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Characterization of the Infectious Bursal Agent
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J. R. MORRIS, F. N. JEROME AND B. S. REINHART the chicken. J. Nat. Cancer Inst. 40: 583-592. Solomon, J. B., 1961a. The onset and maturation of the graft versus host reaction in chickens. J. Embryol. Exp. Morph. 9: 355-369. Solomon, J. B., 1962. A sex difference in the splenomegaly syndrome in chick embryos injected with adult spleen cells or blood. Exp. Cell Res. 28:151157. Solomon, J. B., and D. F. Tucker, 1963. Immunological attack by adult cells in the developing chick embryo. Influence of the vascularity of the host spleen on the homograft rejection by the embryo on splenomegaly. J. Embryol. Exp. Morph. 11:119-134.
Characterization of the Infectious Bursal Agent Y. CHO* AND S. A. EDGAR Department of Poultry Science, Auburn University, Auburn, Alabama, 36830 (Received for publication July 10, 1969)
G
UMBORO disease was first diagnosed as an infectious disease in the fall of 1957 by Cosgrove (1962). Later, two virus agents were isolated from birds believed to be suffering from the Gumboro disease syndrome, Winterfield et al. (1961). The first was isolated from kidneys of affected birds and it was designated the "Gray" agent that caused nephrosis but was believed to be a variant type of infectious bronchitis virus (IBV). The virus was of low virulence for the respiratory tract (Winterfield et ah, 1961; Winterfield and Hitchner, 1962; and Winterfield, 1963). A second agent was isolated later by the same research group from the bursa of affected birds and was designated infectious bursal agent (IBA). Thus, Winterfield et al. (1962)concluded that at least two different agents were involved in Gumboro disease.
The Winterfield group then differentiated the signs and gross pathology of birds infected by the two agents, namely "Gray" agent and IBA. With "Gray" agent infection, the main pathology was in the kidney and there was slight respiratory involvement, but the bursa was not affected. With IBA infection the bursa was the main organ affected. Pathology in the kidneys was secondary, and there was no respiratory involvement. Both agents caused muscle hemorrhages of various gradations (Winterfield et al., 1962). Edgar and Cho (1964) also reported isolation of a virus-like agent from affected birds and transmitted the agent successfully to susceptible 1- to 6-week-old chickens that caused a disease condition similar to that described by Cosgrove (1962), and by the IBA agent of Winterfield et al. (1962).
* In part from the thesis submitted in partial fulfillment of the requirements for the Doctor of Philosophy degree, Auburn University, August, 1967.
Since the isolation of IBA, little has been published on its characterization. Winterfield and Hitchner (1964) reported that the agent was filterable, which led to
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Ebert, J. D., 1959. The Cell, 1st edition. Vol. 1: 619-693. Academic Press, New York and London. Jaffe, W. P., 1965. The bursa of Fabricius and homograft immunity. Poultry Sci. 44: 1615-1616. Jaffe, W. P., and L. N. Payne, 1961. Graft against host reactions in inbred lines of chickens and their crosses. Brit. Poultry Sci. 2:133-143. Morris, J. R., A. E. Ferguson and F. N. Jerome, 1969. Selection for genetic resistance and susceptibility to Marek's disease (MD). Can. J. Ani. Sci. (In press). Purchase, H. G., R. C. Chubb and P. M. Biggs, 1968. Effect of lymphoid leukosis and Marek's disease on the immunological responsiveness of
INFECTIOUS BURSAL AGENT
the belief that it was probably a virus. Benton el al. (1967b) and Cho and Edgar (1968) reported on the size, chemical stability, thermostability, pH stability and ether and chloroform sensitivity of IBA. The purpose of this study was to characterize IBA and differentiate it from other disease producing agents. MATERIALS AND METHODS
The BL60 (infection that resulted in 50% with bursal lesions among inoculated chickens) of unpurified and purified IBA suspensions were determined by the method of Reed and Muench (1938), and was the standard for determining infectivity of all samples of the agent tested. All unfiltered bursal suspensions contained Escherichia coli and/or Proteus spp. Filtrates that passed through 0.3 ix.-membrane filters were subjected to bacteriologic tests by inoculating 0.5 ml. onto blood agar and desoxycholate agar plates to determine whether bacteria had passed through the filters. This procedure tested the integrity of the filters. Particle size of IBA was determined according to Hsuing (1965). Purified suspensions previously referred to were filtered through Gelman triacetate filters of the following pore sizes: 200, 100, and 50 mju. (millimicrons). A Swinny hypodermic adapter with a 10 ml. syringe attached to it was used for filtering instead of a suction funnel (Hsuing, 1965). Filtrates that passed 200, 100 and 50 nux. then were tested in susceptible chickens. Loss of activity was determined by comparing filtrates that passed through the above filters with that which passed through a 300 mp. filter. IBA that passed through a 300 m/i. filter found to be bacteria-free is hereafter referred to as purified. All tests were conducted with purified IBA. Thermostability of IBA was determined according to the method outlined by Quiroz and Hanson (1958). Sealed ampules of IBA were subjected to 60°C. in a water bath for 5,15, 30, or 90 minutes and tested for viability. Stability of IBA held at room temperature was determined by thawing samples and checking for viability at specific periods. One ml. of the suspension was withdrawn immediately and tested in sus-
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In preparation of IBA for study, SO, 5-week-old pathogen-free S. C. White Leghorns reared in isolation were each inoculated intraocularly with 0.05 ml. of a field isolate of IBA. The inoculated birds were sacrificed at 72 hours. The bursa of each inoculated bird was excised carefully and minced with an omnimixer for 5 minutes. This was then suspended in 0.85% sterile saline, 1 g. of tissue in 5 ml. saline. Aseptic techniques were employed throughout all experiments. IBA was freed of bacteria (purified) by centrifugating and filtering. It was freed from cell debris by low speed centrifugation (2,000 r.p.m. for 20 minutes). The supernatant was collected and passed through a No. 2 paper filter in a porcelain Buchner suction funnel. The suspension then was filtered through membrane filters of different porosities (Gelman triacetate filters, pore sizes 5 /*., 1.2 ju., 0.8 p., 0.45 p., 0.3 fi). Suspensions were filtered in an ice bath at 4°C. The preparations were tested for bacterial contamination before and after filtration. The relatively purified IBA suspensions then were pipetted, 1 ml. samples each, into small vials and quickly frozen in dry ice. Samples of the filtrate were tested for infectivity (titer), at 10-fold serial dilutions by intraocular inoculation in susceptible 4-week-old S. C. White Leghorn chickens. All birds were sacrificed at 72 hours after inoculation and examined for lesions of infectious bursal disease (IBD).
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Y. C H O AND S. A. EDGAR
.The sensitivity of I B A to ether was determined according to the technique of Andrews and Hortsman (1949) and Wallbank and Stubbs (1965). I B A suspensions were exposed to reagent grade diethyl ether (1 volume of ether to 4 volumes of IBA suspension) in small rubber-capped bottles. T h e content of each bottle then was mixed b y gentle shaking and allowing it to stand for 18 hours at 4°C. T h e ether was removed from the I B A b y evaporation in open petri dishes for 30 minutes. Ten-fold serial dilutions in sterile saline were tested.
