Epidemiology of Free-Living Ameba Infections1

Small free-living amebas belonging to the genera Acanthamoeba and Naegleria occur world-wide. They have been isolated from a variety of habitats including fresh water, thermal discharges of power plants, soil, sewage and also from the nose and throats of patients with respiratory illness as well as healthy persons. Although the true incidence of human infections with these amebas is not known, it is believed that as many as 200 cases of central nervous system infections due to these amebas have occurred world-wide. A majority (144) of these cases have been due to Naegleria fowleri which causes an acute, fulminating disease, primary amebic meningoencephalitis. The remaining 56 cases have been reported as due either to Acanthamoeba or some other free-living ameba which causes a subacute and/or chronic infection called granulomatous amebic encephalitis (GAE). Acanthamoeba, in addition to causing GAE, also causes nonfatal, but nevertheless painful, vision-threatening infections of the human cornea, Acanthamoeba keratitis. Infections due to Acanthamoeba have also been reported in a variety of animals. These observations, together with the fact that Acanthamoeba spp., Naegleria fowleri, and Hartmannella sp. can harbor pathogenic microorganisms such as Legionella and or mycobacteria indicate the public health importance of these amebas.     

Balamuthia mandrillaris: The multiple nuclei of Balamuthia amebas; their location, activity, and site of development

Multiple nuclei were first noted in the pseudopodia of Balamuthia mandrillaris amebas feeding on mammalian cells. Phase microscope observations of live amebas in vitro reveal that while many amebas have a single nucleus, others have multiple nuclear-like structures, now confirmed as nuclei with hematoxylin and Feulgen stains. In the live cultures, two nuclei located near the tip of an extended pseudopodium were seen to fuse resulting in one larger morphologic unit. Such merging of nuclei has not been previously reported. Other nuclei were located at positions that subsequently became the site for the outgrowth of an additional pseudopod branch. A newly discovered large structure, a polyploid nucleus, was located in the mid-part of the ameba. Nucleoli of uniform size were seen to develop from the central mass of chromatin and each became surrounded by a vesicular component as they moved into the protoplasm as morphologically complete nuclei.     

Lankesteria barretti n. sp. (Eugregarinida,Diplocystidae), a Parasite of the Mosquito Aedes triseriatus (Say) and a Review of the Genus Lankesteria Mingazzini*

SYNOPSIS. Lankesteria barretti n. sp. (Eugregarinida, Diplocystidae) is named from the mosquito Aedes triseriatus in Texas. The young cephalins occur within the midgut epithelial cells. When they reach a length of about 150–200 μ they rupture the host cell and are released into the space between the epithelium and the peritrophic membrane, becoming gamonts. These grow to about 140–310 by 40 μ at the anterior end and 12 μ at the posterior end. When the host pupates they enter the lumen of the Malpighian tubules; pairs join in syzygy by their anterior ends and later more laterally. Each pair forms a spherical gametocyst about 60–100 μ (exceptionally 250 μ) in diameter. A large number of oocysts develop in each gametocyst. The mature oocysts are spindle-shaped, 11 by 5.4–5.7 μ, and contain 8 elongate sporozoites and a refractile residuum. The gametocyst wall breaks down, releasing oocysts in the Malpighian tubules of the host when it is adult. The oocysts pass out in the feces and presumably infect new larvae by ingestion. The cephalins and gamonts of L. barretti differ from those of L. culicis (of Aedes aegypti) in having a relatively anterior instead of a central nucleus and in lacking a noticeable mucron; its longitudinal folds are not as well-developed as in L. culicis, and its paraglycogen granules are larger. The fine structure of L. culicis and L. barretti is described in detail. Their gamonts have a polar ring but no definite conoid. The taxonomy of the genus is reviewed, but its species have been so poorly described that it is impossible to be sure whether they are all really Lankesteria. About 19 species have been described (5 from turbellaria, 8 from tunicates, perhaps 1 from Amphioxus, 1 from the chaetognath Sagitta sp., 1 from Phlebotomus and 3 from mosquitoes).     

