Research Notes Comparison of Disease Susceptibility and Resistance in Three Lines of Chickens Experimentally Infected with Infectious Laryngotracheitis Virus1 DAVID J. POULSEN,2 DEAN R. THUREEN, and CALVIN L. KEELER, JR.3 Delaware Agricultural Experiment Station, Department of Animal and Food Sciences, College of Agricultural Sciences, University of Delaware, Newark, Delaware 19717-1303 ABSTRACT The susceptibility of three F1 hybrid lines of chickens to graded doses of infectious laryngotracheitis virus (ILTV) was investigated. The three F1 hybrid lines, each produced from mating two inbred lines, included the SC (B2B2) and TK (B15B21) lines and the 15I5 × 71 (B2B15) line. Although at 1 d of age all three lines were susceptible to ILTV, SC birds were significantly less susceptible (10%) than TK (80%) or 15I5 × 71 (50%) birds when exposed to 5,000 pfu of virus at 4 wk of age. The ability of each inbred F1 hybrid line to establish a protective immune response to ILTV was also
determined. The SC birds required a smaller immunizing dose of virus (500 pfu) to mount a protective immune response to ILTV than the 15I5 × 71 line (5,000 pfu). A 5,000 pfu immunizing dose did not elicit a protective immune response in the TK line to a 106 pfu challenge dose of ILTV. These results also correlated with the ability to produce ILTV-specific antibodies. This study confirms and expands on observations that lines of chickens differ with respect to their susceptibility and resistance to ILTV.
(Key words: infectious laryngotracheitis, genetic resistance, chicken, B-haplotypes) 1998 Poultry Science 77:17–21
haplotypes have been shown to influence resistance or susceptibility to ILTV infection. Loudovaris et al. (1991b) observed that inbred chickens homozygous for the B113 haplotype were significantly more resistant to ILTV than B114 or B15 homozygous birds. Chickens infected with ILTV produce virus-specific IgG and IgM in their sera, whereas IgG, IgM, and IgA are observed in tracheal washings (Da Silva Martins et al., 1992). In immunologically mature birds (5 wk of age), antibodies can be detected 5 d postinoculation (PI) with peak levels of IgM detected 5 d PI, and levels of IgG and IgA peaking 10 d PI. The IgM response in the trachea is unusual in that it is persistent and cyclical (Da Silva Martins et al., 1992). However, maternal IgG antibody does not confer resistance to infection with ILTV (Cover et al., 1960). In addition, it has been shown that passive transfer of anti-ILTV serum has no effect on reducing the susceptibility of 2-d-old or 4-wk-old chickens to infection (Fahey et al., 1983). Other studies indicate that neutralizing antibodies cannot be routinely recovered from tracheal washings, further suggesting that they do not play a prominent role in recovery from ILTV infection (York et al., 1989). Macrophages have been shown to be susceptible to infection with ILTV (Calnek et al., 1986). Loudovaris et
INTRODUCTION Infectious laryngotracheitis virus (ILTV) causes mild to severe respiratory infections of chickens (Hanson and Bagust, 1991). The virus enters through the upper respiratory tract or conjunctiva and viral replication is generally limited to the nares, oropharynx, and trachea. The virus is seldom detected in extratracheal tissues and viremia is rarely observed. It has been proposed that viral clearance is mediated primarily by cellular immune mechanisms. Fahey et al. (1983) demonstrated that bursectomized chickens, which showed no detectable levels of ILTV-specific serum antibodies, could be protected from viral challenge by vaccination. Furthermore, the absence of mucosal antibody (IgA) does not impair the ability of vaccinated-bursectomized chickens to resist challenge (Fahey and York, 1990). Protection from viral challenge can also be transferred between inbred chickens with immune spleen cells or peripheral blood lymphocytes (Fahey et al., 1984). Selected avian B
Received for publication January 17, 1997. Accepted for publication August 27, 1997. 1Published as Paper Number 1622 in the Journal Series of the Delaware Agricultural Experiment Station. 2Present address: Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratory, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840. 3To whom correspondence should be addressed: Department of Animal and Food Sciences, 040 Townsend Hall, University of Delaware, Newark, DE 19717-1303:
[email protected]
Abbreviation Key: ILTV = infectious laryngotracheitis virus; MDV = Marek’s disease virus; PI = postinoculation.
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al. (1991a) extended these observations to conclude that macrophages from genetically resistant birds expressed higher levels of ILTV antigens than susceptible birds. This finding suggests that macrophages from the resistant inbred lines may be more efficient at processing and presenting ILTV antigens. This observation may also explain the production of higher anti-ILTV antibody titers in chickens of the more resistant lines (Loudovaris et al., 1991b). The purpose of the present study was to expand our knowledge of the influence of genetics on resistance to ILTV by examining the response of three F1 hybrid lines of chickens to the virus. This information is important for developing an appropriate model system to study the avian immune response to ILTV.
