STUDIES OF SOME FAGTORS TO BE CONSIDERED IN EVALUATING THE EFFECT OF CERTAIN CHEMICAL AGENTS ON NEWCASTLE DISEASE VIRUS By CHARLES H. CUNNINGHAM A THESIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR. OF PHILOSOPHY Department of Bacteriology and Public Health Year 1953 ProQuest Number: 10008287 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete m anuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest 10008287 Published by ProQuest LLC (2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 4 8 1 0 6 - 1346 This work is respectfully dedicated to MY FAMILY 349659 AC KNOWLEDGEMENTS The author wishes to express his deep appreciation to Dr. H. J. Staf— seth for his assistance and encouragement in this investigation. Technical assistance by Mrs. Martha P. Spring is gratefully ack­ nowledged. Charles H. Cunningham candidate for the degree of Doctor of Philosophy Final examination: Dissertation: May 18, 1953, 2:00 P. M., Room 101, Giltner Hall Studies of Some Factors to be Considered in Evaluating the Effect of Certain Chemical Agents on Newcastle Disease Virus Outline of Studies: Major subjects: Bacteriology, Virology Minor subjects: Animal Pathology, Physical Chemistry Biographical Items: Born, April 12, 1913, Washington, District of Columbia Undergraduate Studies, University of Maryland, 1930-34 Iowa State College, 1934-38 Graduate Studies, Iowa State College, 1934-37 Michigan State College, 1947-53 Experience: Assistant Professor and Veterinary Inspector, University of Maryland, Maryland State Board of Agriculture, 1938-42, Associate Professor, University of Rhode Island, 1942-45, Associate Professor, Michigan State College, 1945Society Affiliations: The American Association for the Advancement of Science, The New York Academy of Science, The Society of the Sigma Xi, Phi Zeta, American Veterinary Medical Association, Conference of Research Workers in Animal Diseases in North America STUDIES OF SOME FACTORS TO HE CONSIDERED IN EVALUATING THE EFFECT OF CERTAIN CHEMICAL AGENTS ON NEWCASTLE DISEASE VIRUS By Charles H. Cunningham AN ABSTRACT Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Bacteriology and Public Health Year Approved 1953 1 Charles H. Cunningham Studies of the effect of phenol, one per cent, ethyl alcohol, 4-0 per cent, sodium hydroxide, one per cent; sodium hypochlorite, 4-4-0 p.p.m. available chlorine; Roccal, 0 .0 1 per cent; mercuric chloride, 0 .0 1 per cent; and tincture of metaphen, 0.01 per cent, on Newcastle disease virus in proportions of virus:chemical agent of 1:1 and 1:9 at 0 C, 20 C and 37 C for different time intervals indicated a logarithmic decrease in viral activity when undiluted virus-infected allantoic fluid, 0 .5-4 mg nitrogen per ml, l.d.5 0 10 • per 0.1 ml, was employed. Embryonating chicken eggs were used as the indicator host with lethality as the criter­ ion of infectivity of the virus. These findings were substantiated when the chemical agents were tested against different concentrations of the virus at 20 C for 15 minutes. The initial stage of inactivation of the virus was detected earlier with the 1:9 mixtures than with the 1:1 mixtures. The rate of inacti­ vation for the 1 :9 mixtures was greater than that for the 1 :1 mixtures with one exception with phenol, one per cent, where the reverse occurred. Increased temperature augmented the action of ethyl alcohol, sodium hydroxide and sodium hypochlorite. Sodium hydroxide was also efficacious at low temperatures. These studies showed that the number of infective doses of virus, the ratio of virus to chemical agent, the temperature of exposure, and the period of exposure exert an influence on the evaluation of virucidal tests. Extraneous protein material in the virus preparation may affect the action of oxidizing agents and adsorbing compounds. TABLE OF CONTENTS Page INTRODUCTION............................................ 1 HISTORICAL REVIEW ........................................ 7 MATERIALS AND EXPERIMENTAL FROCEDURES ........................ 21 RESULTS ................................................. 25 Effect of Chemical Agents onUndiluted Newcastle Disease Virus. Effect of Chemical Agents onDifferent Concentrations of Newcastle Disease Virus ........................... 25 4-7 DISCUSSION.............................................. 57 SUMMARY................................................. 63 BIBLIOGRAPHY........................................... 64- I INTRODUCTION Newcastle disease is a highly contagious and fatal virus infection of poultry and of wild birds characterized by pneumonic and neurologic disturbances. Certain animals axe susceptible and human infection has been recognized, Kraneveld (1926) first referred to the disease as one prevalent in poultry in the Netherland East Indies. Later in the same year the disease appeared at Newcastle-on-Tyne in England. Doyle (1927) clearly estab­ lished the etiology and nature of the disease and proposed the name of Newcastle disease. Since that time the disease has been encountered throughout the world. Due to the widespread distribution of the disease, the name Newcastle disease is obviously unsuited but has been retained to avoid confusion with the plurality of other names (Doyle, 1933). New­ castle disease is now considered to have been present in California as early as 1935, and perhaps earlier, but it was not until 1944- that the virus of a disease first called a "respiratory-nervous disorder" of chickens (Beach, 1941, 1942; Stover, 1942a, 1942b) and later "avian pneumoencephalitid’was recognized as being immunologically identical with the virus of Newcastle disease (Beach, 1944). Newcastle disease virus (NDV) is classified by Holmes (194^) as Tortor furens. NDV may be found in all internal organs of infected chickens and is excreted from the respiratory and gastro-intestinal tracts (Brandly et al, 1946b). NDV has been detected in the yolk sac of four-day-old chicks, 2 embryos and infertile eggs laid by hens during the active stages of the infection (DeLay, 1947). Egg transmission does not seem to be a factor in the spread of the disease from hen to chick (Bivins et al. 1950; Hofstad, 1949; Prier et al. 1950). Virus has been recovered from the air in pens of infected chickens (DeLay et al, 1948). Chicks hatched from eggs produced by hens recovered from or vaccinated against the disease may have a naturally acquired passive immunity for three or four weeks. Anti­ bodies may be detected in the yolk of such eggs and chicks (Brandly et al. 194&d; Schmittle, 1950; Schmittle and Millen, 194^). HDV may be transmitted to persons handling