Koichi Kadota

Characterization of novel bovine papillomavirus type 12 (BPV-12) causing epithelial papilloma

Bovine papillomavirus type 12 (BPV-12, putative type BAA1) was detected in epithelial papilloma located on the tongue of an infected cow. Then, the whole genome was sequenced, and phylogenetic analysis illustrated that it should be classified as a member of the genus Xipapillomavirus. The viral genome is 7197 base pairs in length and contains five early ORFs (E1, E2, E4, E7 and E8), three late ORFs (L1, L2 and L3), and a long control region that possesses replication regulatory elements. Meanwhile, mRNA of each gene was detected in the papilloma sample. The papilloma was identified as epithelial papilloma by histological and immunohistochemical examination. Based on the genome information and pathological properties, BAA1 was designated as BPV-12 in this study.

Precursor B-1 B cell lymphoma in a newborn calf.

A newborn Holstein female calf had neoplastic lesions in the skin and within the thoracic and abdominal cavities but not in the bone marrow, spleen, thymus, or most lymph nodes. Because the tumor cells were positive for CD79a (B cell marker), CD5 (B-1 cell marker) and terminal deoxynucleotidyl transferase (marker for immature lymphoid precursors), a diagnosis of precursor B-1 B cell lymphoma was made. The diagnosis was strongly supported by the fact that B-1 cells can develop in the fetus, unlike B-2 cells, which are produced after birth. The lymphoma was distinct from the typical calf form of lymphoma of B-2 cell origin, which does not express CD5 and is characterized by generalized lymphadenopathy and involvement of the bone marrow, blood and spleen.

Bovine papillomavirus type 9 induces epithelial papillomas on the teat skin of heifers

Experiments were carried out to investigate whether papillomas could be induced on the teat skin of heifers by intradermal injection with bovine papillomavirus type 9 (BPV-9). Three heifers (#1 and 2, two 0.5-year-old Holsteins; #3, a 1.5-year-old Japanese Black) were injected with BPV-9 and one heifer (#4, a 0.5-year-old Holstein) was mock-infected. Viral DNA load in the inocula was quantified by real-time polymerase chain reaction assay and adjusted to 1.56x10(12) copies per injection. Papillomas appeared at the injection sites in the BPV-9-injected heifers #1, 2 and 3 and grew over the 8 (#1 and 2) and 4 (#3)mo observation period, respectively. However, no papillomas were found in the mock-infected heifer #4. The experimentally induced papillomas were excised and examined. Histologically, the lesions were characterized by hyperplasia of the epidermis with hyperkeratosis and marked acanthosis and were morphologically similar to naturally occurring lesions. BPV-9 DNA and bovine papillomavirus capsid antigen were abundant in the lesions. Therefore, we conclude that BPV-9 is an etiological agent causing epithelial papillomas on the teat skin of heifers.

Adenocarcinoma of the Lung in a Steller Sea Lion (Eumetopias jubatus)

The histological and ultrastructural characteristics of an adenocarcinoma of the lung are described in an about 16-year-old female Steller sea lion with a 1.5 month history of cough and anorexia. The animal had multiple neoplastic nodules in the lungs and diaphragmatic pleura. The bronchial and mediastinal lymph nodes were replaced by neoplastic tissue, and there were several metastatic lesions in the liver and spleen. The lung tumor was characterized by accumulations of encapsulated lesions with central necrosis, and the neoplastic cells showing a papillary growth pattern produced small amounts of mucin. Ultrastructurally, some cells contained basal bodies, and cilia were rarely seen. This neoplasm was considered to be of ciliated bronchial or bronchiolar epithelium origin.

Eosinophilic granuloma with Splendore-Hoeppli material caused by toxigenic Corynebacterium ulcerans in a heifer.

ABSTRACT Raised lesions were present on the left nasal vestibule of a 20-month-old Japanese Brown heifer. The largest mass which caused partial nasal obstruction was removed surgically. Corynebacterium ulcerans was identified in the mass. 16S ribosomal RNA and RNA polymerase beta subunit genes were 100% and 98% identical to other C. ulcerans strains. Histologically, multiple foci of eosinophilic granuloma with Splendore-Hoeppli material were seen. Rod-shaped Gram-positive organisms were detected with metachromatic granules, producing diphtheria toxin with 5, 30 and 48 amino acid differences to another C. ulcerans strain, C. diphtheriae or C. pseudotuberculosis, respectively. The toxin is highly cytotoxic and may be responsible for the formation of abundant Splendore-Hoeppli material. The lesion was therefore judged to be an allergic reaction to bacterial antigens or diphtheria toxin.

