Salmonella typhimurium Infection in Chickens

Evaluation of the Efficacy of Intraperitoneal Immunization in Reducing Salmonella typhimurium Infection in Chickens WENDY I. MUIR,*,1 WAYNE L. BRYDEN,† and ALAN J. HUSBAND* Departments of *Veterinary Anatomy and Pathology and †Animal Science, Faculty of Veterinary Science, University of Sydney, Sydney, NSW, 2006, Australia ABSTRACT Conventional methods of parenteral immunization with killed bacterin vaccines have met with limited success in protecting the avian intestinal mucosa from pathogens such as Salmonella typhimurium. For mucosal vaccines to be successful they must be evaluated for their ability to stimulate local secretory immunoglobulin (SIgA) at the mucosal surface, which acts as the first line of defense against invading pathogens. Previously we have demonstrated the ability of i.p. immunization with nonreplicating antigen in an appropriate adjuvant to induce a primary immune response, which, after an oral booster immunization, stimulates enhanced intestinal IgA responses in chickens. In the experiments reported here we have applied this immunization protocol to vaccinate against S. typhimurium in chickens, and examined the protection provided against subsequent S. typhimurium challenge by placing vaccinated birds on seeded litter with

cohabitant infected birds. Immunized+challenged birds displayed delayed onset of S. typhimurium infection, both at the mucosal surface and within the reticuloendothelial system. Elevated anti-S. typhimurium IgG and IgA titers were detected in serum after vaccination, which markedly increased after challenge, to levels higher than in control+challenged chickens. Anti-S. typhimurium IgA in bile and intestinal scrapings supernatant was also higher in the immunized+challenged birds than in the control+challenged birds 15 d after challenge. This study illustrates the potential for i.p. vaccination to induce a mucosal immune response to S. typhimurium in chickens, which, in the challenge model employed here, provided partial protection against intestinal challenge with the same pathogen and was reflected in deferred onset of bacterial infection and shedding.

(Key words: mucosal immune response, Salmonella typhimurium, intraperitoneal immunization, immunoglobulin A, humoral immunity) 1998 Poultry Science 77:1874–1883

INTRODUCTION Many potentially pathogenic antigens make initial contact with their host at mucosal surfaces, especially in the alimentary tract. The effectiveness of secretory IgA (SIgA) in protecting the mucosa from bacteria by way of immune exclusion was first described by Williams and Gibbons (1972) in mammalian species and subsequently this was confirmed in avian species (Parry et al., 1977; Parry and Porter, 1981). In poultry, vaccination for protection from intestinal disease, such as that induced by Salmonella spp., is desirable due to the production losses encountered, and, in the case of S. typhimurium, the public health risk posed through the potential for vertical transmission.

Received for publication February 9, 1998. Accepted for publication August 3, 1998. 1To whom correspondence should [email protected]

be

addressed:

The importance of local antibody for protection of the intestine from S. typhimurium infection has been demonstrated by Mastroeni et al. (1993) and Michetti et al. (1992) and, therefore, the use of a nonliving vaccine for immunization of poultry against S. typhimurium could be feasible if appropriate routes of delivery and adjuvants are employed. For these procedures to succeed, vaccine formulations and delivery methods that enhance the local mucosal production of SIgA are required. Traditional approaches to developing and administering vaccines to control salmonellosis have met with limited success, partly due to the empirical design of vaccination strategies using killed bacterin immunogens without regard to their relative efficacy in stimulating mucosal immune responses (Barrow, 1993). Immunization via the i.p. route has been shown in

Abbreviation Key: FCS = fetal calf serum; ISS = intestinal scrapings supernatant; pc = post-challenge; SIgA = secretory IgA; TAGA = tryptose agar; TETR = tetrathionate broth; XLD = xylose lysine desoxycholate.

