Local Immunity Against Newcastle Disease Virus in the Newly Hatched Chicken's Respiratory Tract

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1 INFECTON AND IMMUNT, May 1977, p Copyright American Society for Microbiology Vol. 16, No. 2 Printed in U.S.A. Local Immunity Against Newcastle Disease Virus in the Newly Hatched Chicken's Respiratory Tract MERTYN MALKINSONl* AND PARKER A. SMALL, JR. Department ofimmunology and Medical Microbiology, College of Medicine, University of Florida, Gainesville, Florida Received for publication 10 December 1976 Inoculation of 200 mean egg infectious doses (EID50) of lentogenic Newcastle disease virus strain B1 (NDV-B1) into the air sac of 4-day-old specific-pathogenfree chicks provided significant protection against challenge of the air sac with 100 chicken mean lethal doses (LD50) of velogenic NDV-H but no protection against reinfection when the challenge was by the eye. Conversely, inoculation of the eye with 200 EID50 of NDV-B1 provided significant protection against challenge of the eye but not of the air sac with 100 chicken LD50 of NDV-H. Birds that received both antiserum and intraocular immunization were subsequently protected against both eye and air-sac challenge. On the other hand, birds that received antiserum and air-sac immunization were protected only against airsac challenge but not against ocular challenge. Low levels of passively administered antibody did not prevent infection of the eye or air sac but greatly reduced the mortality rate after inoculation of either the vaccine or the challenge viruses. Passively administered antibody also suppressed hemagglutinationinhibiting and virus-neutralizing antibody formation stimulated by air-sac infection but not antibody formation stimulated by ocular infection. These data are consistent with the hypothesis that local immunity is responsible for prevention of infection, since birds were immune to reinfection at one site and simultaneously susceptible at the other site of infection. Immunization of poultry against Newcastle disease is commonly practiced by giving lentogenic virus vaccine by aerosol or as eye drops. Early work showed that newly hatched chicks possessing maternal antiviral antibody, when immunized by eye drop, were subsequently protected against intranasal challenge with velogenic virus as effectively as were antibody-free chickens similarly immunized and challenged (16). It was later demonstrated that when given by aerosol the vaccine protected birds against challenge by the respiratory route but not against an intramuscular challenge (6). These findings were interpreted as evidence for the existence in the fowl of a local immune system associated with the mucous membrane of the respiratory tract. This system has been related more recently to the demonstration of antibodies especially of the immunoglobulin A class (13) that are synthesized and secreted locally in response to topically administered antigens (16). We have attempted to determine whether local infection of the eye and air sacs of 4-dayold chicks could be achieved by using very low I On leave of absence from Kimron Veterinary Institute, Bet Dagan, Israel. doses of vaccine virus, and whether these organs once infected were protected against subsequent challenge with a more virulent strain of Newcastle disease virus (NDV). We have also examined the effect of humoral antibody on localized respiratory tract infection by actively immunizing other chickens in the presence of passively transferred hyperimmune (HI) serum. The results indicate that active immunization protects certain parts of the respiratory tract against local reinfection and that systemic antibody plays a secondary role in preventing infection and reinfection of these organs. MATERIALS AND METHODS Chickens. Newly hatched White Leghorn chickens, originating from specific-pathogen-free breeding flocks, were obtained when 2 days old from Life Sciences, Inc., St. Petersburg, Fla. On arrival, they were housed, in groups of two or three, in a negative-pressure isolation facility. Viruses. Newcastle disease vaccine strains Va-Bl and V-4 Australia (Queensland) were kindly supplied by J. Spalatin, NDV Repository, University of Wisconsin. Challenge strain NDV-H was obtained from G. Gifford, Department of Medical Microbiology, University of Florida. A pool of allantoic fluid 587

