Chorioallantoic Cavity Research Articles

IMPACT OF VITAMIN E AND SELENIUM TREATMENT IN-OVO AND AFTER HATCHING OF BROILER

This study was aimed to investigate the effect of in-ovo injection with vitamin E and selenium during the incubation and post-hatching period. 360 fertilized eggs of Ross 308 broiler breeders were incubated, then distributed on 10th day into three groups (120 eggs/group). 1st group: uninjected eggs, 2nd group: eggs were injected with 0.1 ml deionized water/egg in the chorioallantoic cavity, and 3rd group: eggs were injected with 0.1 ml/egg of Introvit-E-Selen in chorioallantoic cavity. After hatching, 270 chicks were randomly distributed into six groups, and reared until 42 days aged as follows: birds in 1st, 3rd and 5th groups drinks tap water only (free from any addition), while birds of other groups, were reared on drinking water supplemented with 12.5 mg vitamin E and 500 µg sodium selenite/liter water. Results showed a significant increase in hemoglobin, lymphocytes% and serum globulin, and a significant decrease in packed cell volume%, heterophils%, heterophils/lymphocytes ratio, glucose, cholesterol, triglycerides and albumin concentration as compared with control. In conclusion, vitamin E and selenium have enhanced some immunological aspects and reduced stress, as well as a number of hematological parameters of broiler chicks, also, the continuity of Vit.E-Selenium addition led to continuous improvement of physiological parameters, and when it stopped, the values of those parameters were retracted.

In Vitro and In Ovo Host Restriction of Aquatic Bird Bornavirus 1 in Different Avian Hosts.

Aquatic bird bornavirus 1 (ABBV-1) is associated with chronic meningoencephalitis and ganglioneuritis. Although waterfowl species act as the natural host of ABBV-1, the virus has been sporadically isolated from other avian species, showing the potential for a broad host range. To evaluate the host restriction of ABBV-1, and its potential to infect commercial poultry species, we assessed the ability of ABBV-1 to replicate in cells and embryos of different avian species. ABBV-1 replication was measured using multi- and single-step growth curves in primary embryo fibroblasts of chicken, duck, and goose. Embryonated chicken and duck eggs were infected through either the yolk sac or chorioallantoic cavity, and virus replication was assessed by immunohistochemistry and RT-qPCR in embryonic tissues harvested at two time points after infection. Multi-step growth curves showed that ABBV-1 replicated and spread in goose and duck embryo fibroblasts, establishing a population of persistently infected cells, while it was unable to do so in chicken fibroblasts. Single-step growth curves showed that cells from all three species could be infected; however, persistence was only established in goose and duck fibroblasts. In ovo inoculation yielded no detectable viral replication or lesion in tissues. Data indicate that although chicken, duck, and goose embryo fibroblasts can be infected with ABBV-1, a persistent infection is more easily established in duck and goose cells. Therefore, ABBV-1 may be able to infect chickens in vivo, albeit inefficiently. Additionally, our data indicate that an in ovo model is inadequate to investigating ABBV-1 host restriction and pathogenesis.

The First Specific Detection of a Highly Pathogenic Avian Influenza Virus (H5N1) in Ivory Coast

The Virology Laboratory of the Central Laboratory of Animal Diseases in Ivory Coast at Bingerville received samples of wild and domestic avian species between February and December 2006. An RT-PCR technique was used to test for avian influenza (AI) and highly pathogenic AI subtype viruses. Among 2125 samples, 16 were type A positive; of which, 12 were later confirmed to be H5N1. Fifteen of these 16 type A positive samples were inoculated into the chorioallantoic cavity of 11-day-old embryonated hens' eggs for virus isolation. Eight produced virus with hemagglutination titres from 1/64 to 1/512. The 4/16 M-RT-PCR positive samples, which were H5N1 negative, were shown to be H7 subtype negative. The diagnostic efficiency of the laboratory for the surveillance of H5N1 in Ivory Coast was demonstrated. The positive cases of H5N1 were from a sparrowhawk (Accipter nisus); live market poultry and in free-range poultry, where the mortality rate was approximately 20% (2/10) and 96.7% (29/30) respectively. Currently, investigations into intensive poultry farms have proved negative for H5N1. No human cases have been reported this time.

