A lower motor neuron disease in takahe (Porphyrio hochstetteri) is an endoplasmic reticulum storage disease.

AIMS: To investigate the pathogenesis of a disease in takahe (Porphyrio hochstetteri) with intracytoplasmic inclusion bodies in lower motor neurons. METHODS: Four birds aged between 5 and 12 years, from three different wildlife sanctuaries in New Zealand were examined. Of these, only one had signs of spinal dysfunction in the form of paresis. Stained paraffin sections of tissues were examined by light microscopy and immunostained sections of the ventral horn of the spinal cord by confocal microscopy. Epoxy resin sections of the spinal cord from the bird with spinal dysfunction were examined by electron microscopy. RESULTS: Two types of inclusion bodies were noted, but only in motor neurons of the ventral spinal cord and brain stem. These were large globoid eosinophilic bodies up to 5 µm in diameter, and yellow/brown granular inclusions mostly at the pole of the cell. The globoid bodies stained with Luxol fast blue but not with periodic acid Schiff (PAS), or Sudan black. The granular inclusions stained with Luxol fast blue, PAS and Sudan black. Both bodies were slightly autofluorescent. On electron microscopy the globoid bodies had an even electron-dense texture and were bound by a membrane. Beneath the membrane were large numbers of small intraluminal vesicles. The smaller granular bodies were more heterogeneous, irregularly rounded and membrane-bound accumulations of granular electron-dense material, often with electron-lucent vacuoles. Others were more vesicular but contained varying amounts of electron-dense material. The large globoid bodies did not immunostain for lysosomal markers lysosomal associated protein 1 (LAMP1) or cathepsin D, so were not lysosomal. The small granular bodies stained for cathepsin D by a chromogenic method.A kindred matrix analysis showed two cases to be as closely related as first cousins, and another case was almost as closely related to one of them, but the fourth bird was unrelated to any other. CONCLUSIONS: It was concluded that this was an endoplasmic reticulum storage disease due to a specific protein misfolding within endoplasmic reticulum. It was rationalised that the two types of inclusions reflected the same aetiology, but that misfolded protein in the smaller granular bodies had entered the lysosomal system via endoplasmic reticulum autophagy. Although the cause was unclear, it most likely had a genetic aetiology or predisposition and, as such, has clinical relevance.

Animal medical genetics: a historical perspective on more than 50 years of research into genetic disorders of animals at Massey University.

Over the last 50 years, there have been major advances in knowledge and technology regarding genetic diseases, and the subsequent ability to control them in a cost-effective manner. This review traces these advances through research into genetic diseases of animals at Massey University (Palmerston North, NZ), and briefly discusses the disorders investigated during that time, with additional detail for disorders of major importance such as bovine α-mannosidosis, ovine ceroid-lipofuscinosis, canine mucopolysaccharidosis IIIA and feline hyperchylomicronaemia. The overall research has made a significant contribution to veterinary medicine, has provided new biological knowledge and advanced our understanding of similar disorders in human patients, including testing various specific therapies prior to human clinical trials.

Familial episodic ataxia in lambs is potentially associated with a mutation in the fibroblast growth factor 14 (FGF14) gene.

Familial episodic ataxia of lambs is a congenital transient autosomal dominant disorder of newborn lambs, with varying expressivity. Affected lambs show episodes of an asymmetric ataxic gait, base-wide extensor hypertonia of the thoracic limbs and flexor hypertonia of the pelvic limbs. The aim of the study was to determine the genetic variant causing familial episodic ataxia in lambs. Using whole genome sequencing of two half-sib affected lambs, their sire, and their two normal dams, a heterozygous C>T transition at OAR10:77593415 (Oar_v3.1) in exon 1 of the fibroblast growth factor 14 (FGF14) gene (c.46C>T) was identified. The c.46C>T transition resulted in a premature stop codon at position 16 of the 247 amino acid FGF14 protein (p.Q16*). PCR and Sanger sequencing was used to genotype an additional 20 clinically affected animals, demonstrating all lambs carried the c.46C>T variant but 1 clinically more severely affected inbred lamb was homozygous (TT). A further 11 unrelated normal ewes were positionally sequenced, none of which had the variant, while in 18 lambs of unknown status born over 2 years of breeding trials six lambs were found to have the c.46C>T variant, likely clinically unidentified heterozygotes due to the variable expressivity, while 12 did not. In conclusion, familial episodic ataxia of lambs is potentially associated with a c.46C>T variant in the FGF14 gene. Further research is required into the mechanism behind the apparent recovery of lambs.

