Effect of the presence of black pigmentedBacteroides of differing pathogenicity on the phagocytosis ofEscherichia coli by rat polymorphonuclear leucocytes

An investigation was made of the ability of rat polymorphonuclear leucocytes (PMNLs) to phagocytoseEscherichia coli in the presence of two species of black pigmentedBacteroides (B. melaninogenicus andB. intermedius). When both the bacteria were opsonized together in the presence of normal rat serum,B. melaninogenicus andB. intermedius impaired the phagocytosis ofE. coli significantly. However, the phagocytosis of these black pigmentedBacteroides remained unaffected in the presence ofE. coli. The inhibition of phagocytosis was seen only after the initial first hour of incubation. The inhibition of phagocytosis ofE. coli in the presence ofB. intermedius was more than in the presence ofB. melaninogenicus. The above observation confirms the important role played by black pigmentedBacteroides in experimental mixed infections withE. coli as observed by us earlier.     

Signal Transduction during Legionella pneumophila Entry into Human Monocytes

Legionella pneumophila causes Legionnaires’ disease by replication in alveolar macrophages and monocytes. The bacteria are internalized most efficiently by opsonin-dependent, CR3-mediated phagocytosis. This investigation focused on determining the role of actin polymerization and phosphorylation signals in this uptake mechanism. Uptake inhibition assays and confocal microscopic analysis indicated that entry of L. pneumophila activated tyrosine kinase (TK) and protein kinase C (PKC) and induced actin polymerization at the site of bacterial entry. Upon L. pneumophila entry, six major cellular proteins (75, 71, 59, 56, 53, and 52 kDa) were TK phosphorylated in soluble fractions of monocytes, and three of these proteins (52, 53, and 56 kDa) were consistently found in insoluble (i.e., cytoskeletal) fractions of monocytes as well. Tyrosine phosphorylation was suppressed when cells were pretreated with the kinase inhibitor genistein, tyrphostin, or staurosporine. A similar tyrosine-phosphorylated protein pattern was observed with CR3-mediated entry of avirulent L. pneumophila, Escherichia coli, or zymosan into monocytes. This study has shown that PKC and TK signals which activate actin polymerization during the process of phagocytosis are induced upon L. pneumophila entry. In addition, CR3 receptor-mediated phagocytosis into monocytes may involve tyrosine phosphorylation of similar proteins, regardless of the particle being phagocytosed. Therefore, the tyrosine-induced phosphorylation observed during opsonized L. pneumophila entry is not a virulence-associated event.     

The use of high-resolution ¹H nuclear magnetic resonance (NMR) spectroscopy in the clinical diagnosis of Acanthamoeba

Acanthamoeba are opportunistic protozoan pathogens that can produce sight-threatening keratitis and fatal granulomatous encephalitis. The successful prognosis requires early diagnosis and differentiation of pathogenic Acanthamoeba spp. followed by aggressive treatment regimen. In this study, we tested the use of high-resolution ¹H NMR spectroscopy in the clinical diagnosis of Acanthamoeba. Using NMR spectroscopy combined with Pattern Recognition Analysis (PRA), we analysed variations in the biochemical ‘fingerprint’ of invasive and non-invasive Acanthamoeba, its closely related genus, Balamuthia mandrillaris, neuropathogenic Escherichia coli K1 strain E44, a laboratory strain of E. coli K-12, HB101, mammalian cells including human brain microvascular endothelial cells and monkey kidney cells. The findings revealed significant variations in the metabolites of amoebae, mammalian cells and bacteria. Interestingly, ¹H NMR spectra provided distinct biochemical profiles of clinical and non-clinical Acanthamoeba isolates highlighting the potential of ¹H NMR spectroscopy combined with PRA for the development of a novel diagnostic test that could rapidly identify pathogenic Acanthamoeba isolates with high sensitivity and specificity.     

