The NOD-scid IL2rγnull Mouse Model Is Suitable for the Study of Osteoarticular Brucellosis and Vaccine Safety
ABSTRACT
Osteoarticular brucellosis is the most common complication in Brucella-infected humans regardless of age, sex, or immune status. The mechanism of bone destruction caused by Brucella species remained partially unknown due to the lack of a suitable animal model. Here, to study this complication, we explored the suitability of the use of the NOD-scid IL2rγnull mouse to study osteoarticular brucellosis and examined the potential use of this strain to evaluate the safety of live attenuated vaccine candidates. Mice were inoculated intraperitoneally with a single dose of 1 × 104, 1 × 105, or 1 × 106 CFU of B. abortus S19 or the vaccine candidate B. abortus S19ΔvjbR and monitored for the development of side effects, including osteoarticular disease, for 13 weeks. Decreased body temperature, weight loss, splenomegaly, and deformation of the tails were observed in mice inoculated with B. abortus S19 but not in those inoculated with S19ΔvjbR. Histologically, all S19-inoculated mice had a severe dose-dependent inflammatory response in multiple organs. The inflammatory response at the tail was characterized by the recruitment of large numbers of neutrophils, macrophages, and osteoclasts with marked bone destruction. These lesions histologically resembled what is typically observed in Brucella-infected patients. In contrast, mice inoculated with B. abortus S19ΔvjbR did not show significant bone changes. Immunofluorescence, in situ hybridization, and confocal imaging demonstrated the presence of Brucella at the sites of inflammation, both intra- and extracellularly, and large numbers of bacteria were observed within mature osteoclasts. These results demonstrate the potential use of the NOD-scid IL2rγnull mouse model to evaluate vaccine safety and further study osteoarticular brucellosis.
INTRODUCTION
Brucellosis is a zoonotic disease caused by Gram-negative facultative intracellular bacteria of the genus Brucella (1). Among the 12 species currently recognized, B. abortus (cattle), B. melitensis (goats and sheep), and B. suis (swine) are the most pathogenic to humans, with more than half a million new cases of human brucellosis reported annually (2). In animals, brucellosis is usually manifested as infertility and abortions in females and as epididymitis in males (3, 4). In humans, acute brucellosis is frequently associated with nonspecific clinical signs, including undulant fever, headache, sweating, and joint pain requiring long-term antibiotic treatment (5). Clinical signs of chronic infection include endocarditis, orchitis, neurological disorders, and hepatitis (6, 7). Infection in humans occurs through contact with infected animals and animal products (8) with a very low number (10 to 100 CFU) of microorganisms required to induce infection (9). It is well accepted that vaccination is one of the most effective approaches to control brucellosis in animals (10). However, currently available vaccines are not suitable for human use and can induce undesirable side effects in livestock, including residual virulence in pregnant animals leading to abortion and shedding of the organism in milk (11). Osteoarticular brucellosis in humans has been reported in 40 to 80% of infected patients and is the most common complication of Brucella infection (12–14). Peripheral arthritis, sacroiliitis, and spondylitis are the three most commonly reported predilection sites (13, 15), and people of all ages are susceptible in both acute and chronic cases (16). The mechanism driving osteoarticular tropism by Brucella species remains unknown, due in part to the lack for many years of an animal model to study Brucella-induced osteoarticular disease. Immunocompetent mice have proven to be a valuable tool for understanding basic host-agent interactions during Brucella infection (17, 18). However, arthritis is not typically manifested in this host species, and in the few cases observed, osteoarticular damage can take up to 6 months to develop, making it very challenging to use these mice to study this process (17). Recently, immunocompromised mice, including gamma interferon-, CXCR2-, and interleukin-1R-deficient (IFN-γ−/−, CXCR2−/−, and IL-1R−/−, respectively) mice have been used to study osteoarticular brucellosis (19, 20). However, only wild-type B. melitensis 16M and B. abortus 2308 were able to induce pathological changes. Although, RB51 is known to cause infection in humans (11), development of osteoarticular disease was not evident using these models (20). Therefore, an animal model that is highly sensitive to developing osteoarticular disease would be invaluable to screen potential side effects associated with live attenuated vaccines.
