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.

Abstract CASE HISTORY: Cases were obtained through passive surveillance reporting by veterinary pathologists, via the Ministry for Primary Industries Exotic Pest and Disease Hotline. They included ill or dead cows that had evidence of frank haemorrhage, petechial haemorrhages on mucous membranes, wasting or dermatitis of unknown cause, and were reported between 2009–2014. Affected cows (n=16) were from nine seasonally calving dairy farms, aged ≥3 years, and were predominantly in their mid-to-late non-lactating period. A brassica crop was identified in 15/16 cases as part of the current or recent ration. CLINICAL FINDINGS: Eight cows were found dead or died within 2 days of first signs. In eight cases death or euthanasia took place up to 3 weeks after signs were first observed. Cattle clinically examined prior to death (n=11) were generally inappetant, and recumbent or reluctant to move. Five cases had pale mucous membranes, three had petechiae and two were jaundiced. Rectal temperature was normal to sub-normal in eight cases. Evidence of melena or fresh blood at the anus or mouth was found in five cases. In three cases, alopecia and skin thickening was present, predominantly affecting the head and neck. PATHOLOGICAL FINDINGS: Petechiation of mucosal and internal serosal membranes, myocardium, subcutis and skeletal muscle was found in 10 cases. Frank haemorrhage was present in six cases, including haematomas of the subcutis, skeletal musculature, mesentery or omentum, and lumenal haemorrhage of the abomasum and/or intestine. In five cases pale nodules within myocardium and/or kidney, liver or spleen were present. Histopathologically, these were confirmed as granulomatous inflammatory lesions, which were also present within a wide range of tissues. Granulomatous foci typically comprised aggregates of macrophages, lymphocytes, plasma cells, prominent multinucleated giant cells and eosinophils. DIAGNOSIS: Idiopathic multisystemic granulomatous and haemorrhagic disease, occurring sporadically in dairy cattle, in the absence of feeds or feed additives previously associated with comparable syndromes. CLINICAL RELEVANCE: This is the first description of a novel systemic granulomatous and haemorrhagic syndrome seen in adult dairy cattle most often in their non-lactating period. The presentation can mimic important exotic disease differentials in New Zealand including anthrax, haemorrhagic septicaemia (associated with selected Pasteurella multocida strains) or infection with bovine viral diarrhoea virus type 2.

Meckel syndrome (MKS) is an inherited autosomal recessive hepatorenal fibrocystic syndrome, caused by mutations in TMEM67 , characterized by occipital encephalocoele, renal cysts, hepatic fibrosis, and polydactyly. Here we describe an ovine model of MKS, with kidney and liver abnormalities, without polydactyly or occipital encephalocoele. Homozygous missense p.(Ile681Asn; Ile687Ser) mutations identified in ovine TMEM67 were pathogenic in zebrafish phenotype rescue assays. Meckelin protein was expressed in affected and unaffected kidney epithelial cells by immunoblotting, and in primary cilia of lamb kidney cyst epithelial cells by immunofluorescence. In contrast to primary cilia of relatively consistent length and morphology in unaffected kidney cells, those of affected cyst-lining cells displayed a range of short and extremely long cilia, as well as abnormal morphologies, such as bulbous regions along the axoneme. Putative cilia fragments were also consistently located within the cyst luminal contents. The abnormal ciliary phenotype was further confirmed in cultured interstitial fibroblasts from affected kidneys. These primary cilia dysmorphologies and length control defects were significantly greater in affected cells compared to unaffected controls. In conclusion, we describe abnormalities involving primary cilia length and morphology in the first reported example of a large animal model of MKS, in which we have identified TMEM67 mutations.

Defects in primary cilia structure or function result in both common and rare ciliopathies. We have characterized an ovine model of Meckel Gruber Syndrome involving widespread dysplastic changes and cystogenesis in multiple organs. SNPchip screening identified an MKS3 gene defect, while sequencing identified two missense mutations (I680N and I686S) in a conserved region coding for an intracellular loop of meckelin. We report on the nature and extent of renal primary cilia defects associated with this unique model. Columnar, cuboidal and squamous epithelia lined small cysts (1-5mm) distributed throughout the kidney, with extensive interstitial fibrosis. Immunohistochemical and SEM studies revealed a low incidence of dysmorphic epithelia cilia, including many detached near the base and shed into the cysts. Meckelin staining showed extensive vesiculation within epithelia and interstitial fibroblasts, it co-localised to the base and along the length of both attached and shed cilia, and it labeled microvesicles within cysts. In cultured fibroblasts, cilia were highly dysmorphic, ciliary incidence was similar (44% controls; 48% mutants), but ciliary length was significantly longer in mutants (17.8±1.4 cf 8.3±0.7µm; p < 0.05). Golgi markers showed extensive cytoplasmic vesiculation in mutant fibroblasts, while TEM showed Golgi distention, multivesicular bodies, intraciliary vesicles, and extracellular microvesicles. Primary cilia structure was significantly compromised in this large animal model including irregular morphology, length variation, intraciliary vesicles, cilia shedding, Golgi distention, formation of multivesicular bodies, and microvesicle release. Results suggest that disruption to the conserved isoleucines of meckelin compromises protein structure and ciliary function in this model of Meckel Gruber syndrome.

