et al.

IMMUNOLOGY Delayed-Type Hypersensitivity Reaction Induced in Broilers by Killed Staphylococcus aureus1 XIANG Y. ZHU,* ROBERT E. PORTER, JR.,† and PATRICIA Y. HESTER*,2 *Department of Animal Sciences and †Animal Disease Diagnostic Laboratory, Purdue University, West Lafayette, Indiana 47907 ABSTRACT A trial was conducted to determine whether the delayed footpad reaction (DFR) induced by killed Staphylococcus aureus in chickens is a delayed-type hypersensitivity (DTH) reaction. Five criteria were used to assess DTH: 1) DFR with a peak response at 24 to 48 h postchallenge, 2) inhibition of monocyte/macrophage migration, 3) lymphocyte blastogenic response, 4) mononuclear cell infiltration at the challenge site, and 5) passive transfer of DFR by splenic lymphocytes. Broilers were sensitized twice with a s.c. injection in the neck of S. aureus antigen (150 µg/bird) diluted in polyethylene glycol at 3 and 4 wk of age. Controls were s.c. injected with polyethylene glycol. At 6 wk of age, a migration inhibition test was conducted before the birds were challenged intradermally with S. aureus antigen (75 µg/bird) in PBS in the right footpad. The left footpad was injected with PBS. The thickness of the footpad was measured at 0, 4, 24, and 48 h postchallenge to evaluate the DFR. After challenge, blood was collected for the lymphocyte blastogenesis

assay. Birds were euthanatized, and both footpads were removed for histology. The spleens were collected aseptically; splenic lymphocytes were injected i.v. into recipient birds. Sensitized birds showed an increase in the DFR (P < 0.02) and blastogenic response (P < 0.01) compared with nonsensitized birds. Delayed footpad reaction reached a maximum response at 24 h postchallenge. The in vitro migration of monocytes/macrophages from sensitized birds was significantly inhibited (P < 0.01). The histological appearance of S. aureus-injected footpads was characterized by dermal edema and perivascular infiltrates of small lymphocytes and macrophages. Birds that received sensitized splenic lymphocytes had a significantly pronounced DFR following challenge with S. aureus when compared with birds that received nonsensitized lymphocytes (P < 0.0001). These results indicated that the DFR can be used as a standard in vivo test for cell-mediated DTH reaction induced by killed S. aureus antigen in chickens.

(Key words: delayed-type hypersensitivity, delayed footpad reaction, migration inhibition test, lymphocyte blastogenesis, Staphylococcus aureus) 1999 Poultry Science 78:1703–1710

INTRODUCTION Delayed-type hypersensitivity (DTH) is defined as a hypersensitive response mediated by sensitized TDTH cells that release various cytokines. The response generally takes 2 to 3 d to develop, the time required for initial TDTH-cell activation, cytokine secretion to induce localized influxes of macrophages, activation of macrophages, and the subsequent release of their lytic enzymes (Kuby, 1994). Delayed-type hypersensitivity has been demonstrated in chickens following challenge with various antigens including infectious bronchitis virus (Timms et al., 1980), fowlpox virus (Morita, 1973; Dharsana and Spradbrow, 1985), tuberculin (Palladino et al., 1978; Corrier and De-

Received for publication August 21, 1998. Accepted for publication August 3, 1999. 1 Journal Article Number 15,747 of the Purdue University Agricultural Research Programs, West Lafayette, IN 47907. 2 To whom correspondence should be addressed: phester@ purdue.edu

Loach, 1990a), bovine serum albumin (Warner et al., 1971; Palladino et al., 1978), phytohemagglutinin (Corrier and DeLoach, 1990b; Bayyari et al., 1997a,b), bacillus calmetteguerin vaccine (Palladino et al., 1978; Afraz et al., 1994), human gamma globulin (Watabe and Glick, 1983), diphtheria toxoid (Klesius et al., 1977), Salmonella sp. (Warner et al., 1971), Eimeria tenella (Rose, 1977), and Staphylococcus aureus (S. aureus; Cotter et al., 1987). The wattle test has been most commonly used to evaluate in vivo DTH reactions in chickens. It is a valid method of assessing T-cellmediated immunity because of its correlation with the inhibition of the in vitro migration of monocytes/macrophages and stimulation of lymphocyte blastogenesis (Warner et al., 1971; Watabe and Glick, 1983; Dharsana and Spradbrow, 1985; Cotter et al., 1987). The massive

Abbreviation Key: DFR = delayed footpad reaction; dpm = disintegrations per minute; DTH = delayed-type hypersensitivity; MEM = minimal essential medium; MIT = migration inhibition test; PHA = phytohemagglutinin.

