Brucella abortus and Pregnancy in Mice: Impact of Chronic Infection on Fertility and the Role of Regulatory T Cells in Tissue Colonization.

Stealthy intracellular bacterial pathogens are known to establish persistent and sometimes lifelong infections. Some of these pathogens also have a tropism for the reproductive system, thereby increasing the risk of reproductive disease and infertility. To date, the pathogenic mechanism involved remains poorly understood. Here, we demonstrate that Brucella abortus, a notorious reproductive pathogen, has the ability to infect the nonpregnant uterus, sustain infection, and induce inflammatory changes during both acute and chronic stages of infection. In addition, we demonstrated that chronically infected mice had a significantly reduced number of pregnancies compared to naive controls. To investigate the immunologic mechanism responsible for uterine tropism, we explored the role of regulatory T cells (Tregs) in the pathogenesis of Brucella abortus infection. We show that highly suppressive CD4+FOXP3+TNFR2+ Tregs contribute to the persistence of Brucella abortus infection and that inactivation of Tregs with tumor necrosis factor receptor II (TNFR2) antagonistic antibody protected mice by significantly reducing bacterial burden both systemically and within reproductive tissues. These findings support a critical role of Tregs in the pathogenesis of persistence induced by intracellular bacterial pathogens, including B. abortus Results from this study indicate that adverse reproductive outcomes can occur as sequelae of chronic infection in nonpregnant animals and that fine-tuning Treg activity may provide novel immunotherapeutic and prevention strategies against intracellular bacterial infections such as brucellosis.

Brucellosis in the Middle East: Current situation and a pathway forward.

Brucellosis is a bacterial endemic zoonotic disease of global significance with detrimental impacts on public health and food animal production. It is caused by Brucella spp., an expanding group of pathogens able to infect various host species. Bovines and small ruminants, which excrete the bacteria in milk and in reproductive discharges, are major sources of infection for humans and other animals. Contact with contaminated animals and consumption of unpasteurized dairy products are the main routes for human infection. In spite of the considerable progress of knowledge gained and success achieved in brucellosis control in the developed world, this disease continues to be an important burden in the Middle East (ME). Common risk factors implicated in the difficulty and complexity of brucellosis control within the region include (1) social and political instabilities; (2) insufficient resources and infrastructure for appropriate diagnosis, reporting, and implementation of control measures; (3) variation of livestock husbandry systems and their commingling with other livestock and wildlife; and (4) traditional cultural practices, including consumption of unpasteurized dairy products. Development of core interdisciplinary competencies is required for a true One Health-based endeavor against the disease. National awareness and educational programs addressing all population sectors from consumers to decision-makers seem to be the next logical, sustainable, and economically viable approach toward improving disease status in this region. In the present review, we describe the current situation of brucellosis in the ME, focusing on the major limitations and shortcomings regarding disease control. We propose a regional approach toward public awareness of brucellosis as the first step in mitigating the disease and discuss the potential benefits, and components of such a strategy, which can further be used as a model for other endemic zoonotic diseases.

Evaluation of the Efficacy of the Brucella canis RM6/66 ΔvjbR Vaccine Candidate for Protection against B. canis Infection in Mice.

Brucella canis is a Gram-negative, facultative intracellular bacterium and the causative agent of canine brucellosis, a highly contagious disease of dogs that can be transmitted to humans. Unfortunately, no vaccine is available to prevent infection. We recently characterized the kinetics of B. canis infection in the mouse model, establishing the required dose necessary to achieve systemic infection. The objective of this study was to investigate the utility of the mouse model in assessing canine brucellosis vaccine candidates and to subsequently investigate the safety and efficacy of a live attenuated vaccine, the B. canis RM6/66 ΔvjbR strain. Mice vaccinated with a dose of 109 CFU of the vaccine strain by both intraperitoneal and subcutaneous routes were afforded significant protection against organ colonization and development of histopathologic lesions following intraperitoneal challenge. Addition of an adjuvant or a booster dose 2 weeks following initial vaccination did not alter protection levels. Vaccination also resulted in a robust humoral immune response in mice, and B. canis RM6/66 ΔvjbR was capable of activating canine dendritic cells in vitro These data demonstrate that the B. canis RM6/66 ΔvjbR strain shows promise as a vaccine for canine brucellosis and validates the mouse model for future vaccine efficacy studies.IMPORTANCE Canine brucellosis, caused by Brucella canis, is the primary cause of reproductive failure in dogs and represents a public health concern due to its zoonotic nature. Cases in dogs in the United States have been increasing due to the persistent nature of the bacterium, deficiencies in current diagnostic testing, and, most importantly, the lack of a protective vaccine. Current estimates place the seroprevalence of B. canis in the southern United States at 7% to 8%, but with the unprecedented rates of animals moving across state and international borders and the lack of federal regulations in regard to testing, the true seroprevalence of B. canis in the United States may very well be higher. Vaccination represents the most effective method of brucellosis control and, in response to the demand for a vaccine against B. canis, we have developed the live attenuated B. canis RM6/66 ΔvjbR vaccine strain capable of protecting mice against challenge.

Interaction of Brucella abortus with Osteoclasts: a Step toward Understanding Osteoarticular Brucellosis and Vaccine Safety.

