The role of virus dose in experimental bovine leukemia virus infection in sheep.

Twenty-four, six month old lambs were assembled into four groups of five animals each and one group of four animals. All groups were inoculated with lymphocytes from a single donor lamb infected with bovine leukemia virus. The inoculum varied from 250 to 250,000 lymphocytes, in tenfold increments. Animals were exposed by intradermal injection in the neck region immediately anterior to the left shoulder joint. All groups were monitored at 0, 3, 7 and 12 weeks after inoculation using the following procedures: a. Syncytia induction assay for detection of bovine leukemia virus in peripheral blood lymphocytes. b. Agar gel immunodiffusion against the gp51 antigen of bovine leukemia virus for the detection of antibovine leukemia virus gp51 antibody. c. Lymphocyte stimulation test for the assessment of cell-mediated immunity using mitogen, nonfractionated bovine leukemia virus antigen, and partially purified bovine lymphoma tumor-associated antigen for the in vitro activation of lymphocytes from bovine leukemia virus-inoculated and sham-inoculated, control animals. d. Routine hematological techniques for the assessment of total leukocyte and lymphocyte counts. The median infectious dose for lymphocytes from the single bovine leukemia virus-infected donor used in this study was determined to be 2000 cells. The syncytia induction assay detected more infected individuals (13/23) at an earlier time than did the agar gel immunodiffusion assay (10/23). Using either serological or virus isolation techniques, infected animals were first detected at three weeks postinoculation in the group receiving the high-dose inoculum and at seven weeks postinoculation in groups receiving low- or medium-dose inocula.(ABSTRACT TRUNCATED AT 250 WORDS)     

The detection of transmissible gastroenteritis viral antibodies by immunodiffusion.

Precipitating antibodies against transmissible gastroenteritis viral antigens were detected by the immunodiffusion test in two transmissible gastroenteritis viral hyperimmune antisera and in antiserum prepared against haemagglutinating encephalomyelitis virus but not in sera from several species of normal animals, in antisera prepared against a variety of othet viruses and bacteria or sera from swine with bacterial enteritis. When the immunodiffusion test was compared with the virus neutralization test for the detection of transmissible gastroeneritis viral antibodies in 20 swine sera certain samples which contained high titres of virus neutralizing antibodies failed to produce precipitation while other sera were positive in the immunodiffusion test although their virus neutralizing antibody titres were relatively low. Precipitating antibodies were also detected by immunodiffusion in several samples of milk whey from a sow which had been vaccinated with inactivated transmissible gastroenteritis virus.     

Equine infectious anaemia: detection of antibodies using an immunofluorescence test

An indirect immunofluorescence test for detecting antibodies to equine infectious anaemia virus is presented. Using monolayers of equine dermal cells within a defined period after infection, discrete fluorescent spots were observed in the cytoplasm of as many as 95 per cent of the cells. These inclusions appeared as ring-like structures when high titred sera were employed but became spots when the sera were diluted. Cells showing optimal antigen fluorescence were used immediately or after storage at -70 degrees C. The fluorescence test detected lower levels of antibody than the immunodiffusion test, and results were available in less than two hours. It should be useful as a confirmatory assay with the immunodiffusion test.     

Association of parainfluenza-3 virus with bovine macrophages and blood cells: an in vitro study

Bovine blood cells and peritoneal and lung macrophages were exposed in vitro to parainfluenza-3 (PI-3) virus. Residual nonadsorbed PI-3 virus (expressed in percentage of input virus) in the supernate of the various cell fractions 1 hour after incubation at 37 C was as follows: lung macrophages, 11%; peritoneal macrophages, 59%; monocytes, 26%; RBC, 14%; lymphocytes, 28%; and polymorphonuclear cells (PMN), 63%. Lung macrophages, monocytes, lymphocytes, and PMN were monitored over a 72-hour period for hemadsorption of chicken RBC. Hemadsorption increased for lung macrophages and monocytes, whereas it decreased for lymphocytes and PMN. Infective virus could not be recovered from PMN, RBC, lymphocytes, or monocytes for more than 24 hours after PI-3 infection. Recovery of infective PI-3 virus from infected peritoneal and lung macrophages extended over 4 to 8 days, respectively.     

