Paradigm shifts in understanding equine laminitis.

Laminitis, one of the most debilitating conditions of all equids, is now known to be the result of several systemic disease entities. This finding, together with other recent developments in the field of laminitis research, have provoked a rethink of our clinical and research strategies for this condition. First, laminitis is now considered to be a clinical syndrome associated with systemic disease (endocrine disease, sepsis or systemic inflammatory response syndrome, SIRS) or altered weight bearing rather than being a discrete disease entity. Next, laminitis associated with endocrine disease (endocrinopathic laminitis) is now believed to be the predominant form in animals presenting (primarily) for lameness. Third, the designation of laminitis as a primary and severe basement membrane pathology now requires revision. Instead, current data now proposes a variable subclinical phase associated with gross changes in the hoof capsule, with stretching and elongation of the lamellar cells an early and key event in the pathophysiology. These findings have fuelled new mechanistic hypotheses and research directions that will be discussed, together with their implications for future clinical management.

Evaluation of the modified Glasgow Prognostic Score to predict outcome in dogs with newly diagnosed lymphoma.

The modified Glasgow Prognostic Score (mGPS) assigns a numerical value (0-2) from pre-treatment serum concentrations of C-reactive protein (CRP) and albumin to predict patient outcome. CRP and albumin were evaluated in 77 untreated dogs with lymphoma to determine the relationship of mGPS to clinicopathological parameters and whether it could predict progression-free (PFS) and overall survival (OS) in treated dogs. mGPS distribution was significantly associated with clinical stage, substage b, weight loss, gastrointestinal disturbances and lethargy at presentation. On univariate analysis, mGPS was significantly associated with OS and PFS, with shorter median survival times for mGPS 2 compared to mGPS 0 and 1 combined. Hypoalbuminaemia significantly reduced OS and PFS, however increased CRP had no effect. Only clinical stage was significantly associated with OS and PFS on both univariate and multivariate analysis. mGPS has potential prognostic value for canine lymphoma , but further studies are needed.

Lamellar pathology in horses with pituitary pars intermedia dysfunction.

REASONS FOR PERFORMING STUDY: Hoof lamellar pathology in horses with pituitary pars intermedia dysfunction (PPID) has not been described previously. OBJECTIVES: To describe the histomorphometry and pathological lesions in hoof lamellar tissue of animals that had PPID with or without concurrent laminitis, with reference to age-matched controls. We hypothesised that lamellar lesions consistent with laminitis would be associated with PPID, even in animals without current or historical laminitis. STUDY DESIGN: Prospective case-control study. METHODS: Mid-dorsal hoof histological sections were obtained post mortem from the forelimbs of 16 PPID-affected animals either with (n = 6) or without laminitis (n = 10) and 10 age- and breed-matched controls. Sections were examined by a blinded veterinary pathologist. The length and width of 10 primary epidermal lamellae were measured using image analysis software. The morphology and pathology of primary and secondary epidermal lamellae were then typed or graded in axial, middle and abaxial regions. Fasting serum insulin, plasma adrenocorticotropin and blood glucose concentration were measured from blood samples taken prior to euthanasia. RESULTS: All animals with PPID and laminitis had fasting hyperinsulinaemia (median 74.1 miu/l, interquartile range 49.9-349.5 miu/l) whereas PPID animals without laminitis had serum insulin concentrations below the upper limit of the reference range (<20 miu/l). Lamellar pathology in PPID animals with laminitis was variable in severity and unrelated to the reported duration of laminitis (range 2 months-5 years). Most lesions were located abaxially within the lamellar tissue and included increased length and width of the lamellae, chronic abnormal keratinisation, interlamellar epidermal bridging and cell death with more acute lamellar tearing in some cases. The lamellae of PPID animals without laminitis were normal referent to the relevant control group. CONCLUSIONS: Whether PPID and hyperinsulinaemia have a causal inter-relationship or not, it may only be the hyperinsulinaemia that is associated with lamellar morphological alteration and pathology consistent with laminitis.

p62/Sequestosome-1: Mapping Sites of Protein-Handling Stress in Canine Cutaneous Mast Cell Tumors.

