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Complex I (NADH:ubiquinone oxidoreductase) is the first and largest multimeric complex of the mitochondrial respiratory chain. Human complex I comprises seven subunits encoded by mitochondrial DNA and 38 nuclear-encoded subunits that are assembled together in a process that is only partially understood. To date, mutations causing complex I deficiency have been described in all 14 core subunits, five supernumerary subunits, and four assembly factors. We describe complex I deficiency caused by mutation of the putative complex I assembly factor C20orf7. A candidate region for a lethal neonatal form of complex I deficiency was identified by homozygosity mapping of an Egyptian family with one affected child and two affected pregnancies predicted by enzyme-based prenatal diagnosis. The region was confirmed by microcell-mediated chromosome transfer, and 11 candidate genes encoding potential mitochondrial proteins were sequenced. A homozygous missense mutation in C20orf7 segregated with disease in the family. We show that C20orf7 is peripherally associated with the matrix face of the mitochondrial inner membrane and that silencing its expression with RNAi decreases complex I activity. C20orf7 patient fibroblasts showed an almost complete absence of complex I holoenzyme and were defective at an early stage of complex I assembly, but in a manner distinct from the assembly defects caused by mutations in the assembly factor NDUFAF1. Our results indicate that C20orf7 is crucial in the assembly of complex I and that mutations in C20orf7 cause mitochondrial disease.

We report a patient with an autosomal dominant chronic progressive external ophthalmoplegia phenotype associated with multiple mtDNA deletions in muscle from a family in which linkage analysis excluded mutations in DNA polymerase gamma (POLG), adenine nucleotide translocase (ANT-1) or C10orf2 (Twinkle). She presented with prominent Parkinsonism characterized by prolonged benefit from levodopa (L-dopa) and the later development of L-dopa induced dyskinesias and motor fluctuations. Thus L-dopa responsiveness, L-dopa induced dyskinesias and motor fluctuations may also occur in atypical Parkinsonism of mitochondrial disease, just as they may in multiple system atrophy., ((c) 2007 Movement Disorder Society.)

Knowing the etiology of hearing loss in a person has implications for counseling and management of the condition. More than 50% of cases of early onset, nonsyndromic sensorineural hearing loss are attributable to genetic factors. However, deafness is a genetically heterogeneous condition and it is therefore currently not economically and practically feasible to screen for mutations in all known deafness genes. We have developed a microarray-based hybridization biochip assay for the detection of known mutations. The current version of the hearing loss biochip detects nine common mutations in the connexin 26 gene, four mutations in the pendrin gene, one mutation in the usherin gene, and one mutation in mitochondrial DNA. The biochip was validated using DNA from 250 people with apparent nonsyndromic, moderate to profound sensorineural hearing loss. The hearing loss biochip detected with 100% accuracy the mutations it was designed for. No false-positives or false-negative results were seen. The biochip can easily be expanded to test for additional mutations in genes associated with hearing impairment or other genetic conditions.

Background: The mitochondrial DNA mutation A3302G in the tRNA(Leu(UUR)) gene causes respiratory chain complex I deficiency. The main clinical feature appears to be a progressive mitochondrial myopathy with proximal muscle weakness., Objective: To report on clinical and molecular features in 4 novel patients with the A3302G mutation., Design: Case reports., Patients: Four patients (3 of whom are from the same family) with a myopathy caused by the A3302G mitochondrial DNA mutation., Main Outcome Measure: Identification of the A3302G mutation by DNA sequencing., Results: All 4 patients had an adult-onset progressive mitochondrial myopathy with proximal muscle weakness, resulting in exercise intolerance. In 2 unrelated patients, upper limb reflexes were absent with preservation of at least some lower limb reflexes. Other features including hearing loss, recurrent headaches, ptosis, progressive external ophthalmoplegia, and depression were present., Conclusion: While the dominant clinical features of the A3302G mutation were exercise intolerance and proximal muscle weakness, other features of mitochondrial encephalomyopathies, previously not described for this mutation, were present.

