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13 p.-4 fig.-1 tab., Deficiencies in Mannosidase β (MANBA) are associated with neurological abnormalities and recurrent infections. The single nucleotide polymorphism located in the 3′UTR of MANBA, rs7665090, was found to be associated with multiple sclerosis (MS) susceptibility. We aimed to study the functional impact of this polymorphism in lymphocytes isolated from MS patients and healthy controls. A total of 152 MS patients and 112 controls were genotyped for rs7665090. MANBA mRNA expression was quantified through qPCR and MANBA enzymatic activity was analyzed. Upon phytohemagglutinin stimulation, immune activation was evaluated by flow cytometry detection of CD69, endocytic function, and metabolic rates with Seahorse XFp Analyzer, and results were stratified by variation in rs7665090. A significantly reduced gene expression (p < 0.0001) and enzymatic activity (p = 0.018) of MANBA were found in lymphocytes of MS patients compared to those of controls. The rs7665090*GG genotype led to a significant β-mannosidase enzymatic deficiency correlated with lysosomal dysfunction, as well as decreased metabolic activation in lymphocytes of MS patients compared to those of rs7665090*GG controls. In contrast, lymphocytes of MS patients and controls carrying the homozygous AA genotype behaved similarly. Our work provides new evidence highlighting the impact of the MS-risk variant, rs7665090, and the role of MANBA in the immunopathology of MS., This work was supported by the projects PI16/01259 and PI20/01634, integrated into the Plan Nacional de I+D+I, AES 2013–2016 and 2017–2020; funded by the ISCIII and co-funded by the European Regional Development Fund (ERDF) “A way to make Europe”; and by the project PROYECTO/AEI/10.13039/50110001103 from the Agencia Española de Investigación. AGJ holds a Formación de Profesorado Universitario (FPU20/03387) fellowship from Ministerio de Ciencia e Innovación.

A neurological disease was investigated in 3 German Shepherd pups from the same litter that failed to grow normally, appeared stiff, were reluctant to move, and were deaf. They developed intermittent seizures and ataxia and had proprioceptive defects. Histopathology showed severe vacuolation of neurons, astrocytes in nervous tissue, renal tubular epithelial cells, and macrophages in nervous tissue, spleen, and liver. Vacuoles appeared empty with no storage material stained by periodic acid–Schiff (PAS) or Sudan black stains, leading to a diagnosis of a lysosomal storage disease and in particular an oligosaccharidosis. Biochemical and genomic studies showed that this was β-mannosidosis, not previously diagnosed in dogs. A c.560T>A transition in exon 4 of the MANBA gene was found, which segregated in these and other family members in a manner consistent with it being the causative mutation of an autosomal recessive disease. This mutation led to substitution of isoleucine to asparagine at position 187 of the 885 amino acid enzyme, a change expected to have functional significance.

Background The Roma are a European ethnic minority threatened by several recessive diseases. Variants in MANBA cause a rare lysosomal storage disorder named beta-mannosidosis whose clinical manifestation includes deafness and mental retardation. Since 1986, only 23 patients with beta-mannosidosis and biallelic MANBA variants have been described worldwide. Results We now report on further 10 beta-mannosidosis patients of Roma origin from eight families in the Czech and Slovak Republics with hearing loss, mental retardation and homozygous pathogenic variants in MANBA. MANBA variant c.2158-2A>G screening among 345 anonymized normal hearing controls from Roma populations revealed a carrier/heterozygote frequency of 3.77%. This is about 925 times higher than the frequency of this variant in the gnomAD public database and classifies the c.2158-2A>G variant as a prevalent, ethnic-specific variant causing hearing loss and mental retardation in a homozygous state. The frequency of heterozygotes/carriers is similar to another pathogenic variant c.71G>A (p.W24*) in GJB2, regarded as the most frequent variant causing deafness in Roma populations. Conlcusion Beta-mannosidosis, due to a homozygous c.2158-2A>G MANBA variant, is an important and previously unknown cause of hearing loss and mental retardation among Central European Roma.

Hereditary β-mannosidosis causing progressive lysosomal neuropathy and other clinical signs, has been previously described in humans, Nubian goats, and Salers cattle. Here we report the clinicopathological, metabolic, and molecular genetic features of canine beta-mannosidase (MANBA, EC 3.2.1.25) deficiency. A 1-year-old male mix-breed dog from St. Kitts was presented with progressive stumbling, weakness, and regurgitation. Vacuolated lymphocytes were observed on the blood film. Postmortem findings included marked enlargement of nerves, megaesophagus, and internal hydrocephalus. Vacuolated macrophages, neurons, and secretory epithelial cells suggested an oligosaccharide storage disease. Plasma concentration of the β-mannosidosis specific oligosaccharide was approximately 75 fold that of controls. The plasma beta-mannosidase activity was severely reduced to ~5% of controls; five other lysosomal acid hydrolase activities were increased or within their normal reference interval. Genomic sequencing of this dog's MANBA gene identified a homozygous exonic five bp tandem duplication in the penultimate exon of the MANBA gene (c.2377_2381dupTATCA) which results in a reading frame shift, altering the subsequent amino acid sequence and creating a premature stop codon. The truncated beta-mannosidase enzyme is expected to be dysfunctional. This enzyme deficiency causes the accumulation of un-degraded oligosaccharides in cells, which affect the myelination of the peripheral and central nervous systems. This insertion was not encountered in 121 and 80-screened samples from dogs on St. Kitts (all were homozygous for wild-type) and Philadelphia region (wild-type), respectively. In conclusion, canine β-mannosidosis has similar clinicopathological features with some human patients, but milder signs than in ruminants and more severe than in knockout mice. Hence, dogs with β-mannosidosis could become a valuable disease model for the human disease.

