Identification of apolipoprotein E-derived amyloid within cholesterol granulomas of leopard geckos (Eublepharis macularius).

Apolipoprotein E (ApoE) is involved in cholesterol transport among cells and also plays an important role in amyloid formation, co-depositing with amyloid fibrils in various types of amyloidosis. Although the in vivo amyloidogenicity of ApoE has not been previously demonstrated, this study provides evidence of ApoE amyloidogenicity in leopard geckos (Eublepharis macularius), belonging to the class Reptilia. Histologically, amyloid deposits were localized within cholesterol granulomas and exhibited positive Congo red staining, with yellow to green birefringence under polarized light. On mass spectrometry-based proteomic analysis, ApoE was detected as a dominant component of amyloid; of the full length of the 274 amino acid residues, peptides derived from Leu185-Arg230 were frequently detected with non-tryptic truncations. Immunohistochemistry with anti-leopard gecko ApoE antibody showed positive reactions of amyloid deposits. These results show that ApoE is an amyloid precursor protein within the cholesterol granulomas of leopard geckos. Although further investigations are needed, the C-terminal region of ApoE involved in amyloid formation is a lipid-binding region, and there should be a relationship between amyloidogenesis and the development of cholesterol granulomas in leopard geckos. This study provides novel insights into the pathogenesis of ApoE-related diseases.

Disseminated histiocytic sarcoma in a degu (Octodon degus).

A 7-year-and-8-month-old, male degu (Octodon degus) with anorexia, depression, and labored breathing was found to have a thoracic effusion and enlargement of the right testis on radiographic examination. Despite treatment, the animal died. At necropsy, hepatomegaly, splenomegaly, and multifocal nodules on the intestinal serosa and mesentery were observed. Histologically, the foci were densely cellular invasive neoplasms composed of sheets of round to polygonal cells, with occasional multinucleated giant cells. Immunohistochemically, the neoplastic cells were immunopositive for ionized calcium-binding adapter molecule 1, human leukocyte antigen-DR, and CD204. These findings were consistent with disseminated histiocytic sarcoma.

Bilateral renal dysplasia with systemic fibrous osteodystrophy in a four-toed hedgehog (Atelerixalbiventris).

A 4-month-old female four-toed hedgehog (Atelerix albiventris) presented with lethargy, anorexia, dyspnoea and weight loss. Following death, post-mortem computed tomography (CT) and an autopsy were performed. CT revealed that the external surfaces of bones, including the cranial bones and vertebrae, were rough and osteolytic lesions were present multifocally in the ribs and some appendicular bones. On gross examination, both kidneys were severely enlarged and, on cut sections, a few cysts (up to 1 mm diameter) were present in the medulla. The cervical, thoracic and lumbar vertebrae were diffusely enlarged with deformation of the intervertebral discs. Histologically, there were immature glomeruli and tubules and adenomatoid/atypical epithelium in the kidneys. These changes were interpreted as renal dysplasia. In the bones evaluated, the trabeculae were thinner than normal, decreased in number and surrounded by many osteoclasts, with abundant fibrous connective tissue between atrophied trabeculae. These changes were consistent with fibrous osteodystrophy. Although kidney diseases are common in four-toed hedgehogs, there are no reports of congenital renal diseases, including renal dysplasia. To the best of our knowledge, this is the first report of the clinical and pathological features of renal dysplasia with fibrous osteodystrophy in a four-toed hedgehog.

Aortic valve insufficiency caused by a hypoplastic aortic valve in a calf.

A 1-month-old crossbred calf was referred for examination due to marked systolic heart murmurs and poor growth. The heart murmur was most audible on the right side of the cranial thorax. Cardiomegaly was evident on chest radiography, and echocardiography demonstrated aortic regurgitation and decreased fractional shortening. Cardiomegaly, aortic root dilation and cardiac displacement were confirmed by computed tomography. At necropsy, the heart was enlarged, and all three aortic valve leaflets were irregularly shaped. In calves with chronic aortic insufficiency, remodeling displacement of the heart and aorta causes changes in the location and timing of heart murmurs. Therefore, aortic insufficiency cannot be ruled out when a systolic heart murmur can be observed in the right chest wall.

Fibrinogen Aα-chain amyloidosis outbreaks in Japanese squirrels (Sciurus lis): a potential disease model.

