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Studies on the Protective Value of Newcastle-Immune Serum and Gamma Globulin Against Artificially Induced Newcastle Disease of Chickens1
Studies on the Protective Value of Newcastle-Immune Serum and Gamma Globulin Against Artificially Induced Newcastle Disease of Chickens1 J. J. VASINGTON,2 N. C. LAFFER, 3 A. P. HOLST2 AND H. M. DEVOLT 2 Department of Veterinary Science and Department of Microbiology, University of Maryland, College Park, Maryland (Received for publication February 4, 1960)
1 Condensation of a thesis submitted to the Graduate School as a partial fulfillment of the requirements for the Master of Science degree in the Department of Microbiology at the University of Maryland, College Park, Md. Supported in part by funds granted by the U.S. Department of Agriculture for the Investigation of Respiratory Diseases of Poultry (NE-S). Scientific Article No. A-829. Contribution No. 3109 of the Maryland Agricultural Experiment Station (Department of Veterinary Science). 2 Department of Veterinary Science. 3 Department of Microbiology.
lands, several investigations bearing on this problem have been carried out. Lucam (1950) reported on the use of hyperimmune serum produced in turkeys and injected into chickens. Various inoculations were made with immune serum and with virus. As a result, it was concluded that immune serum prolonged the incubation period of the disease but failed to provide complete protection. Schneider (19S2) conducted similar studies with hyperimmune serum from turkeys. Turkeys were hyperimmunized by increasing doses of inactivated virus administered at 5-day intervals. The hemagglutination-inhibition (HAI) serum titer of immunized birds ranged from 1:1200 to 1:2000 when 8 units of virus were used. Immune serum from several birds was pooled and used to inject chickens of various ages. Baby chicks receiving 0.05 ml. and young birds receiving 0.5 ml. were protected against challenge with 106 lethal doses of virus. In a group of 270 chickens (including 28 sick birds) that received simultaneous inoculation of 2 ml. immune serum and 1 ml. vaccine per kilogram of body weight, 24 birds died and 246 remained healthy. Beaudette and Bivins (1953) studied the influence of acquired passive immunity on the response to intramuscular and intranasal administration of Newcastle disease virus. These authors observed that the effect of immune serum followed by intramuscular injection of virus the next day resulted in low levels of HAI
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"VTEWCASTLE disease has constituted a *• ^ serious menace to the poultry industry of the United States for more than a decade. While live-virus vaccines provide a substantial measure of protection against the ailment, they have failed in some respects to measure up to the standards of a perfect immunizing agent. The incidence of Newcastle disease has not decreased in some sections of the land where vaccines have been widely used. In addition, some vaccines fail to provide uniform and lasting immunity. In other instances, complicating respiratory disease has been troublesome in flocks for a time after the application of some vaccines. In view of these facts, studies designed to investigate the possibility of employing other methods of immunization are warranted. In this country, little attention has been given to the possible use of immune serum in combating the ravages of Newcastle disease on the poultry industry. In foreign
PROTECTION AGAINST NEWCASTLE
Schmittle (1952) fractionated Newcastle disease chicken antisera with cold ethanol in accordance with the method described by Nichol and Deutsch (1950), who had obtained a 30 to 35% yield of gamma globulin. Schmittle found that gamma globulins were rich in hemagglutination-inhibition and serum neutralization antibodies. He suggested that considerable activity in the waste fractions indicated a poor quantitative yield of gamma globulin. Dubert et al. (1953) reported the use of cold methanol
for separation of the serum globulin fractions and when analyzed electrophoretically, appeared to indicate a considerably larger yield of gamma globulin. Although they employed rabbit serum, it was suggested that the method might be applicable for other animal sera. Hanson et al. (1950) reported that separation of the gamma globulin component of NDV bovine-antisera against NDV did not alter the activity of NDV antibodies. MATERIALS AND METHODS
The general procedure in conducting studies on the protective value of Newcastle-immune serum and gamma globulin against Newcastle disease of chickens was to inject susceptible chickens with serum and globulin and expose them to the disease artificially by intramuscular injection of Newcastle virus. The susceptibility rate of groups of experimental birds subjected to various experimental procedures was determined on the basis of birds developing symptoms and dying from the disease. Statistical analysis of variations of incubation period, death rate and rate of susceptibility were made by calculation of the chi-square test (Snedecor, 1946). Experimental Chickens Employed. Experimental chickens 4 to 5 weeks of age were employed. These birds were Barred Plymouth Rock-New Hampshire cross-bred chickens of mixed sexes. The average body weight when placed on test was 1.25 lbs. They were hatched from parent stock that had not been vaccinated and had no history of respiratory disease. Before being placed on test representative numbers (5 to 10 birds from groups of 20 to 30) were determined to be negative to the HAI blood test for Newcastle. All experimental birds were confined in wire cages to facilitate observation. Newcastle-Immune Serum Used as a
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titers indicating a neutralization by artificially acquired antibodies. Moynihan et al. (1954) explored the possibility of the use of antiserum of equine origin as a means of conferring passive immunity to chicks that had previously been exposed to Newcastle disease virus by the respiratory route. These investigators reported that a single dose of 0.5 ml. of antiserum administered either 24, 48, or 72 hours after virus exposure was of no value in preventing the development of Newcastle disease in chicks 3 weeks of age. In an attempt to confer an immediate and lasting immunity, Phillips (1956) studied the effect of plasma and yolk in combination with live virus. The plasma was obtained from birds that survived a natural outbreak of the disease and the yolk was obtained from eggs produced by the same flock. The routes of inoculation and immunizing materials were varied. The results of these studies showed that all chickens receiving simultaneous inoculations of the plasma-yolk mixture and virus, and those which received the virus two days later, survived challenge with artificial inoculation. With a four-day interval between protection and challenge, there was a prolonged incubation period following challenge by artificial inoculation. Chickens received no protection when a period of eight days elapsed between immunization and challenge.
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J. J. VASINGTON, N. C. LAFFER, A. P. HOLST AND H. M. DEVOLT
Hemagglutination-Inhibition Tests of Newcastle-Immune Serum and Gamma Globulin. Hemagglutination-inhibition tests (HAI) were conducted on reconstituted gamma globulin samples of fractionated Newcastle-immune serum to determine antibody activity in comparison with unfractionated serum. The beta procedure as described by Cunningham (1956) was employed. Tests of whole serum and gamma globulin fraction were made simultaneously against antigen (chorio-allantoic fluid) prepared with Newcastle virus strain #11914 (California). Eight hemagglutinating (HA) units of virus per 0.25 ml. were used. Electrophoretic Analysis of NewcastleImmune Serum and Gamma Globulin. In order to determine relative homogeniety of the gamma globulin fraction of immune serum, electrophoretic analysis was carried out. Gamma globulin and whole serum were suspended in physiological saline and adMethod of Fractionating Newcastle-Im- justed to a protein content of approximately mune Serum to Obtain Gamma Globulin. 1%. Serum and globulin were dialyzed Gamma globulin was fractionated from against 100 volumes of Veronal buffer whole serum by a modification of the (sodium diethyl barbiturate) at 4°C. in an method of Dubert et al. (1953). Eight ml. Aminco mechanical dialyzer. Veronal buffer of acetate buffer were added to 16 ml. of of pH 8.5 and ionic strength (u) of 0.1 was serum and the temperature of the mixture used. Analysis was carried out by the movlowered to 0°C. in an ice bath. Fifty-six ing boundry method using an Amincoml. of 30% aqueous methanol cooled to Stern electrophoresis apparatus (American — 20°C. were then added slowly (dropwise Instrument Co.). Immune serum and for the first 10 ml.) with constant stirring. gamma globulin were analyzed simultaneThe mixture was allowed to stand for 30 ously in standard clinical cells at 1.6°C; minutes and the precipitate removed by the boundaries were observed with a Thocentrifugation in a refrigerator. The pre- vert-Philpot-Svensson optical system and cipitated globulin from 16 ml. of serum was recorded on photographic film. Analysis was reconstituted in 4 ml. of phosphated saline. conducted for 160 to 180 minutes with a Traces of methanol subsequently were re- potential gradient of 5.6 volts per cm. moved by dialysis against a phosphated Total Nitrogen Determination of Fracphysiological saline solution in a refriger- tionated Lots of Gamma Globulin. As a ator (6°C.) for 24 hours. The dialyzed means of determining the uniformity of gamma globulin solution was then filtered several lots of fractionated gamma globulin, through a Seitz (EK) pad and stored in a total nitrogen content was used as a standdeep freeze refrigerator at — 60°C. until ard. The nitrogen content was determined used. by the micro-Kjeldahl method involving di-
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Source of Gamma Globulin. Newcastle-immune serum used as a source of gamma globulin was salvaged from a slaughter house dressing 9- to 10-week-old chickens. Blood was collected from the drain board in the killing room; the serum was removed from clotted blood and filtered through a Seitz (EK) pad to remove bacterial contamination. Serum was stored in a deep freeze refrigerator ( —60°C.) until used. The birds from which immune serum was obtained had been vaccinated during the first and fourth week with live-virus Newcastle vaccine. In addition during the first week they were vaccinated simultaneously with infectious bronchitis vaccine. Laryngotracheitis vaccine was applied at the age of 5 weeks. Newcastle-immune serum obtained in this manner was tested by the serum neutralization test in embryonating chicken eggs (Cunningham, 1956). It had a neutralizing index (NI) of 10G.
