Wavy changes in the whiskers of domestic cats are correlated with feline leukemia virus infection.

BACKGROUND: Feline leukemia virus (FeLV) is a retrovirus with global impact on the health of domestic cats and is usually examined by serology. In our daily clinical practice, we noticed that cats infected with FeLV often possess wavy whiskers (sinus hairs on the face). To investigate the relationship between wavy whiskers (WW) and FeLV infection, the association between the presence or absence of wavy changes in whiskers and serological FeLV infection was examined in a total of 358 cats including 56 cats possessing WW, using the chi-square test. The results of blood tests from 223 cases were subjected to multivariate analysis (logistic analysis). Isolated whiskers were observed under light microscopy, and upper lip tissues (proboscis) were subjected to histopathological and immunohistochemical analyses. RESULTS: The prevalence of WW was significantly correlated with FeLV antigen positivity in the blood. Of 56 cases with WW, 50 (89.3%) were serologically positive for FeLV. The significant association between WW and serological FeLV positivity was also confirmed by multivariate analysis. In WW, narrowing, degeneration, and tearing of the hair medulla were observed. Mild infiltration of mononuclear cells in the tissues, but no degeneration or necrosis, was found. By immunohistochemistry, FeLV antigens (p27, gp70 and p15E) were observed in various epithelial cells including the sinus hair follicular epithelium of the whisker. CONCLUSIONS: The data suggest that the wavy changes in whiskers, a unique and distinctive external sign on a cat's face, were associated with FeLV infection.

Use of formalin-fixed paraffin-embedded tissue and single-strand conformation polymorphism analysis for polymerase chain reaction of antigen receptor rearrangements in dogs.

PCR for antigen receptor gene rearrangement analysis (PARR) is a new diagnostic method for lymphoid neoplasia. In PARR using formalin-fixed paraffin-embedded tissues (PARR-FFPE), control DNA amplification was successful in only three of five samples. The formalin fixation times of the three samples were shorter than those of the others. Analysis of the formalin fixation time and DNA amplification controls suggested that a formalin fixation time of less than one week is appropriate. Additionally, application of single strand conformation polymorphism (SSCP) for PARR provided clearer results than conventional PARR in 16 unfixed tissues and three FFPE tissues. These results show that PARR-FFPE is viable for tissues with an appropriate formalin fixation time and that application of FFPE and SSCP for PARR are useful for diagnosis and retrospective study of canine lymphoid neoplasia.

Activity of matrix metalloproteinase-2 (MMP-2) in canine oronasal tumors.

Activity of matrix metalloprotease-2 (MMP-2) and the expression of its related molecules were examined in spontaneous canine oronasal tumors. Tissue samples from melanoma and squamous cell carcinoma possessed higher MMP-2 activity, as shown in gelatin zymography, in comparison with acanthomatous epulis and nasal adenocarcinoma. Regional lymph node invasion and distant metastases were more frequently observed in the MMP-2 positive cases. There were no significant differences by RT-PCR examination in the expression of the genes encoding MMP-2, MT1-MMP and TIMP-2 among the tumor histological types. However, the MMP-2/TIMP-2 ratio showed a significantly higher level of the genes in the malignant oral melanoma and squamous cell carcinoma. The MMP-2/TIMP-2 ratio was also positively correlated with MMP-2 activity in gelatin zymography. These results indicate that the MMP-2/TIMP-2 ratio may be of value in evaluating the prognosis in canine oronasal cavity tumors.

Maxillofacial rhabdomyosarcoma in the canine maxillofacial area.

Three dogs had a diagnosis of maxillofacial rhabdomyosarcoma. These dogs were treated with surgery and/or radiotherapy, and had poor clinical responses. The tumor tissues in all three cases were observed around the upper premolar teeth with ulcerative lesions and CT examinations in each case revealed extensive bony involvement into the maxilla. Two cases were subjected to surgical excision of the tissues, followed by an external radiation therapy. The other case was only treated with palliative radiation. Outcomes of the treatment of all the cases were quite poor because of the invasive and refractory nature of the tumor cells, leading to the local recurrence and lung metastasis early in the clinical course. All dogs died within two months of the first admission.

Expression of the MDR1 gene and P-glycoprotein in canine mast cell tumor cell lines.