Development and distribution of rinderpest virus antigen in experimentally infected goats

Three goats, experimentally infected with rinderpest virus were examined for the development and distribution of precipitating antigens in various tissues and secretions using the agar gel immunodiffusion test. Virus antigens were detected in ocular secretions and lymph node biopsies from the second to the fourth and fifth days of pyrexia, respectively, but were not detected in nasal secretions. Precipitating antigens were demonstrated in various lymphoid organs, the lung and abomasum of a goat killed on the fourth day of pyrexia. These findings are discussed in relation to the epidemiology of rinderpest in goats in Africa.     

Radial immunodiffusion enzyme assay for detection of antibodies to pseudorabies virus in swine serum

A radial immunodiffusion enzyme assay for the detection and quantitation of antibodies to pseudorabies virus in swine sera was developed and the methods were standardized. The assay combined the principle of radial immunodiffusion with enzyme-linked immunosorbent assay. Quantitation of pseudorabies virus antibody titers was determined by measuring the diameter of a colored circular zone after overnight incubation of antibody with antigen. The specificity and sensitivity of the radial immunodiffusion enzyme assay were compared with that of the standard virus-neutralization test, and the results were determined to be correlated highly (r = 0.694, P less than 0.0001). The assay also appeared to be highly reproducible and simple to perform.     

Observations on the preparation and stability of infectious bronchitis virus hemagglutination antigen from virus propagated in chicken embryos and chicken kidney cell cultures

A total of 166 infectious bronchitis virus (IBV) hemagglutination (HA) antigen preparations were made during a 30-month period from allanto-amnionic fluid (AAF) from chicken embryos inoculated with 10 different IBV strains (Mass 41, Conn 46, H52, Florida 18288, Ark 99, JMK, T, Holte, EF, SE17). Antigens were prepared by inoculating 9- or 10-day-old embryos with 10(5.0) to 10(6.5) EID50 IBV, harvesting AAF after a 30-hour-postinoculation incubation, and phospholipase C (PLC) treatment of virus concentrated by pelleting from the AAF. Longer (48 hr) incubation times were tried, but production of H52 HA antigen was successful only from AAF harvested after 30 hours of incubation. AAF from JMK-infected embryos had lower infectivity titers and frequently yielded lower HA antigen titers than the other strains. The treatment of AAF with fluorocarbon did not enhance or diminish HA activity but did yield clearer antigens by removing extraneous material. Polyethylene glycol precipitation of virus was an acceptable alternative to pelleting virus at 39,000 X g. Inactivation of IBV with 0.1% betapropiolactone did not affect HA activity, whereas inactivation with 0.1% formalin caused a marked reduction in HA titer. Different buffer formulations including phosphate, tris, or HEPES were tried to optimize the conditions for PLC treatment of virus concentrate, but there were no apparent differences in the antigens prepared in the different buffers. The HA antigen preparations were stored and were stable at 4 C. Antigen titers of greater than or equal to 64 after storage for 20 months or longer were not uncommon. Addition of merthiolate as a preservative had no deleterious effect on HA activity. Antigen stability appeared to be enhanced by incorporating EDTA in buffer for virus pellet recovery and during enzyme treatment. Attempts to produce HA antigens from cell-culture-adapted virus propagated in chicken kidney cells were less satisfactory. An acceptable HA antigen was prepared from only two (Mass 41, SE17) of the seven strains that were tried. Virus propagation in chicken embryos is the better method of the two for IBV HA antigen production. Aside from the need to concentrate virus and treat the concentrate with PLC, there appeared to be considerable latitude in the procedures that can be used to make acceptable IBV HA antigens.     

Diagnosis of enzootic bovine leukosis: a comparison of haematological and immunodiffusion tests.