β-Mannosidase is a lysosomal enzyme from the glycosyl hydrolase family 2 that cleaves the single β(1-4)-linked mannose at the nonreducing end of N-glycosylated proteins, and plays an important role in the polysaccharide degradation pathway. Mutations in the MANBA gene, which encodes the β-mannosidase, can lead to the lysosomal storage disease β-mannosidosis, as well as nystagmus, an eye condition characterized by involuntary eye movements. Here, we present the first structures of a mammalian β-mannosidase in both the apo- and mannose-bound forms. The structure is similar to previously determined β-mannosidase structures with regard to domain organization and fold, however, there are important differences that underlie substrate specificity between species. Additionally, in contrast to most other ligand-bound β-mannosidases from bacterial and fungal sources where bound sugars were in a boat-like conformation, we find the mannose in the chair conformation. Evaluation of known disease mutations in the MANBA gene provides insight into their impact on disease phenotypes. Together, these results will be important for the design of therapeutics for treating diseases caused by β-mannosidase deficiency. DATABASE: Structural data are available in the Protein Data Bank under the accession numbers 6DDT and 6DDU.

International audience; RATIONALE: The first step in the diagnosis of oligosaccharidoses is to evidence abnormal oligosaccharides excreted in urine, usually performed by the poorly sensitive but efficient thin layer chromatography (TLC) method. Developing a tandem mass spectrometry (MS/MS) technique could be of great interest to replace TLC. METHODS: Abnormal underivatized oligosaccharides have been recently studied using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, allowing the unambiguous identification of oligosaccharidoses. Based on this previous work, we developed an advantageous and efficient liquid chromatography (LC)/MS/MS method using a more common triple quadrupole tandem mass spectrometer for oligosaccharides analysis. RESULTS: Oligosaccharidoses (n = 97) and control (n = 240) urine samples were analysed. A specific pattern was obtained for each oligosaccharidosis using this method. In urine, it allows not only the identification of all the oligosaccharidoses previously identified by TLC (fucosidosis, alphamannosidosis, aspartylglucosaminuria, GM1 gangliosidosis, sialidosis, galactosialidosis and Schindler disease), but also extends the field of diagnosis to mucolipidosis type II, Sandhoff disease, and beta-mannosidosis. The same technique was applied to 16 amniotic fluid supernatants from oligosaccharidosis-affected foetuses (n = 16) compared with 37 unaffected. All the affected foetuses could be clearly identified: sialidosis (n = 3), galactosialidosis (n = 4), aspartylglucosaminuria (n = 1), mucolipidosis type II (n = 4) or GM1 gangliosidosis (n = 4). This technique can be applied to early prenatal diagnosis as well as to the oligosaccharidosis screening in the case of non-immune hydrops fetalis. CONCLUSIONS: The method is quick and easy to run, with an LC analysis time of 13 min per sample. The quantitative validation could not be obtained in the absence of a specific standard and of a labelled internal standard for each compound. Even if this LC/MS/MS method is only qualitative, it is very specific and much more sensitive than TLC. It allows the urinary screening of oligosaccharidoses, even mild or late-onset forms, and the screening of antenatal forms in amniotic fluid. Copyright (C) 2017 John Wiley & Sons, Ltd.

β-Mannosidosis is a lysosomal storage disorder characterized by accumulation of disaccharides due to deficiency of the lysosomal enzyme β-mannosidase. The disease is caused by mutations in MANBA and is extremely rare in humans. Although the clinical presentation is heterogeneous, common symptoms include various degrees of developmental delay, behavioral disturbances, hearing loss, and frequent infections. We report a 15-yr-old girl presenting with mild intellectual disability, sensorineural hearing loss, severe behavioral disturbances, dysmorphic traits, and evolving angiokeratomas. Copy-number variation analysis of next-generation sequencing (NGS) data indicated increased coverage in exons 8–11 of MANBA. Low β-mannosidase activity (1 µkatal/kg protein, refv 25–40) established the diagnosis of β-mannosidosis. Whole-genome sequencing (WGS) and cDNA analysis revealed a novel homozygous intragenic inverted duplication in MANBA, where a 13.1-kb region between introns 7 and 11 was duplicated and inserted in an inverted orientation, creating a 67-base nonduplicated gap at the insertion point. Both junctions showed microhomology regions. The inverted duplication resulted in exon skipping of exons 8–9 or 8–10. Our report highlights the importance of copy-number variation analysis of data from NGS and in particular the power of WGS in the identification and characterization of copy-number variants.