PROTECTION AGAINST NEWCASTLE
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rect Nesslerization, a 1:1 H 2 S0 4 digestion gamma globulin and unfractionated serum mixture, and the Nessler reagent of Fisher in various amounts administered 2 weeks (Solution No. FE-32). Optical densities of before challenge. the Nesslerized solutions and ammonium Five groups each containing 20 birds sulfate standards were determined with a were employed. The first 3 groups were inFisher electrophotometer. The factor 6.25 jected with 0.25 ml, 0.50 ml. and 1.0 ml. was used as a protein conversion factor for of gamma globulin respectively. The fourth the nitrogen values obtained. group received 4 ml. of unfractionated imTest of Gamma Globulin for Contami- mune serum. The fifth group remained unnating Virus. Pooled lots of gamma globu- treated and served as a control. All birds lin were tested by the hemagglutination received a challenge dose of 1,000 ELD 50 method and by chicken embryo inoculation units in 0.1 ml. of virus 2 weeks after the for contaminating virus. In addition 0.25 passive immunization. ml. of live Newcastle virus was added to Trial 2. The second trial was conducted 16 ml. of normal (nonimmune) chicken to determine the effect of larger amounts of serum which provided 1.56 X 107 ELD50 immunizing agent injected one week before units per ml. This serum subsequently was challenge. A total of 100 experimental birds fractionated by the methanol method and was employed in 5 groups each containing examined for virus as described above. 20 birds. Method of Challenging Immunized The first two groups were injected with 1 Chickens. Immunized chickens were chal- and 2 ml. of immune gamma globulin relenged by intramuscular injection of New- spectively. The third and fourth groups castle virus strain #11914 (California). were injected with 4 and 8 ml. of immune Challenge virus was inoculated intramuscu- serum respectively. The fifth group was not larly in a volume of 0.1 ml. This virus had immunized and served as a control. All an ELD60 of 10"6-7 (Reed-Muench, 1938). birds were challenged as in the previous exUnless otherwise stated the challenge dose periment with 1,000 ELD 50 units of Newwas 1,000 ELD50 units. castle virus. Trial 3. In the third trial the time of Immunization and Challenge of Groups challenge after passive immunization was of Experimental Chickens to Determine varied. Five groups each containing 20 Protective Value of Immune Gamma Globubirds were employed. The first four groups lin. In studies designed to investigate the received an intramuscular injection of 2.0 protective value of immune gamma globulin ml. of immune gamma globulin. The fifth against artificially induced Newcastle disgroup remained untreated as a control unit. ease, immune gamma globulin was comThe first three groups were challenged with pared with unfractionated immune serum 1,000 ELD50 units of Newcastle virus 3, 2, and the amount of both immunizing agents and 1 week after immunization. The fourth was systematically varied. The time of inoculation was varied with respect to chal- group was challenged with the same virus lenge as well as the number of infective dose simultaneously with immunization. In units of virus used for challenge. A total of addition a hemagglutination-inhibition test 480 chickens were used in 5 trials. All ex- was made 19 days after challenge on the periments were terminated 3 weeks after serum of 5 surviving birds in each group. challenge. Trial 4. Since in trial # 3 , immune gamma Trial 1. The first trial was conducted to globulin administered 3 weeks before chaldetermine the protective value of immune lenge was partially effective against artifi-
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J. J. VASINGTON, N. C. LAFFER, A. P. HOLST AND H. M. DEVOLT
TABLE 1.—Hemagglutination-inhibition titer of Newcastle-immune serum and gammaTiter of gamma globulin fractionation samples1
Titer of unfractionated immune serum
1
2
3
4
5
6
160
640
640
640+
640+
640
640
1
Each sample represents a pooling of gamma globulin from 3 separate protein fractionations.
RESULTS
Hemagglutination-inhibition Titer of Newcastle-Immune Serum and Gamma Globulin Fraction. The hemagglutination-
inhibition titer (HAI) of reconstituted gamma globulin from several fractionation lots of immune serum is shown in Table 1. The HAI titer of unfractionated serum was 160 whereas the titer of gamma globulin fraction quite consistently was 640. Gamma globulin lots # 3 and # 4 were slightly above 640. Since the gamma globulin fraction from 16 ml. of serum was reconstituted in 4 ml. of diluent, the increased titer of the gamma globulin fraction is consistent with the dilution factor. Alpha and beta globulin fractions obtained by the methanol method of serum fractionation were tested for antibody activity by the hemagglutination-inhibition method and found to be negative. Electropkoretic Analysis of Immune Serum and Gamma Globulin. Line diagrams reproduced from photographic films of ascending arm migration boundaries observed with the Thovert-Philpot-Svensson optical system of unfractionated Newcastle-immune serum and its gamma globulin component are shown in Figures 1 and 2 respectively. Single peaks in both figures represent starting peaks. In Figure 1, three peaks of unfractionated serum corresponding to albumin (A), alpha-beta globulin (B) and gamma globulin (C) are shown. Electrophoretic mobility of 7.5 X lO"5 cm.2/volt/sec. was obtained for albumin. Electrophoretic mobility for alpha-beta gloulin was 4.9 X 10-5 cm.2/volt/sec. and 2.5 X 10-5 cm.2/volt/sec. for gamma globulin. Figure 2 shows a single peak for gamma globulin with a mobility of 2.5 X 10-5
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daily induced Newcastle disease, the fourth trial was conducted for the purpose of extending the length of time between immunization and challenge. In this experiment, 90 experimental chickens were used in 6 groups each containing 15 chickens. The first four groups received 2 ml. of immune gamma globulin 6, 5, 4 and 3 weeks respectively before challenge. The fifth group was challenged simultaneously with immunization and the sixth group remained untreated. Trial 5. This experiment was carried out to determine the effectiveness of immune gamma globulin administered simultaneously with increasing doses of Newcastle virus. Five groups of chickens containing IS birds each were used. All groups were injected with 2 ml. immune gamma globulin simultaneously with challenge. The first group received 0.1 ml. of a 10_1 dilution of virus containing 105 ELD50 units of Newcastle virus. The second group received 0.1 ml. of a 10"3 dilution containing 104 ELD50 units, the third group received 0.1 ml. of a 10"5 dilution containing 102 ELD50 units, the fourth group received 0.1 ml. of a 10~7 dilution containing one theoretical ELD 50 unit of virus. A fifth group received 0.1 ml. of a 10"4 dilution of virus containing 103 ELD50 units of virus and was left untreated as a virus-control unit.
