interaction of escherichia coli variants with chicken mononuclear phagocytic system cells

Developmental and Comparative Immunology, Vol. 14, pp. 481-487, 1990 0145-305X/90 $3.00 + .00 Printed in the USA. All rights reserved. Copyright © 1990 Pergamon Press plc

INTERACTION OF ESCHERICHIA COLI VARIANTS WITH CHICKEN MONONUCLEAR PHAGOCYTIC SYSTEM CELLS

Lance Miller,* Muquarrab A. Qureshi and Herman A. Berkhoff'l"

*Department of Poultry Science, and -i-Department of Microbiology, Pathology & Parasitology, College of Veterinary Medicine, North Carolina State University, Raleigh, NC

(Submitted January 1989; Accepted May 1989)

Introduction

Two phenotypic variants of E. coli, Congo Red positive (CR +) and Congo Red negative ( C R - ) , have been identi- fied recently as differing in their ability to produce colisepticemia in chickens. Congo Red positive (CR+) represents the virulent or invasive form of E. coli, while the Congo Red negative ( C R - ) phenotype failed to show any correlation with colisepticemia (1,2). Several immune defense mechanisms contribute to the cure of bacterial infections. A crucial "f i rs t" step during the course of host defense against microbial challenge is phagocytos is and degradation of in- vading microbes by monocytes and macrophages . Avian macrophages have phagocytic and intracellular killing capa- bilities for an extensive array of microorganisms which include various protozoa (3,4), molds (5), and bacteria (6-8).

Several factors , including genetic background of the host, determine the eventual outcome of bacterial-macrophage /monocy te in teract ions (8,9). However, the efficiency of phagocytosis

Address correspondence to Dr. M. A. Qur- eshi, Department of Poultry Science, Box 7608, North Carolina State University, Ra- leigh, NC 27695-7608. Paper No. 1196 of the Journal Series of the N.C. Agricultural Research Service, Raleigh, NC 27695-7643. The use of trade names in this publication does not imply endorsement by the N.C. Agricultural Research Service of the products named nor criticism of similar ones not mentioned.

and intracellular killing by macrophages varies depending upon the type and vari- ations in bacterial strains. Harmon et al. (10) have shown that fully virulent smooth Brucella abortus strain #2308 exhibits a significantly higher intracellular survival rate in bovine mammary gland macrophages in vitro than that of the rough strain #45/20. This study eval- uates the interaction of live C R + and CR - variants of E. coli with the chicken macrophages, both in vitro and in vivo.

Materials and Methods

Animals

Experiments 1 and 2 were conducted utilizing peritoneal macrophages derived from Cornell K-strain (B15B 15) 6-week- old female White Leghorn chickens. Ex- periments 3 and 4 utilized 5-week-old K- strain chickens of mixed sex with six birds averaging 300 g body weight in each treatment group. The chickens were reared in raised wire cages. Feed and water were available ad libitum. The chickens were fed a standard corn-soy starter diet composed of 17.43% crude protein.

Collection of Peritoneal Exudate Cells

Peritoneal Exudate Cells (PECs), as a source of macrophages, were collected according to methods descr ibed by Trembicki et al. (11). Briefly, a single in- traabdominal injection of 3% Sephadex

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482 L. Miller et al.

G-50 (Sigma, St. Louis, MO) suspension in 0.75% sterile saline was given to each bird at the rate of 1 mL per 100 g body weight. Approximately 48 h after injection, the birds were sacrificed and the abdominal cavity flushed with sterile heparin (0.5 U/mL) saline solution. A total of 30 mL of abdominal exudate from each bird was collected in sterile siliconized glass tubes and centrifuged at 1500 rpm and 10°C for 25 min to yield a pellet of exudate cells.

Macrophage Cultures

The PEC pellets were resuspended in RPMI 1640 g rowth medium (Flow, Mclean, VA) with 10% fetal calf serum (FCS) (Gibco, Grand Island, NY) and 1% antibiotics (100 U/mL penicillin, 50 mcg/mL st reptomycin, Gibco). Total n o n e r y t h r o i d PECs in 4 - 6 poo led samples were counted on a hemacytometer and their concentration adjusted to 1 x 106 per mL. One mL of this cell suspension was added to each petri dish (35 mm, Corning, Corning, NY) containing four sterile glass coverslips (Gold Seal, Thomas Scientific, Swedesboro, N J). The petri dishes were incubated at 37°C in a humidified chamber with 5% CO2 for 1 h. The coverslips were then washed with 0.75% sterile saline solution to remove all nonadherent cells and trans- ferred to new petri dishes containing 2 mL of RPMI 1640 growth medium with 10% FCS and no antibiotics. Macro- phages c o n s t i t u t e d a p p r o x i m a t e l y 95-98% of the adherent exudate cells as determined by their morphologic char- acteris t ics after staining with M a y - GrOnwald-Giemsa stain (12).

