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Although equine gestation is unique from the standpoint of fetal gonadal enlargement and regression, the activator of this process is still unknown. The present study aimed to show a possible role of activin during equine gestation. In the first experiment, weekly plasma samples from six pregnant mares were used to measure activin A. In the second experiment, eight pregnant mares carrying female (gestational days 110, 140, 180, and 270) and male fetuses (gestational days 120, 180, 225, and 314) were used for immunohistochemistry of activin receptors (IA, IB, IIA, IIB), and their intracellular mediators (Smad2, Smad3, Smad4). Activin A levels in maternal circulation remained low until fourth weeks of gestation, thereafter, started to increase, and peaked first at 11 weeks of gestation. The second significant peak was observed on the day of parturition. Activin receptors type IA, IB, IIA, and IIB were immunostained in interstitial and germ cells of fetal ovaries and testes along with utero-placental tissues. Smad2, Smad3, and Smad4 were also immunolocalized in all these organs. These results demonstrated the activin-producing capacity of utero-placental tissues, and also evidenced the existence of activin receptors and functional signaling molecules in these organs. The first increment in circulating activin A in maternal circulation coinciding with the timing of initiation of fetal gonadal enlargement suggests that activin from the utero-placental tissues may have a stimulatory role in fetal gonad enlargement and utero-placental development in mares, whereas the second peak could be important to follicular development in the maternal ovary for foal heat.

Immunohistochemical investigations of the expression of surfactant protein A (SP-A) and surfactant protein D (SP-D) in the uterine and placental tissues of 13 pregnant mares were performed using anti-horse monoclonal primary antibodies. Strong positive reactions for both SP-A and SP-D were observed in the trophoblasts in the microcotyledons of the placentae at 182 to 314 days of gestation; in uterine glandular epithelial cells, faint-to-weak reactions were observed during gestation. This study describes, for the first time, the changes in the SP-A and SP-D expression levels in the endometrium of mares during gestation; the SP-A and SP-D expression levels increased after the second trimester of gestation.

Oral and cutaneous tissues are the most frequent origin in canine squamous cell carcinoma (SSC). In SCC, changes in adhesion molecule expression and transition from epithelial to mesenchymal phenotype are thought to be important in development of invasive behavior of neoplastic cells at the leading front of the tumor. We therefore investigated histological invasive front grading and epithelial-mesenchymal transition (EMT) in both oral SCCs and cutaneous SCCs. EMT was assessed by evaluating immunohistochemical expression of E-cadherin, β-catenin, desmoglein, vimentin, and N-cadherin. Regardless of the anatomic location, invasive front grading resulted in higher histological grades than grading of the surface. Most oral SCCs were of significantly higher histologic grade than cutaneous SCCs ( P < .01). Expression of E-cadherin, β-catenin, and desmoglein was significantly lower in oral SCC compared with cutaneous SCC ( P < .01). A significant association was found between invasive front grading and loss of E-cadherin, β-catenin, and desmoglein ( P < .01). Also, vimentin-positive neoplastic cells had low immunoreactivity of these adhesion molecules, and a few of these neoplastic cells were positive for N-cadherin. These results suggest not only E-cadherin and β-catenin but also desmoglein as markers for predicting biological behavior of canine SCC. Depending on their primary sites, EMT correlates with biological behavior and therefore histological grade of canine SCC. We suggest that combining invasive front grading with assessment of immunohistochemical expression of E-cadherin, β-catenin, and desmoglein may allow more accurate prediction of biological behavior of canine SCCs.

Amyloid-producing odontogenic tumors (APOTs) of the facial skin were diagnosed in 3 domestic cats. The neoplasms had the histopathological characteristics of the odontogenic tumor. The neoplastic cells were present in irregular islands, strands, and sheets. The peripheral neoplastic cells of the islands and strands were arranged in a palisading fashion, while the central cells were polyhedral to stellate and randomly arranged. Multiple spherules of homogeneous eosinophilic material were closely apposed to the neoplastic epithelial cells. The spherules stained with Congo red and produced an apple green birefringence under polarization microscopy, indicative of amyloid. Immunohistochemically, amyloid materials of the neoplasms reacted with polyclonal antibodies for ameloblastin, amelogenin, and sheathlin antibodies. Neoplastic epithelial cells also reacted with antiameloblastin, amelogenin, and sheathlin antibodies, with varied intensity. The histopathological and immunohistochemical characteristics of dermal neoplasms of the 3 cats were analogous to those of APOTs reported in the dog and the cat.

Aortic rupture is a well recognized cause of sudden death in thoroughbred horses. Some microscopic lesions, such as those caused by cystic medial necrosis and medionecrosis, can lead to aortic rupture. However, these microscopic lesions are also observed in normal horses. On the other hand, a previous study of aortic rupture suggested that underlying elastin and collagen deposition disorders might be associated with aortic rupture. Therefore, the purpose of this study was to compare the structural components of the tunica media of the aortic arch, which is composed of elastin, collagen, smooth muscle cells and mucopolysaccharides (MPS), in fetal to mature thoroughbred horses. The percentage area of elastin was greatest in the young horses and subsequently decreased with aging. The percentage area of collagen increased with aging, and the elderly horses (aged ≥20) exhibited significantly higher percentage areas of collagen than the young horses. The percentage area of smooth muscle cells did not change with age. The percentage area of MPS was inversely proportional to the percentage area of elastin. The fetuses exhibited a markedly larger percentage area of MPS than the mature horses. We concluded that the medial changes seen in the aortic arch, which included a reduction in the amount of elastin and increases in the amounts of collagen and MPS, were age-related variations.