Development of a fluorescence-based in vivo phagocytosis assay to measure mononuclear phagocyte system function in the rat

Abstract

The mononuclear phagocyte system (MPS) which provides protection against infection is made up of phagocytic cells that engulf and digest bacteria or other foreign substances. Suppression of the MPS may lead to decreased clearance of pathogenic microbes. Drug delivery systems and immunomodulatory therapeutics that target phagocytes have a potential to inhibit MPS function. Available methods to measure inhibition of MPS function use uptake of radioactively-labeled cells or labor-intensive semi-quantitative histologic techniques. The objective of this work was to develop a non-radioactive quantitative method to measure MPS function in vivo by administering heat-killed E. coli conjugated to a pH-sensitive fluorescent dye (Bioparticles®). Fluorescence of the Bioparticles® is increased at low pH when they are in phagocytic lysosomes. The amount of Bioparticles® phagocytosed by MPS organs in rats was determined by measuring fluorescence intensity in livers and spleens ex vivo using an IVIS® Spectrum Pre-clinical In Vivo Imaging System. Phagocytosis of the particles by peripheral blood neutrophils was measured by flow cytometry. To assess method sensitivity, compounds likely to suppress the MPS [clodronate-containing liposomes, carboxylate-modified latex particles, maleic vinyl ether (MVE) polymer] were administered to rats prior to injection of the Bioparticles®. The E. coli particles consistently co-localized with macrophage markers in the liver but not in the spleen. All of the compounds tested decreased phagocytosis in the liver, but had no consistent effects on phagocytic activity in the spleen. In addition, administration of clodronate liposomes and MVE polymer increased the percentage of peripheral blood neutrophils that phagocytosed the Bioparticles®. In conclusion, an in vivo rat model was developed that measures phagocytosis of E. coli particles in the liver and may be used to assess the impact of test compounds on MPS function. Still, the detection of inhibition of splenic macrophage function will require further assay development.     

The phagocytic capacity and immunological potency of human dendritic cells is improved by α2,6‐sialic acid deficiency

Dendritic cells (DCs) play an essential role in immunity against bacteria by phagocytosis and by eliciting adaptive immune responses. Previously, we demonstrated that human monocyte‐derived DCs (MDDCs) express a high content of cell surface α2,6‐sialylated glycans. However, the relative role of these sialylated structures in phagocytosis of bacteria has not been reported. Here, we show that treatment with a sialidase significantly improved the capacity of both immature and mature MDDCs to phagocytose Escherichia coli. Desialylated MDDCs had a significantly more mature phenotype, with higher expression of MHC molecules and interleukin (IL)‐12, tumour necrosis factor‐α, IL‐6 and IL‐10 cytokines, and nuclear factor‐κB activation. T lymphocytes primed by desialylated MDDCs expressed more interferon‐γ when compared with priming by sialylated MDDCs. Improved phagocytosis required E. coli sialic acids, indicating a mechanism of host–pathogen interaction dependent on sialic acid moieties. The DCs harvested from mice deficient in the ST6Gal.1 sialyltransferase showed improved phagocytosis capacity, demonstrating that the observed sialidase effect was a result of the removal of α2,6‐sialic acid. The phagocytosis of different pathogenic E. coli isolates was also enhanced by sialidase, which suggests that modifications on MDDC sialic acids may be considered in the development of MDDC‐based antibacterial therapies. Physiologically, our findings shed new light on mechanisms that modulate the function of both immature and mature MDDCs, in the context of host–bacteria interaction. Hence, with particular relevance to DC‐based therapies, the engineering of α2,6‐sialic acid cell surface is a novel possibility to fine tune DC phagocytosis and immunological potency.     

Effect of extracellular ionic calcium and magnesium on opsonic and non-opsonic phagocytosis of escherichia coli bovine blood polymorphonuclear leucocytes

The effects of extracellular Ca²⁺ and Mg²⁺ concentrations on opsonic and non-opsonic phagocytosis ofEscherichia coli by bovine polymorphonuclear leucocytes (PMN) isolated from blood were evaluated by flow cytometry. Eight cows were used as blood donors. The green fluorescence of blood PMN selectively gated in the forward scatter (FS) - side scatter (SS) dot plot after incubation with fluorescein isothio-cyanate (FITC) - labelledE. coli was used to characterise phagocytosis. Parameters for phagocytosis were percentage fluorescent PMN (% phagocytosis) and mean fluorescence intensity (MFI). The fluorescence of adherent bacteria was quenched with trypan blue to distinguish between adherence and ingestion. Nonopsonic and opsonic phagocytosis were decreased in the absence of extracellular ionic Ca²⁺ and Mg²⁺ compared to physiological levels. Addition of 10 him EGTA to the incubation medium was necessary to block all extracellular Ca²⁺ and resulted in a significant decrease of opsonic phagocytosis, with only 5% phagocytic PMN after quenching. Increasing Ca²⁺ concentrations resulted in a gradual increase in percentage opsonic and non-opsonic phagocytosis and in MFI for opsonic phagocytosis. Ionic calcium plays an important role in phagocytosis (attachment as well as ingestion) by bovine blood PMN in the presence of opsonins, whereas non-opsonic phagocytosis appeared to be less dependent on Ca²⁺. However, reduced serum or milk calcium levels in cows are unlikely to cause a substantial reduction of PMN phagocytosis in vivo.     

