Chronic iron overload causing haemochromatosis and hepatopathy in 21 horses and one donkey.

BACKGROUND: Iron toxicosis is rarely reported in horses and chronic excessive oral iron intake has not been reported to cause clinical symptoms in equids. OBJECTIVES: This case series describes 21 genetically unrelated horses and one donkey with chronic iron overload causing haemochromatosis and hepatopathy. STUDY DESIGN: Case series. METHODS: All equids showing clinical signs compatible with chronic liver disease presented to Utrecht University and diagnosed with iron overload and haemochromatosis based on histopathological evaluation of liver tissue and/or blood transferrin saturation levels of >80% and proof of excess dietary iron intake due to excess iron content in drinking water were included. RESULTS: This study included 22 equids. All tested animals (n = 19) had transferrin saturation >80% and 21 of 22 had increased gamma-glutamyltransferase (γGT). Ultrasonography revealed rounded liver margins in five out of six horses and increased echogenicity in 4/6. Histological examination of liver tissue of 12 animals showed hepatitis, fibrosis and haemosiderin accumulation in macrophages and hepatocytes. Post-mortem examination also revealed haemosiderin accumulation in other organs in all seven examined animals. High iron content in drinking water was identified as the source of iron overload in all cases. All animals were housed under the same conditions for a minimum of 9 years prior to diagnosis of haemochromatosis. Of 22 animals, 13 survived until 1 January 2018, ranging from 17 to 79 months post diagnosis. MAIN LIMITATIONS: Histology of liver tissue was not available for 10 of 22 cases. CONCLUSIONS: Chronic iron overload can lead to haemochromatosis and hepatopathy in equids. Development of disease is slow and clinical signs are nonspecific. Long-term excessive iron intake in equids should be avoided. If animals drink from natural water sources, it is important to test the water for iron content. The Summary is available in Spanish - see Supporting Information.

Controlled release of celecoxib inhibits inflammation, bone cysts and osteophyte formation in a preclinical model of osteoarthritis.

Major hallmarks of osteoarthritis (OA) are cartilage degeneration, inflammation and osteophyte formation. COX-2 inhibitors counteract inflammation-related pain, but their prolonged oral use entails the risk for side effects. Local and prolonged administration in biocompatible and degradable drug delivery biomaterials could offer an efficient and safe treatment for the long-term management of OA symptoms. Therefore, we evaluated the disease-modifying effects and the optimal dose of polyesteramide microspheres delivering the COX-2 inhibitor celecoxib in a rat OA model. Four weeks after OA induction by anterior cruciate ligament transection and partial medial meniscectomy, 8-week-old female rats (n = 6/group) were injected intra-articular with celecoxib-loaded microspheres at three dosages (0.03, 0.23 or 0.39 mg). Unloaded microspheres served as control. During the 16-week follow-up, static weight bearing and plasma celecoxib concentrations were monitored. Post-mortem, micro-computed tomography and knee joint histology determined progression of synovitis, osteophyte formation, subchondral bone changes, and cartilage integrity. Systemic celecoxib levels were below the detection limit 6 days upon delivery. Systemic and local adverse effects were absent. Local delivery of celecoxib reduced the formation of osteophytes, subchondral sclerosis, bone cysts and calcified loose bodies, and reduced synovial inflammation, while cartilage histology was unaffected. Even though the effects on pain could not be evualated directly in the current model, our results suggest the application of celecoxib-loaded microspheres holds promise as novel, safe and effective treatment for inflammation and pain in OA.

Prolactinoma in a Dog.

