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.

An ovine hepatorenal fibrocystic model of a Meckel-like syndrome associated with dysmorphic primary cilia and TMEM67 mutations.

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.

Systemic granulomatous and haemorrhagic syndrome in New Zealand dairy cattle.

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.

Alpha and gamma herpesvirus detection in two herds of farmed red deer (Cervus elaphus) in New Zealand.

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.

Spina bifida with associated malformations of the central nervous system in Dorper-cross sheep.

CASE HISTORY: In 2008, six lambs within a flock of Dorper-cross 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.

Electroencephalographic responses of halothane - anaesthetised calves to slaughter by ventral-neck incision without prior stunning.

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.

Porcine dermatitis and nephropathy syndrome in New Zealand.

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.

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.

Avian cryptococcosis.

Clinical and laboratory findings in 15 unreported cases of avian cryptococcosis from Australia were collated and contrasted with 11 cases recorded in the literature. Cryptococcus species produced localized invasive disease of the upper respiratory tract of captive parrots living in Australia. This resulted in signs referable to mycotic rhinitis or to involvement of structures contiguous with the nasal cavity, such as the beak, sinuses, choana, retrobulbar space and palate. Parrots of widely differing ages were affected and of the seven birds for which sex was determinable, six were male. Cryptococcus bacillisporus (formerly C. neoformans var. gattii) accounted for four of five infections in which the species or variety was determinable, suggesting that exposure to eucalyptus material may be a predisposing factor. In these cases, Cryptococcus appeared to behave as a primary pathogen of immunocompetent hosts. One tissue specimen was available from an Australian racing pigeon with minimally invasive subcutaneous disease; immunohistology demonstrated a C. neoformans var. grubii (formerly C. neoformans var. neoformans serotype A) infection, presumably subsequent to traumatic inoculation of yeast cells into the subcutis. Two similar cases had been reported previously in pigeons domiciled in America. Data for parrots, one pigeon and other birds studied principally in America and Europe (and likely infected with C. neoformans) suggested a different pattern of disease, more suggestive of opportunistic infection of immunodeficient hosts. In this cohort of patients, the organism was not restricted to cool superficial sites such as the upper respiratory tract or subcutis. Instead, infections typically penetrated the lower respiratory tract or disseminated widely to a variety of internal organs. Finally, three captive North Island brown kiwis, one residing in Australia, the other two in New Zealand, died as a result of severe diffuse cryptococcal pneumonia (two cases) or widely disseminated disease (one case). C. bacillisporus strains were isolated from all three cases, as reported previously for another kiwi with disseminated disease in New Zealand.

Bovine and ovine mycoplasmal arthritis and synovitis (abstract).

The pathology of mycoplasmal gonitis in a Friesian cow aged 15 months, and polyarthritis in a 3-week-old East Friesian sheep was described. Both stifle joints of the cow, which was 1/63 calves that developed polyarthritis after being fed milk contaminated with Mycoplasma mycoides mycoides large colony type (MmmLC), were distended with yellow flocculent fluid. The capsular and synovial tissues and their associated tendon sheaths were greatly thickened due to chronic active fibrinopurulent inflammation with ulceration of synovial membranes and progressive organisation of the exudate by granulation tissue and fibrosis. Nodular aggregates of lymphocytes and plasma cells studded the membrane. Articular surfaces were unremarkable. Mycoides cluster organisms were demonstrated on polymerase chain reaction (PCR) analysis of the joint tissues and these were confirmed on culture as MmmLC. The lamb was the offspring of an agalactic ewe whose progeny of the previous 3 years developed polyarthritis. The carpal and hock joints had periarticular nodular swellings and there was mild haemarthrosis of all appendicular skeletal joints. Lesions were characterised as subacute active fibrinopurulent synovitis and periarthritis with haemorrhage. Articular surfaces were normal. Joint tissue was positive by PCR for the mycoides cluster of organisms. Mycoplasma was not isolated in culture.

Gastric Ollulanus tricuspis infection identified in captive cheetahs (Acinonyx jubatus) with chronic vomiting.

Gastritis, vomition and weight loss are common in captive cheetahs (Acinonyx jubatus). Gastric spiral bacteria (Helicobacter spp.) and the very small, viviparous nematode Ollulanus tricuspis, a stomach worm of cats, are believed to be important causes. Three sibling cheetahs at Wellington Zoo, New Zealand, developed chronic vomiting, diarrhoea and debility. Their parents were both South African-born. Response to antibacterial treatment was poor. Endoscopic examinations revealed chronic lymphoplasmacytic gastritis and Ollulanus infection. Treatment with oxfendazole and pyrantel embonate resulted in clinical improvement; however, 1 cheetah, which died 7 months later as a result of a ruptured liver due to hepatic amyloidosis, still had Ollulanus worms present in her stomach. Ollulanus tricuspis is a significant cause of gastritis and vomiting in captive cheetahs, lions and tigers, as well as wild cougars and tigers. The parasite has not yet been found in sub-Saharan Africa. Because of the unusual characteristics of this parasite, the literature on its life history and techniques for diagnosis is reviewed.

Aspergillosis and other causes of mortality in the stitchbird in New Zealand.

Necropsy findings from natural deaths in free living and captive stitchbirds (Notiomystis cincta) were examined over a 3 yr period (November 1991-94) to establish whether disease was an important factor in translocation failures and captive breeding programs undertaken by the New Zealand Department of Conservation. Fresh and fixed material from seven free-living birds and 11 captive birds were examined and were compared with those of a retrospective study of archival material from captive and wild birds collected over a 13 yr period (1979-91). The causes of death in both the present and retrospective study showed a similar pattern with aspergillosis and aspiration pneumonia being the most significant cause of mortality in captive birds. Aspergillosis was diagnosed as the cause of death in 11 of 31 stitchbirds from Mt Bruce; eight of these deaths occurred in the winter months (June-August). The other causes of death in captive birds included trauma, coccidiosis, and sporadic bacterial infections. Hemosiderosis and airsacculitis were common histological findings in most of the wild and captive stitchbirds examined.