Bacterial Preparation

Congo red positive and congo red negative phenotypic variants derived from a common E. coli strain (18344,

Rollins) were grown on congo red agar plates to insure that the desired phenotype was expressed. CR+ or C R - E. coli colonies were washed from plates with 10 mL of sterile phosphate-buffered saline (PBS, pH 7.4).

For in vitro phagocytosis assay, the bacterial pellets from CR + and C R - E. coli var ian ts were r e s u s p e n d e d in RPMI-1640 growth medium supple- mented with 10% FCS without antibiotics. The concentration of each phenotype was adjusted to approximately 5 × 10 7 bacteria per mL using a hemacytometer. The hemacytometer counts were later confirmed by bacteriological plate counts and expressed as colony-forming units (CFUs). For in vivo inoculations, the bacterial pellets were resuspended in PBS and the concentrations adjusted to approximately 4 -5 x 10 6 per mL.

In Vitro Phagocytosis Assay

Macrophage monolayers were incubated with 1 mL of bacterial samples (50:1 bacteria to macrophage ratio) for 1 h in a humidified chamber at 37°C with 5% CO2. The monolayers were then washed with 0.75% sterile saline solution, fixed in methanol, and stained with May-GrOnwald-Giemsa stain (Sigma). The incidence of phagocytic macrophages and mean number of engulfed bacteria per macrophage were scored using a microscope with a 100 x oil ob- jective. Two separate experiments were conducted employing 3-4 petri dishes with three coverslips per dish for each bacterial phenotype.

In Vivo Bacterial Clearance Assay

Each of the six chickens per group was injected via the ulnaris vein with 200 IxL of CR + or CR - bacterial inoculum. Blood samples of equal volume (200 ~L) were drawn from each bird at 0, 10, 25,

E. cofi-macrophage interaction 483

55, 115 and 180 min after inoculation. The whole blood samples were diluted to 1:10 and 1:100 with sterile PBS. Three 10-p~L replicates of the whole blood and the two dilutions for each bird at each time point were pipetted onto Congo Red agar plates. The plates were then incubated at 37°C for 18 h. The number of colony-forming units (CFU) was determined 72 h later to allow for full ex- pression of the CR + and C R - phenotypes. Two separate experiments were performed.

Statistical Analysis

All analyses were conducted using general linear model program from SAS Institute, Inc. (13). The data were sub- jected to one-way analysis of variance and the means were then compared using Student's t test with a = .05.

Results

Interaction o f Two E. coli Phenotypes With the Chicken Mononuclear Cells

Adherent macrophages upon coincu- bation in culture with CR + or C R - variants of E. coli exhibited significantly different bacterial uptake. Depending on the variant (Table 1), there was an overall increase in the percentage of macrophages phagocytizing CR+ bacter ia and an increase in the mean

number of intracytoplasmic CR + bacteria per phagocytic macrophage as c o m p a r e d wi th the C R - v a r i a n t (Fig. 1).

In Vivo Clearance o f CR + and CR - Variants After Intravenous Inoculation

The representative clearance profile as observed in the first clearance experiment is shown in Fig. 2. While both the CR + and C R - phenotypes were even- tually cleared from the circulation, C R - bacteria were cleared in a significantly much shorter time than were the CR + bacteria. There was a significant reduc- tion in the number of CFUs in the first 10 min post inoculation of C R - variant as compared to the CR+ CFUs at the same time point. This difference per- sisted during subsequent sampling times until no CFUs were observed for either E. coli phenotype.

This clearance study was repeated on age-matched chickens obtained from a different hatch. Data from the second clearance experiment exhibited similar clearance profile as observed in the first experiment with a mean of 347, 153, 55, 33, and I 1 CFUs of CR + E. coli at 10, 25, 55, 115, and 180 min post inoculation, respectively. However, clearance of C R - E. coli was even more rapid as compared to the CR + phenotype exhib- iting complete clearance from the blood in the first I0 min post inoculation.

Table 1. In vitro uptake o f two E. coi l variants by chicken peritoneal macrophages

% Phagocyt ic Average # Bacteria/ Macrophages Phagocyt ic Macro-

phage E. coi l M M

Variants Exp 1 Exp 2 Exp 1 Exp 2

C R + 66.19 a 63.75 a 4.81 ° 4.75 a C R - 36.71b 36.26 b 2.96 b 3.31b

=,b Numerical values within columns not sharing a common superscript letter are different by Student's t test at p < 0.05.