Fibrinogen Aα-chain amyloidosis is a hereditary systemic amyloidosis characterized by glomerular amyloid depositions, which are derived from the fibrinogen Aα-chain variant in humans. Despite its unique pathology, the pathogenic mechanisms of this disease are only partially understood. This is in part because comparative pathological studies on fibrinogen Aα-chain amyloidosis are currently unavailable as there is a lack of reported cases in animals other than humans. In this study, mass spectrometry-based proteomic analyses of Japanese squirrels (Sciurus lis) that died in five Japanese zoos showed that they developed glomerular-associated fibrinogen Aα-chain amyloidosis with an extremely high incidence rate (29/38 cases, 76.3%). The condition was found to be age-dependent in the Japanese squirrels, with 89% of individuals over 4 years of age affected. Mass spectrometry revealed that the C-terminal region of the fibrinogen Aα-chain was involved in amyloidogenesis in Japanese squirrels as well as humans. No gene variations were identified between amyloid-positive and amyloid-negative squirrels, which contrasted with the available data for humans. The results indicate that fibrinogen Aα-chain amyloidosis is a senile amyloidosis in Japanese squirrels. The results have also provided comparative pathological support that the amyloidogenic C-terminal region of the fibrinogen Aα-chain is involved in the characteristic glomerular pathology, regardless of the animal species. This study elucidates the potential causes of death in Japanese squirrels and will contribute to future comparative pathological studies of fibrinogen Aα-chain amyloidosis. © 2023 The Pathological Society of Great Britain and Ireland.

First case of hemochromatosis in a sugar glider (Petaurus breviceps).

A 14-year-old, male sugar glider presented with lethargy, anorexia, diarrhea, and paralysis of the hind limbs, and ultrasonography showed possible liver dysfunction. Some medications were administered, but the animal died 10 months after the first presentation, and a necropsy was performed. Histopathologically, hepatocellular degeneration and necrosis, severe deposition of hemosiderin in Kupffer cells and hepatocytes, bridging fibrosis, and regenerative nodules were observed in the liver. Variably amounts of hemosiderin deposition was observed in the heart, lungs, spleen, and kidney. These findings led to the diagnosis of hemochromatosis. No sugar glider cases with hemochromatosis have been reported. The pathological characteristics of hemochromatosis in this species were documented for the first time. The pathogenesis of hemochromatosis in animals remains unclear, but it has been suggested that some commercially available food for sugar gliders containing excessive amounts of iron and vitamin C may induce the disease.

Morphological study of apocrine gland tumors in domestic Richardson's ground squirrels (Urocitellus richardsonii).

The morphology of 58 apocrine tumors from 31 domestic Richardson's ground squirrels (Urocitellus richardsonii) was examined, comparing tumor subtypes with clinical information and histological findings. The squirrels included 23 males (74%) and 8 females (26%). Of the 58 tumors, 36 (62%) were located from the cervical skin to the dorsal skin, 17 (29%) were from the perioral region to the cheek, and the location was unrecorded for the remaining 5 squirrels (9%). Histologically, 49 tumors (84%) were apocrine adenocarcinomas, 6 were apocrine adenomas (10%), and 3 were apocrine cysts (5%). The 49 apocrine adenocarcinomas were subclassified as 16 (33%) cystic papillary type, 16 (33%) tubulopapillary type, 12 (24%) solid type, and 5 (10%) micropapillary invasive type. Fisher's exact test revealed correlations between tumor subtypes and tumor diameters, nuclear atypia, tumor margins, vascular invasion, and periodic acid-Schiff (PAS) positivity. This study showed a predominance of apocrine adenocarcinomas with various histological subtypes. In addition, subclassification of these tumors was useful for predicting the biological behavior of apocrine gland tumors in Richardson's ground squirrels.

Step-by-step protocols for non-viral derivation of transgene-free induced pluripotent stem cells from somatic fibroblasts of multiple mammalian species.

Potentials of immortal proliferation and unlimited differentiation into all the three germ layers and germ cells in induced pluripotent stem cells (iPSCs) render them important bioresources for in vitro reconstitution and modeling of intravital tissues and organs in various animal models, thus contributing to the elucidation of pathomechanisms, drug discovery and stem cell-based regenerative medicine. We previously reported promising approaches for deriving transgene-free iPSCs from somatic fibroblasts of multiple mammalian species by episomal vector or RNA transfection, although the respective step-by-step protocols and the combinatorial usage of these methods, which achieved high induction efficiency, have not been described in the literature so far. Here, we provide a detailed step-by-step description of these methods with critical tips and slight modifications (improvements) to previously reported methods. We also report a novel method for the establishment of iPSCs from the Syrian hamster (also known as golden hamster; Mesocricetus auratus), a unique animal model of hibernation. We anticipate this methodology will contribute to stem cell biology and regenerative medicine research.

