Abstract
Intracellular bacteria provoking zoonoses, such as those of the genus Brucella, present a host cell tropism mostly limited to the monocyte/macrophage lineage, leading to chronic inflammatory reactions, difficult-to-eradicate-infections, and widespread prevalence among ruminants. Eradication of brucellosis has been based on programs that translate into a substantial financial burden for both the authorities and stockbreeders, if not strictly followed. To this end, we sought to create an in vitro cell model that could be utilized as future reference for adequately measuring the number of engulfed brucellae/cell, using peripheral blood-derived sheep macrophages infected with B. melitensis at decimal multiplicities of infection (MOI = 5000-5), to simulate the host cell/microorganism interaction and monitor bacterial loads up to 6 days post-infection. We show that the MOI = 5000 leads to high numbers of engulfed bacteria without affecting macrophages’ viability and that the minimum detection limit of our Real-Time PCR assay was 3.97 ± 5.58 brucellae/cell. Moreover, we observed a time-associated, significant gradual reduction in bacterial loads from Day 2 to Day 6 post-infection (p = 0.0013), as part of the natural bactericidal properties of macrophages. Overall, the work presented here constitutes a reliable in vitro cell model of Brucella melitensis for research purposes that can be utilized to adequately measure the number of engulfed brucellae/cell and provides insights towards future utilization of molecular biology-based methods for detection of Brucella.
Highlights
Intracellular infectious agents, such as bacteria of the genus Brucella, provoke abortions and infertility in ruminant animals [1] and present a host cell tropism mostly limited to the monocyte/macrophage lineage [2]
In an effort to understand the potential reasons that hamper the wider use of Real-Time PCR to detect Brucella infection, we aimed to generate an ovine macrophage infection model, using decimally diluted B. melitensis, to imitate the host cell/microorganism interaction in vitro and monitor the number of incorporated brucellae/cell by simultaneously comparing the classic colony-forming units (CFU) detection method with a molecular-based Real-Time PCR detection method
All results are expressed as means ± standard deviation (SD)
Summary
Intracellular infectious agents, such as bacteria of the genus Brucella, provoke abortions and infertility in ruminant animals [1] and present a host cell tropism mostly limited to the monocyte/macrophage lineage [2]. Brucellosis is difficult to eradicate, since the microorganism may effectively evade antibiotic treatment. Vaccination programs in livestock, monitoring, and slaughtering of seropositive carriers constitute the only available means for eradicating the disease [3]. The available animal vaccine [4] is sometimes ineffective [5] and even infectious for veterinarians [5]. The disease cannot be prevented in humans, since no vaccine currently exists and antibiotic therapy is long-lasting, potentially leading to relapse or re-infection after de novo exposure to the microorganism [6]. Brucella infection in livestock poses severe public health issues, and translates into a substantial financial burden for stockbreeders [7]
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