Inoculation Of Embryonated Eggs Research Articles

English

In Pakistan, the poultry industry is one of the rapidly growing industries. Due to lack of biosecurity measures, this is affected by some important infectious agents such as Avian Influenza virus (H9N2) and Newcastle disease virus (NDV) results in a huge economic loss. So, to control these losses discovery of new anti-viral drugs required to bring into line to fight against these infections. It is a general perception that the active components of medicinal plants have effective results against various infections like the influenza virus. The current therapeutic facilities need to be improved by investigating new antiviral drugs from natural resources to fight against viral infections. The present study was conducted on ethanolic extracts of seven different flowers to examine their antiviral activity against NDV and H9N2 in ovo using chicken embryonated egg inoculation. The spot agglutination and hemagglutination tests showed inhibitory effects of Rosa damascena Miller, Achillea millefolium, Woodfordia fruticosa Kurtz and Bombax ceiba L. against NDV as no agglutination observed. While the extracts of Taxacum officianale Weber, Hyssopus officianalis L. and Chrysanthemum cinerafolium (Trevis.) Vis. showed positive results for both spot agglutination and hemagglutination assay against NDV. However, both spot agglutination and hemagglutination assay showed inhibitory effect of all the flowers extracts against H9N2. The bioactive components such as alkaloids, ethers, terpenoids, etc. of each flower were analyzed through Gas chromatography mass spectrometry (GC-MS). The current results revealed that ethanolic extracts of these flowers possess strong antiviral activity because of their active ingredients. These ingredients should be isolated, commercialized and used for therapeutic purpose. © 2021 Friends Science Publishers

Chicken Meat as a Source of Avian Influenza Virus Persistence and Dissemination

A study was conducted to investigate the persistence of Avian Influenza Virus (AIV) serotype H9N2 in the processed and frozen meat from chickens earlier exposed to this virus. For this purpose a group of chickens were experimentally infected with a field isolate (A/Chicken/Pakistan/NARC-02/2002) of AIV H9N2. After ten days post-inoculation, chickens were euthanized and their carcasses were cut into small pieces and stored at -20°C till further use. Subsequently, after every week selected pieces of frozen chicken were processed for isolation of AIV subtype H9N2 through embryonated egg inoculation. The data indicated that the AI virus was recoverable at different HA titres from various parts of chicken meat which includes neck, wings, breast, oscoxy, legs and bone marrow. It was possible to recover the AI virus using standard in ovo propagation techniques up to six weeks post-storage from most parts of the stored chicken.

Screening of Feral Pigeon (Colomba livia), Mallard (Anas platyrhynchos) and Graylag Goose (Anser anser) Populations for Campylobacter spp., Salmonella spp., Avian Influenza Virus and Avian Paramyxovirus

A total of 119 fresh faecal samples were collected from graylag geese migrating northwards in April. Also, cloacal swabs were taken from 100 carcasses of graylag geese shot during the hunting season in August. In addition, samples were taken from 200 feral pigeons and five mallards. The cultivation of bacteria detected Campylobacter jejuni jejuni in six of the pigeons, and in one of the mallards. Salmonella diarizona 14:k:z53 was detected in one graylag goose, while all pigeons and mallards were negative for salmonellae. No avian paramyxovirus was found in any of the samples tested. One mallard, from an Oslo river, was influenza A virus positive, confirmed by RT-PCR and by inoculation of embryonated eggs. The isolate termed A/Duck/Norway/1/03 was found to be of H3N8 type based on sequence analyses of the hemagglutinin and neuraminidase segments, and serological tests. This is the first time an avian influenza virus has been isolated in Norway. The study demonstrates that the wild bird species examined may constitute a reservoir for important bird pathogens and zoonotic agents in Norway.