Clinical course and pathologic study of retrobulbar histiocytic sarcoma in a central bearded dragon (Pogona vitticeps).

A central bearded dragon (Pogona vitticeps) presented with periorbital swelling and exophthalmos. A retrobulbar mass was detected, and enucleation with the mass was performed. Histologically, the mass was composed of a dense sheet and interlacing bundles of round to polygonal to short spindle-shaped cells with occasional bizarre mononuclear and multinucleated giant cells. Immunohistochemically, the neoplastic cells had various degrees of membranous and/or cytoplasmic granular reactivity to anti-ionized calcium-binding adapter molecule 1 and anti-CD204 antibodies. Ultrastructurally, the neoplastic cells had irregular nuclei and abundant cytoplasm with membrane-bound electron-dense lysosomes and endoplasmic reticula. These findings were consistent with a histiocytic sarcoma. The present study provided a detailed description of retrobulbar histiocytic sarcoma for the first time in a central bearded dragon.

Thymoma originating from the cervical component of the thymus in a degu.

A 5-y-old, male degu (Octodon degus) was presented with a subcutaneous mass in the ventral aspect of the cervical area. The mass was removed surgically. Histologically, the mass was a densely cellular, expansile neoplasm, with compression of thymic tissue to the periphery. The neoplasm consisted of solid sheets of polygonal cells, mixed with fewer small lymphocytes. Rare Hassall bodies were scattered throughout the mass. Polygonal cells were positive for anti-keratin/cytokeratin AE1/AE3 antibody, and small lymphocytes were positive for anti-CD3 antibody. The histopathologic and immunohistochemical findings were consistent with a thymoma. In addition, an autopsy revealed myxosarcoma of the right thoracic wall with metastasis to the lung. To our knowledge, thymoma originating from the cervical component of the thymus has not been documented previously in a rodent species.

Cutaneous papilloma and multicentric squamous cell carcinoma in four-toed hedgehogs (Atelerix albiventris).

Skin lesions possibly caused by Papillomavirus infections in two four-toed hedgehogs are described. In case 1, there was a papillary mass on the right hind limb. Histologically, the mass was consistent with a viral papilloma. In the other case, multifocal papillary masses with erosions and ulcers were found throughout the body, mainly on the extremities. Histology showed continuative lesions composed of acanthosis, Bowenoid in situ carcinoma, and squamous cell carcinoma, with abrupt transitions between the lesions. In both cases, keratinocytes in the granular layer infrequently had features of koilocytes and intranuclear inclusion bodies, and immunohistochemical staining was positive for anti-human papillomavirus antibody. To the best of the authors' knowledge, this is the first pathological documentation of possibly papillomavirus-associated skin lesions in four-toed hedgehogs.

Non-viral derivation of a transgene-free induced pluripotent stem cell line from a male beagle dog.

We previously reported the non-viral derivation of transgene-free induced pluripotent stem cells (iPSCs) from somatic fibroblasts of a female beagle dog using an optimized induction medium and integration-free episomal vectors. Here, we report novel derivation of a male canine iPSC line OF35Y-iPS, which showed standard characteristics of pluripotency such as a strong gene expression profile of pluripotency markers, differentiation potential into all three germ layers, and normal karyotype (78XY). Furthermore, we demonstrated targeted integration of 2A-EGFP into the canine NANOS3 locus. The novel iPSC line would be a useful resource for stem cell research and regenerative veterinary medicine.

Tracheal chondrosarcoma in a blue tegu (Salvator merianae).

An 8-year-and-1-month-old female blue tegu (Salvator merianae) was brought to a clinician with severe cough. The patient died 11 days later despite supportive care, and necropsy was performed by a clinician. The lumen of the distal trachea was almost completely occluded by a milk-white, hard mass measuring 1 cm in diameter. Histopathologically, the mass comprised spindle-shaped to polygonal cells arranged in a loose sheet with abundant production of basophilic cartilaginous matrix, consistent with chondrosarcoma. Reports of tracheal neoplasms in reptiles are limited, and to the best of our knowledge this represents the first pathological documentation of tracheal chondrosarcoma in a reptile.

Clinical, histopathological, and immunohistochemical studies of histiocytic sarcoma in four-toed hedgehogs (Atelerix albiventris): A retrospective study.