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mononuclear cell infiltration at the challenge site and the cellular transfer of DTH reaction were also accepted as major criteria of the DTH reaction (Valentine and Lawrence, 1969; Klesius et al., 1977; Giambrone et al., 1980; Klein, 1990; Stites, 1994). The delayed wattle reaction in chickens (Cotter et al., 1987) and the delayed footpad reaction (DFR) in mice (Taubler, 1968; Easmon and Glynn, 1979; Klein, 1990) have been induced by S. aureus and are considered standard tests in vivo for the DTH reaction (Warner et al., 1971; Klesius et al., 1977; Cotter et al., 1987; Chubb et al., 1988). A DFR in chickens induced by killed S. aureus was established in our previous study (Zhu et al., 1999), but has not yet been confirmed as a cell-mediated DTH reaction. The goal of the present study was to validate the DFR as a measure of cell-mediated DTH reaction. Five criteria were used: 1) DFR with a peak response at 24 to 48 h postchallenge, 2) inhibition of monocyte/macrophage migration, 3) lymphocyte blastogenic response, 4) mononuclear cell infiltration at the challenge site, and 5) passive transfer of DFR reaction by splenic lymphocytes.

MATERIALS AND METHODS Chicks were raised initially in battery brooders and then transferred to larger growing batteries with feed and water available for ad libitum intake. Killed S. aureus (Wood 46 strain) was used in the present study.3 The bacterial protein content was estimated using the Lowry assay (Lowry et al., 1951); the protein concentration was adjusted to 1 mg/mL; and an appropriate dilution was used for the delayed footpad test, migration inhibition test (MIT), and lymphocyte blastogenesis assay.

Experiment 1 Elicitation of the Delayed Footpad Reaction. Fifteen 1-d-old broiler cockerels were assigned to either a treated (n = 8) or a control (n = 7) group. Each treated bird was sensitized at 3 and 4 wk of age with a s.c. injection in the neck of 0.2 mL S. aureus antigen (150 µg/bird) diluted 1:1 (vol:vol) with polyethylene glycol. The control birds were injected with 0.2 mL polyethylene glycol. At 6 to 7 wk of age, blood for the MIT and the blastogenesis assay was taken before and after the elicitation of DFR, respectively. Treated and control birds were injected intradermally in the right footpad with 0.1 mL S. aureus antigen (75 µg/bird) diluted 1:1 with sterile PBS as the eliciting challenge. The left footpad was injected with PBS alone

3

Sigma Chemical, St. Louis, MO 63178. Mitutoyo Corp., Tokyo, Japan. 5 Beckman Model TJ-6, Palo Alto, CA 94304. 6 Gibco Laboratories, Grand Island, NY 14072. 7 Baxter Healthcare Corp., McGaw Park, IL 60085. 8 Wale Apparatus Co., Inc., Hellertown, PA 18055. 9 Corning Glass Works, Corning, NY 14830. 10 Fisher Scientific, Pittsburgh, PA 15219. 11 Southern Precision Instrument Co., Inc., San Antonio, TX 78220. 12 Los Angeles Scientific Instrument Co., Inc., Los Angeles, CA 90039. 4

