Generation of recombinant bovine interferon tau in the human embryonic kidney cell line and its biological activity

The objective of this study was to generate recombinant bovine interferon tau (rbIFNT) in mammalian hosts. The complementary DNA encoding bovine IFNT2 was cloned for the construction of pRcRSV‐bIFNT2 expression vector. The expression vector was transfected to 293 cells. Transfected cells harboring expression vector were selected with G418. Highly expressing clonal line was adapted to serum‐free suspension culture in a spinner flask. The recombinant protein had 24 kDa apparent molecular mass, suggesting being expressed as a glycoprotein, and was purified from serum‐free conditioned medium by the combination of Diethylaminoethanol Sepharose ion exchange and Sephacryl S‐200 HR gel filtration. A total of 7.3 mg rbIFNT was obtained from 13.5 L conditioned medium. Generated rbIFNT was biologically active in terms of antiviral activity measured by the plaque inhibition assay with Madin‐Darby bovine kidney cells and the vesicular stomatitis virus. The recombinant protein was also utilized for immunization to raise antibodies in the rabbit. The generated antibody was capable of use in both Western blotting and the binding assay. The results in the present study suggest that a certain amount of rbIFNT is raised in mammalian hosts by using conventional plasmid vector and its antibody provides useful tools for studies in the biology of bovine IFNT.     

Development and preventative effect against pine wilt disease of a novel liquid formulation of emamectin benzoate

Injection of the poorly water-soluble emamectin benzoate (EB) into pine trunks required the development of an efficient liquid formulation. For injection into big trees in forests a good rate of injection and a high active content were required. Tests on the viscosity and EB-solubilizing ability of 14 various solubilizers in diethylene glycol monobutyl ether (DGMBE) led to the selection of Sorpol SM-100PM as the solubilizer of the formulation. Relationships between the solubilizing ability and amounts of Sorpol SM-100PM and DGMBE relative to that of EB, and between the concentration of the latter and the viscosity or the injection rate of the formulation led to a novel 40 g litre(-1) emamectin benzoate formulation (Shot Wan Liquid Formulation), which was composed of EB (40), Sorpol SM-100PM (120), DGMBE (160) and distilled water (50 g litre(-1)) in methanol. Injection of this formulation at a dose of 10 g EB per unit volume of pine tree prevented over 90% of the trees from wilting caused by pine wood nematode, and this preventative effect continued for 3 years. Neither discolouration of the leaves nor injury around the injection hole on the trees was observed after injection of the formulation.     

Insulin-like growth factor-I as a follicular growth promoter during early pregnancy in thoroughbred mares

To elucidate the physiological role of insulin-like growth factor-I (IGF-I) during early pregnancy in mares, number of ovarian follicles was monitored ultrasonically during different stages of the first trimester of pregnancy in 36 thoroughbred mares. From 9 of 36 mares, blood samples were collected weekly from the mating day till the end of the first trimester of pregnancy and plasma IGF-I profiles were examined with other hormones, like follicle stimulating hormone (FSH), luteinizing hormone (LH), ir-inhibin, progesterone and estradiol-17beta. Plasma IGF-I level fluctuated throughout the studied period with four peaks on the 7th, 28th, 49th and 84th days of pregnancy. Plasma IGF-I showed a positive correlation with plasma FSH (P<0.05), whereas no correlation was found with other hormones during the studied period. Plasma IGF-I had no correlation with the foetal size, but positive correlation with the number of large (> 30 mm) and medium (10-30 mm) follicles. These results suggested that IGF-I might produce from the medium and large follicles during early pregnancy and promote to develop their growth via pituitary FSH mediated effects in the mares.     

Effects of hypothyroidism on gonadal function after transition of short day photoperiod in male golden hamsters (Mesocricetus auratus)

Thyroid hormones permit the annual reproductive transition of seasonal breeders. Although, precise function of thyroid hormones in seasonal breeding is not well understood. In the present study, we examined effects of hypothyroidism on the hypothalamus-pituitary-gonadal axis in adult male golden hamsters after transition of the short-day photoperiod (SD; 8 h light: 16 h dark) condition. We confirmed that hypothyroid, which had been induced by administration of thiouracil in drinking water for 4 weeks, did not have direct effects on testes in male hamsters under the long-day photoperiod. Plasma concentrations of free T3 and T4 decreased 15 weeks after transition of SD condition. Plasma concentrations of testosterone in the hypothyroid group decreased earlier than in the control group after the transition from LD to SD. In animals treated with testosterone after castration, plasma concentrations of LH in the hypothyroid group decreased earlier than in the control group after the transition of SD. On the other hand, pituitary response to GnRH for LH release did not change in castrated hamsters as a result of hypothyroidism. These results suggest that thyroid hormones act the hypothalamus and might be required to maintain GnRH secretion in male golden hamsters.     

Plasma concentrations of immunoreactive (ir)-inhibin, gonadotropins and steroid hormones during the ovulatory cycle of the duck

The present study was undertaken to determine changes in circulating levels of immunoreactive (ir)-inhibin, FSH, LH, estradiol-17beta, progesterone, and testosterone during the ovulatory cycle of Shao ducks. Serial blood samples were taken from two groups of laying ducks for measurement of ir-inhibin, gonadotropins, and steroid hormones at 2 h intervals for 24 h. Plasma concentrations of ir-inhibin did not change significantly during the ovulatory cycle. The highest level of plasma ir-inhibin was observed 6 h prior to ovulation, which coincided with a decreased level of plasma FSH. One FSH surge was found 12 h after ovulation. Estradiol-17beta, progesterone, and testosterone were also determined during the ovulatory cycle. Two peak values were detected for estradiol-17beta 8 h before ovulation and 4 h after ovulation, while progesterone started to increase 4 h before ovulation and reached a peak at ovulation. The highest level of plasma testosterone was detected around the time of ovulation. These results suggest that inhibin may be involved in the control of FSH secretion during the ovulatory cycle. In addition, both LH and progesterone are of importance in the ovulation process of Shao ducks.     

