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We examined the utility of infecting chicken embryos as a means of evaluating the virulence of different Francisella sp. strains and mutants. Infection of 7-day-old chicken embryos with a low dose of F. novicida or F. tularensis subsp. holarctica live vaccine strain (LVS) resulted in sustained growth for 6 days. Different doses of these two organisms were used to inoculate chicken embryos to determine the time to death. These experiments showed that wild-type F. novicida was at least 10,000-fold more virulent than the LVS strain. We also examined the virulence of several attenuated mutants of F. novicida, and they were found to have a wide range of virulence in chicken embryos. Fluorescent microscopic examination of infected chicken embryo organs revealed that F. tularensis grew in scattered foci of infections, and in all cases the F. tularensis appeared to be growing intracellularly. These results demonstrate that infection of 7-day-old chicken embryos can be used to evaluate the virulence of attenuated F. tularensis strains.
Francisella tularensis is a highly infectious, gram-negative bacterial pathogen with a 50% infectious dose of less than 10 cells for most animals (17). Although all strains are highly infectious, there is great variety in the morbidity and mortality that each strain is able to induce in different host animals. F. tularensis subsp. tularensis biovar “type A” is clearly the most virulent biovar in humans and laboratory animals. The type A biovar is found naturally only in North America, where it caused high mortality rates before the availability of antibiotics (18). In one survey 19% of tularemia patients died (12), but it is not known what proportion of these patients were infected with a biovar type A strain. Even with antibiotic treatment infection of humans with this subspecies results in some deaths. Recently, the type A biovar has been separated into two clades (24), but the relative levels of virulence of the two groups have not been studied. F. tularensis subsp. holarctica is found throughout the Northern Hemisphere. Although it is highly infectious in all of the animals that it infects and is fatal to mice (7), this subspecies rarely causes death in humans, although it can cause considerable morbidity. The live vaccine strain (LVS) of F. tularensis subsp. holarctica and F. novicida have been widely used as models of F. tularensis infection, primarily because these bacteria have low virulence in humans and can be handled in BSL2 facilities (3, 14, 16, 19). In the mouse model of infection the LVS has an intradermal 50% lethal dose (LD50) of about 3 × 105 CFU, and F. novicida has an LD50 of about 2 × 103 CFU (25); however, both strains have an intraperitoneal LD50 of less than 10 organisms. Hence, mouse infections with LVS and F. novicida may be approximations of F. tularensis type B and type A infections in humans, respectively.
F. tularensis is thought to grow primarily inside cells during infection of animals. In vitro studies of intramacrophage growth have shown that initially F. tularensis resides in a phagosome, from which it largely escapes between 2 and 4 h after cell entry (8, 20). The F. tularensis-laden phagosome has a relatively neutral pH and accumulates some markers of late endosomes, such as LAMP1 and CD63, while it excludes another late endosome marker, cathepsin D (8). Expression of the F. tularensis protein IglC is required for escape of F. tularensis from the phagosome, but its role is unknown (29). The live vaccine strain of F. tularensis has been shown to induce apoptosis in the J774 mouse macrophage cell line (26) and to inhibit secretion of tumor necrosis factor alpha and interleukin-1 (38). Although the suppression of cytokines probably represents an F. tularensis virulence strategy, the induction of apoptosis likely reflects a defensive response of the host, as caspase-1 knockout mice are more susceptible to F. tularensis infection (30).
A small number of virulence factors have been identified in F. tularensis, and most of these factors affect intramacrophage growth. Inactivation of the mglAB global regulatory genes results in strains whose growth is severely hampered in macrophages (5). Presumably, MglA and MglB are required for transcription of genes encoding effector proteins, especially genes found in the Francisella pathogenicity island (28). There is genetic evidence that the Francisella pathogenicity island-associated genes iglA, iglC, pdpA, and pdpD are required for intramacrophage growth (21, 22, 33). There is biochemical (23) and genetic (36) evidence that a capsule exists and is needed for infectivity and virulence. Defects in the production of lipopolysaccharide can affect intracellular growth (10, 31). The observed in vitro intracellular growth and the requirement for cell-mediated immunity for clearance (1, 4, 9, 14) of an F. tularensis infection suggest that intracellular growth is required for virulence in animals. The observation that mutants defective for growth in macrophages are also less virulent in animals supports this notion (31, 33).