Histiocytic sarcoma was investigated histopathologically and immunohistochemically in 17 four-toed hedgehogs (Atelerix albiventris), along with a review of their clinical data. Cases were histopathologically classified into two types: round-polygonal cell type (6 cases) and spindle cell type (11 cases). Round-polygonal cell type was found in visceral organs such as the spleen, lymph nodes, and more, and most cases of this type were consistent with disseminated histiocytic sarcoma. On the other hand, spindle cell type occurred mainly in skin, and almost all cases were consistent with localized histiocytic sarcoma. The prognosis of patients with round-polygonal cell type appeared worse than that of spindle cell type. Immunohistochemically, neoplastic cells of spindle cell type showed stronger reactivity against human leukocyte antigen-DR than round-polygonal cell type. Neoplastic cells of all cases showed strong reactivity against ionized calcium-binding adapter molecule-1 (Iba-1) and various reactivities against cluster of differentiation (CD) 204. Regardless of morphological classification, most tumor cells were negative for CD163, suggesting that this marker is less effective for the diagnosis of histiocytic sarcoma. The results of this study suggest that Iba-1 is the most effective marker for histiocytic sarcoma.

A fatal case of a captive snowy owl (Bubo scandiacus) with Haemoproteus infection in Japan.

Parasites of the genus Haemoproteus are vector-borne avian haemosporidia commonly found in bird species of the world. Haemoproteus infections are typically considered relatively benign in birds. However, some Haemoproteus species cause severe disease and mortality, especially for captive birds removed from their original habitat. In September 2018, a captive 15-year-old snowy owl (Bubo scandiacus), kept in a zoological garden of Japan, died subacutely after presenting leg dysfunction. This case showed significantly low PCV and elevated AST, ALT, CK, and LDH values. Many megalomeronts with prominent morphological characteristics of Haemoproteus were observed in the left leg muscles. Those megalomeronts exhibited multilocular structures and were internally filled with merozoites. A new lineage of Haemoproteus was detected by subsequent PCR for the cytochrome b (cytb) gene of avian haemosporidia from DNA extracted from several organ tissues. The detected lineage was classified in the subgenus Parahaemoproteus and was similar to those from the wild birds inhabiting the region including the study area, suggesting that this snowy owl likely acquired its infection from wild birds. This is the first report of a fatal case of a captive bird with a locally transmitted Haemoproteus infection in Japan. We considered the pathogenicity of this infection in conjunction with the clinical course and hematology results. We surmise that snowy owls may be particularly susceptible to infection with Haemoproteus parasites, and warming northern temperatures may exacerbate the overall health of these and other high latitude birds. Further research into the prevalence of Haemoproteus in wild birds near zoological gardens and potential biting midge vectors is necessary for the ex situ conservation of introduced birds.

Assessment of reported uterine lesions diagnosed histologically after ovariohysterectomy in 1,928 pet rabbits (Oryctolagus cuniculus).

OBJECTIVE: To assess variations in age, breed, and clinical signs in rabbits with neoplastic or nonneoplastic uterine lesions and to investigate potential relationships between endometrial adenocarcinoma and age at ovariohysterectomy or breed in rabbits. ANIMALS: 1,928 rabbits that underwent ovariohysterectomy for treatment or prevention of possible uterine disease. PROCEDURES: With an online questionnaire distributed to 441 veterinary hospital members of the Japanese Society of Exotic Pet Medicine, data were retrospectively collected regarding age, breed, and findings on physical and histologic examinations for pet rabbits that underwent ovariohysterectomy between January 1, 2009, and April 30, 2018. Rabbits were grouped by reported age, breed, clinical signs, and uterine lesions, and results were assessed across groups. Logistic regression analysis was used to identify potential relationships between endometrial adenocarcinoma and breed or age at ovariohysterectomy in rabbits. RESULTS: The questionnaire response rate was 9.8% (43/441), with data reported for 1,928 rabbits (mixed breed, 600 [31.1%]; Netherland Dwarf, 520 [27.0%]; Holland Lop, 286 [14.8%]; or various other breeds, 522 [27.1%]). The most commonly reported neoplastic and nonneoplastic lesions were endometrial adenocarcinoma (1,035/1,928 [53.7%]) and endometrial hyperplasia (842 [43.7%]), respectively, and the most commonly reported clinical sign was hematuria or serosanguineous vaginal discharge (1,020/1,928 [52.9%]). As age at ovariohysterectomy increased, so did the odds (OR, 1.826; 95% CI, 1.640 to 2.033) of uterine adenocarcinoma. CONCLUSIONS AND CLINICAL RELEVANCE: Results supported ovariohysterectomy in rabbits before 2 years of age as a key preventative measure to mitigate uterine disease, particularly endometrial adenocarcinoma.