Acanthamoeba can propagate on thermophilic Sulfolobus spp.

Acanthamoeba isolation from extreme environments suggests that they may play a role in regulating archaeal densities and contribute to these ecosystems. The purpose of this study was to determine whether Acanthamoeba grow on extremophilic/mesophilic Archaea that are dominant cellular organisms in such environments. Sulfolobus solfataricus P2 and Sulfolobus shibatae were used as representative of Archaea, while Escherichia coli K-12 strain HB101 was used as a positive control for amoeba growth. Acanthamoeba castellanii were inoculated on nonnutrient agar plates containing lawns of Sulfolobus and E. coli. The cultures of Sulfolobus supported A. castellanii growth similar to E. coli K-12, HB101. Overall, the findings revealed that Acanthamoeba feed on Sulfolobus, which may explain amoebae presence in extreme environments. This feeding behavior is important as extremophilic/mesophilic Archaea are known to play a role in biogeochemical cycling of different elements in their natural habitat impacting different ecosystems.     

140-MDa plasmids of Escherichia coli and Shigella flexneri 5 do not influence phagocytosis

Enteroinvasive Escherichia coli (EIEC) and Shigella flexneri possessing a 140-megadalton (MDa) plasmid are capable of invading intestinal epithelial cells and causing dysentery. To determine if this plasmid affected phagocytosis of the organisms by leukocytes, we studied the in vitro phagocytosis of isogenic pairs of EIEC and S. flexneri 5 which differed only in the presence or absence of the 140-MDa plasmid. In addition five EIEC strains containing 140-MDa plasmids as well as one non-enteroinvasive E. coli strain possessing a 120-MDa plasmid were studied. The 140-MDa plasmid did not affect phagocytosis of these bacteria by normal human blood neutrophils or monocytes.     

An inhibitory factor for cell-free protein synthesis from Salmonella enteritidis exhibits cytopathic activity against Chinese hamster ovary cells

A factor inhibiting cell-free protein synthesis was purified from Salmonella enteritidis cell lysate by sequential ammonium sulfate precipitation, chromatography on anion exchange and hydrophobic interaction columns, and polyacrylamide disc gel electrophoresis. The purified factor, which was named SIPS (Salmonella inhibitor of protein synthesis), inhibited in vitro protein synthesis in rabbit reticulocyte lysate and had a molecular mass of 38 kDa, estimated by PAGE under denaturing conditions. SIPS was also cytopathic for Chinese hamster ovary cells. The N-terminal amino acid sequence (20 residues) of SIPS was found to be identical to that of mature L-asparaginase II of Escherichia coli. Indeed, the purified SIPS exhibited asparaginase activity, E. coli L-asparaginase II had cytopathic activity and inhibited in vitro protein synthesis. The results suggest that at least a part of cytotoxicity and inhibition of cell-free protein synthesis caused byS. enteritidis is a property of the bacterial L-asparaginase.     

Stimulating effect of C-reactive protein on phagocytosis of various species of pathogenic bacteria

In an investigation of the relationship between the presence of C-reactive protein (CRP) and the promoting effect of the phagocytosis of leucocytes in an in vitro phagocytosis system containing living pathogenic bacteria, it was found that CRP stimulated phagocytosis of Diplococcus pneumoniae, Staph. aureus, E. coli and Klebsiella aerogenes. It appears that CRP is related to non-specific immunity.     

