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The Use of Periodate-Treated Newcastle Disease Virus as Antigens in the Hemagglutination-Inhibition Test*
The Use of Periodate-Treated Newcastle Disease Virus as Antigens in the Hemagglutination-Inhibition Test* WILLIAM H. MCCOLLUM, E. R. DOLL AND JOHN T. BRYANS Department of Animal Pathology, Kentucky Agricultural Experiment Station, Lexington,
Kentucky
(Received for publication February 25, 1957) INTRODUCTION
T
HE hemagglutinative activity of Newcastle disease virus (NDV) was first described by Lush (1943). The U. S. Department of Agriculture in 1946 circulated a mimeographed sheet describing the use of hemagglutination (HA) and hemagglutination-inhibition (HI) as diagnostic procedures for Newcastle disease. Shortly thereafter, many papers (Brandly et al., 1947; Beach, 1948; Doll et al., 19S0) on these procedures appeared. In every instance one very important variable in the procedure was the time elapsing after mixing the red blood cells (RBC) with the other components of the system before reading the test. This was necessitated because the virus-RBC aggregate was dissociated rapidly by the progressive "enzymic" action of the virus on the receptor substance of the RBC. Patterns formed by such action are indistinguishable from patterns observed in control preparations. The necessity, therefore, of specifying the time of reading the test is obvious. * From the Department of Animal Pathology, Kentucky Agricultural Experiment Station, University of Kentucky, Lexington. The investigation reported in this paper is in connection with a project of the Kentucky Agricultural Experiment Station and is published by permission of the Director. This study was conducted as a segment of Research and Marketing Act Regional Project S-34—Respiratory diseases of fowls. The cooperating agencies under this project are the Alabama, Arkansas, Georgia, Kentucky, North Carolina, Texas, and Virginia Agricultural Experiment Stations. The research reported here was done solely at the Kentucky Station.
The action of periodate in destroying the elution property of NDV without impairing its ability to agglutinate RBC is well known (Hirst, 1950; Granoff et al., 1950; Granoff and Hirst, 1954). This modification of the virus would appear to be extremely desirable as the antigen in the HI procedures, especially where routine and diagnostic work is involved, as the time factor in reading the test is largely eliminated. Since no work has been done on the utilization of this type of antigen routinely, this study was undertaken to determine the value of such antigens for HI determinations. MATERIALS AND METHODS The GB, Roakin and Bl strains of NDV were employed. Allantoic fluid virus was prepared by inoculating 10-11 day chicken embryos intraallantoically with 0.05 ml. of 10-2 dilution of stock allantoic fluid virus. The allantoic fluid was collected from embryos dying after 24 hours. Dialysis was carried out at 4°C. for 48 hours and was accomplished by putting virus in cellophane bags and placing them in 50 or more volumes of saline. Agitation was not used but the saline was changed once or twice during the process. Periodate treatment of the fluids was effected by mixing equal volumes of allantoic fluid and the desired concentration of aqueous potassium periodate (KI0 4 ) at 37°C. and after 10 minutes neutralizing the excess periodate by the addition of a similar volume of 10% glucose. Merthiolate (1-10,000) and thymol (trace) were added to such fluids as preservatives if they were to be kept for any extended length of time. The highest 0.25
855
856
W. H. MCCOLLUM, E. R. DOLL AND J. T. BRYANS
ml. 2-fold dilution of virus causing complete agglutination of 0.2S ml. of 1.0% chicken RBC was regarded as one HA unit. In most instances this titer was obtained by interpolation (McCollum and Brandly, 19S5). The HI test used was the beta procedure and employed 0.2S ml. 2-fold dilutions of serum in physiological saline, 10 HA units of antigen in 0.25 ml. saline, and 0.25 ml. of a 1.0% saline suspension of chicken RBC. The antiserums were obtained from chickens exposed experimentally to either the Roakin or Bl strain of NDV. EXPERIMENTAL AND RESULTS
The presence of a dialyzable substance in allantoic fluid which interferes with the action of periodate on virus has been described (Hirst, 1949). Consequently, the first study was to reinvestigate the action of periodate on the HA activity of dialyzed
and undialyzed NDV infective allantoic fluids. Aliquots of such fluids, infected with GB, Roakin and Bl viruses, were treated with periodate solutions having the following molar concentrations: M/200, M/400, M/800, M/1600, and M/3200. HA titers of these fluids were determined with patterns recorded at 40 minutes and at 2 and 12 hours after the addition of RBC. Results of this study are summarized in Table 1. Only M/200 concentration of periodate exhibited appreciable effect on the stability of the hemagglutinin of undialyzed fluids. Dialyzed fluids, however, were very strikingly affected. Molar/200 concentration destroyed all or nearly all of the hemagglutinin. The other concentrations of periodate, except M/3200, caused some reduction of the HA titer, but in every instance the sedimentation pattern after 12 hours was the same as after 40 minutes. In view of the above results, an experi-
TABLE 1.—The HA activity of dialyzed and undialyzed NDV infected allantoic fluid treated with varying concentrations of potassium periodate for 10 minutes
Viral strain GB
ROAKIN
Bl
Concentration of K104
HA titers* of undialyzed fluid after standing: for 40 min.
