Hisao Izawa

Activation of trout macrophages and production of CRP after immunization with Vibrio angillarum

To examine the mechanism of the protection of rainbow trout (Salmo gairdneri) against Vibrio anguillarum in the early stage of immunization, the activation of macrophages and production of C-reactive protein (CRP) were investigated. Fish immunized with formalin-killed bacteria emulsified in Freunds complete adjuvant (FCA) resisted intraperitoneal challenge with living bacteria seven and ten days after immunization. The activation of macrophages was demonstrated by a significant increase of the chemiluminescent (CL) response and phagocytic activity. These fish also showed a significant increase of the CRP level in sera. Fish immunized with V. anguillarum alone or injected with FCA, however, did not resist the challenge. Though FCA itself increased CRP level and the sera enhanced phagocytic activity, increase of CL activity was weak. These results indicated that the increase of CL activity and opsonising effect of CRP on the phagocytosis of specifically activated macrophages concern to host defense in the early stage of infection.

Isolation and characterization of rainbow trout C-reactive protein

An acute phase serum component, C-reactive protein (CRP), was isolated from the sera of rainbow trout (Salmo gairdneri). The isolation was based on its calcium-dependent binding affinity for pneumococcal C-polysaccharide (CPS) according to the isolation procedure of human C-reactive protein. In SDS-PAGE, the nonreduced CRP showed two subunits with molecular weights of 43,700 and 26,600, respectively, at a molar ratio of 1:1. The reduced CRP showed a single subunit of 26,600. The molecular weight of the native protein was estimated as 66,000 by native gradient PAGE and 81,400 by sedimentation equilibrium analysis using ultracentrifugation. The antigenic determinant on CPS-reactive site was destroyed by periodate oxidation, indicating that rainbow trout CRP is a glycoprotein. CRP levels in rainbow trout serum measured by the CPS-ELISA procedure showed that the rainbow trout CRP could behave as an acute phase reactant, following experimental infection with the fish pathogen Vibrio anguillarum.

Suppression of natural cytotoxic activity of lymphocytes from cattle and sheep during the progress of bovine leukosis

Peripheral blood lymphocytes (PBL) from cattle and sheep exhibited natural cytotoxicity on a fetal lamb kidney (FLK) cell line that was persistently infected with bovine leukemia virus (BLV) by 20-h 51Cr release assay. This cytotoxic activity was tested using PBL from normal cattle and sheep or BLV-infected animals. Although cytotoxic activity was also found in PBL from normal animals and from BLV-infected, but clinically healthy animals, the activity in PBL from animals with persistent lymphocytosis or leukemic animals was markedly decreased. The cytotoxic activity of PBL from 3 sheep sequentially tested before and during the disease was found to decrease gradually with the progress of the disease, and finally no cytotoxic activity was found at the time of death due to leukemia. These results suggest that the natural cytotoxic activity in PBL may have a role in immunosurveillance for progress of the tumor.

Monoclonal antibodies against a paramyxovirus isolated from Japanese Sparrow-Hawks(Accipiter virugatus gularis)

SummaryFive monoclonal antibodies against the haemagglutinin-neuraminidase (HN) molecule of Taka virus, a variant of Newcastle disease virus (NDV), were established to compare the antigenicities of several avian paramyxoviruses including NDV. From the results of the cross haemagglutination-inhibition (HI) test with the monoclonal antibodies, the HN molecule of Taka virus seemed to have at least three different antigenic determinants; one was specific for all NDV strains tested, the second was for Taka virus and Komarov strain of NDV and the third was for Taka virus, Komarov strain, Bangor and Yucaipa. Furthermore, the differences in the ratio of HI to neuraminidase-inhibition titers suggested that the separate active sites involved in haemagglutinin and neuraminidase activities might exist at least in close proximity.

Existence of cytotoxic acitivity against BLV-transformed cells in lymphocytes from normal cattle and sheep☆

Peripheral blood lymphocytes (PBL) from normal cattle and sheep were tested for their cytotoxic activity against several target cells using a 20-hour 51Cr release assay. The following characteristics of the effector cells were observed; 1) PBL from animals showed cytotoxic activity against two sheep cell lines (FLK and SF-28) that were transformed with bovine leukemia virus. However, normal sheep and bovine cells and Molony leukemia virus-induced mouse lymphoma cell line (YAC-1) were not killed by these cells. 2) A time course study showed that the activity was first observed at 4 to 8 hours and reached a maximum at 20 to 30 hours after incubation. 3) Cytotoxic activity was observed in both adherent and nonadherent cell fractions when PBL were passed through a nylon-wool column. This indicated that the effector cells showed some degree of adherence. 4) Treatment of PBL with carrageenan did not change the cytotoxic activity against target cells, indicating that phagocytic capability is not perhaps necessary for cytotoxicity to take place. These results indicate that the effector cells participating in the cytotoxic reaction resembled natural killer cells or natural cytotoxic cells which are present in murine and human systems. However, analysis of the cell surface markers of the effector cells is yet to be done in future studies.