infected birds or working with the virus. In general, the disease is characterized by a superfic­ ial, unilateral, acute, granular conjunctivitis with a mucopurulent dis­ charge (Anderson, 194&; Burnet, 1943; Gustafson and Moses, 1951; Hunter et al, 1951; Ingalls and Mahoney, 1949; Keeney and Hunter, 1950; Thompson, 1950). A systemic syndrome of fever, chills, headache, general malaise, mild leucopenia and relative lymphocytosis indicates that the virus is not always limited to the conjunctiva. Virus has been recovered from the blood, nasal and lachrymal secretions, saliva and urine of affected per­ sons (Hunter et al. 1951). Inclusion bodies have not been conclusively demonstrated in the natural host (Jungherr et al, 1946). complete in one or two weeks without sequelae. Recovery is Medication is without effect on the course of the disease. On the basis of filtration through graded collodion filters, the size of the virus has been estimated to be from 80 mp to 120 mp (Burnet and Ferry, 1934). Spherical as well as sperm-shaped particles have been observed by electron microscopy (Bang, 194&C, 1949; Cunha et al. 1947; 3 Reagan et al, 194-8). The virus is spherical in solutions of physiological concentration but increasing hypertonicity produces filamentous and spermshaped particles that are to be considered as morphological artifacts (Bang, 1948c, 1949). The head-piece of the sperm-shaped particles is about 70 mp x 180 mp (Cunha et al. 1947), 83 x I46 mp (Bang, 1948c), and the tail­ piece about 500 mp (Cunha et al. 1947). 125 mp (Reagan et al. 1948). The diameter is from 100 mp to The virus is a complex of about 67 per cent protein, about 27 per cent lipid and a relatively small amount of nucleic acid, some of which is of the desoxypentose type (Cunha et al. 1947). The virus passes through all grades of Berkefeld, Mandler and Seitz filters and Chamberland L3 and L5 filters (Beaudette, 1943> 1949b, 1950; Beaudette et al. 1949; Brandly, 1950; Brandly et al. 1946b, Doyle, 1927). The 50 per cent end point infective unit of NDV contains an average of gpajn 0f nitrogen, corresponding to 10”^ (Cunha et al. 1947). gram of virus These values suggest an embryo infective unit of 10 particles and verify Bang's (1948a) calculations based on infectivity determinations with four strains of virus. According to Moses (1948), NDV is more resistant to a basic environ­ ment than to an acidic environment. At one week a maximal stability of the virus may be expected within approximately pH 5 to pH 9 (Moses et al. 1947). NDV possesses the ability to agglutinate red blood cells of chickens and other avian species as well as those of certain mammals (Brandly et al. 1946b; Hanson et al. 1950; U.S.D.A., 1946b; Winslow et al. 1950). This hemagglutinative activity of the virus is inhibited by specifically 4 immune serum and may be measured quantitatively. NDV is capable of producing fatal infection of embryonating chicken eggs following injection by any route of inoculation (Bang, 1948a; 194.8b; Brandly et al. 194.6b; Cunningham, 1952a, 1952b; Hanson et al. 194-7; U.S.D.A., 1946a). This characteristic of the virus is utilized as an in­ itial diagnostic criterion for isolation and identification of the virus in tissue specimens from natural outbreaks of the disease. Death of the embryos generally occurs on the third day after inoculation. Dermal pe- techiation and congestion and hemorrhage of the yolk sac may be observed in dead embryos but there are no characteristic gross pathological alter­ ations of the embryo that can be used for diagnosis (Jungherr et al. 194-6). The lethality of NDV is neutralized by specifically immune serum and may be used for a quantitative measurement of the antibody content of the serum (Cunningham, 1951). The heat stability of the hemagglutinative activity and embryo infectivity of certain strains of the virus at 56 C for 30 minutes is vari­ able. With some strains the hemagglutinative activity is more stable than the embryo infectivity and with others the opposite is found. The heat stability of the hemagglutinative activity of the virus may be used as a genetic marker (Hanson et al. 1949; Durusan, 194-9). A serological relationship of mumps and Newcastle disease has been observed but it is suggested that a diagnosis of Newcastle disease in humans be made with caution, especially in the absence of virus isolation (Jungherr et al. 1949). The relationship of NDV with the influenza group (Anderson, 1947; Burnet, 1942; Florman, 194^)> receptor destruction by viruses of the 5 mumps-Newcastle disease-influenza group (Hirst, 1950a, 1950b), modifica­ tion of red blood cells by NDV and use of these cells in serologic studies of infectious mononucleosis and viral hepatitis (Evans, 1950; Kilham, 1950), and NDV hemolysin (Kilham, 1949) have been reported. While these reports indicate certain serologic relationships of the viruses studied and their possible adaptation to diagnosis of human infections, evalua­ tion of the specificity of the reactions must await further investigation. II HISTORICAL REVIEW Many tests of the effect of chemical agents on NDV have been con­ ducted with virus preparations containing varying amounts of tissue ele­ ments and extraneous protein material. Mixtures of the virus and chemi­ cal were incubated for a certain reaction period and then injected into a suitable host for an indication of the infectivity of the virus. The results have been expressed purely on a qualitative basis as to the abil­ ity or inability of the agent to inactivate the virus completely or par­ tially. Evidence of the infectivity of NDV may be detected by injecting chickens or embryonating chicken eggs with the virus. A measurable reac­ tion such as definite symptoms or mortality may be used as an indication of the activity of the virus. When chickens are used, lethality is the usual criterion of viral activity. If lethality is not the sole criterion, respiratory and nervous manifestations of the disease may require long periods of observation. The greatest limitations to the use of chickens are adequate isolation facilities for prevention of cross infection and a constant supply of suitable birds. Embryonating chicken eggs are the medium of choice because lethality may be used as the criterion of viral infectivity. In addition, the use of eggs has the advantages of ready availability and economy, saving in' expense of feed and quarters, mini­ mum danger of cross infection and lack of production of antibodies against the virus injected. Toxic levels of the chemical agent for the indicator host must be