MATERIALS AND METHODS
Chicken Lines The F1 hybrid SC (B2B2) and TK (B15B21) chickens were purchased as fertile eggs from Hy-Line International. The SC line is an F1 hybrid cross of two lines, each fixed for the B2 MHC allele, and the TK line is an F1 hybrid cross of two lines, one fixed for B15 and the other fixed for B21. The F1 hybrid 15I5 × 71 (B2B15) chickens were purchased as fertile eggs from the USDA Avian Disease and Oncology Laboratory. All birds were transferred to negative pressure Horsfall isolation units at day of hatch, where they were maintained throughout the experiments. Feed and water were given for ad libitum consumption.
Susceptibility to ILTV At 1 d, 2 wk, and 4 wk of age, 40 birds from each line (SC, TK, 15I5 × 71) were divided into four groups of 10 chickens each. To determine the dose response of each line to ILTV, chickens were inoculated via the infraorbital sinus route with 100 mL of Dulbecco’s Modified Essential Medium (DMEM) containing either 500, 5,000, or 50,000 pfu of the virulent National Veterinary Services Laboratory (NVSL) standard challenge strain of ILTV, which had been propagated on chicken embryo liver cell monolayers (Keeler et al., 1993). One group served as an uninoculated control. The duration of each trial was 14 d and all birds were examined daily for clinical signs of infection. Laryngotracheitis was diagnosed on the basis of clinical signs that included sinusitis, nasal discharge, conjunctivitis, and tracheal rales. Scoring clinical symptoms is less subjective when the route of inoculation is the infraorbital sinus (sinusitis and conjunctivitis) rather than the trachea (rales, respiratory distress). However, mortality is rarely observed in older birds inoculated via the infraorbital sinus as less mucus and fewer tracheal plugs are formed.
4 Kirkegaard and Perry Laboratories, Gaithersburg, MD 20879-4145.
Chickens were considered positive for laryngotracheitis if they showed clinical signs for 2 d of any 4-d period during the trial. At 14 d PI, the surviving birds from eac h group were euthanatized and tracheas and sinuses were examined for signs of hemorrhage and serofibrinous or caseous exudate.
Vaccination and Challenge with ILTV At 4 wk of age, 40 chickens from each F1 hybrid line were divided into four groups of 10 birds. Chickens were vaccinated via the ocular route with 100 mL of DMEM containing either 50, 500, or 5,000 pfu of the virulent NVSL standard challenge strain of ILTV. One group served as an unvaccinated control. Four weeks postvaccination, all birds were challenged with 106 pfu of the NVSL challenge strain of ILTV via the infraorbital sinus route. Birds were examined daily for clinical signs of infection and were considered positive for laryngotracheitis if they showed clinical signs for 2 d of any 4-d period during the trial. All birds were euthanatized 14 d postchallenge and tracheas and sinuses were examined for the presence of hemorrhage and serofibrinous or caseous exudate. All birds were bled on the day of challenge and 14 d postchallenge via the brachial vein. The serological status of each bird to ILTV was determined using a commercially available ELISA kit.4
Statistical Analysis In response to challenge with ILTV, for each virus dose a test of homogeneity between strains was performed for morbidity and serological data using the Fischer’s exact test (Snedecor and Cochran, 1980).
RESULTS
Susceptibility to ILTV Three F1 hybrid lines of Single Comb White Leghorn chickens, lacking maternal antibodies to ILTV, were evaluated for their susceptibility to ILTV infection at various ages and with different doses of administered virus. All three lines were highly susceptible to ILTV infection in an age- and dose-dependant manner (Table 1). Younger birds were more susceptible to the disease, and mortality was only observed in birds infected at 1 d of age. Mortality of 1 d of age birds averaged 26.7% when challenged with 500 pfu of virus, 43.3% when challenged with 5,000 pfu of virus, and 90% when challenged with 50,000 pfu of virus. No significant difference in mortality was observed among the three lines. The F1 hybrid SC (B2B2) chickens demonstrated a doseand age-dependent decrease in susceptibility to infection when compared to the F1 hybrid TK (B15B21) and 15I5 × 71 (B2B15) lines. At 2 wk of age, SC birds were significantly (P ≤ 0.001) less susceptible to a virus dose of 500 pfu, and at 4 wk of age they were significantly (P ≤ 0.001) less
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RESEARCH NOTE TABLE 1. Susceptibility of inbred chicken lines following challenge with Infectious Laryngotracheitis Virus (ILTV) Virus dose Age
Inbred line
0 pfu
1 d
SC (B2B2) 15I5 × 71 (B2B15) TK (B15B21) SC (B2B2) 15I5 71 (B2B15) TK (B15B21) SC (B2B2) 15I5 × 71 (B2B15) TK (B15B21)
0 0 0 0 0 0 0 0 0
2 wk
4 wk
(0/10)1 (0/10) (0/10) (0/10) (0/10) (0/10) (0/10) (0/10) (0/10)
500 pfu
5,000 pfu
50,000 pfu
100 90 90 30 100 88 20 30 11
100 100 100 70 100 100 10 50 80
100 (10/10) 100 (10/10) 100 (10/10) 75 (6/8) ND2 100 (10/10) 70 (7/10) 100 (10/10) 100 (10/10)
(10/10) (9/10) (9/10) (3/10)3 (10/10) (8/9) (2/10) (3/10) (1/9)
(10/10) (10/10) (10/10 (7/10) (10/10) (10/10) (1/10)3 (5/10) (8/10)
susceptibility to ILTV. (Number of susceptible birds/total number of birds in group × 100). = Not done. 3SC chickens differed significantly (P ≤ 0.001) from either TK or 15I × 7 F birds in the percentage of chickens 5 1 1 with clinical signs of disease for the indicated virus dose and age of the bird. 1Percentage 2ND
susceptible to a virus challenge of 5,000 pfu. At the highest dose of virus (50,000 pfu) no difference in susceptibility was observed among the lines.