Identification and molecular characterization of the infectious bursal disease virus (IBDV) from an outbreak in a broiler flock in midwestern Brazil

In order to identify and characterize the agent of a suggestive clinical case of Gumboro disease (GD) that affected a 34-day-old broiler flock in Buriti Alegre (Goias State, Midwestern Brazil) in the year 2001, we carried out a combination of classic and modern virological methods. Histopathological analysis of the bursa revealed necrosis, presence of depleted follicles, some infiltration of heterophils, edema and formation of cystic cavities that are compatible with lesions observed in GD. Inoculation of embryonated eggs of specific pathogen-free (SPF) chickens with macerated bursa suspension resulted in embryo mortality and lesions which were also compatible with those caused by IBDV. A sample of bursa was submitted to a nested reverse transcriptase-polymerase chain reaction (RT-PCR) procedure to amplify the hypervariable region of the VP2 gene. The amplicon that was obtained from this sample (BR-GO) was digested with the restriction enzymes TaqI, StyI and SspI, but not with SacI, a pattern similar to that observed with very virulent IBDV (vvIBDV) strains. Furthermore, nucleotide sequence analysis revealed alanine, isoleucine, and isoleucine at amino acid positions 222, 256, and 294, respectively, which are also found in vvIBDV strains. Finally, phylogenetic analysis grouped BR-GO isolate with other vvIBDV strains.

Detection and Classification of Infectious Bronchitis Viruses Isolated in Korea by Dot-Immunoblotting Assay Using Monoclonal Antibodies

Dot-immunoblotting assay (DIA) using five monoclonal antibodies (MAbs) to infectious bronchitis virus (IBV) was used to detect and classify the viruses propagated in embryonated chicken eggs. Using a group-specific MAb 3F5, 10 reference strains and 12 Korean isolates of IBV were successfully detected by DIA, and the lowest virus titer of IBV detected by DIA was approximately less than 10(3.8) mean embryo infective dose/ml. For evaluating the diagnostic efficiency, DIA was compared with the conventional infectious bronchitis (IB) diagnostic method. IBV antigens in allantoic fluid from embryonated eggs inoculated with IB-suspected field samples were specifically detected by DIA within only one or two egg passages, whereas the conventional embryonated egg inoculation method required four to seven egg passages for confirming IBV infection. These results indicated that DIA could significantly reduce time and cost for IB diagnosis. For examining the possibility of classifying IBV by DIA, four strain-specific MAbs, 3A4, 2A3, 6F7, and 2C6, were used. According to the MAb reacting patterns to the IBV antigens, the 10 IBV reference strains were classified into six groups; seven strains belonged to three different groups, and the other three strains each belonged to an individual group. In the case of 12 Korean isolates of IBV, they were classified in six groups. Among the six groups, the MAb reacting patterns of three groups matched those of the IBV reference strains, but the others did not. These data suggest that at least three variant serotypes of IBV exist in Korea.

Capillaria hepatica: a cause of septal fibrosis of the liver.

Fine, long, fibrous septa were observed as a late change developing in the acinar zone III of the liver of rats experimentally infected with the helminth Capillaria hepatica. Hepatic septal fibrosis begun 30 days after inoculation of embryonated eggs into the stomach of rats and became clearly evident from the 40th day onwards. Experimental observation was undertaken for 170 days. Septal fibrosis increased progressively with time and was most marked when the parasitic nodules formed around larvae, disintegrating worms and eggs were involving. Septal fibrosis of the liver has not been previously recognized as a manifestation of hepatic capillariasis. The presence of sequestered parasite antigens, probably being slowly released within the liver, appears to be a major factor in the pathogenesis of hepatic septal fibrosis observed in rats with C. hepatica infection.

Laboratory techniques for the diagnosis of chlamydial infections.

