GUMBORO disease was first diagnosed

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1 212 J. R. MORRIS, F. N. JEROME AND B. S. REINHART Ebert, J. D., 15. The Cell, 1st edition. Vol. 1: 1-. Academic Press, New York and London. Jaffe, W. P., 15. The bursa of Fabricius and homograft immunity. Poultry Sci. 44: Jaffe, W. P., and L. N. Payne, 11. Graft against host reactions in inbred lines of chickens and their crosses. Brit. Poultry Sci. 2:1-14. Morris, J. R., A. E. Ferguson and F. N. Jerome, 1. 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, 18. Effect of lymphoid leukosis and Marek's disease on the immunological responsiveness of GUMBORO disease was first diagnosed as an infectious disease in the fall of 157 by Cosgrove (12). Later, two virus agents were isolated from birds believed to be suffering from the Gumboro disease syndrome, Winterfield et al. (11). 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, 11; Winterfield and Hitchner, 12; and Winterfield, 1). 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. (12)concluded that at least two different agents were involved in Gumboro disease. * In part from the thesis submitted in partial fulfillment of the requirements for the Doctor of Philosophy degree, Auburn University, August, 17. the chicken. J. Nat. Cancer Inst. 4: Solomon, J. B., 11a. The onset and maturation of the graft versus host reaction in chickens. J. Embryol. Exp. Morph. : 55-. Solomon, J. B., 12. A sex difference in the splenomegaly syndrome in chick embryos injected with adult spleen cells or blood. Exp. Cell Res. 28:1-7. Solomon, J. B., and D. F. Tucker, 1. 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: Characterization of the Infectious Bursal Agent Y. CHO* AND S. A. EDGAR Department of Poultry Science, Auburn University, Auburn, Alabama, 8 (Received for publication July 1, 1) 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., 12). Edgar and Cho (14) also reported isolation of a virus-like agent from affected birds and transmitted the agent successfully to susceptible 1- to -week-old chickens that caused a disease condition similar to that described by Cosgrove (12), and by the IBA agent of Winterfield et al. (12). Since the isolation of IBA, little has been published on its characterization. Winterfield and Hitchner (14) reported that the agent was filterable, which led to Downloaded from at Penn State University (Paterno Lib) on May 8, 21

2 INFECTIOUS BURSAL AGENT 21 the belief that it was probably a virus. Benton el al. (17b) and Cho and Edgar (18) 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 In preparation of IBA for study, SO, 5-week-old pathogen-free S. C. White Leghorns reared in isolation were each inoculated intraocularly with.5 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.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, r.p.m. for 2 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.,.8 p.,.45 p.,. 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 1-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 of infectious bursal disease (IBD). The BL (infection that resulted in 5% with bursal among inoculated chickens) of unpurified and purified IBA suspensions were determined by the method of Reed and Muench (18), 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. ix.-membrane filters were subjected to bacteriologic tests by inoculating.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 (15). Purified suspensions previously referred to were filtered through Gelman triacetate filters of the following pore sizes: 2, 1, and 5 mju. (millimicrons). A Swinny hypodermic adapter with a 1 ml. syringe attached to it was used for filtering instead of a suction funnel (Hsuing, 15). Filtrates that passed 2, 1 and 5 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 mp. filter. IBA that passed through a 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 (158). Sealed ampules of IBA were subjected to C. in a water bath for 5,,, or 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- Downloaded from at Penn State University (Paterno Lib) on May 8, 21

3 214 Y. CHO AND S. A. EDGAR ceptible chickens, and additional samples were tested at 24, 48,72,, 12, and 144 hours and at 7, 14, and 21 days. Stability of IBA at -2 C. was also determined. An original batch of IBA inoculum was made in January 14 and the titer had been determined at that time. It then was stored at 2 C. In January 17 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 IBA at different ph's. A suspension was adjusted to a ph of 2 by combining equal volumes of undiluted IBA and 1% HC1. A ph of 12 was obtained by combining equal volumes o undiluted IBA and 1%. NaOH. The ph-adjusted IBA suspensions were held at a room temperature of approximately 25 C. for.' minutes (Quiroz^nd Hanson, 158; and Ketler et al, 12), and then tested for infectivity. In the first experiment on. ph stability undiluted IBA suspensions were tested. Serial dilutions of IBA were tested in the second experiment to determine whether a ph of 2 for minutes would lower the infectivity titer of IBA. Untreated IBA suspensions (ph 7.2) of the respective dilutions served as controls..the sensitivity of IBA to ether was determined according to the technique of Andrews and Hortsman (14) and Wallbank and Stubbs (15). IBA suspensions were exposed to reagent grade diethyl ether (1 volume of ether to 4 volumes of IBA suspension) in small rubber-capped bottles. The content of each bottle then was mixed by gentle shaking and allowing it to stand for 18 hours at 4 C. The ether was removed from the IBA by evaporation in open petri dishes for minutes. Ten-fold serial dilutions in sterile saline were tested. Chloroform sensitivity of IBA was measured by the method of Wallbank and Stubb (15), a modification of that by Feldman and Wang (11). Chloroform was added to an IBA 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 1 minutes at 4 C, then centrifuged at 275 X gravity for 5 minutes and the chloroform was removed and discarded. The IBA then was diluted and tested. The stability of IBA exposed to 1% formalin, 1% cresol or 1% phenol was determined according to the method of Quiroz and Hanson (158). Formalin,* cresol, and phenol** were diluted to 2% solutions with sterile distilled water. One part of IBA suspension was combined with 1 part 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, units of penicillin-streptomycin per ml. for minutes at 7 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 4- week-old susceptible S. C. White Leghorns maintained in isolation that were sacrificed and examined for 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 1% infection of test birds with undiluted or 1 _1 dilutions. How- * Formalin 4% formaldehyde, U.S.P., Fisher. ** Cresol and phenol, U.S.P., J. T. Baker Chemical Co. Downloaded from at Penn State University (Paterno Lib) on May 8, 21