Cellular drug resistance to antineoplastic drugs is often due to the presence of a drug efflux pump that reduces intracellular drug accumulation and chemosensitivity. P-glycoprotein (P-gp), which is encoded by the MDR1 gene, is considered to function as an ATP-driven membrane drug efflux pump and appears to play an important role in tumor cell resistance. In the present report, we assessed the expression of MDR1 by RT-PCR in three canine mast cell tumor cell lines, TiMC, CoMS and LuMC, originating from a cutaneous tumor, an oral-mucosal tumor and a gastrointestinal tumor, respectively. P-gp expression was also examined by Western blot analysis, while the functional activity of P-gp was assessed by flowcytometric analysis of intracellular rhodamine-123 (Rhd-123) uptake. The results revealed that MDR1 gene and P-gp were both expressed in CoMS and LuMC cells, whereas neither was present in TiMC cells. In CoMS and LuMC cells, intracellular uptake of Rhd-123 increased in the presence of verapamil, a functional modulator of P-gp. In contrast, TiMC cells did not show any changes in the intracellular accumulation of Rhd-123 after the verapamil addition. These findings suggest that the expressions of MDR1 gene and P-gp probably contribute to cellular drug resistance in canine mast cell tumors.

Tethered cord syndrome with spina bifida aperta in cats: two case reports of different types.

CASE SUMMARY: Two castrated male cats, aged 8 months old (case 1) and 10 months old (case 2), showed a history of progressive paraparesis, an over-reaching pelvic limb gait, urinary incontinence and a palpable dermoid fistula. In case 1, the fistula was connected to the dural sac on the conus medullaris, and the tethered spinal cord was retracted caudally. In case 2, the tubular structure was connected to the dural sac on the thoracic spinal cord, and the tethered spinal cord was retracted dorsally. Tethered cord syndrome secondary to spina bifida aperta was suspected in both cats. Excision of the fistula and release of the tethered spinal cord was performed. A histopathological examination confirmed the diagnosis of a meningomyelocele in case 1 and a meningocele in case 2. Paraparesis improved postoperatively in both cats. However, urinary incontinence in case 1 remained partially unresolved. RELEVANCE AND NOVEL INFORMATION: This is the first report to describe the imaging characteristics, surgical treatments and outcomes of two different types of tethered cord syndrome with spina bifida aperta in cats. Tethered cord syndrome with spina bifida aperta needs to be included in the differential diagnosis of slowly progressive paraparesis in younger cats with or without vesicorectal failure and a palpable dermoid fistula.

Immunohistochemical detection of p27 and p21 proteins in canine hair follicle and epidermal neoplasms.

Trichoblastomas, trichoepitheliomas, and squamous cell carcinomas in the skin of dogs were analysed by immunohistochemistry for the nuclear expression of p27, p21 and proliferating cell nuclear antigen (PCNA). High levels of p27 were present in trichoepitheliomas and trichoblastomas compared with squamous cell carcinomas. Detectable p21 was found in trichoepitheliomas and squamous cell carcinomas, but trichoblastomas had low level of p21 nuclear reactivity. Low levels of PCNA were detected in trichoepitheliomas and trichoblastomas compared with squamous cell carcinomas. The results suggested that nuclear p27 acts as a cyclin-dependent kinase (CDK) inhibitor in trichoepitheliomas and trichoblastomas. Nuclear p21 expression is involved in the induction of epithelial differentiation and seems to be unrelated to CDK inhibition.

Immunoblot analysis of specific antigen bands predictable for Dirofilaria immitis infection in cats.

Serial sera from four mongrel cats experimentally inoculated with infectious larvae of Dirofilaria immitis were analyzed by immunoblot patterns against a phosphate buffered saline-extract of D. immitis. Antigen-specific protein bands detected indicate that the low molecular weight bands of 36, 32, 22, 19 and 14 kDa, are predictable for positive adult worm infection, suggesting diagnostic usefulness for adult D. immitis infection in cats.

Nucleotide sequence of complementary DNA encoding for quaking protein of cow, horse and pig.

Complementary DNA (cDNA) for bovine quaking gene (Bqk), equine quaking gene (Eqk) and porcine quaking gene (Pqk), which are homologous to mouse quaking gene (qkI), were isolated, and their nucleotide sequences were determined. cDNA sequences of Bqk, Eqk and Pqk showed very high homology to that of qkI at nucleotide level; 94.2, 95.7 and 95.6%, respectively. Deduced amino acid sequences for Bqk, Eqk and Pqk perfectly matched to that of qkI. These findings suggest that the quaking gene family is highly conserved during mammalian evolution, and that Bqk, Eqk and Pqk are likely to have important biological functions also in cow, horse and pig.

Infestivity of Demodex canis to hamster skin engrafted onto SCID mice.

We demonstrated that Demodex canis was transferred to skin xenografts of a dog and a hamster onto severe combined immunodeficiency mice. After the transfer of mites, the number of eggs, larvae, nymphs and adult mites per gram of canine and hamster xenografts increased, whereas no live mites were detected on murine allograft. These results indicate that D. canis proliferates in hair follicles of dog and hamster skins but not in murine allograft. Therefore, D. canis may have host preference but not strict host-specificity.