Vaccination and Challenge with ILTV The ability of the three F1 hybrid lines of birds to mount a protective immune response to ILTV was determined as described above. The three lines of birds differed in their abilities to mount a protective immune response (Table 2). The SC birds (B2B2) established a protective response following immunization with 500 pfu of ILTV, whereas the 15I5 × 71 F1 (B2B15) birds required a 10-fold higher dose (5,000 pfu) to establish a protective response. In contrast, TK birds (B15B21) were unable to establish a protective response following immunization at any of the doses examined. Although protection from clinical disease was observed in the SC and 15I5 × 71 F1 birds, virus was readily isolated from the tracheas of both protected and unprotected birds of all three groups, indicating that immunization with ILTV prevents disease but not infection (data not shown). Serum samples obtained prior to and after virus challenge were assayed by ELISA for the presence of
antibodies to ILTV (Table 3). The presence of anti-ILTV antibodies correlated directly with protection against clinical disease (Table 2). Groups that did not exhibit clinical signs of disease had significant (P ≤ 0.001) prechallenge and postchallenge ILTV ELISA titers (> 5,500), whereas susceptible groups had prechallenge ELISA titers below background levels (< 500). In addition, serum samples tested negative for antibodies to several adventitious avian disease agents (NDV, IBV, IBDV, and CAV) (data not shown).
DISCUSSION In chickens, MHC haplotypes have been associated with resistance or susceptibility to a variety of viral disease agents, including avian leukosis (Yoo and Sheldon, 1992), Marek’s disease virus (Briles et al., 1982), Newcastle disease virus (Dunnington et al., 1992), and Rous sarcoma virus (Heinzelmann et al., 1981). Research regarding genetic resistance and susceptibility to avian herpesviruses has focused on Marek’s disease virus (MDV). Genetic resistance to Marek’s disease (MD) is mediated by genes associated with the MHC (B complex) (Briles et al., 1982; Calnek et al., 1989; Bacon
TABLE 2. Susceptibility of immunized chickens following challenge with Infectious Laryngotracheitis Virus (ILTV) Vaccination dose Inbred line (B2B2)
0 pfu
SC 15I5 × 71 (B2B15) TK (B15B21) 1Percentage
50 pfu (12/12)1
100% 100% (10/10) 100% (8/8)
92% (11/12) 100% (10/10) 100% (11/11)
500 pfu (0/12)2
0% 100% (11/11) 100% (10/10)
5,000 pfu 0% (0/11) 0% (0/10) 100% (9/9)3
susceptibility to ILTV. (Number of susceptible birds/total number of birds in group × 100).
2SC chickens differed significantly (P ≤ 0.001) from both TK and 15I × 7 F birds in the percentage of chickens 5 1 1
with clinical signs of disease. 3TK chickens differed significantly (P ≤ 0.001) from both SC and 15I × 7 birds in the percentage of chickens 5 1 with clinical signs of disease.
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POULSEN ET AL. TABLE 3. Antibody titers of chickens before (Pre) and after (Post) Infectious Laryngotracheitis Virus (ILTV) challenge Vaccination dose1 0 pfu Inbred line (B2B2)
SC 15I5 × 71 (B2B15) TK (B15B21)
Pre2 03 88 0
Post2 0 253 0
50 pfu Pre 61 43 15
500 pfu
5,000 pfu
Post
Pre
Post
Pre
Post
285 232 151
6,3494
7,7894
5,607 6,980 925
7,535 7,583 05
0 0
59 77
1Four-week-old birds were vaccinated with the indicated dose of virus as described in Materials and Methods. 2Serum samples were taken immediately before challenge, at 8 wk of age (Pre), and 2 wk after challenge, at 10 wk of age (Post). 3Mean group ELISA titers from all sera in each group. N for each group ranged from 8 to 12 birds (see Table 2). ELISA titers