Osteoarticular disease is a frequent complication of human brucellosis. Vaccination remains a critical component of brucellosis control, but there are currently no vaccines for use in humans, and no in vitro models for assessing the safety of candidate vaccines in reference to the development of bone lesions currently exist. While the effect of Brucella infection on osteoblasts has been extensively evaluated, little is known about the consequences of osteoclast infection. Murine bone marrow-derived macrophages were derived into mature osteoclasts and infected with B. abortus 2308, the vaccine strain S19, and attenuated mutants S19vjbR and B. abortusΔvirB2 While B. abortus 2308 and S19 replicated inside mature osteoclasts, the attenuated mutants were progressively killed, behavior that mimics infection kinetics in macrophages. Interestingly, B. abortus 2308 impaired the growth of osteoclasts without reducing resorptive activity, while osteoclasts infected with B. abortus S19 and S19vjbR were significantly larger and exhibited enhanced resorption. None of the Brucella strains induced apoptosis or stimulated nitric oxide or lactose dehydrogenase production in mature osteoclasts. Finally, infection of macrophages or osteoclast precursors with B. abortus 2308 resulted in generation of smaller osteoclasts with decreased resorptive activity. Overall, Brucella exhibits similar growth characteristics in mature osteoclasts compared to the primary target cell, the macrophage, but is able to impair the maturation and alter the resorptive capacity of these cells. These results suggest that osteoclasts play an important role in osteoarticular brucellosis and could serve as a useful in vitro model for both analyzing host-pathogen interactions and assessing vaccine safety.

Vaccine safety studies of Brucella abortus S19 and S19ΔvjbR in pregnant swine.

Brucellosis in swine is caused by Brucella suis, a bacterial infection of nearly worldwide distribution. Brucella suis is also transmissible to humans, dogs and cattle and is considered a reemerging disease of public health concern. To date, there is no effective vaccine for swine. This prompted us to investigate the potential use of the commercially available vaccine for cattle or the live attenuated vaccine candidate S19ΔvjbR. As the first step, we sought to study the safety of the vaccine candidates when administered in pregnant sows, since one of the major drawbacks associated with vaccination using Live Attenuated Vaccines (LAV) is the induction of abortions when administered in pregnant animals. Fifteen pregnant gilts at mid-gestation were divided into four groups and subsequently vaccinated subcutaneously using different formulations containing 2.0 ±â€¯0.508 × 109 CFU of either S19 or S19ΔvjbR. Vaccination in pregnant animals with the vaccine candidates did not induce abortion, stillbirths or a reduction in litter size. Multiple tissues in the gilts and piglets were examined at the time of delivery to assess bacterial colonization and histopathological changes. There was no evidence of vaccine persistence in the gilts or bacterial colonization in the fetuses. Altogether, these data suggest that both vaccine candidates are safe for use in pregnant swine. Analysis of the humoral responses, specifically anti-Brucella IgG levels measured in serum, demonstrated a robust response induced by either vaccine, but of shorter duration (4-6 weeks post-inoculation) compared to that observed in cattle or experimentally infected mice. Such a transient humoral response may prove to be beneficial in cases where the vaccine is used in eradication campaigns and in the differentiation of vaccinated from infected animals. This study provides evidence to support future efficacy studies of both vaccine candidates in swine.

Characterization of Brucella canis infection in mice.

Canine brucellosis, caused by Brucella canis, is a disease of dogs and represents a public health concern as it can be transmitted to humans. Canine brucellosis is on the rise in the United States and there is currently no vaccine for use in dogs. Mice have been extensively utilized to investigate host-pathogen interactions and vaccine candidates for smooth Brucella species and could serve a similar role for studying B. canis. However, comparatively little is known about B. canis infection in mice. The objective of this study was to characterize the kinetics of colonization and pathogenicity of B. canis in mice in order to evaluate the mouse as a model for studying this pathogen. C57BL/6 mice were inoculated intraperitoneally with 105, 107, or 109 CFU of Brucella canis RM6/66 and euthanized 1-, 2-, 4-, 6-, 9-, and 12-weeks post-inoculation. B. canis induced splenomegaly in mice infected with 109 CFU at 1- and 2 weeks post-inoculation while no gross lesions were observed in other dose groups. Infection at the two higher doses resulted in dose-dependent granulomatous hepatitis and histiocytic infiltration of the spleen and mesenteric lymph nodes by 1-2 weeks. B. canis was cultured from the liver, spleen, uterus, bone marrow, lung, and kidney in all groups with colonization declining at a slow but steady rate throughout the experiment. Clearance was achieved by 9 weeks 105 CFU group and by 12 weeks in the 107 CFU group, while B. canis persisted in the spleen until 12 weeks in the highest dose group. Although B. canis does not demonstrate significant replication in C57BL/6 mice, it has the ability to establish an infection, induce splenomegaly, and persist for several weeks in multiple organs. Moreover, 1 x 107 CFU appears to be a suitable challenge dose for investigating vaccine safety.

The NOD-scid IL2rγnull Mouse Model Is Suitable for the Study of Osteoarticular Brucellosis and Vaccine Safety.

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.