Canine cutaneous mast cell tumors (MCT) are common, frequently malignant neoplasms that are currently graded histologically for provision of prognostic information. Continuing evidence of subsets of MCT within certain grades (with differing survival times) indicate the need for biomarkers that will facilitate better patient stratification and also provide further information on the biological processes involved in progression. We decided to investigate the expression of p62/sequestosome-1 (p62/SQSTM1), a stress-inducible "hub protein" found in all cell types that shuttles rapidly between the nucleus and cytoplasm and is known to play important roles in protein handling and tumorigenesis. The identity of canine p62/SQSTM1 was confirmed in silico and by validation of a commercial antibody using both Western blotting and functional (pharmaceutical-based) analyses in cell culture. Using immunohistochemistry, 3 patterns of p62 expression were identified based on the predominant intracellular localization, that is, nuclear, mixed (nuclear and cytoplasmic), and cytoplasmic. There was a highly significant association with the 2-tier (Kiupel) grade (P < .0001), with all p62-nuclear immunoreactivity being associated with low grade and most p62-cytoplasmic immunoreactivity (93%) with high grade. Most but not all mixed nuclear-cytoplasmic labeling occurred in low-grade MCT; in other (human) tumor types, this pattern has been interpreted as borderline malignant. These data indicate that there is a shift in protein-handling stress from the nucleus to the cytoplasm in association with increasing malignancy in MCT. Studies to identify the processes and drug-able targets involved in this progression are ongoing.

Pathology of Natural Cases of Equine Endocrinopathic Laminitis Associated With Hyperinsulinemia.

Laminitis in equids is a clinical syndrome usually associated with systemic disease. Endocrinopathies recently have been recognized as the most common cause of laminitis, with hyperinsulinemia playing a key role. Descriptions of laminitis-associated lesions have been confusing due to the wide range of experimental models used, failure of adequate clinical documentation for naturally occurring cases, lack of separate analysis of inflammatory and endocrinopathic laminitis, and uncertainty regarding normal morphological variation of lamellae. In this study, lamellar morphology and pathology were described in 14 laminitic horses and ponies that had hyperinsulinemia (>20 mIU/l), with reference to 25 age- and breed-matched controls. The type and severity of lesions noted had no correlation with reported clinical duration and in at least some cases must have preceded it. Lesions were largely localized abaxially within the lamellar tissue and included apoptotic cell death, as well as lamellar fusion, hyperplasia, and partial replacement with aberrant keratin containing nucleated debris and proteinaceous lakes. The lesions resulted in irregular margins between the inner horn and the lamellar tissue. Acute separation originated from the abaxial region, with minimal associated inflammation. Axially, epidermal lamellar tapering was the most frequent morphological observation. The lesions in these chronic cases of laminitis were similar to those described in some inflammatory laminitis models and contained features seen in developmental phases of hyperinsulinemic models. These findings support the theory that repeated episodes of subclinical laminitis occur prior to clinical presentation. In addition, the pathology does not include extensive basement membrane failure seen in some inflammatory models.

The pathogenesis of tendon microdamage in athletes: the horse as a natural model for basic cellular research.

The equine superficial digital flexor tendon (SDFT) is a frequently injured structure that is functionally and clinically equivalent to the human Achilles tendon (AT). Both act as critical energy-storage systems during high-speed locomotion and can accumulate exercise- and age-related microdamage that predisposes to rupture during normal activity. Significant advances in understanding of the biology and pathology of exercise-induced tendon injury have occurred through comparative studies of equine digital tendons with varying functions and injury susceptibilities. Due to the limitations of in-vivo work, determination of the mechanisms by which tendon cells contribute to and/or actively participate in the pathogenesis of microdamage requires detailed cell culture modelling. The phenotypes induced must ultimately be mapped back to the tendon tissue environment. The biology of tendon cells and their matrix, and the pathological changes occurring in the context of early injury in both horses and people are reviewed, with a particular focus on the use of various tendon cell and tissue culture systems to model these events.