F. tularensis infects a wide variety of animals, and several animals, including rabbits, guinea pigs, primates, hamsters, rats, and mice, have been used as models of infection (2, 14-16, 32, 34, 37). Chicken embryos have also been used to test F. tularensis virulence and pathology (6, 35). Recently, researchers have begun to use simple biological systems, such as the nematode Caenorhabditis elegans (11), flies, and insect larvae (39), to examine the virulence properties of bacterial pathogens. Such systems permit large-scale testing that is humane and relatively inexpensive. Our objective in this work was to develop an assay system that allowed us to evaluate the virulence of F. tularensis strains without having to infect animals that have fully developed nervous systems.
The F. tularensis strains used in this study are listed in Table Table1.1. All of the F. novicida strains were derived from the prototype strain U112 (= ATCC 15482), which had been passaged through a mouse and aliquoted for subsequent experiments. The LVS (ATCC 29684) was obtained from the American Type Culture Collection. Strains were grown aerobically at 37°C in either tryptic soy broth or on tryptic soy agar (TSA) supplemented with 0.1% l-cysteine.
F. tularensis strains were grown to the late log phase (optical density at 600 nm, 0.9 to 1.0) and diluted in phosphate-buffered saline (PBS) (Gibco) for injection. The inoculating dose was calculated retrospectively by determining the CFU following dilution and plating on TSA. Fertilized White Leghorn eggs were obtained from the University of Alberta Poultry Research Station. Chicken embryos were incubated at 37°C with high humidity for 7 days prior to infection and throughout the experiment were mechanically tilted to a 45° angle every 40 min. After the initial 7-day incubation the eggs were examined in order to discard those that lacked a viable embryo, a phenomenon that occurred in between 5 and 10% of the fertilized eggs. For inoculation the tops of the egg shells were disinfected with 70% ethanol. A 1-cm-diameter window was made in the air sac end of an egg, and the egg shell membrane was reflected. With a tuberculin syringe, 100 μl of inoculum was injected under the chorioallantoic membrane. After injection the shells were sealed with clear packing tape. Eggs were candled to detect signs of death every 24 h for 6 days. This process consisted of shining a focused bright light at one end of an egg to determine if the network of blood capillaries was intact. Embryos that died within 24 h of inoculation were assumed to have suffered lethal trauma during the inoculation and were removed from the experiment. For the study of the time course of F. tularensis growth, the chicken embryos were killed by incubating the eggs in a −20°C freezer for 1 h, followed by blending of the eggs and plating dilutions of the homogenized egg contents on TSA to determine the bacterial load. All experiments were terminated by the time the embryos were 14 days old. Chicken embryos that were less than 17 days old are not subject to regulatory control in many countries, including Canada.
Embryos were removed from shells and rinsed with PBS, and organs of interest were removed by dissection and fixed overnight with PBS containing 4% paraformaldehyde. The organs were rinsed with PBS, infiltrated with OCT compound (Tissue-Tek 4583), and snap frozen with liquid nitrogen, and then they were transferred to −80°C for storage. Ten-micrometer cryostatic sections were cut and mounted on gelatin-coated slides. The tissue sections were blocked with 5% lamb serum-PBS containing 0.05% Tween 20 at room temperature for 45 min. Primary antibodies were diluted 1:1,000 in 5% lamb serum-PBS and incubated overnight at 4°C with the sections. After several washes in PBS, sections were incubated with Alexa 488 or 568-conjugated (Molecular Probes) goat anti-rabbit or goat anti-mouse secondary antibodies for 2 h at room temperature, rinsed with PBS, and counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (Molecular Probes) After mounting with a coverslip, specimens were examined with a Leica DM-6000 compound microscope.
Digital images were collected, and color was added with OpenLab (version 4.04) or Scion Image (version 4.03). Figures were prepared with Photoshop (Adobe 6.0) by cropping and adjusting the brightness and contrast.
When a small inoculum of F. tularensis subsp. holarctica LVS or F. novicida was introduced into chicken embryos, exponential growth occurred for 4 to 5 days, and the concentration reached about 5 × 108 bacteria per g of egg mass (Fig. (Fig.1).1). F. novicida and LVS appeared to grow at approximately the same rate, and the concentrations leveled off at similar total numbers of bacteria by day 5 postinfection.