ردیابی ویروس برونشیت عفونی در مایع آلانتوئیک به کمک آزمایش هماگلوتیناسیون سریع

Summary To detect the presence of infectious bronchitis virus (IBV) in infected allantoic fluid (AF) of SPF embryonated eggs rapid hemagglutination (HA) activity after treatment with neuraminidase enzyme was used. Twenty IBV suspected materials were inoculated in SPF embryonated eggs via chorioallantoic cavity. Harvested AFs were treated with neuraminidase enzyme and the presence of IBV was detected by inducing hemagglutination of chicken red blood cells. The specificity of rapid HA test was examined with three non-hemagglutinating avian viruses including infectious bursal disease virus, infectious laryngotracheitis virus and fowl pox virus. The sensitivity of the test was compared with reversed transcriptase-polymerase chain reaction. The results showed that this test was specific and had a sensitivity of 100% for IBV detection. The results of this study indicate that HA test using neuraminidase treatment is an accurate, sensitive, specific and inexpensive test for rapid detection of IBV.

Effects of 6-Hydroxydopamine on the antibody response to the hapten dinitrophenyl in the chicken.

Antibody responses to the hapten dinitrophenyl (DNP) and the concentration of catecholamine in chickens received a single injection of 6-Hydroxydopamine (6-OHDA) into the chorioallantoic cavity at the embryonal stage were evaluated. A significantly enhanced anti-DNP IgA response and a markedly decreased level of noradrenaline in the peripheral blood were observed in chickens treated with 400 micrograms of 6-OHDA at 14 days of incubation. These results suggested the immunomodulatory influence of the sympathetic nervous system.

Antibody against the Carboxyl Terminus of the F 2 Subunit of Sendai Virus Fusion Glycoprotein Inhibits Proteolytic Activation

Rabbit antibody, F2C12, was prepared against a synthetic peptide of 13 amino acids corresponding to the C-terminus of the F 2 subunit of Sendai virus F protein. F2C12 was shown to bind specifically to the F 2 subunit, irrespective of proteolytic cleavage of the F protein. FgC12 did not affect the infectivity, hemolytic, and cell fusion activities of activated virus, but F2C12 did inhibit proteolytic cleavage of the precursor F 0 to subunits, F 1 and F 2, by trypsin, factor Xa from chick embryos and Tryptase Clara from rat lungs. When F2C12 was added to the chorioallantoic cavity of chick embryos, cleavage activation of Sendai virus was inhibited. Intranasal administration of F2C12 to Sendai virus-infected rats suppressed activation and multiplication of progeny virus in the lungs. These results indicate that proteolytic cleavage of the F protein, with a single arginine residue at the cleavage site, occurs extracellularly in the allantoic cavity of chick embryos and in the bronchial lumen of rats.

The effects of in ovo mibolerone treatment on the bursa of Fabricius and the humoral immune system of chickens: A dose-response study

The effects of various doses of mibolerone, an analog of testosterone, in the development of the bursa of Fabricius and the humoral immune system were examined by injecting 0.01, 0.1, 1, 10 or 100 μg mibolerone into the chorio-allantoic cavity of 12-day-old chick embryos. Treatment with 0.01 or 0.1 μg of mibolerone caused slight or no bursal atrophy, 1 μg caused substantial bursal atrophy, and 10 or 100 μg caused severe bursal atrophy at the time of hatching and at 6 weeks of age. When mibolerone-treated birds were challenged with sheep red blood cells and killed Brucella abortus at 4 and 5 weeks of age, birds treated with 10 or 100 μg of mibolerone had suppressed humoral responses to sheep erythrocytes and B. abortus whereas birds treated with 0.01 and 0.1 μg of mibolerone had normal humoral immune responses. Birds treated with 1 μg mibolerone had normal humoral immune responses to sheep erythrocytes and B. abortus (except for low IgG responses to B. abortus) despite the fact that the birds had substantially atrophic bursae during their embryonic and post-embryonic development. These results are discussed in relation to an earlier claim that mibolerone given in microgram quantities in feed during the post-hatch period causes rapid development of the humoral immune system.