Mutations in the arylsulfatase E gene, located on the X chromosome, have been shown to cause chondrodysplasia punctata (CDP). A substitution of arginine with serine at amino acid 12 (R12S) was identified in a patient with typical features of mild symmetrical CDP including mild mental retardation. The proband was institutionalized and was found to have seven full and half siblings all of whom were microcephalic. Six siblings are alive and all are mentally retarded. The mother is borderline retarded. The mother and three daughters are carriers of the R12S change, but do not appear to have CDP. A son and three other daughters do not carry the R12S change. Further studies revealed that the mother had phenylketonuria (PKU) and the children maternal PKU. This suggests that the R12S change is not the primary cause of short stature, microcephaly, and mental retardation in this family. The relationship between CDP and PKU, both of which can cause short statue and mental retardation, is discussed., (Copyright 1999 Academic Press.)

Sixteen males and two females with symmetrical (mild) type of chondrodysplasia punctata were tested for mutations in the X chromosome located arylsulphatase D and E genes. We identified one nonsense and two missense mutations in the arylsulphatase E gene in three males. No mutations were detected in the arylsulphatase D gene. Family studies showed segregation of the mutant genes establishing X linked inheritance for these families. Asymptomatic females and males were found in these studies. The clinical presentation varies not only between unrelated affected males, but also between affected males within the same family. We also conclude that clinical diagnosis of chondrodysplasia punctata in adults can be difficult. Finally, our results indicate that brachytelephalangy is not necessarily a feature of X linked symmetrical chondrodysplasia punctata.

We have characterized two mouse genes that code for the E1 alpha subunit of pyruvate dehydrogenase (PDH), Pdha-1 and Pdha-2. The coding regions show a high degree of homology with each other and with the human PDH genes, PDAH1 and PDHA2. Conserved regions include mitochondrial import sequences, phosphorylation sites and a putative TPP binding site. The PDH genes have an analogous chromosomal arrangement to PGK genes in that two isoforms code for a functionally and structurally similar product. Pdha-1 codes for a somatic isoform and maps to the X-chromosome. Pdha-2 is located on an autosome, is intronless and only expressed in spermatogenic cells. Comparison of human and mouse PDH and PGK gene sequences shows that the somatic sequences are more conserved relative to the testis-specific isoforms, and that the mouse PDH E1 alpha genes have experienced a faster rate of DNA change compared to their human counterparts.

The pathological changes in the brains of mice infected with T. gondii were studied at various intervals between 7 days and 22 months post-infection using histology, immunocytochemistry and electron microscopy. Initially, a few single parasites were observed (day 7) but necrotic lesions and microglial and inflammatory nodules rapidly appeared (9-I4 days). The majority of the lesions between days 9 and I4 contained proliferating toxoplasma and early cyst formation but from 2I days onwards the vast majority of nodules contained neither parasites nor Toxoplasma antigen. Intact intracellular cysts persisted throughout the period of study eliciting no host response. A generalized meningoencephalitis developed by day II and persisted with varying degrees of severity throughout the 22 months studied. At first, the inflammatory cells consisted of lymphocytes and monocyte/macrophages but during the chronic phase plasma cells predominated. In chronic infections, the number of microglial/inflammatory nodules was relatively constant with only a few containing toxoplasmic material resulting from recent cyst rupture. A few brains contained small nodules of dystrophic calcification. This study shows that in these asymptomatic animals, the major feature is perivascular cuffing by mononuclear cells and localized microglial/inflammatory nodules. After the development of the chronic state, there is no obvious increase or decrease in the severity of the pathological changes with time.

