A new method for counting of quail leukocytes by flow cytometry

An automatic counting method was developed for fish blood cells using a fluorescent dye, 3, 3-dihexyloxacarbocyanine (DiOC (6)(3)), that selectively stain lipid bilayers in living cells. In the present study, the DiOC(6)(3) method was applied to quail (Cotumix cotumix japonica) blood cells. After quail blood cells were stained with DiOC(6)(3), absolute counts and relative proportions of erythrocytes, granulocytes, monocytes, and lymphocytes plus thrombocytes in whole blood were obtained by means of flow cytometry (FC). The number of each cell types by the FC was in good agreement with those counted microscopically. This method will offer new possibilities for routine blood cell counting for avian medicine.     

Effects of a collagenolytic cell wall component from Fusobacterium necrophorum subsp. necrophorum on rabbit tissue-culture cells

The effects on rabbit tissue-cultured cells of collagenolytic cell wall component (CCWC) from Fusobacterium necrophorum subsp. necrophorum were investigated. Scanning electron microscopy demonstrated that CCWC damaged the cell surfaces of the rabbit granulocytes and hepatocytes but the effects of the cells differed from each other. Granulocytes appeared smooth and morphologically irregular whereas hepatocytes looked rough and had tiny holes in the cell membranes. Differences in cell viability were observed in MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium, inner salt) assay. The findings suggest that cytotoxic activity in vivo may well contribute to the establishment of an initial injury in visceral tissues, and the action of CCWC could increase the chances of survival for an invading F. necrophorum subsp. necrophorum at the first stages of infection.     

Molecular epidemiological survey of benign Theileria parasites of cattle in Japan: detection of a new type of major piroplasm surface protein gene

Benign Theileria species of cattle are found in most parts of the world. The major piroplasm surface protein (MPSP), a conserved protein in all Theileria species, has been used as a maker for epidemiological and phylogenetical studies of benign Theileria species. Parasites with Ikeda- or Chitose-type MPSP genes are dominant in Japan, but we report here mixed infection cases of Theileria parasites with an additional MPSP type parasite infecting cattle in Abashiri District, Hokkaido. The MPSP gene sequence found in the additional type was closely related to MPSP genes of Theileria parasites found in Southeast Asian countries, including Thailand (Narathiwat) and Indonesia (Java). Theileria parasites from the blood sample were also distinguishable from the Ikeda or Chitose type parasites by the small subunit (SSU) rRNA gene sequence analysis, and they are grouped into the SSU rRNA types C/D found in Korea, North America, and Spain. The present finding of mixed infections of cattle with three different types of Theileria makes epidemiological feature of bovine theileriosis in Japan more complex. We have designed a set of primers specific to this MPSP type in order to conduct further epidemiological study.     

Preliminary immunohistochemical study of natriuretic peptide receptor localization in canine and feline heart

We examined the immunohistochemical distributions of natriuretic peptide receptor (NPR)-A, -B and -C that bind with natriuretic peptide hormones A, B and C in four healthy crossbreed young canine and feline cardiac tissues using specific antibodies against human antigens. Cross-immunoreactivities between antigens and antibodies were confirmed using western blot analysis. NPR-A and -C were expressed more strongly in dogs than cats. In both species, these expressions were stronger in the atria than the ventricles, with stronger expression in the left ventricles than the right. NPR-B was largely very weekly or undetected. In canine and feline cardiac tissues, the expressional distribution of NPR-A, -B, and -C closely matched with that of atrial natriuretic peptide, brain natriuretic peptide, and C-type natriuretic peptide as the ligands for corresponding receptors.     