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PROTECTION AGAINST NEWCASTLE A
—
*
albu min
a Ipl a-beta
C —H /V
starting peal
ga Tima
! A = 7 5 X I0' cm-/vo t/sec. B = 4.9 X 10 •cm'/volt/sec. C = 2.5 X lO'crrr/volt/sec.
cm.2/volt/sec. These electrophoretic patterns of serum and gamma globulin with the same mobility for gamma globulin show that a relatively homogeneous solution and a complete fractionation of gamma globulin was obtained by the methanol method. Total Nitrogen Determination. MicroKjeldahl determinations on the various fractionation lots of gamma globulin showed an average of 22 mg. protein per ml. The minimum amount determined was 18 mg./ml. and the maximum 25 mg./ml. Test of Gamma Globulin for Contaminating Virus. Both hemagglutination and chicken embryo inoculation tests for contaminating virus in pooled lots of gamma
starting peak
A = 2.5 X 10 s cnv/volt/sec.
FIG. 2. Electrophoretic pattern of gamma globulin. Ascending arm. Veronal buffer, pH 8.S, n 0.1. Potential gradient 5.6 volts/cm.
TABLE 2.—Protective value of Newcastle-immune serum and immune gamma globulin administered in various amounts two weeks before challenge by intramuscular injection with Newcastle virus
Amount of
Group number
agent injected
1 2 3 4 5
0.25 ml. G.G. 1 0.50 ml. G.G. 1.00 ml. G.G. 4.00 ml. I.S.2 None (control)
1 G.G.—Gamma globulin. 2 1.S.—Immune serum. 8
Incubation Susceptiperiod per group Death rate bilitya (percent) rate (days) (geometric (percent) mean) 4.7 3.8 4.8 4.5 4.2
Includes sick and dead birds.
60 65 55 60 80
75 75 75 60 90
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FIG. 1. Electrophoretic pattern of immune serum. Ascending arm. Veronal buffer, pH 8.S, n 0.1. Potential gradient 5.6 volts/cm.
globulin were negative. Similar tests also were negative when live Newcastle virus was added to unfractionated immune serum which subsequently was subjected to the methanol fractionation process. Immunization and Challenge of Groups of Experimental Chickens to Determine the Protective Value of Immune Serum and Immune Gamma Globulin. The protective value of immune serum and gamma globulin varied with the amount of immunizing agent administered, the time of administration with respect to challenge and the number of infective units of virus employed for challenge. Trial 1. The results obtained when immune gamma globulin and unfractionated immune serum were injected in various amounts 2 weeks before challenge are given in Table 2. The geometric mean incubation period was 4.8 days in the group receiving 1.0 ml. immune gamma globulin and the shortest geometric mean incubation period was 3.8 days in the group receiving 0.50 ml. gamma globulin. A significant difference between the death rate of the 4th group injected with 4 ml. of immune serum (60%) and the untreated group (80%) was observed. A significant difference in the rate of susceptibility between these two groups also was in evidence.
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J. J. VASINGTON, N. C. LAFFER, A. P. HOLST AND H. M. DEVOLT
TABLE 3.—Protective value of gamma globulin and unfractionated immune serum administered in various amounts one week before challenge with NDV Incubation Group numb
A«-ntof
<*
P^
a S injected S d agent
d 0
<<&•> (Arithmetic
Death
S ^ -
,_££,« (percent)
(percent)
rate
5 5 5 0 30
10 10 20 10 50
average) 1 2 3 4 5
4.5 11.0 8.5 10.0 7.0
G.G.—Gamma globulin. I.S.—Immune serum. Includes sick and dead birds.
Trial 2. The results obtained, when the amount of immunizing agent was increased and the time interval between immunization and challenge was shortened to one week, are given in Table 3. In computing the incubation period of the various groups, arithmetic averages are used instead of a geometric mean because of the small number of birds in any group that developed symptoms or died. In the second group, receiving 2.0 ml. and in the fourth group, receiving 8 ml. of immune serum, the average number of days in the incubation period was 11 and 10 respectively. These averages differ significantly from the average incubation period of the untreated group which was 7 days. The percentage death and susceptibility rate in all immunized groups varied significantly from the untreated group. The lowest death and
TABLE 4.—Protective value of 2 ml. of gamma globulin administered at various time ' • before challenge with NDV
1
Group number
Time of immunization before challenge
1 2 3 4 5
3 weeks 2 weeks 1 week simultaneous control
Includes sick and dead birds.
Incubation period per group (days) (geometric mean) 5.4 4.3 7.6 7.0(1 bird) 2.3
Death rate (percent)
Susceptibility1 rate (percent)
Geometric mean HAI serum titers 19 days after challenge
15 20 10 0 55
20 25 15 5 85
194 42 56 128 169
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1 2 8
1.0 ml. G.G.i 2.0 ml. G.G. 4.0 ml. I.S.2 8.0 ml. I.S. None (control)
susceptibility rate in treated groups was zero and 10% respectively as compared with corresponding figures of 30% and 50% in the untreated group. Although the same number of ELD60 units (103) of virus was administered as challenge in this and the foregoing experiment, the lower susceptibility rate in this instance is attributed to the fact that the experiment was conducted in midsummer when the resistance of chickens to Newcastle disease is generally at a higher point. Trial 3. The results obtained when 2 ml. of immune gamma globulin were administered simultaneously and 1, 2 and 3 weeks before challenge are given in Table 4. The geometric mean number of days in the incubation period of the group challenged simultaneously with immunization was 7. The corresponding figure in the group immunized 1 week before challenge was 7.6. Both of these figures differ significantly from the geometric mean number of days in the incubation period of the untreated group which was 2.3. The death and susceptibility rates of all immunized groups differed significantly from the untreated group. The lowest death rate of an immunized group was zero (simultaneous group) and the highest was 20% in the group immunized 2 weeks before challenge as compared with a SS% death rate in the untreated group. The lowest rate of sus-
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PROTECTION AGAINST NEWCASTLE T A B L E 5.—Protective value of 2 ml. gamma _ administered at various time intervals before challenge with NDV
GrouD number
Incubation Time of period per Death immunizing group (days) rate before challenge (geometric (percent) mean) 6 weeks 5 weeks 4 weeks 3 weeks simultaneous control
1
2.9 3.4 3.3 3.4 5.6 3.5
Susceptibility' rate (percent)
40.0 73.3 53.3 40.0 13.3 46.6
46.6 80.0 66.6 60.0 26.6 53.3
Includes sick and dead birds.
Trial 5. The results obtained when 2 ml. of immune gamma globulin were used to immunize 4 groups of experimental chickens challenged with various doses of Newcastle virus ranging from 1 theoretical ELD50 unit to 105 ELD 50 units are given in Table 6. In group 4, receiving 1 theoreti-
T A B L E 6.—Protective value of simultaneous injection and decreasing concentrations of
Group number
1 2 3 4 5 1
A m t . of immunizing a g e n t injected 2 ml. G.G. plus
0.1 0.1 0.1 0.1 0.1
ml. virus ml. virus ml. virus ml. virus ml. virus
10-1 10-3 10-5 10-7 10- 4 (no G.G.)