Isolation of <Emphasis Type="BoldItalic">Acanthamoeba</Emphasis> species in surface waters of Gilan province-north of Iran

We analyzed water samples to determine the prevalence of free-living Acanthamoeba in water sources from Gilan, greater area, Iran. A total of 27 surface water samples were collected from environmental sources, including natural (rivers, lakes, springs, and lagoon) and freshwater source. The samples were filtrated and transferred to non-nutrient agar plates seeded with Escherichia coli and incubated for 2 to 7 days at 30°C or 42°C. The plates were examined by microscopy to morphologically identify Acanthamoeba species. Following DNA extraction, PCR was used to confirm the microscopically identification. A total of 19 out of 27 samples (70.3%) were positive for Acanthamoeba species based on the morphological criteria, and 14 (73.7%) were confirmed by PCR method. The high frequency of Acanthamoeba spp. in different environmental water sources of Gilan is an alert for the public health related to water sources in Iran.     

A three-domain Kazal-type serine proteinase inhibitor exhibiting domain inhibitory and bacteriostatic activities from freshwater crayfish Procambarus clarkii

In crustaceans, Kazal-type serine proteinase inhibitors in hemolymph are believed to function as regulators of the host-defense reactions or inhibitors against proteinases from microorganisms. In this study, we report a Kazal-type serine proteinase inhibitor, named hcPcSPI1, from freshwater crayfish (Procambarus clarkii). We found that hcPcSPI1 is composed of a putative signal peptide, an RGD motif, and three tandem Kazal-type domains with the domain P1 residues L, L and E, respectively. Mainly, hcPcSPI1 was detected in hemocytes as well as in the heart, gills, and intestine at both the mRNA and protein levels. Quantitative real-time PCR analysis showed that hcPcSPI1 in hemocytes was upregulated by the stimulation of Esherichia coli (8099) or became decreased after a white spot syndrome virus (WSSV) challenge. In addition, hcPcSPI1 and its three independent domains were overexpressed and purified to explore their potential functions. All four proteins inhibited subtilisin A and proteinase K, but not α-chymotypsin or trypsin. Recombinant hcPcSPI1 could firmly attach to Gram-negative bacteria E. coli and Klebsiella pneumoniae; Gram-positive bacteria Bacillus subtilis, Bacillus thuringiensis and Staphylococcus aureus; fungi Candida albicans and Saccharomyce cerevisiae, and only domain 1 was responsible for the binding to E. coli and S. aureus. In addition, recombinant hcPcSPI1 was also found to possess bacteriostatic activity against the B. subtilis and B. thuringiensis. Domains 2 and 3 contributed mainly to these bacteriostatic activities. All results suggested that hcPcSPI1 might play important roles in the innate immunity of crayfish.     

Expression and translocation of the chlamydial major outer membrane protein in Escherichia coli

The entire gene encoding the major outer membrane protein (MOMP) from Chlamydia psittaci strain GPIC has been cloned and expressed in Escherichia coli . A tightly regulated T7 promoter is used to control expression of the protein in Escherichia coli. Upon induction of expression, the precursor (pre-MOMP) is synthesized in the cell. This is followed by the appearance of a lower molecular weight protein that comigrates with mature MOMP from chlamydial elementary bodies by both one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis. When E. coli cells expressing MOMP are converted to spheroplasts and subjected to protease treatment, MOMP is quantitatively degraded while cytoplasmic pre-MOMP is protected from degradation. Whole cells subjected to the same protease treatment show no degradation of MOMP. Furthermore, MOMP is not detected in surface-labeling experiments using several MOMP-specific antibodies. These data indicate that pre-MOMP is translocated to the periplasmic space and processed but is not surface exposed in E. coli. Expression of MOMP in this system causes a significant reduction in cell viability. In addition, coexpression in E. coli of MOMP or a MOMP-PhoA fusion with various chaperone proteins does not alter the level of MOMP translocation.     

Differential Activation of Signal Transduction Pathways Mediating Phagocytosis, Oxidative Burst, and Degranulation by Chicken Heterophils in Response to Stimulation with Opsonized Salmonella enteritidis