previously determined to eliminate the influence of this 7 factor in interpretation of the results. Among the problems involved in the evaluation of virucidal agents is that of interpretation of the results obtained in one laboratory in terms of those obtained in another laboratory. Obviously, the difficulty could be largely eliminated by use of a common method in which certain physical factors are standardized. The purpose of the present study is to obtain some information as to the effect of the number of infective doses of the virus, the ratio of virus to chemical agent, the temperature of exposure, and the period of exposure as influencing the evaluation of virucidal tests using embryonating chicken eggs as the indicator host for purposes of standardization. These factors are also to be used in studies of the rate of inactivation of the virus. The agents selected for study were phenol, one per cent, mercuric chloride, 0.01 per cent, ethyl alcohol, 4,0 per cent, Roceal (alkyldimethyl-benzyl-ammonium chloride) 0.01 per cent, Roman cleanser (sodium hypochlorite, 5*25$ by weight), one per cent, and tincture of metaphen (sodium /+-n±tro-anhydro-hydroxy-mercurio-orthocresol) 0.01 per cent. Previous studies (Cunningham, 194-3) showed that in the above concentra­ tions, these agents were not effective against NDV at three minute reac­ tion periods. In greater concentrations generally employed for disinfec­ tion in practice, these agents were effective in inactivating NDV in at least three minutes at room temperature. To study the rate of inactiva­ tion of NDV, it was considered feasible to use the above concentrations of the agents- as their effect on the virus would be extended over a greater period of time than if greater concentrations were used. This would permit a more accurate evaluation of the rate of inactivation.. 8 Doyle (1927) tested the effect of a number of chemical agents on NDV by mixing equal parts of virus-infected chicken saliva, infective in a 10"^ dilution, and allowing the mixtures to react for one hour at room temperature (14-15 C). The mixture was then injected intravenously into susceptible chickens in a dose of one ml. The criterion of infectivity of the virus in the mixtures was death of the chicken. Various dilutions of the chemical agent were employed and the following results from this report show the highest dilutions of the chemical agent in which the virus was either active or inactive: Agent Potassium permanganate Lysol Izal Cresol (Rideal-Walker coefficient 18-20) Carbolic acid Mercuric chloride Oil of cloves Sodium salicylate Copper sulphate Hydrogen peroxide (20 vols.) Virus active Virus inactive 1:10,000 1:5,000 1:1,000 1:1,000 1:5,000 1:1,000 1:500 1:500 1:100 1:100 1:100 1:50 1:50 1:10 1:20 1:20 1:20 1:2 With virus active in a 10*“5*7 dilution (1:500,000) the following results were obtained: Agent Antiformin Formalin Acetone Ether Methyl alcohol Ethyl alcohol Sodium hydroxide Hydrochloric acid Virus active 1:500 1:50 1:5 1:4 1:4 N/lOO N/25 Virus inactive 1:100 1:2 1:5 1:2 1:2 N/50 9 Complete information is lacking as to the procedures employed by Farinas (193^) in his studies of the effect of chemical agents on NDV. Chickens were probably used as the indicator host for virus infectivity. With the exception of the statement that potassium permanganate was tested by oral administration and sodium hydroxide by intravenous inoculation, information is not available as to the potency of the virus, route of ad­ ministration, amount of inoculum, proportions of virus and chemical agent in the mixtures and temperature of exposure. Clenzal, 2.5 per cent, killed the virus in 30 minutes; one and two per cent formalin killed the virus in 30 minutes, but 0.1 and 0.5 per cent were without effect; one per cent chloroform killed the virus in 30 minutes but 0.1 per cent did not kill in 30 minutes at ice-box temperature; hydrochloric acid, N/l, killed the virus in one hour but N/10 and greater dilutions were ineffective. Attempts to test the action of sodium hydroxide were not successful as a W/2,500 solution by intravenous injection in itself was lethal. Potassium perman­ ganate in dilutions from 1:2,000 to 1:10,000 was sufficient to render the virus innocuous. Asplin (194-9) employed embryonating chicken eggs as the indicator host for studies of the effect of chemical agents on NDV. Undiluted virus- infected allantoic fluid was used. The titer was expressed as the log concentration of minimum lethal doses (m.l.d.) per ml. Information was lacking as to the proportion of virus and agent in the mixtures, age of embryos and amount of Inoculum. The mixtures were kept for one hour at the exposure temperature indicated and then stored at refrigerator tem­ perature for the duration of the exposure period. At the end of the ex­ posure period the mixtures were titrated for an indication of the activity 10 of the virus. Sodium carbonate, 4. per cent, was ineffective against c> 10 m.l.d. for one hour and 24- hours at 76 F but at seven days the titer of the virus was reduced from 10® to 10^. Lysol, 2.5 per cent, for one min­ ute at 60 F reduced the infectivity from 10^ to complete inactivity. A one per cent solution completely inactivated 10® m.l.d. in one hour at 75 F but a 0.1 per cent solution reduced the titer from 10^ to 10^". A 0.1 per cent solution at 75 F for 24.hours was capable of reduction of the titer from 10^ to 10^. Phenol, 2.5 per cent, at 68 F reduced the titer from 10^ to 10^ sifter exposure for one hour. After 24.hours the virus was completely inactivated. ry At 68 F a 0.5 per cent solution reduced the titer from 10 to greater than 105 in 24-hours. A proprietary hypochlorite disinfectant in 5 per cent solution, the concentration recommended by the manufacturer for disinfection of grossly ? contaminated material, reduced the titer from 107 to one greater than 10^ at 73 F for one hour. The same concentration at 68 F for three hours com­ pletely inactivated 10^ m.l.d. A one per cent solution at 68 F for one hour was Ineffective but at 24- hours the titer was reduced from 10^ to 105 • Potassium permanganate in 0.4- per cent concentration for one hour at 60 F completely inactivated 10^ m.l.d. but 0.2 per cent under the same conditions reduced the titer from 10^ to one greater than 10^. A propri­ etary permanganate disinfectant at 4-0 P6** cent concentration, the con­ centration recommended by the manufacturer for disinfection of grossly contaminated material, had no effect on the virus for one hour at 65 F as shown by no reduction of the titer from 10^. After 24. hours the titer 