4 INFECTIOUS BURSAL AGENT 2 TABLE 1. A comparison of the infectivity of unpurified versus purified IBA suspensions 1 2 1" " 1-" 1-5 Number of birds infected 1 Unpurified IBA Purified IBA 1 A total of 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. ever, at 1~ 2 only of birds inoculated with the filtrate became infected compared with of inoculated with the original unfiltered preparation; none became infected with a 1" 4 dilution of the purified IBA. Results of the calculations to determine the 5% bursal lesion value (BL 5 ) by the Reed and Muench technique are presented in Table 2. The titer of unpurified IBA was 1 and that of the purified filtrate was Thus, there was evidence of one log less titer in the purification technique employed. Filtrates of purified IBA were inoculated (.5 ml.) onto blood agar and desoxycholate agar plates and incubated at 7 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. One hundred percent of susceptible 4-week-old S. C. White Leghorns became infected regardless of size of filter down to 5 mix. in diameter, the smallest pore tested. Thus, it was determined that the agent was less than 5 m/i. in size. Results of the three experiments to determine thermostability of IBA at C. are summarized in Table 4. All 4-week-old chickens inoculated with a 1 _) dilution of IBA became infected after exposure of IBA to C. in water bath for minutes. The number of test chickens that became infected decreased as dilutions were increased. The BL5 of unexposed TABLE 2. Calculations to determine 5% bursal lesion value (BL5) of IBA referred to in Table I Treatments Jnpurified IBA Purified IBA ID" " 1-5 1" Number birds with no Number birds with Total number birds with no Total number birds with Percentage with Downloaded from at Penn State University (Paterno Lib) on May 8, Proportional distance of unpurified IBA=^ = o< = -5 /1.4 Zo.oi 4Z.O Bursal lesion 5 (BL o) of unpurified IBA was 1" The final titer was l 5 = BL,o per volume of inoculum. Proportional distance of purified IB A = ^-7^= =.5 ou JA) OU Bursal lesion 5 (BL ) of purified IBA was 1" 2 5. The final titer was BL5 of inoculum.

5 21 Y. CHO AND S. A. EDGAR TABLE. Summary of three experiments to determine the particle size of IBA Pore size of filters (m M.) Number birds with bursal /birds tested Uninoculated controls i / / 2 / 1 / 5 / Percentage showing bursal Three experiments, each group contained five, 4-week-old S.C. White Leghorns. 2 Test birds were sacrificed and examined for IBD at 72 hours after inoculation. IBA was versus for IBA exposed to C. for minutes. The difference in titers was not significant. Results of three experiments to determine the viability of IBA exposed to a room temperature of approximately 25 C. for up to 21 days are summarized in Table 5. Stock IBA (1:5 dilution) was tested for infectivity in three groups of five chickens at each of the periods shown, a total of test chickens for each period. IBA exposed to room temperature through days resulted in 1% infection. There was some loss of activity by the fourth day (1 of positive) with a somewhat rapid decline in infectivity by 7 and 14 days. Only 1 of birds became infected with IBA exposed at room temperature for 21 days. It was concluded that purified TABLE 4. Thermostability of ISA at C. Infectivity of IBA Infectivity of exposed to C C. 1 IBA not exposed for (minutes): to C. 45 (No. birds positive) (No. birds positive) 1-1 1~ * 1 1 There were five S.C. White Leghorn males used for each dilution at each exposure period, making a total of birds tested at each period. TABLE 5. Viability of IBA exposed to room temperature (approximately 25 C.) Number days exposure when Dilution of IBA was tested IBA inoculum (No. birds that became infected) 1 1" Five, 4-week-old S. C. White Leghorn males were tested per period in each of three experiments, making a total of birds per test period. IBA is quite stable at room temperature. Results of one experiment involving tests of three samples of the same lot of original unpurified IBA isolate obtained in January 14 is presented in Table. After years at 2 C, the infectivity had not decreased appreciably, with a BL o of 1-1 as compared with 1~ - 5 determined years earlier. Results of two experiments to determine the stability of IBA at different ph's are summarized in Tables 7 and 8. In the first experiment undiluted IBA (1:5 in saline) had lost its infectivity following exposure to ph 12 for minutes, whereas it was still viable after exposure to ph 2 for the same length of time, Table 7. Data of a second experiment, in which serial dilutions of IBA adjusted to ph 2 for minutes and compared with the original IBA (ph 7.2), are summarized in Table 8. There may have been some loss in viability of IBA upon exposure to ph TABLE. Stability of unfiltered IBA suspension after storage at 2 C. for years 1" 2 1~ Number birds that became infected when tested in: There were five, 4-week-old S.C. White Leghorn males tested with each dilution, in each replicate. 2 BL was 1". BL was 1 1. Downloaded from at Penn State University (Paterno Lib) on May 8, 21