(Cotter et al., 1987). The thickness of both footpads at the site of challenge was measured to the nearest 0.01 mm using a Digimatic Caliper4 at 0, 4, 24, and 48 h postchallenge. Changes in the thickness of the footpad were referred to as the DFR and were calculated using the following formula: DFR = thickness of the right footpad (S. aureus) − thickness of the left footpad (PBS) (Corrier and Deloach, 1990a,b). Cell Preparation for the Migration Inhibition Test and Blastogenesis Assay. Ten milliliters of blood were collected from the wing vein with a heparinized (50 U/ mL blood) syringe and centrifuged5 at 500 × g for 15 min at room temperature. The lymphocyte-rich buffy coat was collected by a Pasteur pipette, and the cell suspension was centrifuged at 400 g for 10 min. The cell pellet was collected and washed once in PBS (pH 7.5). The cells were resuspended in serum-free minimal essential medium (MEM)6 for the MIT or in RPMI 1640 medium6 for the blastogenesis assay. The MEM was supplemented with 2 mM L-glutamine, 100 U penicillin G/mL, and 100 µg streptomycin/mL. The RPMI 1640 medium was supplemented with 2 mM L-glutamine, 0.1 mM MEM nonessential amino acids, 1 mM MEM sodium pyruvate, 100 U penicillin G/mL, and 100 µg streptomycin/mL. Cell number was counted, and viability was evaluated using 0.4% trypan blue dye3 exclusion test (Niwano et al., 1990; Kashima et al., 1993). Cell suspensions with more than 95 and 90% viability were used in the MIT (Trifonov et al., 1977; Huynh and Chubb, 1987) and blastogenesis assay (Greaves and Roitt, 1968; Kashima et al., 1993), respectively. Cell concentrations were adjusted to 1 × 107/mL with supplemented MEM for the MIT and 2 × 106/mL with supplemented RPMI 1640 medium for the blastogenesis assay. Monocyte/Macrophage Migration Inhibition Test. The cell suspension was packed into a sterile nonheparinized capillary tube by capillarity. The capillary tubes were sealed at the bottom using S|P Miniseal Capillary Tube Sealant7 and centrifuged on the International Microcapillary Centrifuge (Model MB) at room temperature for 1 min. Using a Hex-Handled Scoring Knife,8 the tube was cut precisely at the fluid-cell interface. The portion of the capillary tube containing the cell pellet was mounted and fixed on a 35-mm diameter tissue culture dish9 by means of autoclaved Dow Corning silicon grease.10 Each dish was filled with 2.0 mL supplemented MEM containing 10% fetal bovine serum with graded concentration of S. aureus antigen as the stimulating dosages. The final concentrations of S. aureus antigen were 15, 30, or 60 µg/mL. Dishes with supplemented MEM plus 10% fetal bovine serum alone served as controls. The culture dishes were incubated at 37 C in a humidified atmosphere of 5% CO2 in air. After 16 h of incubation, the area of migration was projected onto drawing paper with a Bioscope Microprojector11 at 64× magnification. The projected area was measured by LASICO Mechanical Polar Planimeter.12 The planimetric data were divided by the degree of magnification to represent the real area of migration. The percentage

DELAYED-TYPE HYPERSENSITIVITY REACTION

migration inhibition was calculated using the following formula:

[

1−

% migration inhibition =

]