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mammalian species to be an effective strategy to stimulate intestinal SIgA immune responses providing protection at the mucosal surface from invading pathogens (Pierce and Gowans, 1975; Husband, 1978; Sheldrake et al., 1993). We have previously shown that i.p. immunization with tetanus toxoid in Auspharm adjuvant plus Quil A followed by an oral secondary immunization also stimulates the production of antigenspecific IgA responses in the avian intestine (Muir et al., 1995). This response presumably occurs by providing antigen access to the ileal lymphoid aggregates and cecal tonsils via the serosal surface, generating a population of antigen-specific IgA plasma cell precursors that localize at the intestinal lamina propria and undergo clonal expansion after subsequent oral boosting. The studies reported in this paper were designed to assess the ability of i.p. immunization using killed S. typhimurium to interfere with intestinal colonization following homologous challenge. Birds were given primary vaccination by the i.p. route and an oral secondary immunization prior to challenge, which was achieved by exposure to contaminated litter and infected seeder birds. Protection from S. typhimurium challenge was assessed by determining the extent of bacterial colonization of the intestine, spleen, and liver during the 2-wk period after challenge. Immunological responses to both vaccination and exposure to S. typhimurium were determined by evaluating antibody titers in serum, bile, and intestinal secretions at selected times throughout the study.

MATERIALS AND METHODS

Chickens One-day-old male broiler chickens, obtained from Inghams poultry hatchery (Casula, Australia) were housed in brooders with feed and water available for ad libitum consumption.

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The concentration of S. typhimurium in the broth was evaluated by viable plate counts of serial dilutions onto xylose lysine desoxycholate (XLD)(PP2004)2 selective media and tryptose agar (TAGA) plates. For the ELISA assays, S. typhimurium lipopolysaccharide(L-4516)3 was used as the capture antigen.

Vaccine Formulations For preparation of S. typhimurium immunogen, whole bacteria broth cultures were washed twice by centrifugation (11,000 × g for 20 min), resuspended in sterile PBS, pH 7.2, to a final concentration of 2 × 1011 per 0.5 mL, and killed with 0.5% formalin. Inactivation was confirmed by plating onto TAGA and incubating overnight at 37 C. Bacteria were flocculated by adding 3 mL of 10% potassium alum solution and 1.5 mL 7.4% potassium hydroxide solution per 100 mL of vaccine. Primary i.p. immunization doses were prepared from flocculated whole killed S. typhimurium emulsified with an equal volume of Auspharm adjuvant (patent pending) (Husband, 1993) containing the saponin derivative, Quil A4 at 1 mg per dose. Each dose (0.5 mL) contained 1011 killed S. typhimurium. Secondary immunizations were administered orally by gavage as a 0.5 mL dose of 1011 killed S. typhimurium in PBS.

Seeder Chickens Seeder birds were housed in brooders and then in grower cages. At 35 d of age they were confirmed to be free of any pre-existing Salmonella infection by selective enrichment of individual cloacal swabs in tetrathionate broth (TETR) for 24 h at 37 C followed by subculture onto XLD media at 37 C for 24 h. They were then given an oral inoculation with 1012 live S. typhimurium and transferred to a pen (1.5 m2) of fresh litter in an isolation room. Seeder birds commenced shedding S. typhimurium 4 d after oral inoculation.

Ethical Considerations All experimental procedures were approved by the University of Sydney Animal Care and Ethics Committee and complied with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes.

Antigen A recent field isolate of S. typhimurium was used in this experiment. Cultures were grown overnight in Lemco broth at 37 C in a shaking water bath. The viability of the culture was determined by 0.2% trypan blue exclusion.

2Oxoid Australia Pty Ltd., West Heidelberg, Victoria, Australia 3Sigma Chemical Co., St. Louis, MO 63178-9916. 4Superfos Biosector a/s, DK-Vedbaek, Denmark.

3081.

Immunized+Challenged Chickens At 19 d of age, 10 chickens were randomly assigned to three treatment groups according to body weight (control, control+challenged, immunized+challenged), and transferred into wire cages in groups of five. Primary immunization with 1011 killed S. typhimurium was administered i.p. and an oral secondary vaccination was administered 14 d later (Day 33). At 41 d of age, birds were transferred in groups of 10 onto freshly seeded litter in individually ventilated and thermostatically controlled isolation rooms, with 10 seeder birds. Seeder birds were placed on fresh litter 6 d prior to introduction of challenge birds, and seeder and challenge birds were allowed to cohabit in equal numbers for the duration of the experiment. From Day 41, control

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MUIR ET AL.

birds were housed in separate rooms that were maintained under similar conditions to the rooms housing the challenged birds. Before both primary vaccination and challenge with S. typhimurium the absence of any pre-existing Salmonella infection was confirmed in each bird.