2hrs.
12 hrs.
40 min.
2 hrs.
12 hrs
— •— — — — — — — — — — —
35 560 1120 1120 4480 1280
35 560 1120 1120 4480
35 560 1120 1120 4480
—
—
0 560 640 1120 960 1280
0 560 640 1120 960
0 560 560 1120 960
—
—
1920
1920
— •— — — —
— — — — —
0 640 960 1120 1120 1280
0 640 960 1120 1120
0 640 960 1120 1120
—
—
M/200 M/400 M/800 M/1600 M/3200 None
1960 3840 4480 4480 4480 4480
1960
M/200 M/400 M/800 M/1600 M/3200 None
4480 4480 4480 4480 4480 4480
4480
M/200 M/400 M/800 M/1600 M/3200 None
1920 1920 1920 1920 1920 1920
HA titers* of dialyzed fluids after standing for
-t — — — — — — — —
* Highest initial dilution showing 1 + agglutination. When such values were not evident, they were obtained by interpolation. f Complete elution.
857
NEWCASTLE DISEASE ANTIGENS
TABLE 2.—HA liters of untreated, Jormalinized and periodate-treated NDV stored for various periods Viral strain
Treatment
GB Roakin
None None
GB Roakin GB Roakin
HA titers* after storage of preparations for
Storage conditions
1 wk.
1 mo.
4 mos.
10 mos.
18 mos.
- 2 0 C. -20C.
3840 960
2240 2560
5120 1280
3840 1120
4880 1120
0 . 1 % formalin 0 . 1 % formalin
4C. 4C.
560 960
1280 960
2240 960
560 560
240 240
M/800 K104 M/800 KIO4
4C. 4C.
1120 560
1120 1120
1120 1120
480 560
640 1120
* Highest initial dilution showing 1 + agglutination. When such values were not evident, they were obtained by interpolation.
ment was designed to study the suitability of periodate treated NDV as antigens in the HI test. Allantoic fluid pools of GB and Roakin viruses were prepared and each divided into three portions. One portion was distributed in 3 ml. aliquots and stored at — 20°C. Another portion received 0.1 percent final concentration of formalin and stored at 4°C. The remaining fluid was dialyzed against saline and treated with M/800 final concentration of periodate for 10 minutes. HA titers of these preparations (Table 2) and their subsequent employ-
ment as antigens in the HI determinations of serums (Table 3) from chickens experimentally exposed to Roakin or Bl strains of NDV were carried out at 1 week and at 1, 4, 8,10 and 18 months after preparation. The HA titers of the frozen and periodate treated antigens remained unchanged for the 18-month period. The formalinized antigens still agglutinated RBC after 18 months, but there was a considerable decrease of titer. Each antigen produced approximately the same HI titer for a given serum if the values were compared at 40
TABLE 3.—HI titers* of anti-NDV serums utilizing untreated, formalinized and periodatetreated antigens stored for various periods H I titers* of serums utilizing antigens stored for Antigen
Treatment
Serum 1 wk.
1 mo.
4 mos.
10 mos.
18 mos.