11 p was decreased from ICk to 10 * In one per cent solution the titer was C reduced from 10^ to one greater than 10^. A commercial coal-tar disinfectant in 2.5 per cent concentration completely inactivated 10^ m.l.d. in one minute at 60 F. In 1.25 per cent concentration at 75 F for one hour, the concentration recommended by the manufacturer for disinfection of grossly contaminated material, the titer was reduced from 10'7 to 102 . In 0.5 per cent solution the titer was re­ duced from 10^ to 10^ at one hour at 75 F. Complete inactivation occurred in 24.hours. Another commercial coal-tar disinfectant in 2 per cent solution, the concentration recommended by the manufacturer for disinfection of grossly contaminated material, reduced the titer from 10^ to ICp within one hour at 67 F and from 10^ to 10-*- within 24 hours. In one per cent solution at one hour at 67 F' the titer was reduced from 108 to 102 and after 24 hours from 10^ to 10"*". The effect of formalin against the virus was found to be influenced by temperature. Virus exposed to 2 per cent formalin was active after one hour at 65 F but inactive in 12 hours. activated the virus in 12 hours. One per cent formalin also in­ Concentrations of formalin ranging from 0.2 per cent to 0.025 per cent required from 10 to 90 days to inactivate the virus at 34-35 F. Virus exposed to 0.1 per cent formalin at 98 F was Inactivated within six hours. Tilley and Anderson (1947), Cunningham (194^) and Beamer and Prier (1950) studied the effect of several chemical agents on NDV. The criterion of inactivation of the virus was the survival of embryonating chicken eggs during a definite observation period following inoculation with a mixture of the virus and agent. The procedures used in these tests varied in 12 several respects, viz*, concentration of the agents, proportions of the virus and agent in the mixture, reaction period, subsequent dilution of the mixture prior to inoculation of the embryos, amount of inoculum, num­ ber of embryos per test, and the length of the observation period, Not­ withstanding these variations, certain comparisons can be made of the effectiveness of the chemical agents commonly employed in these studies. In Table I are presented data from these studies with the chemical agents employed in the present study. Sodium hydroxide, 2 per cent, inactivated NDV during three and five minute reaction periods. Sodium hydroxide, one per cent, was without effect on NDV for 60 minutes. In 0.1 per cent concentration it was with­ out effect during a three minute reaction period. Phenol, A per cent, partially inactivated the virus, only one of four embryos succumbing to the virus, at five minutes according to Tilley and Anderson. At the 15, 30 and 60 minute periods the virus was completely inactivated. In 3 per cent concentration, phenol completely inactivated the virus in three minutes according to Cunningham. A 2 per cent solution of phenol was without effect during the entire 60 minute period according to Tilley stnd Anderson. The data presented by Beamer and Prier indicate that at five minutes exposure to phenol, 2 per cent, only three of five embryos were killed. At 30 minutes the virus was completely Inactivated. Phenol, one per cent, was nonvirucidal. Ethyl alcohol, 95 per cent, was virucidal in three minutes and in 70 per cent concentration In three and five minutes. When used in 50 per cent concentration, it was without effect at five minutes but at 30 minutes the virus was -almost completely inactivated, one of five embryos being 13 killed. Forty per cent and 25 per cent alcohol were without effect in three minutes. Tincture of metaphen, 0.5 per cent, was considered by Cunningham to be virucidal in three minutes but noneffective in 0.005 per cent concen­ tration. Beamer and Prier reported that In the above concentrations the agent failed to suppress viral activity in 30 minutes. Roccal, 0.1 per cent, inactivated the virus in three and five min­ utes. At 0.01 per cent, it was ineffective according to Cunningham during the three minute reaction period. Tilley and Anderson's data reveal vary­ ing efficacy of 0.1 per cent during the 60 minute reaction period. Mercuric chloride, 0.1 per cent, was virucidal in three minutes but at 0.01 per cent it was Ineffective. Sodium hypochlorite, 0.0525 per cent was without effect in three minutes but 0.2625 per cent and 1.05 per cent were virucidal according to Cunningham. Tilley and Anderson's data indicate that sodium hypochlorite in 200 and 4-00 parts per million (p.p.m.) available chlorine, pH 11.4, was without effect for 60 minutes. In addition to the chemical agents listed in Table I, Beamer and Prier found the following to be apparently effective against NDV in five minutes: liquor cresolis saponatus, 1 :4 0 0 ; tincture of Iodine, undiluted and 1:100; Lugol's solution, 1:1,000; formalin, 2 per cent; potassium permanganate, 1:100 and 1:1,000; merthiolate, 1:1,000. were effective in 30 minutes: The following formalin, 0.5 per cent and liquid green soap, 1:10. Agents that had no apparent effect were potassium dichromate, 1:100, merthiolate, 1:100,000 and hydrogen peroxide, 8 per cent. 14 The following quaternary ammonium compounds were effective in in­ activating the virus in five minutes: Zepharin, undiluted; para-tertiary- octyl-phenoxy-ethoxy-ethyl-dimethyl-benzyl ammonium chloride monohydrate, 1:500 and Roecal, 1:100. The following were ineffective at five minutes but were virucidal at 30 minutes: Zepharin, 1:100; para-di-isobutylphenoxy-ethoxy-ethyl-dimethyl-benzyl ammonium chloride, 1:100 and 1:1,000. According to Tilley and Anderson, the following agents were virucidal: sodium orthophenylphenate, one per cent at five minutes; para-tertiaryoctyl-phenoxy-ethoxy-ethyl-dimethy1-benzyl ammonium chloride monohydrate, 0.1 per cent at five minutes; liquor cresolis saponatus, one per cent, almost complete inactivation at 15 minutes and complete inactivation at 30 minutes. Calcium hypochlorite, 100 and 200 p.p.m. was without effect in 60 minutes, but at 400 p.p.m., pH 8.25, it was virucidal in five minutes. Formalin, 4 per cent, failed to inactivate the virus in 30 minutes but was effective in 60 minutes. Isopropyl alcohol, 50 per cent, ethylene glycol, undiluted, and sodium carbonate, 4 per cent, appeared to be ineffective against the virus. According to Cunningham, the following agents, other than those listed in Table I, were effective against NDV in three minutes: tincture of Zepharin, 0.1 per cent, Phemerol, 3 per cent, Disilyn, 0.02 per cent, liquor cresolis saponatus, 3 per cent, Lysol, 3 per