Sample Collections On Days 41, 43, 45, 47, 49, 51, 54, and 56, cloacal swabs were collected from each bird for detection of intestinal colonization with S. typhimurium. Blood samples were collected from the jugular vein on Days 19, 33, 41, 47, 49, and 56. Serum was prepared and stored at –20 C until assayed. On Day 49, half of the birds from each treatment group, selected at random, were euthanatized with i.v. sodium pentobarbitone. Sections of spleen, liver, and cecal wall plus contents were collected and processed for S. typhimurium isolation and enumeration. From these same birds intestinal scrapings were obtained from the length of the jejunum after the serosal and mucosal surfaces were washed in ice cold PBS, and bile was aspirated from the gall bladder. Intestinal scrapings and bile samples were immediately frozen on dry ice and stored at –80 C for analysis. The intestinal scrapings supernatant (ISS) was collected for antibody determination following heat inactivation for 30 min in a 56 C water bath and ultracentrifuged at 24,000 × g for 60 min (Duncan et al., 1978). On Days 39 and 43 (2 d prior to and 2 d after S. typhimurium challenge) peripheral blood was collected from four randomly selected birds per group, and CD4+ and CD8+ T cell subsets enumerated by flow cytometry. On Days 48 and 55 (7 and 14 d after S. typhimurium challenge) these measures were repeated on two of the birds. Samples of litter were collected on Days 35 (the day the seeder birds were introduced onto the litter), 41 (challenge day), 49, and 56, from which S. typhimurium was isolated and quantified following the technique described for viable organ counts. Three samples of litter, each of 50 g, were collected from each pen; one from near the waterer, another from near the feeder, and one taken at random. These samples were pooled and mixed before analysis.

Isolation of Viable S. typhimurium from Challenged Chickens Cloacal Swabs. Cloacal swabs, collected from individual birds, were selectively enriched for S. typhimurium as previously described.

5Wellcome Diagnostics, Temple, Hill, UK. 6Nunc Immuno Polysorb, Medos Co., Sydney, Australia 2141. 7Kirkegaard and Perry Laboratories, Gaithersburg, MD 20879. 8ICN Biomedicals, Costa Mesa, CA 92626. 9Bethyl Laboratories, Montgomery, TX 77365. 10Bio-Rad, North Ryde, Australia 2113.

Isolation and Enumeration of S. typhimurium from Tissues. Isolation and enumeration of S. typhimurium in the spleen, liver, and cecal wall plus contents was determined following homogenization of 1 g of tissue in a known volume of PBS. Serial dilutions were incubated overnight at 37 C in duplicate on XLD plates, which allowed for detection of greater than 100 cfu/g of tissue. To identify tissues colonized with fewer than 100 cfu/g, a 1-mL aliquot of the original homogenate was incubated in TETR and subcultures were incubated in duplicate on XLD plates for identification but not enumeration of S. typhimurium. The identity of S. typhimurium was confirmed biochemically by testing for the absence of lactose fermentation with o-Nitrophenyl-D-Galactopyranoside discs(DD0130)2 and serologically by slide agglutination using both polyvalent and specific O and H antigen antisera.5

