[333] STUDIES IN NEWCASTLE DISEASE IV. RAPID METHODS OF DIAGNOSIS. purposes. in the field. In both instances the methods described have proven

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1 STUDIES IN NEWCASTLE DISEASE IV. RAPID METHODS OF DIAGNOSIS By R. V. L. WALKER* T1he diagnosis of Newcastle disease is of great importance in the handling of outbreaks in flocks throughout Canada. Laboratory diagnosis is dependent on three definite methods, namely (a) virus isolation, (b) serum neutralization and (c) serologically by the heemagglutination (H) and haemagglutination-inhibition (HI) tube tests. The "H" test is solely to prove the presence of the virus in inoculated embryonated hens' eggs in which inoculi containing suspicious tissue emulsions have proven embryocidal. Serum neutralization and "Hll" tcsts serve the purpose of proving the presence or absence of immune antibodies in the blood circulation system of avian hosts. The phenomenon that some viruses have the ability to agglutinate normal chicken erythrocytes and that immune antibodies inhibit such agglutination was first reported in 1941 by Hirst (1) while studying the influenza virus. The following year Burnett (2) demonstrated that the virus of Newc.3stle disease had similar properties and this has been recognized by the official publication of the United States Bureau of Animal Industry (3) to be used as a standard in all diagnostic laboratories. \Vhile th-ese methods of diagnosis are invaluable, the constant daily demands for such services are time-consuming and require large quantities of sterile laboratory apparatus and glassware and trained technical personnel. In Canada, except for one outbreak of Newcastle disease which reached pandemic proportions, flock outbreaks have otherwise been sporadic. Constant vigilance is required to prevent the infection from becoming widespread and for this purpose the Animal Diseases Research Institute has been required to handle many hundreds of tissue specimens and blood samples. Consequently various rapid diagnostic tests have been studied and in some instances modified for laboratorv and field use. Luginbuhl and Jungherr (4) have described a rapid plate test for detecting antibodies of Newcastle disease in avian and human sera. Zargar and Pomeroy (5) later reported a whole blood plate test for similar diagnostic purposes. in the field. In both instances the methods described have proven satisfactory for screening purposes in poultry flocks. Casual mention was also made by Luginbuhl and Jungherr with reference to a rapid haemagglutination test for the determination of the concentration of Newcastle disease virus in irluids of embryonating eggs by Brandly et al (1945) and Wooding (1948). The constant flow of specimens from the field throughout Canada, during the past three years has required the innoculation and continuous examination of large numbers of embryonating eggs daily. When embryo death has been determined by candling, the egg fluids are harvested. At first the *Division of Animal Pathology, Science Service, Dominion Department of Agriculture, Animal Diseases Research Institute, Hull, Quebec. [333]

2 L334, Comparative Medicine NEWCASTLE DISEASE Vol. XVI, No. 9 [334] Canadian. Journal of September, 1952 serological "H" and "HI" tube tests were employed to ascertain the presence of the virus in these fluids and its ability to be inhibited by specific antiserum. Now, however, rapid test techniques have been developed and successfully employed during the past two years, first being thoroughly checked by the tube tests and at present recognized as standard laboratory techniques. liaemagglutination Test I'horoughly cleaned plates of heavy glass are used and may be marked in squares of approximately 2" with a grease pencil. Eggs containing dead em.nbryos are opened and a few drops of pooled allantoic fluids removed with a sterile Pasteur pipette. To a drop of the egg fluids placed in one of the squares is added a drop of a 5. percent suspension of normal chicken red blood cells. T he mixture is quickly stirred in a rotary motion with a toothpick. A positive reaction with well-pronounced haemagglutination is usually observed within one to two minutes. Further proof of the presence of Newcastle disease virus can be determined by first placing a drop of specific antiserum on the plate to which is added a drop of the egg fluid. After mixing well a drop of tle red cell suspension is then added and mixing continued. A positive result is then determined by inhibition of the haemagglutination. Haernagglutination-Inhibition Test. When blood is submitted from flocks suspected of having been previously exposed to Newcastle disease the rapid test of serum has also proven as significant in routine examinations. To a drop of serum placed on the plate is added a drop of egg fluids containing the virus and thoroughly mixed. The virus is usually freshly harvested egg fluids in which the embryo minimal lethal dose is at least 1-8 and having a haemagglutination titre of 1:64 to 1:128, and diluted 1:5 with normal physiological saline solution. To this is added a drop of a 5. per cent normal chicken red cell suspension and again mixed. Controls are also frequently done with known positive and negative sera. Inhibition of haemagglutination in positive cases is definitely observed between one and two minutes an negative readings presenting haemagglutination are visible within the same time period. The rapid whole blood test as described by Zargar and Pomeroy has also been studied extensively in known recovered and normal birds and appears to be a satisfactory method for screening flocks in the field. However, the use of the live virus antigen was inconsistent with official policies dealing with Newcastle disease in Canada and it was thus necessary to make parallel studies with a formalin-inactivated antigen. The original antigen recommended for use in the whole blood test wa,: Newcastle disease virus 25. cc (egg fluids of high titre) Sodium Citrate solution (25 %) 1. cc Physiological saline solution 74. cc For inactivation it had been determined that the addition of.4 per cent

