess of those likely to be obtained therapeutically in the brain (Carter, 1968).

The taxonomic position of Naegleria places it in the kingdom Protista, phylum Protozoa and class Sarcodina. It is of the order Schizopyrenida and the family Vahlkamphidae due to its ability to transform from trophozoite to flagellate and because of its promitotic nuclear division. Naegleria reproduction involves nuclear division (karyokinesis), in which the nucleolus elongates and divides into two polar masses and the nuclear membrane remains intact, followed by cytoplasmic division (cytokinesis). The genus Naegleria is identified by organisms which are biflagellate and do not possess cytochromes. Within the genus Naegleria there are two species, the pathogen N. fowleri and the nonpathogen N. gruberi (Page, l976). Willaert and Le Ray (l973) have described a third species, N. jadini. Synonyms for N. fowleri are N. aerobia (Singh and Das, l970) and N. invades (Chang, 197l). Synonyms for N. gruberi are Amoeba gruberi, Dimastingamoeba gruberi and N. punctata (Fulton, 1970).

Naegleria fowleri can be differentiated from N. gruberi in many ways. The mitochondria of N. fowleri are dumbbell-shaped rather than oval as in N. gruberi. Naegleria gruberi cysts have numerous conspicuous pores through which excystment occurs while N. fowleri cysts have few inconspicuous pores. Naegleria fowleri cysts are less resistant to drying than are cysts of N. gruberi (Carter, 1970). Naegleria fowleri is pathogenic, grows best at 37 C, although it will grow at 45 C, and is unable to grow in the presence of 0.5% saline. Naegleria gruberi is nonpathogenic, grows best at 25 C and grows well with 0.5% saline in the medium (Singh and Das, 1970). Naegleria fowleri cysts are more sensitive to chlorine than are the cysts of y, gruberi DeJonckheere and Van de Voorde, 1976). These species a1so differ in optimal pH for growth, growth media composition and size of the amebae. Concanavalin A agglutinates fl, gruberi but not H, fowleri (Josephson et a1., 1977). Naegleria iggifli reportedly can be differentiated from E, fowleri by its reduced virulence and inability to grow at 37 C and from y, gruberi by its nonporus cyst wall ( Nillaert and LeRay, 1973).

Researchers have used mice, guinea pigs, monkeys and rabbits in their investigations of experimental primary amebic meningoencephalitis. Probably the most useful laboratory animal model involves the mouse. Mice have been used because investigators have shown that experimentally induced primary amebic meningoencephalitis in mice and natura11y acquired primary amebic meningoencephalitis in humans have a similar incubation period, the disease is essentially confined to the central nervous system, similar clinical and pathological features occur and the outcome is invariable fatal (Carter, 1972; Culbertson, 1971; Duma, 1972 and Martinez et a1., 1973). A150, mice are versatile, inexpensive, easy to handle and small enough to be housed in large numbers in a small area.

Culbertson et al. (1968) inoculated specific pathogen-free mice intranasally (I.N.) with E, fowleri (HB-1 strain) and observed amebic hepatitis and rhinencephatitis. Similar inoculations, intravenously (I.V.) and intraperitoneally (I.P.), showed amebae to be widely disseminated throughout the mouse.

Carter (1972) studied the pathogenicity of E, fowleri administered by a variety of routes. Mice were inoculated by the I.N., I.V., I.P., intramuscular (I.M.), intragastric, subcutaneous, intrahepatic, anterior intracerebral, posterior intracerebral and intrapleural routes. Clinical symptoms and death occurred in all the mice inoculated I.N. and anterior or posterior intracerebrally. A third of the mice died following I.V. or intrahepatic inoculation. No clinical or pathological symptoms of primary amebic meningoencephalitis were found in the mice that were inoculated by the remaining routes.

The flagellate stage of N, aerobia (N. fowleri) was inoculated I.N. into mice in which it produced fatal meningoencephalitis. Brain smears from the infected mice showed only the ameba stage, indicating that the flagellates reverted to amebae after I.N. inoculation. In all likelihood, it was amebae that actually invaded the host and were responsible for death of the mice (Singh and Das, 1972). Similar results were obtained when mice were inoculated I.N. with N. aerobia (N. fowleri) amebae (Singh and Das, 1970).