Includes sick and dead birds.
of 2 ml. gamma NDV
globulin
No. ELD60 units N D V injected
I n c u b a t i o n period per group (days) (geometric mean)
Death rate (percent)
Susceptibility1 rate (percent)
W 10 4 102 1 theoretical 10 s
3.5 3.0 3.5 6.0 4.2
26.6 26.6 26.6 6.6 86.6
40.0 26.6 26.6 6.6 86.6
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ceptibility was 5% in the simultaneous group and the highest was 25% in the group immunized 2 weeks before challenge as compared with susceptibility rate of 85% in the untreated group. The geometric mean HAI serum titer of 5 surviving birds in groups immunized 3, 2 and 1 week before challenge was 194, 42 and 56 respectively. The titer of the group immunized simultaneously with challenge was 128 as compared with a titer of 169 in 5 birds from the untreated group. Challenge virus thus was most highly active in stimulating antibody in the group immunized 3 weeks before challenge. The relatively high serum titer in the group immunized simultaneously with challenge is not explained by these data. Trial 4. The results obtained when the period of time between immunization and challenge was extended from 2 to 6 weeks are given in Table 5. The fifth group, im-
munized simultaneously with challenge had a geometric mean number of days in the incubation period of 5.6 which differs significantly from the geometric mean number of days in the incubation period of the untreated group which was 3.5. Likewise the death and susceptibility rate of the simultaneous groups, 13.3% and 26.6% respectively differed significantly from corresponding figures of the untreated group of 46.6% and 53.3% respectively. The death and susceptibility rates of groups immunized from 3 to 6 weeks before challenge did not differ significantly from corresponding figures in the untreated group. The lowest death rate observed in these groups was 40% recorded for the group immunized 6 and 3 weeks before challenge. The highest rate of susceptibility was 80% in the group immunized 5 weeks before challenge. The relatively poor showing of the groups immunized 3 weeks before challenge as compared with the same period in the previous experiment when the same number of ELD50 units of challenge virus was employed are not explained.
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J. J. VASINGTON, N. C. LAFFER, A. P. HOLST AND H. M. DEVOLT
DISCUSSION
Results obtained in studies on the protective value of Newcastle-immune serum and gamma globulin against artificially induced Newcastle disease, indicate a possible place for serum therapy in controlling the disease. A cursory survey of the slaughter houses of the nation, show that much blood from immunized birds is being discarded. Salvage of Newcastle-immune serum from such sources and safeguarding it against viral contamination might place at the disposal of the industry an additional weapon against this disease. Particularly in the case of virus breaks in valuable stock, serum therapy could prove beneficial. The fact that chickens simultaneously treated with immune gamma globulin and challenged with Newcastle virus showed a relatively high geometric mean hemagglutination-inhibition serum titer as compared with birds treated with immune serum 1
and 2 weeks before challenge presents an interesting problem for further investigation. SUMMARY 1. The methanol method of fractionation of Newcastle-immune serum produced a relatively uniform yield of gamma globulin as indicated by hemagglutination-inhibition activity and protein analysis. 2. Intramuscular injection of 4 to 8 ml. of immune serum and 1 to 2 ml. gamma globulin simultaneosuly with, or 1 to 2 weeks prior to, challenge by intramuscular injection of Newcastle disease virus produced a significant degree of protection under the conditions of these experiments. 3. The protective value of immune globulin was roughly inversely proportional under the conditions of these experiments to the length of time elapsing between passive immunization and challenge. 4. Partial protection was obtained under the conditions of this experiment when 2 ml. of gamma globulin was administered to birds averaging 1.2S lbs. and receiving 105 ELD60 infective units of virus. REFERENCES Beaudette, F. R., and J. A. Bivins, 1953. The influence of passive immunity on the response to intramuscular and intranasal administration of Newcastle disease virus. Cornell Vet. 43: 513— 531. Cunningham, C. H., 1956. A Laboratory Guide in Virology. Burgess Publishing Co., Minneapolis. Dubert, J. M., P. Slizewicz, P. Rebeyrotte and M. Machebocuf, 1953. Nouvelle methode de separation des protiens seriques par le methanol. Application aux serums de lapin et de cheval. Annales de L'Institut Pasteur, 84: 370-375. Hanson, R. P., N. S. Winslow, C. A. Brandley and E. Upton, 1950. The antiviral activity of Newcastle disease immune sera. J. Bacterid. 60: 557-560. Lucam, F., 1950. Recherches sur la sero-prevention, la sero-vaccination et la sero-therapie, en milieu infecte de peste aviare. Revur de Medicine Veterinaire. Moynihan, I. W., R. V. L. Walker, E. P. E. Pow-
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cal ELD50 unit of Newcastle virus the geometric mean number of days in the incubation period differed significantly from the other three groups receiving a larger number of infective doses. The death rate and susceptibility rate of all treated groups varied significantly from the untreated groups challenged with 103 ELD50 units of virus. The death rate in treated groups varied from 6.6% in the group receiving one theoretical infective unit of virus to 26.6% for the other treated groups. The rate of susceptibility varied from 6.6% in the group receiving one theoretical unit of virus to 40% in the group receiving 105 ELD50 units of virus. Both the death and susceptibility rate in the untreated group receiving 10s ELD50 units of virus was 86.6%. Partial protection thus was provided by 2 ml. of immune globulin in chickens challenged with up to 105 ELD50 units of Newcastle virus.
PROTECTION AGAINST NEWCASTLE
ell and D. M. Cooper, 1954. An attempt to passively immunize chicks against the virus of Newcastle disease by the use of antiserum of equine origin. Canad. J. Biol. Med. 43: 62-64. Nichol, J. C, and H. F. Deutsch, 1948. Biophysical studies of blood plasma proteins. VII Separation of gamma-globulin from the sera of various animals. J. Amer. Chem. Soc. 70: 80-84. Phillips, L. V. A., 19S6. Ensayos sobre immunizacion passiva y tratamiento de la enfermedad de newcastle por uso de plasma y vitelo himerinmunes. Vira. Agr. (Lima E. sum.). 33: 111-113.
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Reed, L. J., and H. Muench, 1938. A simple method of estimating fifty percent end points. Amer. J. Hyg. 27: 493-497. Schmittle, S. C, 1952. Studies on Newcastle disease. XI. Newcastle disease antibodies in the serum globulin fractions of the chicken. Proc. 89th Ann. Meet. Amer. Vet. Assoc: 143-147. Schneider, L., 1952. Vaccination and serum treatment against fowl pest. (Newcastle disease). 14th Int. Vet. Congr. 2: 359-362. Snedecor, G. W., 1946. Statistical Methods. The Iowa State College Press, Fourth Edition.
C. W. POPE, 1 A. B. WATTS, ERNEST WILLIAMS 2 AND CLAYTON C. BKUNSON
Louisiana State University, Baton Rouge (Received for publication February 8, 1960)
T
HE seasonal decrease in egg shell quality (Jull, 1924; and Wilhelm, 1940), and the decrease in interior egg quality with increasing age of the bird (Knox and Godfrey, 1938; and Dawson et al., 1951) are problems which confront poultry breeders and commercial poultrymen. At least two theories have been advanced to explain the decline in shell quality. One theory proposes that the decrease is due to increased environmental temperatures. Another theory proposes that the decrease in shell quality is simply an exhaustion effect coming at the end of the laying year. There are considerable data which show that blood calcium and phosphorus levels are associated with egg formation. Riddle and Reinhart (1926), Hughes et al. (1927), Roepke and Hughes (1935), Taylor and 1
Present address: Ralston Purina Co., St. Louis, Mo. 2 Deceased.