cent and one per cent, creolin Pearson, 3 per cent and one per cent; and tincture of iodine, 2.5 per cent and one per cent. The following agents did not completely in­ activate the virus during the three minute reaction period, but they were 15 considered as beirig effective against the virus: Phemerol, one per cent, liquor cresolis saponatus, one per cent; potassium permanganate, 0.1 per cent, and formalin, 10 per cent. The following agents were without effect on the virus during the three minute reaction period: merthiolate, 0.1 per cent and 0.01 per cent, tincture of Zepharin, 0.001 per cent, potassium permanganate, 0.01 per cent, tincture of iodine, 0.1 per cent and 0.01 per cent, formalin, one per cent and 0,01 per cent; and boric acid, 4 per cent. While similarities In the trends of the effectiveness of these agents are evident in all three studies, certain irregularities in the procedures must be considered. Different concentrations of the agents were employed. This is not of major importance except when direct comparisons are to be made. In several agent was used by instances, the same concentration all authors. of It is evident that the a givenchemical authorsattempted to establish facts as to the effectiveness of the agents in concentrations at least those generally employed for disinfection as well as at minimum virucidal concentrations. Differences in the virus:chemical volumetric ratio are obvious. Tilley and Anderson, and Beamer and Prier used virus and agent in equal parts, whereas a 1:9 ratio was used by Cunningham. would result in a marked variation in the number of These differences virus particles at the time of contact with the chemical agents. Cunningham and Tilley and Anderson used undiluted virus-infected allantoic fluid but Beamer and Prier used a 10“3 dilution of the fluid. g The virus used by Cunningham contained 10 infective doses per 0.05 ml. Tilley and Anderson reported that their virus, which was titrated for 16 H to 10° $ infective doses per 0.1 ml. each experiment, contained from 10' These viruses had been originally established and propagated in chicken embryos for 15 passages and four passages, respectively. Beamer and Brier’s virus which contained 10*^ infective doses per 0.1 ml had been isolated from a natural outbreak of the disease in chickens and carried for 17 serial passages in duck embryos. Chicken embryos were used for titration and for the tests. For a quantitative comparison of the three different strains of the virus on a uniform basis of 0.1 ml inoculum per egg, the strain used by Cunningham would have to be considered as containing 2 x 108 or 108 3 (200,000,000) infective doses as titration was done with 0 .0 5 ml inoculum. When the virus and chemical were mixed in a ratio of 1:9, the mixture would be considered to have 1C?*3 (20,000,000) infective doses. Some of the mix­ tures were injected directly into the embryos without further dilution at the end of the reaction period but some were diluted as much as 10”3# The number of virus particles injected into embryos in the various tests ranged from 107*3 (20,000,000) to 10^.3 (20,000).. See Table I. The virus used by Beamer and Prier had an initial infective concen­ tration of lO^O (10,000,000,000) doses but a 10“3 dilution, which would contain 107 (10,000,000) infective doses, was used for mixing with the chemical agents. Since the mixtures were in equal parts, there would be 106-7 (5,000,000) doses of virus. Subsequent dilutions ranged from 10-3*3 (1:2,000) to 10“4 (1:10,000) and the number of virus particles injected ranged from 10^ (2,500) to 102*7 (500). See Table I. ry . Tilley and Anderson employed virus containing from 10' (10,000,000) to 10^ (100,000,000) infective doses. When mixed with equal parts of 17 chemical agent from lO^-7 (5,000,000) to 107*7 (50,000,000) doses were present. Subsequent dilutions ranged from 10"^*7 (1 :5 0 ) to 10’"^#-^ (1:200) which resulted in inocula containing from 10^ *4- (25,000) to 10^*^ (1,000,000) virus particles. See Table I. Summation of these data show that the inocula ranged from 102 *n (500) to 107*3 (20,000,000) or a maximum possible 10^*6 differential. See Table I. The number of eggs employed for each test varied in the three studies. The survival or death of one of the four embryos in Tilley and Anderson^ study would result in a differential of 25 per cent, and 20 per cent, one of five embryos in Beamer and Prier!s work. With Cunningham *s work the differential would be 10 per cent or one of ten embryos. 18 TABLE I THE EFFECT OF CERTAIN CHEMICAL AGENTS ON NEWCASTLE DISEASE VIRUS AS DETERMINED 'BY INFECTIVITY TESTS IN EMBRYONATING CHICKEN EGGS* Chemical Ethyl alcohol Cone. Ratio of virus: chemical 95% 70% 1:9 1:9 1:1 1:1 40% 25% Tincture of metaphen Mercuric chloride Phenol 1:9 1:1 0.005% 1:9 0.005% 1:1 100 10 4% 1:1 1% 1:9 100 100 50 2000 100 5% 1:9 1:9 500 500 1:1 1:1 1:1 2500 2% 0 .1% Roccal 1000 2000 10 2000 1:9 1:9 1:9 0 .1% 0 .1% 100 100 100 100 100 0 100 25 0 0 100 100 100 0 60 50 100 0 0 0 50 100 100 25 25 0 100 100 100 10 1:9 2500 100 1:9 1:1 0 20 0 100 1:9 1:1 0 100 100 100 0 .1% 0 .01% 1:1 1:1 Per cent embryo mortality Reaction period Minutes 3 5 15 30 60 Ref 2500 2500 1:9 1:9 0.5% 0.5% 3% Sodium hydroxide Sub­ sequent dilution 50 25 25 19 TABLE I (Continued) Chemical Sodium hypochlorite Cono. Ratio of virus: chemical 1.05% 0.2625% 0.0525% 1:9 1:9 1:9 Available CI2 p.p.m. 200 1:1 400 1:1 Sub­ sequent dilution 10 50 100 Per cent embryo mortality Reaction period Minutes 5 15 30 60 Ref. 3 0 0 100 (1) (1) (1) 100 100 100 100 100 100 100 100 * Data from (l) Cunningham (194&), Beamer and Prier (1950), and (3 ) Tilley and Anderson (194-7). Eggs used per sample: (l) 10, (2) 5, (3) 4 Infective doses of virus: (l) 10® per 0.05 ml, (2) 10-LU per 0.1 ml (3) 107 to 10s per 0.1 ml Inoculum per egg: (l) 0.05 ml, (2 ) (3) 0.1 ml Temperature of exposure: (l) (2) room temperature, (3 ) 20 C (3 ) (3 ) 20 McCulloch (194-5) focused attention on the fact that there have been marked differences in the procedures employed in studies of the effect of chemical agents on viruses. He suggested that the following factors should be considered for a critical evaluation of virucidal or bactericidal tests: 1. The number of infective doses of the virus. 2. The ratio of virus-containing material to agent. 3. The temperature of exposure. 4.. The period of exposure. 5* The size of the particulate matter containing thevirus. 