The aim of this study was to investigate the suitability of the NOD.Cg-PrkdcscidIl2rγtm1Wjl/SzJ (NOD-scid IL2rγnull, or NSG) mouse as an improved method to further evaluate the safety of potential vaccine candidates. NSG mice are characterized by the lack of functional mature T and B lymphocytes, natural killer cells, and dendritic cells, by undetectable hemolytic complement activity, and by severe impairment in cytokine signaling due to a defect in the IL-2 receptor gamma chain (21), which makes them highly susceptible to infection by Brucella spp. Further, NSG mice can also be used as a humanized mouse model (21–23) to study host-pathogen interactions and mechanisms of disease following human cell transplantation. For this study, we utilized the vaccine candidate B. abortus S19ΔvjbR that was designed in our laboratories by deletion of the LuxR-like transcriptional regulator vjbR gene, which is essential for intracellular survival and virulence in mice (24–26). Prior in vitro and in vivo studies have noted that B. abortus S19ΔvjbR is highly attenuated and confers a significant level of immune protection (24, 25). In the current study, we have investigated NSG mice as a model to determine if there are any potential side effects associated with vaccination when live attenuated vaccine candidates are used in immunocompromised individuals as in pregnancy, chronic infections, childhood, and aging.
RESULTS
Clinical manifestation and survival of NSG mice infected with B. abortus S19 and B. abortus S19ΔvjbR.
NSG mice were inoculated intraperitoneally (i.p.) with different doses ranging from 1 × 104 to 1 × 106 CFU/mouse of (i) B. abortus S19, (ii) B. abortus S19ΔvjbR, or (iii) phosphate-buffered saline (PBS) alone. All animals were monitored daily for the development of any adverse effects associated with vaccination (Table 1). Interestingly, only mice infected with S19 developed clinical signs that started approximately 7 weeks postinoculation, and these consisted of hunched posture, ruffled coat, malaise, hypothermia, and weight loss. By 10 weeks (73 days) postinoculation, 40% of the mice infected with 1 × 104 and 1 × 106 CFU/mouse of B. abortus S19 were euthanized due to the severity of clinical signs (Fig. 1), with the remaining 60% of mice euthanized at week 13 (90 days) and week 12 (86 days) postinoculation, respectively. All mice receiving 1 × 105 CFU/mouse of B. abortus S19 required euthanasia at week 12 (87 days) postinoculation. Interestingly, none of the mice inoculated with B. abortus S19ΔvjbR developed clinical signs (P < 0.001) and exhibited a 100% survival rate (Fig. 1). These results confirm that B. abortus S19ΔvjbR is safer than B. abortus S19 in NSG immunocompromised mice.
TABLE 1
| Parameter | Value for the group | |||
|---|---|---|---|---|
| Naive mouse | B. abortus S19-inoculated mouse | B. abortus S19ΔvjbR-inoculated mouse | ||
| Body temperature | Normal | Hypothermia | Normal | |
| Change in body weight | Gain | Loss | Gain | |
| Presence of splenomegaly | No | Yes | No | |
| Bacterial load | None | High | Low | |
| Osteoarticular changes | None | Major | Minor | |
| Splenic changes | None | Major | Minor | |
| Hepatic changes | None | Major | Minor | |
FIG 1
NSG mice inoculated with B. abortus S19 but not with B. abortus S19ΔvjbR developed hypothermia.
To determine whether inoculation with B. abortus S19 and B. abortus S19ΔvjbR induced body temperature fluctuations in NSG mice as a correlate of the severity of infection, mice were monitored daily for the duration of the experiment. Basal preinoculation body temperatures ranged from 36.3°C to 37.8°C. Mice inoculated with B. abortus S19 developed significant hypothermia from week 7 onwards (P < 0.001) compared to the basal level, and this was used as an indicator for early euthanasia (Fig. 2A to C). Mice inoculated with B. abortus S19ΔvjbR (Fig. 2D to F) or PBS (Fig. 2G) did not show any significant changes in body temperature throughout the study. Similar results have been previously observed in IRF-1−/− mice inoculated with B. abortus S19ΔvjbR (27).