2 588 MALKINSON AND SMALL was prepared for each strain by injecting 9-day-old embryonated eggs and was then frozen at -85 C in 1-ml portions. Ocular vaccination was performed on 4-day-old chicks by delivering, into one eye, 1 drop (approximately 0.01 ml) of infected allantoic fluid (titer of stock virus, 1013 mean egg infective doses [EID50]) with a 27-gauge needle fitted on a 1-ml tuberculin syringe. The volume of viral inoculum was kept to a minimum in an attempt to restrict replication of the virus to the orbit and nasal chamber (9). Air-sac immunization was achieved by entering the cranial thoracic air sac between the ventral ends of sternal ribs 3 and 4 with a 27-gauge needle. A volume of 0.1 ml was inoculated. Inoculation of challenge virus, NDV-H (titer of stock virus, = EID50), was made by the same routes with the same volumes of inoculum. Virus isolation. The trachea was swabbed with cotton tips on day 5 after infection and challenge reinfection. The tips were broken off into 1 ml of dextrose-gelatin-veronal (ph 7.4) containing chloramphenicol. The centrifuged swabbings were either frozen at -85 C or inoculated directly as 0.2-ml volumes into the allantoic cavity of 9-day-old embryonated eggs. The eggs were incubated at 37 C for 6 days and candled daily. Allantoic fluid was obtained from embryos dying between 1 and 6 days after inoculation and from those embryos surviving at 6 days. Hemagglutination (HA) assay was performed on the allantoic fluid (3). If the HA titer was equal to or greater than 8, the embryos were considered to be infected; otherwise, a blind passage was made of the allantoic fluids. Identification of the NDV in selected samples of positive allantoic fluid was confirmed by inhibition of HAI, with a reference Newcastle antiserum. HI serum. A pool of HI serum was prepared from two adult roosters that had been boosted by several intramuscular injections of EID50 of NDV-B1. The HAI titer ofthe pool was 1:512. Prior to intramuscular injection into 4-day-old chicks, the pooled serum was centrifuged (5,000 rpm for 30 min) and sterilized by passage through 0.45-,um membrane filters (Millipore Corp., Bedford, Mass.). A pool of control serum prepared from adult specific-pathogen-free chickens was kindly supplied by W. M. Farrow, Life Sciences, Inc., St. Petersburg, Fla. Serology. (i) HAI test antigen. Infected allantoic fluid was inactivated with S-propiolactone as described by Sullivan et al. (17). The inactivated virus was concentrated with polyethylene glycol solution (11), mixed with an equal volume of glycerol, and stored at 4 C. (ii) Titration of serum HAI antibody. HAI titrations were performed by using a microtiter kit with disposable U-plates; 10 HA units of antigen were used. Serial twofold dilutions of serum in antigen starting at a dilution of 1:2 were prepared by using 25,ul per well. An equivalent volume of 0.5% chicken erythrocytes was added to each well, and the test was read after 30 min of incubation at room temperature. (iii) Virus neutralization test. Neutralizing antibody was titrated by the egg-bit technique (10) with the following modifications. (i) The test was pre- INFECT. IMMUN. formed in U-bottom, 96-hole microplates. Thus, the egg shell was cut into pieces small enough to be placed in the bottom of the hole. Approximately 140 egg bits could be cut from one 12-day-old embryonated egg. (ii) The test virus was V-4 Queensland, and 100 egg-bit mean infectious doses (ID50) per 25 1.l were used (10-4 dilution of stock allantoic fluid) in standard egg-bit medium. (iii) Serial twofold dilutions of serum were made in the virus suspension as described above for the HAI test. One egg bit was then placed in each well, and approximately 0.2 ml of egg bit medium containing 10% fetal calf serum was added to each well. The plates were covered with individual lids, and the stack of plates was placed on an orbital shaker in a 370C walk-in incubator. (iv) After 48 h of rotation at this temperature, the egg bits were removed, and 25 Iu of 0.5% chicken erythrocytes was added to each well. The plates were read for HA 45 min later. RESULTS Titration of vaccine and challenge viruses by the air-sac and ocular routes of infection and the effect of passively administrative antibody on air-sac infection with NDV-B1. To determine the infectivity of the vaccine and challenge strains for the air-sac and ocular routes of inoculation, each virus was titrated in specific-pathogen-free chickens by both routes. It was found that for NDV-B1, 1 EID50 was equal to 1 chicken ID,0 by both routes of infection. For the challenge virus NDV-H, it was found that, when titrated in chickens by the air-sac route or in embryonated eggs, 1 ID50 was the same as 1 mean lethal dose (LD)50. In contrast, 2,000 ELD50 were required to produce 1 chicken LD50 by intraocular infection. In an attempt