Comparison of commercial DNA preparation kits for the detection of Brucellae in tissue using quantitative real-time PCR

<p>Abstract</p> <p>Background</p> <p>The detection of Brucellae in tissue specimens using PCR assays is difficult because the amount of bacteria is usually low. Therefore, optimised DNA extraction methods are critical. The aim of this study was to assess the performance of commercial kits for the extraction of <it>Brucella </it>DNA.</p> <p>Methods</p> <p>Five kits were evaluated using clinical specimens: QIAamp™ DNA Mini Kit (QIAGEN), peqGold™ Tissue DNA Mini Kit (PeqLab), UltraClean™ Tissue and Cells DNA Isolation Kit (MoBio), DNA Isolation Kit for Cells and Tissues (Roche), and NucleoSpin™ Tissue (Macherey-Nagel). DNA yield was determined using a quantitative real-time PCR assay targeting IS<it>711 </it>that included an internal amplification control.</p> <p>Results</p> <p>Kits of QIAGEN and Roche provided the highest amount of DNA, Macherey-Nagel and Peqlab products were intermediate whereas MoBio yielded the lowest amount of DNA. Differences were significant (p < 0.05) and of diagnostic relevance. Sample volume, elution volume, and processing time were also compared.</p> <p>Conclusions</p> <p>We observed differences in DNA yield as high as two orders of magnitude for some samples between the best and the worst DNA extraction kits and inhibition was observed occasionally. This indicates that DNA purification may be more relevant than expected when the amount of DNA in tissue is very low.</p

Взаємодія системи "політика-релігія"

Досліджено феномен суспільних явищ політики і релігії у перерізі їх взаємодії, вивчено історичний досвід такого взаємного впливу. Окреме місце відведено аналізу практичного застосування закону України “Про свободу совісті та релігійні організації”.The article explores the phenomenon of social phenomena politics and religion in the context of their interaction, exploring the historical experience of such mutual influence. A separate analysis is given to the practical application of the Law of Ukraine “On Freedom of Conscience and Religious Organizations”

Comparison of diagnostic tests for the detection of Brucella spp. in camel sera

<p>Abstract</p> <p>Background</p> <p>Brucellosis in livestock causes enormous losses for economies of developing countries and poses a severe health risk to consumers of dairy products. Little information is known especially on camel brucellosis and its impact on human health. For surveillance and control of the disease, sensitive and reliable detection methods are needed. Although serological tests are the mainstay of diagnosis in camel brucellosis, these tests have been directly transposed from cattle without adequate validation. To date, little information on application of real-time PCR for detection of <it>Brucella </it>in camel serum is available. Therefore, this study was performed to compare the diagnostic efficiency of different serological tests and real-time PCR in order to identify the most sensitive, rapid and simple combination of tests for detecting <it>Brucella </it>infection in camels.</p> <p>Findings</p> <p>A total of 895 serum samples collected from apparently healthy Sudanese camels was investigated. Sudan is a well documented endemic region for brucellosis with cases in humans, ruminants, and camels. Rose Bengal Test (RBT), Complement Fixation Test (CFT), Slow Agglutination Test (SAT), Competitive Enzyme Linked Immunosorbant Assay (cELISA) and Fluorescence Polarization Assay (FPA) as well as real-time PCR were used. Our findings revealed that <it>bcsp31 </it>kDa real-time PCR detected <it>Brucella </it>DNA in 84.8% (759/895) of the examined samples, of which 15.5% (118/759) were serologically negative. Our results show no relevant difference in sensitivity between the different serological tests. FPA detected the highest number of positive cases (79.3%) followed by CFT (71.4%), RBT (70.7%), SAT (70.6%) and cELISA (68.8%). A combination of real-time PCR with one of the used serological tests identified brucellosis in more than 99% of the infected animals. 59.7% of the examined samples were positive in all serological tests and real-time PCR. A subpopulation of 6.8% of animals was positive in all serological tests but negative in real-time PCR assays. The high percentage of positive cases in this study does not necessarily reflect the seroprevalence of the disease in the country but might be caused by the fact that the camels were imported from brucellosis infected herds of Sudan, accidentally. Seroprevalence of brucellosis in camels should be examined in confirmatory studies to evaluate the importance of brucellosis in this animal species.</p> <p>Conclusion</p> <p>We suggest combining <it>bcsp31 </it>real-time PCR with either FPA, CFT, RBT or SAT to screen camels for brucellosis.</p

Antibodies against MERS coronavirus in dromedaries, United Arab Emirates, 2003 and 2013

Middle East respiratory syndrome coronavirus (MERSCoV) has caused an ongoing outbreak of severe acute respiratory tract infection in humans in the Arabian Peninsula since 2012. Dromedaries have been implicated as possible viral reservoirs. We used serologic assays to analyze 651 dromedary serum samples from the United Arab Emirates; 151 of 651 samples were obtained in 2003, well before onset of the current epidemic, and 500 serum samples were obtained in 2013. Recombinant spike protein-specific immunofluorescence and virus neutralization tests enabled clear discrimination between MERS-CoV and bovine CoV infections. Most (632/651, 97.1%) dromedaries had antibodies against MERS-CoV. This result included all 151 serum sampl

Camel antibodies for therapeutic and research applications

All camelids and dromedaries in particular have unique antibodies circulating in their blood. Unlike antibodies from all other species these special antibodies are devoid of light chains, and are composed of a heavy chain dimmer only. An immune response is raised in these so-called Heavy-Chain Antibodies (HCAb) following a classical immunisation protocol. These HCAb are easily purified from serum, and were shown to interact with parts of the antigen that are less antigenic to conventional antibodies. Therefore a new class of antibodies is obtained that binds to epitopes that are difficult to target with human or mouse antibodies. Since the antigen binding site of the dromedary HCAb is comprised within one single domain, referred to as VHH or Nanobody (Nb, because of its size in the nm range), we designed a strategy to clone the Nb repertoire of an immunised dromedary and to select the Nbs with specificity for our target antigens. These monoclonal Nbs are well produced in bacteria, are very stable and highly soluble, and they bind the antigen with high affinity and specificity. Currently, we successfully developed such recombinant Nbs as probe in biosensors or to diagnose infections. In addition, the strict monomeric behaviour of Nbs make them ideal for linkage to other molecules exerting special effector function, to generate pluripotent and multidomain man-made drugs to treat diseases like cancer or trypanosomiasis

Glanders & Melioidosis: A Zoonosis and a Sapronosis—“Same Same, but Different”

Glanders, caused by infection with Burkholderia mallei, primarily causes infection in equines, but may be transmitted to humans, and thus qualifies as a true zoonosis. Melioidosis Melioidosis is caused by B. pseudomallei, genetically very similar to B. mallei, but which is an environmental saprophyte capable of infecting humans and a wide range of other animals. Good evidence of animal-to-human, or even human-to-human, transmission of melioidosis is lacking, and so it most appropriately referred to as a sapronosis, or at most a sapro-zoonosis. Although rare in Western countries, both micro-organisms have recently gained much interest because of their potential use as bioterrorism agents Bioterrorism. The increasing recognition of melioidosis in humans and recent outbreaks of glanders Glanders in animals have led to their description as emerging or re-emerging diseases. Laboratory-associated infections with both organisms have also occurred, resulting in their categorisation as Hazard Group 3 pathogens. In this chapter we review the epidemiology of animal and human cases of glanders and melioidosis, the evidence for different modes of transmission, pathogenesis and clinical features, diagnosis and treatment, as well as public health and disease control issues