Pyruvate dehydrogenase E1 alpha deficiency is an X-chromosome-linked disorder, often with fatal consequences. We have searched for genetically useful polymorphisms in or near this gene. No restriction fragment length polymorphisms were detected using a battery of 36 different restriction enzymes and probing with a full-length cDNA fragment, or two single-copy genomic fragments located within intron 8, and 15 kb 3' of the coding region, respectively. The chemical cleavage method was then applied to the detection of base changes in or near the gene. One polymorphism was found in exon 8 of the coding region. However, no base changes were detected in intron 3 or in the part of intron 8 covered by fragment gB2. Three blocks of microsatellite DNA containing variable numbers of CA-repeats were isolated from the 5' end of the gene and characterized. Length polymorphisms in these microsatellite DNAs were analysed using the polymerase chain reaction. Although the three loci are tightly linked, the polymorphisms appear not to be in disequilibrium, making them useful markers in linkage studies of the pyruvate dehydrogenase E1 alpha gene. Of 31 females analysed 12(39%) were heterozygous for at least one length polymorphism of the three (CA)n alleles.

The pyruvate dehydrogenase (PDH) complex converts pyruvate to acetyl CoA, an essential step in aerobic glucose metabolism. We have previously shown that the gene for the E1 alpha subunit of this complex, expressed in somatic tissues, is located on band p22.1 of the human X chromosome. This gene, PDHA1, contains 10 introns and spans approximately 17 kb. An autosomal locus, PDHA2, showing significant cross-hybridization with a PDH E1 alpha cDNA probe, was detected on chromosome 4, in the region q22-q23. We here report the isolation of human testis-specific PDH E1 alpha cDNA clones. The similarity with the X chromosome-linked cDNA coding sequence at the nucleotide level is 84%. Specific amplification using the polymerase chain reaction confirmed the presence of a testis-specific mRNA and indicated that postmeiotic spermatogenic cells express this subunit. In situ hybridization with a unique probe from the 3' untranslated region of the testis-specific cDNA showed that the gene for this form of PDH E1 alpha is localized on chromosome 4 in the region q22-q23. The autosomal human gene was isolated from a chromosome 4-specific genomic library. The transcribed region of this gene is identical to the testis-specific cDNA sequence. It completely lacks introns and possesses characteristics of a functional processed gene.

The ultrastructural changes which occur during the in vitro excystation of the sporozoites of Toxoplasma gondii were examined. The excystation was carried out at 37 degrees C on suspensions of oocysts which had been ground and then treated with an excysting medium containing 0.25% trypsin and 0.75% sodium taurocholate in phosphate buffered saline, pH 7.3. It was found that sporocysts within intact oocysts were unaffected while sporocysts exposed to the medium ruptured. The sporocyst wall consisted of two layers and during excystation the four plates which form the inner layer started to curl inward. At the same time changes were seen at the specialized junctions between these plates. When the junctions finally break, the plates separate. The outer layer of the sporocyst wall is then ruptured at points directly above where the plates were joined. Each of the four portions of the sporocyst wall curled inward to form a tightly wound whorl. The sporozoites can escape through the openings created between the portions of the sporocyst wall.

Toxoplasma gondii undergoing endo-enteric development in the small intestine of the cat affects the ultrastructure of the epithelial cells. The epithelial cells from infected cats have swollen rough endoplasmic reticulum and the mitochondria may be swollen with degenerate cristae. A statistically significant shortening of the microvilli of the epithelial cells was demonstrated. It appeared that the effect on the microvilli length was related to the parasite distribution within the small intestine. These changes occur in both cells with and without parasites present at the level of the sections studied. The abnormalities are quickly rectified after the disappearance of the endo-enteric forms of Toxoplasma.

The Sabin-Feldman dye test was used to detect the presence of Toxoplasma antibodies in two groups of blood donors in central Scotland, one group from a rural area and one from an urban area, and in patients attending a medical outpatients clinic and females attending an antenatal clinic serving a mixed urban and rural area in the midlands of England. Results obtained from these four groups showed that 7.6, 7.8, 35.7 and 14.9% respectively had antibody titres of 1: greater than or equal to 10. A group of travelling people, defined in the Local Government and Planning (Scotland) Act, 1982, as '...persons of nomadic habit of life, whatever their race or origin...', from Scotland were also surveyed and 28% of this group had antibodies of 1: greater than or equal to 10. Individuals in this latter group were reported to have minimal contact with cats because of their lifestyles. The prevalences of the travelling people were analysed by age group and showed no correlation with age, but other groups did show an increasing prevalence with age. The significance of these results is discussed.

