Recent pioneering work opened the way for cloning genes in Paramecium by functional complementation of mutants. We present here the construction and pilot utilization of a new indexed library of Paramecium macronuclear DNA. The library is made of 61,440 clones containing inserts mostly between 6 and 12 kilobases. It has already allowed the complementation cloning of four new genes, and this library has proven to be very useful for rapid hybridization cloning.

The fatty acid composition of four microsporidian species (Glugea atherinae, Spraguea lophii, Glugea americanus, and Pleistophora mirandellae) and their host fishes has been determined using gas chromatography. Twenty-four fatty acids were identified with differences in relative abundance of fatty acids among the four parasites. Certain even-saturated fatty acids were found in a very high proportion: palmitic acid (16:0) represented one-third of total fatty acids in Pleistophora mirandellae. The level of docosahexaenoic acid (22:6ω3) attained 26–28% in Glugea atherinae, Spraguea lophii, and Glugea americanus, but only 8–9% in P. mirandellae. With respect to fatty acid compositions of host organs, some significant differences were evident between marine and freshwater fishes. Palmitic acid was prevalent in the marine fishes, Atherinae boyeri and Lophius piscatorius, and oleic acid (18:1ω9) in the freshwater fish Leuciscus cephalus. The proportion of docosahexaenoic acid in marine fishes was two or three times as great as in freshwater fish Leuciscus. The high polyunsaturated fatty acid content in both parasites and host fishes may be related to the scavenging of these fatty acids by the parasites rather than a microsporidia-specific fatty acid biosynthesis pathway.

Paramecium continues to be used to study motility, behavior, exocytosis, and the relationship between the germ and the somatic nuclei. Recent progress in molecular genetics is described. Toward cloning genes that correspond to mutant phenotypes, a method combining complementation with microinjected DNA and library sorting has been used successfully in cloning several novel genes crucial in membrane excitation and in trichocyst discharge. Paramecium transformation en masse has now been shown by using electroporation or bioballistics. Gene silencing has also been discovered in Paramecium, recently. Some 200 Paramecium genes, full length or partial, have already been cloned largely by homology. Generalizing the use of gene silencing and related reverse-genetic techniques would allow us to correlate these genes with their function in vivo.

Protist organisms (protozoa and fungi) have become increasingly prominent as opportunistic pathogens among persons infected with human immunodeficiency virus (HIV) and among organ transplant recipients—two immunocompromised populations that have increased dramatically in the past two decades. Pneumocystis carinii pneumonia continues to be the most common serious opportunistic infection (OI) among HIV-infected persons in the United States, occurring frequently among persons not previously receiving medical care. Toxoplasmosis, cryptococcosis, cryptosporidiosis, and isosporiasis occur frequently in HIV-infected persons in the developing world. Candidiasis and aspergillosis are common OIs in organ transplant recipients. As these populations of immunosuppressed patients continue to expand worldwide new OIs caused by protist pathogens are likely to emerge.

In recent years the clinical face of the Acquired Immune Deficiency Syndrome has changed significantly as a consequence of use of prophylaxis against Pneumocystis carinii pneumonia and combination antiretroviral therapy. In this context several opportunistic pathogens have emerged as causes of clinically important disease. Many of these infective agents have previously been defined by specific geographical locations. Their clinical presentation frequently mimics other (non) opportunistic infections with which they may co-exist. The diagnosis is frequently delayed as the diagnostic possibility may not be in the clinician's differential diagnosis. Invasive procedures are frequently required in order to secure a diagnosis. Despite treatment, prognosis is often poor. Clinicians should be aware of these opportunistic pathogens in order that a timely diagnosis may be made and appropriate therapy given.

