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SUMMARY In a breeding group of Inca terns (Larosterna inca), 14 birds died without antemortem signs of illness. Other than a poor body condition and a bloody cloaca, no symptoms were observed. Gross necropsy revealed severe segmental hemorrhagic enteritis with intralesional trematodes in most birds. Histopathologic examination revealed infiltration of lymphocytes, plasma cells, and granulocytes in the lamina propria of the duodenum and cross-sections of trematodes in the lumen. The parasites were identified as Ichthyocotylurus erraticus, a trematode of fish-eating birds. The cause of the infestation most likely was the feeding of unfrozen fresh fish. We describe the first case of a lethal I. erraticus infestation in Inca terns.

The hepatic progenitor compartment is of vital importance in liver regeneration when hepatocellular replication is impaired, as it occurs in acute fulminant hepatitis or severe liver fibrosis. It consists of resident progenitor cells in the normal liver, and ductular reaction and intermediate hepatobiliary cells in diseased livers. An histologic and immunohistochemical study was conducted to demonstrate putative hepatic progenitor cells in the normal liver (n = 5) and in a range of hepatic diseases (n = 13) in the cat. Formalin-fixed, paraffin-embedded specimens were stained with HE, the van Gieson stain, and the reticulin stain according to Gordon and Sweet, and immunohistochemically stained for cytokeratin-7 (CK7), human hepatocyte marker 1 (Heparl), and multidrug resistance-binding protein-2/ATP binding cassette C2 (MRP2). The normal feline liver contains a liver progenitor cell morphologically similar to humans and dogs, which resides in the canal of Hering. In acute and chronic feline liver diseases a ductular reaction is present, whether in the parenchyma or in a portal or septal location. The putative progenitor cells could easily be demonstrated by staining for CK7, whereas they were generally negative for Heparl and MRP2. In a parenchymal ductular reaction mitotic figures and cells with an intermediate hepatobiliary phenotype could be demonstrated. This is the first account of hepatic progenitor cells in feline liver.

A 5-year-old male Miniature Schnauzer was presented with unilateral cryptorchidism and signs of feminization. Abdominal ultrasonography revealed an enlarged right testis and a large, fluid-filled cavity that appeared to arise from the prostate. Computed tomography revealed the cavity to be consistent with an enlarged uterine body, arising from the prostate, and showed two structures resembling uterine horns that terminated close to the adjacent testes. The dog had a normal male karyotype, 78 XY. Gonadohysterectomy was performed and both the surgical and the histological findings confirmed the presence of a uterus in this male animal, resulting in a diagnosis of persistent Mullerian duct syndrome (PMDS). The enlarged intra-abdominal testis contained a Sertoli cell tumour. Computed tomography proved to be an excellent diagnostic tool for PMDS

Background/aims: The purpose of this study was to validate spontaneous chronic hepatitis and cirrhosis in dogs as a potential large animal model for fibrotic liver disease in humans by evaluating their molecular pathophysiology. Methods: Transforming growth factor-β1 (TGF-β1) signalling was analysed in liver samples of dogs with acute hepatitis (AH), chronic hepatitis (CH), cirrhosis (CIRR), and a specific form of cirrhosis, lobular dissecting hepatitis (LDH), in comparison with human cirrhotic samples from alcohol abuse (ALC) and hepatitis C (HC). Results: Canine samples were investigated with quantitative real-time PCR (Q-PCR) and Western blotting on TGF-β1 signalling including Smad2/3 phosphorylation. Immunohistochemistry on collagens I and III was performed. Q-PCR showed an increase in TGF-β1 levels and downstream effector gene products in CH, LDH, and CIRR. The same fibrotic diseases also showed an increase in phosphorylated Smad2/3 and a higher deposition of collagens I and III. In contrast, in AH neither active TGF-β1 signalling nor collagen deposition was observed. Western blot analysis on human ALC and HC indicated a high similarity with canine samples in TGF-β1 expression and Smad2/3 phosphorylation. Conclusions: Our results demonstrate that fibrosis in spontaneous dog liver diseases is highly comparable to their human counterparts and might serve as models for anti-fibrotic strategies.

