JAPANESE QUAIL EGG EMBRYO AS A HOST FOR VIRUSES

JAPANESE QUAIL EGG EMBRYO AS A HOST FOR VIRUSES FRANK J. RAUSCHER, JAMES A. REYNIERS, AND MIRIAM R. SACKSTEDER Laboratory of Viral Oncology, National Cancer Institute, U.S. Public Health Service, Bethesda, Maryland, and Germfree Life Research Center, T'ampa, Florida

Received for publication June 23, 1962

MATERIALS AND METHODS

The search for a small species of bird with a rapid breeding cycle, for prolonged maintenance and study in germ-free isolators, was prompted by numerous disadvantages encountered in using standard-breed chickens. The ability to house and care for only 4 to 6 adult leghorns or 8 to 12 bantam chickens in a single isolator (RSU-500), and their relatively long generation time (ca. 270 days), seriously hampered studies of tumorigenesis induced by carcinogens and viruses under germ-free conditions. Use of the Japanese quail (Coturnix coturnix japonica, Temminck and Schlegel) (Peters, 1934; Taka-Tsukasa, 1935; Moreau, 1951) as a laboratory research animal was suggested by Padgett and Ivey (1959) because of "its hardiness, ease of handling, precociousness, and great egg laying ability." The general techniques for

Eggs were collected daily from a laying flock maintained at the Germfree Life Research Center and stored at 23 C prior to weekly shipments to the National Cancer Institute. The eggs averaged 8.5 g in weight. The major and minor axes averaged 3 and 2 cm, respectively. They were speckled in shades of brown or blue, as a result of a thin membrane deposited on them as they were layed (Fig. 1). For chorioallantoic membrane and intravenous routes of inoculation, it was found desirable to remove the colored membrane to facilitate candling. This was done as soon as the eggs were delivered to the laboratory, by rubbing each egg on a slightly moistened steel-wool soap pad. The pigment was easily removed from eggs prior to incubation, but considerably more effort was required in de-

1134

Downloaded from http://jb.asm.org/ on December 16, 2017 by guest

ABSTRACT incubating, hatching, and raising under laboraRAUSCHER, FRANK J. (National Cancer Insti- tory conditions are described in their paper. tute, Bethesda, Md.), JAMES A. REYNIERS, These observations were confirmed and exAND MIRIAM R. SACKSTEDER. Japanese quail tended by Reyniers and Sacksteder (1960). egg embryo as a host for viruses. J. Bacteriol. The latter investigators reported the following 84:1134-1139. 1962.-The egg embryo of Japa- attributes of this host as being advantageous nese quail (Coturnix coturnix japonica, Tem- for maintenance and study under conventional minck and Schlegel) was found to support and germ-free conditions: (i) the hens produced readily the growth of a wide range of viruses fertile eggs as early as 40 days of age, the average known to infect chicken eggs. Methods com- generation time from egg to egg being approximonly used in studying viruses in chicken eggs mately 80 to 90 days; (ii) the hens continued to were modified and adapted to the quail egg. The produce about one egg a day during the entire p)rocedures are described in detail. The sensitiv- year, with an average fertility rate of 60 to 80%; ity of quail eggs to representative viruses of the (iii) average weight of the adult bird was 95 to myxo and pox groups was found to be equal to 110 g, allowing 40 to 50 to be maintained in one that of chicken eggs. Preliminary observations germ-free isolator (RSU-500); and (iv) the newly of the different responses of quail eggs to stand- hatched and the adult birds were highly susard chicken tumor and to quail-adapted Rous ceptible to the oneogenic actions of methylsarcoma virus are described. Viruses known not cholanthrene and of Rous sarcoma virus (RSV). to grow or induce an observable response in Studies of Reyniers and Sacksteder (1960) on chicken eggs (e.g., the agents of mouse hepa- the response of hatched Coturnix to RSV indititis, murine leukemias, and visceral lympho- cated the desirability of an evaluation of the matosis of chickens) also failed to produce such egg embryo as a host for this virus. The present study was carried out for this purpose. responses in quail eggs.