Most species of Penicillium are considered relatively benign with respect to causing human disease. However, one species, P. marneffei, has emerged as a significant pathogen particularly among individuals who live in Southeast Asia and are concurrently infected with the human immunodefiency virus. While environmental and epidemiological studies have yet to resolve the reason for the heightened virulence of P. marneffei, one characteristic does distinguish this fungus from other Penicillium species. Whereas the latter grow as monomorphic moulds bearing typical asexual propagules (conidia), P. marneffei is thermally dimorphic. At room temperature, P. marneffei exhibits the morphology characteristic of the genus. In contrast to other Penicillia, though, P. marneffei grows as a yeast-like entity (arthroconidium) when found in diseased tissue or cultivated at 37 °C. Studies in our laboratory have focused on the differential gene expression between the mould and arthroconidial phases. Many of the genes whose expression differs during mould-to-arthrocondium transition are related to energy metabolism. A better understanding of gene expression during morphogenesis in P. marneffei may help detect unique target sites or cellular processes that can be exploited in the development of antifungal agents or immunomodulation therapies.

Free-living amebae belonging to the genus Acanthamoeba are the causative agents of granulomatous amebic encephalitis, a chronic progressive disease of the central nervous system, and of amebic keratitis, a chronic eye infection. Granulomatous amebic encephalitis occurs more frequently in immunocompromised patients while keratitis occurs in healthy individuals. The recent increased incidence in Acanthamoeba infections is due in part to infection in patients with acquired immune deficiency syndrome, while that for keratitis is due to the increased use of contact lenses. Understanding the mechanism of host resistance to Acanthamoeba is essential since the amebae are resistant to many therapeutic agents. Studies in our laboratory as well as from others have demonstrated that macrophages from immunocompetent animals are important effector cells against Acanthamoeba. We have demonstrated also that microglial cells, resident macrophages of the brain, elicit cytokines in response to A. castellanii. Neonatal rat cortical microglia from Sprague-Dawley rats co-cultured with A. castellanii produced mRNA for the inflammatory cytokines, interleukin 1α, interleukin 1β, and tumor necrosis factor α. In addition, scanning and transmission electron microscopy revealed that microglia ingested and destroyed A. castellanii in vitro. These results implicate macrophages as playing an effector role against Acanthamoeba and suggest immune modulation as a potential alternative therapeutic mode of treatment for these infections.

Immunosuppression is associated with the occurrence of a large variety of infections, several of them due to opportunistic protozoa. The parasitic protozoa of the family Trypanosomatidae vary greatly in their importance as potential opportunistic pathogens. African trypanosomiasis is no more common nor severe during AIDS. The situation with Chagas' disease, however, is much different. Although the process is not clearly understood, there appears to be a reactivation of Trypanosoma cruzi infection, which can lead to severe meningoencephalitis. In persons with AIDS, leishmaniasis is often exacerbated, particularly Leishmania infantum, which causes visceral leishmaniasis in southern Europe. Since 1990, 1,616 cases of visceral leishmaniasis/HIV co-infection have been reported, mainly from southern Europe, and particularly from Spain, southern France, and Italy. The co-infected patients are primarily young adults and belong to the risk group of intravenous drug users. Isoenzymatic identification of 272 isolates showed 18 different L. infantum zymodemes, of which 10 represent new zymodemes hitherto found only during HIV co-infection. New foci of co-infection are emerging in various parts of the world, including Brazil and East Africa. Moreover, since 1995, non-human monoxenous trypanosomatids have been found in AIDS patients, causing both diffuse cutaneous lesions and visceral infections. In countries where visceral leishmaniasis is endemic, particularly in southern Europe, immunosuppressive treatments for organ transplants or malignant diseases often result either in reactivation of asymptomatic visceral leishmaniasis or in facilitation of new infections.

Activation of signal transduction pathways in response to serum complement in Naegleria fowleri amebae was investigated. We examined the activation of protein kinases and changes in the phosphorylation state of proteins in N. fowleri stimulated by normal human serum (NHS). To determine differences in phosphorylation of proteins when amebae were exposed to NHS or heat inactivated serum (HIS) lacking complement, amebae were labeled with [³²P] orthophosphate. An increase in phosphorylation of relatively low molecular weight proteins was noted in N. fowleri incubated in NHS with a concomitant decrease in phosphorylation of high molecular mass polypeptides. To investigate whether serine/threonine or tyrosine kinases were stimulated by NHS, amebae were treated with protein kinase inhibitors H7, staurosporine or genistein, prior to serum exposure and examined for susceptibility to complement. Treatment with each of these inhibitors resulted in increased complement lysis. Incubation of N. fowleri with genistein specifically inhibited tyrosine phosphorylation of proteins stimulated by NHS. A tyrosine kinase activity assay using exogenous polyGlu-Tyr substrate demonstrated differential activation of tyrosine kinases in amebae treated with NHS when compared to treatment with HIS. The results suggest that activation of protein kinases and subsequent protein phosphorylation are important in mediating complement resistance in N. fowleri.

