Biomedical subjects
Publications and source records attributed to E U Canning.
We report the first case of a non-Enterocytozoon bieneusi microsporidial infection in the small intestine of a European AIDS patient with diarrhoea. It is also the first case in which a double infection with two different types of microsporidia has been encountered.
A species of Amblyospora-infecting neurones of Culex pipiens is described. Diplokaryotic meronts, which divided by binary fission, were distinguished at the electron microscope level by their unthickened plasma membranes. Sporonts with an electron-dense surface coat gave rise to eight uninucleate sporoblasts within a sporophorous vesicle, cytoplasmic division occurring at the quadrinucleate or octonucleate stages. Indications that nuclear fusion and chromosome reorganization occurred in merogony and sporogony were obtained by light microscopy but meiosis was not detected at the ultrastructural level. Spores were typical of Amblyospora, being ovoid when fresh, truncate when stained, and having an exospore of two membranous layers subtended by a thick amorphous layer, an electron-lucent endospore, an anisofilar polar filament, and a polaroplast comprised of an anterior region of close-packed lamellae and a posterior region of expanded sacs. The metabolic products in the sporophorous vesicle took the form of large globules, small globules with electron-dense borders, and fine granules. These were depleted in mature sporophorous vesicles, though a surface layer of fine granules on the spores may have been derived from them. Many stages were degenerate and it is suggested that C. pipiens may be an accidental host in which the parasite could develop suboptimally in nervous tissue only. Infections in larvae hatched from eggs in the laboratory indicate that vertical transmission occurs.
The enzyme-linked immunosorbent assay (ELISA) was used to survey human sera for antibodies to Encephalitozoon cuniculi using spores obtained from in vitro cultures as antigen. Sera were obtained from patients with tropical diseases, neurological and renal disorders, patients who were HIV positive and those who had been tested for HIV but found to be negative. Sera from inhabitants of the village of Jali, The Gambia and from healthy blood donors were also examined. Numerous sera from all groups except the blood donors gave positive ELISA reactions at dilutions of 1:400. On titration, those with titres of 1:400 were reclassified as negative. Antibody titres of 1:800 and above were considered to be indicative of past or present infections with E. cuniculi. Many of these ELISA seropositives were also positive by IFAT or PAP. When examined by Western blotting of SDS-PAGE protein profiles of E. cuniculi spores, sera from many patients who had a tropical association reacted with the characteristic profiles shown by known positive mouse and rabbit sera. Others in the tropical group showed antibody binding to some but not all of the immunodominant polypeptides and yet others were negative in spite of their reactivity in the ELISA, IFAT or PAP test. Less agreement between ELISA and Western blotting results was obtained with the other groups of patients, although reactivity with one or more of the major polypeptide bands was sometimes seen. Serum from one blood donor, examined by ELISA and Western blotting, was positive. Differences in the methods of antigen preparation and of epitopes recognized by individuals may account for different reactivities in the tests. It is concluded that infections of E. cuniculi are common in the tropics and that reactivations of these infections might be a hazard to AIDS patients.
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The localisation of monoclonal antibody-derived merozoite antigens of Babesia divergens was examined using immunogold electron microscopy and immunoprecipitation of the monoclonal antibody with both biosynthetically and surface-labelled parasites. Immunogold labelling provided evidence that the antigens are components of the surface coat of the merozoite. Immunoprecipitation with biosynthetically labelled parasites showed the antigens to be of parasite origin, whereas surface labelling confirmed that the antigens form part of the surface coat.
Protozoan infections, against which immunity is predominantly T cell mediated, are likely to be more severe in patients with the acquired immune deficiency syndrome (AIDS) than in immunocompetent hosts. Leishmaniasis, toxoplasmosis and cryptosporidiosis are examples, the last two being particularly common in AIDS patients. Cerebral toxoplasmosis almost always results from recrudescence of latent infections acquired earlier in life. Depletion of T-helper (CD4+) lymphocytes enables bradyzoites to survive if released from cysts in the brain of patients. In the absence of immune pressure bradyzoites revert to tachyzoites and multiply to cause a rapidly developing, necrotizing encephalitis which needs immediate treatment. AIDS patients, especially those who are negative for antibodies to Toxoplasma, should avoid cats, the source of oocysts, and undercooked meat which may contain tissue cysts, as primary infections may become systemic. Cryptosporidium infections are more likely to be primary infections. Sources of infection are other people, farm animals and pets and there is a significant risk from contaminated domestic water supplies. As infections cause a life-threatening secretory diarrhoea in AIDS patients, for which there is not satisfactory treatment at present, such patients should take steps to minimize the risk of infection.