Antibody Detection by ELISA Salmonella typhimurium specific antibody titer in serum, bile, and ISS was determined by an indirect ELISA. ELISA plates(475094),6 were coated with 1 mg per well S. typhimurium lipopolysaccharide(L-4516)3 in carbonate buffer. For IgG analysis, serum was diluted 1:10 and alkaline phosphatase-conjugated goat anti-chicken IgG(1520-06)7 was diluted 1:500 in washing buffer (0.05% Tween 20 and 0.5 M sodium chloride in PBS), and 1 mg/mL p-nitrophenyl phosphate disodium salt (191431)8 in diethanolamine buffer (pH 9.8) was used as the substrate. For IgA analysis, serum was diluted 1:20, bile was diluted 1:100 and ISS was assayed undiluted and horseradish peroxidase-conjugated goat anti-chicken IgA(A30-103P)9 was diluted 1:200 with washing buffer containing 1% bovine serum albumin. Neat ABTS substrate(50-66-00)7 was incubated for 30 min and the reaction stopped with 1% sodium dodecyl sulfate(5085-01).7 Absorbance values were read at 405 nm in a Bio-Rad 3550-UV plate reader.10 A negative buffer blank and a hyperimmune positive reference standard were included in each plate and all samples were analyzed in triplicate. Sample dilutions were adjusted to provide optical density readings on the linear portion of the standard curve, approximately 20% below saturation point. Optical densities were expressed as a percentage of the hyperimmune positive control.

Hyperimmune Antiserum Hyperimmune antiserum was obtained from chickens immunized i.p. with 1011 formalin killed S. typhimurium emulsified in Auspharm adjuvant [patent pending, (Husband, 1993)] containing Quil A4 at 1 mg/dose, at 19 and 33 d of age, and an oral booster at 47 d of age. Blood was collected 7 d later and serum prepared and stored at –20 C.


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TABLE 1. Intestinal colonization with Salmonella typhimurium determined by selective enrichment of cloacal swabs from either control, control+challenged, or immunized+challenged chickens. Data are expressed as the percentage of positive birds from the number (n) of chickens as indicated Days of experiment Vaccination/challenge 41 (challenge day) regimen Control

0 (n = 10) Control+challenge 0 (n = 10) Immunized+challenge 0 (n = 9)

43

45

0

0

40

60

100

100

11

44

67

89

Preparation of Lymphocytes Blood taken from the jugular vein was collected into heparinized tubes and centrifuged at 1,300 × g for 20 min. The buffy coat was aspirated into a lithium heparin rinsed Pasteur pipette, diluted in an equal volume of PBS and then layered onto 10 mL of Ficoll-Paque(17-1440-02).11 Following centrifugation at 900 × g for 30 min the layer at the interface was removed and washed twice in PBS/10% fetal calf serum (FCS) by centrifugation at 300 × g for 5 min before enumeration, and viability assessment by trypan blue exclusion.

Flow Cytometric Analysis Approximately 107 lymphocytes in 100 mL PBS/FCS were incubated with 50 mL optimal dilution of either monoclonal mouse-anti chicken CD4(8210-01) or CD8(822001)12 at 4 C for 20 min. Each sample was washed twice in 2 mL 0.1% sodium azide/PBS, then incubated with 100 mL FITC-conjugated affinity purified-goat anti-mouse immunoglobulin(55519)13 for 20 min at 4 C and washed as before. The cells were fixed in 0.1% paraformaldehyde in PBS, stored at 4 C overnight and analyzed within 24 h of fixing. For each sample, 10,000 cells were subjected to ungated analysis on a FACScan flow cytometer14 using Lysis II software.

Statistical Analysis Statistical analysis of differences between treatment group means was performed on all data following log10 transformation, using a one-way analysis of variance. Between group comparisons were determined using a Tukey’s test (Fokal and Rohlf, 1995). Statistical significance was set at P < 0.05.

11Pharmacia Biotech, Sweden. 12Southern Biotechnology Association Inc., Birmingham, AL 35226. 13Organon Teknika Corp., Cappel Research Products, Durham, NC

27704. 14Becton Dickinson, San Jose, CA 95131.

47

49

(% positive birds) 0 0

51

54

0 (n = 5) 100 (n = 5) 75 (n = 4)

56 0

100 100

0 (n = 4) 100 100 (n = 3)

RESULTS

Isolation of S. typhimurium from Cloacal Swabs Isolation of S. typhimurium from cloacal swabs of the control+challenge birds (Table 1) reflected the trickle challenge to which these birds were exposed, with a graded increase in the number of S. typhimurium-positive birds over a 6-d period. All of the control+challenge birds had positive cloacal swabs on Day 47 [6 d post-challenge (pc)], whereas the first recording of 100% positive cloacal swabs for i.p. plus oral immunized birds occurred on Day 54 [13 d pc]. On all days except Days 54 and 56 the percentage of positive cloacal swabs from i.p. immunized+challenged birds was lower than that of the control+challenged birds.