Infectivity for Chicken Embryos of Tissue-Culture-Modified Infectious Laryngotracheitis Virus

The present study shows the infectivity for chicken embryos of the SPL strain of tissue-culture-modified infectious laryngotracheitis (ILT) virus. The modified virus formed small pocks on the chorioallantoic membrane (CAM) and gave no adverse reaction after administration to chickens. For 1- and 6-day-old embryonated specific-pathogen-free (SPF) eggs, the SPL strain showed higher infectivity than virulent strain NS-175 or wild strains of ILT virus. A high percentage of embryos of day-old eggs inoculated with strains SPL via the yolk sac died within several days, and virus was recovered from yolk and embryos with high titer. In the embryos inoculated at 6 days of age, histopathological lesions characterized by syncytium formation with intranuclear inclusion bodies were observed mainly in the CAM, liver cells, epithelial cells of the oral cavity, and esophagus and rarely in epithelial cells of the uriniferous tubule, bronchus, trachea, choroid plexus, or feather follicles. In the 10-day-old embryonated SPF eggs inoculated via the chorioallantoic cavity, however, the SPL strain demonstrated low pathogenicity compared with strain NS-175 and the two wild strains.

The correlation of fatty acid content of infected cells and virions with Newcastle disease virus (NDV) virulence.

Chick embryo fibroblasts (CEF) infected with virulent strains of Newcastle disease virus (NDV) showed a dramatic increase in total unsaturated fatty acids (UFA). This increase was not seen in cells infected with the avirulent strains of NDV, Sendai virus or influenza A (PR8). The virions of the virulent strains of NDV harvested from the chorioallantoic cavity also had a higher UFA content compared to the avirulent ones. The kinetics of UFA increase in the virulent strains could be correlated with published data on the kinetics of RNA and protein synthesis inhibition, polykaryon formation and membrane permeability changes.

Plasmodium gallinaceum: Response of malarious chicken embryos to injection of serum from hyperimmunized chickens

The injection of serum from chickens hyperimmune to the avian malaria parasite, Plasmodium gallinaceum, or of nonimmune serum into the chorioallantoic cavity of chicken embryos infected with the same parasite resulted in changes in the blood picture of such embryos. In those embryos receiving serum from hyperimmunized birds: The level and rate of parasite increase were depressed; the maturation of the erythroid elements was reduced; the hematocrit values increased only by 27% as compared with 69% in untreated embryos. While hematocrit levels and rates of erythrocytic maturation were depressed in those embryos injected with nonimmune serum, there was no suppression of parasitemia. The results suggest a definite role of immunity in the anemia accompanying malaria.

STUDIES ON GONOCOCCUS INFECTION

Comparable numbers of types 1, 2, 3, and 4 gonococci were placed on the intact chorioallantoic membrane of 236, 10-day old chick embryos. Types 1 and 2 organisms produced infection and could be cultured from chorioallantoic fluid 2 days later significantly more often (69%) than types 3 and 4 organisms (12%, P < 0.001). This confirms in an animal model the same correlation between colony types and infectivity observed in human volunteers and suggests that types 1 and 2 gonococci possess a fundamental virulence characteristic which is absent from types 3 and 4 organisms. Gonococcal infection of the chick embryo chorioallantoic cavity remains a useful model somewhat analogous to localized gonococcal infection in man.

流行性耳下腺炎弱毒生ワクチンに関する研究

The virulent Towata strain of mumps virus was successfully passaged in the chorio allantoic cavity of developing chick embryos, and it was noticed that Towata strain was considerably attenuated after many passages (described in the first report). Attenuated Towata strain of various passage numbers was used in 4 field trials for children in 1967 by the inhalation method. Similar degree of antibody response was obtained in each trial, without any clinical manifestations due to the vaccination. Increase in NT antibody titer was 8 to 18 fold, and 80 per cent of seroconversion was observed in sero-negative cases. The rate of sero conversion of NT antibody was higher than that of HI antibody in paired sera. It was noticed that many children who had low antibody also showed antibody response. In inhalation trials with Enders strain adapted to the chorio allantoic cavity, antibody response almost similar to that of Towata strain was obtained.

Enzyme studies in the embryonate egg and in tissue culture following inoculation with different viruses.