Vl)L. 84, 1962

VIRUS GROWTH IN JAPANESE QUAIL EMBRYO

pigmenting eggs previously incubated for 2 or more days. All eggs were placed in special commercially available egg flats (Georgia Quail Farm, Savannah, Ga.). The flats measure 6 by 11.5 by 1.25 in. and hold 50 eggs. Thus, 200 eggs may be held in approximately the same space occupied by the standard 11.5 by 11.5 by 2 in. flat which holds 30 chicken eggs. The eggs were incubated at 37 to 38 C in the upright position. Relative humidity was maintained at approximately 45 to 55%. The eggs were not turned unless it was planned to allow the chicks to hatch. Previous trials on duplicate lots of the same batch of eggs showed that stationary incubation did not appreciably affect viability of embryos. From October 1959 to September 1961, approximately 9,900 embryos were incubated for experimentation. Of these, 69% were found to be fertile, 76% showed viability of the embryos for at least 7 days of incubation, and a "nonspecific" mortality of 14% was observed within 12 hr after inoculation by various routes. The eggs were candled on the 5th to 7th day of incubation by means of a microscope illuminator (American Optical Co., catalog no. 353), over which was fitted an appropriate-sized black plastic bottle cap in the center of which a 5A-in. diam hole had been drilled. This served as a light-tight seat for the end of the egg and greatly facilitated the ease and efficiency of candling. Live embryos were returned to the incubator, or were marked and small holes were drilled by means of a flexible shaft motor to which was attached a size 6 steel burr. The standard-type egg punch was also tried,

but because of the small size of the egg and of the greater fragility of its shell this procedure was less satisfactory. The holes were drilled large enough to admit a 26-gauge hypodermic needle. The shell membrane of Coturnix eggs was found to be more resistant to puncture than chicken eggs and to adhere more firmly to the chorioallantoic membrane. Except for minor modifications, the techniques of inoculation and of subsequent fluid or tissue harvest were essentially the same as those used with chicken eggs. They are summarized below. Allantoic sac (AS). Embryos (7 or 8 days old) were inoculated with 0.1 ml of inoculum by means of a 27-guage, 5A-in. hypodermic needle attached to a 1-ml tuberculin syringe. The inoculation was made through a hole drilled in the shell approximately }46 in. above the beginning of the air sac, at a point where the chorioallantoic membrane was seen to be relatively free of large blood vessels. The needle was inserted approximately }E in. into the egg. The holes were sealed with flexible collodion to which dye had been added. The color allowed subsequent relocation of the point of inoculation through the pigmented shell and facilitated visual confirmation of a properly sealed egg. Mortality due to trauma of inoculation was less than 2%. Depending on the virus inoculated, surviving embryos were killed by chilling at 4 C for 3 to 4 hr, 2 to 6 days after inoculation. Embryos inoculated with influenza virus were sacrificed 40 to 48 hr after inoculation. Allantoic fluids were harvested by removing the shell from the blunt end of the egg and aspirating the fluids by means of a disposable Pasteur pipette. Yields of allantoic fluids from chilled, infected eggs ranged from 0.7 to 1.6 ml. Yolk sac (YS). Inocula of 0.1 ml were introduced into the yolk sacs of 5- or 6-day-old embryos by means of a 26-guage, 1-in. hypodermic needle inserted to a depth of approximately 34 in. through a hole drilled in the center of the blunt end of the egg. The eggs were sealed and the embryos prepared for harvesting in a manner identical to that described above. The number of embryos which died within 12 hr after inoculation, owing to trauma, averaged about 15%. Embryos were chilled and examined 7 days after inoculation (i.e., the 12th to 13th day of incubation), at which time both the membrane and


FIG. 1. Size and coloration of Coturnix eggs in relation to a white leghorn chicken egg. T'he quail egg shown on the right was partially depigmented by gentle buffing with a steel-wool soap pad.

I1135'

RAUSCHER, REYNIERS, AND) SACKSTEI)ER

1136

the yolk

were

examined for lesions

or

tested for

infective virus.