Differentiation of Entamoeba histolytica: a possible role for enolase

The study of the encystation process of Entamoeba histolytica has been hampered by the lack of experimental means of inducing mature cysts in vitro. Previously we have found that cytoplasmic vesicles similar to the encystation vesicles of Entamoeba invadens are present in E. histolytica trophozoites only in amebas recovered from experimental amebic liver abscesses. Here we report that a monoclonal antibody (B4F2) that recognizes the cyst wall of E. invadens also identifies a 48 kDa protein in vesicles of E. histolytica trophozoites recovered from hepatic lesions. This protein is less expressed in trophozoites continuously cultured in axenical conditions. As previously reported for E. invadens, the B4F2 specific antigen was identified as enolase in liver-recovered E. histolytica, by two-dimensional electrophoresis, Western blot and mass spectrometry. In addition, the E. histolytica enolase mRNA was detected by RT PCR. The antigen was localized by immunoelectron microscopy in cytoplasmic vesicles of liver-recovered amebas. The B4F2 antibody also recognized the wall of mature E. histolytica cysts obtained from human samples. These results suggest that the enolase-containing vesicles are produced by E. histolytica amebas, when placed in the unfavorable liver environment that could be interpreted as an attempt to initiate the encystation process.     

Cellular localization of thiol-proteinase inhibitor in the epidermis of the newborn rat

Summary In cichlid, poecilid and centrarchid fishes luteinizing hormone releasing hormone (LHRH)-immunoreactive neurons are found in a cell group (nucleus olfactoretinalis) located at the transition between the ventral telencephalon and olfactory bulb. Processes of these neurons project to the contralateral retina, traveling along the border between the internal plexiform and internal nuclear layer, and probably terminating on amacrine or bipolar cells. Horseradish peroxidase (HRP) injected into the eye or optic nerve is transported retrogradely in the optic nerve to the contralateral nucleus olfactoretinalis where neuronal perikarya are labeled. Labeled processes leave this nucleus in a rostral direction and terminate in the olfactory bulb. The nucleus olfactoretinalis is present only in fishes, such as cichlids, poecilids and centrarchids, in which the olfactory bulbs border directly the telencephalic hemispheres. In cyprinid, silurid and notopterid fishes, in which the olfactory bulbs lie beneath the olfactory epithelium and are connected to the telencephalon via olfactory stalks, the nucleus olfactoretinalis or a comparable arrangement of LHRH-immunoreactive neurons is lacking. After retrograde transport of HRP in the optic nerve of these fishes no labeling of neurons in the telencephalon occurred. It is proposed that the nucleus olfactoretinalis anatomically and functionally interconnects and integrates parts of the olfactory and optic systems.     

The Fine Structure of Four “Species” of Amoeba*

The fine structure of Amoeba discoides, Amoeba dubia, and Amoeba amazonas was studied and compared with that of Amoeba proteus. The different kinds of amebas showed general similarities but differed in the ultrastructural details of their organelles. With respect to fine structure, A. discoides was indistinguishable from A. proteus, while both A. dubia and A. amazonas had distinctive features. The nuclei of all had a prominent honeycomb-like fibrous lamina, but A. dubia differed from the others in the distribution of nucleoli within the nucleus. The mitochondria of A. amazonas were unusual in having a variable pattern of cristae, some being plate-like and others tubular. Golgi bodies in A. amazonas had a greater proportion of vesicles and a smaller number of cisternae than those of the others, while Golgi bodies in A. dubia had highly flattened cisternae without a lining of filamentous material such as is found in the other types. The plasma membrane of A. dubia also lacked the prominent filamentous cell coat common to A. proteus and other amebas. The relation between the Golgi apparatus and the cell coat and the significance of the degree of development of the cell coat for pinocytosis and other phenomena is considered. The experimental use of these cells, including the formation of hybrids by nuclear transplantation is discussed.     

The Oocysts of Eimeria vermiformis sp. n. and E. papillata sp. n. (Protozoa: Eimeriidae) from the Mouse Mus musclus

SYNOPSIS. Eimeria vermiformis sp. n. and E. papillata sp. n. are described from the mouse Mus musculus. The sporulated oocysts of E. vermiformis are 18–26 by 15–21 μ (mean 23.1 by 18.4 μ); its sporocysts are 11–14 by 6–10 μ (mean 12.8 by 7.9 p). The sporulated oocysts of E. papillata are 18–26 by 16–24 μ (mean 22.4 by 19.2 μ); its sporocysts are 10–13 by 6–9 μ (mean 11.2 by 8.0 μ). A substiedal body is present in E. papillata sporocysts. Patent infections were produced in white laboratory mice with both species. Fourteen species of Eimeria have now been described from the genus Mus.     