GB GB
None None
310 R A081I
1280/-f 1280/-
640/1280/-
1280/1280/-
640/1280/-
640/1280/-
Roakin Roakin
None None
310 R A081I
640/1280/-
640/1280/-
640/1280/-
640/1280/-
640/2560/-
GB GB
0 . 1 % formalin 0 . 1 % formalin
310 R A081I
1280/1280/-
1280/2560/-
5120/10240/-
2560/5120/-
2560/5120/-
Roakin Roakin
0 . 1 % formalin 0 . 1 % formalin
310 R A081I
640/1280/-
640/1280/-
2560/5120/-
1280/2560/-
2560/5120/-
GB GB
M/800 KIO4 M/800 KIO4
310 R A081I
640/1280 1280/1280
640/1280 1280/1280
1280/2560 2560/2560
640/1280 1280/1280
1280/2560 5120/5120
Roakin Roakin
M/800 KIO4 M/800 KIO4
310 R A081I
320/640 640/1280
640/1280 1280/1280 1280/2560 2560/2560
640/640 1280/1280
2560/2560 2560/2560
* Highest dilution of serums showing complete inhibition of agglutination. t Numerator, titer after 40 minutes; denominator, titer after 12 hours (-=complete elution).
858
W. H. MCCOLLUM, E. R. DOLL AND J. T. BRYANS
minutes. There was a tendency, however, to obtain higher titers with the formalinized antigens. After 2 hours complete elution had occurred in all tubes containing untreated or formalinized antigen; whereas, little if any change was apparent after 12 hours in those determinations made with the periodate treated antigen.
out affecting appreciably its agglutinative capacity. Agglutination patterns formed by periodate treated NDV were stable for at least 12 hours; whereas, those formed by untreated and formalinized antigens had disappeared completely within two hours. Consequently, periodate modified antigens appear to be extremely desirable for routine and diagnostic HI determinations.
DISCUSSION
NDV agglutinates chicken RBC by bridging such cells together, causing the formation of a lattice-like matrix. If the ratio of virus and RBC is controlled, the reaction can be observed visually. In most instances, however, the NDV "elutes" or dissociates very rapidly from the RBC leaving the cells unbridged. The present explanation of HA by NDV is that the virus particle attaches itself enzymatically to two or more RBC and that during this stage agglutination is visible. The reason for specifying the time for observing the reaction is therefore obvious. Periodate has the capacity of destroying the elution property of the virus without impairing its adsorptive and agglutinative activity. The advantages of such antigens in diagnostic laboratories or other routine testings is very apparent. The results of this study substantiate the practicality of using antigens modified by periodate as the HA and HI tests can be read from 30 minutes up to at least 12 hours; whereas, in comparative tests utilizing untreated or formalinized antigens, complete elution occurred within 2 hours. SUMMARY
The practicability of utilizing Newcastle disease virus (NDV) modified by potassium periodate as antigens in hemagglutinationinhibition (HI) tests has been studied. Under controlled conditions, periodate destroys the elution property of NDV with-
REFERENCES Beach, J. R., 1948. The application of the hemagglutination-inhibition test in the diagnosis of avian pneumoencephalitis (Newcastle disease). J. Amer. Vet. Med. Assoc. 112: 85-91. Brandly, C. A., R. P. Hanson, S. H. Lewis, N. S. Winslow, H. H. Hoyt and C. M. Nerlinger, 1947. Variables and correlations in laboratory procedures for Newcastle disease diagnosis. Cornell Vet. 37: 324-336. Doll, E. R., M. E. Wallace and W. H. McCollum, 1950. Interpretation of serologic procedures for the diagnosis of Newcastle disease. Amer. J. Vet. Res. 11: 26S-271. Granoff, A., O. C. Liu and W. Henle, 1950. A small hemagglutinating component in preparation of Newcastle disease virus. Proc. Soc. Exp. Biol. Med. 75 : 684-691. Granoff, A., and G. K. Hirst, 1954. Experimental production of combination forms of virus. IV. Mixed influenza A-Newcastle disease virus infections. Proc. Soc. Exp. Biol. Med. 86: 84-88. Hirst, G. K., 1949. The nature of the virus receptors of red cells. IV. Effect of sodium periodate on the elution of influenza virus from red cells. J. Exp. Med. 89: 233-243. Hirst, G. K., 1950. Receptor destruction by viruses of the mumps-NDV-influenza group. J. Exp. Med. 9 1 : 161-175. Lush, D., 1943. The chick red cell agglutination test with the viruses of Newcastle disease and fowl plague. J. Comp. Path. Therap. 2 1 : 157160. McCollum, W. H., and C. A. Brandly, 1955. Hemolytic activity of Newcastle disease virus. Amer. J. Vet. Res. 16: 584-592. U. S. Bureau of Animal Industry, Pathological Division, 1946. The hemagglutination and hemagglutination-inhibition tests for the diagnosis of Newcastle disease. Oct. 21, mimeographed.