Histopathology of insulin-induced laminitis in ponies.

REASONS FOR PERFORMING STUDY: Ponies with laminitis associated with insulin resistance and hyperinsulinaemia lack systemic and/or intestinal inflammatory signs, suggesting a different pathogenesis potentially reflected in differing histopathology. OBJECTIVES: To describe the histological appearance and quantify morphological changes in primary and secondary epidermal lamellae (PEL and SEL) of laminitis lesions from ponies with insulin-induced laminitis. METHODS: Equine hoof lamellar tissue was obtained from 4 control ponies and 5 ponies with laminitis induced following infusion of insulin (1036 ± 55 µU/ml) while maintaining euglycaemia for 55.4 ± 5.5 h. Sections from all 4 hooves were stained and examined by a veterinary pathologist. Measurements of lamellar length (PEL and SEL) were made in mid-dorsal sections of the right forefeet by 2 blinded observers. Immunolabelling for calprotectin was performed using a monoclonal antibody. RESULTS: No lesions were detected in normal ponies. Lesions detected in ponies with laminitis were variable in severity between ponies. Within ponies, SEL lesions were more severe along the axial region of PEL. Lesions included swelling, disorganisation and abnormal keratinisation of epidermal cells, increased mitotic activity and apoptosis. Separation of basement membranes was minimal. Immunostaining revealed inflammatory cells within the lamellar dermis. SEL were significantly elongated in laminitic hooves relative to controls, with the greatest elongation in those attached to abaxial and middle regions of PEL. CONCLUSIONS: Laminitis induced by prolonged infusion of insulin lacked widespread basement membrane disintegration, and increases in epidermal cellular proliferation at axial aspects were marked for this acute stage of disease. POTENTIAL RELEVANCE: Defining equine laminitis entirely in terms of separation of the basement membrane may not be appropriate for laminitis associated with hyperinsulinaemia.

Corneal fibropapillomatosis in green sea turtles (Chelonia mydas) in Australia.

Chelonid corneal fibropapillomatosis has not previously been recorded in Australian waters. During 2008, 724 green sea turtles (Chelonia mydas) were examined in Queensland, Australia at two sites, Moreton Bay (n=155) and Shoalwater Bay (n=569), during annual monitoring. In the same calendar year, 63 turtles were submitted from various sites in southern Queensland for post-mortem examination at the University of Queensland. Four of the 787 animals (0.5%) were found to have corneal fibropapillomas of varying size, with similar gross and microscopical features to those reported in other parts of the world. Two animals with corneal fibropapillomas also had cutaneous fibropapillomas. Clinical assessment indicated that these lesions had detrimental effects on the vision of the turtles and therefore their potential ability to source food, avoid predators and interact with conspecifics. Importantly, these findings represent an emergence of this manifestation of fibropapillomatosis in green sea turtle populations in the southern Pacific Ocean.

A glycogen synthase 1 mutation associated with equine polysaccharide storage myopathy and exertional rhabdomyolysis occurs in a variety of UK breeds.

REASONS FOR PERFORMING STUDY: A glycogen synthase (GYS1) mutation has been described in horses with histopathological evidence of polysaccharide storage myopathy (PSSM) in the USA. It is unknown whether the same mutation is present in horses from the UK. OBJECTIVES: To determine whether the GYS1 mutation occurs in UK horses with histopathological evidence of PSSM and exertional rhabdomyolysis. HYPOTHESIS: The R309H GYS1 mutation is present in a variety of UK horse breeds and that the mutation is commonly associated with exertional rhabdomyolysis. METHODS: DNA was extracted from 47 muscle or blood samples from UK horses with histories of exertional rhabdomyolysis in which muscle biopsy diagnosis had been pursued. The proportions of GYS1 mutation positive cases were compared among histopathologically defined groups. In addition, breeds that carried the GYS1 mutation were identified from a total of 37 grade 2 (amylase-resistant) PSSM cases. RESULTS: Of 47 horses with exertional rhabdomyolysis in which a muscle biopsy diagnosis was pursued, 10 (21%) carried the GYS1 mutation. The mutation was only found in horses with grade 2 PSSM (i.e. not in horses with normal, idiopathic myopathy or grade 1 PSSM biopsy samples). In total, the GYS1 mutation was found in 24/37 (65%) of grade 2 PSSM cases. A variety of breeds, including Quarter Horse, Appaloosa, Warmblood, Connemara-cross, Cob, Polo Pony and Thoroughbred cross carried the mutation. CONCLUSIONS: The GYS1 mutation is an important cause of exertional rhabdomyolysis of UK horse breeds but does not account for all forms of PSSM. POTENTIAL RELEVANCE: Genotyping is recommended in cases of exertional rhabdomyolysis, prior to or in combination with, muscle biopsy. However a significant proportion of horses with histopathological evidence of PSSM and/or exertional rhabdomyolysis have different diseases.