In horse, the characterizations of hyalocytes under the steady state are still unclear. Therefore, we investigated characterizations of hyalocytes in normal equine eyes by their immunohistochemical phenotype, histomorphology and distribution. Thirty-one eyes from 18 horses, divided into 4 groups (G) by age, were used: early (G1) and late gestation (G2) fetuses, 1- to 3-year-old (G3) and 8- to 24-year-old (G4) horses. Equine hyalocytes were histologically classified into 4 types, and they immunohistochemically expressed MHC II and CD163. Hyalocytes were detected on and/or around ciliary processes and pars plana in G2, G3 and G4, but were not located on retina and optic papilla. A significant increase in distribution was found between G2 and both G3 and G4, and the largest distribution was found at ciliary processes in these groups. Equine hyalocytes were characterized as residential ocular macrophage and MHC II antigen-bearing cell, accompanied by a pleomorphic appearance and located in the contiguous ciliary body. Our data provided characterizations of hyalocytes in normal equine eyes and may well contribute to improving the understanding of pathogenesis of equine ocular disease.

Antigen-presenting cells (APCs) in the uveal tract participate in ocular immunity including immune homeostasis and the pathogenesis of uveitis. In horses, although uveitis is the most common ocular disorder, little is known about ocular immunity, such as the distribution of APCs. In this study, we investigated the distribution of CD163-positive and MHC II-positive cells in the normal equine uveal tract using an immunofluorescence technique. Eleven eyes from 10 Thoroughbred horses aged 1 to 24 years old were used. Indirect immunofluorescence was performed using the primary antibodies CD163, MHC class II (MHC II) and CD20. To demonstrate the site of their greatest distribution, positive cells were manually counted in 3 different parts of the uveal tract (ciliary body, iris and choroid), and their average number was assessed by statistical analysis. The distribution of pleomorphic CD163- and MHC II-expressed cells was detected throughout the equine uveal tract, but no CD20-expressed cells were detected. The statistical analysis demonstrated the distribution of CD163- and MHC II-positive cells focusing on the ciliary body. These results demonstrated that the ciliary body is the largest site of their distribution in the normal equine uveal tract, and the ciliary body is considered to play important roles in uveal and/or ocular immune homeostasis. The data provided in this study will help further understanding of equine ocular immunity in the normal state and might be beneficial for understanding of mechanisms of ocular disorders, such as equine uveitis.

Canine mammary tumours (CMTs) are the most common neoplasms affecting female dogs. There is an urgent need for molecular biomarkers that can detect early stages of the disease in order to improve accuracy of CMT diagnosis. The aim of this study was to examine whether caveolin-1 (Cav-1) and matrix metalloproteinase 14 (MMP14) are associated with CMT histological malignancy and invasion. Sixty-five benign and malignant CMT samples and six normal canine mammary glands were analysed using quantitative reverse transcription-polymerase chain reaction. Cav-1 and MMP14 genes were highly expressed in CMT tissues compared to normal tissues. Cav-1 especially was overexpressed in malignant and invasive CMT tissues. When a CMT cell line was cultured on fluorescent gelatin-coated coverslips, localisation of Cav-1 was observed at invadopodia-mediated degradation sites of the gelatin matrix. These findings suggest that Cav-1 may be involved in CMT invasion and that the markers may be useful for estimating CMT malignancy.

Osteosarcoma (OS), the most common bone tumor, includes OS of the head (OSH) and appendicular OS (OSA). In dogs, it is classified into 6 histologic subtypes: osteoblastic, chondroblastic, fibroblastic, telangiectatic, giant cell, and poorly differentiated. This study investigated the significance of the histologic classification relevant to clinical outcome and the histologic and immunohistochemical relationships between pleomorphism and expression of cytoskeletal proteins in 60 cases each of OSH and OSA. Most neoplasms exhibited histologic diversity, and 64% of OS contained multiple subtypes. In addition to the above 6 subtypes, myxoid, round cell, and epithelioid subtypes were observed. Although the epithelioid subtypes were observed in only OSH, no significant difference in the frequency of other subtypes was observed. Also, no significant relevance was observed between the clinical outcome and histologic subtypes. Cytokeratin (CK) was expressed in both epithelioid and sarcomatoid tumor cells in various subtypes, and all CK-positive tumor cells also expressed vimentin. Vimentin and α-smooth muscle actin (SMA) were expressed in all subtypes. A few SMA-positive spindle-shaped tumor cells exhibited desmin expression. Glial fibrillary acidic protein–positive tumor cells were observed in many subtypes, and some of these cells showed neurofilament expression. Although OSH exhibited significantly stronger immunoreactivity for SMA than OSA, no significant difference in other cytoskeletal proteins was observed. Some tumor cells had cytoskeletal protein expression compatible with the corresponding histologic subtypes, such as CK in the epithelioid subtype and SMA in the fibroblastic subtype. Thus, canine skeletal OS is composed of pleomorphic and heterogenous tumor cells as is reflected in the diversity of histologic patterns and expression of cytoskeletal proteins.