Microsporidia are unicellular and obligate intracellular spore-forming parasites. The spore inoculates the host cell with its non-motile infectious content, the sporoplasm, by way of the polar tube–the typical invasive apparatus of the microsporidian spore. Molecules involved in host cell invasion were investigated in Encephalitozoon intestinalis. Mouse polyclonal and monoclonal antibodies were raised against spore proteins and their reactivity was tested by Western-blotting and immunolocalization techniques, including electron and confocal microscopy. The antibodies thus generated could be divided into two major groups. One group reacted to the surface of the parasite at different developmental stages, mostly presporous stages and mature spores, whereas the other group recognized the polar tube. Of the antibodies reacting to the spore wall, one identified an exospore protein at 125 kDa while all others recognized a major doublet at 55–60 kDa, and minor proteins present at the surface of sporogonic stages and in the endospore. All antibodies recognizing spore wall proteins reacted also to the material forming septa in the parasitophorous vacuole. A major polar tube protein at 60 kDa was identified by another group of antibodies.

Lagenophrys dennisi, a peritrich ciliate, was observed attached to the exoskeleton of the crayfish Cambarellus patzcuarensis in Lake Pátzcuaro, Michoacán, Mexico. Lagenophrys dennisi presents a hemispheroidal, suboval or oval lorica in dorsal view, the distinctive lorica aperture consists of a pair of lips highly arched, unthickened, and smooth. Comparison of morphometric characters of the ciliate with previous records is made. Structures such as a “V”-shaped lorica suture, collar ridges, and myoneme are proposed for species identification. An anterior crescentic thickening on the dorsal surface of the lorica was observed under the scanning electron microscope. Lagenophryids were associated with 11 of 13 body parts with antennules and rostrum showing the highest prevalence. Lagenophrys dennisi was also found attached to submerged glass slides. This study represents the first record of L. dennisi on C. patzcuarensis and the first record of its presence in Mexico.

Bacteria were deposited in tubes as compact pellets by centrifuging suspensions of cultured Vibrio at stationary phase. Numbers and protein biomass of flagellates added to these tubes and of the Vibrio, were followed and compared with the growth of the same and other protists on identical, uncentrifuged Vibrio. The flagellates Bodo saliens and Caecitellus parvulus, which could not be seen to multiply in tubes of suspended bacteria, grazed deposited bacteria actively as did the more versatile flagellate Cafeteria roenbergensis. The growth of these flagellates and their consumption of deposited bacteria were very similar to those of the flagellate Pteridomonas danica or the ciliate Uronema marinum fed with suspended bacteria, although deposit-feeders grew more slowly. Gross growth efficiencies (30–60%) of deposit-feeding flagellates were similar to those of the suspension-feeding protists. Caecitellus consumed 55 Vibrio to produce one flagellate, while 4,500 Vibrio were consumed to produce one Uronema. Surface-feeding flagellates are shown to be efficient bacterivores, capable of restricting the numbers of bacteria deposited on surfaces just as other protozoa control numbers of suspended bacteria.