Maturational alterations in gap junction expression and associated collagen synthesis in response to tendon function.

Energy-storing tendons including the equine superficial digital flexor tendon (SDFT) contribute to energetic efficiency of locomotion at high-speed gaits, but consequently operate close to their physiological strain limits. Significant evidence of exercise-induced microdamage has been found in the SDFT which appears not to exhibit functional adaptation; the degenerative changes have not been repaired by the tendon fibroblasts (tenocytes), and are proposed to accumulate and predispose the tendon to rupture during normal athletic activity. The anatomically opposing common digital extensor tendon (CDET) functions only to position the digit, experiencing significantly lower levels of strain and is rarely damaged by exercise. A number of studies have indicated that tenocytes in the adult SDFT are less active in collagen synthesis and turnover than those in the immature SDFT or the CDET. Gap junction intercellular communication (GJIC) is known to be necessary for strain-induced collagen synthesis by tenocytes. We postulate therefore that expression of GJ proteins connexin 43 and 32 (Cx43; Cx32), GJIC and associated collagen expression levels are high in the SDFT and CDET of immature horses, when the SDFT in particular grows significantly in cross-sectional area, but reduce significantly during maturation in the energy-storing tendon only. The hypothesis was tested using tissue from the SDFT and CDET of foetuses, foals, and young adult Thoroughbred horses. Cellularity and the total area of both Cx43 and Cx32 plaques/mm(2) of tissue reduced significantly with maturation in each tendon. However, the total Cx43 plaque area per tenocyte significantly increased in the adult CDET. Evidence of recent collagen synthesis in the form of levels of neutral salt-soluble collagen, and collagen type I mRNA was significantly less in the adult compared with the immature SDFT; procollagen type I amino-propeptide (PINP) and procollagen type III amino-propeptide (PIIINP) levels per mm(2) of tissue and PINP expression per tenocyte also decreased with maturation in the SDFT. In the CDET PINP and PIIINP expression per tenocyte increased in the adult, and exceeded those in the adult SDFT. The level of PINP per mm(2) was greater in the adult CDET than in the SDFT despite the higher cellularity of the latter tendon. In the adult SDFT, levels of PIIINP were greater than those of PINP, suggesting relatively greater synthesis of a weaker form of collagen previously associated with microdamage. Tenocytes in monolayers showed differences in Cx43 and Cx32 expression compared with those in tissue, however there were age- and tendon-specific phenotypic differences, with a longer time for 50% recovery of fluorescence after photobleaching in adult SDFT cells compared with those from the CDET and immature SDFT. As cellularity reduces following growth in the SDFT, a failure of the remaining tenocytes to show a compensatory increase in GJ expression and collagen synthesis may explain why cell populations are not able to respond to exercise and to repair microdamage in some adult athletes. Enhancing GJIC in mature energy-storing tendons could provide a strategy to increase the cellular synthetic and reparative capacity.

Prevalence of equine polysaccharide storage myopathy and other myopathies in two equine populations in the United Kingdom.