A 12-year-old male Yorkshire Terrier was presented because of decreased appetite. Physical examination revealed mammary gland swelling and galactorrhea. Contrast-enhanced computed tomographic scanning of the skull indicated an enlarged pituitary gland, compatible with a pituitary tumor. The serum prolactin concentration was markedly elevated. One week after the start of treatment with the dopamine agonist cabergoline, the serum prolactin concentration normalized and the galactorrhea resolved. Cabergoline was administered for approximately 4 months and then discontinued. Subsequently, serum prolactin concentration increased again, and mammary gland swelling and galactorrhea reappeared. The dog was euthanized 10 months after the first detection of the galactorrhea because of problems not directly related to pituitary disease. Postmortem examination revealed an infiltrative adenoma of the pituitary gland with immunolabeling for prolactin. The clinical and histopathologic findings indicated the diagnosis of a functional prolactinoma in a male dog.

Hydrated nucleus pulposus herniation in seven dogs.

The clinical signs, magnetic resonance imaging (MRI) findings, treatment and follow-up in seven dogs with hydrated nucleus pulposus extrusion (HNPE) are reported. All dogs had tetraparesis or tetraplegia. T2-weighted MRI revealed extradural hyperintense homogeneous material compressing the cervical spinal cord. After conservative treatment (five dogs) or surgical decompression (two dogs), all dogs returned to ambulatory function within 1 month. Follow-up MRI in conservatively treated dogs revealed complete disappearance of the extruded material. Histopathological examination of surgical specimens confirmed that the retrieved material was extruded nucleus pulposus with evidence of early degeneration.

Atlanto-axial malformation and instability in dogs with pituitary dwarfism due to an LHX3 mutation.

BACKGROUND: Canine pituitary dwarfism or combined pituitary hormone deficiency (CPHD) in shepherd dogs is associated with an LHX3 mutation and can lead to a wide range of clinical manifestations. Some dogs with CPHD have neurological signs that are localized to the cervical spine. In human CPHD, caused by an LHX3 mutation, anatomical abnormalities in the atlanto-axial (C1-C2) joint have been described. OBJECTIVES: To evaluate the presence of atlanto-axial malformations in dogs with pituitary dwarfism associated with an LHX3 mutation and to investigate the degree of similarity between the atlanto-axial anomalies found in canine and human CPHD patients with an LHX3 mutation. ANIMALS: Three client-owned Czechoslovakian wolfdogs and 1 client-owned German shepherd dog, previously diagnosed with pituitary dwarfism caused by an LHX3 mutation, with neurological signs indicating a cervical spinal disorder. METHODS: Radiography, computed tomography, and magnetic resonance imaging of the cranial neck and skull, necropsy, and histology. RESULTS: Diagnostic imaging identified abnormal positioning of the dens axis and incomplete ossification of the suture lines between the ossification centers of the atlas with concurrent atlanto-axial instability and dynamic compression of the spinal cord by the dens axis. The malformations and aberrant motion at C1-C2 were confirmed at necropsy and histology. CONCLUSIONS AND CLINICAL IMPORTANCE: The atlanto-axial abnormalities of the dwarf dogs resemble those encountered in human CPHD patients with an LHX3 mutation. These findings suggest an association between the LHX3 mutation in dogs with CPHD and atlanto-axial malformations. Consequently, pituitary dwarfs should be monitored closely for neurological signs.

Skeletal and hepatic changes induced by chronic vitamin A supplementation in cats.

The first aim of this study was to determine whether vitamin D supplementation influenced the effects of high vitamin A intake on new bone formation in adult cats. The second aim was to determine whether high vitamin A intake in cats caused liver pathology and, if so, whether the current upper limit for the dietary intake of vitamin A for healthy adult cats would be safe. Twenty-four healthy adult cats were divided into four groups that received a control diet supplemented with peanut oil (control), or peanut oil containing a 100-fold increase in vitamin A (HA), or a 100-fold increase in vitamin A and a fivefold increase in vitamin D (HAMD), or a 100-fold increase in vitamin A and a 65-fold increase in vitamin D (HAHD) over a period of 18 months. Cats did not show abnormal locomotion or clinical signs of liver failure after 18 months of supplementation but did show subtle skeletal changes and liver pathology, suggesting that the current National Research Council (2006) safe upper limit for vitamin A for cats is too high. The addition of vitamin D did not seem to influence bone pathology. While moderately elevated dietary vitamin D levels (HAMD) seemed to protect cats against the liver pathology caused by the consumption of large amounts of vitamin A, higher dietary levels of vitamin D (HAHD) did not seem to be protective.