The activation of signal transduction pathways is required for the expression of functional enhancement of cellular activities. In the present studies, initial attempts were made to identify the signal transduction factors involved in activating phagocytosis, generation of an oxidative burst, and degranulation by heterophils isolated from neonatal chickens in response to opsonized Salmonella enteritidis (opsonized SE). Peripheral blood heterophils were isolated and exposed to known inhibitors of signal transduction pathways for either 20 min (staurosporin, genistein, or verapamil) or 120 min (pertussis toxin) at 39°C. The cells were then stimulated for 30 min at 39°C with opsonized SE. Phagocytosis, luminol-dependent chemoluminescence (LDCL), and -D glucuronidase release were then evaluated in vitro. The G-protein inhibitor pertussin toxin markedly inhibited (>80%) phagocytosis of opsonized SE. Both the protein kinase inhibitor (staurosporin) and calcium channel inhibitor (verapamil) reduced phagocytosis in a dose response manner. Genistein, a tyrosine kinase inhibitor, had no effect on phagocytosis. Staurosporin had a marked inhibitory effect on LDCL (>90%) while genistein had a dose responsive inhibition on LDCL. Both verapamil (40–45%) and pertussin toxin (50–55%) had a statistically significant, but less biologically significant effect on LDCL. Genistein significantly reduced the degranulation (78–81%) of heterophils by opsonized SE. Staurosporin also reduced degranulation by 43–50%, but neither verapamil nor pertussis toxin had a significant effect on degranulation. These findings demonstrate that distinct signal transduction pathways differentially regulate the stimulation of the functional activities of avian heterophils. Pertussin toxin-sensitive, Ca⁺⁺-dependent G-proteins appear to regulate phagocytosis of opsonized SE, protein kinase C-dependent, tyrosine kinase-dependent protein phosphorylation plays a major role in LDCL, and tyrosine kinase(s)-dependent phosphorylation regulates primary granule release.     

Mechanisms associated with phagocytosis of Arcobacter butzleri by Acanthamoeba castellanii

Acanthamoeba castellanii is a free-living amoeba widely found in environmental matrices such as soil and water. Arcobacter butzleri is an emerging potential zoonotic pathogen that can be isolated from environmental water sources, where they can establish endosymbiotic relationships with amoebas. The aim of this study was to describe the implication of mannose-binding proteins and membrane-associated receptors of glucose and galactose present in the amoebic membrane, during the attachment of Arcobacter butzleri by blocking with different saccharides. Another objective was to describe the signaling pathways involved in phagocytosis of these bacteria using specific inhibitors and analyze the implication of phagolysosome formation on the survival of Arcobacter butzleri inside the amoeba. We infer that the attachment of Arcobacter butzleri to the amoeba is a process which involves the participation of mannose-binding proteins and membrane-associated receptors of glucose and galactose present in the amoeba. We also demonstrated an active role of protozoan actin polymerization in the phagocytosis of Arcobacter butzleri and a critical involvement of PI3K and RhoA pathways. Further, we demonstrated that the tyrosine kinase-induced actin polymerization signal is essential in Acanthamoeba-mediated bacterial uptake. Through phagolysosomal formation analysis, we conclude that the survival of Arcobacter butzleri inside the amoeba could be related with the ability to remain inside vacuoles not fused with lysosomes, or with the ability to retard the fusion between these structures. All these results help the understanding of the bacterial uptake mechanisms used by Acanthamoeba castellanii and contribute to evidence of the survival mechanisms of Arcobacter butzleri.     

Infection of dendritic cells by enterobacteriaceae

Dendritic cells (DC) are potent antigen-presenting cells that play a crucial role in initiation and modulation of specific immune responses. Various pathogens like viruses or bacteria are able to persist inside DC. In this study we investigated the ability of the Gram-negative bacteria Salmonella typhimurium and Escherichia coli to infect DC. DC isolated from peripheral blood of healthy donors were infected with wild-type S. typhimurium and a nonpathogenic E. coli stool isolate. Association of bacteria with DC was assessed by labeling of the bacteria with green fluorescent protein. Both Gram-negative bacteria were associated with DC as evidenced by microscopy and flow cytometry. The intracellular location could be confirmed by lysis of DC and subsequent determination of colony-forming units on agar plates, which showed a rapid decline in viable Gram-negative bacteria 6 h after infection, being by far more pronounced for E. coli than for S. typhimurium. Testing the stimulation of T cells by infected versus uninfected but otherwise identically treated human immature DC in a mitogen-dependent T cell proliferation assay, we found that S. typhimurium, but not E. coli exhibited a suppressive effect on T cell stimulation, being most significant on days 3–5 after infection. Thus, suppression of dendritic cell function was associated with an enteropathogenic bacterium, S. typhimurium, which can cause severe forms of enteritis. The bacteria with normally mild or no gastric symptoms, E. coli, had no influence on stimulation of T cells by DC. Received: 12 January 2000     