Infection of the chorioallantoic cavity of embryonate eggs with PR 8, B/Lee and FM 1 influenza virus strains resulted in changes of LDH, GOT and PHI in the chorioallantoic fluids. All 3 enzyme levels, while markedly increased over that of the controls, were not considered to be strain specific. Fluids of baboon kidney cells (BKC) infected with virulent and avirulent polioviruses showed an increase in PHI activity along with progressive cellular destruction. LDH and GOT values increased only slightly in these cell fluids. Adenovirus type 5 and type 12 infected KB cell cultures failed to release more LDH into the tissue culture fluid than did the controls. While type 5 adenovirus-infected cells released 2–3 times more PHI and GOT into their fluids than the controls, adenovirus type 12 PHI and GOT levels in the culture fluids were only slightly increased. The enzyme release appeared to be in relation to the latent period of these two viruses.

流行性肝炎の病原体に関する研究

MURAKAMI and his collaborator had isolated the hepatitis virus from hepatitis patient and cultured in the chorio-allantoic cavity of chick embryo. The author newly isolated the hepatitis virus from the patient and studied on virological properties of the virus having obtained the purified virus. The following results were obtained.1) In the treatment by heating or by disinfectant, the purified virus showed high resistance to these treatment, and these resistances were thought to be the featuring properties of the virus.2) Also it was observed that the virus was highly resistant to irradiation of ultraviolet rays, and to action of ether.3) The purified virus was highly infections to mouse by peroral administration of it. The pathological changes were observed conspicuously, and moreover the changes induced were just the identical in any case.4) By the serological study, it was observed that the purified virus had an eminent antigenic capacity and reacted with the immune serum obtained by inoculation of the virus to rabbit giving the antigenic titier of 1:64-1:128. Also the virus showed distinctive reaction in the complement fixation test using the convalescent patient's serum from hepatitis; hence, this test might be highly valuable for the serological diagnosis of hepatitis despite of rather low titer than the above.5) The most difficult problem was the identification of neutralizing effect in the neutralization test and “Absättigungsversuch”. For this purpose the author adopted the mitigation of pathological changes caused by the neutralizing effect and observed the marked mitigation of the changes on the test using the virus and corrsponding immune serum; but this evidence was not observed in the test of the virus and uncorresponding serum. The mitigation of the changes was not distinct in the “Absättigungsversuch” possibly by the occurence of combination of the virus and its anti-body in the body of animal instead of in the test tube. It is to be expected the further study on the test procedure and the method of identifying the neutralization effect.

Quantitative studies on the growth of herpes virus in HeLa cells.

SUMMARY: Less than a tenth of the pock-forming particles of the HFEM strain of herpes virus are able to initiate infection in HeLa cells, even after passage in these cells. The pock-forming virus attaches firmly to the cells but most of it remains susceptible to antiserum and presumably does not penetrate the cell. A small proportion of virus initiates infection in the HeLa cells, and, after exposure to more than one HeLa infectious particle/cell, new virus first appears in the cell fraction 12 hr. after infection, and 4 hr. later virus is released into the medium. Virus in the cell fraction can be detected either by disruption of the cells, or simply by removing the cells from the glass with ethylenediaminetetra-acetic acid (EDTA) or saline. This ‘EDTA fraction’ may contain virus released from the cell surface. An attempt was made to determine the number of virus-yielding cells, by inoculation of whole cells on to the chick chorioallantoic membrane. The proportion which yielded virus was lower than would be expected from the input of HeLa-infectious virus. After removal of superficially attached virus with antiserum, it was not possible to detect infective virus during the latent period in the majority of cells which ultimately released virus. Herpes virus is well known as a cause of latent infection, and it is our interest in latency which has prompted further investigation of the growth of this virus. Previous work on herpes virus multiplication has been carried out in the chorioallantoic membrane cavity of fertile hens’ eggs (Scott, Coriell, Blank &amp; Gray, 1953; Wildy, 1954; Yoshino &amp; Taniguchi, 1956a, b) and in explants of rabbit cornea (Scott, Burgoon, Coriell &amp; Blank, 1953). In recent years a more quantitative approach has been possible through the use of cell suspensions and monolayer cultures, and growth studies have been reported by Gostling &amp; Bedson (1956) and Gostling (1956) in chick embryo cells, and more recently by Kaplan (1957) in rabbit kidney cells. We have studied the growth of herpes virus in HeLa cells because of the desirability of propagating cells indefinitely when studying latent infection. HeLa cells are also convenient because they are suitable for isolation of cell clones, and for study of virus release from isolated single cells. This paper describes some aspects of virus growth in monolayer cultures of HeLa cells. Studies on plaque formation in HeLa cells (Farnham, 1958), cytological observations (Ross &amp; Orlans, 1958), chemical changes (Newton &amp; Stoker, 1958), electron microscope studies (Stoker, Smith &amp; Ross, 1958) and single cell experiments (Wildy, P., Stoker, M. G. P. &amp; Ross, R. W., unpublished) will be given in other communications.