The canine hepatic progenitor cell niche: molecular characterisation in health and disease.

Hepatic progenitor cells (HPCs) are an adult stem cell compartment in the liver that contributes to liver regeneration when replication of mature hepatocytes is insufficient. In this study, laser microdissection was used to isolate HPC niches from the livers of healthy dogs and dogs with lobular dissecting hepatitis (LDH), in which HPCs are massively activated. Gene expression of HPC, hepatocyte and biliary markers was determined by quantitative reverse transcriptase PCR. Expression and localisation of selected markers were further studied at the protein level by immunohistochemistry and immunofluorescent double staining in samples of normal liver and liver from dogs with LDH, acute and chronic hepatitis, and extrahepatic cholestasis. Activated HPC niches had higher gene expression of the hepatic progenitor markers OPN, FN14, CD29, CD44, CD133, LIF, LIFR and BMI1 compared to HPCs from normal liver. There was lower expression of albumin, but activated HPC niches were positive for the biliary markers SOX9, HNF1ß and keratin 19 by immunohistochemistry and immunofluorescence. Laminin, activated stellate cells and macrophages are abundant extracellular matrix and cellular components of the canine HPC niche. This study demonstrates that the molecular and cellular characteristics of canine HPCs are similar to rodent and human HPCs, and that canine HPCs are distinctively activated in different types of liver disease.

Delayed endochondral ossification in early medial coronoid disease (MCD): a morphological and immunohistochemical evaluation in growing Labrador retrievers.

Medial coronoid disease (MCD) is a common joint disease of dogs. It has a multifactorial aetiology, but the relationship between known causal factors and the disease has yet to be elucidated. As most of the published literature is clinical and it reports changes associated with advanced disease, it is not known whether the changes reflect the cause or consequences of the condition. The aim of this study was to investigate early micromorphological changes occurring in articular cartilage and to describe the postnatal development of the medial coronoid process (MCP) before MCD develops. Three litters of MCD-prone young Labrador retrievers were purpose-bred from a dam and two sires with MCD. Comparisons of the micromorphological appearance of the MCP in MCD-negative and MCD-positive joints demonstrated that MCD was initially associated with a disturbance of endochondral ossification, namely a delay in the calcification of the calcifying zone, without concurrent abnormalities in the superficial layers of the joint cartilage. Cartilage canals containing patent blood vessels were only detected in dogs <12 weeks old, but the role of these channels in impaired ossification requires further investigation. Retained hyaline cartilage might ossify as the disease progresses, but weak areas can develop into cracks between the retained cartilage and the subchondral bone, leading to cleft formation and fragmentation of the MCP.

Intervertebral disc disease in dogs - part 1: a new histological grading scheme for classification of intervertebral disc degeneration in dogs.

Intervertebral disc (IVD) degeneration is common in dogs and can lead to serious disorders. Current treatments can relieve clinical signs of disease, but do not restore IVD function. The development of regenerative strategies for IVD dysfunction requires detailed knowledge of the pathogenesis of IVD degeneration and its underlying mechanisms. Histological examination of IVDs at different stages of degeneration might provide this knowledge, but as there is currently no histological grading scheme for canine IVD degeneration, the aim of this study, which is the first of a two-part series, was to design and validate an appropriate scheme. Three independent observers evaluated 35 IVDs at different stages of degeneration using the scheme. Glycosaminoglycan contents of the nucleus pulposus and macroscopic grading according to Thompson, which are considered 'gold standards' for IVD degeneration, were used to validate the scheme. Reproducibility was assessed by analysing the inter-observer reliability of all individual variables of the grading scheme, using a weighted κ analysis. Significant correlations were found between Thompson grading and total histological score (r=0.94; P<0.01) and between glycosaminoglycan content and total histological score (r=-0.72; P<0.01). Most individual histological variables showed 'moderate' to 'almost perfect' inter-observer reliability. The high correlation with the gold standards in combination with the high reproducibility indicates that the proposed histological grading scheme is reliable and objective for classification of IVD degeneration in both chondrodystrophic and non-chondrodystrophic dog breeds.