Yolk-sac inoculation of embryonated eggs was superseded 25 years ago by the use of cell cultures (often McCoy) for the isolation of Chlamydia trachomatis. Centrifugation of specimens onto the cell monolayers was shown to increase sensitivity, but little of late has further improved sensitivity which is at least ten-fold greater than that of eggs. However, culture is slow and labour intensive so that non-cultural techniques without these drawbacks have come to dominate. Direct fluorescent antibody (DFA) tests are rapid and have sensitivities that range from 70% to 100% for men and 68% to 100% for women, and specificities that range from 87% to 99% for men and 82% to 100% for women; if the tests are read by competent observers the values are at the top end of the ranges. The detection rate may be enhanced even further by relatively low-speed centrifugation of specimens before staining. Skilled reading is not a feature of enzyme immunoassays (EIAs) which according to the literature have sensitivities that range from 62% to 97% for men and 64% to 100% for women, and specificities that range from 92% to 100% for men and 89% to 100% for women. However, comparison against poor reference tests is responsible for most of the higher values and the clinician should not be misled into believing that EIAs have excellent sensitivity; the lower values in the ranges are closer to reality. Furthermore, EIAs that are being designed for use by general practitioners should be regarded with the greatest caution since lack of sensitivity means that chlamydia-positive patients will go undetected. The polymerase chain reaction (PCR) is not bedevilled by insensitivity but it is no more sensitive than the most sensitive cell culture or DFA tests. PCR is unsuitable for routine diagnosis but has a place as a research tool. For men, examination of "first-catch" urine samples by the best of the non-cultural procedures provides an acceptable non-invasive approach to diagnosis; for women, the value of examining urine may be less, but needs to be thoroughly tested. However, there is little doubt that a Cytobrush used to obtain cervical specimens holds no practical advantage over a swab. Serological tests are reliant on the provision of paired sera for making a diagnosis; high antibody titres in single sera may be suggestive of an aetiological association in deep-seated chlamydial infections (epididymitis, arthritis, salpingitis, etc), but unequivocal interpretation is unusual, particularly in an individual case, since the distinction between a current and past infection is problematical.

Observations et isolements de pseudopicornavirus a partir de dindonneaux malades

Forty-nine sick turkeys (from 31 flocks), 2- to 6-week-old, were examined. They had enteritic and respiratory signs. Direct electron microscopy, inoculation of embryonated eggs by the yolk sac route and inoculation of young SPF turkeys with materials from affected turkeys showed in several cases presence of picornalike virus. Two strains of virus were isolated.

In vivo susceptibility of the Legionnaires disease bacterium to ten antimicrobial agents

The susceptibility of the Legionnaires disease bacterium to various antimicrobial agents was determined by inoculation of embryonated eggs via the yolk sac. When administered prophylactically, the minimal dose of drug preventing all deaths due to the infection was as follows: rifampin, 0.02 mg; gentamicin, 0.25 mg; streptomycin, 0.39 mg; erythromycin, 0.62 mg; sulfadiazine, 1.56 mg; chloramphenicol, 2.50 mg; and cephalothin, 20.0 mg. Smaller amounts delayed deaths, and larger or equal amounts rendered the embryos free of infection. Oxytetracycline in the largest tested amount, 5.0 mg, protected 80% of the embryos from death, and as little as 0.31 mg delayed death. Chlortetracycline (0.50 mg) and ampicillin (10.0 mg) were ineffective. The six most effective drugs were studied in an experiment in which they were administered at various times after infection in doses that were twice the minimal prophylactic dose preventing all deaths. In this therapeutic experiment, rifampin, and erythromycin allowed 100% survival when given even 72 h after infection; gentamicin, streptomycin, sulfadiazine, and chloramphenicol did so when given 48 h after infection. All six drugs increased mean survival time when administered 72 h after infection.

Ascaris suum: Hatching of embryonated eggs in swine

Larvae of the nematode Ascaris suum were recovered in posterior small intestinal and colonie mucosal scrapings of pigs 4 to 29 hr after inoculation of embryonated eggs. Larvae were also recovered in the mucosal scraping from ligated intestinal segments at 4 to 19 hr after injection of embryonated eggs into the segments. Location of the ligated segment in the small intestine did not appear to affect hatching of eggs or migration of the larvae into the mucosal surface. Embryonated eggs hatched in ligated cecums and larvae were recovered in the livers of pigs after 2.5 and 3.5 days. Larvae were recovered in washings from Thiry-Vella intestinal loops at 5 to 23 hr after embryonated eggs were inoculated into the loop. Hatching of eggs occurred in intestinal loops in pigs unexposed to eggs and in pigs given multiple doses of eggs either orally or into the intestinal loop.