β-Mannosidosis (OMIM # 248510 ) is an autosomal-recessive lysosomal storage disorder caused by deficiency of the lysosomal enzyme β-mannosidase (MANBA, E.C. 3.2.1.25). The disorder has been reported in goat, cattle and man. The human disorder is rare and only 20 cases in 16 families have been reported. We have sequenced the exons and exon–intron borders in a European patient with infantile onset of β-mannosidosis. The patient was compound heterozygous for a silent mutation (c.375A > G) in exon 3 causing alternative splicing, and a missense mutation (c.1513T > C, p.Ser505Pro) in exon 12. The alternative splicing event deleted four nucleotides from the transcript and was predicted to result in premature termination of translation. In order to evaluate the consequence of the missense mutation, we inserted the human β-mannosidase gene into an expression vector, performed site-directed mutagenesis and expressed the normal and mutant enzyme in COS-7 cells. We also included the previously reported β-mannosidosis-associated missense mutations c.544C > T (p.Arg182Trp) and c.1175G > A (p.Gly392Glu), which were found in patients presenting a milder phenotype. Cells transfected with the wild-type construct showed a 33-fold increase in β-mannosidase activity compared to mock-transfected cells, whereas cells transfected with the mutant constructs showed no detectable increase in activity. We propose that the milder phenotype described in some β-mannosidosis patients with missense mutations in the MANBA gene is not due to residual β-mannosidase activity, but rather caused by epigenetic and/or environmental factors.

Beta-mannosidase, a lysosomal enzyme which acts exclusively at the last step of oligosaccharide catabolism in glycoprotein degradation, functions to cleave the unique beta-linked mannose sugar found in all N-linked oligosaccharides of glycoproteins. Deficiency of this enzyme results in beta-mannosidosis, a lysosomal storage disease characterized by the cellular accumulation of small oligosaccharides. In human beta-mannosidosis, the clinical presentation is variable and can be mild, even when caused by functionally null mutations. In contrast, two existing ruminant animal models have disease that is consistent and severe. To further explore the molecular pathology of this disease and to investigate potential treatment strategies, we produced a beta-mannosidase knockout mouse. Homozygous mutant mice have undetectable beta-mannosidase activity. General appearance and growth of the knockout mice are similar to the wild-type littermates. At >1 year of age, these mice exhibit no dysmorphology or overt neurological problems. The mutant animals have consistent cytoplasmic vacuolation in the central nervous system and minimal vacuolation in most visceral organs. Thin-layer chromatography demonstrated an accumulation of disaccharide in epididymis and brain. This mouse model closely resembles human beta-mannosidosis and provides a useful tool for studying the phenotypic variation in different species and will facilitate the study of potential therapies for lysosomal storage diseases.

Beta-mannosidase deficiency results in beta-mannosidosis, a severe neurodegenerative lysosomal storage disease identified in cattle, goats, and humans. To more fully understand the molecular pathology of this disease, the mutation associated with bovine beta-mannosidosis was identified by sequence analysis of cDNA from an affected calf. A transition mutation of G to A at position 2574 of the cDNA coding sequence creates a premature stop codon near the 3' end of the protein coding region. To aid commercial breeders of Salers cattle, a PCR-based test was developed to detect the mutation for beta-mannosidosis carrier screening. Application of this test also revealed the presence of two beta-mannosidase pseudogenes. Portions of the pseudogenes were amplified with allele-specific primers and then sequenced. One pseudogene was highly homologous (99% sequence identity) to the expressed cDNA sequence over the 1292 bp that were sequenced, while the other showed more divergence (83% sequence identity) in the 477 bp that were sequenced. Both are processed pseudogenes that are not expressed. The severity of the bovine beta-mannosidosis phenotype suggests that the 22 C-terminal amino acids of beta-mannosidase play an important role in the function of this enzyme.

β-Mannosidosis is a lysosomal storage disorder caused by deficiency of β-mannosidase. Thirteen families with cases of β-mannosidosis have been described including one case previously reported by our group. We present clinical and biochemical data in a new adult case, and the molecular analyses in both this new case and the one previously reported. We detected four novel mutations: p.R182W, p.G392E, p.W466X and c.1848delA. Discrepancies between genomic DNA and cDNA results when detecting this last deletion suggested a nonsense-mediated decay cell process (NMD).

Background β-Mannosidosis (OMIM 248510) is a rare inborn lysosomal storage disorder caused by the deficient activity of β-mannosidase, an enzyme encoded by a single gene (MANBA) located on chromosome 4q22-25. To date, only 20 cases of this autosomal recessive disorder have been described and 14 different MANBA mutations were incriminated in the disease. These are all null mutations or missense mutations that abolish β-mannosidase activity. In this study, we characterized the molecular defect of a new case of β-mannosidosis, presenting with a severe neurological disorder. Methods Genomic DNA was isolated from peripheral blood leukocytes of the patient to allow MANBA sequencing. The identified mutation was engineered by site-directed mutagenesis and the mutant protein was expressed through transient transfection in HEK293T cells. The β-mannosidase expression and activity were respectively assessed by Western blot and fluorometric assay in both leukocytes and HEK293T cells. Results A missense disease-associated mutation, c.1922G>A (p.Arg641His), was identified for which the patient was homozygous. In contrast to previously described missense mutations, this substitution does not totally abrogate the enzyme activity but led to a residual activity of about 7% in the patient's leukocytes, 11% in lymphoblasts and 14% in plasma. Expression studies in transfected cells also resulted in 7% residual activity. Conclusion Correlations between MANBA mutations, residual activity of β-mannosidase and the severity of the ensuing neurological disorder are discussed. Whether the c.1922G>A mutation is responsible for a yet undescribed pseudodeficiency of β-mannosidase is also discussed.