to trauma of inoculation varied between 15 and

30'%. Surviving embryos were sacrificed 4 to 5 days after inoculation. From 0.1 to 0.8 ml of amniotic fluid could be harvested from individual eggs. Pools of allantoic fluids from eggs inoculated with mumps virus were found to be uniformly as infectious as pools of amniotic fluid. Consequently, both allantoic and aminiotic fluids were harvested and pooled for subsequent titration or serial passage. Chorioallantoic membrane (CAM). The false air sac method of preparing the CAM for inoculation was used. The CAM of 6- to 11-day-old embryos can be "dropped" with relative ease. The 7-day-old embryo was chosen as the standard host for this route, because of a slightly greater efficiency in dropping the membrane and to allow as many observation days as possible before hatching. The shell pigment was removed from an area on the side of the egg to allow positive identification of a "dropped CAM" on candling. Holes were drilled through the shell both at the center of the blunt end and on the depigmented side at a site relatively free of large blood vessels. The eggs were placed in a horizontal position, swabbed lightly with 95c%, ethyl alcohol, and the shell membrane was pierced through the hole at the blunt end. A drop of warm (38 C) sterile saline was placed on the side hole for lubricating the membrane as it dropped, and the underlying shell membrane was carefully pierced. The false air sac was then created by gentle aspiration through the hole at the blunt end. The latter procedure was performed while candling. Inocula of 0.05 ml were tolerated by the eggs with little or no leakage. Both holes were sealed with flexible

collodion, and the eggs were returned to the upright position prior to continued incubation. An average of 12% of the embryos of inoculated eggs died "nonspecifically" within 12 hr. Surviving embryos were chilled and examined 7 days after inoculation. Intravenous. Embryos (9 days old), the shells of which had previously been depigmented, were candled to determine the position of a large stationary blood vessel. A triangular opening was made through the shell immediately over the vessel by means of a carborundum wheel attached to the flexible shaft drill. The shell membrane was made translucent with a drop of warm (38 C) sterile mineral oil. Inocula of 0.025 to 0.05 ml were introduced into the veins by means of a 30-gauge, 54-in. hypodermic needle attached to a Y4-ml tuberculin syringe. The inoculation was done under a fluorescent magnifying lamp. Embryos exhibiting excessive bleeding after withdrawal of the needle were discarded. The triangular shell openings were sealed with warm alcohol-sterilized glass cover slips, using liquified petrolatum and melted paraffin (1:4) as adhesive. "Nonspecific" deaths within 12 hr of inoculation ranged between 5 and 18%. Surviving embryos were either chilled and examined on the 17th day of incubation or were allowed to hatch for further observation and subsequent necropsy. Intravenous inoculation was found to be a feasible and useful technique with Coturnix embryos. However, it should be noted that up to 50% of the embryos had to be discarded because of inaccurate drilling and inoculation, immediate hemorrhaging, or subsequent "nonspecific" mortality. Serial passage. A 10-s dilution of virus was used for the first passage into Coturnix eggs of those agents known to grow well in chicken eggs. A serial passage line of mumps virus was initiated with undiluted, infected chicken-egg allantoic fluid. Pools of fluids or tissues from individual eggs, demonstrated by means of hemagglutination (HA), death, or pock formation to have been infected, were used for the first three passages of each virus. Thereafter, fluids or tissues from embryos were pooled without testing each egg contributing to the pool. The first passage into Coturnix eggs of those agents not known to grow in chicken eggs was initiated by inoculating dilutions 10-1 and 10-4


Amniotic sac (AmS). Embryos (7 days old) received 0.05-ml inocula by means of a 27-gauge, yg-in. hypodermic needle. Before inoculation, the eggs were buffed and the pigment removed in the general area to he drilled. The eggs were candled on the day of inoculation, and a mark for drilling was made over the location of the embryo eye. The inoculation was made during candling after jabbing the embryo with the needle point. Previous test inoculations with dye showed that in about 85% of eggs the inocula were deposited within the amniotic sac. Mortality due

J. BACTERIOL.

VOL.


84, 1962

1137

TABLE 1. Summary of attempts to passage different viruses in Japanese quail eggs Obvious growth or No. of serial growth Obvious response in eggs passages in

~~~~~~~~Log

or

Virus

Chicken

Influenza Influenza Influenza Influenza

A (PR8) ................ B (Lee) ................. C ....................... D (Sendai) ...... .......

Mumps (Habel) ...................

Lymphomatosis (RPL12).......... Mouse hepatitis (Manaker) ....... Murine leukemia (Moloney)....... Mouse leukemia (Schwartz) ....... Murine leukemia (Rauscher) ......

+ + + + + -

+ + + +

+ + +

+ + +

+ + -

-

-