An ELISA, using Encephalitozoon cuniculi spores as antigen, was used to determine the prevalence of specific anti-E cuniculi IgG in a group of stray dogs. In a preliminary survey 51 of 248 sera were classified as positive with titres of 1:400 to 1:3200. The 18 sera with titres of 1:400 were reclassified as negative when no IgG binding to the spores could be detected by comparison with a standard curve of anti-E cuniculi IgG. The remaining 33 sera (13.3 per cent) were classified as low, moderate or strong positives. Comparison of total IgG and specific IgG showed that specific IgG was greatly increased in the moderately and strongly positive sera. E cuniculi may be of importance as one cause of fading puppy syndrome when transmitted transplacentally, and as a complicating infection in human immunodeficiency diseases.
A Babesia divergens merozoite antigen was purified by affinity chromatography using a monoclonal antibody. Silver staining of SDS-PAGE gels revealed 2 bands of Mr 50-60 kDa and Mr 24-29. It is proposed that the Mr 24-29 kDa band represents the native protein and that it is complexed, perhaps as a dimer or perhaps to host protein to give the Mr 50-60 kDa band. Peptide analysis of the antigen followed by Western blotting with the monoclonal antibody revealed only an Mr 20-24 kDa band. After affinity purification the antigen was precipitated with acetone and inoculated subcutaneously, together with complete Freund's adjuvant, into gerbils. Two inoculations were given 3 weeks apart, after which the immunized animals and a group of control animals were challenged with 5 x 10(7) parasitized red cells from a virulent microaerophilus stationary phase (MASP) culture of an homologous strain of B. divergens. Parasitaemias rose rapidly in control animals and all were dead by the fifth day. Two of the vaccinated animals survived the challenge. Parasitaemias in the remaining two rose slowly but both animals succumbed on the seventh day. Western blotting of merozoites and of the purified antigen, using sera from the surviving animals showed strong reactivity with the Mr 24-29 kDa band.
The Weybridge strain of Babesia divergens became less virulent after 18 months in culture and was believed to be attenuated. Inoculations with the attenuated line using as many as 5 x 10(8) infected erythrocytes failed to raise a normal infection in Mongolian gerbils (Meriones unguiculatus) whereas, with the line that had been passaged from culture through gerbils at bimonthly intervals, inoculations of 10(7) infected erythrocytes gave rise to fatal infections. Gerbils were immunised with the attenuated line using doses ranging from 5 x 10(5) to 5 x 10(7) infected erythrocytes. Parasitaemias in the infected gerbils did not exceed 0.7 per cent after immunisation. All animals were protected when challenged with the virulent line four weeks later. Control animals died four days after challenge infection.
Nuclear division and chromosome cycle of microsporidia are reviewed in the light of recent speculation that the group diverged as an early branch in the eukaryotic line of descent. Microsporidia are primitive eukaryotes with simple cytoplasmic organisation, lacking mitochondria, peroxisomes and a classical Golgi apparatus. The ribosomes resemble prokaryotic ribosomes in size and in having sequences complementary to the eukaryotic 5.8S rRNA contained within their large (23S) ribosomal subunit, rather than a separate 5.8S molecule. Nuclei may be isolated or closely appressed as a diplokaryotic pair which divide synchronously. Mitosis is intranuclear: there are no centrioles but spindle termini are electron dense plaques in pores in the nuclear envelope. Some genera have isolated nuclei throughout the life cycle, while others have diplokaryotic nuclei throughout: meiosis is not known in either of these categories of genera. In contrast, certain polymorphic species, which are transmitted horizontally between copepods and mosquitoes and vertically between generations of mosquitoes, alternate between stages with isolated nuclei and stages with diplokaryotic nuclei. Haploid spores in copepods are infective to mosquito larvae, in which gametogenesis and plasmogamy occur to give diplokaryotic (diploid) stages. Stages remain diplokaryotic through transovarial transmission to the next generation, when an unusual form of meiosis is initiated in both nuclei of the diplokaryon (as indicated by synaptonemal complexes), and mingling of chromosomes occurs when the two nuclei fuse during pachytene.