Anti-S. typhimurium Antibody Titers All birds had low S. typhimurium serum IgG titers on Day 19 (Figure 1), and titers remained low in the control birds for the duration of the experiment. On Day 33, immediately before secondary immunization, i.p. primed birds had a significantly higher mean anti-S. typhimurium serum IgG titer (P < 0.05) than either of the unvaccinated groups of birds. On Day 41, S. typhimurium-specific IgG had risen slightly, remaining significantly higher than the control group mean titer (P < 0.05). Following S. typhimurium challenge, mean serum IgG of birds that had been immunized i.p. plus oral gradually increased until Day 49, with a dramatic increase between Days 49 and 56 (P < 0.01), compared to the control birds. The control+challenged birds experienced a continual increase in serum IgG from Day 41 until Day 56. On Days 47 and 56 their mean IgG titer was significantly higher (P < 0.05) than that of the control birds. Serum anti-S. typhimurium IgA antibody in birds receiving an i.p. primary immunization was elevated on Day 33 relative to Day 19 and were higher than both of the control groups (P < 0.05) (Figure 1). However, on Day 41, 7

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MUIR ET AL.

d after the booster immunization, the titer was lower than the Day 33 titer. Following S. typhimurium challenge, serum IgA titers in the i.p. plus oral vaccinated birds increased, remaining significantly higher than the mean titer of the control birds on Day 47 (P < 0.05) and on Day 56 (P < 0.001). Salmonella typhimurium-specific serum IgA titers also increased in the control+challenged birds following challenge, being significantly higher than the mean titer of the control birds on Day 47 (P < 0.05), with the highest mean titer on Day 56 (P < 0.05), compared with the control group. However, on Day 56 the average serum

anti-S. typhimurium IgA titer of control+challenged birds was only half that observed in the i.p. plus oral immunized+challenged birds.

Intestinal Scrapings Supernatant and Biliary anti-S. typhimurium IgA Both ISS and biliary anti-S. typhimurium specific IgA titers were negligible on Day 49, irrespective of vaccination and S. typhimurium challenge (Figure 2). However, on Day 56 the mean titers in the i.p. immunized+challenged

FIGURE 1. Serum anti-Salmonella typhimurium IgG antibody (top panel) and IgA antibody (bottom panel), following immunization and challenge with S. typhimurium, in control (control), control+challenged (control/chall), and immunized+challenged (IP/chall) birds as determined by ELISA. Data are expressed as percentage of a hyperimmune control serum titer. Histograms are the mean of data from the number of chickens indicated in Table 1, and vertical bars are standard errors. Columns with different letter superscripts are significantly different from other observations for that time point, where lower case letters indicate P < 0.05 and upper case letters indicate P < 0.01.


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of all challenged birds. The number of S. typhimurium recovered from the cecum increased relative to Day 49 levels in both challenged groups, but fewer S. typhimurium were recovered from the immunized+challenged birds compared with the control+challenged group. On Days 49 and 56 no S. typhimurium was isolated from any tissues of the control birds.

Peripheral Blood T Cell Subsets On Day 39 the frequency of CD4+ and CD8+ T cells in peripheral blood was similar for all groups of birds (Figure 3). However, on Day 43 the number of CD4+ lymphocytes increased in both the control and immunized+challenged groups, but remained similar to the Day 39 measure for the control+challenged birds. Similarly, CD8+ counts increased in control birds, whereas all other groups experienced slight decreases. By Day 48 the number of cells in each subset more closely approximated the Day 39 measures of each group. On Day 56 an increase was observed in the number of CD4+ lymphocytes in the control+challenged birds, with a smaller increase in CD8+ cells.

