Embryo Passage Research Articles

Emergence Pattern of Egg-Adapted Avian Encephalomyelitis Virus by Alternating Passage in Chickens and Embryos

The chance of inducing complete egg-adaptation was slight in alternating passage in chickens and embryos with chicken-brain-passaged viruses of wild avian encephalomyelitis viruses: egg-adaptation was accomplished by use of an incipient egg-adapting mutant, but failed with other passaged virus.

Vaccination and Revaccination with a Holland (H) Strain of Infectious Bronchitis Virus

One-day-old chickens possessing maternally derived immunity to Massachusetts-type infectious bronchitis virus (IBV) were vaccinated by the eyedrop route with a Holland (H) IBV strain of the Massachusetts type. Vaccination was done with the 72nd and 92nd passages in chicken embryos. Attenuation of the virus was done in the authors' laboratory. Four weeks postvaccination the immunity was challenged with 8 different IBV serotypes. A broad spectrum of protection from respiratory signs was obtained in each of the challenged groups (compared with unvaccinated controls). With 5 of the challenge viruses, protection against tracheal virus shed was good (70% or greater). Revaccination at 4 weeks old by the aerosol route with the 53rd and 72nd passages did not greatly enhance protection. However, the virus-serum-neutralizing (VN) titers with Massachusetts-type IBV as an indicator were high, and an anamnestic response was demonstrated at 8 weeks of age. The VN titers observed at 4 weeks old were generally low and did not correlate necessarily with the protection engendered.

Virulence of established vaccine strains and artificially passaged field strains of Mycoplasma mycoides subsp mycoides

The virulence of Mycoplasma mycoides for mice, which appears to parallel virulence for cattle, was assessed in respect of four vaccine strains well known for their use in the control of contagious bovine pleuropneumonia. The T1 strain was slightly more virulent than V5, and both were much more virulent than F and KH3J, which could not be distinguished from each other. There was no significant difference between cultures of the T1 strain supplied by three different vaccine-producing laboratories, The mousevirulence test was also used to study the effect of serial passage in artificial systems on virulent field strains of M mycoides; two such strains underwent no appreciable change in virulence during many passages in chick embryos or in mice, although the same strains, and two others, were readily attenuated by repeated subculture in broth. With one strain of M mycoides there was evidence that the total number of subcultures in broth over a given period was more important than the duration of each subculture.

Immunity to Infectious Bronchitis Virus from Spray Vaccination with Derivatives of a Holland Strain

Study was made of the protection of chickens from spray vaccination with different modifications of a Holland (H) strain of infectious bronchitis virus (IBV). Noted in each trial were the virus-serum-neutralizing antibody titers induced and the virulence of the vaccine virus at different passage levels. Protection against respiratory signs was good from challenge with a homologous virus serotype as well as with 5 heterologous IBV isolates. This occurred from vaccinating day-old chicks possessing homologous IBV antibodies as well as vaccinating susceptible 4-week-old chickens. The tracheal virus shed-rate following challenge was considered low (20% or less) with 4 of the challenge viruses. The challenge virus shed-rate was somewhat higher from vaccination at one day old than from vaccination at 4 weeks; the virus-serum-neutralization titers were lower, or even insignificant, with early-age vaccination. The immunogenicity and virulence of the vaccine virus was apparently reduced in the higher embryo passages employed. None of the vaccine derivatives was considered virulent under these conditions.

Characterization of a New Infectious Bronchitis Virus Isolate. III. Cell-Culture Adaptation of Clark 333

The Clark 333 strain of infectious bronchitis virus (IBV) was substantially resistant to primary chicken cell-culture adaptation. More than 40 serial embryo passages were required before the virus would produce cytopathic alterations upon cell-culture inoculation. The cytopathic effect was characteristic of the effect of reported for IBV. Adaptation was not accomplished by alternating serial passages in embryo and cell-cultured systems. A careful monitoring of cell-culture fluid infectivity by embryo inoculation was found to be essential because absence of adaptation is accompanied by a loss in virus titer (3 to 5 serial passages). Helpful additional aids were immunofluorescence and cytopathology.

Further studies on the pathogenicity and immunogenicity of an embryo‐adapted strain of Eimeria tenella

The pathogenicity, immunogenicity and reproduction of a strain of E. tenella, serially passed in embryos was studied. A strain which had completed 62 embryo passages no longer regained pathogenicity after nine passages in chickens. The reproduction of the strain in chickens, judged by oocyst production was poor, but increased after nine serial passages in chickens. The embryo passaged strain was immunogenic and a single dose of oocysts gave substantial protection against challenge with the pathogenic strain 14-42 days later. Histological studies confirm earlier studies that the loss of pathogenicity accompanying repeated embryo passage resulted in the gradual disappearance of large second generation schizonts. It is also possible that the large sub-epithelial second generation schizonts gradually change to a smaller type as they develop in epithelial, as opposed to sub-epithelial cells.