Animal medical genetics: a perspective on the epidemiology and control of inherited disorders.

This perspective considers genetic disorders of domestic animal populations, in particular their epidemiology and control. Inherited disorders of animals share the same basic molecular biology as those of human beings, but they differ in their epidemiology due largely to the breed structure of the various species, human control of breeding and a greater influence of the founder effect, particularly due to extensive use of a limited number of sires, and inbreeding. Control of genetic disorders in animals is also more practical through extensive screening for disease, or heterozygous animals within defined breed populations, followed by exclusion of affected or carrier animals from breeding. This is assisted by the fact that, within a breed, many inherited monogenic disorders are associated with a single mutation. However some of the more important disorders may be inherited in a non-Mendelian manner, being influenced by multiple genes as well as environmental factors. These aspects are discussed and contrasted with similar aspects in human medical genetics.

Traumatic brain injury, axonal injury and shaking in New Zealand sea lion pups.

Trauma is a common cause of death in neonatal New Zealand sea lion pups, and subadult male sea lions have been observed picking up and violently shaking some pups. In humans, axonal injury is a common result of traumatic brain injury, and can be due to direct trauma to axons or to ischaemic damage secondary to trauma. 'Shaken baby syndrome', which has been described in human infants, is characterised by retinal and intracranial subdural haemorrhages, and has been associated with axonal injury to the brain, spinal cord and optic nerve. This study identifies mechanisms of traumatic brain injury in New Zealand sea lion pups, including impact injuries and shaking-type injuries, and identifies gross lesions of head trauma in 22/36 sea lion pups found dead at a breeding site in the Auckland Islands. Despite the high frequency of such gross lesions, only three of the pups had died of traumatic brain injury. Observational studies confirmed that shaking of pups occurred, but none were shown to die as a direct result of these shaking events. Axonal injury was evaluated in all 36 pup brains using ß-amyloid precursor protein immunohistochemistry. Immunoreactive axons were present in the brains of all pups examined including seven with vascular axonal injury and two with diffuse axonal injury, but the severity and pattern of injury was not reliably associated with death due to traumatic brain injury. No dead pups had the typical combination of gross lesions and immunohistochemical findings that would conform to descriptions of 'shaken baby syndrome'. Axonal injury was present in the optic nerves of most pups, irrespective of cause of death, but was associated with ischaemia rather than trauma.

Familial episodic ataxia in lambs.

HISTORY: A similar episodic neurological disorder occurred in new born lambs on two unrelated properties involving disparate breeds of sheep. Because of the number of lambs born, cross-breeding and the fact it occurred in some mating groups and not others, a dominant mode of inheritance was, initially and separately, suspected in each case. The sires of affected lambs were apparently normal. Whereas one was New Zealand Romney, the other was a composite breed with East Friesian genetics, but both rams originated from the same source property. To investigate the pathogenesis of the disorder these two rams were acquired and mated with unrelated sheep, under experimental conditions in a more controlled environment. CLINICAL FINDINGS: A proportion of lambs born to both sires exhibited a similar neurological disorder. Some lambs were noted to be abnormal at birth, both on home properties and in the experimental flock. They tended to adopt a head and neck extended posture and were slow to get to their feet and suckle when they then became more or less normal. When forced to move, they and other more robust lambs elicited an asymmetric gait, base-wide extensor hypertonia (hypometria) of thoracic limbs and flexor hypertonia (hypermetria) of pelvic limbs. In some there was nystagmus. After several metres of asymmetric ataxic gait they would fall to one side, sometimes adopting a sitting position. Recovery usually occurred in one to several minutes. As lambs aged, it became more difficult to elicit the episodes of dysfunction and by 6 months of age they appeared normal. DIAGNOSIS: The disorder was diagnosed as a dominant familial episodic cerebellovestibular ataxia inherited as a dominant trait, with incomplete penetration of observed clinical signs and variable expressivity. CLINICAL RELEVANCE: A proportion of affected lambs are likely to die in the neonatal period so the specific nature of the disorder may go unrecognised. Because of incomplete penetrance and varying expressivity, many of the lambs carrying this mutation will survive without showing clinical signs and may enter breeding flocks, where the disorder may be perpetuated and contribute to neonatal deaths.