FIGURE 2. Intestinal scrapings supernatant (ISS, top panel) and biliary (bottom panel) anti-Salmonella typhimurium IgA antibody titer on Days 49 and 56, for control (control), control+challenged (control/chall), and immunized+challenged (IP/chall) birds determined by ELISA. Data are expressed as percentage of a hyperimmune control serum titer. Histograms are the mean of data from the number (n) of chickens indicated in Table 2 for Day 49 and Table 3 for Day 56 observations, and vertical bars are standard errors.

birds had increased considerably with less notable increases in the bile of the control+challenged birds. However, there were no statistically significant differences.

Isolation and Enumeration of S. typhimurium in Tissues On Day 49 (8 d pc) no S. typhimurium was isolated from the liver of any birds, regardless of their immune status (Table 2). In contrast, S. typhimurium was isolated from the spleen and cecal wall plus contents in all challenged birds. However, the mean number of colony-forming units per gram of tissue was less in the immunized+challenged birds than in the control+challenged birds. By Day 56, Table 2, S. typhimurium was less frequently isolated from the spleen of both the control+challenged and the immunized+challenged birds and fewer bacteria were recovered from spleen compared with observations on Day 49. Fewer birds were positive and fewer bacteria were present in the spleen and liver of i.p. plus oral immunized+challenged birds than in the control+ challenged birds. Irrespective of vaccination status, S. typhimurium was isolated from the cecal wall plus contents

Isolation of S. typhimurium from the Litter The number of S. typhimurium in litter for each treatment group throughout the course of the experiment are presented in Table 3. Salmonella typhimurium was not detected in the litter of any pen at Day 35, before the seeder birds were introduced. Similarly, S. typhimurium was not isolated from the litter of the control birds throughout the course of the experiment. However, both challenge pens had similar numbers of bacteria per gram of litter on the day of challenge and this continued to increase until the termination of the experiment. On Days 49 and 56, fewer S. typhimurium were isolated from the litter of the i.p. immunized+challenged birds than from the control+challenged pens.

DISCUSSION We have reported previously that i.p. immunization of chickens, using an oil emulsion formulation, invokes enhanced IgA responses after oral challenge (Muir et al., 1995). A number of studies including Truscott (1981) and Barrow et al. (1990) have investigated the potential for repeat oral immunization with killed S. typhimurium to protect chicks from a subsequent oral challenge of the bacteria. The failure of this immunization regimen to facilitate protection is not unexpected when the inability of oral delivery of nonreplicating antigen to stimulate intestinal SIgA is appreciated (Muir et al., 1995; Widders et al., 1996). However, both of these studies have demonstrated the significant antigen-specific IgA antibody titers induced following i.p. immunization of

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MUIR ET AL. TABLE 2. Isolation and enumeration of Salmonella typhimurium in spleen, liver, and cecal wall and contents of either control, control+challenged, or immunized+challenged chickens on Day 49 and 56. Data are expressed as the percentage of positive chickens from the number (n) of chickens, and the number of colony-forming units per gram of tissue ± standard error Isolation of S. typhimurium from

Vaccination/challenge regimen

Spleen (% positive)

Day 49 Control (n = 5) Control+challenge (n = 5) Immunized+challenge (n = 4) Day 56 Control (n = 4) Control+challenge (n = 5) Immunized+challenge (n = 3)

0

Liver (cfu/g) 0

(% positive)

Cecal wall and contents (cfu/g)

(% positive)

0

0

0

(cfu/g) 0

100

70 ± 8

0

0

100

3,510 ± 1,027

100

56 ± 6

0

0

100

900 ± 501

0

0

0

0

0

0

40

20 ± 11

80

40 ± 9

100

4,940 ± 3,063

33

17 ± 14

67

33 ± 14

100

1,828 ± 1,434

FIGURE 3. CD4+ and CD8+ T cell subset distribution determined by flow cytometry in control (control), control+challenged (control/chall), and immunized+challenged (IP/chall) birds at Day 39 (top left panel), Day 43 (bottom left panel), Day 48 (top right panel), and Day 55 (bottom right panel). Data are expressed as mean cells per microliter for four chickens per group on Days 39, 43 and for two chickens per group on Days 48 and 55. Vertical bars are standard errors of the means.