International audience; Beta-mannosidosis (OMIM 248510) is an inborn lysosomal storage disorder caused by deficiency of beta-mannosidase activity. This enzyme is encoded by a single gene (MANBA), located on chromosome 4q22-25. This autosomal recessive disorder is characterized by a wide range of symptoms including mental retardation, behavioural problems, hearing loss, recurrent respiratory infections, angiokeratoma, facial dysmorphism, skeletal deformation, seizures, hypotonia, demyelinating polyneuropathy, and hepatosplenomegaly. The age of symptom onset is variable. We describe a 14-year clinical follow-up of a patient with beta-mannosidase deficiency with symptoms of mental retardation, progressive spasticity and cerebellar ataxia, a clinical spectrum that so far has never been reported in beta-mannosidosis. A novel mutation in the MANBA gene was found in our patient. Evoked potentials were in favour of a demyelinating pathology of the central nervous system. Serial MRI showed generalized cortical and subcortical atrophy in the absence of white matter changes suggesting an additional axonal pathophysiological component.

A storage disease characterised by widespread vacuolation of cells throughout the body was diagnosed in a newborn calf of the Salers breed. Extraction and analyses of water soluble material showed that the storage material was oligosaccharide in nature and was probably derived from the core region of the heterosaccharide moiety of glycoproteins. Glycosidase analyses showed that the disease was due to a deficiency of acidic beta-mannosidase.

In caprine beta-mannosidosis, an inherited dysmyelinating disorder, the myelin deficit shows substantial variation throughout the nervous system. In this study morphometric analysis of optic nerve and corpus callosum sections at selected developmental stages was conducted in order to investigate development and persistence of myelin sheaths, the population of axons ensheathed, and the extent of myelin deficits and glial cell abnormalities. The results show that the myelin deficit is severe at very early stages of development and persists to about the same extent into postnatal life. The corpus callosum, much more severely involved than the optic nerve, contains a substantially smaller percentage of myelinated axons when compared to control. In both regions, larger axons are preferentially myelinated. In the corpus callosum before myelination begins, many glial cells appear abnormal, suggesting an early cellular defect. In the postnatal, myelin-deficient corpus callosum, there is a substantial decrease in glial cell density as compared to control, with abnormal appearance of many of the remaining cell profiles. These results define developmental characteristics of the dysmyelination in caprine beta-mannosidosis and document both the early appearance and the persistence of glial cell body and myelin abnormalities.

Background Human β-mannosidosis, a rare disorder of oligosaccharide catabolism, results from a deficiency of β-mannosidase activity. So far, mutational analysis has been performed in only seven families and revealed 11 mutations in the MANBA gene which encodes the enzyme β-mannosidase. Objectives We report here a 36-year-old Arab female with β-mannosidosis who presented with mental retardation and multiple angiokeratomas. We describe in this patient a novel null mutation and review the previously reported MANBA gene mutations and their clinical correlations. Methods Histopathology, ultrastructural analysis, and enzyme assays were performed. Sequencing of cDNA and genomic DNA analysis was conducted in a search for a mutation in the MANBA gene. Results Histopathology of a skin biopsy specimen from the patient showed the characteristic findings of angiokeratoma. Electron microscopy showed cytoplasmic vacuolation. Enzymatic activity of β-mannosidase in the patient's serum, leukocytes, and fibroblasts was less than 1% of control values. Sequencing of the MANBA cDNA revealed a G→A transition in exon 6 at nucleotide position c.693, resulting in the formation of a stop codon (W231X). Limitations Only one family was studied. Conclusions A new case of human β-mannosidosis is presented and the first MANBA gene mutation from Arab ancestry is reported. Reviewing the reported MANBA gene mutations does not reveal a clear genotype–phenotype correlation. The importance of angiokeratoma corporis diffusum as the clue to the diagnosis of β-mannosidosis and other lysosomal storage diseases is emphasized.

Beta-mannosidosis is a lysosomal disorder which is caused by a deficiency of beta-mannosidase. This disorder was first described in goats. Twelve human cases have already been reported. We present the first case in Japan in whom the diagnosis was reached from angiokeratoma corporis diffusum. Futhermore, mental retardation, hearing loss, and renal failure were also detected. Pseudoxanthoma elasticum was also present, but whether it is a complication of beta-mannosidosis or not remains unknown. The activity level of beta-mannosidase in the patient's plasma was only 2% of the normal range, while that in the patient's mother was 40%. We suggest that beta-mannosidosis should be one of the differential diagnoses when lysosomal enzyme disorders are suspected in cases of angiokeratoma corporis diffusum.