A total of 657 cattle from three different herds were tested for the presence of specific antibodies against EBL antigens by means of the immunodiffusion test and the results compared with the classification according to the lymphocyte counts. All animals with pathologically raised lymphocyte counts showed a positive immunodiffusion test reaction. In the majority of these both antigens, 'p24' and 'gp', gave positive results; in a smaller number antibodies against the gp antigen only were detected, but never against the p24 antigen only. It is concluded that for field testing programmes the use of the gp antigen is sufficient. In future experiments, however, both antigens should be employed, because the lack of one of them might perhaps prove to be correlated with the future course of the disease. The absence of specific antibodies, together with a normal number of lymphocytes, in young animals should, however, not lead to the conclusion that they are free of the disease.     

家蚕浓核病病毒简易提纯与早期诊断的研究

剖取浓核病蚕的新鲜肠组织,在pH7.2的磷酸级冲液中捣碎,经用氯仿反复抽提澄清,硫酸铵盐析,结合3000r/min常温低速离心分离纯化本病毒,得到了紫外吸收高峰260nm、电镜观察病毒粒子纯净的病毒悬液,将此病毒液制备抗血清,得到了效价在1024特异性强的抗血清.用此抗血清对浓核的病蚕进行各种早期诊断时,免疫双扩散、对流免疫电泳法,一般在感染后24—36小时检出,间接荧光抗体法12—24小时就能检出.具有特异性强、灵敏度高、检出早的特点,在当前富有实用意义.     

Experimental rabies in skunks: persistence of virus in denervated muscle at the inoculation site.

Striped skunks (Mephitis mephitis) were inoculated into the denervated abductor digiti quinti muscle with street rabies virus. They were killed at various times after inoculation and several tissues were examined by immunofluorescence and light microscopy. Muscle at the inoculation site was examined electron microscopically. Rabies antigen was detected in muscle fibers first on day 7 and persisted until day 28. Light and electron microscopic lesions at the inoculation site included atrophic and degenerating muscle fibers and a few focal and regional endomysial accumulations of macrophages, lymphocytes and plasma cells. Scattered myocytes contained bodies of matrix, virions and anomalous tubular structures on electron microscopic examination. The results indicate that replication of rabies virus may occur in infected muscle fibers at the inoculation site until 28 days after exposure. This could contribute to variations in the incubation period for the first two to three months after exposure. However, the results do not support the contention that virus is contained in striated muscle cells throughout the long incubation periods.     

Hemorrhagic enteritis: virus distribution and sequential development of antibody in turkeys

Turkeys poults were inoculated intraperitoneally with hemorrhagic enteritis virus (HEV) at 4-1/2 weeks of age. Antibody response and sequential development of viral antigen in various tissues were monitored. An enzyme-linked immunosorbent assay (ELISA) was developed to study antibody production, and immunoperoxidase staining was used to determined sites of localization of the viral antigens in tissues. Results of ELISA and immunodiffusion tests were compared. ELISA detected antibody from day 3 post-infection (p.i.), and gel diffusion detected antibody from day 5 p.i. Peak ELISA antibody titer appeared from day 14 p.i. HEV antigen was detected from 2-6 days p.i. in the spleen, liver, intestine, kidney, and bone marrow; peak titers in the spleen were on day 3 p.i. Virus was not detected after day 6 p.i.     

Effect of incubation temperature or maternal antibody on virus growth and mortality of embryonated chicken eggs inoculated with the B1 strain of lentogenic Newcastle disease virus

The effects of incubation temperature on virus growth and mortality patterns were studied in antibody-free chicken eggs inoculated with the B1 strain of Newcastle disease virus. Embryo mortality patterns did not differ much between 37, 39, and 41 C of incubation, although virus growth curves were higher at 39 C and 41 C than at 37 C. Below 35 C, embryo mortality was delayed, although from 48 hours after incubation the virus growth curves were similar to those at 37 C. In eggs with and without maternal antibody, virus titers were maximum 48 hours after incubation, although slightly higher in antibody-free eggs than in eggs with antibody whether the eggs were alive or dead. Eggs with maternal antibody had delayed mortality patterns.     

