Clinical evaluation of growth hormone secretion in cattle using insulin tolerance test

Growth hormone secretion was evaluated in cattle. Clinically healthy bovine growth hormone (bGH) concentrations were 10.7 +/- 1.6 ng/ml in Holstein and 7.8 +/- 3.9 ng/ml in Japanese black cattle. The bGH concentration alternated at three-hour intervals, and tended to be higher at midnight and lower in the morning and before feeding. Insulin tolerance test (ITT) at an insulin dosage of 0.25 U/kg showed a significant increase of bGH concentration to 331 +/- 153% at 60 to 90 min after injection. In ITT applied to five under-growth calves of Japanese black cattle, the basal bGH concentrations were lower and peak values after insulin injection were shown to be significantly low. The ITT is useful for the clinical examination of bGH secretion in cattle.     

Establishment of a potency test by ELISA for a rabies vaccine for animal use in Japan

The ELISA we developed was able to determine the antigen content and was suitable for a potency test, and we described a relative potency assay method which determines the potency of test vaccines by comparing the ELISA value of a test vaccine to that of a reference vaccine. In the present study, we standardized the reference vaccine used for determining the potencies of test vaccines, and established a potency test by ELISA. We evaluated the proposed reference vaccine by the neutralizing antibody responses in dogs after vaccination, by the challenge protection test in guinea pigs (GP potency test), which is the earlier official potency test used in Japan, and by the NIH potency test, which is widely used throughout the world. The results showed that a 4-fold dilution of the proposed reference vaccine induced sufficient immunity in dogs. A 3-fold dilution of the proposed reference vaccine passed the GP potency test. The international units (IU) calibrated by the NIH potency test were 3.7 IU/dose. From the results and the WHO recommendation that veterinary rabies vaccines should have a potency of at least 1.0 IU/dose, we determined to dilute the proposed reference vaccine by 3 fold and regarded it as the reference vaccine. Finally, we confirmed that there is a good agreement between the results of the potency test by ELISA and the results of the GP potency test. The establishment of the potency test by ELISA has made it possible to monitor the potency in the production process and has contributed to the stable production of the vaccine.     

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.     

Polymerase chain reaction (PCR) for the detection of herpesvirus in tortoises

A polymerase chain reaction (PCR)-based method using a herpesvirus consensus primer was assessed for the identification of herpesviral infections in tortoises. A single band of about 230 bp was detected in PCR products from two out of twenty swabs taken from the oral cavity, three out of three paraffin-embedded tissue sections from the liver (two cases) and oral mucosa (one case), and one out of two fresh tissue samples from the oral mucosa. Nucleotide sequencing of these PCR products indicated that the herpesvirus present in these tortoises might belong to the alphaherpesvirinae. PCR using swabs and biopsy tissues was a sensitive and highly specific method for the diagnosis of herpesviral infections in tortoises.     

Evaluation of the drug sensitivity and expression of 16 drug resistance-related genes in canine histiocytic sarcoma cell lines

Canine histiocytic sarcoma (HS) is an aggressive tumor type originating from histiocytic cell lineages. This disease is characterized by poor response to chemotherapy and short survival time. Therefore, it is of critical importance to identify and develop effective antitumor drugs against HS. The objectives of this study were to examine the drug sensitivities of 10 antitumor drugs. Using a real-time RT-PCR system, the mRNA expression levels of 16 genes related to drug resistance in 4 canine HS cell lines established from dogs with disseminated HS were determined and compared to 2 canine lymphoma cell lines (B-cell and T-cell). These 4 canine HS cell lines showed sensitivities toward microtubule inhibitors (vincristine, vinblastine and paclitaxel), comparable to those in the canine B-cell lymphoma cell line. Moreover, it was shown that P-gp in the HS cell lines used in this study did not have enough function to efflux its substrate. Sensitivities to melphalan, nimustine, methotrexate, cytarabine, doxorubicin and etoposide were lower in the 4 HS cell lines than in the 2 canine lymphoma cell lines. The data obtained in this study using cultured cell lines could prove helpful in the developing of advanced and effective chemotherapies for treating dogs that are suffering from HS.     

Development of an enzyme-linked immunosorbent assay to detect an immunomodulatory alpha-D-glucan-protein complex, MPG-1, in basidiomycete Tricholoma matsutake and related processed foods

We previously isolated a novel immunomodulatory alpha-(1,4)(1,6)(1,2)- d-glucan-protein complex (MPG-1) from mycelia of Tricholoma matsutake in basidiomycetes. In the present study, we raised a polyclonal antibody by immunizing rabbits with MPG-1 and constructed a sandwich enzyme-linked immunosorbent assay (ELISA) system to examine the distribution of MPG-1 among edible mushrooms and related processed foods. The system detected MPG-1 quantitatively at concentrations of more than 10 ng/mL, with a coefficient of variation of less than 10% by intra-assay and interassay precision. Analysis with the system of chemically modified MPG-1 suggested that the sugar moiety was mainly involved in the detection. The system detected MPG-1 in the extracts of the fruiting bodies of T. matsutake but not in those of 34 other basidiomycete species. Moreover, a significant amount of MPG-1 was detected in the extracts of their cultured mycelia. The antigenic structure of MPG-1 was relatively stable in terms of pH and temperature. MPG-1 was detected in processed foods supplemented with T. matsutake. These results suggest that MPG-1 is distributed predominantly in T. matsutake species and that the ELISA system can detect it in processed foods supplemented with T. matsutake.