beta-Mannosidase and endo-beta-mannanase are involved in the mobilization of the mannan-containing cell walls of the tomato seed endosperm. The activities of both enzymes increase in a similar temporal manner in the micropylar and lateral endosperm during and following germination. This increase in enzyme activities in the micropylar endosperm is not markedly reduced in seeds imbibed in abscisic acid although, in the lateral endosperm, endo-beta-mannanase activity is more suppressed by this inhibitor than is the activity of beta-mannosidase. Gibberellin-deficient (gib-1) mutants of tomato do not germinate unless imbibed in gibberellin; low beta-mannosidase activity, and no endo-beta-mannanase activity is present in seeds imbibed in water, but both enzymes increase strongly in activity in the seeds imbibed in the growth regulator. For production of full activity of both beta-mannosidase and endo-beta-mannanase in the endosperm, this tissue must be in contact with the embryo for at least the first 6 h of imbibition, which is indicative of a stimulus diffusing from the embryo to the endosperm during this time. These results suggest some correlation between the activities of beta-mannosidase and endo-beta-mannanase, particularly in the micropylar endosperm, in populations of tomato seeds imbibed in water, abscisic acid and gibberellin. However, when individual micropylar endosperm parts are used to examine the effect of the growth regulators and of imbibition in water on the production of the two enzymes, it is apparent that within these individual seed parts there may be large differences in the amount of enzyme activity present. Micropylar endosperms with high endo-beta-mannanase activity do not necessarily have high beta-mannosidase activity, and vice versa, which is indicative of a lack of co-ordination of the activities of these two enzymes within individuals of a population.

SummaryBackground There are seven well-known lysosomal storage diseases that produce angiokeratoma corporis diffusum clinically. β-Mannosidosis (MANB1; OMIM248510), first reported in humans in 1986, is a rare hereditary lysosomal storage disease caused by a deficiency of the enzyme β-mannosidase. Since then, 13 cases of β-mannosidase deficiency in ten families have been described. A human β-mannosidase mutation has been reported only by Alkhayat et al. in 1998. Objectives To clarify its pathogenesis we did electron microscopic, biochemical and molecular biological investigations of a Japanese patient with β-mannosidosis. Methods Ultrastructural analyses, enzyme assays, cell culture and mRNA and genomic DNA were sequenced to find mutations in the β-mannosidase gene. Results Electron microscopy of skin biopsy specimens from the patient showed cytoplasmic vacuolation of lysosomes in blood and lymph vessels, endothelial cells, fibroblasts, secretory portions of eccrine sweat glands, neural cells and basal keratinocytes in the epidermis. This vacuolation was also observed in cultured keratinocytes and fibroblasts. Assays of seven enzyme activities in plasma and cultured skin fibroblasts showed a marked decrease of β-mannosidase activity. Sequencing the β-mannosidase cDNA revealed a four-base (ATAA) insertion between exons 7 and 8, resulting in a frameshift at codon 321 and termination at codon 325. Analysis of the patient's genomic DNA revealed a novel homozygous A(+1)G splice site mutation in intron 7. Conclusions To our knowledge, this is the first case of β-mannosidosis reported in Japan and the second report in which a gene mutation is identified. The biological importance of β-mannose moieties in glycoproteins in basal keratinocytes is suggested.

In this study the clinical signs and pathology of five plant poisonings and a mycotoxicosis affecting the nervous system of domestic ruminants in southern Africa are described. For comparative purposes, an inherited storage disease (bèta-mannosidosis) and a drug-induced neurotoxicosis (closantel overdose) are also presented. The aims of this thesis were: (1) to document the pathology of those conditions where detailed descriptions were lacking, (2) to study the light and transmission electron microscopical lesions from a differential diagnostic perspective, and, (3) to consider the possible causative mechanisms of the nervous lesions. A novel lysosomal storage disease in goats induced by the plant Ipomoea carnea is reported. Analysis of plant material confirmed the presence of swainsonine and two glycosidase inhibitors, namely, calystegine B2 and calystegine C1. The storage disease is analogous to the lysosomal storage disease in ruminants induced by ingestion of locoweeds (Astragalus and Oxytropis spp.) and poison peas (Swainsona spp) and closely mimics inherited a-mannosidosis in animals. ?-Mannosidosis is documented in Hereford calves, a breed not previously known to be afflicted by this inherited disorder. The condition was biochemically confirmed by an absence of lymphocyte ?-mannosidase activity in one of the affected calves. ‘Maldronksiekte’ in cattle, caused by ingestion of the plant Solanum kwebense, is characterised by degeneration, necrosis and loss of cerebellar Purkinje cells and, to a lesser extent, neurons in cerebellar nuclei. Affected Purkinje neurons were either swollen, pale and finely vacuolated or shrunken and more eosinophilic than normal. In the present study, some Purkinje cells and neurons in cerebellar nuclei stained strongly with Canavalia ensiformis (ConA) agglutinin and weakly with Triticum vulgaris (WGA) and succinyl-WGA (S-WGA) agglutinins. The neuronal lesions were not caused by apoptosis as tested by a commercial deoxyuridine triphosphate nick-end labeling (TUNEL) method. Based on the selective involvement and nature of lesions in cerebellar Purkinje cells, ‘maldronksiekte’ is classified as an acquired cerebellar cortical degeneration and not a lysosomal storage disease. Four toxic myelinopathies are described; three are induced by plants (Helichrysum argyrosphaerum, Ornithogalum prasinum and O. saundersiae and Crotalaria sparthioides) and one is caused by overdosage of the anthelmintic compound closantel. The four conditions are characterized by indistinguishable spongy changes (status spongiosis) in the central nervous system caused by splitting of myelin lamellae at the intraperiod line consistent with myelin oedema. The brain lesions in toxicity with Crotalaria sparthioides in cattle are typical for hepatic encephalopathy secondary to cirrhosis. The pyrrolizidine alkaloid senkirkine was isolated from toxic plant material. Primary toxic retinal degeneration was present in poisoning by Helichrysum argyrosphaerum and in closantel poisoning. Finally, the pathology of acute and chronic Aspergillus clavatus intoxication in cattle is studied. In acute cases there is chromatolysis and cytoplasmic vacuolation particularly of motor neurons in the ventral horns throughout the spinal cord. Electron microscopy revealed disintegration of Nissl bodies, abundant free ribosomes and increased numbers of mitochondria and dense bodies comparable to the axonal reaction. In chronic cases fed sorghum beer residues, extensive, bilaterally symmetrical Wallerian degeneration in ascending and descending tracts throughout the length of the spinal cord were seen. This neuromycotoxicosis is classified as a neuronopathy/axonopathy. A histological feature of all the conditions that are described in this thesis is the presence of vacuolation of varying severity in neurons and/or neuropil in the central nervous system of affected animals. Apart from the storage diseases, the pathogenesis of the nervous lesions is largely unknown.