Russell (1935), Correll and Hughes (1935) and Deobald et al. (1938) reported that the blood calcium level greatly increases as birds come into production. Deobald et al. (1938) also reported that total blood phosphorus showed a concurrent change similar to that of calcium. Conrad (1939) observed that blood calcium levels decreased by 25 to 30 percent as environmental temperatures increased from 70° to 90°F. This experiment was designed to study the effects of the length of time in production, intensity of production and stage of egg formation on certain egg quality measurements and blood constituents of laying hens. MATERIALS AND METHODS
Eighty-four New Hampshire pullets of a laying strain were used in this study. They were hatched in late March and housed on October 1 when they were approximately six months of age. Individual laying cages in a conventional-type laying
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The Effect of the Length of Time in Production and Stage of Egg Formation on Certain Egg Quality Measurements and Blood Constituents of Laying Hens
1 Condensation of a thesis submitted to the Graduate School as a partial fulfillment of the requirements for the Master of Science degree in the Department of Microbiology at the University of Maryland, College Park, Md. Supported in part by funds granted by the U.S. Department of Agriculture for the Investigation of Respiratory Diseases of Poultry (NE-S). Scientific Article No. A-829. Contribution No. 3109 of the Maryland Agricultural Experiment Station (Department of Veterinary Science). 2 Department of Veterinary Science. 3 Department of Microbiology.
lands, several investigations bearing on this problem have been carried out. Lucam (1950) reported on the use of hyperimmune serum produced in turkeys and injected into chickens. Various inoculations were made with immune serum and with virus. As a result, it was concluded that immune serum prolonged the incubation period of the disease but failed to provide complete protection. Schneider (19S2) conducted similar studies with hyperimmune serum from turkeys. Turkeys were hyperimmunized by increasing doses of inactivated virus administered at 5-day intervals. The hemagglutination-inhibition (HAI) serum titer of immunized birds ranged from 1:1200 to 1:2000 when 8 units of virus were used. Immune serum from several birds was pooled and used to inject chickens of various ages. Baby chicks receiving 0.05 ml. and young birds receiving 0.5 ml. were protected against challenge with 106 lethal doses of virus. In a group of 270 chickens (including 28 sick birds) that received simultaneous inoculation of 2 ml. immune serum and 1 ml. vaccine per kilogram of body weight, 24 birds died and 246 remained healthy. Beaudette and Bivins (1953) studied the influence of acquired passive immunity on the response to intramuscular and intranasal administration of Newcastle disease virus. These authors observed that the effect of immune serum followed by intramuscular injection of virus the next day resulted in low levels of HAI
1418
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"VTEWCASTLE disease has constituted a *• ^ serious menace to the poultry industry of the United States for more than a decade. While live-virus vaccines provide a substantial measure of protection against the ailment, they have failed in some respects to measure up to the standards of a perfect immunizing agent. The incidence of Newcastle disease has not decreased in some sections of the land where vaccines have been widely used. In addition, some vaccines fail to provide uniform and lasting immunity. In other instances, complicating respiratory disease has been troublesome in flocks for a time after the application of some vaccines. In view of these facts, studies designed to investigate the possibility of employing other methods of immunization are warranted. In this country, little attention has been given to the possible use of immune serum in combating the ravages of Newcastle disease on the poultry industry. In foreign
PROTECTION AGAINST NEWCASTLE
Schmittle (1952) fractionated Newcastle disease chicken antisera with cold ethanol in accordance with the method described by Nichol and Deutsch (1950), who had obtained a 30 to 35% yield of gamma globulin. Schmittle found that gamma globulins were rich in hemagglutination-inhibition and serum neutralization antibodies. He suggested that considerable activity in the waste fractions indicated a poor quantitative yield of gamma globulin. Dubert et al. (1953) reported the use of cold methanol
for separation of the serum globulin fractions and when analyzed electrophoretically, appeared to indicate a considerably larger yield of gamma globulin. Although they employed rabbit serum, it was suggested that the method might be applicable for other animal sera. Hanson et al. (1950) reported that separation of the gamma globulin component of NDV bovine-antisera against NDV did not alter the activity of NDV antibodies. MATERIALS AND METHODS
The general procedure in conducting studies on the protective value of Newcastle-immune serum and gamma globulin against Newcastle disease of chickens was to inject susceptible chickens with serum and globulin and expose them to the disease artificially by intramuscular injection of Newcastle virus. The susceptibility rate of groups of experimental birds subjected to various experimental procedures was determined on the basis of birds developing symptoms and dying from the disease. Statistical analysis of variations of incubation period, death rate and rate of susceptibility were made by calculation of the chi-square test (Snedecor, 1946). Experimental Chickens Employed. Experimental chickens 4 to 5 weeks of age were employed. These birds were Barred Plymouth Rock-New Hampshire cross-bred chickens of mixed sexes. The average body weight when placed on test was 1.25 lbs. They were hatched from parent stock that had not been vaccinated and had no history of respiratory disease. Before being placed on test representative numbers (5 to 10 birds from groups of 20 to 30) were determined to be negative to the HAI blood test for Newcastle. All experimental birds were confined in wire cages to facilitate observation. Newcastle-Immune Serum Used as a
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titers indicating a neutralization by artificially acquired antibodies. Moynihan et al. (1954) explored the possibility of the use of antiserum of equine origin as a means of conferring passive immunity to chicks that had previously been exposed to Newcastle disease virus by the respiratory route. These investigators reported that a single dose of 0.5 ml. of antiserum administered either 24, 48, or 72 hours after virus exposure was of no value in preventing the development of Newcastle disease in chicks 3 weeks of age. In an attempt to confer an immediate and lasting immunity, Phillips (1956) studied the effect of plasma and yolk in combination with live virus. The plasma was obtained from birds that survived a natural outbreak of the disease and the yolk was obtained from eggs produced by the same flock. The routes of inoculation and immunizing materials were varied. The results of these studies showed that all chickens receiving simultaneous inoculations of the plasma-yolk mixture and virus, and those which received the virus two days later, survived challenge with artificial inoculation. With a four-day interval between protection and challenge, there was a prolonged incubation period following challenge by artificial inoculation. Chickens received no protection when a period of eight days elapsed between immunization and challenge.
1419
1420
J. J. VASINGTON, N. C. LAFFER, A. P. HOLST AND H. M. DEVOLT
Hemagglutination-Inhibition Tests of Newcastle-Immune Serum and Gamma Globulin. Hemagglutination-inhibition tests (HAI) were conducted on reconstituted gamma globulin samples of fractionated Newcastle-immune serum to determine antibody activity in comparison with unfractionated serum. The beta procedure as described by Cunningham (1956) was employed. Tests of whole serum and gamma globulin fraction were made simultaneously against antigen (chorio-allantoic fluid) prepared with Newcastle virus strain #11914 (California). Eight hemagglutinating (HA) units of virus per 0.25 ml. were used. Electrophoretic Analysis of NewcastleImmune Serum and Gamma Globulin. In order to determine relative homogeniety of the gamma globulin fraction of immune serum, electrophoretic analysis was carried out. Gamma globulin and whole serum were suspended in physiological saline and adMethod of Fractionating Newcastle-Im- justed to a protein content of approximately mune Serum to Obtain Gamma Globulin. 1%. Serum and globulin were dialyzed Gamma globulin was fractionated from against 100 volumes of Veronal buffer whole serum by a modification of the (sodium diethyl barbiturate) at 4°C. in an method of Dubert et al. (1953). Eight ml. Aminco mechanical dialyzer. Veronal buffer of acetate buffer were added to 16 ml. of of pH 8.5 and ionic strength (u) of 0.1 was serum and the temperature of the mixture used. Analysis was carried out by the movlowered to 0°C. in an ice bath. Fifty-six ing boundry method using an Amincoml. of 30% aqueous methanol cooled to Stern electrophoresis apparatus (American — 20°C. were then added slowly (dropwise Instrument Co.). Immune serum and for the first 10 ml.) with constant stirring. gamma globulin were analyzed simultaneThe mixture was allowed to stand for 30 ously in standard clinical cells at 1.6°C; minutes and the precipitate removed by the boundaries were observed with a Thocentrifugation in a refrigerator. The pre- vert-Philpot-Svensson optical system and cipitated globulin from 16 ml. of serum was recorded on photographic film. Analysis was reconstituted in 4 ml. of phosphated saline. conducted for 160 to 180 minutes with a Traces of methanol subsequently were re- potential gradient of 5.6 volts per cm. moved by dialysis against a phosphated Total Nitrogen Determination of Fracphysiological saline solution in a refriger- tionated Lots of Gamma Globulin. As a ator (6°C.) for 24 hours. The dialyzed means of determining the uniformity of gamma globulin solution was then filtered several lots of fractionated gamma globulin, through a Seitz (EK) pad and stored in a total nitrogen content was used as a standdeep freeze refrigerator at — 60°C. until ard. The nitrogen content was determined used. by the micro-Kjeldahl method involving di-
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Source of Gamma Globulin. Newcastle-immune serum used as a source of gamma globulin was salvaged from a slaughter house dressing 9- to 10-week-old chickens. Blood was collected from the drain board in the killing room; the serum was removed from clotted blood and filtered through a Seitz (EK) pad to remove bacterial contamination. Serum was stored in a deep freeze refrigerator ( —60°C.) until used. The birds from which immune serum was obtained had been vaccinated during the first and fourth week with live-virus Newcastle vaccine. In addition during the first week they were vaccinated simultaneously with infectious bronchitis vaccine. Laryngotracheitis vaccine was applied at the age of 5 weeks. Newcastle-immune serum obtained in this manner was tested by the serum neutralization test in embryonating chicken eggs (Cunningham, 1956). It had a neutralizing index (NI) of 10G.