Kentucky
(Received for publication February 25, 1957) INTRODUCTION
T
HE hemagglutinative activity of Newcastle disease virus (NDV) was first described by Lush (1943). The U. S. Department of Agriculture in 1946 circulated a mimeographed sheet describing the use of hemagglutination (HA) and hemagglutination-inhibition (HI) as diagnostic procedures for Newcastle disease. Shortly thereafter, many papers (Brandly et al., 1947; Beach, 1948; Doll et al., 19S0) on these procedures appeared. In every instance one very important variable in the procedure was the time elapsing after mixing the red blood cells (RBC) with the other components of the system before reading the test. This was necessitated because the virus-RBC aggregate was dissociated rapidly by the progressive "enzymic" action of the virus on the receptor substance of the RBC. Patterns formed by such action are indistinguishable from patterns observed in control preparations. The necessity, therefore, of specifying the time of reading the test is obvious. * From the Department of Animal Pathology, Kentucky Agricultural Experiment Station, University of Kentucky, Lexington. The investigation reported in this paper is in connection with a project of the Kentucky Agricultural Experiment Station and is published by permission of the Director. This study was conducted as a segment of Research and Marketing Act Regional Project S-34—Respiratory diseases of fowls. The cooperating agencies under this project are the Alabama, Arkansas, Georgia, Kentucky, North Carolina, Texas, and Virginia Agricultural Experiment Stations. The research reported here was done solely at the Kentucky Station.
The action of periodate in destroying the elution property of NDV without impairing its ability to agglutinate RBC is well known (Hirst, 1950; Granoff et al., 1950; Granoff and Hirst, 1954). This modification of the virus would appear to be extremely desirable as the antigen in the HI procedures, especially where routine and diagnostic work is involved, as the time factor in reading the test is largely eliminated. Since no work has been done on the utilization of this type of antigen routinely, this study was undertaken to determine the value of such antigens for HI determinations. MATERIALS AND METHODS The GB, Roakin and Bl strains of NDV were employed. Allantoic fluid virus was prepared by inoculating 10-11 day chicken embryos intraallantoically with 0.05 ml. of 10-2 dilution of stock allantoic fluid virus. The allantoic fluid was collected from embryos dying after 24 hours. Dialysis was carried out at 4°C. for 48 hours and was accomplished by putting virus in cellophane bags and placing them in 50 or more volumes of saline. Agitation was not used but the saline was changed once or twice during the process. Periodate treatment of the fluids was effected by mixing equal volumes of allantoic fluid and the desired concentration of aqueous potassium periodate (KI0 4 ) at 37°C. and after 10 minutes neutralizing the excess periodate by the addition of a similar volume of 10% glucose. Merthiolate (1-10,000) and thymol (trace) were added to such fluids as preservatives if they were to be kept for any extended length of time. The highest 0.25
855
856
W. H. MCCOLLUM, E. R. DOLL AND J. T. BRYANS
ml. 2-fold dilution of virus causing complete agglutination of 0.2S ml. of 1.0% chicken RBC was regarded as one HA unit. In most instances this titer was obtained by interpolation (McCollum and Brandly, 19S5). The HI test used was the beta procedure and employed 0.2S ml. 2-fold dilutions of serum in physiological saline, 10 HA units of antigen in 0.25 ml. saline, and 0.25 ml. of a 1.0% saline suspension of chicken RBC. The antiserums were obtained from chickens exposed experimentally to either the Roakin or Bl strain of NDV. EXPERIMENTAL AND RESULTS
The presence of a dialyzable substance in allantoic fluid which interferes with the action of periodate on virus has been described (Hirst, 1949). Consequently, the first study was to reinvestigate the action of periodate on the HA activity of dialyzed
and undialyzed NDV infective allantoic fluids. Aliquots of such fluids, infected with GB, Roakin and Bl viruses, were treated with periodate solutions having the following molar concentrations: M/200, M/400, M/800, M/1600, and M/3200. HA titers of these fluids were determined with patterns recorded at 40 minutes and at 2 and 12 hours after the addition of RBC. Results of this study are summarized in Table 1. Only M/200 concentration of periodate exhibited appreciable effect on the stability of the hemagglutinin of undialyzed fluids. Dialyzed fluids, however, were very strikingly affected. Molar/200 concentration destroyed all or nearly all of the hemagglutinin. The other concentrations of periodate, except M/3200, caused some reduction of the HA titer, but in every instance the sedimentation pattern after 12 hours was the same as after 40 minutes. In view of the above results, an experi-
TABLE 1.—The HA activity of dialyzed and undialyzed NDV infected allantoic fluid treated with varying concentrations of potassium periodate for 10 minutes
Viral strain GB
ROAKIN
Bl
Concentration of K104
HA titers* of undialyzed fluid after standing: for 40 min.