A 2-year-old male peach-faced lovebird ( Agapornis roseicollis ) with a subcutaneous sarcoma on the right carpus was treated by surgical amputation. Three months after surgery, lung metastases causing clinical signs of dyspnoea were diagnosed radiographically and subsequently the bird was euthanased. At necropsy, a tumour firmly attached to the right testis, kidney and lung was found, and several tumours were present in the lung parenchyma. Histopathology revealed a mesenchymal growth pattern in the carpal subcutis and lung neoplasms, and an infiltrating epithelial pattern in the abdominal one. Immunohistochemistry for muscle actin, keratin, neurone-specific enolase and chromogranin confirmed the different cell lineage of the neoplasms, thus leading to the diagnosis of a fibrosarcoma in the subcutis with pulmonary metastases, and a carcinoma of indeterminate origin in the cranial abdomen.

In a 10-year-old castrated male shorthaired German pointer polyuria was associated with slight hypokalemia, hypophosphatemia and alkalosis, as well as elevated plasma concentrations of a glucocorticoid-inducible iso-enzyme of alkaline phosphatase. Repeated measurements of urinary corticoids and normal suppressibility of the hypothalamus-pituitary-adrenocorticial axis excluded glucocorticoid excess. Urine osmolality (Uosm) did not increase during administration of the vasopressin analogue desmopressin. At the time water deprivation had caused Uosm to rise from 300 to 788 mOsm/kg, there was also plasma hypertonicity. During hypertonic saline infusion the osmotic threshold for vasopressin release was increased. The combination of elevated plasma aldosterone concentrations and unmeasurably low plasma renin activity pointed to primary hyperaldosteronism. As initially computed tomography (CT) did not reveal an adrenocortical lesion, the dog was treated with the aldosterone antagonist spironolactone. This caused Uosm to rise in a dose-dependent manner. However, well-concentrated urine was only achieved with doses that gave rise to adverse effects. Once repeated CT, using 2-mm-thick slices, had revealed a small nodule in the cranial pole of the left adrenal, unilateral adrenalectomy was performed which resolved the polyuria completely. Also the plasma concentrations of kalium, aldosterone and renin activity returned to within their respective reference ranges. The adrenocortical nodule had the histological characteristics of an aldosteronoma, with the non-affected zona glomerulosa being atrophic. In this dog with primary hyperaldosteronism the polyuria was characterized by vasopressin resistance and increased osmotic threshold of vasopressin release, similar to the polyuria of glucocorticoid excess. The possibility is discussed that the polyuria of glucocorticoid excess is actually a mineralocorticoid effect.

Summary In a 12‐year‐old male shorthaired cat with attacks of hypo‐kalaemic muscular weakness in spite of oral potassium supplementation, highly elevated plasma aldosterone concentrations in combination with low plasma renin activity pointed to primary hyperaldosteronism. Ultrasonography and computed tomography revealed a large left‐sided adrenal tumour growing into the phrenicoabdominal vein and the caudal vena cava. The tumour and its intravascular extension were surgically removed, but the subsequent stenosis of the caudal vena cava caused congestion and renal failure. At autopsy pulmonary micrometastases of the aldosteronoma were found.

A 15-year-old Dutch Warmblood mare presented with respiratory distress and anorexia. Clinical examination revealed liquothorax and an abdominal mass. At necropsy, an infiltrating tumour was found in the visceral trunk, continuing through the diaphragm into the mediastinum. Histopathology, immunohistochemistry and electronmicroscopy showed empty channels lined by atypical endothelial cells, and neoplastic cells in a disorganized pattern, consistent with the diagnosis of a lymphangiosarcoma.