Immunohistochemical and ultrastructural detection of intestinal spirochetes in Thoroughbred horses

Studies of equine intestinal spirochetes have long focused on intestinal contents alone, but intestinal spirochetosis has been reported recently in a 21-month-old Thoroughbred colt in Japan. To define the clinical and pathological significances of intestinal spirochetosis in several horses, an epizootiologic survey with histologic, immunohistochemical, and ultrastructural methods was conducted for Brachyspira antigen–containing intestinal spirochetes in 12 diseased or injured Thoroughbred horses, aged from 35 days to 17 years. Brachyspira antigen–containing spirochetes were found in 7 of 12 horses (58.3%) and were more frequent in the cecum than in other parts of the bowel. It was not clear whether the infection was clinically related to diarrhea or dysentery, but histopathology revealed a close association between the bacterial infection and epithelial hyperplasia. Crypt epithelium consisted mainly of goblet cells and showed frequent mitosis throughout its length. Inflammatory cells and congestion were also present. There were numerous spirochetes in the crypts, and some invaded the cecal and colonic epithelia and underlying lamina propria. Ultrastructurally, the spirochetes were divided into 4 types. Three types were identified in degenerative epithelial cells or intracellularly. Brachyspira antigen–containing intestinal spirochetes invading the mucosa were capable of causing epithelial hyperplasia in the cecum and colon in the horses. The findings in this study will increase awareness of the importance of intestinal spirochetosis and may also be helpful for diagnosis and treatment of this condition.

Plasma Cell Myeloma Producing IgG, IgM, and IgA Immunoglobulins in a Cow

A diagnosis of plasma cell myeloma was made in a 6-year-old Holstein cow that showed continuous nosebleed, progressing emaciation, and hyperglobulinemia. Necropsy revealed enlargement of the caudal mesenteric lymph nodes. Lymph nodes and spleen were infiltrated by neoplastic plasma cells. Renal lymph nodes, bone marrow, and kidneys also were affected. Amyloid-like material that was negative for Congo red was present in most of the affected lymph nodes. Congo red–positive amyloid material was noted in spleen, kidneys, liver, and adrenal glands. Neoplastic plasma cells exhibited immunohistochemical reactions for λ-light chain and 3 classes of heavy chain but not for κ-light chain. Nonamyloid deposits were labeled with antisera against λ-light chain and heavy chains. Plasmacytoid cells, which stained positively for IgG, IgM, or IgA, were intermingled in all lesions. Production of 3 types of immunoglobulins may be caused by heavy-chain class switching, constantly occurring in certain proportions.

Intranuclear coccidiosis caused by Cyclospora spp. in calves

Intranuclear coccidiosis in 3 calves is described. Two calves with no genetic relationship were kept in the same pen, and the third calf was on another farm. The animals suffered from watery diarrhea or severe emaciation. Histologic examination showed epithelial desquamation (cases 1 and 2) or atrophy (case 3) of the jejunal villi. Coccidial meronts or merozoites were present in the nuclei of the majority of villus epithelial cells. There were rare intranuclear macrogametocytes, macrogametes, microgametocytes, microgametes, and oocysts in cases 1 and 2, but these were more easily observed in case 3. Parasite 18S ribosomal RNA sequences from case 1 showed 99.5% sequence identity with Cyclospora sp. Guangzhou 1, which has been found in fecal samples of cattle from China. In addition to the molecular results in one of the cases, the fact that the microgametocytes and oocysts were noticeably smaller in size than those of Eimeria alabamensis in all cases indicates the close association between Cyclospora and the 3 cases described herein.

Detection of bovine papillomavirus type 2 DNA in calf conjunctival myofibroblastoma

An 8-month-old male Japanese Black calf was referred for the evaluation of a slow-growing conjunctival mass in the right eye. A superficial keratectomy was performed followed by recurrence on two occasions. No metastases were found in surrounding tissues. Histological, immunohistochemical, and ultrastructural investigation revealed that both the primary and the recurrent lesions were benign, conjunctival, myofibroblastomas. Interestingly, bovine papillomavirus type 2 (BPV-2) DNA was detected in both myofibroblastoma lesions. Archival bovine myofibroblastomas from the vulva and neck were also analyzed for papillomaviral genomes. BPV-2 DNA was also amplified from these lesions. To the best of our knowledge, this is the first report describing a potential causal relationship between BPV-2 infection and conjunctival myofibroblastoma.

Eosinophilic granuloma with Splendore-Hoeppli material caused by Mannheimia granulomatis in a calf.

A large subcutaneous mass, formed on the left lower jaw of a 4-month-old Japanese Black male calf, was partially excised for histological and bacteriological examinations. Antibiotic treatment resulted in a good prognosis. Bacteria isolated from the excised material were characterized by weak hemolysis and positive reactions for catalase and oxidase, and were 99% identical to Mannheimia granulomatis strains. The presence of the leukotoxin gene product was demonstrated by polymerase chain reaction amplification. Histological examination showed that the excised material was composed of dense fibrous connective tissue with sparsely distributed eosinophilic granulomas or abscesses. These foci frequently contained Splendore-Hoeppli material with rod-shaped Gram-negative bacteria. Except for the absence of lymphangitis and the presence of basophils and mast cells, the histology of this lesion resembled that of lechiguana associated with coinfection of M. granulomatis and Dermatobia hominis. Leukotoxin was demonstrated by immunohistochemistry within Splendore-Hoeppli material and was judged to be responsible for its formation.