-

~~~~~quail

16 17 10 10 4 17 16 12 6 4 3 1 2 6 6 6 8

AS AS AS AS

8.86 8.26

10.13 8.26 3.42 8.99 5.65 8.05 5.31 9.63 9.10 7.42

AMS, AS AS CAM CAM CAM CAM CAM CAM

AS, AS, AS, AS, AS,

titer per ml of highest passage quail material in eggs Quail Chicken

CAM, CAM, CAM, CAM,

8.98 7.55 9.30 7.55 4.61 9.01 5.74 8.21 5.60 9.40 9.00 7.36

iv

YS, iv YS, iv YS, iv

CAM

of each virus into separate groups of eggs. ring on the CAM's of 8 to 10 eggs was added to Equal amounts of fluids or tissue extracts were the log dilution of the inoculum. The resulting pooled from both groups of eggs. Such pools titers were expressed in terms of 1.0 ml. Death were likewise diluted 10-1 and 10-4 for subse- was used as the index of infection for those emquent passages and inoculated into separate bryos inoculated with the virus of vesicular groups of eggs. Three to six blind passages were stomatitis and infectious avian bronchitis. The performed for each of these viruses by each log LD50 values were computed by the method of Reed and Muench (1938), and virus titers route of inoculation listed in Table 1. Infectivity titrations. Depending on the virus were expressed in terms of log LD50 units per 1.0 being titrated and on the number of eggs avail- ml. able, 8 to 12 eggs were used in each virus dilution RESULTS group. Allantoic fluids from eggs previously eggs to 17 different of Coturnix The response inoculated with the viruses of influenza, mumps, in 1. It will be Table viruses is summarized for tested were using disease HA, or Newcastle Coturnix or chicken erythrocytes or both. seen that this host readily supported the growth Blood derived by heart puncture was pooled of the viruses of influenza A, influenza B, infrom at least three birds, and the red cells were fluenza C, influenza D, Newcastle disease, sedimented and washed three times in 0.85% mumps, vaccinia, laryngotracheitis, fowl pox, NaCl. The final concentration of cells employed vesicular stomatitis, and infectious avian bronwas 1%. HA tests for infectivity and for hemag- chitis. The dilution end points of these viruses glutinin titer of Newcastle disease virus were inoculated into quail eggs, and the amounts of done at 4 C. All other HA tests were carried recoverable infectivity, were essentially the out at room temperature. Infectivity tests were same as those following infection of chicken done in conventional depression plates, and eggs. Table 2 presents a quantitative comhemagglutinin titers were performed in Kahn parison of various biological characteristics of three myxoviruses before and after 16 serial tubes. in Coturnix embryos. Serial passage passages and The viruses of Rous sarcoma, fowl pox, alter or modify those virus did not demonstrably The follows. as titrated were laryngotracheitis log of the average number of gross lesions occur- properties compared.


Newcastle disease (Blacksburg). Laryngotracheitis .......... ....... Fowl pox ......................... Infectious bronchitis .............. Vaccinia .......................... Vesicular stomatitis .............. Rous sarcoma (Bryan) ............

+ + + + + + +

Quail

eggs

Route of inoculation

1138

RAUSCHER, REYNIERS, AND SACKSTEDER

J. BACTERIOL.

TABLE 2. Comparison of various characteristics of three myxoviruses before and after serial passage through quail eggs Characteristic

Bb___

_

Chicken Quail Chicken RBC Quail RBC Mice Mice Mice

Influenza A (PR8)

PR8__

Influenza B (Lee) (Lee)

Oa

Q16a

0

Q16

0

Q16

9.02 9.14

9.13 8.94

9.26 8.94

8.86 8.98

8.43 8.67

7.68 7.34

1,024 1,024 1,024 2,048 512 512 1,024 512 2,048 1:4.6 1:4.1 1:5.3

512 512 1:5.0

1,024 6.65 3.01

6.82 2.78

-

-

-

-

-

a Refers to the number of serial passages the virus had undergone in quail eggs. Reciprocal of virus dilution in which hemagglutination was complete. Arithmetic dilution of virus which, after intracerebral inoculation, caused 50% of the mice to convulse when suspended by their tails and twirled. d Log dilution of virus which, after intranasal instillation, caused a minimal average of 25% lung consolidation in 50% of recipient mice.