migration area with antigen × 100. migration area without antigen

A mean migration inhibition of 20% or more was considered to be significant (Rocklin et al., 1970; Morita and Sockawa, 1972; Vlaovic et al., 1975; Huynh and Chubb, 1987; Chubb et al., 1988). Twenty-one MIT were conducted in the sensitized group (7 birds × 3 in vitro S. aureus dosages of 15, 30, or 60 µg/mL), and 15 MIT were conducted in the nonsensitized group (5 birds × 3 in vitro S. aureus dosages). However, two samples in the nonsensitized group were lost, for a total of 13 MIT. Lymphocyte Blastogenesis Assay. Peripheral blood lymphocytes were cultured in quadruplicate in 96-well flat-bottomed tissue culture microplates.10 Each well contained 100 µL 2 × 106 cells/mL lymphocyte suspension, 10% fetal bovine serum, and 100 µL S. aureus antigen, 100 µL supplemented RPMI 1640 medium as a negative control, or 100 µL phytohemagglutinin (PHA)3 as a positive control. The final concentrations of S. aureus antigen were 15, 30, or 60 µg/mL. The final concentrations of PHA were 25 or 50 µg/mL. The culture microplates were incubated at 37 C for 48 and 72 h in a humidified atmosphere of 5% CO2 in air. Because peak reactions of S. aureus antigen and PHA were both reached after 48 h of incubation, 48 h of incubation were used in all subsequent tests. After incubation, each well was pulsed with 50 µL 20 µCi/mL 3H-methyl-thymidine13 dissolved in prewarmed supplemented RPMI 1640. The cultures were incubated for an additional 18 h (Timms, 1979; Timms et al., 1980). Cells were harvested onto glass fiber filters14 using a PHD cell harvester (Model 290),14 and the radioactivity was measured in a Tri-Carb Liquid Scintillation Analyzer (model 1600 TR)15 for 5 min. Background disintegrations per minute (dpm) from a blank control vial was subtracted from dpm obtained from S. aureus or PHA stimulated and nonstimulated cultures. Results were expressed as a stimulation index using the following formula: stimulation index mean dpm of S. aureus or PHA stimulated culture . = mean dpm of unstimulated culture Six sensitized birds and five nonsensitized birds were used in the lymphocyte blastogenesis assay. Two in vitro antigen concentrations of PHA were employed (25 and 50 µg/mL) for a total of 12 samples in sensitized birds and

13 Amersham Life Science, Arlington, IL 60005. Specific activity, 47.0 Ci/mmol. 14 Cambridge Technology Inc., Watertown, MA 02172. 15 Packard Instrument, Meriden, CT 06450.

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10 samples in nonsensitized birds. Three in vitro antigen concentrations of S. aureus were employed (15, 30, and 60 µg/mL) for a total of 18 samples in sensitized birds and 15 samples in nonsensitized birds.

Experiment 2 Birds. Twenty-five 1-d-old broiler cockerels were assigned to either a treated (n = 13) or a control (n = 12) group. The same protocol was used as in Experiment 1 with respect to sensitization, elicitation of the DFR, and the MIT. Histopathology. Both footpads were excised 3 d after challenge from sensitized birds with positive DFR and fixed in 10 mL 4% paraformaldehyde solution/g of tissue. Sections were cut at 5 µm, stained with hematoxylineosin, and evaluated by light microscopy (American Registry of Pathology, 1968). Preparation of Splenic Lymphocytes. Spleens were collected aseptically from treated and control birds and soaked for 10 min in PBS containing 1000 U penicillin/ mL and 1000 µg streptomycin/mL. After washing twice with sterile PBS, the splenic capsule was removed using forceps; spleen tissue was minced and collected into a centrifuge tube containing PBS. Tissue debris was sedimented by centrifuging at 1 × g for 5 min (Hovi et al., 1978); supernatant was collected and centrifuged at 400 × g for 10 min. The cell pellet was collected and washed twice in PBS. The splenic lymphocyte suspensions were obtained by pooling splenic lymphocytes from sensitized birds or control birds. Cells were counted (Kashima et al., 1993), and concentration was adjusted to 3 × 107/mL with PBS for the passive transfer of the DFR to recipient birds. Passive Transfer of the Delayed Footpad Reaction. Fifteen 7-wk-old broiler cockerels were divided into two recipient groups. Eight recipient birds were injected i.v. (brachial vein) with splenic lymphocytes from treated donor birds that had been sensitized with killed S. aureus previously and had positive DFRs; seven recipients were injected i.v. with splenic lymphocytes from control donor birds. Splenic donor cells were not matched for MHC with recipient birds. Each recipient received 1 mL pooled splenic cells (3 × 107 cells/bird). Immediately after cell transfer, all recipient birds were challenged in the right footpad with 0.1 mL S. aureus antigen (75 µg/bird in PBS; Huynh and Chubb, 1987). The left footpad was injected with 0.1 mL PBS. The footpad thickness was measured 0, 4, 24, and 48 h after challenge.

Statistical Analysis Footpad thickness was analyzed by one-way ANOVA with a split plot in time to access changes in thickness at 0, 4, 24, and 48 h following the eliciting challenge of S. aureus antigen (Steel and Torrie, 1980). The General Linear Model procedure of the SAS system was used (SAS Institute, 1988). If a significant treatment × time postchallenge interaction occurred, the means were partitioned by Newman-Kuels’ multiple range test (Steel and Torrie, 1980).