Chloroform sensitivity of IBA was measured by the method of Wallbank and Stubb (1965), a modification of t h a t by Feldman and Wang (1961). Chloroform was added to an I B A suspension in a small flask with a screw cap (1 volume of chloroform to 4 volumes of IBA suspension). The mixture was agitated slowly on a magnetic stirrer for 10 minutes at 4 ° C , then centrifuged at 275 X gravity for 5 minutes and the chloroform was removed and discarded. The I B A then was diluted and tested. T h e stability of I B A exposed to 1% formalin, 1 % cresol or 1 % phenol was determined according to the method of Quiroz and Hanson (1958). Formalin,* cresol, and phenol** were diluted to 2 % solutions with sterile distilled water. One part of I B A suspension was combined with 1 p a r t of 2 % formalin, 2 % cresol, or 2 % phenol at room temperature (25° C.) for 1 hour. These treated suspensions of IBA were tested then for infectivity in the manner described earlier. Finally, the sensitivity of IBA to 5,000 units of penicillin-streptomycin per ml. for 60 minutes at 37°C. was determined. Ten-fold serial dilutions of this treated IBA were also tested. All,IB A suspensions were tested for infectivity by intraocular inoculation of 4week-old susceptible S. C. White Leghorns maintained in isolation t h a t were sacrificed and examined for lesions of IBA at 72 hours after inoculation. RESULTS AND DISCUSSION
Results of three experiments to determine the infectivity of unpurified versus purified, filtered, bacteria-free IBA sussuspensions are summarized in Table 1. There was 100% infection of test birds with undiluted or 10 _ 1 dilutions. How* Formalin—40% formaldehyde, U.S.P., Fisher. ** Cresol and phenol, U.S.P., J. T. Baker Chemical Co.
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ceptible chickens, and additional samples were tested at 24, 4 8 , 7 2 , 96, 120, and 144 hours and at 7, 14, and 21 days. Stability of I B A at - 2 0 ° C . was also determined. An original batch of I B A inoculum was made in J a n u a r y 1964 and the titer had been determined at t h a t time. I t then was stored at • — 20°C. I n January 1967 three random sample bottles were thawed, serial dilutions were prepared and titered, and the infectivity of the suspensions for the two periods of storage were compared. Two experiments were conducted to determine the stability of I B A at different p H ' s . A suspension was adjusted to a p H of 2 by combining equal volumes of undiluted IBA and 1 % HC1. A p H of 12 was obtained by combining equal volumes o£ undiluted IBA and 1%. N a O H . T h e pH-adjusted IBA suspensions were held at a room temperature of approximately 25°C. for.'60 minutes (Quiroz^nd Hanson, 1958; and Ketler et al, 1962), and then tested for infectivity. I n the first experiment on. p H stability undiluted IBA suspensions were tested. Serial dilutions of I B A were tested in the second experiment to determine whether a p H of 2 for 60 minutes would lower the infectivity titer of IBA. Untreated I B A suspensions (pH 7.2) of the respective dilutions served as controls.
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INFECTIOUS BURSAL AGENT TABLE 1.—A comparison of the infectivity of unpurified versus purified IBA suspensions Number of birds infected1 Unpurified IBA
Purified IBA
15 15 15 9 6 0
15 15 9 3 0 0
2
10° 10"1 10-2
10"3 10-" 10-5
ever, at 10~2 only 9 of 15 birds inoculated with the filtrate became infected compared with 15 of 15 inoculated with the original unfiltered preparation; none became infected with a 10"4 dilution of the purified IBA. Results of the calculations to determine the 50% bursal lesion value (BL50) by the Reed and Muench technique are presented in Table 2. The titer of unpurified IBA was 10 36 and that of the purified filtrate was 102-5. Thus, there was evidence of one log less titer in the purification
TABLE 2.—Calculations to determine 50% bursal lesion value (BL50) of IBA referred to in Table I
Treatments
Dilutions
Jnpurified IBA
ID"2 10- 3 10-" 10-5
Purified IBA
10"1 10-2
10- 3 10-4
Number birds Number birds Total number Total number birds birds with no with with no with lesions lesions lesions lesions 0 6 9 15 0 3 9 15
15 9 6 0 15 9 3 0
0 6 15 30 0 3 12 27
30 15 6 0 27 12 3 0
71 43-50 21.43 = Proportional distance of unpurified I B A = ^ „ = o< 0-5 /1.43—Zo.oi
4Z.O0
Bursal lesion 50 (BL6o) of unpurified IBA was 10"3-5The final titer was l0 3 5 = BL,6o per volume of inoculum. Proportional distance of purified IB A = ^ - 7 ^ = — = 0 . 5 oU—JA) OU
Bursal lesion 50 (BL60) of purified IBA was 10"2 5. The final titer was 102-6 BL50 of inoculum.
Percentage with lesions 100 71.43 28.57 0 100 80 20 0
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1 A total of 15 birds were inoculated with each dilution in three experiments. Test birds were susceptible 4-week-old S. C. White Leghorn males. 2 One part infected bursa to 4 parts of saline.
technique employed. Filtrates of purified IBA were inoculated (0.5 ml.) onto blood agar and desoxycholate agar plates and incubated at 37°C. for 48 hours. No bacteria were recovered, whereas E. coli and/or Proteus spp. were present in unfiltered bursal suspensions. IBA used in the remaining experiments was purified (bacteria-free) and will be referred to as IBA unless designated otherwise. Results of three experiments to determine particle size of IBA are summarized in Table 3. One hundred percent of susceptible 4-week-old S. C. White Leghorns became infected regardless of size of filter down to 50 mix. in diameter, the smallest pore tested. Thus, it was determined that the agent was less than 50 m/i. in size. Results of the three experiments to determine thermostability of IBA at 60°C. are summarized in Table 4. All 4-week-old chickens inoculated with a 10 _) dilution of IBA became infected after exposure of IBA to 60°C. in water bath for 90 minutes. The number of test chickens that became infected decreased as dilutions were increased. The BL50 of unexposed
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TABLE 3.—Summary of three experiments to determine the particle size of IBA Pore size of filters (mM.) Uninoculated controlsi 300 200 100 50
Number birds with bursal lesions/birds tested 0/15 15/15 15/15 15/15 15/15
Percentage showing bursal lesions 0 100 100 100 100
TABLE 5.—Viability of IBA exposed to room temperature (approximately 25°C.) Dilution of IBA inoculum • 10"
Number days exposure when IBA was tested 0
1
2
3
4
5
6
7
• 14 21
(No. birds that became infected)1 15 15 15 15 15 14 12 9 5
1
1
Five, 4-week-old S. C. White Leghorn males were tested per period in each of three experiments, making a total of 15 birds per test period.