To the Editor: Toxoplasma gondii, a zoonotic protozoan parasite for which felids are the only definitive hosts, can infect humans and other warm-blooded animals. Transmission usually occurs orally from oocysts shed by felids in water and on food, through tissue cysts in undercooked meat, or transplacentally. In particular, young cats shed oocysts that can sporulate and become infectious within a day, depending on temperature and humidity. Sporulated oocysts can survive in moist soil for months to years (1). In September 2014, the number of dead squirrels reported to the Dutch Wildlife Health Centre and the Dutch Mammal Society increased suddenly. The red squirrel (Sciurus vulgaris) is the only species of squirrel endemic to the Netherlands. Members of the public claimed that squirrels were “dropping dead from trees.” Subsequently, the public was encouraged to report and submit dead squirrels. A total of 187 animals were reported through October 2014, of which 37 were submitted for necropsy. Necropsy included macroscopic examination; cytologic analysis of liver, spleen, lungs, and intestinal contents stained with hemacolor (Merck, Darmstadt, Germany); and histologic examination of samples of various organs fixed in formalin, embedded in paraffin, cut into 4-μm sections, and stained with hematoxylin and eosin. For 8 adult animals, body condition (based on degree of fat storage and muscle development) was good; 12 juveniles were in poor condition. Typically, the trachea contained foam, and lungs were hyperemic and edematous. The liver was enlarged and pale, and the spleen was enlarged. In 13 animals, numerous small crescent-shaped organisms, with eccentrically placed nuclei consistent with tachyzoites of T. gondii, were identified by cytology in lung, liver, and spleen (2). Main histopathologic findings were pulmonary interstitial lymphoplasmocytic and neutrophilic infiltrates with edema and numerous intra-alveolar macrophages (17/20) and multifocal lymphoplasmocytic infiltrates with necrosis in the liver (13/20). Extensive splenic necrosis was occasionally observed (4/20). Intestines contained mild plasmacytic infiltrates. Numerous tachyzoites consistent with T. gondii were present in alveolar macrophages and epithelial cells, splenic macrophages, and hepatocytes. Duplicate slides were stained immunohistochemically by using polyclonal antibodies against T. gondii following a standard ABC protocol (3). Organisms stained for T. gondii in liver, spleen, lungs, and intestine. Toxoplasma was not detected in any brain. DNA was isolated (DNeasy Blood and Tissue Kit; QIAGEN, Hilden, Germany) from tissues of 14 squirrels and tested by quantitative PCR (1); T. gondii DNA was detected in 13. We successfully sequenced the T. gondii GRA6 gene for 11 squirrels and identified sequences to clonal type II T. gondii previously identified in sheep from the Netherlands (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"GU325790","term_id":"283580620","term_text":"GU325790"}}GU325790) (4). Incidental findings in the animals tested were encephalitis (2/20), coccidiosis (5/20), trauma (6/20), myocarditis (4/20), nephritis (1/20), lymphadenitis (1/20), and intestinal (3/20) and external (5/20) parasites. The remaining 17 animals showed >1 of the following pathologic conditions: hemorrhages consistent with trauma (12/17), mild to severe intestinal coccidiosis (12/17), pneumonia (3/17), splenitis (1/17), Taenia martis cysticerci (1/17), and external parasites (8/17). Immunohistochemistry results for all 17 were negative for T. gondii. On the basis of necropsy and molecular findings, we conclude that 20 of 37 examined squirrels died of disseminated T. gondii type II infection. These animals included adults and juveniles and were not restricted to specific geographic areas (Figure). The remaining animals died of trauma (12/17) or other causes (5/17). Figure Spatial distribution of wild red squirrels (Sciurus vulgaris) investigated for Toxoplasma gondii and classified by cause of death, the Netherlands, 2014. Red squirrels are susceptible to T. gondii, and infection can lead to death. However, in our sample, the proportion of squirrels that died of toxoplasmosis (>50%) was higher than in other studies (≈16%) (5–7). The apparent increase in squirrel deaths and unexpectedly high proportion of fatal T. gondii infections suggests a toxoplasmosis outbreak among red squirrels. Possible explanations for this surge in cases include increased exposure to the parasite, increased susceptibility to infection, or increased virulence of the pathogen. Clonal T. gondii type II, the strain most frequently involved in human cases and endemic to Europe and North America, was identified. An increased virulence of the pathogen could not be proven (8). On the basis of lymphoid hyperplasia in the spleen and lymph nodes, affected squirrels had no signs of immunosuppression. Thus, the most likely explanation is increased exposure to the parasite. Sources of infection for red squirrels are not known; however, oocysts shed in cat feces may contaminate the nuts, fungi, shoots, and berries that constitute the diet of the squirrel. Stray, unspayed cats are common in the Dutch countryside. More than 3 million domestic cats (Felis domesticus) exist in the Netherlands, including several tens of thousands of free-roaming cats that reproduce (9). Determining the exact source of infection is important because humans also harvest wild fruits, nuts, and fungi from these areas. This outbreak highlights that contamination of the environment with T. gondii oocysts is of concern not only from a public health viewpoint but from a biodiversity perspective as well (1,10).