Intraocular extraskeletal osteosarcoma in a rabbit (Oryctolagus cuniculus).

An 8-year-and-9-month-old male, lop-eared rabbit (Oryctolagus cuniculus) presented with gradual enlargement of the left eye to 4 × 4 × 4 cm and exophthalmos. The animal died 3 months later, and necropsy was performed. On gross pathology, the intraocular tissue was effaced and occluded by a hard, light-gray mass. Histologically, the mass comprised spindle-shaped to angular cells arranged in interlacing bundles with abundant production of osteoid, bone and cartilage, consistent with osteosarcoma. Limited cases of intraocular neoplasm have been reported in pet rabbits. To the best of our knowledge, this represents the first pathologic documentation of intraocular osteosarcoma in a rabbit.

The utility of DLA typing for transplantation medicine in canine models.

Transplantation medicine is used for the treatment of severe canine diseases, and the dog leukocyte antigen (DLA) is considered to be important in graft rejection. However, the utility of direct sequencing of both DLA classes I and II has not been assessed thoroughly. Eight healthy beagles with identified DLA genes were divided into two sets of four dogs, each including one donor and three recipients for skin transplantation. The following recipients were selected: one dog with a complete match, one with a haploidentical match, and one with a complete mismatch of the DLA gene with the donor. Full-thickness skin segments were obtained from each donor and transplanted to the recipients. A mixed lymphocyte reaction (MLR) assay was performed and analyzed by flow cytometry. Skin grafts of DLA haploidentical and mismatched pairs were grossly rejected within 14 days, whereas in fully matched DLA pairs, survival was as long as 21 days. Histopathological evaluation also showed moderate to severe lymphocytic infiltration and necrosis in DLA mismatched pairs. As seen in the MLR assay, the stimulation index of DLA mismatched pairs was significantly higher than that of fully matched DLA pairs in both sets (P<0.001). The allogeneic transplantation results suggested that it is possible to prolong transplant engraftment by completely matching the DLA genotype between the donor and recipient. Additionally, the MLR assay may be used as a simplified in vitro method to select donors.

Eucoleus garfiai (Gállego et Mas-Coma, 1975) (Nematoda: Capillariidae) infection in wild boars (Sus scrofa leucomystax) from the Amakusa Islands, Japan.

We examined lingual tissues of Japanese wild boars (Sus scrofa leucomystax) captured in the Amakusa Islands off the coast of Kumamoto Prefecture. One hundred and forty wild boars were caught in 11 different locations in Kamishima (n = 36) and Shimoshima (n = 104) in the Amakusa Islands, Japan between January 2016 and April 2018. Lingual tissues were subjected to histological examinations, where helminths and their eggs were observed in the epithelium of 51 samples (36.4%). No significant differences in prevalence were observed according to maturity, sex or capture location. Lingual tissues positive for helminth infection were randomly selected and intact male and female worms were collected for morphological measurements. Based on the host species, site of infection, and morphological details, we identified the parasite as Eucoleus garfiai (Gállego et Mas-Coma, 1975) Moravec, 1982 (syn. Capillaria garfiai). This is the first report from outside Europe of E. garfiai infection in wild boars. Phylogenetic analysis of the parasite using the 18S ribosomal RNA gene sequence confirmed that the parasite grouped with other Eucoleus species, providing additional nucleotide sequence for this genus. Since wild boar populations are widely distributed in Japan, continuing surveys on the epidemiology of the parasite and identifying possible intermediate host candidates are crucial for elucidating the transmission route of the parasite.

SQUAMOUS CELL CARCINOMAS WITH PULMONARY METASTASIS AND ENDOCARDIOSIS IN THE SIBERIAN CHIPMUNK ( EUTAMIAS SIBIRICUS): TWO NECROPSY CASES.

Reports of neoplastic diseases in the Siberian chipmunk ( Eutamias sibiricus) are limited. The authors describe herein two necropsy cases of Siberian chipmunks with squamous cell carcinoma; both of which showed self-biting behaviors on presentation to the clinician. In both cases, the neoplasms comprised trabeculae and islands of polyhedral cells, supported by moderate amounts of fibrous stroma. Anisocytosis and anisokaryosis were moderate to prominent. Neoplastic cells were occasionally keratinized, with a keratin pearl formation. In one case, valvular endocardiosis of the left and right atrioventricular valves with secondary pulmonary and hepatic lesions was incidentally diagnosed. This report provides the first documentation of squamous cell carcinoma and endocardiosis in the Siberian chipmunk.