Eimeria tenella: Reproduction, pathogenicity and immunogenicity of a strain maintained in chick embryos by serial passage

Techniques were developed for the recovery and sporulation of oocysts from embryos, and a strain of Eimeria tenella was successfully passaged through 42 batches of embryos. This was done after the poor yields of oocysts at the 5th and 9th passage had been built up by passaging the strain in groups of chickens. After 28 embryo passages the strain appeared to have a slightly lower pathogenicity for embryos and perhaps also to chickens, but after 37 to 42 passages it was substantially less pathogenic for both chickens and embryos without a corresponding loss of immunogenicity for chickens. Pathogenicity appeared to be largely regained during the second passage of the “adapted” strain in chickens.

Plaque morphology of some South American strains of foot-and-mouth disease virus and the effects of polyionic compounds on plaque formation

The plaque forming ability of attenuated and field strains of FMDV of types A and O Vallee and C Waldmann were compared in BHK cell cultures. It was found that the vaccine strains attenuated by chick embryo passage produced smaller plaques. Reactor viruses recovered from vaccinated animals generally produced larger plaques than the vaccine and field strains. Type O vaccine, reactor and field viruses produced plaques of less variation in size and morphology. Field viruses of types A and C produced non-clear plaques which are readily distinguishable from the corresponding vaccine and reactor viruses.

The culture of dysentery bacillus in chick embryo

Shigella dysenteriae (type Shiga) has been cultured in chick embryo using Tanaka's method in which a hen's egg is settled in a longitudinal, standing position, that is, with its dull point up and its sharp one down.After several chick embryo passage uses the virulence of Shigella dysenteriae increased in the chick embryo, and then it increased in mice.It has been demonstrated that after several passage uses in certain species of animals, virulence increased in other species of animals as well as the passage animal.The above-mentioned fact explains the mechanism of some epidemic infections.

The inhibitor-destroying activity of influenza A virus strains of differing neurovirulence for mice.

Most strains of influenza A virus contain sialidases which are distinct from those of the host cell (Laver, 1963; Kelly & Grieff, 1965) and are presumed to be coded by the virus genome. However, the neurotropic variant nws, derived from the A0 strain ws (Stuart-Harris, 1939), did not elute readily after adsorption to red blood cells, or destroy the haemagglutination-inhibiting (HI) activity which various sialo-mucoids exhibit against sensitive test strains of influenza virus (Burnet & Lind, 1951). In contrast, the parent ws strain was active in both these tests. A decrease in viral sialidase activity may thus be a genetic marker or determinant of neurovirulence. On the other hand, these two viral characteristics could have arisen separately during the long series of chick embryo and animal passages by which nws was selected from ws, and have merely been accidentally associated in early allantoic fluid pools of this strain.

Tetratrichomonas gallinarum in the blood of chickens infected with spirochetes.

SYNOPSIS. Tetratrichomonas gallinarum was found in the blood of 3 chickens experimentally infected with Borrelia anserina. The blood infection was successfully reproduced in healthy chickens and was accompanied by evidence that the trichomonads multiplied in the blood, ingested red blood cells, and were themselves phagocytized by leucocytes. T. gallinarum was maintained in serial passage in chicken embryos as well as in the usual trichomonad culture media.

The Effect of Scald - Water Temperature on the Histologic Appearance of Chicken Skin

1. Adler, H. E. A comparison of some characteristics of Mycoplasma var. mycoides and Mycoplasma gallisepticum. Am. J. Vet. Res. 25, 243-245, 1964. 2. Hofstad, M. S., and L. Doerr. A chicken meat infusion medium enriched with avian serum for cultivation of an avian pleuropneumonia-like organism, Mycoplasma gallinarum. Cornell Vet.) 46 (3), 439-446. 3. Jungherr, E. L., R. E. Luginbuhl, and R. E. Jacobs. Pathology and serology of air sac infection. Proc. Book AVMA, 303-311, 1953. 4. United States Department of Agriculture, Agricultural Research Service. Standard methods for testing avian sera for the presence of Mycoplasma galliselpticum antibodies. Pamphlet. 5. van Herick, E. L., and M. D. Eaton, An unidentified pleuropneumonialike organism isolated during passage in chick embryos. J. Bact. 50, 47-55, 1945.