Martinez et al. (1973) inoculated mice with amebae of two different strains of E, fowleri, (LEE-1 and CJ-1). After the onset of clinical symptoms (ruffed fur, circling, hunching), the disease progressed rapidly to death. Examination of brain tissue showed that both grey and white matter were affected and characterized by hemorrhage, edma, disintegration of neural structures with wide-spread invasion by amebae. Amebae were observed adjacent to arterioles and eapillaries. The nasal and olfactory mucosa was extensively infiltrated by motile amebae.

Cerva, (l97l) inoculated mice intracerebrally and I.N. with Naegleria (Vitek strain). After intracerebral inoculation, all of the experimental mice died. Shortly before death the mice showed symptoms of infection such as reduction of activity, uneven coat, disturbed equilibrium and finally loss of coordination. The mice that were inoculated I.N. died a few days after those inoculated intracerebrally. Histological examination of both groups of mice showed necrosis of much of the brain tissue and hemorrhage of the frontal lobes and also destruction of the mucous membranes of the I.N. inoculated mice.

Guinea pigs inoculated I.M. and subcutaneously with E, aerobia (fl, fowleri) exhibited generalized loss of weight and strength. Hind- quarter I.M. injections caused enlargement of the regional lymph nodes. There was no amebic involvement of the brain; however, hepatosplenomegaly, enlarged kidney and amebic lesions of the intestines did occur (Culbertson et al., 1968).

Červa, (l97l) inoculated guinea pigs I.N. with high, medium and low doses of N. fowleri. Guinea pigs given the low dose developed an elevated body temperature for an extended period of time and over half of the guinea pigs died. In the two higher dose groups, a rise in body temperature was noted only a few days before death. In a similar experiment Singh and Das (l972) inoculated two guinea pigs I.N. with y, aerobia (fl, fowleri). Fatal meningoencephalitis developed soon afterwards.

Phillips (l974) inoculated adult, germ-free guinea pigs I.N., intraorally, into the conjunctival sac and into skin lesions. Most of the guinea pigs inoculated I.N. died with meningoencephalitis. However, guinea pigs which were inoculated by the other routes remained in good health and were free of tissue damage at autopsy. Histological examination of the guinea pigs that had succumbed to g, fowleri infection (I.N. inoculated) showed destruction of the cerebellum, hemorrhagic meningitis, destruction of the frontal lobes, degradation of the meninges and a hemorrhagic condition of the anterior brain.

Monkeys have been inoculated I.N., I.V. and intrathecally with y, fowleri. Those receiving amebae I.N. or I.V. exhibited no evidence of Naegleria infection or central nervous system involvement. Monkeys that died as a result of E, fowleri inoculation intrathecally developed extensive lesions in the cerebellum with only small hemorrhages in the cerebrum. Amebae were isolated from the brains, spinal cords, lungs and liver. The monkeys that survived intrathecal inoculation exhibited fever, anorexia, leukocytosis, elevated levels of serum enzymes, and varying degrees of central nervous system involvement (Wong et al., l975).

Naegleria fowleri (HB-l strain) amebae have been injected into the marginal ear vein of adult rabbits. Rabbits that died exhibited extensive brain and liver damage (Culbertson et al., l968).

Investigators have concluded that the natural route of invasion for y, fowleri is from the nasal mucosa through the cribiform plate and into the brain. Therefore, the logical way to inoculate experimental animals would be by I.N. installation. Unfortunately, it is often difficult to administer a consistently accurate dose I.N. The reasons are that (1) when under ether anesthesia the mice tend to sneeze out a portion of the inoculum, (2) some of the inoculum may remain on the external nares and (3) a portion of the inoculum may flow into the nasopharynx and be swallowed or, even worse, be aspirated and contribute to possible pneumonia. So, although it is possible to calculate the dose given, it is difficult to determine the number of amebae retained by the host.

One way to avoid losing amebae and yet obtain results similar to I.N. installation would be to inoculate the mice I.V. The lateral tail veins are readily accessible for inoculation, the entire calculated inoculum is retained by the mouse and hematogenous spread carries amebae to the brain. The aim of my thesis is to examine the course of infection for mice inoculated I.V. with N. fowleri.

The overall results of this study are that mice inoculated I.V. with N. fowleri died from meningoencephalitis similar to that observed for mice inoculated I.N. Although amebae were detected in liver, lung, spleen and kidney, pathological involvement of these tissues appeared to be minimal.

Comments

Scanned, with permission from the author, from the original print version, which resides in University Archives.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

1-23-2018

Included in

Microbiology Commons

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