Development, characterization, and lethal effect of monoclonal antibodies against hemocytes in an adult female tick, Ornithodoros moubata (Acari: Argasidae)

In the present study, 19 monoclonal antibodies (mAbs) against adult Ornithodoros moubata hemocytes were established, and the reactivity of the hemocytes to these mAbs was examined by an indirect fluorescent antibody test (IFAT), Western blot and immunoprecipitation analyses. It was shown that the reactivities of the hemocytes to the mAbs varied among morphologically similar hemocyte types, and most mAbs produced in the present study showed the multiple band reactivity. However, the presence of shared epitopes among peptide subunits of the same protein or entirely different proteins are not common, so their reactivity could not be explained in detail. These results suggest that there are morphologically similar but functionally differentiated hemocytes. Therefore, in addition to morphological classification, the molecular-based classification of the hemocytes is also required. In order to assess the lethal effect of blood meal containing each mAb, artificial feeding was performed. The OmHC 31 showed the strongest lethal effect on adult female O. moubata. In conclusion, anti-hemocyte mAbs produced in this study are useful not only for the immunological classification of hemocytes but also for the immunological control of the tick.     

Insulin-like growth factor-I stimulated DNA replication in mouse endometrial stromal cells

Much evidence has suggested that sex steroid hormone-induced growth of uterine cells is mediated by polypeptide growth factors synthesized in uterine tissues. The present study aimed to clarify the effect of insulin-like growth factor-I (IGF-I) on the proliferation of mouse endometrial stromal cells obtained from immature mice. IGF-I and IGF-I receptor (type I) mRNAs were detected in the endometrial stromal cells. IGF-I increased bromodeoxyuridine (BrdU) uptake in the endometrial stromal cells, indicating an increase in DNA replication. E2 increased IGF-I mRNA levels in the endometrial stromal cells. IGF-I receptor is a tyrosine kinase receptor, and treatment with genistein, a tyrosine kinase inhibitor, reduced IGF-I-induced BrdU-uptake in the endometrial stromal cells. IGF-I signaling pathways involve mitogen-activated protein (MAP) kinase and phosphatidylinositol-3 kinase (PI-3 kinase). Treatment with 10(-7) M of the MAP kinase inhibitor PD098059 and 10(-5) M of the PI-3 kinase inhibitor LY294002 decreased IGF-I-induced BrdU-uptake in the endometrial stromal cells. However, LY294002 (10(-5) M) also decreased the BrdU-uptake in the absence of IGF-I treatment. These results suggest that endometrial IGF-I is involved in the proliferation of endometrial stromal cells in a paracrine or autocrine manner, and that the MAP kinase pathway is involved in DNA replication of endometrial stromal cells.     

A case report of traumatic neuroma of the cervical spinal cord in a dog

Traumatic neuroma of the cervical spinal cord was diagnosed in a 14-year-old male mixed-breed dog. A gross view showed two intradural extramedullary masses, measuring 1 and 0.6 cm in length and 0.7 and 0.4 cm in diameter, attached to the left side of the spinal cord at the level of the sixth and seventh cervical vertebrae. Microscopically, the cervical spinal masses comprised interlacing fascicles of axons and Schwann cells surrounded by collagenous stroma. Immunohistochemically, the fascicles were stained positively for neurofilament and S-100 proteins. Ultrastructurally, variably sized myelinated fibers and onion bulb-like structures were observed. To our knowledge, this is the first report of a traumatic neuroma in the cervical spinal cord of a dog.     

Impressions and purchasing intentions of Japanese consumers regarding pork produced by ‘Ecofeed,’ a trademark of food‐waste or food co‐product animal feed certified by the Japanese government

Impressions and purchasing intentions of Japanese consumers regarding pork produced by ‘Ecofeed’, a trademark of food‐waste or co‐product animal feeds certified by the Japanese government, were investigated by a questionnaire on the Internet. ‘Ecofeed’ did not elicit specific impressions as compared to domestic, imported, Kurobuta (in Japan), and specific pathogen‐free (SPF) pork. Purchasing intent for ‘Ecofeed’ pork was the second lowest of the five pork products. Knowledge and purchasing experience regarding ‘Ecofeed’ pork was the lowest of the five pork products. Respondents were classified into four categories according to their impressions of ‘Ecofeed’ pork. The largest category of respondents did not have any specific impression of ‘Ecofeed’ pork and had little knowledge of pork farming. A category that had a positive impression for ‘Ecofeed’ pork had high knowledge of the pork farming system. In order to establish ‘Ecofeed’ pork in Japan, our results suggest that information disclosure and education about ‘Ecofeed’, its certification system, environmental benefits and the current self‐efficiency ratio of animal feed, are needed.