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Activation of bovine neutrophils by Brucella spp

https://doi.org/10.1016/j.vetimm.2016.05.011Get rights and content

Abstract

Brucellosis is a globally important zoonotic infectious disease caused by gram negative bacteria of the genus Brucella. While many species of Brucella exist, Brucella melitensis, Brucella abortus, and Brucella suis are the most common pathogens of humans and livestock. The virulence of Brucella is largely influenced by its ability to evade host factors, including phagocytic killing mechanisms, which are critical for the host response to infection. The aim of this study was to characterize the bovine neutrophil response to virulent Brucella spp. Here, we found that virulent strains of smooth B. abortus, B. melitensis, B. suis, and virulent, rough, strains of Brucella canis possess similar abilities to resist killing by resting, or IFN-γ-activated, bovine neutrophils. Bovine neutrophils responded to infection with a time-dependent oxidative burst that varied little between Brucella spp. Inhibition of TAK1, or SYK kinase blunted the oxidative burst of neutrophils in response to Brucella infection. Interestingly, Brucella spp. did not induce robust death of bovine neutrophils. These results indicate that bovine neutrophils respond similarly to virulent Brucella spp. In addition, virulent Brucella spp., including naturally rough strains of B. canis, have a conserved ability to resist killing by bovine neutrophils.

Introduction

Brucellosis is a bacterial zoonosis, caused by intracellular bacteria of the genus Brucella, and remains a major agricultural, global health, and economic problem in many parts of the world (World Health Organization, 2012). Globally, brucellosis is also considered one of 8 neglected zoonotic diseases by the World Health Organization. Brucella spp. are closely genetically related, but infect a wide variety of domestic livestock, including goats, cattle, camels, sheep and pigs (Godfroid et al., 2005, Heller et al., 2012). Seropositive animals have higher rates of abortion, stillbirth, infertility and calf mortality along with lower milk production and longer intervals between calving (McDermott et al., 2013). In addition to domestic animals, Brucella spp. also infect wildlife such as bison, elk, and feral swine in the United States which provide a potential reservoir of re-introduction of brucellosis into domestic livestock (Musser et al., 2013, Rhyan et al., 2013). Beyond its effects on agriculture, brucellosis remains a global human health problem, particularly for the Mediterranean rim, Middle East, Central Asia, Africa, and South America, and there are estimated to be over 500,000 new human cases of brucellosis annually (Pappas et al., 2006). Humans are usually infected via ingestion of unpasteurized milk products or raw meat from infected animals (Zinsstag et al., 2005).

Brucella abortus is the primary agent of brucellosis in cattle globally. However, Brucella melitensis is emerging as an important pathogen of cattle worldwide (Alvarez et al., 2011, Mick et al., 2014, Muendo et al., 2012, Szulowski et al., 2013, Wareth et al., 2014). Brucella suis has also been shown to cause disease in cattle, including a recent outbreak in the U.S. that was presumed to be acquired from a local population of feral swine (Fretin et al., 2013, Musser et al., 2013, Szulowski et al., 2013). Brucella canis isolates naturally possess a rough LPS (Moreno et al., 1984), and B. canis is generally thought to be less virulent than B. melitensis, B. abortus, and B. suis (Moreno and Moriyon, 2006). However, B. canis has been reported to infect humans (Lucero et al., 2010, Lucero et al., 2005) and to be able to replicate and cause a persistent infection in mice (Chacon-Diaz et al., 2015). In addition, there have been reports of cattle testing positive for B. canis in Korea (Baek et al., 2012, Kang et al., 2009). Thus, while B. abortus is the main cause of bovine brucellosis, other Brucella spp. also have the ability to infect cattle and potentially cause disease.

