Microsporidiosis in mammals.

Microsporidia are small, single-celled, obligately intracellular parasites that have caused significant agricultural losses and interference with biomedical research. Interest in the microsporidia is growing, as these organisms are recognized as agents of opportunistic infections in persons with AIDS and in organ transplant recipients. Microsporidiosis is also being recognized in children and travelers, and furthermore, concern exists about the potential of zoonotic and waterborne transmission of microsporidia to humans. This article reviews the basic biology and epidemiology of microsporidiosis in mammals.

Screening of compounds for antimicrosporidial activity in vitro.

Relatively few effective compounds are available for treating microsporidiosis in humans. In this study, several compounds were assayed for activity against Encephalitozoon intestinalis (Cali, Kotler et Orenstein, 1993) and Vittaforma corneae Shadduck, Meccoli, Davis et Font, 1990 in vitro. Of the benzimidazoles tested, albendazole was most effective and the MIC50 values were 8.0 ng/ml and 55.0 ng/ml for E. intestinalis and V. corneae, respectively. Fumagillin and its analogue, TNP-470 were nearly equally effective against both E. intestinalis and V. corneae. The MIC50 values of fumagillin were 0.52 ng/ml and 0.81 ng/ml, and the MIC50 values of TNP-470 were 0.35 ng/ml and 0.38 ng/ml for E. intestinalis and V. corneae, respectively. In addition, 12 of 44 purines and pteridines with putative tubulin binding activity that were synthesized at Southern Research Institute (SRI), inhibited microsporidial replication by more than 50% at concentrations that were not toxic to the host cells. Several chitin synthesis/assembly inhibitors inhibited growth of the microsporidia in vitro but were toxic for the host cells making it difficult to interpret the results. One exception was lufenuron, which caused no significant toxicity to the host cells and expressed approximate MIC50 values of 2.95 micrograms/ml and 6.3 micrograms/ml against E. intestinalis and V. corneae, respectively. These results warrant further studies on albendazole, fumagillin, TNP-470, lufenuron, and the selected SRI purines and pteridines for developing therapeutic strategies for microsporidiosis.

Microsporidia of the genus Trachipleistophora--causative agents of human microsporidiosis: description of Trachipleistophora anthropophthera n. sp. (Protozoa: Microsporidia).

Trachipleistophora anthropophthera n. sp., was found at autopsy in the brain of one and in the brain, kidneys, pancreas, thyroid, parathyroid, heart, liver, spleen, lymph nodes, and bone marrow of a second patient with AIDS. The parasite is similar to the recently described T. hominis Hollister, Canning, Weidner, Field, Kench and Marriott, 1996, in having isolated nuclei, meronts with a thick layer of electron dense material on the outer face of their plasmalemma and sporogony during which spores are formed inside a thick-walled sporophorous vesicle. In contrast to T. hominis, this species is dimorphic as it forms two kinds of sporophorous vesicles and spores: Type I--round to oval polysporous sporophorous vesicle, 7-10 microns in size, usually with eight spores (3.7 x 2.0 microns), thick endospores, subterminal anchoring disc and anisofilar polar filaments forming seven thicker and two thinner terminal coils. This type of sporophorous vesicle is associated with 25-30 nm filaments extending into the host cell cytoplasm. Type II--smaller, bisporous sporophorous vesicle (4-5 x 2.2-2.5 microns) with two, nearly round, thin-walled spores, 2.2-2.5 x 1.8-2.0 microns in size, having 4-5 isofilar coils. No outside filamentous elements are associated with the bisporous sporophorous vesicle. Both types of sporophorous vesicles were common in the infected brain tissue and could be found within the same cell. The newly described species, together with T. hominis and previously reported Pleistophora-like parasites from human muscle, likely represent a group of closely related human microsporidia.

Biology of microsporidian species infecting mammals.