Application of phosphoenolpyruvate into canine red blood cell cryopreservation with hydroxyethyl starch.

Phosphoenolpyruvate (PEP) is a phosphorylated glycolytic intermediate that can penetrate the RBC membrane and be metabolized to 2,3-DPG and ATP. In this study, we evaluated the effects of PEP treatment on canine red blood cells (RBCs) cryopreserved with 12.5% (w/v) HES. RBCs were incubated for 30, 60, and 90 min at 37 degrees C with PEP solution containing 60 mM mannitol, 30 mM sodium chloride, 25 mM glucose, 1 mM adenine and 50 mM PEP (340 m osm/kg), pH 6.0 and then cryopreserved in liquid nitrogen with 12.5% (w/v) HES for 2 weeks. 2,3-DPG and saline stabilities of the PEP treated groups were increased and osmotic fragility indices were significantly decreased compared to the untreated control group. There were no differences in 2,3-DPG levels within the PEP treated groups with different PEP incubation times. These results suggest that PEP treatment may be beneficial for the cryopreservation of canine RBCs with HES.

Molecular survey of Mycoplasma haemofelis and 'Candidatus mycoplasma haemominutum' infection in cats in Yamaguchi and surrounding areas.

A molecular survey of hemoplasma (Mycoplasma haemofelis and 'Candidatus Mycoplasma haemominutum') in Yamaguchi Prefecture and surrounding areas was performed by using molecular methods. PCR-RFLP with HindIII revealed that 2 cats were infected with M. haemofelis, and 16 with 'C. Mycoplasma haemominutum' among 102 randomly selected cats. Partial 16S rRNA gene sequences of M. haemofelis and 'C. Mycoplasma haemominutum' determined in this study showed percent similarities of 98.3-99.8% and 96.4-100%, respectively, with those from other countries. Hemoplasma infections were more frequently detected in free-roaming cats than inside cats. Also, the status of FeLV infection was another significant risk factor for hemoplasma infection.

Expression of a tumor-associated antigen, RCAS1, in canine mammary tumors.

Receptor-binding cancer antigen expressed on SiSo cells (RCAS1), one of novel cancer cell-surface antigens, is strongly expressed in invasive cancers. RCAS1 inhibits the in vitro growth of lymphocytes such as T cells and natural killer (NK) cells, and induces apoptotic cell death. We investigated the expression of RCAS1 in canine mammary tumor cell lines and tumor cells by immunohistochemistry, and also in situ deoxyribonucleic acid (DNA) fragmentation in tumor-infiltrating lymphocytes (TILs) by the terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labeling (TUNEL) method. All canine mammary tumor cell lines expressed RCAS1 at both the messenger ribonucleic acid (mRNA) and protein level. Immunohistochemically, RCAS1 was negative in 100% of normal mammary glands, but was expressed in 100% of malignant tumors examined. In most malignant mammary tumors, RCAS1 was localized in the cytoplasm with no polarity of expression. In benign mammary tumors, it was detected on the luminal surface of the tumor cell. RCAS1 expression or localization was significantly correlated with malignancy. In situ DNA fragmentation of CD3-positive TILs was observed in RCAS1-expressing tumors. RCAS1-expressing tumors, indicating a possible induction of apoptotic cell death in TILs through RCAS1 expression. These observations suggest that RCAS1 probably plays an important role in tumor progression and escape from immune surveillance in canine mammary tumors.

Molecular cloning of canine RCAS1 cDNA.

Receptor-binding cancer antigen expressed on SiSo cells (RCAS1) is a novel cancer cell-surface antigen, strongly expressed in invasive cancers. RCAS1 inhibited the in vitro growth of immunocytes, and induced apoptotic cell death. The cloning of canine RCAS1 cDNA was carried out and identified from the mammary gland tumor of a dog. A canine RCAS1 cDNA of 864 bp in length has an open reading frame of 642 bp nucleotides encoding a protein of 213 deduced amino acids. The predicted amino acid sequence of canine RCAS1 showed 96.2% and 96.7% homologies with those of human and mouse RCAS1 respectively. Canine RCAS1 has an N-terminal transmembrane segment and a coiled-coil structure in the C-terminal protein, which are highly conserved in mouse and human RCAS1.

A comparative study of the effects of glycerol and hydroxyethyl starch in canine red blood cell cryopreservation.