The presence of actin in nuclei: a critical appraisal.

To assess the significance of actin associations with nuclei, we have examined Amoeba proteus nuclei for the presence of labeled actin under a variety of circumstances without (in most instances) isolating nuclei or breaking up cytoplasms prior to the extraction of proteins.We first established that: the 42,000 dalton proteins (presumed to be actin) present in cytoplasm and non-isolated nuclei are identical electrophoretically; the putative actin of amebas has the same size and almost the same isoelectric point as rat muscle actin; and the peptide “fingerprints” of putative ameba actin and rat actin are very similar after tryptic digestion. We therefore concluded that the 42,000 dalton protein of ameba is actin.We determined that: the concentrations of actin in the cytoplasm and nucleus of amebas are the same; actin is readily lost from nuclei that are released from lysed cells; shortly after a ³⁵S-labeled nucleus is transplanted into unlabeled cytoplasm, or an unlabeled nucleus is transplanted into ³⁵S-labeled cytoplasm, the concentration of ³⁵S-actin in nucleus and cytoplasm is the same; and when cells containing ³⁵S-actin are subjected to long chase periods on unlabeled food, the concentrations of ³⁵S-actin in nucleus and cytoplasm fall in parallel. These observations taken together suggest that actin is not tightly associated with nuclei. Rather, actin may associate with nuclei for the trivial reason that the nuclear envelope is no barrier to free movement of that protein between the two compartments.We conclude that the mere presence of actin in nuclei is insufficient grounds for assuming that it has any role in nuclear functions, such as, for example, chromosome condensation.     

An ecological study of amebas from a small stream in Northern Florida

Summary An ecological study of amebas in the Hogtown Creek, which serves as a water-shed for the northwest quadrant of the city of Gainesville, Florida, indicates that the species which inhabit the creek are those typical of slow-flowing, moderate to warm freshwater streams of slightly alkaline nature. The Creek is essentially an unpolluted rural stream at its source; but receives both industrial and civil pollution in its course through the city. In the course of its flow some seven miles beyond the major entry of pollution, it recovers, and becomes again a rural stream with little significant pollution. Amebas disappear from the creek at the point where industrial pollution enters, and are found again in small numbers at two to four miles downstream. Thereafter, in the course of another three miles, amebas appear in greater numbers. Active amebas were not found whenever the pH of the stream became acid, at whatever station. None of the species known to be acidophilous were seen, with the exception of Difflugia oblonga, which may be ubiquitous. Some indication is noted that certain amebas prefer waters of certain temperature range, particularly within the genus Difflugia. In that genus D. lobostoma, D. tuberculata, D. urceolata, D. amphora, D. oviformis and D. acuminata were present more often in waters above 21° C.; whereas D. oblonga, D. globulosa, D. corona, D. gramen, and D. elegans were more often present in waters at temperatures below 21° C. Both naked and testate rhizopods were more numerous at or near pH 7.6; and in general both naked and testate amebas were more numerous at temperatures slightly below 21° C. It is suggested that any species of ameba may have a more or less restricted range of tolerance to any particular physical factor in the environment, with a presumed optimum for each factor, resulting in the appearance and disappearance of various species as the factors vary.Supported by Research Grant NIH-E-1158.     

Developmental changes in intestinal globule leukocytes of normal rats

Among vertebrates, telencephalo-pontine systems exist only in birds and mammals. However, three nuclei in the diencephalon and mesencephalon of teleost fishes have been indicated — analogous to the pons — to represent relay stations between telencephalon and cerebellum. Since two of these nuclei (dorsal preglomerular nucleus, dorsal tegmental nucleus) have only been described in the highly derived, electrosensory mormyrids, we investigated telencephalic connections in two nonelectrosensory teleosts, the goldfish Carassius auratus and the freshwater butterflyfish Pantodon buchholzi, and cerebellar connections only in the latter species, since for C. auratus these connections are already established. Horseradish peroxidase tracing reveals that C. auratus has a dorsal tegmental nucleus and a paracommissural nucleus both of which are telencephalo-recipient and project to the cerebellum, and that P. buchholzi has a dorsal preglomerular nucleus with such connections. These results extend our knowlegde of the distribution and, therefore, the phylogeny of telencephalo-cerebellar systems in teleosts. Similar to tetrapods, teleosts appear to have developed telencephalo-cerebellar systems several times independently.     