Microsporidia (phylum Microspora) are obligate intracellular protozoan parasites that infect a wide range of vertebrate and invertebrate hosts. Over 1000 species have been classified into approximately 100 genera, and at least 13 species have been reported to infect mammals. Phylogenetically, the microsporidia are early eukaryotes because they have a true nucleus, possess prokaryote-like ribosomes, and lack mitochondria. The species that infect mammals are relatively small, measuring 2.0-7.0 microns long and 1.5-5.0 microns wide. The mature organism is the spore, which is enclosed by a chitinous coat, making it relatively resistant to the environment. Infections often occur by fecal-oral or urinary-oral transmission, although vertical transmission is quite common in the carnivores. Host cells become infected through a process of germination in which the spore propels its contents through the everting and unwinding polar filament into the host cell. The polar filament is unique to the microsporidia. With a few exceptions, microsporidiosis is typically chronic and subclinical in immunologically competent hosts. Young carnivores infected with microsporidia, however, develop severe and sometimes lethal renal disease, and immunodeficient laboratory animals (e.g. athymic and SCID mice) develop ascites and die from microsporidiosis. This review describes the morphology, life cycle, taxonomy, and host-parasite relationships of the species of microsporidia that infect mammals.

Animal models of human microsporidial infections.

Two new models have been described for Enterocytozoon bieneusi, non-human primates and immuno-suppressed gnotobiotic pigs, but there still is no successful cell culture system. The intestinal xenograft system holds promise as an animal model for Encephalitozoon intestinalis. Encephalitozoon hellem is easily propagated in mice, and also may be an important cause of spontaneous disease of psittacine birds. Encephalitozoon cuniculi occurs spontaneously in a wide variety of animals and can be induced experimentally in athymic mice. This is a useful experimental system and animal model, but the infection is relatively rare in man. Mammalian microsporidioses first were recognized as spontaneous diseases of animals that later confounded studies intended to elucidate the nature of diseases of humans. Much was learned about both experimental and spontaneous animal microsporidial infections that subsequently has been applied to the human diseases. In addition, new diseases have appeared, in both animals and humans, for which models are being developed. Since there are now animal models for almost all the known human microsporidioses, information on pathogenesis, host defenses, and effective treatments may become available soon. The microsporidioses provide a good example of the value of comparative pathology. Dr. Payne: Joe Payne. How much accidental infection has occurred with adjacent laboratory animals? Dr. Shadduck: A hard question. The organisms are thought to spread horizontally, and there is some pretty good evidence for that in rabbits. One assumes that this also is the explanation for the occurrence in infected kennels. Horizontal transmission probably occurs via contaminated urine, at least in the case of rabbits and dogs. Experimentally, horizontal transmission has been difficult to demonstrate in mice. Relative to the danger in people, I don't know how to answer that. I have always treated this as one of those things where you should be careful, but you shouldn't get paranoid. So, we have handled infected cell cultures and animals as if they were potentially infectious for man, but not as if they were something as hot as the human AIDS virus, for example. With the increasing number of reports in humans, I think it is clear that one would never want anybody who was at risk of being immunocompromised to work with these organisms. Dr. Fenkel: Are there other questions? Dr. Mysore: How do the parasites spread within the infected hosts? Dr. Shadduck: The usual answer is hematogenously via infected macrophages, but data that actually support that statement are rare. One does see infected macrophages in tissues, so it is not unreasonable to think that some of them escape and lodge in other tissues. But that has never actually been formally demonstrated. Dr. Nakeeb: Is E. bieneusi a human pathogen? Dr. Shadduck: The answer depends on which paper you read and what approach the authors took. There are papers in which the authors argue that the organism is not a cause of clinical disease in AIDS patients, but the general belief today is that the parasite does cause diarrhea and enteritis. I think the evidence for pathogenicity is quite strong for the various species of the Encephalitozoon, based on the severity and distribution of the lesions.

Clarifying the relationship of veterinary medical education and moral development.