FIG 2
NSG mice inoculated with B. abortus S19 but not with B. abortus S19ΔvjbR demonstrated weight loss.
Body weight was monitored daily for the duration of the experiment. Except for one animal from a group inoculated with a dose of 104 CFU, all mice inoculated with B. abortus S19 at 1 × 105 and 1 × 106 CFU/mouse exhibited a substantial decrease in body weight (Fig. 3A to C) in a dose-dependent manner, and this was associated with high bacterial colonization (P < 0.05). No significant differences in body weights were noted for mice inoculated with B. abortus S19 at 1 × 104 CFU/mouse, B. abortus S19ΔvjbR, or the control group (Fig. 3D to G).
FIG 3
B. abortus S19-inoculated NSG mice depicted high bacterial colonization and inflammatory response.
The extent of bacterial colonization of different organs was evaluated to determine if the degree of colonization was correlated with the manifestation of clinical signs as well as the induction of an inflammatory response. Spleens, livers, and lungs were collected at different time points, weighed, and homogenized in 1 ml of PBS, and bacterial CFU counts were determined by plating samples onto tryptic soy agar (TSA) medium. Mice inoculated with B. abortus S19, regardless of the dose, exhibited an overwhelming bacterial burden in the spleen, liver, and lung. In contrast, mice inoculated with S19ΔvjbR exhibited significantly less bacterial growth (P < 0.05) in the spleen (except at the dose of 104 CFU/mouse), liver, and lung (Fig. 4C to E). Mice inoculated with B. abortus S19 also developed splenomegaly with a significant increase in spleen weight (P < 0.05), whereas mice inoculated either with B. abortus S19ΔvjbR or PBS had no change in splenic weight (Fig. 4A). None of the groups showed a significant difference in liver weights (Fig. 4B).
FIG 4
Evaluation of pathological changes in NSG mice following Brucella inoculation.
To determine the extent of any gross and histologic changes, a full postmortem examination was performed in all Brucella-inoculated mice. Gross changes were most prominent in the tail, spleen, and liver of mice inoculated with B. abortus S19. B. abortus S19-inoculated mice developed swelling and deviation of the tail vertebrae (Fig. 5A). The spleens were markedly pale and enlarged, and the livers were pale. Interestingly, NSG mice inoculated with S19ΔvjbR or PBS did not show any apparent gross changes. Histologically, all mice inoculated with B. abortus S19 demonstrated severe inflammation of the tail in a dose-dependent manner characterized by the presence of large numbers of macrophages and neutrophils, massive bone resorption, and intervertebral disk erosion with fibrosis (Fig. 5B, bottom panel). As expected, due to the immunocompromised status of NSG mice, the spleens of the PBS-inoculated group did not exhibit normal architecture or well-defined white and red pulp with lack of lymphoid follicle formation (Fig. 6A). Interestingly, the spleens from the B. abortus S19-inoculated group had a marked histiocytic and granulomatous splenitis (Fig. 6A, bottom panel) associated with bacterial colonization (Fig. 6B, bottom panel). Livers exhibited a multifocal, random histiocytic hepatitis with the presence of microgranulomas (Fig. 7A, bottom panel) associated with bacterial colonization (Fig. 7B, bottom panel). In contrast, mice inoculated with B. abortus S19ΔvjbR did not show any significant changes in bone pathology, except with the presence of scattered neutrophils observed in the medullary cavity (Fig. 5B, middle panel). The spleens of the S19ΔvjbR-inoculated group exhibited mild neutrophilic infiltration (Fig. 6A) associated with the presence of bacterial colonies (Fig. 6B) but in smaller numbers than in the B. abortus S19-inoculated group. No histologic changes were observed in the liver of S19ΔvjbR-inoculated mice (Fig. 7A, middle panel).
FIG 5
FIG 6
FIG 7
Identification of Brucella in NSG mice tissues via IHC, IF, and FISH techniques.