Spore Morphology and Taxonomy of Polypodiaceae

The examination of spores belonging to eight species representing five genera of Polypodiaceae reveals that they are typically free, anisopolar and monolete (bilateral). In a majority of cases the spores are yellow and have a short laesura—about half the length of the greater equatorial axis of the spore. Generally, the spores are elliptical in polar view and plano-convex to concavo-convex in profile view but in cross section they vary in shape. The average measurements in equatorial plane range from 44 μ to 71 μ whereas in the polar direction they vary from 27 μ to 47 μ. The exine is thin (about 2.5 μ and nearly smooth (laevigose-scabrous-foveolose) in Microsorium and Phymatodes but in the other investigated genera, it is thicker, possessing different types of projections. P: E varies from 4.6 to 5.5: 8. Oil globules are present in the spores of some species.

Available data on morphology of spores of Polypodiaceae have been tabulated in two tables on a comparative basis and their importance in taxonomy and phylogeny of some genera have been discussed.     

Ecologic Aspects of Protozoan Infections in Antarctic Fishes1

ABSTRACT. Plankton and fishes are abundant in Antarctic waters. Benthic invertebrates and fishes of the continental shelf are well-known, but the abyssal benthos below the highly productive open ocean is largely unsampled. The fishes are adapted (with antifreeze properties) to temperatures that are often or always below the freezing points of their body fluids. All the major groups of helminth parasites are found in or on these fishes. The few records of protozoa include Cryptobia, haemogregarines, a monoflagellate, the myxosporan Neoparvicapsula, and (in this paper) Ceratomyxa. Myxidium, Zschokkella, and a coccidian. Most of the protozoa were obtained from nototheniid fishes. No protozoa and few, if any, other parasites were recovered from 173 midwater fishes collected from outside of the continental shelf. Differences in infections in different localities and depths are due to many ecologic factors needing much more study of their relations to parasitism. These factors include temperatures, salinities, densities of fish populations, food and feeding habits, migrations of adult and immature fishes, availability of potential intermediate hosts, and marine “snow.”     

Testing the Mean Vector and the Correlation Coefficient in Life-Testing Under Bivariate Normality

In life-testing situations under bivariate normality of (X, U), a few smallest or a few largest Y-observations may not be available. Tests for μ = 0 (mean vector) and o = 0 (correlation coefficient) are developed from the available Y-observations and their concomitant X-observations. The robustness of these tests to departures from normality is investigated.     

The praeoptico-hypophysial system,Nucleus tuberis lateralis and the subcommissural organ of Gasterosteus aculeatus after changes in osmotic stimuli

Summary The histology and cytology of the praeoptico-hypophysial system, nucleus tuberis lateralis and subcommissural organ in Gasterosteus aculeatus were analyzed after the fishes had been put in waters of different salinity (see Tables 1–4).These three structures are all identical in fishes which are accustomed to fresh water and to 32 salt-water. A transference to hypertonic water causes changes in the neurosecretory system which suggest the existence of an antidiuretic principle in the neurosecretory substance. No such relationship is found when the fishes are put into hypotonic water. Further, no connection is found between either the secretion production in nucleus tuberis lateralis or in the subcommissural organ and variations in osmotic value.Aided by grants from the Swedish Natural Science Research Council.     

Characterization of comparative genome‐derived simple sequence repeats for acanthopterygian fishes