We determined small subunit ribosomal DNA sequences from three parabasalid species, Trichomitus batrachorum strain R105, Tetratrichomonas gallinarum, and Pentatrichomonas hominis belonging to the Trichomonadinae subfamily. Unrooted molecular phylogenetic trees inferred by distance, parsimony, and likelihood methods reveal four discrete clades among the parabasalids. TheTrichomonadinae form a robust monophyletic group. Within this subfamily T. gallinarum is closely related to Trichomonas species as supported by morphological data, with P. hominis and Pseudotrypanosoma giganteum occupying basal positions. Our analysis does not place T. batrachorum within the Trichomonadinae. Trichomitus batrachorum (strains R105 and BUB) and Hypotrichomonas acosta form a well-separated cluster, suggesting the genus Trichomitus is polyphyletic. The emergence of T. batrachorum precedes the Trichomonadinae-Tritrichomonadinae dichotomy, emphasizing its pivotal evolutionary position among the Trichomonadidae. A third cluster unites the Devescovinidae and the Calonymphidae. The fourth clade contains the three hypermastigid sequences from the genus Trichonympha, which exhibit the earliest emergence among the parabasalids. The addition of these three new parabasalid species did not however resolve ambiguities regarding the relative branching order of the parabasalid clades. The phylogenetic positions of Tritrichomonas fœtus, Monocercomonas sp., Dientamoeba fragilis, and the unidentified Reticulitermes flavipes gut symbiont 1 remain unclear.

Photosynthetic pico- and nanoplankton dominate phytoplankton biomass and primary production in the oligotrophic open ocean. Species composition, community structure, and dynamics of the eukaryotic components of these size classes are poorly known primarily because of the difficulties associated with their preservation and identification. Molecular techniques utilizing 18S rRNA sequences offer a number of new and rapid means of identifying the picoplankton. From the available 18S rRNA sequence data for the algae, we designed new group-specific oligonucleotide probes for the division Chlorophyta, the division Haptophyta, and the class Pelagophyceae (division Heterokonta). Dot blot hybridization with polymerase chain reaction amplified target rDNA and whole-cell hybridization assays with fluorescence microscopy and flow cytometry were used to demonstrate probe specificity. Hybridization results with representatives from seven algal classes supported the phylogenetic affinities of the cells. Such group- or taxon-specific probes will be useful in examining community structure, for identifying new algal isolates, and for in situ detection of these three groups, which are thought to be the dominant algal taxa in the oligotrophic regions of the ocean.

The small subunit-rRNA genes of 18 myxozoans from Lake Sasajewun, Algonquin Park were amplified and digested with restriction endonucleases for riboprinting analysis. Identical riboprints were not found between the myxosporeans and the actinosporeans. The distinct riboprinting patterns observed among these myxozoans indicate considerable genetic diversity within this group. Identical riboprints were found between Myxobolus pendula and Myxobolus pellicides, and between triactinomyxon ‘C’ and Triactinomyxon ignotum. Parsimony analysis of the riboprints demonstrated that neither the myxosporeans nor the actinosporeans formed a monophyletic group. Some species of Myxobolus are more closely related to forms of triactinomyxon, echinactinomyxon or raabeia than to other Myxobolus species. These results are consistent with the two-host life cycle hypothesis of myxozoans that myxosporeans and actinosporeans are alternating stages of the same organisms.

A new species of Cryptosporidium is described from the feces of domestic cattle, Bos taurus. Oocysts are structurally similar to those of Cryptosporidium muris described from mice but are larger than those of Cryptosporidium parvum. Oocysts of the new species are ellipsoidal, lack sporocysts, and measure 7.4 × 5.5 μm (range, 6.0–8.1 by 5.0–6.5 μm). The length to width ratio is 1.35 (range, 1.07–1.50). The colorless oocyst wall is < 1 μm thick, lacks a micropyle, and possesses a longitudinal suture at one pole. A polar granule is absent, whereas an oocyst residuum is present. Oocysts were passed fully sporulated and are not infectious to outbred, inbred immunocompetent or immunodeficient mice, chickens or goats. Recent molecular analyses of the rDNA 18S and ITS1 regions and heat-shock protein 70 (HSP-70) genes demonstrate this species to be distinct from C. muris infecting rodents. Based on transmission studies and molecular data, we consider the large form of Cryptosporidium infecting the abomasum of cattle to be a new species and have proposed the name Cryptosporidium andersoni n. sp. for this parasite.