Intracisternal enzyme replacement therapy in lysosomal storage diseases: dispersal pathways, regional enzyme concentrations and the effect of posttreatment posture.

AIMS: To investigate routes of dispersal of enzyme, its regional uptake and the effect of posture when replacement enzyme is administered directly into the cerebrospinal fluid (CSF). METHODS: Dispersal pathways of particles and solutes were investigated using intracisternal injections of india ink with visual assessment, and a contrast medium (Iohexol) with computer tomography (CT). Replacement enzyme was measured at 46 loci within the central nervous system (CNS) in four groups of dogs subjected to different post-injection postural changes. RESULTS: India ink and CT studies showed dispersal pathways for CSF to be mainly via cisterns and sulci. Replacement enzyme reached all areas of the CNS tested, although mean concentrations varied 49-fold over different areas of the brain. Posttreatment posture had only modest effects on enzyme uptake in limited anatomical sites. CONCLUSIONS: Dispersal of solutes after injection is rapid and initially enhanced by the injection process. Preferential pathways for CSF flow in the subarachnoid spaces of the brain involve cisterns and sulci. The splenial and suprasplenial sulci in particular appear important conduits for dispersal to more dorsal and rostral areas of the brain. Expansion and contraction of these sulci during brain pulsation is considered important to the forward flow of solutes in CSF through these compartments. Following intracisternal enzyme replacement therapy, enzyme reached all areas of the brain, but there was considerable disparity of enzyme uptake with some areas recording much higher levels than others. Posttreatment posture made only modest differences to enzyme uptake.

Mucopolysaccharidosis type VI in a Miniature Poodle-type dog caused by a deletion in the arylsulphatase B gene.

AIM: To investigate and characterise an inborn error of metabolism in a dog with skeletal and ocular abnormalities. METHODS: A 2.5-year-old small male Miniature Poodle-like dog was presented with gross joint laxity and bilateral corneal opacities. Clinical examination was augmented by routine haematology, serum chemistry, radiographs, pathology, enzymology and molecular genetic studies. Euthanasia was requested when the dog was 3 years of age because of progressively decreasing quality of life. RESULTS: Radiology revealed generalised epiphyseal dysplasia, malformed vertebral bodies, luxation/subluxation of appendicular and lumbosacral joints with hypoplasia of the odontoid process and hyoid apparatus. These clinical and radiographic findings, together with a positive urinary Berry spot test for mucopolysaccharides, and metachromatic granules in leucocytes, were indicative of a mucopolysaccharidosis (MPS), a lysosomal storage disease. Histological lesions included vacuolation of stromal cells of the cornea, fibroblasts, chondrocytes, macrophages and renal cells. The brain was essentially normal except for moderate secondary Wallerian-type degeneration in motor and sensory tracts of the hind brain. Dermatan sulphate-uria was present and enzymology revealed negligible activity of N-acetylgalactosamine-4-sulphatase, also known as arylsulphatase B, in cultured fibroblasts and liver tissue. A novel homozygous 22 base pair (bp) deletion in exon 1 of this enzyme's gene was identified (c.103_124del), which caused aframe-shift and subsequent premature stop codon. The "Wisdom pure breed-mixed breed" test reported the dog as a cross between a Miniature and Toy Poodle. CONCLUSIONS: The clinicopathological features are similar to those of MPS type VI as previously described in dogs, cats and other species, and this clinical diagnosis was confirmed by enzymology and molecular genetic studies. This is an autosomal recessively inherited lysosomal storage disease. CLINICAL RELEVANCE: The prevalence of MPS VI in Miniature or Toy Poodles in New Zealand and elsewhere is currently unknown. Due to the congenital nature of the disorder, malformed pups may be subject to euthanasia without investigation and the potential genetic problem in the breed may not be fully recognised. The establishment of a molecular genetic test now permits screening for this mutation as a basis to an informed breeding policy.