In order to identify the distribution of Brucella antigen in different tissues, immunohistochemical (IHC) analysis was performed using bright-field (Fig. 8A) and fluorescence microscopy (Fig. 8B). Formalin-fixed paraffin-embedded tissue sections (FFPE) were immunolabeled with polyclonal rabbit anti-Brucella abortus primary antibody. The tail vertebrae from mice inoculated with B. abortus S19 demonstrated positive, strong immunostaining scattered throughout the section. Distribution of Brucella antigen in the medullary cavity, subchondral bone, and within necrotic areas was evident (Fig. 8A, bottom panel). In contrast, only one of five mice inoculated with 1 ×106 CFU/mouse of B. abortus S19ΔvjbR demonstrated a weak positive signal of Brucella antigen in the tail vertebrae, while the remaining mice had no detectable immunoreactive signal (Fig. 8A, middle panel). As an alternative to increased sensitivity and specificity of IHC, we performed immunofluorescence (IF) staining (28) of Brucella antigen that further supported Brucella distribution of antigens in the tail of S19- or S19ΔvjbR-inoculated mice (Fig. 8B, middle and bottom panels). In addition, fluorescence in situ hybridization (FISH) using a Bru-996 Alexa-Fluor 555-labeled DNA probe that specifically hybridizes Brucella 16S rRNA confirmed B. abortus S19 distribution within the lesions (Fig. 8C).
FIG 8
B. abortus S19 colonizes osteoclasts in NSG mice.
IHC and IF staining demonstrated the abundance of Brucella antigen in multinucleated osteoclasts (arrow) and, to a lesser degree, in other cell types as well as in extracellular space surrounding the affected area in B. abortus S19-inoculated mice (Fig. 8A and B, bottom panels). To further confirm the association of Brucella for osteoclasts, double immunofluorescence staining (29) was performed on paraffin sections of tail vertebrae to simultaneously identify osteoclasts and Brucella. As osteoclasts express a high level of the enzyme cathepsin K (30), a polyclonal rabbit anti-cathepsin K primary antibody was used to identify osteoclasts along with a polyclonal rabbit antibody against Brucella for Brucella identification. Using fluorescently labeled secondary antibodies, we demonstrated that Brucella colocalized with osteoclasts (Fig. 9A). The colocalization of Brucella in different depths of osteoclasts was analyzed by using confocal microscopy. Confocal images of Z sectioning demonstrated Brucella distribution at various cellular depths inside these cells (Fig. 9B and Movie S1 in the supplemental material). The right panel indicates the color intensity plot of Brucella distribution throughout the section. Quantitative analysis of the different depths of Z sections revealed a maximum density of Brucella in the center of osteoclasts (Fig. 9C). When we compared fluorescence intensities, B. abortus S19ΔvjbR signal was significantly less (P < 0.001) than that of B. abortus S19 (Fig. 9D).
FIG 9
DISCUSSION
Brucellosis is one of the most common worldwide bacterial zoonotic diseases during which osteoarticular complications may arise in up to 80% of human cases (12, 13). The study of Brucella-induced osteoarthritis has been challenging due to the lack of a suitable laboratory animal model as well as the impracticality of using natural hosts. Although few mouse models (IFN-γ−/−, CXCR2−/−, and IL-1R−/−) have been used to understand the mechanism of osteoarticular brucellosis, they required either a virulent biosafety level 3 (BSL3) Brucella strain to induce detectable bone damage or injection directly into the joints (17, 19, 20). The development and characterization of an NSG immunocompromised mouse bearing a mutated interleukin-2 receptor gamma chain (IL2rγnull) have facilitated its use as a humanized mouse model to study human hemopoietic stem cell engraftment and infectious diseases caused by Epstein-Barr virus, dengue virus, Salmonella enterica serovar Typhi, and Plasmodium falciparum (22, 31–36). In the present study, we sought to investigate the potential of using an NSG mouse model as a means to study the side effects (if any) associated with vaccination and its use to study osteoarticular brucellosis. The currently available B. abortus S19 vaccine, which is essential for the control of bovine brucellosis, is a live attenuated vaccine that is not suitable for use in humans (37) due to its known side effects. Very recently, Xie et al. in their meta-analysis have clearly shown various adverse effects