I B A is quite stable a t room temperature. Results of one experiment involving tests of three samples of the same lot of original unpurified I B A isolate obtained IBA was 102-38 versus 102-24 for I B A ex- in J a n u a r y 1964 is presented in Table 6. posed to 60°C. for 90 minutes. T h e differ- After 3 years at — 2 0 ° C , t h e infectivity had n o t decreased appreciably, with a ence in titers was n o t significant. -313 as compared with 10~3-5 Results of three experiments to deter- BL6o of 1 0 mine t h e viability of I B A exposed to a determined 3 years earlier. Results of two experiments to deterroom temperature of approximately 25°C. for u p to 21 days are summarized in Table mine t h e stability of I B A a t different 5. Stock I B A (1:5 dilution) was tested for p H ' s are summarized in Tables 7 a n d 8. infectivity in three groups of five chickens I n t h e first experiment undiluted I B A at each of the periods shown, a total of 15 (1:5 in saline) h a d lost its infectivity foltest chickens for each period. I B A ex- lowing exposure to p H 12 for 60 minutes, posed to room temperature through 3 whereas it was still viable after exposure days resulted in 1 0 0 % infection. There to p H 2 for the same length of time, Table was some loss of activity b y t h e fourth 7. D a t a of a second experiment, in which day (13 of 15 positive) with a somewhat serial dilutions of I B A adjusted to p H 2 rapid decline in infectivity b y 7 and 14 for 60 minutes and compared with the days. Only 1 of 15 birds became infected original I B A ( p H 7.2), are summarized in with I B A exposed a t room temperature Table 8. There m a y have been some loss for 21 days. I t was concluded t h a t purified in viability of I B A upon exposure to p H TABLE 4.—Thermostability of ISA at 60°C.
Infectivity of Dilutions IBA not exposed to 60°C. 1
10-12 10~ 10-° 10-* 1
Infectivity of IBA exposed to 60CC. for (minutes):
T A B L E 6.—Stability of unfiltered IBA suspension after storage at — 20°C. for 3 years
Dilutions
15 30 45 60 90
(No. birds positive) (No. birds positive) 15 15 15 15 15 15 10 8 10 9 9 8 3 4 3 3 3 3 0 0 1 0 0 0
There were five S.C. White Leghorn males used for each dilution at each exposure period, making a total of 15 birds tested at each period.
10"2 10~3 10-4 10-6
Number birds that became infected when tested in:1 19642
19673
15 9 6 0
15 7 3 0
1 T h e r e were five, 4-week-old S.C. W h i t e L e g h o r n males tested w i t h each dilution, in each replicate. 2 BL 6 0 w a s 1 0 " . 3 BL 6 0 w a s 10 3 1 3 .
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1 Three experiments, each group contained five, 4-week-old S.C. White Leghorns. 2 Test birds were sacrificed and examined for IBD lesions at 72 hours after inoculation.
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INFECTIOUS BURSAL AGENT
TABLE 7.—Infectivity of IBA after exposure to a pH of 2 or a pH of 12 for 60 minutes
Treatments
Number birds that became infected, replicates:
Uninoculated Inoculated IBA pH 7.2 Inoculated IBA pH 2 Inoculated IBA pH 12
1
2
3
0 5 5 0
0 5 4 0
0 5 5 0
™ t, infect 0 15 14 0
1 There were five, 4-week-old S. C. White Leghorn males in each treatment of each replicate, examined for IBA lesions 72 hours after inoculation.
Results of an experiment to test t h e stability of I B A treated with certain other chemicals are summarized in Table 10. I B A treated for 60 minutes with 1 % TABLE 8.—Infectivity
of IBA exposed to
pH 2 for 60 minutes Dilutions tested 1 IO-1 10"2 10-'
io-<
IBA at pH7.22
IBA a t pH23
Number positive Number positive 15 15 10 7 3 2 0 0
1 There were 15, 4-week-old S.C. White Leghorn males inoculated with each dilution, 5 birds in each of 2three replicates. BL60 of untreated IBA was 102-88. 3 BL60 of IBA exposed to a pH of 2 was 10207.
Dilutions tested
Number of birds infected when inoculated with IBA:1 Untreated Chloroform3 control treated
Untreated control
Ether2 treated
io-' 10-2 10-s 10-4
15 7 2 0
14 7 1 0
15 8 3 0
15 6 1 0
BLra
1Q207
101->S
102-23
101-88
i Tested in five, 4-week-old S. C. White Leghorn males per dilution in each of three experiment. 2 Time of exposure was 18 hours at 4°C 3 Time of exposure was 10 minutes at 4°C.
formalin, 1 % cresol or 1 % phenol lost infectivity completely. Thus, i t was evident t h a t I B A is highly sensitive t o these compounds. T h e sensitivity of I B A t o penicillinstreptomycin was tested and results are summarized in Table 11. T h e infectivity of IBA-treated with penicillin-streptomycin for 60 minutes a t 37°C. was n o t decreased. Actually, a slightly higher percentage of t h e 15 chickens inoculated with penicillin-streptomycin-treated I B A became infected with 1 0 - 2 and 1 0 - 3 dilutions than became infected with respective dilutions of untreated IBA. Benton et al. (1967b) found t h a t I B A was n o t susceptible t o 0 . 5 % phenol in 1 TABLE 10.—Sensitivity of IBA to
three chemical compounds Number of birds infected, replicates:2 '_ 1 2 3 Uninoculated 0 0 0 IBA-untreated 5 5 5 1% formalin-treated IBA 0 0 0 1% cresol-treated IBA 0 0 0 1% phenol-treated IBA 0 0 0 T, . ., 1 reatment1
1 Undiluted 10° IBA suspensions for each treatment were 1:1 dilutions. Untreated IBA was mixed with saline. IBA suspensions treated with compounds were exposed for 60 minutes. 2 Five, 4-week-old S.C. White Leghorn males were inoculated with each preparation. Test birds were sacrificed and examined for lesions of IBA 72 hours after inoculation.
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2 because only 7 of 15 and 2 of 15 test birds became infected following inoculations with 10~2 and 1 0 - 3 dilutions, respectively; a t the same dilutions 10 of 15 and 3 of 15 test birds became infected after inoculation with I B A having a p H of 7.2. D a t a of three experiments to test t h e sensitivity of I B A to ether and to chloroform are summarized in Table 9. T h e titer of I B A n o t exposed to 2 0 % ether was 10 2 0 7 , b u t t h a t treated with ether for 18 hours was 101-93. T h e titer of I B A , not exposed t o 2 0 % chloroform was 102-23, b u t t h a t exposed to chloroform for 10 minutes was 101-88. These tests revealed t h a t I B A was quite resistant to ether and to chloroform, commonly used in characterizing viruses.