Chordomas of the tip of the tail in 6 ferrets were examined using histopathological, histochemical and immunohistochemical procedures. Histopathologically, round neoplastic cells containing numerous cytoplasmic vacuoles of varying sizes, categorized as “physaliphorous cells”, were observed in the amorphous eosinophilic or pale basophilic myxoid stroma. Physaliphorous cells were arranged in lobules and in a “chordoid” or “cobblestone” manner. The neoplasms were diagnosed as benign chordoma without local invasion and metastasis. Histochemically, the cytoplasm of small neoplastic cells was positive for periodic acid-Schiff stain and alcian blue (AB) pH 2.5 and pH 1.0 stains, but negative for hyaluronidase digestion-AB pH 2.5 stain. All neoplastic cells were strongly stained with colloidal ion, negative for high iron diamine AB pH 2.5 and toluidine blue pH 2.5 stains, and positive for Mayer’s mucicarmine stain. Immunohistochemistry using antibodies directed against low-molecular-weight cytokeratins (CK18, CK19 and CK20), vimentin and mucin core protein (MUC5AC) revealed that neoplastic cells had both epithelial and mesenchymal elements. The expression of low-molecular-weight cytokeratins suggests that neoplastic cells acquired the properties of glandular epithelial cells and produced epithelial mucus. Furthermore, the expression of cytokeratins, vimentin, S100 protein, brachyury and epithelial membrane antigen indicates that the neoplasms were equivalent to the classic type of human chordoma. Therefore, immunohistochemistry using these antibodies can be useful for the characterization of ferret chordoma.

To contribute to early diagnosis and treatment of gastric ulcer of foals, we examined the gastric mucosa of healthy and affected foals using an endoscope. In healthy foals, the characteristic changes in the development of the squamous mucosa were seen mainly in the squamous mucosa, and maturation of the squamous mucosa in the greater curvature (GC-S) occurred more slowly than that of the squamous mucosa in the lesser curvature (LC-S). Epithelial desquamation in the LC-S and GC-S was observed between 6 and 90 days but was not observed in the LC-S at about 60 days, whereas it was observed in the GC-S until 90 days old. These findings suggest that there is a difference in the development of the gastric mucosa by region and that desquamation continues over a term longer than studies have reported in the past. In the affected foals, the minimum age at which gastric ulcer was observed was 4 days old. Gastric ulcers formed predominantly in the squamous mucosa (LC-S and GC-S) of foals with an immature mucosa before the weaning period, and the peak incidence occurred between 61 and 90 days old. The differences in the ulceration sites were considered to depend on the difference in the development (maturation) stage of the squamous mucosa. The grading score of the gastric ulcer increased with the growth of the affected foals. The gastric ulcer might be enhanced greatly by stress in the weaning period.

Simultaneous removal of bilateral thyroid tumors was performed while preserving the parathyroid gland in six dogs. At least one external parathyroid gland was identified in all dogs. In five cases, the external parathyroid gland and its blood supply were preserved intact. In one dog, the vessels supplying the external parathyroid gland had been invaded by the tumor, and the gland was thus removed and reimplanted into the sternohyoid muscle. That dog required postoperative treatment with oral calcium gluconate and vitamin D3. Local tumor recurrence was not observed in any of the cases. The mean survival time was 920 days. We found that the external parathyroid gland could be identified and preserved in most dogs undergoing total thyroidectomy.

A male Thoroughbred fetus was aborted on day 251 of pregnancy. Gross and histological examinations detected systemic granulomatous lesions in many superficial and visceral lymph nodes and organs including the liver, tonsils, lungs, thymus, spleen, right thyroid gland and gastrointestinal tract, and suppurative placentitis, pyogranulomatous amnionitis and intralesional acid-fast bacilli were also detected. An examination of the DNA base sequence of the β subunit of RNA polymerase demonstrated that Mycobacterium avium strain 104 had infected several organs. To the best of our knowledge, this is the first report of equine fetal mycobacterial infection in Japan.

A 3-year-old male black-tailed prairie dog (Cynomys iudovicianus) was presented with a dome-shaped cutaneous mass over the left femur. Microscopically, the mass was encapsulated and composed of proliferating spindle cells arranged in haphazard, interlacing bundles. There were vascular structures within the mass and some spindle cells had transitioned from the peripheral regions of the vascular wall. Immunohistochemically, the cells expressed vimentin, α-smooth muscle actin, desmin and heavy caldesmon. Based on these findings, the mass was diagnosed as a cutaneous angioleiomyoma. To the best of our knowledge, this is the first case of cutaneous angioleiomyoma in a black-tailed prairie dog.

A 10-year-old Japanese Black cow presented with a swelling of the right femur, and a hard, large mass occupied the pelvic cavity. The mass strongly adhered to the visceral surface of the ischium and had posteriorly invaded among the right femoral muscles. Histologically, the mass was composed of neoplastic osteoblasts and exhibited osteoid and immature trabecular bone production. In the region where the mass adhered to the ischium, neoplastic cells were continuously proliferating into the medullary cavity. Tumor emboli were observed in the small vessels of the femoral muscles and lungs. Based on these findings, the mass was diagnosed as an osteosarcoma and considered to have arisen from the ischium.