Activity and responsiveness to novel stimulation were assessed in three groups of mice infected with Toxoplasma. One group was infected when adult; two groups were infected congenitally, one born to dams infected during gestation, the other to dams chronically infected prior to mating. Each mouse was tested in a box, the floor of which was marked off into 16 equal squares, and its activity was measured over ten minutes by counting the number of times the mouse entered each square. Infected mice were more active. In addition, infected mice showed a smaller relative preference for the more novel central area of the box, especially towards the end of the observation period. These differences were independent of emotionality (as measured by defaecation counts), general health (as measured by subjective health ratings and body weight) and the number of Toxoplasma tissue cysts in specified brain regions. We suggest that differences arise from pathological changes caused by proliferating toxoplasms in the brains of the infected mice; an immunopathological reaction due to the presence of tissue cysts in the brain may also be involved. Other possible factors contributing to observed deficits in behaviour are also discussed. We suggest that such deficits may render Toxoplasma-infected mice more susceptible to predation by the domestic cat, the definitive host of Toxoplasma.

The structural organization of the X-linked gene for the E1 alpha subunit of the human pyruvate dehydrogenase complex has been determined by restriction endonuclease mapping and DNA sequence analysis of overlapping genomic clones. The gene is approximately 17 kilobase pairs long. It contains 11 exons ranging from 61 to 174 base pairs and introns ranging from 600 base pairs to 5.7 kilobase pairs. All the splice donor and acceptor sites conform to the GT/AG rule. The transcription initiation site was determined by S1 nuclease mapping. The DNA sequence around this site is very GC-rich. A "TATA box"-like sequence and a "CAAT box"-like sequence are present 24 and 113 bases upstream from the cap site, respectively. Also upstream from the cap site are several sets of inverted repeats, direct repeats, several sequences resembling the transcription factor Sp1 binding site, a glucocorticoid-responsive element, and two cAMP receptor binding sites.

The mucosal surfaces of villi from the small intestine of cats infected with Toxoplasma gondii were studied with the scanning electron microscope and compared with those from uninfected control cats. In uninfected cats villi were predominantly leaf shaped and were lined with ridges; goblet cell openings could be seen. The enterocytes had a hexagonal surface outline and were dome-shaped. Infected cats had both normal and abnormal villi. Injured villi were shortened and attained a broad leaf shape, often with blunt edges. Enterocytes containing oocysts were enlarged, and microvilli were resolvable only on these surfaces. Ruptured cells from which parasite discharge had occurred were seen. Oocysts were observed and possessed a smooth coat.

The macrogametes of Toxoplasma gondii develop within the epithelial cells of the cat ileum. As they develop the nucleus enlarges and dense patches of chromatin which were present in the nucleoplasm, disappear. Polysaccharide granules and lipid globules appear in the cytoplasm and increase in number during development. The wall-forming bodies of Type I (WFB I) appear before the wall-forming bodies of Type II (WFB II); WFB I are smaller, more osmiophilic and more numerous than the WFB II. The WFB I appear to form from vesicles produced by the smooth endoplasmic reticulum, and the WFB II form within the lacunae of the rough endoplasmic reticulum. Double membraned vacuoles appear to form from the nuclear membranes but the function of these is unknown. Throughout development the macrogamete retains a normal pellicle which possesses numerous micropores. The first evidence of oocyst wall formation is the appearance of participate matter in the parasitophorous vacuole which precipitates to form Layer 1 of the oocyst wall. Layers 2 and 3 are unit membranes which form between Layer 1 and the pellicle. During this development the organism has an organelle complement similar to that of the macrogamete. Layers 4 and 5 form between Layer 3 and the pellicle. Layer 4 is osmiophilic and its formation is accompanied by the disappearance of WFB I. Layer 5 is less osmiophilic than Layer 4 and its formation is accompanied by the disappearance of WFB II. The two innermost layers (Layers 4 & 5) in the oocyst wall of Toxoplasma are similar to those found in Isospora spp. and Eimeria spp. Toxoplasma seems to be unusual in that, firstly, it possesses an additional 3 layers and, secondly, all 5 layers are formed outside the pellicle of the original macrogamete.