OBJECTIVE: To clarify the relationship between veterinary medical education and moral development in response to 2 previous studies that presented conflicting evidence that the experience of veterinary medical education may inhibit moral development. DESIGN: The Defining Issues Test (DIT) was used to survey the moral reasoning of veterinary medical students at the beginning and end of their education. SAMPLE POPULATION: First and fourth-year veterinary medical students. PROCEDURE: The moral reasoning of 98 veterinary medical students was assessed at the beginning of their first semester of veterinary medical education and again, 4 years later, at the end of their last semester to determine whether their moral reasoning scores would reflect the expected maturity-related increases usually found at this age range and education level. RESULTS: The DIT scores ranged from 8.3 to 70.0 for first-year students and from 16.7 to 76.7 for fourth-year students. The first-year mean was 44.0 and the fourth-year mean was 45.4. The mean change of +1.45 points was not significant. Statistical analysis did not reveal any significant correlation between the moral reasoning scores and age; however, there was a significant correlation between the moral reasoning scores and gender, with females scoring higher on the first and second test. The difference in the rate of change between tests by gender was not significant. CONCLUSION: This study appears to confirm the findings of an earlier study suggesting veterinary medical education inhibits an increase of moral reasoning in veterinary medical students.

Identification and characterization of three Encephalitozoon cuniculi strains.

Microsporidia are increasingly recognized as causing opportunistic infections in immunocompromised individuals. Encephalitozoon cuniculi is probably the most studied mammalian microsporidian that infects insects and mammals, including man. In this study, 8 E. cuniculi isolates were compared and were found to fall into 3 strains. Strain type I includes the rabbit type isolate, as well as isolates from an additional rabbit, a dwarf rabbit, and a mouse. Strain type II includes 2 murine isolates and strain type III includes 2 isolates obtained from domestic dogs. By SDS-PAGE, the 3 strains differ primarily in the molecular weight range of 54-59 kDa where strain type I displays an apparent broad singlet at 57 kDa, strain type II displays an apparent doublet at 54 and 58 kDa, and strain type III displays an apparent broad band at 59 kDa. Antigenic differences were detected in the molecular weight regions of 54-58 kDa as well as 28-40 kDa by Western blot immunodetection using murine antisera raised against E. cuniculi, Encephalitozoon hellem, and the Encephalitozoon-like Septata intestinalis. Polymerase chain reaction (PCR) products containing only small subunit rDNA sequences from the different E. cuniculi isolates formed homoduplexes whereas PCR products containing intergenic rRNA gene sequences formed heteroduplexes in mobility shift analyses. Fok I digestion of the PCR products containing the intergenic rRNA gene region resulted in unique restriction fragment length polymorphism patterns, and DNA sequencing demonstrated that in the intergenic spacer region, the sequence 5'-GTTT-3' was repeated 3 times in strain type I, twice in strain type II, and 4 times in strain type III. This study indicates that there exist at least 3 E. cuniculi strains which may become important in the epidemiology of human E. cuniculi infections. Furthermore, as additional E. cuniculi isolates are characterized, these strains will be named or reclassified once the criteria for taxonomy and phylogenetic tree construction for microsporidia become better defined.

Small subunit ribosomal DNA phylogeny of various microsporidia with emphasis on AIDS related forms.

Phylogenetic analysis of the small subunit ribosomal DNA of a broad range of representative microsporidia including five species from humans (Enterocytozoon bieneusi, Nosema corneum, Septata intestinalis, Encephalitozoon hellem and Encephalitozoon cuniculi), reveals that human microsporidia are polyphyletic in origin. Septata intestinalis and E. hellem are very similar to the mammalian parasite E. cuniculi. Based on the results of our phylogenetic analysis, we suggest that S. intestinalis be designated Encephalitozoon intestinalis. Furthermore, analysis of our data indicates that N. corneum is much more closely related to the insect parasite Endoreticulatus schubergi than it is to other Nosema species. This finding is supported by recent studies which have shown a similarity between E. schubergi and N. corneum based on the origin and development of the parasitophorous vacuole. Thus these opportunistic microsporidian parasites can originate from hosts closely or distantly related to humans. Finally, the phylogeny based on small subunit ribosomal DNA sequences is highly inconsistent with traditional classifications based on morphological characters. Many of the important morphological characters (diplokaryon, sporophorous vesicle, and meiosis) appear to have multiple-origins.

Detection of microsporidia by indirect immunofluorescence antibody test using polyclonal and monoclonal antibodies.