Intracisternal enzyme replacement therapy in lysosomal storage diseases: routes of absorption into brain.

AIMS: The research concerns enzyme replacement therapy in lysosomal storage diseases with central nervous system involvement. The principle aim was to understand the routes of entry of enzyme into the brain when delivered directly into the cerebrospinal fluid (CSF) via the cerebellomedullary cistern. METHODS: Pathways for absorption of replacement enzyme were investigated in dogs with mucopolysaccharidosis IIIA (MPSIIIA) following intracisternal injections of human recombinant N-sulphoglucosamine sulphohydrolase (rhSGSH, EC3.10.1.1) by light and confocal microscopy using chromogenic and fluorescent immune probes. RESULTS: Enzyme entered the brain superficially by penetration of the pia/glia limitans interface, but the main route was perivascular along large veins, arteries and arterioles extending onto capillaries. It further dispersed into surrounding neuropil to be taken up by neurones, macrophages, astrocytes and oligodendroglia. Enzyme also entered the lateral ventricles adjacent to the choroid plexus, probably also by the tela choroidea and medullary velum, with further spread throughout the ventricular system and spinal canal. There was secondary spread back across the ependyma into nervous tissue of brain and spinal cord. CONCLUSIONS: Enzyme mainly enters the brain by a perivascular route involving both arteries and veins with subsequent spread within the neuropil from where it is taken up by a proportion of neurones and other cells. Penetration of enzyme through the pia/glia limitans is minor and superficial.

Coat-colour dilution and hypotrichosis in Hereford crossbred calves.

AIM: To investigate cases of coat-colour dilution and hypotrichosis in a group of Hereford x Friesian crossbred calves, and to define the underlying molecular genetics of the disorder. METHODS: The investigation was predicated on the hypothesis that this disorder was similar to a known dominantly inherited disorder of calves of black breeds crossed with Simmental cattle, for which there were candidate gene mutations. Sequence analyses of PCR amplicons from exon 1 and exon 11 of the premelanosome protein 17 gene (PMel17) were carried out. Restriction enzyme digestions of amplicons were followed using electrophoresis of digested fragments. RESULTS: It was shown that an affected calf and its Hereford sire were heterozygous for a three-base deletion in exon 1 of the PMel17 gene. These two animals were also heterozygous for a second mutation in exon 11 of the PMel17 gene. Four other related animals were likewise heterozygous for both mutations in the sire's herd of origin. CONCLUSIONS: Coat-colour dilution and hypotrichosis in Hereford crossbred calves in New Zealand is the same genetic disorder as that previously described in Simmental crossbred calves, and is linked to mutations in the PMel17 gene.

Pathology of mucopolysaccharidosis IIIA in Huntaway dogs.

Dogs with mucopolysaccharidosis (MPS) IIIA were bred within an experimental colony. As part of characterizing them as a model for testing therapeutic strategies for the analogous disease of children, a pathologic study was undertaken. By histology, there were variably stained storage cytosomes within neurons, including many that stained for gangliosides. On ultrastructure examination, these cytosomes contained either moderately dense granular material, tentatively interpreted as precipitated glycosaminoglycan; a variety of multilaminar bodies, interpreted as being associated with secondary accumulation of gangliosides; or a mixture of both types. In the liver, storage vesicles also contained excess glycogen as a secondary storage product. In various tissues, there were large foamy macrophages. In the brain, many of these were in juxtaposition with neurons, and, on ultrastructure examination, they contained storage cytosomes similar to those in neurons. However, the neuron in association with such a macrophage frequently showed little such material.

Segmental axonopathy of Merino sheep in New Zealand.

