23 research outputs found
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: QIAampâą DNA Mini Kit (QIAGEN), peqGoldâą Tissue DNA Mini Kit (PeqLab), UltraCleanâą Tissue and Cells DNA Isolation Kit (MoBio), DNA Isolation Kit for Cells and Tissues (Roche), and NucleoSpinâą 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
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
Antimicrobial resistance of Campylobacter isolates from small scale and backyard chicken in Kenya
Background Thermophilic Campylobacter species are a major cause of bacterial
foodborne diarrhoea in humans worldwide. Poultry and their products are the
predominant source for human campylobacteriosis. Resistance of Campylobacter
to antibiotics is increasing worldwide, but little is known about the
antibiotic resistance in Campylobacter isolated from chicken in Kenya. In this
study, 35 suspected Campylobacter strains isolated from faeces and cloacal
swabs of chicken were tested for their susceptibility to seven antibiotics
using a broth microdilution assay and molecular biological investigations.
Results Overall, DNA of thermophilic Campylobacter was identified in 53
samples by PCR (34 C. jejuni, 18 C. coli and one mix of both species) but only
35 Campylobacter isolates (31 C. jejuni and 4 C. coli) could be re-cultivated
after transportation to Germany. Isolates were tested for their susceptibility
to antibiotics using a broth microdilution assay. Additionally, molecular
biological detection of antibiotic resistance genes was carried out. C. jejuni
isolates showed a high rate of resistance to nalidixic acid, tetracycline and
ciprofloxacin of 77.4, 71.0 and 71.0 %, respectively. Low resistance (25.8 %)
was detected for gentamicin and chloramphenicol. Multidrug resistance in C.
jejuni could be detected in 19 (61.3 %) isolates. Resistance pattern of C.
coli isolates was comparable. Resistance to ciprofloxacin was confirmed by
MAMAâPCR and PCRâRFLP in all phenotypically resistant isolates. The tet(O)
gene was detected only in 54.5 % of tetracycline resistant C. jejuni isolates.
The tet(A) gene, which is also responsible for tetracycline resistance, was
found in 90.3 % of C. jejuni and in all C. coli isolates. Thirteen
phenotypically erythromycin-resistant isolates could not be characterised by
using PCRâRFLP and MAMAâPCR. Conclusions To the best of our knowledge, this
study is the first report about resistance to antibiotics in thermophilic
Campylobacter originating from chicken in Kenya. Campylobacter spp. show a
high level of resistance to ciprofloxacin, nalidixic acid and tetracycline but
also a remarkable one to chloramphenicol and gentamicin and they are multidrug
resistant. Resistance to antibiotics is a global public health concern. In
Kenya, resistance surveillance needs further attention in the future. Efforts
to establish at least a National Laboratory with facilities for performing
phenotypic and genotypic characterization of thermophilic Campylobacter is
highly recommended
Prevalence of Coxiella burnetii in clinically healthy German sheep flocks
<p>Abstract</p> <p>Background</p> <p>Current epidemiological data on the situation of <it>Coxiella (C.) burnetii </it>infections in sheep are missing, making risk assessment and the implementation of counteractive measures difficult. Using the German state of Thuringia as a model example, the estimated sero-, and antigen prevalence of <it>C. burnetii </it>(10% and 25%, respectively) was assessed at flock level in 39/252 randomly selected clinically healthy sheep flocks with more than 100 ewes and unknown abortion rate.</p> <p>Results</p> <p>The CHECKITâą Q-fever Test Kit identified 11 (28%) antibody positive herds, whereas real-time PCR revealed the presence of <it>C. burnetii </it>DNA in 2 (5%) of the flocks. Multiple-locus variable number of tandem repeats analysis of 9 isolates obtained from one flock revealed identical profiles. All isolates contained the plasmid QpH1.</p> <p>Conclusions</p> <p>The results demonstrate that <it>C. burnetii </it>is present in clinically inconspicuous sheep flocks and sporadic flare-ups do occur as the notifications to the German animal disease reporting system show. Although <it>C. burnetii </it>infections are not a primary veterinary concern due to the lack of significant clinical impact on animal health (with the exception of goats), the eminent zoonotic risk for humans should not be underestimated. Therefore, strategies combining the interests of public and veterinary public health should include monitoring of flocks, the identification and culling of shedders as well as the administration of protective vaccines.</p
Genotyping and antibiotic resistance of thermophilic Campylobacter isolated from chicken and pig meat in Vietnam
Background Campylobacter species are recognized as the most common cause of
foodborne bacterial gastroenteritis in humans. In this study nine
Campylobacter strains isolated from chicken meat and pork in Hanoi, Vietnam,
were characterized using molecular methods and tested for antibiotic
resistance. Results The nine isolates (eight C. jejuni and one C. coli) were
identified by multiplex PCR, and tested for the presence or absence of 29 gene
loci associated with virulence, lipooligosaccharide (LOS) biosynthesis and
further functions. flaA typing, multilocus sequence typing and microarray
assay investigation showed a high degree of genetic diversity among these
isolates. In all isolates motility genes (flaA, flaB, flhA, fliM),
colonization associated genes (cadF, docB), toxin production genes (cdtA,
cdtB, secD, secF), and the LOS biosynthesis gene pglB were detected. Eight
gene loci (fliY, virB11, Cje1278, Cj1434c, Cj1138, Cj1438c, Cj1440c, Cj1136)
could not be detected by PCR. A differing presence of the gene loci ciaB (22.2
%), Cje1280 (77.8 %), docC (66.7 %), and cgtB (55.6 %) was found. iamA, cdtC,
and the type 6 secretion system were present in all C. jejuni isolates but not
in C. coli. flaA typing resulted in five different genotypes within C. jejuni,
MLST classified the isolates into seven sequence types (ST-5155, ST-6736,
ST-2837, ST-4395, ST-5799, ST-4099 and ST-860). The microarray assay analysis
showed a high genetic diversity within Vietnamese Campylobacter isolates which
resulted in eight different types for C. jejuni. Antibiotic susceptibility
profiles showed that all isolates were sensitive to gentamicin and most
isolates (88.8 %) were sensitive to chloramphenicol, erythromycin and
streptomycin. Resistance rates to nalidixic acid, tetracycline and
ciprofloxacin were 88.9, 77.8 and 66.7 %, respectively. Conclusions To the
best of our knowledge, this study is the first report that shows high genetic
diversity and remarkable antibiotic resistance of Campylobacter strains
isolated from meat in Vietnam which can be considered of high public health
significance. These preliminary data show that large scale screenings are
justified to assess the relevance of Campylobacter infections on human health
in Vietnam
Harmonizing methods for wildlife abundance estimation and pathogen detection in Europe-a questionnaire survey on three selected host-pathogen combinations
__Background:__ The need for wildlife health surveillance as part of disease control in wildlife, domestic animals and humans on the global level is widely recognized. However, the objectives, methods and intensity of existing wildlife health surveillance programs vary greatly among European countries, resulting in a patchwork of data that are difficult to merge and compare. This survey aimed at evaluating the need and potential for data harmonization in wildlife health in Europe. The specific objective was to collect information on methods currently used to estimate host abundance and pathogen prevalence. Questionnaires were designed t