TABLE 9.—A summary of three experiments to determine the infectivity of IBA after treatment with ether or chloroform
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Y. CHO AND S. A. EDGAR
TABLE 11.—Effect
of penicillin-streptomycin on IBA1 2
Number of birds infected : Dilutions of IBA 10- 1 10"2 10"3 10-4
Control untreated
Penicillin-streptomycin treated 3
15 8 2 0
15 9 3 0
hour, whereas in this study it was killed by 1% phenol in the same length of time. Results of other tests common to the two studies were similar. Purified IBA in the absence of bacteria was quite stable at room temperatures because some was viable for at least 21 days in liquid state. This is not unusual for viruses because, according to Smith et al. (1964) polio virus (Picorna) may retain its infectivity for weeks in feces at room temperature. Benton et al. (1967a) also reported that an IBA-contaminated building remained infective for 122 days after infected birds had been removed. The characteristics of the IBA in the study presented here are most like the Picorna virus group described by Cabasso (1965) and Hamparian et al (1963) except that IBA was more heat stable than the Picorna viruses. However, more work must be done before IBA can be definitely classified as belonging to the Picorna group. SUMMARY AND CONCLUSIONS
IBA, purified by passing through graded membrane filters, was found to pass through 50 m/*. pores. It was stable at 60°C. for 90 minutes and was still infectious at a room temperature of approximately 25°C. for 21 days. Unpurified IBA
ACKNOWLEDGEMENTS
The financial support of the Southeastern Poultry and Egg Association, Decatur, Georgia; Alabama Flour Mills, Decatur, Alabama; Adams Egg Farm, Edwards, Mississippi; and the MacMillan Feed, Inc., Decatur, Indiana; is gratefully acknowledged. REFERENCES Andrews, C. H., and D. M. Horstmann, 1949. The susceptibility of viruses to ethyl ether. J. Gen. Microbiol. 3: 290-297. Benton, W. J., M. S. Cover and J. K. Rosenberger, 1967a. Studies on the transmission of the infectious bursal agent (IBA) of chickens. Avian Dis. 11: 430-438. Benton, W. J., M. S. Cover, J. K. Rosenberger and R. S. Lake, 1967b. Physiochemical properties of the infectious bursal agent (IBA). Avian Dis. 11:438-445. Cabasso, V. J., 1965. The emerging classification of animal viruses—a review. Avian Dis. 9:471-489. Cho, Y., and S. A. Edgar, 1968. The infectious bursal agent and pathology of the disease. Poultry Sci. 47: 1661. Cosgrove, A. S., 1962. An apparently new disease of chickens—avian nephrosis. Avian Dis. 6:385-389. Edgar, S. A., and Y. Cho, 1964. Gumboro disease in poultry. Highlights of Agr. Res. V. 11, No. 3, p. 5., Agr. Exp. Sta., Auburn Univ., Auburn, Ala. Feldman, H. A., and S. S. Wang, 1961. Sensitivity of various viruses to chloroform. Proc. Soc. Exptl. Biol. Med. 106: 736-738. Hamparian, V. V., M. R. Hilleman and A. Ketler, 1963. Contributions to characterization and classification of animal viruses. Proc. Soc. Exptl. Biol. Med. 112: 1040-1050. Hanson, R. P., D. L. Filmer and C. Quiroz, 1960. Properties of Fahey-Crawley virus. Avian Dis. 4: 79-85.
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1 A summary of three experiments. Five, 4-weekold S.C. White Leghorn males were inoculated with each dilution in each experiment. 2 BL50 of control IBA was 1 0 2 " versus 102 3I in the antibiotic treated IBA. 3 IBA treated with penicillin-streptomycin for 60 minutes.
had lost very little potency after 3 years at — 20°C. The agent was resistant to treatment with 20% ether (18 hours) or 20% chloroform (10 minutes), but it was not infective after exposure to 1% formalin, cresol or phenol for 1 hour. The infectivity of IBA was not affected by exposure to penicillin-streptomycin for 1 hour. The agent is believed to be a virus and most nearly related to the Picorna group.
INFECTIOUS BURSAL AGENT
754-755. Winterfield, R. W., 1963. Some newly identified infectious diseases. Twelfth Ann. New Hampshire Poultry Health Conf. p. 1-5. Winterfield, R. W., A. S. Cosgrove and S. B. Hitchner, 1961. Avian nephrosis (Gumboro disease). L & M News and Views, 2 (8). Winterfield, R. W., and S. B. Hitchner, 1962. Etiology of an infectious nephritis-nephrosis syndrome of chickens. Am. J. Vet. Res. 23:12731279. Winterfield, R. W., S. B. Hitchner, G. S. Appleton and A. S. Cosgrove, 1962. Avian nephrosis, nephritis and Gumboro disease. L & M News and Views, 3: 1-3. Winterfield, R. W., and S. B. Hitchner, 1964. Gumboro disease—anew threat to thepoultry industry. Poultry Digest, May, p. 206-207.
Seasonal Decline in Fertility of Turkey Eggs1'2 J. A. HARPER AND G. H. ARSCOTT Department of Poultry Science, Oregon State University, Corvattis, Oregon 97331 (Received for publication July 11, 1969)
K
NOX and Marsden (1954) reported while egg numbers in the Beltsville Small White turkey increased by selection in eight generations from 80 to 146 per hen, the fertility (Olsen and Marsden, 1952) was satisfactory only from January through April. This reduced fertility prevents high reproductive efficiency when selecting for genetic improvement in persistency of egg production. Lorenz et al. (1959) suggest inherited differences and age as factors accounting for some of the variations in sperm viability of individual hens and that, "sperm have a shorter life in old hens' oviducts than in those of young hens." More recently Van Krey et al. (1967) have shown late season declines 1 Oregon Agricultural Experiment Station Technical Paper 2715. 2 Supported in part by a grant from the National Turkey Federation.
in fertility due to non-retention of spermatozoa within the uterovaginal site. The research reported herein was conducted to determine the effect of age, sex difference and semen characteristics on seasonal decline in fertility of the turkey. PROCEDURE Late May hatched Medium White (Wrolstad) turkeys were brooded in confinement to eight weeks, then range reared and 160 to 180 females housed in November each year in a wire-sided pole house. Ninety males of the same hatches were maintained as breeders in woodshavings covered range lots equipped with shelters. A minimum 14 hours of light daily was provided the males on December 15 and the females on January 1. The ration fed from time of lighting was the OSU Station all-in-one breeder diet.
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Hsuing, G. D., 1965. Use of ultrafiltration for animal virus grouping. Bacterid. Rev. 29: 477-486. Ketler, A., V. V. Hamparian and M. R. Hileman, 1962. Characterization and classification of ECHO 28-rhinovirus-coryzavirus agents. Proc. Soc. Exptl. Biol. Med. 110: 821-831. Quiroz, C. A., and R. P. Hanson, 1958. Physicalchemical treatment of inocula as a means of separating and identifying avian viruses. Avian Ms. 2: 94-98. Reed, L. J., and H. Muench, 1938. A simple method of estimating fifty per cent end points. Am. J. Hyg. 27: 493^97. Smith, D. T., N. F. Conant and J. R. Overman, 1964. Microbiology. 13th Ed. Appleton-Century Crafts, New York, p. 956. Wallbank, A. M., and E. L. Stubb, 1965. Sensitivity of strain R avian erythroblastosis virus to ether and chloroform. Proc. Soc. Exptl. Biol. Med. 120:
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J. R. MORRIS, F. N. JEROME AND B. S. REINHART the chicken. J. Nat. Cancer Inst. 40: 583-592. Solomon, J. B., 1961a. The onset and maturation of the graft versus host reaction in chickens. J. Embryol. Exp. Morph. 9: 355-369. Solomon, J. B., 1962. A sex difference in the splenomegaly syndrome in chick embryos injected with adult spleen cells or blood. Exp. Cell Res. 28:151157. Solomon, J. B., and D. F. Tucker, 1963. Immunological attack by adult cells in the developing chick embryo. Influence of the vascularity of the host spleen on the homograft rejection by the embryo on splenomegaly. J. Embryol. Exp. Morph. 11:119-134.