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Data obtained from the lymphocyte blastogenesis assay and the MIT were analyzed by Student’s t-test to determine whether differences existed between the treated and control groups.

RESULTS Delayed Footpad Reaction. Birds sensitized twice and subsequently challenged with killed S. aureus antigen had a pronounced DFR when compared with nonsensitized but challenged birds (overall mean of 0.89 ± 0.15 vs 0.33 ± 0.15, P < 0.02, Experiment 1; 0.59 ± 0.09 vs 0.22 ± 0.09, P < 0.005, Experiment 2). These data are not presented in the table or figures. The DFR of sensitized birds increased following challenge with S. aureus, reaching a maximum response at 24 h postchallenge and declining at 48 h postchallenge (P < 0.001; Figure 1), whereas the thickness of the footpad of nonsensitized birds showed no significant change within 48 h postchallenge (Figure 1A, Experiment 1) or showed only a slight but significant increase, after challenge with killed S. aureus (Figure 1B, Experiment 2). The DFR of sensitized birds was greater (P < 0.05) than that of nonsensitized birds at 4, 24, and 48 h postchallenge in Experiments 1 (Figure 1A) and 2 (Figure 1B). Migration Inhibition Test. There was a difference (P < 0.0001) between the monocyte/macrophage migration of sensitized and nonsensitized birds in the presence of S. aureus antigen (Table 1). Eighteen out of 21 samples from sensitized birds showed a significant migration inhibition as indicated by migration area and percentage migration inhibition, whereas only one of 13 samples from nonsensitized birds had significant migration inhibition. Lymphocyte Blastogenesis Assay. Compared with nonsensitized birds, S. aureus antigen significantly stimulated the lymphocyte blastogenic transformation in previously sensitized birds as evidenced by the stimulation index (P < 0.0001; Figure 2). As a positive control, PHA stimulated DNA synthesis of lymphocytes in sensitized and nonsensitized birds, although the stimulation index in sensitized birds was higher (P < 0.0001). Histopathology of the Delayed Footpad Reaction. Microscopic examination of tissue sections from footpads of S. aureus-sensitized birds revealed dermal edema with perivascular infiltrates of lymphocytes, macrophages, and lesser numbers of heterophils at the site of challenge with S. aureus (Figure 3). Tissue sections prepared from PBS-injected left footpads showed no signs of an inflammatory reaction. Passive Transfer of the Delayed Footpad Reaction. Birds that received splenic lymphocytes from S. aureussensitized birds had a greater increase in the thickness of the footpad than birds that received splenic lymphocytes from nonsensitized birds at 4, 24, and 48 h postchallenge with S. aureus antigen (P < 0.05; Figure 4). The overall increase in footpad thickness for recipient birds receiving sensitized vs nonsensitized splenic lymphocytes was 0.82 ± 0.05 mm and 0.41 ± 0.05 mm, respectively (P < 0.0001).

FIGURE 1. The interacation of treatment × hours postchallenge (P < 0.001, Experiment 1, Fig. 1A; P < 0.0003, Experiment 2, Fig. 1B) on the delayed footpad reaction (DFR) of sensitized and nonsensitized broilers challenged with killed Staphylococcus aureus. Each data point is the least squares mean ± SEM of eight observations of sensitized birds and seven observations of nonsensitized birds for Figure 1A and of 13 observations of sensitized birds and 12 observations of nonsensitized birds for Figure 1B. Mean comparisons were made over time (hours postchallenge). Means within sensitized birds or nonsensitized birds with no common superscripts are different (P < 0.05). DFR = S. aureus response, right footpad − PBS response, left footpad.