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Two rhesus monkeys were each infected with 2.1 x 10(6) sporozoites of Plasmodium cynomolgi bastianellii; one was treated with 1.0 mg of pyrimethamine base per kg body weight for 5 d after sporozoite inoculation. A further 2 monkeys were each infected with 9.75 x 10(6) sporozoites of the same parasite; one was treated with 10 mg of proguanil per kg body weight for 4 out of 5 d after inoculation. The treated monkeys showed a delayed primary parasitaemia and relapses. In sections of liver biopsies taken 7.5 d after sporozoite inoculation, all monkeys showed numerous hypnozoites. However, there were no full grown schizonts and only rare retarded schizonts in the treated monkeys, in contrast to the untreated monkeys which had many mature or nearly mature schizonts. Later biopsies confirmed the continued presence of hypnozoites in all monkeys.
Immunogens derived from microaerophilous stationary phase (MASP) cultures of Babesia divergens grown in bovine erythrocytes were used to inoculate the laboratory host of B. divergens, the Mongolian gerbil, Meriones unguiculatus. Animals inoculated subcutaneously twice with preparations of freeze-thawed merozoites in complete Freund's adjuvant were fully protected against homologous challenge, as were gerbils immunised with a non-viable preparation of parasite-enriched lysed infected bovine erythrocytes. Animals which had been infected with small numbers of parasitised erythrocytes from cultures cooled to 4 degrees C, allowed to recover, then challenged, also survived. All three groups had high antibody titres which dropped immediately after challenge and then rose again. Gerbils given culture supernatants containing soluble merozoite protein coat antigens were partially protected only after receiving a third inoculation. Non-immunised animals all died 4 days after challenge.
A monoclonal antibody-producing hybridoma cell line has been raised against merozoites of Babesia divergens. This antibody is strongly reactive against merozoites in an enzyme-linked immunosorbent assay (ELISA) and is also positive in an indirect fluorescent antibody test (IFAT) on preparations of live merozoites. The antibody recognizes an antigen, of molecular weight (Mr) 50-60 K by Western blot analysis on SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gels. Merozoite neutralization assays show that the antibody significantly inhibits merozoite invasion of bovine erythrocytes.
An enzyme-linked immunosorbent assay (ELISA) was used to detect antibodies to Encephalitozoon cuniculi in man, using spores derived from tissue culture as antigen. Negligible cross-reactions were found with other microsporidia of vertebrate or of invertebrate origin and there was no cross-reaction with Toxoplasma gondii, using ELISA, immunoperoxidase or immunofluorescence staining. A high prevalence of antibodies to E. cuniculi was found in patients suffering from schistosomiasis, malaria and neurological and psychiatric disorders, but not in healthy individuals.
Primary cultures of Rhesus monkey were infected with sporozoites of P. c. bastianelli. Despite a high level of contamination, two schizonts were seen at days 6 and 11.
Unikaryon legeri (Dollfus, 1912) Canning and Nicholas, 1974, has been reexamined by electron microscopy from material collected in Portugal. It parasitises metacercariae of Meigymnophallus sp. in Cardium edule. A sporophorous vesicle forms around the sporonts, arising as a blister that separates from the electron-dense surface coat of the sporont. Sporogony is disporoblastic, giving rise to 2 spores that are retained in pairs within the sporophorous vesicle. Unikaryon piriformis, which is the type species of the genus and is also hyperparasitic in platyhelminth larvae, has not been examined by electron microscopy, and it is not known whether this species also produces sporophorous vesicles. If it does, then all that will be required is a simple addition of this character to the definition; if not, U. legeri will have to be transferred to a new genus and reclassified with other disporoblastic genera that sporulate in sporophorous vesicles.
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