Indirect immunofluorescence and immunodiffusion tests in the detection of antibodies to foot-and-mouth disease virus

The antibody response detected by indirect immunofluorescence (IIF) as well as that directed against 140 S and virus infection associated antigen (VIA), as detected by agar immunodiffusion, was studied in three mammal species susceptible to Foot and Mouth Disease Virus, after challenge with living virus, immunization and hyperimmunization with inactivated virus, and immunization followed by challenge. By spot indirect immunofluorescence, antibodies were detected only in animals undergoing an active infection, and were not detected in immunized or hyperimmunized animals. This behaviour was similar to that of the anti-VIA antibodies in the same groups of animals and differed from that of anti-140 S antibodies. It appeared that spot indirect immunofluorescence for the detection of VIA antigen is comparable to the immunodiffusion test, but the speed of IIF and the possibility of handling many samples make it more practical.     

Isolation and trypsin-enhanced propagation of turkey enteric (bluecomb) coronaviruses in a continuous human rectal adenocarcinoma cell line

Turkey enteric coronavirus (TCV) from intestinal contents of diarrheal poults was isolated and serially propagated in HRT-18 cells, an established cell line derived from a human rectal adenocarcinoma. In these cells, TCV induced cytopathic changes, including polykaryocytosis, which depended on trypsin in the medium and incubation at 41 C. Viral antigens could be demonstrated in the cytoplasm by immunofluorescence, and extracellular virus was detected by an ELISA and negative electron microscopy. The cell-free virus had characteristics of TCV: shape, surface projections, buoyant density of 1.18 to 1.20 g/ml in sucrose, and hemagglutination of rat RBC. The one-step growth curve was complete by postinoculation hours 14 to 16, and maximal titers reached 9 to 9.5 log10 TCID50/ml during 5 passages, after which the titer remained stable. Electron microscopic examination of infected cell monolayers revealed budding of typical coronavirus particles through intracytoplasmic membranes and accumulation of complete virus in cytoplasmic vesicles. Late in the infection, aggregated progeny vial particles were detected near the outer surface of infected cells. One-day-old turkey poults inoculated orally with tissue culture-adapted TCV isolates developed mild to severe diarrhea.     

Syncytium-induction inhibition test with complement for detection of antibodies against bovine leukemia virus.

A modified syncytium-induction inhibition test which is more sensitive than the immunodiffusion test, was developed using rabbit complement. In this test, fetal lamb kidney cells continuously infected with bovine leukemia virus were used as effector cells, and the CC81 cat cells transformed with murine sarcoma virus, were used as indicator cells. The syncytium-induction inhibition effect of anti-bovine leukemia virus serum was enhanced significantly by the addition of rabbit complement. The syncytium-induction inhibition titers had a statistically significant correlation with the immunodiffusion titers and were four to 64 times higher than immunodiffusion titers. In 12 experimentally infected cattle, the syncytium-induction inhibition test detected the antibodies earlier than the immunodiffusion test and continuously detected them when immunodiffusion antibody changed to negative. In the 81 sera from naturally infected herds, 35 (43.2%) were positive by the immunodiffusion test and 55 (67.9%) by the syncytium-induction inhibition test.     

Detection of duck plague virus by reverse passive hemagglutination test

A reverse passive hemagglutination (RPHA) test was developed to detect duck plague virus (DPV). The technique used sheep erythrocytes stabilized with formaldehyde and pyruvaldehyde and coated with immunoglobulin G (IgG) containing anti-DPV antibody prepared from antiserum produced in sheep. Optimum coating of stabilized erythrocytes occurred at 25 C and pH 4.0 with a concentration of IgG of 20-40 micrograms/ml and a 90-min incubation period. The coated cells were stable for 40 days when stored at 4 C or for at least 4 months (the longest period tested) when frozen at -70 C or -196 C. The RPHA test was conducted at 25 C and read after 3 hours. The high specificity of the test is indicated by the absence of cross-reactions with heterologous virus strains, with specimens prepared from normal duck livers, and with normal chicken embryo chorioallantoic fluid, as well as by the inhibition of hemagglutination only with DPV antiserum. The RPHA test detected six strains of DPV in all virus-containing specimens as well as the immunofluorescence (IF) test did; however, conventional plaque assays (PA) failed to detect virus in five specimens that contained three non-plaque-forming strains of DPV. The mean quantity of DPV that could be detected in the RPHA test was 25 plaque-forming units or 65 fluorescent units per ml. Although the RPHA test was less sensitive than either the PA or the IF test, there was a positive correlation in the titers of DPV antigens between all three tests. The RPHA test is a rapid, simple procedure that is sufficiently sensitive for diagnostic detection of DPV in acute infections, especially in tissues of ducks dying of the disease.     