PROTECTION AGAINST NEWCASTLE
1421
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rect Nesslerization, a 1:1 H 2 S0 4 digestion gamma globulin and unfractionated serum mixture, and the Nessler reagent of Fisher in various amounts administered 2 weeks (Solution No. FE-32). Optical densities of before challenge. the Nesslerized solutions and ammonium Five groups each containing 20 birds sulfate standards were determined with a were employed. The first 3 groups were inFisher electrophotometer. The factor 6.25 jected with 0.25 ml, 0.50 ml. and 1.0 ml. was used as a protein conversion factor for of gamma globulin respectively. The fourth the nitrogen values obtained. group received 4 ml. of unfractionated imTest of Gamma Globulin for Contami- mune serum. The fifth group remained unnating Virus. Pooled lots of gamma globu- treated and served as a control. All birds lin were tested by the hemagglutination received a challenge dose of 1,000 ELD 50 method and by chicken embryo inoculation units in 0.1 ml. of virus 2 weeks after the for contaminating virus. In addition 0.25 passive immunization. ml. of live Newcastle virus was added to Trial 2. The second trial was conducted 16 ml. of normal (nonimmune) chicken to determine the effect of larger amounts of serum which provided 1.56 X 107 ELD50 immunizing agent injected one week before units per ml. This serum subsequently was challenge. A total of 100 experimental birds fractionated by the methanol method and was employed in 5 groups each containing examined for virus as described above. 20 birds. Method of Challenging Immunized The first two groups were injected with 1 Chickens. Immunized chickens were chal- and 2 ml. of immune gamma globulin relenged by intramuscular injection of New- spectively. The third and fourth groups castle virus strain #11914 (California). were injected with 4 and 8 ml. of immune Challenge virus was inoculated intramuscu- serum respectively. The fifth group was not larly in a volume of 0.1 ml. This virus had immunized and served as a control. All an ELD60 of 10"6-7 (Reed-Muench, 1938). birds were challenged as in the previous exUnless otherwise stated the challenge dose periment with 1,000 ELD 50 units of Newwas 1,000 ELD50 units. castle virus. Trial 3. In the third trial the time of Immunization and Challenge of Groups challenge after passive immunization was of Experimental Chickens to Determine varied. Five groups each containing 20 Protective Value of Immune Gamma Globubirds were employed. The first four groups lin. In studies designed to investigate the received an intramuscular injection of 2.0 protective value of immune gamma globulin ml. of immune gamma globulin. The fifth against artificially induced Newcastle disgroup remained untreated as a control unit. ease, immune gamma globulin was comThe first three groups were challenged with pared with unfractionated immune serum 1,000 ELD50 units of Newcastle virus 3, 2, and the amount of both immunizing agents and 1 week after immunization. The fourth was systematically varied. The time of inoculation was varied with respect to chal- group was challenged with the same virus lenge as well as the number of infective dose simultaneously with immunization. In units of virus used for challenge. A total of addition a hemagglutination-inhibition test 480 chickens were used in 5 trials. All ex- was made 19 days after challenge on the periments were terminated 3 weeks after serum of 5 surviving birds in each group. challenge. Trial 4. Since in trial # 3 , immune gamma Trial 1. The first trial was conducted to globulin administered 3 weeks before chaldetermine the protective value of immune lenge was partially effective against artifi-
1422
J. J. VASINGTON, N. C. LAFFER, A. P. HOLST AND H. M. DEVOLT
TABLE 1.—Hemagglutination-inhibition titer of Newcastle-immune serum and gammaTiter of gamma globulin fractionation samples1
Titer of unfractionated immune serum
1
2
3
4
5
6
160
640
640
640+
640+
640
640
1
Each sample represents a pooling of gamma globulin from 3 separate protein fractionations.
RESULTS
Hemagglutination-inhibition Titer of Newcastle-Immune Serum and Gamma Globulin Fraction. The hemagglutination-
inhibition titer (HAI) of reconstituted gamma globulin from several fractionation lots of immune serum is shown in Table 1. The HAI titer of unfractionated serum was 160 whereas the titer of gamma globulin fraction quite consistently was 640. Gamma globulin lots # 3 and # 4 were slightly above 640. Since the gamma globulin fraction from 16 ml. of serum was reconstituted in 4 ml. of diluent, the increased titer of the gamma globulin fraction is consistent with the dilution factor. Alpha and beta globulin fractions obtained by the methanol method of serum fractionation were tested for antibody activity by the hemagglutination-inhibition method and found to be negative. Electropkoretic Analysis of Immune Serum and Gamma Globulin. Line diagrams reproduced from photographic films of ascending arm migration boundaries observed with the Thovert-Philpot-Svensson optical system of unfractionated Newcastle-immune serum and its gamma globulin component are shown in Figures 1 and 2 respectively. Single peaks in both figures represent starting peaks. In Figure 1, three peaks of unfractionated serum corresponding to albumin (A), alpha-beta globulin (B) and gamma globulin (C) are shown. Electrophoretic mobility of 7.5 X lO"5 cm.2/volt/sec. was obtained for albumin. Electrophoretic mobility for alpha-beta gloulin was 4.9 X 10-5 cm.2/volt/sec. and 2.5 X 10-5 cm.2/volt/sec. for gamma globulin. Figure 2 shows a single peak for gamma globulin with a mobility of 2.5 X 10-5
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daily induced Newcastle disease, the fourth trial was conducted for the purpose of extending the length of time between immunization and challenge. In this experiment, 90 experimental chickens were used in 6 groups each containing 15 chickens. The first four groups received 2 ml. of immune gamma globulin 6, 5, 4 and 3 weeks respectively before challenge. The fifth group was challenged simultaneously with immunization and the sixth group remained untreated. Trial 5. This experiment was carried out to determine the effectiveness of immune gamma globulin administered simultaneously with increasing doses of Newcastle virus. Five groups of chickens containing IS birds each were used. All groups were injected with 2 ml. immune gamma globulin simultaneously with challenge. The first group received 0.1 ml. of a 10_1 dilution of virus containing 105 ELD50 units of Newcastle virus. The second group received 0.1 ml. of a 10"3 dilution containing 104 ELD50 units, the third group received 0.1 ml. of a 10"5 dilution containing 102 ELD50 units, the fourth group received 0.1 ml. of a 10~7 dilution containing one theoretical ELD 50 unit of virus. A fifth group received 0.1 ml. of a 10"4 dilution of virus containing 103 ELD50 units of virus and was left untreated as a virus-control unit.