During a screening for monoclonal antibodies (MAbs) to the microsporidian Encephalitozoon hellem, three murine hybridoma cell lines producing strong enzyme-linked immunosorbent assay (ELISA) reactivities were cloned twice, were designated C12, E9, and E11, and were found to secrete MAbs to the immunoglobulin M isotype. On subsequent ELISAs, the three MAbs reacted most strongly to E. hellem, and they reacted somewhat less to Encephalitozoon cuniculi and least to Nosema corneum, two other microsporidian species. The MAbs produced values of absorbance against microsporidia that were at least three times greater than reactivities obtained with control hybridoma supernatants or with uninfected host cell proteins used as antigens. By Western blot immunodetection, the three MAbs detected three E. hellem antigens with relative molecular weights (M(r)s) of 62, 60, and 52 when assayed at the highest supernatant dilutions producing reactivity. At lower dilutions, the MAbs detected additional proteins with M(r)s of 55 and 53. By using indirect immunofluorescence antibody staining, the MAbs, as well as hyperimmune polyclonal murine antisera raised against E. cuniculi and E. hellem, were able to detect formalin-fixed, tissue culture-derived E. cuniculi and E. hellem and two other human microsporidia, Enterocytozoon bieneusi and Septata intestinalis, in formalin-fixed stool and urine, respectively. E. bieneusi, however, stained more intensely with the polyclonal antisera than with the MAbs. Neither the MAbs nor the hyperimmune murine polyclonal antibodies detected Cryptosporidium, Giardia, Trichomonas, or Isospora spp. At higher concentrations, the polyclonal antisera did stain N. corneum and yeast cells. The background staining could be absorbed with Candida albicans. These results demonstrate that polyclonal antisera to E. cuniculi and E. hellem, as well as MAbs raised against E. hellem, can be used for indirect immunofluorescence antibody staining to detect several species of microsporidia known to cause opportunistic infections in AIDS patients.

Experimental microsporidiosis in immunocompetent and immunodeficient mice and monkeys.

Microsporidia cause opportunistic infections in AIDS patients and commonly infect laboratory animals, as well. Euthymic C57B1/6 mice experimentally infected with intraperitoneal injections of 1 x 10(6) Encephalitozoon cuniculi Levaditi, Nicolau et Schoen, 1923, Encephalitozoon hellem Didier et al., 1991, or Nosema corneum Shadduck et al., 1990 displayed no clinical signs of disease. Athymic mice, however, developed ascites and died 8-16 days after inoculation with N. corneum, 21-25 days after inoculation with E. cuniculi, and 34-37 days after inoculation with E. hellem. All athymic mice displayed hepatomegaly, dilated intestine and accumulation of ascites fluid. Granulomatous lesions are primarily located in the liver, lung, pancreas, spleen, and on serosal surfaces of abdominal organs. The murine microsporidiosis model also was used to examine immune response that inhibit microsporidia growth in vitro. Recombinant murine interferon-gamma (mIFN-gamma, 100 mu/ml) alone or in combination with lipopolysaccharide (LPS; 10 ng/ml) could activate thioglycollate-induced peritoneal murine macrophages to destroy E. cuniculi. The production of the nitrogen intermediate, NO2-, correlated with parasite destruction. Inhibition of NO2- generation by addition of the L-arginine analogue, NG-monomethyl L-arginine (NMMA), inhibited microsporidia killing, as well. Since microsporidiosis is becoming an important opportunistic infection in AIDS patients, a microsporidiosis model is being developed using SIV/DeltaB670-infected rhesus macaque monkeys (Macaca mulatta). SIV-infected immunocompetent monkeys given E. cuniculi or E. hellem per os developed specific antibodies, and microsporidia could be detected sporadically by calcofluor or antibody fluorescence staining of stool and urine sediment smears. As immunodeficiency progressed, monkeys developed diarrhoea, cachexia, and anorexia, and organisms were detected in urine and stool with greater frequency. Immunodeficient SIV-infected monkeys died approximately 27 days after receiving E. hellem by intravenous inoculation, and approximately 110 days after receiving E. hellem per os. Lesions typical for SIV-infection were observed in both groups of monkeys and microsporidia were detected in kidney and liver of the intravenously-injected monkeys. The murine microsporidiosis model provides an efficient means for studying protective immune responses to microsporidiosis, and may prove useful for screening immunological and chemotherapeutic agents. The pathogenesis of Encephalitozoon microsporidiosis in SIV-infected monkeys appears to parallel encephalitozoonosis in AIDS patients, suggesting that simian microsporidiosis may provide a useful model for evaluating diagnostic methods and therapeutic strategies during various stages of progressing immunodeficiency.