6 INFECTIOUS BURSAL AGENT 217 TABLE 7. Infectivity of IBA after exposure to a ph of 2 or a ph of 12 for minutes Number birds that Treatments became infected, replicates: t, infect 1 2 Uninoculated Inoculated IBA ph Inoculated IBA ph Inoculated IBA ph 12 1 There were five, 4-week-old S. C. White Leghorn males in each treatment of each replicate, examined for IBA 72 hours after inoculation. 2 because only 7 of and 2 of test birds became infected following inoculations with 1~ 2 and 1 - dilutions, respectively; at the same dilutions 1 of and of test birds became infected after inoculation with IBA having a ph of 7.2. Data of three experiments to test the sensitivity of IBA to ether and to chloroform are summarized in Table. The titer of IBA not exposed to 2% ether was 1 27, but that treated with ether for 18 hours was The titer of IBA, not exposed to 2% chloroform was 1 2-2, but that exposed to chloroform for 1 minutes was These tests revealed that IBA was quite resistant to ether and to chloroform, commonly used in characterizing viruses. Results of an experiment to test the stability of IBA treated with certain other chemicals are summarized in Table 1. IBA treated for minutes with 1% TABLE 8. Infectivity of IBA exposed to ph 2 for minutes tested 1 IO- 1 1" 2 1-' io-< IBA at IBA at ph7.2 2 ph2 Number positive Number positive There were, 4-week-old S.C. White Leghorn males inoculated with each dilution, 5 birds in each of three replicates. 2 BL of untreated IBA was BL of IBA exposed to a ph of 2 was TABLE. A summary of three experiments to determine the infectivity of IBA after treatment with ether or chloroform tested io-' s 1-4 BLra Untreated control 7 2 1Q27 Number of birds infected when inoculated with IBA: 1 Ether2 treated >S Untreated control Chloroform treated 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 Time of exposure was 1 minutes at 4 C. formalin, 1% cresol or 1% phenol lost infectivity completely. Thus, it was evident that IBA is highly sensitive to these compounds. The sensitivity of IBA to penicillinstreptomycin was tested and results are summarized in Table 11. The infectivity of IBA-treated with penicillin-streptomycin for minutes at 7 C. was not decreased. Actually, a slightly higher percentage of the chickens inoculated with penicillin-streptomycin-treated IBA became infected with 1-2 and 1 - dilutions than became infected with respective dilutions of untreated IBA. Benton et al. (17b) found that IBA was not susceptible to.5% phenol in 1 TABLE 1. Sensitivity of IBA to three chemical compounds Number of birds infected, T,.., replicates: 2 1 reatment 1 '_ 1 2 Uninoculated IBA-untreated % formalin-treated IBA 1% cresol-treated IBA 1% phenol-treated IBA 1 Undiluted 1 IBA suspensions for each treatment were 1:1 dilutions. Untreated IBA was mixed with saline. IBA suspensions treated with compounds were exposed for minutes. 2 Five, 4-week-old S.C. White Leghorn males were inoculated with each preparation. Test birds were sacrificed and examined for of IBA 72 hours after inoculation. Downloaded from at Penn State University (Paterno Lib) on May 8, 21

