Detection of antibodies against porcine parvovirus in swine sera by enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay (ELISA) was developed for detection of antibody against porcine parvovirus in swine sera. The antigen used for the assay was partially-purified virus treated with fluorocarbon and shown to contain 7 proteins by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Of these proteins 83-, 64- and 60-K proteins reacted in Western immunoblotting with swine serum after infection with porcine parvovirus. Antibody responses were demonstrated by ELISA in pigs subcutaneously-infected with porcine parvovirus as by hemagglutination-inhibition (HI) test and Western immunoblotting reaction with the 83-, 64- and 60-K viral proteins. The results of ELISA on random swine-serum samples were well-correlated with those of the HI test. These findings indicate the usefulness of the ELISA as a serological tool for porcine parvovirus infection.     

Modification of three avian viruses passaged in Chinese hamster lung cells (Don) in pathogenicity to chicken embryo

The Beaudette 42 strain of avian infectious bronchitis virus, Sato strain of Newcastle disease virus, and Uchida strain of avian reovirus were passaged in Chinese hamster lung cells (Don), and some properties were examined. The Don-passaged strains showed a difference in replication in Don and chicken embryo kidney cells in one-step growth curve examinations and a partial modification in pathogenicity to chicken embryos; nevertheless, neutralization tests revealed no serological alteration.     

Establishment of a novel feline leukemia virus (FeLV)-negative B-cell cell line from a cat with B-cell lymphoma

There is significant lack of basic hematologic and immunological data in adult sows. Therefore, aim of this study was to provide respective reference intervals. 32 clinically healthy multiparous Large White sows aged 33.5 ± 9.6 months and all of them two months postpartum were included in this study. Mean erythrocyte count was 5.5 ± 0.7 × 10(6)/μl and total leukocyte count was 12.1 ± 2.1 × 10(3)/μl. Proportion of lymphocytes was 44.7 ± 10.2% and of neutrophils 41.6 ± 11.0%. The ratio of na?ve T helper (Th) cells to memory Th cells was 1:3.1 and the ratio of Th cells to cytotoxic T cells (CTLs) was 1:4.2. Proportions of regulatory T cells, NK cells, and CD21(+) B cells were lower (3.1, 2.6, and 6.0%) than those of memory Th cells ranging from 8.8 to 27.5% depending on the activation status and CTLs with 37.3%. γδ T cells were found at comparably high numbers (19.1%). Flow cytometric measurement of intracellular cytokines in PBMCs revealed marginal levels for IL-1β, IL-2, IL-4, IL-6, IL-10, and IL-12p35, but remarkable levels for TNF-α and IFN-γ. Highest mRNA levels were found for IL-1, IL-10, and TNF-α, with TNF-α showing the least inter-individual variation.     

Staphylococcus pseudintermedius exfoliative toxin EXI selectively digests canine desmoglein 1 and causes subcorneal clefts in canine epidermis

Staphylococcal exfoliative toxins are known to digest desmoglein (Dsg) 1, a desmosomal cell–cell adhesion molecule, thus causing intraepidermal splitting in human bullous impetigo, staphylococcal scalded skin syndrome and swine exudative epidermitis. Recently, a novel exfoliative toxin gene (exi), whose sequence shares significant homology with previously identified exfoliative toxins, was isolated from Staphylococcus pseudintermedius. Little is known about the pathogenic involvement of this toxin in canine pustular diseases such as impetigo. The aim of this study was to determine whether EXI, the product of the exi gene, digests canine Dsg1 and causes intraepidermal splitting in canine skin. An exi gene was isolated from chromosomal DNA of an S. pseudintermedius strain obtained from a pustule of a dog with impetigo, and was used to produce a recombinant EXI by Escherichia coli expression. When purified recombinant EXI was injected intradermally into normal dogs, it caused the development of vesicles or erosions with superficial epidermal splitting. In addition, the EXI abolished immunofluorescence for Dsg1, but not for Dsg3, at the injection sites. Moreover, the EXI directly degraded baculovirus‐secreted recombinant extracellular domains of canine Dsg1, but not that of canine Dsg3, in vitro. The EXI also degraded mouse Dsg1α and swine Dsg1, but not human Dsg1, mouse Dsg1β and Dsg1γ. Conversely, recombinant SIET, previously designated as S. intermedius exfoliative toxin, did not cause intraepidermal splitting or degradation of any Dsgs. These findings indicate that EXI has a proteolytic activity that digests canine Dsg1, and this characteristic might be involved in the pathogenesis of intraepidermal splitting in canine impetigo.     