A 6-year-old Friesian stallion was examined because of signs of exercise intolerance, stiff gait and symmetrical hind weakness, and increased serum liver enzymes. On presentation, the horse showed muscle atrophy of the hindquarters. Neurological investigation showed no abnormalities. Laboratory findings revealed a prolonged prothrombin time and increased levels of alkaline phosphatase (AF), aspartate aminotransferase (ASAT), gamma-glutamyl-transferase (GGT), lactate dehydrogenase (LDH), and bile acids. Histological evaluation of the liver revealed severe cirrhosis and intracytoplasmic greyish brown granules in almost all hepatocytes, sinusoidal Kuppfer cells, and macrophages. These granules stained strongly for copper. Treatment to slow hepatic fibrosis was advised and included oral prednisolone administration for at least 1 month. A diet to support liver function was formulated by a nutritional specialist, and vitamin E was advised as dietary supplement to support neuromuscular function. Soon after diagnosis, the animal showed signs of intravascular haemolysis, with the presence of Heinz bodies in peripheral blood smears, and haemoglobinuria. On the basis of this haemolytic crisis and the poor prognosis of the chronic hepatic disease, the horse was euthanized at the owners' request. Although we could not establish the cause of the hepatic copper accumulation, this case report highlights that excessive copper in the liver should be considered in the differential diagnosis of hepatic cirrhosis and Heinz body anaemia in the horse.

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.

To the Editor: A Q fever epidemic among humans started in the Netherlands in 2007 and peaked in 2009 (1). Epidemiologic evidence linked the epidemic to abortions and deliveries among Coxiella burnetii–infected dairy goats and dairy sheep (1,2). However, questions arose about whether C. burnetii infection in free-living wildlife might be another source of Q fever in humans. C. burnetii has a wide host range (3), but to our knowledge no studies had addressed its occurrence in nondomestic animals in the Netherlands (4). The main objective of this study was to look for evidence of C. burnetii infection in carcasses of free-living roe deer (Capreolus capreolus) in the Netherlands, where C. capreolus is the most common species of wild ruminant. Additional objectives were to 1) analyze characteristics, location, and time of death of case-animals for more information on the infection in roe deer and 2) determine the genotype of C. burnetii strains from roe deer and compare them with the genotype of strains from domestic animals and humans for evidence of spillover. The sample consisted of 79 roe deer that were euthanized or found dead in 9 of the 12 provinces in the Netherlands during January 2008–May 2010. All animals had undergone postmortem examination, and tissue samples were frozen until testing. Tissues tested were lung (n = 46), spleen (n = 50), bone marrow (n = 50), liver (n = 74), and kidney (n = 75), as available. We extracted DNA by using the DNeasy Blood and Tissue Kit (QIAGEN, Hilden, Germany). A duplex quantitative PCR targeting the IS1111a element was used with an internal control gene, as described (2). Tissues with cycle threshold (Ct) values 1 year of age, 15 (30%) had positive results, compared with 2 (15%) of 13 deer

Hepatocyte growth factor (HGF) is crucial for the development and regeneration of the liver and offers a possible new therapeutic strategy for the treatment of canine liver disease. In this study, the in vitro and in vivo bioactivity of recombinant canine HGF (rcHGF) produced with a baculoviral expression system in insect cells was measured. In vitro rcHGF induced mitogenesis, motogenesis, and phosphorylated the HGF receptor c-MET and its downstream mediators PKB and ERK1/2 in two canine epithelial cell lines. After a partial hepatectomy (phx) in dogs, rcHGF increased phosphorylation of c-MET, PKB and ERK1/2. A moderate increase was seen with the cell cycle protein PCNA in rcHGF treated livers, but no HGF-induced increase in liver weight was seen 7 days after phx. Furthermore, rcHGF treated livers showed lower levels of the key mediator of apoptosis, caspase-3, at 7 days after phx. It is concluded that rcHGF is a biologically active protein in vitro and in vivo and the baculoviral expression system supplies sufficient amounts of rcHGF for future clinical studies in dogs.