Toxoplasma gondii, the causal agent of toxoplasmosis, is a ubiquitous protozoan parasite which has the potential to infect man and all warm-blooded animals. It has as its definitive host the cat. Infections acquired by the human adult are generally subclinical. However, if acquired for the first time during pregnancy, the parasite can invade the developing foetus. Under these circumstances infection can result in abortion, stillbirth or production of live-born offspring with severe multiple organ involvement. Alternatively, offspring can be symptomless at birth but may manifest neurological and ocular sequelae of toxoplasmosis in later life. The degree of clinical involvement is thought to be dependent upon the time of acquisition of infection by the pregnant female. Recent investigations have been concerned with behavioural aspects of predator-prey relationships between cats and rodents. Since these aspects affect the spread of toxoplasmosis in the environment, they have strong epidemiological and ecological implications. Toxoplasma infection in the environment, they have strong epidemiological and ecological implications. Toxoplasma infection in mice is associated with subtle alterations in exploratory behaviour which are almost certainly maladaptive, rendering infected mice more susceptible to predation by cats. Consequently the behavioural abnormalities in mice associated with Toxoplasma infection would be of major importance for the continuation of the life-cycle of the parasite. Ingestion of Toxoplasma-infected mice leads to oocyst production in cats and this opens up the well-known pathway whereby Toxoplasma infection reaches the human species.

The endogenous forms of Isospora felis were observed within the epithelial cells of the small intestine of the cat. They were situated within a parasitophorous vacuole which was limited by a multimembranous wall. The ultrastructural features of microgametogenesis were studied at 8 days post-infection. The initial phase of microgamont development consisted of cytoplasmic growth accompanied by a number of nuclear divisions. The gamont was enclosed by a pellicle and its surface area was greatly increased by deep invaginations. In the later stages of development the numerous nuclei were situated close to the pellicle. Each nucleus has peripherally condensed chromatin. Formation of the microgametes occurred as protrusions from the microgamont surface. Two basal bodies, the dense portion of a nucleus and a mitochondrion entered each protrusion. The microgametes matured while still attached to the gamont from which they finally budded off into the parasitophorous vacuole leaving a large residual cytoplasmic mass. The mature microgamete was found to consist of an elogate nucleus which overlaps with a mitochondrion towards the anterior end of the organism. The anterior portion contains a dense perforatorium and two basal bodies with attached flagella. In addition a number of microtubules (5-9) were found to run longitudinally from the basal body region.

Dehydrogenases of glycolysis, Krebs' cycle and pentosephosphate shunt are present in the metabolic machinery of toxoplasma endozoites (strain SS-119), thus suggesting a mixed oxidative metabolism in these. The enzymes are most commonly localized in the perinuclear area of the cytoplasm. Of special interest is the increased activity of some enzymes (especially lactate dehydrogenase) (fig HC) in the infected cells seen as definite layers of the stuff surrounding each parasite. This may correspond to the "overcoating" by mitochondrial material observed by Jones and Hirsch (1972) with the electron microscope. The importance of host-parasite relationships in the existence of the obligate intracellular parasite is discussed.

cDNA clones corresponding to the entire length of mRNA for the alpha subunit of human pyruvate dehydrogenase (EC 1.2.4.1), the E1 component of the pyruvate dehydrogenase complex, have been isolated from liver cDNA libraries. Two classes of cDNA clones were obtained and these correspond to two forms of pyruvate dehydrogenase E1 alpha mRNA. Both mRNA species have been demonstrated in a variety of human tissues and cultured fibroblasts. The cDNA sequence has been determined and, from it, the protein sequence of the human E1 alpha subunit was deduced. The protein is synthesized with a typical mitochondrial import leader sequence and the peptide bond at which this sequence is cleaved after transport into the mitochondrion has been determined by direct amino acid sequencing of the mature E1 alpha subunit. The human pyruvate dehydrogenase E1 alpha subunit contains identical phosphorylation sites to those found in the corresponding porcine protein. Preliminary studies of pyruvate dehydrogenase E1 alpha mRNA in cultured fibroblasts from patients with severe pyruvate dehydrogenase deficiency have revealed considerable heterogeneity as would be expected from protein studies.