1423
PROTECTION AGAINST NEWCASTLE A
—
*
albu min
a Ipl a-beta
C —H /V
starting peal
ga Tima
! A = 7 5 X I0' cm-/vo t/sec. B = 4.9 X 10 •cm'/volt/sec. C = 2.5 X lO'crrr/volt/sec.
cm.2/volt/sec. These electrophoretic patterns of serum and gamma globulin with the same mobility for gamma globulin show that a relatively homogeneous solution and a complete fractionation of gamma globulin was obtained by the methanol method. Total Nitrogen Determination. MicroKjeldahl determinations on the various fractionation lots of gamma globulin showed an average of 22 mg. protein per ml. The minimum amount determined was 18 mg./ml. and the maximum 25 mg./ml. Test of Gamma Globulin for Contaminating Virus. Both hemagglutination and chicken embryo inoculation tests for contaminating virus in pooled lots of gamma
starting peak
A = 2.5 X 10 s cnv/volt/sec.
FIG. 2. Electrophoretic pattern of gamma globulin. Ascending arm. Veronal buffer, pH 8.S, n 0.1. Potential gradient 5.6 volts/cm.
TABLE 2.—Protective value of Newcastle-immune serum and immune gamma globulin administered in various amounts two weeks before challenge by intramuscular injection with Newcastle virus
Amount of
Group number
agent injected
1 2 3 4 5
0.25 ml. G.G. 1 0.50 ml. G.G. 1.00 ml. G.G. 4.00 ml. I.S.2 None (control)
1 G.G.—Gamma globulin. 2 1.S.—Immune serum. 8
Incubation Susceptiperiod per group Death rate bilitya (percent) rate (days) (geometric (percent) mean) 4.7 3.8 4.8 4.5 4.2
Includes sick and dead birds.
60 65 55 60 80
75 75 75 60 90
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FIG. 1. Electrophoretic pattern of immune serum. Ascending arm. Veronal buffer, pH 8.S, n 0.1. Potential gradient 5.6 volts/cm.
globulin were negative. Similar tests also were negative when live Newcastle virus was added to unfractionated immune serum which subsequently was subjected to the methanol fractionation process. Immunization and Challenge of Groups of Experimental Chickens to Determine the Protective Value of Immune Serum and Immune Gamma Globulin. The protective value of immune serum and gamma globulin varied with the amount of immunizing agent administered, the time of administration with respect to challenge and the number of infective units of virus employed for challenge. Trial 1. The results obtained when immune gamma globulin and unfractionated immune serum were injected in various amounts 2 weeks before challenge are given in Table 2. The geometric mean incubation period was 4.8 days in the group receiving 1.0 ml. immune gamma globulin and the shortest geometric mean incubation period was 3.8 days in the group receiving 0.50 ml. gamma globulin. A significant difference between the death rate of the 4th group injected with 4 ml. of immune serum (60%) and the untreated group (80%) was observed. A significant difference in the rate of susceptibility between these two groups also was in evidence.
1424
J. J. VASINGTON, N. C. LAFFER, A. P. HOLST AND H. M. DEVOLT
TABLE 3.—Protective value of gamma globulin and unfractionated immune serum administered in various amounts one week before challenge with NDV Incubation Group numb
A«-ntof
<*
P^
a S injected S d agent
d 0
<<&•> (Arithmetic
Death
S ^ -
,_££,« (percent)
(percent)
rate
5 5 5 0 30
10 10 20 10 50
average) 1 2 3 4 5
4.5 11.0 8.5 10.0 7.0
G.G.—Gamma globulin. I.S.—Immune serum. Includes sick and dead birds.
Trial 2. The results obtained, when the amount of immunizing agent was increased and the time interval between immunization and challenge was shortened to one week, are given in Table 3. In computing the incubation period of the various groups, arithmetic averages are used instead of a geometric mean because of the small number of birds in any group that developed symptoms or died. In the second group, receiving 2.0 ml. and in the fourth group, receiving 8 ml. of immune serum, the average number of days in the incubation period was 11 and 10 respectively. These averages differ significantly from the average incubation period of the untreated group which was 7 days. The percentage death and susceptibility rate in all immunized groups varied significantly from the untreated group. The lowest death and
TABLE 4.—Protective value of 2 ml. of gamma globulin administered at various time ' • before challenge with NDV
1
Group number
Time of immunization before challenge
1 2 3 4 5
3 weeks 2 weeks 1 week simultaneous control
Includes sick and dead birds.
Incubation period per group (days) (geometric mean) 5.4 4.3 7.6 7.0(1 bird) 2.3
Death rate (percent)
Susceptibility1 rate (percent)
Geometric mean HAI serum titers 19 days after challenge
15 20 10 0 55
20 25 15 5 85
194 42 56 128 169
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1 2 8
1.0 ml. G.G.i 2.0 ml. G.G. 4.0 ml. I.S.2 8.0 ml. I.S. None (control)
susceptibility rate in treated groups was zero and 10% respectively as compared with corresponding figures of 30% and 50% in the untreated group. Although the same number of ELD60 units (103) of virus was administered as challenge in this and the foregoing experiment, the lower susceptibility rate in this instance is attributed to the fact that the experiment was conducted in midsummer when the resistance of chickens to Newcastle disease is generally at a higher point. Trial 3. The results obtained when 2 ml. of immune gamma globulin were administered simultaneously and 1, 2 and 3 weeks before challenge are given in Table 4. The geometric mean number of days in the incubation period of the group challenged simultaneously with immunization was 7. The corresponding figure in the group immunized 1 week before challenge was 7.6. Both of these figures differ significantly from the geometric mean number of days in the incubation period of the untreated group which was 2.3. The death and susceptibility rates of all immunized groups differed significantly from the untreated group. The lowest death rate of an immunized group was zero (simultaneous group) and the highest was 20% in the group immunized 2 weeks before challenge as compared with a SS% death rate in the untreated group. The lowest rate of sus-
142S
PROTECTION AGAINST NEWCASTLE T A B L E 5.—Protective value of 2 ml. gamma _ administered at various time intervals before challenge with NDV
GrouD number
Incubation Time of period per Death immunizing group (days) rate before challenge (geometric (percent) mean) 6 weeks 5 weeks 4 weeks 3 weeks simultaneous control
1
2.9 3.4 3.3 3.4 5.6 3.5
Susceptibility' rate (percent)
40.0 73.3 53.3 40.0 13.3 46.6
46.6 80.0 66.6 60.0 26.6 53.3
Includes sick and dead birds.
Trial 5. The results obtained when 2 ml. of immune gamma globulin were used to immunize 4 groups of experimental chickens challenged with various doses of Newcastle virus ranging from 1 theoretical ELD50 unit to 105 ELD 50 units are given in Table 6. In group 4, receiving 1 theoreti-
T A B L E 6.—Protective value of simultaneous injection and decreasing concentrations of
Group number
1 2 3 4 5 1
A m t . of immunizing a g e n t injected 2 ml. G.G. plus
0.1 0.1 0.1 0.1 0.1
ml. virus ml. virus ml. virus ml. virus ml. virus
10-1 10-3 10-5 10-7 10- 4 (no G.G.)
