MANY studies on genetic resistance to

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1 Sire and Dam Effects on Hemagglutination Titers in Avian Eggs Following Inoculation with Newcastle Disease Virus 1 G. RETA, 2 B. B. BOHREN AND H. E. MOSES Purdue University Agricultural Experiment Station, Lafayette, Indiana (Received for publication March 25, 1963) INTRODUCTION MANY studies on genetic resistance to disease have been conducted on poultry and other animals. A genetic basis for resistance has been found for most diseases studied in all species observed. The literature on the subject has been adequately summarized by Hutt (1958). Only a few studies have been conducted on genetic resistance to the virus of Newcastle disease in poultry. Godfrey (1952) observed strain, breed, and cross differences in resistance to a natural outbreak of a virus infection, presumably Newcastle disease, as measured by pauses in egg production. Diagnosis of the disease was based on clinical signs and the virus was not isolated. Francis and Kish (1955) found differences in mortality and days to death between families of New Hampshire chicks when challenged with a standard dose of Newcastle disease virus. Cole and Hutt (1961) also observed distinct differences in mortality between two strains of White Leghorns following wing-web inoculation at 16 weeks of age with Roakin strain live-virus vaccine. No reports on the genetic resistance of avian embryos to Newcastle disease virus, or parental effects on the increase in virus concentration in eggs following egg inoculation with Newcastle were found. 1 Journal Paper No of the Purdue University Agricultural Experiment Station. 2 Present Address, School of Veterinary Medicine, Ciudad Universitaria, Mexico City, Mexico. The purpose of the present study was to investigate parental effects on hemagglutination titers of avian eggs following inoculation under standardized procedures with strain Bi Newcastle disease virus. MATERIALS AND METHODS The eggs used in this study were obtained from the North Central Region Poultry Breeding Laboratory, from pens maintained for the production of the Cornell Randombred control stock. Five females and one male were randomly placed in each of five individual pens. These birds had been vaccinated for Newcastle disease with La Sota strain in the drinking water at four days of age, and again at 18 weeks of age. These birds had shown no clinical signs of disease. The birds were 56 weeks old when the first eggs were collected for incubation. Two settings, each consisting of fourteen days collection of eggs from these pens, were made. Each setting of eggs was for one replicate of the experiment. The date of lay, dam and sire numbers were recorded on the shell of the eggs. The eggs were held at F. until three hours prior to setting, at which time they were removed to room temperature of 70 F. During this time they were numbered on one side with a code number and the eggs were trayed at random. For each setting, all the eggs were placed in a Jamesway Incubator (Model 252) which was maintained at a temperature of 99J F. with a wet bulb reading of 85 F. 1182

2 SIRE AND DAM EFFECTS ON VIRUS TITERS 1183 After 10 days of incubation, the eggs were candled and the infertile eggs and early dead embryos were removed. The viable eggs from the birds to be tested were inoculated in the allantoic sac with 0.1 cc. of a virus dilution containing 10 4 embryo lethal doses. Eighteen hours after the inoculation, 0.2 ml. of allantoic fluid were taken from each embryo in a random order, the procedure being similar to that described by Green and Freyman (1949). The fluid removed was mixed with 0.8 ml. of buffered saline solution and stored at 7 C. until the hemagglutination tests were carried out. In the second replicate, 18 hours following inoculation all eggs were removed from the incubator and chilled at 4 C. overnight, after which samples were taken from each egg. This procedure was an attempt to reduce the random error due to continued virus growth during the two hours required for the collection of samples as in the first replicate. The erythrocytes required for the hemagglutionation tests were obtained by standard procedures from a single male white Leghorn for each replicate. A suspension of 0.5 percent erythrocytes was used in the tests. The Bi strain of Newcastle disease virus used in this study, was obtained from the North Central Regional Newcastle Virus Repository, Department of Veterinary Science, University of Wisconsin. Prior to the experimental work, four passages of virus were made in 10-day-old chicken embryos from a commercial