A Chicken-Embryo-Lethal Strain of Duck Hepatitis Virus

The mortality of chicken embryos inoculated with field strains of duck hepatitis virus (DHV) has varied (5,9) and remained irregular after 35 passages in chicken embryos (2). Nevertheless, the tendency of increasing embryo mortality through serial passages of DHV in chicken embryos has been demonstrated (1,6,8). An English strain of DHV, TN90, which was the chicken embryo 90th-passage virus, has been reported to kill most 10-day-old embryos within 48 hours (1). One other DHV strain of chicken embryo origin that usually killed 7-day-old embryos in 2 to 4 days when diluted 106-fold was also reported (7), but the history of this strain was not available. Nevertheless, it is obvious that both were highly embryo-lethal laboratory strains. Because of the low mortality rate and irregular mortality 417

Nonpathogenic Rickettsias Related to the Spotted Fever Group Isolated from Ticks, Dermacentor Variabilis and Dermacentor Andersoni from Eastern Montana

Summary Forty strains of a rickettsia, referred to as the EM rickettsia, which was avirulent for guinea pigs and which retained this property after serial passage in chick embryos were isolated from Dermacentor variabilis collected in two counties of eastern Montana. The EM rickettsia possessed properties common to agents of the spotted fever group. However, although it appeared to be a variant of Rickettsia rickettsii, it could not be identified with any of the four previously distinguished type strains of this species. The EM rickettsia was also isolated from Dermacentor andersoni collected in one of these localities. EM isolates were compared with an avirulent U strain of R. rickettsii which had been recovered from D. andersoni from the Bitterroot Valley, Montana. They differed primarily in their respective capacities to produce cross-reacting antitoxin against the virulent R strain of R. rickettsii. Guinea pigs injected with 104 doses of EM rickettsias developed high titers of CF antibody but little, if any, antitoxin, against the R strain. In contrast, the U strain produced high titers of both antitoxin and CF antibody for the R strain. Guinea pig hyperimmune serum against the EM rickettsia neutralized toxins of three strains of R. rickettsii representing the R, S, and T types, respectively, but did not neutralize toxins of Rickettsia conori, Rickettsia siberica, or the rickettsia from the tick Amblyomma maculatum. A high rate of infection with EM strains was found in the tick populations examined. This rickettsia was recovered from about 6 to 8% of D. variabilis from each of five localities studied. With one exception all strains isolated in eastern Montana were of the EM type. One isolate which possessed the characteristics of strains carried by the tick Haemaphysalis leporis palustris was isolated from a lot of ten D. variabilis from the eastern Montana area. The T strain commonly isolated from D. variabilis from eastern United States was not encountered in the study area.

A new living vaccine against fowl plague disease.

IN a previous investigation1 three attenuated substrains from a pea fowl strain were obtained by serial passages mainly in pigeon embryos. One of these substrains (PEC2 P11 C30)—that is, pea fowl strain subjected to 2 passages in chick-embryos, 11 in pigeon-embryos and 30 in chick-embryos successively—was subjected for further attenuation to more than 400 passages in pigeon embryos using either the intra-allantoic or the intra-yolk route of inoculation. During these passages 33 desiccated batches were prepared from the pigeon embryos. These batches were titrated in chick-embryos, fowls, and afterwards tested for safety and potency in laboratory and field experiments.

A host-induced modification of hemagglutinating virus of Japan (HVJ, Sendai virus) in its hemolytic and cytopathic activity

Host-induced changes of HVJ in hemolytic activity and in growth characteristics in mouse lung cells were investigated. HVJ grown in the allantoic cavity of the chick embryo is hemolytic, but the virus loses this property after a single passage in the mouse lung or after a single growth cycle in cultivated mouse lung cells. This nonhemolytic virus becomes hemolytic after a single chick embryo passage. Similar changes in virus growth characteristics were also observed. When HVJ was adapted to growth in monolayers of mouse lung cells, virus was produced within 24 hours of infection, but cytopathic effects were delayed, often for several days. After one passage in the chick embryo, however, this virus reproduced in the tissue culture with concomitant destruction of the cells. After a single passage in tissue culture the cytopathic virus returned to the noncytopathic condition.

The Effects of Rapid Embryo Passage upon the Infectious Bronchitis virus

T HAS been well established that the virus of infectious bronchitis (IB) of chickens changes its immunogenic properties upon repeated embryo cultivation.5'89 It is equally well known that the Beaudette strain 42 of virus, when propagated in embryonating chicken eggs, has been prone to develop titers of 10-5-0 to 10-7.0, which makes end-point titrations of immune sera impossible without resorting to a diluted serum method. This investigation was conducted to determine whether rapid embryo passage [i.e., passage of the infectious bronchitis virus (IBV) at the peak of the logarithmic phase of growth] would change the characteristics of a virulent strain of virus in regard to virus concentration, embryo mortality rate, and pathogenicity for the host. In addition, it was expected that rapid embryo passage of the Beaudette strain 42 of IBV would result in an increase of the EID0o. IBV was first reported by Beaudette and Hudson' to be readily propagated in embryonating chicken eggs. Continued serial passages of the virus in embryos increased the virulence of the virus for embryos and was accompanied by a decrease in virulence for chickens.