Yolk sac tumors (YSTs) are rare neoplasms of germ cell origin. In humans, the tumors primarily occur in the testes or ovaries, but occasionally develop at other sites. The neoplastic cells of YSTs form many histological patterns resembling embryonal structures, and the World Health Organization classification lists 11 such patterns: reticular, macrocystic, endodermal sinus, papillary, solid, glandular-alveolar, myxomatous, sarcomatoid, polyvesicular vitelline, hepatoid, and parietal. Among domestic animals, only 2 cases of YST, which were of testicular and abdominal cavity origin, have been reported in calves. In both cases, neoplastic cells had epithelial properties and disseminated metastases in the abdomen. In the present study, the enlarged testis of a newborn calf, which was subsequently diagnosed as YST and exhibited myxomatous, reticular, and polyvesicular vitelline histological patterns, is described. There was no metastasis in this case, and histological and immunohistochemical features varied from previous cases of YST.

A 29-year-old stallion presented with bilateral blindness following the chronic purulent nasal drainage. The mass occupied the right caudal nasal cavity and right paranasal sinuses including maxillary, palatine and sphenoidal sinuses, and the right-side turbinal and paranasal septal bones, and cribriform plate of ethmoid bone were destructively replaced by the mass growth. The right optic nerve was invaded and involved by the mass, and the left optic nerve and optic chiasm were compressed by the mass which was extended and invaded the skull base. Histologically, the optic nerves and optic chiasm were degenerated, and the mass was diagnosed as lymphoma which was morphologically and immunohistochemically classified as a diffuse large B-cell lymphoma. Based on these findings, the cause of the blindness in the stallion was concluded to be due to the degeneration of the optic nerves and chiasm associated with lymphoma occurring in the nasal and paranasal cavities. To the best of our knowledge, this is the first report of the equine blindness with optic nerve degeneration accompanied by lymphoma.

A 33-year-old red-crowned crane (Grus japonensis) had a diffuse intestinal thickening from the duodenum to colon. Microscopically, neoplastic cells were arranged in sheets and occasionally nests or cords without gland or squamous differentiation. Metastatic tumor cells were found in the lungs, heart, kidneys and adrenal glands. Immunohistochemically, the neoplastic cells were strongly positive for pan-cytokeratin and cytokeratin 8 and 18 and only partly positive for E-cadherin antibodies. Immunostaining for CD3 was positive in normal lymphocytes, and NSE was also positive in normal nerve fibers. But, the neoplastic cells were not immunoreactive to CD3 and NSE. Based on the histological features and the epithelial characteristics in the immunohistochemical stain, the present case was diagnosed as undifferentiated carcinoma originating from the intestine. Interestingly, the neoplastic cells showed a unique growth pattern; they never invaded the submucosa or muscularis throughout the intestine, whereas they spread lymphogenously or hematogenously to other organs.

A 2-year-old Thoroughbred horse presented with acute onset of colitis, and the intussuscepted jejunum was surgically resected. A transmural mass protruding into the lumen was found at the leading edge of the intussusceptum. Based on histological and immunohistochemical examinations, the mass was diagnosed as diffuse large B-cell lymphoma with metastasis to the mesenteric lymph nodes. Anatomical localization of the mass in the intussusception and absence of other obvious underlying diseases indicated that the intussusception had occurred in association with the mass. To our knowledge, this case is the first report of equine intussusception associated with focal intestinal lymphoma.

Contrast-enhanced magnetic resonance (MR) imaging with a new liver-specific contrast agent gadolinium-ethoxybenzyl-diethylenetriamine penta-acetic acid (Gd-EOB-DTPA; EOB·Primovist®) was studied in 14 normal beagles and 9 dogs with focal liver lesions. Gd-EOB-DTPA accumulates in normally functioning hepatocytes 20 min after injection. As with Gd-DTPA, it is also possible to perform a dynamic multiphasic examination of the liver with Gd-EOB-DTPA, including an arterial phase and a portal venous phase. First, a reliable protocol was developed and the appropriate timings for the dynamic study and the parenchymal phase in normal dogs using Gd-EOB-DTPA were determined. Second, the patterns of these images were evaluated in patient dogs with hepatic masses. The optimal time of arterial imaging was from 15 s after injection, and the optimal time for portal venous imaging was from 40 s after injection. Meanwhile, the optimal time to observe changes during the hepatobiliary phase was from 20 min after injection. In patient dogs, 11 lesions were diagnosed as malignant tumors; all were hypointense to the surrounding normal liver parenchyma during the hepatobiliary phase. Even with a low-field MR imaging unit, the sequences afforded images adequate to visualize the liver parenchyma and to detect tumors within an appropriate scan time. Contrast-enhanced MR imaging with Gd-EOB-DTPA provides good demarcation on low-field MR imaging for diagnosing canine focal liver lesions.

Adiaspiromycosis is a pulmonary infection caused by the soil fungi, Emmonsia crescens and E. parva. It primarily affects small mammals and can range from an asymptomatic condition to fatal disseminated disease. We detected a granuloma containing fungal spherules, which were morphologically consistent with the adiaspores of E. crescens in the lungs of a female Hokkaido sika deer. This is the first reported case of adiaspiromycosis involving a cervid in the world.

We report an atypical mycobacterial infection in an Indian flap-shelled turtle, Lissemys punctata punctata, that died in an aquarium in Japan. At necropsy, the turtle showed multiple white nodules on the capsular surface and parenchyma of various organs such as the liver, spleen, intestine, and lung. Histologically, granulomatous inflammation surrounding a central zone of necrosis was observed. Sections stained by the Ziehl-Neelsen method revealed numerous acid-fast bacilli in the cytoplasm of macrophages and in the central area of necrosis. The organisms were identified as a mycobacterial species by PCR and nucleotide sequence analysis and revealed 98-100% homology to M. ulcerans. This is, to our knowledge, the first report of mycobacteriosis due to M. ulcerans in a turtle.