flock used for general work in the laboratory. Pooled allantoamnionic fluid obtained from the embryos of the fourth passage was the source of the inocula for the experimental eggs. The procedure followed for the hemagglutination test was essentially that proposed by Brandly, Moses, Jungherr and Jones (1946). Briefly, this procedure utilized progressive doubling dilutions in buffered salt solution, of the virus samples from the eggs, starting with one-in-five (1:5) dilution and doubling as necessary, until the end-point of agglutinating activity was attained. For each egg, a series of 10 round-bottom agglutination tubes (13X100 mm.) containing nine dilutions of virus from 1:5 up to and including 1:1,280, and a control tube containing no virus material. The test was read when the erythrocytes in the control tube had sedimented to form a central button-like deposit. Usually this occurred about 45 to 60 minutes after setting up the test series. At that time, any tube resembling the control was considered as a negative test. Agglutination, or a positive test, was indicated by a thin uniform deposit over the entire bottom and shoulders of the tube. The end-point was considered to be the highest dilution showing complete agglutination. For statistical analysis each egg was given a score from zero to nine, corresponding to the dilution level of the endpoint. Thus, a score of one means that there was agglutination only at the dilution 1:5, while a score of five, for example, indicates that the highest dilution showing agglutination was 1:80. In this manner, each increment in score corresponds to a doubling of the virus concentration in the allantoic fluid. The model for the statistical analysis was based on the hierarchical or nested design with unequal subclass numbers. The deviation of any egg from the mean (M) can then be expressed as due to the effects of the kih egg (k=l,, «,-,-) produced by the jih dam (j=l,, rf,-) mated to the ith sire (i=l, -, s) in the model Yijk ti-\-si-\-dj{i)-\-bk(,i}) -\-ekaj).

3 1184 G. RETA, B. B. BOHREN AND H. E. MOSES The term 8*(,7) refers to genetic segregation effects from the sire and dam and is completely random, while the term e^,-,-) is a purely environmental random effect and would include the effect of any uncontrolled factors which are randomized over the embryos produced by each sire and dam combination. The analysis does not permit the separate estimation of these two terms and they are jointly estimated as ov or the random error variation between eggs from the same sire and dam. The form of the analysis of variance and the mean squares are as follows: Source of variation Degrees of freedom Sires s 1 Dams in sires d s Eggs in dams in sires N d Total N-\ Mean squares (S-CT)/{s-\) (DS)/(ds) (T-D)/(N-d) ' Expected mean squares ^E+fe^B+fao^s <T*E+kl<T'D <r*e (Note: N =X{ 2/ nij and <Z=Si di). The computation formulae for the uncorrected sums of squares are, Total sum of squares (7 1 ) =2i 2/ Sit Y^ijk, (S» Sy Si Correction term (CT) =- N Si (Sj St Sire sum of squares (S) = Sy nij YijkY Yijk)' SiS;(SiF,V^ Dam sum of squares (D) = nij and The coefficients in the expected mean squares were derived as shown by Steel and Torrie (1960) and in replicate one i = 5.12, ^2 = 5.75, and 3= In replicate two, i = 6.72, 2 = 7.65, and 3 = A dam was included in the analysis if she had one or more eggs scored for hemagglutination titer. In replicate one, 22 dams with one to seven embryos each and a total of 116 embryos scored, were used. In the second replicate, there were 21 dams having one to ten scored embryos each and a total of 147 embryos. Because of the different number of dams represented in the two replicates and because of the unequal numbers of observations in the two replicates, analyzed separately. each replicate was RESULTS AND DISCUSSION The distribution of the hemagglutination scores for all eggs in both replicates is shown in Figure 1. This is a typical multinomial distribution with some skewness. The mean was 5.9 which is only slightly above that expected on the basis of a preliminary test which showed a mean score about midway between five and six after 18 hours of incubation following inoculation. Replicate two was distinctly more skewed than replicate one. It had a mean score of 6.3 compared to a mean score of 5.5 for replicate one. There also appears to be an excess of the very low scores. While a slightly shorter incubation time might have given a less skewed distribution, it is 60 So ho a 3 30 Scores Dil«levels t , 1, 10 $ - 1 t t t 61i0-1 «1280 Hemagglutination Titer Scores FIG. 1. Distribution of the HA titers from replicates 1 and 2.