associated with the existing licensed Brucella vaccines, including Brucella abortus S19, Brucella melitensis Rev1, and Brucella abortus RB51. Some of these adverse effects associated with Brucella abortus S19 vaccination include arthropathy, arthralgia, and myalgia (38). As a control, we utilized B. abortus S19 vaccine to compare with our vaccine candidate, B. abortus S19ΔvjbR. The present study demonstrated that within 13 weeks of B. abortus S19 vaccine strain inoculation, NSG immunocompromised mice had to be euthanized due to overwhelming infection. Mice developed gross and histopathological changes and osteoarticular lesions resembling chronic human brucellosis, making this model suitable to study not only vaccine safety but also osteoarticular brucellosis. NSG mice inoculated with B. abortus S19 displayed signs of illness, a low survival rate, splenomegaly, and high bacterial loads in the spleen, liver, and lung. It is well known that Brucella has a tropism to reticuloendothelial tissues such as spleen and liver (39). The high bacterial colonization was associated with marked histiocytic and neutrophilic inflammation, which is typically observed in patients with brucellosis (40–45). In contrast, NSG mice inoculated with the vaccine candidate B. abortus S19ΔvjbR exhibited milder clinical and pathological changes associated with Brucella infection, indicating that the B. abortus S19ΔvjbR vaccine seems to be a safer choice. These results support our previous findings that demonstrated the safety of ΔvjbR mutants in wild-type and IRF-1−/− mice (24, 25, 27). The previous study showed that NSG mice are not capable of inducing an inflammatory immune reaction against infectious agents (46). Interestingly, in spite of having decreased immunity, NSG mice were capable of mounting inflammation against Brucella infection in this study. This may be attributed to the presence of neutrophils, monocytes, macrophages, and dendritic cells in NSG mice (21). Taken together, these observations suggest that the NSG mouse model might be a more sensitive predictor of vaccine safety for brucellosis, especially in immunodeficient individuals. The most frequent clinical sign associated with brucellosis in humans is undulant fever and weight loss (3, 5). Fever or hyperthermia is a physiological response to an inflammatory process and infection that aims to enhance host survival (47). In this study, NSG mice inoculated with B. abortus S19 showed a decrease in body temperature or hypothermia, which is considered a sign of septicemia and poor health (48–50). In mice, interestingly, the vaccine strain B. abortus S19ΔvjbR did not induce hypothermia in NSG mice, a result that is similar to what we have previously reported (27). While normal weight gain is considered a sign of the healthy animal, weight loss is considered a sign of disease (3, 51). In this study, weight loss was recorded in the B. abortus S19-infected NSG mice in a dose-dependent manner; however, this was not the case in NSG mice infected with the B. abortus S19ΔvjbR vaccine candidate. This corroborates the observation that the B. abortus S19ΔvjbR vaccine seems to be safer than the B. abortus S19 vaccine.
Once the clinical signs of brucellosis were characterized, we focused our attention to the bone lesions as well as immunolocalization of bacterial antigen in the affected areas (52). Although a recent report demonstrated that NSG mice have a mild reduction in trabecular bone mass, they do not display any apparent abnormalities in bone development or bone homeostasis (53). Grossly and histologically, we found that the bones in NSG mice are normal despite their immune status. In the present study, NSG mice infected with B. abortus S19 developed severe diskospondylitis, which is a common finding in chronically infected Brucella patients (52). Importantly, when animals were inoculated with the vaccine candidate B. abortus S19ΔvjbR, no arthritic lesions were observed. Importantly, Brucella antigen distribution as demonstrated by immunohistochemistry, immunofluorescence, and FISH techniques revealed a direct relationship between bacterial virulence and pathological changes in the affected tissues. This suggests that the severity of bone damage is dependent not only on the inflammatory cell response but also the concentration of Brucella antigen. Indeed, B. abortus S19-infected NSG mice showed a dose-dependent increase in antigen accumulations and damage in the tail.