Influenza virus増殖に及ぼす孵化鶏卵漿尿液のpH並びにInhibitorの影響

Influences of pH and inhibitor of chorioallantoic fluid on the multiplication and infectivity of influenza virus were observed in detail with indivisual eggs infected. PR8 strain of influenza A virus was employed throughout this study. When incubated at 37°C, in control eggs, the pH of chorioallantoic fluid falled rapidly since about the 14th incubation day. The values of pH of the chorioallantoic fluid corresponding to the harvest on the first time were higher than those corresponding to that on the second time or more of the successive harvestion from day, to day when observation was made with eggs in same incubation day. And the difference between the two values was about 0.5.While, in the infected eggs with the virus, the distinct descending of the pH of the fluid as observed in the control eggs was not recognizable. When the virus was infected to the eggs the pH of whose chorioallantoic fluid was in rapidly descending stage, the characteristic re-ascending curve of pH was recognized. The pH value under 6.0 seemed to act as inhibiting the multiplication of the virus; however, even such low pH value did not demonstrate a absolute negative significance for the infection of the virus.The inhibitors from chorioallantoic fluid, egg white and egg yolk had no influences on the multiplication of the virus. It appeared likly that the inhibitors from chorioallantoic fluid and egg white were closely analogous to mucoprotein from their attitude to the action of RDE and heating. To the contrary, the inhibitor from egg yolk was considered to be derived from the other substance.Both the wave height of polarographic catalytic protein wave of chorioallantoic fluid due to the sulfosalicylic acid filtrate test and the inhibiting titer of the same fluid for the hemagglutination of the virus were raised as the incubation day was progressed, and a definite relationship was observable between them. The inhibitor of mucoprotein nature was proved to exist also in the infected fluid and in this connection no significant difference was recognized between normal and infected fluid. The eggs on the 17th incubation day were able to be rendered to show as high a hemagglutinating titer as that on the 12th incubation day to the inoculation of the virus, when a certain volume of physiological saline was added to the chorioallantoic cavity and thus the volume of the fluid was increased.

インフルエンザウイルスの赤血球凝集能と感染能及免疫能との関連性

We studied the hemagglutinating activity, infectivity and immunizing ability of influenza virus FM1 (egg-adapted strain and mouse-adapted strain). The mouse-adapted strain FM1 had infectivity, immunizing ability and toxicity against chick embryo proportion to its hemagglutinating activity, but not against mice.After we repeated the adsorption of virus in virus-suspension with chick red cells, we inoculated the 10-1-10-5 dilution of that non-hemagglutinating supernatant into the chorioallantoic cavity of eggs, and, after 48 hours' incubation, we recognized the virus-production by their hemagglutination. So that there may be the non-hemagglutinating but infective influenza virus.

急性灰白髄炎ウイルスの孵化鶏卵内培養

The culture of poliomyelitis virus in the developing chick embryo has been hitherto considered to be a difficult matter. However, in 1952, Cox and others displayed its culturability by inoculating the yolk sack and the chorio-allantoic cavity of the developing chick embryo with the MEFI strain of this virus, with subsequent transmission of the cultured virus to M. rhesus monkeys. Their work proved the possibility of the cultivation of poliomyelitis virus not only by tissue culture but also in the developing chick embryo.We have already observed that the growth of some virus in the chick embryo can be indirectly demonstrated by following the growth of another challenge virus subsequently inoculated. Here, we have made inoculations of the Lansing strain of poliomyelitis virus in the chorio-allantoic cavity and the yolk sack of the 6 day old chick embryo, and a challenge inoculation with the mumps virus (Enders strain and Fujimura strain) was made in the chorio-allantoic cavity or in the amnion cavity and have succeeded in determining the growth aspect of the polio virus by observing the degree of growth of the mumps virus. This observation is based on the so-called interference of the growth of the two different viruses, and we have made investigations on the various factors which come to play upon the incidence of this phenomena.