Inoculation of RSV onto the CAM of Coturnix eggs resulted in the production of lesions typical for this virus. Repeated dual titrations of standard chicken tumor virus in both chicken and Coturnix eggs were found to yield virtually identical end points. The RSV-quail egg system, however, was found to differ in several respects from that of chicken eggs. Coturnix embryos failed to support serial passage of the standard virus. Twelve attempts to transmit RS V for more than three serial passages were unsuccessful. This was true regardless of the age of the embryo at the time of infection, or of the amount of virus used to initiate each passage series. Conversely, the virus was easily propagated through ten serial passages on two separate occasions, in conventional or germ-free adult quail. Dual infectivity titrations of tumor tissue from each passage in both strains of embryos showed 5 to 7 log pock-forming units of extractable virus for each host. Extracts of tumors from the 10th serial passage of RSV in adult quail were used to initiate a series of passages in Coturnix eggs. Unexpectedly, this line could be propagated serially through 12 passages in quail eggs. Such passage was accompanied by a gradual increase in infective titer when assayed in quail eggs, with a concomitant gradual loss of infectivity for chicken eggs until the 5th passage, when extracts of infected quail CAM were no longer infective for chicken eggs. This quail egg-adapted line of RSV is being characterized as to its anti-

genicity, host range, and sensitivity to antiserum prepared against standard chicken tumor RSV in studies now in progress. Lastly, the data in Table 1 show that viruses not known to induce an observable response in chicken eggs (e.g., mouse hepatitis, murine leukemia, visceral lymphomatosis of chickens) also did not produce such responses in Coturnix eggs. DISCUSSION

The findings that embryos of Japanese quail may be manipulated in a manner similar to that of chicken eggs, and that they are susceptible to at least 12 different viruses, show this host to be suitable for virus studies. The relatively small size of the egg, as well as of the hatched quail, the ease of maintenance of the quail, and their rapid generation time make them more suitable as an avian host in studies requiring long periods of observation in parent or progeny (e.g., vertical transmission studies). The sensitivity and uniformity of the individual egg responses to those viruses tested (e.g., RSV and influenza A virus) have been shown to equal or exceed that of chicken eggs. Individual eggs from three different flocks of quail collected throughout the year were shown to have an average variation of less than twofold in terms of pocks on the CAM or in terms of ID5o values in allantoic fluid. Less than 5% of Coturnix embryos were found to be totally resistant to in-


Infectivity (log ID50) Infectivity (log 1D50) Hemagglutinationb Hemagglutinationb Neurotoxicity (TCD50)c Neuropathic effect (LD 50) Pneumonia (ED50)d

Newcastle disease (Rb)

Host

VOL. 84, 1962


the Germfree Life Research Center (Reyniers and Sacksteder, 1960). LITERATURE CITED MOREAU, R. E. 1951. The British status of the quail and some problems of its biology. Brit. Birds 44:257. PADGETT, C. A., AND W. D. IVEY. 1959. Coturnix quail as a laboratory research animal. Science 129:267-268. PETERS, J. L. 1934. Check-list of birds of the world. Harvard University Press, Cambridge, Mass. REED, L. J., AND H. MUENCH. 1938. A simple method of estimating 50% end points. Am. J. Hyg. 27:493-497. REYNIERS, J. A., AND M. R. SACKSTEDER. 1960. Raising Japanese quail under germfree and conventional conditions and their use in cancer research. J. Natl. Cancer Inst. 24:14051416. TAKA-TsUKAsA, P. 1935. The birds of Nippon, vol. 1, p. 204-238. Witherby, London.


fection by RSV. Conversely, numerous reports by other investigators have shown that 10 to 15% of chicken eggs from most commercial flocks are completely refractory to strong doses of this virus. Whether Japanese quail are susceptible to some viruses of the avian leukosis complex has not been unqualifiedly determined. Studies are in progress to elucidate this important consideration. Fertile Coturnix eggs are available at present for those investigators desirous of starting and maintaining a laying flock (Germfree Life Research Center, Tampa, Fla.). At least three commercial suppliers of laboratory chicks and eggs have begun developing laying flocks for subsequent supply embryos of various age (Shammrock Farms, East Brunswick, N.J.; Truslow Chicks, Chestertown, Md.; Westbrook Game Farms, Brandon, Fla.). All of these production flocks were begun with the T-2 laboratory line of Japanese quail developed at

1139