DISCUSSION Staphylococcus aureus is an important pathogen in humans. As a result, mice and rabbits have been used extensively by medical researchers to study the pathogenesis of S. aureus. When these laboratory animals are repeatedly exposed to S. aureus and then challenged with intradermal injection of the same organism, they display cell-mediated DTH reaction (Klein, 1990). Hypersensitive animals are more susceptible to infection with S. aureus than are nonsensitized controls (Taubler, 1968; Banerjee et al., 1971; Easmon and Glynn, 1975a,b; Adlam and Easmon, 1983). In mice, the footpad test has been used commonly to evaluate cell-mediated DTH reaction, and the response

DELAYED-TYPE HYPERSENSITIVITY REACTION TABLE 1. In vitro migration inhibition of monocyte/macrophage from Staphylococcus aureus sensitized and nonsensitized broilers (Experiment 1) Chicken

Migration area1

Migration inhibition2 (%)

Sensitized Nonsensitized

(mm2) 9.2 ± 1.8B 30.4 ± 4.5A

60.8 ± 6.4A −19.2 ± 7.9B

MIT positive3 /no. tested 18/21 1/13

A,B Means within a column with no common superscripts are different (P < 0.0001). 1 Data are expressed as means ± SEM.

[

Migration inhibition (%) = 1 −

2

]

migration area with antigen × migration area without antigen

100. 3 Significant migration inhibition test (MIT) ≥ 20% migration inhibition.

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with S. aureus. The eliciting challenge of S. aureus combined with certain stress conditions may lead to the outbreaks of staphylococcosis. In chickens, the wattle test is the most commonly used method to assess in vivo DTH reactions (Warner et al., 1971; Watabe and Glick, 1983; Cotter et al., 1987; Corrier and DeLoach, 1990a). The delayed wattle reaction to killed S. aureus antigen in White Leghorn chickens reached the maximum response at 24 to 48 h postchallange (Cotter et al., 1987). The results of the DFR in the present study (Figure 1) were consistent with those previous studies (Taubler, 1968; Cotter et al., 1987). Antigen-sensitized lymphocytes secrete a cytokine called migration inhibitory factor in response to rechallenge with the same antigen (McCarthy and Remold, 1992). Migration inhibitory factor inhibits the migration of normal monocytes/macrophages. Our results were

usually peaks at 24 to 48 h postchallenge with antigen (Taubler, 1968; Klein, 1990). The DTH reaction may also play a role in the pathogenesis of staphylococcosis in chickens. Birds are in an environment in which S. aureus has been consistently isolated from their skin, feet, and respiratory tracts as well as from the air in the poultry houses (Cooper and Needham, 1976; Witte et al., 1977; Devriese, 1980; Sauter et al., 1981). Thus, birds could be repeatedly exposed to S. aureus and sensitized through multiple challenges of the respiratory tract or the footpad. The eliciting challenge would most likely be through the footpad, but could occur through other sources such as vaccinating equipment contaminated

FIGURE 2. Stimulation index of Staphylococcus aureus or phytohemagglutinin (PHA)-induced blastogenesis of lymphocytes from Staphylococcus aureus sensitized (n = 12 for PHA and n = 18 for S. aureus) and nonsensitized broilers (n = 10 for PHA and n = 15 for S. aureus, Experiment 1). Means within S. aureus or PHA stimulation with no common superscripts are different (P < 0.0001). Stimulation index mean dpm of S. aureus or PHA-stimulated culture = , mean dpm of unstimulated culture where dpm = disintegrations per minute.

FIGURE 3. Footpad biopsy 3 d after intradermal challenge with 75 µg/bird of Staphylococcus aureus antigen in PBS in the right footpad (a) or PBS in the left footpad (b). The right footpad shows dermal edema with perivascular infiltrates of lymphocytes (arrow), macrophages, and lesser numbers of heterophils. The left footpad is normal. Each chicken was sensitized with 150 µg S. aureus antigen in polyethylene glycol 3 wk prior to footpad testing. Magnification 200×. Bar scale = 10 µm.