6. The nature of the fluid in which the virus is suspended. a. pH b. buffer capacity c. reducing properties d. osmotic index e. surface tension Ill MATERIALS AND EXPERIMENTAL PROCEDURES A strain of NDV (Accession 51-52 308) which had been originally isolated from lung and tracheal material from a natural outbreak of the disease In chickens was used. This virus was established and cultivated through 8 serial passages in the allantoic cavity of embryonating chicken eggs and was capable of killing all embryos by the end of the second day after inoculation. The virus preparation employed consisted of pooled allantoic fluid from embryos dead on the second postinoculation day. Por­ tions of the pooled virus were placed in 30 ml capacity screw cap vials and stored at -30 C. At the time of use, the virus was thawed at room temperature, centrifuged to sediment the yellow, insoluble precipitate formed on freezing and thawing, and the supernatant fluid was transferred by pipette to another vial. The virus-infected supernatant fluid contained 0.54 mg. nitrogen per ml as determined by the macro Kjeldahl method (Assoc­ iation of Official Agricultural Chemists, 1950), and 10^*^ lethal doses per 0.1 ml as determined by titration in eggs (Cunningham, 1952). The study was divided into two parts: (l) the effect of the various chemical agents on undiluted virus in proportions of virus: chemical agent of 1:1 and 1:9 at 0 C, 20 C and 37 C during various periods of ex­ posure, and (2) the effect of the chemical agents on serial ten-fold dilutions of virus ranging from undiluted through a 10-6 dilution in propor­ tions of virus:chemical agent of 1:9 at 20 C for 15 minutes. 22 All chemical agents and the virus were at thermal equilibrium at the contact period. The chemical agents, which had been prepared in distilled water, were dispensed for the first part of the study in two ml amounts from two ml serological pipettes for the 1:1 mixture in 13 x 100 mm Pyrex tubes and in 4.5 ml amounts from five ml measuring pipettes for the 1:9 mixture. The virus was then added to the chemical agents from two ml ser­ ological pipettes, two ml for the 1:1 mixture and 0.5 ml for the 1:9 mix­ ture. The ingredients were thoroughly mixed by aspirating and expelling the mixture 20 times with the pipette. The samples tested at 0 C were placed in crushed ice in a "Thermos" laboratory vessel. An electric, thermostatically controlled water bath was used for the tests at 20 C and 37 C. .The level of the water in the baths was above the level of the virus-chemical mixture in the tubes to insure thermal equilibrium through­ out the reaction period. For the second part of the study, serial ten-fold dilutions of the virus through the 10”^ dilution were prepared in distilled water using one ml of virus to nine ml of distilled water. The mixtures were prepared in the same manner as the 1:9 mixtures previously described for the first part of the study. At the end of each reaction period, 0.1 ml of the mixture was trans­ ferred with a one ml serological pipette to 9.9 ml of distilled water to dilute the chemical agent to a concentration nontoxic for the embryo. The ingredients were mixed by aspirating and expelling the mixture with the pipette 20 times. Some of the agents in the concentrations employed could be injected directly into eggs while others required further dilution. 23 Previous studies (Cunningham, 194.8) had shown that the following dilutions were sufficient to render the agents nontoxic for embryos: Agent Concentration Tincture of metaphen Ethyl alcohol Mercuric chloride Roccal Sodium hypochlorite Phenol Sodium hydroxide Dilution 0,5% 40% 0.01% 0,1% 0.0525% 1% 1% 10 10 100 100 These dilutions cover a 100-fold range and for uniformity of inoculum a 10“^ dilution was made prior to injection of embryos. Ten eggs were used for each test at the various reaction periods. Injection was via the allantoic cavity with 0.1 ml inoculum per egg. All incubation of eggs was at 99-99*5 F in an electric, forced-draft incubator (Jamesway Model 252) in which the eggs were automatically turned every two hours. The postinoculation incubation period was seven days. Eggs were candled daily. Embryo mortality during the first 24 hours was con­ sidered to be due to nonspecific causes and these eggs were not included in the final results. Titration of the virus for the first part of the study was accomplished by preparing serial ten-fold dilutions of the virus using nutrient broth as the diluent (0.5 ml virus: 4.5 ml diluent). Five eggs were employed per dilution and each was inoculated with 0,1 ml of the respective dilu­ tions via the allantoic cavity (Cunningham, 1952). bated for seven days following inoculation. The eggs were incu­ Mortality rates were used in computing the titer which was expressed as the lethal dose according to the method of Reed and Muench (1938). (l.d.5 0 ) The l.d.5 0 was cal­ culated to the centile and rounded off to the decile. The number of 24 lethal doses of virus was considered to be the antilog of the reciprocal of the l.d. 5 0 Titration of the virus for the second part of the study was accom­ plished by using the virus dilutions prepared for mixture with the chem­ ical agents. Ten eggs were used per dilution. The procedures and cal­ culation of the l.d.^o were the same for both parts of the study. IV RESULTS The results of the tests of the infectivity of NDV in mixtures of certain chemical agents in proportions of virus:chemical agent of 1:1 and 1:9 at 0 C, 20 C and 37 C at different time intervals are summarized for the first part of the study in Table II. The criterion of viral infectivity was embryo mortality. Death of all embryos was recorded as 100 per cent, indicating that the virus was not affected by the chemical agent. Survival of all embryos was recorded as 0 per cent, indicating that the chemical agent dompletely inactivated the virus. For one of the tests the databest fit (l) a regression line, Y a a + bX, and for the other tests (2) alogarithmic curve, log Y - a + bX, as computed from the following respective equations: (1) Na-h IbX a £Y a £ X + b £ X 2 = £XY (2) Na + £bX = £log Y a £X + b £X2 = £Xlog Y Effectof Chemical Agents In all on Undiluted Newcastle Disease Virus testsin the first part of the study where the effect of the various chemicals on undiluted virus was investigated, it was assumed that the virus in the mixtures was capable of killing all embryos immediately following contact of the virus with the chemical agents. The data for some tests showed that the virus was capable of killing all embryos through­ out the entire series of reaction periods. In some tests, some degree of 26 inactivation of the virus occurred. For an indication of the rate of in­ activation of the virus in the latter tests, the last period at which 100 per cent mortality occurred was given the value 0.0X. The data were used throughout the first period at which inactivation was complete (0.0 per cent mortality) or the terminal reaction period if complete inactivation had not occurred prior to this time. At 0 C, inoculum from the mixture of equal parts of NDV and phenol, one per cent remained infective for all embryos for 60 minutes. Inoculum containing NDV and phenol in proportions of 1:9 was infective for all em­ bryos for 4-0 minutes but at 50 minutes the embryo mortality was 90 per cent. At 60 minutes, only 20 per cent of the embryos were killed. These data indicate that for J+0 minutes NDV exhibited similar resistance to phenol in proportions of 1:1 and 1:9. During the next ten minutes there was a slight inactivation of the virus in the 1:9 mixture followed by a precipi­ tous decrease in viral activity during the next ten minutes. These data are presented graphically in Figure 1. The straight line regression equa­ tion Y * 110 + (-4*.0)X best fits the data for the 1:9 mixture during the 4,0 to 60 minute periods. For the mixture of equal parts, the equation log Y - 2.0 + 0.0X fits the data. When I3DV and phenol were mixed in equal portions at 20 C, the virus killed all embryos in 30 minutes. During the next ten minutes the infec­ tivity of the virus was markedly reduced so that at 4-0 minutes only ten per cent of the embryos were killed. At 50 minutes, ail embryos survived but ten per cent were killed at the 60 minute period. the virus in the 1:9 mixture occurred at a rapid rate. Inactivation of Only 60 per cent of the embryos were killed ten minutes after contact of NDV with the phenol. 27 TABLE I I PER CENT EMBRYO MORTALITY FOLLOWING INOCULATION WITH MIXTURES OF NEWCASTLE DISEASE VIRUS AND CERTAIN CHEMICAL AGENTS 0C Temperature 20 C 37 C Proportion of virus; chemical agent Reaction period - mins. 1:1 1:9 10 20 30 40 50 60 100 100 100 100 ICO 100 Phenol, 1%, pH 6.460 100 100 100 10 100 100 10 100 100 0 10 0 0 90 10 10 20 10 20 30 4-0 50 60 100 100 100 100 100 100 15 30 4-5 60 75 90 105 120 90 90 50 4.0 30 60 10 30 1:1 Ethyl alcohol. 100 100 100 100 100 100 100 100 100 100 100 100 Sodium hydroxide. 30 11.1 30 10 20 10 30 10 4.0 20. 20 10 30 10 0 20 Sodium hypochlorite, 44.0 10 15 100 0 100 30 10 100 4-5 10 100 60 0 100 75 0 100 90 0 100 10$ 10 100 120 p.p.m 100 100 100 100 100 100 100 100 1:9 1:1 100 100 100 1:9 1C4 50 20 100 pH 6.9 88.8C 100 88.& 60 80 100 100 100 60 60 37.51 10 20 0 0 70 , pH 13. 0 20 0 0 0 20 0 0 10 0 0 0 0 0 0 0 0 22 .2° 10 0 0 0 10 0 0 Virus l.d.5 0 chlorine, pH 10 11.le 100 0 100 0 90 55.5d 11.le 0 AO 20 20 10 30 10 - 28 TABLE II (C o n tin u e d ) Temperature 20 G 0c 37 C Proportion of virus:chemical agent Reaction period - mins. 1 :1 15 30 45 60 75 90 100 100 100 100 100 100 15 30 45 60 75 90 105 120 100 100 100 100 100 100 100 100 1 :9 1 :1 1 :9 1 :1 1 :9 100 100 100 90 100 80 0 0 10 0 10 10 Roccal, 0.01% , pH 6 .1 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 Mercuric chloride, 0.01% , pH 3 .5 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 Tincture of metaphen, 0.01% ,, pH 8 .4 15 30 45 60 75 90 105 120 a One 100 100 100 .100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 Per cent calculated from 8/9 mortality rate b Two aribryos died from nonspecific causes Per cent calculated from 3/8 mortality rate. c One ;rabryo died from nonspecific causes, Per cent calculated from 2/9 mortality rate, jmbryo died from nonspecific causes, Per cent One d calculated from 5/9 mortality rate, e One jmbryo died from nonspecific causes, Per cent calculated from 1/9 mortality rate. 29 XI o o ^«? o iH o h O o CO O rH • • OJ cv w o 02 rH CT1 •* •• rH i—I O I T3 •OH U 0> sO a o •H -op o c VO (c D; PS o « o• I* O '— + + o o O O CO • rH 02 H O M H C bO o rH 02 rH *• O•* —1 O P -4- fi rH O' i—1i —I •H 10 }> cd o ft £ O 0 m s P o « o *H -P •V P O • f t vO o u ix; f t f t p • •H O *>, !> I—ICO c^i •*,_* • CO -P 1—1 o cd o rH **—^ •ft o I —t o Cd 100 o o (J\ o 00 o £> o vO o Jft o ~ o &q ^ T T e^-j:OTJ!I oAiqin© q.u©o c\i 0 u o *t r4 Uh 32 mixture but occurred at a later time. Comparison of the data for the different temperatures shows that inactivation occurred more rapidly at 20 C than at 0 C, When eggs were inoculated with mixtures of virus:ethyl alcohol, 4-0 per cent, in proportions of 1:1 and 1:9* all embryos died throughout the 60 minute period at 0 C, Figure 3. When similar mixtures were exposed at 20 C for 60 minutes, the virus was sufficiently active in the 1:1 mix­ ture to kill all embryos. The equation log Y = 2.0 + 0.0X fits these data for 0 C and 20 C. Inoculum from the 1:9 mixture killed 88.8 per cent of the embryos (eight of nine embryos) at the ten and 20 minute periods. During the 30 to 60 minute interval, the mortality ranged from 60 to 80 per cent; 60 per cent at 30 minutes, 80 per cent at 40 minutes, 60 per cent at 50 minutes and 70 per cent at 60 minutes. The equation log Y ~ 1.98 + (-0.003)X best fits these data. At 37 C the mixture of equal parts of virus and alcohol killed all embryos in 4° minutes, but at 50 minutes only 60 per cent of the embryos were killed. Figure 4 * The equation log Y *- 2.0 + (-0,02)X fits these data. The 1:9 mixture killed 37.5 per cent of the embryos (three of eight embryos) at the ten minute interval, ten per cent at 20 minutes and 20 per cent at 30 minutes. Inoculum after 40 and 50 minutes exposure was innocuous. The curve log Y = 2.035 + (-0.043)X fits these data. These results show that the. effect of ethyl alcohol, 40 per cent, NDV in proportions of 1:1 and 1:9 at 0 C for 60 minutes could not be detected. At 20 C and 37 C, inactivation was more rapid in the 1:9 mistures than in the 1:1 mixtures. Thermal influence was evident in that 33 to to o o v O I—I o UN O TO un CO o o Xo CO o p u *H o o o to o o * CD O2 CO Cd TP o CO * 02 02 rH o <1> p CQ cd •H ^3 O o CD O i — I -P -P f—I to O P ( —I £5 cd cd o c•o « CD i—i ^ np § § o •H P o *H •»! 1 •* • |—4 HD Q n h mp-iCo rH *N O HD o ' — rP "Bp Q o o h D O, tCn o CO CD o o Com U o ♦• rH rH 0 • o P -P O CD *H -P cm rH O «a O ►>>03 H- + W cd Cm rH O CO o 02 ^q.T"[Bq.Jcoi[i oj£j:qiii0 quso J ia j O i—t £ bQ Pm *rl 34 00•»c erf •CO O O i—! •• • rH rH vO WPH 0«H U• "nO "Orf rH x *\ * O o v Q C \?o• o o r-i rC TsR O ^ O• I' ps •H o > o 0) »H o CO > O -H S 03 O m ♦hO rH O O CO r ^ j erf 0 ) <+H CO O rH *H r^TJ -P Op erf P OHOO rH O UA MOO O CV CV CA O rH CV 3 U UA "Cf «D qjO o OA UA rH O CQ 0) -P •r-j g li O CV x i rH O (D UA P-, O t—i c o »H -P o O CA cd 0) — -vO CV OA tO MO O O• o *o UA £> CV O MO CO o o o CV *cv• O MO UA ' O CD CV CO cd TO CD c to cd •H o o o rH P CO cd cd o £ ** (D rH is; rH CJ a o cd ^ rH O «• • rH OA i—1 cm O iU a, 0) TO .M o rH Jh V —' 'B i>> a> Pi _i $ o •H TO o ■§ O K** C PJ •+ 5 g P3 P *H •H TO i> o to C h o