Chronological Observations of Feather Pulp Lesions in Chickens Inoculated with Marek's Disease Virus

Observations of the feather pulp from chickens inoculated with Mareks disease (MD) virus revealed diverse lesions similar to those in the peripheral nerves of chickens infected with MD. These lesions were categorized as follows: R1-type lesions = from minimal perivascular to diffuse infiltration of small lymphocytes mixed with a few medium lymphocytes or blast cells; R2-type lesion = edema and cellular infiltration consisting of plasma cells and small lymphocytes; and T-type lesion = tumorous proliferation of lymphoid cells predominantly composed of medium lymphocytes or blast cells. Chronological observations of the feather pulp biopsies taken at 2-week intervals from individual chickens revealed the lesion progression R1-type leads to T-type, or R1-type leads to R2-type. The former progression was usually seen in birds showing evidence of both persistent nuclear-inclusion (NI) formation in the feather-follicle epithelium (FFE) and development of lymphoid tumors in the viscera. The latter progression was seen exclusively in chickens that showed transient NI formation in the FFE, regardless of the incidence of MD.

Induction of lymphosarcoma in sheep inoculated with bovine leukaemia virus

Five sheep were experimentally inoculated with BLV in order to study the humoral immune response in animals infected with bovine leukaemia virus (BLV). During experimental periods of 46 months, 2 sheep died with leukaemia and one sheep showed splenomegaly and proliferation of tumour cells. The other 2 sheep were clinically normal. All of the inoculated sheep developed antiviral antibodies 1 month after inoculation and BLV could be re-isolated in lymphocytes 2 to 3 months after inoculation. Antibody against glycoprotein antigen (gp51) of BLV appeared earlier than the antibody against protein antigen (p24) and antibody titres of the former were higher than those of the latter during the course of the experiment. The complement dependent antibody cytotoxicity test was performed for the detection of antibody against BLV-related cell membrane antigen with 2 different kinds of target cells; FLK cells which are foetal lamb kidney cells chronically infected with BLV and SF-28 cells which are sheep fibroblasts transformed with BLV in vitro. All 5 sheep developed cytotoxic antibodies against both types of cells. In sera from two leukaemic sheep, cytotoxic antibody titres against SF-28 cells gradually decreased 30 months after inoculation and finally became negative one to 3 months before they died of leukaemia. However, these leukaemic sheep persistently produced antibodies against gp51 and p24.

Serological and genetical relationships of three herpesvirus strains from salmonid fish

SummarySerological and genetical relationships of three salmonid herpesviruses [strain H-83, OO-7812 strain ofOncorhynchus masou virus (OMV) andHerpesvirus salmonis] were investigated. Although the serological relationships of theses viruses were not distinctly demonstrated by cross-neutralization test, cross enzyme-linked immunosorbent assay revealed that strain H-83 and OMV appeared to be serologically more closely related to each other than toH. salmonis. Strain H-83 was genetically similar to OMV but distinct fromH. salmonis by restriction endonuclease cleavage analysis and DNA-DNA hybridization test. These results indicated that strain H-83 should be classified under the same group as OMV, andH. salmonis belong to another group of salmonid herpesviruses.

Effect of platelet-derived factor on expression of bovine leukemia virus genome.

SummaryPlasma of cattle infected with bovine leukemia virus (BLV) contains a factor, plasma blocking factor (PBF), that inhibits the expression of viral genome in cultured lymphocytes from BLV infected cattle. When platelet lysate was added to this culture, BLV antigen became detectable in the culture and there are some factors (PDF) in platelet lysate which have inhibitory activity against PBF. The PDF was present in platelet from BLV-free cattle as well as BLV-infected cattle at relatively high titer. The effect of platelet lysate against PBF on the expression of BLV genome seemed to be irreversible.

Marek's Disease in Field Chickens: Correlation Between Incidence of Marek's Disease and Nuclear-Inclusion Formation in the Feather-Follicle Epithelium

The present study confirmed that Mareks disease (MD)-associated nuclear-inclusion (NI) formation in the feather-follicle epithelium (FFE) is related to mortality from MD; it also presented useful data on the epidemiology of MD in HVT-vaccinated field chickens. Incidence of NI formation in the FFE of chickens on six rearing farms varied greatly by age and flock, but most of the field flocks showed biphasic peaks of incidence of NI in chickens consisting of a small peak at an early age (usually at 2-4 weeks of age) and a large peak between 13 and 16 weeks of age. MD tended to occur in chickens over 20 weeks old, and almost all MD-affected chickens showed NI formation persistently in the FFE, usually between 13 and 20 weeks of age. Chickens that were healthy at the end of observation showed either transient NI formation, usually between 13 and 16 weeks of age, or no detectable NI formation. Incidence of NI formation in the FFE of chickens at 19-20 weeks of age was related to mortality from MD: chickens with NI formation had a MD mortality rate of 66.7%, whereas chickens without NI formation had MD mortality of only 0.8%.