Characterization of the Infectious Bursal Agent Y. CHO* AND S. A. EDGAR Department of Poultry Science, Auburn University, Auburn, Alabama, 36830 (Received for publication July 10, 1969)
G
UMBORO disease was first diagnosed as an infectious disease in the fall of 1957 by Cosgrove (1962). Later, two virus agents were isolated from birds believed to be suffering from the Gumboro disease syndrome, Winterfield et al. (1961). The first was isolated from kidneys of affected birds and it was designated the "Gray" agent that caused nephrosis but was believed to be a variant type of infectious bronchitis virus (IBV). The virus was of low virulence for the respiratory tract (Winterfield et ah, 1961; Winterfield and Hitchner, 1962; and Winterfield, 1963). A second agent was isolated later by the same research group from the bursa of affected birds and was designated infectious bursal agent (IBA). Thus, Winterfield et al. (1962)concluded that at least two different agents were involved in Gumboro disease.
The Winterfield group then differentiated the signs and gross pathology of birds infected by the two agents, namely "Gray" agent and IBA. With "Gray" agent infection, the main pathology was in the kidney and there was slight respiratory involvement, but the bursa was not affected. With IBA infection the bursa was the main organ affected. Pathology in the kidneys was secondary, and there was no respiratory involvement. Both agents caused muscle hemorrhages of various gradations (Winterfield et al., 1962). Edgar and Cho (1964) also reported isolation of a virus-like agent from affected birds and transmitted the agent successfully to susceptible 1- to 6-week-old chickens that caused a disease condition similar to that described by Cosgrove (1962), and by the IBA agent of Winterfield et al. (1962).
* In part from the thesis submitted in partial fulfillment of the requirements for the Doctor of Philosophy degree, Auburn University, August, 1967.
Since the isolation of IBA, little has been published on its characterization. Winterfield and Hitchner (1964) reported that the agent was filterable, which led to
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Ebert, J. D., 1959. The Cell, 1st edition. Vol. 1: 619-693. Academic Press, New York and London. Jaffe, W. P., 1965. The bursa of Fabricius and homograft immunity. Poultry Sci. 44: 1615-1616. Jaffe, W. P., and L. N. Payne, 1961. Graft against host reactions in inbred lines of chickens and their crosses. Brit. Poultry Sci. 2:133-143. Morris, J. R., A. E. Ferguson and F. N. Jerome, 1969. Selection for genetic resistance and susceptibility to Marek's disease (MD). Can. J. Ani. Sci. (In press). Purchase, H. G., R. C. Chubb and P. M. Biggs, 1968. Effect of lymphoid leukosis and Marek's disease on the immunological responsiveness of
INFECTIOUS BURSAL AGENT
the belief that it was probably a virus. Benton el al. (1967b) and Cho and Edgar (1968) reported on the size, chemical stability, thermostability, pH stability and ether and chloroform sensitivity of IBA. The purpose of this study was to characterize IBA and differentiate it from other disease producing agents. MATERIALS AND METHODS
The BL60 (infection that resulted in 50% with bursal lesions among inoculated chickens) of unpurified and purified IBA suspensions were determined by the method of Reed and Muench (1938), and was the standard for determining infectivity of all samples of the agent tested. All unfiltered bursal suspensions contained Escherichia coli and/or Proteus spp. Filtrates that passed through 0.3 ix.-membrane filters were subjected to bacteriologic tests by inoculating 0.5 ml. onto blood agar and desoxycholate agar plates to determine whether bacteria had passed through the filters. This procedure tested the integrity of the filters. Particle size of IBA was determined according to Hsuing (1965). Purified suspensions previously referred to were filtered through Gelman triacetate filters of the following pore sizes: 200, 100, and 50 mju. (millimicrons). A Swinny hypodermic adapter with a 10 ml. syringe attached to it was used for filtering instead of a suction funnel (Hsuing, 1965). Filtrates that passed 200, 100 and 50 nux. then were tested in susceptible chickens. Loss of activity was determined by comparing filtrates that passed through the above filters with that which passed through a 300 mp. filter. IBA that passed through a 300 m/i. filter found to be bacteria-free is hereafter referred to as purified. All tests were conducted with purified IBA. Thermostability of IBA was determined according to the method outlined by Quiroz and Hanson (1958). Sealed ampules of IBA were subjected to 60°C. in a water bath for 5,15, 30, or 90 minutes and tested for viability. Stability of IBA held at room temperature was determined by thawing samples and checking for viability at specific periods. One ml. of the suspension was withdrawn immediately and tested in sus-
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In preparation of IBA for study, SO, 5-week-old pathogen-free S. C. White Leghorns reared in isolation were each inoculated intraocularly with 0.05 ml. of a field isolate of IBA. The inoculated birds were sacrificed at 72 hours. The bursa of each inoculated bird was excised carefully and minced with an omnimixer for 5 minutes. This was then suspended in 0.85% sterile saline, 1 g. of tissue in 5 ml. saline. Aseptic techniques were employed throughout all experiments. IBA was freed of bacteria (purified) by centrifugating and filtering. It was freed from cell debris by low speed centrifugation (2,000 r.p.m. for 20 minutes). The supernatant was collected and passed through a No. 2 paper filter in a porcelain Buchner suction funnel. The suspension then was filtered through membrane filters of different porosities (Gelman triacetate filters, pore sizes 5 /*., 1.2 ju., 0.8 p., 0.45 p., 0.3 fi). Suspensions were filtered in an ice bath at 4°C. The preparations were tested for bacterial contamination before and after filtration. The relatively purified IBA suspensions then were pipetted, 1 ml. samples each, into small vials and quickly frozen in dry ice. Samples of the filtrate were tested for infectivity (titer), at 10-fold serial dilutions by intraocular inoculation in susceptible 4-week-old S. C. White Leghorn chickens. All birds were sacrificed at 72 hours after inoculation and examined for lesions of infectious bursal disease (IBD).
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Y. C H O AND S. A. EDGAR
.The sensitivity of I B A to ether was determined according to the technique of Andrews and Hortsman (1949) and Wallbank and Stubbs (1965). I B A suspensions were exposed to reagent grade diethyl ether (1 volume of ether to 4 volumes of IBA suspension) in small rubber-capped bottles. T h e content of each bottle then was mixed b y gentle shaking and allowing it to stand for 18 hours at 4°C. T h e ether was removed from the I B A b y evaporation in open petri dishes for 30 minutes. Ten-fold serial dilutions in sterile saline were tested.