Discussion

There is the quest ion of whether chicken macrophages play a role in the differential virulence of CR + and C R - variants. Our in vitro studies showed that chicken macrophages phagocytize the pathogenic CR+ phenotype more efficiently than the nonpathogenic C R - phenotype. Thus, macrophages may rec- ognize some differential antigenic deter-


Figure 1. Photomicrograph of Sephadex-elicited chicken peritoneal macrophages (a) normal (b) fed with CR+ bacteria. The presence of several intracytoplasmic vacuoles and bacteria in phago- somes is indicated. Magnification is 1000 x.

minant(s) on the CR+ variant which may be absent from the C R - phenotype. There is precedent for this as- sumption that certain structures asso-

ciated with outer bacterial cell walls do, in fact, enhance bacterial uptake by macrophages, namely pili on E. coli (14). In contrast, pili on Neisseria gonococci

E. coli-macrophage interaction 485

10 4

~o 03 1 o | o

o 102

o

2 01 o 1

100

/~ -~ C R +

~ \ -~ c . - \

L,,o

10 20 30 40 50 60 70 80 90 100 110 120 130 t 4 0 150 t 6 0 170 180

Mlnutes af ter IntraveoQug InJec.tl~n

Figure 2. Time course of the clearance of Congo Red Positive (CR+) and Congo Red Negative (CR-) bacteria from chicken blood. The numbers in parentheses indicate mean CFUs of three replicate samples at each sampling time.

inhibit phagocytosis (15) and the presence of sulfatide groups on the outer surface of Mycobacterium tuberculosis blocks macrophage activation (16). De- pressed uptake of C R - variant by chicken macrophages could be due to the presence of similar molecule(s) on the C R - variant which renders macrophages refractory to phagocytosis.

Indeed, when the bacterial washes from CR + and C R - bacterial colonies were analyzed electrophoretically, a dis- tinct protein band around 66KD position was consis tent ly found in C R - and missing in CR+ samples (unpublished observation). It is, therefore, hypothe- sized that the presence of some differential molecule(s) on the outer surface of C R - may in some way inhibit C R - variant binding to target organs which, in turn inhibits infection in exposed chickens. On the contrary, the enhanced uptake of CR + variant by macrophages might, in fact, act as a source of persis- tent E. coli infection in chickens. It has been shown that several bacteria appear to have developed survival mechanisms which prevents killing after they are

phagocytized by macrophages. Myco- bacterium intracellulare (17), Mycobac- terium tuberculosis (16), and Mycobac- terium leprae (18) are examples of or- g a n i s m s w h i c h c a n s u r v i v e in macrophage cytoplasm. Based on the results of our enhanced CR+ uptake, we propose that after initial infection with the CR+ variant, macrophages phagocyt ize C R + bacter ia in large numbers and eliminate them from the system via degradative mechanisms. However, after initial infection, certain "infected macrophages" might serve as carriers for those CR+ E. coli which survive in the cytoplasm. Later, under stressful conditions, rapid intracellular replication of CR+ E. coli may occur which results in the colisepticemic con- dition and initiation of a new disease cycle. This possibility seems to be in line with the pattern of deaths seen in classic air sac epizootics in broiler flocks, that is an increased number of deaths at 3 to 4 weeks, with a second peak at 6 to 7 weeks (19,20).

The fact that the CR + E. coli were phagocytized by macrophages more ef-


fectively in vitro but were cleared rela- tively late f rom the circulation points towards an inverse functional relationship be tween macrophages and mononuclear phagocyt ic cells in the circulation (e.g., monocytes , th rombocytes , heterophils). A similar inverse relationship for macrophage versus m onocy t e funct ional activity has been described in mice (21). We have shown previously that chicken c o n g e n i c l i n e s w h i c h a r e h igh re - s p o n d e r s for p e r i t o n e a l m a c r o p h a g e function (8) exhibit depressed monocyte chemotact ic ability towards bacterially derived chemoat t rac tants (9).

The d e l a y e d c l e a r a n c e o f v i ru l en t ( C R + ) E. coli in our s tudy is compa- rable to the findings of Arp and Cheville (22) who reported that nonpathogenic E. coli were rapidly cleared f rom the circulation whereas the pathogenic E. coli re- sisted clearance. The possibility of differential recognition of these variants by macrophages in spleen and liver, the pri- mary organs involved in clearing significant numbers of bac te r ia during early bac te remia (23), cannot be ruled out. In addition, C R + variants may be less suscept ible to coat ing by humora l fac tors s u c h as c o m p l e m e n t or a n t i b o d i e s , thereby allowing prolonged persistence.

In conclusion, it would be interesting to determine how the two E. coil phenotypes would interact with chicken macrophages if they were previously opson- ized with specific ant ibodies . Whether mac rophages are acting as carr iers of pa thogenic E. coli is ano ther quest ion which needs to be answered. Never the- less, this study suggests that the E. coli var ian t s are still po ten t ia l pa thogens both with respect to being pr imary etio- logical agents causing frank clinical disease or rendering the immune sys tem preoccupied with such commonly occur- ring microorganisms . Such an interaction will cer ta inly c o m p r o m i s e the immune sys tem adverse ly and make the ch ickens suscept ible to other oppor tu- nistic infections.

Acknowledgements--We thank K. Gjessing, R. Hunter and J. Mason for their excellent technical assistance in this study. Secretarial assistance of M. Cummings is gratefully ac- knowledged. Supported by grants from the North Carolina Biotechnology Center (IDG 887108) and the North Carolina Agricultural Research Service (NCO6038) of North Caro- lina State University.

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interaction of escherichia coli variants with chicken mononuclear phagocytic system cells

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