Hydroxyethyl starch (HES) is a nonpenetrating extracellular cryoprotectant. In contrast to glycerol, it does not require labor-intensive removal from thawed red blood cells (RBCs) prior to transfusion. In this study, we compared glycerol and HES, and assessed HES as a substitute for glycerol in cryopreserved canine RBCs. The RBCs were preserved for 2 months in liquid nitrogen using a 20% (w/v) glycerol solution, and variable concentrations of HES solution. We evaluated the two cryoprotectants by the percentage of post-thaw hemolysis from the total free hemoglobin, saline stability, osmotic fragility, and by observing the erythrocyte morphology using a scanning electron microscope after thawing. The optimal concentration of HES was 12.5% (w/v) for the cryopreservation of canine RBCs. The thaw hemolysis, saline stability, and osmotic fragility index were 25.6 +/- 4.7%, 87.8 +/- 6.9%, and 0.445 +/- 0.024% NaCl respectively. These parameters resemble the results of RBCs frozen in a 20% (w/v) glycerol solution, which are 24.7 +/- 5.2%, 99.2 +/- 0.1%, and 0.485 +/- 0.023% NaCl respectively. From a morphological point of view, 12.5% (w/v) HES showed the best cryoprotection of RBCs compared to the other concentrations of HES. These results suggest that HES could be a possible substitute for glycerol for the cryopreservation of canine RBCs.

Detection of the anti-P53 antibodies in dogs with tumors.

To detect the anti-P53 antibodies of dogs with tumors, a GST-recombinant canine (rc) P53 fusion protein was expressed and purified. Immunoblot analysis was performed using this GST-rcP53 fusion protein as an antigen and serum samples from dogs suffering from tumors as primary antibodies. Out of 16 serum samples obtained from various tumor cases, four samples showed reaction with GST-rcP53. In contrast, serum from other 12 dogs with tumors, four dogs with non-neoplastic diseases and two control healthy dogs (as controls) did not show any reaction with GST-rcP53 in immunoblotting. The p53 gene mutation and the P53 protein expression were examined, using the tumor tissues to explore the relationship between the existence of the GST-rcP53 bands, gene mutations of p53 and the accumulation of P53 protein. One case, which showed a clear GST-rcP53 band, had a point mutation of the p53 cDNA and showed nuclear accumulation of P53 protein. These results suggest that the anti-P53 antibodies are also produced in tumor dogs with p53 gene mutations.

Evaluation of cytokine messenger RNA expression in peripheral blood mononuclear cells from dogs with canine demodicosis.

Using RT-PCR and semi-quantitative PCR, mRNA expression for canine interferon (IFN)-gamma, interleukin (IL)-2, IL-4, IL-5, IL-10, tumor necrosis factor (TNF)-alpha and transforming growth factor (TGF)-beta in peripheral blood mononuclear cells (PBMCs) was examined in dogs with or without demodicosis. mRNA expression for IFN-gamma as well as TNF-alpha in dogs with demodicosis (localized (LD) and generalized (GD)) was slightly lower than those in dogs without demodicosis (healthy controls). Expression of IL-5 mRNA in dogs with demodicosis was higher than that in control dogs, but there were no significant differences in IL-4 and IL-10 mRNA expression levels among the three groups. On the other hand, expression levels of TGF-beta mRNA in dogs with GD were higher than those in control dogs and dogs with LD. The expression levels of IL-5 and TGF-beta mRNA decreased in all three dogs with GD which showed resolution of the clinical signs. Taken together, these results suggest that the Th2-like response in PBMCs from dogs with demodicosis is up-regulated, and that subsequent increased expression of IL-5 and TGF-beta mRNA in dogs with GD is reversible after treatment. Therefore, these cytokines, particularly IL-5, might be a useful clinical index of the clinical course in demodicosis. Also, increased TGF-beta mRNA expression might be a key factor for revealing the difference in the mechanism of onset between LD and GD.

Transorbital echoencephalography in cattle.

The purpose of this study was establishment of the criteria of transorbital echoencephalography in cattle and experimental applications to bovine practice. Quantitative investigations using magnetic resonance (MR) imaging revealed that this examination could be applied to cattle under 3 months of age. The method of transducer positioning was established in Japanese Black (J.B.) and filial (F1) cattle (turning caudally at an angle of about 16 degrees and dorsally at an angle of about 23 degrees) or in Holstein cattle (turning caudally at an angle of about 20 degrees and dorsally at an angle of about 21 degrees). Examinations in clinical calves revealed that the cerebral parenchyma and the lateral ventricle could be detected antemortem or postmortem. In this study, the diagnoses of hydrocephalus or hydranencephaly was possible using antemortem transorbital echoencephalography. Transorbital echoencephalography was especially useful as the imaging method for bovine hydranencephaly.