Experimental Infections of Wild Birds with the Viruses of Eastern Equine Encephalitis, Newcastle Disease and Vesicular Stomatitis

Received for publication April 7, 1959. * On leave from the Central Institute of Hygiene, Zagreb, Yugoslavia. Paper NS 270 from the Department of Veterinary Science, University of Wisconsin, Madison, published with the approval of the director of the Wisconsin Agricultural Experiment Station_ sin. The crows, hawks and owl were captured in southern Wisconsin by personnel of the Wisconsin Conservation Department. All birds appeared healthy at the beginning of the experiments. The eastern equine encephalitis virus (EEEV) used was originally isolated in 1956 from the brain of a horse which died during an encephalitis epizootic in southeastern Georgia. Allantoic fluids from the thirteenth and twenty-third serial passages in chicken embryos were used in these experiments. The vesicular stomatitis virus (VSV) used was the Wisconsin strain (New Jersey serotype) isolated from a cow in 1949 and transmitted serially in chicken embryos. The Newcastle disease virus (NDV) was Texas GB strain isolated from an outbreak of severe nervous-respiratory disease in chickens in 1948 and maintained by serial passage in chicken embryos. The routes of viral exposure used in the experiments were oral, subcutaneous, intracardial and intracerebral. Dosage varied according to the route of exposure employed and the estimated susceptibility of the individual bird. Birds were visually examined at least once daily after viral exposure to record clinical signs of illness. Body temperatures were not measured, since it was felt that the excitement produced by handling would greatly influence thermometer readings. Blood was drawn on alternate days after infection and examined for evidence of viremia. Fecal samples obtained by cloacal washing were collected daily from crows exposed to the viruses by ingestion of infected chicken embryos. Immune response was judged on the basis of virus neutralization (VN) tests on paired pre-exposure and convalescent serums. Convalescent serums were usually collected on the fourteenth day post exposure. Neutralization indices of 50 or higher in convalescent serums were considered indicative of infection and in birds which possessed no antibody before experimental exposure, this was recorded as a positive immune response. Most of the birds which did not die as a result of the infections were killed by bleeding on the fourteenth day post exposure and tissues, usually brain, lung, liver and spleen, were examined for histopathology and viral content. Tissues were prepared for histopathological examination by

Characteristics of the Asian strain of influenza A.

SummaryThe amniotic sac of the chick embryo was much more effective than monkey kidney cultures for isolation of the Asian strain of influenza A, and virus was obtained from at least 60% of serologically diagnosed illnesses. Both monkey kidney culture and chick embryo propagated virus agglutinated human, fowl, sheep and monkey erythrocytes at 4° or 24°C, but titers were lower with sheep red blood cells. Asian virus multiplied readily in monkey kidney cultures, but several passages were required for adaptation to the allantoic sac. Some strains during early egg passages were not inhibited by non-specific substances in normal sera, and some were initially non-reactive with antibody, especially in rde treated sera. Increased sensitivity to inhibitors and antibody occurred with additional chick embryo passages, but remained lesser than the tissue culture line. Asian virus propagated in monkey kidney cultures was preferable to the egg line for determinations of hemagglutination-inhibition and neutralizing anti...

Avian Vibrionic Hepatitis

TN DECEMBER 1955, a vibrio was isolated from the liver of an adult White Leghorn. The history of the flock revealed that production had been 15 to 20 percent below the expected level for several months. Also there was an increasing number of cull birds manifesting pale, dry and shrunken combs. On necropsy, the surface of the liver was found to be studded with hemorrhages varying from two to three millimeters in size. The ovary had regressed and only a small cluster of pea size ova remained. The kidneys were pale and slightly enlarged. A small block of liver tissue approximately 0.5 cm. square was aseptically collected, suspended in about five times its volume of nutrient broth and triturated. Four, six-day embryos were inoculated into the yolk sac with 0.2 ml liver suspension. All the embryos died the second day. Yolk smears from these embryos, stained with Gram stain revealed Gram negative vibrios. Yolk from subsequent embryo passages was lyophilized. Propagation of the organism was accomplished by inoculating a yolk suspension of the vibrio on to chicken infusion agar and incubating under 10 percent carbon dioxide tension. Growth was obtained more readily with yolk than allantoic fluid. At this time, no relationship was suspected between the hepatitis agent isolated by Delaplane