In this study the clinical signs and pathology of five plant poisonings and a mycotoxicosis affecting the nervous system of domestic ruminants in southern Africa are described. For comparative purposes, an inherited storage disease (bèta-mannosidosis) and a drug-induced neurotoxicosis (closantel overdose) are also presented. The aims of this thesis were: (1) to document the pathology of those conditions where detailed descriptions were lacking, (2) to study the light and transmission electron microscopical lesions from a differential diagnostic perspective, and, (3) to consider the possible causative mechanisms of the nervous lesions. A novel lysosomal storage disease in goats induced by the plant Ipomoea carnea is reported. Analysis of plant material confirmed the presence of swainsonine and two glycosidase inhibitors, namely, calystegine B2 and calystegine C1. The storage disease is analogous to the lysosomal storage disease in ruminants induced by ingestion of locoweeds (Astragalus and Oxytropis spp.) and poison peas (Swainsona spp) and closely mimics inherited a-mannosidosis in animals. ?-Mannosidosis is documented in Hereford calves, a breed not previously known to be afflicted by this inherited disorder. The condition was biochemically confirmed by an absence of lymphocyte ?-mannosidase activity in one of the affected calves. ‘Maldronksiekte’ in cattle, caused by ingestion of the plant Solanum kwebense, is characterised by degeneration, necrosis and loss of cerebellar Purkinje cells and, to a lesser extent, neurons in cerebellar nuclei. Affected Purkinje neurons were either swollen, pale and finely vacuolated or shrunken and more eosinophilic than normal. In the present study, some Purkinje cells and neurons in cerebellar nuclei stained strongly with Canavalia ensiformis (ConA) agglutinin and weakly with Triticum vulgaris (WGA) and succinyl-WGA (S-WGA) agglutinins. The neuronal lesions were not caused by apoptosis as tested by a commercial deoxyuridine triphosphate nick-end labeling (TUNEL) method. Based on the selective involvement and nature of lesions in cerebellar Purkinje cells, ‘maldronksiekte’ is classified as an acquired cerebellar cortical degeneration and not a lysosomal storage disease. Four toxic myelinopathies are described; three are induced by plants (Helichrysum argyrosphaerum, Ornithogalum prasinum and O. saundersiae and Crotalaria sparthioides) and one is caused by overdosage of the anthelmintic compound closantel. The four conditions are characterized by indistinguishable spongy changes (status spongiosis) in the central nervous system caused by splitting of myelin lamellae at the intraperiod line consistent with myelin oedema. The brain lesions in toxicity with Crotalaria sparthioides in cattle are typical for hepatic encephalopathy secondary to cirrhosis. The pyrrolizidine alkaloid senkirkine was isolated from toxic plant material. Primary toxic retinal degeneration was present in poisoning by Helichrysum argyrosphaerum and in closantel poisoning. Finally, the pathology of acute and chronic Aspergillus clavatus intoxication in cattle is studied. In acute cases there is chromatolysis and cytoplasmic vacuolation particularly of motor neurons in the ventral horns throughout the spinal cord. Electron microscopy revealed disintegration of Nissl bodies, abundant free ribosomes and increased numbers of mitochondria and dense bodies comparable to the axonal reaction. In chronic cases fed sorghum beer residues, extensive, bilaterally symmetrical Wallerian degeneration in ascending and descending tracts throughout the length of the spinal cord were seen. This neuromycotoxicosis is classified as a neuronopathy/axonopathy. A histological feature of all the conditions that are described in this thesis is the presence of vacuolation of varying severity in neurons and/or neuropil in the central nervous system of affected animals. Apart from the storage diseases, the pathogenesis of the nervous lesions is largely unknown.

References 3. Lindsay DS, Dubey JP: 1989, Immunohistochemical diagnosis of Neospora caninum in tissue section. Am J Vet Res 50:19811. Dubey JP, Beattie CP: 1988, Toxoplasmosis of animals and man. 1983. CRC Press, Boca Raton, FL. 220 pp. 4. Munday BL, Mason RW, Hartley WJ, Presidente PJA, Obendorf 2. Dubey JP, Speer CA, Fayer R: 1989, Sarcocystosis of animals D: 1978, Sarcocystis and related organisms in Australian wildand man. CRC Press, Boca Raton, FL. 215 pp. life: 1. Survey findings in mammals. J Wild1 Dis 14:417-433.