AIM: To investigate an axonopathy of Merino sheep that caused progressive hindlimb ataxia and slight to moderate paresis, with the purpose of understanding its pathogenesis. METHODS: Tissues were fixed in buffered paraformaldehyde or paraformaldehyde and glutaraldehyde, processed into wax and epoxy resin, respectively, and examined by light and electron microscopy. Fresh frozen spinal cord and trigeminal nerve roots were subjected to homogenisation, centrifugation and two-dimensional electrophoresis. Selected protein spots were identified using matrix-assisted laser desorption ionisation (MALDI) mass spectrometry. RESULTS. By light microscopy, there were large pale foamy spheroidal axonal swellings affecting peripheral as well as central axons. By electron microscopy, these were shown to contain many membrane-bound vesicles. The main abnormalities in expressed proteins involved cytoskeletal elements and myosin heavy chain, the latter interpreted as associated with the molecular motor myosin Va. CONCLUSIONS: The disorder is the same as that described in Merinos in Australia as segmental axonopathy, and believed to have an inherited aetiology. The lesions and protein changes indicate abnormalities of the cytoskeleton, its relationship with the myelin sheath, and myosin Va molecular motor. The consequence appears to be abnormal axonal transport and inability to maintain the integrity of axons and their myelin sheaths.

Congenital polycystic kidney disease in lambs.

AIM: To describe the pathology and inheritance of a congenital polycystic kidney disease (PKD) of sheep. METHODS: Mode of inheritance of PKD was investigated by evaluation of results of the disorder from planned matings in two consecutive years within subsets of a flock that had a high prevalence of PKD in lambs. Gross pathological and histopathological studies were based on tissues derived from this study. Haematoxylin and eosin (H&E)-stained paraffin sections of kidney, liver, extrahepatic biliary and pancreatic ducts, pancreas and epididymis were used to describe the lesions. RESULTS: Twenty-five lambs affected by PKD, of both sexes, were born, numbers in accord with those expected for an autosomal recessive disorder in the population studied. In all cases for which tissues were available, the renal, bile ductal (intrahepatic and extrahepatic), pancreatic and epididymal tissues had widespread dysplastic changes and associated cyst formation. CONCLUSIONS: The findings of renal cysts in conjunction with cysts in other organs are unifying features in many of the human and animal forms of PKD and suggest a related pathogenic and genetic base consistent with an autosomal recessive disorder.

Cerebellar cortical abiotrophy in Wiltshire sheep.

AIM: To investigate the nature of a neurological disease in Wiltshire sheep. METHODS: Three affected lambs were examined, humanely killed and necropsied. Selected neurological tissues were examined by light and electron microscopy. RESULTS: Primary neurological lesions were confined to the cerebellum and were characterised by loss of Purkinje cells and the presence of large hypertrophied dendrites of surviving Purkinje cells. These contained stacks of smooth endoplasmic reticulum. There was hyperplasia and cell swelling of Bergmann glia. Mild Wallerian-type degeneration affected white matter in the cerebellum and spinal cord. CONCLUSION: The cerebellar lesions were of a degenerative and reactive rather than hypoplastic nature. These, and the history, suggest a genetic cause with putative inheritance as an autosomal recessive trait. Accordingly, the disorder is described as a cerebellar abiotrophy.

Genetic disorders of sheep in New Zealand: a review and perspective.

Genetic disorders of sheep that have occurred in New Zealand are reviewed and discussed with regard to phenotype, inheritance and, where known, genotype. Inbreeding was a major factor in the emergence of some of them. The various disorders reflect a continuum, ranging from simple monogenic diseases or malformations due to dysfunctional gene products, those monogenic disorders dependant on environmental interactions, malformations due to homeotic gene dysfunctions, and multifactorial diseases for which genetic factors are associated with disease susceptibility. Chromosomal aberrations, although of limited importance, have contributed to an understanding of the physical chromosome map and derivative linkage map of sheep.

Myophosphorylase deficiency (glycogen storage disease Type V) in a herd of Charolais cattle in New Zealand: confirmation by PCR-RFLP testing.