to define the role of humoral antibody on the infectivity of the vaccine virus, NDV-B1 was further titrated by the air-sac route in 4-day-old chicks injected intramuscularly with HI serum at the time of infection. The protective effect of the antiserum against mortality, but not against infection of the respiratory tract, is shown in Table 1. Infection and cross-reinfection of the eye and air sac of normal and passively immunized 4-day-old chicks. Approximately day-old chicks were injected (in their air sacs) with a single dose of EID50 of NDV-B1. At the time of immunization, half of these were injected intramuscularly with normal serum, and the rest received HI serum. Two similar sets of hatchmates were immunized intraocularly with the same dose of virus. Control groups consisted of nonimmunized chicks and chicks that were not injected with HI serum. Five days after injection, tracheal swabs were taken from six chickens in each group. Vaccine virus was recovered from all but one of the infected birds, and the proportion of birds shed-

3 VOL. 16, 1977 TABLE 1. Effect ofpassively transferred serum on the survival and virus recovery of 4-day-old chicks inoculated by the air-sac route with NDV-Bl a Viral dos- Mor- Virus reage Serum treatment tal- isola- (EID50) ityb tione Normal 4/6 2/2 Hyperimmune 0/6 4/ Normal 1/6 3/5 Hyperimmune 0/6 3/6 10' 3 Normal 1/6 3/5 Hyperimmune 0/6 5/6 10" 3 Normal 0/6 4/6 Hyperimmune 0/6 0/6 a Titer = 1093 EID50. b Number of chickens dying/number of chickens inoculated. c From tracheal swabs taken 5 days post-immunization (number positive/total). ding virus was unaffected by treatment with HI serum (Table 2). On day 14 after immunization, each treatment group, including the controls, was divided into three subgroups of which the first and second were reinfected with challenge virus intraocularly and into the air sac, respectively; the third subgroup was not challenged. The dose of challenge virus for each route of reinfection was 100 chicken LD50. Blood samples were taken from each bird immediately prior to reinfection and on days 5, 10, 13, and 17 thereafter. Tracheal swabs were taken from the surviving birds on day 5 after challenge. A bird was considered to have become reinfected if one or more of the following criteria were met: virus was isolated from the trachea; a fourfold or greater rise in neutralizing antibody titer was detectable within 17 days of reinfection; death preceded by characteristic signs of Newcastle disease occurring during this period. Protection against reinfection with virulent virus was best demonstrated by the groups infected and reinfected homotopically and was independent of passive immunization with antiserum (Table 2). The exception was the group treated with intraocular vaccine and HI serum; these birds were equally well protected against homotopic and heterotopic reinfection. Passive antibody when given alone on day 0 failed to protect the birds against infection with virulent virus, although mortality was markedly reduced. In the absence of any form of prophylactic treatment, reinfection by either route produced 100% mortality in the control groups. Immunosuppression by passively trans- LOCAL IMMUNITY OF CHICKS AGAINST NDV 589 ferred antibody. The immunosuppressive effect of injecting HI serum at the time of air-sac vaccination was reflected in the differences between the mean HAI and virus-neutralizing antibody titers of the passively immunized and normal serum recipients 14 days after immunization. The results of the experiments shown in Tables 1 and 2 are combined in Table 3. Significantly reduced antibody titers were recorded in the groups receiving the three highest doses of vaccine virus. In marked contrast to these results, no immunosuppression was demonstrated after intraocular immunization. The mean titer of the group receiving HI serum was slightly higher than those injected with normal serum (P = 0.06). The antibody present in the control, uninfected chickens receiving HI serum undoubtedly represents residual passive antibody. It is unclear, however, why the group receiving the lowest viral dosage and HI serum had no residual antibody, or why the group inoculated with EID5O and normal serum, of which four birds subsequently shed virus, had no humoral antibody response. DISCUSSION Local immunity can be demonstrated by showing that of two potentially infectable sites within an animal, one becomes immune while the other is susceptible. If systemic immunity was the first line of defense, then both sites would be immune. Hence, the demonstration that most groups of chickens