Measurement of systemic and local respiratory cell-mediated immunity after influenza infection in chickens

The detection of ChIFN production after ex vivo antigenic-stimulation of T-lymphocytes has been evaluated for the first time, as a tool to assess cell-mediated immunity (CMI) after avian influenza (AI) infection in 10-day-old SPF chickens. Preliminarily, recall antigens have been produced either by concentrating and inactivating the whole virus or by dissociating the viral proteins. Biologically and structurally intact forms of the viral proteins were isolated by non-ionic detergents while heating, chemical agents and ionic detergent used for virus inactivation altered the antigenic viral components. The n-octyl-B-D-gluco-pyranoside treatment at low temperature was very efficient to produce AI antigenic proteins used for evaluation of ChIFN production after ex vivo antigenic-stimulation of splenic and peripheral lymphocytes. In addition, protocols to isolate lymphocytes from the respiratory tract - the trachea and the lung - have been adapted for local CMI evaluation after similar ex vivo recall assay. Specific AI CMI in the spleen, the blood and the lung was detected for 5 weeks after low pathogenic AI (LPAI) infection in chickens, while further development is needed for tracheal CMI measurement.     

Modulation of function of bovine polymorphonuclear leukocytes and lymphocytes by high temperature in vitro and in vivo.

Function of polymorphonuclear leukocytes (PMNL) and proliferation of lymphocytes after stimulation with mitogens were evaluated in vitro at incubation temperatures of 38.5 and 42 C, and after in vivo heat stress of lactating Holstein cows. Cytochrome-c reduction and random migration of PMNL were reduced when cells were preincubated or incubated at 42 C, but high incubation temperature had little or no effect on phagocytosis and killing of Escherichia coli. Proliferation of lymphocytes was reduced when cells were incubated for 60 hours at 42 C after stimulation with phytohemagglutinin, pokeweed mitogen, or concanavalin A. After stimulation with phytohemagglutinin, lymphocytes were most sensitive to high temperature during the first 24 hours of the 60-hour culture period. High incubation temperature had little effect on viability of cells. In vivo heat stress had no significant effect on responses of PMNL in vitro, but the decrease in proliferation of lymphocytes in vitro at high temperature was less when cells were obtained from heat-stressed cows. Total leukocyte counts in blood and somatic cell counts in milk were higher in heat-stressed cows. Results indicate that: exposure to high temperature in vitro can depress responses of PMNL and lymphocytes; apparent adaptive mechanisms induced by in vivo heat stress provide protection from effects of high temperature seen in vitro; and evidence could not be found to support the hypothesis that reduction in immune function is the basis for increases in the incidence of mastitis during the summer.     

Effect of exogenous corticosteroids on circulating virus and neutralizing antibodies in striped bass (Morone saxatilis) infected with infectious pancreatic necrosis virus

Corticosteroids have been reported to induce immunosuppression in fish exposed to many types of bacterial antigens. We document a similar phenomenon in fish exposed to infectious pancreatic necrosis virus (IPNV). Fingerling striped bass that were injected with the steroid triamcinolone acetonide (100 mg/kg body weight) 24 hours before receiving intraperitoneal inoculation with IPNV became viremic 3 days post inoculation (dpi) and virus was still detected in the buffy coat cells 14 dpi. In contrast, viremia could not be detected after 7 dpi in fish that received virus but not steroids. Circulating virus neutralizing antibodies were first detected in steroid treated fish at 10 dpi compared to 7 dpi for the virus injected fish and titers were consistently lower in the steroid group. Steroid treatment of chronic IPNV-carriers did not induce detectable viremia nor alter circulating antibody levels in chronic IPNV-carriers. None of the striped bass demonstrated clinical signs of viral disease.