Effect of Mechanical Stretching on Expressions of Muscle Specific Transcription Factors MyoD, Myf-5, Myogenin and MRF4 in Proliferated Myoblasts

We examined expression of four important members of myogenic regulatory factors (MRFs) in the myoblasts both at mRNA and protein levels, which were subjected to mechanical stretching in in vitro condition. Our results showed that MyoD expression existed both in the stretch and in the control group at all time periods of the mechanical stimulus. Myf-5 expressed only at early stage of the stretch group. Although mRNA and protein expressions of myogenin and MRF4 were detected both in the stretch and in the control group at 12 h after the stretching, their expressions were only shown in the stretch group at 24 h after the mechanical stimulus. However, at 36 and 48 h, none of the MRFs examined except MyoD appeared in both groups. Our results suggest that the MRFs are up-regulated upon mechanical stimulus and each member plays a different major role for either proliferation or differentiation of the myoblasts.     

First report of triploidy in Ailanthus altissima,an invasive tree species

Ailanthus altissima is a woody plant that has become invasively naturalized in many countries. Using nuclear SSR markers, we studied specimens of this species collected throughout Japan and found an individual (labeled Aa-1) that was not diploid. We examined pollen morphology and ploidy level of Aa-1 by optical microscopy and flow cytometry (FCM), respectively. Some pollen grains were morphologically abnormal and much larger than normal. FCM analysis demonstrated that Aa-1 was triploid. Formation of abnormal pollen indicates that Aa-1 may well have reduced fertility compared to normal diploids. If this were the case, vegetative progeny Aa-1 could be used as ornamentals without attendant risks of future naturalization.     

Enhancement of reactive oxygen species production from canine blood leukocytes by human recombinant interleukin-12

A novel biological activity of human recombinant interleukin-12 (rhIL-12) on canine peripheral blood mononuclear cells (PBMC) was investigated in vitro. Canine PBMC were cultured in the presence or absence of rhIL-12 for 3 days. The reactive oxygen species (ROS) production induced by opsonized-zymosan (OZ) was then measured by a luminol-dependent chemiluminescense assay and demonstrated that the ROS production was enhanced after culture with rhIL-12. A nitro blue tetrazolium test and flowcytometry analysis revealed that canine lymphocytes, eosinophils, and monocytes were capable of ROS production, but that monocytes had the highest capacity. These results suggest that rhIL-12 enhances ROS production from canine monocytes.     

Proliferative potential of endometrial stromal cells, and endometrial and placental expression of cyclin in the bovine

The objective of the present investigation was to study proliferative activity of fibroblast-like endometrial stromal cells in bovine endometrial caruncular (CAR) and intercaruncular (ICAR) areas that have distinct functions during implantation. Endometrial stromal cells were derived from CAR and ICAR of nonpregnant cows, and their proliferative potential was analyzed in an in vitro cell culture system. In addition, expression of four types of cell cycle regulatory molecules was analyzed by RT-PCR in samples of CAR, ICAR, cotyledon (COT) and fetal membrane of both artificially inseminated (AI) and somatic nuclear transferred (NT) cows on days 30 and 60 of gestation. The proliferation growth curve showed that the cells derived from CAR had higher proliferative activity than that of ICAR-derived cells. No morphological differences were found between the cells derived from CAR and ICAR at population-doubling levels (PDL) of the two. Most of the cells derived from ICAR of nonpregnant cows exhibited expanded shape with no further proliferation at PDL 6 with a lack of cyclin E expression. Of the regulatory molecules, cyclin D1, CDK2 and CDK4 were expressed in both CAR and ICAR cells derived from both nonpregnant, and AI cows on days 30 and 60 of gestation. The expression of cyclin E in both AI and NT cows was confined to COT and fetal membrane on day 30 of gestation. Cyclin E expression on day 60 of gestation in AI cows was observed in all but ICAR areas. In marked contrast, however, cyclin E expression on day 60 of gestation in NT cows was confined to COT, suggesting that poor placentation in these cows is possibly associated with a lack of cyclin E expression. These results suggest that CAR-derived stromal cells have higher proliferative potential, which may be related to cyclin E expression during implantation.     

A severe hepatic disorder with myelodysplastic syndrome, treated with cytarabine ocfosfate, in a dog

An 8-year-old female Shih Tzu was presented with weight loss and vomiting. Alanine aminotransferase was high and abdominal radiographs revealed hepato- and splenomegaly. Mild anaemia, neutrophilia with left shift, eosinophilia, a thrombocytosis with dysplastic features of eosinophils and platelets, were detected. The animal was initially considered to have hepatitis and was treated accordingly, but clinical signs persisted. Histological examination of liver biopsy samples showed disruption of the hepatic lobule, with extensive infiltration by haemopoietic cells. Further investigation of the bone marrow suggested a diagnosis of myelodysplastic syndrome. The animal was treated with cytarabine ocfosfate, a prodrug of cytosine arabinoside, and appeared to recover.