Comparative pathology of microsporidiosis.

The obligate intracellular protozoan parasites belonging to the phylum Microspora are ubiquitous. They parasitize insects and all five classes of vertebrates. Only one genus infects mammals and birds but at least four genera affect humans. Two genera have been isolated from human specimens and both infect experimental animals. Some genera (eg, Enterocytozoon and Pleistophora) seem to be limited to a very few tissues but others (Encephalitozoon and the Encephalitozoon-like genus Septata) can infect multiple organs. Lesions range from classic microgranulomas to foci of infected cells unaccompanied by any inflammatory response. The most commonly occurring microsporidia infection of man (Enterocytozoon bieneusi) is characterized by infection of enterocytes of the villus tips of the small intestine, accompanied by villus blunting, crypt hyperplasia, sloughing of infected villus tip cells, and increased numbers of intraepithelial lymphocytes.

Experimental infection of athymic mice with the human microsporidian Nosema corneum.

Athymic mice (BALB/c nu/nu/Ola/Hsd) were experimentally infected intraperitoneally with Nosema corneum spores. Infection was monitored in the first and second weeks post-infection. The liver, spleen, kidney, intestine, lung, heart, brain and eye were collected. Quantification of infection in each organ using three different techniques gave approximately the same pattern of infection. Infection increased with time. Histological observations were made on the sites of infection in each organ. All organs were infected, the liver being the most heavily infected. The eye was infected in the retina in contrast to the cornea which was the site of infection in the original host. The present study of N. corneum in athymic mice has shown that this system could also be used to study host-parasite relationships and serve as a model for testing therapeutic agents. Previously the only microsporidian serving as a suitable model for human microsporidiosis was Encephalitozoon cuniculi.

Ribosomal DNA sequences of Encephalitozoon hellem and Encephalitozoon cuniculi: species identification and phylogenetic construction.

A segment of ribosomal DNA, about 1,350 base pairs long, was amplified from the microsporidian species Encephalitozoon hellem, isolated from AIDS patients, and Encephalitozoon cuniculi. The amplified DNA segment extends from position 530 in the small ribosomal RNA subunit to position 580 in the large ribosomal RNA subunit. A comparison of sequence data from this region for Encephalitozoon hellem and Encephalitozoon cuniculi shows relatively high sequence similarity, supporting the placement of these two organisms in the same genus. At the same time, sequence differences between the two organisms confirm that they are not the same species. Three separate isolates of E. hellem were sequenced in the highly variable intervening spacer region. The sequence was identical for all three isolates. Within the amplified DNA segment, regions were sequenced which yield highly variable, moderately variable and highly conserved sequence information, appropriate for comparison with other species in the phylum Microspora at all taxonomic levels. We suggest that sequence data from these regions be included in future species descriptions for the purposes of species identification and phylogenetic analysis. Restriction digests of the amplified region are presented and give a rapid method for distinguishing between the two Encephalitozoon species.

Hepatic lesions in rabbits infected with Encephalitozoon cuniculi administered per rectum.

Microsporidia have been recognized recently as opportunistic pathogens in acquired immunodeficiency syndrome patients. In an attempt to develop an animal model of enteric microsporidiosis, adult (5 to 6 months old) male Flemish Giant rabbits from a closed New York colony were administered 5 x 10(3), 5 x 10(5), and 5 x 10(7) Encephalitozoon cuniculi per rectum. Rabbits given 5 x 10(5) and 5 x 10(7) E. cuniculi had moderate granulomatous periportal infiltrates, characterized by the presence of numerous macrophages, epithelioid cells and a few multinucleated giant cells, lymphocytes, and plasma cells. Inflammatory cells also were seen infiltrating the tunica adventitia and tunica media of hepatic portal veins and branches of the hepatic artery. This study demonstrates that administration of E. cuniculi per rectum to rabbits results in infection that is characterized by high frequency and severity of hepatic lesions.