CASE HISTORY:In September 2004 two hinds on Farm 1 were observed with epiphora and keratoconjunctivitis, and corneal scarring. A low pregnancy rate in some hinds had been recorded that year. In the same year six yearling deer were observed on Farm 2 with keratitis, uveitis and corneal scarring.CLINICAL AND PATHOLOGICAL FINDINGS:On Farm 1, conjunctival swabs and blood samples were collected from the hinds with ocular lesions, and from 24 other hinds. The two affected hinds were immunosuppressed with dexamethasone for 7 days. Conjunctival, nasal and vaginal swabs were collected daily before euthanasia and necropsy on the eighth day. Subsequently, another five non-pregnant hinds were similarly immunosuppressed and necropsied, and the reproductive tracts of 20 non-pregnant hinds were collected following slaughter. Semen samples were collected from four stags implicated with reproductive failure. On Farm 2, conjunctival swabs were collected from six hinds with ocular lesions and from 14 unaffected deer. Viral culture, consensus primer PCR and sequencing for specific herpesviruses was carried out on conjunctival swabs, buffy coat from blood samples, semen and reproductive tracts. Necropsy samples were also examined using gross pathology and histopathology. On Farm 1, a type 2 rhadinovirus (CvRhV) was detected in the conjunctiva of one hind with keratoconjunctivitis using PCR. Following immunosuppression, gross vesicular and histological vaginal lesions typical of infection with alphaherpesvirus were observed in samples of vaginal tissue from the same hind. Buffy coat, vaginal and lumbar spinal nervous tissues were also positive for cervid herpesvirus 1 (CvHV-1) using PCR. Herpesviruses were not detected in reproductive tracts, ocular or semen samples of the other deer. CvRhV was detected in buffy coats from four other hinds and in a conjunctival swab from one hind, all without ocular lesions, using PCR. On Farm 2, conjunctival swabs from two deer with keratitis were culture positive for CvHV-1. Two culture-negative conjunctival samples from deer without ocular lesions were positive for CvHV-1 by PCR. In two other affected animals, presence of CvRhV was confirmed by PCR and sequencing.DIAGNOSIS:Infection with CvHV-1 associated with keratitis and vulvovaginitis, and CvRhV infection in deer with and without ocular lesions.CLINICAL RELEVANCE:CvHV-1 is a likely cause of keratoconjunctivitis and possibly reproductive tract pathology in deer. Investigation of ocular lesions and reproductive failure in farmed deer should include CvRhV and CvHV-1.

CASE HISTORY: In 2008, six lambs within a flock of Dorpercross sheep were born with musculoskeletal and neurological disease. Clinical signs included hindlimb weakness, and urinary incontinence. CLINICAL FINDINGS: All lambs had focal, inverted areas of alopecic skin over the caudal sacrum, and short, often kinked tails. Four affected lambs were subject to euthanasia, and necropsied. On gross examination, the arches of sacral vertebrae were absent, and spinal nerves and meninges were adherent to the overlying subcutis. Other gross lesions included narrow, elongated skulls, herniation of the occipital lobes into the caudal fossas, hydrocephalus, and syringomyelia. One lamb had coning of the cerebellar vermis, but cerebellar herniation through the foramen magnum was not identified. DIAGNOSIS: Spina bifida, with associated malformations of the central nervous system. CLINICAL RELEVANCE: Examination of breeding records suggested either an autosomal recessive or partially penetrant autosomal dominant pattern of inheritance. Because of the associated tail lesions it is proposed that the pathogenesis of this syndrome involves a defect in development of the tail bud (secondary neurulation), that tethering of the spinal cord resulted in the clinical signs, and abnormal pressure of the cerebral spinal fluid resulted in the defects in the skull and brain.