Chloroform sensitivity of IBA was measured by the method of Wallbank and Stubb (1965), a modification of t h a t by Feldman and Wang (1961). Chloroform was added to an I B A suspension in a small flask with a screw cap (1 volume of chloroform to 4 volumes of IBA suspension). The mixture was agitated slowly on a magnetic stirrer for 10 minutes at 4 ° C , then centrifuged at 275 X gravity for 5 minutes and the chloroform was removed and discarded. The I B A then was diluted and tested. T h e stability of I B A exposed to 1% formalin, 1 % cresol or 1 % phenol was determined according to the method of Quiroz and Hanson (1958). Formalin,* cresol, and phenol** were diluted to 2 % solutions with sterile distilled water. One part of I B A suspension was combined with 1 p a r t of 2 % formalin, 2 % cresol, or 2 % phenol at room temperature (25° C.) for 1 hour. These treated suspensions of IBA were tested then for infectivity in the manner described earlier. Finally, the sensitivity of IBA to 5,000 units of penicillin-streptomycin per ml. for 60 minutes at 37°C. was determined. Ten-fold serial dilutions of this treated IBA were also tested. All,IB A suspensions were tested for infectivity by intraocular inoculation of 4week-old susceptible S. C. White Leghorns maintained in isolation t h a t were sacrificed and examined for lesions of IBA at 72 hours after inoculation. RESULTS AND DISCUSSION
Results of three experiments to determine the infectivity of unpurified versus purified, filtered, bacteria-free IBA sussuspensions are summarized in Table 1. There was 100% infection of test birds with undiluted or 10 _ 1 dilutions. How* Formalin—40% formaldehyde, U.S.P., Fisher. ** Cresol and phenol, U.S.P., J. T. Baker Chemical Co.
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ceptible chickens, and additional samples were tested at 24, 4 8 , 7 2 , 96, 120, and 144 hours and at 7, 14, and 21 days. Stability of I B A at - 2 0 ° C . was also determined. An original batch of I B A inoculum was made in J a n u a r y 1964 and the titer had been determined at t h a t time. I t then was stored at • — 20°C. I n January 1967 three random sample bottles were thawed, serial dilutions were prepared and titered, and the infectivity of the suspensions for the two periods of storage were compared. Two experiments were conducted to determine the stability of I B A at different p H ' s . A suspension was adjusted to a p H of 2 by combining equal volumes of undiluted IBA and 1 % HC1. A p H of 12 was obtained by combining equal volumes o£ undiluted IBA and 1%. N a O H . T h e pH-adjusted IBA suspensions were held at a room temperature of approximately 25°C. for.'60 minutes (Quiroz^nd Hanson, 1958; and Ketler et al, 1962), and then tested for infectivity. I n the first experiment on. p H stability undiluted IBA suspensions were tested. Serial dilutions of I B A were tested in the second experiment to determine whether a p H of 2 for 60 minutes would lower the infectivity titer of IBA. Untreated I B A suspensions (pH 7.2) of the respective dilutions served as controls.
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INFECTIOUS BURSAL AGENT TABLE 1.—A comparison of the infectivity of unpurified versus purified IBA suspensions Number of birds infected1 Unpurified IBA
Purified IBA
15 15 15 9 6 0
15 15 9 3 0 0
2
10° 10"1 10-2
10"3 10-" 10-5
ever, at 10~2 only 9 of 15 birds inoculated with the filtrate became infected compared with 15 of 15 inoculated with the original unfiltered preparation; none became infected with a 10"4 dilution of the purified IBA. Results of the calculations to determine the 50% bursal lesion value (BL50) by the Reed and Muench technique are presented in Table 2. The titer of unpurified IBA was 10 36 and that of the purified filtrate was 102-5. Thus, there was evidence of one log less titer in the purification
TABLE 2.—Calculations to determine 50% bursal lesion value (BL50) of IBA referred to in Table I
Treatments
Dilutions
Jnpurified IBA
ID"2 10- 3 10-" 10-5
Purified IBA
10"1 10-2
10- 3 10-4
Number birds Number birds Total number Total number birds birds with no with with no with lesions lesions lesions lesions 0 6 9 15 0 3 9 15
15 9 6 0 15 9 3 0
0 6 15 30 0 3 12 27
30 15 6 0 27 12 3 0
71 43-50 21.43 = Proportional distance of unpurified I B A = ^ „ = o< 0-5 /1.43—Zo.oi
4Z.O0
Bursal lesion 50 (BL6o) of unpurified IBA was 10"3-5The final titer was l0 3 5 = BL,6o per volume of inoculum. Proportional distance of purified IB A = ^ - 7 ^ = — = 0 . 5 oU—JA) OU
Bursal lesion 50 (BL60) of purified IBA was 10"2 5. The final titer was 102-6 BL50 of inoculum.
Percentage with lesions 100 71.43 28.57 0 100 80 20 0
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1 A total of 15 birds were inoculated with each dilution in three experiments. Test birds were susceptible 4-week-old S. C. White Leghorn males. 2 One part infected bursa to 4 parts of saline.
technique employed. Filtrates of purified IBA were inoculated (0.5 ml.) onto blood agar and desoxycholate agar plates and incubated at 37°C. for 48 hours. No bacteria were recovered, whereas E. coli and/or Proteus spp. were present in unfiltered bursal suspensions. IBA used in the remaining experiments was purified (bacteria-free) and will be referred to as IBA unless designated otherwise. Results of three experiments to determine particle size of IBA are summarized in Table 3. One hundred percent of susceptible 4-week-old S. C. White Leghorns became infected regardless of size of filter down to 50 mix. in diameter, the smallest pore tested. Thus, it was determined that the agent was less than 50 m/i. in size. Results of the three experiments to determine thermostability of IBA at 60°C. are summarized in Table 4. All 4-week-old chickens inoculated with a 10 _) dilution of IBA became infected after exposure of IBA to 60°C. in water bath for 90 minutes. The number of test chickens that became infected decreased as dilutions were increased. The BL50 of unexposed
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Y. C H O AND S. A. E D G A R
TABLE 3.—Summary of three experiments to determine the particle size of IBA Pore size of filters (mM.) Uninoculated controlsi 300 200 100 50
Number birds with bursal lesions/birds tested 0/15 15/15 15/15 15/15 15/15
Percentage showing bursal lesions 0 100 100 100 100
TABLE 5.—Viability of IBA exposed to room temperature (approximately 25°C.) Dilution of IBA inoculum • 10"
Number days exposure when IBA was tested 0
1
2
3
4
5
6
7
• 14 21
(No. birds that became infected)1 15 15 15 15 15 14 12 9 5
1
1
Five, 4-week-old S. C. White Leghorn males were tested per period in each of three experiments, making a total of 15 birds per test period.