Within mammalian hosts, Brucella spp. can infect a variety of cells, with a particular preference for professional phagocytes (Copin et al., 2012). In sheep and goats, Brucella antigen has been detected in macrophages and neutrophils within mammary glands, sedimented milk, lung, kidney and liver (Ilhan and Yener, 2008, Meador et al., 1988). Macrophages and neutrophils are also thought to transport brucellae from systemic circulation into mammary glands and milk (Meador et al., 1989), and transport Brucella through the lymphatics (Ackermann et al., 1988). In addition, neutrophil recruitment is associated with a variety of Brucella-induced pathologies in both ruminants and in humans (Lacey et al., 2016). Necrotic, neutrophilic, placentitis with perivascular infiltrates is the most common microscopic change in cattle experimentally infected with Brucella (Xavier et al., 2009). In aborted bovine fetuses, pleuropneumonia and pericarditis with neutrophilic infiltrates are most common pathologic finding (Lopez et al., 1984, Xavier et al., 2009).

The data described above indicate that neutrophilic infiltration is associated with Brucella-induced pathology in ruminants and that neutrophils can be a target cell of Brucella infection. Interactions between intracellular bacteria and their in vivo target cells is critical to the outcome of infection. However, little is known about the role of the interplay between neutrophils and Brucella, relative to the extensive studies performed on Brucella-macrophage interactions. It is known that smooth strains of B. abortus are more resistant to bovine neutrophil killing than rough strains (Riley and Robertson, 1984b). While bovine neutrophils readily phagocytose B. abortus, they are relatively ineffective at killing Brucella. This is thought to be due to a suboptimal respiratory burst by bovine neutrophils (Riley and Robertson, 1984a, Riley and Robertson, 1984b). Later studies showed that B. abortus actively suppresses the respiratory burst of neutrophils. This effect may be dependent on LPS, as the generation of H2O2 and nitric oxide is decreased when neutrophils are exposed to the LPS of Brucella (Iyankan and Singh, 2002).

It has been recently reported that B. abortus rapidly induces death of human neutrophils (Barquero-Calvo et al., 2015), while others have shown that Brucella components, including heat-killed B. abortus, prolonged the lifespan of human neutrophils (Zwerdling et al., 2009). However, it is unknown if Brucella infection causes death of bovine neutrophils. In addition, human neutrophils have been shown to be better at killing B. abortus, than B. melitensis (Young et al., 1985), indicating that the neutrophil response to infection may be Brucella-species specific. As several Brucella spp. can cause infect cattle, here we sought to investigate the Brucella species-specific response of bovine neutrophils to infection.

Section snippets

Bacterial strains and growth conditions

All experiments with live Brucella were performed in biosafety level-3 facilities at the University of Missouri. B. abortus 2308 and B. melitensis 16 M, were obtained from Montana State University (Bozeman, MT). B. suis 1330 was obtained from BEI Resources (Manassas, VA), and clinical isolates of B. canis from diagnostic samples at the Veterinary Medical Diagnostic Laboratory at the University of Missouri. All Brucella strains were grown from freezer stocks on Brucella agar plates at 37 °C/5% CO2

Virulent strains of B. abortus, B. melitensis, B. suis, and B. canis have a similar ability to resist killing by bovine neutrophils

Others have previously reported that human neutrophils are better able to kill B. abortus than B. melitensis (Young et al., 1985), indicating that the neutrophil response to infection may be Brucella-species specific. Here we investigated the ability of bovine neutrophils to kill virulent Brucella. To do this, we infected bovine neutrophils with virulent smooth B. abortus 2308, B. melitensis 16 M, and B. suis 1330 along with an isolate of naturally rough B. canis at an MOI of 100. At one and two

Conclusions

In summary, we found that both smooth and rough species of virulent Brucella have a similar ability to resist killing by both resting and activated bovine neutrophils. Virulent Brucella spp. also induced minimal cell death of, and a similar inflammatory response by, bovine neutrophils. The activation of bovine neutrophils was also dependent on SYK and TAK1, suggesting C-type lectin receptors might be involved in the neutrophil response to Brucella. Future studies will determine what receptors

Funding

This work supported by the USDA National Institute of Food and Agriculture, Animal Health Projects 100588 and 1008622, and funds from the University of Missouri, College of Veterinary Medicine

Acknowledgements

We would like to thank Dr. Thomas Reilly (University of Missouri Veterinary Medical Diagnostic Laboratory) for the B. canis strains used in this study.

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