were immune to homotopic challenge while simultaneously remaining susceptible to heterotopic infection suggests that local immunity is a major factor in the defense of chickens against Newcastle disease infection. In this study, by using low dosage levels of virus, replication was confined to epithelial surfaces without any apparent systemic spread, especially to the other site of infection. Burnstein and Bang (9) had previously shown that limited infection of the upper respiratory tract could be established by placing very small volumes of viral inoculum into the eye and that recovery of virus from the trachea was exceptional. There is some debate concerning the effect of passively acquired antibody on the development of immunity, especially of young chickens after nonparenteral immunization. Data both for and against immunosuppression and lack of protection against reinfection have been presented (6). More recently, workers have concerned themselves with examining the parameters involved in the virus-host relationship, viz., amount and avidity of passively acquired

4 590 MALKINSON AND SMALL INFECT. IMMUN. TABLE 2. Effects ofpassively transferred serum, routes of immunization, and reinfection on mortality, virus isolation, and antibody response of 4-day-old chicksa Roueoimunia- VN anti- Virus Increased VirUSb b Route of chal- Mortal-.i,s in VN antion and serum isolation pro- lenge (reec- y io tibody titreatment duction" dcn tion) tion ter Air sac, HI serum 6/6 Eye 0/5 1/5 Air sac 0/5 0/5 None 0/3 0/3 Air sac, normal se- 5/6 13/13 Eye 1/5 1/4 rum Air sac 0/5 0/5 None 0/3 0/3 Eye, HI serum 6/6 Eye 0/6 0/6 Air sac 0/6 1/6 None 0/4 0/4 Eye, normal serum 6/6 14/16 Eye 0/6 0/6 Air sac 2/6 0/4 None 0/4 0/4 HI serum only 0/6 Eye 1/5 4/5 Air sac 1/5 4/5 None 0/3 0/3 Normal serum only 0/6 0/13 Eye 5/5 Air sac 5/5 None 0/3 4/5 5/51 P < O.Olf 1/5 1/5J 0/3 0/3 1/5 1/5j 0/3 0/3 2/6 2/6 1/6 2/6 0/4 0/4 1/6 1/6 P < /4 6/6 0/4 0/4 4/4 5/5 4/4 5/5 0/3 0/3 a Immunized with EID50 NDV-B1 and challenged with 102 chicken LD50 NDV-H. bfive days post-inoculation; number positive/total. c Virus-neutralizing (VN) antibody titer of 1:2 or greater on day of challenge; number responding/total. d Fourfold or greater rise in titer. etotal number of birds showing mortality, virus isolation, or increase in antibody titer. f Values were compared by the chi-square test. TABLE 3. Effect ofpassively transferred serum on antibody production of chickens 14 days after inoculation via the eye or air sac with NDV-Bl virus Viral dosage Air saca Eye (EID50) HIb Normal HI Normal * d * c e (N =6) (N =2) *4.42 ± 0.41 *6.3 ± (N =6) (N= 5) j1y.3f *3.92 ± 0.57 *5.54 ± ± ± 0.56 (N= 13) (N= 13) (N= 16) (N= 16) 1011i 3.92 ± ± (N =6) (N =5) 10" : <1.0 <1.0 (N =6) (N = 6) None *2.4 ± 0.48 *<1.0 (N= 13) (N= 13) a Route of inoculation. b Serum treatment. c Asterisk indicates that the value is significantly different by the Student t-test (P s 0.05). d HI antibody titer; log2 geometric mean ± standard deviation. N, Number tested. e Not tested. f All values for this infectious dosage are virus-neutralizing antibody titers; log2 geometric mean - standard deviation. 5/5 5/5 0/3 Totale

5 VOL. 16, 1977 LOCAL IMMUNITY OF CHICKS AGAINST NDV 591 antibody, dosage, strain, and route of administration of the vaccine virus (5). However, the mulating evidence for local antibody produconstrated in these experiments. Based on accu- consensus of opinion is that humoral antibody tion, a hypothesis has been presented to explain levels give a poor indication of the resistance of the differences in the immune defense mechanisms operative at the two anatomical extremi- the mucous membrane to infection and reinfection (7). In this report, recovery of vaccine or ties of the avian respiratory tract, i.e., the eye challenge virus from the throats of passively and air sac. immunized chickens was the rule rather than the exception, and the secondary antibody response that occurred after heterotopic chal- ACKNOWLEDGMENTS lenge strengthened the evidence that reinfection of the respiratory tract had taken place We gratefully acknowledge the technical assistance of Donna Birdsell and Joseph Kowalski. This work was supported by Public Health Service research grant Al from the National Institute of Allergy despite the presence of humoral antibody. We do not fully understand why passively and Infectious Diseases. acquired antibody failed to immunosuppress antibody synthesis after intraocular infection LITERATURE CITED but did so after air-sac infection. These findings 1. Albini, B., G. Wick, M. E. Rose, and E. Orlans may be related to the histological structure of Immunoglobulin production in chicken Harderian these two organs and the facility with which glands. Int. Arch. Allergy Appl. Immunol. 