Detecting Marek's Disease Herpesvirus: Evaluation of Biological Systems

Systems were evaluated for isolating and identifying Marek's disease herpesvirus (MDHV) from purposely contaminated samples of poultry virus vaccines. The sensitivity and specificity of duck embryo fibroblasts (DEF) and chick kidney (CK) cell cultures for detecting MDHV were compared with the inoculation of embryonated eggs and day-old chicks. Duck embryo fibroblast cell cultures were more sensitive for detecting MDHV and less susceptible to the cytopathogenic effect (CPE) of vaccine viruses than either CK cell cultures or embryonated eggs. The inoculation of day-old chicks appeared less sensitive than DEF cell cultures when infection was determined by culture of kidneys or by use of a radial diffusion test for MDHV feather follicle antigen at 14 days postinoculation. At 21 days postinoculation, chicks appeared as sensitive as DEF cell cultures when infection was determined by kidney culture and more sensitive than cell cultures when the radial diffusion test was employed to determine infected birds. Inoculation of chicks was the only satisfactory method for isolating MDHV from samples of avian pox virus vaccines. Fluorescent-antibody (FA) staining confirmed the identity of MDHV isolates made in DEF cell cultures. The FA procedure was used to examine 28 samples of commercial virus vaccines for MDHV. This virus was not detected as a contaminant in any of the samples.

Embryonic Response to Eimeria tenella Infection

The chorioallantoic membranes (CAM) of chicken embryos were infected with Eimeria tenella by inoculation of sporozoites into the allantoic cavity. Embryonic mortality during the hemorrhagic phase of the infection (4th to 8th days postinoculation) was highly dose dependent. Mortality among embryos produced by strains of fowl differing in resistance to cecal coccidiosis was compared with the susceptibility of 11-day-old chicks produced by the same matings. Although significant differences in embryonic resistance were found among different strains of fowl, in ovo inoculation with E. tenella was an ineffective technique for detecting genetic differences in resistance to cecal coccidiosis. The results of experiments designed to study the effect of in ovo inoculation on the resistance of 11-day-old chicks indicated that mortality following inoculation with 105 E. tenella oocysts was much higher among chicks derived from inoculated eggs than among chicks from noninoculated eggs. Although these results suggested that inoculation of embryonated eggs induced partial immunological tolerance to an antigen(s) associated with the parasite, other possible causes of the observed phenomenon could not be positively ruled out. Eimeria tenella, the parasitic agent of cecal coccidiosis in the fowl, is a rigidly host-specific parasite which confines its endogenous development to the cecal pouches and adjacent areas of the intestine. Long (1965) first demonstrated that E. tenella sporozoites, inoculated into the allantoic cavity of the chick embryo, initiate an apparently "normal" infection within the chorioallantoic membrane (CAM). Intravenous inoculation failed to produce infections, while intraamniotic inoculations resulted in sudden deaths associated with multiple hemorrhages. Subsequently, four species of Eimeria (E. brunetti, E. mivati, E. necatrix, and E. tenella) were found to develop in the CAM of chicken embryos (Long, 1966). Jeffers and Wagenbach (1969) reported significantly greater mortality among female embryos than among male embryos during the acute hemorrhagic embryonic response to E. tenella infection. The objective of the present study was to further characterize the embryonic response to E. tenella infection. General pathological effects of the infection were examined and a Received for publication 2 January 1970. * Supported in part by Research Grant A1-04101 from NIAID, U. S. Public Health Service, and by North Central Regional Project NC-89. Published with the approval of the Director of the Wisconsin Agricultural Experiment Station, College of Agriculture, Madison. t Present address: Parasitology Section, Hess and Clark, Ashland, Ohio 44805. I Present address: Department of Biology, Carleton College, Northfield, Minnesota 55057. dose-response curve was obtained for embryos of a representative strain of fowl. The response of embryos from mating groups selected for differential genetic susceptibility to cecal coccidiosis was studied in order to detect possible associated genetic differences in embryonic susceptibility to in ovo inoculation with E. tenella. A strong positive relationship would suggest that inoculation of embryonated eggs with E. tenella might be a rapid and economical method for detecting genetic differences in the postnatal resistance of different strains of fowl. Other experiments were designed to study the effect of in ovo inoculation on the resistance of chicks to challenge with E. tenella oocysts. Burnet and Fenner (1949) theorized that an individual may become immunologically unresponsive to antigens to which it was exposed during embryonic development. Immunological tolerance to certain bacterial and viral antigens has been induced in the chicken embryo (Buxton, 1954; Friedman and Gaby, 1960; Hanson and Cannefax, 1967; Rubin et al., 1962; Solomon, 1968). MATERIALS AND METHODS The oocyst cultures used for all experiments were obtained through serial passage of a pure line E. tenella strain which has been maintained in this laboratory for many years. Bacteriologically sterile oocyst suspensions were prepared using a Clorox digestion procedure described by Wagenbach et al. (1966). Sporocysts, released from the oocysts by agitation in the presence of sterile 4 mm diameter glass beads, were added to an excysting medium consisting of 0.25% trypsin and 5.0%

鶏の伝染性喉頭気管炎に関する研究

In March, 1962, on a poultry farm in Osaka, a certain respiratory disease occurred in a 50-day-old chicken flock. The outstanding symptoms were gasping, rale, and coughing. Some chickens showed gasping, with the head extended and the beak open. The fatality rate was 20 per cent in the flock. The mucous membrane of larynx and trachea was covered with a yellowish caseous exudate or blood-stained exudate. In some chickenss blood covered the mucosa.Histopathologically, intranuclear inclusion bodies were found in the tracheal epithelial cells. By intratracheal inoculation of the tracheal material, the same respiratory symptoms were produced and serial passage through the trachea was possible. A certain virus was isolated by inoculation of embryonated eggs in CAS and on CAM. There were white grayish areas or pocks produced by the virus and intranuclear inclusion bodies in sections of the membrane. In chicken inoculation of the virus, intranuclear inclusion bodies were found in groups of tracheal epithelial cells.Neutralization test was carried out, by inoculation on CAM, with the virus and serum from chickens inoculated with the isolated virus. The result was successful.The sinus of the chicken was sensitive to the isolated virus.In conclusion, the virus isolated on CAM was infectious laryngotracheitis virus. Therefore, the respiratory disease affecting this chicken flock was diagnosed as infectious laryngotracheitis.

Pityriasis rosea. An etiologic study.

Since the initial description of pityriasis rosea as a separate clinical entity by Gibert in 1860,¹the causal relationship of an infectious agent has received occasional consideration. Attention has been directed toward the possible involvement of fungal or bacterial organisms,^2-4but to date the role of these micro-organisms in the etiology of the disease continues to remain obscure. Surprisingly few studies relating to viruses have been undertaken in this connection. Markham⁵and, later, Felsher⁶reported unsuccessful attempts to detect the presence of viral agents in materials from cases by inoculation of embryonated eggs. In view of the recent advances in techniques for the isolation of viral agents, it was considered worthwhile again to approach the problem through the use of tissue cultures. It is the purpose of this communication, therefore, to describe the results of studies undertaken with in vitro cell cultures prepared from human, simian,

A MUTANT OF HERPES SIMPLEX VIRUS POSSESSING AN ATTENUATED PATHOGENICITY FOR BABY MICE OBTAINED BY EGG PASSAGE OF A NEWLY ISOLATED STRAIN