The phenotype of bovine beta-mannosidosis (beta-mannosidase deficiency), recently identified in Salers cattle, is similar to the caprine form of the disease (Abbitt et al., 1991). This investigation was designed to characterize accumulated kidney oligosaccharides in bovine beta-mannosidosis. Oligosaccharides were extracted from the kidney of an affected Salers calf and purified by chromatographic techniques. The amount of accumulating oligosaccharides in 1 g of wet tissue was about 21 mumol. Structures of derivatized oligosaccharides were characterized by high-performance liquid chromatography, mass spectrometry, methylation analysis and sequential exoglycosidase digestions. The major accumulating oligosaccharides were Man beta 1-4GlcNAc and Man beta 1-4GlcNAc beta 1-4GlcNAc. Oligosaccharides accumulating in minor amounts were Man beta 1-4GlcNAc beta 1-4Man beta 1-4GlcNAc, Man alpha 1-6Man beta 1-4GlcNAc beta 1-4GlcNAc and Man beta 1-4GlcNAc beta 1-4Man beta 1-4GlcNAc beta 1-4GlcNAc. As in caprine beta-mannosidosis, oligosaccharides with terminal beta-mannose residues and cleaved as well as uncleaved chitobiose linkages were identified in bovine beta-mannosidosis kidney. The accumulating oligosaccharides in tissue were thus identical in bovine and caprine beta-mannosidosis; however, the source of the novel oligosaccharides remains to be determined.

Central nervous system myelin deficiency is a consistent feature of caprine beta-mannosidosis, an autosomal recessive neurovisceral lysosomal storage disease. To investigate the possibility of an intrinsic oligodendrocyte defect in beta-mannosidosis, oligodendrocyte-enriched glial cultures from the cerebral hemisphere white matter of two affected and six control goats were compared with respect to culture yield and morphology. Fewer oligodendrocytes were cultured per gram of white matter from affected animals than from control animals. Galactocerebroside-positive oligodendrocytes from all animals were similar morphologically at all stages of culture by phase contrast and fluorescence microscopy. These findings are consistent with in vivo morphological observations and suggest that differentiated oligodendrocytes from affected animals do not show morphological abnormalities in culture. However, increased numbers of galactocerebroside-negative bipolar cells, which may be glial progenitor cells, were present in cultures from affected animals. This observation suggests the possibility of a defect in differentiation to mature oligodendrocytes, with persistence of the undifferentiated glia during late stages of development.

Sir: [3-mannosidosis is a lysosomal storage disorder caused by deficiency of the enzyme ~-mannosidase. We would like to report a girl (parents were first cousins), who besides a [3-mannosidase deficiency had an ethanolaminuria. She was born at term. Birth length and weight are unknown. Her development during the 1st year was unremarkable. She walked at 15 months. In that period the parents became worried about speech development. Because of recurrent upper respiratory tract infections the adenoids were removed and tympanotubes inserted. Otological examination revealed a hearing-loss of 45 DB. She was admitted to our clinic because of absent speech development and anorexia. Physical examination at the age of 5 years 10 months showed a slightly built girl: height and weight below 3rd percentile, head circumference 10th percentile. Motor and mental development were seriously retarded. There were no joint contractures, but she stood with slightly flexed hips and knees. No hepatosplenomegaly was present. Electroencephalography demonstrated a non-specific abnormality without c~production. A CT-scan of the brain was normal. Oligosaccharides in urine, analysed according to Blom et al. [1] and Dorland et al. [3], showed a strong band which could be identified as the 13-mannosidosis disaccharide mannosyl-13(1-4)-N-acetylglucosamine. ~-Mannosidase activity was measured with minor modifications of the method described by Panday et al. [4]. The activity of 13-mannosidase in leucocytes was 4.7nmol/h per milligram protein (normal: 160-360). In plasma and cultured fibroblasts the activity of 13-mannosidase was not detectable (normal respectively: 160-300 nmol/h per milliliter, 115-455nmol/h per milligram protein). Heparan sulphamidase was normal. Furthermore we found an ethanolaminuria: 607-1010gmol/mmol creatinine (normal 35-130). The ethanolamine concentration in the serum was also elevated ranging from 38-70 gmol/1 (normal: traces). All hitherto described patients with [3-mannosidosis had mental retardation. Most of them had hearing loss with delayed development of expressive language. Striking is their susceptibility to respiratory and dermal infections. There is no uniform facial or skeletal dysmorphism. The significance or ethanolaminuria in our patient is not known. Wenger et al. [6] described a combined deficiency of 13-mannosidase and heparan sulphamidase. Vietor et al. [5] reported two sibs with ethanolaminuria and generalized storage disease, cardiomegaly, cerebral dysfunction and early death. It was argued that a marked increase in ethanolamine excretion could result from a deficiency of kinase enzyme involved in the synthesis of membrane phospholipid phosphatidylethanolamine. As suggested by Cole et al. [2] ethanolamine may result from accelerated breakdown of ethanolamine phospholipids (e.g. from myelin) without compensatory re-utilization in the synthesis of new lipids. However, there were no clinical signs of myelin disease in our patient. References