I B A is quite stable a t room temperature. Results of one experiment involving tests of three samples of the same lot of original unpurified I B A isolate obtained IBA was 102-38 versus 102-24 for I B A ex- in J a n u a r y 1964 is presented in Table 6. posed to 60°C. for 90 minutes. T h e differ- After 3 years at — 2 0 ° C , t h e infectivity had n o t decreased appreciably, with a ence in titers was n o t significant. -313 as compared with 10~3-5 Results of three experiments to deter- BL6o of 1 0 mine t h e viability of I B A exposed to a determined 3 years earlier. Results of two experiments to deterroom temperature of approximately 25°C. for u p to 21 days are summarized in Table mine t h e stability of I B A a t different 5. Stock I B A (1:5 dilution) was tested for p H ' s are summarized in Tables 7 a n d 8. infectivity in three groups of five chickens I n t h e first experiment undiluted I B A at each of the periods shown, a total of 15 (1:5 in saline) h a d lost its infectivity foltest chickens for each period. I B A ex- lowing exposure to p H 12 for 60 minutes, posed to room temperature through 3 whereas it was still viable after exposure days resulted in 1 0 0 % infection. There to p H 2 for the same length of time, Table was some loss of activity b y t h e fourth 7. D a t a of a second experiment, in which day (13 of 15 positive) with a somewhat serial dilutions of I B A adjusted to p H 2 rapid decline in infectivity b y 7 and 14 for 60 minutes and compared with the days. Only 1 of 15 birds became infected original I B A ( p H 7.2), are summarized in with I B A exposed a t room temperature Table 8. There m a y have been some loss for 21 days. I t was concluded t h a t purified in viability of I B A upon exposure to p H TABLE 4.—Thermostability of ISA at 60°C.
Infectivity of Dilutions IBA not exposed to 60°C. 1
10-12 10~ 10-° 10-* 1
Infectivity of IBA exposed to 60CC. for (minutes):
T A B L E 6.—Stability of unfiltered IBA suspension after storage at — 20°C. for 3 years
Dilutions
15 30 45 60 90
(No. birds positive) (No. birds positive) 15 15 15 15 15 15 10 8 10 9 9 8 3 4 3 3 3 3 0 0 1 0 0 0
There were five S.C. White Leghorn males used for each dilution at each exposure period, making a total of 15 birds tested at each period.
10"2 10~3 10-4 10-6
Number birds that became infected when tested in:1 19642
19673
15 9 6 0
15 7 3 0
1 T h e r e were five, 4-week-old S.C. W h i t e L e g h o r n males tested w i t h each dilution, in each replicate. 2 BL 6 0 w a s 1 0 " . 3 BL 6 0 w a s 10 3 1 3 .
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1 Three experiments, each group contained five, 4-week-old S.C. White Leghorns. 2 Test birds were sacrificed and examined for IBD lesions at 72 hours after inoculation.
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INFECTIOUS BURSAL AGENT
TABLE 7.—Infectivity of IBA after exposure to a pH of 2 or a pH of 12 for 60 minutes
Treatments
Number birds that became infected, replicates:
Uninoculated Inoculated IBA pH 7.2 Inoculated IBA pH 2 Inoculated IBA pH 12
1
2
3
0 5 5 0
0 5 4 0
0 5 5 0
™ t, infect 0 15 14 0
1 There were five, 4-week-old S. C. White Leghorn males in each treatment of each replicate, examined for IBA lesions 72 hours after inoculation.
Results of an experiment to test t h e stability of I B A treated with certain other chemicals are summarized in Table 10. I B A treated for 60 minutes with 1 % TABLE 8.—Infectivity
of IBA exposed to
pH 2 for 60 minutes Dilutions tested 1 IO-1 10"2 10-'
io-<
IBA at pH7.22
IBA a t pH23
Number positive Number positive 15 15 10 7 3 2 0 0
1 There were 15, 4-week-old S.C. White Leghorn males inoculated with each dilution, 5 birds in each of 2three replicates. BL60 of untreated IBA was 102-88. 3 BL60 of IBA exposed to a pH of 2 was 10207.
Dilutions tested
Number of birds infected when inoculated with IBA:1 Untreated Chloroform3 control treated
Untreated control
Ether2 treated
io-' 10-2 10-s 10-4
15 7 2 0
14 7 1 0
15 8 3 0
15 6 1 0
BLra
1Q207
101->S
102-23
101-88
i Tested in five, 4-week-old S. C. White Leghorn males per dilution in each of three experiment. 2 Time of exposure was 18 hours at 4°C 3 Time of exposure was 10 minutes at 4°C.
formalin, 1 % cresol or 1 % phenol lost infectivity completely. Thus, i t was evident t h a t I B A is highly sensitive t o these compounds. T h e sensitivity of I B A t o penicillinstreptomycin was tested and results are summarized in Table 11. T h e infectivity of IBA-treated with penicillin-streptomycin for 60 minutes a t 37°C. was n o t decreased. Actually, a slightly higher percentage of t h e 15 chickens inoculated with penicillin-streptomycin-treated I B A became infected with 1 0 - 2 and 1 0 - 3 dilutions than became infected with respective dilutions of untreated IBA. Benton et al. (1967b) found t h a t I B A was n o t susceptible t o 0 . 5 % phenol in 1 TABLE 10.—Sensitivity of IBA to
three chemical compounds Number of birds infected, replicates:2 '_ 1 2 3 Uninoculated 0 0 0 IBA-untreated 5 5 5 1% formalin-treated IBA 0 0 0 1% cresol-treated IBA 0 0 0 1% phenol-treated IBA 0 0 0 T, . ., 1 reatment1
1 Undiluted 10° IBA suspensions for each treatment were 1:1 dilutions. Untreated IBA was mixed with saline. IBA suspensions treated with compounds were exposed for 60 minutes. 2 Five, 4-week-old S.C. White Leghorn males were inoculated with each preparation. Test birds were sacrificed and examined for lesions of IBA 72 hours after inoculation.
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2 because only 7 of 15 and 2 of 15 test birds became infected following inoculations with 10~2 and 1 0 - 3 dilutions, respectively; a t the same dilutions 10 of 15 and 3 of 15 test birds became infected after inoculation with I B A having a p H of 7.2. D a t a of three experiments to test t h e sensitivity of I B A to ether and to chloroform are summarized in Table 9. T h e titer of I B A n o t exposed to 2 0 % ether was 10 2 0 7 , b u t t h a t treated with ether for 18 hours was 101-93. T h e titer of I B A , not exposed t o 2 0 % chloroform was 102-23, b u t t h a t exposed to chloroform for 10 minutes was 101-88. These tests revealed t h a t I B A was quite resistant to ether and to chloroform, commonly used in characterizing viruses.