The aim of this study was to determine the prevalence of equine polysaccharide storage myopathy (EPSM) in two populations of horses in the UK. Biopsy specimens from 94 horses presented to an abattoir (population 1), and 46 horses with neuromuscular disorders presented to a university referral hospital (population 2) were obtained over a period of 4years. Histological sections were examined by a veterinary pathologist for lesions including abnormal polysaccharide inclusions in myofibres. In population 1, a diagnosis of EPSM was made in 8% and non-specific myopathy in 33% of horses. In population 2, a diagnosis of EPSM was made in 22%, equine motor neurone disease (EMND) in 15% and non-specific myopathy in 37%. Within each population there was no difference in age, sex or breed distribution and muscle disease diagnosis. However, populations differed from each other in age and breed distributions and muscle disease diagnosis. EPSM was found in draft, Warmblood and related breeds and was diagnosed for the first time in cob-types. EMND was reported in 7/46 horses presented for neuromuscular disease and weakness, representing an important diagnosis in the UK. This study showed a high prevalence of EPSM and other myopathies in typical breeds of horses in the UK.

The pathology of bronchointerstitial pneumonia in young foals associated with the first outbreak of equine influenza in Australia.

REASONS FOR PERFORMING STUDY: The first outbreak of equine influenza virus (EIV) infection was confirmed in Australia in 2007. Some EIV-positive young foals died with bronchointerstitial pneumonia, an rare disease process in this age group that is often postulated to be caused by viral infection. OBJECTIVES: The aim of this study was to describe post mortem lesions in EIV-infected foals. METHODS: Post mortem examinations were conducted on 11 young foals (age 2-12 days) submitted to the Scone Veterinary Hospital, NSW over a 2-month period in 2007. The foals had presented with or developed fatal pneumonia, and were known or suspected to be EIV-positive. Equine influenza virus nucleic acid was detected in tissue specimens using an Influenza A group reactive real-time reverse transcriptase PCR assay. RESULTS: Grossly there was diffuse or extensive pulmonary consolidation. Histological changes included: bronchiolar and alveolar necrosis; neutrophilic infiltration; hyaline membrane formation; and hyperplasia and squamous metaplasia of airway epithelium. Tissues for 10 foals were EIV-positive, with a positive nasal swab from the remaining animal. CONCLUSIONS: This is the first detailed pathological description of bronchointerstitial pneumonia associated with EIV infection in young foals. It is also the first series of such cases in which a causative agent has consistently been detected. POTENTIAL RELEVANCE: Given the findings in this outbreak, and a previous outbreak in the UK in 1965 involving a similarly naive population, veterinary clinicians and pathologists should be aware that EIV can cause fatal bronchointerstitial pneumonia in young foals that do not have maternal immunity. The lesions did not differ from those previously reported in foals of various ages with bronchointerstitial pneumonia of other or undefined causes, indicating that this is most likely a stereotypical response to a variety of insults. Therefore, tissue specimens should be obtained from cases of pneumonia in young foals for virological and bacteriological testing.

Effects of exercise on tenocyte cellularity and tenocyte nuclear morphology in immature and mature equine digital tendons.

REASON FOR PERFORMING STUDY: The injury-prone, energy-storing equine superficial digital flexor tendon (SDFT) of the mature performance horse has a limited ability to respond to exercise in contrast with the noninjury-prone, anatomically opposing common digital extensor tendon (CDET). Previous studies have indicated low levels of cellular activity in the mature SDFT, but in foal tendons the tenocytes may still have the ability to adapt positively to increased exercise. OBJECTIVES: To measure tenocyte densities and types in histological sections from the SDFT and CDET of horses from controlled long-term, short-term and foal exercise studies. METHODS: Specimens were collected from mid-metacarpal segments of the CDET and SDFT for each horse and processed for histology; central and peripheral regions of the SDFT cross-section were analysed separately (SDFTc, SDFTp). Tenocyte nuclei were counted in a total area of 1.59 mm(2) for each tendon region in each horse. Each nucleus was classified as type 1 (elongate and thin), type 2 (ovoid and plump) or type 3 (chondrocyte-like); type 1 cells are proposed to be less synthetically active than type 2 cells. RESULTS: No significant differences were noted between exercise and control groups in any of the studies, with the exception of an exercise-related reduction in the proportion of type 1 tenocytes for all tendons combined in the long-term study. There were tendon- and site-specific differences in tenocyte densities and proportions of type 1 and 2 cells in all 3 studies. CONCLUSIONS AND POTENTIAL RELEVANCE: There was no indication that exercise increased tenocyte density or proportions of the (theoretically) more active type 2 cells in immature horses (short-term and foal studies), perhaps because the training regimens did not achieve certain threshold strain levels. In the foal study these findings can still be interpreted positively as evidence that the training regimen did not induce subclinical damage.