Disorders of sexual development and associated changes in the pituitary-gonadal axis in dogs.

Normal sexual differentiation depends on completion of chromosomal sex determination, gonadal differentiation, and development of the phenotypic sex. An irregularity in any of these three steps can lead to a disorder in sexual development (DSD). We examined nine dogs with DSD by abdominal ultrasonography, laparotomy, histologic examination of the gonads, and reproductive tract, cytogenetic analysis, and mRNA expression of the SRY gene. We also determined the plasma concentrations of luteinizing hormone (LH), estradiol-17ß, and testosterone before and after administration of gonadotropin-releasing hormone (GnRH) and compared these results with those obtained in anestrous bitches and male control dogs. The gonads of three dogs with DSD contained both testicular and ovarian tissue, while in the other six only testicular tissue was found. Each of the dogs had a uterus. Based on gynecologic examination, cytogenetic analysis, and the histology of the gonads, seven of the nine dogs appeared to be XX sex reversals. Three of these were XX true hermaphrodites and four were XX males; the other two dogs had incomplete XY gonadal dysgenesis. All seven XX sex-reversed dogs were found to be negative for the SRY gene by polymerase chain reaction. The basal plasma luteinizing hormone (LH) concentration was significantly higher in dogs with DSD than in anestrous bitches but not significantly different from that in male dogs. The basal plasma LH concentration increased significantly after GnRH administration in all dogs with DSD. The basal plasma estradiol concentration was significantly higher in dogs with DSD than in anestrous bitches but not significantly different from that in male dogs. The basal plasma testosterone concentration was lower in dogs with DSD than in male dogs. In all dogs with DSD both the basal and GnRH-induced plasma testosterone concentrations were above the upper limit of their respective ranges in the anestrous bitches. In conclusion, the secretion of LH and estradiol in these dogs with DSD, all of which had testicular tissue in their gonads, was similar to that in male control dogs. These results indicate that the basal and/or GnRH-stimulated plasma testosterone concentration might be used to detect the presence of testicular tissue in dogs with DSD.

Lymphocytic hypophysitis in a dog with diabetes insipidus.

An 8-year-old male German longhaired pointer was referred for diabetes insipidus responsive to treatment with desmopressin. The dog had polyuria and polydipsia, exercise intolerance and a dull hair coat. Plasma concentrations of thyroid-stimulating hormone, thyroxine, growth hormone (GH) and insulin-like growth factor-1 were decreased; plasma adrenocorticotropic hormone (ACTH) was slightly elevated and plasma α-melanocyte-stimulating hormone (MSH) was within the reference range. Computed tomography revealed a heterogeneously contrast-enhancing pituitary mass compressing the hypothalamus. Transsphenoidal hypophysectomy was performed and microscopical examination of the surgical biopsy samples revealed hypophysitis without evidence of pituitary adenoma. The hypophysitis was characterized by marked lymphocytic infiltration of the adenohypophysis that contained a mixed population of neuroendocrine cells expressing GH, ACTH or α-MSH. The lymphocytes were identified as T cells, resulting in a final diagnosis of lymphocytic hypophysitis strongly resembling human primary lymphocytic hypophysitis.

Gluten-dependent antibodies in horses with inflammatory small bowel disease (ISBD).