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4 L33Canadian I Journal of NEWCASTLE DISEASE Vol. XVI, No. 9 Comparative Medicin6 September 1952 formalin to the live virus material rendered it non-viable in three days. Thus, in place of the live virus antigen, an inactivated virus was substituted in the formula. While this inactivated antigen proved satisfactory when freshly prepared, it was noted that a heavy precipitate (non-bacterial) formed in thle bottles within about 1 days. Further studies demonstrated that the virus material could be inactivated in the same 3-day period with.25 per cent formalin and subsequently no precipitate was observed. Thus in later production of this antigen virus inactivation was produced with.25 per cent formalin. For application of the whole blood test, on a clean glass plate, marked in squares of 1 Y2 to 2 inches is placed fairly large drops (. 1 cc) of the antigen. From the punctured wing vein of the birds is obtained a wire loopful (.2 cc) of blood and added to the antigen. ( The wire loop is similar to that used in obtaining blood for the pullorum rapid test). The antigen-whole blood combination is then well mixed and readings can be made in approximately two minutes. Blood from birds which have no antibodies will exhibit the characteristic haemagglutination, whereas those having been previously exposed to the infection and carrying antibodies will demonstrate inhibition of the haemagglutination. In a comparative survey of this inactivated virus antigen with the live virus antigen, no differences have been encountered in demonstrating positive and negative reactions. In comparative studies with the rapid tests and the tube test Table #1 graphically presents the results obtained with twenty-one birds in an experimental group. Birds listed as Nos. 1 to 7 had received an inoculation of inactivated virus vaccine about 2 days prior to the test. Those listed as Nos. 8 to 11 were normal birds that had at no time been exposed to the virus. The sera of the group listed as Nos. 12 to 21 were obtained from game birds that had been exposed to and recovered from Newcastle disease in a flock outbreak. In this group "HI" tube tests had been conducted on a number of occasions to check the persistence of immune antibodies. Bird #793 always gave a negative reading while the remainder gave high antibody titres. It will be noted from Table #1 that complete agreement exists in the rapid tests and tube tests except for bird #791 (a game bird imported from the United States), in which the serological test exhibits the presence of antibodies, whereas similar readings were not obtained in either the whole blood or blood serum rapid tests. Since the completion of the experimental studies, the inactivated virus antigen has been produced regularly during the past two years and distributed throughout Canada for screening purposes. The results have been valuable in checking poultry flocks, and in instances where any suspicion exists from the

5 Canadian Journal of NEWCASTLE DISEASE Vol. XVI, No.9 FQT71 Comparative Medicint September, 1952 L whole blood tests, flock samplings are then obtained for serological study in the laboratory. SUMMARY A rapid plate haemagglutination test for the detection of Newcastle disease virus has proven satisfactory for use in the laboratory. Inhibition of the agglutination is also demonstrable with specific antisera. A study of a rapid plate whole blood test for detection of Newcastle disease antibodies producing inhibition of haemagglutination has been conducted, particularly to compare a formalin-inactivated antigen with live virus antigen. In a survey of blood sera from twenty-one birds, in only one instance did the result of the haemagglutination-inhibition tube test disagree with the observations of the rapid plate test. The results obtained with a formalin inactivated virus antigen proved comparable to a live virus antigen for the detection of Newcastle disease antibodies in whole blood tests by the rapid plate method. BIBLIOGRAPHY (1) HIRST, C. K. The Agglutination of Red Cells by Allantoic Fluids of Chick Embryos with Influenza Virus. Science 94: 22-23, (2) BURNETT, F. M. Affinity of Newcastle Virus to Influenza Group. Aust. J. Exptl. Biol. and Med. Sci., 2: 81-88, (3) UNITED STATES DEPT. OF AGRICULTURE,AGRIC. RES. ADM. B.A.I., PATHOLOGICAL DIVIsIoN. The hemagglutination and hemagglutination-inhibition tests for the diagnosis of Newcastle disease. 21 Oct (4) LUGINBUHL, R. E., and JUNGHERR, E. A plate hemagglutination-inhibition test for Newcastle disease antibodies in avian and human serums. Poult. Sci., 28: , (5) ZARGAR, S. L. and POMEROY, B. S. A rapid whole blood plate test for the diagnosis of Ne castle disease. Jour. Am. Vet. Med. Assoc., 115: 354, 1949.