Beta-mannosidosis, an inherited defect of glycoprotein catabolism associated with deficiency of tissue beta-mannosidase and accumulation of Man(beta 1-4)GlcNAc and Man(beta 1-4)GlcNAc(beta 1-4)GlcNAc, appeared in four of 13 offspring of a single pair of clinically normal, related Nubian goats. Neurological examinations revealed that all four affected goats were unable to rise or walk. All had facial dysmorphism, dome-shaped skulls, small palpebral fissures, carpal contractures, hyperextension of the pastern joints, proximal muscle atrophy, intermittent ocular oscillations resembling pendular nystagmus, marked intention tremor, and deafness. With intensive care, three affected kids were hand-reared and then killed at 1, 7, and 21 days of age. Macroscopically, there were paucity of myelin in the cerebral and cerebellar hemispheres and ventricular dilatation. Microscopically, the extent and distribution of cytoplasmic vacuolation, myelin paucity, axonal spheroids, and filamentous expansions were evaluated in the cerebrum, cerebellum, brainstem, spinal cord, and peripheral nerves of the four affected kids and two age-matched, clinically normal kids. Widespread cytoplasmic vacuolation correlated with the previously reported accumulation of oligosaccharides in the brain and kidney and the deficiency of tissue beta-mannosidase. beta-Mannosidosis, not yet identified in man or other species, is characterized by distinctive neonatal clinical, pathological and biochemical features which differentiate it from the alpha-mannosidosis and other inherited diseases of glycoprotein catabolism.

Four oligosaccharide fractions were isolated and purified from the kidney of goats affected with beta-mannosidosis by repeating Bio-Gel P-2 column chromatography. The structural characterization of the purified oligosaccharide fractions (oligosaccharides A, B, C1,2, and D) included sugar composition analysis by gas chromatography, sugar sequence analysis by mass spectrometry of their permethylated alditols, and by methylation analysis as well as anomeric configuration studies by exoglycosidase digestions. Oligosaccharides A and B were the major oligosaccharides accumulating in the kidney and were elucidated as Man beta 1-4GlcNAc and Man beta 1-4GlcNAc beta 1-4GlcNAc, respectively (Matsuura, F., Laine, R. A., and Jones, M. Z. (1981) Arch. Biochem. Biophys. 211, 485-493). Oligosaccharide C1,2 was a mixture of two tetrasaccharides and oligosaccharide D was a pentasaccharide. The proposed structures are: oligosaccharide C1, Man beta 1-4GlcNAc beta 1-4Man beta 1-4GlcNAc; oligosaccharide C2, Man alpha 1-6Man beta 1-4GlcNAc beta 1-4GlcNAc; oligosaccharide D, Man beta 1-4GlcNAc beta 1-4Man beta 1-4GlcNAc beta 1-4GlcNAc. Tetrasaccharide C1 and pentasaccharide D are heretofore undiscovered oligosaccharides. There is no precedent for these structures in glycoproteins or other glycoconjugates. One possibility which accounts for the presence of oligosaccharide C1 and D is that a bisecting N-acetylglucosamine (the beta-N-acetylglucosamine residue linked at the C-4 position of the beta-mannosyl residue of the trimannosyl core of the asparagine-linked sugar chains) is linked by a beta-mannosyl residue. Moreover, the detection of oligosaccharides containing two N-acetylglucosamine residues at the reducing terminus, together with those containing a single N-acetylglucosamine residue, is further corroboration of species-specific differences in glycoprotein catabolic pathways (Hancock, L. W., and Dawson, G. (1984) Fed. Proc. 43, 1552) or in glycoprotein structures.

A rapidly fatal neurovisceral storage disease was discovered in both male and female offspring of clinically normal Nubian goats. Microscopic examination of fixed tissues revealed extensive demyelination and ubiquitous distribution of lysosomal storage vacuoles containing dispersed floccular material and membranous fragments. Urine was found to contain elevated levels of both mannose and N-acetylglucosamine, suggestive of an oligosaccharide storage disease. Brain was found to contain 2.2 mumol/g of a trisaccharide Man(beta 1-4)GlcNAc(beta 1-4)GlcNAc; (Jones, M. Z., and Laine, R. A. (1980) Fed. Proc. 39, 2521 and Jones, M. Z., and Laine, R. A. (1981) J. Biol. Chem. 256, 5181-5184). A profound deficiency of beta-D-mannosidase activity was found in a number of tissues from affected goats; obligate heterozygotes showed a partial enzyme deficiency. Other lysosomal hydrolase activities were normal or elevated over normal, including alpha-D-mannosidase, confirming that this was a hitherto undescribed inborn error of glycoprotein catabolism.

Both p-nitrophenyl and 4-methylumbelliferyl β-man-nopyranoside are suitable substrates for estimation of β-mannosidase activity in the plasma of goats. Assays using the latter substrate were more sensitive than those using p-nitrophenyl β-D-mannopyrano-side, but the values obtained with both substrates were similar. Application of the p-nitrophenyl β- mannopyranoside assay to Anglo Nubian goats showed that kids affected with β-mannosidosis had the lowest β-mannosidase activity and that their parents had lower activities than most goats.

The clinical features of caprine beta-mannosidosis were evaluated in 10 newborn goats, one stillborn goat and one goat fetus. The phenotypic abnormalities observed in all 10 live affected animals included an inability to rise from a recumbent position, moderate to marked intention tremor, eye movements resembling pendular nystagmus, clinical deafness, bilateral Horner's syndrome, carpal contractures, pastern joint hyperextension, thickened skin and to a varying degree, a dome-shaped skull. Subjective evaluation suggested that most animals had a decreased muscle mass. Together, these characteristics represent a common phenotype which is expressed at birth in caprine beta-mannosidosis.