The wild-type strain of F. novicida is highly virulent in mice and also appears to be highly virulent in chicken embryos, and 100% lethality was observed with 30 to 200 CFU (Fig. (Fig.2).2). Infections with different doses and repetitions of experiments with different lots of eggs demonstrated that the time to death due to infection generally correlated with the infectious dose and was consistent for different experiments. Some aberrations were seen, like the results obtained with the intermediate dose given in experiment 2, and these aberrations may have been due to the natural variation in embryos or to the inexact nature of the inoculation. All of the embryos inoculated with phosphate-buffered saline lived until the termination of the experiment on day 6 postinfection (data not shown).
In the mouse F. novicida is about 100-fold more virulent than LVS when both organisms are delivered via intradermal injection. Infection of chicken embryos revealed an even larger difference in virulence between the two organisms, and F. novicida was at least 10,000-fold more lethal than F. tularensis LVS (Fig. (Fig.22 and and33).
The virulence of the strains listed in Table Table11 was evaluated by performing a series of infection experiments using different numbers of bacteria as inocula and determining the time to death of the chicken embryos. First, a pilot experiment was performed for each mutant to determine the approximate lethal dose, and then at least two repetitions of experiments were carried out using inoculum concentrations in the sublethal to lethal range. For each inoculating dose seven eggs were infected. Mutants of F. novicida U112 which had previously been shown to have defects in virulence or growth in macrophages displayed a range of virulence levels in chicken embryos. In total, the mutant strains were about 100-fold less virulent to more than 100,000-fold less virulent (Fig. (Fig.4)4) than the wild-type F. novicida U112 strain. Mutant GB2, which has a defect in the global regulator MglA, and mutant NZ9, which has a substitution in the pathogenicity island gene pdpA, are both avirulent in mice and were found to be unable to kill any of the six chicken embryos that were infected with inocula larger than 106 CFU. The previously described mutants SC66, CG57, and CG69 exhibited intermediate levels of virulence. Strain SC66, which has a defect in O-antigen production, required an inoculum that was about 100 times that of its parent strain, strain U112, to induce 100% lethality. A purA mutant, CG57, was shown to be attenuated about 10,000-fold compared to wild-type F. novicida. Finally, mutant CG69, which has an insert in the gene encoding the heat shock-induced protease ClpB, was shown to be attenuated about 106-fold compared to the wild-type strain.
Infected chicken embryos were surveyed by microscopy to localize tissues that support F. tularensis growth. Embryos that had been infected with the U112 strain or LVS were sectioned, and bacteria were visualized with strain-specific antisera or monoclonal antibodies (Fig. (Fig.55 and and6).6). Two days after infection of the embryos, the immunoreactive foci were small and widely dispersed (Fig. 6A to C). Immunoreactive material was found in all of the tissues examined, including the heart, liver, kidney, and bone marrow. For both the U112 strain and LVS immunoreactivity was found inside individual chicken cells in which distinctive granular DAPI staining dominated the cytoplasm. Four days after infection, immunoreactive foci were abundant and were present throughout embryonic tissues. Bacterial cells appeared to be contained within chicken cells, and the infected cells were typically in clusters (Fig. 5A to F and 6D to I). Often, lesions had necrotic tissue at the center that was surrounded by a ring of infected cells. In some instances the host cell nucleus was pyknotic, which is typical of an apoptotic response (Fig. (Fig.5G5G).
These studies indicate that the chicken embryo system is a useful system for evaluating the virulence of F. tularensis strains. F. tularensis grew vigorously in chicken embryos and induced death within 1 week after inoculation. The bacteria were intracellular, and the structure of the lesions suggested that the initial infections spread by localized infection of adjacent cells. The use of chicken embryo infection is not intended to replace mammalian models of tularemia, which involve complex immune responses and approximate human tularemia. However, despite its flaws, F. tularensis infection of chicken embryos provides a rapid, inexpensive test to determine differences in levels of bacterial strain virulence that does not induce pain in animals. This procedure could be combined with in vitro intracellular assays to screen mutants, allowing researchers to plan subsequent animal experiments so that the minimal number of animals is used.
This work was supported by grant 5R01 AI056215-02 from the National Institute of Allergy and Infectious Diseases.
Editor: D. L. Burns