Immunolocalization of inhibin/activin subunits in the shiba goat fetal, neonatal, and adult testes

The objective of this study was to investigate the cellular immunolocalization of inhibin alpha and inhibin/activin (betaA and betaB) subunits in the fetal, neonatal and adult testes of Shiba goats. The testes were obtained from a fetus at 90 days, a neonate at 15 days, and two adult Shiba goats (both of 3 years old). The sections of testes were immunostained by the avidin-biotin-peroxidase complex method (ABC) using polyclonal antisera raised against porcine inhibin alpha, inhibin/activin betaA, and inhibin/activin betaB. Inhibin alpha and inhibin/activin (betaA and betaB) subunits were expressed in Leydig cells, but not in the Sertoli cells of the fetus with a weak immunostaining. An increase in the number of positive cells and a more intense immunohistochemical signal for inhibin alpha and inhibin/activin (betaA and betaB) subunits were observed in the Leydig cells of neonatal testes. Moreover, inhibin alpha, betaA, and betaB subunits were expressed in the Sertoli cells and Leydig cells of adult testes, respectively. These results suggest that Shiba goats testes have the ability to synthesize inhibins in the fetus, neonate, and adult, and the cellular localization of inhibin/activin subunits showed age-related changes in fetal, neonatal, and adult testes of Shiba goats.     

Congenital hepatic arteriovenous fistula with intrahepatic portosystemic shunt and aortic stenosis in a dog

Examination of a 2-month-old male golden retriever presented to the hospital revealed malnutrition, ascites, cardiac murmur and hyperammonemia. Identification of subaortic stenosis and hepatic arteriovenous fistula was made through ultrasonography and angiocardiography. In addition, intrasurgical mesenteric portography showed an intrahepatic portosystemic shunt. The dog did not show portal hypertension and secondary multiple extrahepatic portosystemic shunts. Surgical correction was attempted after medical treatment. The hepatic artery branch which was connected to the hepatic arteriovenous fistula was separated, and completely ligated using silk ligature. However, the separation of the intrahepatic shunt blood vessel was unsuccessful and the dog died 15 hr postoperatively.     

Lymphoid apoptosis in acute canine distemper

The relationship between the canine distemper virus (CDV) infection and apoptosis in the canine lymphoid tissues was investigated using immunostaining for single stranded DNA (ssDNA), TdT-mediated dUTP-biotin nick end-labeling (TUNEL) method, and electron microscopy. Twenty-six lymphoid tissues from 8 spontaneously CDV-infected dogs and 1 non-infected dog were used, and lesions were classified into 4 groups according to frequency of the CDV-antigen. Histologically, the degree of lymphoid depletion tended to depend on amount of CDV antigen. The numbers of ssDNA- and TUNEL-labeling cells were significantly high in the lymphoid tissues with abundant viral antigen. However, ssDNA- and TUNEL-positive lymphocytes were also frequently found even in the lymphoid tissues where there was only a small amount of CDV-antigen in sinus histiocytes. The incidence and distribution of apoptotic cells in the CDV-antigens-negative lymphoid tissues from infected dogs were equal to those from a non-infected dog. Double labeling immunostaining using a ssDNA and a CDV nucleocapsid protein (CDV-NP) antibody revealed that there were ssDNA positive but CDV-NP negative cells besides those stained doubly positive. Ultrastructurally, lymphocytes in the CDV-infected lymphoid tissues frequently had characteristic morphological features of apoptosis such as apoptotic bodies. All these results suggest that CDV leads to lymphocytic apoptosis directly or indirectly, resulting in severe lymphoid depletion and immunosuppression in acute or subacute phase of CDV infection.     

Method for estimation of toxic endotoxin in inactivated salmonella vaccine in D-galactosamine-sensitized mice

We developed a method to estimate the content of the toxic endotoxin in inactivated Salmonella vaccine in D-galactosamine-sensitized mice. Ten-fold serially diluted vaccines were injected intraperitoneally into D-galactosamine-sensitized mice. Lethality in the mice was judged 3 days after the injection. The best result was obtained when C3H/HeN mice were used for the test. Correlation was observed between the endotoxin content measured by Limulus amoebocyte lysate assay and the LD50 in the mouse safety test (r=0.81). These results suggested that this test could be applied to the estimation of endotoxin content in inactivated vaccines of Salmonella.     

Establishment of a chicken monoclonal antibody panel against mammalian prion protein

A panel of chicken monoclonal antibodies (mAbs) was developed against prion protein (PrP), the sequence of which is a highly conserved molecule among mammals. A portion of the splenocytes from chickens immunized with recombinant mouse PrP was fused with the chicken B cell line, MuH1. The remaining splenocytes were used to generate the recombinant mAbs by phage display. A total of 36 anti-PrP mAbs, 2 from cell fusion and 34 from phage display were established. The specificity of these mAbs was determined by Western blot and ELISA using various PrP antigens including recombinant PrPs, synthetic PrP peptides and PrPs from brains or scrapie-infected neuroblastoma cell line. These mAbs were classified into three main groups, protease K (PK)-sensitive (Group I), PK cleavage site proximal (Group II) and PK-resistant (Group III), based on their abilities to recognize PrP following PK-treatment. Some mAbs were found to selectively recognize different glycoforms of PrP as well as the metabolic fragments of PrP. Furthermore, we found that PrP recognition by chickens differed from that by PrP-knockout mouse. These results indicate that these newly generated PrP antibodies from chickens will help to research the PrP and to establish the diagnosis of prion disease.