It has been attempted in this laboratory for the past years to isolate and passage herpes simplex virus by the chorioallantoic inoculation of embryonated eggs and pursue the changes in pathogenicities for various host animals occurring during the course of the passage. Among the pathogenicities examined were included those for baby mice intracerebrally or intraperitoneally inoculated, since Kilbourne and Horsfall (1951) stated that one-day old mice were equally susceptible to herpes virus inoculated by these two routes. A strain designated SK was studied most extensively.A fact came to light thereby that the SK strain after passaged through eggs about 50 times was hardly lethal to baby mice when given by the intraperitoneal route, whereas the classical HF strain experiencing more than 450 egg passages still possessed a high infectivity to baby mice even by the peripheral inoculation. This paradoxical result stimulated a quantitative analysis of the infectivities to baby mice of SK strain in comparison with HF strain, and it was eventually disclosed that a mutant had appeared incidentally in an early egg passage of SK strain, which subsequently had replaced the parent type virus after several more passages. Details of those experiments are reported in the present communication.

Experimental combined viral and bacterial infection (influenza C and hemophilus influenzae, type B) in embryonated eggs.

Influenza C virus, J.J. strain, is readily propagated following intra-amniotic inoculation of embryonated eggs on the 14th day of incubation. The resulting infection is inapparent in that there is no obvious interference with normal embryonic development and no evidences of injury can be detected by the light microscope. Hemophilus influenzae, type b thrives luxuriantly in the amniotic fluid of embryonated eggs inoculated by the intra-amniotic route on the 15th day of incubation. The effects of the establishment of the bacterial infection in the embryo are noted by the occurrence of death, bacteriemia or characteristic inflammatory lesions in the form of purulent sinusitis, pharyngitis, tracheo-bronchitis and meningo-encephalitis. These lesions may occur singly or in various combinations. The incidence and severity of disease manifestations in infected embryos depends on the proportion of encapsulated and virulent bacilli in the inoculum, the number of infectious doses and the growth rate of the bacteria in the surrounding amniotic fluid. Combined viral and bacterial infection established by intra-amniotic inoculation with influenza C virus on the 14th followed by Hemophilus influenzae, type b on the 15th incubation day brings about a significant increase in the incidence and severity of disease manifestations in the embryos. Selective survival and marked acceleration of the growth rate of encapsulated and virulent elements of the bacterial population is promoted in the virus-infected embryos. The disease process appears to be entirely attributable to the bacterial component. There seems to be relatively little or no effect on influenza C virus by the accompanying proliferation of Hemophilus. The exact nature of the virus-induced influences which enhance the pathogenicity of the bacteria and favor the establishment of the infectious process under these circumstances remains to be determined.

Studies on herpes simplex virus in tissue culture. I. Characteristics of virus growth.

The Z strain of herpes simplex virus as well as three freshly isolated strains of the same virus caused a complete degeneration of the fibroblastic outgrowth in roller tube cultures of human embryonic lung. Infectivity titrations made in such tissue cultures regularly gave higher values than the lethality titers obtained by yolk sac inoculation of embryonated eggs. Furthermore, the virus amounts found in the fluid phase of roller cultures were comparable to those obtained in the allantoic fluid of eggs. Three strains of herpes simplex virus have so far been isolated directly in tissue culture from herpetic vesicles. However, more work is needed, involving isolation experiments from pathological specimens simultaneously in tissue cultures and other hosts before the relative sensitivity of the tissue culture method for primary isolation purposes can be evaluated.

Cultivation of Toxoplasma in Embryonated Egg. An Antigen Derived from Choriolallantoic Membrane

Inoculation of embryonated eggs with toxoplasma resulted in a generalized parasitic disease of this host with a fatal termination. The agent could be maintained by serial passage of embryo brain. Chorioallantoic membranes from infected eggs contained a stable, specific soluble antigen which fixed complement with toxoplasma immune animal serum. Human sera from both proven and suspected cases of toxoplasmosis which were known to contain neutralizing antibody against the parasite also fixed complement with chorioallantoic membrane antigen.