Brucella as an intracellular pathogen resides inside phagocytic and nonphagocytic cells (54). Confocal microscopy revealed that although B. abortus S19 was located both extracellularly and intracellularly, large numbers of bacteria were observed inside mature osteoclasts at different cellular depths. Osteoclasts are multinucleated bone-resorbing cells that differentiate from monocyte/macrophage lineage under the effect of two osteoclastogenic cytokines that are required for their survival and differentiation: receptor activator of nuclear factor κB (NF-κB) ligand (RANKL) and macrophage colony-stimulating factor (MCSF) (55–58). Osteoclasts degrade bone matrix through secretion of several osteolytic enzymes and acids that solubilize bone components (59, 60), and previous studies have reported that activated osteoclasts play a pivotal role in bone destruction, such as an inflammatory bone loss and rheumatoid arthritis (61). In light of the significant bone destruction observed in NSG mice, it is possible that Brucella may use these cells as a replicative niche to spread or sustain the infection. Therefore, future studies are required to investigate whether mature osteoclasts are involved in Brucella infection and therefore the progression of Brucella-induced bone destruction.
Collectively, our results revealed that NSG mice can be used as a more sensitive tool to study potential side effects associated with vaccination of live attenuated vaccine candidates. Interestingly, we observed that it could be used not only to study a potential side effect of vaccination including osteoarticular disease but also, most importantly, as a tool to understand the host-antigen interaction that can cause bone damage. While mice inoculated with B. abortus S19 developed symptoms of brucellosis, S19ΔvjbR-inoculated mice did not show significant clinical changes, supporting the safety of the S19ΔvjbR vaccine candidate.
MATERIALS AND METHODS
Bacterial strains.
Strains used in this experiment included B. abortus S19 (National Veterinary Services Laboratory [NVSL], Ames, IA) and B. abortus S19ΔvjbR (engineered for a previous study) (24). Tryptic soy agar (TSA; Difco, Becton, Dickinson) was used to grow the bacteria at 37°C with 5% (vol/vol) CO2 for 3 days. Bacteria were harvested from the plates using phosphate-buffered saline (PBS), pH 7.2 (Gibco), and adjusted to a final concentration of either 1 × 104, 1 × 105, or 1 × 106 CFU/0.1 ml/mouse (i.p.) based on a Klett colorimeter meter reading against a standard curve. Viable counts were retrospectively confirmed by serial dilution of Brucella and plating onto TSA medium.
Animal resource, housing, and care.
Six- to 8-week-old female NOD.Cg-PrkdcscidIl2rγtm1Wjl/SzJ (NOD-scid IL2rγnull [NSG]) mice were obtained from The Jackson Laboratory (Bar Harbor, ME). Mice were housed under specific-pathogen-free conditions and acclimated for 2 weeks prior to bacterial inoculation. All experimental procedures and animal care were performed in compliance with the institutional animal care guidelines.
Measurement of body temperature and body weight.
Body temperature was measured as described previously (27). Briefly, implantable temperature transponders (IPTT-300) and a handheld reader (DAS-7007; Bio Medic Data Systems, DE) were used according to the manufacturer’s instructions. Before bacterial inoculation, the transponders were placed subcutaneously on the left lateral side of the flank using an insertion device. The handheld reader was used to measure the body temperature by putting the detector 5 to 10 cm from the implanted chip site. The basal body temperature and body weight were recorded for each mouse for 3 days prior to inoculation and daily for the duration of the experiment.
Evaluation of virulence of B. abortus S19 and B. abortus S19ΔvjbR in NSG mice.
Six groups of mice (n = 5) were inoculated intraperitoneally (i.p.) with (i) B. abortus S19, (ii) B. abortus S19ΔvjbR, or (iii) PBS alone. Each group was given one of three different doses: 1 × 104, 1 × 105, and 1 × 106 CFU/mouse. Mice were monitored daily for any clinical signs associated with inoculation. Animals showing abnormal posture, reduced mobility, ruffled coat, or a body temperature below 32°C were immediately euthanized via carbon dioxide asphyxiation. At 13 weeks postinoculation, all animals that survived were euthanized to determine bacterial burden and associated gross and histopathological findings. Spleens, livers, and lungs were collected, weighed, and mechanically homogenized in 1 ml of PBS to determine the number of CFU/organ. A total of 100 μl of a serial dilution of tissue homogenate was plated in duplicate on TSA medium and incubated at 37°C for 3 days to quantify bacterial CFU.
Evaluation of histopathological changes in NSG mice inoculated with B. abortus S19 and B. abortus S19ΔvjbR.