Influence of cortisone, piromen and ACTH on susceptibility of embryonated eggs to fowl-pox virus.

Summary and Conclusions1. The pre-and post-treatment of embryonated eggs with 1.25 mg of cortisone by the chorioallantoic and allantoic cavity routes significantly decreased the susceptibility of the chorioallantois to infection with the fowl-pox virus as measured by EID50 determinations and marked reduction in development of virus lesions. However, the incubation of diluted virus with cortisone (1.25 mg/ml), prior to inoculation onto the chorioallantois, did not alter the EID50, nor was the development of the virus lesions impaired. 2. The pre- and post-treatment of embryonated eggs with various concentrations of Piromen or constant quantities of ACTH (1.25 mg) by the chorioallantoic and allantoic cavity routes failed to alter the susceptibility of the chorioallantois to infection with the fowl-pox virus. Likewise, the incubation of Piromen with virus at room temperature failed to alter the infectivity of the fowl-pox virus. 3. Simultaneous treatment of eggs with cortisone and Piromen or ACTH resulted in...

エクトロメリア・ウイルス (仙台株) の分離

The present paper deals with an ectromelia virus, which was isolated from liver of hybrid mice, during several attempts to obtain the causative agent of Izumi fever.At first, blood specimen taken from a patient diagnosed as Izumi fever, was inoculated intraperitoneally to mice. Subsequent passages were performed 3 times successively, by intraperitoneal injection of resected liver. At the 4th transfer some characteristic changes of liver were noticed in all tested animals. But other viscera, such as lung or spleen were not so affected. Moreover the surface tinge of the liver was so similar to that of described as mouse hepatitis virus rather than that of ectromelia virus, so the picking of hepatitis virus latent in tested mice was anticipated. Subsequent transfers were performed further and fertile egg culture was also examined.By the inoculation of mice liver suspensions into chorioallantoic cavity, CCA activity of fluids was proved.Further the serological identification of the HA assured that the agent is NPV. NPV is an agent which is related to influenza viruses and was isolated in this laboratory in 1952 from the case of newborn pneumonitis. Afterwards the latency in mice was also ascertained in this laboratory. However the liver damage observed in this experiment was so conspicuous, so the reason of this liver changes deemed probably not owing to NPV.So the contamination of both NPV and hepatitis virus was assumed and the separation of both virus was intended. Chicken cell adsorption of the chorioallantoic harvests at low temperature was repeated and the supernate was combined with anti-NPV rooster serum. After the above treatment, the supernate injection to mice, caused the death of mice representing the same characteristic changes of liver, But now, the further transfer in fertile eggs could not produce any HA. Thus the removing attempt of the NPV from this agent was successful.But after the removing of the NPV, further examination proved that the virus is related rather to ectromelia virus than hepatitis virus. By foot pad inoculation, amputation of hind legs was occured. Spleen necrosis and presence of inclusion bodies in monocytes of ascites was ascertained by intraperitoneal injection. Pock formation on the chorioallantoic membrane became also evident. So the original virus was confirmed to be an ectromelia virus. Some characteristics of the virus were summarized as follows: LD 50 for mice weighing 12g in average was 10.-8.5 Some decreases of titer were seen after filtration with Berkefeld N candle. The virus was precipitated by protamine sulfate.The distribution of virus in several viscera after intraperitoneal injection was also determined and the high concentration was proved in liver, spleen and intestines. Other incculation procedures, such as intranasal instillation, also caused the death of mice, but prolongation of survival time was observed. Contrary to my expectation, histopathological changes and the virus distribution in each viscera by other inoculation routes was not different in principle from that observed by intraperitoneal inoculation, Young guinea pig was also susceptible against this virus.Electronmicrographic studies were made in detail and the results were compared with that of standard ectromelia virus (Tama-strain). Though the same picture with standard virus was obtained after the treat ment with dil. sod. hydroxide, native picture was clearly different from each other.From several results described above, the virus isolated thus in this laboratory from hybrid mice was assumed to be an ectromelia virus, although some differences in electronmicrographic figures were noticed.