2hrs.
12 hrs.
40 min.
2 hrs.
12 hrs
— •— — — — — — — — — — —
35 560 1120 1120 4480 1280
35 560 1120 1120 4480
35 560 1120 1120 4480
—
—
0 560 640 1120 960 1280
0 560 640 1120 960
0 560 560 1120 960
—
—
1920
1920
— •— — — —
— — — — —
0 640 960 1120 1120 1280
0 640 960 1120 1120
0 640 960 1120 1120
—
—
M/200 M/400 M/800 M/1600 M/3200 None
1960 3840 4480 4480 4480 4480
1960
M/200 M/400 M/800 M/1600 M/3200 None
4480 4480 4480 4480 4480 4480
4480
M/200 M/400 M/800 M/1600 M/3200 None
1920 1920 1920 1920 1920 1920
HA titers* of dialyzed fluids after standing for
-t — — — — — — — —
* Highest initial dilution showing 1 + agglutination. When such values were not evident, they were obtained by interpolation. f Complete elution.
857
NEWCASTLE DISEASE ANTIGENS
TABLE 2.—HA liters of untreated, Jormalinized and periodate-treated NDV stored for various periods Viral strain
Treatment
GB Roakin
None None
GB Roakin GB Roakin
HA titers* after storage of preparations for
Storage conditions
1 wk.
1 mo.
4 mos.
10 mos.
18 mos.
- 2 0 C. -20C.
3840 960
2240 2560
5120 1280
3840 1120
4880 1120
0 . 1 % formalin 0 . 1 % formalin
4C. 4C.
560 960
1280 960
2240 960
560 560
240 240
M/800 K104 M/800 KIO4
4C. 4C.
1120 560
1120 1120
1120 1120
480 560
640 1120
* Highest initial dilution showing 1 + agglutination. When such values were not evident, they were obtained by interpolation.
ment was designed to study the suitability of periodate treated NDV as antigens in the HI test. Allantoic fluid pools of GB and Roakin viruses were prepared and each divided into three portions. One portion was distributed in 3 ml. aliquots and stored at — 20°C. Another portion received 0.1 percent final concentration of formalin and stored at 4°C. The remaining fluid was dialyzed against saline and treated with M/800 final concentration of periodate for 10 minutes. HA titers of these preparations (Table 2) and their subsequent employ-
ment as antigens in the HI determinations of serums (Table 3) from chickens experimentally exposed to Roakin or Bl strains of NDV were carried out at 1 week and at 1, 4, 8,10 and 18 months after preparation. The HA titers of the frozen and periodate treated antigens remained unchanged for the 18-month period. The formalinized antigens still agglutinated RBC after 18 months, but there was a considerable decrease of titer. Each antigen produced approximately the same HI titer for a given serum if the values were compared at 40
TABLE 3.—HI titers* of anti-NDV serums utilizing untreated, formalinized and periodatetreated antigens stored for various periods H I titers* of serums utilizing antigens stored for Antigen
Treatment
Serum 1 wk.
1 mo.
4 mos.
10 mos.
18 mos.