47: Allan, W. H The effect of neonatal vaccination humoral antibody can transude into them. Local aggregates of lymphoid tissue in the eye nal antibody. Vet. Rec. 92:645. against Newcastle disease in the presence of mater- (Harder gland) and nasal chamber are capable 3. Anonymous Methods for examination of poultry of antibody production if the antigen is given biologics. U.S. National Academy of Sciences, Washington, D.C. locally (1, 15). Tears containing HAI and VN 4. Bang, B. G., and F. B. Bang Localized lymphoid antibody are secreted soon after the eye is inoculated with inactivated NDV (12). In contrast. organ systems in chickens. Am. J. Pathol. 53:735- tissues and plasma cells in paraocular and paranasal the histological structure of the air-sac wall is Beard, C. W., and M. Brugh Immunity to Newcastle disease. Am. J. Vet. Res. 36: far simpler. It is composed of a single avascular layer of squamous epithelium without glandular tissue or lymphoid aggregates (18). It would of the route of administration of Newcastle disease 6. Beard, C. W., and B. C. Easterday The influence seem that, although lachrymal secretions were virus on host reponse. I. Serological and virus isolation studies. J. Infect. Dis. 117: not assayed for antibody content in these experiments, passively administered antibody did of the route of administration of Newcastle disease 7. Beard, C. W., and B. C. Easterday The influence not block the local immune response to intraocular immunization, but interfered with the immunity. J. Infect. Dis. 117: virus on host reponse. II. Studies on artificial passive 8. systemic synthesis of antibody after air-sac infectionistics. Lab. Invest. 28: Bienenstock, J., N. Johston, and D. Y. E. Perey Bronchial lymphoid tissue. I. Morphologic character- The group given passive antibody and immunized intraocularly was also exceptional by upper respiratory tract and a mild (vaccine) strain of 9. Burnstein, T., and F. B. Bang Infection of the Newcastle disease virus. I. Initiation and spread of being protected against heterotopic challenge. the infection. Bull. Johns Hopkins Hosp. 102:127. In this case, the elevated antibody response of 10. Fazekas de St. Groth, S., and D. 0. White Comparison of the infectivity of influenza viruses in two this treatment group could conceivably have been high enough to protect against air-sac host systems: the allantois of intact eggs and surviving allantois-on-shell. J. Hyg. 56: challenge. The mean antibody titer was no different from that of the group immunized and of certain myxoviruses with polyethylene glycol. Na- 11. Kanarek, A. D., and G. W. Tribe Concentration subsequently shown to be protected against airsac challenge (Table 3: 5.54 versus 5.19; degrees 12. Katz, D., and A. Kohn Antibodies in blood and ture (London) 214:927. of freedom, 27; P = secretions of chickens immunized parenterally and 0.8). This result is consistent with the observations that maternal anti- 14th Congress of the International Association of Bio- locally with killed Newcastle disease virus vaccine. body does not interfere with aerosol or intraocular immunization of newly hatched chicks (2). 33: logical Standardization, Dev. Biol. Stand. 13. Lebacq-Verheyden, A.-M., J.-P. Vaerman, and J. F. The contrast between the histological complexity of the lining of the upper portion of the lian IgA in chicken serum and secretions. Immunol- Heremans A possible homologue of mamma- respiratory tract with its local aggregates of ogy 22: lymphoid tissue (8) and the structural simplicity of the air sacs is further reflected in the vaccine (intranasal type) in parentally immune and 14. Markham, F. S., C. A. Bottorff, and H. R. Cox The conjunctival application of Newcastle disease diversification of response to infection as dem- susceptible chicks. Cornell Vet. 43:

6 592 MALKINSON AND SMALL 15. Mueller, A. P., K. Sato, and B. Glick The chickens lacrimal gland, gland of Harder, caecal tonsil, and accessory spleens as sources of antibody-producing cells. Cell. Immunol. 2: Parry, S. H., and I. D. Aitken Immunoglobulin A in the respiratory tract of the chicken following exposure to Newcastle disease virus. Vet. Rec. 63: INFECT. IMMUN. 17. Sullivan, J. F., E. Gill, and A. M. Somer Immune response of chickens to beta-propiolactone-killed Newcastle disease vaccines. Am. J. Vet. Res. 19: Walsh, C., and J. McLelland The ultrastructure of the avian extrapulmonary respiratory epithelium. Acta Anat. 89: