Evaluation of oral, subcutaneous, and nasal administration of ...

Evaluation of Oral, Subcutaneous, and Nasal Administration of Salmonella enteritidis-Immune Lymphokines on the Potentiation of a Protective Heterophilic Inflammatory Response to Salmonella enteritidis in Day-Old Chickens Michael H. Kogut, Kenneth Genovese, Rita B. Moyes, and Larry H. Stanker

ASTRACT We have previously reported that the prophylactic administration of factor(s) from T-cell supernatants derived from Salmonella enteritidisimmune chickens (ILK) have a favorable effect in controlling or eliminating salmonellosis in neonatal poultry. Experimentally, we have used the intraperitoneal injection as the standard method of administering ILK to neonatal poultry. However, this method is neither easy, practical, nor economical for the poultry industry. In the present study, we evaluated the effectiveness of oral (PO), intranasal (IN), and subcutaneous (SC) administration of ILK for ease of delivery, induction of protective resistance against Salmonella enteritidis (Se) organ invasion, and the ability to activate peripheral blood heterophils in day-old chickens. In the first experiments, delivery of ILK PO, IN, and SC significantly (P < 0.01) increased the resistance of day-old chickens to Se organ invasion. The level of protection was equivalent to that induced by the IP route. Administration of a comparable protein control (bovine serum albumin, BSA) by the 3 routes induced no protective effect against Se organ invasion. Likewise, a significant increase was found in the number of circulating heterophils within 4 h of administration of the ILK by all routes. In the 2nd experiment, the function of the heterophils from ILK-treated birds was compared with that of the control cells in adherence, chemotaxis, and phagocytosis assays. The heterophils from birds given ILK IP,

SC, PO, or IN had significantly (P < 0.01) increased functional activities when compared to the activities of the heterophils from the control birds. These studies indicate that the delivery of ILK either orally or parenterally, routes which can be used by the poultry industry, can confer protection to chickens against a localized enteric Se organ invasion by potentiating the systemic heterophilic innate response.

tion etait equivalent 'a celui induit par la voie IP. L'administration d'une quantite comparable de proteine temoin (albumine bovine serique) par les memes voies n'a pas eu d'effet protecteur contre l'invasion par Se. Une augmentation significative du nombre d'heterophiles circulant fut observee 4 h ou moins apres l'administration d'ILK quelle que soit la voie d'administration. Lors de la seconde experience, I'activite des heterophiles provenant d'oiseaux traites 'a l'ILK fut comparee a celles provenant d'oiRESUME seaux temoins en ce qui a trait ia l'adherence, le chimiotaxisme et la II a ete prece'demment rapporte' phagocytose. Les hete'rophiles proque l'administration prophylac- venant d'oiseaux traites ia l'ILK tique de facteur(s) provenant du etaient significativement plus actifs surnageant de lymphocytes T (P < 0,01) que les heterophiles propreleves de poulets immuns contre venant des oiseaux temoins. Ces Salmonella enteritidis (ILK) avait experiences demontrent que l'admiun effet benefique pour controler nistration d'ILK par voie orale ou ou eliminer la salmonellose chez les parenterale peut conferer aux poussins. L'injection intraperi- poulets une protection contre une toneale fut utilisee experimentale- invasion par Se en augmentant la ment comme voie d'administration reponse des heterophiles systemique. d'ILK aux poussins. Par contre, (Traduit par docteur Serge Messier) cette methode s'averant difficile, peu pratique et peu economique 'a utiliser par I'industrie avicole, INTRODUCTION I'administration orale (PO), intranasale (IN) et sous-cutanee (SC) Immunological research on the cond'ILK furent comparees pour leur trol of intestinal and tissue colonizafacilite d'administration, l'induc- tion of poultry by invasive Salmonella tion de protection envers une infec- enteritidis (Se) has largely focused on tion invasive par S. enteritidis (Se), the development of live, attenuated et la capacite d'activer les hete- vaccines (1). Regardless of the effirophiles sanguins chez des poussins cacy of a vaccine, at least 7-10 d are ages de 1 jr. Lors des experiences required for the stimulation of the initiales, I'administration d'ILK acquired immune response for protecPO, IN et SC a augmente de faqon tion. Unfortunately for the poultry significative (P < 0,01) la resistance industry, neonatal poultry are most de poussin de 1 jr 'a une infection susceptible to Salmonella infections invasive par Se. Le degre de protec- during the first 4 d post-hatch, after

USDA, Agricultural Research Service, Food Animal Protection Research Laboratory, College Station, Texas, USA (Kogut, Stanker); Department of Pathobiology, College of Veterinary Medicine (Genovese), Department of Biology (Moyes), Texas A & M University, College Station, Texas, USA. Received May 7, 1997.

Can J Vet Res 1998; 62: 27-32

27

which they become increasingly more resistant to infection (2). During this first 4 d post-hatch, we have hypothesized that immunopotentiation of the innate defense mechanism(s) would prevent Salmonella organ infectivity. Our laboratory has been evaluating the practicality of potentiating the endogenous innate host defenses of poultry using immune and inflammatory cytokines. Cytokines, chemical messengers secreted by various immune and non-immune cells, are some of the most effective mediators of natural host defenses (3-8). Specifically, we have found that the prophylactic administration of cytokines derived from T-cells (ILK) isolated from Salmonella enteritidis-immune chickens have a favorable effect in controlling or eliminating salmonellosis in neonatal poultry (9,10). This resistance was associated with a dramatic peripheral blood leukocytosis within 4 h after the injection of the ILK (11) followed by a marked infiltration of bactericidal inflammatory heterophils in the lamina propria of the ceca (9). Heterophils, the avian equivalents to the mammalian neutrophil, are highly phagocytic, polymorphonucleated white blood cells which are important mediators of innate resistance in poultry; especially in young birds that have not yet developed an acquired immune response (12,13). Under normal conditions, invasion of the intestinal mucosa by Salmonella spp. initiates the recruitment of heterophils to the lamina propria which control bacterial numbers in the bird until the development of acquired immunity. Administration of ILK into neonatal chicks induces a significant increase in the production and release of heterophils into the peripheral blood and also augments the effector functions of these phagocytes (1 1,14). Thus, infections by salmonellae are not only controlled but eliminated from chicks following the administration of ILK. Experimentally, we have used the intraperitoneal injection as the standard method of administering ILK to neonatal poultry. However, this method is neither easy nor practical for the poultry industry. We have demonstrated that in ovo injection was a successful and practical method of early delivery of ILK to young poultry (15). 28

However, it is important and necessary to evaluate other practical methods of administering ILK to neonatal poultry for the control of pathogens. In the present study we evaluated the effectiveness of other routes of administration of ILK for ease of delivery, induction of protective resistance to Se organ invasion, and the ability to activate peripheral blood heterophils in day-old chickens.

MATERIALS AND METHODS

for Salmonella, were immunized by oral gavage with 108 colony forming units (CFU) of Se. On days 14 and 21 after the initial immunization, the hens were further boosted by oral gavage with the same number of viable Se. PREPARATION OF LYMPHOKINES

Lymphokines from the T-lymphocytes of Se-immune chickens (immune lymphokines - ILK) were prepared, concentrated, and stored as described in detail previously (9,10). PERIPHERAL BLOOD COUNTS

Total and differential blood counts were conducted for each bird in the One-day-old Leghorn chicks (HyLine experiments by previously described W-36) were obtained from a commermethods All blood counts were (11). cial hatchery and randomly placed in performed individual unaware by an electrically heated commercial of the various treatment groups. The brooder batteries (Petersime Incubaabsolute numbers of lymphocytes, tor Co., Gettysburg, Ohio, USA) located within a biological hazard iso- large mononuclear cells (LMNs, lation unit on the research farm of the monocytes and blast cells), and heterophils per cubic millimeter were College of Veterinary Medicine, calculated for each animal from the Texas A&M University. Upon arrival, total and differential leukocyte counts. paper liners from the chick transport containers were cultured for the pres- ORGAN INVASION BY Se ence of Salmonella using described Specimens of liver were collected procedures (16). Chicks were profrom each bird and culaseptically vided ad libitum access to water and a tured individually. Organ samples balanced unmedicated corn-soybean ration. The feed ration contained or were incubated for 18 h at 37°C in exceeded the levels of critical nutri- tetrathionate broth. After incubation, ents recommended by the National the broth was streaked on BGA plates Research Council (17). Before use, containing 25 g/mL of NO and the feed ration was cultured for the 20 g/mL of NA, incubated for an presence of Salmonella using a stan- additional 24 h at 37°C, and examined dard culture method (16). Salmonella for the presence of lactose-negative, were not detected in the feed or from NO-NA-resistant colonies (16). the paper liners. ISOLATION OF PERIPHERAL BLOOD EXPERIMENTAL ANIMALS

INFECTIVE MATERIAL

HETEROPHILS

A poultry isolate of Se was obtained from the National Veterinary Services Laboratory, Ames, IA and was selected for resistance to novobiocin-nalidixic acid (NO-NA) and maintained on nutrient agar. Salmonella enteritidis challenge inocula were prepared in sterile phosphate-buffered saline (PBS). The viable cell concentration of the inoculum was determined by colony counts on brilliant green agar (BGA; Difco Laboratories, Detroit, Michigan, USA) plates.

Avian heterophils were isolated from the peripheral blood of day-old Leghorn chickens as described previously (14).

IMMUNIZATION FOR LYMPHOKINE PRODUCTION

Forty-two-week-old layer hens (HyLine W-36), determined to be seronegative and feces-culture-negative

HETEROPHIL ADHERENCE ASSAY

Tissue culture chamber slides (8-well, Nunc, Naperville, Illinois, USA) were coated overnight at 4°C with 200 ,uL of a 1% BSA (Sigma) solution in PBS. Before adherence, each well was rinsed with RPMI + 5% FCS. One hundred microliters of heterophils (1 x 106 cells) were then added to each chamber in quadruplicate and the chamber slide was subsequently incubated for 30 min at 37°C. After incubation, non-adherent cells were removed by gently rinsing the

chambers 2 times with warm RPMI. istration of ILK by the different For the 3rd rinse, the plastic chambers routes enhanced the resistance of the were removed from the slide and the chicks to Se organ invasion and slide was gently rinsed. The slide was induced the correlated peripheral allowed to air dry and then was fixed blood heterophilia described previand stained using the Diff-Quik sys- ously (11,22). One-day-old Leghorn tem (CMS, Dallas, Texas, USA). chicks were randomly divided into Adherence was assessed microscopi- 9 treatment groups of 25 chicks/ cally by counting cells contained in group: (A) Se challenge controls, (B) 5 fields/chamber using an ocular grid. ILK injected IP + Se challenge, (C) The data is expressed as the percent- 1% bovine serum albumin in PBS age of deviation from the adherence (BSA, protein control) injected IP + of heterophils from the control birds. Se challenge, (D) ILK injected subcutaneously (SC) + Se challenge, (E) HETEROPHIL CHEMOTAXIS ASSAY BSA injected SC + Se challenge, (F) Modified Boyden blind well ILK gavaged orally (PO) + Se chalchemotaxis chambers (Nucleoprobe, lenge, (G) BSA PO + Se challenge, Cabin John, Maryland, USA) were (H) ILK given intranasally (IN) + Se used to quantitate heterophil random challenge, and (I) BSA IN + Se chalmigration and chemotactic movement lenge. Chicks were given 0.5 mL of (14). Pooled chicken serum diluted in either ILK or BSA (protein = Hank's balanced salt solution (HBSS) 123 ,ug/mL) IP, SC, or PO. For IN or recombinant human IL-8 (rHIL-8, administration in order to maintain 10 g/mL) diluted in HBSS were the identical protein concentration placed in the bottom wells of the blind administered per chick, the standard well chemotaxis chamber and were ILK and BSA preparations were furused as positive chemotactic controls. ther concentrated 5-fold by ultrafiltraRandom migration of the heterophils tion using YM-10 membranes. Chicks was determined in control chambers were then given 0.1 mL of either ILK which contained HBSS alone. All or BSA into the right nare. Thirty solutions were incubated at 37°C for minutes after the ILK administration, at least 30 min before the assay in all birds were orally challenged with order to standardize the temperature. 5 X 104 cfu of Se. At 4 h postFifteen oil immersion fields per filter challenge, 5 chicks from each group were scored for the numbers of het- were killed by decapitation and blood erophils migrating through the filter. samples collected. Total and differenValues were expressed as the total tial blood counts were determined as number of cells migrating in described above. At 24 h post15 (lOOX) fields per filter. challenge, the remaining 20 chicks from each group were killed and HETEROPHIL PHAGOCYTOSIS ASSAY organs (liver) cultured for Se accordPhagocytosis of S. enteritidis by the ing to the NPIP guidelines (16). Each heterophils was determined by using experiment was conducted 5 times duplicate, sterile 15 mL screw-capped over a 6 wk period of time. Data from conical polypropylene centrifuge these replicate experiments were tubes that contained 2 X 106 het- pooled for presentation and statistical erophils and 2 X 107 bacteria in a total analysis. volume of 1 mL HBSS. The tubes were centrifuged (450 x g, 15 min, DESIGN OF STUDY 2 room temperature) in order to maxiDay-old Leghorn chickens were mize contact and then allowed to randomly divided into 5 treatment incubate at 37°C for 30 min to allow groups of 30 chicks/group: (A) vehifor phagocytosis (14). The results are cle (pyrogen-free saline) only, (B) expressed as the phagocytic index ILK given IP, (C) ILK injected SC, (PI), where PI = the percentage of het- (D) ILK given PO, and (E) ILK erophils that contain bacteria X the administered IN Chickens were average number of bacteria per administered with 0.5 mL of either vehicle or ILK except for the IN route ingesting heterophil) X 100. where 0.1 mL of ILK was placed in DESIGN OF STUDY 1 the right nare of each chick. Four The purpose of these experiments hours after administration of either was to determine whether the admin- vehicle or ILK, the birds were killed

by decapitation and the blood from each bird was collected in a 10 mL Vacutainer tube containing EDTA (Becton Dickinson, Rutherford, New Jersey, USA). The anti-coagulated blood from all 30 chicks was pooled and the heterophils were isolated from each treatment group. Each heterophil functional assay was conducted 3-4 times with data from these replicate experiments pooled for presentation and statistical analysis. STATISTICAL ANALYSIS

Absolute heterophil number data are presented as the mean of 15 chicks per group. Data for replicate experiments were pooled for the present

studies. Differences between ILKadministered, BSA-administered, and saline-injected groups were determined by 1-way analysis of variance using the SigmaStat statistical analysis software program (Jandel Scientific Software, San Rafael, California, USA). Chi-square analysis was used to determine significant differences between groups in Se organ colonization. Analysis of correlation to determine the relationship between peripheral blood heterophil counts and Se organ invasion was done using the SigmaStat statistical software program. Differences in heterophil functional assays between groups were determined by analysis of variance. Significant differences were further separated using Duncan's multiple range test. The probability level for significance was P < 0.01.

RESULTS STUDY 1

Administration of ILK subcutaneously, orally, and intranasally significantly (P < 0.01) increased the resistance of day-old Leghorn chickens to Se organ invasion (Figure 1A). The level of protection was equivalent to that induced by the positive control (IP) route of administration. Administration of an equivalent concentration protein control (BSA) by either route induced no protective effect in the neonatal chickens against Se organ invasion. As seen previously with IP injections, ( 11,22), a significant increase in the number of circulating 29

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TABLE I. Chemotaxis of heterophils following the administration of Salmonella enteritidisimmune lymphokines (ILK) by different routes Chemotactic Chemotactic Source of Randommigration movement movement (chicken serum) heterophils (HBSS) (rHIL-8) 41.8 ± 6.9a 106.2 ± 7.2a 162.3 ± 8.Oa Saline-injected IP ILK given: IP 1375.0 ± 176.5b 1817.4 ± 119.lb 79.0 ± ll.lb SC 98.3 ± 4.Ob 1717.3 ± 93.9b 1626.7 ± 149.Ob 96.3 ± 6.3b PO 1667.7 ± 126.6b 1832.4 ± 191.1b IN 84.8 ± 7.7b 1741.2 ± 106.8b 1939.8 ± 74.3b Values expressed as the mean number of heterophils ± SD Within columns, means followed by different superscripts are significantly different (P < 0.01) Each data point represents the mean of 4 replicate experiments

TABLE II. Phagocytosis of Salmonella enteritidis (Se) by heterophils isolated from chickens given either saline or Salmonella enteritidis-immune lymphokines (ILK) 4 h previously

FIGURES 1A and 1B. Relationship between the resistance to Salmonella enteritidis (Se) organ invasion and the number of circulating heterophils induced by Se-immune lymphokines (ILK). Results represent the inverse relationship between the percentage of birds positive for Se organ invasion (IA) and the number of peripheral blood heterophils (IB). Asterisks represent significant differences (P < 0.01) between the ILKinjected chickens and either the saline- or bovine serum albumin (BSA)-injected chickens. Each bar represents the mean SD of 5 replicate experiments.

FIGURE 2. Percent increase in adherence of Salmonella enteritidis-immune

(ILK)-treated heterophils

lymphokines heterophils

over

from

saline-injected controls. Asterisk represignificant differences (P < 0.01) between the adhesion of the heterophils from the ILK-injected chickens and the salineinjected chickens (the mean number of hetsents

erophils/chamber ences

were

heterophils

was

37 ± 12). No differ-

found in adherence between from chickens

receiving

ILK

by

the different routes. Each bar represents the mean

±

SD of 3

replicate experiments.

PMNs occurred within 4 h of adminis-

by all routes into day-old chicks (Figure iB). Addition0.981, ally, a strong correlation (r tration of the ILK

=

30

% heterophils Average number of Source of S. enteritidis per containing S. enteritidis heterophils ingesting heterophils Phagocytic index SalineinjectedIP 13.8 ± 1.0a 3.86 ± 0.10a 46.9a ILK given: IP 80.4 ± 3.3b 7.27 ± 0.27b 584.5b SC 68.2 ± 3.4c 9.52 ± 0.88c 649.9b 83.8 ± 7.4b PO 6.20 ± 0.62b 519.6b IN 8.47 ± 0.29c 82.6 ± 4.7b 699.6b Values expressed as mean ± SD Within columns, means followed by different superscripts are significantly different (P < 0.01) Each data point represents the mean of four replicate experiments.

P < 0.01) was observed between the number of circulating heterophils and the protection induced by the IP injection of ILK. Similar correlations were found between circulating heterophil numbers and the protection induced by ILK administered subcutaneously (r = 0.926, P < 0.01), orally (r = 0.931, P < 0.01), or intranasally (r = 0.895,

heterophils from all ILK-treated chickens was significantly greater (P < 0.01) than that found with the heterophils from the saline-injected control heterophils. Heterophils from all ILK-treated chickens, regardless of method of delivery, had a 10-fold increase (P < 0.01) in chemotactic movement to both chicken serum and P