Background Hepatic fibrosis is a common outcome of hepatic injury in both man and dog. Activated fibroblasts which develop myofibroblastic characteristics play an essential role in hepatic fibrogenesis, and are comprised of three subpopulations: 1) portal or septal myofibroblasts, 2) interface myofibroblasts and 3) the perisinusoidally located hepatic stellate cells (HSC). The present study was performed to investigate the immunohistochemical characteristics of canine portal myofibroblasts (MF) and HSC in the normal unaffected liver as a basis for further studies on fibrogenesis in canine liver disease. Results In the formalin-fixed and paraffin embedded normal canine liver vimentin showed staining of hepatic fibroblasts, probably including MF in portal areas and around hepatic veins; however, HSC were in general negative. Desmin proved to react with both portal MF and HSC. A unique feature of these HSC was the positive immunostaining for alpha-smooth muscle actin (α-SMA) and muscle-specific actin clone HHF35 (HHF35), also portal MF stained positive with these antibodies. Synaptophysin and glial fibrillary acidic protein (GFAP) were consistently negative in the normal canine liver. In a frozen chronic hepatitis case (with expected activated hepatic MF and HSC), HSC were negative to synaptophysin, GFAP and NCAM. Transmission electron microscopy (TEM) immunogold labelling for α-SMA and HHF35 recognized the positive cells as HSC situated in the space of Disse. Conclusion In the normal formalin-fixed and paraffin embedded canine liver hepatic portal MF and HSC can be identified by α-SMA, HHF35 and to a lesser extent desmin immunostaining. These antibodies can thus be used in further studies on hepatic fibrosis. Synaptophysin, GFAP and NCAM do not seem suitable for marking of canine HSC. The positivity of HSC for α-SMA and HHF35 in the normal canine liver may eventually reflect a more active regulation of hepatic sinusoidal flow by these HSC compared to other species.

Background We analyzed two spontaneous dog diseases characterized by subnormal portal perfusion and reduced liver growth: (i) congenital portosystemic shunts (CPSS) without fibrosis and (ii) primary portal vein hypoplasia (PPVH), a disease associated with fibrosis. These pathologies, that lack inflammation or cholestasis, may represent simplified models to study liver growth and fibrosis. To investigate the possible use of those models for hepatocyte growth factor (HGF) treatment, we studied the functionality of HGF signaling in CPSS and PPVH dogs and compared this to aged-matched healthy controls. Results We used quantitative real-time polymerase chain reaction (Q-PCR) to analyze the mRNA expression of HGF, transforming growth factor β1 (TGF-β1), and relevant mediators in liver biopsies from cases with CPSS or PPVH, in comparison with healthy control dogs. CPSS and PPVH were associated with a decrease in mRNA expression of HGF and of MET proto-oncogene (c-MET). Western blot analysis confirmed the Q-PCR results and showed that intracellular signaling components (protein kinase B/Akt, ERK1/2, and STAT3) were functional. The TGF-β1 mRNA levels were unchanged in CPSS whereas there was a 2-fold increase in PPVH indicating an active TGF-β1 pathway, consistent with the observation of fibrosis seen in PPVH. Western blots on TGF-β1 and phosphorylated Smad2 confirmed an activated pro-fibrotic pathway in PPVH. Furthermore, Q-PCR showed an increase in the amount of collagen I present in PPVH compared to CPSS and control, which was confirmed by Western blot analysis. Conclusion The pathophysiological differences between CPSS and PPVH can adequately be explained by the Q-PCR measurements and Western blots. Although c-MET levels were reduced, downstream signaling seemed to be functional and provides a rational for HGF-supplementation in controlled studies with CPSS and PPVH. Furthermore both diseases may serve as simplified models for comparison with more complex chronic inflammatory diseases and cirrhosis.

Summary A 22‐year‐old Dutch Warmblood mare was referred to Utrecht University with progressive left hind limb paresis and hyporeflexia. The preliminary clinical diagnosis was the neurological form of equine herpes virus (EHV‐1) infection. Within 1 day of admission, the mare became recumbent and deteriorated rapidly. Postmortem examination revealed an adenocarcinoma of the caecum, with metastases in all regional lymph nodes and extending from the lumbar nodes into the vertebral canal, causing spinal cord compression and destruction of the left 4th and 5th lumbar nerves.

Background Hepatocellular carcinoma (HCC) is one of the most worldwide frequent primary carcinomas resulting in the death of many cirrhotic patients. Unfortunately, the molecular mechanisms of this cancer are not well understood; therefore, we need a good model system to study HCC. The dog is recognized as a promising model for human medical research, namely compared with rodents. The objective of this study was to establish and characterize a spontaneous canine tumor cell line as a potential model for studies on HCC.