4 SIRE AND DAM EFFECTS ON VIRUS TITERS 1185 felt that the deviation from the desired normal distribution is not sufficient to interfere seriously with the analysis of variance. The results of the analysis of variance are shown in Table 1. It is noted that the mean squares were larger in replicate two than in replicate one, in spite of the change in procedure in replicate two, designed to reduce the error variance. Clearly, both the sires and the dams have an effect on the hemagglutination titer scores of their progeny eggs as indicated by the statistically significant mean squares for each in both replicates. Additional information as to the relative magnitude of the effects of the sires and dams on their progeny scores and some insight into the cause of these effects can be obtained from estimates of the variance components for sires (<TS 2 ), dams (CTD 2 ) and eggs (<TE 2 ). These estimates were obtained by equating the observed mean squares (Table 1) to the expected mean squares and solving for the components. The results are shown in Table 2. The theoretical compositions of these components have been presented by many workers (see Lerner, 1958; pages 57-65). Assuming that the population is in genetic equilibrium for the sex-linked genes and that the sexes are equally represented among the embryos, of the sex-linked genetic variance would be contained in the TABLE 1. Analysis of variance of hemagglutination titer scores Source of variation Sires Dams in sires Eggs in dams in sires Total Degrees of freedom Replicate Mean squares 16.35" 2.99** 1.21 * Significant at the.05 probability level. ** Significant at the.01 probability level. Degrees of freedom Replicate 2 Mean squares 29.50* 6.97** 2.51 TABLE 2. Estimates of the variance components Component as 2 O\D 2 BE? Total Replicate Replicate sire variance component, \ in the dam variance component, and in the full-sib variance component or between eggs. Thus, the sire component (<rs 2 ), as estimated here, would be expected to contain one-quarter of the additive genetic variance plus a small part of the additive genetic interactions plus of the variance due to sex-linked genetic effects. The dam component (ere 2 ) would contain one-quarter of the additive genetic, dominance and sex-linked variances plus portions of all types of genetic interaction variance, plus all of the variance due to maternal effects of the dam on her eggs. In view of the composition of the components, the fact that the dam components are smaller than the sire components in both replicates is striking. It could be argued that the sex-linked effects contained in the sire components are larger than the combined sex-linkage, maternal effect and non-additive genetic variances contained in the dam components. On the other hand, it could be argued that the parental immunity induced by vaccination of the adults for Newcastle disease was being uniformly transmitted to the eggs, overriding the genetic variation among the hens, and thereby reducing the size of the variance component due to the dam. It would seem more likely that the hens would not be equally capable of transmitting immunity to their eggs, and as a maternal effect, the occurrence of parentally contributed passive immunity in the eggs would be expected to increase

5 1186 G. RETA, B. B. BOHREN AND H. E. MOSES the magnitude of the dam component. Furthermore, Brandly, Moses and Jungherr (1946) were unable to demonstrate antiviral activity in the allantoic fluid of eggs from hens, either vaccinated for or recovered from Newcastle disease, until the fifteenth day of incubation of the eggs. The small number of sires and dams observed in this experiment prevents definite conclusions regarding the importance of sex-linkage, but the absence of large maternal effects on the titer scores of the eggs at this stage of incubation is clear. Other studies of older embryos would be of interest along this line. The second striking feature of the variance components is the large relative size of the sire and dam components. Assuming that only additive genetic variance was present, either the sire or the dam component would be an estimate of onequarter of the genetic variance. An estimate of the heritability of the scores (h 2 ) or the proportion of the total variation in scores due to genetic variation can be obtained as 2(<r fl»+<r fl *) W- o's 2 +0\D 2 +<r.e 2 In the first replicate the estimate of heritability was 0.81 and in the second replicate the estimate was The average of the estimates in both replicates was 0.77, indicating that about 77 percent of the variation from egg to egg in titer score was accounted for by genetic variations among the embryos. The precise relationship of the hemagglutination titer scores to the growth of Newcastle disease virus in the inoculated egg may be debated. However, it is quite reasonable to interpret the hemagglutination titer scores as indicators of virus concentration in the allantoic fluid. Eggs with high concentrations of