AIM: To investigate whether the electroencephalographic (EEG) responses to slaughter by ventral-neck incision without prior stunning may be perceived as painful in halothane-anaesthetised calves.METHODS: Fourteen Angus steers were minimally anaesthetised with halothane, using an established anaesthesia protocol. EEG indices were recorded bilaterally for 5 minutes prior to and 5 minutes following ventral-neck incision. A single incision was made in the ventral aspect of the neck, severing all tissues ventral to the vertebral column including the major blood vessels supplying and draining the head. Changes in the median frequency (F50), 95% spectral edge frequency (F95) and total power of the EEG (Ptot) were used to investigate the effects of ventral-neck incision. At the completion of the experiment, brains of calves were examined histologically.RESULTS: During the 30 seconds following ventral-neck incision, the F95 and Ptot showed significant changes (p<0.05) compared with pre-treatment values. The F50 increased significantly from recordings from the right side of the cranium. No gross or histological abnormalities were detected in the brains following slaughter.CONCLUSIONS: This study is the first investigation of the noxiousness of slaughter by ventral-neck incision, using EEG spectral analysis. It demonstrated that there is a period following slaughter where ventral-neck incision represents a noxious stimulus.

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.

Abstract CASE HISTORY: An outbreak of fatal or debilitating disease characterised by wasting and cutaneous lesions that usually necessitated euthanasia, occurred over a period of 4 months in pigs 14–16 weeks of age from a commercial ‘farrow-to-finish’ unit in the North Island of New Zealand. During the outbreak, 67 deaths were attributed to the condition, accounting for 2–3% of total production. The outbreak preceded the diagnosis of post-weaning multisystemic wasting syndrome (PMWS) on this farm. CLINICAL FINDINGS: Sick pigs were thin, depressed, and pyrexic, and most had multiple extensive erythematous crusting cutaneous lesions that were most prominent in the region of the hindquarters, forequarters and head. Those with cutaneous lesions affecting the hindlimbs had subcutaneous oedema and enlarged superficial inguinal lymph nodes. Diarrhoea was commonly present. PATHOLOGICAL FINDINGS AND DIAGNOSIS: In addition to the skin lesions, the main gross pathological changes were renal petechiation and lymphadenomegaly. The cutaneous and renal lesions resulted from necrotising and neutrophilic vasculitis, and thrombosis and infarction of adjacent tissues. Vascular lesions were variably present in the spleen, liver, lymph nodes and intestines. Renal tubules were distended by sero-haemorrhagic fluid and proteinaceous casts. Lymphoid tissue changes typical of porcine circovirus type 2 (PCV2)-associated disease included depletion of mature lymphocytes, histiocytic infiltrations and circoviral inclusion bodies. Lesions associated with PCV2 in non-lymphoid tissues included granulomatous or non-suppurative interstitial pneumonia, interstitial nephritis, gastroenteritis, and hepatitis with hepatocellular apoptosis. DIAGNOSIS: Porcine circovirus-associated disease (PCVAD) represented by concurrent porcine dermatitis and nephropathy syndrome (PDNS) and emergent PMWS. CLINICAL RELEVANCE: This is the first published description of PDNS and of combined PDNS and PMWS in pigs in New Zealand. In addition, the prevalence of PDNS, which is usually a sporadic disorder, was inconsistent with a diagnosis of PDNS alone.

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.

Extract Among the puzzling aspects of the recently described autosomal recessive polycystic kidney disease (ARPKD) of sheep (Johnstone et al 2005 Johnstone, AC, Davidson, BI, Roe, AR, Eccles, MR and Jolly, RD. 2005. Congenital polycystic kidney disease in lambs. New Zealand Veterinary Journal, 53: 307–314. [Taylor & Francis Online], [Web of Science ®] , [Google Scholar]) is the multiplicity of organ systems affected and the phenotypic variations that occur between the various animal species. It has been shown in studies of other inherited polycystic kidney disease syndromes that the normal gene products of a mutant are likely to be involved in ciliary structure and function. The studies that have led to this recognition can be expected to eventually provide the basis for a better understanding of the functioning of this organelle, and the pathogenesis of lesions in the related diseases. It is well known that cilia are important in the perception of light, olfactory stimuli and sound and that motile cilia provide cell motility (e.g. sperm) and transport of mucus and other fluids. Recent research has indicated that the involvement of the primary cili-um/basal body complex is of central importance in the detection and cellular response to extracellular movement of fluid, critical phases of embryonic development, cell cycle regulation and maintenance of cell polarity. Confocal microscopic studies of renal cyst epithelium in ovine ARPKD have shown that only 30% of cells have a cilium and that these are often truncated (McGlashan et al 2005 McGlashan, SR, Poole, CA, Stayner, C, Johnstone, AC, Eccles, MR and Jensen, CG. 2005. “Primary cilia in fibrosis associated with two models of polycystic kidney disease”. In Proceedings of the 45th Annual Conference of the American Society for Cell Biology December 10–14 [Google Scholar]). The observation, although preliminary, strongly supports the argument that the mutant gene in this disease of sheep normally encodes for a protein essential for primary ciliary function.

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.

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.

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.