\ »> i—ItO 0 • 93 H (XO -W i —I o o-^ O 9^ ^ UAt>» «H * rP '-"3 0 rH 9 93 *H -vf- z> o •H « 0 i —I o vO P 0 ft O *H bD i —OI i —I i —1 v\ ■SX a I 0 »H g 0 > P erf •H 0 ep 9O3 *rH0o 0 93 0 C H tlOtiD o o rH rH rH (H i —I0s •• O to o •• rH ir \ O o o~\ ** *« i—I rH i—IO'' o H- O CH O CV iqUnjoin OiCvTqurs q.u30 uaj o o C Figure 7. Effect of sodium hypochlorite (440 p.p.m. available chlorine), pH 10.9, on Newcastle disease virus, l.d.cq 10° •, in proportions of virus:sodium hypochlorite of 1:1 and 1:9 at 0 C and 20 C. I c\i 0 a to that in the same mixture at 0 C, Fifteen minutes after contact, 22.2 per cent of the embryos (two of nine embryos) were killed by the virus. At 30 minutes ten per cent of the embryos died but at 4-5 through 75 min­ utes none of the embryos were killed. Ten per cent died at 90 minutes and none at 105 and 120 minutes. Using the data through the 4-5 minute reaction period, the mortality at 90 minutes not being considered signif­ icant, the equation log I = 2.03 + (-0.042)X fits the data well and in­ dicates the logarithmic regression of activity of the virus. It is obvious in the 0 C and 20 G tests that the rate of inactivation with the 1:9 mixtures was much greater than with the 1:1 mixtures. At 37 C the effect of thermal influence was quite marked. Figure 8. Fifteen minutes after contact of the virus and the agent in the 1:9 mixture, 11.1 per cent of the embryos (one of nine embryos) died and none were killed at the 30 and 4-5 minute periods. Sixty minutes after contact, 11.1 per cent of the embryos died. No mortality occurred at the 75 min­ ute interval but 20 and ten per cent mortality was produced at the 90 and 105 minute periods, respectively. Using the data through the 30 minute exposure as an indication of the period of inactivation, the curve log Y = 2.014+ (-0.066)X fits these data well. It is assumed that the variability in mortality during the latter stages of exposure was due to nonspecific causes. With the mixture of equal parts of virus and agent, all embryos died during the first 30 minutes. Ninety per cent were killed at the 45 minute period, 55.5 per cent (five of nine embryos) at 60 minutes, 40 per cent at 75 minutes, 20 per cent at 90 minutes, and 30 and ten per cent, respectively, at the 105 and 120 minute periods. The equation log Y = 2.04 + (-0.0103)X fits the data well throughout the entire period of inactivation. 42 CD ot 4c3 d i —I -4f3t •H cd f > cd *H H >• T O B• < D? CO CO P Cd < D Ph CO ' H Td *hf-t i—I 2>x£) O• o • O xO rH O o cv h 1 tr \ 4 &c?8 o c -t ° + CV c v o CO Ox CD -P P 53 •rj U"\ g uO o uO o I I I— CD * r- I— 1 P o o 43 O h •H P O to -P ch •"--V cd CD -p •H c IT\ o ''t •H O C\ o ft Ox•» O •H xQ P O o rH -P cd CO

O -P O CD •H cd i—1 £3 -P 43 P Ox O P o .• * P3 * o g 1—1 p CO o 53 •H <+H 3 £3 o to CD •tlD H P i— 1 •• rH • Pc •CO o o 0 rH CD -P 0 o P CD O VT\ P cd P h f3 P£0

UNO O ON o xO x 4’ G n On O • nO E> • c-N i 1 nd fl TO Cd — *» • i— IC \i UN Pi O •H t> o C\2 0 TO »v O ON cd o 0 O TO *H nd -P rH ON •• cd •* i —Ii —I UN rH CO CD -P P P •H O e i no o 0 rH On -P TOi—I cd o nd £ P 0 cd rH p •• —1 •'Ch 0 i—1 o P U nO i—1 o P i [~J ocd ON o •H Pi O -P O O h r--•'n■P»* *H O i U UN !> o o • • o o o sO O -t- + o o o tNi • • nd C \i c \ i ii II cd M O fciOMtUD o o o rH rH UN o ON UN rH O • 100 o On O to o £> O vO o un NN CO iH P o p • *h 3 > rH PH O cd rH O' • • «• iH rH o cd 0 pd O c ON o p CD •H d 1 T3 O o *H v£> U P Pi G UA o m"•H -P O cd CD O Pi rA > < >< o o o o•o• ■cda o.°. C\i Oi o ii rt O M t>H cv W U0 -vo o O H i —1 O rH O'' *• •• rH iH o • I TO o G •4 cd ■& • rH 00 G ♦» *H rH Pi o OA Cvi *H P -P rH H P Pi • •H CO to I I m p vD •Prii I >

o

O vO O x r\ O o CM ^q.TX^Q.Jom o.&iqnra q.UQ0 .19j O t—1 o ho hO CD O C o •H +-* 02 rO C . +3 ir \ i — I o to 43 ON 02 to o O CO oAiqui© q.ueo aaj o Jh 0 2 o *o to Figtire 13. Effect of certain chemical agents on different concentrations of Newcastle disease virus, l.d.^p 1 0 '*^ at 20 C for 15 minutes in proportions of virus:chemical agent of 1:9. 51 OJ 52 log Y - 2.0 + 2.OX and indicate that mercuric chloride completely in­ activated as many as 1 5 8 ,5 0 0 doses of virus. Sodium hydroxide, one per cent, and Roccal, 0 .0 1 per cent, exerted a rapid effect on NDV and at the same rate. Figures IX and 15. When 1 q9 .2 £oses 0f virus were mixed with these chemical agents, there was ten per cent mortality of the embryos followed by no mortality with 1 0 ^ * 2 or fewer doses. These data, while insufficient for analysis of the rate of inactivation, suggest a logarithmic regression as there was a 90 per cent reduction of embryo mortality from the time of contact of the virus and chemical agent to the sampling period. These results indicate that these chemical agents inactivated as many as 158 ,5 0 0,0 00 doses of virus. Sodium hypochlorite, XX° p.p.m. available chlorine, completely in­ activated the virus in all concentrations of virus employed from lO^ * 2 through 1 0 ^*2 ^th the exception of the lO^-*2 concentration. This latter concentration in which ten per cent mortality occurred is not considered significant in evaluating the results. These data indicate that sodium hypochlorite, XX0 p.p.m. available chlorine, completely inactivated 1 >585 ,0 0 0 ,0 0 0 period. doses of the virus from the time of contact to the sampling The rate of inactivation was probably a logarithmic regression. Tincture of metaphen, 0.01 per cent, did not inactivate the virus in concentration of 109- 2 through 1 0 ^ .2 as evidenced by 100 per cent embryo mortality in these dilutions. With 10^ *2 doses of virus the mortality was 30 per cent. Using the dilution factor of 10~1 as previously described for evaluating rates of inactivation, this would indicate that only 1 0 ° • 2 doses of virus were injected into the eggs. Comparison of the 30 per cent mortality obtained with this concentration of WDV and tincture of metaphen 100 cm cm •H o o to O rH CM U~\ T O•COh •H Pi •P o cd £ CM 02 o • sO XI PL, £2 02 g ft £> ^ o 0 0 S3 Cf-t o o •H -P 0 P • -p a •H rH P *H o CCS S > o ch S3 o O ft • •H O -p u csj ft ft -P .. S3 *H 0 0 0 •H O J U0 -S O~ *± t ig to 02 CO Pi ° -P £ o -P -H C6 0 02 Ch •O ON P i O •H -P cd 2S3 3 0 02 O h O 02 O U 1»H f tm 0 i —I ch ch xJ ch S 3o o o 0 02 -P ft -P 0 0 bo cd02 «N i —cd Io♦ oo •H rH e ^ • 0 oo ft! 02 v o »* o * rH