Includes sick and dead birds.
of 2 ml. gamma NDV
globulin
No. ELD60 units N D V injected
I n c u b a t i o n period per group (days) (geometric mean)
Death rate (percent)
Susceptibility1 rate (percent)
W 10 4 102 1 theoretical 10 s
3.5 3.0 3.5 6.0 4.2
26.6 26.6 26.6 6.6 86.6
40.0 26.6 26.6 6.6 86.6
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ceptibility was 5% in the simultaneous group and the highest was 25% in the group immunized 2 weeks before challenge as compared with susceptibility rate of 85% in the untreated group. The geometric mean HAI serum titer of 5 surviving birds in groups immunized 3, 2 and 1 week before challenge was 194, 42 and 56 respectively. The titer of the group immunized simultaneously with challenge was 128 as compared with a titer of 169 in 5 birds from the untreated group. Challenge virus thus was most highly active in stimulating antibody in the group immunized 3 weeks before challenge. The relatively high serum titer in the group immunized simultaneously with challenge is not explained by these data. Trial 4. The results obtained when the period of time between immunization and challenge was extended from 2 to 6 weeks are given in Table 5. The fifth group, im-
munized simultaneously with challenge had a geometric mean number of days in the incubation period of 5.6 which differs significantly from the geometric mean number of days in the incubation period of the untreated group which was 3.5. Likewise the death and susceptibility rate of the simultaneous groups, 13.3% and 26.6% respectively differed significantly from corresponding figures of the untreated group of 46.6% and 53.3% respectively. The death and susceptibility rates of groups immunized from 3 to 6 weeks before challenge did not differ significantly from corresponding figures in the untreated group. The lowest death rate observed in these groups was 40% recorded for the group immunized 6 and 3 weeks before challenge. The highest rate of susceptibility was 80% in the group immunized 5 weeks before challenge. The relatively poor showing of the groups immunized 3 weeks before challenge as compared with the same period in the previous experiment when the same number of ELD50 units of challenge virus was employed are not explained.
1426
J. J. VASINGTON, N. C. LAFFER, A. P. HOLST AND H. M. DEVOLT
DISCUSSION
Results obtained in studies on the protective value of Newcastle-immune serum and gamma globulin against artificially induced Newcastle disease, indicate a possible place for serum therapy in controlling the disease. A cursory survey of the slaughter houses of the nation, show that much blood from immunized birds is being discarded. Salvage of Newcastle-immune serum from such sources and safeguarding it against viral contamination might place at the disposal of the industry an additional weapon against this disease. Particularly in the case of virus breaks in valuable stock, serum therapy could prove beneficial. The fact that chickens simultaneously treated with immune gamma globulin and challenged with Newcastle virus showed a relatively high geometric mean hemagglutination-inhibition serum titer as compared with birds treated with immune serum 1
and 2 weeks before challenge presents an interesting problem for further investigation. SUMMARY 1. The methanol method of fractionation of Newcastle-immune serum produced a relatively uniform yield of gamma globulin as indicated by hemagglutination-inhibition activity and protein analysis. 2. Intramuscular injection of 4 to 8 ml. of immune serum and 1 to 2 ml. gamma globulin simultaneosuly with, or 1 to 2 weeks prior to, challenge by intramuscular injection of Newcastle disease virus produced a significant degree of protection under the conditions of these experiments. 3. The protective value of immune globulin was roughly inversely proportional under the conditions of these experiments to the length of time elapsing between passive immunization and challenge. 4. Partial protection was obtained under the conditions of this experiment when 2 ml. of gamma globulin was administered to birds averaging 1.2S lbs. and receiving 105 ELD60 infective units of virus. REFERENCES Beaudette, F. R., and J. A. Bivins, 1953. The influence of passive immunity on the response to intramuscular and intranasal administration of Newcastle disease virus. Cornell Vet. 43: 513— 531. Cunningham, C. H., 1956. A Laboratory Guide in Virology. Burgess Publishing Co., Minneapolis. Dubert, J. M., P. Slizewicz, P. Rebeyrotte and M. Machebocuf, 1953. Nouvelle methode de separation des protiens seriques par le methanol. Application aux serums de lapin et de cheval. Annales de L'Institut Pasteur, 84: 370-375. Hanson, R. P., N. S. Winslow, C. A. Brandley and E. Upton, 1950. The antiviral activity of Newcastle disease immune sera. J. Bacterid. 60: 557-560. Lucam, F., 1950. Recherches sur la sero-prevention, la sero-vaccination et la sero-therapie, en milieu infecte de peste aviare. Revur de Medicine Veterinaire. Moynihan, I. W., R. V. L. Walker, E. P. E. Pow-
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cal ELD50 unit of Newcastle virus the geometric mean number of days in the incubation period differed significantly from the other three groups receiving a larger number of infective doses. The death rate and susceptibility rate of all treated groups varied significantly from the untreated groups challenged with 103 ELD50 units of virus. The death rate in treated groups varied from 6.6% in the group receiving one theoretical infective unit of virus to 26.6% for the other treated groups. The rate of susceptibility varied from 6.6% in the group receiving one theoretical unit of virus to 40% in the group receiving 105 ELD50 units of virus. Both the death and susceptibility rate in the untreated group receiving 10s ELD50 units of virus was 86.6%. Partial protection thus was provided by 2 ml. of immune globulin in chickens challenged with up to 105 ELD50 units of Newcastle virus.
PROTECTION AGAINST NEWCASTLE
ell and D. M. Cooper, 1954. An attempt to passively immunize chicks against the virus of Newcastle disease by the use of antiserum of equine origin. Canad. J. Biol. Med. 43: 62-64. Nichol, J. C, and H. F. Deutsch, 1948. Biophysical studies of blood plasma proteins. VII Separation of gamma-globulin from the sera of various animals. J. Amer. Chem. Soc. 70: 80-84. Phillips, L. V. A., 19S6. Ensayos sobre immunizacion passiva y tratamiento de la enfermedad de newcastle por uso de plasma y vitelo himerinmunes. Vira. Agr. (Lima E. sum.). 33: 111-113.
1427
Reed, L. J., and H. Muench, 1938. A simple method of estimating fifty percent end points. Amer. J. Hyg. 27: 493-497. Schmittle, S. C, 1952. Studies on Newcastle disease. XI. Newcastle disease antibodies in the serum globulin fractions of the chicken. Proc. 89th Ann. Meet. Amer. Vet. Assoc: 143-147. Schneider, L., 1952. Vaccination and serum treatment against fowl pest. (Newcastle disease). 14th Int. Vet. Congr. 2: 359-362. Snedecor, G. W., 1946. Statistical Methods. The Iowa State College Press, Fourth Edition.
C. W. POPE, 1 A. B. WATTS, ERNEST WILLIAMS 2 AND CLAYTON C. BKUNSON
Louisiana State University, Baton Rouge (Received for publication February 8, 1960)
T
HE seasonal decrease in egg shell quality (Jull, 1924; and Wilhelm, 1940), and the decrease in interior egg quality with increasing age of the bird (Knox and Godfrey, 1938; and Dawson et al., 1951) are problems which confront poultry breeders and commercial poultrymen. At least two theories have been advanced to explain the decline in shell quality. One theory proposes that the decrease is due to increased environmental temperatures. Another theory proposes that the decrease in shell quality is simply an exhaustion effect coming at the end of the laying year. There are considerable data which show that blood calcium and phosphorus levels are associated with egg formation. Riddle and Reinhart (1926), Hughes et al. (1927), Roepke and Hughes (1935), Taylor and 1
Present address: Ralston Purina Co., St. Louis, Mo. 2 Deceased.
Russell (1935), Correll and Hughes (1935) and Deobald et al. (1938) reported that the blood calcium level greatly increases as birds come into production. Deobald et al. (1938) also reported that total blood phosphorus showed a concurrent change similar to that of calcium. Conrad (1939) observed that blood calcium levels decreased by 25 to 30 percent as environmental temperatures increased from 70° to 90°F. This experiment was designed to study the effects of the length of time in production, intensity of production and stage of egg formation on certain egg quality measurements and blood constituents of laying hens. MATERIALS AND METHODS
Eighty-four New Hampshire pullets of a laying strain were used in this study. They were hatched in late March and housed on October 1 when they were approximately six months of age. Individual laying cages in a conventional-type laying
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The Effect of the Length of Time in Production and Stage of Egg Formation on Certain Egg Quality Measurements and Blood Constituents of Laying Hens