Complement component 5 does not interfere with physiological hemostasis but is essential for Escherichia coli-induced coagulation accompanied by Toll-like receptor 4

There is a close cross-talk between complement, Toll-like receptors (TLRs) and coagulation. The role of the central complement component 5 (C5) in physiological and pathophysiological hemostasis has not, however, been fully elucidated. This study examined the effects of C5 in normal hemostasis and in Escherichia coli-induced coagulation and tissue factor (TF) up-regulation. Fresh whole blood obtained from six healthy donors and one C5-deficient individual (C5D) was anti-coagulated with the thrombin inhibitor lepirudin. Blood was incubated with or without E. coli in the presence of the C5 inhibitor eculizumab, a blocking anti-CD14 monoclonal antibody (anti-CD14) or the TLR-4 inhibitor eritoran. C5D blood was reconstituted with purified human C5. TF mRNA was measured by quantitative polymerase chain reaction (qPCR) and monocyte TF and CD11b surface expression by flow cytometry. Prothrombin fragment 1+2 (PTF1·2) in plasma and microparticles exposing TF (TF-MP) was measured by enzyme-linked immunosorbent assay (ELISA). Coagulation kinetics were analyzed by rotational thromboelastometry and platelet function by PFA-200. Normal blood with eculizumab as well as C5D blood with or without reconstitution with C5 displayed completely normal biochemical hemostatic patterns. In contrast, E. coli-induced TF mRNA and TF-MP were significantly reduced by C5 inhibition. C5 inhibition combined with anti-CD14 or eritoran completely inhibited the E. coli-induced monocyte TF, TF-MP and plasma PTF1·2. Addition of C5a alone did not induce TF expression on monocytes. In conclusion, C5 showed no impact on physiological hemostasis, but substantially contributed to E. coli-induced procoagulant events, which were abolished by the combined inhibition of C5 and CD14 or TLR-4.     

Membrane-bound proteinase 3 and PAR2 mediate phagocytosis of non-opsonized bacteria in human neutrophils

The molecular mechanisms underlying the non-opsonic phagocytosis of bacteria by neutrophils are poorly understood. We previously reported the efficient uptake of Streptococcus sanguinis by human neutrophils in the absence of opsonins. To characterize the phagocytosis receptor, protein lysates from neutrophils and HL-60 cells were subjected to affinity chromatography using epoxy beads coated with S. sanguinis. Denaturing electrophoresis of the eluted proteins and subsequent mass spectrometry revealed that one of the proteins eluted from neutrophils was proteinase 3 (PR3). Enzymatic cleavage of the glycosylphosphatidylinositol linker of NB1, a co-receptor for membrane-bound PR3 (mPR3), significantly reduced the phagocytosis of S. sanguinis. In addition, the neutralization of mPR3 with antibody reduced both binding and phagocytosis of S. sanguinis. Treatment of neutrophils with a serine proteinase inhibitor indicated that protease activity is required for phagocytosis. Thus, we studied whether protease-activated receptor 2 (PAR2) is involved in signal transmission from mPR3 during this process. Indeed, neutralizing antibodies against PAR2 inhibited phagocytosis and S. sanguinis-induced calcium mobilization desensitized PAR2. Furthermore, the phagocytosis of S. sanguinis and the concomitant activation of Rho family GTPases were inhibited by the intracellular calcium chelator, BAPTA-AM. Collectively, mPR3 acts as a non-opsonic phagocytosis receptor for bacteria probably by activating PAR2 in neutrophils.     

Phagocytic and oxidative burst activity of chicken thrombocytes to Salmonella,Escherichia coli and other bacteria

The potential role of chicken thrombocytes in immune responses to Salmonella, Escherichia coli and other bacteria was investigated by in vitro assays of phagocytosis and respiratory burst activity. Thrombocytes were found to phagocytose bacteria, but were found to be less phagocytic than heterophils. Oxidative burst activity was generated upon challenge of thrombocytes with various Salmonella strains, E. coli, three other bacterial species, and zymosan A. These findings indicate that thrombocytes may play a role in innate immunity to bacteria in the chicken.