The ultrastructure of the late sporoblast and the initiation of sporozite formation in E. brunetti was examined in oocysts which had been allowed to sporulate for 24 hours at 27 degrees C. The late sporoblast was ellipsoidal in shape and possessed two nuclei, and with associated Golgi bodies, situated at either end of the organism. Its cytoplasm contained numerous polysaccharide granules, electron translucentral region of the plaques. Sporozoite formation was similar to the initiation of merozoite formation in the schizont. Endodyogeny did not appear to be involved in the process of sporozoite formation.t vacuoles, dense bodies, mitochondria, and some strands of rough endoplasmic reticulum. The sporoblast, at this stage, was enclosed by two unit membranes. Sporozoite formation was initiated by the appearance of a dense plaque at either end of the organism in the vicinity of a nucleus, adjacent to the limiting membrane of the sporoblast. A conoid was present in the central region of the plaques Sporozoite formation was similar to the initiation of merozoite formation in the schizont. Endodyogeny did not appear to be involved in the process of sporozoite formation.

Male mice were exposed to a black arm and the stem of a Y-maze; entrance to a white arm was blocked by a transparent door. Toxoplasma-infected mice were significantly less active and tended to produce fewer faecal boluses than uninfected controls. In a subsequent free-choice trial, in which both arms were black, the uninfected mice spent significantly more time in the novel (previously blocked) arm; the infected mice showed no preference for either choice arm. Possible explanations are discussed. In particular, it seems that Toxoplasma-infected mice may be less responsive to novel stimuli. This suggestion has important implications for our understanding of one of the major ways in which Toxoplasma passes from host to host. If Toxoplasma infections impair responsiveness to novel stimuli, then infected mice are more likely to be taken by predators.

A study of the eyes of adult mice infected in utero with Toxoplasma gondii is reported. The histopathological features of the ocular inflammatory response in the infected mice ranged from minimal damage to complete destruction of the retinal tissue. Notable features such as retinal vasculitis and an almost uniform and highly selective destruction of the photoreceptor layer of the retina suggest a similarity between experimental autoimmune retinitis and the disease process in the retinas of our Toxoplasma-infected mice. We suggest that our mouse model could provide a simple and inexpensive tool for the investigation of immuno-pathological processes in the retina resulting from congenital Toxoplasma infection. The model has the advantage of low post-natal mortality coupled with high ocular morbidity. Furthermore, its aetiology is probably analogous to that of human ocular toxoplasmosis, in that the foetus becomes infected in utero via a mother whose primary infection is acquired during gestation.

A histopathological study of toxoplasmic retinochoroiditis in 39 eyes of mice infected in utero with Toxoplasma gondii and sacrificed at 16 weeks post-partum showed a wide variation in the pattern of tissue destruction. The changes in individual eyes were graded from mild to severe; Toxoplasma cysts were present in the retina and optic nerve in each grade. In the least affected eyes, Toxoplasma cysts were rarely seen and the disease was limited to a low grade uveitis and retinal lymphocytic perivasculitis. In the more severely affected eyes, there was focal, sectorial or total retinal destruction with secondary degeneration in the lens. In some eyes inflammatory destruction of the outer retina was associated either with a paucity of cells, or with lymphocytic infiltration or with plasma cell infiltration; giant cell granulomatous reactions were rare. In the most severely affected eyes the retina was necrotic and calcified. The findings illustrate the complexity of toxoplasmic retinochoroiditis and suggest that autoimmunity may play a part in the disease process.