Simple sequence repeats (SSRs) have become one of the most popular molecular markers for population genetic studies. The application of SSR markers has often been limited to source species because SSR loci are too labile to be maintained in even closely related species. However, a few extremely conserved SSR loci have been reported. Here, we tested for the presence of conserved SSR loci in acanthopterygian fishes, which include over 14 000 species, by comparing the genome sequences of four acanthopterygian fishes. We also examined the comparative genome‐derived SSRs (CG‐SSRs) for their transferability across acanthopterygian fishes and their applicability to population genetic analysis. Forty‐six SSR loci with conserved flanking regions were detected and examined for their transferability among seven nonacanthopterygian and 27 acanthopterygian fishes. The PCR amplification success rate in nonacanthopterygian fishes was low, ranging from 2.2% to 21.7%, except for Lophius litulon (Lophiiformes; 80.4%). Conversely, the rate in most acanthopterygian fishes exceeded 70.0%. Sequencing of these 46 loci revealed the presence of SSRs suitable for scoring while fragment analysis of 20 loci revealed polymorphisms in most of the acanthopterygian fishes. Population genetic analysis of Cottus pollux (Scorpaeniformes) and Sphaeramia orbicularis (Perciformes) using CG‐SSRs showed that these populations did not deviate from linkage equilibrium or Hardy–Weinberg equilibrium. Furthermore, almost no loci showed evidence of null alleles, suggesting that CG‐SSRs have strong resolving power for population genetic analysis. Our findings will facilitate the use of these markers in species in which markers remain to be identified.     

Haemogregarina faiyumensis n. sp. in the Toad Bufo regularis in Egypt*

SYNOPSIS. Haemogregarina faiyumensis n. sp., a parasite of toads, Bufo regularis, in Faiyuni Province, Egypt, U.A.R., is described. In a survey of 689 toads from various localities in Cairo, Giza and Faiyurn provinces, only 3 out of 13 toads from Kom O Shim near Faiyum were infected. This species, known only by blood forms (most probably gametocytes) of two different staining reactions, is 13-17 μ long and 4-5 μ wide, with an average of 15.5 × 4.5 μ. The nucleus is typically subcircular and 3-5 × 3–5 μ, with an average of 4.5 × 3.9 μ.     

A Fluorescence Microscopic Study of the Retina of the River Lamprey (Lampetra fluviatilis) and the Atlantic Hagfish, (Myxine glutinosa)

During the last few years many facts have been revealed about the neural structure in the retina of several vertebrate groups, particularly with regard to the monoamine containing neurons. A number of investigations have been made on mammals, birds and teleost fishes by the Falck-Hillarp method for the cellular demonstration of such amines. The present study has paid attention to two cyclostomes, the river lamprey, Lampetra fluviatilis, and the atlantic hagfish, Myxine glutinosa.     

Pathogenic and Non-pathogenic Entamoeba: Pore Formation and Hemolytic Activity1

Pore-forming activity in planar lipid bilayers and liposomes of extracts from differentially pathogenic Entamoeba and the-capacity of trophozoites and subcellular fractions to lyse human red blood cells (hrbc) were investigated. In all amebas studied, the two activities paralleled each other. They were high in E. histolytica irrespective of the virulence of the particular strain, but low in non-pathogenic E. histolytica-like amebas of human origin as well as in E. invadens, which is pathogenic for reptiles, and in E. moshkovskii isolated from sewage. We conclude that the capacities to insert pores and to lyse are not sufficient for virulence although they may be necessary. The subcellular distribution of the hemolytic activity of E. histolytica and its sensitivity to a variety of inhibitors and activators differ from those of other known amebic cytotoxic activities including pore formation. Therefore, there may be an additional constituent of E. histolytica involved in the cytotoxicity of the parasite.     

Platyamoeba stenopodia. n. g., n. sp., a Freshwater Amoeba*

SYNOPSIS. Platyamoeba stenopodia n. g., n. sp., isolated from a lake in Alabama, is an amoeba with flattened form, usually more than 2.5 × as long as broad, the anterior half hyaline and the posterior half granular, with a single vesiculate nucleus at the posterior edge of the hyaline region. It forms a transitory floating stage with slender, hyaline pseudopods. Its cyst is spherical, uninucleate, with a smooth inner wall and a thinner, closely applied outer wall, which is often slightly wrinkled. Length of locomotive amoeba 15–36 μ diameter of cyst 7–11.5 μ Reproduction by binary fission with mesomitotic nuclear division, the nuclear membrane disappearing in prophase. The genus Platyamoeba is erected for amoebae including this organism; the type species is P. placida.     

Studies on the phytoplankton of the River Ganges,Varanasi, India Part IV “Phytoplankton in relation to fish population”

Summary A detailed and systematic study of the algae present in the guts of several fishes from the river and their percentage composition was carried out. Only a few fishes like Hilsa ilisha, Gadusia chapra and Barbus stigma showed herbivorous and omnivorous habits. Definite grazing of the phytoplankton by fishes was clearly envisaged. Their comparative preference for diatoms was evident.