BACKGROUND: Equine inflammatory small bowel disease (ISBD) is an idiopathic pathologic condition seeming to increase in prevalence. OBJECTIVE: To investigate the potential role of gluten in equine ISBD. ANIMALS & METHODS: Antibodies known to be important in the diagnosis of human coeliac disease (CD): IgA antibodies to human recombinant and guinea pig tissue-transglutaminase (TGA), native gliadin (AGA), deamidated-gliadin-peptides (DGPA), and primate and equine endomysium (EMA) were assessed in blood samples from three different groups of horses: ISBD affected (n = 12) on a gluten-rich diet and controls either on gluten-rich (n = 22) or gluten-poor (n = 25) diets. Significant differences (p < 0.05) between groups were assessed using the Wilcoxon test. RESULTS: Both ISBD-affected horses and gluten-rich controls had significantly (p < 0.0004) higher hrTGA titers than gluten-poor controls. However, ISBD horses did not show significantly increased levels of any of the CD related antibodies when compared to gluten-rich controls. Nevertheless, markedly increased antibody levels (TGA, EMA and DGPA) were found in one of the ISBD horses. The introduction of a gluten-free ration in this 14-year-old warmblood stallion resulted after 6 months in the reduction of antibody levels and clinical recovery associated with improved duodenal histopathology. CONCLUSION: To the best of our knowledge, this is the first study assessing gluten-related antibodies in horses and results suggest a potential pathogenic role of gluten in at least some cases of equine ISBD. Clinical importance and impact for human medicine: Given serology and concurrent clinical findings, this study warrants further investigations into the immunologic basis of possible gluten-sensitive enteropathy in horses and analogy with human disease.

Expression of Ki-67, PCNA, and p27kip1 in canine pituitary corticotroph adenomas.

Pituitary-dependent hypercortisolism (PDH), which is caused by adrenocorticotropic hormone (ACTH)-secreting pituitary adenomas, is a common endocrinopathy in dogs. Dogs with non-enlarged pituitaries harboring a microadenoma have a better prognosis than those with enlarged pituitaries. The aim of this study was to investigate the expression of the proliferation markers Ki-67 and proliferating cell nuclear antigen (PCNA) and the cell-cycle inhibitor p27kip1 in corticotroph adenomas in enlarged and non-enlarged pituitaries. The expression of Ki-67, PCNA, and p27kip1 was analyzed by immunohistochemical staining of 17 pituitary adenoma samples harvested during pituitary surgery in dogs with PDH. The labeling index was calculated by counting the number of immunopositive cells per 1,000 cells. The mean (+/- standard deviation) labeling index for Ki-67 was 8.4%+/-14.2% for the group with enlarged pituitaries, and 8.8%+/-5.5% for the group with non-enlarged pituitaries; that for PCNA was 35.5%+/-12.2% and 37.0%+/-15.5%; and that for p27kip1 was 29.3%+/-22.6% and 42.5%+/-27.9%, respectively. No significant differences in Ki-67, PCNA, and p27kip1 labeling indices were found between enlarged and non-enlarged pituitaries. However, a trend toward significance was observed when comparing the expression of p27kip1 in enlarged pituitaries versus normal pituitary tissue. It is concluded that Ki-67 and PCNA are not useful as proliferative markers for studying the pathobiology of pituitary corticotroph adenomas in dogs.

Histopathological effects of chronic aqueous exposure to bis(tri-n-butyltin)oxide (TBTO) to environmentally relevant concentrations reveal thymus atrophy in European flounder (Platichthys flesus).

Although the use of tributyltin in antifouling paints has been banned, this compound is still a serious pollutant of the marine environment. This paper describes a unique study in which European flounder (Platichthys flesus) were chronically (8 months) exposed to bis(tri-n-butyltin)oxide (TBTO) in the water under controlled laboratory conditions. Residue levels in selected tissues (liver, muscle) and general health status indices were measured and the effects on several organs (gills, liver, mesonephros, ovary/testis, spleen, and gastrointestinal tract) were examined histopathologically. Additionally, morphometric analysis of the thymus was performed. The major finding is that exposure of flounder to 5 microg TBTO/l over a period of 8 months, resulting in body burdens comparable to high field levels, induced significant reduction of thymus volume, possibly affecting immunocompetence of the animals. Chronic exposure of European flounder to tributyltin is therefore likely to affect the general health status of this species in heavily polluted aquatic environments.