Keratoacanthoma causing beak deformity in a budgerigar (Melopsittacus undulatus).

This report describes a keratoacanthoma causing abnormal maxillary beak growth in a 6-year-old male budgerigar (Melopsittacus undulates). Although the bird was still capable of eating, it had recently been demonstrating signs of respiratory distress and euthanasia was recommended. On histological examination, the neoplasm was dramatically effacing the normal structure of the maxilla and infiltrating into the rostral nasal sinuses. The tumour consisted of many cyst-like proliferations of well-differentiated squamous epithelial cells with central keratinization. Contiguity of the tumour cells with the stratum germinativum of the beak was noted in one microscopic field. This tumour type has not previously been reported in the avian beak; however, this case shows some similarities to subungual keratoacanthomas occurring in human patients and nailbed keratoacanthomas occurring in dogs and cats.

Solitary extramedullary plasmacytoma of the canine larynx.

An 11-year-old, female, spayed cocker spaniel was presented with dysphonia caused by a solitary laryngeal mass. Excisional biopsy was performed, and a diagnosis of plasmacytoma was made on the basis of histological examination. Further investigations showed no signs of systemic involvement. Coarse fractionated radiation therapy failed to control the tumour. Therapy was successfully instituted with a conventional combination chemotherapy protocol over a period of 14 months. The dog remains disease free 30 months after diagnosis. Most solitary, extramedullary plasmacytomas in dogs arise in the gastrointestinal tract, with fewer reports in other sites. The larynx is an uncommon sight of involvement in any species, and to the authors' knowledge, this is the first report of this tumour type in the canine larynx. In contrast to the therapeutic benefits reported in humans, the combination of surgery and radiation therapy was unsuccessful in this case, although sustained remission was gained following chemotherapy.

Metabolic bone disease in wild collared doves (Streptopelia decaocto).

The records of 666 casualty collared doves examined at a wildlife hospital in south-west England over a period of five years were reviewed. Signs of metabolic bone disease were recorded in 51.2 per cent of the juvenile birds but in only 9.6 per cent of the adults. The incidence of the condition was highest between December and February and decreased almost to zero between June and August. Histological lesions in 11 of the juvenile doves were consistent with vitamin D deficiency, possibly as a result of inadequate exposure to uvb light during the short winter days.

Patch test responses to Malassezia pachydermatis in healthy basset hounds and in basset hounds with Malassezia dermatitis.

The effects of the patch test application of Malassezia pachydermatis extracts were evaluated in seven healthy basset hounds and in seven basset hounds with Malassezia dermatitis. Antigens (4 and 0.4 mg/ml) and saline controls were applied for 48 h using filter paper discs in Finn chambers. One healthy basset hound and five affected hounds showed positive patch test reactivity to the yeast antigens. Positive patch test reactions were characterized histologically by mild epidermal hyperplasia and mild to moderate perivascular, periadnexal and interstitial infiltrates of neutrophils and CD3+ lymphocytes. Immediate intradermal test reactivity to M. pachydermatis antigens was seen in one healthy and one affected hound, whereas delayed intradermal test reactivity was seen in six healthy hounds and five affected hounds. This study indicates that patch test reactivity to M. pachydermatis antigen may occur in healthy basset hounds, and in contrast to delayed intradermal test reactivity, is more frequent in basset hounds with Malassezia dermatitis.