TABLE 9.—A summary of three experiments to determine the infectivity of IBA after treatment with ether or chloroform
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Y. CHO AND S. A. EDGAR
TABLE 11.—Effect
of penicillin-streptomycin on IBA1 2
Number of birds infected : Dilutions of IBA 10- 1 10"2 10"3 10-4
Control untreated
Penicillin-streptomycin treated 3
15 8 2 0
15 9 3 0
hour, whereas in this study it was killed by 1% phenol in the same length of time. Results of other tests common to the two studies were similar. Purified IBA in the absence of bacteria was quite stable at room temperatures because some was viable for at least 21 days in liquid state. This is not unusual for viruses because, according to Smith et al. (1964) polio virus (Picorna) may retain its infectivity for weeks in feces at room temperature. Benton et al. (1967a) also reported that an IBA-contaminated building remained infective for 122 days after infected birds had been removed. The characteristics of the IBA in the study presented here are most like the Picorna virus group described by Cabasso (1965) and Hamparian et al (1963) except that IBA was more heat stable than the Picorna viruses. However, more work must be done before IBA can be definitely classified as belonging to the Picorna group. SUMMARY AND CONCLUSIONS
IBA, purified by passing through graded membrane filters, was found to pass through 50 m/*. pores. It was stable at 60°C. for 90 minutes and was still infectious at a room temperature of approximately 25°C. for 21 days. Unpurified IBA
ACKNOWLEDGEMENTS
The financial support of the Southeastern Poultry and Egg Association, Decatur, Georgia; Alabama Flour Mills, Decatur, Alabama; Adams Egg Farm, Edwards, Mississippi; and the MacMillan Feed, Inc., Decatur, Indiana; is gratefully acknowledged. REFERENCES Andrews, C. H., and D. M. Horstmann, 1949. The susceptibility of viruses to ethyl ether. J. Gen. Microbiol. 3: 290-297. Benton, W. J., M. S. Cover and J. K. Rosenberger, 1967a. Studies on the transmission of the infectious bursal agent (IBA) of chickens. Avian Dis. 11: 430-438. Benton, W. J., M. S. Cover, J. K. Rosenberger and R. S. Lake, 1967b. Physiochemical properties of the infectious bursal agent (IBA). Avian Dis. 11:438-445. Cabasso, V. J., 1965. The emerging classification of animal viruses—a review. Avian Dis. 9:471-489. Cho, Y., and S. A. Edgar, 1968. The infectious bursal agent and pathology of the disease. Poultry Sci. 47: 1661. Cosgrove, A. S., 1962. An apparently new disease of chickens—avian nephrosis. Avian Dis. 6:385-389. Edgar, S. A., and Y. Cho, 1964. Gumboro disease in poultry. Highlights of Agr. Res. V. 11, No. 3, p. 5., Agr. Exp. Sta., Auburn Univ., Auburn, Ala. Feldman, H. A., and S. S. Wang, 1961. Sensitivity of various viruses to chloroform. Proc. Soc. Exptl. Biol. Med. 106: 736-738. Hamparian, V. V., M. R. Hilleman and A. Ketler, 1963. Contributions to characterization and classification of animal viruses. Proc. Soc. Exptl. Biol. Med. 112: 1040-1050. Hanson, R. P., D. L. Filmer and C. Quiroz, 1960. Properties of Fahey-Crawley virus. Avian Dis. 4: 79-85.
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1 A summary of three experiments. Five, 4-weekold S.C. White Leghorn males were inoculated with each dilution in each experiment. 2 BL50 of control IBA was 1 0 2 " versus 102 3I in the antibiotic treated IBA. 3 IBA treated with penicillin-streptomycin for 60 minutes.
had lost very little potency after 3 years at — 20°C. The agent was resistant to treatment with 20% ether (18 hours) or 20% chloroform (10 minutes), but it was not infective after exposure to 1% formalin, cresol or phenol for 1 hour. The infectivity of IBA was not affected by exposure to penicillin-streptomycin for 1 hour. The agent is believed to be a virus and most nearly related to the Picorna group.
INFECTIOUS BURSAL AGENT
754-755. Winterfield, R. W., 1963. Some newly identified infectious diseases. Twelfth Ann. New Hampshire Poultry Health Conf. p. 1-5. Winterfield, R. W., A. S. Cosgrove and S. B. Hitchner, 1961. Avian nephrosis (Gumboro disease). L & M News and Views, 2 (8). Winterfield, R. W., and S. B. Hitchner, 1962. Etiology of an infectious nephritis-nephrosis syndrome of chickens. Am. J. Vet. Res. 23:12731279. Winterfield, R. W., S. B. Hitchner, G. S. Appleton and A. S. Cosgrove, 1962. Avian nephrosis, nephritis and Gumboro disease. L & M News and Views, 3: 1-3. Winterfield, R. W., and S. B. Hitchner, 1964. Gumboro disease—anew threat to thepoultry industry. Poultry Digest, May, p. 206-207.
Seasonal Decline in Fertility of Turkey Eggs1'2 J. A. HARPER AND G. H. ARSCOTT Department of Poultry Science, Oregon State University, Corvattis, Oregon 97331 (Received for publication July 11, 1969)
K
NOX and Marsden (1954) reported while egg numbers in the Beltsville Small White turkey increased by selection in eight generations from 80 to 146 per hen, the fertility (Olsen and Marsden, 1952) was satisfactory only from January through April. This reduced fertility prevents high reproductive efficiency when selecting for genetic improvement in persistency of egg production. Lorenz et al. (1959) suggest inherited differences and age as factors accounting for some of the variations in sperm viability of individual hens and that, "sperm have a shorter life in old hens' oviducts than in those of young hens." More recently Van Krey et al. (1967) have shown late season declines 1 Oregon Agricultural Experiment Station Technical Paper 2715. 2 Supported in part by a grant from the National Turkey Federation.
in fertility due to non-retention of spermatozoa within the uterovaginal site. The research reported herein was conducted to determine the effect of age, sex difference and semen characteristics on seasonal decline in fertility of the turkey. PROCEDURE Late May hatched Medium White (Wrolstad) turkeys were brooded in confinement to eight weeks, then range reared and 160 to 180 females housed in November each year in a wire-sided pole house. Ninety males of the same hatches were maintained as breeders in woodshavings covered range lots equipped with shelters. A minimum 14 hours of light daily was provided the males on December 15 and the females on January 1. The ration fed from time of lighting was the OSU Station all-in-one breeder diet.
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Hsuing, G. D., 1965. Use of ultrafiltration for animal virus grouping. Bacterid. Rev. 29: 477-486. Ketler, A., V. V. Hamparian and M. R. Hileman, 1962. Characterization and classification of ECHO 28-rhinovirus-coryzavirus agents. Proc. Soc. Exptl. Biol. Med. 110: 821-831. Quiroz, C. A., and R. P. Hanson, 1958. Physicalchemical treatment of inocula as a means of separating and identifying avian viruses. Avian Ms. 2: 94-98. Reed, L. J., and H. Muench, 1938. A simple method of estimating fifty per cent end points. Am. J. Hyg. 27: 493^97. Smith, D. T., N. F. Conant and J. R. Overman, 1964. Microbiology. 13th Ed. Appleton-Century Crafts, New York, p. 956. Wallbank, A. M., and E. L. Stubb, 1965. Sensitivity of strain R avian erythroblastosis virus to ether and chloroform. Proc. Soc. Exptl. Biol. Med. 120:
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