Multiple tissues were evaluated to determine histopathological changes. Spleen, liver, and bone tissues were collected and fixed in 10% neutral buffered formalin for 5 days and then stored in 70% ethanol. Bone specimens were decalcified in 15% formic acid for approximately 3 days. Tissues were rinsed in tap water, gradually dehydrated in alcohol, cleared in xylene, and embedded in paraffin. Paraffin sections (5 μm) were stained with hematoxylin and eosin (H&E). Histopathological changes between groups were evaluated by a board-certified veterinary pathologist.
Immunohistochemical detection of B. abortus in tissue sections.
Immunohistochemistry (IHC) was performed on 5-μm-thick formalin-fixed paraffin-embedded (FFPE) tissue sections from different experimental groups. Following deparaffinization in xylene, sections were rehydrated in multiple serial dilutions of ethanol (100 to 50%) and washed in distilled water. Antigen recovery was performed using sodium citrate (pH 6) at 80°C in a water bath for 10 min for bone sections and for 20 min for the spleen and liver. Endogenous peroxidase removal was performed using Bloxall (SP-6000; Vector Laboratories, USA) for 10 min, followed by blocking of nonspecific binding using normal goat serum for 20 min at room temperature. Blocked sections were incubated with polyclonal rabbit anti-Brucella abortus primary antibody (1:4,000) (bs-2229R; Bioss Antibodies, USA) diluted in PBS (pH 7.4) for 60 min at room temperature. Following 5 min of washing in PBS-Tween 20 (PBST) (pH 7.4, 0.05% Tween 20 in 10 mM PBS), biotinylated goat anti-rabbit secondary antibody was added to the sections and incubated for 30 min at room temperature. Sections were then rinsed in PBST (pH 7.4) twice for 5 min each, followed by incubation with avidin-biotin complex for 30 min at room temperature (Vectastain ABC kit; Vector Laboratories, USA), according to the manufacturer’s instructions. The color was developed using diaminobenzidine (DAB) substrate (BDB2004; Biocare Medical, USA) at room temperature for 3 min, and samples were rinsed with distilled water and counterstained with hematoxylin (Sigma-Aldrich) for 1 min. Stained tissue sections were mounted with aqueous mounting medium and imaged using a bright-field microscope.
Immunofluorescence and confocal microscopic analysis of B. abortus in tissue sections.
Formalin-fixed paraffin-embedded tissue sections (5 μm) of mice from different treatment groups were processed as described above. Following a 1-h incubation with polyclonal rabbit anti-Brucella abortus primary antibody (1:1,000) (bs-2229R; Bioss Antibodies, USA) at room temperature, fluorescently tagged goat anti-rabbit IgG(H+L) Alexa Fluor 488 (ab150077; abcam, USA) secondary antibody (1:2,000) was added to stain Brucella. At the same time, Texas Red X-phalloidin (T7471; Thermo Fisher Scientific, USA) was added (1:300) to stain F-actin and incubated overnight at 4°C. Mounting medium with 4,,6, -diamidino-2-phenylindole (DAPI) was added for nuclear staining, and the slides were analyzed by an Olympus microscope DP73 and Zeiss LSM 780 confocal microscope. Confocal Z images were collected at a 0.25-μm gap on a Zeiss LSM 780 confocal microscope equipped with a Plan-Apo 40×/1.40 numerical aperture (NA) oil objective. Image processing and quantitative data analysis were performed using Fiji.
Double immunofluorescence staining was also performed using polyclonal rabbit anti-cathepsin K antibody as a marker to identify osteoclasts in the tail vertebral tissue sections of mice inoculated with B. abortus S19. Formalin-fixed paraffin-embedded sections were deparaffinized in xylene, rehydrated with a serial dilution of ethanol alcohol (100 to 50%), and washed with distilled water. Antigen retrieval was achieved for 10 min using sodium citrate (pH 6) at 80°C in a water bath. Tissue sections were then rinsed with PBS-Tween 20 (PBST) (pH 7.4, 0.05% Tween 20) twice for 5 min between each step. Normal goat serum was added for 20 min to block nonspecific binding. Polyclonal rabbit anti-mouse cathepsin K antibody (IgG isotype, ab19027; Abcam, USA) (1:200) diluted in PBS (pH 7.4) was applied overnight at 4°C. The slides were then washed with PBST twice for 5 min. Tissue sections then incubated with goat anti-rabbit IgG(H+L)-Alexa Fluor 555 (ab150078; Abcam, USA) fluorescent secondary antibody (1:2,000) diluted in PBS (pH 7.4) for 30 min at room temperature. To stain B. abortus antigen on the same sections using polyclonal rabbit anti-Brucella abortus antibody, the nonspecific binding step was repeated by adding normal goat serum, followed by incubation for 30 min at room temperature; then IF steps similar to those described above to identify B. abortus antigen were exactly followed, and the slides were analyzed by an Olympus microscope DP73 using DAPI, fluorescein isothiocyanate (FITC), and tetramethyl rhodamine isocyanate (TRITC) filters and Fiji software.
