Role of Brucella Toll/Interleukin-1 Receptor (TIR) Domain Containing Protein (∆TcpB) Deletion Mutant in Protective Immunity against Brucellosis

Brucellosis is an important zoonotic disease affecting about 500,000 people annually. The development of safer and more efficacious Brucella Live attenuated vaccines addresses safety concerns that include the identification of reproducible and reliable surrogates of protection and mechanisms to bolster longevity. Brucella encodes a toll interleukin receptor domain containing protein (TcpB/Btp-1). These proteins subvert host innate immunity by abrogating NF-κB mediated cytokine production, by binding to and/or causing the degradation of signaling molecules TIRAP (MAL) and MyD88. TcpB has also been shown to directly reduce the CTL killing activity of infected host cells. In the current study, we investigated the effect of deleting tcpB from Brucella on invitro and invivo immune responses. We also evaluated an in vitro murine Brucella growth inhibition co-culture assay to determine the capacity of immune splenocytes from mice exposed to the tcpB mutant or wild type to control the growth of Brucella melitensis in murine bone marrow derived macrophages. A tcpB knockout constructed by gene replacement in the Brucella abortus S19 genetic background was used to vaccinate C57BL/6 mice assessed for development of CD4+ memory T cells. Mice vaccinated with the mutant displayed an elevated Th1 response, compared to the parental S19 and non- vaccinated controls, as manifested by multiple factors. These include; elevated IFN-γ early post vaccination, and a significant elevation of memory CD4+CD44+CD62L+ within CD4+T cell population in splenocytes derived from mice vaccinated with the mutant. S19 and S19ΔtcpB strains induced a significant increase in the IgG2a levels post vaccination. Consistent with a shift to a Th1 response, S19ΔtcpB induced a higher response later in vaccination. Splenocytes obtained from mice vaccinated with the S19ΔtcpB mutant exhibited significantly higher levels of killing activity compared to cells derived from S19 vaccinated mice and PBS controls. Consistent with enhanced immune protection, fewer bacteria were recovered from the spleens of mice vaccinated with the S19ΔtcpB mutant, which had reduced inflammatory lesions consistent with reduced bacterial burden. These results provide strong evidence that tcpB deletion improves immunogenicity, longevity and protective efficacy of S19 and that ex vivo co- cultivation may be employed to predict potential vaccine efficacy

The Case for Live Attenuated Vaccines against the Neglected Zoonotic Diseases Brucellosis and Bovine Tuberculosis

Vaccination of humans and animals with live attenuated organisms has proven to be an effective means of combatting some important infectious diseases. In fact, the 20th century witnessed tremendous improvements in human and animal health worldwide as a consequence of large-scale vaccination programs with live attenuated vaccines (LAVs). Here, we use the neglected zoonotic diseases brucellosis and bovine tuberculosis (BTb) caused by Brucella spp. and Mycobacterium bovis (M. bovis), respectively, as comparative models to outline the merits of LAV platforms with emphasis on molecular strategies that have been pursued to generate LAVs with enhanced vaccine safety and efficacy profiles. Finally, we discuss the prospects of LAV platforms in the fight against brucellosis and BTb and outline new avenues for future research towards developing effective vaccines using LAV platforms

Efficient detection of symptomatic and asymptomatic patient samples for Babesia microti and Borrelia burgdorferi infection by multiplex qPCR.

BACKGROUND:Tick-borne infections have been increasing steadily over the years, with co-infections with Borrelia burgdorferi and Babesia microti/divergens emerging as a serious health problem. B. burgdorferi is a spirochetal bacterium that causes Lyme disease while protozoan pathogens belonging to Babesia species are responsible for babesiosis. Currently used serological tests do not always detect acute Lyme disease or babesiosis, and fail to differentiate cured patients from those who get re-infected. This is a major problem for proper diagnosis particularly in regions endemic for tick-borne diseases. Microscopy based evaluation of babesiosis is confirmatory but is labor intensive and insensitive such that many asymptomatic patients remain undetected and donate blood resulting in transfusion transmitted babesiosis. RESULTS:We conducted multiplex qPCR for simultaneous diagnosis of active Lyme disease and babesiosis in 192 blood samples collected from a region endemic for both diseases. We document qPCR results obtained from testing of each sample three times to detect infection with each pathogen separately or together. Results for Lyme disease by qPCR were also compared with serological tests currently used for Lyme disease when available. Considering at least two out of three test results for consistency, 18.2% of patients tested positive for Lyme disease, 18.7% for co-infection with B. burgdorferi and B. microti and 6.3% showed only babesiosis. CONCLUSIONS:With an 80% sensitivity for detection of Lyme disease, and ability to detect co-infection with B. microti, multiplex qPCR can be employed for diagnosis of these diseases to start appropriate treatment in a timely manner

Diagnosis of <i>B</i>. <i>burgdorferi</i> infection by C6 Lyme ELISA.

<p>Histogram showing the Lyme Index distribution produced by C6 Lyme ELISA conducted on all 192 patient samples. An index ≤ 0.90 is a negative Lyme diagnosis (118 samples), an index of 0.91–1.09 is an equivocal result (7 samples), and an index ≥ 1.10 is a positive Lyme diagnosis (67 samples).</p

Diagnosis of Lyme disease by qPCR and two-tier serology.

<p>Diagnosis of Lyme disease by qPCR and two-tier serology.</p

Venn diagram summarizing the agreement in positive diagnoses of Lyme disease using different diagnostic methods.

<p><i>B</i>. <i>burgdorferi</i> infection can be detected by qPCR at a higher rate than C6 Lyme ELISA and 2-tier serological tests. There is significant overlap among all three tests.</p

Diagnosis o Lyme disease by qPCR and serological tests.

<p>Diagnosis o Lyme disease by qPCR and serological tests.</p

Additional file 1: of A novel quantitative PCR detects Babesia infection in patients not identified by currently available non-nucleic acid amplification tests

A novel quantitative PCR detects Babesia infection in patients not identified by currently available non-nucleic acid amplification tests. Table S1. Analyses of patient blood samples tested by qPCR, FISH, IFA and microscopic examination of Giemsa-stained blood smears. (DOCX 17 kb

Live attenuated vaccines.

<p>Live attenuated vaccines.</p

Approaches to LAV generation.

<p>Approaches to LAV generation.</p