The endotheliotropism of equine herpesvirus-1 (EHV-1) leads to encephalomyelitis secondary to vasculitis and thrombosis in the infected horse central nervous system (CNS). To identify the host factors involved in EHV-1 infection of CNS endothelial cells, we performed functional cloning using an equine brain microvascular endothelial cell cDNA library. Exogenous expression of equine major histocompatibility complex (MHC) class I heavy chain genes conferred susceptibility to EHV-1 infection in mouse NIH3T3 cells, which are not naturally susceptible to EHV-1 infection. Equine MHC class I molecules bound to EHV-1 glycoprotein D (gD), and both anti-gD antibodies and a soluble form of gD blocked viral entry into NIH3T3 cells stably expressing the equine MHC class I heavy chain gene (3T3-A68 cells). Treatment with an anti-equine MHC class I monoclonal antibody blocked EHV-1 entry into 3T3-A68 cells, equine dermis (E. Derm) cells and equine brain microvascular endothelial cells. In addition, inhibition of cell surface expression of MHC class I molecules in E. Derm cells drastically reduced their susceptibility to EHV-1 infection. These results suggest that equine MHC class I is a functional gD receptor that plays a pivotal role in EHV-1 entry into equine cells.

The aim of present study was to clarify the post-natal profile of follicle-stimulating hormone (FSH), luteinizing hormone (LH), immunoreactive (ir)-inhibin, progesterone, testosterone, and estradiol-17β, and their relationships in Thoroughbred colts. Six hundred and thirty-six colts were used for the study. Single plasma samples from each animal were harvested from the blood drawn through jugular venipuncture. The subjects were born with high amounts of progesterone, testosterone, and estradiol-17β, all of which dropped significantly and remained at lower levels till the end of 6 months. FSH decreased transiently after birth until day 12 and then gradually increased to peak at day 100 which then maintained in lesser levels towards the end of the studied period. LH was highest during birth which decreased until day 26 and then increased slowly to sub-birth levels up to day 90. Animals were born with high amounts of ir-inhibin. It dropped slowly and halved by day 20 and then decreased towards rest of the studied period. The increase in FSH is negatively correlated with the declining ir-inhibin levels. The early increase in FSH can be the indication of early post-natal maturation of the hypothalamic pituitary testicular axis that ultimately might be responsible for priming the testes for future development.

Changes in follicle-stimulating hormone (FSH), luteinizing hormone (LH), prolactin, immunoreactive(ir)-inhibin, testosterone, estradiol-17β, and insulin-like growth factor (IGF)-I in Thoroughbred stallions along with changes in prolactin secretion in geldings were studied. The correlations of day-length with changes in the concentrations of these hormones were also studied. Five stallions and thirteen geldings were employed to draw blood samples in monthly basis and radioimmunoassay was performed to measure these hormones. All hormones showed a seasonal pattern, the levels being highest during the breeding season and lowest during the winter months. Most of the hormones were at their highest concentration during the month of April, the mid of spring in northern hemisphere. The concentration of circulating IGF-I also demonstrated seasonality, the peak lying on the month of April. The plasma concentration of prolactin also increased during the breeding season. This phenomenon was similar both in stallions and geldings although geldings had lower concentration than that of stallions. The changes in concentration of prolactin in stallions and geldings correlated more towards the day-length than towards the temperature. These results clearly indicate the seasonality of pituitary and gonadal hormones of Thoroughbred stallions, the activity being highest during the month of April and May of the breeding season.

A lymphoplasmacytic lymphoma was diagnosed in a 12- year-old domestic cat that had a primary cutaneous mass involving the stomach, liver, kidneys, heart, abdominal wall, diaphragm, bone marrow and several lymph nodes. Histopathologically, the most characteristic feature of this tumor was the heterogeneity of cell components, such as small lymphocytes, well-differentiated plasma cells and plasmacytoid transformed lymphocytes. Amyloid was deposited in the skin, stomach, and several lymph nodes. Immunohistochemically, neoplastic small lymphocytes were positive for CD20, and well-differentiated plasma cells and plasmacytoid transformed lymphocytes were positive for λ-Ig light chains and MUM1/IRF-4. These results emphasize the importance of lymphoplasmacytic lymphoma as a differential diagnosis of extramedullary cutaneous plasmacytoma in cats.

The amyloid of canine amyloid-producing odontogenic tumor (APOT) was evaluated biochemically and immunohistochemically. The N-terminal amino-acid sequence of purified amyloid protein from a canine APOT was strikingly similar to the sequence in both rat ameloblastin and porcine sheathlin. Immunohistochemically, the amyloid in APOT from 9 dogs was strongly reactive with anti-rat ameloblastin, anti-porcine sheathlin, and anti-canine APOT amyloid and weakly reactive with anti-porcine amelogenin but negative for antibodies to cytokeratins, vimentin, desmin, α-smooth muscle actin, amyloid A, glial fibrillary acidic protein, or S100 protein. The neoplastic epithelial cells of APOT were focally reactive with antibodies to ameloblastin, sheathlin, amelogenin, and canine APOT amyloid. The similarity in amino-acid sequence of the amyloid protein of canine APOT to that of enamel proteins, such as ameloblastin, sheathlin, and amelogenin, and the expression of these antigens in both APOT amyloid and in the neoplastic cells suggest that the amyloid of canine APOT is derived from enamel proteins secreted by ameloblasts.