GB GB
None None
310 R A081I
1280/-f 1280/-
640/1280/-
1280/1280/-
640/1280/-
640/1280/-
Roakin Roakin
None None
310 R A081I
640/1280/-
640/1280/-
640/1280/-
640/1280/-
640/2560/-
GB GB
0 . 1 % formalin 0 . 1 % formalin
310 R A081I
1280/1280/-
1280/2560/-
5120/10240/-
2560/5120/-
2560/5120/-
Roakin Roakin
0 . 1 % formalin 0 . 1 % formalin
310 R A081I
640/1280/-
640/1280/-
2560/5120/-
1280/2560/-
2560/5120/-
GB GB
M/800 KIO4 M/800 KIO4
310 R A081I
640/1280 1280/1280
640/1280 1280/1280
1280/2560 2560/2560
640/1280 1280/1280
1280/2560 5120/5120
Roakin Roakin
M/800 KIO4 M/800 KIO4
310 R A081I
320/640 640/1280
640/1280 1280/1280 1280/2560 2560/2560
640/640 1280/1280
2560/2560 2560/2560
* Highest dilution of serums showing complete inhibition of agglutination. t Numerator, titer after 40 minutes; denominator, titer after 12 hours (-=complete elution).
858
W. H. MCCOLLUM, E. R. DOLL AND J. T. BRYANS
minutes. There was a tendency, however, to obtain higher titers with the formalinized antigens. After 2 hours complete elution had occurred in all tubes containing untreated or formalinized antigen; whereas, little if any change was apparent after 12 hours in those determinations made with the periodate treated antigen.
out affecting appreciably its agglutinative capacity. Agglutination patterns formed by periodate treated NDV were stable for at least 12 hours; whereas, those formed by untreated and formalinized antigens had disappeared completely within two hours. Consequently, periodate modified antigens appear to be extremely desirable for routine and diagnostic HI determinations.
DISCUSSION
NDV agglutinates chicken RBC by bridging such cells together, causing the formation of a lattice-like matrix. If the ratio of virus and RBC is controlled, the reaction can be observed visually. In most instances, however, the NDV "elutes" or dissociates very rapidly from the RBC leaving the cells unbridged. The present explanation of HA by NDV is that the virus particle attaches itself enzymatically to two or more RBC and that during this stage agglutination is visible. The reason for specifying the time for observing the reaction is therefore obvious. Periodate has the capacity of destroying the elution property of the virus without impairing its adsorptive and agglutinative activity. The advantages of such antigens in diagnostic laboratories or other routine testings is very apparent. The results of this study substantiate the practicality of using antigens modified by periodate as the HA and HI tests can be read from 30 minutes up to at least 12 hours; whereas, in comparative tests utilizing untreated or formalinized antigens, complete elution occurred within 2 hours. SUMMARY
The practicability of utilizing Newcastle disease virus (NDV) modified by potassium periodate as antigens in hemagglutinationinhibition (HI) tests has been studied. Under controlled conditions, periodate destroys the elution property of NDV with-
REFERENCES Beach, J. R., 1948. The application of the hemagglutination-inhibition test in the diagnosis of avian pneumoencephalitis (Newcastle disease). J. Amer. Vet. Med. Assoc. 112: 85-91. Brandly, C. A., R. P. Hanson, S. H. Lewis, N. S. Winslow, H. H. Hoyt and C. M. Nerlinger, 1947. Variables and correlations in laboratory procedures for Newcastle disease diagnosis. Cornell Vet. 37: 324-336. Doll, E. R., M. E. Wallace and W. H. McCollum, 1950. Interpretation of serologic procedures for the diagnosis of Newcastle disease. Amer. J. Vet. Res. 11: 26S-271. Granoff, A., O. C. Liu and W. Henle, 1950. A small hemagglutinating component in preparation of Newcastle disease virus. Proc. Soc. Exp. Biol. Med. 75 : 684-691. Granoff, A., and G. K. Hirst, 1954. Experimental production of combination forms of virus. IV. Mixed influenza A-Newcastle disease virus infections. Proc. Soc. Exp. Biol. Med. 86: 84-88. Hirst, G. K., 1949. The nature of the virus receptors of red cells. IV. Effect of sodium periodate on the elution of influenza virus from red cells. J. Exp. Med. 89: 233-243. Hirst, G. K., 1950. Receptor destruction by viruses of the mumps-NDV-influenza group. J. Exp. Med. 9 1 : 161-175. Lush, D., 1943. The chick red cell agglutination test with the viruses of Newcastle disease and fowl plague. J. Comp. Path. Therap. 2 1 : 157160. McCollum, W. H., and C. A. Brandly, 1955. Hemolytic activity of Newcastle disease virus. Amer. J. Vet. Res. 16: 584-592. U. S. Bureau of Animal Industry, Pathological Division, 1946. The hemagglutination and hemagglutination-inhibition tests for the diagnosis of Newcastle disease. Oct. 21, mimeographed.