virus, when tested, would agglutinate the erythrocytes at high dilution levels, and thus have high titer scores. Therefore, high titer scores are interpreted as indicating high concentrations of virus in the allantoic fluid, while the low titer scores are conversely indicative of low virus concentrations. It is therefore concluded that genetic differences between eggs in the concentration of Newcastle disease virus in the allantoic fluid have been demonstrated. This experiment provides no information on the physiological mechanism by which these differences were produced. SUMMARY Five randomly selected hens were mated at random to each of five randomly chosen males. A total of 116 pedigreed eggs from 22 hens was used as replicate one, and 147 eggs from 21 hens were used in a second replicate from the same parents. The eggs were inoculated with 0.1 cc. of a dilution of strain Bi Newcastle disease virus containing 10 4 embryo lethal doses of virus. After 18 hours of incubation, a 0.2 cc. sample of allantoic fluid was removed from each egg for testing the hemagglutination titer. A hemagglutination titer score was given to each egg depending on the highest dilution at which agglutination occurred, relative to a negative control tube. Eggs containing large concentrations of virus would thus have high scores indicative of rapid viral growth in the egg. Analysis of variance of the scores in a hierarchal design showed that both the sires and dams made statistically significant contributions to the variation in titer scores, and thus the variation in rate of viral growth in their eggs. The variance components for sires and dams were estimated in both replicates. The sire and dam components were averaged as the best estimate of one-fourth of the genetic

6 SIRE AND DAM EFFECTS ON VIRUS TITERS 1187 variance and were used to estimate heritability. The heritability estimates were 0.81 in replicate one, and 0.72 in replicate two. The average of these is 0.77, indicating that about 77 percent of the egg-toegg variation is due to genetic variation among the embryos. In both replicates the sire component was slightly larger than the dam component, suggesting the possibility of some sex-linked effects. The small numbers involved preclude definite conclusions on this point. On the other hand, the small size of the dam component clearly indicates the absence of maternal effects on the antiviral activity of the eggs at this age, such as, for example, the maternal transmission of viral antibody activity to the eggs. REFERENCES Brandly, C. A., H. E. Moses and E. L. Jungherr, Transmission of antiviral activity via the egg and the role of congenital passive immunity to Newcastle disease in chickens. Am. J. Vet. Res. 7: THERE are reports that eggs handled with modern equipment which rotates the eggs can cause a lowering of internal quality. This loss of quality has been attributed to the amount and severity of rotation of the egg, as it passes over the handling equipment. Slowing down the equipment supposedly eliminates the problem. Little can be found in the literature to support or dispute these conjectures. Pennington (1949) states that Brandly, C. A., H. E. Moses, E. L. Jungherr and E. E. Jones, The isolation and identification of Newcastle disease virus. Am. J. Vet. Res. 7: (See also, National Academy of Sciences, National Research Council: Methods for the examination of poultry biologies. Pub. 70S, Washington, D. C, 1959.) Cole, R. K., and F. B. Hutt, Genetic differences in resistance to Newcastle disease. Avian Diseases, 5: Francis, D. W., and A. F. Kish, Familial resistance to Newcastle disease in a strain of New Hampshires. Poultry Sci. 34: Godfrey, G. F., Evidence for genetic variation in resistance to Newcastle disease in the domestic fowl. J. Heredity, 43: Green, R. H., and M. W. Freyman, A method of obtaining influenza virus growth curves in individual eggs. Proc. Soc. Exp. Biol. Med. 71: 476^78. Hutt, F. B., Genetic Resistance to Disease in Domestic Animals. Comstock Publishing Associates, Cornell University Press, Ithaca, New York. Lerner, I. M., The Genetic Basis of Selection. John Wiley & Sons, Inc., New York. Steel, G. R., and H. J. Torrie, Principles and Procedures of Statistics with Special Reference to the Biological Sciences. McGraw-Hill Book Company, New York. (See page 126.) The Effect of Rotation on the Internal Quality of Chicken Eggs G. E. REHKTJGLER AND R. C. BAKER Departments of Agrictdtural Engineering and Poultry Husbandry, Cornell University, Ithaca, New York (Received for publication April 3, 1963) gentle turning of eggs before the candle breaks some of the mucin fibers and increases the amount of thin white. Since there is some confusion relative to this subject, it seemed important to investigate under controlled conditions whether rotation by egg handling equipment does cause a decrease in egg quality. It also seemed important to investigate to what extent the change in the internal quality is dependent upon speed and the