Ectopic bone formation in the left atrioventricular valves and cardiac fibroskeleton, with systemic circulatory disturbance, is reported in a 4-year-old crossbred heavy horse. Microscopically, there was fibrosis, chondral metaplasia and mature bone, with bone marrow within the left atrioventricular cusps and in the annuli of the aortic and right atrioventricular valves.

Bovine viral diarrhea virus (BVDV) causes fetal brain malformations in ruminants when the fetuses are infected transplacentally in mid-pregnancy. In both cytopathic and non-cytopatic virus infections, viral lytic infection in actively replicating cells and interruption of vascular integrity have been suggested as the pathogenesis, but functional disturbance of infected neural developing cells has been unclear. In this study, we examined the effect of infection with non-cytopathic BVDV2 on the differentiation of neural stem/precursor cells isolated from the bovine fetus. In the process of differentiation to three types of neural cells, neurons, astrocytes and oligo-dendrocytes, virus infection significantly and selectively inhibited the differentiation of neural stem/precursor cells into the astrocytic lineage. This inhibition is possibly important for the pathogenesis of congenital brain malformations associated with non-cytopathic BVDV infection.

At necropsy, an 11-year-old Japanese Black cow with anemia, leukocytopenia, and progressive hind limb ataxia had marked diffuse splenomegaly and multiple masses in the thoracic vertebrae. Histologically, neoplastic erythrophagocytic histiocytes were in the splenic red pulp, vertebral masses, and blood vessels of the liver and lungs. The spinal cord was compressed by the vertebral masses. Clinicopathological, macroscopic, and histologic findings were consistent with hemophagocytic histiocytic sarcoma. Vertebral involvement with spinal cord compression and resultant hind limb ataxia is an unusual presentation for this tumor, which has been described mainly in dogs and cats.

Objective—To evaluate the histologic diversity of epidermal laminae in hooves from horses without clinical signs of laminitis. Sample Population—Formalin-fixed samples of stratum internum obtained from the mid region of the dorsal aspect of the hoof wall from the forelimbs of 35Thoroughbred cadavers (including foals [n = 9], yearlings [5], 2 year olds [6], racing horses [5], and mares [10]). Procedures—Paraffin-embedded laminar tissues were stained with H&E for the evaluation of architectural variety of primary epidermal laminae (PEL) and secondary epidermal laminae (SEL). For detection of cytokeratin (CK) expression in epidermal laminae, immunohisto-chemical staining was performed by use of anti-CK14 and anti-CK8.12 antibodies. Results—The morphology of the PEL, SEL, and tips of PEL was classified into 3, 5, and 3 patterns, respectively. Differences in the predominant type of SEL depended on their location with respect to the laminar interface. In SEL attached to the sides of PEL, the basal cells were immunoreactive to CK14 and CK8.12, which was interpreted as a normal pattern. In some SEL at the tips of PEL, the suprabasal cells expressed CK14, CK8.12, or both, which constituted a hyperplastic pattern. Conclusions and Clinical Relevance—The histologic diversity of epidermal laminae from hooves of Thoroughbreds was attributable to the combined morphology of PEL and SEL. Detection of hyperplastic changes in the laminar interface does not justify a diagnosis of laminitis because such changes can develop independent of clinical disease. The classification system used here should aid investigators in making a more accurate histologic evaluation of laminae.

Histopathological examination of clinically long-standing lesions with durations of one year or more in the extremities of two cattle revealed the presence of sarcomas with distant metastases. In case 1, neoplastic cells were fusiform to pleomorphic, stained for no specific differentiation markers, and diagnosed as undifferentiated sarcoma. Neoplastic growth in case 2 was composed of spindle to histiocytoid cells and a significant number of multinucleated giant cells, both of which were immunoreactive to histiocyte markers, and diagnosed as giant cell malignant fibrous histiocytoma. Neoplastic cells of both cases were immunohistochemically positive for nitric oxide-related antigens, which were recognized as markers of inflammation-induced carcinogenesis in human and laboratory animals.

Epithelioid cells are found in lymph nodes in cases of metastatic carcinoma; however, epithelioid cells with benign features have also been discovered incidentally in lymph nodes. Epithelioid cells were observed in mediastinal lymph nodes of cattle without cancer during routine diagnostic necropsy. To explain this finding, the authors evaluated the prevalence and histopathologic, immunohistochemical, and ultrastructural features retrospectively using mediastinal lymph nodes from 110 cattle and found that 66 of the lymph nodes contained epithelioid cells. In all 66 nodes, most of the epithelioid cells were individual or aggregated in nodal sinuses; in 21 nodes, some epithelioid cells formed tubular structures. The individual and aggregated epithelioid cells were mostly considered to be mesothelial in origin by immunohistochemical and ultrastructural examination; presumably, they entered the lymph nodes via lymphatic vessels, because they were found in afferent lymphatic vessels and nodal sinuses. Although the presence of epithelioid cells in lymph nodes prompts suspicion of metastatic carcinoma, the epithelioid cells in these bovine lymph nodes did not disrupt nodal architecture, lacked atypia or mitotic figures, and did not invade nodal parenchyma.