INTRAPERITONEAL IMMUNIZATION REDUCES S. TYPHIMURIUM INFECTION TABLE 3. Enumeration of Salmonella typhimurium in the litter of pens of control, control+challenged, or immunized+challenged chickens. Data are the number of colony-forming units per gram of litter ± standard error from the number (n) of litter samples S. typhimurium Days of experiment

n

35 41 49 56

2 2 2 2

Control

Control+ challenge

Immunized+ challenge

0 0 0 0

(cfu/g) 0 175 ± 56 3,850 ± 354 7,550 ± 2,022

0 200 ± 0 2,265 ± 799 6,125 ± 1,414

chickens. However, whether i.p. immunization in the chicken induces an intestinal immune response sufficient to protect the mucosa against S. typhimurium infection has not been previously reported. The study described here demonstrates that i.p. vaccination with whole killed S. typhimurium in an oil adjuvant, followed by an oral secondary immunization stimulates a specific SIgA response in the intestine and following challenge, the onset of intestinal invasion is delayed in vaccinated birds compared with unimmunized birds. The appearance of serum anti-S. typhimurium IgG and IgA titers indicated that vaccinated birds were immunologically primed following i.p. plus oral immunization, which facilitated further increases in serum antibody titers following exposure to S. typhimurium. The kinetics of this systemic serological response are similar to observations from infected chickens (Hassan et al., 1991). A marked increase in mean serum anti-S. typhimurium IgG and IgA titers of immunized chickens was observed between Days 49 and 56, concurrent with an elevation in the number of S. typhimurium isolated from the cecum and liver. Although these findings support the hypothesis of Cooper et al. (1992) that increased serum antibody titers indicate bacterial invasion of the tissues, an inconsistency exists in the spleen data where, on Day 56 S. typhimurium was isolated from fewer birds in lower numbers compared with Day 49 observations. The slight reduction in serum IgA titers in immunized birds observed between Day 33 (secondary oral immunization) and Day 41, the day of S. typhimurium challenge, is difficult to explain and had not been previously observed in any other group of birds vaccinated in a similar manner. The SIgA response following i.p. plus oral immunization was correlated with a reduction in the rate of S. typhimurium invasion with fewer bacteria isolated from colonized tissues compared with control+challenged chickens. The overall increase in S. typhimurium in the cecum and liver of all challenged birds on Day 56 compared to Day 49, may have been due to increased challenge pressure, reflected in the increased concentration of S. typhimurium in the litter probably resulting

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from the continual presence of the seeder birds on the litter for the duration of the experiment. Therefore, the challenge may have been much greater than would normally occur and the extent to which this immunization regimen reduces shedding by vaccinated birds may be more accurately determined if the experiments were repeated with seeder birds removed from the litter at the time of challenge. Interestingly, anti-S. typhimurium IgA titers in ISS and bile at Day 49 were similar in all groups of birds regardless of immunization+challenge status. However, by Day 56 an increase in mean bile and ISS antibody titers was observed in all challenged birds, with immunized+challenged birds exhibiting the highest antigen-specific IgA titers. This result also supports the proposal that the vaccinated birds were immunologically primed and more able to produce a mucosal immune response after bacterial challenge. The simultaneous increases in serum, bile, and ISS anti-S. typhimurium IgA titers on Day 56 may be partially explained by the contribution from serum of IgA to bile in chickens (Rose et al., 1981), which supplements the concentration of IgA at the mucosal surface, reflected in the increased ISS IgA titers. Measurement of T cell subsets in peripheral blood were undertaken because of the reported association of CD4+ T helper cells with the induction of cytokine secretion that Kaufmann (1993) has proposed is essential for the establishment of acquired resistance to intracellular bacteria, through the stimulation of antimicrobial activity in infected cells. In chickens infected with S. typhimurium, Lessard et al. (1995) observed increased lymphocyte proliferative responses and cytotoxic activity of natural killer cells at both 8 and 20 d after infection and Lee et al. (1981, 1983) detected delayed type hypersensitivity responses at both 2 and 5 wk after infection. The increase in CD4+ and CD8+ T cell populations on Day 56 in the control+challenged birds in this study may correspond with increased activation of the cell-mediated immune system in response to systemic infection with S. typhimurium. In contrast, at this same time point these subsets in immunized birds were similar to levels observed in control birds, which may indicate limited activation of these components of cell-mediated immunity following S. typhimurium challenge, perhaps the result of a less extensive systemic infection in immunized chickens. Under the high challenge pressure employed in this study, the mucosal response generated following i.p. immunization was insufficient to provide absolute protection against infection. Although a delay in colonization was observed, it did not prevent infection in immunized chickens. Although higher titers of SIgA may improve protection, Ziprin et al. (1989) consider cell mediated immunity to be pivotal for the induction of resistance to salmonellosis, particularly in newly hatched chicks, and this should be assessed in future studies. However, if should be noted that Michetti et al.