The ultrastructural changes of Eimeria brunetti which occur during microgametogony in the small intestine of the domestic fowl are described. The development of the trophozoite into the microgametocyte was accompanied by cytoplasmic growth and nuclear division. The developing microgametocytes could be differentiated from developing schizonts because of ultrastructural differences in both cytoplasm and nuclei. The surface area of the microgametocyte was increased by deep invaginations of the limiting membrane. At this developmental stage, the nuclei were situated adjacent to this membrane and protrusions which developed, initiated microgamete formation. Two centrioles were positioned between each nucleus and the limiting membrane and were transformed into the basal bodies of the flagella. The flagella grew out from the basal bodies which then entered the protrusions. A mitochondrion and the dense chromatin-containing portion of the nucleus also entered each protrusion. The microgametes matured while they were attached to the residual cytoplasm of the microgametocyte; they were finally budded off into the parasitophorous vacuole. The microgamete consists of an elongated nucleus which overlaps a mitochondrion towards the arterior of the cell. At this end, a dense plaque (perforatorium) is found together with the 2 basal bodies and their attached flagella. 5 microtubules were apparent and ran longitudinally from the basal body region. In cross section they were arranged in a row of 4 with 1 diagonally opposite; only 2 of these microtubules extended to the posterior tip of the microgamete.

Clinical examination of the eyes of mice with congenital toxoplasmosis provided a good indication of the extent of underlying histopathological damage in the eye, but was of little value for predicting neuropathological severity. Furthermore, the dye-test titre was not related either to the degree of ocular damage or to the severity of brain damage.

The brains of mice with congenital ocular toxoplasmosis were examined histologically. Toxoplasma tissue cysts and a subacute/chronic meningo-encephalitis were present in the brains of all infected mice. The findings were very similar to those seen in human congenital toxoplasmosis. The murine model of congenital toxoplasmic encephalitis described in this paper will be valuable in the study of the pathogenesis, natural history and treatment of this condition.

The ultrastructure of the immature sporocyst and the initiation of sporozoite formation in T. gondii was examined in oocysts which had been allowed to sporulate for 12 or 24 hours at 27 degrees C. The sporocyst was ellipsoidal in shape and possessed a two layered sporocyst wall. A nucleus, with associated Golgi bodies, was situated at either end of the organism. The cytoplasm of the sporocyst also contained a number of polysaccharide granules, lipid globules, mitochondria and a few strands of rough endoplasmic reticulum. Sporozoite formation was initiated by the appearance of two dense plaques at either end of the organism in the vicinity of a nucleus, adjacent to the limiting membrane of the organism.

The initial stages of sporulation in oocysts of Toxoplasma gondii were examined in samples sporulated at 27 degrees C for 0, 6, 12, 16 and 24 hours. The initial zygote was roughly spherical and was limited by a single unit membrane. A few micropores of the inactive type were present on this membrane. The cytoplasm contained a large nucleus with a nucleolus, a number of polysaccharide granules, lipid globules, mitochondria, and Golgi bodies together with a few strands of rough endoplasmic reticulum. After the initiation of sporulation little change was noted in the cytoplasm except for an increase in protein synthesis as evidenced by the augmentation of the amount of rough endoplasmic reticulum and the appearance of polyribosomes. Nuclear division occurred twice giving rise to four nuclei which were situated close to the cell periphery and well separated from each other. At this multinucleate stage a second limiting membrane was formed. The cytoplasmic mass then divided to form the two sporoblasts. This was accomplished by an invagination of the limiting membranes in combination with internally formed membranes. The two binuclear sporoblasts were roughly spherical. They were limited by two unit membranes and contained the same cytoplasmic organelles as described for the zygote.

The host parasite relationship in the brains of asymptomatic mice chronically infected with Toxoplasma gondii was examined at 3, 6 and 12 months post-infection (PI) using electron microscopy. The parasites were located in large numbers within tissue cysts which ranged in size from 10-50 microns in diameter. The cysts were predominantly found in the grey matter. The toxoplasms were enclosed by a cyst wall consisting of a membrane, with irregular invaginations, and an underlying layer of homogeneous osmiophilic material. A detailed examination of 50 cysts revealed that all the cysts were present within intact host cells irrespective of their size or the period PI. The majority of host cells could be positively identified as neurons by the presence of synapses. No extracellular cysts were observed. It is probable that the intracellular location of the cysts protects them from recognition and attack by the host immune system.