Canine thyroid C-cell carcinomas (CTCCs) are malignant tumors derived from calcitonin-producing C-cells of the thyroid gland. This study aimed to investigate the histological diversity of CTCCs from the viewpoint of stroma variations and to investigate their components by histological and immunohistochemical analyses including semiquantitative analysis of the density of microvessels (MVs) and α-SMA-positive cell count. Moreover, we examined whether the variations correlated with the Ki-67 index and expressions of glucose transporter 1 (GLUT-1) and monocarboxylate transporter 1 (MCT-1). Three stroma types (reticular, R, nest, N, and trabecular, T) were observed in CTCCs, and 21 cases were divided into 3 variations based on their combinations: mixed R and N (R/N) (n=7), simple N (n=7) and mixed T and N (T/N) (n=7). Immunohistochemically, stroma types depended on morphological features of α-SMA/fibronectin/laminin/collagen type IV-positive stroma cells. The density of MVs in R/N tended to be highest, and the density of those in N was significantly higher than the density of those in T/N (P=0.028). The α-SMA-positive cell count for N tended to be the lowest among the 3 variations. The Ki-67 index for R/N was significantly higher than those of the other variations (vs. N, P=0.007; vs. T/N, P=0.03), and that for T/N tended to be higher than that for N. Although there were no significant differences, GLUT-1 and MCT-1 expressions tended to be low in N. We concluded that stroma variations reflect tumor cell proliferation and expressions of GLUT-1 and MCT-1 in CTCCs.

The objective of this study was to find serum indicators of gastric ulcers in foals. By using two-dimensional electrophoresis of serum proteins, three distinct spots were detected in samples from foals with gastric ulcers detected endoscopically. One of them appeared with high frequency and was identified by partial digestion with trypsin and subsequent nano-electrospray ionisation-tandem mass spectrometry (nanoesi-ms/ms) analysis as an alpha(1)-antitrypsin. Western blot analysis, using an antibody against human alpha(1)-antitrypsin, revealed at least two bands, of molecular weight 58 kDa and 55 kDa, in the sera. The 55 kDa band was detected in 44 of 47 serum samples from foals with gastric ulcers, but in only three of 22 serum samples from healthy foals.

The prevalences of Trichinella T9 in trapped raccoons (Procyon lotor) and several other potential mammalian reservoirs in Hokkaido, Wakayama, and Nagasaki Prefectures were investigated. Muscle samples were collected from 2003 to 2006 from 1,080 raccoons, 113 raccoon dogs including 2 species (Nyctereutes procyonoides albus and N. p. viverrinus), 41 wild boars (Sus scrofa leucomystax), 14 martens (Martes melampus), 10 badgers (Meles meles), 5 Siberian weasels (Martes sibirica coreana), 7 mink (Mustela vison), and 1 red fox (Vulpes vulpes japonica). The samples were digested, and the prevalence and mean intensity of infection with the Trichinella muscle larvae were determined. The prevalence and intensity of the muscle larvae were 0.9% and 93.3 larvae/g (range 0.4-201.8) in raccoons, and 1.6% and 61.6 larvae/g in raccoon dogs, respectively. The infected animals were captured in different areas in Hokkaido Prefecture. These results confirmed that raccoons, which have been introduced from North America since the 1970s, are involved in the sylvatic cycle of Trichinella in Japan. In raccoons, the muscle density of Trichinella T9 larvae was highest in the tongue, and larvae were not found in the heart muscle or diaphragm. This is the first report of Trichinella T9 infection of feral raccoons in Japan.

The purpose of this investigation was to evaluate the clinical usefulness of the total radiographic bone aluminum equivalency (RBAE) method for the measurement of bone mineral content (BMC) in the third metacarpal bone (McIII) of the young Thoroughbred by examining: a) changes in BMC and bone metabolism marker levels in young Thoroughbreds being trained in the growth period; b) correlation of BMC and bone metabolism marker levels; and c) relationships of BMC and bone metabolism marker levels with the state of occurrence of bone disorders. The total RBAE method by the ortho system was chosen for this study. Ninety-one 2-year-old Thoroughbreds that showed no abnormalities on physical examination before the study were evaluated for BMC, and 39 were selected at random for evaluation of the bone metabolism marker levels. BMC of the normal horses showed no change between Days 0 and 90 of the study, but significantly increased on Day 180 (p

The objective of this study was to investigate the cellular immunolocalization of inhibin α and inhibin/activin (βA and βB) subunits and steroidogenic enzymes in neonatal equine ovaries. The ovaries were obtained from two equine neonates of 3 days and 18 days of age. The sections of ovarian tissues were immunostained by the avidin-biotin-peroxidase complex method (ABC) using polyclonal antisera raised against porcine inhibin α, inhibin/activin βA and inhibin/activin βB and bovine adrenal cholesterol side-chain cleavage cytochrome P450 (P450scc), human placental 3β-hydroxysteroid dehydrogenase (3βHSD), porcine testicular 17α-hydroxylase cytochrome P450 (P450c17), and human placental aromatase cytochrome P450 (P450arom). Inhibin α and inhibin/activin βA subunits, but not inhibin/activin βB subunits were expressed in the ovarian interstitial cells of equine neonates. P450scc and P450c17 were also identified in the ovarian interstitial cells and P450arom with weak immunostaining. However, 3βHSD was not found in the ovarian interstitial cells of the equine neonates. These results suggest that equine neonatal ovaries have the ability to synthesize inhibin A and steroid hormones, which may play some important physiological roles in the developing ovaries of equine neonates.