AIM: To describe a disease of muscle in Charolais calves and confirm the putative diagnosis of inherited myophosphorylase deficiency. METHODS: Variously stained paraffin sections of muscle prepared from affected calves were used to describe the lesions. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) test was developed and applied to affected calves, their sires, dams and other individuals. RESULTS: The lesions were those of rhabdomyolysis of skeletal muscles and sub-sarcolemmal spaces in normal fibres. The PCRRFLP test confirmed the expected mutation for phosphorylase deficiency of Charolais cattle in two affected calves. In addition, sires, dams and other closely-related individuals of four affected calves tested as heterozygous for the mutation. Other apparently unrelated animals also tested as heterozygous. CONCLUSIONS: The diagnosis of myophosphorylase deficiency was confirmed. The PCR-RFLP test is suitable for use in controlling this recessively-inherited disorder as it can diagnose heterozygous individuals that are otherwise clinically normal.

Microphthalmia in Texel lambs.

AIM: To describe an outbreak of congenital microphthalmia in Texel lambs in New Zealand. METHODS: Affected eyes were fixed in 10% formol saline, processed routinely for histopathology and stained with haematoxylin and eosin, Holme's silver and haematoxylin with and without luxol fast blue, and luxol fast blue alone. RESULTS: Macroscopically, the globes and optic nerves were hypoplastic. Histologically, all embryonic components of the eye were present, but the lens, ciliary body, iris and retina were dysplastic. Axons of the hypoplastic optic nerve were poorly myelinated. CONCLUSIONS: Congenital microphthalmia occurs in Texel sheep in New Zealand, and is likely inherited as an autosomal recessive trait.

Neuronal ceroid lipofuscinosis in Merino sheep.

OBJECTIVE: To characterise neuronal ceroid lipofuscinosis (NCL) in Merino sheep. DESIGN: A prospective clinical, pathological, biochemical and genetic study. PROCEDURE: NCL cases were studied from a medium-wool Merino flock, the stud of origin of its replacement rams, and an experimental flock established at the University of Sydney. RESULTS: Behavioural changes and visual impairment were first detected at 7 to 12 months of age and progressed, with associated motor disturbances and at later stages seizures, to premature death by 27 months of age. At necropsy there was severe cerebrocortical atrophy associated with neuronal loss, astrocytosis and the presence in neurons of eosinophilic intracytoplasmic storage bodies with the characteristics of a lipopigment. In the retina there was progressive loss of photoreceptor cells. Storage bodies isolated from fresh brain, liver and pancreas formed electron-dense aggregates and coarse multilamellar and fine fingerprint profiles ultrastructurally, and consisted mainly of the hydrophobic protein, subunit c of mitochondrial ATP synthase. A homozygosity mapping approach localised the gene causing the disease in Merino sheep to the chromosomal region (OAR7q13-15) associated with NCL in South Hampshire sheep. CONCLUSION: NCL in Merino sheep is a subunit c-storing disease, clinically and pathologically similar to NCL in South Hampshire sheep. We propose that the disease in both breeds represents mutation at the same gene locus in chromosomal region OAR7q13-15.

Mitochondrial dysfunction in the neuronal ceroid-lipofuscinoses (Batten disease).

There are at least eight genetic entities known as the ceroid-lipofuscinoses in humans which share clinical and pathological features that have caused them to be grouped together under the eponym of Batten disease. They present pathologically as lysosomal storage diseases but are also characterised by severe neurodegeneration. Although the biochemical defects appear primarily centred on lysosomes and defects in proteolysis, the link between this and pathogenesis of neuronal death is poorly understood. The pathogenesis of neurodegeneration has been studied particularly in two animal models these being the English setter dog and the New Zealand Southhampshire sheep (OCL6). In these, and some of the human entities, there is evidence of mitochondrial dysfunction. This includes the accumulation of subunit c of ATP synthase as a component of storage material in at least six of eight genetic forms of the disease; structural abnormalities of mitochondria and selective loss of neurons in areas of the brain that are particularly metabolically active. Direct evidence of dysfunction comes from mitochondrial function tests in fibroblasts and, in animal models, isolated liver mitochondria. Supporting evidence of mitochondrial dysfunction was shown by disturbances in proportions of energy-rich phosphates in fibroblasts in some of these diseases. If these various defects were reflected in neurons, then it would support the hypothesis that neuron death was associated with energy-linked excitotoxicity.