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FIGURE 4. The interaction of treatment × hours postchallenge (P < 0.0001) on the delayed footpad reaction (DFR) of recipient broilers injected i.v. with splenic cells (SPC) from donor broilers and subsequently challenged with killed Staphylococcus aureus (Experiment 2). Each data point is the least squares mean ± SEM of eight observations of birds that received sensitized SPC and seven observations of birds that received nonsensitized SPC. Means within hour postchallenge with no common superscripts are different (P < 0.05). DFR = S. aureus response, right footpad − PBS response, left footpad.

consistent with those of previous experiments using MIT as a criterion of cell-mediated DTH reaction, although in those experiments different antigens, such as tuberculin, diphtheria toxoid, infectious bronchitis virus, and bovine serum albumin, were used in the sensitization of chickens and inhibition of monocyte/macrophage migration (Morita and Sockawa, 1972; Trifonov et al., 1977; Timms, 1979; Chubb et al., 1988). In vitro blastogenic transformation of previously sensitized lymphocytes, measured by DNA synthesis, has been demonstrated in chickens following stimulation with either nonspecific mitogens or specific antigens. Blastogenic transformation correlates reasonably well with in vivo cell-mediated DTH skin tests (Haynes et al., 1980). Our results of peripheral blood lymphocyte blastogenesis stimulated by killed S. aureus antigen are in agreement with those of previous investigations, in which phytohemagglutinin, concanavalin A, pokeweed mitogen, lipopolysaccharide, avian leukosis virus, Marek’s disease virus, Newcastle disease virus, infectious bronchitis virus, tuberculin, diphtheria toxoid, and Pasteurella multocida were used as stimulants of lymphocyte transformation (Lee, 1971; Ghumman and Bankowski, 1976; Maheswaran et al., 1976; Meyers et al., 1976; Klesius et al., 1977; Visco and Buening, 1977; Hovi et al., 1978; Timms et al., 1980; Regnier and Kelley, 1981). Histological examination of the swollen footpad in the current study showed a perivascular infiltration of mononuclear cells, predominated by small lymphocytes, with some heterophils and macrophages at a greater distance from the blood vessels (Figure 3). This massive small lymphocyte infiltration at the challenge site is one of the

most characteristic features of the DTH reaction (Anderson, 1971; Warner et al., 1971; Klesius et al., 1977; Giambrone et al., 1980; McCorkle et al., 1980; Regnier and Kelley, 1981; Klein, 1990; Stites, 1994). The dense accumulations of these cells at the injection site, along with some edema caused by the seepage of large amounts of exudate from the bloodstream, are responsible for the footpad swelling. In the present study, the DFR was successfully transferred from sensitized birds with positive DFR to nonsensitized recipient birds via i.v. injection of splenic lymphocytes (Figure 4). Similar results have been reported by George and Vaughan (1962), Rocklin et al. (1970), Easmon and Glynn (1975a), Klein (1990), and Binns et al. (1996). Steps were not taken to ensure a MHC match between donors and hosts. If the DFR observed in the recipient treated group was due to MHC mismatches, then it should have been evident in the control group. The treated host group’s DFR response was always significantly greater than the control recipient group (4, 24, and 48 h postchallenge, Figure 4), indicating that the DFR had been passively transferred by the sensitized splenocytes. Easmon and Glynn (1975a) demonstrated that the DTH reaction in mice induced by repeated s.c. injection of S. aureus was characterized by footpad swelling at 48 h with a mononuclear cell infiltration at the challenge site and could be transferred to noninfected recipients with living lymphocytes from infected animals, but not by serum (antibody) from infected donors or T lymphocytes from noninfected donors. Antibody involvement in the initiation and expression of the S. aureus-induced DTH reaction has been excluded in our previous study and in studies from other researchers, which have indicated that bursectomy of chickens at hatching did not impair their ability to develop DTH reaction (Jankovic et al., 1963; Warner et al., 1971; Theis and Thorbecke, 1973; Zhu et al., 1999). Each criterion for the DTH reaction to killed S. aureus using the footpad model in chickens was met. These criteria were 1) DFR with a peak response at 24 h postchallenge, 2) inhibition of monocyte/macrophage migration, 3) lymphocyte blastogenic response, 4) mononuclear cell infiltration at the challenge site, and 5) passive transfer of the DFR reaction by splenic lymphocytes. Therefore, we conclude that the DFR can be used as a standard in vivo test for cell-mediated DTH reaction induced by killed S. aureus in chickens.

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