7 218 Y. CHO AND S. A. EDGAR TABLE 11. Effect of penicillin-streptomycin on IBA 1 of IBA 1-1 1" 2 1" 1-4 Number of birds infected : 2 Control untreated 8 2 Penicillin-streptomycin treated 1 A summary of three experiments. Five, 4-weekold S.C. White Leghorn males were inoculated with each dilution in each experiment. 2 BL 5 of control IBA was 1 2 " versus 1 2 I in the antibiotic treated IBA. IBA treated with penicillin-streptomycin for minutes. 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. (14) polio virus (Picorna) may retain its infectivity for weeks in feces at room temperature. Benton et al. (17a) 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 (15) and Hamparian et al (1) 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 5 m/*. pores. It was stable at C. for minutes and was still infectious at a room temperature of approximately 25 C. for 21 days. Unpurified IBA had lost very little potency after years at 2 C. The agent was resistant to treatment with 2% ether (18 hours) or 2% chloroform (1 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. 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, 14. The susceptibility of viruses to ethyl ether. J. Gen. Microbiol. : Benton, W. J., M. S. Cover and J. K. Rosenberger, 17a. Studies on the transmission of the infectious bursal agent (IBA) of chickens. Avian Dis. 11: Benton, W. J., M. S. Cover, J. K. Rosenberger and R. S. Lake, 17b. Physiochemical properties of the infectious bursal agent (IBA). Avian Dis. 11: Cabasso, V. J., 15. The emerging classification of animal viruses a review. Avian Dis. : Cho, Y., and S. A. Edgar, 18. The infectious bursal agent and pathology of the disease. Poultry Sci. 47: 11. Cosgrove, A. S., 12. An apparently new disease of chickens avian nephrosis. Avian Dis. :85-8. Edgar, S. A., and Y. Cho, 14. Gumboro disease in poultry. Highlights of Agr. Res. V. 11, No., p. 5., Agr. Exp. Sta., Auburn Univ., Auburn, Ala. Feldman, H. A., and S. S. Wang, 11. Sensitivity of various viruses to chloroform. Proc. Soc. Exptl. Biol. Med. 1: Hamparian, V. V., M. R. Hilleman and A. Ketler, 1. Contributions to characterization and classification of animal viruses. Proc. Soc. Exptl. Biol. Med. 112: 14-. Hanson, R. P., D. L. Filmer and C. Quiroz, 1. Properties of Fahey-Crawley virus. Avian Dis. 4: Downloaded from at Penn State University (Paterno Lib) on May 8, 21

8 INFECTIOUS BURSAL AGENT 21 Hsuing, G. D., 15. Use of ultrafiltration for animal virus grouping. Bacterid. Rev. 2: Ketler, A., V. V. Hamparian and M. R. Hileman, 12. Characterization and classification of ECHO 28-rhinovirus-coryzavirus agents. Proc. Soc. Exptl. Biol. Med. 11: Quiroz, C. A., and R. P. Hanson, 158. Physicalchemical treatment of inocula as a means of separating and identifying avian viruses. Avian Ms. 2: 4-8. Reed, L. J., and H. Muench, 18. A simple method of estimating fifty per cent end points. Am. J. Hyg. 27: 4^7. Smith, D. T., N. F. Conant and J. R. Overman, 14. Microbiology. 1th Ed. Appleton-Century Crafts, New York, p. 5. Wallbank, A. M., and E. L. Stubb, 15. Sensitivity of strain R avian erythroblastosis virus to ether and chloroform. Proc. Soc. Exptl. Biol. Med. 12: KNOX and Marsden (154) reported while egg numbers in the Beltsville Small White turkey increased by selection in eight generations from 8 to 14 per hen, the fertility (Olsen and Marsden, 152) 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. (15) 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. (17) have shown late season declines 1 Oregon Agricultural Experiment Station Technical Paper Supported in part by a grant from the National Turkey Federation Winterfield, R. W., 1. Some newly identified infectious diseases. Twelfth Ann. New Hampshire Poultry Health Conf. p Winterfield, R. W., A. S. Cosgrove and S. B. Hitchner, 11. Avian nephrosis (Gumboro disease). L & M News and Views, 2 (8). Winterfield, R. W., and S. B. Hitchner, 12. Etiology of an infectious nephritis-nephrosis syndrome of chickens. Am. J. Vet. Res. 2: Winterfield, R. W., S. B. Hitchner, G. S. Appleton and A. S. Cosgrove, 12. Avian nephrosis, nephritis and Gumboro disease. L & M News and Views, : 1-. Winterfield, R. W., and S. B. Hitchner, 14. Gumboro disease anew threat to thepoultry industry. Poultry Digest, May, p Seasonal Decline in Fertility of Turkey Eggs 1 ' 2 J. A. HARPER AND G. H. ARSCOTT Department of Poultry Science, Oregon State University, Corvattis, Oregon 71 (Received for publication July 11, 1) 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 1 to 18 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 and the females on January 1. The ration fed from time of lighting was the OSU Station all-in-one breeder diet. Downloaded from at Penn State University (Paterno Lib) on May 8, 21