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(1992) demonstrated that monoclonal SIgA at the intestinal mucosal surface protected mice from an oral challenge with S. typhimurium. The delay in the onset of S. typhimurium fecal shedding by immunized birds is consistent with the reduced number of bacteria per gram of litter in these pens, compared with the unimmunized+challenged birds on both Days 49 and 56. On the day of challenge (Day 41) both pens had similar numbers of bacteria per gram of litter, but, the rate of increase following the introduction of challenge birds was more rapid with the control+challenged birds. However, any benefits that may have resulted from reduced bacterial shedding in vaccinated birds may have been masked by the continual presence of the seeder birds. The advantages of the delay in infection observed in i.p. immunized birds may be further enhanced if used in conjunction with improved levels of husbandry and sanitation. The extent to which the mode of immunization described here may contribute to the interruption of vertical transmission of S. typhimurium via the egg should be addressed in future studies. As the reproductive tract forms a part of the common mucosal immune system (McDermott and Bienenstock, 1979), an improved mucosal immune response should include an increase in local antibody production in the oviducts and cloaca, acting to restrict the opportunity for pathogens to gain access to the egg during its formation and transit. Further, i.p. immunization, in addition to stimulating local immunity in the intestine, is a recognized mode of systemic immunization. Therefore, a systemic IgG response may provide additional opportunity for establishing passive protection for hatching chicks via transfer of maternal IgG to the egg, as McCapes et al. (1967) observed in turkey poults originating from hens vaccinated with a S. typhimurium bacterin. Although the immunization strategy utilized in this study reduced but did not prevent intestinal colonization with S. typhimurium, it may be more effective if repeated i.p. injections are used. However, i.p. may only be a practical approach with breeder birds. Alternatively, i.p. immunization with a killed bacterin may be used in sequence with an attenuated live vaccination, a strategy that both Suphabphant et al. (1983) and Vielitz et al. (1992) have observed demonstrates benefits over using either vaccine alone. This study illustrates the induction of a substantial mucosal and systemic immune response to S. typhimurium in birds immunized i.p. plus oral with whole killed bacteria. Following a trickle challenge with S. typhimurium immunized chickens displayed a vigorous immune response in serum, with the generation of anti-S. typhimurium IgA antibody in ISS and bile occurring 15 d (Day 56) later. Immunized chickens demonstrated reduced intestinal colonization and lower levels of bacterial invasion compared with control+challenged chickens. However, as infection was delayed but not

prevented, these results raise the question of whether SIgA alone, is a sufficient first line of defense against challenge with S. typhimurium, and indicate that concurrent activation of both humoral and cellular arms of immunity may be necessary for complete resistance to challenge in chickens.

ACKNOWLEDGMENTS The authors wish to thank P. Widders, Victorian Institute of Animal Science, Mickelham Rd., Attwood, Victoria, 3049, Australia, for the donation of S. typhimurium isolates, and, the technical staff at the Poultry Unit, Department of Animal Science, University of Sydney for their invaluable assistance. The work described in this paper was supported by grants from the Australian Chicken Meat Research and Development Council and the Egg Industry Research and Development Council. WIM was a recipient of a Junior Research Fellowship from this source.

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