FISH to detect B. abortus S19 and B. abortus S19ΔvjbR.
Fluorescence in situ hybridization (FISH) was performed on 4-μm-thick paraffin-embedded sections of tail vertebral tissue collected from mice inoculated with Brucella abortus S19 and Brucella abortus S19ΔvjbR as described previously (62). Tissue sections were deparaffinized in xylene, rehydrated in serial dilution of ethanol alcohol (100 to 70%), and washed in distilled water. Once the tissue sections were air dried, a DNA Bru-996–Alexa 555 probe (5′-CCACTAACCGCGACCGGGATG) was added to hybridize with the bacterial 16S rRNA gene (63) at a concentration of 5 ng/μl with hybridization buffer. For nonspecific hybridization, a Non338-Alexa 555 probe (5′-CGACGGAGGGCATCCTCA) was used under the same condition (64). Hybridization was carried out in a chamber overnight at 46°C for 14 h in hybridization buffer (20 mM Tris, 0.9 M NaCl, 0.1% SDS, and 40% formamide), pH 7.2. Then the slides were washed with washing buffer (20 mM Tris, 0.9 M NaCl), pH 7.2, at 48°C for 20 min, rinsed in distilled water, and allowed to air dry. Antifade mounting medium with DAPI was added (65), and the slides were analyzed with an Olympus DP73 microscope using DAPI and TRITC filters and Fiji software.
Statistical analysis.
All data analyses were performed using GraphPad Prism, version 6.0 (San Diego, CA, USA). A nonparametric two-way analysis of variance (ANOVA) test was used to compare body weights and temperatures of different groups. Tukey’s multiple-comparison test was used to generate P values. A Mantel-Cox test was used using GraphPad Prism, version 6.07 (San Diego, CA, USA), to determine significant differences of the survival curve. P values of <0.05 were considered significant.
ACKNOWLEDGMENTS
This work is supported by a faculty start-up grant from Texas A&M University and grant KO1TW009981 from NIH to A.M.A.-G. A research scholarship to O.H.K. was provided by the College of Veterinary Medicine, University of Baghdad, Iraq. T.A.F. was supported by NIH grant RO1HD084339.
T.A.F.’s spouse, A. Rice-Ficht, is not an investigator in this study but, as a managing partner of NanoRelease Technologies (NRT), LLC Inc., has a 95% equity interest in NRT, a company involved in vaccine delivery platforms. The terms of this arrangement have been reviewed and approved by TXAgriLife Research and Texas A&M University in accordance with their conflict of interest policies.
We thank R. Barhoumi for confocal microscopy, A. Ambrus for immunohistochemistry, and L. W. Stranahan for additional reading.
Supplemental Material
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Information & Contributors
Information
Published In
Infection and Immunity
Volume 87 • Number 6 • June 2019
eLocator: 10.1128/iai.00901-18
Editor: Craig R. Roy, Yale University School of Medicine
Copyright
© 2019 American Society for Microbiology. All Rights Reserved.
History
Received: 19 December 2018
Returned for modification: 12 February 2019
Accepted: 27 March 2019
Published online: 21 May 2019
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Craig R. Roy
Editor
Yale University School of Medicine
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2019.
The NOD-scid IL2rγnull Mouse Model Is Suitable for the Study of Osteoarticular Brucellosis and Vaccine Safety. Infect Immun
87:10.1128/iai.00901-18.
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