The changes in testicular and pituitary functions in response to human chorionic gonadotropin (hCG) in the breeding and non-breeding season were investigated and compared in the Thoroughbred stallion. Five mature Thoroughbred stallions ranging in ages from 7 to 21 years were injected in May (breeding season) and October (non-breeding season). All animals received an intramuscular injection of 5,000 IU hCG in the experiments. Peripheral blood samples were collected in heparinized tubes from the jugular vein for hormonal assays just before the injection (Day 0) and at a daily interval for five days following the injections (Days 1, 2, 3, 4 and 5). Basal levels of immunoreactive (ir) inhibin, testosterone, estradiol-17β, FSH and LH were higher in the breeding season than in the non-breeding season. There were significant differences in estradiol-17β, FSH and LH between the two seasons. Ir-inhibin exhibited significant increases on Day 2 after the hCG treatment in the non-breeding season, though there was no change in the breeding season. Plasma levels of testosterone showed remarkable increases after the hCG injection in both seasons. The peak levels of plasma testosterone were observed on Days 2 and 3 in the breeding and non-breeding seasons. Plasma estradiol-17β was significantly higher than the basal level on Day 3 in the non-breeding season, whereas there was no change in the breeding season. Plasma levels of FSH declined from Day I to Day 3, then recovered to the basal level (Day 0) on Day 4 after hCG treatment in the breeding season, whereas there was no change in the non-breeding season. Circulating LH showed a significant decrease on Day 3 compared to the based level in the breeding season, but no significant change in the non-breeding season after a treatment of hCG. A significant negative correlation was observed between testosterone and FSH in the breeding season. In conclusion, hCG treatment stimulates secretion of testicular hormones and these testicular hormones temporarily suppress secretion of gonadotropins from the pituitary gland. These results also suggested that Leydig cells of thoroughbred stallion testes have LH receptors in the non-breeding season, the same as in the breeding season.

To elucidate the function of inhibin/activin subunits in developmental fetal adrenal glands, we investigated immunolocalization of inhibin α and inhibin/activin (βA and βB) subunits protein in the equine fetal adrenal glands during mid to late gestation. Fetal adrenal glands of three equines were obtained at 217, 225 and 235 days of gestation. The sections of fetal adrenal glands were immunostained by the avidin-biotin-peroxidase complex method (ABC) using polyclonal antisera raised against porcine inhibin α, inhibin/activin βA and inhibin/activin βB. Inhibin/activin βB was identified in cortex cells of fetal adrenal glands throughout 217-235 days of gestation, but inhibin α and inhibin/activin βA were not observed in fetal adrenal glands in this period. These results suggest that equine fetal adrenal glands have the ability to synthesize activin B but not inhibins and activin A. Also, inhibin/activin βB may play a regulatory role in developing equine fetal adrenal glands.

To elucidate the relationship between steroidogenic hormones and developing adrenal glands, we investigated the immunolocalization of steroidogenic enzymes in equine fetal adrenal glands during mid-late gestation. Fetal adrenal glands were obtained from three horses at 217, 225 and 235 days of gestation. Steroidogenic enzymes were immunolocalized using polyclonal antisera raised against bovine adrenal cholesterol side-chain cleavage cytochrome P450 (P450scc), human placental 3beta-hydroxysteroid dehydrogenase (3betaHSD), porcine testicular 17alpha-hydroxylase cytochrome P450 (P450c17) and human placental aromatase cytochrome P450 (P450arom). Histologically, cortex and medulla cells were clearly observed in the three fetal adrenal gland tissue samples. P450scc and P450c17 were identified in cortex cells close to medulla cells and in some medulla cells in the fetal adrenal glands. P450arom was present in both cortex and medulla cells in the fetal adrenal glands. However, 3betaHSD was not found in any of the equine fetal adrenal gland tissue samples. These results suggest that equine fetal adrenal glands have the ability to synthesize androgen and estrogen, which may play an important physiological role in the development of equine fetal adrenal glands.

The relationship among circulating levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), immunoreactive (ir) inhibin, progesterone, testosterone and estradiol-17β were investigated in fillies from birth to 6 months of age. Six hundred ninety-four Thoroughbred female foals were used for evaluating the changes in these hormones. Blood samples were collected by jugular venipuncture for hormonal assays. The results show that after birth, progesterone, testosterone and estradiol-17β levels rapidly drop within 48 hr, and remain at this lower level until six months of age. On the other hand, plasma concentrations of ir-inhibin begin to decrease at 3 days after birth, falling by more than half in the first 20 days after birth, and then continue to slowly decline until six months of age. Circulating levels of FSH show a small increase within 48 hr after birth, then decline to the lowest levels at 30 days after birth, followed by a rise to more or less stable levels at six months of age, whereas LH concentrations rapidly decrease after birth, dropping to a lower level at 10 days after birth, then remain at the lower value until six months of age. These results demonstrate that, after birth, in a filly, there is a notable decline in steroid hormones which then remain at these relatively stable lower levels until six months of age. The steady increase in basal FSH negatively correlates with the decline in circulating inhibin. This slow increase in the basal levels of FSH may stimulate follicular development during the early stages of puberty in a filly.