67Gallium citrate scintigraphy to assess metastatic spread in a dog with an oral melanoma.

Gallium scintigraphy was used to evaluate therapeutic response in a 10-year-old, male, Dutch sheepdog, suffering from an oral melanoma. Treatment was performed with a combination of carboplatin and hypofractionated radiation. Nineteen weeks after radiation therapy, the left submandibular lymph node was surgically removed because of metastatic disease. Thirty weeks after radiation therapy, 67Gallium scintigraphy was performed to assess for residual disease and metastasis. Increased uptake in the right submandibular lymph node area was noted and identified as a melanoma metastasis on cytology. Surgical excision was performed. Twenty-one weeks later, the dog was euthanased because of advanced pulmonary metastases. This report of a case of oral melanoma illustrates the advantages of 67Gallium scintigraphy in monitoring for the presence of metastatic disease and effectiveness of therapy.

Case report: epitheliotropic lymphoma in a zebrafish (Danio rerio).

A proliferative disorder of lymphocytes was observed in a macroscopically normal young adult wild-type zebrafish (Danio rerio) during routine histological screening in a two-generation toxicity study. Proliferating lymphocytes were observed to invade the gill arches, infiltrate the cranial skeletal muscle and inner ear, and accumulate at distant sites in the frontal epidermis. The test compound, tetrabromobisphenol A (TBBPA, a flame retardant), is not known as a carcinogen and no tumours were detected in any of the 53 other fish in the study, including tank mates. Although neoplastic lymphoid proliferation in the thymus region is occasionally observed, we have never seen epitheliotropism in zebrafish during other similar exposure studies or brood stock. Our findings indicate that epitheliotropic lymphoma can occur spontaneously in zebrafish but at a low incidence.

Collapse following gastrointestinal bleeding secondary to a congenital duodenal diverticulum in two littermate boxer pups.

Two littermates, a young male and female boxer, were admitted to the Utrecht University's Department of Clinical Sciences of Companion Animals within a three month period. Both dogs suffered from anaemia caused by chronic intestinal blood loss, vomiting and weight loss. In both cases, there was no response to conservative medical management. Eventually, the dogs suffered significant gastrointestinal haemorrhage that resulted in collapse. Gastroduodenoscopy and exploratory surgery showed a duodenal diverticulum in both dogs. This is the first report that describes this congenital anomaly in two siblings.

Telomerase reverse transcriptase (TERT) expression and proliferation in canine brain tumours.

Telomerase is a ribonucleoprotein enzyme complex that synthesizes telomere DNA. It is detected in 85-90% of malignant tumours in humans, but not in most somatic cells. Because telomerase plays a critical role in cell immortality, it represents an important target for anticancer therapies. We have previously shown that the dog is a potentially useful model for evaluating telomerase-based therapeutics. In this present study we analysed 93 canine brain tumours for telomerase reverse transcriptase (TERT) expression by immunohistochemistry. TERT immunoreactivity was detected in 16 of 50 grade 1 (32%) and 29 of 43 grade 2 tumours (67.4%), demonstrating a statistically significant association with histological grade (P = 0.00012). A subset of 51 tumours was also assessed for MIB-1 expression. The MIB-1 labelling index (LI) was found to correlate significantly with tumour grade, with a mean MIB-1 LI of 1.5% for grade 1 tumours, as compared with a mean MIB-1 LI of 21.7% for grade 2 tumours (P << 0.001). The MIB-1 LI was also significantly associated with TERT expression in all brain tumours (P << 0.001). These data further support the dog as a model for the preclinical development of telomerase-based therapeutics in brain tumours.